1-200review of Microbiology and Immunology (PDFDrive) - 1-200

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Reviewof
MicrobiologyandImmunology
Sixth Edition
ApurbaSankarSastryMD(JIPMER)DNBMNAMS PDCR
Hospital Infection Control
OfficerOfficerin-
charge,HICCAntimicrobialStewar
dshipLeadAssistantProfessor
DepartmentofMicrobiology
JawaharlalInstituteofPostgraduateMedicalEducation&Research(JIPMER)Pu
ducherry,India
Sandhya BhatKMDDNBMNAMS PDCR

ViceDean(Research)
AssociateProfessor
DepartmentofClinicalMicrobiologyPondicherryI
nstituteofMedicalSciences(PIMS)Puducherry,In
dia
TheHealthSciencesPublisher
NewDelhi|London|Panama
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ReviewofMicrobiologyandImmunology
FirstEdition:2011
SecondEdition:2012
ThirdEdition:2014
FourthEdition:2015
FifthEdition:2016
SixthEdition:DigitalVersion2018
ISBN:978-93-86322-39-5
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Dedicatedto
Our Beloved Parentsand
Family Members
Andaboveall,LordGaneshawhogaveustheknowledgeandinspiration
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PrefacetotheSixthEdition
IMPORTANCEOFMICROBIOLOGYFORPGENTRANCES
Microbiologyisoneofthehigh-scoringsectionsand,hence,isthekeysubjectforPGentrances.
• Thoughitmightlooktoughbeforeyoustart,butasyougothroughitgradually,youwillsurelyrealizethatrepeatedrevisionscanta
keyoutothatlevelatwhichyoucananswerallthepossibleMCQsinanyentrance.
• The beauty of this subject is if you are thorough in Microbiology, you can solve many infection-related MCQs of
Medicine,PSM,Pediatrics,etc.
MICROBIOLOGY MICROBIOLOGY INFECTION
ENTRANCE MCQs INFECTION MCQs ENTRANCE MCQs MCQs
AIIMSNov2016 21/200 39/200 JIPMERNov2016 25/250 44/250
AIIMSMay2016 29/200 43/200 JIPMERMay2016 21/250 37/250
AIIMSNov2015 28/200 41/200 JIPMERNov2015 21/250 31/200
AIIMSMay2015 16/200 31/200 JIPMERMay2015 14/250 27/200
PGINov2016 19/250 31/250 JIPMERNov2014 28/200 42/250
PGIMay2016 21/250 36/250 APPG2015 21/200 35/200
PGINov2015 15/250 25/250 TNPG2015 25/250 39/250
PGIMay2015 18/250 32/250 MHPG2015 38/300 52/300
ALLINDIA2016 20/300 DNB 2016 18/300
(According to Syllabus) (Accordingto Syllabus)
EachChapterContains
• ChapterReview:Givesapreliminaryoverallideaabouthowachaptercanbefinishedfast.
• MCQswithdetailedexplanations:Gone tothedepthcoveringall theimportantaspectsindetail.
• Separatesectionofrecent2016entranceexaminationquestions.
• Includesimage-basedMCQsatthebeginningofthebook.
• Biomedicalwastemanagementrules2016included.Chan
gesDoneComparedtothePreviousEditionChapter
Review(Theoryportion)Part
• HasbeenthoroughlyupdatedfromHarrison19thedition,Park23rdeditionandApurbaSastry’sEssentialsofMedical
Microbiology,1steditionandApurbaSastry’sEssentialsofMedicalParasitology,1stedition.
• Allrecentinformation,suchasZikavirus,EBOLAvirus,Polioeradication,Denguevaccine,Vaccine-
derivedPolioViruses(VDPVs),MERS-CoV,andupdatesinbacterialdrugresistanceetc.havebeenincorporated.
• SeparateAnnexuresectioncontainingimportantexam-orientedrapid-firetopics.
• Image-basedquestionbankisfurtherstrengthenedwithnewimages(>500imagesareincluded).
MCQPart
• Recent questions included from AIIMS 2016 (Nov and May), JIPMER 2016 (Nov and May), PGI 2016 (Nov and May),
CMCVellore (2016)andRecent questionsfromothernational-levelexaminations.
• State entrance MCQs conducted in 2016 are also included. As no state PGMEE was carried out separately in 2017 and
thesame may be continued in future;so students should give more focus to MCQ pattern of All India/NEET exam;
rathergivingimportancetopreviousstateexamMCQs.
OneWeapon-TwoTargets:2ndYearMBBSExamsandPGEntrance
Thisrevisededitionispreparedinsuchamannerthatitwillhelpthe2ndyearMBBSstudentstopreparefortheirMBBSexamaswellasfortheP
Gentrances.Thechapterreviewpartofeachchapterisrevisedandupdatedinsuchawaythatbystudyingthisbook,thestudentscaneasilysolv
ethelongessays,shortnotesofMBBSexamsaswellasMCQsofvariousPGentrances.
Apurba Sankar
SastrySandhya
BhatK
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GoldenTipsforYourExamPreparation
StudyMethodology–AntegradevsRetrograde
Studentswillalwaysbeinadilemmawhethertofollowantegradeorretrogrademethodologyforpreparation.
• Antegrademethodistime-
consumingbutcoversthetopicsinasystematicway,whileretrogrademethodfailstocoverunaskedMCQsandrecentupdates.Lo
ng-termmemoryisusuallypoorforthefollowersofretrogrademethod.
• Ourbookmaintainsaperfectbalancebetweenboththemethods.
TipsforYourPreparation
Target-orientedLabor
• Onlylabor:Givesyou30–40%success
• Target-orientedlabor:Provides70%success
• Thatmeans:Youshouldknowwheretoreadandhowmuchtoreadandnottowastetimereadingunnecessarythingswhichareleas
tasked.
RepeatedRevisions
Repeatedrevisionsratherthanreadingextensivelywithoutanyrevision—
CrucialFactorSurveyconductedatvariouscoachingcentersandmedicalcolleges:
Revision 1st 2nd 3rd 4th 5th 6th 7th
Performance in Exam 25% 28% 32% 35% 50% 55% 60%
RankinAllIndia Nil Nil 5000 4700 2500 1800 <1000
Methods toImproveYour Memory

• Trytocorrelatethethingsandrememberratherthanpurelymuggingup
• Groupstudyorcouplestudy
• Recallingeverynight
• Boosterrevisions:Shouldbedonebeforeyoutotallyforgetthematter(i.e.inshort-interval)
• Mnemonics:Goodbutshouldbelimited.
RegularlyAssessYourself
Mostofthestudentsassesstheirpreparationdirectlyattheexamhallwhichisabsolutelyworstmethodofassessing.Youshouldassessyourst
andardondailybasisandmodifyyourpreparationstyleaccordingtotherequirement.Youcandothatby:
• Groupstudy:Comparingyourselfwithyourfriends.
• Self-assessmentbyrecallingeverynight(lasttwohourspostdinner)
• Grandtest:Assesswhetherstudyingisreflectedintheperformanceornot.YoucancompareyourselfwithstudentsthroughoutIn
dia.
RoleofGrandTest
• Helpsinassessingyourself
• Anytrialsanderrorscanbeattemptedingrandtestsandwhicheverexperimentissuccessfulcanbeexecutedinmainexam
• Tolearntimeadjustment
• Toenhanceguessingability
• Toimproveself-confidence
• Tocompareyourperformancewithothers.
Weekdays(Self-study)vsWeekendStudy(CoachingCenter)
• Coachinginstituteistheplacewhereyouwillbetrainedwiththeentrance-orientedimportantaspectsofthesubjects.
• However,studentsattendingcoachinginstitutesaregetting50to60dayslessfortheirpreparationascomparedtotheotherstudentswh
oprepareathome.
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GoldenTipsforYourExamPreparation vii
• Youshouldneverwastethetimegap(i.e.weekdays)betweenclasses.
• Youwillnevergettimetoreviseifyouhavenotcoveredthelastsubjectbeforethenextsubjectstarts.
Last100Days
• Accelerateneartheslugovers:Last100daysofstudyareverycrucial—
becausethestudents’surveyhasshownthat80%ofwhatyouwillanswercorrectlyintheexamdependsuponthelast2to3months’study
.
• Neverleaveanysubject:Bemasterinyourareabut,atthesametime,coveratleastaverageoftheuncoveredareaasMCQswillbeaskedf
romallthesubjects.
• Sleepingwellthepreviousnight:Increasesyourefficiencyatleastby10%.
• Donotforgettheimportanceoftime:Oncelost,itcanneverberecycled.
BeanEarlyRiser
As wisely quoted by Benjamin Franklin “Early to bed and early to rise makes a man healthy, wealthy, and wise.”.
Rememberwhat you read during early morning (3 am-6 am), will stimulate your memory cells maximum and will be retained
longer.More so, there will be no disturbance as compared to late night reading where other friends/TV shows/parties or the
wholedaytirednessetc.willdisturbyoualot.
MyTricktoGetupEalry
• Sleepearly(10pm)
• Keepthreealarms,5mingap
• Keepalarmawayfromcot
• Brushandmaketeabeforegotoread
• Neverstartafreshchapter,asyouwillhavestartingproblemwhichwillinducesleep
• Alwaysreadachaptercontinuedfromyesterday'sreading.
WhileWritingPGExam..........TheFollowingThingsare tobeKeptinMind
• Timemanagement
• Guessability:
○ Bycorrelatingthethings
○ Byrulingouttheoptions
• Guessonlyin50–50situation(twoorthreeoptionsareruledout)
• Neverguesswhenyoucanruleoutonlyoneorzerooptions.
FINALLY,WHILECHOOSINGAPGSEAT
StudentsChooseaPGSeat—either‘byChoice’or‘byChance’
• Bychoice(similartolovemarriage):TakeyourdreamPGseat(thishappensonlywhenyougetadesiredrank).Onlyafewblessedstu
dentsfallunderthiscategory.
• Bychance(similartoarrangedmarriage):TaketheavailablePGseat(thishappenswhenyougetarank,butnotgoodenoughtogetyourch
oicesubject).Mostofthestudentswillfallunderthiscategory.
Ifyouhavearankbutnotgoodenoughtogetyourchoicesubject,youhavetwooptions:
• Takewhatisavailable
• Waitforthenextyear.
Thisisagainacontroversialsituation.Manyhaveopinionsinboththecategories.
• Somesaythatyoucanenjoythesubjectonlyifitisyourdreamsubject.
• Somesaythatyoushouldnotwastetimeinwaitingasthereisnoguaranteethatyouwilldobetterinthenextexamandinthecurrent
onlinemultisessionexampattern,thesituationishighlyuncertain.
BestWaytoSolvethisissueis
Trytoseethedifferencebetweenlovemarriageandarrangedmarriage.
• Thereisnoguaranteethatinlovemarriage,youwillnotfightandyouwillhaveapeacefullife.
• Loveisthereinarrangedmarriagealso,butitiscreatedaftermarriage.
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viii ReviewofMicrobiologyandImmunology
Ouradvice as timeis precious:

• Neverwaitforlongtogetyourdesiredsubject.
• Developinterestinthesubjectthatyouchoose.
• DojusticewithyoursubjectinyourthreeyearsofPGcourse.
• And,in turn, thesubject will giveyou name, fameand prosperity throughout yourlife.
• Ifyoudislikethesubject,thenitwillendupindivorce(i.e.youwillleavethecourseorwilllivethroughout).
STRATEGYTOCHOOSEAPGSEAT
Preparea‘prioritylist’ofsubject-collegecombinations,basedonyourrankandlast-yearcounseling.
CollegePriority to bediscussion Prepared
• Thisisequallyimportantbecausewhateversubjectyoutake,youwillbemasterofthatifyoudoinAIIMS/PGI/JIPMER/
CMCkindofcollege.
• Evenifthesubjectisoflowpriority,itwillgiveyoumoresatisfactionasyouwillbeeasilyfamousinyourlocalityandcountry.
DEMO: Whowillfeel moresatisfiedinthe jobandmorefamous intheworld?
• Astate-levelcricketplayerofIndia,whoisstrugglingtogetachancetoplayinthenational-
levelteam(e.g.doingmedicinefromstatecollege);or
• Bangladeshcricketteamcaptain(e.g.doingpathologyfromPGI).
SubjectPriorityistobePreparedBasedontheFollowingFacts
Basedonthenatureofwork,timeofworkandyourdesiretoearnnameandmoney,i.e.:
• For9amto5pmjob(facultyjobisthebest):
○ Subjectpreferenceshouldbebasedonjobvacancy
○ Allsubjectswillhavethesamesalary,butnightdutywillbethereonlyinafewclinicalsubjects.
(Amedicinefacultysometimesgetsfrustratedbyseeingadermatologyfaculty)
For 7 am to 9 pm job (private practice is the best): Income is partly based on subject and partly on your luck.
So,hereyoucangiveimportancetocertainsubjects,likeRD,Paed,Ortho,etc.But,remember,alotofriskisinvolvedinpracticean
dbepreparedforthat.
• Femalesshouldkeepinmindthattheyhavetomanagefamilyinfuture.So,justfortheinfatuationsake,donotgoforOBG.Beprepa
redthatyouwillhavedutiesandlotofprofessionalresponsibilitiesbeforeoptingforOBG.
• IfchoosingRD,anesthesia:Bepreparedthatthereisnopatientinteraction.Donotcrylater.
• If choosingmedicine/surgery: Bepreparedthat DM/MChisa MUST.So,a lotof hardworkis
expectedinfutureandsettlementisalwayslate.
• Ifchoosingpsychiatry/radiotherapy:Bepreparedthatthepatient’scomplianceispoor.So,youmay/
maynotgetthejobsatisfactionofcuringapatient.
• Ifchoosingparaclinicalandnonclinical:Bepreparedthatthereisnopatientexposure,butonehastheadvantageofnoduty,s
amesalaryifworkingasfacultyand,mostimportantly,noneedtoprepareagain.
Remember,ForSatisfaction
• BEINGHAPPYWITHWHATGODHASGIVENISMOREIMPORTANTTHANCRAVINGFORWHATISNOTGI
VENTOYOU.
• WHATYOURFAMILYWILLTHINKISMOREIMPORTANTTHANWHATYOURFRIENDS/
NEIGHBORSWILLTHINK.
WishYou“ALLTHEBESTFORTHESUCCESSANDTHEBRIGHTFUTUREAHEAD”.
Keep in touch with us through the FBdiscussion group andpersonal touch via FB messangeror mail.
ApurbaSankarSastry
(drapurbasastry@gmail.com)
SandhyaBhatK
(sandhyabhatk@gmail.com)
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Acknowledgments
Our efforts bore fruit with the successful completion of this project ‘Review of Microbiology and Immunology’ 5th
edition.However,therearemanyotherswhosharetherewardofthiseffortsimplybecauseitwouldneverhavebeenthisgoodwithoutt
heirhelp.Itgivesusgreatpleasuretoacknowledgethecontributionofthosewhoguided,supportedandstoodbyusthroughthearduou
sjourneyofcompletingthistediouswork.
• WewouldliketosincerelythankourbelovedDirector,JIPMER,Puducherry,whogaveusconstantinspirationandsupportforwrit
ingthisbook.
• WewouldalsoliketoexpressourgratitudetotheDirector,PondicherryInstituteofMedicalSciences(PIMS),Puducherryforhise
ncouragement.
• Wearethankfultoallthefaculties,residentsandotherstaffofJIPMERandPondicherryInstituteofMedicalSciences(PIMS),Puduc
herryfortheirhelpandencouragement.
• Weofferourheartygratitudetoallourpaternalandmaternalrelativesandallourcousins.
Wealsoofferourgratitudetosomeofthestudentswhoconstantlygavetheirinputsandsupportduringthecorrectionandeditingof
thebook.
1. DrRamyaRaghavan,JIPMER
2. DrPrasannaBhat,JIPMER
3. DrSuryaprakash,StanleyMedicalCollege,Chennai
4. SalmanMapara,GrantMedicalCollegeandSirJamshedjeeJeejeebhoyGroupofHospitals
5. ManishChoudhary,KasturbaMedicalCollege,Manipal
6. SunilChillalshetti, Belgaum Institute ofMedical Sciences
7. AkshiMalhotra,JIPMER
8. ManjulaGunasekaran, PSGInstitute ofMedical Sciencesand Research
9. PriyankaPatra,SKNMCpune
10. PraveenG,MelmaruvathurAdhiparasakthiInstituteofMedicalSciencesandResearch,TamilNadu
11. AartiChitkara,SirMaharajaAgrasenMedicalCollege,Haryana
12. AshwathVh,AdichunchanagiriInstituteofMedicalSciences,Karnataka
13. RajaSumanDatta,GSLMedicalCollege,Rajahmundry,AndhraPradesh
WearegratefultoShriJitendarPVij(GroupChairman),MrAnkitVij(GroupPresident),MsChetnaMalhotraVohra(AssociateDirector)
, Ms Payal Bharti (Project Manager), Mr Ravinder (Typesetter), Mr Himanshu Shekhar Lal (Proofreader) and all theother
members of Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India for giving us this wonderful opportunity ofwriting
this book and their excellent support throughout the journey, especially during the editing work on Apurba
Sastry'sMicrobiologydiscussion.
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Contents
Annexure xiii-xv
Image-basedQuestions
Section1:General Microbiology
Chapter1.1History,Taxonomy,MorphologyandPhysiologyofBacteriaandMicrobialPathogenicity 3
Chapter1.2SterilizationandDisinfection 20
Chapter1.3CultureMediaandMethods,IdentificationofBacteriabyConventional,AutomatedandMolecularMethods 32
Chapter1.4BacterialGeneticsandAntimicrobialResistance 41
Section2:Immunology
Chapter2.1Immunity 51
Chapter2.2Antigen,Antibody,Antigen-AntibodyReaction,Complement 58
Chapter2.3StructureofImmuneSystemandImmuneResponse 79
Chapter2.4Hypersensitivity 93
Chapter2.5Autoimmunity,Immunodeficiency,Transplantation,andImmunoprophylaxis 99
Section3:SystemicBacteriology
Chapter3.1Staphylococcus 115
Chapter3.2Streptococcus,EnterococcusandPneumococcus 125
Chapter3.3NeisseriaandMoraxella 138
Chapter3.4CorynebacteriumandBacillus 146
Chapter3.5Anaerobes:ClostridiumandNon-SporingAnaerobes 159
Chapter3.6Mycobacteria 174
Chapter3.7Enterobacteriaceae(E.Coli,Klebsiella,Proteus,Shigella,Salmonella,Yersinia) 196
Chapter3.8Vibrio 217
Chapter3.9PseudomonasandOtherNonfermentersandHaemophilus,Bordetella,Brucella(HBB) 229
Chapter3.10Spirochetes 245
Chapter3.11Rickettsia,ChlamydiaandMycoplasma 261
Chapter3.12MiscellaneousBacteria 277
Section4:Virology
Chapter4.1GeneralPropertiesofViruses 297
Chapter4.2HerpesvirusesandOtherDNAViruses 307
Chapter4.3MyxovirusesandRubella 328
Chapter4.4Arboviruses,PicornavirusesandRabiesVirus 345
Chapter4.5HepatitisViruses 372
Chapter4.6HIVandOtherRetroviruses 388
Chapter4.7MiscellaneousViruses 404
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xii ReviewofMicrobiologyandImmunology
Section5:Mycology
Chapter5Mycology 417
Section6:Parasitology
Chapter6.1GeneralParasitology 447
Chapter6.2IntestinalAmebae,Free-LivingAmebaandBalantidiumColi 451
Chapter 6.3 Flagellates 459
Chapter6.4PlasmodiumSpeciesandBabesia 468
Chapter6.5CoccidianParasites 479
Chapter6.6CestodesandTrematodes 484
Chapter6.7Nematodes 497
Section7:AppliedMicrobiology
Chapter7.1ClinicalMicrobiology(InfectiveSyndromes) 515
Chapter7.2HospitalAcquiredInfection,BiomedicalWaste,BacteriologyofWater,AirandMilk 531
RecentQuestions2016 539
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Annexure
POLIOVACCINEUPDATE2016
IPVandbOPVinIndia
• IPVintroducedinIndia:fromNovember2015
• BivalentOPVwillbeintroducedinIndia:fromApril2016
IPVinIndia
• Government of India introduced IPVunder universal immunization program
• FromNovember2015
• OnedoseofIPV
• Inaphasedmanner
• Inthefirstphase,IPVhasbeenintroducedinsixhigh-
riskstates:Assam,Gujarat,Punjab,Bihar,MadhyaPradesh,andUttarPradesh
• This IPVdoseis extra;givenover andabovethe TrivalentOPV.
IndianGovernmentActionPlan:SwitchoveroftOPVtobOPVandIPV
• FromNovember2015toMarch2016:ThreedosesoftrivalentOPVplusonedoseofIPValongwiththethirddoseofOPVat14weeks.
• FromApril2016:BivalentOPVthreedosesplusonedoseofIPValongwiththethirddoseofbOPVat14weeks.
IMPORTANTNATIONALREFERENCECENTERSFORINFECTIOUSDISEASESININDIA
• Tuberculosis:
○ TRC:TuberculosisResearch Centre,Chennai
○ LRSInstituteofTuberculosis&RespiratoryDiseases,Delhi
○ NationalInstituteofTuberculosis,Bengaluru
○ NationalJALMAInstituteofLeprosyandOtherMycobacterialDiseases,Agra
• Leprosy:NationalJALMAInstituteofLeprosyandOtherMycobacterialDiseases,Agra
• Salmonella:
○ Nationalsalmonellareferencecentreforidentificationofunusualserotype:CentralResearchInstitute(CRI),Kasauli
○ Referencecentreforsalmonellaofanimalorigin:IndianVeterinaryInstitute,Izatnagar
○ Referencecentreforphagetyping:LadyHardingeMedicalCollege,Delhi
• BCGvaccinepreparedinIndia:BCGVaccineLaboratory,Guindy,Chennai
• Yellowfevervaccine:CentralResearchInstitute(CRI),Kasauli
• Plaguevaccine:HaffkineInstitute,Mumbai
• ReferencecentreforphagetypingofStaph.auerusinIndia:MaulanaAzadMedicalCollege,Delhi
• Cholera:NationalInstituteofCholeraandEntericDiseases(NICED),Kolkata
• VDRL antigen is prepared inIndia: Institute of Serology, Kolkata
• NationalInstituteofVirology,Pune:PreparediagnostickitsforHepatitisviruses,JE,Dengue,ChikungunyaandRotavirus
• NARI(NationalAIDSResearchInstitute):Pune
• LeptospirosisReferenceLaboratory:RegionalMedicalResearchInstitute(RMRI),PortBlair
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xivReviewofMicrobiologyandImmunology
INCUBATIONPERIODOFIMPORTANTORGANISMS
MMR+2Pox Hepatitisviruses Malaria STD

Mumps 19d HAV 15–45d P.vivax 14d Syphilis 9–90d
Measles 10d HBV 30–180d P.falciparum 12d Chancroid 1–14d
Rubella 14d HCV 15–160d P.malariae 28d Herpes 2–7d
Smallpox 12d HDV 30–180d P.ovale 17d Donovaniasis 1–4wks
Chickenpox 15d HEV 14–60d Filariasis 8–16 LGV 3d–6wk
months
Arboviruses Otherviruses Leishmaniasis 1–4 Gas gangrene
months
Dengue 5–6d Rabies 1–3m DPT Cl.perfringens 10–48hr
Chikugunya 5–6d Polio 7–14d Diphtheria 2–6d Cl.septicum 2–3d
KFD 3–8d HIV 10 Pertussis 7–14d Cl.novyi 5–6d

yrs(Averag
e)
Japaneseencephalitis 5–15d Infectious mononucleosis 4–8wks Tetanus 6–10d Plague
Yellowfever 3–6d Influenza 18–72hrs Leprosy 3–5yrs Bubonic 2–7d
&Septicaemi
c
Cholera 1–2d Pneumonic 1–3d
Typhoid 10–14d
ARTHROPOD-BORNEDISEASES
Mosquito Female:anopheles:Malaria
Aedes:Dengue,Chikungunya,YellowfeverCulex:JE,
Filaria(Lymphatic)
Louse Trenchfever(Bartonellaquintana),
Epidemic relapsing fever (Borrelia
recurrentis),Epidemic typhus, Pediculosis
Flea Bubonic
plague,Endemic
typhus,Hymenolepisdim
inuta
HardTick Ticktyphus,
Arbo(KFD,RSSE,Crimean-Congohemorrhagicfever,Coloradotickfever),Babesia,Tularemia
SoftTick Endemic relapsing fever (B. duttoni),Q-

fever(in animals)
Mite Scrub
typhus,Rickettsi
al pox,Scabies
Cyclops Guinea worm
disease,Fishtapeworm(D.lat
um)
Black fly Onchocerciasis
Reduviidbug Chaga’sdisease
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Annexurexv
EXAMPLESOFSPECIALMEDIA(E—ENRICHED,EN—ENRICHMENT,S—SELECTIVE,D—
DIFFERENTIALMEDIA)
Organism Medium
Entericpathogens—for Hektoenentericagar(S) Xylose-lysin-deoxycholate agar
Salmonella, Shigella (S)Deoxycholate citrate agar (S)Eosin methylene blue agar
(S)MacConkey(D andS) Salmonella Shigella agar(S)
WilsonblairforSalmonella(S)SeleniteFbroth(En),Tetrathionatebroth(En)
Bloodculture—forblood- Castaneda’sbiphasicmedia(E):Brain-heartinfusionagarslopeandbroth
bornepathogens
Vibriocholerae(likesalkalinegr TCBS (Thiosulfate Citrate Bile Sucrose agar)
owthmedium) (S)Mansour’sGelatinTaurocholateTrypticaseagar(S)Alka
linebilesalt agar(S)
APW:AlkalinePeptoneWater(En)
S.aureus Mannitol salt agar
(S)Milk salt agar
(S)Ludlam’smedium(S)
Streptococcus Crystalvioletbloodagar(S)
Neisseria Chocolateagar(E),Thayer-Martinmedia(S),
ModifiedNewYorkmedium(S)
Corynebacterium Loeffler’sserummedium(E)
Potassiumtelluriteagar(S)
Bacillusanthracis PLET:PolymyxinLithium EDTAThallousacetate (S)
Bacillus cereus MYPA:Mannitoleggyolkphenolredpolymyxinagar(S)
Anaerobes Thioglycollate (En)
Robertsoncookedmeatbroth(En)
Listeria PALCAMagar(S)
Pseudomonas Cetrimideagar(S),King’smedia(forpigment)
Burkholderia Ashdown’smedium
Haemophilus Blood agar with staph streak
(E)Chocolateagar(E)
Levinthal’smedium(E),Fildesagar(E)
Bordetella Reganlowmedia(E)
Bordet Gengou Glycerin potato blood agar
(E)Lacey’sDFPmedia (S)
Mycobacterium LowensteinJensen,Dorsetegg(S)
Leptospira EMJH (E), Fletcher’s (E), Korthof’s (E)
Campylobacter Skirrow’s,Butzler,CampyBAP(S)
Legionella BCYE(Bufferedcharcoalyeastextract)(E)
Reiter’streponema SmithNoguchimedia
Urinarypathogen MacConkeyagar(DandS)
CysteinLactoseElectrolyte-deficientagar(CLEDagar)(DandS)
Organism Transportmedia
Streptococcus Pike’smedia
Neisseria Amies,Stuart’smedia
Vibrio VR,Autoclaved sea water,CarryBlair
Entericpathogen CarryBlairmedium
Shigella, Salmonella Bufferedglycerolsaline
Bordetella ModifiedStuart’s(withcasaminoacid)
Mischulow’scharcoalagar
Dacronorcalciumalginateswabused
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SECTION 1
GeneralMicrobiology
CHAPTEROUTLINE
1.1 History,Taxonomy,MorphologyandPhysiologyofBacteriaandMicrobialPat
hogenicity
1.2 SterilizationandDisinfection
1.3 CultureMediaandMethods,IdentificationofBacteriabyConventional,Auto
matedandMolecularMethods
1.4 BacterialGeneticsandAntimicrobialResistance
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History,Taxonomy,Morphologyand CHAPTER
ofBacteriaandMicrobialPathogenicity
1.1
HISTORY
LouisPasteur
LouisPasteurisalsoknownasfatherofmicrobiology.Hehasmanycontributionstomicrobiology:
1. Hehasproposedtheprinciplesoffermentationforpreservationoffood Louis Pasteur proposed:
2. He introducedsterilizationtechniquesanddevelopedsteamsterilizer, Principlesoffermentation
Pasteurizationofmilk
hotairovenandautoclave. Sterilization techniques
3. Hedescribedthemethodofpasteurizationofmilk Germtheoryofdisease
4. He had also contributed for designing the vaccines against several diseases such
asanthrax,fowlcholeraandrabies
5. Hedisprovedthetheoryofspontaneousgenerationofdiseaseandpostulatedthe‘germtheo
ry of disease’. He stated that disease cannot be caused by bad air or vapor, but it
isproducedbythemicroorganismspresentinair.
6. Liquidmediaconcept-Heusednutrientbrothtogrowmicroorganisms.
7. HewasthefounderofthePasteurInstitute,Paris.
RobertKoch
RobertKochprovidedremarkablecontributionstothefieldofmicrobiology:
1. Heusedofsolidmediaforcultureofbacteria-
EilshemiusHesse,thewifeofWaltherHesse,oneofKoch’sassistantshadsuggestedtheuseofagar
assolidifyingagent. ExceptionstoKoch’spostulates:
2. Healsointroducedmethodsforisolationofbacteriainpureculture. Mycobacteriumleprae
Treponemapallidum
3. Describedhangingdropmethodfortestingmotility. Neisseriagonorrhoeae
4. Discoveredbacteriasuchastheanthraxbacilli,tuberclebacilliandcholerabacilli.
5. Introducedstainingtechniquesbyusinganilinedye.
6. Koch’s phenomenon: Robert Koch observed that guinea pigs already infected
withtubercle bacillus developed a hypersensitivity reaction when injected with
tuberclebacilli oritsprotein.ThisreactioniscalledKoch’sphenomenon.
7. Koch’s Postulates:
AccordingtoKoch’spostulates,amicroorganismcanbeacceptedasthecausativeagentofaninfectiousdiseaseonlyifthefollowingconditionsarefulfilled:
Themicroorganismshouldbeconstantlyassociatedwiththelesionsofthedisease.
Itshouldbepossibletoisolatetheorganisminpureculturefromthelesionsofthedisease.
Thesamediseasemustresultwhentheisolatedmicroorganismisinoculatedintoasuitablelaboratoryanimal.
Itshouldbepossibletore-isolatetheorganisminpureculturefromthelesionsproducedintheexperimentalanimals.
Anadditionalfifthcriterionwasintroducedsubsequentlywhichstatesthatantibodytothecausativeorganismshouldbedemonstrableinthe patient’s serum.
Exceptions to Koch’s postulates: It is observed that it is not always possible to apply these postulates to study all the human diseases.Therearesome bacteriathat donotsa
MycobacteriumlepraeandTreponemapallidum:Theycannotbegrowninvitro;howevercanbemaintainedinanimals.
Neisseriagonorrhoeae:Thereisnoanimalmodel;however,bacteriacanbegrowninvitro.
MolecularKoch’spostulates:ItwasamodificationofKoch’spostulates(byStanleyFalkow).Hestatedthatgene(codingforvirulence)ofamicroorganismshould satisfy allthe criteri
PaulEhrlich
1. Hewasthefirsttoreporttheacid-fastnatureoftuberclebacillus.
2. Hedevelopedtechniquestostaintissuesandbloodcells.
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4ReviewofMicrobiologyandImmunology
3. HeproposedatoxinantitoxininteractioncalledasEhrlichphenomenonandalsointroduced
methodsofstandardisingtoxinandantitoxin
4. Heproposedthe‘sidechaintheoryforantibodyproduction’.
ImportantDiscoveries: 5. Hediscovered‘salvarsan’,anarsenicalcompound(magicbullet)fortreatmentofsyphilis,he
AVLeeuwenhoek:Simplemicroscope nceknownasfatherofchemotherapy.
Ernst Ruska: Electronmicroscope 6. Thebacteria‘Ehrlichia’wasnamedafterhim.
E.Jenner: Smallpox vaccine
Karry B Mullis: PCR OtherImportantContributorsinMicrobiology
1. AntoniePhilipsvanLeeuwenhoek:Discoveredsingle-
lensmicroscopeandnamedorganismsas‘Littleanimalcules’.
2. EdwardJenner:Developedthefirstvaccineoftheworld,thesmallpoxvaccinebyusing
cowpoxvirus.
3. JosephLister:Heisconsideredtobethefatherofantisepticsurgery.Heusedcarbolicaciddurings
urgery.
4. HansChristian Gram: Hedeveloped ‘Gram stain’.
6. ErnstRuska:Hewasthefounderofelectronmicroscope.
7. AlexanderFleming:Hediscoveredtheantibioticpenicillin.
8. ElieMetchnikoff:Hedescribedphagocytosisandtermedphagocytes.
9. Kleinberger:HedescribedtheexistenceofLformsofbacteria.
10. BarbaraMcClintock:Shedescribedtransposons.
11. WalterGilbertandFrederickSanger:werethefirsttodevelop(1977)themethodof
DNAsequencing.
12. KarryBMullis:Discoveredpolymerasechainreaction(PCR).
Table1.1.1:Discoveryofimportantmicroorganisms
Table1.1.2:Commonnamesofbacterianamedafterthediscoverers
Discoverer Organism Discoverer Organism
CommonnameOgston Staphylococcusaureus
Scientific name YersinandKitasatoCommonname Yersiniapestis
Scientific name
Neisser
Kleb-Loefflerbacillus Neisseriagonorrhoeae
Corynebacteriumdiphtheriae SchaudinnandHoffman
Whitmorebacillus Treponemapallidum
Burkholderiapseudomallei
Weichselbaum
PreiszNocardbacillus Neisseriameningitidis
Corynebacteriumpseudotuberculosi DanielCarrion Batteybacillus Bartonellabacilliformis
Mycobacteriumintracellulare
Loeffler s Corynebacteriumdiphtheriae d’Herelle Bacteriophages
Section
KochWeekbacillus Haemophilusaegyptius Eaton’sagent Mycoplasmapneumoniae

Frenkel Streptococcuspneumoniae W.H.Welch Clostridiumperfringens
Johne’sbacillus Mycobacteriumparatuberculosis Pfeiffer’sbacillus Hemophilusinfluenzae
Bruce
GaffkyEberthbacillus Brucella melitensis
SalmonellaTyphi A.EpsteinandY.Barr Epstein-Barr virus
Kitasato Clostridiumtetani Hansen Mycobacteriumleprae
BACTERIALTAXONOMY
Cavalier-
Smith’ssixkingdomsclassification(1998)isthemostrecentandwidelytaxonomicclassification.
It divides organisms into 6 kingdoms Bacteria, Protozoa, Chromista,
Plantae,FungiandAnimalia.
PrincipleusedforBacterialClassification
1. Phylogenetic classification: This is a hierarchical classification representing a
branchingtree-like arrangement; one characteristic (or trait) is being employed for division
at eachnodeofthetree
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History,Taxonomy,MorphologyandPhysiologyofBacteriaandMicrobialPathogenicity 5
2. Adansonian (orphonetic)classification:To avoidtheuse ofweighted

characteristics,MichelAdansonproposedaschemethatclassifiesorganismsbasedongivingequ
alweighttoeverycharacteroftheorganism.Thisprincipleisusedinnumerictaxonomy.
BacterialClassification:
3. Molecular classification:Basedongeneticrelatedness(guanine+cytosinecontent)of Phylogenetic: Based onweightedcharacteristics
differentorganisms. Adansonian (or phonetic):Basedongivingequalweighttoev
Molecular: Based on geneticrelatedness
MICROSCOPY
Agoodmicroscopeshouldhavethreeproperties:
1. Good resolution: Resolution power refers to the ability to produce separate images
ofclosely placed objects so that they can be distinguished as two separate entities.
Theresolutionpowerof:
• Unaidedhumaneyeisabout0.2mm(200µm)
• Lightmicroscopeisabout0.2µm
Resolutionpowerof:
• Electronmicroscopeisabout0.5nm
Unaidedhumaneye-0.2mm
Resolutiondependsonrefractiveindex.Oilhasahigherrefractiveindexthanair. Lightmicroscope-0.2µm
2. Goodcontrast:Thiscanbefurtherimprovedbystainingthespecimen. Electronmicroscope-0.5nm
3. Goodmagnification:Thisisachievedbyuseofconcavelenses.
Bright-FieldorLightMicroscope
Thebright-fieldorlightmicroscopeformsadarkimageagainstabrighterbackground.
DarkFieldMicroscope
•Principle: In dark field microscope, the object appears bright against a dark
background.Thisismadepossiblebyuseofaspecialdarkfieldcondenser Numerical aperture = n sin α(nisrefractiveindexofmed
ResolvingPower=0.61
•Applications: It is used to identify the living, unstained cells and thin bacteria ×wavelength/Numericalaperture
likespirocheteswhichcannotbevisualizedbylightmicroscopy.
PhaseContrastMicroscope
Itisusedtovisualizethelivingcellsbycreatingdifferenceincontrastbetweenthecellsandwater.I
tconvertsslightdifferencesinrefractiveindexandcelldensityintoeasilydetectablevariationsinl
ightintensity.Itisusefulforstudying:
• Microbial motility
• Determiningtheshapeof livingcells
• Detectingbacterialcomponentssuchasendosporesandinclusionbodies. TypeofLightUsedinMicroscopes:
Transmittedlight:Usedin
FluorescenceMicroscope brightfield
Principle: When fluorescent dyes are exposed to ultraviolet rays (UV) rays, they Reflected light: Used in darkfield
Polarized light: Used indifferential interferencecontrast m
becomeexcited and are said to fluoresce, i.e. they convert this invisible, short wavelength
GeneralMicrobiolog
rays intolightoflongerwavelengths(visiblelight).
Applications:Epifluorescencemicroscopehasthefollowingapplications:
• Autofluorescence,whenplacedunderUVlamp,e.g.Cyclospora
• Microbescoatedwithfluorescentdye,e.g.Acridineorangeformalariaparasites(QBC)
andAuraminephenolforM.tuberculosis.
•Immunofluorescence: It uses fluorescent dye tagged antibody to detect cell
surfaceantigens or antibodies bound to cell surface antigens. Thereare three types: Fluorescence microscopeApplications:
Autofluorescence,e.g.
direct IF,indirectIF,andFlowcytometry. Cyclospora
Microbes coated withfluorescent dye: QBC forPlasmodiu
ElectronMicroscope Immunofluorescence:Florescent dye taggedantibody/antig
ItwasinventedbyErnstRuskain1931.Itdiffersfromlightmicroscopebyvariousways(tablegive
nbelow).TherearetwotypesofEM:
1. TransmissionEM(MCtype,examinetheinternalstructure,resolution0.5nm,gives2dimensiona
lview)
2. ScanningEM(examinethesurfaces,resolution7nm,gives3dimensionalview)
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Table1.1.3:Differencesbetweenlightmicroscopeandelectronmicroscope
Features Lightmicroscope Electronmicroscope
Highestpracticalmagnification About1,000–1,500 Over100,000
Best resolution 0.2µm 0.5nm
Radiationsource Visiblelight Electronbeam
Electronmicroscope: Mediumoftravel Air Highvacuum
>100,000magnification
Specimen mount Glass slide Metalgrid(usuallycopper)
0.5nmresolution
Radiation source-Electronbeam Typeoflens Glass Electromagnet
Highvacuummedium Sourceofcontrast Differentiallightabsorption Scatteringofelectrons
Specimen mounted on Metalgrid
Electromagnetlens Focusing mechanism Adjustlensmechanically Adjustcurrentto themagneticlens
PrincipleofTransmissionElectronMicroscope
Specimenpreparation:Cellsaresubjectedtothefollowingstepstoprepareverythinspecimens(20t
o100nmthick)
• Fixation:Cellsarefixedbyusingglutaraldehydeorosmiumtetroxideforstabilization.
• Dehydration:Specimenisthendehydratedwithorganicsolvents(e.g.acetoneorethanol).
• Embedding: Specimen is embedded in plastic polymer and then, is hardened to form
asolid block. Most plastic polymers are water insoluble; hence complete dehydration
ofspecimenismustbeforeembedding.
• Slicing:Specimenisthencutintothinslicesbyultramicrotomeknife,andslicesare
mountedonametalslide(copper).
Freeze-
etchingtechnique:Itisanalternatemethodforspecimenpreparationtovisualizetheinternalorgane
lleswithinthecells.Cellsarerapidlyfrozenthenwarmed
→ fractured by a knife exposing the internal organelles → subjected to sublimation
→shadowedbycoatingwithplatinumandcarbon.
MeasurestoincreasethecontrastofEMinclude:
• Stainingbysolutionsofheavymetalsaltslikeleadcitrateanduranylacetate
• Negativestainingwithheavymetalslikephosphotungsticacidoruranylacetate.
• Shadowing:Specimeniscoatedwithathinfilmofplatinumorotherheavymetalat
45°anglesothatthemetalstrikesthemicroorganismononlyoneside
STAININGTECHNIQUES
Stainingisnecessarytoproducecolorcontrastandtherebyincreasethevisibilityoftheobject.
Beforestaining,thefixationofthesmeartotheslideisdone:
• Heatfixationisusuallydoneforbacterialsmearsbygentlyflameheatinganair-
driedfilmofbacteria
Section
• Chemicalfixationsuchasethanol,aceticacid,mercuricchloride,formaldehyde,methan
ol and glutaraldehyde.This isuseful for examinationof bloodsmears.
StainingTechniquesUsedinMicrobiology
1. Simple stain: Basic dyes such as methylene blue or basic fuchsin are used as
simplestains.Theyprovidethecolorcontrast,butimpartthesamecolortoallthebacteriaina
smear.
2. Negative staining, e.g. India ink or nigrosin. The background gets stained black
whereasunstained bacteria stand out in contrast. This is very useful in the
demonstration ofbacterialcapsuleswhichdonottakeupsimplestains.
3. Impregnationmethods(e.g.silver):Usedfordemonstrationofthinstructureslike
bacterialflagellaandspirochetes
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4. Differential stain: Two stains are used which impart different colors which help
indifferentiatingbacteria,e.g.
• Gram stain: Differentiates bacteria into Gram-positive (appear violet) and Gram-
negative(appearpink)groups
○ Primarystainbycrystalviolet(orgentianvioletormethylviolet)foronemin-ute.
○ MordantbyGram’siodineforoneminute.
○ Decolorization by acetone (for 1–2 sec) or ethanol (20–30 sec) with
immediatewash. Decolorizer removes the primary stain from Gram-negative
bacteriawhile theGram-positive bacteriaretain theprimary stain.
○ CounterstainorSecondarystainsbysafraninordilutedcarbolfuchsin;isadd-
edfor30sec.
• Acid-faststain:Acid-
fastorganisms(tablebelow)resistdecolorizationtomineralacids.Thisisduetopresence
ofmycolicacidsinthecellwall.(detailsinchapter3.6).
• Albertstain:Differentiates bacteriahaving metachromatic granules (e.g.
Corynebacteriumdiphtheriae)fromotherbacteriathatdonothave(referchapter3.4).
5. OtherSpecialStainingMethods:
• Spore staining: Acid fast stain (using 0.25% sulfuric acid) and Malachite green
stain(SchaefferandfultonmethodmodifiedbyAshby)methodsareused;however,phaseco
ntrastmicroscopeofunstainedwetfilmisthebestmethod. Special Staining Methods:
• Lipidsstainedby:SudanBlackstain Spore staining: Acid fast stain(0.25% H2SO4) and Schaeff
Lipids stained by: Sudan Blackstain
• Carbohydrate(Glycogen)stainedbyIodinestain Carbohydrate stained by:Iodine stain
• Flagellarstain:Tannicacidstaining(Leifsonmethod) Flagellarstain:Tannicacid(Leifson method)
6. MicroscopyofBacteriainLivingState
• Unstained(wet)preparations:Usedfor:
○ Checkingbacterialmotility(e.g.inhangingdropandwetmountpreparations)
○ Fordemonstrationofspirochetes(e.g.indarkfieldorphasecontrastmicros-copy).
• Vitalstains:Differentiatethelivingcellsfromdeadcells:
○ Insupravitalstaining,livingcellsthathavebeenremovedfromanorganismarestai
ned;whereasintravitalstainingisdonebyinjectingstainintothebody.
○ Examplesofvitalstainsareeosin,propidiumiodide,trypanblue,erythrosineandn
eutralred.
Table1.1.4:Acidfast organisms
Acidfastbacteria Acidfastparasites
Mycobacteria–M.tuberculosis/M.leprae/NTM Cryptosporidium,CyclosporaandIsospora
Nocardia,RhodococcusandLegionellamicdadei Tineasaginataeggandscolex
GeneralMicrobiolog
Others:BacterialSporeandSpermhead HookletsofhydatidcystandSchistosomamansonieggs
MORPHOLOGYOFBACTERIA
Table1.1.5:Characteristicsofprokaryotesandeukaryotes
Characteristics Prokaryotes Eukaryotes
Majorgroups Bacteria,bluegreenalgae Fungi,parasites,otheralgae,plantsandanimals
Nucleus Diffuse Welldefined
Nuclearmembrane Absent Present
Nucleolus Absent Present
Ribonucleoprotein Absent Present
Celldivision Binary fission Mitosis, Meiosis
Contd...
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Contd...
Characteristics Prokaryotes Eukaryotes
Chromosome One, circular Many,liner
ExtrachromosomalDNA Foundinplasmid Foundinmitochondria
Sterolsincell membrane Absentexceptinmycoplasma Present
Cellularorganelles Absent (except ribosome) Present
Ribosome 70sinsize 80sinsize
Siteofrespiration Mesosome Mitochondria
Pinocytosis Absent Present
Table1.1.6:ShapeofbacteriaandtheirGramstainingproperty
Gram-positivecocciarrangedin Gram-negativecocciarrangedin
Cluster BacterialCellWall
Staphylococcus Pairs,lensshaped Meningococcus
Chain Thecellwallisatoughandrigidstructure,surroundingthebacterium.Apartfromprovidingprote
Streptococcus Pairs, kidney shaped Gonococcus
Pairs,lanceolateshaped
Gram-positive cocci arrangedin: ction and conferring rigidity,Gram-negativebacilliarrangedin
Pneumococcus certain parts of cell wall (e.g. LPS) are immunogenic
Cluster:Staphylococcus
Tetrads andactasvirulencefactor.
Micrococcus Pleomorphic(various shapes) Haemophilus,Proteus
Chain:Streptococcus •Sarcina
Peptidoglycan is main component of the cell wall which makes it rigid. It is
Octate
Pairs,lanceolateshaped- Thumbprintappearance Bordetellapertussis
Pneumococcus composedof alternate units of N-acetyl muramic acid (NAM) and N-acetyl glucosamine
Pair or in short chain, Enterococcus Curved Campylobacter (Gull-
Tetrads:Micrococcus
spectacleeyedshaped (NAG)molecules; cross linked to each other via tetrapeptide side chains and
wingshaped)andHelicobact
Octate:Sarcina pentaglycinebridges. er
Gram-positivebacilliarrangedin
Pair,spectacleeyedshaped-
• Gram-positivebacteriahasathickerpeptidoglycanandcontainsteichoicacid
Spirally coiled, rigid Spirillum
Enterococcus
Chain(bamboostickappearance) •Bacillusanthracis
Section
Gram-negative bacteria-peptidogly can layer

Spirally coiled, flexible is thin, and it contains additional
Spirochetes
Chineseletterarrangement C.diphtheriae
partssuchas(1)Outermembrane,
Commashaped(fishinstream) Vibriocholerae
Pallisadearrangement Other(2)Lipopolysaccharide(LPS)whichinturnconsistsof(i)LipidAorendotoxin,
Corynebacteriumspecies Chain Streptobacillus
(ii)Corepolysaccharide,(iii)Osidechain
Branching GPB Actinomycetes
Table1.1.7:DifferencesbetweenGram-PositiveandGram-Negativecellwall
Characters Gram-Positivecellwall Gram-Negativecellwall

Peptidoglycanlayer Thicker(15–80nm) Thinner(2nm)
At3rdpositionoftetrapeptidesidechain L-Lysinepresent Mesodiaminopimelicacidpresent
Pentaglycinebridge Present Absent
Lipidcontent Nilorscanty(2–5%) Present (15–20%)
Contd...
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Contd...
Characters Gram-Positivecellwall Gram-Negativecellwall

Lipopolysaccharide Absent Present (endotoxin)
Teichoicacid Present Absent
Varietyofaminoacids Few Several
Aromaticaminoacids Absent Present
OtherPartsofBacterialCell
IntracytoplasmicInclusions
Theyarethestoragesitesofnutrients/bacteria,formedinnutritionaldeficiencyconditions:
GramPositivecellwall:
• Organicinclusionbodies,examplesincludeglycogengranulesandpoly- Thicker Peptidoglycan
hydroxylbutyrategranules. Absent:LPS,AromaticAAandlipids
• Inorganicinclusionbodies,examplesinclude: Present:Teichoicacid
○ PolymetaphosphateorvolutinormetachromaticgranulesinC.diphtheriae.
○ SuflurgranulesfoundinActinomyces.
Nucleoid
Bacteriadonothaveatruenucleus;butthegeneticmaterialislocatedinanirregularly
shapedregioncalledthenucleoid.
• Thereisnonuclearmembraneornucleolusandlacksbasicproteins.
•
Possessasinglehaploidchromosome,comprisingofsupercoiledcirculardsDNA(excep
ttwochromosomesinVibrio). Capsulatedbacteria:
• BacterialDNAdividesbysimplebinaryfission. Pneumococcus
• ThenucleoidcanbeseenbyelectronmicroscopyoronstainingwiththeFeulgenstain Meningococcus
• Bacteriaalsopossessextra-chromosomalDNAcalledplasmids. Haemophilusinfluenzae
Klebsiellapneumoniae
Pseudomonasaeruginosa
Capsuleand SlimeLayer Bacillusanthracis
Somebacteriapossessalayerofamorphousviscidmateriallyingoutsidethecellwallcalledglycoc Capsulatedfungus:
alyx which may be well organized (capsule) or unorganized loose material (slime Cryptococcus
layer).SomebacteriamaypossessbothcapsuleandslimelayerasinStreptococcussalivarius.
Table1.1.8:Examplesofcapsulatedorganisms
Thecapsulehasvariousfunctions
Capsulatedbacteria Composition Capsulatedbacteria Composition
• Pneumococcus
Actsbyinhibitingphagocytosisandcomplement-mediatedlysis
Polysaccharide Bacillusanthracis Polypeptide (glutamate)
• Meningococcus
Biofilmformationandtherebyhelpsinadherencetodamagedtissuesandplastic
Polysaccharide Streptococcuspyogenes(somestains) Hyaluronicacid
GeneralMicrobiolog
surfaces(e.g.medicaldevices)
Haemophilusinfluenzae Polysaccharide Bacteriodes fragilis Polysaccharide
• Sourceofnutrientsandenergyforthebacteria
• Klebsiellapneumoniae Polysaccharide Capsulatedfungus
Capsulesasvaccine,e.g.Pneumococcus,MeningococcusandHaemophilusinfluenzae
Pseudomonasaeruginosa Polysaccharide
serotype-bandS.TyphiViVaccine. Cryptococcusneoformans Polysaccharide
Demonstrationofcapsuleby
•
NegativestainingbyIndiainkandnigrosinstain:Capsuleappearsasaclearrefractilehaloaro
undthebacteria;whereasboththebacteriaandthebackgroundappearblack.
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• M’FaydeancapsulestainforBacillusanthracisbyusingpolychromemethylenebluestain.
• Serological test:Capsular materialisantigenic andcanbe demonstratedbymixing itwitha
specificanticapsularserum:
○ Quellungreactionfor Streptococcuspneumoniae
○ Latexagglutinationtestbyusingspecificanticapsularantibodiescoatedonlatex.
Flagella
Flagellaarethread-likeappendages,protrudingfromthecellwall,composedofprotein
subunitscalledflagellin.Ithasthreeparts:filament,kookandbasalbody.
• Size-Theymeasure5–20µminlengthand0.01–0.02µminthickness
• Theyareorgansoflocomotion,confermotilitytothebacteria.
Arrangementofflagella
• Monotrichous(singlepolarflagellum)e.g.Vibriocholerae,PseuodmonasandCamplylobacter
• Lophotrichous(multiplepolarflagella)e.g.Spirillum.
• Peritrichousovertheentirecellsurface)e.g.SalmonellaTyphi,Escherichiacoli.
• Amphitrichous(singleflagellumatboththeends)e.g.Alcaligenesfaecalis,Spirillum(Note:
Spirillumhastuffofflagellaatoneorboththeends(Amphi>lophotrichous)
Flagellacanbedemonstratedby
• Directdemonstrationofflagella
○ Tannicacidstaining(Leifson’smethodandRyu’smethod)
○ Darkground,phasecontrastorelectronmicroscope
• Indirectmeansbydemonstratingthemotility:
○ CragietubemethodandHangingdropmethod
○ Semisolidmedium,e.g.mannitolmotilitymedium.
Typesofmotility: Table1.1.9:Varioustypesofmotility
Tumbling:Listeria
Gliding:Mycoplasma FimbriaeorPili
Typesofmotility Bacteriashown Typesofmotility Bacteriashown
Stately:Clostridium ManyGram-negativeandsomeGram-positivebacteriapossessshort,fine,hair-
Tumblingmotility Listeria Dartingmotility Vibrio
Darting:Vibrio,Campylobacter
cholerae,Camp
likeappendagesthatarethinnerthanflagellaandnotinvolvedinmotility,calledfimbriaeorpili.T
Swarming: Proteus, C.tetani
heymeasure0.5µmlongand10nminthickness:
Gliding motility Mycoplasma ylobacter
Corkscrew,lashing,flexionextension:Spirochete
Swarming motility Proteus,Clostridiumtetani
• Stately
Piliaremade-upofproteincalledpilin
motility Clostridium
Corkscrew,lashing,flexionext Spirochete
Section
• Theyareantigenic;however,theantibodiesagainstfimbrialantigensarenotprotective.
ension
• Functions:Fimbriaearecalledtheorganofadhesion.Thispropertyenhancesthevirulenceofbac
teria.
○ Certainfimbriaecalledsexpilialsohelpinbacterialgenetransfer.
○ Fimbriaearenotrelatedtomotility,canbefoundbothinmotileaswellasinnon-
motileorganisms.
• Types
○ Commonpili:Theseareofsixtypes
○ SexorF(fertility)pili:Helpinbacterialconjugation,e.g.asfoundinGonococcus
○ ColI(colicin)pili.
• Detectionoffimbriae
○ DirectdemonstrationoffimbriaebyElectronmicroscopy
○ Indirectmethodstoknowthepresenceoffimbriaeare:
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▪ Hemagglutination: Many fimbriated bacteria (e.g. Escherichia coli,

Klebsiella)strongly agglutinate RBCs. The hemagglutination can be specifically
inhibitedbyD-mannose.
▪ Surfacepellicle,e.g.insomeaerobicfimbriatedbacteriaformathinlayeratthe
surfaceofabrothculture.
AtypicalformsofBacteria
•
Involutionforms:Swollenandaberrantformsofbacteria(e.g.GonococciandYersiniapestis
)inagingcultures
• Pleomorphicbacteria:Differentshapeandsizeofindividualcells,e.g.Proteusand
Yersiniapestis
•
LformorCellwalldeficientforms:Whenbacterialoosecellwall,theybecomesphericalcalledL
form.
○ DiscoveredbyE.Klieneberger,whilestudyingStreptobacillusmoniliformis
○ Itisnamedafteritsplaceofdiscovery,i.e.ListerInstitute,London
○ Lformsmediatespersistenceofpyelonephritisandotherchronicinfections.
○ LformsinGram-positivebacteriaarecalledProtoplastsandinGram- Involution forms:
negativebacteriaarecalledSpheroplasts. Swollen and aberrant forms ofbacteria
Forexample,Gonococciand
○ Yersiniapestis
Seeninagingcultures.
Mycoplasmadonothaveatruecellwall;thepeptidoglycanlayerisreplacedbysterol.I
tispostulatedthatMycoplasmamayrepresentstableLformsofyettobeunidentifiedpa
rentbacteria.Butmanyresearchersdonotconsiderthis.
BacterialSpores
Sporesarehighlyresistantresting(ordormant)stageofthebacteriaformedinunfavorableenvir
onmentalconditions as a result of the depletion of exogenous nutrients.
•
Structureofaspore:Frominnermosttowardstheoutermost,thelayersarecore→cortex→
coat→exosporium
•
Sporicidalagents:Sporesarehighlyresistant.Onlylimitedsterilizationmethodsareavaila
bletokillthespores
• Usedasindicatorforpropersterilization.
○ Sporesof Geobacillus stearothermophilusare used as sterilization control for auto-
clave.
○ Sporesofnon-
toxigenicstrainsofClostridiumtetaniareusedassterilizationcontrolforhotairoven.
• Usedasagentsofbioterrorism;e.g.SporesofBacillusanthracisinthe2001USAattack.
GeneralMicrobiolog
GenerationTime
PHYSIOLOGYOFBACTERIA(BACTERIALGROWTHAND Generation time:
NUTRITION)
Timerequiredforabacteriumtogiverisetotwodaughtercellsunderoptimumcondition. Escherichiacoli:20minutes
Mycobacterium tuberculosis:
• ForEscherichiacoliandmostoftheotherpathogenicbacteria,-itis20minutes;
14hours
• ForMycobacteriumtuberculosis:Itis14hours Mycobacterium leprae: 12–13days
• ForMycobacteriumleprae:Itis12–13days
BacterialCount
• Totalcount:Indicatestotalnumberofbacteria(liveordead)inthespecimen.Thisisdoneby
countingthebacteriaundermicroscopeusingcountingchamber.
• Viablecount:Measuresthenumberofliving(viable)cellsinthegivenspecimen.Viable
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countmaybeobtainedby:
○ Pourplatemethod(bestmethod)
○ Surfaceviablecountbyspreadingmethod
○ SurfaceviablecountbyMilesandMisramethod.
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BacterialGrowthCurve
Fig.1.1.1:Bacterialgrowthcurve
When a bacterium is inoculated into a suitable liquid culture medium and incubated,
itsgrowthfollowsa definite course. When bacterial count of such culture is
determinedat different intervals and plotted in relation to time, a bacterial growth curve is
obtainedcomprisingoffourphases(Table1.1.10givenbelow).
Table1.1.10:Phasesofbacterialgrowthcurve
Lag phase Log phase Stationary phase Declinephase

Bacteriadivide No Yes Yes No
Bacterialdeath No No Yes Yes
Totalcount Flat Raises Raises Flat
Viablecount Flat Raises Flat Falls
Special features Preparatoryphase Uniformlystained, Gram variable Produceinvolutionforms
Accumulation Metabolicallyactive Produce: Granules, spores,
ofenzymesand Small size exotoxin,antibiotics,bacteriocin
metabolites
Attains maximum size
atendoflagphase
FactorsAffectingGrowthofBacteria
Section
1. Oxygen:Onbasisoftheiroxygenrequirementsbacteriaareclassifiedas:
Bacterialgrowthcurve: • Obligateaerobes:Growonlyinthepresenceofoxygen(e.g.Pseudomonas,M.tuberculosis,Bac
Lag phase: ↑enzymes andmetabolites, Max cell size illus,BrucellaandNocardia)
Log phase: Cells uniformlystained,active • Facultativeanaerobes:Theyareaerobesthatcanalsogrowanaerobically(e.g.mostofthepat
Stationary phase: Producegranules, spores, exotoxin,antibiotics,bacteriocin
Decline phase: Produceinvolutionforms hogenicbacteria,e.g.E.coli,S.aureus,etc).
• Facultativeaerobes: They are anaerobes that can also grow aerobically (e.g.
Lactobacillus)
• Microaerophilicbacteria:Cangrowinthepresenceof5–10%ofoxygen(e.g.
Campylobacter,Helicobacter,Mycobacteriumbovis).
• Obligateanaerobes:Growonlyintheabsenceofoxygen.Oxygenislethaltothesebacteria(e.g.
Clostridium)
• Aerotolerantanaerobe:Theycantolerateoxygenforsometime,butdonotuseit(Clostridium
histolyticum).
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2. Carbondioxide:Capnophilicbacterianeed5–
10%ofCO2.E.g.Brucellaabortus,Streptococcuspneumoniae,etc.
3. Temperature:Basedon optimaltemperature neededfor growth,bacteriacan begrouped
into:
• Psychrophiles:Growbelow20°C,e.g.saprophytes.
• Mesophiles:Growbetween25°Cand40°C,e.g.mostofthepathogenicbacteria
• Thermophiles:Growaboveof55°C- 80°C,e.g.Bacillusstearothermophilus
4. pH:MostpathogenicbacteriagrowbetweenpH7.2-pH7.6.Veryfewbacteria(lactobacilli) can
grow at acidic pH below pH 4, while bacteria such as Vibrio cholerae
arecapableofgrowingatalkalinepH(8.2–8.9).
5. Light:Bacteria(exceptphototrophs)growwellindark.Theyaresensitivetoultravioletrays
and other radiations in light. Photochromogenic mycobacteria produce
pigmentsonlyonexposuretolight.
6. Otherfactors:OsmoticEffect,MechanicalandsonicStresses,Moistureanddesiccation.
MICROBIALPATHOGENICITY
Microbialpathogenicitydependsuponthesumtotalofseveralfactorsasdescribedbelow.
1. Routeoftransmissionofinfection
2. Infectivedoseoftheorganism:Minimuminoculumsizethatiscapableofinitiatinganinfect
ion.(tablegivenbelow)
Low infective dose Large infective dose
Shigella: Very low (as low as 10 Escherichiacoli(106–108bacilli)
bacilli)Cryptosporidium parvum: very low (10 to 30 Salmonella(102–105bacilli)
oocysts)EscherichiacoliO157:H7 (<10bacilli) Vibriocholerae(106–108bacilli)
EntamoebaandGiardia: fewcysts
Campylobacterjejuni(500bacilli)
3. Evasionofthelocaldefenses
4. Adhesion:ByFimbriaeorpili,otheradhesins,biofilmformation
5. Invasion:Virulencefactorsthathelpininvasioninclude:
• VirulencemarkerantigenorinvasionplasmidantigensinShigella
• Enzymes:Invasion of bacteria is enhanced by many enzymes: Hyaluronidase,
Collagenase,Streptokinase(fibrinolysin)andIgAproteases
• Antiphagocyticfactors:Capsule
• Cellwallproteinssuchas:ProteinAofStaphylococcusaureusandMproteinof
Streptococcuspyogenes
• Cytotoxins:Interfere with chemotaxis or killing of phagocytes. For example,
GeneralMicrobiolog
S.aureusproduceshemolysinsandleukocidinsthatlyseanddamageRBCsandWBCs.
Mechanismofintracellularsurvival Organism
Inhibitionofphagolysosomefusion Legionellaspecies,Mycobacteriumtuberculosis,Chlamydia
Resistancetolysosomalenzymes SalmonellaTyphimurium,Coxiella,Mycobacteriumleprae,Leishmania
Adaptation to cytoplasmic replication Listeria,RickettsiaandFrancisellatularensis
ExamplesofObligateintracellularorganisminclude:
• Bacteria:Mycobacteriumleprae,Rickettsia,ChlamydiaandCoxiella
• Allviruses
• Fungi:Pneumocystisjirovecii
• Parasite:Toxoplasma,Cryptosporidium,Plasmodium,Leishmania,Babesia,Trypanosomacruzi
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Table1.1.11:Differencesbetweenbacterialendotoxinsandexotoxins
Endotoxins Exotoxins
Lipopolysaccharidesinnature Proteininnature
PartofGram-negativebacterialcellwall SecretedbothbyGram-positiveandnegativebacteria;diffuseintosurroundingmedium
Releasedbycelllysis,Notbysecretion Activelysecretedbythebacteria
Highlystable Heatlabiledestroyedat60oC
Modeofaction–Induces↑IL-1andTNF Mostlyenzymelikeaction
Nonspecific(fever,shock,etc.) Specificactiononparticulartissues
Nospecificaffinityfortissues Specificaffinityfortissues
Onlylargedosesarefatal Morepotent,evensmallerdosesfatal
Poorlyantigenic Highlyantigenic
Neutralizationbyantibodiesisineffective Neutralizedbyspecificantibodies
Noeffectivevaccineisavailableusingendotoxin Toxoidformsareusedasvaccine,e.g.tetanustoxoid
Table1.1.12:Exotoxinsandtheirmechanismsofaction
Toxins(Exotoxins) Mechanism
EnterotoxinandTSSTofS.aureus Actassuperantigen;stimulateTcellnon-
Streptococcalpyrogenicexotoxin specifically,toreleaseoflargeamountsofcytokines.
DiphtheriatoxinandExotoxin-AofPseudomonas Inhibitsproteinsynthesis(byinhibitingelongationfactor-2)
Anthrax toxin ↑cAMP intargetcell,edema
αtoxinofClostridiumperfringens Lecithinaseandphospholipaseactivity→causesmyonecrosis
Tetanustoxin(tetanospasmin) Decrease in neurotransmitter (GABA and glycine) release from
theinhibitoryneurons→Spastic paralysis
Botulinum toxin Decreaseinneurotransmitter(acetylcholine)releasefromneurons→Flaccidparalysis
Heat labile toxin of ETEC Activationofadenylatecyclase→↑cAMPintargetcell→Secretorydiarrhea

andCholeratoxin(V.cholerae)
Heatstabletoxin ↑cGMPintargetcell→Secretorydiarrhea
Verocytotoxin(EHEC)and Inhibitproteinsynthesis(byinhibitingribosome)
Shigatoxin(Shigelladysenteriaetype-1)
Section
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MULTIPLECHOICEQUESTIONS
HISTORY 10. Swarmingisseenin: (NEETPatternBased)

a. Clostridiumtetani
1. Syphilisdiscoveredinwhichyear? (TNPG2015) b. Clostridiumperfringens
a. 1838 b. 1905 c. Clostridiumbotulinum
c. 1906 d. 1921 d. Clostridiumdifficile
2. All areKoch’spostulatesexcept: (JIPMER2014) 11. Whichisaeukaryote:(PGIDec2008,LatestMCQ2013)
a. A microorganism should be constantly a. Mycoplasma b. Bacteria
associatedwiththelesionsofthedisease c. Fungus d. Chlamydia
b. It should be possible to isolate the bacterium in
12. Roleof bacterial capsule is: (JIPMERMay2015)
pureculturefromthelesions
a. Support b. Transport
c. Inoculationofsuchpurecultureintolaboratoryanima
c. Antiphagocytic d. Adhesion
lsshouldreproducethelesions
d. Administrationofbroadspectrumantimicrobialagen 13. Capsulatedorganism: (PGIJune2003,DNBDec2011)
tdependablyeradicatestheorganismsandcuresthedi a. Candida b. Klebsiella
seases c. Proteus d. Cryptococcus
e. Histoplasma
3. Corynebacteriumdiphtheriaeisalsocalled:
(NEETPatternBased) 14. WhichisnotpresentinGram-negativebacteria:
a. Koch’sbacillus b. Kleb-Loefflerbacillus (TNPG2014)
c. Rouxbacillus d. Yersin bacillus a. Peptidoglycan b. Teichoic acid
c. LPS d. Porinchannels
4. Leeuwenhoek is associated with: (DNBDEC2012)
a. Telescope b. Microscope 15. Craigie’stube method is used to differentiate:
c. Stains d. Immunization a. Motileandnonmotilestrains (JIPMER2014)
b. Virulentandavirulentstrains
5. Theoryofwebofcausationwasgivenby?
c. Capsulatedandnoncapsulatedstrains
(DNBDEC2012)
d. Roughandsmoothstrains
a. McMohanandPugh b. Pettenkoffer
c. Johnsnow d. LouisPasteur
6. LouisPasteurisassociatedwithallEXCEPT:
STAININGTECHNIQUESANDMICROSCOPY
(DNBDEC2012) 16. Factoraffectingelectronmicroscopeis?
a. Vaccinationofsmallpox a. Eyepiece (AIIMSNov2016)
b. Germtheory b. Wavelength of the light
c. Pasteurization c. Typeofglasslensused
d. Vaccinationofrabies d. Thicknessofthesample
7. Bestchemicaldisinfectanttodisinfectstethoscopeis: 17. Resolvingpowerofmicroscopedependsonallexcept:
GeneralMicrobiolog
a. Isopropylalcohol (RecentQuestion2015) (AIIMSNov2016)
b. Ethyleneoxides a. Refractiveindexof medium
c. Halogenatedcompound b. Wavelength
d Steamplasmasterilization c. Diameterofaperture
d. Focallengthofobjectivelens
18. Whatisthereasonfordecolourisationingram-
BACTERIALCELLBIOLOGYANDSTRUCTUR
8. Whichisnotaprokaryote? (JIPMERNov2015)
negativebacilliinGramstaining(JIPMERMay2016)
a. Mycoplasma b. Rickettsiae
E a. Due to thetwo dyesused instaining
c. Shigella d. Entamoeba
b. Lipid content
9. Nonmotile: (RecentMCQ2014,NEETPatternBased) c. Cellmembraneintegrity
a. Shigella b. E.coli d. Teichoicacid
c. Proteus d. Vibrio
19. CompositionofZNstainareallEXCEPT:
(RecentQuestions2014)
a. Basicfuchsin b. Acidfuchsin
c. Phenol d. Alcohol
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20. Acidfastbacilli–stainedby: (TNPG2015)

28. Correctsequenceofbacterialgrowthcurve:
a. Ziehl–Neelsen b. Albert
(PGIDec2007)
c. Neisser d. Ponder
a. Logphase–LagPhase–Stationaryphase–
21. CorrectsequenceinGramstaining?(LatestMCQ2013) Declinephase
a. MethylvioletIodineAcetoneCarbolfuchsin b. LagPhase–Logphase–Stationaryphase–
b. CarbolfuchsinIodineAcetoneMethylviolet Declinephase
c. MethylvioletAcetoneIodineCarbolfuchsin c. Stationaryphase–Logphase–LagPhase–
d. MethylvioletCarbolfuchsinAcetoneIodine Declinephase
22. Notusedingramsstaining: (DNBDEC2012) d. Lag phase – Exponential phase – Log phase –
a. Methylene blue b. Crystalviolet Deathphase
c. Iodine d. Safranin e. Exponentialphase–Lagphase–Deathphase–
23. Indianinkstainingispositiveforwhichofthefollowing Stationary
cocci: (PGINov2014,TNPG2014) 29. Populationdoublingtimeincoliformbacilliis:
a. Pneumococcus b. Staphylococcus (MHPG2014)
c. Meningococcus d. Gonococcus a. 20seconds b. 20minutes
e. Enterococcus c. 20hours d. 20days
24. WhichofthefollowingaboutGramstainingismostcorrec 30. Phototropismmeans: (RecentQuestions2014)
t: (LatestQuestion2013) a. Growingtowards the sunlight
a. Heatingisdonetopromoteentryofstainintocell b. Obtainingenergyfromsunlight
wallofbothGram+veand–vebacteria c. Reflectingenergyfromlightsource
b. Ifobservedstainingtechniqueandmicroscopybefore d. None ofthe above
decolorization,bothGram+veand–
vebacteriaappearsame MICROBIALPATHOGENESIS
c. Teichoic acid is present in the cell wall of Gram–
vebacteria 31. WhichorganismgrowsinalkalinepH?
(NEETPatternBased)
BACTERIALGROWTHANDNUTRITION a. Klebsiella b. Vibrio
c. Pseudomonas d. E.coli
25. Whicheventtakes placelag phaseof growthcurve? 32. Obligateintracellularorganismis:
(RecentQuestion2015) (NEETPatternBased,AI2005)
a. Bacterialcellnumberincrease a. Mycoplasma b. Chlamydia
b. Bacterialcellsizeincrease c. Cryptococcus d. Helicobacter
c. Sporulation 33. Trueaboutexotoxin: (RecentMCQ2014)
26. Whichofthefollowingismicroaerophilic: a. LPSinnature b. Heat stable
(AIIMS May 2009) c. Notantigenic d. Toxoidcanbeprepared
a. Campylobacter 34. Allthefollowingaretrueregardingexotoxinexcept:
b. Vibrio a. Highlyantigenic (JIPMER2014)
c. Bacteroides b. Heatstable
d. Pseudomonas c. Neutralizedbyantibody
27. The termviable notcultivable isusedfor: d. Activeinveryminutedoses
Section
(PGIDec2007) 35. Allthefollowingstatementsaretrueregardingendotoxi

a. M.leprae nsexcept: (JIPMER,May2015)
b. M.tuberculosis a. Neutralizedbyspecificantibodies
c. Treponemapallidum b. Heatstable
d. Salmonella c. Somaticantigen
e. Staphylococcus d. Poorlyantigenic
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EXPLANATIONS
HISTORY
1. Ans.(b)(1905)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep5,Ananthanarayan9th/p371
• Treponemapallidum,theagentofsyphiliswasdiscoveredon1905bySchaudinnandHoffmann.
2. Ans.(d)Administrationofbroadspectrumantimicrobialagentdependablyeradicatestheorganismsandcuresthedis-
easesRefertextRef:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep4,Ananthanarayan9/ep5
3. Ans.(b)(Kleb-Loeffler’s...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep5,Ananthanarayan9/ep236
• CorynebacteriumdiphtheriaeisalsocalledKlebs-Loefflerbacillus
4. Ans.(b)(Microscope)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep3,Ananthanarayan9/ep3,8/
ep3AntonyvanLeeuwenhoekfromHollandwasthefirstpersontoobserveanddescribemicroorganismsaccuratelybyusinglenses
andwasconsideredasthefounderofsimplemicroscope(1694).
• ZachariasJansenandhisfather–describedthefirstcompoundmicroscope
• ErnstRuska:InventedElectronMicroscopein1931.
5. Ans.(a)(McMohanandPugh)Ref:Park21/ep32
Conceptofcausationofdisease:
• Spontaneousgenerationoflife:Lifecanbecreatedbychemicalreactions.ItwasdisprovedbyLouisPasteur.
• Germtheoryoflifeorsinglecausetheory(LouisPasteur)-onetoonerelationshipbetweencausativeagentand
disease, i.e. [Agent→Man→ Disease].It sufferedcritics asNot everyone exposedto anagent developsdisease.
• Multifactorialcausation:ProposedbyPettenkoferandMunich.
• Theoryofwebofcausation:SuggestedbyMacMahon,Pugh,andIpsen(1960)intheirbookEpidemiologicprinciplesand methods’
that disease is due to the result of complex interrelationship between all the predisposing factors
withvariablerelativerisk.
6. Ans.(a)(Vaccinationof..)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep3,Ananthanarayan9/ep4,8/ep4
• Vaccinationof smallpox by using cowpoxwas proposed by Edward Jenner.
• LouisPasteurpreparedthe vaccinesfor– Anthrax,Rabies,Cholera (CAR)
• ForothercontributionsofLouisPasteur-Refertext.
7. Ans(a)(Isopropylalcohol)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep42
Low-leveldisinfectantsuchasIsopropylalcoholshouldbeenoughasstethoscopeisanoncriticalitemaccordingto
Spaulding’sclassification.
GeneralMicrobiolog
BACTERIALCELLBIOLOGYANDSTRUCTURE
8. Ans(d)(Entamoeba)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep9
Parasitesandfungiareeukaryotes(Entamoebaisaparasite);whereasbacteriaandbluegreenalgaeareprokaryotes.
9. Ans.(a)(Shigella)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep298,Ananthanarayan9/ep284
• Shigella,KlebsiellaareNonMotileamongGramnegativeEnterobacteriaceaeorganisms.
10. Ans.(a)(C.tetani)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep310,Ananthanarayan9/ep259
• Swarmingisexhibitedby-Cl.tetani,Proteus,VibrioparahemolyticusandVibrioalginolyticus
11. Ans.(c)(fungus)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep9,Ananthanarayan9e/p427,8/ep425
• Prokaryotes–includebacteriaandbluegreenalgae
• Eukaryotes–includefungi,algae(otherthanbluegreen),protozoa,helminthsandslimemoulds
• Seethetablefromthetextfordetail.
12. Ans.(c>d)(Antiphagocytic>Adhesion)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep21
• Themainroleofbacterialcapsuleistoescapefromphagocytosis.Capsulealsohelpsinbiofilmformationandadhesion.
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13. Ans.(b)and(d)(KlebsiellaandCryptococcus)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep21
• Listofcapsulatedbacteria-Refertext
14. Ans.(b)(Teichoicacid)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep18,Ananthnarayan9/ep15
• Referchapterreview.
15. Ans.(a)Motile..Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep319,Ananthanarayan9/ep293
STAININGTECHNIQUESANDMICROSCOPY
16. Ans(d)(Thicknessofthesample)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p13
• InordertogetvisualizedbyEM,thespecimenshouldbeverythin(20to100nmthickness).
17. Ans(d)(Focallengthofobjectivelens) Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p10
• Resolvingpowerofmicroscopedependsonwavelengthandnumericalaperture(thelatterisinturndependsonrefractiv
eindexandangularaperture).
18. Ans.(b)(lipidcontent)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p15
Gramnegativebacteriagetsdecolorizedeasilyduetofollowingfactors-1)Thinpeptidoglycanlayerandlesscrosslink-
ing,2)Presenceoflipidrichlipopolysaccharidelayerwhichgetseasilydestroyedbydecolorizer.
19. Ans.(b)(Acidfuchsin)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep16,Ananthanarayan9/
ep14ZNstainhasthreecomponents:
• Primarystain:Basicfuchsin+Phenol
• Decolorizer:Sulfuricacidoracid-alcohol
• Counterstain:MethyleneblueorMalachiteGreen
20. Ans.(a)(Ziehl–Neelsen)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep16,Ananthanarayan9/
ep13Referchapterreviewofchapter1.1
21. Ans.(a)(Methyl..)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep14-15,Ananthanarayan9/ep13,8/
ep15Referchapterreview
22. Ans.(a)(Methyleneblue)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep14-15,Ananthanarayan9/ep13
• Gramstainhasfoursteps(referchapterreview)
• Methyleneblueisusedacounterstainforacidfaststain
23. Ans.(a)(c)(Pneumococcus,Meningococcus)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep21Indiainkstainisusedtodemonstratethecapsule.Amongcocci,Pneumococcus,Meningococcusarecapsulated.
24. Ans.(b)(Ifobservedstaimingtechniquemicroscopybeforedecolorization,bothGram+veand–vebacteriaappearsame)
Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep15,Ananthanarayan9/ep13.
• In Gramstain:
○ Underdecolorization:Everythingwilllookgram-positive(violet)
○ Overdecolorization:Everythingwilllookgram-negative(pink)
Section
• Heating(Intermittentheating)isdoneinacidfaststain(notforgramstain)
BACTERIALGROWTHANDNUTRITION
25. Ans.(b)(Bacterialcellsizeincrease)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep27
26. Ans.(a)(Campylobacter)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep28,Ananthanarayan9/ep24
• Microaerophilic:Requiresmallamountofoxygen(5%)e.g.Campylobacter,HelicobacterandMycobacteriumbovis.
• Fordetailexplanation:Refertext
27. Ans.(a)and(c)(M.lepareandTreponemapallidum)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep4,397Alreadyexplained
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28. Ans.(b)(Lag-Log-Stationary-Decline)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep27
• Fordetailexplanation-refertext
29. Ans.(b)(20minutes)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep26,Ananthanarayan9/
ep22Populationdoublingtimeorthegenerationtimeof-E.coli-20min,M.tuberculosis-20hrandM.leprae-20days
30. Ans.(b)(Obtainingenergy...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p28,Ananthanarayan9/
ep23Referchapterreviewfordetail.
MICROBIALPATHOGENESIS
31. Ans.(b)(Vibrio)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep28,Ananthanarayan9/ep25,8/ep24
• Vibrio survives inalkaline pH of8.6
32. Ans.(b)(Chlamydia)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
33. Ans(d)(Toxoidcan...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep93,Ananthanarayan9/ep75Refertext
34. Ans.(b)(Heatstable)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep93,Ananthanarayan9/ep75
35. Ans(a)(Neutralizedbyspecificantibodies)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep93
GeneralMicrobiolog
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CHAPTER
SterilizationandDisinfection 1.2
DEFINITIONS
Sterilization: It is the process by which all living microorganisms including viable spores,
Definitions: aredestroyed with reduction of at least 10 6log colony forming units (CFU) of
Sterilization: Destroys allmicrobesincludingspore
microorganismsandtheirspores.
Disinfection: Destroys allmicrobesexceptspore
Disinfection: It refers to a process that destroys or removes most if not all
Asepsis: Chemical agentsappliedtobodysurfaces
Decontamination (or Sanitiza-tion): Makes items as safe tohandle. but not bacterial spores with reduction of at least 10 3 log CFU of
pathogenicorganisms
microorganismbutnotspores.
Asepsis: It is a process where the chemical agents (called antiseptics) applied to body
surfaces(skin) will kill or inhibit the pathogenic microorganisms (and also commensals)
present onskin.
Decontamination (or Sanitization): It refers to the reduction of pathogenic microbes to
alevelatwhichitemsareconsideredassafetohandlewithoutprotectiveattirewithreductionofatleast
1logCFUofmicroorganismbutnotspores.
FactorsInfluencingEfficacyofSterilant/Disinfectant
Theefficiencyofasterilant/disinfectantantimicrobialagentisaffectedbyatfollowing
factors:
1. Organismload:Largermicrobialpopulationrequiresalongertimetodiethanasmallerone.
2. Natureoforganisms:Itgreatlyinfluencestheefficacyofthedisinfectants
3. Concentrationofthechemicalagentandtemperatureofthephysicalagent
4. Natureofthesterilant/disinfectant:
• Microbicidalability,Rapidityofaction,Residualactivity.
• Abilitytoactinpresenceoforganicmattersuchaspus,blood,andstool.
5. Durationofexposure:Moreistheexposuretime,betteristheefficacy.
6. pH:HeatkillsmorereadilyatanacidicpH
7. Biofilmformation:Preventstheentryofdisinfectants.
Table1.2.1:Classificationofsterilization/disinfectionmethods.
A.Physicalmethods
1. Heat
Dry heat: Flaming, Incineration and Hot air
ovenMoistheat:
a. Temperature<100°C,e.g.pasteurization,waterbathandinspissation
b. Temperatureat100°C,e.g.boiling,steamingandtyndallization
c. Temperature>100°C,e.g.autoclave
2. Filtration:Depthfiltersandmembranefilters
3. Radiation
Ionizingradiation:Y-rays,X-raysandcosmicrays
Non-ionizingradiation:Ultraviolet(UV)andinfraredrays
4. Ultrasonicvibration
B.Chemicalmethods
1.Alcohols:Ethylalcohol,isopropylalcohol
2.Aldehydes:Formaldehyde,glutaraldehyde,Ortho-phthaladehyde
3.Phenoliccompounds:Cresol,lysol,chlorhexidine,chloroxylenol,hexachlorophene
4.Halogens:Chlorine,iodine,iodophors
5.Oxidisingagents:Hydrogenperoxide,Peraceticacid
Contd...
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SterilizationandDisinfection21
Contd...
B.Chemicalmethods
6.Salts: Mercuricchloride, copper salts
7.Surfaceactiveagents:Quaternaryammoniumcompoundsandsoaps
8.Dyes:Anilinedyesandacridinedyes
9.Gassterilization:Lowtemperaturesteamformaldehyde,Ethyleneoxide(ETO)andBeta-
propiolactone(BPL)
PHYSICALMETHODSOFSTERILIZATION/DISINFECTION Mechanism of action of heatsterilization:

Dryheatkillstheorganismsby
HeatSterilization/Disinfection (CODE):
Charring
MechanismofAction Oxidativedamage
• DryheatkillstheorganismsbyCharring,Oxidativedamage,Denaturationofbacterialprotei Denaturationofbacterialprotein
nandElevatedlevelsofelectrolytes(CODE). Elevated levels of electro-lytes.
Moist heat kills the microor-ganismsbydenaturationandco
• Moistheatkillsthemicroorganismsbydenaturationandcoagulationofproteins.
DryHeat(Hotairoven)
• Holdingtemperaturerequired:160°Cfor2hours
• Materialssterilized:Hotairovenisbestmethodforsterilizationof:
○ Glasswarelikeglasssyringes,petridishes,flasks,pipettesandtesttubes.
○ Surgicalinstrumentslikescalpels,scissors,forceps,etc.
○ Chemicalssuchasliquidparaffin,fats,glycerol,andglovedustpowder,etc.
• Sterilizationcontrol:
○ Spores(106)ofnontoxigenicstrainsofClostridiumtetaniorBacillussubtilissubsp.
niger
○ ThermocouplesandBrowne’stube.
MoistHeatataTemperaturebelow100ºC
• Pasteurization:Usedforperishablebeverageslikefruitandvegetablejuices,beer,anddair
yproductssuchasmilk.
○ Twomethods:Holdermethod(63ºCfor30min)andFlashmethod(72ºCfor20sec
followedbycoolingto13ºC). Pasteurization: Used forbeverages and dairy pro
○ All nonsporing pathogens are killed except Coxiella burnetii which may survive Holdermethod(63°Cfor30
min)
inholdermethod.
Flash method (72°C for 20 secfollowedbycoolingto13°C).
• Waterbath:Usedfordisinfectionofserum,bodyfluids,andvaccines(60°Cforonehour) All nonsporing pathogens arekilledexceptCoxiellaburnetii
• Inspissation(Fractionalsterilization):
○ Itisaprocessofheatinganarticleon3successivedaysat80–85ºCfor30min
○ Usedforsterilizationofeggbased(LJandDorset’seggmedium)andserumbased
GeneralMicrobiolog
media(Loeffler’sserumslope).
MoistHeatataTemperatureof100ºC
• Boiling: Boiling of the items in water for 15 minutes may kill most of the
vegetativeformsbutnotthespores.
• Steaming: Koch’s or Arnold’s steam sterilizers are used to provide temperature of
100°Cfor 90 minutes. It is useful for those media which are decomposed at high
temperature ofautoclave.Itkillsmostofthevegetativeformsbutnotthespores.
• Tyndallization or intermittent sterilization: Involves steaming at 100°C for 20 min for
3consecutive days. It is used for sterilization of gelatin and egg, serum or sugar
containingmedia.Itkillsmostofthevegetativeformsincludingspores.
MoistHeatataTemperatureabove100ºC(Autoclave)
• Principle: Autoclave functions similar to a pressure cooker. At normal pressure,
waterboilsat100°Cbutwhenpressureinsideaclosedvesselincreases,thetemperatureatwhich
waterboilsalsoincreases.
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• Sterilizationconditions:121°Cfor15minatpressureof15psi(mostcommonlyused).
• Uses of autoclave: Autoclave is useful for surgical instruments and culture media
andthose materials which cannot withstand the higher temperature of hot air oven or
SterilizationConditions:
Hotairoven:160°Cfor2hours mediacontainingwaterthatcannotbesterilizedbydryheat.
•
Autoclave: 121°C for 15 min atpressureof15psi.Sterilizationcontrol:
○ Biologicalindicator-SporesofGeobacillusstearothermophilus(bestindicator)
○ ThermocoupleandindicatorslikeBrowne’stube,Autoclavetapes:
Filtration
Filtration is an excellent way to remove the microbial population in solutions of heat-
labilematerialslikevaccine,antibiotics,toxin,serumandsugarsolutionaswellasforpurification
ofairinlaminarairflowsystems.
Therearetwotypesoffilters;depthandmembranefilters.
Depth filters: They are porous filters that retain particles throughout the depth of the
filter,ratherthanjustonthesurface.Theyareusedforindustrialapplicationssuchasfiltrationoffo
odandbeverage,andchemicals,butarenottofilterbacteria.Examples include:
• Candlefiltersmadeupofdiatomaceousearth(Berkefeldfilters),unglazedporcelain(Chamb
erlandfilters)
• Asbestosfilters(SeitzandSterimatfilters)
• Sinteredglassfilters
AirFiltration: Membranefilters:Theyarewidelyusedfiltersforbacterialfiltration.Theyareporous;retain
alltheparticlesonthesurfacethataresmallerthantheirporesize.
HEPAfilter removes 99.97%ofparticlesofsize≥0.3µm.
ULPA filters: Removes fromthe air 99.999% of particles ofsize≥ 0.12µm.
• Madeupofcelluloseacetate,cellulosenitrate,polycarbonate,polyvinylidenefluoride
• Poresize:Membranefiltershaveanaverageporediameterof0.22µm(MCused)
• Filtrationofair:Airfiltersaremembranefiltersusedtodeliverbacteria-freeair.Examples:
○ Surgicalmasks(thatletairinbutkeepmicroorganismsout).
○ Inbiologicalsafetycabinetsandlaminarairflowsystems;twofiltersareused
▪ HEPA filters (High-efficiency particulate air filters): HEPA filter removes
99.97%ofparticlesofsize≥0.3µm.
▪ ULPA filters (Ultra-lowparticulate/penetrationairfilters):Removesfromtheair
99.999%ofparticlesofsize ≥0.12µm.
• SterilizationcontrolincludesBrevundimonasdiminutaandSerratiamarcescens.
Radiation
Ionisingradiations:
• Examplesinclude,X-rays,gammarays(fromCobalt60source),andcosmicrays.
• Mechanism:ItcausesbreakageofDNAwithouttemperaturerise(hencecalledascoldsterili
zation).
• Itdestroyssporesandvegetativecells,butnoteffectiveagainstviruses.Itisusedfor:
○ Disposableplastics,e.g.rubberorplasticsyringes,infusionsetsandcatheters.
Section
○ Catgutsutures,boneandtissuegraftsandadhesivedressings,antibioticsandhor
mones.
• AdvantagesofIonizingradiation:
○ Highpenetratingpower,
○ Rapidityofactionand
○ Temperatureisnotraised
• Sterilizationcontrol:EfficacyofionisingradiationistestedbyusingBacilluspumilus.
Nonionizingradiation:
• Examplesofnonionizingradiationincludeinfraredandultravioletradiations.
• Theyarequitelethalbutdonotpenetrateglass,dirtfilms,water.
• Dose:250–300nmwavelengthfor30min
• Usedforsterilizationofcleansurfacesinoperationtheatres,laminarflowhoodsaswell
asforwatertreatment.
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CHEMICALAGENTSOFSTERILIZATION/DISINFECTION
Table1.2.2:Classificationofchemicaldisinfectantsbasedontheirefficacy
Level Bacterial Tubercle Nonenveloped Enveloped Vegetative
ofdisinfecta spores bacilli viruses Fungi viruses bacteria
nt
Low No No No +/- Yes Yes
leveldisinfec
tant
Intermediate No Yes Yes Yes Yes Yes
leveldisinfectant
High Maybe Yes Yes Yes Yes Yes
leveldisinfec
tant
Chemicalsterilants Yes Yes Yes Yes Yes Yes
Alcohols
• Theyactonbacteria,fungi,someenvelopedviru(e.g.HIV);butnotspores.
• Theyactbydenaturingproteinsandpossiblybydissolvingmembranelipids.
• Ethylalcoholisusedassurgicalspirit(70%)inhandrubsasantiseptics.
• Isopropylalcohol:Usedforclinicalthermometers.
Aldehydes
Theycombinewithnucleicacidsandproteinsandinactivatethem,probablybycrosslinkingandal Glutaraldehyde:
kylatingthemolecules.Theyaresporicidalandcanbeusedaschemicalsterilants. Used as 2% concentration (2%cidex)for 20min
1. Formaldehyde:Itisbestusedfor: It has to be activated byalkalinisationbeforeuseOnceactiva
• Preservationofanatomicalspecimen
• Formaldehydegasisusedforfumigationofclosedareassuchasoperationtheaters
• Preparationoftoxoidfromtoxin.Itistoxic,irritantandcorrosivetometals.
2. Glutaraldehydeislesstoxic,lessirritantandlesscorrosive,henceisbestusedtosteri-
lizeendoscopesandcystoscopes:
• Itisusedas2%concentration(2%cidex)for20min.
• Ithastobeactivatedbyalkalinizationbeforeuse.Onceactivated,itremainsactiveonlyfo
r14days.
3. Ortho-Phthalaldehyde(0.55%):Itcanalsobeusedforsterilizationofendoscopesand
cystoscopesandhasmanyadvantagesoverglutaraldehyde:
• Itdoesnotrequireactivation
• Lowvaporproperty
• Betterodor
• Morestableduringstorage GeneralMicrobiolog
• ↑mycobactericidalactivity.
PhenolicCompounds
Phenolicsasdisinfectants:Cresol,xylenol,Lysolandortho-
phenylphenolareusedasdisinfectantsinlaboratoriesandhospitals.
• Allhavetheabilitytoretainactivityinpresenceoforganicmatter.
• Theyaretoxicandirritanttoskin,henceusedasdisinfectantsbutnotasantiseptics.Phenolicsasa
ntiseptics:Certainphenolicsarelessirritanttoskin,persistinskinforlongerperiodandarewidelyuse
dasantiseptics.Ingeneraltheyaremoreactiveagainstgram-positivethangram-negativebacteria.
• Chlorhexidine:Itisanactiveingredientofsavlon(chlorhexidineandcetrimide)
• Chloroxylenol:Itisanactiveingredientofdettol.
• Hexachlorophane:Asitcancausebraindamage,henceitsuseasantisepticisrestrictedonlyt
oastaphylococcaloutbreak.
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Halogens
Iodine:Itisusedasaskinantisepticandkillsmicroorganismsbyoxidizingcellconstituentsand
iodinating cell proteins, e.g. Tincture of iodine (2%) and Iodophor (iodine complexed
withanorganiccarrier)e.g.Betadine.
Chlorine:Itisthemostcommonlyuseddisinfectant:
• For municipal water supplies and swimming pools and is also employed in the
dairyandfoodindustries
• Aslaboratorydisinfectant
• As bleaching agent: to remove the stain from clothes. (Common uses of chlorine
aregivenintablebelow).
• Preparations:Itmaybeavailableas:(i)chlorinegas,(ii)sodiumhypochlorite
(householdbleach,5.25%),or(iii)calciumhypochlorite(bleachingpowder)
• Mechanisms:Allpreparationsyieldhypochlorousacid(HClO)whichcausesoxidative
destruction ofvegetative bacteriaand fungi,but not spores.
• Disadvantages:
○ Organicmatterinterfereswithitsaction,henceexcesschlorinealwaysisadd-
edtowatertoensuremicrobialdestruction
○ Carcinogenic
○ Needdailypreparation
○ TheyarenotactiveagainstGiardiaandCryptosporidium,
○ Sodiumhypochloriteiscorrosiveandshouldbehandledcautiously.
OxidisingAgents
HydrogenPeroxide(H2O2)
• Modeofaction:Itisachemicalsterilant,actsbyliberatingtoxicfreehydroxylradicalswhichat
tackmembrane,lipid,DNA,andothercellularcomponents.
• ConcentrationofH2O23–
6%isideal,exceptforcatalaseproducingorganismsandsporeswhichrequire10%ofH2O2.
• Usedtodisinfectventilator,softcontactlenses,andtonometerbiprisms.VaporizedH 2O2
isusedforplasmasterilization.
• Advantage:1.Itactsperfecteveninpresenceoforganicmatter2.Lowtoxicity
3.Environmentallysafe.
PeraceticAcid
Itisachemicalsterilant;oftenusedinconjunctionwithH 2O2,todisinfecthemodialyzersandin
plasma sterilization. It is also used for sterilizing endoscopes. However, it may
corrodesteel,iron,copper,brassandbronze.
PlasmaSterilization
Plasmasterilization:
Section
Activeagent:H2O2and/or:peraceticacid Thisisrecentlyintroducedsterilizationdevice(e.g.SterradandPlazlyte)usedforcreatingplas
Bestforheatlabilesurgicalinstruments as lowmastate,soastomaintainauniformvacuuminsidethechamber.
tempmaintained(< 50°C)
Sterilization control:Geobacillusstearothermophilus.
• Chemicalsterilantssuch asH2O2aloneor amixture ofH2O2and peraceticacid
• Active agent is Ultraviolet (UV) photons and radicals (e.g., O and OH): Kill
microorganismsandspores.
• Lowtemperatureismaintained(<50°C),Sobestforheatlabilesurgicalinstruments.
• Sterilizationcontrol:Geobacillusstearothermophilus,Bacillussubtilissubsp.niger.
HeavyMetalSalts
Heavymetallicsaltsareoflimiteduseincertainareas:
• Silversulfadiazineisusedonburnssurfaces.
• Silvernitrate(1%)solutionusedforeyesofinfantstopreventophthalmianeonatorum.
• Coppersulfate isan effectivefungicide (algicide)in lakes andswimming pools.
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• Mercury salts such as mercuric chloride, thiomersal and mercurochrome were

knownantisepticsinpast.Thiomersal(merthiolate)isusedaspreservativeinvaccinesandsera.
• Mechanism of action: Heavy metals combine with bacterial cell proteins, often with
theirsulphydryl groups, and inactivate them. They may also precipitate cell proteins.
Manyheavymetalsaremorebacteriostaticthanbactericidal.
SurfaceActiveAgents
Theylowerthesurfacetensionbetweentwoliquidsorbetweenaliquidandasolid.Surfactants
may act as detergents, wetting agents, and emulsifiers because they have
bothpolarhydrophilicandnonpolarhydrophobicends.
1. Cationicsurfactants(Quaternaryammoniumcompounds):
• Theydisruptmicrobialmembranesandmayalsodenatureproteins.
• They kill most bacteria (gram-positives are better killed than gram-negatives)
butnotM.tuberculosisorspores.
• NontoxicbutareinactivatedbyacidicpH,organicmatter,hardwaterandsoap.
• Cationicdetergentsareoftenusedasdisinfectantsforfoodutensilsandsmallinstrumen
tsandasskinantiseptics.
• Examplesinclude:
○ Acetyltrimethylammoniumbromide(cetavlonorcetrimide)
○ Alkyltrimethylammoniumsalts
○ BenzalkoniumchlorideandCetylpyridiniumchloride
2. Anionicsurfactants,e.g.soaps,havestrongdetergentbutweakantimicrobialproperties.They
areactiveatacidicpH.
3. Theamphotericsurfactants:Theyhavebothdetergentandantimicrobialactivity.
• TheyareactiveoverawiderangeofpH,butisreducedinpresenceoforganicmatter.
• E.g.‘Tegocompounds’:Usedasantisepticsindentalpractice,butcauseallergicreactions.
Dyes
Anilineandacridinedyeshavebeenusedextensivelyasskinandwoundantiseptics. Cationic surfactants(Quaternary ammoniumcom
Acetyltrimethylammonium
1. Anilinedyes:E.g.crystalviolet,gentianviolet,brilliantgreenandmalachitegreen: bromide(cetavlonorcetrimide)
• They are more active against gram-positive bacteria than gram-negative and Alkyltrimethylammonium salts
havenoactivityagainstM.tuberculosis. Benzalkonium chloride andCetylpyridiniumchloride
• Theyarenon-toxicandnon-irritanttothetissues. Anionicsurfactants,e.g.soaps
Theamphotericsurfactants,e.g.Tegocompounds.
• Theiractivityisreducedinpresenceoforganicmaterialsuchaspus.
• Theyinterferewiththesynthesisofpeptidoglycancomponentofthecellwall.
• Thesedyesareusedinthelaboratoryasselectiveagentsinculturemedia(e.g.malachitegree
ninLJmedium)
GeneralMicrobiolog
2. Acridinedyes:Theseincludeacriflavine,euflavine,proflavineandaminacrine:
• Theyareaffectedverylittlebythepresenceoforganicmaterial.
• Moreactiveagainstgram-positivebacteriabutarenotasselectiveastheanilinedyes.
• Theyinterferewiththesynthesisofnucleicacidsandproteinsinbacterialcells.
GaseousSterilization
EthyleneOxide(ETO)
Ethyleneoxidesterilizerisoneofthewidelyusedgaseouschemicalsterilantsinpresentdays.
• Ithashighpenetrationpower,hasbothmicrobicidalandsporicidalactivity;actsby
combiningwithcellproteins.
• However,itishighlyinflammable,irritant,explosiveandcarcinogenic.Henceitisusuallysupplie
dina10to20%concentrationmixedwithinertgases.
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•
Sterilizationcondition:5to8hoursat38°Cor3to4hoursat54°C.
•
Sterilizationcontrol:Bacillusglobigii.
•
Use:Forsterilizationofmanyheatsensitiveitemssuchasdisposableplasticpetridishesandsyrin
Prionsarethemostresistantstructure. Recommendedmethodsare:
Autoclavingat134°Cfor1–1.5 ges,heart-lungmachine,sutures,catheters,respiratorsanddentalequipments.
hour, Thedecreasingorderofresistanceofmicroorganismstodisinfectantorsterilizingagentsisasfollows:
Treatmentwith1NNaOHfor1hour Prions (highest resistance) > Cryptosporidium oocysts > Coccidian cyst > Bacterial spores >Mycobacteria>Otherparasitecysts(Giard
0.5%sodiumhypochloritefor2hours.
Sporicidalagentsinclude:
EFGH:Ethyleneoxide,Formaldehyde,Glutaraldehyde,Hydrogenperoxide.
3P:Peraceticacid,O-PhthalicacidandPlasmasterilization.
AutoclaveandHotairoven.
TestingofDisinfectants: TESTINGOFDISINFECTANTS
Phenol coefficient (RidealWalker)test
ChickMartintest 1. Phenolcoefficient(RidealWalker)test:
Capacity(Kelsey-Sykes)test • Determined by the dilution of the disinfectant in question which sterilizes
In-use (Kelsey and Maurer)test. thesuspension of Salmonella Typhi in a given time divided by the dilution of
phenolwhichsterilizesthesuspensioninthesametime.
• Phenolcoefficientof>1istakenassatisfactory.
• Drawbacks:
○ Onlythephenoliccompoundscanbeassessed
○ Itdoesnotassessdisinfectantabilitytoactinpresenceoforganicmatter.
2. ChickMartintest:Modifiedridealandwalkertestinwhichthedisinfectantsactinthe
presenceoforganicmatter(e.g.driedyeast,feces,etc.)tosimulatethenaturalconditions.
3. Capacity (Kelsey-Sykes) test: It tests the capacity of a disinfectant to retain its
activitywhenrepeatedlyusedmicrobiologically.
4. In-
use(KelseyandMaurer)test:Itdetermineswhetherthechosendisinfectantiseffectiveinactua
luseinhospitalpractice.
Table1.2.3:Biologicalsterilizationindicator
Hotairoven Clostridiumtetaninontoxigenicstrain,B.subtilissubsp.niger
Autoclave Geobacillus stearothermophilus
Filtration Brevundimonasdiminuta,Serratia
Ionizing radiation Bacilluspumilus
Section
Ethyleneoxide Bacillusglobigi
Plasma sterilization Geobacillus stearothermophilus, Bacillus subtilissubsp.niger
Table1.2.4:Methodsofsterilization/disinfectionusedindifferentclinicalsituations
Material Methodofsterilization/disinfection
Clinicalthermometer Isopropylalcohol
Paraffin,glasssyringe,flask,slide,oil,grease,fat,glycerol Hotairoven
OT, entryway, ward, laboratory Formaldehydegas>UV>BPL
fumigation,Preservationofanatomicalspecimen,woolenblank
et
Cystoscope,bronchoscope Orthophthaldehyde>glutaraldehyde2%(cidex)
Heartlungmachine,respirator,dentalequipments Ethyleneoxide
Contd...
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Contd...
Material Methodofsterilization/disinfection
Vaccine,sera,antibiotic,sugarsolution,antibioticandbodyfluids Filtration
Sharpinstruments Cresol
Milk Pasteurization
Plasticsyringe,catgutsuture,swab,catheter,boneandtissuegrafts,adhesivedressing Ionizing radiation
Culturemedia,metalinstruments,glasswareandallsuturematerialsexceptcatgut Autoclave
Metallicinoculationwire RedhotbyBunsenburner
Infectivemateriallikesoileddressing,bedding,animalcarcasses Burning (incineration)
Metallicsurgicalinstruments Autoclave,infra-redradiation
Water Chlorineashypochlorite0.2%
Skin Tinctureiodine,spirit(70%ethanol),savlon
Contactlenses H2O2
Table1.2.5:Commondisinfectantsandtheirspectrumofaction
Germicide Bacteria Inactivated
and Level andenvelope Unenveloped M. tubercu- by
theirconcentra ofdisinfecta dviruses Fungi viruses losis Spore organicma
tions nt tter
Glutaraldehyde (2%) High/CS + + + + + –
Formaldehyde(3–8%) High/CS + + + + + –
H2O2(3–25%) High/CS + + + + + +/–
Chlorine(100– High + + + + +/– +
1000ppm)
Isopropyl Intermediate + + +/– + – +/–
alcohol(60–95%)
Phenol (0.4–5%) Intermediate + + +/– + – –
Iodophor (30–50 ppm Intermediate + + + +/– – +
offreeiodine)
Quaternary– Low + +/– – – – +
ammonium(0.4–1.6%)
Table1.2.6:Spaulding’sclassificationofmedicaldevicesaccordingtothedegreeofriskforinfectioninvolved
Medical device Definition Examples Recommendedmethod
Criticaldevice Enteranormallysterilesite Surgicalinstruments,cardiacandurinarycatheters, Heat based
GeneralMicrobiolog
implants, eye and dentalinstruments sterilization,Chemical
sterilant
orhighleveldisinfectant
Semicritical device Come in contact with Respiratory therapy Highleveldisinfectant
mucousmembranes or minor equipments,anesthesiaequipments,
skinbreaches endoscopes,laryngoscope,rectal/vaginal/
esophagealprobes
Noncriticaldevices Comes in contact with BPcuff,ECGelectrodes,bedpans,crutches,stethoscope Intermediatelevel
intactskin , thermometer orlowleveldisinfectant
Noncriticalsurfaces Less direct contact Surfacesofmedicalequipments,examinationtable, Lowleveldisinfectant
withpatient computers
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STERILIZATION
9. Heart lungmachineis beststerilizedby:
1. Sterilizationaccuracyisassessedbyusing: a. Cidex (JIPMER,May2015)
(PGINov2016) b. Ethyleneoxide
a. Clostridiumperfringenes c. Isoprophylalcohol
b. Geobacillusstereothermophilus d. Formaldehyde
c. Staphylococcusaureus 10. AnupperGIendoscopewasperformedinaTBsuspect. To
d. Clostridiumbotulinum reuse the instrument it should be sterilizedby:
e. Bacillus subtilissubsp.Niger (RecentQuestion2015)
2. Bestmethodofserasterilizationis: (APPG2014) a. 2%Glutaraldehyde
b. Hotairoven
a. Filtration b. Autoclaving
c. Autoclave
c. Radiation d. Heating
11. Bestdisinfectantforendoscopesis:
3. Which of the following is used to test the efficiency of
a. Hypochlorite (JIPMERNov2014)
sterilizationinanautoclave?
b. Formaldehyde
(AIIMSNov2011,AIIMSNov2010,MHPG2014) c. Glutaraldehyde
a. Clostridiumtetani d. Chlorhexidine
b. Bacillusstearothermophilus
12. Prionsarebestkilledby: (NEETPatternBased)
c. Bacilluspumilus
a. Autoclavingat121°C
d. Bacilluscereus
b. 5%formalin
4. SporeofwhichbacteriaisusedassterilizationcontrolofP c. Sodiumhydroxidefor1hr
lasmasterilization: (AIIMSNov2010) d. Sodiumhypochloridefor10min
a. B.subtilis b. B.pumilis
13. Sterilizationoffiberopticisdoneby:
c. Cl.tetani d. B.stearothermophilus
(DNBDEC2012,AIIMSNov2003)
5. Conditionrequiredforautoclaveis? (DNBJune2012) a. Glutaraldehyde b. Chlorine
a. 121°Ctemperaturefor20min c. Autoclave d.Phenol
b. 121°Ctemperaturefor15min 14. AccordingtoSpauldingclassification,laparoscope
c. 100°Ctemperaturefor60min andarthroscopicinstrumentsareunder:
d. 100°Ctemperaturefor90min a. Criticalitem (RecentQuestion2015)
6. Whichofthefollowingismostresistanttosterilization? b. Semicriticalitem
a. Cysts (AI2012,2008) c. Noncriticalitem
b. Prions 15. Whichisfalseaboutspauldingclassification?
c. Spores
(AIIMSNov2010)
d. Viruses
a. Noncriticalitemsalsoincludedinclassification
7. Choosethecorrectonesforthedecreasingorderofresist b. Semicriticalitems—contactwithmucusmembrane
ancetosterilization: (PGIDec2007)
Section
c. Semicriticalitems—needslowdisinfectant
a. Prions,Bacterialspores,Bacteria d. Cardiaccatheter,e.g.ofcriticalitems
b. Bacterialspores,Bacteria,Prions 16. Phenolcoefficientindicates:
c. Bacteria,Prions,Bacterialspores (DNBDEC2012,JIPMER2009)
d. Bacterialspores,Prions,Bacteria a. Efficacyofadisinfectant
b. Dilutionofadisinfectant
DISINFECTION c. Quantityofadisinfectant
d. Purityofadisinfectant
8. LowLeveldisinfectantis: (TNPG2015) 17. Sputumcanbedisinfectedbyallexcept:
a. Benzalkoniumchloride (AIIMSMay2012,PGIDec2008)
b. Isopropylalcohol a. Autoclaving
c. Glutaraldehyde b. Boiling
d. Hydrogen peroxide c. Cresol
d. Chlorhexidine
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18. Surgicalbladeissterilizedby? (DNBJune2009)

21. Boilingof milk isan example for: (TNPG2015)
a. Autoclave b. Gammaradiation
a. Concurrentdisinfection
c. Hotairoven d. Steaming
b. Precurrentdisinfection
19. Sporicidalagentsare: c. Terminaldisinfection
(PGJune2009,June2006,JIPMER2010) d. Sterilization
a. Glutaraldehyde
22. Whichofthefollowingareusedforsterilizationofsurgica
b. Ethyleneoxide
linstrument: (PGIMay2013)
c. Formaldehyde
a. Ethyleneoxide b. Gammaradiation
d. Benzalkoniumchloride
e. Chlorine c. Autoclaving d.Glutaraldehyde
e. Hotairoven
20. Savloncontains: (AIIMSMay2010)
a. Cetrimide+Chlorhexidine 23. Allofthefollowingaretheteststochecktheefficiency
b. Cetrimide+Butylalcohol ofdisinfectantExcept: (TNPG2014,Kerala2016)
c. Cetrimide+Chlorhexidine+Butylalcohol a. ChickMartintest b. RiedelWalkertest
d. Cetrimide+Cetavlon c. HughLeifsontest d. Kelsey Sykestest
GeneralMicrobiolog
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EXPLANATIONS
STERILIZATION
1. Ans.(b,e)(Geobacillus..,B.subtilissubsp.Niger)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p40
• GeobacillusstereothermophilusandBacillussubtilissubsp.Nigerareusedasbiologicalindicatorsinsterilization.
2. Ans.(a)(Filtration)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep35,Ananthanarayan9/ep33
Filtrationisthebestmethodofsterilizationofheatlabileliquidssuchasseraandsolutionsofsugarsorantibiotics,toxinsandvaccines.Ith
elpstoremovebacteria.
3. Ans.(b)(Bacillusstearothermophilus)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep34,Ananthanarayan9/
ep32,8/ep34
BiologicalSterilizationIndicator:(Detaillist—Refertext)
• Autoclave:B.stearothermophillus
• Hotairoven:Clostridiumtetaninontoxigenicstrain
4. Ans.(d)(B.stearothermophilus)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep34,Ananthanarayan8/
ep32,MackieMcCartney’spracticalmicrobiology14/ep817
BiologicalSterilizationIndicatorofplasmasterilization:B.stearothermophillus,B.subtilissubspeciesniger (Detaillist—Refertext)
5. Ans.(b)(121°Ctemperature.......)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep34,Ananthanarayan9/ep31-
32,8/ep32,MackieMcCartney’spracticalmicrobiology14/ep817
• Autoclave:Recommendedcondition-121–124°Ctemperaturefor15minat1.1barpressure
• Alternate:134–138°Ctemperaturefor3minat2.2barpressure
• Hotairoven:160°Ctemperaturefor120minor180°Ctemperaturefor30min
6. Ans.(b)(Prions)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep31,PatrickMurray9/ep69
• Prionsarethehighestresistancestructure
• Refertexttoknowdetail
7. Ans.(a)(Prions,Bacterialspores,Bacteria)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep31,PatrickMurray9/ep69
Refertexttoknowdetail
DISINFECTION
8. Ans.(a)(Benzalkoniumchloride)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep41
• Isopropylalcohol,Phenol:Intermediateleveldisinfectant
• QuaternaryammoniumcompoundslikeBenzalkoniumchloride:Lowleveldisinfectant
• Glutaraldehyde,formaldehyde,Hydrogenperoxide,Chlorine:Highleveldisinfectant.
Section
9. Ans.(b)(Ethyleneoxide)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep41
• Heartlungmachines,respiratorsanddentalequipmentsarebeststerilizedbyEthyleneoxide.
10. Ans(a)(2%Glutaraldehyde)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep41
11. Ans(c)(Glutaraldehyde)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep412%GlutaraldehydeandOphthaldehydearerecommendeddisinfectantsforendoscopes.
12. Ans.(c)(Sodiumhydroxidefor1hour)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep524,Ananthanarayan9/ep34,8/
ep36,37,MackieMcCartney’spracticalmicrobiology14/ep827
• Prionsaresterilizedby:0.5%Hypochloritefor2hror1NNaOHfor1hourorAutoclavefor134°Cfor1–1.5hour
13. Ans.(a)(Glutaraldehyde)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep37,Ananthanarayan9/ep34
• Afiberopticisaflexible,transparentfibermadeofhighqualityextrudedglass(silica)orplasticusedforlaryngoscopes.
• FiberopticlaryngoscopesarebeststerilizedbyGlutaraldehydeorortho-ophthaldehyde.
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14. Ans.(a)(Criticalitem)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep42
15. Ans.(c)(Semicriticalitems—needslowdisinfectant)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep42
• Semicritical items are those that come in contact with mucous membranes or minor skin breaches and they needs high
tointermediateleveldisinfectantforsterilization.
• ForthedetailofSpaulding`scriteriaofdevices–Refertext
16. Ans.(a)(Efficacyofadisinfectant)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep40
• Testing efficacy of disinfectants by Phenol coefficient (Rideal Walker ) test - Phenol coefficient is determined by
thedilutionofthedisinfectantinquestionwhichsterilizesthesuspensionofS.Typhiinagiventimedividedbythedilutionofphenolw
hichsterilizesthesuspensioninthesametime.
• FordetailofTestingefficacyofdisinfectants—Refertext
17. Ans.(d)(Chlorhexidine),Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep30
• Thecommonestpathogensuspectedtobepresentinsputum—
Tuberclebacilli.Soithastobedisinfectedbyanappropriatetechnique.
• Chorhexidineisaskinantiseptic,usedforburnsorhanddisinfection.Itisnotmycobactericidal.
• Sputumcanbedisinfectedby:
○ Autoclaving:Idealmethod
○ Othermethodsinclude:Burningofthematerial,Cresol5%orPhenolorBoiling
18. Ans.(a)(Autoclave)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep41
• Steamisthepreferredmethodforsterilizingcriticalmedicalandsurgicalinstrumentsthatarenotdamagedbyheat,steam,pressur
e,ormoisture...................................................CDCGuideline,2008
• Metalinstrumentsandsurgicalbladesarebettersterilizedbyautoclave.
19. Ans.(a),(b),(c)(Glutaraldehyde,Ethyleneoxide,Formaldehyde)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology
1/ep37,Ananthanarayan9/ep34
• Listofsporicidalagent.Referchapterreview
• Benzalkoniumchlorideisasurfaceactiveagent,widelyusedaswettingagents,detergentsandemulsifiers.
20. Ans.(a)(Cetrimide+Chlorhexidine)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep39,Park22/ep119
• Savloncontains—Cetrimide3%(Cetavlon)+Chlorhexidine0.3%(Hibitane)
21. Ans.(b)Precurrentdisinfection.Ref:Park23/ep127,22/ep119
Alreadyexplained.
22. Ans. (c) (d) (e) (Autoclaving, Glutaraldehyde, Hot air oven)Ref: Guideline for Disinfection and Sterilization in
HealthcareFacilities,CDC,2008
• For the
sterilizationofsurgicalinstruments:Mostmedicalandsurgicaldevicesaremadeofmaterialsthatareheatstableandthereforecan
besterilizedbestbyautoclave(moistheat)
• Heatlabileinstruments(e.g.plastics)canbesterilizedbyEthyleneoxidegasorothernew,low-
temperaturesterilizationsystems(e.g.hydrogenperoxidegasplasma,peraceticacidimmersion,ozone)
• Dry-
heatsterilizerssuchashotairovenshouldbeusedonlyformaterialsthatmightbedamagedbymoistheatorthatareimpenetrableto
GeneralMicrobiolog
moistheat(e.g.,powders,petroleumproducts,sharpinstruments)
• Ionizingradiation:TherearenoFDA-approvedionizingradiationsterilizationprocessesforuseinhealthcarefacilities.
23. Ans.(c)(HughLeifsontest)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep39,Ananthanarayan9/ep36
• HughLeifsontestisusedtodifferentiatemicrococcifromstaphylococci
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tureMediaandMethods,Identification CHAPTER
ofBacteriabyConventional,Automated
and Molecular Methods 1.3
CULTUREMEDIA
Thebasicconstituentsofculturemediaare:
• Peptone:Mixtureofpartiallydigestedproteins
• Agar:Itisusedforsolidifyingtheculturemedia.Ithasnonutritionalproperty.
○ Itispreparedfromseaweeds(redalgaeofspeciesGelidiumandGracilaria).
○ Agarispreferredovergelatine,asitisbacteriologicallyinert,anditmeltsat98°Cand
usuallysolidifiesat42°C
Concentrationofagar:
○ Concentrationofagar:
▪ Forsolidagar:1–2%(Japaneseagar2%orNewZealandagar1.2%)
For solid agar preparation1–2% (Japanese agar 2% orNewZealandagar1.2%)
Forsemisolidagar0.5% ▪ Forsemisolidagar:0.5%
For solid agar to inhibitProteusswarming6% ▪ ForsolidagartoinhibitProteusswarming:6%
• Others:Meatextract,Yeastextract,Bloodandserum,WaterandElectrolytes(NaCl).
Simple/BasalMedia
Theycontainminimumingredientsthatsupportthegrowthofnon-
fastidiousbacteria.Examplesinclude:
1. Peptonewater:Itcontainspeptone(1%)+NaCl(0.5%)+water
2. Nutrientbroth:Itismade-upofpeptonewater+meatextract(1%)
3. Nutrientagar:Itismade-upofnutrientbroth+2%agar
Thebasalmediaareusedfor:
• Testingthenon-fastidiousnessofbacteria
Simple/Basal Media:
•
Peptone water: It containspeptone(1%)+NaCl(0.5%)Theyserveasthebaseforthepreparationofmanyothermedia
+water • Nutrientbrothisusedforstudyingthebacterialgrowthcurve
Nutrientbroth:Itismadeupofpeptone water +• meatNutrientagaristhepreferredmediumfor:
extract(1%).
Nutrient agar: It is made up ofnutrientbroth+2%agar
○ Performingthebiochemicaltestssuchasoxidase,catalaseandslideagglutination
○ TostudythecolonycharacterandPigmentdemonstration.
EnrichedMedia
Whenabasalmediumisaddedwithadditionalnutrientssuchasblood,serumoregg,itiscalled
enriched medium.They also supportthe growth offastidious bacteria, e.g.:
1. Bloodagar:Preparedbyadding5–10%ofsheepbloodtothemoltennutrientagarat45°C.
Itis usedto testthehemolytic propertyof thebacteria
DifferentialMedia: 2. Chocolateagar:Itistheheatedbloodagar,bloodisaddedtothemoltennutrientagarat
MacConkeyaAgar 70°C.Itismorenutritiousthanbloodagar,andevensupportsHaemophilusinfluenzae.
CLEDagar 3. Loeffler’sserumslopeisusedforisolationofCorynebacteriumdiphtheriae.
4. Bloodculturemedia:Usedforbloodculture.Theyareoftwotypes:
○ Monophasicmediumismade-upofbrainheartinfusion(BHI)broth
○ Biphasicmediumhasaliquidphase(BHIbroth)andasolidagarslope(BHIagar).
EnrichmentBroth
Liquidmediathatallowcertainorganism(pathogens)togrowandinhibitothers(normalflora):
• SeleniteFandTetrathionatebrothusedforSalmonellaandShigella
• Alkalinepeptonewater(APW)usedforVibriocholerae.
SelectiveMedia
Solidmediathatallowcertainorganism(pathogens)togrowandinhibitothers(normalflora):
1. LowensteinJensen(LJ)mediumisusedforisolationofMycobacteriumtuberculosis
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CultureMediaandMethods,IdentificationofBacteriabyConventional,AutomatedandMolecularMethods 33
ThiosulphateCitrateBilesaltSucrose(TCBS)agarusedforisolationofVibriospecies
FortheisolationofentericpathogenssuchasSalmonellaandShigellafromstool:
DCA(DeoxycholateCitrateAgar)
XLD(XyloseLysineDeoxycholate)agar
4. Potassiumtelluriteagar(PTA)isusedforisolationofCorynebacteriumdiphtheriae.
5. WilsonBlairbismuthsulphitemedium:ItisusedforisolationofSalmonellaTyphi.
TransportMedia
Theyareusedforthetransportofspecimenscontainingdelicateorganismorwhenthedelayisexpecte
d.Bacteriadonotmultiplyinthetransportmedia,theyonlyremainviable.
Table1.3.1:Transportmediausedforcommonbacteria
Organism Transportmedia
Streptococcus Pike’smedium
Neisseria Amiesmedium,Stuart’smedium
Vibriocholerae VR (Venkatraman-Ramakrishnan) medium, Cary Blair medium and
Autoclavedseawater
Shigella, Salmonella Bufferedglycerolsaline,andCaryBlairmedium
DifferentialMedia
Thesemediadifferentiatebetweentwogroupsofbacteriabyusinganindicator.
1. MacConkeyagar:ItdifferentiatesorganismsintoLForlactosefermenters(producepink
colonies, e.g. E. coli and Klebsiella) and NLF (produce colorless colonies, e.g.
ShigellaandSalmonella).
2. CLEDagar(Cysteinelactoseelectrolyte-deficientagar):ThisissimilartoMacConkeyagar,
differentiates between LF and NLF. It is used as an alternative to combination
ofbloodagarandMacConkeyagar,fortheprocessingofurinespecimens:
• Advantagesover MacConkey agar: It is less inhibitory than MacConkey agar,
supportsgram-positivebacteria(exceptβhemolyticStreptococcus)andCandida.
• Advantageoverbloodagar:ItcanpreventtheswarmingofProteus.
AnaerobicCultureMedia
Anaerobicmediacontainreducingsubstanceswhichtake-
upoxygenandcreatelowerredoxpotentialandthuspermitthegrowthofobligateanaerobessuchas
Clostridium.Examplesare:
1. Robertson’scookedmeat(RCM)broth:Itcontainschoppedmeatparticles(beefheart),which
GeneralMicrobiolog
provideglutathioneandunsaturatedfattyacids.
2. Otheranaerobicmediainclude:
• BHISagar:Brainheartinfusionagarwithsupplements(vitaminKandhemin)
• Thioglycollatebroth,Anaerobicbloodagar
• Neomycinbloodagar,EggyolkagarandPhenylethylagar.
CULTUREMETHODS
VariousAerobicCultureMethods
1. Streakculturebyusingloopwithintermittentheating:Itistheroutinelyusedmethod.
2. Lawnorcarpetculture:UsefulforCarryingoutantimicrobialsusceptibilitytestingbydisk
diffusion method, Bacteriophage typing and producing large amount of
bacterialgrowthforpreparationofbacterialantigensandvaccines.
3. Stroke culture (zigzag fashion by straight wire): Used for citrate, urease and TSI
(triplesugarirontest)
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4. Stab culture: Stabbing the semisolid agar butt by a straight wire. It is used for:
(i) maintainingstockcultures,(ii)OFtest(iii)Mannitolmotilitymedium,(iv)Nutrient
agarsemisolidbutts,(v)TSI(herebothstrokeandstabculturesaremade).
5. Liquidculture:
• Uses:Liquidculturesareusefulfor:(i)bloodculture,(ii)forsterilitytesting.
• Advantages:Usedfor:(i)Whenbacterialoadisless,(ii)specimens(e.g.blood)containing
antibiotics it is neutralized by dilution in the medium, (iii) when
Aerobicculturemethods: largeyieldsofbacteriaarerequired,(iv)fordemonstrationofbacterialgrowthcurve
Streak culture • Disadvantages:Liquidculturesdonotprovideapureculturefromamixedinoculum.
Lawnorcarpetculture 6. Pour-plateculture:Quantitativeculturemethod,usedtoestimateviablebacterialcount.
Stroke culture
Stab culture IncubatoryConditions
Liquidculture
Pour-plate culture • Mostofthepathogenicorganismsgrowbestat37°C.
• Candle jar: Itprovides capnophilicatmosphere (3–5%CO 2). Thisis usefulfor
Brucellaabortus,Streptococcus, pneumococcus and gonococcus.
• Microaerophilicbacteria suchas Campylobacterand Helicobacterrequire 5% oxygen.
AnaerobicCultureMethods
1. Productionofvacuum
2. Bydisplacementandcombustionofoxygen:Thisprincipleisusedin:
AnaerobicCultureMethods: • McIntoshandFilde’sanaerobicjar(Itinvolvesevacuationofairandreplacementwithhydro
Productionofvacuum gengasmanually)
By displacement andcombustionofoxygen: • AnoxomatSystem(principleissame,butdonebyautomatedinstrument)
McIntosh and Filde's an-aerobicjar
Anoxomat System
3. GaspakSystem(Absorptionofoxygenchemically,e.g.usingalkalinepyrogallol).Indicators
GaspakSystem ofanaerobiosisare:
Anaerobic Glove Box • Chemicalindicator:Reducedmethyleneblue
Byreducingagents • Biologicalindicator:Pseudomonas
PRAS media 4. AnaerobicGloveBox(oranaerobicchamber)
5. Byreducingagents:Suchasglucose,thioglycollate,meat,cysteineandascorbicacid.
6. PRASmedia (Prereduced, Anaerobically Sterilized).
PreservationofMicroorganisms
• Short-termmethods:(i)Sub-culturing,(ii)Immersingthecultureinglycerol,orsterile
distilledwater,(iii)Freezingat–20°Cand(iv)Drying(formouldsandspores).
• Long-termmethods:ByUltratemperaturefreezingandLyophilization(freeze-drying).
IDENTIFICATIONOFBACTERIA
Identificationofbacteriacanbedoneby:(i)conventional(cultureandidentificationby
biochemicalreactions),(ii)automatedculturetechniques(iii)molecularmethods.
AutomatedCultureTechniques
Section
Conventionalculturemethodsoftenyieldpoorresultsbecauseoflowbacterialload.Therefore,
various automated blood culture techniques have been in use since last
decade.Advantages:Themajoradvantagesofautomatedbloodculturetechniquesare:
• Continuous automatedmonitoring(onceinevery 15–20min bytheinstrument).
• Otheradvantages:Moresensitive,↑yield,rapid,lesslaborintensive
Disadvantages:(i)highcost(ii)inabilitytoobservethecolonymorphologyasliquidmediumisused,
(iii)noseparatedetectioninmixedcultures,(iv)radioactivehazardsforBACTEC.
MOLECULARMETHODS
Nucleicacidamplificationtechniques(NAATs)include:
• Polymerasechainreaction(PCR)anditsmodificationincludingRealtimePCR
• Ligasechainreaction(LCR)andTranscription-mediatedamplification(TMA)
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Table1.3.2:Automatedculturesystemsindiagnosticbacteriology
Forbacterialculture Principle used
1.BACTEC Inwasradiometrybased,butlaterchangedtofluorescentbaseddetectiontechnique
2.BacT/Alert CO2liberatedfrombacteria,causesapHchange,detectedbycolorimetry
3.ESPculture system CO2liberatedfrombacteria,causesapressurechange,detectedbymanometry
For bacterial identification • Phoenixbacterialidentificationsystem
• MALDI–TOF(Matrix-assistedlaserdesorption/ionizationtime-of-flight),e.g.VITEKMS
• VITEK2bacterialidentificationandantimicrobialsensitivitysystem
• MicroscanWalkawaysystem
ForM. tuberculosis MGIT(MycobacterialGrowthIndicatorTube)
• Nucleicacidsequencebasedamplification(NASBA)
• Stranddisplacementamplification(SDA).
PolymeraseChainReaction(PCR)
PCR is a technology in molecular biology used to amplify a single orfew copies of a piece of
DNAtogeneratemillionsofcopiesofDNA.ItwasdevelopedbyKaryBMullis. PCRinvolvesthreebasicsteps:
DNA extraction from theorganism
Principle:PCRinvolvesthreebasicsteps.
AmplificationofextractedDNA
1. DNAextractionfromtheorganism Gelelectrophoresisof
2. AmplificationofextractedDNA:TheextractedDNAissubjectedtorepeatedcycles(30–35 amplifiedproduct
numbers) of amplification in a thermocycler which takes about 3–4 hours.
Eachamplificationcyclehasthreesteps:
• Denaturationat95°C:ThisinvolvesseparationthedsDNAintotwoseparatessDNA.
• Primerannealing(55°C):Primerisashortoligonucleotidecomplementarytoasmall
sequenceofthetargetDNA.ItannealstothecomplementarysiteonssDNA.
• Extensionoftheprimer(72°C):ThisstepiscatalyzedbyTaqPolymeraseenzyme. Eachamplificationcyclehas
3. Gelelectrophoresisofamplifiedproduct:TheamplifiedDNAiselectrophoretically threesteps:
migratedaccordingtotheirmolecularsizetoformbands;seenunderUVrays. Denaturationat95°C
Primerannealing(55°C)
Advantages:PCRhasthefollowingadvantagescomparedtotheconventionalculture: Extension of the primer(72°C)
• Moresensitive:ItcanamplifyveryfewcopiesofaspecificDNA,soitismoresensitive.
• Morespecific:ByuseofprimerstargetingspecificDNAsequenceoftheorganism
• Detectstheorganism:(i)eitherfromsample,(ii)toconfirmcultureisolate.
• Detecttheorganismsthatarehighlyfastidiousornoncultivablebyconventionalculturemet
hods.
• Detectgenescodingdrugresistance(e.g.MecAgenedetectioninStaphylococcusaureus)
• Detectsgeneticdiseasessuchassicklecellanemia,phenylketonuria,etc.
GeneralMicrobiolog
Disadvantages:
• ConventionalPCRdetectsonlytheDNA,butnottheRNA(detectedbyRT-PCR).
• Qualitative,notquantitative(QuantitationisdonebyrealtimePCR).
• Viability:PCRcannotdifferentiatebetweenviableornonviableorganisms.
• FalsepositiveamplificationmayoccurduetocontaminationwithenvironmentalDNA.
• Falsenegative:byPCRinhibitorspresentinsomespecimenssuchasblood,feces,etc.
ModificationofPCR
1. ReversetranscriptasePCR(RT-PCR):ForamplifyingRNA,RT-PCRisdone.
• AfterRNAextraction,thefirststepisadditionofreversetranscriptaseenzymethatcover
ts RNA into DNA. Then, the amplification of DNA and gel
documentationstepsaresimilartothatdescribedforconventionalPCR.
Modification of PCR:
• UsefulfordetectionofRNAvirusesor16SrRNAgenesoftheorganisms. ReversetranscriptasePCR
2. NestedPCR:TheamplifiedproductsofthefirstroundPCRissubjectedtoanotherroundofamplif NestedPCR
icationusingasecondprimertargetingadifferentgeneofsameorganism. Multiplex PCR
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• More sensitive:Double roundofamplification yieldshighquantity ofDNA.

• Morespecific:Useoftwoprimerstargetingsameorganismsmakesmorespecific.
• Disadvantage:ThereismorechanceofcontaminationofthePCRtubes,whichmayleadt
Bacteriophagetypingisdone
for: ofalsepositiveresults.
Staphylococcusaureus 4. MultiplexPCR:ItusesmorethanoneprimerwhichcandetectmanyDNAsequencesof
SalmonellaTyphi severalorganismsinonereaction.
Vibriocholerae • Syndromicapproach:Todiagnoseinfectioussyndromecausedbymorethanoneorgani
Brucella
Corynebacteriumdiphtheriae
sm.
• Contamination chances of reaction tubes with environmental DNA.
Real-timePCR(rt-PCR)
RealtimePCR,thoughexpensive,buthasmanyadvantagesoveraconventionalPCR:
• Quantitative,hencecanbeusedformonitoringtreatmentresponse,e.g.inHIVorHBV.
• Takeslesstime:Astheamplificationcanbevisualizedevenwhentheamplificationcycle
isgoingon.
Bacteriocintypingisdonefor: • Contaminationrateisextremelyless.
Shigella sonnei(colicin typing) • Sensitivityandspecificityofrt-PCRassaysareextremelyhigher.
Klebsiella(klebocin typing),
E.coli(colicin typing) DetectionofamplificationproductsofrealtimePCR
Proteus(proticin typing),
Pseudomonas(pyocin typing) • Nonspecificmethodsuse SYBR green dye that stains any nucleic acidnonspecifically.
• Specific methods use fluorescent labelled DNA probesuch as (i) TaqMan or
hydrolysisprobe, (ii) Molecular beacon and (iii) FRET (Fluorescence Resonance Energy
Transfer)probe.
Biotypingisdonefor: MICROBIALTYPING
C.diphtheriae
Microbialtypingreferstocharacterizationofanorganismbeyonditsspecieslevel.Itisusedtodetermi
Vibriocholerae
netherelatednessbetweendifferentmicrobialstrainsofthesamespeciesandtherebyhelpsto(i)inves
tigateoutbreaks,(ii)determinethesourceandroutesofinfections.
Genotypic methods are more reliable and have better reproducibility and
discriminativepowerthanphenotypicmethods,howevertheyareexpensive.
Table1.3.3:Typingmethods
Phenotypicmethods Genotypingmethods
Bacteriophagetyping:Basedontheirsusceptibilitytobacteriophages.Itisdonefor NonAmplificationbasedmethods
• StaphylococcusaureusandSalmonellaTyphi 1. Plasmidprofileanalysis
• Vibriocholerae,BrucellaandCorynebacteriumdiphtheriae 2. Chromosomal DNAanalysis
Bacteriocintyping:Basedontheabilityofastraintoproduceparticularbacteriocinwhichinhibitsthegrowthofa 3. RFLP(Restrictedfragmentlengthpoly
setofselectedindicatorstrains.It isdonefor: morphism)
• Shigella sonnei(colicin typing) 4. Ribotyping(RFLPanalysisofribo
• Klebsiella(klebocin typing),E.coli (colicin typing) somalDNA)
Section
• Proteus(proticin typing),Pseudomonas (pyocin typing) 5. Pulsefieldgelelectrophoresis

(PFGE)-Gold standard method
Biotyping:Basedondifferentbiochemicalpropertiesoftheorganism.Itisdonefor Amplificationbasedmethods
• C.diphtheriae(gravis,intermediusandmitis 1. PCR-RFLP
• VibriocholeraeO1(classicalandElTor)andYersiniapestis 2. AmplifiedFragmentLength
Antibiogramtyping:Basedontheirresistancepatterntodifferentantimicrobials.Itisthemostcommonlyusedtypi Polymorphism (AFLP)
ng method. 3. Sequencing-based methods
Auxotyping:Basedonnutritionalrequirementoftheorganism(e.g.Gonococcus) 4. Microarrays
Morphotyping:Basedondifferent typeofcolonies inculture(e.g.Pseudomonas)
Serotyping:Basedontheantigenicpropertyofanorganism.
• Streptococcus(Lancefieldgrouping)
• Basedoncapsularantigen,e.g.pneumococcus,meningococcusandH.influenzae
• Basedonsomaticantigen-E.coli,Shigella,SalmonellaandVibriocholerae.
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CULTUREMEDIA/METHODS 9. Bloodagarisanexample: (DNBJune2012)

a. Enrichedmedia
1. Antibioticsensitivitytestingisusuallydoneon: b. Enrichmentmedia
(RecentQuestion2015) c. Nutrientmedia
a. Bloodagar d. Specialmedia
b. Chocolateagar
10. Whichoneofthefollowingistrue:
c. MacConkeyagar
(AIIMSNov2006,AI2007)
d. MuellerHintonagar
a. Agarhasnutrientproperties
2. CLEDmediumisusedinpreferencetoMacConkeyagarin b. Chocolate medium is selectivemedium
relationtocultureoforganisminvolvedinurineinfectio c. Additionofselectivesubstancesinasolidmediumiscal
n.Thereason is (AIIMSMAY2016) ledenrichmentmedia
a. ItpreventsswarmingofProteus d. Nutrientbrothisbasalmedium
b. DifferentiatesLFandNLF
11. SmithNoguchi’smediaisusedfor: (DNB2007)
c. PromotesStaphylococcus,StreptococcusandCandida
a. Salmonella
d. IdentifiesPseudomonas
b. Klebsiella
3. Whichoffollowingculturemediacombinationis/ c. Spirochetes
aretrueexcept: (PGINov2014) d. Bacillus
a. Thayer-Martinmedia:Gonorrhea
12. To prevent swarming the % agar in Nutrient agar
b. Chocolateagar:Enrichedmedia
hastobeincreasedatleast to%.
c. Lowenstein-Jensenmedium:Mycobacteriumtuber-
(TNPG2014)a. 2% b.4%
culosis c. 6% d. 8%
d. Mueller-Hintonagar:Corynebacteriumdiphtheriae
e. MacConkey’s agar: Nonlactose fermenters form
colourlesscolonies MOLECULARANDAUTOMATEDMETHODS
4. TSIis: (RecentQuestions2014) 13. PCRisusedto: (AIMSNov2013)
a. Selective b. Enrichment a. Detecttargetplasmids
c. Enriched d. Composite b. AmplifysmallamountofDNA
5. SelectivemediaforShigella: (RecentQuestions2014) c. SealthecutendsofDNA
a. Wilsonblair b. TCBS d. Cleavethebacterialplasmid
c. DCA d.Bloodagar 14. Reversetranscriptaseis: (PGIJune2011)
6. Recommendedtransportmediumforstoolspecimen a. DNAdependentRNApolymerase
suspectedtocontainentericpathogensis: b. RNAdependentDNApolymerase
GeneralMicrobiolog
(TNPG2015,NEETPatternBased) c. DNAdependentDNApolymerase
a. Amie’smedium d. RNAdependentRNApolymerase
b. Bufferedglycerolsalinemedium e. RNApolymerase
c. MacConkeymedium 15. Enzyme(s)usedinpolymerasechainreactionis/are:
d. Bloodagar a. Restrictionendonuclease (PGIJune2011)
b. DNApolymerase
7. Agar conc.In nutrientagaris: (NEETPatternBased)
c. Alkaline phosphate
a. 2% b. 4%
d. RNApolymerase
c. 1% d. 3%
e. Reversetranscriptase
8. Robertsoncookedmeatbrothisanexample:
16. AllofthefollowingarerequiredinPCRexcept:
a. Enrichedmedia (NEETPatternBased)
a. Deoxyribonucleotides (AIIMSNov2011)
b. Enrichmentmedia
b. Thermostableenzyme/DNApolymerase
c. Nutrientmedia
c. Dideoxyribonucleotides
d. Anaerobic media
d. Magnesium/ssDNA/TemplateDNA
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17. Chromosomalmutationcanbeidentifiedbyallexcept:
19. ForPCR,Thermusacquaticusplantisusedtoprepare:
a. Singlestrandpolymorphism (AIIMSNov2011)
a. DNApolymerase (RecentQuestion2013)
b. Agarosegelelectrophoresis
b. RNApolymerase
c. DenaturatingGradientgelelectrophoresis
c. Primers
d. Dideoxynucleotidetrailsequencing
d. RestrictionendonucleaseII
18. Northernbloting isused forseparation of:
20. DNAhybridizationiscalled: (RecentQuestion2013)
a. DNA (DNBJune2012)
a. Southernblot
b. RNA
b. Northernblot
c. Proteins
c. Easternblot
d. None
d. Westernblot
Section
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EXPLANATIONS
CULTUREMEDIA/METHODS
1. Ans.(d)(MuellerHintonagar)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep83
2. (c)(PromotesStaphylococcus,Streptococcus&Candida)Reference:ApurbaSastry'sEssentialsofMedicalMicrobiology/
p46
CLEDagar(Cysteinelactoseelectrolyte-
deficientagar):ThisissimilartoMacConkeyagar,differentiatesbetweenLFandNLF.Itisusedasanalternativetocombinationofblooda
garandMacConkeyagar,fortheprocessingofurinespecimens.
• AdvantagesoverMacConkeyagar:ItislessinhibitorythanMacConkeyagar,supportsthegrowthofgram-positive
bacteria (exceptβhemolyticStreptococcus)andCandida.
• Advantageoverbloodagar:ItcanpreventtheswarmingofProteus.
3. Ans.(d)(Mueller-Hintonagar)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep44-46
• Thayer-Martinmedia:Gonorrhea(Selectivemedia)
• Chocolateagar:Enrichedmedia
• Lowenstein-JensenMedium:Mycobacteriumtuberculosis(selectivemedia)
• Muller-Hintonagar:Forantibioticsusceptibilitytest
• MacConkey’sagar:Differentialmedia,differentiatesLF(pinkcolony)andNLF(colourlesscolonies).
4. Ans.(d)(Composite)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep57,Ananthanarayan9/ep52
• TSIisapopularcompositemedium,whichindicatedwhetherbacteriafermentsglucoseonly,orlactoseandsucrosealso,wit
h/withoutgasformation,besidesindicatingH 2Sproductionaswell.
5. Ans.(c)(DCA)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep306,Ananthanarayan9/ep285
• Deoxycholatecitrateagar (DCA)andXylose lysinedeoxycholate (XLD)areselective mediaforShigella.
6. Ans.(b)(Buffered...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep45Ananthanarayan9/ep42,8/ep39
Recommendedtransportmediumforstoolspecimenis:
• Bufferedglycerolsalinemedium(whenSalmonellaorShigellaissuspected)
• VRmedium-(whenVibriocholeraeissuspected).
7. Ans.(a)(2%)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep43,Ananthanarayan9/ep39,8/ep39
• 2%ofagarisroutinelyusedformakingsolidmedia.
8. Ans.(d)(Anaerobic...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep46,Ananthanarayan9/ep43,8/ep39
• Robersoncookedmeatbrothisusedforcultureofanaerobicorganisms.
GeneralMicrobiolog
9. Ans.(a)(Enriched...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep45,Ananthanarayan9/ep40,8/ep39
• Enrichedmediacontainsextranutritionalfactorslikeblood,serum,eggsothatitsupportsthefastidiousorganisms,
e.g.Bloodagar,Chocolateagar,Loeffler’sserumslope.
10. Ans.(d)(Nutr.....)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep44Ananthanarayan9/ep40,8/ep39
• Nutrientbroth,PeptonewaterandNutrientAgararethebasalmedia.
• Usedtopreparesolidmedia,butithasnonutritivevalue
• Chocolatemediumisenrichedmedium
• Additionofselectivesubstancesinasolidmediumiscalledselectivemedium
11. Ans.(c)(Spirochetes)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep375,Ananthanarayan9/ep42
• SmithNoguchi’smedium:IsusedforNonpathogenicTreponemes
• PathogenicTreponemescannotbegrowninartificialculturemedium.
12. Ans.(c)(6%)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep43,Ananthanarayan9/ep42
Agarconcentrationroutinelyused:(i)Insolidmedia2%,(ii)Semisolidmedia0.5-1%,(iii)Toinhibitswarming6%
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MOLECULARANDAUTOMATEDMETHODS
13. Ans.(b)(Amplifysmall...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep60,Ananthanarayan9/ep65
• Thepolymerasechainreaction(PCR)isabiochemicaltechnologyinmolecularbiologytoamplifyasingleorafew
copiesofapieceofDNA,generatingthousandstomillionsofcopiesofaparticularDNAsequence.
14. Ans.(b)(RNAdependentDNApolymerase)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep62
• ReversetranscriptasehasRNAdependentDNApolymeraseactivityanditusesthegenomeRNAasatemplateand1stsynthesizessinglestr
andedDNAbyreversetranscriptionofgenomicRNA,thenfinallyadoublestrandedDNA.
• TheHIVandotherretrovirusespossessreversetranscriptase.
15. Ans.(b)(DNA...)ApurbaSastry'sEssentialsofMedicalMicrobiology1/ep62
• Taq Polymerase is a DNA Polymerase that amplifies the target gene into millions of copy without denaturation. It
isone of the basicrequirements of PCR alongwith other requirements likeprimers, Nucleotide and Mg ++ion.
AboutOtherOptions
• Restrictionendonuclease:RequireforRFLP(RestrictedFragmentLengthPolymorphism)
• Reversetranscriptase:RequireforReversetranscriptasePCR(RT-PCR)
• Alkalinephosphate:RequiredforELISA.
• RNApolymerase:Notrequired.
16. Ans.(c)(Dideoxyribonucleotides)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep61,Ananthanarayan9/e
p65,8/ep70-71,BaileyandScott’sDiagnosticMicrobiology12/ep127-131
• DideoxyribonucleotidesisnotusedinPCR.
RequirementofPCR
• TaqPolymerase:ObtainedfromThermusaquaticusplantwhichcanwithstandthehightemperature.
• Primers-shortoligonucleotidecomplementarytoasmallsequenceofthetargetDNA
• Deoxynucleotide,Mg++ion
• TargetDNApresentinthesample.
17. Ans.(b)(Agar..):Ref:ApurbaSastry’sEssofMedMicrobiology1/ep62,Prescott’sMicrobiology6/ep251-54,Wikipedia
• Agarose Gel electrophoresis is used to separate the DNA by charge or by size. It is usually performed to visualize the
amplifiedDNAafterPCR,butmaybeusedasapreparativetechniquepriortouseofothermethodssuchasmassspectrometry,RFLP,PCR,
cloning,DNAsequencing,orSouthernblottingforfurthercharacterization.
• Please remember, it is just a method of visualizing the DNA after separating by size. So it is always used as a part of
anymolecularmethodtovisualizetheDNA.Butaloneitcannotbeusedtodetectanymutations.
18. Ans.(b)(RNA)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep77,Ananthanarayan9/ep65,8/ep69
• Northernblotting is usedfor detection of- RNA
• Southernblottingisusedfordetectionof-DNA
• Westernblottingisusedfordetectionof-Protein
19. Ans.(a)(DNApol...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep61,MackieMcCartney14/
ep227TheheatstableDNApolymeraseenzymeusedforPCRisderivedfromplantThermusacquaticus.
Section
20. Ans.(a)(Southernblot)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep77,Ananthanarayan9/ep65
• DNAhybridizationiscalledSouthernblot
• RNAhybridizationiscalledNorthernblot.
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CHAPTER
BacterialGeneticsand
AntimicrobialResistance 1.4
BACTERIALGENETICS
Bacterialgeneticsdealswiththestudyofheredityandvariationseeninbacteria.Allheredi-
tarycharacteristicsofthebacteriaareencodedintheirDNAwhichispresentinchromsomeaswel
linextrachromosomalgeneticmaterialasplasmid.
Plasmid
PlasmidsaretheextrachromosomaldscircularDNAmoleculesthatexistinfreestateinthecytopl
asmofbacteriaandalsofoundinsomeyeasts:
• Notessentialforlife:Bacteriamaygainorloseplasmidduringtheirlifetime.
TypeofPlasmidsbasedonfunction:
• Numbers:Theymaybepresentsinglyorinmultiplenumbersupto>40plasmidspercell.
FertilityorF-plasmids
• Capableofreplicatingindependently Resistance(R)plasmids
• Episome:PlasmidmayintegratewithchromosomalDNAofbacteriaandsuchplasmidsareca Colplasmids
lledepisomes. Virulenceplasmids
• Curing:Theprocessofeliminatingtheplasmidsfrombacteriaisknownascuring. Metabolic Plasmids
ClassificationofPlasmids
1. Basedonabilitytoperformconjugation:
• Conjugativeplasmidsorself-transmissibleplasmids
• Nonconjugativeplasmidsornontransmissibleplasmids.Theycannottransferthemsel
ves.
2. Basedoncompatibility:CompatibleplasmidsandIncompatibleplasmids.Onlycompatibl
eplasmidscanstaytogetherinsideacell.
3. Basedonfunction:Therearefivemainclassesofplasmids:
• FertilityorF-plasmids:Codeforsexpilithatformstheconjugationtube.
• Resistance(R)plasmids,whichcontaingenesthatcoderesistancetovariousantibiotics.
• Colplasmids:Containgenesthatcodeforbacteriocins.
• Virulenceplasmids:Codesforvirulencefactorsliketoxins,adhesins.
• Metabolicplasmids:Theyenablethehostinvariousmetabolicactivities.
4. Plasmid as vector: By their ability to transfer DNA from one cell to another,
plasmidshave become important vectors in genetic engineering. Plasmids contain
certain
siteswheregenescanbeinsertedartificiallybyrecombinantDNAtechnology.Suchplasmids
canbeusedforproteinproduction,genetherapy,etc.
HorizontalGeneTransferinBacteria
Genetransferinbacteriacanbebroadlydividedinto:
• Verticalgenetransfer(transmissionofgenesfromparentstooffspring)
• Horizontalgenetransfer(transmissionofgenesfromonebacteriumtoanotherbacterium).This
occursby:
Transformation
TransformationistheprocessofrandomuptakeoffreeornakedDNAfragmentfromthe
surrounding medium by a bacterial cell and incorporation of this molecule into
itschromosomeinaheritableform.
• It has been studied so far only in certain bacteria: Streptococcus, Bacillus,
Haemophilus,Neisseria,AcinetobacterandPseudomonas.
• TheGriffith experiment (1928) on miceusing pneumococci strains provided thedirect
evidenceoftransformation.
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Transduction
Transductionisdefined astransmission ofa portionofDNA fromone bacteriumto another
byabacteriophage.
Typesoftransduction
1. Generalizedtransduction:Itinvolvestransferofanypartofthedonorbacterialgenomeintothe
recipientbacteria.
2. Restrictedorspecializedtransduction:Here,onlyaparticulargeneticsegmentofthebacteria
lchromosomethatispresentadjacenttothephageDNAistransduced.
Roleoftransduction
• InadditiontochromosomalDNA,transductionisalsoamethodoftransferofepisomesandpl
asmids.
• Drugresistance,e.g.plasmidcodedpenicillinresistanceinstaphylococci.
• Treatment:Asamethodofgeneticengineeringinthetreatmentofsomeinbornmetabolicdefects.
Lysogenic Conversion
Duringthetemperateorlysogeniclifecycle,thephageDNAremainsintegratedwiththebacterialc
hromosomeasprophage.
• Theprophageactsasanadditionalchromosomalelementwhichencodesfornewcharacters
tothedaughtercells.
• Impartstoxigenicitytothebacteria:PhageDNAmaycodeforvarioustoxinsabbreviatedasAB
CDE:
AandCofStreptococcuspyrogenicexotoxin(SPE),BotulinumtoxinCandD,Choleratoxin,Di
phtheriatoxinandE.coli(Verocytotoxin).
Conjugation
Conjugationreferstothetransferofgeneticmaterialfromonebacterium(donorormale)toanoth
erbacterium(recipientorfemale)bymatingwitheachotherandformingtheconjuga-
tiontube.ItwasdiscoveredfirstbyLederbergandTatum.
• F+×F–
Mating:WhentheF+cell(containingaplasmidcalledFfactororfertilityfactor)comescloset
otheF–
cell(lackingFfactor),theFfactorformsconjugationtube,throughwhichtheFfactoristransm
ittedtoF–cellultimatelymakingF–cellintoF+cell.
• HFRConjugation:Ffactorbeingaplasmid,itmayintegratewithbacterialchromosomeandb
ehaveasepisome.
○ SuchdonorcellsareabletotransferchromosomalDNAtorecipientcellswithhighfrequency
incomparisontoF+cells,therefore,namedasHfrcells(highfrequencyofrecombination).
○ During conjugationofHfrcellwithanF-cell,onlyfewchromosomalgenesalongwith
only a part of the F factor get transferred. Hence, F- recipient cells do
notbecomeF+cells.
TypesofConjugation: • F′Conjugation:TheconversionofanF+cellintoanHfrcellisreversible.
Section
F+ × F- Mating ○ WhentheFfactorrevertsfromtheintegratedtofree-state,itmaysometimescarrywith it
HFRConjugation some chromosomal DNA from adjacent site of its attachment. They
F′Conjugation arenamedasF’factor(Fprimefactor).
○ WhenF’cellconjugateswitharecipient(F-),ittransfers,alongwiththeFfactor,theho
stDNAincorporatedwithit.TherecipientbecomesF’cell.Thisprocessiscalledsexdu
ction.
• Conjugation plays an important role in the transfer of plasmids coding for
antibacterialdrugresistance[resistancetransferfactor(RTF)]andbacteriocinproduction[
Colicinogenic(Col)factor].
• Rfactor(ortheresistancefactor)isaplasmidwhichhastwocomponents.
○ Resistance transfer factor (RTF) is the plasmid responsible for conjugational trans-
fer(similartoFfactor)
○ Resistancedeterminant(r):Codesforresistancetoonedrug.AnRfactorcanhavesever
alrdeterminants.
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BacterialGeneticsandAntimicrobialResistance 43
ANTIMICROBIALRESISTANCE
Antimicrobialresistancecanbeoftwotypes;intrinsicandacquired.
• IntrinsicResistance:Itistheinnateabilityofabacteriumtoresistaclassofantibiotics
• AcquiredResistance:Itistheemergenceofresistanceinbacteria,byacquiringthedrugresistantg
eneseitherby–(i)mutationalorby(ii)transferabledrugresistance
Table1.4.1:Intrinsicantimicrobialresistance
Organism Intrinsicresistanceagainst
Anaerobicbacteria Aminoglycosides
Aerobicbacteria Metronidazole
Gram-negativebacteria Vancomycin
Klebsiella species Ampicillin
Pseudomonas Sulfonamides,trimethoprim, tetracycline, orchloramphenicol
Enterococci AminoglycosidesandAllcephalosporins
Table1.4.2:Mutationalvstransferabledrugresistance
Mutationaldrugresistance Transferabledrugresistance
Resistancetoonedrugatatime Multipledrugresistance atthe sametime
Lowdegreeresistance Highdegreeresistance
Resistancecanbeovercomebycombinationofdrugs Cannotbeovercomebydrugcombinations
Virulenceofresistancemutantsmaybelowered Virulencenotdecreased
Resistanceisnottransferabletootherorganisms;butspreadto Resistanceistransferabletootherorganisms-
offspringsbyverticalspread only Spread by: horizontal spread (conjugation, or rarely by
transduction/transformation)
MechanismofAntimicrobialResistance
Bacteriadevelopantimicrobialresistancebyseveralmechanisms.
1. Decreasedpermeabilityacrossthecellwall:Certainbacteriamodifytheircellmembranepori
nchannels;therebypreventingtheantimicrobialsfromenteringintothecell.Thisstra-
tegyhasbeenobservedinmanygram-
negativebacteriasuchasPseudomonas,EnterobacterandKlebsiellaspeciesagainstdrugssuchas
imipenem,aminoglycosidesandquinolones.
2. Effluxpumps:Certainbacteriapossesseffluxpumpswhichmediateexpulsionofthe
drug(s) from the cell, soon after their entry; thereby decreasing the
intracellularaccumulationofdrugs.Thisstrategyhasbeenobservedin:
• EscherichiacoliandotherEnterobacteriaceaeagainsttetracyclines,chloramphenicol
• Staphylococciagainstmacrolidesandstreptogramins
• StaphylococcusaureusandStreptococcuspneumoniaeagainstfluoroquinolones.
3. By modification of the antimicrobial target sites within the bacteria: This strategy
GeneralMicrobiolog
hasbeenobservedin:
• MRSA(MethicillinresistantStaphylococcusaureus):(seechapter-3.1fordetails).
• VRE(VancomycinresistantEnterococci)(Seechapter-3.2fordetails)
• StreptomycinresistanceinMycobacteriumtuberculosis:Duetomodificationofriboso
malproteinsor16SrRNA.
• RifampicinresistanceinMycobacteriumtuberculosis:DuetomutationsinRNApolymer
ase.
• Quinoloneresistance(seeninS.aureusandS.pneumoniae):DuetomutationsinDNAgyr
aseenzyme. MechanismofAntimicrobialResistance:
Decreasedpermeabilityacrossthecell wall
4. Byenzymaticinactivation:Certainbacteriacaninactivatetheantimicrobialagentsbyproduci Effluxpumps
ngvariousenzymessuchas: By modification of theantimicrobial target sites withinthe
• Aminoglycoside modifying enzymes like (acetyltransferases, Byenzymaticinactivation
adenyltransferases,andphosphotransferases,producedbybothgram-
negativeandgram-positivebacteria): Theydestroythestructureof aminoglycosides.
• Chloramphenicolacetyltransferase:ItisproducedbymembersofEnterobacteriaceae
• βlactamaseenzymeproduction.
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Beta-LactamaseEnzymes
β-lactamaseenzymesarecapableofhydrolysingtheβ-lactamrings(theactivesite)ofβ-
lactamantibiotics;therebydeactivatingtheirantibacterialproperties.
• Itisobservedinbothgram-positiveandgram-negativebacteria
• Theyareplasmidcoded,andtransferredfromonebacteriumtoothermostlybyconjugation,
(except in Staphylococcus aureus where they are transferred by
transduction).Betalactamasescanbeclassifiedintwoways:
○ Ambler’sclassification(structuralormolecularclassification)—Seetable
○ BushJacobyMedeirosclassificationorfunctional(phenotypic)—
Advancedandcomplexclassification
Table1.4.3:Ambler classification ofbeta-lactamases
ClassA-ESBL(Extendedspectrumβ-lactamases)
OrganismsproducingESBLenzymesareresistanttoallPenicillinsand1st,2ndand3rdgenerationcephalosporinsandmonobactams,howeverremain sensitive to
carbapenems andcephamycins
• Resistancecanbeovercomebyβ-lactam+β-lactamaseinhibitor(e.g.sulbactumorclavulanicacid)
• DetectedbyCombinationdisktest(Ceftazidimeandcetftazidime+clavulanicacid),Threedimensionaltest(bestmethod)
ClassB-MBL(Metallobeta-lactamase)
TheseorganismsareresistanttoallthoseantibioticstowhichAmpCbeta-lactamaseproducersareresistantplustheyareresistantto
carbapenems.
• Resistancecannotbeovercomebyβ-lactam+β-lactamaseinhibitorcombination
• DetectedbyEDTAdisksynergytest,modifiedHodgetest
ClassC-AmpCbeta-lactamase
TheseorganismsareresistanttoallthoseantibioticstowhichESBLproducersareresistantplustheyareresistanttocephamycins(e.g.cefoxitinandcefotetan ). But they
aresensitive to carbapenems.
• Resistancecannotbeovercomebyβ-lactam+β-lactamaseinhibitorcombination
• DetectedbyAmpCdisktestusingcefoxitindisk
ClassD-oxacillinase
Resistancecanbeovercomebyβ-lactam+β-lactamaseinhibitorcombination
AntimicrobialSusceptibilityTesting
Antimicrobialsusceptibilitytestingmethodsinclude:
• Diskdiffusionmethods:Kirby-BauerdiskdiffusionmethodandStokesdiskdiffusion
AntimicrobialSusceptibilityTestingMethods:
Discdiffusionmethods method:
Dilutiontests
○ Mueller-Hinton agar(MHA)isconsideredas thebestmedium
EpsilometerorE-test
Automated methods ○ LawnculturemethodisusedtoinoculatetheorganismontoMHA
Molecular methods ○ Controlstrains:ATCC(AmericanTypeCultureCollection)strainsareused
○ ReportingisdoneaccordingtoCLSI(ClinicalandLaboratoryStandardsInstitute)
guidelines
• Dilutiontests:BrothdilutionmethodandAgardilutionmethod:
Section
○ Here,theantimicrobialagentisseriallydiluted,eachdilutionistestedwiththetestorgan
ism forantimicrobialsusceptibility testandtheMIC iscalculated.
○ MIC(minimuminhibitoryconcentration)isthelowestconcentrationofanantimi-
crobial agent thatwill inhibitthe visiblegrowth ofa microorganism.
• Epsilometer or E-test: This is a quantitative method detecting MIC by using the
principlesofboththedilutionanddiffusionofantibioticintothemedium.
• AutomatedmethodssuchasVITEK2,PhoenixSystemandMicroScanWalkAway
system
• Molecularmethods(PCRdetectingdrugresistantgenes)e.g.MecAgeneforMRSA.
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BACTERIALGENETICS b. Ifresistanceisplasmidmediated,itisalwaystransferred
vertically
1. Bacteria used in Griffith experimentis:(PGINov 2016) c. Alterationoftargetseeninpneumococcalresistance
a. Streptococcuspyogenes d. CompleteremovaloftargetiscauseofresistancetoVan
b. CapsulatedStreptococcuspneumoniae comycin
c. Staphylococcusaureus 10. Multipledrug resistance is spread by: (TN2008)
d. MRSA a. Transformation b. Transduction
e. Non-capsulatedStreptococcuspneumoniae c. Mutation d. Conjugation
2. PickthetruestatementregardingPlasmids: 11. Apatientiskeptonceftriaxoneandamikacin,ESBLKlebsi
a. Nonself-replicative (JIPMERMay2015) ellainfection.Whatwillyoudonext?
b. ActsasMessengerRNA (AIIMSNov 2010)
c. InvolvedinConjugationaltransferbetweenstrains a. Continuewithsameantibioticbutinhigherdose
d. Involvedintransformation b. Changeceftriaxoneandaddceftazidime
3. Nontoxigenic C. diphtheriae changes to toxigenic c. Startimipeneminplaceofceftriaxone
C.diphtheriaebythehelpofbacteriophage...bywhichme d. RemoveAmikacin
thodthisconversionoccur? (AIIMSNov2015) 12. DrugagainstESBLproducingPseudomonas:
a. Transfection (AIIMSNov2014,JIPMERNov2014)
b. Transduction a. Ceftriaxone+Piperacillin
c. Conjugation b. Ceftriaxone+Tazobactam
d. RecombinantTechnology c. Piperacillin+Tazobactam
4. MovementofDNAfromonebacteriatoanotherconnecti d. Ceftriaxone+Piperacillin+Tazobactam
ontubeorpilusiscalled:(JIPMER2014,2012) 13. MIC(minimuminhibitoryconcentration)canbecalculat
a. Transformation b. Transduction ed by all of the following antibiotic
c. Conjugation d. Lysogenicconversion sensitivitymethodsexcept: (RecentMCQ2013)
5. MechanismofdirecttransferoffreeDNA: a. Etest
(NEETPatternBased) b. Agardilutionmethod
a. Transformation b. Conjugation c. KirbyBauer’sdiskdiffusionmethod
c. Transduction d. None d. Brothdilutionmethod
6. Phagemediate transferof cDNAintohostisknownas: 14. Forantibioticsensitivitytest,theorganismbroth
(DNBDec2012) preparedshouldmatchwith: (RecentMCQ2013)
a. Transduction b. Transformation a. McFarlandstandard0.5
c. Transmission d. Conjugation b. McFarlandstandard1
7. Horizontaltransmissionof‘R’factorisby: c. McFarlandstandard2
(JIPMER2009) d. McFarlandstandard3
GeneralMicrobiolog
a. Transduction b. Transformation 15. Betalactamaseisproducedby: (RecentMCQ2013)
c. Conjugation d. Fusion a. E.coli b. Gonococcus
c. Staphylococcusaureus d. Alloftheabove
16. Whichofthefollowingdisease(s)is/arenottoxin
ANTIMICROBIALRESISTANCE mediated: (PGINov2014)
8. Mostcommonmethodofbacteriaresponsiblefordrugre a. Diphtheria b. Tetanus
sistance: (PGINov2016) c. Pertussis d. Anthrax
a. Conjugation b. Transduction e. Syphilis
c. Transformation d. Enzymeinactivation 17. A strain of E. coli isolated from urine is resistant
e. Mutation tothirdgenerationcephalosporins.Themechanismofdev
9. Nottrueaboutbacterialdrugresistancemechanism: elopmentofresistanceis: (JIPMERNov2014)
(AI2012,AIIMSNov2011,AIIMSMay2012) a. ExtendedspectrumBeta-Lactamases
a. Most commonmechanism is production b. Decreasedpermeability
ofneutralizingenzymes c. Activeeffluxof Beta-Lactamagents
d. Alteration ofPBP
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EXPLANATIONS
BACTERIALGENETICS
1. Ans.(b,e)(CapsulatedS.pneumoniae,Non-capsulatedS.pneumoniae)Ref:ApurbaSastry'sEssentialsofMedicalMicro-
biology/p71
Capsulated dead S.pneumoniae + non-capsulated live S.pneumoniae → Transformation of gene coding capsule from
deadtolivepneumococci→Resultsincapsulatedlivepneumococci
2. Ans(c)(Involvedinconjugational...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep67
Plasmidsareselfreplicativeextra-chromosomalelementsfrequentlytransferredbyconjugation.
3. Ans(b)TransductionRef:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep73
• Transductionisthetransferofbacterialgenesfromonebacterialtootherbybacteriophage
• LysogenicConversionwouldhavebeenabetteranswerhere.ItistheprocessbywhichthephageDNAisintegratedtobacteria
lDNAandremainsaslysogenicphage.Insuchcase,certainphagegene(e.g.genecodingfordiphtheriatoxin)impartstoxigeni
citytothebacteria.
4. Ans.(c)ConjugationRef:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep73,Ananthanarayan9/ep61
5. Ans.(a)(Trans...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep70,Ananthanarayan9/ep59,8/ep63
• TransformationistheprocessofthetransferoffreeDNAitselffromonebacteriumtoanother.
6. Ans.(a)(Transduction)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep671,Ananthanarayan9/ep59
Refertext
7. Ans.(c)(Conjugation)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep73,Ananthanarayan9/ep60
• Conjugationistheprocesswherethereistransferofgeneticelementsfromonebacterium(male)toanother(female)alongse
xpilusorconjugationtube-Horizontalgenetictransfer.
ANTIMICROBIALRESISTANCE
8. Ans(a,d)(Conjugation,Enzymeinactivation)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p82-83.
Transferrableresistancebyconjugationisthemostcommonmethodoftransferofbacterialresistantgenes.Enzymeinacti-
vationisthemostcommonmechanismsofbacteriadrugresistance.
9. Ans.(b)
(Ifresistanceisplasmidmediated,itisalwaystransferredvertically)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep82,Ananthanarayan9/ep63,8/ep67,Harrison18/ep1157
• Ifresistanceischromosomallymediated,itisusuallytransferredverticallyfromparenttodaughterbacteria.
• Ifresistanceisplasmidmediated,itisusuallytransferredbyhorizontalroutemainlybyconjugation.
AboutOtherOptions
Section
• Clinically,enzymaticdruginactivationisthemostcommonmechanismforacquiredmicrobialresistancebybacteria
• Mostcommonmechanismofbacterialdrugresistance:
• Pneumococcalresistance:ismainlyduetoAlterationoftarget,i.e.Penicillinbindingprotein(PBP)
• ResistancetoVancomycinisduetocompleteremovaloftargetDalanyl-
Dalaninepresentthebacterialcellwallisthetargetsite for Vancomycin, which binds there and inhibits it and thus
inhibits the cellwall synthesis.
ThefourMajorMechanisms ofAntibacterialResistance:Refer chapterreview.
10. Ans.(d)(Conjugation)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep73,Ananthanarayan9/ep60-61
• Resistance(R)factorsareextrachromosomalplasmidsresponsibleforspreadofmultipledrugresistanceamongbacteria.
• TheyarecirculardoublestrandedDNAcarrygenesforvarietyofenzymesthatcandestroyantibiotics
• Rfactorconsistsof2components:Resistancetransferfactor(RTF)andresistantdeterminant(r).
• Theresistancetransferfactorisresponsibleforconjugationaltransferwhileeachrdeterminantcarriesresistanceforoneofthesev
eralantibiotics.
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11. Ans.(c)(Startimipeneminplaceofceftriaxone)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology1/ep83
• ESBL(Extendedspectrumbetalactamases)areresistanttoallPenicillinand1st/2nd/3rdcephalosporinandmonobactam
• Whichcanbeovercomebyadditionofβlactamaseinhibitorlikeclavulinicacid
• Otheralternatewhichcanbegivenare:
○ CarbapenamslikeImipenemandmeropenem
○ Cephamycins(likecefoxitinandcefotetan)
○ Differentclassofantibioticslikeaminoglycoside
12. Ans.(c)(Piperacillin+Taz...)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology1/ep83
ExtendedSpectrum-β-
Lactamases(ESBL)producingPseudomonascanbetreatedwithanantipseudomonialβlactam(e.g.piperacillin)plusβlactamas
einhibitorsuchastazobactamcombinationtherapy.
13. Ans.(c)(KirbyBauer’sdiskdiffusionmethod)Ref:Ananthanarayan8/ep619&9/ep635
• Kirby Bauer’s disk diffusion method is used to know the zone of inhibition of the streaked organism surrounding
thedisk by which we can know whether the organism is sensitive or resistant to the antibiotic disk. However, we
cannotknowtheMIC.
• MIC(Minimalinhibitoryconcentration)oftheantibioticisdefinedasthelowestconcentrationofanantimicrobial
agentthatwillinhibitthevisiblegrowthofamicroorganism.
• MICiscalculatedby:(i)Agardilutionmethod,(ii)Brothdilutionmethodand(iii)Epsilometer(Etest)
14. Ans. (a)(McFarlandstandard0.5) Ref: MackieMcCartney14/ep851-852
• Inmicrobiology,McFarlandstandardsareusedasareferencetoadjusttheturbidityofbacterialsuspensionssothatthenumberofb
acteriawillbewithinagivenrange.
• A0.5McFarlandstandardispreparedbymixing0.05mLofbariumchloridedihydratewith 9.95 mL of
1%sulfuricacidanditsequivalentto150millionno.ofbacteria/mLinabroth.
• Forantibioticsensitivitytest,theorganismbrothpreparedshouldmatchwith-0.5McFarlandstandard.
15. Ans.(d)(Alloftheabove)Ref:Ananthanarayan9/ep62,233
Betalactamaseenzymesareplasmidcoded,producedbybothgrampositiveandgramnegativeorganisms.
16. Ans.(e)(syphilis…)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep93SyphilisiscausedbyT.pallidum.Referchapterreview.
17. Ans.(a)(ExtendedspectrumBeta-Lactamases)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep83
OutofseveralmechanismsofbetalactamresistanceinE.coli,Beta-
LactamaseproductionistheMOSTCOMMON;particularlyExtendedspectrumBeta-Lactamases(ESBL).
GeneralMicrobiolog
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SECTION 2
Immunology
CHAPTEROUTLINE
Immunity
Antigen,Antibody,Antigen-AntibodyReaction,Complement
StructureofImmuneSystemandImmuneResponse
Hypersensitivity
Autoimmunity,Immunodeficiency,Transplantation,andImmun
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CHAPTER
Immunity 2.1
INNATEIMMUNITY
Theterm‘immunity’isdefinedastheresistanceofferedbythehostagainstmicroorganism(s)oranyfo
reignsubstance(s).Immunitycanbebroadlyclassifiedinto:
1. Innateimmunity:presentrightfromthebirth
2. Acquired/Adaptive immunity:acquired duringthe courseofthe life.
Table2.1.1:Differencesbetweeninnateandacquiredimmunity
Innate immunity Acquired/Adaptiveimmunity
Resistancetoinfectionthatanindividualpossessesfrombirth Resistancetoinfectionthatanindividualacquiresduringhislifet
ime
Immuneresponseoccursinminutes Immuneresponseoccursindays
Priorexposuretotheantigenisnotrequired Developsfollowingtheantigenicexposure
Diversityislimited,actsthrougharestrictedsetofreactions Morevaried andspecialized responses
Immunologicalmemoryresponsesareabsent Immunologicalmemoryresponsesarepresent
Respond to microbial antigens that are not specific to some microbe,rather Respondtospecificmicrobialantigens
shared by many microbes called microbes-
associatedmolecularpatterns(MAMP)
Hostcellreceptors(patternrecognitionreceptors)arenonspecific, Hostcellreceptorsarespecific,e.g.Tcell receptors
e.g.Toll-likereceptor andBcellimmunoglobulinreceptors
Componentsofinnateimmunity Componentsofacquiredimmunity:
• Anatomicalbarriers,suchasskinandmucosa • Tcell
• Physiologicalbarriers(e.g.bodytemperature) • B cell
• Classicalcomplementpathway
• Phagocytes(neutrophils, macrophagesandmonocytes)
• Antigenpresentingcells
• Naturalkiller(NK)cells
• Cytokines(IL-2,IL-4,IL-5,IFN-γ)
• OtherClassesoflymphocytes-γδTcells,NK-Tcells,B-1cellsand
marginal-zone B cells
Typesofacquiredimmunity:
• Mast cells
Itcanbeclassifiedintwoways:
• Dendriticcells
• Complementpathways:alternateandmannosebindingpathways • Activeandpassiveimmunity
• Feverandinflammatoryresponses • Artificialandnaturalimmunity
• Normalresidentflora
• Cytokines-TNF,certaininterleukin(IL-1,IL-6,IL-8,IL-12,IL-16,IL-18),IFN-
α,βandTGF-β
• Acutephasereactantproteins(APRs)
FactorsInfluencingInnateImmunity
• DependsontheSpecies,Race,Individual(geneticinfluence)
• Age,HormonalinflueneandNutrition.
TollLikeReceptors
TheyaresonamedbecausetheyaresimilartoTollreceptorspresentinthefruitfly-Drosophila,where
it is the main receptor for induction of innate immunity by bind to particular
MAMPmoleculesonmicrobialsurfaces.Theyareof13types,outofwhichimportantonesare: Tolllikereceptors:
• TLR-2bindstobacterialpeptidoglycan Theyaretheprinciplehostcellreceptorsofinnateimmunity
• TLR-3bindstodsRNAofviruses
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• TLR-4bindstoLPSofGram-negativebacteria
• TLR-5bindstoflagellaofbacteria
• TLR-7&8bindtossRNAofviruses
• TLR-9bindstobacterialDNA.
AcutePhaseReactantProteins(APRs)
Theyaretheproteinssynthesizedbyliveratsteadyconcentration,buttheirsynthesiseitherincreases
ordecreasesexponentiallyduringacuteinflammatoryconditions.
Thoughliveristheprimarysite,APRscanalsobesynthesizedbyvariousothercellssuchasendoth
elialcells,fibroblasts,monocytesandadipocytes.
PositiveAPRsaretheproteinswhoselevelsincreaseduringacuteinflammation.Examples:
• SerumAmyloidA
• C-Reactiveprotein
• Complementproteins:Complementfactors(C1–C9),factorB,D,andproperdin
• Coagulation protein,e.g.fibrinogen,vonWillebrand factor
• Proteinaseinhibitors,e.g.α1antitrypsin
• α1acidglycoprotein
• Mannosebindingprotein
• Haptoglobin
• Metalbindingproteins,e.g.ceruloplasmin
Negative APRs: They are the proteins whose levels are decreased during acute
inflammationthus creating a negative feedback that stimulates the liver to produce positive
APRs. ExamplesofnegativeAPRsinclude:albumin,transferrin,andantithrombin.
RoleofAPRs:Theyhaveawiderangeofactivitiesthatcontributetothehostdefense:
• APRshavevariousantimicrobialandanti-inflammatoryactivities(e.g.complement)
• Metal binding proteins can chelate various metals such as iron, copper, etc. making
themunavailableforthebacteria.
C-ReactiveProtein(CRP)
CRP is an example of APR that rise in acute inflammatory conditions including
CRP bacterialinfections.Itbelongstobetaglobulinfamily.
Normallevel<0.2mg/dl. • CRPissonamedbecauseitprecipitateswithC-carbohydrate(polysaccharide)antigenof
Detectionlimitoflatex Pneumococcus. However, it is not an antibody against the C-carbohydrate antigen
agglutinationtest–0.6mg/dl ofPneumococcus;itisnonspecific,canberaisedinanyinflammatoryconditions.
Insignificantincrease
• Commonestmarkersofacuteinflammation,usedinmostdiagnosticlaboratories.
(<1mg/dl),e.g.inheavyexercise, common cold,andpregnancy
Moderate increase (1–10mg/dl), e.g. bronchitis,cystitis, malignancies,pancreatitis, myocardialinfarction.
CRPLevel
Marked increase (> 10 mg/dl), e.g. acute bacterial in-fections, major trauma andsystemic vasculitis.
ThenormallevelofCRPis<0.2mg/
dl.However,itincreasesbyseveralfoldsinacuteinflammatoryconditions.
• InsignificantincreaseofCRP(<1mg/
dl):Occursinconditionssuchasheavyexercise,commoncold,andpregnancy
Section
• Moderateincrease(1–10mg/
dl):Occursinconditionssuchasbronchitis,cystitis,malignancies,pancreatitis,myocardialinfarc
tion.
• MarkedincreaseofCRP(>10mg/
dl):Occursinconditionssuchasacutebacterialinfections,majortraumaandsystemicvasculitis.
CRPcanbedetectedby
• PrecipitationmethodusingCcarbohydrateantigen(obsolete,notinusenow)
• Latex(passive)agglutinationtestusinglatexparticlescoatedwithanti-CRPantibodies.
○ It isthemost widelyusedmethodemployed worldwide.
○ DetectionlimitofCRPbylatexagglutinationtest0.6mg/dl.
• Highly sensitive CRP (hs-CRP) test: Minute quantities of CRP can be detected by
variousmethods(e.g.nephelometry,enzymeimmunoassays).Thisisusefulinassessingther
isktocardiovasculardiseases.
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Immunity53
ACQUIREDORADAPTIVEIMMUNITY
Acquiredimmunityisdefinedastheresistanceagainsttheinfectingforeignsubstancethatanindividu
alacquiresoradaptsduringthecourseofhislife.
• Itisoftwotypes:Activeimmunityandpassiveimmunity.
• Activeimmunitycanagainbedividedinto:
○ Primaryimmuneresponse(whichdevelopsafterfirstmicrobialexposure)
○ Secondaryimmuneresponse(whichdevelopsaftersubsequentmicrobialexpo-sure).
Table2.1.2:Differencesbetweenactiveandpassiveimmunity
Activeimmunity Passive immunity
Producedactivelybyhostimmunesystem Immunoglobulinsreceivedpassively
Inducedby: Acquiredby:
• Infection(natural) • MothertofetusIgGtransfer(natural)
• Vaccination(artificial) • Readymadeantibodytransfer(artificial)
Longlasting Lastsforshorttime
Lagperiodpresent NoLagperiod
Memorypresent NoMemory
Boosterdosesareuseful Subsequentdosesarelesseffective
Negativephasemayoccur NoNegativephase
Inimmunodeficiencyindividualsnotuseful Usefulinimmunodeficientindividuals
Table2.1.3:Differencesbetweenprimaryandsecondaryimmuneresponse
Primaryimmuneresponse Secondaryimmuneresponse
Immuneresponseagainstprimaryantigenicchallenge Immuneresponseagainstsubsequentantigenicchallenge
Slow,sluggish(appearlate)andshortlived Prompt,powerfulandprolonged(longlasting)
Lagperiodislonger(4–7days) Lagperiodisabsentorshort(1–3days)
Nonegativephase Negativephasemayoccur
AntibodyproducedinlowtiterandisofIgMtype. Antibody produced in high titer and is of IgG
Antibodiesaremorespecificbutlessavid typeAntibodiesarelessspecificbutmore avid
Antibodyproducingcells:NaiveBcells Antibodyproducingcells:MemoryBcells
BothTdependentandTindependentantigensareprocessed. OnlyTdependentantigensareprocessed.
Immunolog
Primary immune responseImmune response against
Slow,sluggish(appearlate)
andshortlived
Lagperiodislonger
(4–7days)
Nonegativephase
Antibody produced in low titerandisofIgM type.
Antibodiesaremorespecific
butlessavid
Antibody producing cells-NaiveB cells
BothTdepdandTindepdAgareprocessed.
Fig.2.1.1:Primaryandsecondaryimmuneresponses
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OTHERTYPESOFIMMUNITY
Local(ormucosal)Immunity
Localormucosalimmunityistheimmuneresponsethatisactiveatthemucosalsurfacessuchasintesti
nalorrespiratoryorgenitourinarymucosa:
• ItisusuallymediatedbyatypeofIgAantibodycalledsecretoryIgA.
• Localimmunitycanonlybeinducedbynaturalinfectionorbylivevaccination,e.g.afterOPV(
Local or mucosal immunityImmune response that is active atthe mucosal surfaces
Mediatedbysecretory IgA. butnotbykilledvaccines).
Induced by natural infection orbylivevaccination
HerdImmunity
Herd immunity is defined as the overall immunity of a community (or herd) towards
apathogen:
• Herd immunity plays a vital role in preventing epidemic diseases. If the herd
immunityisgood,thatmeanslargepopulationofthecommunityareimmunetowardsapatho
gen.Hence,epidemicsarelesslikelytooccuranderadicationofthediseasemaybepossible.
• Elementsthatcontributetocreateastrongherdimmunityare:
○ Occurrenceofclinicalandsubclinicalcasesintheherd
○ Ongoing immunisationprogramme
○ Herdstructure,i.e.typeofpopulationinvolved
○ Type of pathogen: Herd immunity may not be strong in a community against
allthepathogens.
Herd immunity developsfollowingvaccinationagainst:
DiphtheriaandPertussis • Herdimmunitydevelopsfollowingeffectivevaccinationagainstsomediseaseslike:
vaccine ○ DiphtheriaandPertussisvaccine
Measles, Mumps and Rubella(MMR) vaccine ○ Measles,MumpsandRubella(MMR)vaccine
Polio(Oralpoliovaccine) ○ Polio(Oralpoliovaccine)
Smallpox vaccine
○ Smallpoxvaccine.
AdoptiveImmunity
ItistheprocessoftransferofCMIfromoneindividualtoother.
• ItoccursfollowinginjectionofimmunologicallycompetentT-
lymphocytesknownasTransferfactor.
• Itis useful for treatmentwhen the CMI islow, e.g. in lepromatous leprosy.
Section
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Immunity55
1. Tolllikereceptors:correctstatementis:
8. Allofthefollowingareapartoftheinnateimmunityexcep
a. Antigenspecific (RecentQuestion2015)
t: (AIIMSMay2005)
b. Actsbycytokinerelease a. Complement
c. Partofadaptiveimmunity
b. NKcells
2. Nottrueaboutinnateimmunity:(NEETPatternBased) c. Macrophages
a. Notinfluencedby hormones d. Tcells
b. Dependentongeneticconstitution
9. Activeimmunitycanbeinducedby: (PGIMay2013)
c. Identicaltwinshavesamedegreeofresistance
a. Toxoids
d. Notinfluencedbyexposuretoantigen
b. Subclinicalinfection
3. Allaretrueaboutinnateimmunityexcept: c. Antitoxin
a. Actsasfirstlineofdefense (NEETPatternBased) d. Immunoglobulins
b. Complementsareexamples e. Antigenexposure
c. Nonspecific
d. Noteffectedbygeneticinfluences 10. True aboutpassiveimmunity: (PGIMay2013)
a. Cannotbegivenwithactiveimmunity
4. Transferfactorisanexampleof:(NEETPatternBased)
b. Lastfor4–5daysonly
a. Artificialactiveimmunity
c. Itcanbegivenbeforediseaseoccurrence
b. Naturalactiveimmunity
d. CanbetransferredbyantibodiesfromanotherHost
c. Adoptiveimmunity
e. Takeslongertimetodevelop
d. Artificialpassiveimmunity
11. Superantigenisproducedby:
5. Componentsofinnateimmunity: (PGINov2010)
a. Staphylococcusaureus (RecentQuestion2013)
a. Tlymphocyte b. Blymphocyte
b. Streptococcuspneumoniae
c. Complements d. NKcells
c. Pseudomonas
e. Integrins
d. Clostridium
6. Innateimmunityisstimulatedbywhichpartofbacteria?
(DNBDec2011)
a. Carbohydratesequenceinthecellwall
b. Flagella
ACUTEPHASEREACTANTSANDCRP
c. Bacterialcellmembrane 12. SpanofCreactiveproteinhalf-life: (TNPG2014)
d. Nucleus
a. 18hrs
7. Innateimmunityactiveagainstviralcells: (AI2007) b. 2hrs
a. NKcells b. CytotoxicTcells c. 12hrs
c. B cells d. MemoryBcell
d. 15hrs
Immunolog
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EXPLANATIONS
1. Ans.(b)(Actsby.........)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep98
TLRisreceptorofinnateimmunity,antigennonspecific.
2. Ans.(a)(Notinfluencedbyhormones)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep97,Ananthanarayan9/ep78,8/
ep84,Kuby’sImmunology6/ep53
• Innateimmunityisinfluencedbyhormones:Endocrinedisorderslikediabetesareassociatedwithenhancedsusceptibilityt
oinfectionduetoalteredinnateimmunity.
• Innateimmunityisdependentongeneticconstitutionoftheindividual.Homogenousidenticaltwinsexhibitsimilardegreeo
finnateimmunity.
• Innateimmunityisnonspecific,itisnotinfluencedbyexposuretoantigen.
3. Ans.(d)(Noteffectedbygeneticinfluences)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep97,Ananthanarayan9/ep78,8/ep83,Kuby’sImmunology6/ep53
• Innateimmunityreferstotheresistancetoinfectionthatanindividualpossessfrombirthbyitsgeneticorconstitutionalmakeup.
• Otheroptionsarecorrect-forexplanation,refertext.
4. Ans.(c)(Adoptive.)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep103,Ananthanarayan9/ep81,8/ep89,150
Adoptiveimmunity
• AcquiredbyinjectionofimmunologicallycompetentT-lymphocytesknownasTransferfactor
• UsedfortreatmentwhentheCMIislow,e.g.Lepromatousleprosy.
5. Ans.(c)(d)(e)(Complements,NKcells,Integrins)ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep97,Ref:Anan-
thanarayan9/ep79,8/ep83,Kuby’sImmunology6/ep53,Harrison18/ep2654
• Complementpathways(alternateandmannosebinding,NKcells,andPatternrecognitionreceptorslikeIntegrinsarecomponent
sofInnateimmunity
• ForthedetaillistofcomponentsofInnateimmunity-Refertext.
6. Ans.(a)(Carbohydratesequenceincellwall)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep97,Harrison18/ep2654
• Patternrecognitionreceptorsareimportantcomponentofinnateimmunity
• Toll-
likereceptorsare,e.g.ofPRRProteinFamilywhichbindtocarbohydrateantigensonbacterialandviralsurfaces.Examples
ofmicrobial ligandsthat bindto Patternrecognition receptorson hostcells include:
• Bacterialandviralcarbohydratesresiduesoncellwall
• TerminalmannoseandCarbohydrateonHLAmolecules
• Lipopolysaccharide(LPS)
• ViralDNABacterialmuramyldipeptide
Section
7. Ans.(a)(NKcells)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep97,Ananthanarayan9/ep137,8/ep131
• NaturalkillercellsarecomponentsofinnateimmunityKuby’sImmunology6/ep53
• Naturalkillercellspossessspontaneouscytotoxicitytowardsvirusinfectedcellsandmalignantcellsandtheircytotoxicityis
notantibodydependentnorMHCrestrictedAnanthanarayan8/ep131
• AboutOtheroptions:CytotoxicTcells,BcellsandMemoryBcellarecomponentsofadaptive/
acquireimmunityKuby’sImmunology6/ep53
• ComponentsofInnateimmunity-refertext.
8. Ans.(d)(Tcells)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep97,Kuby’sImmunology6/ep53
• AlternatepathwaysofComplement,NKcellsandMacrophages(asphagocytes)arethecomponentsofinnateimmunity
• Bcell,Tcell,ClassicalcomplementandAntigenpresentingcellarecomponentsofadaptive/acquireimmunity.
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Immunity57
9. Ans.(a)(b)(e)(Toxoids,Subclinicalinfection,Antigenexposure)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiol-ogy1/
ep102,Ananthnarayan9/ep81-82
• Activeimmunity can be inducedby any substance that actively stimulates the immune system to produce
antibody.Vaccines,toxoids,infectionorantigenexposurecaninduceactiveimmunity
• Antitoxinandimmunoglobulinsare,e.g.ofPassiveimmunity.
10. Ans.(c)(d)
(Itcanbegivenbeforediseaseoccurrence,canbetransferredbyantibodiesfromanotherHost)Ref:ApurbaSastry’sEssentials
ofMedicalMicrobiology1/ep103,Ananthanarayan9/ep82-83
• Immunoglobulinscanbegivenalongwithvaccineinpostexposureprophylaxis,e.g.Rabiesimmunoglobulins
• Passiveimmunitylastfordaystomonths
• Immunoglobulinsshouldbegivenimmediatelyaftertheexposurebutbeforethediseaseoccurrence
• PassiveimmunitycanbetransferredbetweenindividualsbySerumtherapy(antibodies)
• Passiveimmunityworksimmediately
11. Ans.(a)(Staphylococcusaureus)Ref:Stite’sMedicalImmunology10/ep152
ACUTEPHASEREACTANTSANDCRP
12. Ans.(a)(18hrs)Journal:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep101,C-reactiveprotein:acriticalup-
date,JClinInvest.2003.
Theplasmahalf-
lifeofCRPisabout19hoursandisconstantunderallconditionsofhealthanddisease,sothatthesoledeterminantofcirculatingCRPconce
ntrationisthesynthesisrate.
Immunolog
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CHAPTER
Antigen,Antibody,Antigen-Antibody
Reaction,Complement 2.2
ANTIGEN
Antigenisdefinedasanysubstancethatsatisfiestwodistinctimmunologicproperties:
Antigen has two distinctproperties: 1. Immunogenicity: Ability of an antigen to induce immune response in the body
Immunogenicity:Ability ofAg (bothhumoraland/orcellmediated).
to induce immune response(CMI/AMI)
• B-cells+antigen→effectorB-cells(plasmacell)+memoryB-cells
Immunological reactivity:Ability of Ag to combine withthe final products of the abovetworesponses(i.e.,antibodiesand/or T-cell-surfacereceptors).
• T-cells+antigen→effectorT-cells(helperorcytotoxicT-cell)+memoryT-cells
2. Antigenicity (immunological reactivity): Ability of an antigen to combine
specificallywith the final products of the above two responses (i.e., antibodies and/or T-
cell-surfacereceptors).
All molecules having immunogenicity property, also show antigenicity, but the
reverseis nottrue(e.g.haptens– whichareantigenic,but notimmunogenic).
Epitope
Epitopeorantigenicdeterminantisthesmallestunitofantigenicity:
• ItisdefinedasasmallareapresentontheantigenthatiscapableofsensitizingT-andB-
cellsandreactingwithspecificsiteofT-cellreceptororanantibody.
• Thespecificsiteofanantibodythatreactswiththecorrespondingepitopeofanantigeniscall
edasparatope.
Epitopesmaybegroupedintotwotypes:
1. Sequentialorlinearepitope:Presentasasinglelinearsequenceoffewaminoacid.
2. Conformationalornonsequentialepitopesarefoundontheflexibleregionofcomplexantigen
shavingtertiarystructures.
Ingeneral,T-cellsrecognizesequentialepitopes,whileB-cellsbindtotheconformational.
Haptens
Haptensarelowmolecularweightmoleculesthat:
• Lackimmunogenicity(cannotinduceimmuneresponse),but:
• Retainantigenicityorimmunologicalreactivity(i.e.canbindtotheirspecificantibodyorT-
cellreceptor).
Hapten-carrier conjugateproduceantibodiesagainst:
Epitopesofhapten However,Haptenscanbecomeimmunogenicwhencombinedwithalargerproteinmoleculecalled‘ca
Unalteredepitopesonthecarrierprotein
rrier’.
New epitopes formed bycombined parts of both thehaptenandcarrier
Itisobservedthathapten-carrierconjugateinduceantibodiesspecificfor:
• Epitopesofhapten
• Unalteredepitopesonthecarrierproteinand
• NewepitopesformedbycombinedpartsofboththehaptenandcarrierHapt
ensmaybeclassifiedascomplexorsimple:
• Complex haptens contain two or more epitopes; they can react with specific
antibodiesandthehapten-
antibodycomplexcanbevisualizedbyvariousmethodssuchasprecipitationreaction.
• Simplehaptensusuallycontainonlyoneepitope(univalent).Suchhaptenscanbindtothe
antibodies but the hapten antibody complex cannot be not visualized as it is
believedthatprecipitationtohappen,itrequirestheantigentohaveatleasttwoormoreepitopes.
FactorsInfluencingImmunogenicity
Therearevariousfactorsthatinfluenceimmunogenicityofanantigen:
1. Sizeoftheantigen:Largeristhesize(e.g.hemoglobin),moreistheimmunogenicity.
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Antigen,Antibody,Antigen-AntibodyReaction,Complement 59
2. Chemicalnature:Proteinsarestrongerimmunogensthancarbohydratesfollowedbylipidand
nucleicacids.
3. Susceptibilityofantigentotissueenzymes—
Chemicalnatureofantigen:
ItincreasesimmunogenicitybyexposingmoreepitopesoftheAg. • Proteins are
4. Structuralcomplexityoftheantigenincreasesimmunogenicity. strongerimmunogens
5. Foreignnesstothehost:MoreistheforeignnessofAg,moreistheimmunogenicity. thancarbohydrates followed by
lipidandnucleicacids.
6. Genetic factor
7. Optimaldoseofantigencanonlyinduceimmuneresponse.Atoolittledosefailstoelicitimmu
neresponseandatoolargedosecausesimmunologicalparalysis.
8. Routeofantigenadministration
9. Repeateddosesofantigensoveraperiodoftime
10. Multipleantigens:Oneantigenmaydiminish(duetoantigeniccompetition)orenhance(dueto
adjuvantlikeaction)theimmunogenicityofotherantigen.
11. Heterophilenatureoftheantigens(explainedbelow).
12. Adjuvant(explainedbelow)
Adjuvantexamples:
Adjuvant Alum
Mineraloil(liquidparaffin)
Thetermadjuvantreferstoanysubstancethatenhancestheimmunogenicityofanantigen.They Freund'sadjuvant
are usually added to vaccines to increase the immunogenicity of the vaccine LPSofBordetellapertussis
Otherbacterialproducts
antigen.Examplesofadjuvantinclude: Mycobacteriumbovis
• Alum(aluminumhydroxideorphosphate) Toxoid(DTandTT)
• Mineraloil(liquidparaffin) Nonbacterialproducts:Silicaparticles, beryllium sulfate,squ
• Freund’sincompleteadjuvant:Itisawater-in-oilemulsion;withAgintheaqueousphase.
• Freund’scompleteadjuvantisthemixtureofFreund’sincompleteadjuvantandkilledtubercl
ebacilliintheoilphase.
• LPSofBordetellapertussisactsasanexcellentadjuvantfordiphtheriaandtetanustoxoids.
• Otherbacteriaortheirproducts:
○ Mycobacteriumbovis
○ Toxoid(DTandTTactasadjuvantforHaemophilusinfluenzae-bvaccine)
• Nonbacterialproducts:Silicaparticles,berylliumsulfate,squalene,andthimerosal.
Heterophile antigens:Applications
HeterophileAntigens Weil-Felixreaction
Paul-Bunnell test
Heterophile antigens share epitopes with each other. Antibody produced against antigen Coldagglutinationtest
ofonespeciescanreactwiththeotherandviceversa: Streptococcus MG
Forssmannantigen
• Weil–
Felixreactionisdonefordiagnosistyphusfever.Antibodiesagainstrickettsialantigensaredetect
edbyusingcrossreactingProteusantigens.
• Paul-
Bunnelltestisdoneforinfectiousmononucleosis(causedbyEBV).Here,sheepredbloodcell(
RBC)antigensareusedtodetectcross-reactingantibodiesinpatient’ssera.
• Cold agglutination test and Streptococcus MG test are done for primary atypical Immunolog
pneumonia.Here,antibodiesagainstMycoplasmapneumoniaearedetectedbyusinghuman
ObloodgroupRBCandStreptococcusMGantigensrespectively.
• Forssmannantigenisuniversalheterophileantigen,presentinallanimals,plantsandbacteria,bu
tabsentinrabbits.
BiologicalClassesofAntigens
Depending on the mechanisms of inducing antibody formation, antigens are classified asT-
celldependent(TD)andT-cellindependent(TI)antigens.
T-dependent(TD)Antigens
Most normal antigens are T-cell dependent, they are processed and presented by
antigenpresenting cells (APCs) to T-cells. The activated T-cells secrete cytokines that in turn
stimulatetheB-cellstoproduceantibodies.
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T-independent(TI)Antigens
There are a few antigens such as bacterial capsule, flagella and LPS that do not need the help
ofT-cells and APCs. They directly bind to Ig receptors present on B-cells and stimulate B-
cellspolyclonallyleadingtohypergammaglobulinemia.
T-independentantigen T-dependentantigen
Structurallysimple:LPS,capsularpolysaccharide,flagella Structurallycomplex:proteininnature
Dosedependentimmunogenicity Immunogenicoverwiderangeofdose
Nomemory Memorypresent
Noantigenprocessing Antigenprocessingstepisneeded
Slowly metabolized Rapidlymetabolized
ActivateB-cellspolyclonally Activate B-cells monoclonally
ActivatebothmatureandimmatureB-cells ActivatematureB-cellsonly
B-cellsstimulatedagainstTindependentantigendonotundergo- B-cellsstimulatedagainstTdependentantigenundergoAffinitymaturationand
AffinitymaturationandClassswitchover Classswitch over
Antibodyresponse isrestricted to IgMandIgG3 Antibodiesofallclassescanbeproduced
Superantigens
Superantigens are the third variety of biological class of antigens, recently described in
thelastdecade.Theuniquefeatureofsuperantigensis,theycanactivateT-
Superantigens:
cellsdirectlywithoutbeingprocessedbyantigenpresentingcells(APCs).
•
ActsdirectlyonTcell,withoutAPCinvolvement The variable β region of T-cell receptor (vβ of TCR) appears to be the receptor
ReceptorofsuperAg-variable forsuperantigens.
βregionofT-cellreceptor
• Theydirectlybridgenon-specificallybetweenMHC-IIofAPCsandT-cells.
ExamplesofSuperantigens
• Bacterialsuperantigen:
○ Staphylococcaltoxin:TSST,Exfoliativetoxin,Enterotoxins
○ Streptococcaltoxin:Streptococcalpyrogenicexotoxin(SPE)-AandC
○ Mycoplasmaarthritidismitogen-I
○ Yersiniaenterocolitica
○ Yersiniapseudotuberculosis
• Viral:EBV,CMV,Rabiesnucleocapsid,HIVencodednef(negativeregulatoryfactor)
• Fungalsuperantigen:Malasseziafurfur
Section
Fig.2.2.1:Modeofactionofvariousantigens
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Antigen,Antibody,Antigen-AntibodyReaction,Complement61
ANTIBODY
StructureofImmunoglobulins
Antibodyorimmunoglobulinisa‘Y-
shaped’heterodimer;composedoffourpolypeptidechains:twolight(L)chainsandtwoheavy(H)
chains:
• AllfourHandLchainsareboundtoeachotherbydisulfidebonds,andbynoncovalentinteracti
ons such as salt linkages, hydrogen bonds,and hydrophobic bonds.
• Chainshavetwoends:Anaminoterminalend(NH3)andacarboxylterminalend(COOH).
• TherearefiveclassesofHchains(γ,α,µ,δandε)andtwoclassesoflightchains(κandλ).
• Anyantibodycontainsonlyonetypeoflightchainandonetypeofheavychain.
• Based on the constant region of the heavy chains (γ, α,µ,δ and ε); Ig has been
classifiedintofivetypes;lgG,IgA,IgM,IgDandIgErespectively.
• EachHandLchaincomprisesoftworegions:Variableregionandconstantregion
• Within the variable region, there are some zones or hot spots called as
hypervariableregions or complementarity determining regions that show higher
variability. There arethreehotspotsintheLandfourintheHchain.
• Paratopeisthesiteonthehypervariableregionsthatmakeactualcontactwiththeepitopeofanant
igen.
• Hingeregion:
○ It is the junction formed between constant region of heavy chains of IgG, IgA
andIgD.ItisabsentinIgEandIgM.
○ Thisregionisrichinprolineandcysteine.Thehingeregionisquiteflexible,thushelpst Enzymatic digestion:
heantibodyinreachingtowardstheantigen. Papain digestion: generatesTwoFabandoneFcfragment
Pepsin digestion: generatesOne F(ab')2and Many smallerf
• Enzymaticdigestion: Mercaptoethanol digestion:generates four fragments (twoH
○ Papain digestion: Papain cleaves the Ig molecule at a point above the hinge
region;resulting in three fragments: Two Fab fragments (Ag binding fragment) and
one Fcfragment(crystallisablefragment)
○ Pepsin digestion: Pepsin cleaves the Ig molecule at a point below the hinge
region;resultinginformationof-OneF(ab’)2fragmentandManysmallerfragments
○ Mercaptoethanol digestion: Generates four fragments (two H and 2 L chains) as
itcleavesonlydisulphidebondssparingthepeptidebonds.
• Immunoglobulinchainscodedbydifferentchromosomes
○ Heavychains:Itiscodedbychromosome-14
○ Lightchainkappa:Itiscodedbychromosome-2
○ Lightchainlambda:Itiscodedbychromosome-22.
○ Afterthesynthesis,germlinerecombination(rearrangement)ofchainsoccurs.
FunctionsofImmunoglobulins
• Antigenbinding(byFabregion)
Immunolog
Immunoglobulin chains codedbydifferentchromo
• Effectorfunctions(byFcregion)
Heavychainsiscodedby
○ Fixationofcomplement:Antibodycoatingthetargetcellbindstocomplementthroug chromosome-14
hitsFcreceptorwhichleadstocomplementmediatedtargetcelllysis Lightchainkappaiscodedbychromosome-2
○ Bindingtovariouscelltypes:Phagocyticcells,lymphocytes,platelets,mastcells,NKcell, Light chain lambda is coded bychromosome-22
eosinophils and basophils bear Fc receptors (FcR) that bind to Fc region
ofimmunoglobulins.
PropertiesofVariousImmunoglobulin
IgGAntibody:
• IgGishighestforDHS(decreasingorderforDHSis—GAMDEi.e.highestisIgGandlowestIgE):
○ Dailyproduction,
○ Halflife(23days),
○ Serumconcentration
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• Foursubtypes: IgG1-4(Decreasing orderfor DHSisIgG1>IgG 2> IgG3> IgG4)

○ IgGisResponsiblefor:
○ Precipitation,
○ Neutralization,
○ NKcellbinding(toperformADCC)
○ Classicalcomplementbinding(IgM>IgG3>IgG1>IgG2)(IgG4doesnotfixcomplement)
○ Coagglutinationbybindingto S.aureusproteinA(ExceptIgG3)
○ Opsonization
• IgGappearslate,soindicatespast/chronicinfection
• IgGavidityincreaseswithtime–So,detectionoflessavidityIgGindicatesrelativelyrecentinfection
• Secretedinplacenta(MaximumplacentaltransferIgG1,minimumIgG2)
• Secretedinbreastmilk
IgGisResponsiblefor:
• HelpsinphagocytosisbybindingtoFcRonphagocytes(ExceptIgG2)
Precipitation
Neutralization
NKcellbinding(toperformADCC) IgEAntibody:
Classicalcomplementbinding • ItistheonlyHeatlabileantibody
CoagglutinationbybindingtoS.aureus protein
• ALowestforDHS
(ExceptIgG3)
Opsonization
• Responsiblefor-TypeIhypersensitivityreaction
• Homocytotropic(Speciesspecific)antibody
• AlsocalledasReaginantibody
• Raisedinhelminthicinfections
IgAAntibody:
IgAisthesecondmostabundantantibody(2ndhighestforDHS).Itisoftwotypes:
• SerumIgA: Predominantly inmonomeric form.
• SecretoryIgA(SIgA):Itisdimeric(valencyfour);botharejoinedbyJchain.Inaddition,thereisanotherjoiningsegme
ntcalledsecretorycomponent(synthesizedbymucosalepithelium).
• SecretoryIgAisresponsibleforMucosal/localimmunity.
IgAalsoexistintwosubclasses/isotypes:IgAismainlyfoundinserum.IgA2predominatesinsecretions.
IgDAntibody:
• SurfaceimmunoglobulinonthesurfaceofB-cells
• Possesshighestcarbohydratecontent
IgMAntibody:
• IgMishighestforMIS:
○ Molecularweight(900,000),
○ Intravasculardistribution(bloodAntibody)(80%),
IgM is responsible for (ormediates): ○ Sedimentationcoefficient(19),
Agglutination • Pentamericinnaturewith10valency
Hemolysis • IgM(andIgD)actassurfaceimmunoglobulinonthesurfaceofB-cells
Opsonization • IgMisthefirstantibodytoappearfollowinginfection,indicatesrecentinfection
Classicalcomplementpathwaybinding • IgMisthefirstantibodytoappearinintrauterinelifealso(20weeks):Indicatescongenitalinfection
• IgMisresponsible for (or mediates):
○ Agglutination,
○ Haemolysis,
Section
○ Opsonization,
○ Classicalcomplementpathwaybinding
Example:
• Antibodyintyphoid,
• ReaginAntibody(syphilis)
• NaturalantibodyofABO,Rhsystem.
AbnormalImmunoglobulin: AbnormalImmunoglobulin
BenceJonesproteins 1. BenceJonesProteins:Theyareproducedinmultiplemyeloma(lightchaindisease).
Waldenstrom’smacroglobulinemia
○ Thecancerousplasmacellsproduceexcessoflightchain(BenceJonesproteins)whicha
Heavycthaindisease
Cryoglobulinemia reaccumulatedinpatient’sserumandexcretedinurine.
○ Suchproteinshaveauniquepropertyofgettingcoagulatedat50°Candredissolv-
ingagainat70°C.
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2. Waldenstrom’s Macroglobulinemia: It is lymphoma affecting B-cells producing

excessIgM. It has been seen in multiple myeloma. Somatic mutations in MYD 88 gene occur
inover90%ofpatients.
3. Heavy Chain Disease: It is characterized by an excessive production of heavy
chainsthat are short and truncated. Four types of heavy chain disease have been
recognizedbased on H chain involved—alpha (Seligmann’s disease), gamma
(Franklin’s disease),muanddeltachaindisease.
4. Cryoglobulinemia:Itisaconditionwherethebloodcontainscryoglobulins;atypeofIg that
becomes insoluble (precipitate) at low temperatures but redissolves again if
thebloodisheated:
○ CryoglobulinsusuallyconsistofIgMdirectedagainsttheFcregionofIgG.
○ TheyhavebeenassociatedwithmultiplemyelomaandhepatitisCinfection.
IgSpecificityorAntigenicDeterminantsofIg
Isotypes
ThefiveclassesofIgs(lgG,IgA,IgM,IgDandIgE)andtheirsubclassesarecalledasisotypes,theyvar
yfromeachotherintheaminoacidsequencesoftheconstantregionoftheirheavychains.Suchvari
ationiscalledasisotypicvariation.
Idiotypes
The unique amino acid sequence present in paratope region (in V Hand VLregions) of
onememberofaspeciesactsasantigenicdeterminanttoothermembersofthesamespecies:
• Such antigenic determinants are called as idiotopes. Immunoglobulins vary from
eachotherintheiridiotopespresentinvariableregion,whichiscalledasidiotypicvariation.
• Idiotypesinanindividualarisecontinuouslyfrommutations(somatichypermutations)in the
genes of variable region. Hence, idiotypes may act as foreign to the host
itself;howeverdonotevokeautoimmuneresponsebecausetheyarepresentinsmallnumbers.
Allotypes
The antigenic determinants present in the isotype genes in the constant region of H and
Lchains,encodedbymultipleallelesarecalledasallotypes: Epitope: Antigenic determinantof Ag against which Ab is
Paratope: Specific site ofAb that reacts with thecorrespon
• Althoughallmembersofaspeciesinheritthesamesetofisotypegenes,multipleallelesexist Idiotope: Antigenic determinantof Ab against which Ab is
for some of the allele genes. Hence, allotypes are present in the constant region ofIg Isotope: Classes andsubclasses of Ab, which varyinconsta
moleculesof thesame class,in some,butnot all,members ofa species.
• Theyhavebeencharacterized—kappalightchain,γandαheavychains.
• Anti allotype specific antibodies may also be developed following blood transfusion
orbymaternalpassageofIgGintothefetus.
MonoclonalAntibody
Monoclonalantibodies(mAb)aredefinedastheantibodiesderivedfromasinglecloneofplasm
Immunolog
a cell; all having the same antigen specificity— i.e. produced against a single
epitopeofanantigen.
HybridomatechniqueisusedtoproducemAb(NobelprizeGwardeeKohlerandMilstein):
• CloneofmousesplenicB-cellstimulatedagainstasingleepitopeofantigenisfusedwithan
immortal cell, e.g. myeloma cell (capable of multiplying indefinitely) to produce
ahybridomacell.Thishybridomacellhastwouniqueproperties:
○ ProducesmAbofsameantigenspecificity(duetoB-cellcomponent)
○ Multipliesindefinitelyproducingcloneofidenticalcells(duetomyelomacell)
• However, in the reaction chamber, along with hybridoma cells, there will be
someunfused mouse B-cell and unfused myeloma cells which are removed by sub-
culturingthefluidonaspecialmediumcalledHATmedia.
• HATmediumcontainshypoxanthine,aminopterinandthymidine.
○ Purinesynthesisinmammaliancell(e.g.splenicB-
cell)occursbyeitherdenovoorsalvagepathways.
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○ Aminopterinblocksthedenovopathwaysothatthecellhastoperformthesalvagepath
waytosynthesizepurinesforitssurvival.
○ Salvagepathwayrequirestwoimportantenzymes:HGPRT(hypoxanthineguaninepho
sphoribosyltransferase)andthymidinekinase.
Fate of three type of cells onHATmedia:
UnfusedsplenicB-cells:Can ○ Soanycell(e.g.myelomacell)thatlacksHGPRTcannotgrowonHATmedium.
growbutdonotsurvivelongastheyarenotimmortal.
• FateofthreetypeofcellsonHATmedia:
Unfused myeloma cells:Cannot grow as they lackHGPRTenzymetoperformthesalvagepathway.
Hybridoma cells: Can growandsurvive long. ○ UnfusedsplenicB-cells:Cangrowbutdonotsurvivelongastheyarenotimmortal.
○ Unfusedmyelomacells:CannotgrowastheylackHGPRTenzymetoperformthesalvage
pathway.
○ Hybridomacells:Cangrowandsurvivelong.
• Selectionofindividualhybridomacells:ByradioimmunoassayorELISAusingthespecifi
cantigenfragments
TypesofMonoclonalAntibodies
• MousemAbcontains100%mousederivedproteins.Themouseproteinsbeingforeign;cani
nduceimmuneresponseinhumansproducinghumananti-
mouseantibody(HAMA);thatinturneliminatethemAbfasterfromthebody.
• ChimericmAbispreparedbyrecombinationof34%mouseproteins(variableregion)and66%h
Typesofmonoclonalantibodies: umanproteins(constantregion).
MousemAbcontains100%
mousederivedproteins. • Humanized mAb: Here, only the antigen binding site is mouse derived (10%) and
theremainingpartofmAbishumanderived.
Chimeric mAb: 34% mouseproteins (variable region) and66%humanproteins(constantregion).
Humanized mAb: Ag bindingsite is mouse• derived
HumanmAb:100%ofaminoacidsarehumanderived.ItisthebestacceptedmAb.
(10%)and the remaining humanderived.
Human mAb: 100% of aminoacidsarehumanderived.
ANTIGENANTIBODYREACTIONS
Antigen(Ag):Antibody(Ab)reactionsarecharacterizedbythefollowinggeneralproperties:
• Specific:Involvesspecificinteractionofepitopeofanantigenwiththecorrespondingparato
peofitshomologousantibody.
• Non-covalentinteractionsexistbetweenantigenanditsantibodysuchas:
○ Hydrogenbonds
○ Electrostaticinteractions
○ Hydrophobicinteractions
○ vanderWaalforces
Noncovalent interactions ofAg-AbReaction:
• Strength:Thestrengthorthefirmnessisinfluencedbytheaffinityandavidity
Hydrogenbonds
Electrostaticinteractions ○ Affinity:Itreferstosumtotalofnon-
Hydrophobicinteractions covalentinteractionsbetweenasingleepitopeofanantigenwithitscorrespondingparato
vanderWaalforces pepresentonantibody.Itcanbemeas-uredby:
(i)byequilibriumdialysisand(ii)bysurfaceplasmonresonancemethod
○ Avidity:Itisatermusedtodescribetheaffinitiesofallthebindingsiteswhenmul-
tivalentantibodyreactswithacomplex antigencarryingmultipleepitopes.
Section
Marrack’sLatticeHypothesis
When the sera containing antibody is serially diluted (in normal saline), gradually
theantibody level decreases. To such a set of test tubes containing serially diluted sera,
when afixedquantityofantigenisadded,then:
• Inthemiddletubes,Ag-
Abreactionoccursatitsbest,becausetheamountofantigenandantibodyareequivalenttoeachot
Marrack’sLatticeHypothesis: her(zoneofequivalence).
Zoneofequivalence:AgandAblevelequal
Prozone:ExcessAntibody
• Intheearliertesttubes,antibodiesareexcess,hencetheAg-
Prozone:ExcessAntigen Abreactiondoesnotoccur:Thisiscalledasprozonephenomenon.
• Inthelatertesttubes,antigenisexcess,hencetheAg-
Abreactionfailstooccur:Thisiscalledaspostzonephenomenon.
ThislatticehypothesisholdstrueforanyAg-Abreactions.
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TYPESOFANTIGEN-ANTIBODYREACTIONS
• Conventional techniques: Precipitation, Agglutination reaction, Complement PrecipitationReaction:
fixationtestandNeutralizationtest Usessolubleantigen
• Newer techniques: ELIS, IFA, RIA, CLIA, Immunohistochemistry, Rapid tests Abismainly IgG type
(Lateralflow assay or ICT and Flow through assay), Western blot and Immunoassays
usingelectronmicroscope.
PrecipitationReaction
When a soluble antigenreacts with itsantibody in the presenceof optimal
temperature,pHandelectrolytes(NaCl),itleadstoformationoftheantigen-
antibodycomplexintheformof:
• Insolubleprecipitatebandwhengelcontainingmediumisusedor
• Insoluble floccules when liquid medium is used.
A. PrecipitationinLiquidMedium
• Ringtest:StreptococcalgroupingbyLancefieldtechnique,andAscoli’sthermoprecipita-
tiontestdoneforanthrax.
• Slide flocculationtest: VDRLand RPRtestsused fordiagnosis ofsyphilis.
• Tubeflocculationtest:Kahntestusedpreviouslyforsyphilis.
B. PrecipitationinGel(Immunodiffusion)
Ithasmanyadvantagesoverliquidmedium:
(i)Clearvisiblebandsareformed,whichcanbepreservedforlongertime,
(ii)Individualantigensfromamixturecanbedifferentiated,e.g.
• Singlediffusioninonedimension(Oudinprocedure)
• Doublediffusionsinonedimension(Oakley–Fulthorpeprocedure)
• Single diffusion intwo dimensions (Radialimmunodiffusion)
• Doublediffusionsintwodimensions(Ouchterlonyprocedure),e.g.Elek’stest(diphtheriatoxin)
andEikentest(E.colitoxin).
C. Precipitationin Gelin PresenceofElectricCurrent

ThemovementofAgandAbcanbemadefasterifimmunodiffusioningeliscarriedoutinpresenceo
felectriccurrent.Examplesinclude:
• Electroimmunodiffusion(EID) AgglutinationReaction:
Usesparticulateorinsolubleantigen
• CIEP(Countercurrentimmunoelectrophoresis)
Abismainly IgM type
• Rocketelectrophoresis. More sensitive, easy tointerpret
AgglutinationReaction
Whenaparticulateorinsolubleantigenismixedwithitsantibodyinthepresenceofelectrolytes
atasuitabletemperatureandpH,theparticlesareclumpedoragglutinated. Immunolog
Agglutinationismoresensitivethanprecipitationtestandtheclumpsarebettervisualizedandinterpr
eted.
DiagnosticApplicationsofAgglutinationTests
Slideagglutination:Toconfirmtheidentificationandserotypingofbacterialcoloniesgrownincultu
re.
Tubeagglutinationisroutinelyusedfor:
• Typhoidfever(Widaltest):DetectsAbagainstbothH(flagellar)andO(somatic)Ag
• Acutebrucellosis(Standardagglutinationtest)
• Bloodgrouping(ABOandRhgrouping)
• CoombstestorAntiglobulintest:DetectsincompleteRhantibodies:
○ DirectCoombstest:DetectsboundRhantibodiesinfetus/baby’sserum
○ IndirectCoombstest:DetectsfreeRhantibodiespresentinmaternalserum.
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• Heterophileagglutinationtests:
○ Typhusfever(Weil–Felixreaction)
CoombstestorAntiglobulintest: ○ Infectiousmononucleosis(PaulBunnelltest)
DetectsincompleteRhAb- ○ Mycoplasmapneumonia(Cold agglutinationtest).
Direct Coombs test: DetectsboundRhantibodiesinfetus/baby’sserum
Microscopicagglutinationtest(MAT)forleptospirosis
Indirect Coombs test: Detectsfree Rh antibodies present inmaternal serum
Indirectorpassiveagglutinationtest:AntigeniscoatedoncarrierssuchasLatexorRBCstodetectAb
inserum.Examples:
• Indirecthemagglutinationtest(IHA)
• Latexagglutinationtest(LAT)forantibodydetection’e.g.ASO.
Reversepassiveagglutinationtest:AntibodyiscoatedoncarrierssuchasLatexorRBCstodetectAgi
nserum.Examples:
• RPHA(Reversepassivehemagglutinationassay),e.g.HBsAgdetection
• Latexagglutinationtestforantigendetection,e.g.CRP,RAfactor,capsularantigeninCSFands
treptococcalgrouping.
• Coagglutinationtest(here,S.aureusproteinAisusedascarrier).
ComplementFixationTest
CFTdetectscomplementfixingantibodiesinpatient’sserum.Itisnowalmostobsolete:
• WassermantestwasthemostpopularCFT,usedforthediagnosisofsyphilis.
• CFTwasalsowidelyusedfordetectionofantibodiesinRickettsia,Chlamydia,Brucella,Mycop
lasmainfectionsandsomeviralinfectionssuchasarboviruses,rabies,etc.
• Indirectcomplementfixationtest:Detectscertainavian(e.g.duck,parrot)andmammalian(e.g.
horse, cat) serumantibodies which cannotfix guinea pig complement.
• Conglutinationtest:ToperformCFTusingnonhemolyticcomplements,e.g.horsecompleme
nts.
Complementsarealsousedforvariousserologicaltests,otherthanCFTsuchas:
• Treponemapallidumimmobilizationtest(fordetectingantibodiestoT.pallidum)
• Sabin-FeldmandyetestfordetectingToxoplasmaantibodies.
• VibriocidalantibodytestforV.cholerae.
NeutralizationTest
Neutralizationtestsarealsolesscommonlyusedinmoderndays.Examplesinclude:
Neutralizationtests,e.g.
Viralneutralizationtest • Viralneutralizationtest:Detectsviralneutralizingantibodies
Plaque inhibition test-forbacteriophages • Plaqueinhibitiontest:Doneforbacteriophages.
Toxin-antitoxin neutralizationtest: • Toxin-antitoxinneutralizationtest:
Schick test for ○ SchicktestforCorynebacteriumdiphtheriae.
C.diphtheriae.
Nagler'sreactionfor
○ Nagler’sreaction:Duetoα-toxinofClostridiumperfringens
C.perfringens ○ ASLOdetection in past (now it is doneby latex agglutination)
ASLOdetection • Hemagglutinationinhibition (HAI)test.
Section
Hemagglutination inhibition(HAI) test

NEWERTECHNIQUESOFANTIGENANTIBODYREACTION
The newer techniques use a detector molecule to label antibody or antigen which in
turndetects the corresponding antigen or the antibody in the sample by producing a visible
effect.Mostofthenewertechniquesusethesameprinciple,buttheydifferfromeachotherbythety
peoflabeledmoleculeusedandthetypeofvisibleeffectproduced.
Abbreviation Immunoassaymethod Molecules used for labeling Typeofvisibleeffect

ELISA Enzyme linked Enzyme Colorchangeisdetectedbyspectrophotometer
immunosorbentassay
IFA ImmunofluorescenceAssay Fluorescentdye Emitslight,detectedbyfluorescencemicroscope
Contd...
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Contd...
Abbreviation Immunoassaymethod Molecules used for labeling Typeofvisibleeffect
RIA Radioimmunoassay Radioactiveisotope Emitsβandγradiations,detectedbyβandγ
counters
CLIA Chemiluminescence-linked im- Chemiluminescentcompounds Emitslight,detectedbyluminometer
munoassay
IHC Immunohistochemistry EnzymeorFluorescentdye Color change (naked eye)
orFluorescence microscope
WB Westernblot Enzyme Colorband(nakedeye)
Rapidtest Immunochromatographic test Colloidalgoldorsilver Colorband,(nakedeye)
Flowthroughassay ProteinAconjugate Colorband,(nakedeye)
IEM Immunoferritinelectro Electron dense molecules Appearsasblackdotunderelectronmicroscope
nmicroscopy (e.gferritin)
ELISA(EnzymeLinkedImmunosorbentAssay)
ELISAissonamedbecauseoftwoofitscomponents:
• Immunosorbent: It is an absorbing material used (e.g. polystyrene, polyvinyl),
thatspecifically absorbstheantigenorantibodypresentinserum.
• Enzyme is used to label one of the components of immunoassay (i.e. antigen or
antibody).ELISA is the method of choice in big laboratories as large number of samples can be
testedtogetherusingthe96wellmicrotiterplate.Butitisnotpreferredinsmalllaboratories:
• Itiseconomical,takes2-3hours(rapidteststake10–20min)
• ELISAisthemostsensitiveimmunoassay,thusisthepreferredscreeningtestatbloodbanks
andtertiarycaresites.
• Itsspecificityusedtobelow.Butnow,withuseofmorepurifiedrecombinantandsynthetican
tigens, and monoclonalantibodies, ELISA has becomemore specific.
• ItneedsexpensiveequipmentssuchasELISAwasherandreader.
ELISAtype Usedfordetectionof Enzyme is labeled with
DirectELISA Antigen Primaryantibody
Indirect ELISA Antibodyorantigen Secondaryantibody
Sandwich ELISA Antigen Primary antibody in sandwich direct
ELISASecondaryantibodyinsandwichindirectELIS
A
Competitive Antigenorantibody Secondaryantibody
ELISA
ELISPOT Cellsproducingantibodyorcytoki Primaryantibody
ne
Note: Primary antibody is directed against the antigen, Secondary antibody is an anti-human (or otherspecies)Ig
directedagainstFc regionofanyhuman/otherspecies Ig. Immunolog
ImmunofluorescenceAssay
ItisatechniquesimilartoELISA,butdiffersbysomeimportantfeatures:
• Fluorescentdyeisusedinsteadofenzymeforlabelling
• Detectscellsurfaceantigensorantibodiesboundtocellsurfaceantigens,unlikeELISAwhich
detectsfreeAgorAb.
TypesofImmunofluorescenceAssays:
• DirectImmunofluorescenceAssay:Detectscellscarryingsurfaceantigens
• IndirectImmunofluorescenceAssay:ThisdetectsantibodiesboundtocellsurfaceAg
• Flowcytometry:Thisisalaser-basedfluorescencetechnologydetects4properties:
○ CellCounting, e.g. CD4 T-cell count
○ Differentiatingbetweentwocells,e.g.CD4andCD8T-cells
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○ Scatteringofcells:
▪ Forwardscatterisproportionaltosizeofthecell;largercellshavegreaterfor-
wardscatter.
▪
Flow cytometry detect fourimportant properties: Sidescatterisproportionaltointracellularcomplexity;cellswithmoregranules
CellCounting,e.g.CD4 (e.g.neutrophils)havemoresidescatterthancellswithasimplecytoplasm(lymphocytes).
T-cellcount ○ Sortingoutofcellsfromamixture.
Differentiatingbetweentwocells,
e.g.CD4andCD8T-cells
Scatteringofcells:
WesternBlot
Forward scatter: Refers tosizeofthe cell Westernblotdetectsspecificproteins(antibodies)inasamplecontainingmixtureofantibodies
Side scatter: Refers tointracellularcomplexity
each targetedagainst differentantigens ofsame microbe:
Sorting out of cells from amixture
• ItissonamedforitssimilaritytoSouthernblot(detectsDNAfragments)andNorthernblot(detect
smRNAs).
• TheEasternblotisthelatestadditiontothelist;itisamodificationofWesternblot,whichdetectst
hecarbohydrateepitopespresentonproteinsorlipids.
Westernblotcomprisesofthreebasiccomponents:
1. SeparationofcomplexproteinantigenmixtureintofragmentsbySDSPAGE(sodiumdodecyl
sulfate-polyacrylamidegelelectrophoresis)
WesternblotThreeSteps:
2. NCMblotting:Theantigenfragmentsinthegelaretransferred(blotted)totheNitrocellulose
Separation of Ag mixture intofragmentsbySDSPAGE
membrane(NCM)
NCMblotting:Agfragmentsinthe gel are transferred to theNCM
Enzyme immunoassay:Detects specific Ab 3. Enzymeimmunoassay:Detectsspecificantibodiesseparatelyinpatient’sserumcontaining
separatelyin sample against AgfragmentsblottedonNCM
mixtureofantibodies.
RapidTest
Rapidtestsarerevolutionaryinthediagnosisofinfectiousdiseases:
• They are very simple to perform (one step method), rapid (result obtained in 10–20
min),requireminimaltraining,doesnotneedanysophisticatedinstruments.
• These tests are also called Point of care (POC) tests, because unlike ELISA and
otherimmunoassays, the Point of Care tests can be performed independent of
laboratoryequipmentanddeliverinstantresults.
Rapidtests:Twotypes
Twoprinciplesofrapidtestsareavailable:
Lateral flow assay orImmunochromatographic test(ICT)
Flowthroughassay • LateralflowassayorImmunochromatographictest(ICT):(i)colloidalgoldorsilverisused
for labelling, (ii) the sample flows laterally through the NCM. Example
includesantigendetectioninmalaria
• Flowthroughassay:(i)ProteinAisusedforlabelling,insteadofgoldconjugateand
(ii) the sample flows vertically through the NCM as compared to lateral flow in
ICT.ExampleincludesHIVtridottest.
COMPLEMENT
Complementareagroupofproteinsnormallyfoundinserumininactiveform,butwhenactivatedt
heyaugmenttheimmuneresponses:
• Constituteabout5%ofnormalserumproteins
Section
• Antigennonspecific:Theirleveldoesnotincreasefollowingeitherinfectionorvaccination.
• Speciesnonspecific
• Heatlabile:56°Cfor30minutes
• BindstoFcregionofantibody:IgM(bindsstrongly)followedbyIgG3→1→2
• SiteofSynthesis ofComplements: Mainlyby liver,also byGIT, macrophageandspleen.
ComplementPathways
Therearethreepathwaysofcomplementactivation(Referfigure2.2.2):
1. Classicalpathway:ThisisanAbdependentpathway,triggeredbytheAg-Abcomplex.
2. Alternativepathway:ThisisanAbindependentpathway,triggeredbytheantigendirectly.
3. Lectinpathwayresemblesclassicalpathway,butitisAbindependent.
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StagesofComplementActivation
Therearefourmainstagesintheactivationofanyofthecomplementpathways:
1. Initiationofthepathway
2. FormationofC3convertase
3. FormationofC5convertase
4. Formationofmembraneattackcomplex(MAC)
Allthethreepathways(figure)differfromeachotherintheirinitiationtillformationofC3convertase.T
hen,theremainingstagesareidenticalinallthepathways.
BiologicalRoleofComplement
• Targetcelllysis:MACmakesporesorchannelsinthetargetcellmembrane
• Inflammatoryresponse:Complementby-
productssuchasC3a,C4aandC5aactasanaphylatoxinsandchemotactic.
• Opsonization:C3bandC4bactasmajoropsonins
• MediatehypersensitivityreactionIIandIII
• Removestheimmunecomplexesfrombloodtospleen-byC3b BiologicalRoleofComplement:
• Immuneadherence:CR2actsasEBVreceptors Targetcelllysis
Inflammatoryresponse-C3a,
• Kininlikeactivity(↑vascularpermeability):C2b
C4aandC5a
• Viralneutralization Opsonization-C3bandC4b
MediateHSNIIandIII
Table2.2.1:Differencesbetweenthreecomplementpathways Removes the immunecomplexes from blood tospleen-byC
Classical Alternativepathway Lectin pathway Immuneadherence-CR2actsasEBV receptors
Kininlikeactivity-C2b
Activator (initiator) Antigena EndotoxinI Carbohydrate residue Viralneutralization
ntibodyc gA, IgD, ofbacterial cell wall
omplex Cobra (mannosebinding protein) that
venomNephritic bindstohostlectinantigen.
factor
1st C1 C3b C4
complementactiv
ated
C3convertase C14b2a C3bBb MBL/MASP-C4b2a
C5convertase C14b2a3b C3bBb3b MBL/MASP-C4b2a3b
(C3convertase+3b)
Complementlevelintheser AllC1-C9:Low C1,C4,C2- C1-
um NormalOthers-Low NormalOther
s-Low
Immunity Acquired Innate Innate
Table2.2.2:Complementdeficiencydiseases
i.Complementproteindeficiencies Pathway(s) involved Disease/pathology
C1,C2,C3,C4 C1,C2,C4-Classicalpathway SLE,glomerulonephritisandpyogenicinfections
C3-Commondeficiency
Immunolog
Properdin, Factor D Alternativepathway Neisseriaandpyogenicinfection
Membraneattackcomplex(C5-C9) Commondeficiency DisseminatedNeisseriainfection
ii.Complementregulatoryproteindeficiencies Diseases
C1esteraseinhibitor Overactiveclassicalpathway Hereditaryangioneuroticedema
ii.Complementregulatoryproteindeficiencies Diseases
DAF (Decay accelerating factor) Deregulated C3 PNH(Paroxysmalnocturnalhemoglobinurea)
andCD59 convertaseIncreasedRBClysis
Factor I Deregulated classical pathway with Immune complex disease; recurrent
overconsumptionofC3 pyogenicinfections
Factor H DeregulatedalternativepathwaywithincreasedC Immunecomplexdisease;pyogenicinfection
3convertaseactivity
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Fig.2.2.2:Complementpathways
Section
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ANTIGEN 9. Antibodydiversityisdueto: (PGIDec2008)

a. Generearrangement
1. Smallestunitofantigenicity? (JIPMERNov2015) b. Genetranslocation
a. Epitope c. Antigenicvariation
b. Paratope d. CD40molecules
c. Complexhapten e. Mutation
d. Simplehapten
10. Allofthefollowingstatementsabouthybridoma
2. Superantigensareproducedbyallthefollowing technologyaretrueexcept: (AIIMSNov2008)
pathogens,except: (AI2010,APPG2014,similarMCQ a. Specificantibodyproducingcellsareintegratedwith
inPGIJune2005,JIPMER2014) myelomacells
a. Staphylococcusaureus b. MyelomacellsinsalvagepathwaygrowswellinHATm
b. Enterococcusfaecalis edium
c. Rabiesvirus c. Aminopterin,afolateantagonist,inhibitsdenovopath
d. HIV way
3. Allofthefollowingstatementsaboutcarbohydrate d. HGPRTaseandthymidylatesynthetasearerequiredfors
antigenaretrueexcept: (AIIMSNov2008) alvagepathway
a. Ithaslowerimmunogenicity 11. Inwhichof the following(s), the two allele are
b. Memoryresponseisseen inheritedtogether: (PGINov2014)
c. CausepolyclonalB-cellstimulation a. Idiotype b. Genotype
d. DoesnotrequirestimulationbyT-cells c. Phenotype d. Allotype
4. Heterophileantibodyisfoundin: e. Isotype
a. Weil-Felixtest (NEETPatternBased) 12. WhenPapaincleavesIgGantibody,itproduces:
b. Widaltest (RecentQuestion2013,DNB2010)
c. Standardagglutinationtest a. 2Faband1Fc b. 2Faband2Fc
d. All c. 1Faband1Fc d.1Faband2Fc
5. Themain aimof anadjuvant isto increase:
a. Distribution (NEETPatternBased) IgG
b. Absorption
13. Trueaboutanimmunoglobulin:(NEETPatternBased)
c. Antigenicity
a. IgGhasmaxconc.inserum
d. Metabolism
b. IgMhasmaxconc.inserum
c. IgAhasmaxconc.inserum
ANTIBODY d. IgEhas maxconc. in serum
6. What is the percentage of immunoglobin in 14. IgthathelpsinOpsonization: (NEETPatternBased)
plasmaprotein? a. IgA b. IgG
(RecentQuestion2015)a. 5to10% b.10to15%
c. IgD d. IgE Immunolog
c. 15to 20% d. 25to 30% 15. Theserumconcentrationofwhichofthefollowing
humanIgGsubclassismaximum: (AI2005)
7. Variableportionofanimmunoglobulin:
a. IgG1 b. IgG2
a. Aminoterminal (NEETPatternBased)
c. IgG3 d. IgG4
b. Carboxyterminal
c. Acidterminal 16. WhichIgGcancrossplacentamostefficiently?
d. Amoxyterminal (RecentQuestion2015)
a. IgG1 b.IgG2
8. Vaccinationisbasedontheprincipleof: (AI2012)
c.IgG3 d.IgG4
a. Agglutination
17. Whichofthefollowingimmunoglobulinscancross
b. Phagocytosis
placenta? (DNBDEC 2012,PGI June2001)
c. Immunologicalmemory
a. IgA b. IgM
d. Clonal detection
c. IgG d. IgD
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18. Trueaboutantibody:
29. Whichantibodyiselevatedinprimaryimmuneresponse
(PGIMAY2013,APPG2012,SimilarMCQ)
? (RecentQuestion2013)
a. IgMisproducedinprimaryresponse
a. IgA b. IgM
b. IgDprotectsmucosa
c. IgG d. IgE
c. IgEismainantibodyinsecondaryresponse
d. IgGismainantibodyinsecondaryresponse 30. Whichisthefirstantibodyiselevatedinfetallife?
e. IgAprotectsbodysurface (RecentQuestion2013)
a. IgA b. IgM
19. Whichantibodydeficiencycausesrecurrentinfectionby
c. IgG d. IgE
organismswithpolysaccharidecapsule?
(RecentQuestion2013)
a. IgA b. IgG1 IgA
c. IgG2 d. IgM
31. Synthesisofanimmunoglobulininmembraneboundors
20. Whichofthefollowingantibodiesshowsanamnestic ecretoryformisdeterminedby:(AIIMSMay2012)
response? (RecentQuestions2014) a. Oneturntotwoturnjoiningrule
a. IgA b. IgM b. Classswitching
c. IgG d. IgD
c. DifferentialRNAprocessing
21. OfalltheIgGsubclassessmallestis: d. Allelicexclusion
(RecentQuestion2015)
32. ImmunoglobulininPeyer’spatchis:(DNBJune2011)
a. IgG1 b. IgG2
a. IgM b. IgG
c. IgG 3 d. IgG4
c. IgA d. IgD
IgM 33. Immunoglobulinpresentinlocalsecretionsis:
(DNBDEC2012,DNBDEC2009)
22. WhichantibodiesarefoundinCryoglobulinemia? a. IgG b. IgA
(RecentQuestion2015) c. IgM d. IgD
a. IgG b.IgM 34. TheIgwhichactivatesalternatecomplementpathway:
c.IgE d.IgA (JIPMER2014,2013)
23. Coldantibodyis: (RecentQuestion2015) a. IgG b. IgE
a. IgA b. IgG c. IgA d. IgM
c. IgE d. IgM
24. Activatorofclassicalpathwayofcomplement? IgE
a. IgA b.IgG(NEETPatternBased)
c. IgM d. IgE 35. Immunoglobulinthatisinactivebyheatingis?
25. Pentameric antibodywitha Jchainis? (DNBDEC2012)
(NEETPatternBased,DNBDec2010,DNBJune2009) a. IgG b. IgA
a. IgA b. IgG c. IgM d. IgE
c. IgM d. IgE 36. Immunoglobulinthatiselevatedin
helminthicinfection?
26. WhichimmunoglobulinactsasreceptoronB-cell?
(WBPG2016,MHPG2015,NEETPatternBased)
(DNBDec2010)
a. IgG b. IgA
Section
a. IgG b. IgA c. IgM d. IgE

c. IgM d. IgE
37. IgEissecretedby: (PGI2005)
27. Inutero infection leads to raise of a. Mastcell b. Basophil
whichimmunoglobulinfirst? (AI2003,DNBDEC2012) c. Eosinophils d. Plasmacells
a. IgG b. IgA e. Neutrophils
c. IgG d. IgM
28. Rheumatoidarthritisisbestdiagnosedby:
(AIIMS Nov 2011) (Modified repeat of PGI 1998)
ABNORMALIMMUNOGLOBULIN
a. Anticitrullineantibody 38. Whichprecipitatesat50to60°Cbutredissolveonheating
b. IgGantibody : (AI2012;AI2000)
c. IgAantibody a. BenceJonesproteins
d. IgMantibody b. Heavychain
c. Bothlightand heavychains
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ANTIGENANTIBODYREACTION 50. Heterophileagglutinationis/areusedinalltestexcept:

(PGIMAY2013)
39. Whichoneistheexampleofgelprecipitationtest? a. Widaltest b. Weil-Felixreaction
(PGINov2016) c. Paulbunnel test d. ELISA
a. Immunoelectrophoresisb. VDRL e. Coldagglutinationtest
c. WIDAL d.Coomb’stest 51. Regardingtolatticeformationwhichistrue?
e. Elek’stest (RecentQuestion2015)
40. Overallstrengthofantigenwithitsantibodybondis a. Associatedwithprecipitationbutnotagglutination
referredtoas? (RecentQuestion2015) b. Associatedwithagglutinationbutnotprecipitation
a. Affinity b. Avidity c. Associatedwithboth
c. Antigenicity d. Neitherassociatedwithprecipitationnorwithaggluti
nation
41. Rosewallertestis: (RecentQuestion2015)
52. Antigen antibody reaction is seen maximum in?
a. PassiveagglutinationtestforRheumatoidarthritis
a. Excessantibody (AIIMSMay10)
b. Activeagglutinationforrheumatoidarthritis
b. Excessantigen
c. Doneforsyphilis
c. Antigenandantibodyareequal
42. TypeofAg-AbreactionseeninVDRL? d. Antigenandantibodyarelow
(RecentQuestion2015) 53. Whichofthefollowingisaneutralizationtest?
a. Agglutination b. Passiveagglutination a. Kahntest (RecentQuestion2015)
c. Flocculation d. Gelprecipitation b. ASLO
43. Naeglerreactionisexampleof: (NEETPatternBased) c. Hemagglutinationtest
a. Precipitation b. CFT
c. Agglutination d. Neutralization COMPLEMENTSYSTEM
44. Wassermantestis: (RecentQuestion2015)
54. Complementwhichleadtokillingoforganismandprotec
a. Agglutinationtest b. Precipitationtest
tsus? (AIIMSMAY2016)a.
c. Neutralizationtest d. Complementfixation C2345 b.C56789
45. All of the following interaction occurs between c. C34567 d. C3456
antigenantibodyreactionexcept: 55. Powerfulactivatorofclassicalcomplementpathway:
a. Ionicbond (RecentQuestion2013) (NEETPatternBased)
b. Covalentbond a. IgA b. IgG
c. Hydrogenbond c. IgM d. IgD
d. vanderWaalsforces 56. Complementformedinliver: (NEETPatternBased)
46. PaulBunneltestisexampleof: (NEETPatternBased) a. C2, C4 b. C3, C6, C9
a. Agglutination b. Precipitation c. C5,C8 d. C1
c. Neutralization d. CFT 57. Whichofthefollowingactsasachemoattractant?
(MHPG2015,DNBDEC2012)
47. Whichareexampleofagglutinationtest:
a. C3a b. C3b
(NEETPatternBased)
c. C5b d. LTB4
a. Widaltest b. VDRLtest
58. Whichofthefollowingbestdenotesclassicalcomplemen
Immunolog
c. Kahntest d. Ascoli’stest
tpathwayactivationinimmunoinflammatorycondition
48. Whichofthefollowingactsasanopsonin? : (AIIMS2004)
(DNBJune2011) a. C2,C4,C3decreased
a. C3a b. C3b b. C2 andC4 normal,C3is decreased
c. C5a d. LTB4 c. C3 normal and C2 C4decreased
49. Thefollowingmethodsofdiagnosisutilizelabeled d. C2,C4,C3allareelevated
antibodiesexcept: (AIIMSMay2005) 59. Which compliment binds with Fc portion of IgM
a. ELISA(EnzymeLinkedImmunosorbentAssay) inClassicpathwayorwhichofthefollowingcomplement
b. Hemagglutinationinhibitiontest components attaches to the crystallizable fragment
c. Radioimmunoassay ofIgM? (RecentQuestions2014)
a. C1 b. C2
d. Immunofluorescence
c. C3 d. C4
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EXPLANATIONS
ANTIGEN
1. Ans.(a)(Epitope)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep105
Epitopeorantigenicdeterminantisthesmallestunitofanantigenthatbindstoparatopeofanantibody.
2. Ans.(b)(Enterococcusfaecalis)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiologyp108,Journal-
SuperantigenIJMM2004Vol.22Iss.4
Refertext
3. Ans.(b)(Memoryresponseisseen)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiologyp109,Ananthanarayan9/ep90
• CarbohydrateantigenlikeLipopolysaccharide(LPS)isanexampleofTIndependentAntigen
• TIndependentAntigen:
○ DirectlystimulatesB-cellforantibodyproductionwithoutparticipationofT-cell.
○ DosedependentimmunogenicwithLimitedantibodyresponse-IgMandIgG3
○ Lackmemoryresponse
○ LPScancausepolyclonalB-cellactivation.
4. Ans.(a)(Weil-Felixtest)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiologyp106,Ananthanarayan9/ep90,8/ep93
• Weil-
Felixtestisanexampleofheterophileagglutinationtest.RicketssialantibodiesaredetectedbyusingProteusOx2,Ox19andOxKan
tigens.
5. Ans.(c)(Antigenicity)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology1/ep107,Ananthanarayan9/ep150
• Adjuvant is anysubstance thatenhances immunogenicityof Antigen.
ANTIBODYSTRUCTUREANDPROPERTIESOFANTIBODY
6. Ans.(c)
(15to20%)Ref:InternetSourcesImmunoglubulinsconstitu
te20%oftotalserumprotein.
7. Ans.(a)(Amino...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiologyp114,Ananthanarayan9/ep94,8/ep96
• Variableregionend—Aminoterminal(NH4)
• Constantregionend—carboxyterminal(CHO)
8. Ans. (c) (Immunological memory) Ref: Apurba Sastry’s Essentials of Medical Microbiology p149, Ananthanarayan 9/e
p83Vaccination leads to production of memory cells against the immunogens which play an important role in prevention of
theinfectionbyproducingantibodiesonsubsequentexposureoftheorganism.
9. Ans. (a), (e) (Gene rearrangement, mutation) Ref: Apurba Sastry’s Essentials of Medical Microbiology p118, Kuby’s Im-
Section
munology6/ep123
SomatichypermutationandRecombinationofV-(D)-
Jsegmentsjoining(Generearrangement)areoneoftheimportantmechanismsinvolved for in Antibodydiversity.
AntibodyDiversity
Thereare1010antibodiesthatcanbegeneratedagainstvariousantigenicstimuli.Thisanti
bodydiversityispossibledueto:
• PresenceofMultiplegerm-linegenesegments
• RecombinationofV-(D)-JSegmentsjoining
• Junctionalflexibility
• P-regionnucleotideaddition(P-addition)
• N-regionnucleotideaddition(N-addition)
• Somatichypermutation
• Combinatorialassociationoflightandheavychains.
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10. Ans.(b)
(Myelomacells...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiologyp117'Myelomacellsl
acksHGPRTenzyme,hencetheycannotgrowwellinHATmedium.'FordetailofHybridomatec
hnology,referchapterreview
11. Ans. (d) (Allotypes) Ref: Apurba Sastry’s Essentials of Medical Microbiology p115, Ananthanarayan 9/e
p96ALLOTYPES,orallelicvariantswithintheconstantregions,areknowntoexistforsomeoftheseisotypes,andareinheritedina
Mendelianco-dominantfashioninallelicmanner(“allelictype”).
12. Ans.(a)(2Faband1Fc)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p,Ananthanarayan9/ep95
• Papaincleavesantibodyto:2Faband1Fc
• Pepsincleavesantibodyto:2(Fab)’
IgGIMMUNOGLOBULIN
13. Ans.(a)(IgGhas...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p113,Ananthanarayan9/ep96,8/ep98
• DecreasingorderofSerumlevelofvariousimmunoglobulinisGAMDE:ThatishighestisIgGandlowestisIgE
• DecreasingorderofSerumlevelofIgGsubtypes:IgG1>2>3>4
14. Ans.(b)(IgG)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep113,Ananthanarayan9/ep96
• Mostimportantopsonization-C3bandFc(IgG)
• Examples ofopsoninmoleculesinclude:
○ Antibodies:IgGandIgM
○ Components ofthecomplementsystem:C3b,C4b,and iC3b
○ Mannose-bindinglectin(initiatestheformationofC3b).
15. Ans.(a)(IgG1)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep113,Ananthanarayan9/ep97,8/ep98
• SerumlevelofIgGsubtypes:IgG1-65%,IgG2-23%,IgG3-8%,IgG4-4%
16.Ans.(a)(IgG1)Ref:Journal:Vaccine.2003Jul28;21(24):3365-9
17. Ans.(c)(IgG)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep113,Ananthanarayan9/ep96,8/ep98
• IgGissecretedinplacenta,breast
• Amongthefoursubclasses:IgG2-poorlycrossesplacenta.
18. Ans.(a)(d)(e)
(IgMisproducedinprimaryresponse,IgGismainantibodyinsecondaryresponse,IgAprotectsbodysurface)Ref:ApurbaSast
ry’s EssentialsofMedicalMicrobiology 1/ep113,Ananthanarayan9/e/p96-98
Referchapterreviewfordetail.
19. Ans.(c)(IgG2deficiency)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep113,Jawetz25/
ep130,RobinsPathology7/ep144-147
IgG2subclassisthepredominantantibodyraisedagainstthepolysaccharisecapsularantigensanditsdeficiencyisassociatedwit
hrecurrentpyogenicinfectionduepyogeniccapsulatedbacteria.
20. Ans.(c)(IgG),Ref:Journal:AnevaluationofmodifiedWidaltestinthediagnosisofentericfever,JIndian MedAssoc.1989 Immunolog
Anamnesticresponseindicatessecondaryimmuneresponsewhichmaybenon-specificandmediatedbyIgG.
21. Ans.(d)(IgG4)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep112
IgMIMMUNOGLOBULIN
22. Ans.(b)(IgM)Ref:InternetSources
CryoglobulinsusuallyconsistofIgMdirectedagainsttheFcregionofIgG.
23. Ans.(d)(IgM)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep113
24. Ans.(c)(IgM)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep114,Ananthanarayan9/ep98,8/ep99
• ClassicalcomplementisactivatedbybothIgGandIgM.However,IgMisapowerfulactivatorthanIgG.
• IgMispentamerichavingavalencyof10,thefivemoleculesofIgMarejoinedtogetherbyJ-chain.
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• Immunoglobulinactsasreceptoron B-cell(i.e.surfaceimmunoglobulin)-IgM andIgD
27. Ans.(d)(IgM)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep114,Ananthanarayan9/ep98,8/ep99
• IgMisphylogeneticallyoldestimmunoglobulinandtheearliesttobesynthesizedbythefetus,beginningbyabout20weeks.
28. Ans.(a)>(d)(Anticitrullineantibody,IgMantibody)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep185
• Anticitrullineantibodies
○ Autoantibodiesthatarefrequentlydetectedinthebloodofrheumatoidarthritispatients.
○ Researchsuggeststhatinthejointsofpatientswithrheumatoidarthritis,proteinsmaybechangedtocitrullineaspartoft
heprocessthatleadstoinflammationoftherheumatoidjoint.
○ Whenthecitrullineantibodyisfoundinapatient’sblood,thereisa90-95%likelihoodthatthepatienthasrheu-
matoidarthritis.
• RheumatoidarthritisisalsodiagnosedbydetectingRF(Rheumatoidfactor)
• RFisaIgMantibodydirectedagainstFcportionofIgGantibody.
• RFisdetectedbypassiveagglutinationtestslike:
○ Rose-Waalertest
○ Latexagglutinationtest
29. Ans.(b)(IgM)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p114,Ananthanarayan9/ep146
• IgM:Elevatedinprimaryimmuneresponse
• IgG:Elevatedinsecondaryimmuneresponse
30. Ans.(b)(IgM)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p114,Ananthanarayan9/ep98
• IgM:Firstantibodyiselevatedinfetallife
• IgG:Onlyantibodythatcrossesplacenta.
IgAIMMUNOGLOBULIN
31. Ans.(c)(Differential...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p114,Kuby’sImmunology6/ep130-131
• 'DifferentialRNAprocessingofacommonprimarytranscriptdetermineswhetherthesecretedormembraneformofan
immunoglobulinwillbeproduced'…….Kuby6/ep131
Membrane-boundorsecretedformofimmunoglobulin:
• MaturenaiveB-cellsproduceonlymembrane-
boundantibody(IgDorIgM),whereasdifferentiatedplasmacellsproducesecretedantibodies(IgA).
• Membrane-boundorsecretedformofimmunoglobulinissynthesizedbyalternateRNAsplicing.
32. Ans.(c)(IgA)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep114,
• Peyer’spatchesarecomposedof30-40lymphoidfolliclespresentinintestinalsubmucosa
• TheyarerequiredforintestinalsecretoryimmunoglobulinAresponseswhichprovideslocalorintestinalimmunityormucosalim
munity.
Section
• Peyer’spatchesalsocontainIntraepithelialCD8lymphocytecontainingTCRδreceptors
33. Ans.(b)(IgA)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p114,Ananthanarayan9/ep97,8/ep99-100
• Refertext
34. Ans.(c)(IgA)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p114,Ananthanarayan9/ep121-24
IgEIMMUNOGLOBULIN
35. Ans.(d),(IgE)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep114,Ananthanarayan9/ep98,8/ep100
• Onlyheat labileimmunoglobulin is– IgE
36. Ans.(d),(IgE)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep114,Ananthanarayan9/ep99,8/ep100
• HelminthicinfectionischaracterizedbyanincreaseofIgEantibodies.
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37. Ans.(d),(Plasma...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep114,Ananthanarayan9/ep99,8/ep100
• IgE,likeotherimmunoglobulinsissecretedbyplasmacells.
• IgEaftersecretingfromplasmacell,getsboundtomastcellbyFcportion.WhenantigencomesandbindstofabregionofIgE,itinturn
stimulatesmastcellandmastcelldegranulationoccurs.(TypeIhypersensitivityreaction)
ABNORMALIMMUNOGLOBULIN
38. Ans.(a)(Bence-Jones...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep115,Ananthanarayan9/ep99
Bence-Jones Protein:
• Abnormalimmunoglobulinfoundinurine
• Coagulatesat50°C,redissolvesat70°C
• ElevatedinMultiplemyeloma
ANTIGENANTIBODYREACTION
39. Ans.(a,e)(immunoelectrophoresis,Elek’stest)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p124
• Refertextfordetail
• VDRLisaflocculationtestandWidalandCoomb’stestsareagglutinationtests.
40. Ans.(b)(Avidity)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep121
• Affinityisstrengthbetweensingleepitopeofanantigenanditsparatopeoftheantibody.
• Avidityisthesumoftotalstrengthbetweenallepitopesofamultivalentantigenandtheirparatopes.
41. Ans.(a)(Passive...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep126
42. Ans.(c)(Flocculation)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep123
43. Ans.(d)(Neutralization)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep129,Ananthanarayan9/ep112
• Naeglerreactionisanalfatoxinantialfatoxinneutralizationtest
44. Ans.(d)(Complementfixation)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep128
45. Ans. (b) (Covalent bond) Ref: Apurba Sastry’s Essentials of Medical Microbiology 1/e p121, Ananthanarayan 9/e
p103Theantigenantibodyreactionisreversibleandtheantigenandantibodymoleculesareboundtogetherbyweakerbondssuc
h as ionic bond, van der Waal’s forces and hydrogen bond rather than stronger bond like covalent bond
46. Ans.(a)(Agglutination)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep126,Ananthanarayan9/ep109,8/ep111,120,
• PaulBunneltestisanheterophileagglutinationtestdoneininfectiousmononucleosis.
47. Ans.(a)(Widaltest)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep126,Ananthanarayan9/ep109,8/ep111,120,
• Widaltestisanagglutinationtest
Immunolog
• VDRL:Slideflocculationtest(Precipitationtest)
• Kahntest:Tubeflocculationtest(Precipitationtest)
• Ascoli’sthermoprecipitationtest:RingprecipitationtestdoneforAnthraxantigendetection
48. Ans.(b)(C3b)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep170,Ananthanarayan9/ep112,8/ep111,120
• Opsonins (like Fc of IgG and complement factors like C3b, C4b and ic3b) play a very important role in uptake of bacteria by
bindingto the specific molecules on the surface of bacteria thus facilitating the engulfment by the phagocyte (which bear the
receptors fortheopsonins).
49. Ans.(b)(Hemagglutinationinhibitiontest)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep129,Ananthanarayan9/
ep112,
• HemagglutinationinhibitiontestisdetectedbyinhibitionofhemagglutinationofantiRBCantibodypresentinpatient’sserum
withrespectiveRBCantigen.
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Methodsofdiagnosisutilizinglabeledantibodies:
• Labeledantibodiesareusedfor:
○ Antibodydetection:Labeledantihumangammaglobulinisusedtodetecthumangammaglobulinpresentinpatient’sse
rum.
○ Antigendetection:Labeledspecificantibodiesareusedtodetectcorrespondingantigensfromthesample.
50. Ans.(a)(d)(Widal,ELISA)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p126,Ananthanarayan9/
ep90Referchapterreviewtoknowthelistofheterophileagglutinationtests
51. Ans.(c)(Ass.withboth)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep122
52. Ans.(c)(Antigenandantibodyareequal)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep122,Ananthanarayan9/
ep105,8/ep104
• Antigenantibodyreactionresultswhenalargelatticeisformedconsistofalternateantigenandantibodymoleculeswhichoccurint
hezoneofequivalence.
53. Ans.(b)(ASLO)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep129
COMPLEMENTSYSTEM
54. Ans(b)C56789Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p140
• C56789isknownasmembraneattackcomplex,causesmicrobiallysis.
• OnlyIgMfollowedbyIgG3>1>2canfixtoclassicalpathwayofcomplements.
56. Ans.(b)(C3...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep138,Ananthanarayan9/ep122,8/ep122
• SiteofComplementsynthesis:Liver:C3,C6,C9,GIT-C1,Macrophage-C2,C4,Spleen-C5,C8
57. Ans.(a)(C3a)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep142,Ananthanarayan9/ep122,8/ep122
RoleofByproductsofcomplements:
• ChemotaxisandAnaphylaxis-C5aandC3a
• Opsonization- C3b
• Kininlikeactivity(↑vascularpermeability)-C2b
58. Ans.(a)(C2,C4,C3...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep142-3,Ananthanarayan9/ep121
C2,C3,andC4allareutilizedinClassicalpathway,hencetheirserumlevelwillbelow.
Classicalpathway:
• Starts by Activationof C1by antigenantibody complex
• ActivatedC1 inturn activatesC4 to C4a(anaphylotoxin) andC4b
• C14binturnactivatesC2(inpresenceofMgion)toformC2aandC2b(kininlike,increasesvascularpermeability)
• C14b2aisknownasC3convertasewhichactivatesC3toC3a(chemotacticandanaphylotoxin)andC3b
• C14b2a3bisknownasC5convertasewhichactivatesC5toC5a(chemotacticandanaphylotoxin)andC5b
• LatecomponentsaddtoC5b–
toformC56789(membraneattackcomplex)whichformsporesoncellmembranecausingcelllysisandalsoactivatesbystan
Section
dercellstomakethemsusceptibletolysis.
59. Ans.(a)(C1),Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p113,Ananthanarayan9/
ep123Referchapterreviewfordetail
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CHAPTER
StructureofImmuneSystemand
ImmuneResponse 2.3
STRUCTUREOFIMMUNESYSTEM
1. LymphoidOrgans:Consistofcentralandperipherallymphoidorgans
○ CentralorPrimarylymphoidorgans,examplesincludethymusandbonemarrow Structureofimmunesystem:
○ PeripheralorSecondarylymphoidorgans,examplesincludelymphnode,spleen,andMALT LymphoidOrgans
LymphoidCells
2.LymphoidCells:ConsistoflymphocytessuchasT-cells,B-cellsandNKcells Othercellsofimmunesystem
Cytokines
3.Othercellsofimmunesystem:Includephagocytes,suchasmacrophageandmicrophages(neutrophil,eosinophil
andbasophil),dendriticcells,masT-cellsandplatelets.
4.Cytokines:Solubleproductssecretedfromvariouscellsofimmunesystem
CentralLymphoidOrgans
BoneMarrow
TheprogenitorT-andB-cellsoriginateinbonemarrow.FurtherdevelopmentofB-
cellsoccursinbonemarrowitself;whereastheprogenitorT-cellsproliferateinthymus.
Thymus
Thymusisdevelopedintheembryoniclife(thirdmonth)fromthird/fourthpharyngealpouch.
It is highly active at birth, reaches its peak size at puberty, and then it
degenerates.Structure:Thymushastwolobes,eachisdifferentiatedintoanoutercortexandani
nnermedulla.
• Cortex is densely populated and contains: Numerous thymocytes (thymic
lymphocytes),epithelial cells andnurse cells (specialized epithelial cells with long
membrane extensionsthatsurroundmanythymocytes)
• Medullaissparselypopulatedandcontains:Fewthymocytes,epithelialcells,dendriticcells
andHassall’scorpuscles(concentriclayersofdegeneratingepithelialcells)
• Thymic hormones such as thymulin, thymopoietin and thymosin are produced
whichhelpinT-celldevelopment.
MaturationofT-cells:
• DN cell: T-cell precursors after entering into the thymus transform into double
negative(DN)T-cells(CD4–CD8–)
• DPcells:ThentheDNcellsacquireCD4andCD8tobecomeDoublepositive(DP)T-
cells.TheDPcellshavethefollowingfates.
○ Death by neglect: Majority (95%) do not specifically recognize their MHC mol-
eculesandaredestroyed Fate of Double positiveT-cells:
Majority(95%):Undergodeath
○ Positiveselection:Theremaining5%ofDPT-cellsundergoPositiveselection
byneglect
○ Negativeselection:Outof5%ofDPT-cellsthatarepositivelyselected,2–5%areself- The remaining 5%: UndergoPositiveselection,outofwhich
reactingandhencetheyarenegativelyselected,i.e.destroyed.(Centraltoler-ance) 2–5%:UndergoNegativeselection
• Mature T-cells: The remaining DP T-cells (2–5%) loose either CD4 or CD8 to 2–5%: Become MaturesinglepositiveT-cells.
formMatureT-cells(e.g.CD4+helperT-cellsandCD8+cytotoxicT-
cells).Theyacquirethymus specific antigens and then are released into the circulation
and migrate to theperipheral lymphoid organs where theyrespond to the antigenic
stimulus.
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PeripheralLymphoidOrgans
LymphNode
Lymphnodesaresmallbeanshapedorgans;dividedintothreeparts:cortex,medulla(bothare B-cell
areas) and paracortex (T-cell area). Cortex contains lymphoid follicles which
aremainlyoftwotypes.
• Primary lymphoid follicles: They are present before the antigenic stimulus; they are
smallerandcontainrestingB-cells.
• Secondary lymphoid follicles: They are formed following contact with an antigen,
T and B cell area:Lymphnode: containactivatedB-cells(i.e.plasmacellsandmemoryB-
T-cellarea:Paracorticalarea cells).Theyaredividedintocentralarea(germinalcenter)andperipheralzone(mantlearea).
B-cellarea:Cortex,medulla
Spleen: Spleen
T-cellarea:Periarteriolarlymphoidsheath
B-cellarea:Marginalzone Spleenisthelargestsecondarylymphoidorgan.Itisdividedintotwocompartments;central
whitepulpandouterredpulp.
• White pulp has two parts: (i) Periarteriolar lymphoid sheath (T-cell area), (ii)
Marginalzone(B-cellarea)
• Redpulpcontainsthesinusoids,filledwithRBCs.TheolderanddefectiveRBCsare
destroyedhere.
Defectinspleen:Asspleenisthesiteofdestructionofmostofthemicrobes,abnormalitiesofspleenors
plenectomy,oftenleadstoanincreasedincidenceofbacterialsepsiscausedprimarilybycapsulatedba
cteria.
MucosaAssociatedLymphoidTissue(MALT)
M-cell MALTarepresentliningthemucosaofintestine,respiratory,andurogenitaltract.
Specializedflattenedepithelial
cells MALTinintestinearethebeststudiedMALT,presentindifferentlayersofintestinalwall:
Donothavemicrovilli. • SubmucosacontainsPeyer’spatches,composedoflymphoidfollicles
Many microbes enterintestine via M-cells such
• asSalmonella,Shigella,Vibrio,andPoliovirus.
Laminapropriacontainslooseclustersoflymphocytesandmacrophages.
• Epitheliallayercontains:
○ Fewspecializedlymphocytescalledintraepitheliallymphocytes(γδT-cells)
○ M cells: They are specialized flattened epithelial cells that do not have
microvilli.TheyactastheportalofentryofanumberofmicrobessuchasSalmonella,Shigell
a,Vibrio,andPoliovirus.
○ TheyarelinedbySecretoryIgAwhichprovidelocalormucosalimmunity.
FunctionofLymphocytes Lymphocytes
Naivecell-Transformstoeffector cell on primaryexposuretoantigen
Effectorcell-Eliminateantigen Lymphocytes can be of three types: T lymphocytes, B lymphocytes and NK (natural
Memorycell-Transformstoeffector cell on secondaryexposuretoantigen
killer)cells.
T- and B-cells can also be classified into naive lymphocytes (prior antigenic
contact)andlymphoblasts(followingantigeniccontact)whicheventuallydifferentiateintoeffec
Section
torcellsormemorycells.
Table2.3.1:Differencesbetweennaivecell,effectorcellandmemorycell
Naivecell Effectorcell Memorycell
Location Secondarylympho Inflamedtissuesand Both the locations of naive
(presentmostlyin) idorgans mucosal surfaces andeffectorcell
Cellcycle Dormant(G0phase) Active Dormant(G0phase)
Morphology Smalllymphocyte Largelymphocyte Smalllymphocyte
Lifespan Short Short Long
Function Transformstoeffectorcellonprim Eliminateantigen Transformstoeffectorcellonsecondaryexposuretoantige
ary exposure to n,occursfastwithoutlagperiod
antigen;occursslowduetolagperi
od
Contd...
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StructureofImmuneSystemandImmuneResponse81
Contd...
Surfacemarkers
CD127(IL-7R) High Low High
CD45isoform CD45RA CD45RO CD45RO
CD25(IL-2Rα)on T-cells No Yes Yes
CD27onB-cells No Yes Yes
B-cellsproducingIgtypes IgMandIgD IgG, IgA, IgE IgG, IgA, IgE
andtheiraffinity Lowaffinity Highaffinity Highaffinity
TLymphocytes
TherearetwotypesofeffectorT-cellsCD4 +helperT-cellsandCD8+cytotoxicT-cell.Rare
subtypesareTREGcellsandγδT-cells
RegulatoryT-cells(TREGcells,formerlyknownassuppressorT-cells):
• Theyprovidetolerancetoself-
antigens(peripheraltolerance),andpreventthedevelopmentofautoimmunedisease.
• Surfacemarkers:TREGcellspossesssurfacemarkerssuchasCD4,CD25andFoxp3
• DeficiencyofFoxp3receptorsleadstoasevereformofautoimmunediseaseknownas
Immunedysregulation,Polyendocrinopathy,EnteropathyX-linked(IPEX)syndrome.
γδT-cells:
Theyconstitute5%oftotalT-cells,expressγ/δchainsofTCRchains;insteadofα/βchains.
• TheylackbothCD4andCD8molecules.
• TheydonotrequireantigenprocessingandMHCpresentationofpeptides.
• Theyarepartofinnateimmunityastheγδreceptorsexhibitlimiteddiversityforthe
antigen.
• Theyareusuallyfoundinthegutmucosa,asintraepitheliallymphocytes(IELs).
• ThefunctionofγδT-cellsisnotknown,theymayencounterthelipidantigensthatenter
throughtheintestinalmucosa.
Table2.3.2:DifferencesbetweenT-cellandB-cell
Property T-cell B-cell γδT-cells:
Origin Bone marrow Bone marrow 5%oftotalT-cells
LackbothCD4andCD8
Maturation Thymus Bone marrow DonotrequireAPC
Peripheralblood 70–80%oftotallymphocytes 10–15%oftotallymphocytes Partofinnateimmunity
Found in the gut mucosa, asintraepithelial lymphocytes(IE
Antigen T- B-cellreceptor-Surface IgM orIgD Mayencounterthelipidantigensinintestine.
recognitionreceptors cellreceptorscomplexedwithCD3 complexedwithIgα/Igβ
CDmarkers CD3,4,8 CD19,21,24
Thymus specificAg Present Absent
Microvillionthe surface Absent Present
BLymphocytes Immunolog
B-cellsproliferatethroughvariousstages,firstinbonemarrow,theninperipherallymphoid
organs.
B-celldevelopmentinbonemarrowisdescribedbelow.
Pro B-
cells(proge Mature
nitorB- B-cells
PreB-cells(precursorB-cells) ImmatureB-cells
cells)
Events Only expressa µ heavy chain is formedSurrogate Light chain genes rearrangement Express
heterodimerIgα light chain is occursMembraneIgM expression bothmembran
/Igβ formedPreBCRsynthesis occurs Toleranceto selfAg isproducedby: eIgMandIgD
Allelicexclusionoccurswhichallowstheexpressionofonlyoneo Receptor
utofthetwoallelescodingforanIgchain editingNegativesel
ection
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Developmentinperipherallymphoidorgans:
ImmatureB-cellsmigratefrombonemarrowtoperipherallymphoidorgans(lymphnodeand spleen)
where they transform into mature or naive B-cells. Following antigenic stimulus,the mature B-
B-celldevelopmentstages: cells transform into activated B-cells (lymphoblasts) which further
Pro B-cells (progenitor B-cells)
differentiateintoeithereffectorB-cells,i.e.plasmacells(majority)ormemoryB-cells.
→ Pre B-cells (precursor B-cells)
→Immature B-cells→ Mature
B-cells NaturalKiller(NK)Cells
NKcellsarelargegranularlymphocytesthatconstitute10–
15%oftotallymphocytes,describedlaterunderCMI.
OtherCellsofImmuneSystem
Othercellsofimmunesysteminclude:Macrophages,dendriticcells,Granulocytes(e.g.
neutrophils,eosinophilandbasophils)andmast-cells.
Macrophages differ frommonocytesby: Macrophage

5–10foldslargerthan
Monocytes/macrophagesoriginatefrombonemarrow,fromaseparategranulocyte-monocyte
monocytes
Contain more lysozymes,organelles, enzymes progenitor
andcytokinescells. Monocytes are the largest blood cells present in blood. They
Possess greater phagocyticactivity donotdivideandwithin8hourstheymigratetotissues.
Have a longer life in tissues(months to years)
Macrophagesdifferfrommonocytesinthefollowing:
• 5–10foldslargerthanmonocytes
• Containmorelysozymes,organelles,enzymesandcytokines
• Possessgreaterphagocyticactivityandhavealongerlifeintissues(monthstoyears)Two
majorfunctions ofmacrophage arePhagocytosisand Antigenpresentation.
Table2.3.3:Typesofmacrophages
Bodysites Macrophage designation Bodysites Macrophage designation
Peripheralblood Monocytes Inflammation site Epithelioid
Tissues Macrophages cellsMultinucleated
cell(Langhansgiant-
Liver Kupffercells cells)
Brain Microglial cells Connectivetissues Histiocytes
Kidney Mesangial cells Placenta Hofbauercell
Lungs Alveolar macrophages Lymphoidfollicle Tingiblebodymacrophage
Bone Osteoclasts
Section
Table2.3.4:Distributionandfunctionofdendriticcells
Typesofdendriticcells Site Function
Langerhanscells Skinandmucosa Antigenpresentation
Interstitialdendriticcells Organs
(lungs,liver,spleen, ExpresshighMHC-IIandB7molecules
etc.)
Interdigitatingdendriticcells Thymus
Circulatingdendriticcells Bloodandlymph
Folliculardendriticcells Lymphnodes B-
cellsmaturation,anddifferentiation;MHC-
II and B7 molecules
absent;CoatedwithAg-Abcomplex
Note:Langerhanscellisadendriticcell;whereasLanghansgiantcellisamacrophage.
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MajorHistocompatibilityComplex
• MHC molecules or human leukocyte antigens (HLA) serve as a unique
identificationmarkerforeveryindividualasthegeneticsequenceofMHCgenesisdifferentfo MHCclassI
reveryindividual. Presentonallnucleatedcells(exceptsperms)&platelets
• Theyalsodeterminethehistocompatibilitybetweenthedonorgraftandtherecipient. Presents the endogenous orintracellular Ag (viral/tumor) t
• Inhumans,HLAcomplexcodingforMHCproteinsarelocatedinshortarmofchromosome-6.
• ThegenesareclusteredinthreeregionsnamedasMHCregion-I,IIandIII
• MHCIandIIhelpinantigenpresentationtoT-cells:
○ MHCIpresentsintracellularantigenonviral/tumorcellstocytotoxicT-cells
○ MHC IIpresents extracellularantigen onAPCsto helperT-cells
• MHCIIIdoesnothelpinAgpresentation,butcodeforvariousproteinssuchascomplementfa
ctors(C2,C4,C3convertase,factorBandproperdin),heatshockprotein,TNF-
αandβandsteroid21-hydroxylases.
MHCclassII
PresentonallAPCs
Table2.3.5:DifferencesbetweenMHCclassIandMHCclassIImolecules Presents the extracellular AgtoCD4T-cells
MHCclassI MHCclassII
Presenton All nucleated cells (except sperms) Antigen presenting
andplatelets cells(APCs)
Peptideantigenis presentedtoCD8T-cells presented to
CD4T-cells
Nature of Endogenousorintracellular(viral/tumorAg) Exogenous
peptideantigen
Peptideantigen(size) 8–10aminoacidlong 13–18aminoacidlong
Antigenpresentation ByCytosolicpathway ByEndocyticpathway
Peptide-binding site α1/α2groove α1/β1groove
CD4orCD8bindingsite α3bindstoCD8moleculesonTCcells β2 bindstoCD4onTH
cells
Immunolog
Fig.2.3.1:StructureofMHCmolecules
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Table2.3.6:Sourcesandfunctionsofcytokines
Cytokine Cytokinesecretingcells TargeT-cellsandfunctions
Interleukins (IL)
IL-1 Produced by all 1. THcells-IL1producedbyAPCsstimulatesTHcellsactivationandproliferation:
nucleatedcells(MainlybyAPCssu ○ Promotes IL2 secretion byTHcells
chasMacrophages, ○ InducesIL-2receptorexpressiononTHcells
monocytesdendritic cell, B-cells ○ Induces↑MHC-IIexpressiononAPCs
andendothelialcell) 2. B-cell:PromotesB-celldevelopmentandmaturation
3. Liver:Inducessynthesisofacutephasereactantproteins
4. Hypothalamus:inductionoffever
5. Macrophageandneutrophilactivation:↑expressionofICAM
IL-2 TH1 cells Induces proliferation of activated TH cells, TC cells and some NK
cells(PreviouslycalledT-cell growthfactor)
IL-3 THcell, NKcell, Mastcell 1. Stimulateshematopoiesis(actsasmulti-CSF)
2. Mastcelldegranulation-↑histaminesecretion
IL-4 TH2 cells 1. THcellsPromoteTH2cellactivityandinhibitT H1cell
2. B-cell:PromoteB-cellsactivationandproliferationandinduceB-cellclassswitchover
toproduceIgE,IgG4,IgG1;previouslycalledB-cellgrowthfactor
3. MacrophageandAPCs:Induce↑MHC-IIexpression
IL-5 TH2 cells Promoteeosinophilgrowthanddifferentiation
IL-6 TH2 cells, macrophages IL-
1andTNFlikeeffects(synergisticeffect)PromotesBcellprolif
erationandantibodyproduction
IL-7 Bone marrow/thymic stromal cells ServesasagrowthfactorforT-cellandB-cellprecursors.
IL-8 Macrophages,endothelialcells Attractsneutrophils,NKcells,eosinophilsandbasophils.
IL-9 THcells Hematopoieticandthymopoieticeffects
IL-10 TH2 cells ReducescytokineproductionbyTH1cell.
IL-11 Bone marrow stromal cells Hematopoietic effect (B-cell and platelet
development)Liver:Inducesynthesisofacutephasereactantprotein
IL-12 Macrophages Promote TH 1 cell induction and inhibit TH 2 activity; promotes CMI responses, NK cellstimulatory
factor
IL-13 TH2 cells Mimic IL-4 function
IL-17 CD4+activatedmemoryTcell H Initiatesandmaintainsinflammation
Interferons (IFN)
IFN-α Leukocytes Antiviralactivity
IFN-β Fibroblasts Antiviralactivity
IFN-γ TH andTC cells,NKcells 1. Macrophage:Activatestherestingmacrophagesintoactivatedmacrophage
2. B-cells:ActivateB-cellstoproduceIgG
3. Promotesinflammationofdelayedtypeofhypersensitivity(alongwithTNF-β)
4. TH2cell:InhibitsTH2cellproliferation
Tumornecrosisfactors(TNF)
TNF-α Macrophage 1. IL-1likeeffect
2. Tumorcells:Promotevascularthrombosisandtumornecrosis
3. Inflammatory cells: Induce cytokine secretion
Section
4. Induceslipolysis,causesextensiveweightlossassociatedwithchronicinflammation
TNF-β TH1cellandTCcell Tumorcells:SimilareffectlikeTNF-α
Macrophage:Enhancephagocyticactivity
Colonystimulatingfactor(CSF)
GM-CSF Fibroblasts, endothelium,T- Macrophageandgranulocytegrowthstimulation
cells,macrophages
G-CSF Bone marrow stromal Granulocytegrowthstimulation
cells,macrophages
M-CSF Fibroblasts,endothelium Macrophagegrowthstimulation
Others
TGF-β Macrophages, masT- 1. InhibitTandBcellproliferationandhematopoiesis
cellsTandB-cells,platelet 2. Promotewoundhealing
3. Promotesclass switching ofB-cells to the IgAclass
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IMMUNERESPONSES
Immune response refers to the highly coordinated reaction of the cells of immune system
andtheirproducts.IthastwoarmsHumoralorAntibodyMediatedImmuneresponse(AMI)andC
ell-MediatedImmuneresponse(CMI)
• Bothe CMI andAMI interdependent,regulated by thehelper T(T H) cells
• ThereiscommonpathwayfirstbeforeCMI/AMI;whichinvolvesantigenpresentation
tohelperT-cellsfollowedbyactivationofhelperT-cells.
• ActivatedhelperT-cellsdifferentiateintoeitherT H1orTH2subsets.InductionofTH1cells secrete
cytokines that stimulate CMI whereas if T H2 derived cytokines induce theB-
cellstoproduceantibodies.
Antigen-PresentingCells(APCs) Antigen-presenting cells(APCs)

ProfessionalAPCs:
APCsimpliestocellsthatpresenttheantigenicpeptidealongwithMHCclassIItoT Hcells.Theymay Macrophages
begroupedinto: Dendriticcells
• ProfessionalAPCs:e.g.Macrophages,DendriticcellsandB-cells B-cells
Non-professionalAPCs:e.g.,
• NonprofessionalAPCs:e.g.Fibroblasts(skin),Thymicepithelialcells,Pancreaticbeta
Fibroblasts (skin)
cells,Vascularendothelialcells,Glialcells(brain)andThyroidepithelialcells. Thymicepithelialcells
Pancreaticbetacells
Table2.3.7:Antigen ProcessingPathways Vascularendothelialcells
Property Cytosolicpathway Endocytic pathway Glial cells (brain)
Thyroidepithelialcells
Antigenprocessed Endogenous Exogenous
Antigeniscomplexedwith MHC I molecules MHC II molecules
Antigenispresentedto TC cells TH cells
HelperT-cells(ActivationandDifferentiation)
ActivationofTHcellsrequiresgenerationofthesespecificsignals: THcellsActivation-
Requiresspecificsignals:
1. Antigen-specificsignal:InvolvesbindingofantigenicpeptideonAPCstoTCRofT Hcells.
Antigen-specific signal: Ag onAPCs toTCR ofTHcells.
2. CD4moleculesofTHcellsalsointeractwithβ2domainofMHC-II. CD4sof THcellswithβ2
3. CostimulatorysignalinvolvesbindingofCD28moleculeonTHAPC cellstoB7moleculeson domainofMHC-II.
s. Costimulatory signal: CD28 onTHcells to B7 onAPCs.
4. Cytokinesignal:IL-1secretedfromAPCsactsonTHcells. Cytokinesignal:IL-1actson
THcells
Followingsignaltransduction,naiveT HcellsdifferentiatesintoeffectorandmemoryT-cells.
EffectorT cellsfurtherdifferentiateintoeitherT1orT2subsets,regulatedbyIL-12which
H H H
promotesTH1subsetproliferation.TH1andTH2derivecytokinesmediatesvariousfunctions.
Table2.3.8:TH1cytokinesandtheirfunctions
TH1cytokinesandtheirfunctions
Immunolog
IL-2 PromotesactivationofTHandTCcellsandNKcell
IFN-γ 1. Activatestherestingmacrophagesintoactivatedmacrophage
2. ActivatesB-cellstoproduceIgG
3. Promotesinflammationofdelayedtypeofhypersensitivity(alongwithTNF-β)
4. InhibitsTH2cellproliferation
TNF-β Enhancesphagocyticactivityofmacrophage
TH2cytokinesandtheirfunctions
IL-4 1. InhibitsTH1celldifferentiation
2. StimulatesB-cellstoproduceIgEandalsoIgG4andIgG1
IL-5 1. Enhancesproliferationofeosinophils
2. IL-4andIL-5togetherprovideprotectionagainsthelminthicinfectionsandmediateallergicreaction
IL-6 PromotesB-cellproliferationandantibodyproduction
IL-10 InhibitsTH1celldifferentiation
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Cell-MediatedImmuneResponse
CMI provides immunity against: (i) microbes residing in intracellular milieu (both
obligateandfacultative)(ii)tumorcells,(iii)Mediatedelayed/typeIVhypersensitivity(iv)plays
keyrole intransplantationimmunityandgraft-versushostreaction.
ComponentsofCMI:
Agspecific:TCCell EffectorcellsofCMIinclude:
AgNonspecific: • Antigenspecificcells:(i)CytotoxicT-cells(principalmediatorofCMI)
NKcell • Antigennonspecific cells: (ii) NK cells, (iii) Cells performing ADCC (NK cells,
ADCCperformingcells:NKcells, Neutrophils, Eosino-philsandMacrophages
macrophages,neutrophilandeosinophils)
CytotoxicTLymphocytes
CD8TCcellsaretheprincipaleffectorcellsofCMI.ActivationofNaiveTCcellsrequiresthese
specificsignals.
1. Antigen-specific signal:TCRof naiveTCcellsbinds toMHCI-peptide complexoftarget
cells.
2. CD8ofTCcellsalsointeractswithdomainofMHC-I.
3. Costimulatorysignal:CD28ofnaiveTCcellsinteractswithB7moleculeontargetcells.
TccellsActivation:
4. Cytokinesignal:IL-2(secretedbyTH1cell)actsonTCcells
Requiresspecificsignals:
TheactivatedTCcellsproducetwotypesoflethalenzymes;called(i)perforins(forporeson
Antigen-specific signal: Ag ontargetcellstoTCRofT ccells.
CD4ofTHcells:withα3 thetargetcells)and(ii)granzymes(destroythetargetcells).
domainofMHC-I.
NaturalKillerCells
Costimulatory signal: CD28 onTccellstoB7ontargetcells.
Cytokinesignal:IL-2actson
Tccells NKcellsarelargegranularlymphocytesthatconstitute10–
15%ofperipheralbloodlymphocytes:
• Theyarederivedfromaseparatelymphoidlineage.NKcellsarecytotoxicbutantigen
nonspecific.
• Theyarepartofinnateimmunity,actasfirstlineofdefenseanddonotrequireprior
contactwiththeantigen.
• MechanismofNKcellmediatedcytotoxicity:
○ Receptor interaction: When activation receptors (e.g. NKR-P1, CD16) present
onNKcellsareengagedwithligandspresentonthetargetcells;NKcellsbecomeacti
vated.
NKcellmediatedcytotoxicityoccurs by:
○ Targetcelldestruction:issimilartothatofTCcells,i.e.viasecretingperforinsandgranz
Receptorinteractionfollowedby ymes.However,theNKcellsenzymesareconstitutivelyexpressed(i.e.theyare
Targetcelldestruction cytotoxic all thetime, even withoutexposure to the antigen).
Table2.3.9:DifferencesbetweenNKcellsandTCcells
Property NKcells TCcells
Surface markers CD16,D56 CD3,CD8
MHC restriction No MHC-I restricted
Memory No Yes
Section
Immunity PartofInnate immunity,Ag nonspecific Acquired immunity,Agspecific

Targetcell VirusinfectedcellsandTumorcells SameasNKcells
Mechanism Perforinsandgranzymes(Constitutive) Same as NK cells,
ofdestruction butinducible
Immune response CMI CMI
Antibody-DependentCell-MediatedCytotoxicity(ADCC)
Anumberofnonspecificcytotoxiccellsexpressreceptors(FcR)ontheirsurfacethatcanbind
totheFcregionofanyIg:
• ThesecellscanbindtoFcportionoftheantibodycoatedonthetargetcells,andsubsequentlyc
auselysisofthetargetcellbyreleasingvariouscytotoxicfactorssuchas:
○ NKcellssecreteperforins,andgranzymes
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○ Neutrophilsreleaseslyticenzymes
○ Eosinophilscanreleaselyticenzymesandperforins;protectsagainsthelminths
○ MacrophagesproducelyticenzymesandTNF
• Althoughthesecytotoxiccellsarenonspecificfortheantigen,thespecificityoftheantibodydirect
sthemtowardsthespecifictargetcells.Thistypeofcytotoxicityisreferredtoasantibody-
dependentcell-mediatedcytotoxicity(ADCC).
Assessment/DetectionofCMI
(i)Mixed-lymphocytereaction(MLR),(ii)Cell-mediatedlympholysis(CML),(iii)Thegraft-
versushostreaction(GVH)inexperimentalanimals.
Humoral/AntibodyMediatedImmuneResponse(AMI)
AMIprovidesprotectiontothehostbysecretingantibodies;thatpreventinvasionofmicrobesprese
ntonthesurfaceofthehosT-
cellsandintheextracellularenvironmentbuthasnoroleagainstintracellularmicrobes.
AMIoccursthroughthefollowingthreesequentialsteps:
1. ActivationofB-cellscarryingmicrobialantigen(B-
cellsactasAPCs)andpresentingtoTHcells.Thisrequiresthefollowingsignals:
• Signal-1isinducedbythecrosslinkingofmIgonBcellmembranewiththemicrobial
antigen.
• Signal-2providedbybindingofCD40onB-cellwithCD40L(ligand)onactivatedT Hcells. B-cellsActivation:
• Signal-3CytokinesproducedbytheactivatedT HcellsactonB-cells Requiresthreesignals:
2. DifferentiationofB-cellsintoeffectorcells(plasmacells)andmemorycells.Thisoccurs mIg on B-cell with themicrobialantigen
CD40 on B-cell with CD40L(ligand)onactivatedTHcells.
ingerminalcentreofsecondarylymphoidfollicles.
Cytokinesproducedbythe
activatedTHcellsactonBcells
Following Ag contact, the naive B-cells transforms to →Centroblasts → undergosomatic hypermutation and affinity maturation to form Centr
Table2.3.10:CytokinesrsponsibleforIgclass/subclassswitchover
Cytokine(s) Igclassproduced
IFN-γ IgG2aorIgG3
Immunolog
IL-5+TGF-β IgAorIgG2b
IL-4 IgEorIgG1orIgG4
IL-2,4,5 IgM
IL-4,5,6+IFN-γ IgG
3. Effector functions: Production of secreted antibodies by plasma cells which in

turncounter act with the microbes in many ways such as neutralization,
opsonization,complementactivation,ADCC,mucosalimmunity,etc.
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LYMPHOIDORGAN 8. MostpotentstimulatorofNaiveT-cells:
1. Allareperipherallymphoidorganexcept: a. Maturedendriticcells (AI2011,AIIMSNov08)
a. LN (PGIMay10) b. Folliculardendriticcells
b. Spleen c. Macrophages
c. MALT d. B-cells
d. Thymus 9. AllofthefollowingarefunctionsofCD4helpercells,excep
e. Bonemarrow t: (AI2009)
2. T-celldependentregioninlymphnode: a. Immunogenic memory
a. Cortex (NEETPatternBased) b. Produceimmunoglobulins
b. Medulla c. Activatemacrophages
c. Paracorticalarea d. Activatecytotoxiccells
d. Mantle layer 10. TruestatementregardingNKcellsareallexcept:
(PGIMay2015)
a. Alsocalledlargegranularlymphocyte
CELLSOFIMMUNESYSTEM b. Cankillvirusinfectedcell
3. Whichisnotanexampleofantigenpresentingcells(APC) c. Formsfirstlineofdefence
? (AIIMSMAY2016) d. Cankilltumourcell
a. Mcells e. Noroleincellmediatedimmunity
b. Thymocytes 11. NullcellsthatlackcharacteristicsofB-orT-
c. Langerhancells cellsconstitutewhatpercentageofperipherallymphocyte
d. Macrophages s:
4. Trueregardingthymusis: (JIPMERNov2014) (JIPMER2014,2013)
a. MaturethymocytesexpressCD4andCD8 a. 0–1 b.2–3
b. Thymocytes whose T-cell receptor bind with c. 5–10 d.15–20
highaffinitytoself- 12. A New agent was developed to increase the
antigensandMHCComplexesareclonallydeleted recognitionof foreign antigens by Antigen presenting
c. Mature thymocytes express surface IgMand IgD cells. TrueregardingthephysiologicalaspectsofAPCs:
d. CD 4 and CD 8 double positive cells are (JIPMERNov2014)
completelyeliminatedbyaprocessofnegativeselecti a. AntigenispresentedviaMHC–Icomplexes
on b. APCsarerequiredbeforearesponsetovirusesaregenera
5. Whichofthefollowingfeaturesisnotsharedbetween‘T- ted
cells’and‘B-cells’? (AI2012,AIIMSNov2012) c. Direct antibodystimulation stillrequiresAPC’s
a. Positiveselectionduringdevelopment d. AntigenprocessedbyAPCarerecognizedbyCD4+TCel
b. ClassIMHCexpression ls
c. Antigenspecificreceptors 13. Macrophagesaremajorsourceof: (DNBJune2010)
d. Alloftheabove a. IL-1 b. IL-5
Section
6. Cellmediatedimmunityisbyvirtueof: c. IL-7 d. IFN-Y

a. HelperT-cell (DNBJune2011)
b. SuppressorT-cells MHCCOMPLEX
c. CytotoxicT-cells
d. Allabove 14. TheroleplayedbyMHC1and2isto:
7. CommonbetweenB-andT-cells: (PGI June 2007) (AIIMSNov2013,AIIMSMay2014,AIIMSNov2014)
a. Originfromsame celllineage a. Stimulateinterleukinproduction
b. Sitedifferentiation b. Immunoglobulinclassswitchover
c. Antigenicmarker c. TransducethesignaltoT-
d. PerformBothhumoralandcellularimmunity cellfollowingantigenbinding
d. PresentingtheantigenforrecognitionbyT-
e. Furtherdifferentiationseen
cellantigenbindingreceptors
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15. MHCmoleculesarecodedby: (NEETPatternBased)

19. MHCIIIcodesfor: (RecentMCQ2013)
a. Chromosome-6
a. Complements
b. Chromosome-7
b. Interleukin
c. Chromosome-8
c. Prostaglandins
d. Chromosome-9
d. Interferon
16. GenecodingforMHC-Iinclude: (NEETPatternBased)
a. A,B,C
b. DR
CYTOKINES
c. DP
d. DQ
20. Whichofthefollowingisnotpyrogenicinnature?
17. PeptidebindingsiteonMHC- a. IL1 (AI2012)
IforpresentingprocessedantigentoCD8T- b. IL18
cellsisformedby: c. TNFalpha
(AI2010) d. Interferonalpha
a. Junctionofproximaldomainsmade-
21. IL–2producedby: (RecentMCQ2014,AI2000)
upofAlphasubunit
a. T-cells
b. Junctionofdistaldomainsmade-upofAlphasubunit
b. B-cells
c. Junctionofproximaldomainsmade-upofbetasubunit
c. Monocytes
d. JunctionofdistalProximaldomainsmade-
d. Neutrophils
upofbetasubunit
22. Mostimportantchemicalmediatorsofgramnegativesep
18. MHCIIarepresentedon: (PGINov10)
ticemia: (RecentMCQ2013)
a. Macrophages b. Dendritic cells
a. IL-2B
c. Lymphocytes d. Eosinophils
b. TNF-α
e. Platelet
c. TGB-β
d. IL-20
Immunolog
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EXPLANATIONS
LYMPHOIDORGAN
1. Ans.(d),(e)(Thymus,Bonemarrow)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep145,Ananthanarayan9/
ep128,8/ep123,132,Kuby’sImmunology6/ep295
LymphoidOrgan:
• Central/Primary:ThymusandBursaoffabricus(birds)/BoneMarrow(human)
• Peripheral/Secondary:Spleen,LymphNode,MALT(GITandRespiratorymucosa).
2. Ans.(c)(Paracortical...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep146-7,Kuby’sImmunology6/ep45-46
Lymphnode Spleen
T-cellarea-Paracorticalarea T-cellarea-PeriarteolarlymphoidsheathB-
B-cellarea-Cortexandmedulla cellarea-marginal zone
CELLSOFIMMUNESYSTEM
3. Ans:(b)(Thymocytes)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p162
ThymocytesaretheTcellspresentinthymus;andTcellsNEVERactasAPC;infactAPCspresenttheantigentoTcells.
4. Ans.(b)(Thymocyteswhose..)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep150
CD4andCD8doublepositive(DP)T-cellsarenoteliminatedcompletely.Theyhavethreefates:
• Positiveselection:The5%ofDPT-
cells,whoseαβreceptorsarecapableofrecognizingtheirMHCmoleculesarepositivelyselected.ThisresultsinMHCrestricti
on.
• Deathbyneglect:MajorityofDPcells(95%)failpositiveselectionbecausetheydonotspecificallyrecognizetheirMHC
molecules.
• Negative selection: The survived cells that undergo positive selection (5%) are MHC restricted. However, some of
thesesurvivingcells(2–
5%)reacttotheselfantigensandtherefore,theyareselectedtobekilledbyapoptosisandremoved(negativelyselection).
• TheremainingDPT-cells(2–5%)havingαβtypeTCRselectivelylooseeitherCD4orCD8andbecomesinglepositive
matureT-cells
AboutOtheroptions:
• Maturethymocytesaresinglepositive;i.e.theyexpresseitherCD4orCD8;butneverboth.
• MatureBcells(notthymocytes)expresssurfaceIgMandIgD.
5. Ans.(a)(Positiveselectionduringdevelopment)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep150
• Duringembryoniclife,fordeletionofselfreactingclones,T-cellsundergopositiveselectionfollowedbynegativeselectionwhereasB-
cellsundergoonlynegativeselection.
Section
AboutOtherOptions
• ClassIMHCmoleculesareexpressedonallnucleatedcells.BothTandB-
cellsbeingnucleated,sotheyexpressclassIMHC.WhereasclassIIMHCareexpressedonallAPCs(sopresentonB-
cellsbutnotonT-cells).
• Antigenspecificreceptor:
○ AntigenspecificreceptoronT-cell-TCR(T-cellreceptor)
○ AntigenspecificreceptorinB-cell-BCR(B-cellreceptor),i.e.surfaceimmunoglobulinslikeIgMandIgD
6. Ans.(d)(Alloftheabove)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep162,Ananthanarayan9/ep152
• CellmediatedimmunityreferstospecificimmuneresponsemediatedbyeffectorT-cells(bothT-helper,Cytotoxic
T-cellsandSuppressorT-cells)generatedagainstanantigen.
7. Ans.(a),(e)
(Originfromsamecelllineage,Furtherdifferentiationseen)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep145,Kuby’sImmunology6/ep29,34
• BothTandB-cellsoriginatefromsamelymphoidprogenitorcelllineageinbonemarrow.
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• Siteofdifferentiation–Tcell-inThymusandBcell-Inbonemarrow
• AntigenicmarkerarealsodifferentforT&B-cells(Refertext)
• Immuneresponse-Tcellmediates-cellularimmunityandBcellmediates-humoralimmunity
• Furtherdifferentiation:
○ TcelldifferentiatedtoTH(TH1andTH2)andTccells
○ Bcelldifferentiatedtoplasmacells
8. Ans.(a)(Maturedendriticcells)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep155
• MaturedendtriticcellspossesshigherlevelofMHC-II&costimulatoryB-7molecules,hencetheyarethemostpotent
stimulatorofT-cells.
• Detail-Refertext
9. Ans.(b)(Produceimmunoglobulins)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep162-3
• WhenrestingThcellbindstoaMHCclassII-antigencomplexonAPC,thatinitiatestheactivationofThcell.
• TheactivatedThcelldividesmanytimestoproduceeffectorThcellandmemoryThcell.
• EffectorThcellfurtherdividestoTh1and2andperformthefollowingactions:
○ ActivationandproliferationofTccell
○ Regulatemonocytemacrophagesystem
○ Helps in activation of B-cell to produce plasma cell that secretes immunoglobulins, (but never produce immuno-
globulins).
10. Ans(a),(b),(c),(d)(Also...,Can...,Forms...,Can...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep166
NKcellisanimportantcomponentofCMIalongwithTCcellandADCC.
ReferchapterreviewfordetailofNKcell.
11. Ans.(c)(5-10)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p166,Ananthnarayan9/ep137
12. Ans(d)(AntigenprocessedbyAPC..)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep162
Exogenous(foreign)antigensarefirstphagocytosedbyAPCs,thentheyareprocessedbyAPCsandpresentedonthesurfaceofAPC
salongwithMHCIItoberecognizedbyCD4+TCells.
13. Ans.(a)(IL-1)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep153-4,Kuby’sImmunology6/ep209
Macrophageproducesvarioussecretorymolecules:
• Enzymes-Lysozyme,proteases,elastases,collagenases,plasminogenactivator,
• Cytokines-IL-1, 8,12,TNF-α, TGF-β,prostaglandins
• Plateletderivedgrowthfactor(PDGF),Plateletactivatedfactor(PAF),
• CSF(colonystimulatingfactor),ACE(AngiotensinConvertingEnzyme)
• Complements-C2,C4
• ReactiveO2species-H2O2,NO,OH
MHCCOMPLEX
14. Ans.(d)(PresentingtheantigenforrecognitionbyT- Immunolog
cellantigenbindingreceptors)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep156,Ananthanarayan9/ep140
ThemajorfunctionofMHCIandIImoleculeistopresentthepeptideantigentotheT-cellreceptorsofT-cells.
• MHCImoleculepresentsthepeptideantigentoT ccells.
• MHCIImoleculepresentsthepeptideantigentoThelpercells.
15. Ans.(a)(Chromosome-6)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep156-7,Ananthanarayan9/ep139
• MHCgenes are locatedon short arm ofChromosome-6
16. Ans.(a)(A,B,C)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep156-7,Ananthanarayan9/ep139
• ClassIMHC geneincludes- A,B,C
• ClassII MHC gene includes –DP, DQ, DR
• Class IIIMHC geneincludes-genes forcertaincomplement factors,Heat shockprotein,TNF
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17. Ans.(b)(Junctio...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1e/p156-7,Kuby’sImmunology6/ep194
BindingsitesofMHCI&IImolecules:
• Theantigenbindingsite:
○ ForMHCI-groovebetweenα1andα2
○ ForMHCII-groovebetweenα1andβ1
○ BothethesegrooveslieinthedistalpartofMHCmolecules
• Theco-receptorbindingsite:
○ ForMHCI-α3domainbindstoCD8ofTccell
○ ForMHCII-β2domainbindstoCD4ofTHcell
○ BothethesegrooveslieintheproximalpartofMHCmolecules.
18. Ans.(a)(b)(Macrophages,Dendriticcells)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p156-7,Anan-
thanarayan9/e,p142,8/ep134
• MHCImoleculesarepresentonallnucleatedcells(RBCandplateletsarenonnucleated,soMHCImoleculesarenotfound)
• MHCIImoleculesaremorerestrictedindistribution,presentonmacrophages,dendriticcellsandB-cells.
19. Ans.(a)(Complements)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/e,p156-7,Ananthanarayan9/ep140
• MHCIIIcodesforComplementsC2,C4,C3convertase,factorB,properdin,HSPandTNF.
CYTOKINES
20. Ans.(b)(IL18)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep160,Kuby’sImmunology6/ep279
Journal-CirculatingCytokinesasMediatorsofFever,MihaiG.NeteaetalClinInfectDis.(2000)31(Supplement5):S178-S184.
• CytokinesIL-1,TNF-andIL-6actsonthehypothalamustoinduceafeverresponse…….Kuby’sImmunology6/ep279
• IFN-αisalsoapotentinduceroffever
• BacterialLPSstimulatesmacrophagesandendothelialcellstoreleaseTNFandIL1whichinturnactivatesIL-6.Finally
ProstaglandinE2(PGE2)isstimulatedwhichisthemainmediatoroffever.
21. Ans.(a)(T-cell)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep160,Kuby’sImmunology6/
ep307Actionofcytokines:Referchapterreview
22. Ans.(b)(TNF-α)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep160,Stite’sMedicalImmunology,10/e
p152
• Bacterialsepticshockapparentlydevelopsbecausebacterialcell-wallendotoxinsstimulatemacrophagestooverproduce
IL-1andTNF-αtohigherlevelsthatcausesepticshock
• TNFαistheprinciplecytokinesinvolvedingram-negativesepticshock.
• TNFαisprimarilyresponsibleforShwartzmanreaction,inwhichrepeatedinjectionofLPS(obtainedfromgram-
Section
negativecellwall)intoasolidtissueleadstohemorrhagicinfarction.
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CHAPTER
Hypersensitivity 2.4
Hypersensitivityorallergyreferstotheinjuriousconsequencesinthesensitizedhost,
followingsubsequentcontactwithspecificantigens.
Table2.4.1:GellandCoombsclassificationofhypersensitivityreactionsandtheirfeatures
Type–I Type–II Type–III Type–IV
Immuneresponsealtered Humoral Humoral Humoral Cellmediated
Immediateordelayed Immediate Immediate Immediate Delayed
Antigencontacttosymptomsinterval 2to 30min 5–8hrs 2–8hrs 24to72hrs
Antigen Soluble Cellsurfacebound Soluble Solubleorbound
Mediator IgE IgG Ag-Ab complex TDTHcell
Effectormechanism Mastcelldegranulation 1. ADCC Complementac Macrophageactivatio
2. Complement tivation n leads
mediatedcytol andinflammato tophagocytosis or
ysis ryresponse cellcytotoxicity
Desensitizationtotheallergen Easy,butshortlasting Easy,butshortlasting Easy,butshortlas Difficult
ting butsustained
Typicalmanifestations 1. Anaphylaxis 1. Transfusionr 1. Arthus reaction 1. Tuberculintest
2. Asthma eactions 2. Serum sickness 2. Granulomaformati
3. Atopicdermatitis 2. Rh 3. Glomerulone- on intuberculosis
incompatibility phtiritis andleprosy
3. Hemolytic 4. Rheumatoida 3. Contactdermatitis
anemia rthritis
TYPE-IHYPERSENSITIVITYREACTION
Mechanism
Type-IHypersensitivityreactionoccursthroughtwophases:
Sensitizationphase:Primingdoseofantigen(allergen)→processedbyAPC→antigenicpeptidepresentedtoT-cell→
TH2activated→secretesIL4→ActsonB-cell→IgEproduced→MastcellscoatedwithIgE(Fc)atmucosalsites.Effectorphase:Shocking(subsequent)doseof
Table2.4.2:MediatorsofType-1hypersensitivityreaction
Primary Mediators Action
Histamine,HeparinandSerotonin ↑Vascularpermeability,↑Smooth-musclecontraction
Eosinophil chemotactic factor (ECF-A) Eosinophil chemotaxis
Neutrophilchemotacticfactor(NCF-A) Neutrophilchemotaxis
Proteases Bronchial mucus secretion;
Degradationofblood-vesselbasementmembrane
Secondary Mediators Action
Platelet-activating factor Platelet aggregation and
degranulation;Contractionofpulmonarysmoothmuscle
s
Contd...
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Contd...
Secondary Mediators Action
Leukotrienes ↑Vascularpermeability;
(slowreactivesubstanceofanaphylaxis,SRS-A) Contractionofpulmonarysmoothmuscles
Prostaglandins ↑Vasodilation;Contractionofpulmonarysmoothmuscles;Plateletaggregati
on
Bradykinin ↑Vascularpermeability;Smooth-musclecontraction
Cytokines Systemicanaphylaxis;↑Expressionofcelladhesionmolecules(CAMs)on
(IL-1andTNF-α) venular endothelialcells
Table2.4.3:CommonallergensassociatedwithtypeIhypersensitivityreaction
Food Nuts,egg,peas,seafood,beans,milk
Plantsandpollens Ryegrass,ragweed,timothygrass
Proteins Foreign serum vaccines
Drugs Penicillin,sulfonamides,localanestheticsandsalicylates
Insect bite products Venomofbee,wasp,ant,cockroachcalyxanddustmites
Others Moldspores,animalhairanddander
ExamplesofTypeIHypersensitivityReaction
• ExperimentstodemonstratetypeIhypersensitivityreaction:P-
Kreaction,SchultzDalephenomenonandTheobaldsmithphenomenon
•
Experiments to demonstratetypeI HSN Systemicanaphylaxis
P-K reaction • Localizedanaphylaxis(atopy)suchas:
SchultzDalephenomenon ○ Allergicrhinitis(orhayfever)
TheobaldSmithphenomenon
○ Asthma
○ Food allergy
○ Atopicurticarial
○ Atopicdermatitis(allergiceczema)
○ Drugallergy
○ Whealandflarereaction.
• Parasiticdiseases/tests:
○ Casonitest(hydatiddisease)
○ TropicalPulmonaryEosinophilia(TPE)
○ Loeffler’spneumonia(Ascaris)
○ Grounditch(Hookworm)
DetectionoftypeIHSN ○ Leakageofhydatidfluid
Skinpricktest ○ Cercarialdermatitis/swimmer’sitch(Schistosoma).
RIST:detectsthetotalserumIgE
Section
RAST: Detects allergenspecific serum IgE

DetectionofTypeIHypersensitivity
• Skinpricktest
• Radioimmunosorbenttest(RIST):ItquantitativelydetectsthetotalserumIgE
• Radioallergosorbenttest(RAST):ItquantifiestheserumlevelofallergenspecificIgE.
Treatmentof TypeIHypersensitivityReaction
HyposensitizationtotreattypeIHSN • Avoidanceofcontactwithknownallergens
Repeated exposure to increasedsubcutaneousdosesofallergenscan reduce or eliminate theallergic response to the sameallergen
• Hyposensitization:Repeatedexposuretoincreasedsubcutaneousdosesofallergenscan
reduceoreliminatetheallergicresponsetothesameallergen.
• HumanizedMonoclonalanti-IgE-ItcanbindandblockIgE.
• Drugs:Severaldrugsareusefulinsuppressingtype-
1responsesuchasantihistamines,adrenaline,cortisone,theophyllineandcromolynsodiu
m.
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Hypersensitivity95
TYPE-IIHYPERSENSITIVITYREACTION
Intype-IIreactions,thehostinjuryismediatedbyantibodies(IgGorrarelyIgM)which
interactwithvarioustypesofantigenssuchas:
• Hostcell surface antigens (e.g. RBC membrane antigens like blood group and Rh
antigens)
• Extracellularmatrixantigensor
• Exogenous antigens absorbed on host cells (e.g. a drug coating on RBC
membrane).AfterAg-Abbindingoccurs,theFcregionofantibodyinitiatesthetype-
IIreactionsbythefollowingthreebroadmechanisms:
Antibody(Fc)ActivatingComplementSystem
Bycomplementdependentcytolysis(duetoMAC),inflammation(byC5a,C3a),opsonization(byC3ba
ndC4b)
Itisseeninfollowingconditions: Antibody dependent cellulardysfunction(ADCD).
• Transfusionreaction(ABOincompatibility) Grave’sdisease
• Erythroblastosisfetalis Myastheniagravis
• Autoimmunehemolyticanemia,agranulocytosis,orthrombocytopenia Goodpasturesyndrome
Perniciousanemia
• Druginducedhemolyticanemia Rheumaticfever
• Pemphigusvulgaris MyocarditisinChagasdisease
• Hyperacutegraftrejection.
Antibody(FCPortion)InteractingwithFcReceptorsonTargetCells
• Antibodydependentcellularcytotoxicity(ADCC)
• Opsonization.
AntibodyDependentCellularDysfunctionorADCD
AutoantibodyMediated:
• Activationof receptor,e.g.Grave’sdisease
• Inhibitionofreceptor,e.g.Myastheniagravis
• OtherexamplesofADCD:
○ Goodpasturesyndrome(antibodyproducedagainsttypeIVcollagen)
○ Perniciousanemia(antibodydirectedagainstintrinsicfactor)
○ Rheumaticfever(antibodyagainststreptococcalantigenscrossreactingwithheart)
○ MyocarditisinChagasdisease.
TYPE-IIIHYPERSENSITIVITYREACTION
Type-IIIhypersensitivityreactionsareasaresultofexcessformationofimmunecomplexes(Ag-
Abcomplexes)whichinitiateaninflammatoryresponsethroughactivationofcomplementsyste
mleadingtotissueinjury: Parasitic example of type IIIHSN:
Nephroticsyndromein
Immunolog
• LocalizedorArthusreaction Plasmodium malariae
○ Inskin:(i)followinginsectbitesor(ii)duringallergicdesensitization Katayama fever inschistosomiasis
○ Inlungs(i)Farmer’slung(Saccharopolysporaspecies),(ii)Bird-Fancier’sdisease African trypanosomiasis
• GeneralizedorSystemictypeIIIReactions,e.g.Serumsickness.
Table2.4.4:DiseasesassociatedwithgeneralizedtypeIIIhypersensitivityreactions
Connectivetissuedisorders:Duetoautoantibodiesformingimmunocomplexeswithself-antigens
• SLE(Systemiclupuserythematosus):Anti-DNAAb
• Rheumatoidarthritis:Ab againsthumanimmunoglobulin
• PAN(Polyarteritisnodosa)
Parasiticdiseases:Resultingfromimmunocomplexdeposition
• NephroticsyndromeinPlasmodiummalariae
• Katayamafever inschistosomiasis
• African trypanosomiasis
Contd...
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Contd...
Bacterialdiseasesresultingfromimmunocomplexdeposition
• Streptococcuspyogenes:Poststreptococcalglomerulonephritis
• Mycobacteriumleprae(Leprareactiontype2)
In Rheumatoid arthritis:ThereiscomplexofbothtypeIIIandIV hypersensitivity Reaction. ButType III is a more appropriateanswerthantypeIV.
Viraldiseaseswithimmunocomplexdeposition
• HepatitisB(arthritis)
• HepatitisC(arthritis)
• Infectiousmononucleosis(EpsteinBarrVirus)
• Dengue(arthritis)
TYPE-IVHYPERSENSITIVITYREACTION
Others:
• Subacutebacterialendocarditis
Type-IVhypersensitivityreactionsdifferfromothertypesinvariousways:
• Serum sickness
• Itisdelayedtype(occursafter48–72hoursofantigenexposure)
TypeIVHSNdifferfromothertypes by: • Cellmediated:CharacteristiccellscalledTDTHcellsaretheprincipalmediators
Delayedtype(occursafter • Tissueinjuryoccurspredominantlyduetoactivatedmacrophages.
48–72hoursofAgexposure)
Cellmediated(TDTHcells)
Tissueinjury-duetoactivatedmacrophages.
MechanismofType-IVReactions
• Sensitization phase (occurs 1–2 weeks following Ag exposure): APCs process
andpresent the antigenic peptides to TH cells. T H cells are differentiated to TH1 cells
whichfurtherdifferentiatestoformTDTHcells
• Effectorphase:TheTDTHcells,onsubsequentcontactwiththeantigen,secretevarietyof
Leprareactions cytokinessuchas:
Lepra reaction type I, e.g. ofHSNIV ○ Interferonγ
LeprareactionstypeII,e.g.of
○ IL-2
HSNIII
○ MCAF(Monocytechemotacticandactivatingfactor)
○ TNF-β
○ MIF(migrationinhibitoryfactor)
○ IL-3
○ GM-CSF(granulocyte-monocyte colony stimulatingfactor).
Cytokinesinturnperformvariousfunctionswhichmaybeeitherprotectivetypeortissuedamage
type.
Table2.4.5:Infections/conditionsassociatedwithtypeIVhypersensitivityreactions
Intracellular bacteria: Intracellular fungi: Noninfectiousconditions:
• Mycobacteriumleprae • Pneumocystisjirovecii • Diabetesmellitustype1
• M.tuberculosis • Candidaalbicans • Multiple sclerosis
• Listeriamonocytogenes • Histoplasmacapsulatum • Peripheralneuropathies
Section
• Brucellaabortus • Cryptococcusneoformans • Hashimoto’sthyroiditis

• Crohn’sdisease
• Chronictransplantrejection
• Graft-versus-hostdisease
Intracellularviruses: SkinteststodemonstrateDTH: Granulomaformationseenin:
• Herpessimplexvirus • Tuberculintest(Mantouxtest) Tuberculosis,sarcoidosis,schistosomiasisandothertr
• Variola(smallpox) • Lepromintest ematode infections
• Measles virus • Montenegro test (leishmaniasis) Otherexample:
• Frietest–doneinLGV LeprareactiontypeI
Contactdermatitis:Followingexposuretocontactantigens:
Nickel,poisonivy,poisonoak,picrylchloride
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Hypersensitivity97
TYPE-IHYPERSENSITIVITY 8. 2daysafterpenicillinadministration,antibodiesdevelo
ped that resulted in hemolysis. Which type
1. Asthmaisduetowhichtypeofhypersensitivityreaction? ofhypersensitivityreactionisthis?(AIIMSNov2014)
(RecentQuestion2015) a. Type-I b.Type-II
a. Type1 b.Type2 c. Type-III d.Type-IV
c. Type 3 d.Type4
2. Schultzdalephenomenon,thefollowingistruestatemen
TYPE-IIIHYPERSENSITIVITY
t: (TNPG2015)
a. Anaphylaxisinvitro 9. Serumsickness is which type of reaction?
b. Testtodetectatopy (AIIMSMAY2016)
c. TypeVhypersensitivityreaction
a. Type1 b. Type2
d. Testforallergiccontactdermatitis
c. Type3 d. Type4
3. TypeIhypersensitivityismediatedbywhichofthefollow
10. Whichofthefollowingisanimmunecomplexmediated
ingimmunoglobulins?
disease? (PGIMay2016)
(NEETPatternBasedKerala2016)
a. SLE
a. IgA b. IgG
c. IgM d. IgE b. Bronchialasthma
c. Rheumatoidarthritis
4. Anaphylaxis ismediatedby: (PGIMay2013)
d. Good pasture
a. 5-hydroxytryptamine
e. Tuberculinskintest
b. Heparin
c. Prostaglandin 11. TypeIIleprareactionisanexamplehypersensitivitytyp
d. Anaphylotoxinsfromcomplementactivation e: (JIPMER2013,2014)
e. Plateletactivatingfactor a. I b. II
5Allergicrhinitisisanexampleof: (RecentMCQ2013) c. III d. IV
a. TypeIhypersensitivityreaction 12. Thehypersensitivityreactioninvolvedinthehyperacut
b. TypeIIhypersensitivityreaction erejectionofrenaltransplantis:
c. TypeIIIhypersensitivityreaction (AIIMS May 2005)
d. TypeIVhypersensitivityreaction a. TypeI b. TypeII
c. TypeIII d. TypeIV
TYPE-IIHYPERSENSITIVITY
TYPE-IVHYPERSENSITIVITY
6. Lysisofredcellsinautoimmuneanemiasandhemolyticd
isease of thenewborn are examples of: 13. WhichofthefollowingistypeIVhypersensitivityreactio
a. TypeIhypersensitivity (MHPG2015) n? (NEETPatternBased;PGIJune2005)
b. TypeIIhypersensitivity a. Arthusreaction
c. TypeIIIhypersensitivity b. Serumsickness Immunolog
d. Type IV hypersensitivity c. Schwartzmanreaction
7. Type2hypersensitivityreactionseenin: d. Granulomatousdisease
a. Myastheniagravis (RecentQuestion2013)
14. Skintestsareusedforwhichhypersensitivityreactions?
b. Goodpasturesyndrome
(PGI June 2007)
c. Sarcoidosis
a. I b. II
d. Grave’sdisease
c. II d. IV
e. Hyperacutegraftrejection
e. V
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EXPLANATIONS
TYPE-IHYPERSENSITIVITY
1. Ans.(a)(Type1)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep174
2. Ans.(a)(Anaphylaxis...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep172,Ananthanarayan9/ep164
• SchultzdalephenomenonisanexampleoftypeIhypersensitivity(Anaphylaxisinvitro)
3. Ans.(d)(IgE)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep174,Ananthanarayan9/ep162,8/ep163
• Type1hypersensitivityreactionisdependentonIgEantibody(alsoknownasReaginantibodyorcytotropicantibody).
4. Ans.ALL(a)(b)(c)(d)(e)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep172-5,Ananthanarayan9/
ep164Referchapterreviewtoknow thelistofmediators ofanaphylaxis(i.e.type-I hypersensitivityreaction)
5. Ans.(a)(TypeIhy...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep172-5,Ananthanarayan9/ep165
• Allergicrhinitis orHay feveris, e.g.of TypeI hypersensitivityreaction.
TYPE-IIHYPERSENSITIVITY
6. Ans.(b)(TypeIIhypersensitivity)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep177Referchapterreview.
7. Ans.(a)(b)(d)
(Myastheniagravis,Goodpasturesyndrome,Grave’sdisease)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep176-
7,Ananthanarayan9/ep16
Referchapterreviewforexplanation.
8. Ans.(b)(Type-II)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep176-
7Penicillininhighdosescaninduceimmune mediated hemolysis via the hapten mechanism in which antibodies are
targeted against the combination
ofpenicillininassociationwithredbloodcells.Complementisactivatedbytheattachedantibodyleadingtotheremovalofred
bloodcellsbythespleen.This isanexampleofdruginducedtype-II hypersensitivityreaction.
TYPE-IIIHYPERSENSITIVITY
9. Ans:(c)(type3)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p179
Refertext
10. Ans(a,c)(SLE,RA)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p176-79
• SLEandRheumatoidarthritis–TypeIII(Immunecomplexmediatedhypersensitivity)
• Bronchialasthma(HSNtypeI),Goodpasture(HSNtypeII)andTuberculintest(HSNtypeIV)
• Rheumatoidarthritis,thereiscomplexofbothtypeIIIandIVhypersensitivityReaction.ButTypeIIIisamoreappropriateans
werthantypeIV.(KubyandRoitt’simmunology).
Section
11. Ans.(c)(III)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep179,Ananthanarayan9/ep165
12. Ans.(c)(TypeIII)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep178,Ananthanarayan9/ep166,8/ep162
• HyperacutegraftrejectionisanexamplesofTypesIIIHypersensitivityreactionwhereaschronicgraftrejectionandGraft-versus-
hostdiseaseisTypesIVHypersensitivityreaction.
TYPE-IVHYPERSENSITIVITY
13. Ans.(d)(Granu...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p180,Ananthanarayan9/ep166,8/ep162
• Granulomatousdisease–typeIVHypersensitivityreaction
• Arthusreaction,SerumsicknessandSchwartzmanreaction–typeIIIHypersensitivityreaction.
14. Ans.(a),(d)(TypeI,IV)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep180,Ananthanarayan9/ep162
• TypeIskintest:Casoniskintest
• TypeIVskintest:Tuberculin,Leprominandpatchtest.
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Autoimmunity,Immunodeficiency,Tr CHAPTER
ansplantation,andImmunoprophylaxis
2.5
AUTOIMMUNITY
Autoimmunity is a condition in which the body’s own immunologically competenT-cells
orantibodiesactagainstitsself-antigensresultinginstructuralorfunctionaldamage.
ImmunologicalTolerance Centraltolerance(Mechanism):
In thymus: Removes the selfreacting T-cells by negatives
Immunological tolerance is a state in which an individual is incapable of developing In bone marrow: Removes theself reacting
animmune response against his own tissue antigens. It is mediated by two broad mechanisms B-cells by negative selectionandreceptorediting.
—centraltoleranceandperipheraltolerance.
CentralTolerance
Thisreferstothedeletionofself-
reactiveTandBlymphocytesduringtheirmaturationincentrallymphoidorgans
• Inthymus:RemovestheselfreactingT-cellsbynegativeselection
• InbonemarrowforB-cells:RemovestheselfreactingB-
cellsbynegativeselectionandreceptorediting
PeripheralTolerance
Thisreferstoseveralback-upmechanismsthatoccurintheperipheraltissuestocounteracttheself-
reactiveT-cellsthatescapecentraltolerance.Itisprovidedbyseveralmechanisms.
1. Ignorance:Theself-reactiveT-cellsmightneverencountertheself-
antigenwhichtheyrecognizeandtherefore remain in a stateof ignorance.
2. Anergy:Byblockingco-stimulatorysignalbybindingofB7moleculesonAPCtoCTLA-
4moleculesonT-cells
3. Phenotypicskewing:Self-reactive T-cellsstimulatedbyself-
antigenssecretenonpathogeniccytokines
4. ApoptosisofSelf-reactiveT-cellsbyactivation-inducedcelldeath(AICD)
Peripheral tolerance(Mechanism):
5. RegulatoryT-cells(Tregcells)candownregulatetheself-reactiveT-cells Ignorance
6. Dendriticcells(DCs):Whencertaindendriticcells,suchasimmatureDCsandtolerogenicDCs Anergy (CTLA-4 mediated)
capture the self-antigen for processing, they down regulate the expression of molecules of co- Phenotypic skewing
stimulatory ligands, such as CD40 and B7 molecules or act indirectly by induction of Apoptosis of self-reactiveT-cells
regulatoryT-cells. RegulatoryT-cellsmediated
Dendritic cells (immature DCsandtolerogenic) mediated
7. Sequestration of self-antigen in immunologically privileged sites, e.g. corneal proteins, Sequestrationofself-antigen.
testicularand brain antigens.
MechanismsofAutoimmunity
• BreakdownofCTLA-4mediatedT-
CellAnergy:Seeninmultiplesclerosis,rheumatoidarthritisandpsoriasis
• Failure ofAICD(activation-inducedcelldeath):Seenin SLE
• Lossof Tregcells
• ProvidingT-cellhelptostimulateself-reactingB-cells
• ReleaseofSequesteredAntigens(spermatozoaandocularantigens)duetoinjurytoorgans
• MolecularMimicry,e.g.inpoststreptococcalacuterheumaticfeverandglomerulo-
nephritis
• PolyclonalLymphocyteActivation:MediatedbySuperantigens,EBVandHIV
• Exposureofcrypticself-epitopes
• Epitopespreading
• Bystanderactivation.
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Table2.5.1:Autoimmunediseases
SingleOrganorCellTypeAutoimmuneDiseases
Disease Self-antigen present on Typeofimmuneresponseandimportantfeatures
Autoimmuneanemias
Autoimmune RBCmembraneproteins Auto-
hemolyticanemia antibodiestoRBCantigenstriggerscomplementmediatedlysisorantibody-
mediatedandopsonizationoftheRBCs
Drug induced Drugs alter the red cell DrugssuchaspenicillinormethyldopainteractwithRBCssothatthecells
hemolyticanemia membraneantigens becomeantigenic
Perniciousanemia Intrinsic factor (a membrane-bound Auto-antibodiestointrinsicfactorblocktheuptakeofvitaminB12;leads
protein on gastric parietalcells) tomegaloblasticanemia
Idiopathic Platelet membrane Auto-antibodies against platelet membrane antigens leads to

ThrombocytopenicPurpura proteins(glycoproteinsIIb- ↓plateletcount
IIIaorIb-IX)
Goodpasturesyndrome Renal and lung Auto-antibodies bind to basement-membrane antigens on kidneyglomeruli
basementmembranes and the alveoli of the lungs, followed by complementmediated injury leads
to progressive kidney damage and pulmonaryhemorrhage
Myasthenia gravis Acetylcholine receptors Blocking type of autoantibody directed against Ach receptorspresent
on motor nerve endings, leads to progressive weakening ofthe skeletal
muscles
Graves’disease Thyroid-stimulating AntiTSH- (stimulates thyroid follicles,leads to
hormone(TSH) receptor autoantibodyhyperthyroidst
ate)
Hashimoto’sthyroiditis Thyroidproteinsandcells Autoantibodies and TDTHcells targeted against thyroid antigen
leadstosuppression ofthyroid gland:
• Seeninmiddleagedfemales
• Hypothyroidstateisproduced(↓productionofthyroidhormones)
Post- Kidney Streptococcalantigen:antibodyglomer complexes are deposited on
streptococcalglom ularbasementmembrane
erulonephritis
Insulin-dependent Beta cells present in islets TDTHcellsandauto-antibodiesdirectedagainstpancreaticbetacells
diabetesmellitus ofLangerhansofpancreas cause↓productionofinsulin
Spontaneous infertility Sperm antigens released due Autoantibodiesdirectedagainstspermantigensleadtoinfertility
totesticularinjury
Addison’sdisease Adrenal cells Autoimmunedestructionoftheadrenalcortex,causedbyautoantibodies
against the enzyme 21-hydroxylase leads to
chronicprimaryadrenalinsufficiency(↓steroidhormonesproduction)
SystemicAutoimmuneDiseases
Systemic Autoantibodies are • Ageandsex:Women(20–40yearsofage)arecommonlyaffected;female to
lupuserythema producedagainst various tissue male ratio is10:1.
Section
tosus antigenssuch as DNA, nuclear • Immunecomplexes(selfAg-

protein,RBCandplateletmembrane autoAb)areformed;whicharedepositedinvarious organs
s. • Majorsymptoms:Fever,butterflyrashoverthecheeks,arthritis,pleuris
y,andkidney dysfunction
Rheumatoidarthritis Here, a group of • Ageandsex:Women(40–60yearsofage)affected
autoantibodiesagainst the host IgG • Auto-antibodiesbindtocirculatingIgG,formingIgM-IgGcomplexes that
antibodiesare produced called RA are deposited in the joints and can activate thecomplement cascade.
factor.It is an IgM antibody • Main feature: Arthritis (chronic inflammation of the joints, beginsat
directedagainst the Fc region of synovium; most common joints involved are Small joints of
thehands,feet andcervical spine)
IgG.ACPA(Anticitrullinatedpeptid
• Otherfeatures:Hematologic,cardiovascular,andrespiratorysystemsarea
eantibodies)arealsoproduced lsofrequently affected
Sjögrensyndrome Ribonucleoprotein(RNP)anti- Auto-antibodies to the RNP antigens SS-A (Ro) and SS-B (La); leadsto
gensSS-A (Ro)andSS-B immune-mediated destruction of the lacrimal and salivary
(La)presentonsalivarygland,lacrimalg glandsresultingindryeyes(keratoconjunctivitissicca)anddrymouth(xerosto
land,liver,kidney,thyroid mia)
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Autoimmunity,Immunodeficiency,Transplantation,andImmunoprophylaxis 101
Contd...
SingleOrganorCellTypeAutoimmuneDiseases
Disease Self-antigen present on Typeofimmuneresponseandimportantfeatures
Scleroderma(Syste Nuclear antigens such as Helper T-cell (mainly) and autoantibody
micSclerosis) DNAtopoisomerase and mediated.Excessive fibrosis of the skin, throughout the
centromerepresent in heart, lungs, bodyTwotypes:
GIT,kidney,etc. 1. Diffusescleroderma:AutoantibodiesagainstDNA
topoisomeraseI(anti-Scl70)iselevated
2. Limitedscleroderma↑Anticentromereantibody,characterizedby
CREST syndrome: Calcinosis, Raynaud
phenomenon,esophagealdysmotility,sclerodactyly,andtelangiectasia
Seronegative Spondyloar- Sacroiliac joints and Commoncharacteristics:Theypresentasrheumatoidarthritislikefeatures,butdiff
thropathies othervertebrae er from itby:
Several types: • AssociationwithHLA-B27
• Ankylosing spondylitis • Pathologicchangesbeginintheligamentousattachmentstothebonerather
• Reitersyndrome than inthe synovium
• Psoriaticarthritis • Involvementofthesacroiliacjoints,and/
• Spondylitiswithinflammatory orarthritisinotherperipheraljoints
Boweldisease • AbsenceofRFs(hencethename'seronegative')
• Reactivearthritis • Auto-Abandimmunecomplexmediated
Multiplesclerosis Brain (white matter) Self-reactive T-cells produce characteristic inflammatory lesionsin
brain that destroys the myelin sheath of nerve fibers; leads
tonumerousneurologicdysfunctions
IMMUNODEFICIENCYDISORDERS
Immunodeficiencyisastatewherethedefensemechanismsofthebodyareimpaired,leadingto
enhanced susceptibility to microbial infections as well as to certain forms of cancer. It
isbroadlyclassifiedasprimaryorsecondary.
• Primaryimmunodeficiencydiseasesresultfrominheriteddefectsaffectingimmune
systemdevelopment.
• Secondary immunodeficiency diseases are secondary to some other disease process
thatinterferes with the proper functioning of the immune system (e.g. infection,
malnutrition,aging, immunosuppression, autoimmunity, or chemotherapy). They are
more commonthanprimaryimmunodeficiencydiseases.
Table2.5.2:Primaryimmunodeficiencydiseases
Humoralimmunodeficiency(B-celldefects)
1 Brutondisease(X-linkedagammaglobulinemia)
Itischaracterizedbyfailureofpre-B-cellstodifferentiateintoimmatureB-cellsinthebonemarrow-duetoabsenceofanenzymecalledBruton’s tyrosinekinase
leadingto total absenceofB-cells,plasmacells andallclassesofIg:
• The B-cell maturation stops at pre B cell stage; after the synthesis of heavy-chain without forming the light chains. Hence
thecytoplasmofpreBcell may haveincompleteimmunoglobulins.
Immunolog
• Xlinked,seenprimarilyinmales;rarelyinfemales.Onset-after6monthsoflife.
• Secondaryinfection:Recurrentbacterialinfections,viruses(enteroviruses)andparasites(Giardialamblia)
• Autoimmunediseases(suchasSLEanddermatomyositis)alsooccurinupto20%ofcases.
2 Commonvariableimmunodeficiency
Theclinicalmanifestations(secondaryinfection,autoimmunediseases)aresuperficiallysimilartothoseofBrutondiseases;butdiffer
inthefollowingaspects:
• Bothsexesareaffectedequally
• Onsetofsymptomsismuchlater,inthesecondorthirddecadeoflife.
• ItisalsoB-celldevelopmentdefect;B-cellsmaybepresentincirculationinnormalnumbers,buttheyappeardefectiveintheirabilityto
differentiateintoplasmacellsandsecrete immunoglobulins.
• Thediagnosisisusuallyoneofexclusion(afterothercausesofimmunodeficiencyareruledout);thebasisoftheimmunoglobulindeficiencyisvariable
(hence the name).
• ThedefectintheantibodyproductionhasbeenvariablyattributedtointrinsicB-celldefects,deficientT-cellhelp,orexcessive
T-cellsuppressoractivity.
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3 Isolated IgAdeficiency
Itisthemostcommonofalltheprimaryimmunodeficiencydiseases,affectsabout1in700whiteindividuals:
• IgA deficiency leads to defective mucosal immunity; predispose patients to recurrent sinopulmonary infections and diarrhea.
Thereisalsoasignificant autoimmunediseases.
• Pathogenesis is due to a block in the terminal differentiation of IgA-secreting B-cells to plasma cells, which in turn is due to alteredT-cell
production of cytokines that drive IgA responses (e.g. TGF-β and IL-5) or due to intrinsic B-cell defect. The levels of
otherimmunoglobulinsareusuallynormalorevenexcess.
4 Hyper-IgMsyndrome
Hyper-IgMsyndromeisanX-linkeddisorder;resultsduetoadefectinisotypeclassswitchoverofB-
cells,duetomutationsineitherCD40LorCD40genes;preventtheT-andB-cellinteraction;neededforclass switchover:
• HyperIgMlevelswithlackofsynthesisofotherclassesofantibodies
• LackofotherclassesofIgleadstosecondaryinfection,autoimmunediseases
5 Transienthypogammaglobulinemiaofinfancy
ThisoccursduetoanabnormaldelayintheinitiationofsynthesisIgG(orsometimeIgAorIgM):
• IgG synthesisusuallystartsby2 monthsofage.Butinsome infants,itisdelayedleading todefectinopsonizationorcomplementactivationresulting
inrecurrentotitismedia andrespiratoryinfections.
• However,spontaneousrecoveryoccursusuallyby18–24monthsofage.
• Interestingly,theseinfantsshowanormalantibodyresponseagainstvaccines.
Cellularimmunodeficiencies(T–celldefects)
1 DiGeorgesyndrome(Thymichypoplasia)
ItresultsfromacongenitaldefectinthymicdevelopmentleadingtodefectinT-cellmaturation:
• Infantsareextremelyvulnerabletoviral,fungal,intracellularbacterialandprotozoaninfections.
• Pathogenesis:In90%ofcases,thereoccursadeletionaffectingchromosome22q11→leadstomalformationofthirdandfourth
pharyngealpouchesinembryoniclife
• Asaresult,allthestructuresthatdevelopfromthirdandfourthpharyngealpouchessuchasthymus,parathyroidglands,andportionsofthe face
andaorticarchbecomedefective.
• Thus,inadditiontothethymicdefects,theremaybeassociated:
○ Parathyroidglandhypoplasiaresultinginneonataltetanyandhypocalcemia
○ Anomaliesoftheheartandthegreatvessels(Fallot’sTetralogy).
○ Characteristicfacialappearance
• B-cellsandserumimmunoglobulinlevelsaregenerallyunaffected.
• Treatment:Thymustransplantation
2 Chronicmucocutaneouscandidiasis
ItrepresentsanimpairedCMIagainstCandidaalbicansleadstosuperficialinfectionsoftheskin,mucosaandnails:Thereisno↑riskofinfectio
nsbutitisoftenassociatedwithendocrinopathiesandautoimmunedisorders.
Transferfactortherapy,alongwithamphotericinBhasbeenreportedtobeeffective.
3 Purine nucleoside phosphorylase (PNP) deficiency
Itisarareautosomalrecessivedisorder(chromosome14),characterizedbydeficiencyofanPNPenzyme:
• PNPisakeyenzymerequiredforpurinedegradation;catalyzestheconversionofguanosinetohypoxanthine.
• Itsdeficiencyleadstoelevateddeoxy-GTPlevelsresultinginT-celltoxicity.However,B-cellsarenotaffected.
• T-celldepletionpredisposestoincreasedsusceptibilitytoinfectionandautoimmunedisorders.
Combinedimmunodeficiencies(BandT-celldefects)
1 Severecombinedimmunodeficiencies
SCIDrepresentsgroupsofgeneticallydistinctsyndromes;allhavingincommondefectsinbothhumoralandCMI.
• TypesofgeneticdefectinSCIDinclude:
○ Mutation in cytokine receptors (IL-7 mainly and others like IL-2, IL-4, IL-9, and IL-15)- MC type defect(50–60%), X-linked,seen
inmales
Section
○ TheremainingcasesofSCIDareinheritedasautosomalrecessivemanner;include:
▪ Adenosinedeaminase(ADA)deficiency
▪ Recombinase-activatinggenes(RAG)mutation
▪ Jak3 (intracellular kinase) mutation
▪ ClassIIMHCdeficiency(barelymphocytesyndrome)
• Infections:Affectedinfantsaresusceptibletosevererecurrentinfectionsbyawidearrayofpathogens
• Treatment:Bonemarrowtransplantationandgenetherapyreplacingthemutatedgenes.
2 Wiskott-Aldrichsyndrome
ItisanX-linkedrecessivedisease,characterizedbyimmunodeficiencywiththrombocytopenia,eczema:
• TheseverityofWASincreaseswithage.
• ItfirstmanifestsitselfbydefectiveresponsestobacterialpolysaccharidesandbylowerIgMlevels.IgGlevelsareusuallynormal.
ParadoxicallythelevelsofIgAandIgEareoftenelevated.
• OtherTandBcellresponsesarenormalinitially,butwithincreaseofage,therearerecurrentbacterialinfectionsandagraduallossofhumoralandcellular
responses.
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• PatientsarealsopronetodevelopnonhodgkinB-celllymphomas.
• Patientsmaypresentwithbloodydiarrheasecondarytothrombocytopenia.
• Pathogenesis:MutationinthegenecodingWASproteinpresentinprecursorlymphoidcellsofbonemarrow.Itisacytoskeletalglycoprotein(sialophorin
orCD43),required for actinpolymerization.
3 Ataxiatelangiectasia:Thesyndromeischaracterizedby:
• Difficultyinmaintainingbalancewhilewalking(cerebellarataxia)
• Appearanceofbrokencapillaries(telangiectasia)intheeyesandchoreoathetoidmovements(ininfancy).
• DeficiencyofIgAandsometimesIgE
• Profoundsinopulmonaryinfections
Theprimarydefectappearstobeinakinaseinvolvedinregulationofthecellcycle. Therelationshipbetweentheimmunedeficiency
andtheotherdefectsinataxiatelangiectasiaremainsobscure.
4 Nezelofsyndrome
Itisanautosomalrecessiveconditioncharacterizedbycellularimmunodeficiencyresultingfromthymushypoplasia:
• Insomepatients,B-cellsarenormal,whereasinothersaB-celldeficiencyissecondarytotheT-celldefect.
• Affectedindividualssufferfromchronicdiarrhea,viralandfungalinfections,andageneralfailuretothrive.
Disordersofphagocytosis
1 Chronicgranulomatousdisease
Pathogenesis involves inherited defects in the gene encoding NADP oxidase of phagocyte which breaks down hydrogen peroxide
→leadstodecreasedoxidativeburstwhichpredisposesto(i)recurrentcatalaseproducingbacterialinfections,(ii)↑inflammatoryreactionsthatresult
ingingivitis,swollen lymphnodes,etc.
CGDisageneticdiseasethatrunsinfamilyintwoforms:
• InX-linkedform(morecommon,70%):membranecomponentofphagocyteoxidaseisdefective.
• Inautosomalrecessiveform:cytoplasmiccomponentofphagocyteoxidaseisdefective.
• Nitrobluetetrazoliumreductiontest(NBT)ispositive
2 Myeloperoxidase deficiency
Deficiencyineitherquantityorfunction,ofmyeloperoxidase,anenzymeproducedbyneutrophils.Patientspresentwithimmunedeficiencyandrecurrentinfecti
ons,especiallywithCandidaalbicans.
3 Chediak-Higashisyndrome:Itisanautosomalrecessivedisease,characterizedby:
• Defectivefusionofphagosomesandlysosomesinphagocytes→leadsto↑susceptibilitytopyogenicinfections
• Abnormalitiesinmelanocytesleadingtoalbinism(lackofskinandeyepigment)
• Abnormalitiesincellsofthenervoussystem(associatedwithnervedefects)
• Plateletsabnormalities,causingbleedingdisorders
• Aggressivebutnonmalignantinfiltrationoforgansbylymphoidcells.
Pathogenesis:DuetoamutationinaproteincalledLYSTwhichisbelievedtoregulatelysosomaltrafficking:
• Themutationimpairsthetargetingofproteinstosecretorylysosomes,whichmakesthemunabletolysebacteria.
• Phagocytescontaingiantgranulesbutdonothavetheabilitytokillbacteria.
4 Leukocyte adhesion deficiency is rare autosomal recessive disorder, characterized by a defect in the adhesion of leukocytes
whichresultsinpoorleukocytechemotaxisparticularlyneutrophil,inabilitytoformpusandneutrophilia.Thusitpredisposestovariousbacterialandfungal
infections.LADisoftwo types.
• LAD1:Mutationsinβ2integrinsubunit(CD18),oftheleukocytecelladhesionmolecule(inchromosome21).
• LAD2:Mutationsinfucosyltransferaserequiredforsynthesisofsialylatedoligosaccharide
5 Lazyleukocytesyndrome
Itisanidiopathicconditionduetodefectinneutrophilchemotaxiswhichresultsinincreasedpyogenicinfectionssuchasgingivitis,abscessformation,
pneumoniaandneutropenia.
6 Job’ssyndromeorHyper-
Immunolog
IgEsyndrome:Rare,characterizedbyeczema,recurrentstaphylococcalskinabscesses,recurrentlunginfections(pneumatocele),
eosinophiliaandhighserum levelsofIgE.
Mechanism:Duetoadefectinneutrophilchemotaxis.Mostcasesaresporadic,butsomefamilialcaseshavealsobeenreportedwhichmaybe:
• Autosomaldominantcases:LinkedtomutationsintheSTAT3gene
• Autosomalrecessivecases:DuetomutationsinDOCK8gene.
7 Tuftsindeficiency
Tuftsinisatetrapeptide(Thr-Lys-Pro-Arg)producedprimarilyinthespleen,bythecleavageoftheFc-portionoftheheavychainofIgG.
Itstimulatesphagocytosis.Tuftsindeficiencyresultsin↑susceptibilitytocapsulatedorganisms.
8 Shwachman’sdisease
Itisararecongenitaldisordercharacterizedbyneutropenia,exocrinepancreaticinsufficiency,bonemarrowdysfunction,skeletal
abnormalities,andshortstature
Disordersofcomplement(Describedinchapter2.2)
1 Complementcomponentdeficiencies
2 Complementregulatoryproteindeficiencies
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Table2.5.3:Riskofsecondaryinfectionsinvariousimmunodeficiencies
Pathogen T-celldefect B-celldefect Granulocyte defect Complementdefect

Bacteria Bacterial sepsis Streptococci, Staphylococci, Neisseria,
Staphylococci,Haemophilus Pseudomonas Toxoplasma
influenzae Nocardia Otherpyogenic
Infections
Viruses CMV,EBV,Severevaricella, Enterovirusencephalitis -
Chronicinfectionswith
respiratoryandintestinalviruses
Fungi Candida,Pneumocystisjirovecii - Candida,Aspergillus
Parasites - Giardiasis -
Special features Aggressivediseasewith Recurrentsinopulmonary -
opportunisticpathogens,failure infections,Sepsis,chronic
toclearinfections meningitis
TRANSPLANTIMMUNOLOGY
Basedonthegeneticrelationshipbetweenthedonorandtherecipient:
• Autograftisself-
tissuetransferredfromonepartofthebodysitetoanotherinthesameindividual,e.g.skingrafts
• Isograftorsyngeneicgraftistissuetransferredbetweengeneticallyidenticalindividuals(e.g.mo
nozygotictwins)
• Allograftistissuetransferredbetweengeneticallynon-
identicalmembersofthesamespecies(e.g.mosttransplants)
• Xenograftistissuetransferredbetweendifferentspecies(e.g.thegraftofababoonheartintoama
n).
Table2.5.4:Typesofgraftrejection
Graftrejection Timetakenforrejection Immunemechanismsinvolved
Hyperacute Minutestohours Preformedantibodies(AntiABOand/oranti-HLA)
Acute Weekstomonths CytotoxicT-cellmediated
Immune mechanisms of graftrejection:Chronic Months to years ChronicDTHmediatedandAntibodymediated
Hyperacute:PreformedAb
mediated Second set rejection of the graft is always faster than the first set rejection, i.e. if, in a
Acute:Tccellmediated recipientthat has rejected a graft by the first set response, another graft from the same
Chronic:DTHmediatedandAb mediated.
donor istransplanted,itwillberejectedinanacceleratedfashion.
MechanismofGraftRejection
Theprocessofgraftrejectioncanbedividedintotwostages:
1. Sensitization phase: Involves alloantigen (mainly graft MHC molecules) presentation
Section
torecipient’sT-cellseitherbydirectorindirectpathways:
• Direct pathway: The MHC molecules on graft’s APCs are directly presented to
therecipient’shelperT-
cells.Thispathwayisresponsibleformostoftheacutegraftrejectionsmediatedbycytotoxic
T-cells.
• Indirect pathway: This is similar to that for recognition of any foreign antigen by
thehost APCs. The graft alloantigens are processed and presented by recipient
APCsto recipient’s helper T-cells. This pathway is responsible for most of the
chronicrejectionmediatedbyhelperT-
cellsviaspecializedformofchronicDTHreaction.
2. Effector phase: This involves a variety of effector mechanisms leading to
immunedestructionofthegraftsuchas:
• Cell-mediatedreactionsinvolvingdelayed-typehypersensitivityT-
cellsandcytotoxicT-cells.
• Antibodymediatedmechanisms:ComplementmediatedlysisandADCC.
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LaboratoryTeststoDetermineHistocompatibility
Priortotransplantation,variouslaboratorytestsshouldbecarriedouttoassessthehistocompatibil
ity:
• ABObloodgroupcompatibilitytestingbybloodgroupingandcrossmatching.
• Immunosuppressivetherapy
• HLAtyping:Inthistest,donor’santigensexpressedonthesurfaceofleukocytesortheirgene
to that of recipientare matched. The HLAcompatibility is determined by:
○ Phenotypic method: Microcytotoxicity (serology) and Mixed lymphocyte reaction
(tissuetyping)
○ Genotypicmethods,suchas:
▪ PCR detecting HLAgenes and PCR-RFLP
▪ Variable number tandem repeat (or VNTR) typing and STR (Short tandem repeat) typ-
ing
▪ DNASequence based typing and Karyosomeanalysis.
Graft-Versus-HostReaction
GVH reaction is a condition, where graft mounts an immune response against the host
(i.e.recipient)andrejectsthehost,incontrarytotheusualsituationwheretherecipientmountsan GVHreactionoccurswhen:
immuneresponseagainstthegraftantigens. The graft must containimmunocompetentT-cells
The recipient should possesstransplantation antigens thata
GVHreactionoccurswhenthefollowingthreeconditionsarepresent: The recipient may beimmunologicallysuppressed.
• ThegraftmustcontainimmunocompetentT-
cells(e.g.stemcellsorbonemarroworthymustransplants)
• Therecipientshouldpossesstransplantationantigensthatareabsentinthegraft.
• The recipient may be immunologically suppressed and therefore cannot mount
immuneresponseagainstthegraft.
GVH reaction occurs in two forms. Acute or fulminant (occurs < 100 days) and chronic
GVHdisease(occursafter100days)
• The acute GVH disease is characterized by selective damage to the liver
(hepatomegaly),skin (rash), mucosa, and the intestine (diarrhea) mediated graft’s
immunocompetentT-cell.
• ChronicGVHdiseasealsoattackstheaboveorgans,butinaddition,itcausesdamagetothecon
nectivetissuesandexocrineglands.
• Experimentally,GVHreactioncanbeproducedinmice,calledRuntdisease.
• Treatment:Glucocorticoids.
TumorAntigens
Twotypesoftumorantigenshavebeenidentifiedontumorcells:
1. Tumor-specific transplantation antigen(TSTA): Tumor-associated antigens(TATAs): Immunolog
Theyarepresentonlyontumorcellsandareabsentinnormalcells. Oncofetalantigens
Carcinoembryonic antigen(CEA)
TSTAareinducedontumorcellseitherbychemicalorbyphysicalcarcinogens,andalsobyvir
Carbohydrateantigens(CA
alcarcinogens. 125,CA19-9)
• Inchemically/physicallyinducedtumors,theTSTAistumorspecific.Different Prostatespecificantigenand
2macroglobulin.
tumorspossessdifferentTSTA,eventhoughinducedbythesamecarcinogen.
• Incontrast,theTSTAofvirusinducedtumorsisvirusspecific;alltumorsproduced
byoneviruswouldpossessthesameantigen.
2. Tumor-
associatedantigens(TATAs):Inadditiontotumorcells,theymayalsobeexpressedbynorm
alcellsbutataverylowlevel,e.g.Oncofetalantigens,Carcinoembryonicantigen(CEA),Carbohyd
rateantigens(CA125,CA19-9),prostatespecificantigenandβ2macroglobulin.
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IMMUNOPROPHYLAXIS
Table2.5.5:Characteristicsofkilledandlivevaccines
Characteristic KilledVaccine LiveVaccine
Numberofdoses Multiple Single*
Needforadjuvant Yes No
Durationofimmunity Shorter Longer
Effectivenessofprotection Lower Greater
Mimicsnaturalinfection Lessclosely More closely
Immunoglobulinsproduced IgG IgAandIgG
Mucosalimmunity Absent Induced
Cell-mediatedimmunity Poor Induced
Reversebacktovirulentform No Possible
Feco-oral spread No Possible
Interferencebyothermicroorganismsinhost No Possible
Stabilityatroom temperature High Low
Immunodeficiencyandpregnancy Safe Unsafe
*ExceptionisOPV,whichisgiveninmultipledosesatspacedintervalstoachieveeffectiveimmunit
y
Table2.5.6:Examplesofvaccines
LiveVaccines Killed/Inactivatedvaccine
Bacterial Viral Bacterial Viral
BCG vaccine Oralpoliovaccine(OPV,Sa Typhoidvaccine Injectablepoliovaccine(IPVorSalk
bin vaccine) vaccine)
Typhoral Liveattenuatedinfluenza Choleravaccine Killedinfluenzavaccine
vaccine
Epidemict Yellowfever17Dvaccine Pertussis vaccine Rabiesvaccine
yphus
Chickenpoxvaccine Plague vaccine HepatitisA
Japanese B Japanese B
encephalitis(14-14-2 encephalitis(Nakayama
strain) strain)
Measles vaccine Combinedvaccine
Mumps vaccine Bacterial Viral
Rubellavaccine DPTvaccine MMR vaccine
(mumps,measles, rubella)
Section
HepatitisAvaccine Pentavalentv
accine
(DPT+Hib+Hep
B)
Rotavirusvaccine Toxoidvaccine Subunitvaccine
DT HepatitisB
(Diphtheriatoxo
id)
Meningococcal,Pneumo TT HPV(Humanpapillomavirus)vac
coccal,Haemophilusinfl (Tetanustoxo cine
Cellularfraction uenzaetypeb(Hib) id)
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Table2.5.7:NationalImmunizationSchedule(NIS)forinfants,childrenandpregnantwomen(India)
Vaccine When to give Dose Route Site
ForPregnantWomen
TT-1 Earlyinpregnancy 0.5ml Intramuscular Upperarm
TT-2 4weeksafterTT-1* 0.5ml Intramuscular Upperarm
TT-Booster Ifreceived2TTdosesinapregnancywithint 0.5ml Intramuscular Upperarm
he last3years*
For Infants
BCG Atbirthorasearlyaspossibletilloneyearofage 0.1ml(0.05ml Intradermal Leftupperarm
until1monthage)
HepatitisB Atbirthorasearlyaspossiblewithin24hours 0.5ml Intramuscular Anterolateral side of
midthigh
OPV-0 Atbirthorasearlyaspossiblewithinthe 2drops Oral Oral
first15days
OPV1,2and3 At6weeks,10weeksand14weeks 2drops Oral Oral

DPT1,2and3 At6weeks,10weeksand14weeks 0.5ml Intramuscular Anterolateral side of
midthigh
HepatitisB1,2and3 At6weeks,10weeksand14weeks 0.5ml Intramuscular Anterolateralsideofmidthigh
Measles 9completed months-12 months (give upto 0.5ml Subcutaneous Rightupperarm
5yearsifnotreceivedat9-12monthsage)
VitaminA(1st dose) At9months withmeasles 1ml(1 lakhIU) Oral Oral
ForChildren
DPTbooster 16–24months 0.5ml Intramuscular Anterolateralsideofmidthigh
OPV Booster 16–24months 2drops Oral Oral
Measles(2nddose) 16–24months 0.5ml Subcutaneous Rightupperarm
Japanese Encephalitis** 16–24monthswithDPT/OPVbooster 0.5ml Subcutaneous Leftupperarm
VitaminA*** 16 months with DPT/OPV booster. 2ml(2 lakhIU) Oral Oral
(2ndto9thdose) Thenonedoseevery6monthsuptotheageof5years
DPTBooster 5–6years 0.5ml Intramuscular Upperarm

TT 10yearsand16years 0.5ml Intramuscular Upperarm
* Give TT-2 or Booster doses before 36 weeks of pregnancy. However, give these even if
morethan36weekshavepassed.GiveTTtoawomaninlabor,ifshehasnotpreviouslyreceivedTT.
**SA14-14-
2Vaccine,inselectedendemicdistrictsofUttarpradesh,Karnataka,WestBengalandAssam
***The2ndto9thdosesofVitaminAcanbeadministeredtochildren1–5yearsold.
Table2.5.8:Passiveimmunoprophylaxis Immunolog
Immunoglobulin preparations Source Indications
Diphtheriaantitoxin Equine Treatmentofrespiratorydiphtheria
Tetanusimmuneglobulin(TI Equine,Human TreatmentoftetanusasPEP,forpeoplenotadequatelyimmunizedwithtetanustoxoid
G)
Botulinumantitoxin Equine,Human Treatmentofbotulism
Varicella-zosterimmuneglobulin(VZIG) Human PEPforimmunosuppressedcontactsofacutecasesornewborncontacts
CMV-IG Human PEPinhematopoieticstemcellandkidneytransplantrecipients
Rabiesimmuneglobulin(RIG) Equine,Human TreatmentofrabiesandPEPinpeoplenotpreviouslyimmunizedwithrabiesvaccine
Contd...
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Contd...
Immunoglobulin preparations Source Indications

HepatitisBimmuneglobulin(HBIG) Human PEPfor-PercutaneousormucosalorsexualexposureandNewbornofmotherwithHBsAg+ve
HepatitisA Human PostexposureprophylaxisforFamilycontactsandTravelers

immuneglobulin(HAIG)
Rubella Human Womenexposedduringearlypregnancy
Measles Human Infantsorimmunosuppressedcontactsofacutecasesexposed<6dayspreviously
Rhisoimmunization(RhIG) Human Treatment–

Rh-vemotherondeliveryofaRh+vebaby
Section
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NATIONALIMMUNISATIONSCHEDULE 8. Chronicgranulomatousdisorderisduetodefectin:
(NEETPatternBased)
1. Whichvaccinecanbegiventopregnantwomen: a. B-cell b. NADPHoxidase
a. HepatitisB (RecentQuestion2013,PGIDec2006) c. IgA d. T-cell
b. Meningococcus 9. PurineNucleoside phosphorylasedeficiency:
c. Rabies (NEETPatternBased)
d. Measles a. Humoralimmunitydeficiency
e. BCG b. Acquiredimmunitydeficiency
2. According to national immunisation schedule, c. SCIDs
whichof the following is recommended for a child of d. Cellmediatedimmunitydeficiency
5-yearof age? (AIMSNov2013) 10. DecreasedIgM,bleedingtendencywitheczemais
a. PentavalentvaccineandvitaminA seenin: (AI2012)
b. DTbooster a. Wiskott-Aldrichsyndrome
c. DT, OPV andvitamin A b. Chronicgranulomatousdisease
d. DPTboosterandvitaminA c. Job’ssyndrome
3. Liveattenuatedvaccine: (RecentQuestion2015) d. ChediakHigashisyndrome
a. Mumps b. Hepatitis B 11. In a 5-year-old boy who has history of
c. Salkvaccine d. Typhoral pyogenicinfectionsbybacteria withpolysaccharide-
richcapsules,whichofthefollowinginvestigationsshou
AUTOIMMUNITY ldbe done? (AI2012,AIIMSMay2012)
a. IgAdeficiency
b. IgG1deficiency
4.Whichofthefollowing is a immunologically c. IgG2deficiency
privilegedsite? (AIIMSNoc2015) d. IgAandIgG2deficiency
a. Seminiferoustubule 12. Adenosinedeaminasedeficiencyisseeninthefollowi
b. Areapostrema ng: (NEETPatternBased)(AI2005,2001)
c. Opticnerve a. Commonvariableimmunodeficiency
d. Loopofhenle
5. Whichisnotanautoimmunedisorder? (TNPG2015) b. Severecombinedimmunodeficiency
a. Myastheniagravis b. Sicklecell anemia c. Chronicgranulomatousdisease
c. Graves’disease d. SLE d. Nezelofsyndrome
6. Autoimmunitycanbecausedduetoallofthefollowing 13. Thecommonestprimaryimmunodeficiencyis:
except: (AIIMSMay2005) (PGIJune2005)
a. Thepressureofforbiddenclones a. Commonvariableimmunodeficiency
b. Expressionofcrypticantigens b. IsolatedIgAimmunodeficiency
c. NegativeselectionofT-cellsinthethymus c. Wiskott–Aldrichsyndrome
d. Inappropriate expression ofthe MHC proteins d. AIDS
Immunolog
14. WhichisfoundinDiGeorge’ssyndrome (PGI2001)
a. Tetany
IMMUNODEFICIENCY
b. Eczema
7. All are true about severe combined c. MucocutaneousCandidiasis
immunodeficiencyexcept: (PGIMay2015) d. AbsentB-andT-cells
a. B & T cell deficiency e. TotalabsenceofT-cells
b. Adenosinedeaminasedeficiencymayoccur 15. Disordersofphagocytosisareallexcept:
c. Affectedchildcansurvivesbeyondadolescencewitho a. Job'ssyndrome (PGIMay2013)
uttreatment b. Chediak-Hegashisyndrome
d. CantransmiteitherasX- c. Myeloperoxidase deficiency
linkedorautosomalrecessivedefect
d. Wiskott-AldrichSyndrome
e. Personsusceptibletorecurrentandsevereinfections
e. Tuftsindeficiency
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TRANSPLANTATIONIMMUNOLOGY 18. Allograftrejectionisanexampleof: (JIPMER2014)

a. GVHD
b. Delayed(cellmediated)hypersensitivity
16. A woman with infertility receives an ovary c. Immediatehypersensitivity
transplantfrom her sister who is an identical twin. d. Acuterejection
What type ofgraftis? (AI2005) 19. Skin transplant was done from sister to brother.
a. Xenograft b. Autograft Afterfew years, brother to sister skin transplant was
c. Allograft d.Isograft done,butrejectionoccurred.Whatisthisphenomenacalle
17. Hyperacutegraftrejectionisseenin: d?
a. Bonemarrow (RecentMCQ2013) a. Schwartzmanreaction (RecentMCQ2013)
b. Kidney b. TheobaldSmithphenomena
c. Liver c. EichwaldSilmsereffect
d. Heart d. SchultzDalephenomena
Section
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EXPLANATIONS
NATIONALIMMUNISATIONSCHEDULE
1. Ans.(a),(b),(c)(HepatitisB,Meningococcus,rabies)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep205Park22/
ep98/,21/ep98
AlllivevaccinesuchasMeaslesandBCGarecontraindicatedinpregnancy
2. Ans.(d)(DPTboosterandvitaminA)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep205,Park22/
ep115AccordingtonationalimmunizationscheduleinIndia:
• DPTschedule:1st/2nd/3rddoseat6/10/14weeksandtwoboostersat16–24monthsand5–6yr
• VitaminA:1stdoseat9month(alongwithmeasles),2nddoseat16–
24months(alongwithDPTbooster)andthe3rdto9thdosegivenevery6monthtill5yrs.
• OPVschedule:Zerodoseatbirth,then1st/2nd/3rddoseat6/10/14weeksandboosterat16–24months.
3. Ans.(d)(Typhoral)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep203
AUTOIMMUNITY
4. Ans.(a)(Seminiferoustubule)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep184
Seminiferoustubule,spermatozoa,lensprotein,placentaandthefetusaretheimmunologicallyprivilegedsite.Brainisnolongerc
onsideredimmunologicallyprivileged.
5. Ans.(b)(Sickelcellanemia)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep185Referchapterreviewofchapter2.5.
6. Ans.(c)(NegativeselectionofT-cellsinthymus)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep150,Har-rison17/
ep2071-2073
NegativeselectionofT-cellisaprocessbywhichselfreactiveT-cellsarekilledinthymus–
thisisoneofthemechanismofpreventingdevelopmentofAutoimmunity.
TheotheroptionsarethemechanismsofpreventingAutoimmunity:
• ForbiddencloneswithselfreactivityariseasaresultoffailureofnegativeselectionofautoreactiveT-cell.
• Crypticantigensaretheantigenicepitopes,whicharenormallynotexposed,getsexposedtotheimmunesystemasaresultofi
njuryorinfectionandleadstodevelopmentofimmuneresponse.
• InappropriateexpressionofMHCproteinsalsoleadstodevelopmentofself-destructiveimmuneresponse.
IMMUNODEFICIENCY
7. Ans.(a,b,d,e)
(B&Tcell..,Adenosinedeaminase..,Cantransmit..,Personsusceptible..)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology
Immunolog
1/ep190
InSevereCombinedImmunodeficiencies(SCID),bothCMIareAMIareaffected.Itrunsas:
• Xlinked:Mutationincytokinereceptor
• Autosomalrecessive:Itoccursas:(i)Adenosinedeaminase(ADA)deficiency,(ii)Jak3mutation,
(iii)RAGmutationand(iv)ClassIIMHCdeficiency
• Theaffectedinfantsaresusceptibletosevererecurrentinfectionsbyawidearrayofpathogens,includingCandida,Pneumoc
ystis,cytomegalovirusandPseudomonas.
• PrognosisofSCIDispoor.Bonemarrowtransplantationisthemainstayoftreatment.
8. Ans.(b)(NADPHoxidase)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep191,Ananthanarayan9/ep172
Chronicgranulomatousdisease:
• Duetodefectinphagocytosis (resultsfromabsenceof NADPHoxidase)
• ↑Recurrentcatalase+vepyogenicinfection(catalase–veorganismsarehandlednormally)
• Screeningtestused-Nitrobluetetrazolium(NBT)reductiontestisnegative.
9. Ans.(d)(Cellmediatedimmunitydeficiency)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep190
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• Purine Nucleoside phosphorylase deficiency is an autosomal recessive inherited trait, leads to low CMI &
↑Recurrentinfection(Candidiasis),hypoplasticanemia&↓uricacid.
10. Ans. (a) (Wiskott-Aldrich syndrome) Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep190,Ananthanarayan9/
ep172,8/ep158,Harrison17/ep2060
Wiskott-Aldrichsyndrome(ReferChapterreviewfordetail)
11. Ans.(c)(IgG2deficiency)Ref: Apurba Sastry’sEssentials of MedicalMicrobiology 1/e p188,Jawetz 25/e p130.
• IgG2subclassisthepredominantantibodyraisedagainstthepolysaccharidecapsularantigensanddeficiencyofIgG2subcla
ss is commonly associated with recurrent pyogenic infection due to bacteria with polysaccharide capsule like
Str.pneumoniaeorH.influenzae.
Accordingtothememoryrecallofsomeotherstudents….
Thequestionaskedas-'Boywithhistoryofrecurrentsinopulmonaryinfectionsbybacteriawithpolysaccharide-richcapsules'.
• Forthisquestion…theanswercouldbe.'BothIgG2&IgAdeficiency'
• IgAdeficiency:Dueto historyofrecurrentsinopulmonaryinfections
○ IgAisthemajorimmunoglobulininmucosalsecretionsandweakenedmucosaldefensespredisposepatientstorecurre
ntsinopulmonaryinfectionsanddiarrhea… Robins7/ep144-147
• IgG2deficiency:Duetoinfectionsbybacteriawithpolysaccharide-richcapsules.
12. Ans.(b)(Severecombinedimmunodeficiency)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep190,Anan-
thanarayan9/ep174,8/ep158.
• Severecombinedimmunodeficiencyresultsfromvariousmechanisms:MCmechanismisX-
linked(mutationinIL7receptor).OthermechanismincludeAdenosinedeaminase(ADA)deficiency.
13. Ans.(b)(IsolatedIgAimmunodeficiency)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep189,Anan-
thanarayan9/ep173,8/ep158andHarrison17/ep2058
• Amongimmunodeficiencydiseases,primaryimmunodeficiencyisrelativelyuncommon,whencomparedtosecondaryimmuno
deficiency(common).
• ThemostcommonisisolatedIgAdeficiency,occursinapproximately1in600individuals(inEuropeandNorth
America)anditisreportedinabout0.2%ofnormalpopulations.
• Nextmostcommon disorder:Common variableimmunodeficiency, characterizedbypan hypogammaglobulinemia.
• Bothoftheseimmunodeficiencystatesoftenbecomeclinicallyevidentinyoungadults.
• Themoresevereformsofprimaryimmunodeficiencyarerelativelyrare,havetheironsetearlyinlife,andfrequently
resultindeathduringchildhood.
14. Ans.(a),(c)(TetanyandMucocutaneousCandidiasis)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
15. Ans.(d)(Wiskott-AldrichSyndrome)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep190Referchapterreviewforthedetaillistofdisordersofphagocytosis.
TRANSPLANTATIONIMMUNOLOGY
16. Ans.(d)(Isograft) Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep193,Ananthanarayan9/ep183,8/
ep178Referchapterreviewfordetail.
Section
17. Ans.(b)(Kidney)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep194,Ananthanarayan9/ep184,166
• Hyperacutegraftrejectionisseeninkidneyandskingraftingandispreformedantibodymediated(TypeIIIHypersensitivity
reaction)
18. Ans.(b)(Delayed)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep180,Ananthanarayan9/ep184
19. Ans.(c)(EichwaldSilms......)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep194,Ananthanarayan9/ep185
• Whiletransplantsbetweenmembersofahighlyinbredstrainofanimalsaresuccessful,anexceptionisseenwhenthedonorisamale
andtherecipientafemale.
• Suchgraftsarerejectedasthegraftedmaletissue(XY)willhaveantigensdeterminedbytheYchromosomewhichwillbeabse
ntinthefemale(XX)recipient.
• Graftsfromthefemaletothemalewillsucceed.
• ThisunilateralsexlinkedhistoincompatibilityisknownastheEichwald-Silmsereffect.
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SECTION 3
SystemicBacteriology
CHAPTEROUTLINE
3.1 Staphylococcus
3.2 Streptococcus,EnterococcusandPneumococcus
3.3 NeisseriaandMoraxella
3.4 CorynebacteriumandBacillus
3.5 Anaerobes:ClostridiumandNon-SporingAnaerobes
3.6 Mycobacteria
3.7 Enterobacteriaceae(E.Coli,Klebsiella,Proteus,Shigella,Salmonella,Yersinia)
3.8 Vibrio
3.9 PseudomonasandOtherNonfermentersandHaemophilus,Bordetella,Brucella(HBB)
3.10 Spirochetes
3.11 Rickettsia,ChlamydiaandMycoplasma
3.12 MiscellaneousBacteria
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CHAPTER
Staphylococcus 3.1
Grampositivecoccicanbeclassifiedinto:MicrococcaceaeandStreptococcaceaeFamily
Family Micrococcaceae family Streptococcaceae family
Examples Staphylococcus,Micrococcus Streptococcus,Pneumococcus,Enterococcus
Catalase Catalasepositive Catalasenegative
ArrangementinGram Stain GPC in cluster GPCinchain(Streptococcus)
(Staphylococcus)GPCintetrad GPC in pair (Pneumococci, Enterococci)
(Micrococcus) (ascelldivisiontakesplaceinsingleplane)
(ascelldivisiontakesplaceinmultipleplanes)
Grampositivecocci
Catalasetest
Catalasetestdifferentiates
Staphylococcus (positive)
Streptococcus (negative)
Catalase+ve(staph/micrococcus) Catalase–ve(strepto/enterococcus/pneumococcus)
Hemolysis
Staph. Micrococcus
OF test Fermentative Oxidative β-Strepto Nonhemolytic α-Pneumococcus
Furazolidone Sensitive Resistant groupAorB enterococcus str.viridans
Bacitracin Resistant Sensitive
STAPHYLOCOCCUSAUREUS
Staphylococcus aureusiscatalasepositive,coagulasepositive, facultativeanaerobe,non-motile,
non-sporingandoccasionallycapsulated. S.aureuscellwallproteins
• InGreek,Staphylemeansbunchofgrapes. ProteinA:ResponsibleforCoagglutinationreaction
• StaphylococcuswasdiscoveredbySirAlexanderOgstonandS.aureuswasnamedbyRosenba Clumping factor/Boundcoagulase:Responsibleforslide coa
ch.
VirulenceFactorsofS.aureus
Cellwallfactors Activity
Peptidoglycan Morethicker,
Conferscellrigidityandinducesinflammatoryresponse
Teichoicacid Helpsinadhesiontomucosalsurfacesandpreventopsonisation
Clumping Responsibleforslidecoagulasereaction
factor/Boundcoagulase
ProteinA Antiphagocytic,anticomplementary,chemotactic
Bindsto FcregionofIgGleavingFabregionfreetobindtoanAntigen-BasisofCoagglutinationreaction
Toxins Activity
Membraneactivetoxins
A.Hemolysins
αHemolysin Inactivatedat70°C;reactivatedparadoxicallyat100°C(duetodenaturationofaheatlabileinactivatorat100°C)Leucocidal,Cytotoxi
c,dermonecrotic,lethal
Contd...
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Contd...
Toxins Activity
βHemolysin Sphingomyelinase
LysessheepRBC,butnothumanorrabbitRBCExhibitshot-
coldphenomenon
γHemolysin Bicomponentprotein,
LysesrabbitsheepandhumanRBCs
δHemolysin Detergentlike,
Lysesrabbit,sheepandhumanRBCs
B. Two components F and
Leucocidins/Panton SDamagePMNandmacrophages
valentine (PV)toxins AssociatedwithCommunityacquiredMRSA
Synergohymenotropictoxins:Bicomponenttoxinssuchasγ andPVtoxinactsynergisticallyandarecalled
asSynergohymenotropic toxins
Other toxins
Epidermolytic MainlybelongtophagegroupII
toxin(Exfoliative Scalded skin syndrome (Nikolsky’s sign-epidermal layer separated)Severe-
toxin) Ritter disease (newborn), toxic epidermal necrolysis (TEN) (adult)Milder-
Pemphigusneonatorum,bullousimpetigo
Enterotoxins Produced by 50% of clinical
isolatesCausefoodpoisoning
IncubatoryPeriod:1-6hr dueto preformedtoxin
Siteofaction:Thetoxinstimulatesthevagusnerveandvomitingcenterofthebrain.Italsostimulatestheintestinalperistalticac
tivity.
Heatstable(notdestroyedafterheatingfood)
Mostcommonfooditemsinvolvedaremilkproducts,bakeryfood,custards,potatosalad,orprocessedmeats.
Multipleantigenictype(A–E,G–I,R-TandV)(MC-typeA)
Toxicshocksyndromet MoststrainsbelongtophagegroupI
oxin EnterotoxinsF(pyrogenicexotoxinC)isthemostcommonTSST,followedbyEnterotoxinB,C
Riskfactor:Useofvaginaltamponbymenstruatingfemales(however,malesandnonmenstruatingfemalesalsogeteffected
rarely)
AntiTSST1Antibodyisprotective
Manifestations:Rash,fever,hypotensionandMultiorganfailure
Diagnosis:DetectionofTSSTbylatexagglutinationtestandenzymeimmunoassay.DetectionofTSSTgenes1and2byPCR
Treatment:Clindamycin(reducestoxinsynthesis)
Extracellularenzymes:
• SpecifictoS.aureus:Coagulase,heatstablethermonuclease,DNase,phosphatase
• Presentinmoststaphylococci:Protease,lipase,staphylokinase(fibrinolysin),hyaluronidase
Pathogenesis
StaphylococcusaureusistheMCagentofthefollowingconditions:
• Skinandsofttissueinfections
Section
• Botryomycosis (mycetoma-like condition)

• Tropicalpyomyositis–S.aureus,(acutebacterialmyositis–GroupAStreptococcus)(Overall-S.aureus)
• Osteomyelitisandsepticarthritis(MCsite-knee)
• Postoperativeparotitis
• Paronychia
• Pyomyositis(skeletalmuscleinfection):IntropicsandHIVinfectedpeople(OverallMCagent-S.aureus,exceptinacutebacterialmyositis–
GroupAStreptococcus isthe MC agent)
• Pneumatocele-Shaggy,thin-walledcavitiesinlungs)inneonates
• Abscess:Psoasabscessandepiduralabscess
• Surgicalwoundinfection
• Folliculitis,furuncle,carbuncleandHidradenitissuppurativa
• Mastitisandbreastabscess(innursingmothers)
• Toxin-mediateddiseases:Toxicshocksyndrome,foodpoisoning,scalded-skinsyndrome
Contd...
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Staphylococcus117
Contd...
InfectionsassociatedwithCommunity:AssociatedMethicillinResistantS.aureus(CA-MRSA)
WhileskinandsofttissuesarethemostcommonsitesforCA-MRSAstrains;5–10%ofstrainsareinvasiveandcancausevariousinvasive
infections,suchas:
• Necrotizingpneumonia
• SepsiswithWaterhouse:Friderichsensyndromeorpurpurafulminans(S.aureusisrarecause;mostcommonlycausedbymeningococci).
• Necrotizingfasciitis(S.aureusisararecause,Streptococcuspyogenesisthemostcommoncause)
Endocarditis:
• MCcauseofNativevalveendocarditis:OverallorHospitalacquired-S.aureus,Communityacquired-S.viridans
• MCcauseofProstheticvalveendocarditis
○ Earlyprostheticvalveendocarditis(<12months):Staphylococcusepidermidis
○ Lateprostheticvalveendocarditis(>12months):Viridansstreptococci
○ OverallMCcauseofprostheticvalveendocarditis.Staphylococcusepidermidis
• MCcauseofEndocarditis inIV drugusers:
○ Rtsided–Staphylococcusaureus,
○ Ltsided–Enterococcus>Staphylococcusaureus
○ Overall–Staphylococcusaureus
• MCcauseofSubacuteendocarditis–Viridansstreptococci
LaboratoryDiagnosis
• Directsmearmicroscopy:Puscellswithgrampositivecocciincluster
• Culture:
○ Nutrientagar:Goldenyellowpigmentedcolonies(pigmentsmadeupofbeta
carotene)
○ Bloodagar:Colonieswithnarrowzoneofβ-hemolysis
○ Selectivemedia:
▪ Mannitolsaltagar(yellowcoloniesduetomannitolfermentation)
▪ Salt milkagar
▪ Ludlam’smedium
• Culturesmearmicroscopy:Gram-positivecocciinclusters
• Biochemicalidentification
○ Catalasetest-positive
○ Testsdifferentiatingstaphylococcifrommicrococci-Oftest(showsfermentative
pattern)
○ TestsdifferentiatingS.aureus(positive)fromCoNs(negative)
▪ Coagulasetest(slideandtube)-positive
▪ Heatstablethermonucleasetest-positive
▪ DNasetest-positive Enzymesspecificto
S.aureus(absentinCoNS):
▪ Phosphatasetest-positive Coagulase
▪ Mannitolsugarisfermented Heatstablethermonuclease
▪ Blackcoloredcoloniesonpotassiumtelluriteagar DNasetest
SystemicBacteriolog
▪ Gelatinliquefaction-positive Phosphatase test
▪ ProteinAdetection
• TypingofS.aureus:
○ MCmethodfortypingofS.aureus–Phagetyping(patternmethod)
○ Nationalreferencecentreforphagetyping—inMaulanaAzadMedicalCollege,Delhi.
○ EpidemicstrainofS.aureusisPhagetype80/81.Itcausesoutbreaksinhospitals.
Tubecoagulase Slidecoagulase
Duetocoagulaseenzyme Dueto clumpingfactor
Coagulasetest:
RequiresCRFinplasma DoesnotrequiresCRFinplasma
BothTubeandSlidecoagulasepositive:
Doneintube Doneonslide S.aureus,S.hyicusand
Test+ve:coagulumformed/plasmaclotted Test+ve:clumpsformed S.intermedius
OnlySlidecoagulasepositive:
S.lugdunensis–ve S.lugdunensis +ve S.lugdunensis
S. schleiferi +ve S.schleiferi–ve OnlyTubecoagulasepositive:
S. schleiferi
Bothtubeandslidecoagulasepositive:S.aureus,S.hyicusandS.intermedius
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TreatmentofStaphylococcusaureusInfections
SinceS.aureusrapidlydevelopsdrugresistance,antibioticsshouldbecautiouslychosen
Parenteraltherapyforseriousinfections:
Sensitivetopenicillin DOC:PenicillinG
Sensitive to methicillin DOC:Nafcillinoroxacillin
Resistant to DOC:Vancomycin(15–20mg/kgbd)
methicillin(MRSA) Alternatedrugs:Seetextbelow
Empiricaltherapy (if MRSAstatusnotyet known): Vancomycinwithorwithoutanaminoglycoside
VancomycinisindicatedonlyifMRSAriskishighorconditionisserious,e.g.cardiac
implant
Oraltherapyforskinandsofttissueinfections
Sensitive to methicillin Dicloxacillin,cephalexin
Resistanttomethicillin(MRSA) Clindamycin
Alternatedrugs:Cotrimoxazole,doxycycline,linezolid
DrugResistanceinS.aureus
ResistanceinS.aureustoβlactamantibiotics
S.aureusshowsresistancetoβlactamantibioticsinvariousways:
CommunityassociatedMRSA(CA-MRSA):
MediatedbymecAgene 1. Productionofβlactamaseenzyme:βlactamaseorpenicillinseenzymescleavethe
subtypeIV,V,VI. βlactamring:
MorevirulentandexpressPV • Thisresistanceisplasmidcoded,canbetransferredbetweenS.aureusstrains by
toxin. transduction.
Cause invasive skin and softtissue infections, such asnecrotizingfasciitis
• Itisproducedby>90%ofstrainsofS.aureus.
• Thisresistancecanbeovercomebyadditionofβlactamaseinhibitorssuchasclavulanic
acidorsulbactam.
2. ByalterationsofPBP:ItisshownbyMRSAstrains(seebelow)
MethicillinResistantStaphylococcusaureus(MRSA)
MRSAismediatedbymecAgene;whichisachromosomallycoded.Italterspenicillinbindingprotein(PBP)presenton
S.aureuscellmembranetoPBP-2a:
PBPisanessentialproteinneededforcellwallsynthesisofbacteria.βlactamdrugsbindandinhibitthisprotein,therebyinhibitingcellwallsynthesis.
ThealteredPBP2aofMRSAstrainshaslessaffinityforβlactamantibiotics;henceMRSAstrainsareresistanttoallβlactamantibiotics.
BORSAstrains(BorderlineOxacillinresistantS.aureus):Occasionallyanon-mecAgenemediatedlowlevelresistancetooxacillin is observedin some strain
ThereisanincreasingtrendofMRSArateoverlastfewdecades.Thoughitvariesfromplacetoplace,overallabout30–40%strainsofS.aureusareMRSA.
MRSArateinIndiais30-40%.ItislowestinScandinaviancountries
Section
TypesofMRSA:MRSAareeithercommunityorhospitalassociated.
CommunityassociatedMRSA(CA-MRSA) HospitalassociatedMRSA(HA-MRSA)
ItismediatedbymecAgenesubtypeIV,V,VI Itismediated bymecAgenesubtypeI,II,III
Theyareusuallymorevirulentandexpressseveraltoxinssuch Theyaremultidrugresistant(but theirvirulenceislow)
asPV toxin.
Theycauseinvasiveskinandsofttissueinfectionssuchasnecrotizingfasciitis Theycauseperioperativewoundinfectionsinhospitalsandnosocomialoutbreaks(hos
pitalstaffarethe majorcarries)
Note: CA-MRSA andHA-MRSA terminologies arebecoming artificial nowadays;as many CA-MRSAstrains have
beenisolatedinhospitalsandviceversa.
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Staphylococcus119
DetectionofMRSA
Antimicrobialsusceptibilitytest:Diskdiffusiontestcanbedonebyusingcefoxitinoroxacillindisks.
Cefoxitinistherecommendeddisktobeused.
Ifoxacillindiskisused,thencertainconditionstobemaintainedsuchas—usingmediacontaining2–4%NaCl,incubationat30°Cfor24hours.
Oxacillin screenagar: Addingoxacillin 6µg/ml andNaCl(2–4%) tothe medium.
PCRdetectingmecAgene
LatexagglutinationtestdetectingPBP-2a
TreatmentofMRSA
VancomycinisthedrugofchoiceforMRSA.
Alternate drugs include:
Teicoplanin,linezolid,quinupristin-dalfopristin,tigecycline,oritavancin
Daptomycin(forendocarditisandcomplicatedskininfections),
Mupirocin2%ointment(fornasalcarriersofMRSA)
However,evensimpleorallyeffectivedrugssuchastetracycline,erythromycinorcotrimoxazolemayalsobeeffective.These can be indicated in non-ser
Allβlactamdrugsshouldbeavoided.However,5thgenerationcephalosporins,suchasCeftobiprole,ceftaroline,
ceftolozanehaveshownsomeactivityagainstMRSA.
ResistancetoVancomycin(VRSAandVISA)
Erroneousandoveruseofvancomycinmayleadtoemergenceofresistancetovancomycin,
whichmaybeoftwotypes:
• VRSA(Vancomycin Resistant S. aureus): High grade resistance with MIC≥ 16 µg/ml
• VISA(VancomycinIntermediateS.aureus):LowgraderesistancewithMIC4-8µg/ml
• Epidemiology:VRSAisveryrare.InIndia,itisreportedfromfewplacessuchasHyderabad,Kolka
taandLucknow.However,VISAismorefrequentlyreported.Mechanisms:
○ VRSA is mediated by vangene. A The vanA gene is believedto be acquired froma
vancomycin-resistantstrainofEnterococcusfecalisbyhorizontalconjugaltransfer.
○ VISAisduetoincreaseincellwallthicknessofS.aureus.
• TreatmentofVRSA/VISA:Linezolid,telavancin,daptomycinandquinupristin/
dalfopristinaretheeffectivedrugs.VancomycinandTeicoplaninarenoteffective.
S.aureuscarriers
• About25–50%ofhealthypopulationarecarriersofS.aureus.
Resistancetovancomycin:
• MCsiteofcolonization:AnteriornaresandSkin,(perineum,axilla,groins) VRSA: MIC ≥ 16 µg/ml, Due toVangene
• MCwayofspreadofinfectioninhospital-throughthehandsofhospitalstaff VISA:MIC4-8µg/ml,Dueto
• Mosteffectivewaytopreventthehospitalinfection–handwashing cellwallthickeing
SystemicBacteriolog
• DOCfornasalcarriersofMRSA:Mupirocin2%ointment.
COAGULASENEGATIVESTAPHYLOCOCCUS(CoNS)
Theyaremostlythenormalfloraofskin.
StaphylococcusEpidermidis
• MCCoNS—Accountsfor60–70%ofCoNS
• Producespolysaccharideglycocalyx(slime)(Biofilmproduction)
• Adheretoanyimplantedforeignbodieslikevalvularshunts,prostheticdevices
• Infections:
○ Endocarditiswithinsertionofvalvularprosthesis
○ VentricularshuntinfectionsandStitchabscess.
S.epidermidisInfections:
StaphylococcusSaprophyticus Endocarditiswithinsertionofvalvularprosthesis
Ventricularshuntinfections
• It causesUTIinyoungsexually activefemales. Stitchabscess
• Itisresistanttonovobiocin.
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1. Howtodifferentiatemicrococciandstaphylococci?
10. ProteinAofStaphylococcusaureusispartofbacterial?
(RecentQuestion2015) a. Genome(RecentQuestions2014)
a. Catalase b. Cellwall
b. Oxidase c. Limitingmembrane
c. Gramstaining d. Plasmid
2. PneumatocelesinchestX- 11. SynergohymenotropictoxinsofStaphylococalconsistsof:
rayarecharacteristicallyseeninpneumoniadueto: (PGIJune2011)
(APPG2015) a. atoxin b. toxin
a. Streptococcuspneumoniae c. toxin d. dtoxin
b. Staphylococcusaureus e. Panton–Valentinetoxin
c. Streptococcuspyogenes 12. A person had infection due to gram positive
d. Hemophilusinfluenzae organismtreated with methicillin and then culture
3. ToxicepidermalnecrosisandScaldedSkinSyndromear sensitivityshows resistance to it. Hence all can be
eassociatedwithwhichtoxin? (WestBengal2016) given in MRSAexcept: (AIIMSNov2011)(AI2012)
a. ExfoliativetoxinofS.aureus a. Cotrimoxazole
b. TSSTofS.aureus b. Cefaclor
c. EnterotoxinofS.aureus c. Vancomycin
d. SPEofS.pyogenes d. Ciprofloxacin
4. SurrogatemarkerforMRSAdetectionis? 13. Mostcommoncauseofpyomyositisis:(DNBDec2011)
(RecentQuestions2014) a. Streptococcuspyogenes
a. Cefotaxime b. Ceftazidime b. Pseudomonas
c. Cephazolin d. Cefoxitin c. Staphylococcsaureus
5. AllthefollowingaretrueaboutStaphylococcusaureusexce d. E.Coli
pt: (JIPMER2014,2013) 14. Preformedtoxinproducesdiarrheainwhichorganism?
a. Coagulasepositive a. Staphylococcusaureus (DNBJune2010)
b. Catalasenegative b. Vibriocholera
c. DNAsepositive c. Salmonella
d. Indolenegative d. Escherichiacoli
6. CephalosporinwithantiMRSAactivity: 15. In a postoperative ward, 4 out of 10 patients
(AIIMSNov2014) developedwound discharge, which on culture was
a. Ceftriaxone b. Aztreonam found to
c. Cefazolin d. Ceftobiprole bepositiveforcoagulasepositivegrampositivecocci.Ant
imicrobialsusceptibilityshowedthatthestrainwas
7. Drug(s)usedinMRSAis/are: (PGINov2014) resistant to Methicillin. On surveillance cultures,a
a. Linezolid b. Cephalothin health care personnel attending to the patients
c. Vancomycin d. Meropenam wasfound to be a nasal carrier for the same agent.
e. Piperacillin + Tazobactum Thefollowingaretrueregardingtheagentresponsiblefo
Section
8. Staphaureuscauses: (NEETPatternBased) rtheoutbreak: (AI2011)

a. Erythrasma a. Themajorrouteofspreadcausingtheoutbreakisair
b. Acnevulgaris borne
c. Chancroid b. Theresistancetomethicillinisplasmidmediated
d. Bullousimpetigo c. Theorganismhasanalterationinitspenicillinbinding
9. WhatisthebestwaytocontroltheoutbreakofMRSAinfec proteins.
tioninahospitalward? d. Itwillbesensitivetotreatmentwithantibioticscontain
ingamoxicillin+clavulinicacidcombination.
(JIPMERMay2015,Nov2014,AIIMS2012)
a. Vancomycinisgivenempiricallytoallthepatients 16. Toxicshock syndrome is caused by:
b. Frequentfumigationofwards (JIPMER2011and2010,PGIDec2007)
a. Streptococcuspyogenes
c. Wearingmaskbeforeinvasiveprocedure
b. Staphylococcusaureus
d. Washing of hands before and after treating the
c. Strept.albicans
patients
d. Enterococcusdurans
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Staphylococcus121
17. FalsestatementaboutStaphylococcusepidermidis?
22. The following are characteristic features
(NIMHANS2016)
ofstaphylococcusfoodpoisoningexcept: (AIIMS2004)
a. Itissensitivetopenicillin
a. Optimumtemperaturefortoxinproductionis37°C
b. Itis Gram+ve CONS
b. Intradietic toxin is responsible for intestinal
c. Itsecretsslimelayeraroundit
symptoms
d. Itcausesinfectionthroughcathetersandcardiacimpla
c. Toxincanbedestroyedbyboilingfor30minutes
nts.
d. Incubationperiodis1-6hours
18. AllaretrueaboutPBPexcept: (AI2010)
23. Staph.aureuscausesvomitingin6–
a. PBPislocalizedinoutercellwall
8hours.Themechanismofactionis: (AIIMSMay2002)
b. PBPisessentialforcellwallsynthesis
a. StimulationofcAMP
c. PBPactsascarboxypeptidaseandtranspeptidase
b. Vagalstimulation
d. AlterationinPBP–basisofMRSA
c. StimulationofcGMP
19. All true aboutS.aureus except: (AI2010) d. ActsthroughgangliosideGMreceptor
a. MCsourceofinfection–
24. Whichofthefollowingorganismisimplicatedinthecausa
crossinfectionfrominfectedpersonsinhospital
tionofbotryomycosis? (PGI2001)
b. 30%ofgeneralpopulationishealthycarriers
a. Staphylococcusaureus
c. TSSandepidermolysinareSuperantigens
b. Staphylococcusalbus
d. MRSA-chromosomallymediated
c. Pseudomonasaeruginosa
20. Apatienthasprostheticvalvereplacementandhedevelo d. Streptococcuspneumoniae
psendocarditis8monthslater.Organismresponsibleis: e. Streptococcuspyogenes
(AIIMSNov2010)
25. MCphagetypeofStaphylococcusaureus:
a. 79/80 (RecentQuestionof2013)
b. StreptococcusViridans
b. 3A/3B
c. Staphylococcusepidermidis
c. 80/81
d. HACEK
26. A25-year-
21. A 25-year-old man with 3 weeks fever presented
oldfemalepresentedtothehospitalonthethirddayofme
withtricuspidvalvevegetation.Patientisintravenousdr
nstruationwithcomplaintsofhighfever,vomitingandar
ugabuser.Mostcommoncauseofendocarditis in
ashonhertrunkandextremities.Oninvestigationsheha
thispatientis: (AIIMSNov2009)
dleucocytosisandanegativebloodculture.Sheisdiagno
a. Staph.aureus
sedas:
b. Candidaalbicans
a. Staphylococcalfoodpoisoning (MHPG2014)
c. Pseudomonas
b. Scaldedskinsyndrome
d. Strep. Viridian
c. Toxicshocksyndrome
d. Varicellazosterinfection
SystemicBacteriolog
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EXPLANATIONS
1. Ans. (b) (Oxidase) Ref: Apurba Sastry’s Essentials of Medical Microbiology 1/e
p211Micrococciarepositiveformodifiedoxidasetest,andstaphylococciareoxidasenegative
.
2. Ans(b)(S.aureus)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep215
Pneumatoceleisshaggy,thin-walledcavitiesinlungsofneonates(commonly)andS.aureusistheMCcause.
3. Ans(a)(ExfoliativetoxinofS.aureus)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep213
ToxicepidermalnecrosisandSSSS(StaphylococcalScaldedSkinSyndrome)areassociatedwithepidermolyticorexfoliativetoxinofS.a
ureus.
4. Ans. (d) (Cefoxitin) Ref: Apurba Sastry’s Essentials of Medical Microbiology 1/e p219, CLSI Guideline
2014SurrogatemarkerforMRSAdetection:Cefoxitin(Best),OxacillinandMethicillin
5. Ans.(b)(Catalasenegative)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep212,Ananthanarayan9/ep205
6. Ans.(d)(Ceftobiprole)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep218ClinDermatol.2008;9(4):245-54.
• Fifth-generationcephalosporinssuchasceftobiproleandceftarolineareeffectiveagainstMRSA.
• Ceftobiprolehasadditionalactivityagainstpenicillin-
resistantStreptococcuspneumoniae,Pseudomonasaeruginosa,andVancomycinresistantenterococci(VRE).
7. Ans.(a)(c)(Linezolid),(Vancomycin)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep218
• MRSAstrainsareresistanttoallβlactamdrugs.
8. Ans.(d)(Bullousimpetigo)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep215Ananthanarayan9/ep202
• BullousImpetigoisacutaneousconditionthatcharacteristicallyoccursinthenewborn,presentingwithbullae.
• BullousimpetigoiscausedbyStaphylococcusaureus,whichproducesexfoliativetoxins,whereasnon-
bullousimpetigoiscausedbyeitherStaphylococcusaureus,orStreptococcuspyogenes.
9. Ans.(d)(Washingof...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiologyp609,Harrison18/ep1166,19/ep959
• Handwashingisthemosteffectivewaytopreventhospitalacquiredinfections.
• Prevention of the spread of S. aureus infections in the hospital setting involves handwashing and careful attention
toappropriateisolationprocedures…….Harrison18/ep1166.
10. Ans.(b)(Cellwall)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep212,Ananthanarayan9/
11. Ans.(c)(e)(toxin,PantonValentinetoxin)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep212
• GammahemolysinandPantonValentinetoxinaremembraneactivetoxinsofStaph.aureusthatarecomposedoftwocompo
nents (bicomponenttoxins) andtogethercalled asSynergohymenotroic toxins.
12. Ans.(b)(Cefaclor)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep218
Section
• MRSAstrainsareresistanttoallβlactamantibioticsasitresultsfromalternationofpenicillinbindingprotein.(Fordetail-
referchapterreview).
13. Ans.(c)(Staphyloco...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep215,Harrison19/ep957,18/ep1164
• Pyomyositis is an unusual infection of skeletal muscles that is seen primarily in tropical climates but also occurs
inimmunocompromised and HIV-infected patients.Pyomyositis presents as fever, swelling, and pain overlying
theinvolvedmuscle.
• Tropicalpyomyositis–MCcauseisS.aureus,(acutebacterialmyositis:MCcauseisGroupAStreptococcus)
(Overall:MCcauseofpyomyositisisS.aureus).
14. Ans.(a)(Staphy...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep213,Harrison19/ep959,18/ep1165
• Example of Preformed toxin i.e. toxin secreted in food:S.aureus enterotoxin, Bacillus cereus emetic type of
enterotoxinandbotulinumtoxin.
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Staphylococcus123
15. Ans.(c)(Theorga...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep219,Ananthanarayan9/ep205,8/ep197
• Coagulasepositivegrampositivecocci:IndicatesStaphylococcusaureus
• ResistanttoMethicillin:IndicatesMRSA(MethicillinresistantStaphylococcusaureus)
• Healthcarepersonnelofthehospitalfoundtobenasalcarrierforthesameagent:Indicatesthattheinfectionhasspread
tothepatientfromhealthcarepersonnelofthehospital
• “TheMCwayofspreadofinfectioninhospital:Throughthehospitalstaff(notfromotherpatientfromthehospital)”
• MechanismofResistanceofMRSA:DuetochromosomallymediatedMecAgenewhichcodesforalteredPBP2a(PenicillinBindingp
roteinorreceptor2a)whichhaslessaffinityforβlactamdrugs.
AboutOtherOptions
• Themajorrouteofspreadcausingtheoutbreakisthroughthehandsofhospitalstaff
• TheresistancetoMethicillinis chromosomallymediated
• SinceMRSAcodedalteredPBP2a(PenicillinBindingprotein2a)haslessaffinityforβlactamdrugs,soMRSAstrainsareresi
stanttoallβlactamdrugs.Evencombinationofβlactamandβlactamaseinhibitorwillnotwork.
16. Ans.(a),(b)(Streptococcuspyogenes,Staphyloco...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep213
• StaphTSST1(enterotoxinF/pyrogenicExotoxinC):causesvaginaltamponassociatedTSS
• StaphenterotoxinB,CNonmenstrualcasesofTSS
• StreptococcuspyrogenicexotoxinA,B,C:CanalsocauseTSS.
Alsoknow:
• StreptococcalTSS:Bacteraemic,↑softtissuenecrosis(necrotizingfasciitis),butrashlesscommon
• StaphylococcalTSS:Rashmorecommon,bacteraemialesscommon,lesstissuenecrosis
17. Ans.(a).ItissensitivetoPenicillin)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p220Most
staphylococci are resistant to Penicillin due to beta lactamase production.
18. Ans.(a)(PBPislocalizedinoutercellwall)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep219
• PBP(PenicillinBindingproteinorreceptor)islocalizedinoutercellmembrane(notinthecellwall).
• PBPactsastranspeptidaseorcarboxypeptidaseenzyme(essentialforcellwallpeptidoglycansynthesis).
• PenicillinbindstoPBPandinhibitstranspeptidationthusinhibitingpeptidoglycansynthesis.
• InMRSA–MecAgenealtersPBPtoPBP2awhichhaslessaffinityforβlactamdrugs.
19. Ans.(a)
(MCsourceofinfection:Crossinfectionfrominfectedpersonsinhospital)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology
1/ep219,609,Harrison19/ep959,18/ep1160
• MCsourceofinfection:Crossinfectionfromhandsofhospitalstaffs
• Mosteffectivewaytopreventthehospitalinfection:handwashing
• 25–30%ofgeneralpopulationishealthycarriersofS.aureus
• MCsiteofcolonization:anteriornares,Skin(perineum,axilla,groins)
• ExampleofStaphylococcalsuperantigen:TSStoxinandEpidermolytictoxinandenterotoxin
• MRSA:ChromosomallymediatedwhilePenicillinaseproductionisplasmidmediated.
SystemicBacteriolog
20. Ans.(c)(Staphylococ...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep220,Harrison19/ep960,18/
ep1053Refertext(chapterreview)forexplanation.
21. Ans.(a)(S.aureus)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep215,Harrison19/ep958,18/
ep1053,Refertext(chapterreview)forexplanation.
22. Ans.(c)(Toxincanbe...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep212-13
Staphylococcalenterotoxinisheatstable.Socookingthecontaminatedfoodandleavingatroomtempforsometimeleadsto
toxinaccumulation.
AboutOtherOptions
Optiona:OptimumtemperaturefortoxinproductionissameasOptimumtemperatureforS.aureusgrowthi.e.at37ºC.Soitseemst
obeacorrectstatement.
Remember:Optimumtemperatureforpigmentproductionis22ºCandforMRSAexpressionis37ºCOption b:
St.aureusenterotoxinisintradieticinnaturei.e.itispreformed,secretedinthedietOptiond:IPofS.aureusfood
poisoningis1–6hourduetopreformedtoxin
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23. Ans.(b)(Vagalstimulation)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep213,Jawetz25/ep188
Jawetz25/
ep188:'Theemeticeffectofenterotoxinisprobablytheresultofcentralnervoussystemstimulation(vomitingcenter)afterthetoxinactsonneural
receptorsinthegut.'
Harrison18/ep1165:'Thetoxinstimulatesthevagusnerveandthevomitingcenterofthebrain.Italsoappearstostimulateintestinal
peristalticactivity.'
24. Ans.(a),(c),(e)(S.aureus,Pseudomonas,Strept.Pyogenes)Ref:JagdishChander’sMycology,3/ep158/table11.6
AgentofBotryomycosis:
• S.aureus(MCagent)
• Others:Streptococcus,Pseudomonas,E.coli,Proteus,CoNS,Peptostreptococcus,Streptococcusspp
25. Ans.(c)(80/81)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep218,Ananthanarayan8/
ep203Staphylococcusaureusphagetype80/81isthemostcommontypeassociatedwithhospitalinfections.
26. Ans.(c)(Toxic...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep213,Ananthanarayan9/e/
p202Referchapterreview.
Section
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Streptococcus,Enterococcus CHAPTER
andPneumococcus
3.2
FamilyStreptococcaceaearecatalasenegativegram-positivecocci,arrangedinpairsorchains(duetosingleplaneofdivision).
Streptococcus,EnterococcusandPneumococcusaretheimportantmembersofthisfamily.
However,accordingtothemolecularstructure,Enterococcusisnowreclassifiedunder
separatefamilyEnterococcaceae. TypingofStreptococcus:
Serogrouping(Lancefield’s):Based oncarbohydrateantig
Serotyping (Griffith typing):Based on M protein (>100
CLASSIFICATION emmgenotyping:Basedon
genecodingforMprotein,
>124emm
Onthebasisofhemolysis,streptococcicanbedividedinto3groups
αHemolytic:(Partialorgreenhemolysis),e.g.StreptococcusViridans,Streptococcuspneumoniae
βHemolytic:(Completeoryellowishhemolysis),e.g.βhaemolyticStreptococcus
γHemolytic:(nohemolysisisseen),e.g.Enterococci
Lancefield’sgrouping(forβhaemolyticstreptococci)
Basedoncarbohydrateantigenincellwall,theβhaemolyticstreptococciarefurtherdividedinto20serogroups:GroupAtoVexceptIandJ.
CarbohydrateantigenextractedbyHCl(Lancefield’smethod),Formamide(Fuller’smethod),Enzymatic(Maxted’s)orautoclaving.
StreptococcusgroupA(S.pyogenes)isfurthersubdividedbasedon
Griffithtyping:BasedonMprotein(>100Mserotypes)or
emmtyping:BasedongenecodingforMprotein,>124emmgenotypesidentified.
STREPTOCOCCUSPYOGENES(GROUPA)
VirulenceFactors
Cell • Innerthickpeptidoglycanlayer(conferscellwallrigidity,inducesinflammatoryresponsea
wallanti ndhasthrombolytic activity)
gens • C-carbohydrateantigen:Presentasmiddlelayerandisgroupspecific
• Outerlayerofprotein(M,T,R)andlipoteichoicacid(helpsinadhesion)
• M protein:
○ Mediatesadherencetoepithelialcells,inhibitsphagocytosis
○ Bindstofibrinogenandneutrophilsleadingstoreleaseofinflammatorymediatorsth
atinducevascularleakage(streptococcaltoxicshock).
○ MproteinisfurtherdividedintoClassIandClassII.AntibodiestoclassIM
proteinareresponsibleforpathogenesisofrheumaticfever.
Capsule • Expressedbymucoidstrains,made-upofhyaluronicacid.
• Capsuleisanti-phagocytic,helpsinadhesion;butitisnotantigenic.
Contd...
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Contd...
SPE • 3Types(SPEA,BandC):TypeAandCare,e.g.ofSuperantigens
(Streptococcalp • TypeAandCbacteriophagecoded,Btoxinchromosomalmediated
SPE is associated with thepathogenesis of:
yrogenicexotoxi • Pathogenic role: Associated with the pathogenesis of scarlet fever,
Scarlet fever
n) necrotizingfasciitisandstreptococcal toxic shock syndrome.
Necrotizingfasciitis
StreptococcalTSS. orErythrogenict • Dick test: Intradermal injection of SPE produces erythema only in
oxin thosechildrenwhoaresusceptible todevelopscarletfever.
• SchultzCharltonreaction(blanchingofrashafterinjectionofantiSPE
antibodies):Usedfordiagnosingscarletfeverinpast
Hemolysins StreptolysinOandStreptolysinS(seetablebelow)
Streptokinase Fibrinolysin(activatesplasminogen)
Rapidspread:Bypreventingtheformationoffibrinbarrier.Therapeuticallyusedintre
atment ofcoronarythrombosis.
DNase AlsocalledDeoxyribonucleaseorStreptodornanse(4types:A,B,C,D)
• Diagnostic use: Anti-DNase B > 300–350 U is useful for the
retrospectivediagnosis of skin infections (pyoderma) and acute glomerulonephritis
whereASOtiter islow
• Therapeuticuse:Preparation containing streptodornaseandstreptokinase can
beusedtoliquefythethickexudatesinempyemacases.
Otheren • Hyaluronidase (spreading factor): Expressed by noncapsulated strains, suchas M
zymes type 4 and 22. It breaks down the hyaluronic acid of the tissues, thus helpsinthe
spreadofinfectionalongtheintercellularspace
• Serumopacityfactor:Lipoproteinaseenzymeinnature
• NADase,C5apeptidaseandSpyCEP(inactivatesIL-8)
Streptolysin-O (SL-O) Streptolysin-S (SL-S)

Oxygen labile (hence named streptolysin- Oxygen stable
O )Heatlabile Serumsoluble(hencenamedstrept
Antistreptolysin-O antibodies(ASO):
Raisedinmostofthe olysin-S)
streptococcalinfections Hemolysisisseenonlyindeepcolonies(pourplate)asitisinactivatedinthe Causes hemolysis on
Used as a standard markerfor retrospective diagnosis ofstreptococcalinfections
presenceofoxygen thesurfaceofbloodagarplate
Except in glomerulonephritisand pyoderma; where ASOtiterislow.
Itiscytotoxicforneutrophils,plateletsandcardiactissue Ithasleucocidalactivity
Stronglyantigenic Notantigenic
Antistreptolysin-O antibodies (ASO) are raised in most of Not useful for
thestreptococcal infections and are used as a standard marker serologicaldiagnosis of
Section
forretrospective diagnosis of streptococcal infections (except streptococcalinfections

inglomerulonephritisandpyoderma;whereASOtiterislow)
Manifestations
Streptolysin-Oisstructurallyandfunctionallysimilar to:
• TetanolysinofClostridiumtetani
Streptococcuspyogenescausesbothsuppurativeandnonsuppurativemanifestations.
• PneumolysinofS.pneumoniae
Suppurative Manifestations
• ThetatoxinofClostridiumperfringens
Scarlet fever: • ListeriolysinOofListeria
Respiratoryinfections:
CausedbyS.pyogenes,Nowrare,characterized• by:• Pharyngitis/sorethroat(MCcause,20–40%ofallcases)
CereolysinofBacilluscereus
PharyngitisandSandpaper
• Pneumoniaandempyema
rashes, strawberry tongue
Pastia’slines:prominent Scarletfever(MC cause): Nowrare, characterizedby:
rashesinskin folds • PharyngitisandSandpaper rashes,strawberry tongue
Pathogenesis is due to SPEtoxin (Dick test +ve).
• Pastia’slines-prominentrashesinskinfolds
• PathogenesisisduetoSPE toxin (Dicktest +ve)
Contd...
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Streptococcus,EnterococcusandPneumococcus127
Contd...
Suppurative Manifestations
Skinandsofttissueinfections:
• Impetigo(pyoderma):(MCcause)
○ Seeninchildren,poorhygiene,warmclimate
○ Characterizedbypustularlesionsthatbecomehoneycomblikecrusts,nofever,painless.
○ AssociatedwithhigherMtypes,andnephritogenicstrains.
• Cellulitisanderysipelas(MCcause):
○ Tender,brightred,swollenandinduratedpeaud’orangetextureofskin(duetoinvolvementofthesuperficial
lymphatics)alongwithfever andchills.
○ MCsite-malarareaoftheface,seeninolderpeople.
Deepsofttissueinfections:
• Necrotizingfasciitisorstreptococcalhemolyticgangrene-S.pyogenesisMCcause(60%),itis
rapidlyspreading,henceS.pyogenesisalsocalledflesheatingbacteria
• Toxicshocksyndrome(staphylococcalTSSisMC,butbacteremiaisMCinstreptococcalTSS)
• Streptococcalmyositis(S.aureusisMCcauseofmyositis)
Complications:
• Puerperalsepsis (Group BStreptococcus isMC cause),
• Others:Otitismedia,Quinsy,Ludwig’sangina,pneumonia(postviral),osteomyelitis,meningitis
NonsuppurativeComplications
Streptococcal antigens show molecular mimicry with human antigens. Due to Nonsuppurative complicationsof S.pyogenes:
antigeniccross reactivity, antibodies produced against previous streptococcal infections Acute rheumatic fever
cross reactwith human tissues to produce lesions. This accounts for a number of Post streptococcalglomerulonephritis (PSGN)
Guttatepsoriasis
nonsuppurativecomplicationssuchas: Reactivearthritis
• Acuterheumaticfever PANDAS
• Poststreptococcalglomerulonephritis(PSGN)
• Guttatepsoriasis
• Reactivearthritis
• PediatricAutoimmuneNeuropsychiatricDisordersAssociatedwithStreptococcuspyogene
s(PANDAS)
Antigenic cross reactivity between streptococcal antigens and the corresponding
humanantigens
StreptococcalAg MammalianAg
CellwallMprotein(ofserotypesM1,M5,M6,andM19) Myocardium(tropomyosin andmyosin)
CellwallCcarbohydrate Cardiacvalves
Cytoplasmicmembrane Glomerularvascularintima
Peptidoglycan Skinantigens
SystemicBacteriolog
Hyaluronicacid Synovial fluid
Differencesbetweenacuterheumaticfeverandpoststreptococcalglomerulonephritis
Acute rheumatic Poststreptococcal
Feature fever(ARF) glomerulonephritis(PSGN)
Priorhistoryofinfection Pharyngitis strains Mainlypyodermalstrains,orrarely
with pharyngitisstrains
Serotype Mostofthe strainsof Pyodermalstrains:49,53–55,59–61and
S.pyogenes Pharyngitisstrains:1,12
Immune response Marked Moderate
Complementlevel Unaltered Low(duetodepositioninglomeruli)
Genetic susceptibility Present Absent
Repeatedattacks Common Uncommon
Penicillinprophylaxis Indicated Notindicated
Contd...
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Contd...
Acute rheumatic Poststreptococcal
Feature fever(ARF) glomerulonephritis(PSGN)
Serology:
Course Progressive
ASO Ab: Titer > 200 Todd unit/ml in most streptococcalinfectionsexceptinpyodermaandPSGN Spontaneous resolution
Anti-DNase-B Ab – Titer >300–350units/mlisdiagnosticofPSGNandpyoderma.
Prognosis Variable Good
Hypersensitivityreaction TypeII TypeIII
LaboratoryDiagnosisofStreptococcusPyogenes
• Transportmedium:Pike’smedium
• Directsmearmicroscopy:Puscellswithgram-positivecocciinshortchains
• Culture:
○ Bloodagar:Pinpointcolonywithawidezoneofβ-hemolysis
○ Selectivemedia:CrystalvioletbloodagarandPNF(polymyxinB,neomycin,fusid
icacid)media
○ Liquid media: Granular turbidity with powdery deposit
• Biochemicalidentification:Catalasenegative,BacitracinsensitiveandPyrrolidonylArylamid
ase(PYR)testispositive
• Typing:
○ Lancefieldgrouping:ShowsgroupAStreptococcus
○ TypingofgroupAStreptococcus:Griffithtypingandemmtyping
• Serology:ASOantibodiesandAnti-DNaseBantibodies
Treatment of Necrotizingfasciitis: ○ ASOantibodiestiteriselevated>200Toddunit/mlinmoststreptococcalinfec-
Surgicaldebridement(mostcrucial)plus tionsexceptinpyodermaandPSGN.
PenicillinGplus ○ Anti-DNase-BAb–Titer>300–350units/mlisdiagnosticofPSGNandpyoderma.
Clindamycin
○ OtherantibodieselevatedareAntihyaluronidaseandantistreptokinaseantibodies.
Treatmentofstreptococcalinfection
Penicillinisthedrugofchoiceforalltypeofstreptococcalinfections
Conditions Treatmentrecommended
Pharyngitis BenzathinepenicillinG,IMsingledose
ororalpenicillinVfor10days
Erysipelas/Cellulitis Mild- Procaine
penicillinSevere-
Penicillin G
Necrotizingfasciitis Surgicaldebridement(mostcrucial)+PenicillinG+Clindamycin
Pneumoniaandempyema PenicillinG+drainageofempyema
StreptococcalTSS PenicillinG+Clindamycin+immunoglobulin(toSPE)
Rheumaticfever Benzathine penicillin G, IM single
dose;ororalPenicillinV for10days
Long-termmaintenancetherapywithpenicillinGmonthly:
• For5yrsoruntil21yrsofage,(without carditis)
Section
• For10yrs (with carditis)

Rheumaticfever: • upto40yrsofage/lifelong(withresidualheartdisease)
Treatment: BenzathinepenicillinG,IMsingledose;ororalPenicillinVfor10days
PSGN Gmonthly
Long-term maintenancetherapy, with penicillin BenzathinepenicillinG,IMsingledose;
For5yearsoruntil21 ororalpenicillinVfor10days
yearsofage,(without Treatmentofasymptomaticcarriers
carditis)
Pharyngeal carrier Penicillin V + rifampicin
For10years (with carditis)
Rectalcarriers
Up to 40 years of age/lifelong(withresidualheartdisease) Vancomycin+rifampicin
Prophylaxis
Long-termmaintenancetherapywithpenicillin(alternative-
sulfadiazineorerythromycininpenicillin allergy) is required for children who develop early
signs of rheumatic fever. Thisprevents streptococcalreinfectionandfurther damagetoheart.
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GROUPBSTREPTOCOCCUS(S.AGALACTIAE)
Pathogenesis: Approximately 30% of women are vaginal or rectal carriers of group
BStreptococcus. Hence, the infection is common in neonates and in pregnancy. It is a
majorcauseof:
• Neonatalsepsisandmeningitis:Neonatalsepsiscanbeoftwotypes-
earlyonsetandlateonsettype
• Puerperalsepsisandperipartumfever
• Infectionsinelderlypeoplewithunderlyingillness,suchasdiabetesmellitusormalignancy:
Cellulitisandsofttissueinfections,UTI,pneumonia,andendocarditis.
Group B StreptococcusCauses:
Laboratory Diagnosis: It can be differentiated from Group A Streptococcus by
Neonatalsepsisand
followingbiochemicaltests(seetable) meningitis
• CAMPpositive,Hippuratehydrolysistestpositive,BacitracinresistantandPYRtestisnegati Puerperal sepsis andperipartumfever
ve Infections in elderly peoplewithunderlyingillness
• Orangepigmentproduction-enhanced inIslam’smedium.
• βhemolyticcolonies,whicharemucoidandslightlylarger(2mm).
• Ithasacapsularpolysaccharide,whichcanbetypedintonineserotypes.Earl
yandlateonsetGroupBStreptococcusdiseaseinneonates
Characteristics Early-onset disease Late-onset disease

Ageofonset 0–6daysofbirth 7–90daysofbirth
Increased risk following Prematurityandprolongedlabor Notassociated
obstetriccomplications
Mode of transmission During or before birth from the Contact with a
tothebaby colonizedmaternalgenitaltract colonizedmotherandnursingpers
onnel
Common Pneumonia and/or respiratory Bacteremia and
clinicalmanifestati distresssyndromefollowed bymeningitis meningitis(most common)
ons
Commonserotypes Ia,III,V,II,Ib IIIpredominates
Characters S. pyogenes S.agalactiae

Lancefieldgroup GroupA Group B
Bacitracin sensitivity test Sensitive Resistant
PYR test Positive Negative
Hippuratehydrolysistest Negative Positive
CAMPtest Negative Positive
SystemicBacteriolog
β hemolytic colonies 0.5–1mm,pinpoint Mucoid,larger(2mm)
ENTEROCOCCUS
Theenterococciwereinitiallygroupedundergroup-DStreptococcus,butlater,ithasbeen
reclassifiedasaseparategenusEnterococcusunderfamilyEnterococcaceae.
• Enterococci are the part of normal flora of human GIT. At the same time, they are
alsoincreasingly important agents of human disease especially in hospitals mainly
becauseoftheirresistancetoantibiotics.
• E. faecalis is the most common species found in clinical specimens; whereas E. faecium
ismoredrugresistantthanE.faecalis.
VariousClinicalManifestationsInclude
• Urinarytractinfections(cystitis,urethritis,pyelonephritisandprostatitis)
• Bacteremiaandmitralvalveendocarditis(inIVdrugabusers)
• Intra-abdominal,pelvic,andsofttissueInfection
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• Late-onsetneonatalsepsisandmeningitis
• Infectiononburnsurface.
LaboratoryDiagnosis
Enterococcishowthefollowingcharacteristicsthathelpintheidentification:
AllcocciareNonmotileexcept: • Theyaregram-positiveovalcocciarrangedinpairs(spectacleeyedappearance)
Enterococcusgallinarum • Nonmotilecocci(exceptE.gallinarumandE.casseliflavus)
Enterococcus casseliflavus • Bloodagar: It produces nonhemolytic, translucent colonies (rarely produces α
orβhemolysis)
• MacConkeyagar:Itproduces minutemagentapink colonies.
• Bileaesculinhydrolysistestispositive
• PYRtestispositive
• Growthoccursinpresenceof:
○ 6.5%NaCl,40%bileandpH9.6
○ Heattolerancetest:Theyarerelativelyheatresistant,cansurvive60°Cfor30minu
tes.
Treatment
Most strains of enterococci are resistant to penicillins, aminoglycosides and
sulfonamides.They showintrinsicresistance tocephalosporinsand cotrimoxazole.
• Resistance is overcome by combination therapy with penicillin and
aminoglycoside(due to synergistic effect) and is the standard therapy for life-
threatening
enterococcalinfections;howeverinUTI,monotherapywithampicillinornitrofurantoiniss
ufficient.Resistancetothiscombinationtherapymayalsodevelop.
• Vancomycinisusuallyindicatedinresistantcasesbutresistancetovancomycinhasalsobeen
reported.
VancomycinResistantEnterococci(VRE)
Vancomycinresistanceinenterococcihasbeenincreasinglyreportednowadays.
• VRE is mediated by Van gene, which codes for altered target site for vancomycin in
thecellwall(i.e.D-alanyl-D-alaninesidechainofpeptidoglycanlayerisalteredtoD-alanyl-D-
serineorD-alanyl-D-
lactate)andthisalteredsidechainshavelessaffinityforbindingtovancomycin.
• Vangenehas9genotypes.Importantonesare:VanAtoVanE.
○ StrainswithVanAgeneshowhighlevelresistancetobothglycopeptides-vancomy-
cinandteicoplanin.
○ Strains with VanB gene show low level resistance to vancomycin, but sensitive
toteicoplanin.
○ E.gallinarumandE.casseliflavuspossessVanCgeneswhichischromosomalcoded(othe
r genotypes transposon coded), and they show intrinsic resistance to both gly-
Section
Pathogenic Viridansstreptococci: copeptides.

S.mutans:Causesdental • ScreeningofpatientsforVREiscarriedoutby:RectalswabculturingonBileesculin
cariesandplaques azideagarwith6μg/mlvancomycin
S.sanguis: Causes subacutebacterialendocarditis
S.milleri group: Producessuppurativeinfections
VIRIDANSSTREPTOCOCCI
Viridansstreptococciareαhemolytic,commensalofmouth:
• S.mutans:Causesdentalcariesandplaques
• S.sanguis:Causessubacutebacterialendocarditis
• S.millerigroup:Producessuppurativeinfections,differinhemolyticpattern(maybeα,βorγ
hemolytic).
Treatment:Usuallysensitivetopenicillin(exceptneutropenicpatientswithbacteremia;wherevanco
mycinisgiven).
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STREPTOCOCCUSPNEUMONIAEORPNEUMOCOCCUS
VirulenceFactorsandPathogenesis VirulenceFactorsof
S.pneumoniaepossessesanumberofvirulencefactorssuchas: S. pneumoniae:
Capsule (detected by Quellungreaction)
• Capsularpolysaccharide:Itprotectsthecoccifromphagocytosis: C carbohydrate antigen:CRPisnamedafterthis
○ Itistypespecific(>90capsularserotypesarerecognized),serotypesaredetected Pneumolysin
byQuellungreaction. Autolysin:Responsibleforbilesolubility and draughtsmanc
○ Quellungreaction:Capsularswellingoccurswhencoloniesareaddedwithtype-
specificantiserumandmethylenebluedye.
○ Itdiffuses(soluble)intoculturemedia,tissueandexudates,hencealsocalledsolu-
blespecificsubstance.
• C-carbohydrate antigen (C-polysaccharide or C-substance): It is species specific.
CRP(C-reactive protein) present in sera of patients with acute inflammation. It is so
namedbecause;itprecipitateswithpneumococcalC-antigen.
• Pneumolysin:Itisamembranedamagingtoxin,inhibitsneutrophilchemotaxis.
• Autolysin: It is an amidase enzyme that cleaves peptidoglycan leading to autolysis
ofcells. This property is responsible for bile solubility and draughtsman appearance
ofpneumococcalcolonies.
ClinicalManifestation
S.pneumoniaeisthemostcommoncauseof:
• Lobarpneumonia
• Pyogenicmeningitisinallages(exceptinneonates)
• Noninvasivemanifestationssuchasotitismediaandsinusitis. S.pneumoniae is the mostcommoncauseof:
Lobarpneumonia
Other invasive manifestations: S. pneumoniae can cause osteomyelitis, septic Pyogenic meningitis in all ages(exceptinneonates)
Noninvasivemanifestations,such as otitis media andsinusi
arthritis,endocarditis, pericarditis, primary peritonitis, rarely, brain abscess and
hemolytic-
uremicsyndrome.Empyemaandparapneumoniceffusionmayoccurascomplicationsofpneumonia.
Epidemiology
• Sourceofinfectioninhumansisupperrespiratorytractofcarriers(lessoftenpatients).
• Carrierrate>90%ofchildrenof6monthsto5yrsofageharborS.pneumoniaeinthenasopharynx.
• Modeoftransmissionisbyinhalationofcontaminateddropletnuclei.
Riskfactors:
SystemicBacteriolog
• Children(<2yrs)
• Splenectomy, sickle cell disease and other hemoglobinopathies: As spleen is the site
ofdestructionofcapsulatedbacteria,theconditionswheretheopsonizationandclearanceofcirc
ulatingbacteriabythespleenishampered,thereisincreaseriskofpneumococcalinfection.
• Underlying comorbid diseases, such as chronic lung, heart, kidney and liver Nature of infecting serotypes ofPneumococcus:
In children: Serotypes 4, 6B,9V,14,18C,19F,and23Fareco
disease,cochlearimplants,diabetesmellitusandimmunosuppression(e.g.HIV). Inadults:Serotypes1–8arecommon
• Underlyingviralupperrespiratorytractinfections(e.g.Influenza). Virulent serotypes: Serotype 3followed by 7 are more viru
• Natureofinfectingserotypes:
○ Inchildren:Serotypes4,6B,9V,14,18C,19F,and23Farecommon
○ Inadults:Serotypes1–8arecommon
○ Virulent serotypes: Serotype 3 followed by 7 are more virulent and they
producemucoidcolonies.
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LaboratoryDiagnosis
S.pneumoniaecanbedifferentiatedfromViridansstreptococcibyvariousfeatures:
Properties Pneumococcus Viridansstreptococci
Morphology Lanceolateorflameshaped Round/oval
Arrangement Gram-positivecocciinpairs Gram-positivecocciinlongchains
Capsule Present Absent
Onbloodagar Draughtsmanorcaromcoincolony Convexshapedcolony
Liquidmedium Uniformturbidity Granular turbidity
Bile solubility Solubleinbile Insolubleinbile
Inulin fermentation Fermenter Nonfermenter
Optochin Sensitive Resistant
Mice Pathogenicity Pathogenic Nonpathogenic
Treatment
Penicillin G remains the drug of choice; cephalosporins, such as ceftriaxone can be
TreatmentofPneumococcalInfections: givenalternatively.
PenicillinGremains theDOC Oralamoxicillinisrecommendedforchildrenwithacuteotitismedia.
Ceftriaxone can be givenalternatively.
Oral amoxicillin isrecommended for children withacuteotitismedia.
Prevention(CapsularPolysaccharideVaccines)
Twotypesofpneumococcalvaccinesareavailable.
23-ValentPneumococcalPolysaccharideVaccine(PPV23)
PPV23includescapsularpolysaccharideof23serotypesofpneumococci.Itgivesprotectionfora
bout5years.
Indication:Itisrecommendedforpeoplewith
• Aspleniaorsplenicdysfunction • Diabetesmellitus
• Sicklecelldiseaseorceliacdisease • Cochlearimplants
• Chroniclung,heart,kidneyandliverdisease • Cerebrospinalfluidleaks
• Immunocompromisedpatients(HIV) • Ageabove65years
ContraindicationtoPPV-23include:
Contraindications to PPV-23include:
Malignancies Malignancies, pregnancy and children of < 3years: As capsular antigens are examples of T-
Pregnancy independent antigen, they are poorly immunogenic to children of < 3 years. Hence, PPV-
Childrenof<3years
23isnotusefulamongchildrenof<3years.
Section
7-ValentPolysaccharideConjugateVaccine(PCV-7)
It consists of capsular polysaccharide of 7 serotypes added to a protein conjugate. It
mainlyincludesthe childhoodserotypes (suchas 6B, 9V,14, 19F,23F, and 18C):
• Whenaproteinconjugateisadded,itincreasestheimmunogenicityofcapsularantigen(acta
sadjuvant),hencecanbegiventochildrenof<3years.
• Schedule:4dosesadministeredat2,4,6,and12–15monthsofage.
• As, resistance to antibiotics is most often noted in pneumococcal serotypes 6, 9, 14,
19,and23;henceuseofPCV-7hasshowntodecreasepneumococcalresistance.
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STREPTOCOCCUSANDPNEUMOCOCCUS 8. Treatmentforstreptococcalnecrotizingfasciitis:
a. Surgicaldebridement (PGIJune2008)
1. Boy presented with skin ulcer on leg. Culture b. Penicillin
revealsbetahemolyticStreptococci.Culturefromschool c. Clindamycin
children with sore throat some days back also
d. Metronidazole
revealedbetahemolyticStreptococci.Whatisthecharact
e. Vancomycin
eristicwhichcantellboththestrainsaresameordifferen
t? 9. GroupAhemolyticpharyngitisisdueto:
a. C-carbohydrateAg (AIIMSNov2010) a. Localinfection (DNBJune2010)
b. Mprotein b. Systemictoxicity
c. Emuprotein c. Attachmenttomucosa
d. MecAgene d. Localtoxins
2. Lancefield grouping of streptococci isdone by using:
(RecentQuestion2013,AIIMSNov2007,June1998) GROUPBSTREPTOCOCCUS
a. MProtein
b. GroupCpeptidoglycancellwall 10. Achildpresentswithsepsis.Bacteriaisolatedshowed
c. GroupCcarbohydrateantigen hemolysisonbloodagar,resistanttobacitracinandapos
d. Stainingproperties itiveCAMPtest.Themostprobableorganismis:
(RecentQuestion2013,AI2001,2010)
3. Streptokinaseistakenfromwhichsource:
a. S.pyogenes b. S.agalactiae
a. Streptococcusequisimilis (RecentQuestion2015)
c. Enterococcus d. Pneumococcus
b. Streptococcusbovis
c. Streptococcuscanis 11. Neonatalmeningitisacquiredduringpassagethroughbi
rthcanal isdue to: (MHPG2015,TN2002)
GROUPASTREPTOCOCCUS a. Streptococcusagalactiaeb. S.equisimilus
c. S.pyogenes d. Pnemococci
4. Streptococcustoxinwhichisresponsibleforconnectivetiss
uebreakdown? (RecentQuestions2014)
a. Hyaluronidase
ENTEROCOCCUS
b. StreptolysinO 12. Vancomycinresistanceinenterococcusisdueto?
c. StreptolysinS (RecentQuestion2015)
d. Streptococcuspyogenicexotoxin a. Thickeningofcellwall
5. FalseregardingStreptococcuspyogenes: b. Alteredtargetsite
(Recentquestions2014,NEETPatternBased) 13. Whichgroupofstreptococcusgrowat60°C:
a. Causesnecrotizingfascitis (NEETPatternBased)
SystemicBacteriolog
b. Betahemolytic
a. A b. B
c. Mproteinisvirulencefactor
c. C d. D
d. Resistanttobacitracin
14. A patient admitted to an ICU is on central venous
6. Whichstreptococcalantigencrossreactswithsynovialflui
linefor the last one week. He is on ceftazidime and
d? (AI2008)
amikacin.After7daysofantibiotics,hedevelopsaspikeof
a. Carbohydrate(groupA)
feverandhisbloodcultureispositiveforgrampositiveco
b. Cellwallprotein
cciinchains,whicharecatalasenegative.Followingthis,
c. Capsularhyaluronicacid
vancomycin was started but the culture
d. Peptidoglycan
remainedpositive for the same organism even after 2
7. Achildpresentswithinfectiveskinlesionoftheleg.Cultu weeks oftherapy. The most likely organism causing
reshowedhemolyticcolonieswhichweregram
infectionis:
+vecocciinchains.Thetesttoconfirmtheorganismis:
(AIIMSNov2011,May2006,Nov2006,AI2007)
(Recentquestions2014,AI2012,AIIMSNov2006;
AIIMSNov2011,AIIMSMay2007, AI2007)
b. Viridansstreptococci
a. Bilesolubility b. Optochinsensitivity
c. Enterococcusfaecalis
c. Bacitracinsensitivity d. Catalasepositive
d. Coagulasenegative Staphylococcus
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15. Which of the following organisms, when isolated

a. Novobiocin b. Optochin
inthe blood, requires the synergistic activity of
c. Bacitracin d. Oxacillin
penicillinplus anaminoglycoside for
appropriatetherapy: 22. The following
a. Enterococcusfaecalis (AIIMSNov2004) statementsaretrueregardingStreptococcuspneumonia
b. Staphylococcusaureus eexcept: (AI2011)
c. Staphylococcuspneumoniae a. Itisbile-sensitive
d. Bacteroidesfragilis b. ThecapsuleofS.pneumoniaeallowsestablishmentofi
nfection
c. Itisanetiologicalagentofpneumoniaandotitismedia
STREPTOCOCCUSVIRIDANS d. Pneumococcalmeningitisistheleastvirulentofthemajor
16. ApatientofRHDdevelopedinfectiveendocarditisafter bacterialmeningitides
dental extraction. Most likely organism causingthisis: 23. 65 years old patient presented to the emergency
(AIIMSNov01) withhigh grade temperature and increased respiratory
a. Streptococcusviridans rate.Hecomplainedofpaininthechestandhaddevelope
b. Streptococcuspneumoniae dcoughwithexpectoration.Hissputumwassenttothela
c. Streptococcuspyogenes boratoryforgramstrainingwhichshowedthe presence
d. Staphylococcusaureus of pus cells and gram-positive cocci inpair. The
culture on the blood agar medium was alsopositive.
PNEUMOCOCCUS Which of the following laboratory tests willhelp to
differentiate the specific pathogen from
17. Patientispresentedwithcoughwithrustysputum.Onex theothercommensalgram-positivecocci?
amination, lower lobe consolidation and a. Bacitracinsensitivity (AIIMSNov2009)
bronchialbreathsoundswereheard.ItgivesapositiveQ b. Catalasetest
uellungreaction.WhatistheprobableGramstainingapp c. Bilesolubility
earance? (JIPMERMay2016) d. Coagulasetest
a. Gram-vebacilli b. Gram+vecocci
24. ‘C’inCreactiveproteinstandsfor: (AI2011)
c. Gram+vebacilli d. Gram-vediplococci
a. CapsularpolysaccharideinPneumococcus
18. Mostcommonorganismcausingacuteotitismediaina4- b. Concanavalin-a
8yearchild? (NIMHANS2016) c. Calretinin
a. Streptococcuspneumoniae d. C-carbohydrateantigen
b. Moraxellacatarrhalis
25. Inasplenectomizedpatientthereisincreaseofinfection
c. Staphylococcusaureus
byalltheorganismsexcept:
d. Shigella
(NEETPatternBased,PGI2000,SGPGI2005)
19. Most common cause of meningitis in alcoholics: a. Pneumococci b. Klebsiella
(RecentQuestion2015) c. H.influenzae d.Staph.aureus
a. Pneumococcus b. H.influenzae
26. Mostcommoncausativeorganismforlobarpneumoniais:
20. MechanismofdevelopmentofresistancetopenicillininS (AIIMS2004)
treptococcuspneumoniaeis: (JIPMERNov2014) a. Staphylococcusaureus
a. ProductionofBetalactamase b. Streptococcuspyogenes
b. Mutationsintheproteinsonthebacterialsurface c. Streptococcuspneumoniae
c. Changesinthemembranepermeabilitytopenicillin d. Haemophilusinfluenzae
Section
d. Productionofanalternativepenicillinbindingprotein
27. Themostcommonorganismcausingacuteotitismediais:
21. A person presents with pneumonia. His sputum (MHPG2014)
wassent for culture. The bacterium obtained was a. H.influenza
grampositivecocciinchainsandalphahaemolyticcoloni b. S.pneumoniae
esonsheepagar.Whichofthefollowingwillhelpinconfir c. M.catarhalis
mingthediagnosis: (AIIMSMay2012) d. S. aureus
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EXPLANATIONS
STREPTOCOCCUSANDPNEUMOCOCCUS
1. Ans.(a)(C-carbohydrateAg)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/rp227,Ananthanarayan9/ep209
• βhemolyticStreptococciwasobtainedfromtwocases-Boywithskinulcerandchildrenwithsorethroat.
• ToknowtherelatednessbetweenBetahemolyticStreptococci,Lancefieldgroupingisdone
• LancefieldgroupingisbasedonC-carbohydrateAg.
ClassificationofStreptococcus-Refertext(chapterreview)forexplanation.
2. Ans.(c)(GroupCcarbohydrateantigen)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep221,Ananthanarayan
9/ep209
• βhemolyticStreptococcusisclassifiedtogroupA-VbasedonCcarbohydrateantigen(Lancefieldclassification)
• MproteinisusedtofurtherclassifygroupAStreptococcus(Griffithtyping).
3. Ans. (a)(Streptococcusequisimilis)Ref:InternetSources
• Streptococcusequisimilisisusedasourceforpreparationofstreptokinase.
GROUPASTREPTOCOCCUS
4. Ans.(a)(Hyaluronidase)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep223,Ananthanarayan9/ep213
Referchapterreview.
5. Ans.(d)(Resistanttobacitracin)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep227,Ananthanarayan9/e
p213
• Streptococcuspyogenes(GroupA)issensitivetobacitracinwhereasStr.agalactiae(GroupB)isresistanttobacitracin.
6. Ans.(c)(Capsul...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep225,Ananthanarayan9/ep212
• Capsularhyaluronicacidcrossreactswithsynovialfluid
• StructuralcomponentsofStreptopyogenescrossreactswithhumantissues-Refertext(chapterreview).
7. Ans.(c)(Bacitracin...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep227,Ananthanarayan9/ep215
• Hemolyticcolonywithgram-positivecocciinchainissuggestiveofβhemolyticStreptococcus.
• Optionc:BacitracinsensitivityisusedtodifferentiatebetweenβhemolyticStreptococciGroupA-Bacitracinsensitive
andGroupb:Bacitracinresistant
• Optionaandb:BilesolubilityandOptochinsensitivityareusedtodifferentiatePneumococcusandS.Viridans
• Optiond:CatalasetestisusedtodifferentiateStreptococcusandStaphylococcus.
SystemicBacteriolog
8. Ans.(a),(b),(c)(Surgicaldebridement,Penicillin,Clindamycin)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology
1/e p225,Harrison19e/p96618/ep1176,
• TreatmentofNecrotizingfasciitis:Surgicaldebridement(mostcrucial)+PenicillinG+Clindamycin
• Ifsingleoptiontobeselected:ThenAnswershouldbeSurgicaldebridement
• Harrison17/ep886states:‘Drainageanddebridementarecentraltothemanagementofnecrotizingfasciitis;antibiotic
treatmentisausefuladjunct,butsurgeryislife-saving’.
9. Ans.(c)(mucosalattachment)Ref:Harrison19/ep964,18/ep1172
ThecapsularpolysaccharidemayalsoplayaroleinGAScolonizationofthepharynxbybindingtoCD44,ahyaluronicacid–
bindingproteinexpressedonhumanpharyngealepithelialcells.
GROUPBSTREPTOCOCCUS
10. Ans.(b)(S.agalactiae)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep229,Ananthanarayan9/ep216,8/e
p207
Alreadyexplained
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• PointsfavoringtoS.agalactiae:
○ Septicemiainachild
○ βhemolysisonbloodagar
○ ResistanttobacitracinandapositiveCAMPtest
• S.pyogenes:sensitivetobacitracinandCAMPtestisnegative.
11. Ans.(a)(Streptococcusagalactiae)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep229,Ananthanarayan
9/ep216
Referchapterreview.
ENTEROCOCCUS
12. Ans.(b)(Alteredtargetsite)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep231
13. Ans.(d)(D)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep230,Ananthanarayan8/ep206
GroupDStreptococcilikeEnterococcicangrow>60°C.
14. Ans.(c)(Enterococcusfaecalis)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep231
ThisiscaseofVRE(VancomycinresistantEnterococci)
Pointsinfavor:
• Gram+vecocciinchainandCatalase–vepointstowardsStreptococcaceaefamily
• Resistanttoaminoglycoside,cephalosporinsandvancomycin
• S,aureusandCONSarecatalase+ve,hencetheyareruledout.
15. Ans.(a)(Enterococcusfaecalis)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep231,
ResistanceinEnterococci–Referchapterreview
STREPTOCOCCUSVIRIDANS
16. Ans.(a)(Streptococcus...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep231,Harrison,18/ep1052-53
• MCcauseofNativevalveendocarditis:S.aureus
• MC cause of subacuteendocarditis: MC agentStreptococcus Viridans
• MC Streptococcus Viridans causing endocarditis: S.sanguis
• Followingtoothextraction,transientbacteremiaoccursandStreptococcusViridansgetslodgedintopredamagevalve.
• So,prophylacticantibioticisimplementedbeforetoothextraction.
PNEUMOCOCCUS
17. Ans(b)(Gram+vecocci)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p234
• Historyoflobarconsolidation–indicateslobarpneumonia
Section
• Quellungtestpositive-indicatespneumococcalpneumonia
• PneumococciareGrampositivediplococci,lanceolateshaped
18. Ans(a).(Streptococcuspneumoniae)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology/p232
• StreptococcuspneumoniaeistheMCcauseofacuteotitismediainchildren.
19. Ans.(a)(Pneumococcus)Ref:Community-AcquiredBacterialMeningitisinAlcoholicPatients,http://
www.ncbi.nlm.nih.gov/pmc/articles/PMC2817003/
20. Ans(d)(Productionofanalternativepenicillinbindingprotein)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology
1/ep234.
Alterationsinthetargetenzymesforβ-lactamantibiotics,thepenicillin-bindingproteins(PBPs),havebeenrecognizedasa
majorresistancemechanisminStreptococcuspneumoniae.
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21. Ans.(b)(Opt...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep234,Ananthanarayan9/ep222,8/ep220
• α-hemolyticcoloniesandgram-positivecocci:Isolatedfromasputumofapatientwithfeverandrespiratorydistress
-Suggestiveof:
○ PneumococcalPneumoniaor
○ Pneumoniadue toStr. viridans
• TodifferentiatePneumococcusfromStr.viridans—Optochinsensitivitytestisdone.
22. Ans.(d)(Pneumococcalmeningitisistheleastvirulentofthemajorbacterialmeningitis)
Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep234,Ananthanarayan9/ep223,8/p219-222
Amongthemajorbacterialmeningitides,Pneumococcusisoneofthemostvirulentorganism.
AboutOtherOptions
Pneumococcusis:
• Capsulatedprinciplevirulencefactor
• Bilesoluble
• MCcauseoflobarpneumoniaandotitismedia.
23. Ans.(c)(Bilesolubility)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep234,Ananthanarayan9/ep222
Pointsinfavor:
• Feverand↑respiratoryrate,chestpainandcoughwithexpectoration:SuggestiveofPneumonia
• GrampositivecocciinpairwasisolatedfromSputum:SuggestiveofPneumococcus
• TesttodifferentiatePneumococcusfromcommensallikeStreptViridans:Bilesolubility
• RefertextfordifferentiatingpropertiesbetweenPneumococcusandS.viridans.
Itcan be differentiatedfrom other commensal insputum, i.e. Strept Viridans:

• Optochinsensitivity(Ethylhydrocuprein)
• Bilesolubility
Outofthis,BilesolubilityisabetterOptionbecause:
• FewstrainsofStreptViridansalsocanbesensitivetoOptochinwhilefewstrainsofPneumococcuscanberesistant.
• BilesolubilityisaconstantpropertyofPneumococcus,henceisofdiagnosticimportance...Ananthanarayan8/ep219
24. Ans.(d)(C-carbohydrateantigen)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep234,Ananthanarayan9/e
p222
C-reactiveprotein:Referchapterreview
25. Ans.(d)(Staph.aureus)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep235,170,Ananthanarayan9/ep18,200
• Capsulatedorganismcanresistkillingbypreventingphagocytosis.
• Spleenhasanimportantroleinremovingthesecapsulatedorganismswhereopsonizationtakesplace.
• Certaincomplements(opsonins)canattachtotheseorganismsandoncedeliveredtospleen,theiruptakeisfacilitatedbyco
mplementreceptorslikeCR1,2.etc.presentonsplenicphagocytesurface(ProcessknownasOpsonization).
• Hence,inasplenectomizedpatientthereisincreaseriskofinfectionbycapsulatedorganisms.
SystemicBacteriolog
• Amongthe options,all are capsulatedexcept S. aureus.
26. Ans.(c)(Streptococcuspneumoniae)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep233,Ananthanarayan
9/ep222
• StreptococcuspneumoniaeistheMostcommoncausativeorganismforlobarpneumonia
• Adult:MCserotype associated:Type 1-8
• Children:Type6,14,19,23arefrequentlyassociatedserotypes
• MostVirulent: Type3.
27. Ans.(b)(S.pneumoniae)Ref:EarInfections/CDCWebsite
• StreptococcuspneumoniaefollowedbyHaemophilusinfluenzaeandMoraxellacatarrhalisarethemostcommonbacteriali
solatesfromthemiddleearfluidofchildrenwithacuteotitismedia.
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CHAPTER
NeisseriaandMoraxella 3.3
GramnegativecocciincludeNeisseria,MoraxellacatarrhalisandVeillonella
Neisseriaearecatalaseandoxidasepositive,non-motile,aerobicgram-negativediplococci:
•
N.meningitidis: Fermentsglucoseandmaltose Twospeciesarepathogenictohumans:N.meningitidisandN.gonorrhoeae
N.gonorrheae: Ferments onlyglucose • Othersarecommensalsofintestine,genitaltractororalcavity.
N.meningitidis N.gonorrhoeae
Capsulated Noncapsulated
Lensshaped/half-moonshaped Kidney shaped
(diplococciwithadjacentsidesflattened) (diplococciwithadjacentsidesconcave)
Fermentsglucoseandmaltose Fermentsonlyglucose
Rarelyhaveplasmids Usuallypossessplasmids,codingfordrugresistancegenes
Existinbothintraandextracelularforms Predominantly,existinintracelularform
Colony—circular Colony—varyinsizewithirregularmargin
Habitat—Nasopharynx Habitat—genitaltract(urethra,cérvix),rarelypharynx.
NEISSERIAMENINGITIDIS(MENINGOCOCCUS)
VirulenceFactors
• Capsularpolysaccharide:Itpreventsthebacteriafromphagocytosis,canbetypedinto13serog
roups:
Meningococcal Diseasepattern:
Epidemic(Africa): DuetoA ○ Only5serogroupsaccountforthemajorityofcases:A,B,C,Y,andW135;
andalsobyW135 ○ Othercapsularserotypesandnoncapsulatedstrains(16%ofisolatesarenotcapsu-lated)
Outbreaks:Duetoserogroup colonizethenasopharynxofasymptomaticcarriers.
C • Outermembraneproteins:OMPisusedtoclassifyserogroupstoserotypes.
Hyperendemicdisease:Dueto • Lipopolysaccharideandendotoxin:Causesendothelialinjuryleadsto:
serogroup B
Sporadiccases:Duetoall,i.e. ○ Increasedvascularpermeabilityleadingtolossoffluidandshock
A,B,C,Y,andW135 ○ Intravascularthrombosisleadingtodisseminatedintravascularcoagulation(DIC)
○ Waterhouse-Friderichsensyndrome
○ Myocardialdysfunction
• IgAproteases—CleavemucosalIgA
• Transferrinbindingprotein.
• Adhesins:MediatedbyOPAproteinandpili.
Epidemiology
Worldwide,nearly5lakhcasesofmeningococcaldiseaseoccureachyear,and10%ofthosedie.
• Diseasepattern:Thereareseveralpatternsofthediseasenoted:
○ Epidemicsoccursmainlyinsub-SaharanAfrica—DuetogroupA(mainly)andW135.
○ Outbreaks—mainlyduetoserogroupC(insemi-closedcommunitiessuchas
schools,militarycamps,etc.).
○ Hyperendemicdisease(>10casesper100,000population)DuetoserogroupB.
○ Sporadiccases(0.3–5casesper100,000population)canoccurduetoall,i.e.A,B,C,
Y,and W135.
• Highprevalenceareaissub-SaharanbeltofAfrica(fromEthiopiatoSenegal)
• Seasonalvariationisseencommonlyinwinterandspring(coldanddryclimate)
• Age:Meningitisiscommoninearlychildhood(3monthsto5years).
• Riskfactorsthatpromotecolonizationinclude:
○ Overcrowdingandsemi-closedcommunitiessuchasschools,militaryandrefugee
○ Travellers(Hajjpilgrims)andsmoking
○ ViralandMycoplasmainfectionoftherespiratorytract
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NeisseriaandMoraxella 139
• Riskfactorsthatpromotesdiseaseinclude:
○ Deficiencyofterminalcomplementcomponents(C5–C9)
○ Hypogammaglobulinemia Risk factors that
○ Hyposplenism. promoteMeningococcal
Infectionsdisease:
Pathogenesis • Deficiencyofterminal
complement components
• Source:MCsourceofinfectionishumannasopharyngealcarriers(mainlychildren).
(C5–C9)
• Carrierratemayvaryfrom5–10%(duringinterepidemicperiod)upto70–80%(duringepidemic). • Hypogammaglobulinemia
• Modeoftransmissionisbydropletinhalation • Hyposplenism
• Spreadofinfection:Fromnasopharynx,meningococcireachthemeningeseitherby:
○ Hematogenousroute(mostcommon)or
○ Bydirectolfactorynervespreadthroughcribriformplateor
○ Rarelythroughconjunctiva.
• Casefatalityratiois80%(fallsto10%ifearlytreatmentisstarted).
ClinicalManifestations
Asymptomaticcolonizationisthemostcommonpresentation.Variousmanifestationsinclude
• Rashes:Anonblanchingrash(petechialorpurpuric)developsin>80%ofcases.
• Septicemia:Itisattributedtoendotoxininducedendothelialinjury
• Waterhouse-
Friderichsensyndrome:Itisasevereformoffulminantmeningococcemia,characterizedbylarge
purpuricrashes(purpurafulminans),shock,DIC,bilateraladrenalhemorrhageandmulti-
organfailure.
• Pyogenicmeningitis:Commonlyaffectsyoungchildren(3–5yearsofage).
• Chronic meningococcemia: Occurs rarely and characterized by petechial rash,
fever,arthritis,andsplenomegaly.
Meningococcal Infections:
• Postmeningococcal reactive disease: Immune complexes develop 4–10 days later, lead Rashes
tomanifestationslikearthritis,rash,iritis,pericarditis,polyserositisandfever. Septicemia
Waterhouse Friderichsensyndrome
LaboratoryDiagnosis Pyogenic meningitis
• Specimen: Chronicmeningococcemia
○ Forcases:Blood andCSF Post meningococcal reactivedisease
○ Forcarriers:Nasopharyngealswab
• CSFexamination:
○ Firstportioniscentrifugedandusedfor:
▪ Capsular antigen detection
▪ Biochemical analysis: ↑CSF pressure, ↑protein and ↓glucose in CSF
▪ Gramstaining:Puscellswithgram-negativediplococci,lens-shaped
○ Secondportion:Forcultureonbloodagar,chocolateagar
○ ThirdportionisenrichedinBHIbrothandincubatedfor7days
• Nasopharyngeal swabculture:OnThayerMartin medium
• Biochemicaltests:
SystemicBacteriolog
○ Oxidaseandcatalasepositive
○ Fermentglucoseandmaltosebutnotsucrose
• Serogrouping:bylatexagglutinationtest:
• Serology:AntibodiestocapsularAg(ELISA),Usefulinretrospectivediagnosisofdisease
• Moleculardiagnosis:BymultiplexPCR.
Treatment
• DOCfortreatment→Ceftriaxoneandcefotaxime
• DOC for carriers and prophylaxis→ Ceftriaxone (DOC), others: Rifampicin and cipro-
floxacin. DirectexaminationofCSF:
CapsularAgdetection
Vaccine Biochemicalanalysis:↑CSFpressure,↑proteinand
↓glucoseinCSF
Polyvalentvaccinecontaining→FourgroupsA,C,Y,W135 Gram staining: Pus cells withgram-negativediplococci,len
• NovaccineforGroupB:
○ AsGroupBcapsuleismadeupSialicacidresiduewhichisencephalitogenicand
poorly immunogenic
○ However,Outermembranevesicles(OMVs)basedvaccinestrailsaregoingon.
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• Dose50µgsingledose,immunitystartsin10days,lastsfor3years
• VaccineisindicatedtotravellerslikeHajjpilgrimage
• Contraindicationspregnancyandbelow3year(capsulebeingTindependent,ispoorlyimm
unogenic<3year).
TreatmentofMeningococcalInfections:
DOCfor treatment→
Ceftriaxoneandcefotaxime
NEISSERIAGONORRHOEAE(GONOCOCCUS)
DOC for carriers andprophylaxis→ Ceftriaxone(DOC),others:Rifampicinandciprofloxacin
N.gonorrhoeaeisnoncapsulated,Gramnegativekidneyshapeddiplococci.
VirulenceFactors
• Piliorfimbriae:Principalvirulencefactor,helpsinadhesionandinhibitphagocytosis.
• Outermembraneproteins:
○ Porin(proteinI):Theyformmembranechannels(pores)
▪ Therearetwomajorserotypes:PorB.1AandPorB.1Bserotypes.
No vaccine for Group BMeningococcus:
▪ PorB.1Astrainsassociatedwithlocalanddisseminatedgonococcalinfections(DGI)
AsGroup B capsule ismade ▪ PorB.1Bstrainsusuallycauselocalgenitalinfections.
upSialicacidresiduewhichisencephalitogenic and○poorlyimmunogenic
Opacity-associatedprotein(ProteinII):Ithelpsinadhesion,andinvasion
• Transferrin-bindingandlactoferrinbindingprotein:Itisrequiredforuptakeofiron
• IgA1protease:It degradesmucosal IgAantibody
• Lipooligosaccharide(LOS):ItdiffersfromLPSofEnterobacteriaceaebylackingthe
repetitiveO side chain.
TypingofGonococci
• Serotypingisbasedonprotein-I(porin).
• Auxotyping:Typingisbasedonnutritionalrequirementsofthestrains,e.g.AHUauxotypene
edsarginine,hypoxanthineanduracilasgrowthfactors.
Gonorrheaisavenerealdiseasereportedsinceancienttime.
1. Inmales:
• Acuteurethritisisthemostcommonmanifestation.Purulenturethraldischarge(the
word‘gonorrhea’isderivedfromflowofseedresemblingsemen)
• Theusualincubationperiodis2–7days.
• Complications:Epididymitis,prostatitis,edemaofthepenis,andbalanitis.
• Infectionmayspreadtoperiurethraltissuescausingabscesswithsinusformation
(knownaswater-canperineum).
2. Infemales:Gonococcalinfectionislesssevereinfemaleswithmoreasymptomaticcarriage:
• Mucopurulentcervicitisisthemostcommonpresentation.
• Vulvovaginiti seen in prepubertal girls and postmenopausal women, but not
inadultfemalesastheadultvaginaisresistanttogonococcalinfection(duetoitslowpHa
ndthickstratifiedsquamousepithelium).
• Salpingitisandpelvicinflammatorydiseasemayleadtosterility.
• Fitz-Hugh-Curtis syndrome: It is a rare, characterised by peritonitis and
associatedperihepatitis.
Section
3. Inboththesexes:
• Anorectalgonorrhea(asacuteproctitis):Rectalisolatesareusuallydrugresistant.
Gonococcal infection: • Pharyngealgonorrhea(spreadbyorogenitalsex)
Inmales:AcuteurethritisisMC • Oculargonorrhea.
In females: Less severewithmore carriage 4. Inneonates(Ophthalmianeonatorum):
• Characterizedbypurulenteyedischarge,occurswithin2–5daysofbirth.
• Transmissionoccursduringbirthfromcolonizedmaternalgenitalflora.
• Treatment:Silvernitratesolutionintotheeyesofnewborn(Crede’smethod).
5. Disseminatedgonococcalinfection(DGI):Occurs in0.5–3% ofuntreatedpersons:
• DGIischaracterizedbypolyarthritisandrarelydermatitisandendocarditis.
• ItismostcommonlyassociatedwithPorB.1AserotypesandAHUauxotypes.
• Menstruationandcomplement(C5–C9)deficiencyareriskfactorsforDGI
6. InHIV-infectedpersons:EnhancesthetransmissionofHIV.
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NeisseriaandMoraxella141
LaboratoryDiagnosis
Sample
• Males:Urethraldischarge Disseminated gonococcalinfection (DGI):
• Females:Endocervicalswab(highvaginalswabnotrecommended). Occursin0.5–3%ofuntreated
persons
• ForDGI:Bloodcultureandsynovialfluidculture.
Characterizedbypolyarthritisand rarely dermatitis andendo
Transportmedia AssociatedwithPorB.1A
serotypes andAHUauxotypes
• Charcoalimpregnatedswabs/medium(Stuart/Amiesmedia) Risk factors: Menstruationand complement (C5-C9)defici
• Forlongerholdingperiod:CO2generatingsystem(JEMBECsystem)
Gramstaining
• Formales:Gramstainingofurethraldischargeismoresensitive(90%):Basedonwhich
treatmentcanbestarted
• Forfemales:Gramstainingislesssensitive(50–60%)duetopresenceofcommensal
Neisseriaspp.ingenitaltract.So,Endocervicalcultureisrecommended.
Endocervicalculture:Gonococciaredifficulttogrowthanmeningococci:
• Culturemediainacutegonorrhoeae:ChocolateagarandMueller-Hintonagar
• Selectivemediaareusefulinchroniccases:
○ Thayer:Martinmedia
○ ModifiedNewYorkcitymedium
○ Martin:LewisMedia
Treatment
Gram staining of urethraldischarge:
• DOC:Single-doseregimenofCeftriaxoneandcefotaxime For males: More sensitive
• Treatboththepartners,regimenshouldalsoincludeazithromycinforChlamydia (90%),basedonwhich
• Moststrainsareresistanttopenicillinduetopenicillinaseproduction.SuchstrainsarecalledasP treatmentcan bestarted
PNGstrains(PenicillinaseproducingstrainsofN.gonorrhoeae) For females: Less sensitive(50–60%) due to presence ofco
Properties Gonococcal urethritis (GU) Nongonococcalurethritis(NGU)

Agent N.gonorrhoeae Bacterial:
• Chlamydiatrachomatis-MCcauseofNGU
• Ureaplasmaurealyticum
• Mycoplasmahominis
• Some cases may be due to
gonococcalinfection,thecoccipersistingasLforms
andhenceundetectablebyroutinetests
• Viruses:Herpesvirusandcytomegalovirus
• Fungi:Candidaalbicans
• Parasites:Trichomonasvaginalis
Onset 48hours Longer(>1week)
Urethraldischarge Purulent(flowlikeseed) Mucous to mucopurulent
SystemicBacteriolog
Complication DGI Reiter’ssyndrome:Characterizedby
Water-canperineum conjunctivitis,urethritis,arthritisandmucosallesions
OthercomplicationsarecommontobothGUandNGUsuchas: Selective media are useful inchronicgonococcalc

• Males:Epididymitis,prostatitis,seminalvesiculitisandbalanitis Thayer Martin media
ModifiedNewYorkcity
• Females:SalpingitisandpelvicinflammatorydiseaseandFitz-Hugh-Curtissyndrome
medium
Diagnosis • Gram stain, For Chlamydia: Culture on McCoy and HeLa MartinLewisMedia.
• CultureonThayer- celllines
Martinmedia Trichomonas: Detection of
trophozoiteCandida:Detectionofbuddingyeastcellsi
ndischarge
PCR:CanbedoneforHSVorChlamydia
Treatment Ceftriaxone ForChlamydia:Doxycycline
ForTrichomonas:Metronidazole
ForCandida:Clotrimazole(asvaginalcreamor
tablet)
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MENINGOCOCCUS a. Educatestudentsaboutmeningococcaltransmission
andtakepreventivemeasures
1. Vaccineavailableagainstwhichoftheserotypeof b. ChemoprophylaxistoallagainstbothgroupBand
meningococcus? (RecentQuestion2015) groupC
a. Serotypebonly c. Vaccineprophylaxisofcontactsofxavier
b. Serotypeaandconly d. Vaccineprophylaxisofcontactsofyogendra
c. Serotypebandconly
8. Conjugatevaccinesareavailableforthepreventionofinv
d. Serotype a,b andc only
asivediseasecausedbyallofthefollowingexcept:
2. Patientgivesh/olowgradefever,skinrashand a. Hinfluenzae (AIIMS2004)
increasedWBCcounts.Whatwillyoudonext? b. Streppneumoniae
a. Bloodculture (JIPMERNov2015) c. N.meningitidis(groupC)
b. CT scan for meningococci d. N.meningitidis(groupB)
c. Serology
3. A 16-year-old male patient presents with
headache,fever and neck stiffness for the past 24
GONOCOCCUS
hours. 9. Proteindegradingenzymesecretedby:
Similarhistorywaspresentoneyearback.CSFanalysissh (JIPMER, May 2015)
owsWBCcount–400/ml,with90%neutrophils.Gramsta a. Pneumococci
iningshowsgramnegativediplococci.Theimmunesyste b. Staphylococcusaureus
maffectedinthisconditionis:(JIPMERNov2014)
c. Gonococci
a. Blymphocytes
d. Enterococci
b. Tlymphocytes
c. Immunoglobulins 10. 19 years old male patient comes to the
d. Complement system emergencydepartmentwithcomplaintsofurethraldisc
harge,1 week after having unprotected sex. Gram
4. Waterhouse-Friedrichsen syndrome is a complication
stainingreveals numerous neutrophils, some gram
seenininfectionwith:
negativeintracellulardiplococci.Hewastreatedwithcef
(NEETPatternBased,TNPG2014)
triaxone 250 mg I.M; 5 days later he returns
a. Neisseriagonorrhoeae
withthesamecomplaints.Whatisthediagnosis?
b. Neisseriameningitidis
a. Chlamydia trachomatis (JIPMERNov2014)
c. Escherichiacoli
b. PenicillinresistantNeisseriagonorrhoeae
d. Mycobacteriumtuberculosis
c. ReinfectionwithNeisseriagonorrhoeae
5. WhichamongthefollowingdifferentiatesNeisseriamen d. Ureaplasmaurealyticum
ingitidisfromNeisseriagonorrhoeae?
11. TrueaboutNeisseriagonorrhoeae?
(NEETPatternBased,DNBDec2010) a. Kidneyshaped (RecentQuestions2014)
a. Itisoxidasepositive b. IsolatedinPIKESmedium
b. Itfermentsglucose
c. Itisnottransmittedthroughsexualcontact
Section
c. Itfermentsmaltose
d. ProteinIIisusefulfortyping
d. Itreducesnitrates
12. Gonococcicanbeidentifiedby? (DNBDec2011)
6. VirulencefactorsforMeningococci: (PGIDec2007)
a. GrowthonMacconkeymedium
a. Capsule
b. Growthat22°C
b. Pili
c. Bythefermentationofglucose
c. Endotoxin
d. Growthin45to60%bile
d. Coagulase
e. M. protein 13. ThevirulencefactorofNeisseriagonorrhoeaeincludes
allofthefollowingexcept: (AIIMSMay2003)
7. Xavier and Yogender stay in the same hostel of
sameuniversity. Xavier develops infection due to a. Outermembraneproteins
group b. IgAprotease
BMeningococcus.AfterfewdaysYogenderdevelopsinfe c. Mproteins
ction due to Group C Meningococcus. All of d. Pili
thefollowingaretruestatementsexcept: (AI2002)
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14. Whichisthetruestatementregardinggonococcalurethr
a. Mannitolsalt agar
itis? (PGIDec2001)
b. Thayer-Martinmedia
a. Symptomsaremoresevereinfemalesthaninmales
c. Potassiumtelluriteagar
b. Rectumandprostateareresistanttogonococci
d. TCBS
c. Mostpatientspresentwithsymptomsofdysuria
d. Singledoseofciprofloxaciniseffectiveintreatment
e. Commonlyleadstoarthritis
MORAXELLA
15. Apatienthashistoryofsexualintercoursewithacommer
cial sex worker 3 days back, has 16. Causeofangularconjunctivitisandalloptionswasof
developedgenitaldischargeresembling‘flowofseed’.W moraxella: (RecentQuestion2015)
hatmedium should be used for culture of the a. Moraxellacatarrhalis
dischargematerial? (RecentQuestion2013,
b. Moraxellalacunata
PGIJune2005))
SystemicBacteriolog
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EXPLANATIONS
MENINGOCOCCUS
1. Ans.(b)(Serotypeaandc)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep238
2. Ans(a)(Bloodculture)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep237
DefinitivediagnosisofmeningococcalmeningitisisdonebyCSForbloodculture.Serology(antibodydetection)onlyhelps
inretrospectivediagnosis.
3. Ans(d)(Complementsystem)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep144
• ThisismostprobablyacaseofpyogenicmeningitisduetoMeningococcus.
• Latecomplement(C5-C9)deficiencyisanimportantriskfactorformeningococcalinfection
4. Ans.(b)(N.meningitidis)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep237,Ananthanarayan9/ep229
Referchapterreviewfordetail.
5. Ans.(c)(Itferments...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep237,Ananthanarayan9/ep228
• MeningococcuscanfermentglucoseandmaltosewhereasGonococcuscanfermentonlyglucose.
6. Ans.(a),(b),(c)(Capsule,Pili,Endotoxin)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep236
VirulencefactorofMeningococcus:
• Refertext(chapterreview)forexplanation:
• Optiond: Coagulase is a Virulence factorfor Staphylococcus
• Optione:MproteinisaVirulencefactorforStreptococcus.
7. Ans.(c)(VaccineprophylaxisofcontactsofXavier)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep239,Ananthanarayan9/ep230,Harrison19/ep1001,18/ep1219
• MeningococcalPolyvalent vaccinecontains à A,C,Y,W135, immunitylasts for 3years
• NovaccineavailableforGroupB:
• Asitscapsuleispoorlyimmunogenic--α-2,8-N-
acetylneuraminicacidisexpressedonthesurfaceofneuralcellsinthefetussuchthat theBpolysaccharide isperceivedas
‘self’andtherefore isnotimmunogenic inhumans.
• Alsoremember,vaccineisnoteffectiveinchildrenbelow3yearsofagebecausecapsularpolysaccharidebeingTindependen
tAntigen,ispoorlyimmunogenic<3years.
8. Ans.(d)(N.meningitidis...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep239,Ananthanarayan9/ep230
Alreadyexplained:
GONOCOCCUS
9. Ans(c)(Gonococci)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep239
Section
• GonococciandMeningococciproducedIgA1proteasethatsplitsandinactivatesIgA.
10. Ans(b)(Penicillinresistant...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep241,Harrison19/ep1005-07
It’sacaseofdrugresistanceofinfectedstrain,notacaseofreinfection.
• H/ourethraldischargewith↑neutrophils,andgramnegativeintracellulardiplococci-SuggestiveofGonococcus.
• DOCforNeisseriagonorrhoeaeinfectionis-ceftriaxone
• However some strains fail to respond to beta lactams due to – i) production for beta lactamase; they are called as
PPNG(PenicillinaseproducingNeisseriagonorrhoeae)orii)maybeduetoalteredpenicillin-bindingprotein2.
(ResistancetoceftriaxoneisduetoalteredPBP2)
• Reinfection may be seen only with infected women with antibody to gonococcal OMP called Rmp (reduction
modifiableprotein);becauseRmpantibodiesblocktheeffectofbactericidalantibodiestoporinandLOS.ReinfectionisNOTs
eenfollowingtreatment.
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11. Ans.(a)(Kidney...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep239,Ananthanarayan9/ep230-31.
Referchapterreview.
12. Ans.(c)(Bythe...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep239,Ananthanarayan9/ep234
• GonococcuscannotgrowonMacConkeymedium
• Gonococcuscangrowat37°Cbutnotat22°C
• Gonococcuscanfermentglucose
• Gonococcuscannotgrowinpresenceof40%bile(ItisapropertyofEnterococcus)
13. Ans.(c)(Mpro...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep239,Ananthanarayan9/ep231
VirulencefactorforGonococcus–Refertextfor(chapterreview)explanation.
14. Ans.(c)(Mostpatientspresentwithsymptomsofdysuria)Ref:Harrison18/ep1222-23,19e/p1005
‘AcuteurethritisisthemostcommonclinicalmanifestationofgonorrheainmalesandmajorsymptomsareUrethraldischargeanddysuria,usually
withouturinaryfrequencyorurgency’………Harrison18/ep1222-23
AboutOtherOptions
• Fluoroquinolonesofferedtheadvantageofantichlamydialactivitywhenadministeredfor7days
• Third-generationcephalosporinshaveremainedhighlyeffectiveassingle-dosetherapyforgonorrhea
• AdultvaginaisresistanttoGonococcus,soinfectionislesssevereinfemale
• Inmales,itcaninvolverectumandprostateandepididymitis.
(neverinvolvetestes).AboutdetailofDGI(gonococcalarthritis)-Referchapterreview
15. Ans.(b)(Thayer-Martin...)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology1/ep240,Ananthanarayan9/ep233
• Genitaldischargeresembling‘flowofseed’.........SuggestiveofGonococcalurethritis
• SelectivemediaforcultureofGonococcus:Thayer-Martinmedium.
MORAXELLA
16. Ans.(b)(M.lacunata)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep242
SystemicBacteriolog
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CHAPTER
CorynebacteriumandBacillus 3.4
CORYNEBACTERIUM
Corynebacterium are club-shaped gram-positive, noncapsulated, non-sporing, non-
motilerods.
Corynebacteriumdiphtheriae(orKlebs-Loefflerbacillus)showstwoadditionalfeatures:
Volutingranulescanalsobepossessed by:
Corynebacteriumxerosis • Chinese letter or cuneiform arrangement in smear (V or L shaped)- due to
Gardnerella vaginalis bacterialcells divide and daughter cells tend to lie at acute angles to each other. This
Spirillium type of celldivisioniscalledsnappingtypeofdivision.
FewMycobacterium species • Metachromaticgranulespresentatendsorpolesofbacilli(alsocalledpolarbodiesor
Enterobacteraerogene
Few yeasts BabesErnstbodiesorvolutingranules):
○ Theyarestoragegranulescomposedofpolymetaphosphates.
○ Theyarebetterstainedwithspecialstains,suchasAlbert’s,Neisser’sandPonder’s
stain.
○ Granulesarewelldevelopedonenrichedmedia,suchasbloodagarorLoeffler’s
serumslope.
○ Volutingranulescanalsobepossessedby:
▪ Corynebacteriumxerosis
▪ Gardnerellavaginalis
▪ Spirillium
▪ FewMycobacteriumspecies
▪ Enterobacteraerogene
▪ Fewyeasts.
VirulenceFactor(Diphtheria Toxin)
MechanismofAction
DTistheprimaryvirulencefactorresponsibleforthediphtheria:
• Ithastwofragments:FragmentA(activeunit)andB(bindingunit)
DiphtheriaToxin(Mechanism): • FragmentBbindstothehostcellreceptors(suchasepidermalgrowthfactor)andhelpsinent
Fragment B binds to the hostcell receptors
ryoffragmentA.
Fragment A causes→ ADPribosylation of elongationfactor 2(EF2) → ↓ of EF2→ ↓proteinsynthesis→celldeath.
•
Mechanism: Similar toexotoxinAof Pseudomonas. Fragment A is internalized into the host cells and then causes→ ADP ribosylation
ofelongationfactor2(EF2)→inhibitionofEF2→irreversibleinhibitionoftranslationstepof
proteinsynthesis→celldeath.
• MechanismofDTissimilartoexotoxinAofPseudomonas.
ToxinProductionisDependenton
• Phagecoded: DT is coded by β corynephage carrying tox gene.
• Ironconcentration:Toxinproductiondependsonoptimumironconcentration(0.1mgperliter).
HigherlevelsofironinhibittoxinsynthesisbyupregulatingDTrepressorgeneinthebacterialchr
DiphtheriaToxinproductionisdependent on: omosome.
Phage coded
• DTrepressorgene(DtxR)isanirondependentnegativeregulatorofDTproductionand
Iron concentration
DTrepressorgene ironuptakeinC.diphtheriae.
Biotypes • Biotypes:AmongthethreebiotypesofC.diphtheriae,allstrainsofgravis,95–
Other species: DT is alsoproduced by C.ulcerans andC.pseudotuberculosis
99%strainsofintermediusand80–
85%ofmitisstrainsaretoxigenic.However,toxinsproducedbydifferentbiotypesareantige
nicallysimilar.
• Otherspecies:DTisalsoproducedbyC.ulceransandC.pseudotuberculosis.
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Corynebacteriumand Bacillus 147
ToxoidisusedforVaccination
• Diphtheriatoxinisantigenicandantitoxinsareprotectiveinnature.However,asitis
virulent,cannotbegivendirectlyforvaccination. ParkWilliam8strainof
• Toxincanbeconvertedtotoxoidwhichisusedforvaccination.Toxoidisaformoftoxin,where C.diphtheriae
thevirulenceislost,retainingitsantigenicity. Used as a source of toxin forpreparationofvaccine
• Toxoidformationispromotedbyformalin,acidicpH andprolongedstorage.
• ParkWilliam8strainofC.diphtheriaeisusedasasourceoftoxinforpreparationofvaccine.
• Lfunit:DTisexpressedasLoeffler’sflocculatingunit.1Lfunitistheamountoftoxinwhichfloc
culatesmostrapidlywithoneunitofantitoxin.
PathogenicityandClinicalManifestations
Diphtheriaistoxemiabutneverabacteremia:
• Bacilliarenoninvasive,presentonlyatlocalsite(pharynx),secretethetoxinswhich
spreadbybloodstreamtovariousorgans.
• Itisthetoxinwhichisresponsibleforalltypesofmanifestationsincludinglocal(respiratory)
andsystemiccomplications(excepttheskinlesionswhichmaybecausedduetotheorganis
m).
RespiratoryDiphtheria
Thisthemostcommonformofdiphtheria.Tonsilandpharynx(faucialdiphtheria)arethemost
Pseudomembrane:
common sites followed by nose and larynx and rarely non-respiratory mucosa, such Toughleatherygreyishwhite
asconjunctivaorvagina.Incubationperiodisabout3–4days: coat
• Faucialdiphtheria:Toxinelicitsaninflammatoryresponsethatleadstonecrosisoftheepith Composed of an innerband of fibrin surroundedbyneutrop
So named, as it is adherentandbleedsonremoval
eliumandexudateformationleadingtoformationofpseudomembrane(tough leathery
greyish white coat composed of an inner band of fibrin surrounded
byneutrophils,RBCsandbacteria).
• Pseudomembraneissonamed,asitisadherenttothemucosalbaseandbleedsonremovalincontr
asttothetruemembranewhichcanbeeasilyseparated(e.g.asinCandida).
• Extension of pseudomembrane: In severe cases, it may extend into larynx and medium-
sizedbronchialairwayswhichmayresultinfatalairwayobstructionleadingtoasphyxia,w
hichmandatesimmediatetracheostomy.
• Bull-neckappearance:Characterizedbymassivetonsillarswellingandneckedema.
CutaneousDiphtheria
• Characterized by punched-out ulcerative lesions with necrosis, orrarely
SystemicBacteriolog
pseudomembrane.
• Cutaneousdiphtheriaisduetotheorganismitselfandisnottoxinmediated.
• Skinlesionscanalsobecausedbynontoxigenicstrains.
• Thereisincreasingtrendofcutaneousdiphtherianowadays,especiallyinvaccinated
children,becauseantitoxinspresentinvaccinatedpeoplecannotpreventthedisease.
SystemicComplications
Neurologicmanifestations(cranialnerveinvolvement,Peripheralneuropathy,ciliaryparalysis Laboratory diagnosis ofdiphtheriais necessary o
Confirmationofclinical
)andmyocarditisarelatetoxicmanifestations,occurringafterweeksofinfection. diagnosis
Initiating the control measures
LaboratoryDiagnosis Epidemiological purposes(Not to start treatment)
Thediagnosis of diphtheria is based on clinical signs and symptoms plus laboratory

confirmation.
• Becauseofriskofrespiratoryobstruction,specifictreatmentshouldbeinstitutedimmediat
elyonclinicalsuspicionofdiphtheriawithoutwaitingforlaboratoryreports.
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• Laboratorydiagnosisisnecessaryonlyfor:
○ Confirmation of clinical diagnosis
○ Initiatingthecontrolmeasures
○ Epidemiologicalpurposes
• Laboratorydiagnosisconsistsofisolationofthebacilliandtoxindemonstration.
Isolation of the DiphtheriaBacillus

• Specimen:Throatswab(oneortwo)containingfibrinousexudatesandaportionof
Culturemedia: membrane
Enrichedmedium:Suchas
Loeffler’sserumslope • Directsmearmicroscopy:
Detectsgrowthearly(6–8 ○ Gramstain:C.diphtheriaeappearsasirregularlystainedclubshapedgram-
hrs) positivebacilli arranged in Chinese letter or cuneiform arrangement (V or L
Best medium formetachromatic granulesproduction shaped). It
Selectivemedium:Suchas
PTAandTinsdale medium
isdifficulttodifferentiatethemfromothercommensalcoryneformsfoundintheres-
Bestmediaforisolation piratorytract.
Blackcoloniesappearonly ○ Albert’s stain is more specific for C.diphtheriae, where they appear as green
after48hours bacilliwithbluishblackmetachromaticgranules.
• Culturemedia:
○ Enrichedmedium:suchasLoeffler’sserumslope:
▪ Advantages:(i)detectsgrowthearly(6–8hrs),(ii)bestmediumformetachro-
maticgranulesproduction.
▪ Disadvantage:Asitisanenrichedmedium,ifincubatedbeyond6–8hrs,itsup-
portsgrowthofotherthroatcommensalsalso.
○ Selectivemedium:suchasPotassiumtelluriteagar(PTA)andTinsdalemedium:
▪ Advantage: Throat commensals are inhibited, hence they are best media
forisolationofC.diphtheriaefromcasesaswellascarriers;asthenormalflorawillbe
inhibited.
▪ Disadvantage:Theblackcoloniesappearonlyafter48hoursofincubation.
• Biochemicalidentification:
○ Hiss’sserumsugarmedia:C.diphtheriaefermentsglucoseandmaltose(byallbio-
types)andstarch(byonlygravis)
○ Pyrazinamidasetest:NegativeforC.diphtheriae,C.ulceransandC.pseudotuberculosis
○ Ureasetest:NegativeforC.diphtheriae;butC.ulceransandC.pseudotuberculosisareureasep
ositive.
○ Corynebacteriumiscatalasepositivebutoxidasenegativeandnonmotile.
Toxindetection:
ToxinDemonstration
In vivo tests (Guinea pigsinoculation) As the pathogenesis is due to diphtheria toxin, mere isolation of bacilli does not
In vitro tests: completethediagnosis.Toxindemonstrationshouldbedonefollowingisolation,whichcanbeoft
Elek'sgelprecipitationtest
Detectionoftoxgene-byPCR wotypes;invivoandinvitro:
Detectionofdiphtheriatoxin • Invivotests(Guineapigsinoculation)by(i)subcutaneoustest,(ii)Intracutaneous
byELISA inoculation
Section
Cytotoxicityproducedoncelllines
• Invitrotests:
○ Elek’sgelprecipitationtest
○ DetectionoftoxgenebyPCR
○ DetectionofdiphtheriatoxinbyELISAorimmunochromatographictest(ICT)
○ Cytotoxicity producedon celllines.
TypingofC.diphtheriae
• Biotyping (McLeod’s classification): C.diphtheriae can be typed into four biotypes, such
asgravis, intermedius, mitis and belfanti based on various properties. Biotype belfanti is
anitratenegativevariantofmitisbiotype.
• Other methods: Serotyping, Bacteriophage typing, Bacteriocin typingand
Moleculartypingmethods,suchasPFGE.
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Corynebacteriumand Bacillus149
C.diphtheriaebiotypes
Properties Gravis Intermedius Mitis
Morphology Short,nogranules Longbarred,poorgr Longcurved,prominentgra
anules nules
ColoniesonPTA Daisyhead Frog’seggcolony Poachedeggcolony
Starch fermentation +ve –ve –ve
Toxigenicstrains 100% 95–99% 80–85%
Virulence Severe Moderate Mild
Occurrence Epidemic Epidemic Endemic
Complications Paralyticand Hemorrhagic Obstructive
hemorrhagic
Hemolysis Variable Nonhemolytic Hemolytic
Epidemiology
Diphtheria is an endemic disease known from ancient time. However, there is
decliningtrendofdiphtheriacasesinmostdevelopedaswellasafewdevelopingcountriesincludi
ngIndiaduetowidespreadvaccinationcoverage.
• Sourceofinfection:Carriers(95%)aremorecommonsourceofinfectionthancases(5%).
• Carriersmaybetemporary(persistsforamonth)orchronic(persistsforayear).Nasalcarrie
rsaremoredangerousduetofrequentsheddingthanthroatcarriers.Incidenceofcarrierrate
variesfrom0.1to5%.
• Transmissionisviatheaerosolroute,orrarelybycontactwithinfectedskinlesions.
• Reservoir:Humansaretheonlyreservoir
• Age:Diphtheriaiscommoninchildrenaged1–5years.Withwidespreadimmunization,a
shift in age has been observed from preschool to school age. Newborns are
usuallyprotectedduetomaternalantibodies.
Treatment
Treatmentshouldbestartedimmediatelyonclinicalsuspicionofdiphtheria:
• AntidiphtheritichorseserumorADS(antitoxin):Itisthetreatmentofchoiceasit
neutralizesthetoxin. Antibiotics are useful indiphtheriaonly:
• Antibiotics:Penicillinor erythromycinisthe drugof choice.Antibioticplays Ifgivenearly(<6hrsof
infection),beforethetoxinrelease
aminorroleasitisofnouseoncethetoxinissecreted.However,antibioticsareuseful: Topreventfurtherreleaseof
○ Ifgivenearly(<6hrsofinfection),beforethetoxinrelease toxinbykillingthebacilli
○ Preventfurtherreleaseoftoxinbykillingthebacilli Totreatmentforcutaneous
○ Treatmentforcutaneousdiphtheria. diphtheria
• Treatmentofcarriers:Drugofchoiceiserythromycin
SystemicBacteriolog
VaccineorADS Antibiotic
Carrier Noteffective Effective
ClinicalDiphtheria ADSis treatmentofchoice Not
Vaccineisgivenforprevention effective(Exceptearly
stage)
CutaneousDiphtheria Noteffective Effective
Prophylaxis(Vaccination)
• Activeimmunizationisdonebydiphtheriatoxoidthatinducesantitoxinproductionin
thebody.
• Protectivetiterofantitoxinis>0.01Unit/ml.
• Herdimmunityof>70%isrequiredtopreventepidemicspreadofdiphtheria.
• However,vaccineisnoteffectivefor:
○ Preventionofcutaneousdiphtheria
○ Eliminationofcarrierstage
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• Typesofvaccine:
○ Singlevaccine:Diphtheriatoxoid(alumorformalprecipitated)
○ Combinedvaccine:
▪ DPT:ContainsDT(diphtheriatoxoid),Pertussis(wholecell)andTT(tetanustoxoid
)
dT ▪ DaPT:ContainsDT,TTandacellularpertussis(aP)
ContainsTTandadultdose(2Lf)ofdiphtheriatoxoid. ▪ DT:ContainsDTandTT
Giventoadults>12years ▪ dT:Containsadultdose diphtheriatoxoid(d)and TT.
DPTVaccine
• DPTisthepreparationofchoiceforvaccinatinginfants,because:
○ Infantscanbeimmunizedsimultaneouslyagainstthreeimportantchildhooddis-
eases: diphtheria,tetanusand pertussisbysingleinjection.
○ PertussiscomponentactsasadjuvantandincreasesimmunogenicityofDTandTT.
• TherearetwotypesofDPT:
○ Plainformoltoxoid(orfluidtoxoid):Toxoidispreparedbyincubatingtoxinwith
formalin
○ Adsorbed(alumadsorbed):Alumactsasadjuvantandincreasestheimmunogenic-
ityoftoxoid.
• AdministrationofDPT:
○ Schedule: Under national immunisation schedule of India, total five doses are
given;three doses at 6, 10 and 14 weeks of birth followed by two booster doses at
16–24monthsand5years.
○ Site: DPT is given deep intramuscularly (IM) at anterolateral aspect of thigh, (glu-
teal regionis notpreferredas fatmay inhibitDPTabsorption)
○ Thiomersal(0.01%)isusedaspreservative
○ Storage: DPT should be kept at 2–8°C, if accidentally frozen then it has to be dis-
carded.
○ Dose:1dose(0.5ml)contains:
▪ Glaxo:25Lf(DT),5Lf(TT),20,000million(Pertussiskilledbacilli)
▪ Kasauli:30Lf(DT),10Lf(TT),32,000million(pertussiskilledbacilli)
○ dT:ItcontainsTTandadultdose(2Lf)ofdiphtheriatoxoid.dTisgiventoadults
>12years(3dosesat0,1month,and1year).
• ReactionsfollowingDPTadministration:
○ Mild:Feverandlocalreaction(swellingandindurations)areobservedcommonly.
○ Severe:WholecellkilledBordetellapertussisisencephalitogenic.Itisassociatedwithn
eurologicalcomplications.Hence,DPTisnotrecommendedafter6yrsofage.
NondiphtheriaeCorynebacteriumspecies
C.ulcerans:Mimicsrespiratory ○ AbsolutecontraindicationtoDPT:
Section
diphtheria ▪ Hypersensitivitytopreviousdose
C. pseudotuberculosis (PreiszNocard bacilli): Affect▪ sheepandhorse
Progressiveneurologicaldisorder
C. minutissimum: Causeserythrasma,whichgivescoralredcolorunderwoodlamp.
Acelluarpertussiscomponent(aP)isdevoidofneurologicalcomplicationandisgivensafely
C. jeikeium: Multidrug resistantspecies, causes opportunisticinfectioninhuman.
C.parvum: Example ofimmunomodulator toolderchildren.
Schicktest:Itisatoxin-antitoxinneutralizationtest,obsoletenowadays:
• Itwasinuselongbackwhenthevaccinewasintroducedinitially.
• Thetest was being done on the people before starting immunization to identify
susceptible individuals.
• Susceptibleindividualsusedtodeveloperythemaandindurationontestarmfollowingintra
dermal inoculation of toxin. Vaccine was administered only to those
susceptiblepeople.
• Sincenowsafertoxoidpreparationsareavailable,Schicktestisnotperformed.
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NondiphtheriaCorynebacterium
• Clinicaldiphtheriae:C.ulceransandC.pseudotuberculosiscanalsoproduceDTandproduce
clinicaldiphtheriae.However,theyareureasepositiveandusuallyaffectanimals.
○ C.ulcerans:Mimicsrespiratorydiphtheria,transmittedbycowmilk
○ C.pseudotuberculosis(PreiszNocardbacilli):Affectsheepandhorse
• C.minutissimum:Causescutaneouslesioncallederythrasma,whichgivescoralredcolor
underwoodlamp.
• C.jeikeium:Multidrugresistantspecies,causesopportunisticinfectioninhuman.
• C.parvum,e.g.ofimmunomodulator.
BACILLUS
Sporeformingbacillibelongtotwogenera:
• Bacillus:Theyareobligateaerobes;havingnonbulgingspores
• Clostridium:Theyareobligateanaerobeswithbulgingspores.
BacillusAnthracis
B.anthracisisthecausativeagentofanimportantzoonoticdiseasecalledanthrax.Italsogainedimp
ortancerecentlybecauseofitsabilitytobeusedasbiologicalweapon.
AnthraxToxin:Tripartitetoxin,consistingof:
VirulenceFactorsandPathogenesis Edemafactor:↑hostcellcyclic
AMP.
Pathogenesisofanthraxisduetotwoimportantvirulencefactors;anthraxtoxinandcapsule.
Protectivefactor:Helpsin
binding
Anthraxtoxin Lethal factor: It causes celldeath
• Itisatripartitetoxin,consistingofthreefragments:
○ Edemafactor:Itistheactivefragment;actsasadenylcyclase→increaseshostcellcyclicA
MP.
○ Protectivefactor:Itisthebindingfragmentthatbindstothehostcellreceptor.
○ Lethalfactor:Itcausescelldeath,inhibitsmitogenactivatedproteinkinasepathway.
• Thesefragmentsarenottoxicindividually,butincombination,theyproducelocal
edemaandgeneralizedshock.
• Toxinsynthesisiscontrolledbyaplasmid(pX01).Lossofplasmidmakesthestrainavirulent.
ThiswasprobablythebasisoforiginalanthraxvaccinepreparedbyPasteur.
• Liveattenuatedsporevaccine(Sterne,Mazucchi):preparedbydeletingthecapsule
genes.
Anthraxcapsule
SystemicBacteriolog
B.anthracishas a polypeptide capsule made-up of polyglutamate (in contrast to the AnthraxTransmission:
polysaccharidecapsulepresentinothercapsulatedbacteria). Cutaneous(MCmode)
Byinhalationofspores
• Capsuleisplasmid(pX02)coded. Ingestionofcarcasses
• Itinhibitscomplementmediatedphagocytosis. Indirectly through fomites
PathogenesisandClinicalManifestations
Anthraxisprimarilyazoonosis.Herbivorousanimalssuchascattle,sheepandlessoften
horsesandpigsareaffectedmorecommonlythanthecarnivorousanimals.
HumanTransmission:Humanbeingsacquireinfectionby:
• Cutaneousmode:Bysporesenteringthroughtheabradedskin;seeninpeoplewithoccupatio
nalexposuretoanimals(mostcommonmode)
• Byinhalationofspores
• Ingestionofcarcassesofanimalsdyingofanthraxcontainingspores(manifestedas
bloodydiarrhea)
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Typesofhumananthrax:Mainlythreetypes:
i. Cutaneous,
ii. Pulmonaryand
iii. Intestinalanthrax
Cutaneousanthrax Pulmonaryanthrax
Also called Hideporter’sdisease Woolsorter’sdisease
(as it commonly occurs in dock workerscarrying (as it is seen in workers of
loads of hides and skins on their barebacks) woolfactory,duetoinhalationofdustfromi
CutaneousAnthrax: nfectedwool)
CalledasHideporter’s Transmission Cutaneous exposure to spores (enter Inhalationofspores
disease
throughabradedskin)
Feature:Malignantpustule
Characterized Malignant pustule: Hemorrhagic
by • Painless coal-black, necrotic pneumonia:Bacilli spread by
escharsurrounded by non-pitting lymphatics orblood,leadingto:
induratededema. • Bacteremia
• The name anthrax, which means • Hemorrhagicmediastinitis
coal,comesfromtheblackcoloroftheeschar. • Hemorrhagicmeningitis
• However,itisanonmalignantcondition.
Occupations Dockworker,butcher,abattoirandfarmer Woolfactory
Occurrence MostCommon(95%) Rare
PulmonaryAnthrax:
CalledWoolsorter’sdisease Prognosis Self-limiting,rarelybecomesfatalifuntreated. Fatal
Feature: Hemorrhagicpneumonia Bioterrorism Rarelyassociatedwithbioterrorism Most common form to be
associatedwithbioterrorism
B.anthracisasanAgentofBioterrorism
• B. anthracishas been a major agent of bioterrorism andbiologic warfare such as
outbreaksinSverdlovskin1979andinUnitedStatesin2001
• Pulmonaryanthraxisthemostcommonformseeninbioterrorismoutbreaks.
• Itoccursviainhalationofanthraxsporesfromcontaminatedanimalproducts.
LaboratoryDiagnosis(AnthraxvsAnthracoidBacilli)
B.anthraciscanbedifferentiatedfromotherBacillusspecies(AnthracoidBacilli)bythepresenceo
ffollowingproperties:
LabDiagnosisofAnthrax:
• B.anthracisisNonmotileandCapsulated
Gram staining: Bamboo stickappearance
Medusaheadappearancecolony • MCFadyeanreaction:Polypeptidecapsuleisseenasamorphouspurplematerialsurroundin
Gelatinstab:Appearas g bluebacilliwhenstainedwith polychromemethyleneblue.
invertedfirtreecolony • Gramstaining:chainofbacilliarrangedinbamboostickappearance.
Solid media with penicillin:String of pearl•appearancecolonies
OnAgarplate:Medusaheadappearancecolony(underlowpowermicroscope)
Blood agar: Nonhemolyticcolonies • Gelatinstab:Appearasinvertedfirtreeappearance
• Solidmediawithpenicillin:Stringofpearlappearanceinculturesmear
• Bloodagar:nonhemolyticcolonies
• Selective medium:PLETmedia
• DFA(Directfluorescentantibodytest):Detectscapsularantigen.Itisusedforconfirmation
Section
ofdiagnosisduringbioterrorismoutbreaks.
• Ascoli’sthermoprecipitintest:Itisaringprecipitationtest,detectsanthraxantigens.
• Sporescanbedemonstratedbyphasecontrastmicroscopeoruseofspecialstainssuch
ashotmalachitegreen(Ashby’smethod)or0.25%sulfuricacid(sporesareacidfast).
• LipidgranulescanbedemonstratedbySudanblackB(Burdon’smethod).
Guidelines for Diagnosis of Anthrax during Bioterrorism Attacks (CDC,

2001)Following2001USAattack,CDChaspreparedguidelinesforidentificationofB.anthracisdurin
gbioterrorism.
• Forpresumptive identification of anthrax: Any large gram-positive bacillus with
morphologyandculturalpropertiessimilartoanthraxbacillus
• Forinitialconfirmation,thetestsdoneareLysisbygammaphageandDFA
• FurtherconfirmationisdonebyPCR.
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Treatment
Anthraxcanbesuccessfullytreatedifthediseaseispromptlyrecognizedandappropriate
therapyisinitiatedearly.
• Antibioticsfortreatment:Consistsofciprofloxacinordoxycycline,plusclindamycin,and/
orrifampin,for60days.
• Antibioticsforpost-exposureprophylaxis:
○ Ciprofloxacinfor60days and
○ Doxycycline, for60 days orAmoxicillin, for 60 days
• Raxibacumab:Itisamonoclonalantibodythatneutralizesanthraxtoxin(protectiveantigen).Itis
intendedfortheprophylaxisandtreatmentofinhaledanthrax.
Prevention
• Liveattenuated,noncapsulatedsporevaccine(Sternvaccine):Itisextensivelyusedin
animals,butnotforhumanuse.
• Alumprecipitatedtoxoidvaccine:Itispreparedfromtheprotectiveantigen.Itissafeandeffectivef
orhumanuse.
OtherBacillusSpecies
BacillusCereus
Itisanormalhabitantofsoil,alsowidelyisolatedfromfooditems,suchasvegetables,milk,
cereals,spices,meatandpoultry.
Manifestations:
• Foodpoisoning:Itproducestwotypesoftoxins;diarrhealtoxinandemetictoxin
• Oculardisease:Causesseverekeratitisandpanophthalmitisfollowingtraumatothe
eye. B.cereus(Emetictypetoxin):
• Otherconditions:Itrarelycausessystemicinfections,includingendocarditis,meningitis,osteo Preformed toxin
myelitis,andpneumonia;thepresenceofamedicaldeviceorintravenousdruguse.Laboratorydiag IP:1–5hours
Heatstable
nosis:B.cereusismotile,noncapsulatedandnotsusceptibletogammaphage.
MC food: Rice (Chinese friedrice)
Itcanbeisolatedfromfecesbyusingselectivemediasuchas; MCfeature:Vomiting,abdominalcramps
• MYPA(Mannitol,eggyolk,phenolred,polymyxinandagar) MCserotype:1,3,5
• PEMBA(polymyxinB,eggyolk,mannitol,bromothymolblueandagar)
Treatment:B.cereusissusceptibletoclindamycin,erythromycin,vancomycin.Itisresistant
topenicillin(byproducingβ-lactamase)andtrimethoprim.
B.cereus Diarrhealtype Emetic type
Incubationperiod 8–16hours 1–5hours
Toxin Secretedinintestine Preformed toxin
(SimilartoClostridiumperfringens (formedindiet,similarto
SystemicBacteriolog
enterotoxin) S.aureusenterotoxin)
Heatlabile Heatstable
Fooditemscontaminated Meat,vegetables,driedbeans,cereals Rice(Chinesefriedrice)
Clinicalfeature Diarrhea,fever,rarelynausea Vomiting,abdominalcramps
Serotypeinvolved 2,6,8,9,10,12 1,3,5
BacillusThuringiensis
Itmayoccasionallyproducefoodpoisoning.Itisalsousedaslarvicidalagentformosquitocontrol.
Bacillus usedas SterilizationControl

• B.stearothermophilusandB. subtilis: Both are used as biological controls for autoclave and
plasmasterilization.
• B.pumilusisusedasbiologicalcontrolforionizingradiation.
• B.globigiiisusedasbiologicalcontrolforethyleneoxide.
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CORYNEBACTERIUMDIPHTHERIAE 9. Inacompletelyandadequatelyimmunizedchildagainst
Diphtheria, the throat swab was collected. Itshowed
1. Achildwithbullneckandmembraneovertonsil.What is the presence of Corynebacterium diphtherialike
mechanism of the toxin responsible for organisms on Albert staining. These organismscan
thiscondition? (JIPMERMay2016) have one of the following properties on
a. IncreasedcAMP furtherlaboratoryprocessing: (AIIMSNov2004)
b. InhibitsGABAneurons a. ItcangrowonPotassiumtelluritemedium
c. InhibitsElongationFactor-2 b. ItwouldshowapositiveElek’sgelprecipitationtest
2. Which Corynebacterium carries toxin similar to c. Itcanbepathogenictoexperimentalguineapigs
thatcausingdiphtheria toxin? (RecentQuestion2015) d. Itcanproducecytotoxicityintissuecultures
a. C.auris 10. A 12-year-old child presents with fever and
b. C.jivelai cervicallymphadenopathy.Oralexaminationshowsagr
c. C.ulcerans eymembrane on the right tonsil extending to the
3. TrueaboutCorynebacteriumdiphtheriae: anteriorpillar.Whichofthefollowingmediumwillbeide
(AIIMSMay2007/Nov2007.DNBDec2010,AI alforthe culture of the throat swab for a rapid
2000/2011,NEETPatternBased) identificationofthepathogen?
a. All types producetoxin (NEETPatternBased,AIIMSNov2002)
b. Toxinproductionisdependentuponcriticalconcentr a. Nutrientagar
ationofiron b. Bloodagar
c. Codedbyplasmid c. Loffler’sserumslope
d. InhibitcAMP d. Lowenstein-Jensenmedium
4. Proteinsynthesisisinhibitedbythetoxin(s)of: 11. Metachromaticgranulesseenin: (PGI2000)
a. Pertussis (AIIMS2000,PGIMay2012) a. C.diphtheriae
b. Cholera b. Mycoplasma
c. Pseudomonas c. Gardnerella
d. Diphtheria d. Chlamydia
e. Shigatoxin e. Staphylococcus
5. Metachromaticgranulesareseenin: (DNBDec2011) 12. PositiveSchick’stestindicatesthatpersonis:
a. Corynebacterium b. E.coli (NEETPatternBased,AI2002,AIIMS2000,AIIMSNov
c. Yersinia d. Pseudomonas 2007,MP2007,Kolkata2003)
a. Immunetodiphtheria
6. Thetypeofdiphtheriawithhighestmortalityis:
b. Hypersensitivetodiphtheria
a. Pharyngeal (JIPMER2009)
c. Susceptibletodiphtheria
b. Nasal
d. Carrierofdiphtheria
c. Laryngeal
d. Conjunctival 13. Herdimmunityover…….
Section
%isrequiredtopreventepidemicspreadofdiphtheria:
7. Trueaboutdiphtheriais: (SGPGI2008)
(DNB2000)a. 50 b.55
a. Causecranialnervepalsiesin2ndand3rdweeks
c. 60 d. 70
b. Treatmentwitherythromycin
14. DOCfordiphtheriacarrier: (DNB03)
c. Itisgram-negativeorganism
a. Erythromycin
d. Passive immunization is harmful and should not be
b. Tetracycline
tried
c. Penicillin
8. ThespecialstainusedtoidentifyC.diphtheriaeina d. DPT
throatswabis: (MHPG2015)
15. Diphtheriacarriersarediagnosedby: (MP2000)
a. Albert’sstain
a. Throatculture
b. Giemsastain
b. Gram’sstaining
c. Gram’sstain
c. Albert’sstain
d. Indiaink
d. Schick’stest
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BACILLUSANTHRACIS 24. Allaretrueaboutcutaneousanthraxexcept:

(JIPMER2009)
16. Wool sorter diseaseis: (RecentQuestion2015) a. Extremelypainful
a. Cutaneousanthrax b. Thewholeareaiscongestedandedematous
b. Pulmonaryanthrax c. Centralcrustationwithblackeschar
c. Intestinalanthrax d. Satellitenodulearoundinguinalregion
17. Eschar isformed bywhichof thefollowing organism? 25. Apersonworkinginanabattoirpresentedwithapustule
(RecentQuestions2014) on his hand that progressed to form an ulcer.Smear is
a. B.henselae b. B.anthracis taken from the ulcer and sent to laboratoryfor
c. Staphaureus d. E.coli investigation. Which of the following is the
beststaintodeterminethecausativeagentoftheulcer?
18. Anabattoirworkerpresentedwithamalignantpustule
(AIIMSNov2012,AI2007,AIIMSNov2006)
on his hand that progressed to form an
a. Trichromemethyleneblue
ulcer.Smearwastakenfromtheulcerandsenttolaborato
b. Carbolfuschin
ryforinvestigation.Thediagnosisis:(AIIMSNov2012)
c. Acidfaststain
a. Cutaneousanthrax
d. Calcoflourwhite
b. Carbuncle
26. Bacillusanthracis,falsestatementis:
c. Ulceratingmelanoma
a. Gram-negative (RecentQuestion2013)
d. Infectedrodentulcer
b. Bacilli in longchain
19. Bacillususedtotestefficacyofsterilizationbyautocla c. Nonhemolytic colony in blood agar
veis: (DNBDec2010) d. Medusaheadappearanceonnutrientagar
a. Bacillussubtilis 27. Medusaheadcolonyisseenin:(RecentQuestions2014)
b. Bacilluspumilis a. Bordetellapertussis b. Bacillusanthracis
c. Bacillusstearothermopilus c. Bartonellahenselae d. Bacteroidesspecies
d. Coxiellaburnetti
20. Trueaboutanthrax: (PGI June 2009) BACILLUSCEREUS
a. Cause by Gram-positive bacilli
b. Soilreservoir 28. Bacilluscereusfoodpoisoningisassociatedwithwhichf
c. Sporeformationtakesplaceinsidethebody ood? (WestBengal2016)
d. Morecommonincarnivorousthanherbivores a. Firedrice b. Bakedpotato
e. Penicillinis drug ofchoice c. Dairyproducts
21. Invertedfirtreeappearanceischaracteristic: 29. CharacteristicofBacilluscereusfoodpoisoningis:
a. Bacillusanthrax (JIPMER2004) a. PresenceofFever (AIIMS 2010)
b. Haemophilusinfluenzae b. PresenceofPainabdomen
c. Yersiniapestis c. AbsenceofVomiting
d. Brucella d. AbsenceofDiarrhea
22. Whichofthefollowingistrueregardinganthrax: 30. IncubationperiodforB.cereus: (PGIJune2008)
a. 1–6hrs b. 8–16 hrs
SystemicBacteriolog
(PGIDec2001)
c. 24hrs d. >24hrs
a. McFadyeanreactionshowscapsule
e. Weeks
b. Humansareusuallyresistanttoinfection
c. Lessthan100sporescancausepulmonaryinfection 31. Chineserestaurantsyndromeaftereatingfriedrice
d. Gramstainshowsorganismwithbulgingspores andvanillasauceisdueto:(APPG2014,MHPG2014)
e. Sputummicroscopyhelps indiagnosis a. Clostridiumperfringens
b. Bacilluscereus
23. AnthraxbacillidiffersfromAnthracoidbacilliby c. Staphylococcusaureus
being: (PGMEE2006) d. Clostridiumbotulinum
a. Noncapsulated
32. Coralredcolorunderwoodlampexamination.Whatisth
b. Strictaerobe ediagnosis? (JIPMERNov2015)
c. Nonmotile a. Ecthyma b. Erythema
d. Haemolyticcoloniesonbloodagar c. Erythrasma
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EXPLANATIONS
CORYNEBACTERIUMDIPHTHERIAE
1. Ans(c)(InhibitsElongationFactor-2)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p244
• Achildwithbullneckandmembraneovertonsil….suggestiveofdiphtheria
• DiphtheriatoxininhibitsElongationfactor-2,whichleadstoinhibitionoftranslationstepofproteinsynthesis.
2. Ans.(c)(C.ulcerans)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep244
3. Ans.(b)
(Toxinproductionisdependentuponcriticalconcentrationofiron)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep244,Ananthanarayan8/ep238-39
• Optimumconc.ofiron(0.1mg/L)isrequiredforexpressionofdiphtheriatoxin.
• AmongthebiotypesofC.diphtheriae,allmostallstrainsofgravis,95–99%strainsofintermediusand80–85%strains
ofmitisaretoxigenic.
4. Ans.(c)(d)(e)(Pseudomonas,Diphtheria,Shiga)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep239,244
Mechanismofactionofexotoxin:
• ToxinthatincreasescAMP:Pertussistoxin,Choleratoxin,heatlabiletoxinofE.coli,Anthraxtoxin(edemafactor)
• ToxinthatincreasescGMP:HeatstabletoxinofE.coli
• Toxinthatinhibitsproteinsynthesis:
○ Byinhibitingribosome:ShigatoxinandVerocytotoxinofE.coli
○ Byinhibitingelongationfactor2-DiphtheriatoxinandPseudomonastoxin
• Neurotoxin:
○ BlocksthereleaseofglycineandGABA:Tetanospamsin
○ Blocksthereleaseofacetylcholine:botulinumtoxin
5. Ans.(a)(Corynebacterium)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep243,Ananthanarayan9/ep236
• MetachromaticGranuleisproducedByC.diphtheriae,C.xerosis(Referchapterreviewforthedetail)
6. Ans.(c)(Laryngeal)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep244,Harrison19/ep979,18/ep1189Formation
of extensive pseudomembrane over larynx carries the risk of airways obstruction.
7. Ans.(a)(Cause…)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep245,Harrison19e/p979,18/ep1190
• Manifestationsmayappearduringthefirstorsecondweekofillness,typicallybeginningwithdysphagiaandnasal
dysarthriaandprogressingtoothersignsofcranialnerveinvolvement.
Aboutotheroptions
Optionb:Promptadministrationofdiphtheriaantitoxiniscriticalinthemanagementofrespiratorydiphtheria.
• Antibioticshavenoroleoncethetoxinisformedandaremainlyusedtopreventtransmissiontoothersusceptible
contacts.
Section
• ProcainepenicillinGgivenfortreatmentanderythromycingivenforcarriers.
Option c:C.diphtheriaeisagram+vebacilli
Optiond:Passiveimmunizationiscriticalinthemanagementofrespiratorydiphtheria.
LABDIAGNOSISOFDIPHTHERIA
8. Ans(A)(Albert’sstain)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep243
9. Ans.(a)(ItcangrowonPotassiumtelluritemedium)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep246
• ThischildisacarrierforC.diphtheriae.
• Adequatevaccinationcanpreventthediseasebutnotthecolonization.
‘Massimmunizationmightresultindisappearanceoftoxigenicstrainsfrommanypopulationsbuthasnoeffectoncarriageofnontoxige
nicstrains.’
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• Sincethechildiscompletelyvaccinated,antitoxinlevelinserumcanalwaysneutralizethetoxinbutitcannotprevent
thecolonizationofnontoxigenicstrain.
• So, the organism can be grown on Potassium tellurite medium but since it might be a nontoxigenic strain, so any
testtodetectthetoxigenicitywillbenegative.
10. Ans.(c) (Loffler’s…) Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep246,Ananthanarayan9/ep237
• RapididentificationofC.diphtheria:Loffler’sserumslope(6–8hour)
• Bestmediaforisolationincarriers/contacts/convalescent:PTA(48hour)
• Metachromaticgranulesbestdevelopson:Loffler’sserumslope
11. Ans.(a),(c)(C.diphtheriae,Gardnerella)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep243,Anan-thanarayan9/
ep237
• Fordetail:Referchapterreview
12. Ans.(c)(Susceptible…)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep248,Ananthanarayan9/ep241
• Positivetestmeansthatpersonissusceptibletodiphtheria:Soimmunizationisrequired.
• Shicktest:Fordetail,Referchapterreview
13.Ans.(d)(70%)Ref:,Park23/ep161,22/ep152
• Herdimmunityover70%isrequiredtopreventepidemicspreadofdiphtheria.
14. Ans.(a)(Erythromycin)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep248,Park23/ep161,22/ep153
• DOCor carriersof diphtheria:Erythromycin
15. Ans.(a)(Throatculture)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep246,Park23/ep161,22/ep152
• Carriersharborthediphtheriabacilliinthroat,hencecanbeeffectivelydiagnosedbythroatculture.
BACILLUSANTHRACIS
16. Ans.(b)(Pulmonaryanthrax)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep251
17. Ans.(b)(B.anthracis)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep252,Ananthanarayan9/ep245
18. Ans.(a)(Cutaneousanthrax)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep251,Ananthanarayan9/ep247
• Malignantpustuleisthecharacteristicfeatureofcutaneousanthrax
19. Ans.(c)(Bacillus…)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep255,Ananthanarayan9/ep32
IndustrialApplicationofBacillusspecies:Refertext
20. Ans.(a),(b)(CausebyGram-positivebacilli,Soilreservoir)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep251-
53,Ananthanarayan8/ep243-248
• Optiona:BacillusanthracisisaGram-positivebacilli
• Optionb:Reservoirisanimalandsoil.Animalgetsinfectionbyinhalationofsporesinsoil
SystemicBacteriolog
• Optionc:Sporeformationnevertakesplaceinsidethebody
• Pleaseremember‘sporeformationtakesplaceinunfavorableconditionsandhumanbodyisafavorableenvironment for
the organism like Bacillus anthracis causing anthrax and Clostridium perfringens causing gasgangrene.’
• Optiond:Anthraxismorecommoninherbivoreslikesheepandcattlethancarnivorous
• Option e: Jawetz 24/e p205 and 25/e p167 says- ‘Ciprofloxacin is recommended for treatment; penicillin G, along
withgentamicinorstreptomycin,haspreviouslybeenusedtotreatanthrax.’
21. Ans.(a)(Bacillusanthracis)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep253,Ananthanarayan9/ep245
• InvertedfirtreeappearanceingelatinstabcultureseenbyBacillusanthracis
• BacillusanthracislaboratoryfindingsRefertext.
22. Ans.(a),(b),(c),(e)(McFadyeanreactionshowscapsule,Humansareusuallyresistanttoinfection,Lessthan100sporescan cause
pulmonary infection, Sputum microscopy helps in diagnosis) Ref: Apurba Sastry’s Essentials of Medical
Micro-biology/p250-53,Ananthanarayan9/ep247-48,8/ep243
• Optionb:Greenwood16/ep226says‘HumansarerelativelyresistanttoinfectionwithBanthracis’
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• Optionc:Harrison17/ep1344and18/
ep1769says‘WhileanLD50of10,000sporesisagenerallyacceptednumberforanthraxithasalsobeensuggestedthatasfewasonet
othreesporesmaybeadequatetocausediseaseinsomesettings.’
• Optiond:Gramstainshowsorganismwithnonbulgingspores.
• BulgingsporesareseenforClostridium
• Optione:Microscopyofthesamplelikelesion,blood,sputumrevealsgram-positivebacilliinchain.
23. Ans.(c)(Nonmotile)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep254,Ananthanarayan9/ep249
• Anthraxbacillusiscapsulated,nonmotileandproducenonhemolyticcolonyonbloodagar.
• BothAnthraxbacilliandAnthracoidbacilliarestrictaerobe.
• AlsoReferthetableonthetext.
24. Ans.(a)(Extremelypainful)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep251,Ananthanarayan9/
ep247CutaneouslesionsinAnthraxarepainless.
25. Ans.(a)(Tric...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep251-52,Ananthanarayan9/ep248,8/ep246
• ThisisacaseofCutaneousanthrax(Pointsinfavorabattoiroccupation,pustuleandulceronhand)
• ProvisionaldiagnosisofAnthrax:demonstrationofCapsulebypolychromemethyleneblue(M’fadyeanreaction)
• Confirmeddiagnosisbyimmunofluorescencemicroscopy
• Cutaneousanthrax(Hideporter’sdisease):MC(95%),butusuallyselflimiting
• Occupationassociated:Dockworker,butcher,abattoir,farmer.
26. Ans.(a)(Gram-negative)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep253,Ananthanarayan9/ep244
Bacillusanthracisisgram-positivebacilli.
27. Ans.(b)(Bacillusanthracis)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep253,Ananthanarayan9/
BACILLUSCEREUS
28. Ans(a)(Firedrice)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep255
29. Ans.(b)(Presence…)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep255,Ananthanarayan9/ep249
• FeverisnotaconsistentfeatureofB.cereusfoodpoisoning.
• AbdominalcrampsmaybeseeninbothemeticanddiarrhealtypeofB.cereusfoodpoisoning.
• Fordetailedexplanationrefertext(chapterreview).
30. Ans.(a),(b)(1–6,8–16)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep255,Ananthanarayan9/
ep249Emetictype:IP1–5hour
Diarrhealtype:IP8–16hour
31. Ans.(b)(Bacilluscereus)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep255,Ananthanarayan9/p249
• Chineserestaurantsyndromemeans:Foodpoisoningwithin1–6hrofconsumptionofChinesefriedrice…………
MostprobableagentisBacilluscereus.
32. Ans(c)(Erythrasma)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep249
Section
Erythrasmaisaskin lesioncausedbyC.minutissimumand underwoodlamp examination,itgives coralredcolor.
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Anaerobes:Clostridiumand CHAPTER
Non-SporingAnaerobes
3.5
Anaerobicbacteriadonothavecytochromesystemforoxygenmetabolismandhenceare
unabletoneutralizetoxicoxygenmetabolites.Theycanbeclassifiedas:
• Obligate anaerobes: They cannot grow in presence of oxygen as they completely
lacksuperoxide dismutase and catalase and are susceptible to the lethal effects of oxygen.
Forexample,Clostridiumandnon-sporinganaerobes Anaerobicbacteria:
• Aerotolerant anaerobes: They do not utilize oxygen for growth, but tolerate its Obligate anaerobes: Cannotgrowinpresenceofoxygen
Aerotolerant anaerobes: Donotutilizeoxygenforgrowth,bu
presence.Thisisbecausetheypossesssmallamountsofsuperoxidedismutaseandperoxidas
e
(butlackcatalase)whichmayneutralizethetoxicoxygenradicals.Examplesinclude
ClostridiumhistolyticumandBacteroides.
CLOSTRIDIUM
Clostridiaareobligateanaerobicgram-positivebacilli,havingbulgingspores:
• Clostridiaaresaprophytesfoundinsoil,organicmatter,andalsoinintestineofanimals
includinghumans
• Onlyfewinfecthumans,suchasC.perfringens,C.tetani,C.botulinumandC.difficile
• Industrialimportance:Someclostridia,suchasC.acetobutylicumandC.butyricumare
usedtopreparechemicals,suchasacetoneandbutanol.
• Theyaremotile(exhibitstatelymotility)exceptC.perfringens,C.ramosumandC.tetaniVI.
• TheyarenoncapsulatedexceptC.perfringensandC.butyricum.
• Mostoftheclostridiabearasub-terminalsporesexcept:
○ C.tetani:Producessphericalandterminalspore(drumstickappearance)
○ C.tertium:Producesovalandterminalspore(tennisracketappearance)
○ C.bifermentans:Producescentralandovalspore.
CLOSTRIDIUMPERFRINGENS
C.perfringens(previously,C.welchii)isasaprophyteandcommensalinthelargeintestineofhuma
nbeingsandanimals:
• Itiscapsulated,nonmotile,gram-positivebacillus
• Itbearssub-terminalbulgingspores;butthegasgangrenestrainsdonotproducespores.
VirulenceFactors
C.perfringensisinvasiveaswellastoxigeniC.Thetoxinsandtheothervirulencefactorscanbegrou
pedinto:
• Fourmajortoxins:Alpha(α),beta(β),epsilon(ε)andiota(ι).
Alphatoxin(orlecithinaseorphospholipaseC)istheprinciplevirulencefactorforgasgangr
eneandfoodpoisoning.
• Eightminortoxins:Gamma(γ),delta(δ)lambda(λ),kappa(κ),theta(θ),eta(η),mu(μ)andnu(υ)
• Theyalsoproduceheatlabileenterotoxin.
• SolublesubstancesproducedbyC.perfringensareneuraminidase,histamine,burstingfacto
r(producemusclelesions)andcirculatingfactor(inhibitphagocytosis).
C.perfringensinfectionsaremostlypolymicrobialinvolvingotherclostridiaspecies.Variousman
ifestationsinclude:
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1. ClostridialWound Infection
MacLennanhasclassifiedthemas:(i)Simplewoundcontamination,(ii)Anaerobiccellulitis,
(iii)Anaerobicmyositis(gasgangrene)
Enteritis necroticans (gasgangrene of the bowel):
Ischemicnecrosisofthe
jejunum and gas in tissueplane 2. ClostridialEntericInfection
Also known as pigbel in PapuaNew Guinea•and darmbrand inGermany.
Foodpoisoning:CausedbyC.perfringenstypeAenterotoxin(codedbygenecpe)
CausedbyC.perfringenstype
Cproducingβ toxin. • Enteritisnecroticans(gasgangreneofthebowel):
○ Ischemicnecrosisofthejejunumandgasintissueplane
○ AlsoknownaspigbelinPapuaNewGuineaanddarmbrandinGermany.
○ Caused by C. perfringens type C producing β toxin.
• Necrotizingenterocolitis:ResemblesenteritisnecroticansbutassociatedwithC.perfringen
stypeA,hasbeenfoundinpreviouslyhealthyadults.
• Gangrenousappendicitis
3. OtherClostridialInfections
• Bacteremia:C.perfringensfollowedbyC.tertiumandC.septicum.
• Skinandsoft-
tissueinfections:C.perfringens,C.histolyticum,C.septicum,C.novyi,andC.sordelliicause
necrotizing infections of the skin and soft tissues.
• Infectionoftheendometriumleadingtotoxicshocksyndrome(C.sordellii).
• Meningitisandbrainabscess
• Panophthalmitis(duetoC.sordelliiorC.perfringens).
GasGangrene
Gasgangrene(by(Oakley)isdefinedasarapidlyspreading,edematousmyonecrosis,occurringin
associationwithseverelycrushedwoundscontaminatedwithpathogenicclostridia,
particularlywith C.perfringens.It isalwayspolymicrobial:
• Establishedagents:C.perfringens(MC,60%)andC.novyiandC.septicum(20–40%).
• Probable agents: C. histolyticum, C. sporogenes, C. fallax, C. bifermentans, C. sordellii,
C.aerofoetidum andC.tertium.
PathogenesisofGasGangrene
Thedevelopmentofgasgangrenerequires:
• Anaerobicenvironment:Crushinginjuriesofmuscles(roadtrafficaccidents)orforeignbodies(b
ulletinjuries)
• Contaminationofwoundwithclostridialsporespresentinthesoil(duringwarorroadtrafficaccid
ent)orclothes.
• Rarely,spontaneousnon-
traumaticgasgangreneoccursviainvasionofbowelclostridia;occursinpeoplewithgastrointest
inalpathologies(e.g.colonicmalignancy).
Incubation period for gasgangrene:
10-48hrsforC.perfringens IncubationPeriodofGasGangrene
2-3daysforC.septicum
Theincubationperiodisvariable,dependinguponthenatureofinjury,infectivedoseandclostridi
5-6daysforC.novyi
Section
alspeciesinvolved:
• 10–48hrsforC.perfringens,2–3daysforC.septicumand5–6daysforC.novyi
GasGangreneisClinicallyCharacterizedBy
• Suddenonsetofexcruciatingpainattheaffectedsite
• Rapiddevelopmentofafoul-smellingthinserosanguineousdischarge
• Gasbubbles(crepitus)inthemuscleplanesandbrawnyedemaandinduration
• Shockandorganfailuredeveloplater
• Associatedwithhighermortalityrate(50%).
LaboratoryDiagnosis
• Specimen:Necrotictissues,musclefragmentsandexudatesfromdeeperwound.
• Directmicroscopy:
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Anaerobes:ClostridiumandNon-SporingAnaerobes161
○ Thick,stubby,boxcar-shapedgram-positivebacilliwithoutspore–
suggestiveofC.perfringens
○ Gram-positivebacilliwithspore-suggestiveofotherClostridiumspecies
• CultureMedia:Robertsoncookedmeatbroth(RCM),eggyolkagar,etC. IdentificationofC.perfringens:
• IdentificationofC.perfringens: Thick,stubby,boxcar-shapedgram-positive bacilli withouts
○ Targethemolysis(doublezonehemolysis) Targethemolysis(doublezonehemolysis)
Nagler’sreactionPositive
○ Nagler’sreaction:Opalescencesurroundingthestreaklineoneggyolkagar,inhib- ReverseCAMPtest:Positive
itedbyaddinganti-alphatoxin.
○ ReverseCAMPtest:Positive.
Treatment
• Earlysurgicaldebridementisthemostcrucialstepinthemanagementofgasgangrene.
• Antibiotics:Penicillin+clindamycinarerecommendedfor10–14days.
• Hyperbaricoxygen:Itmaykilltheobligateanaerobicclostridia,suchasC.perfringens Treatmentofgasgangrene:
• Passiveimmunizationwithantiαtoxinantiserum. Earlysurgicaldebridement(main stay)
Antibiotics: Penicillin +clindamycin
Hyperbaricoxygen
CLOSTRIDIUMTETANI Antiαtoxinantiserum
C.tetaniisobligateanaerobic,gram-
positivebacilluswithterminalroundspores(drumstickappearance):
• Itisthecausativeagentof‘tetanus’manifestedbyskeletalmusclespasmandautonomic
nervoussystemdisturbance.
• C.tetaniiswidelydistributedinsoil,hospitalandintestineofmanandanimals.
VirulenceFactors
C.tetaniproducestwoexotoxins:Tetanolysinandtetanospasmin:
• Tetanolysinisaheatlabile,oxygenlabilehemolysin.Itplaysnoroleinthepathogenesis.
• Tetanospasminortetanustoxin(TT)isaneurotoxinresponsibleforthepathogenesisoftetan Tetanospasminortetanustoxin(TT):
us: Actspresynapticallyatthe
○ Itisoxygenstablebutheatlabile;codedbyplasmid. inhibitory neuron terminals andprevents release of inhibit
○ Mechanism of action: Toxin acts pre-synaptically at the inhibitory neuron Strychnine poisoningsimilar mechanism but actspostsynap
terminalsand prevents release of inhibitory neurotransmitter GABA and glycine →
leads tospasticmusclecontraction.
○ Strychninepoisoninghasasimilarmechanismexceptthatitactspost-synaptically.
ModeofTransmission
Tetanusbacillienterthrough:
• Injurylikeroadtrafficaccidents,unsterilesurgery/abortion/
SystemicBacteriolog
delivery,otitismedia(otogenictetanus)
• Itisnoninfectious:Thereisnopersontopersonspread
• Incubationperiodisabout6–10days.ShortertheIP,graveristheprognosis.
• Musclesofthefaceandjawareoftenaffectedfirst(duetoshorterdistancesforthetoxintoreachthe
nerveterminals).
• 1stsymptom:Increaseinthemassetertoneleadingtotrismusorlockjaw,followedbymusclep Complicationsoftetanus:
ainandstiffness,backpain,anddifficultyinswallowing. Risussardonicus
Opisthotonosposition
• Inneonates,difficultyinfeedingistheusualpresentation
• Asthediseaseprogresses,painfulmusclespasmdevelopswhichmaybe:
○ Localized:involvestheaffectedlimb
○ Generalizedpainfulmusclespasm:leadstodescendingspasticparalysis.
• Hands,feetaresparedandmentationisunimpaired
• Deeptendonreflexesareexaggerated
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• Autonomicdisturbanceismaximalduringthesecondweekofseveretetanus-
characterizedbyloworhighbloodpressure,tachycardia,intestinalstasis,sweating,increasedtr
achealsecretionsandacuterenalfailure.
Complications
• Risussardonicus:Characteristic,abnormal,sustainedspasmofthefacialmusclesthatappe
arstoproducegrinning.
• Opisthotonospositionofthebodyoccursduetogeneralizedspasticcontractionof
extensormuscles.
• Respiratorymusclesspasm-maycauseairwayobstruction.
Warmclimate,ruralareawithfertilesoilisassociatedwithincreasedrisk.
LaboratoryDiagnosis
Treatmentshouldbe startedimmediatelybasedonclinicaldiagnosis.Laboratorydiagnosis
helpsonlyinconfirmation.
• Specimen:Excisedtissuebitsfromthenecroticdepthsofwounds.
• Gramstaining:
○ Revealgram-positivebacilliwithterminalandroundspores(drumstickappear-ance)
○ HowevermicroscopyaloneisunreliableasitcannotdistinguishC.tetanifrommor-
phologicallysimilarclostridialikeC.tetanomorphumandC.sphenoides.
• Culture:Cultureismorereliablethanmicroscopy:
○ InRCMbroth:C.tetani,beingproteolyticturnsthemeatblackandproducesfoulodor.
○ Bloodagar:C.tetaniproducescharacteristicswarminggrowth.
• ToxigenicityTest:Fordemonstrationoftoxinproduction
○ Invitrohemolysisinhibitiontest:detectstetanolysin
○ Invivomouseinoculationtest:detectstetanospasmin
Treatment
• Passiveimmunization(tetanusimmunoglobulin):Itisthetreatmentofchoice:
○ Twopreparationsareavailable:
▪ HTIG(Humantetanusimmunoglobulin):250IU(protectsfor30days)
▪ ATS(antitetanusserum,equinederived):1500IU(protectsfor7–10days)
○ HTIGispreferredasATSisassociatedwithsideeffects,suchasserumsicknessandanaph
ylactoidreactions
• Combinedimmunization(Bothactiveandpassiveimmunization):Indicatedinnon-
vaccinatedperson
• Antibiotics:Antibioticshasminorroleastheycannotneutralizethetoxinoncereleased:
○ However,theyareuseful(i)Inearlyinfectionbeforeexpressionofthetoxin(before6hrs),
(ii)Topreventfurtherreleaseoftoxin
○ Metronidazoleisthedrugofchoice.Penicillincanbegivenalternatively.
Section
PreventionbyActiveImmunization(Vaccine)
Tetanus toxoid (TT) is used for active immunization. It is available either as (i)
MonovalentvaccineasTTand(ii)CombinedvaccineasDPT(detailsarediscussedinchapter3.4)
• Primaryimmunizationofchildren:UndernationalimmunizationscheduleofIndia,total
seven doses are given; three doses of DPT at 6, 10 and 14 weeks of birth followed
Tetanus Toxoid Immunizationschedule: bytwoboosterdosesofDPTat16–
Primaryimmunizationof
24weeksand5yearsfollowedbytwoadditionaldosesofTTat10yrsand16yrs.
children:totalsevendoses
•
Adult immunization: FourdosesofTTisgiven Adultimmunization:
○ Itisindicatedifprimaryimmunizationisnotadministeredinchildhood.
○ Four doses of TT is given; 2 doses of TT at 1 month interval followed by 2
boosterdosesat1yrand6yrs.
• Site:TTisgivendeepIMatanterolateralaspectofthigh(children)andindeltoid(adults).
• Protectivetiteroftetanusantitoxinis≥0.01unit/ml.
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PreventionofTetanusAfterInjury
All types of wounds need surgical toilet followed by immunization which depends on
thewoundtypeandimmunizationstatusoftheindividual.
Recommendationforpreventionoftetanusafterinjury
Simple wound
Immunity Wound<6hrs,clean,non- Complicated
Category penetrating,no/negligible tissue wounds(Other
damage wounds)
CategoryA Nothingrequired Nothingrequired
CategoryB Toxoid1dose Toxoid1dose
CategoryC Toxoid1dose Toxoid1dose+HTIG
CategoryD Toxoidcompletedose Toxoidcompletedose+HTIG
• CategoryA:TakencompletecourseofTT/boosterwithinthepast5years
• CategoryB:TakencompletecourseofTT/boosterwithinthepast>5years<10years
• CategoryC:TakencompletecourseofTT/boosterwithinthepast>10years
• CategoryD:Not Takencomplete courseof TT/boosteror immunitystatusis unknown.
PreventionofNeonatalTetanus
Neonatal tetanus is defined by WHO as ‘child loses ability to suck and cry between day
3and28oflifeandbecomesrigidandhasspasms’.Itisalsoknownas‘8thdaydisease’asthesympt
omsusuallystartsafter1week:
• Mostcommonreason:Unhygienicpracticesduringdeliveries,suchasinfectedumbilicalstump
s due to application of cow dung, rarely bycircumcision or by ear piercing.
• Seasonal:Morecommonin July,Augustand Septembermonths. Neonataltetanuseliminationisbased on:
• Neonataltetanuscanbepreventedby: Neonataltetanusrate
○ Discouraginghomedeliveriesandpromotinghospitalorattendeddeliveries. <1/1000livebirths
○ Following aseptic clean practices are followed during deliveries: clean hand , TTcoveragetopregnantwomen:> 90%
cleansurface, clean blade for cutting cord, clean cord tie, clean cord stump, clean Attendeddeliveries:>75%
towelandcleanwater.
○ TT(2doses)aregiventoallpregnantwomenduring2ndtrimesterat1monthgap.
• Neonataltetanuselimination isbasedon:
○ Neonataltetanusrate<1/1000livebirths
○ TTcoveragetopregnantwomen>90%
○ Attendeddeliveries>75%.
CLOSTRIDIUMBOTULINUM
Clostridiumbotulinumproducesbotulinumtoxinandcausesbotulism.
Pathogenesis(BotulinumToxin) SystemicBacteriolog
C.botulinumisnoninvasiveandthepathogenesisisduetoproductionofpowerfulneurotoxin‘botulin
umtoxin’(BT),probablythemosttoxicsubstanceknowntobelethaltomankind:
• Serotype: Based onlight chain,there are eightserotypes-A, B,C1, C2, D,E, Fand G:
○ SerotypesA,B,Ecommonlycausehumandisease;mostseverebeingserotypeA.
○ Allserotypesproduceneurotoxin;exceptC2whichproducesanenterotoxin MechanismofBotulinumtoxin:BT blocks the release
○ Botulinumtoxin TypeC and Dare bacteriophagecoded
• BTisproducedintracellularly,notsecretedandappearsoutsideonlyafterautolysisof
bacterialcell.
• BTissynthesizedasprotoxin,convertedintoactiveformbyproteolyticenzymes.
• Mechanism:BTblocksthereleaseofacetylcholineinneuromuscularjunction,whichleadst
oflaccidparalysis.
• Therapeuticuses:AsBTproducesflaccidparalysisitcanbeusedtherapeuticallyforthetreatme
ntofspasmodicconditions,suchasstrabismus,blepharospasmandmyoclonus.
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• BTisalsoproducedbyotherclostridia,suchasC.butyricum,C.barattiandC.argentinense.
• Recovery:Blockingofacetylcholinereleaseispermanent,buttheactionisshortlasting
astherecoveryoccursin2–4months,oncethenewterminalaxonssprout.
Themanifestationsofbotulismareduetodecreasedacetylcholineincranialnerveand
parasympatheticnerveterminals.Commonsymptomsinclude:
• 5Ds-
Botulism(Symptoms) Diplopia,dysphasia,dysarthria,descendingsymmetricflaccidparalysisand↓deeptendonrefle
5Ds-Diplopia, dysphasia,dysarthria, descendingsymmetric
xes flaccid paralysisand↓deeptendonreflexes
Constipation • Constipation
• Respiratorymuscleparalysismayleadtodeath
• Thereisnosensoryorcognitivedeficits.
TypesofBotulism
1. Foodbornebotulism:Resultsfromconsumptionoffoodscontaminatedwithpreformedbotuli
numtoxin;commonlyduetoconsumptionofhomemadecannedfood.
2. WoundbotulismresultsfromcontaminationofwoundswithC.botulinumspores.Itpresentslik
efoodbornebotulismexceptforabsenceofgastrointestinalfeatures.
3. Infantbotulism:Byingestionofcontaminatedfood(usuallyhoney)withsporesofC.botulin
uminchildren≤1yearofage.Sporesgerminateinintestinereleasingthetoxin:
• Manifestationsincludeinabilitytosuckandswallow,weakenedvoice,ptosis,andflopp
yneck,extremeweakness(calledfloppychildsyndrome)
• Itisusuallyself-limiting,rarely,progresstogeneralizedflaccidity.
4. Iatrogenicbotulism:Resultsfrominjectionofoverdoseofthetoxin.
CLOSTRIDIUMDIFFICILE
Clostridiumdifficileistheagentof‘pseudomembranouscolitis’whichoccursalmostexclusively in
association with prolonged antimicrobial use. It was so named due to
unusualdifficultiesinvolvedintheisolationofC.difficile.
RiskfactorforC.difficile
infection:
Prolongedhospitalstay Pathogenesis
Prolongedantimicrobial Clostridiumdifficileinfection is associatedwith the following riskfactors:
Cephalosporinsandotherantibiotics
Toxinproduction • Prolongedhospitalstay:Sporesfromhospitalenvironmentgetscolonizedincolonofpatie
nts.
• Prolongedantimicrobialusecanresultindisruptionofthenormalcolonicflora:
○ Cephalosporins(e.g.ceftriaxone)arefrequentlyresponsibleforthiscondition.
○ Otherantibiotics,suchasclindamycin,ampicillinandfluoroquinolones(ciprofloxa-
cin)
○ However, all antibiotics, including vancomycin and metronidazole (which are
theDOCinC.difficileinfection)havebeenfoundtocarryariskofinfection,ifgivenforpro
longedduration.
• Toxinproduction:Pathogenesisistoxinmediated.C.difficile(nontoxigenicstrains)may be a
Section
part of normal intestinal flora, however only the toxigenic strains can
causepseudomembranouscolitis:
○ Itproducestwotoxins:ToxinA(enterotoxin)andToxinB(cytotoxin).Bothareim-
portantforpathogenesis.
○ Infants do not develop symptoms because they lack suitable mucosal toxin recep-
tors.
• Otherriskfactorsincludeolderage,underlyingillness,intestinalsurgery,useof
electronicrectalthermometersandantacidtreatment.
• DiarrheaisthemostcommonmanifestationcausedbyC.difficile.Othermanifestations
includefever, abdominal painand leukocytosis. Blood instool is uncommon.
• Pseudomembraneformationovercolonicmucosawitharelapseseenin15–30%ofcases.
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LaboratoryDiagnosis
Laboratory diagnosis of C. difficile infection depends on isolation of the bacilli followed
bytoxigenicitytesting: TreatmentofC.difficile
• Stool culture on selective media, such as CCFA (cefoxitin cycloserine fructose agar) infection:
orCCYA (cefoxitin cycloserine egg yolk agar). Stool culture is highly sensitive but not Initial episode, mild tomoderate cases: Oralmetronida
Recurrent episodes or severecases:VancomycinisDO
specific.Toxindemonstrationismoremeaningful. Severe complicated orfulminant infection: Thecombin
• Toxindemonstration:Toxinscanbedetectedbyvariousmethods:
○ Cellculturecytotoxintest:Itishighlyspecificbutnotassensitiveasstoolculture;
itistimeconsuming.
○ Enzyme immunoassay for toxinA and/toxins B instool is rapid, butnot sensitive.
○ PCRforC.difficiletoxinBgeneinstoolitishighlyspecificandsensitive.
• Colonoscopyishighlyspecificifpseudomembranesareseen,butsensitivityislow.
• HistopathologyofcolonicpseudomembraneisalsohighlyspecifiC.
Treatment
• Initial episode,mild tomoderatecases: Oralmetronidazole isthe drugofchoice
• Recurrentepisodes or severe cases:Vancomycin is the drug of choice
• Severecomplicatedorfulminantinfection:VancomycinplusIVmetronidazole
NON-SPORINGANAEROBES
Classification ofNon-sporinganaerobes
Gram-positivecocci Peptostreptococcus, Peptococcus

Gram-negative cocci Veillonella
Gram-positivebacilli Bifidobacterium,Propionibacterium,EubacteriumLactobacillus,Mobiluncus,Actinomyces
Gram-negativebacilli Bacteroides,Prevotella,PorphyromonasFusobacterium,Leptotrichia
Spirochete Treponema,Borrelia
Anaerobesasapartofnormalflora
Anatomicalsite Commonanaerobicnormalflora
Mouth (saliva) Anaerobiccocci,Actinomyces,Bifidobacterium,P.melaninogenica,Spirochetes
Stomach Lactobacillus
Jejunum/ileumandColon Anaerobiccocci,Bacteroidesfragilis,Fusobacterium,BifidobacteriumP.melaninogenica
Vagina Anaerobiccocci,Lactobacillus,P.melaninogenica,Bifidobacterium
Skin Propionibacterium
SystemicBacteriolog
Commondisease
Mobilincus:Bacterial vaginosis (alsocaused byGardnerella)
LeptotrichiaorFusobacterium fusiformis:Agent ofVincent’sangina(alsocausedby Borreliavincentii)
Anaerobiccocci:
Puerperalsepsisandotherfemalegenitaltractinfection(alsobyB.fragilisandPrevotella)Skinandsofttissueinfections.
Bacteroidesfragilis:
• Non-sporinganaerobe,capsulatedgram-negativebacilli
• MCcommensalinhumanintestine
• MCanaerobetocauseinfection,causesabdominalinfection
• Endotoxinislesstoxicthanthatofaerobicgram-negativebacilli
Fusobacteriumnecrophorum:AgentofLemierre’ssyndrome(isaformofthrombophlebitis)
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GENERAL 8. Besttreatmentforcontaminatedwoundwithnecrotic
materialis: (RecentQuestion2013,AIIMSNov2013)
1. Trueaboutobligateanaerobes: (JIPMERMay2016) a. Anti-gasgangreneserum
a. Inabsenceofoxygenitgrowswell b. Debridement
b. Inabsenceofoxygen,itdoesnotgrow c. Antibiotics
c. Inpresenceofoxygen,itgrowsbutdoesnotutilize d. Tetanustoxoid
theoxygen
9. Regarding Clostridium perfringens gas gangrene,
2. GasgangreneandTetanuscausedbywhichgroupof falseis: (PGIJune2008,AIIMSMay2009)
bacteria: (JIPMER,May2015) a. Common causeofgasgangrene
a. Campylobacter b. Naglerreactionpositive
b. Clostridium c. Mostcommontoxinis Hyaluronidase
c. Citrobacter d. FoodpoisoningstrainofCl.Perfringensproducesheat
d. Cardiobacterium resistantspores
3. Nonmotileclostridiais: (JIPMER2010,AI2009) 10. Gastrointestinalenteritisnecroticansiscausedby:
a. Cl.perfringens a. Clostridiumdifficile (PGIDec2000)
b. Cl.novyi b. Clostridiumperfringens
c. Cl.botulinum c. Botulinum
d. Cl.difficile d. Campylobacterjejuni
4. Ovalbulgingterminalsporesseenin: e. Pseudomonas
(AI2008,PGIDec08) 11. Trueaboutgasgangrene: (PGIMay2013)
a. Cl.tertium a. Underlying skin and muscles are normal
b. Cl.welchii b. Causedbytetanospasmintoxin
c. Cl.perfringens c. Musclerigidityandspasmsarecharacteristic
d. Cl.histolyticum d. MostcommonorganismimplicatedisC.perfringens
e. Passiveimmunizationdoesnothelp
5. Amaleispresentedwithleftmidthighcrushedinjury.Wh
ichisthemostessentialsteptopreventgasgangreneinthi CLOSTRIDIUMTETANI
spatient? (JIPMERMay2016)
12. WhichistheprinciplevirulencefactorinClostridium
a. Wounddebridement
tetani? (RecentQuestion2015)
b. Antigas gangreneserum
a. Tetanolysin
c. Antitetanus
b. Tetanospasmin
d. Hyperbaric oxygen
13. Apatientispresentedwithtrismuswithopisthotonuspo
6. Whichofthefollowingmostlikelycausenecrotizing
sition.Theprobablecausativeagentis:
Section
enteritis? (RecentQuestion2015)
a. Clostridium tetani (RecentQuestion2015)
a. Clostridiumbotulinum
b. Clostridiumperfringens
b. Clostridiumtetani
c. Clostridiumdifficile
c. ClostridiumperfringensA
d. ClostridiumperfringensC 14. Mechanismofactionoftetanospasmin:
a. InhibitionofGABArelease (NEETPatternBased)
7. Trueaboutgasgangrene:(PGIMay2015)
b. InhibitioncAMP
a. -toxinismaincauseofthetoxaemiaassociated
c. InactivationofAchreceptors
withgasgangrene
d. InhibitionofcGMP
b. CausedbymainlyClostridiumintestinale
c. Lowoxygentensionintissueisimportantpreconditio 15. Whichofthefollowingisfalsefortetanus? (AI2011)
n a. Producesheatresistantspores
d. Devitalizedtissuepredisposestogasgangrene b. Persontopersontransmissiondoesnotoccur
e. Notoccurifdeadtissueisnotpresent c. Incubationperiodis6–10days
d. Threedosesofprimaryvaccinationisprotective
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16. Siteofactionoftetanustoxin: (JIPMER2007)

24. WrongaboutCl.tetani: (AIIMSNov2010)
a. Presynapticterminalofspinalcord
a. Mainreservoir-Soil,humanandanimalintestine
b. Postsynapticterminalofspinalcord b. MC modeoftransmission- Traumaand
c. Neuromuscularjunction contaminatedwound
d. Musclefibers c. Herdimmunity-notuseful
17. Trueabouttetanus: (PGIDec2004,Kerala2016) d. MCseason–winteranddry
a. Gram–ve spore forming organism 25. Forneontataltetanuselimination,theincidenceshould
b. ProducesTelanolysinandTetanospasmin be….: (DNB08)
c. Trismusandneckstiffnessareearlysign a. Lessthan0.5/1000livebirth
d. Generalizedtonic-clonicseizureoccursonhyper b. Lessthan1/1000livebirth
stimulation c. Lessthan0.1/1000livebirth
e. Wounddebridementisnecessary d. Lessthan10/1000livebirth
18. A25-year- 26. Periodofcommunicabilityfortetanus: (UP07,RJ01)
boyispresentedwithdeepinjuryandabrasionsonthelef a. 7days b. 14days
tshoulder,thighandlegwithimmunization status c. 21 days d. None
unknown. What is to be givennow? (JIPMERNov2015) 27. A person has got a non penetrating wound 11
a. DTaPonly b. DTaP+Ig hourback.HehadtakencompletecourseofTT1yearback
c. dTonly d. dT + Ig .Whattreatmentisrecommendednext? (MP09)
19. A10-year- a. Fullcourseoftetanustoxoid
oldboyfollowingaroadtrafficaccidentpresentstotheca b. Singledoseoftetanustoxoid
sualtywithcontaminatedwoundovertheleftleg.Hehasr c. Nothing required
eceivedhiscompleteprimaryimmunizationbeforepres d. HumanTetglobulinandsingledoseoftoxoid
choolageandreceivedabooster of DT at school entry 28. Anadultwithnoimmunizationhistorypresentswith a
age. All of the followingcanbedoneexcept:(AIIMS2001) clean non penetrating wound 2 hrs back.
a. InjectionofTT Whatmeasurehastobetaken? (Karnataka2011)
b. Injectionofhumanantiserum a. Fullcourseoftetanustoxoid
c. Broadspectrumantibiotics b. Singledoseoftetanustoxoid
d. Wounddebridement and cleaning c. Nothing required
d. HumanTetglobulinandsingledoseoftoxoid
20. Apersonhasreceivedcompleteimmunizationagainst
tetanus 10 years ago. Now he presents with aclean
wound without any lacerations from an CLOSTRIDIUMBOTULINUM
injurysustained2.5hoursago.Heshouldnowbegiven:
(AI2001,Karnataka2011) 29. Trueaboutfoodbotulism: (RecentQuestion2015)
a. Fullcourseoftetanustoxoid a. Symptomsresemblesstrychninepoisoning
b. Singledoseoftetanustoxoid b. MostcommonbotulinumtoxinserotypeistypeC
c. HumanTetglobulin andD
d. HumanTetglobulinandsingledoseoftoxoid c. Thebacteriainvadestheintestine
d. BotulinumtoxinactsbyblockingA.chrelease
SystemicBacteriolog
21. Thecausativeorganismcanbeisolatedinwhich
30. True about Botulinumtoxin: (PGIMay2015)
amongthefollowingconditions: (PGIDec2001)
a. Interferewithadrenergictransmission
a. Serumintoxicshocksyndrome
b. InterferewithCholinergictransmission
b. Meningococcalrash
c. Increasereleaseofsynapticvesicles
c. Rheumaticvalvulitis
d. Inhibitreleasefromsynapticvesicles
d. CSF intetanus
e. ActalsoonCNS
e. Diphtheriticmyocarditis
31. Botulismismostcommonlydueto:
22. Allaredonetopreventneonataltetanusexcept:
(NEETPatternBased)
a. TwoTTdosestoallpregnant (AI2007) a. Egg b. Milk
b. TTtoallfemalesinthereproductiveage c. Meat d. Cannedvegetables
c. TTtoallnewborne
32. Botulisimcauses: (NEETPatternBased)
d. Penicillininjectiontoallnewborne
a. Descendingflaccidparalysis
23. ThreedosesofTTprovideimmunityfor: (DPG2006) b. Descendingspasticparalysis
a. 1yr b.5yr c. Ascendingparalysis
c. 10yr d. 15yr d. Ascendingspasticparalysis
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33. Botulinum affects allexcept:

40. Clostridiumdifficileinfectionoccursafter:
(AI 2007, NEET Pattern Based)
a. Afterprolongantibiotictherapy (PGIJune2008)
a. Neuromuscularjunction
b. Pantoprazoleincreasestherisk
b. Preganglionicjunction
c. Associatedwithuseofrectalthermometer
c. Postganglionicnerves
d. Increasedwithproportionofhospitalstay
d. CNS
41. Pseudomembranouscolitis,allaretrueexcept:
34. An18-year-
(AIIMSMay2007,AI2000)
oldmalepresentedwithacuteonsetdescending
a. ToxinAisresponsibleforclinicalmanifestation
paralysis of 3 days duration. There is
b. ToxinBisresponsibleforclinicalmanifestation
alsohistoryofblurringofvisionforthesameduration.On
c. Bloodinstoolsisacommonfeature
examination, the patient has quadriparesis
d. Summitlesionsisanearlyhistopathologicalfinding
withareflexia. Both the pupils are nonreactive. The
mostprobablydiagnosisis: (AIIMS2006) 42. ApatientofAcutelymphocyticleukemia
a. Poliomyelitis b. Botulism withfeverandneutropeniadevelopsdiarrheaafteradmi
c. Diphtheria d. Porphyria nistration of amoxycillin therapy, which of
thefollowing organism is most likely to be the
35. Thefollowingstatementsaretrueregardingbotulism
causativeagent: (AIIMSNov2005)
except: (AIIMSMay2003)
a. SalmonellaTyphi
a. Infantbotulismiscausedbyingestionofpreformed
b. Clostridiumdifficile
toxin
c. Clostridiumperfringens
b. ClostridiumbotulinumA,B,CandFcausehuman
d. Shigellaflexneri
disease
c. The gene for botulinum toxin is encoded by a 43. DOCforpseudomembranousenterocolitis:
bacteriophage a. OralVancomycin (AIIMS Nov2014)
d. Clostridiumbarattimaycausebotulism b. Penicillin
c. OralAmpicillin
36. TrueaboutClostridiumtertium: (PGI June2008)
d. Clindamycin
a. Gramvariable
b. Terminalspore
c. Producesexotoxin NONSPORINGANAEROBES
d. Causessepticemicarthritis
37. AmongthetoxinproducedbyClostridiumbotulinum, 44. With reference to Bacteroides fragilis the
followingstatementsaretrue,except:
thenonneurotoxiconeis: (JIPMER2000)
(AIIMSNov2012,11,06,May2006,03,AI2007)
a. A b. B
a. B. fragilis is the most frequent anaerobe
c. C1 d. C2
isolatedfromclinicalsamples
e. D
b. B.fragilisisnotuniformlysensitivetometronidazole
c. ThelipopolysaccharideformedbyB.fragilisisstructur
CLOSTRIDIUMDIFFICILE allyandfunctionallydifferentfromtheconventionale
38. Causeofclostridiumdifficileassociateddiarrhea: ndotoxin
d. Shockanddisseminatedintravascularcoagulation
a. Trauma (NEETPatternBased)
arecommoninBacteroidesbacteremia
b. Dairyproducts
c. Friedrice 45. A patient presents with frontal abscess. Foul
d. Antibiotic use smellingpusisaspirated.Pusshowsredfluorescenceon
Section
ultravioletexamination.Themostlikelyorganismcausi
39. Pseudomembranouscolitisiscausedby:
ngthefrontalabscessis: (AIIMSMay2002)
(DNBJune2012,Kerala2016)
a. Bacteroides
a. Clostridiumperfringens
b. Peptostreptococcus
b. Clostridiumdifficile
c. Pseudomonas
c. Clostridiumtetani
d. Acanthamoeba
d. Clostridiumbotulinum
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EXPLANATIONS
GENERAL
1. Ans.(a)(Inabsenceofoxygenitgrowswell)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p28
• Obligateanaerobesgrowonlyinabsenceofoxygenasoxygenislethaltothem.
• Aerotolerantanaerobestolerateoxygenforsometimeandgrowinpresenceofoxygen,butdonotutilize.
2. Ans.(b)(Clostridium)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep256,Ananthanarayan9/ep251
• GasgangreneiscausedbyClostridiumperfringensandTetanusiscausedbyClostridiumtetani.
3. Ans.(a)(Cl.perfringens)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep256,Ananthanarayan9/ep252
• AllClostridiaaremotileexceptCl.perfringensandCl.tetanitypeVI.
• Clostridiashowstatelytypeofmotility.
• AllClostridiaarenoncapsulatedexceptCl.perfringensandCl.butyricum.
4. Ans.(a)(Cl.tertium)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep256,Ananthanarayan9/
ep252Allclostridiaproducebulgingspore(sporearewiderthanthebacilli)exceptCl.bifermentansandCl.sordelli
• C.tertiumproducesOvalandterminalspore(tennisracketshaped)
• TypeofsporeproducedbyClostridiumspecies:ReferChapterreview:
5. Ans(a)(Wounddebridement)Ref:ApurbaSastry'sEssentialsofMedicalMicrobiology/p260
Earlysurgicaldebridementisthemostcrucialstepinthemanagementofgasgangrenetoremovealldevitalisedtissuessoastorem
oveconditionsthatproduceanaerobiosis.
6. Ans.(d)(C.perfringensC)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep258Necrotizingenteritisiscausedbyβ-toxinofTypeCClostridiumperfringens.
7. Ans(a,c,d,e)(a-toxin...,low...,devitalized...,notoccur...)Ref:Harrison19th/p990-
02,ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep258
 -toxinistheprinciplevirulencefactorofgasgangrene
• GasgangreneismainlycausedClostridiumperfringens
• The development of gas gangrene requires an anaerobic environment and contamination of a wound with C.
perfringens.Devitalized tissue, foreign bodies, and ischemia reduce locally available oxygen levels and favor
outgrowth of C.perfringens.….Harrison19/ep992
SystemicBacteriolog
• Intheabsenceofdevitalizedtissue,thepresenceofclostridiadoesnotnecessarilyleadtoinfection.
8. Ans.(b)(Debridement)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep260,Harrison19/ep990,18/ep1205
• Contaminated wound with necrotic material is suggestive of higher risk of clostridial infection and gas
gangrene.
• Treatmentofgasgangrene:ReferChapterreview
9. Ans.(c)(Most...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep258,59,Ananthanarayan9/ep254
• Most common toxin is ‘α toxin’not ‘Hyaluronidase’
• Cl.perfringensproduces4Major(Lethal)Toxins–alfa,beta,iota,epsilon
• CommonestcauseofgasgangreneisClostridiumperfringenstypeA(60%)
AboutOtherOptions
Option b: Nagler reaction positive for Cl. perfringens and Cl.
bifermentansFordetailofNaeglerreaction:ReferChapterreview
Optiond:FoodpoisoningstrainofCl.perfringens(mostlyTypeA)characterizedbytheirmarkedheatresistantsporesbut
feebleproductionofandthetatoxin.
Insteadtheyproduceheatlabileenterotoxin(similartoLTofE.coli)
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Alsoremember
‘GasgangreneproducingCl.perfringensstrainsdonotproduceSporesintissue/media.
10. Ans.(b)(Cl.perfringens)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep258,Ananthanarayan9/ep255,56
Enteritisnecroticans(PigbelinNewGuinea):
• CausedbyCl.Perfringenstype-Cstrainswithheatresistantspores
• Sporesgerminatesinintestineproducingbetatoxin
• Usuallyfollowingapigmeatdietalongwithatrypsininhibitorslikesweetpotato
11. Ans.(d)(Mostcommon...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep258,Ananthanarayan9/ep254
• MostcommonorganismimplicatedinGasgangreneisClostridiumperfringensfollowedbyC.novyiandC.septicum
• Underlyingskinandmusclesaregangrenous
• Gasgangreneiscausedby α-toxin
• PainandcrepitusinmusclearecharacteristicofGasgangrene
• Passiveimmunization–Antiα-toxin(antigasgangreneserum)isofgreatvalueforthetreatmentofGasgangrene.
CLOSTRIDIUMTETANI
12. Ans.(b)(Tetanospasmin)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep261
13. Ans.(a)(Clostridiumtetani)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep261
14. Ans.(a)(Inhibition...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep261,Ananthanarayan9/ep262
• Mechanism of action of tetanospasmin is: Acts presynaptically and inhibits of glycine and GABA release that leads
tospasticcontractionofmuscles.
15. Ans.(d)(Threedosesofprimary...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep263
AccordingtoNationalimmunisationschedule:
• Primarycourseofimmunisationincludes:
• ‘3dosesofDPTat4–8weekapartfollowedbyboosterofPDTat16–24monthfollowedbyboosterofDPTat5–
6yearfollowedbybooster ofTTat 10and 16year.’ Park 23/ep312, 22/ep115,21/e p113
AboutOtheroptions:
• C.tetaniproducesheatresistantspores
• Persontopersontransmissiondoesnotoccurintetanus
• Incubationperiodoftetanusis6–10days.
16. Ans.(a)(Presynaptic...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep261,Ananthanarayan9/ep261
• Tetanospasmin:BlocksreleaseofinhibitorytransmittersglycineandGABAatCNSandPresynapticterminalofspinalcord→
leadstospasticparalysis
• ThemanifestationsofTetanusandstrychninepoisoningaresimilarexceptTetanospasminactsatPresynapticterminalwhereasst
rychnineactsatpostsynapticterminal.
17. Ans.(b),(c),(d),(e)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep261,Ananthanarayan9/ep261
Section
• Optiona:Cl.tetaniisGram+vesporeformingorganism
• Optionb:TetanolysinandTetanospasminaretheprincipletoxinproducedbyCl.tetani
• Optionc:1stsymptom-↑massetertone(trismus/lockjaw)then→descendingtetanus
• Optiond:‘Tetanuspatientshouldbeisolatedtoprotectthemfromnoiseandlightwhichmayprovokeconvulsion’
• Optione-Prophylacticmeasuresinclude-Woundtoilet,antibioticandmostimportantlyimmunization.
18. Ans(b)(DTaP+Ig)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep263DTaP(completeimmunization)+HumanTetanusIgisrecommendforthiscondition.
• HumanTetanusIg-becauseit’sadeepinjury
• DTaP(completeimmunization)-becausenoh/
oimmunization.Acellularpertussis(aP)isgiveninsteadofkilledpertussisvaccineafter5yearofage.
19. Ans.(b)(Injection...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep262,Park23/ep312,22/ep286-87
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• Treatmentoftetanusconsistsof(Wounddebridement+Antibiotic+immunization)
• Fortreatmentofwoundinjury,wehavetoknowthetypeofwoundandtheimmunecategorythatthepatientbelongsto.
• Thishistoryissuggestiveof:
○ 10-year-oldchildTakencompleteimmunizationwithboosterofDTatschoolentryage—
indicatestakencompleteimmunization>5to<10yearbackSobelongstoImmunitycategoryB
○ Typeofwound-contaminated(otherwoundcategory)
○ TreatmentrequiredforCategoryBwithcontaminated(otherwound)Toxoid1dose
• Antibiotichasroletoeradicatethesourceoftoxin(Noroleafter6hourwhenthetoxinisalreadyformed)
• HTIghasroleincategoryCandDpersons,i.e.whenthecompleteimmunizationtaken>10yearortheimmunizationisnotcom
plete/unknown.
Treatment:Alltypeofwoundsneedsurgicaltoiletfollowedby:Immunization.(Referchapterreview)
20. Ans.(b)(Toxoid1dose)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep263,Park23/ep314,22/ep286
• Typeofwound:Cleanwoundwithoutanylacerationsfromaninjurysustained2.5hoursago
• PatientbelongstoImmunitycategoryCcompleteimmunizationtaken10yearsago
• So,Treatmentrequiredfor CategoryCwith simplewoundis:Toxoid 1dose
21. Ans.(a),(b)(SeruminToxic...,Meningococcalrash)Ref:Topley10/ep813,Harrison19/ep992,18/ep1117
• Optiona:LabtestfordiagnosisofToxicShockSyndromeincludesisolationofS.aureusfrommucosalornormallybodysite(Topley10/
ep813)andisolationofStr.pyogenesfrommucosalornormallybodysitelikeblood(Harrison19/ep992)
• Optionb:‘Meningococcimaysometimebedemonstratedinpetechiallesionsbyculture.’Ananthanarayan8/ep226
• RheumaticfeverisduetocrossreactingantigenbetweenStreptMproteinandhumanmyocardium.
• Diphtheriaandtetanusbothcancausetoxemiabutneverbacteremia.
22. Ans. (d)(Penicillininjectiontoall newborne) Ref:Park23/e p312,22/ep286
• ATSisrecommendedtoallneonatesbornetounimmunizedmother.(Detail-referchapterreview)
23. Ans(c)(10years)Ref:Park23/ep312,22/ep286
• Individualssustainingtetanus-
pronewoundsshouldbeimmunizediftheirvaccinationstatusisincompleteorunknownoriftheirlastboosterwasgiven>10years
earlier
• TwodosesofimmunizationprovidesimmunityforseveralyearsPark22/ep285
24. Ans. (d)(MCseason–winter anddry) Ref:Park23/e p312,22/ep284-85
• Tetanusshoesseasonalvariation,inIndia,>50%ofcasesoccurin-JulytoSeptember
• Mainreservoir:Soil,humanandanimalintestine
• MCmode of transmission:Trauma and contaminatedwound
• Herdimmunitynotusefulfortetanus
25. Ans(c)(Less...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep263,Park23/ep312,22/ep286,21e/p287
26. Ans(d)NoneRef:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep263,Park23/ep312,22/ep284
• TetanusisNOTinfectiousfrommantoman.
27. Ans(b)(Singledoseoftetanustoxoid)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep263,Park22/ep286
SystemicBacteriolog
• ItisacaseofSimplewound(Nonpenetratingwound1hrback)andcategoryC(h/ocompletecourseofTT11yr).
• TherecommendationforsimplewoundandcategoryCisSingledoseoftetanustoxoid
28. Ans(a)(Fullcourse...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep263,Park23/ep312,22/ep286
• It’sacaseofsimplewound(cleanandnonpenetrating)andcategory-D(unimmunized)
• TherecommendationforsimplewoundandcategoryDisFulldoseoftetanustoxoid
CLOSTRIDIUMBOTULINUM
29. Ans.(d)(Botulinumtoxin…)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep264
• MCbotulinumtoxinisserotypeA,B,andE.MostsevereistypeA
• Tetanusresemblesstrychninepoisoning
• C.botulinumisnoninvasive.Diseaseistoxinmediated.
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30. Ans (b, d)(Interfere…, inhibit...) Ref:Harrison 19th/p987-88, Apurba Sastry’s Essentials of Medical Microbiology 1/e
p264Botulinum toxin blocks the release of Acetyl choline at synapses of NM junction (MC site), peripheral ganglia
andparasympatheticnerveending;howeverithasnoactiononCNS.
31. Ans.(d)(Cannedvegetables)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep264,Park22/ep217
• Themostcommonfoodassociatedwithbotulismarehomepreservedfoodssuchashomecannedvegetables,smokedorpick
ledfish,homemadecheeseandsimilarlowacidfood.
32. Ans.(a)(Descendingflaccidparalysis))ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep264
• Botulismblocksacetylcholinerelease,hencecausesDescendingflaccidparalysis
33. Ans.(d)(Central...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep264,Harrison19/ep987,18/ep1200
• ‘Botulinumneurotoxin,actsonperipheralcholinergicnerveterminals,butnotonCNS:
34. Ans.(b)(Botulism)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep264,Harrison19/ep987,18/ep1201
Pointsinfavor:
Descendingparalysis,blurringofvision,quadriparesis(flaccidparalysis),areflexiaandnonreactivepupils
Alsoknow:
• CausesofDescendingparalysis:Tetanus,botulism,polio,diphtheria
• Tetanuscausesspasticparalysiswhereasbotulismcausesflaccidparalysis
35. Ans.(a)(Infant...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep264;Harrison19/ep988,18/ep1200
• ‘Infant botulismoccurs duetoingestion ofspores,toxinreleased inside
Whereasfoodbornebotulismoccursduetopreformedtoxinmixedwithcannedfood’
36. Ans.(a),(b),(d)(Gram...,terminal...,causessepticarthritis)ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep264
37. Ans.(d),(C2)Ref:Ananthanarayan9/ep264,8/ep262,ApurbaSastry’sEssentialsofMedicalMicrobiology1/
ep264Allsubtypes(A–G)ofBotulinumtoxinarepharmacologicallysimilar(neurotoxin),exceptC2(enterotoxin)
Cl.tertium:
• Nonexotoxin-producing,Aerotolerant,Gramvariable
• Ovalandterminalspore–tennisracketshaped
CLOSTRIDIUMDIFFICILE
• Resembleslactobacilli
• Itisararehumanpathogen
38. Ans.(d)(Antibioticuse)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep265,Ananthanarayan9/ep265
• Causes:NecrotizingFasciitisandGangrene,septicemia,septicarthritis
• ProlongedbroadspectrumantibiotictherapyisthemostimportantriskfactorforClostridiumdifficileassociated
diarrhea.
39. Ans.(b)(Clostridium...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep265,Ananthanarayan9/ep265
Section
• PseudomembranouscolitisiscausedbyClostridiumdifficile
40. Ans. (a), (b), (c), (d) (After prolong antibiotic therapy, Pantoprazole increases the risk, Associated with use of rectal ther-
mometer, Increased with proportion of hospital stay) Ref: Apurba Sastry’s Essentials of Medical Microbiology 1/e
p265,Harrison19/ep857,18/ep1091
RiskfactorsforPseudomembranouscolitis:Referchapterreview.
41. Ans.(c)(Blood...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep265,Harrison19/ep858,18/
ep1092Says:StoolsarealmostneverGrosslybloodyandrangefromunformedtowateryormucoidinconsistency,withacharacteristicodor.
AboutOtheroption:
• Cl.difficileToxins:A→EnterotoxinandB→Cytotoxin
• Botharerequiredformanifestation.
• Intheearlieststage,Summitlesion,i.e.tinysuperficialintercryptalerosionsmaybefound.Topley10/ep1120
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42. Ans.(b)(Clostridiumdifficile)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep265,Harrison19/
ep85818/ep1091,Ananthanarayan9/ep265,8/ep263
• Pointsfavoringdiarrheaafteradministrationofamoxicillintherapy
43. Ans.(a)(Oral...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep266,Harrison19/ep86018/ep1093-
94EtiologicalagentforpseudomembranousenterocolitisisClostridiumdifficile.
TreatmentofClostridiumdifficileinfection:Referchapterreview
NONSPORINGANAEROBE
44. Ans.(d)(Shock...)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep268;Harrison18/ep1332,1338
‘B.fragilisLPSare100–1000timeslessbiologicallypotentthanendotoxinassociatedwithaerobicgram-negativebacteria.
ThisaccountsforthelowerfrequencyofDICandpurpurainBacteroidesbacteremiathaninfacultativeaerobicgram-
negativebacillarybacteremia.’
AboutOtheroptions:
Optiona:‘Bacteroidesarethemostcommonanaerobesisolatedinclinicalspecimen.’Ananthanarayan9/ep269,8/ep267
Optionb:‘Metronidazoleisactiveagainstgram-
negativeanaerobes,includingtheB.fragilisgroup;resistanceisrarebuthasbeenreported’Harrison18/ep1332,1338,17/ep810
45. Ans.(a)(Bacteroides)Ref:ApurbaSastry’sEssentialsofMedicalMicrobiology1/ep268,Ananthanarayan9/ep269
• Frontalabscesswithfoulsmellingpus:Pointstowardsanaerobicinfection
• Fluorescenceonultravioletexamination:IndicatesPrevotellamelaninogenicawhichisatypeofBacteroidesinfection.
Alsoknow,theAnaerobic/AerobicRatioinhumanGITflora-(Harrison18/ep1332,1338,17/ep903)1:1(stomach,
ileum,jejunum,saliva)
103:1(TerminalileumandcolonandGingivalcrevices)10:1(Fema
le genitaltract)
SystemicBacteriolog
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CHAPTER
Mycobacteria 3.6
Mycobacteria are obligate aerobic weakly gram-positive, straight or slightly curved
bacilli,sometimes show branching filamentous forms resembling fungal mycelium. They
have thefollowingminimumproperties:
Acidfastnessis dueto: 1. Acidfastness:Thisisdueto:
Presence of mycolic acids inthecell wall • Presenceofmycolicacidsinthecellwalland
Integrityofthecellwall • Integrityofthecellwall.
2. Guanine plus cytosine (G + C) content of DNA is 61–71 mol
%Mycobacteriaareclassifiedinto:
• M.tuberculosiscomplex
• M.leprae
• Nontuberculousmycobacteria(NTM).
MYCOBACTERIUMTUBERCULOSISCOMPLEX
M.tuberculosiscomplexconsistsofgeneticallyrelatedspeciesthatcausetuberculosisinman:
• M.tuberculosis:Itisthemostcommoncauseoftuberculosisinman
• Others:M.bovis,M.caprae,M.africanum,M.microti,M.pinnipediiandM.canetti
Pathogenesis
About one third of world population is infected with M.tuberculosis, of which 5–10%
developclinical disease. India accounts for one fourth (25%) of global TB cases with prevalence
andincidenceratesof256and185casesperonelakh.
ModeofTransmission
• Byinhalationofdropletnuclei(<5–10μmsize)directlyfromcasesofpulmonary
tuberculosis
• Inoculation(rare):Directskincontactwithaninfectedperson
• Ingestion(rare):Swallowingofsputum(ininfants)orunpasteurizedmilk.
RiskFactorsFavoringtheTransmissionandDiseaseProgressionInclude
• Sputumpositivepatientstransmitmoreefficiently
• Bacillaryload:Atleast104bacilli/mlinsputumisrequiredforaneffectivetransmission
Atleast104bacilli/mlMTBloadinsputumis neededfor:
• Adultpatientswithcavitarylesionsinlunghavemorebacillaryload
Effectivetransmission
Patient to become smearpositive • Overcrowdinginpoorlyventilatedrooms
• LowcellmediatedimmunityasoccursinHIVinfectedpeople
• Otherco-morbidconditionssuchaspostsilicosis,post-
transplantation(renal,cardiac),jejuno-ileal bypass, gastrectomy, chronic renal
failure/hemodialysis, diabetes, IV drugabuse,smoking,etc
• Age:Lateadolescenceandearlyadulthoodperiodsaremoreprone.
• Sex:Riskishigherinwomenat25–34years,whileatolderages,menhavegreaterrisk.
TheSequenceofPathogenicEventsthatTakePlaceisasFollows:
1. Dropletnucleicontainingtuberclebacillifrominfectiouspatientsareinhaled.
Intracellular Survival of MTB isdue to:
Impairsphagosome-lysosome
2. Majorityaretrappedintheupperairwaysandexpelledoutbytheciliaryactionofthemucosal
fusion to bacterial cell walllipoarabinomannan cells;onlyafraction(usually<10%)ofsmalldropletsreachthealveoli.
3. Adhesiontomacrophages:Mycobacterialsurfacelipoarabinomannan(LAM)bindstocompl
ementreceptorsandmannosereceptorspresentonthesurfaceofmacrophages.
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Mycobacteria175
Phagocytosisbymacrophagesisenhancedbycomplement(C3b)mediatedopsonization.
Survival inside the macrophages: This is due to bacterial cell-wall lipoarabinomannanwhich impairs phagosome-lysosome fusion by inhibiting i
Tuberculosis(TB)isclassifiedaspulmonary(80%)andextrapulmonaryforms(20%).
PulmonaryTuberculosis
ItisclassifiedintoPrimaryPTBandPost-primary/secondaryPTB
Features Primary PTB Post-primary/secondary PTB

Resultsdueto Initialexogenousinfectionwithtuberclebacilli • Exogenous reinfection
• Endogenousreactivationoflatentprimarylesion
Agegroupaffected Children Adults
Parts of the Subpleurallesion(upperpartoflowerlobeandlower Apical and posterior segments of the upper lobes (areas of
lungscommonlyaffec partofupperlobe) highoxygentension)
ted
Lesions formed at Fibrotic nodular lesions are Calcifiednodulesareformed(Assmannfocus)
theinitialsites formed(Ghon focus) EarlyhematogenousseedlingintheapexoflungscalledasSimon’sfocus
Lymphnode Ghon focus with hilar Lymphnodeinvolvementisunusual

lymphadenopathy(calledasprimarycomplex
)
Clinicalfeature It may be asymptomatic or may present Lesionsundergoingnecrosisandtissuedestruction,leadingtocavity
withfever, cough, hemoptysis, chest pain, formation.
nightsweating,weightloss Symptomsaresimilar,butmorepronounced.
Fate Inthe majority ofcases: Inmajorityofcases:Thenecroticmaterialbreaksintotheairways,leadingto:
• Lesionshealspontaneously. • Bronchogenicspreadtothesameoroppositelung
• Primarycomplexbecomescalcified • Expectorationofbacterialadensputum
(Rankecomplex) • Hematogenousspreadleadingtoseedlingofbacilliinvariouspa
Rarely, develops to progressive rtsofthe body
primaryTBwhichspreadsbylocalinvasionandbyl • Spontaneoushealingisrare
ymphatics
ExtrapulmonaryTuberculosis(EPTB)
EPTB results from hematogenous dissemination of tubercle bacilli to various
organs.ThoughEPTBconstitutesabout15to20%ofallcasesofTB,inHIV-
positivepatientsthefrequencyismuchhigheraccountingfor20–50%ofallcasesoftuberculosis.
Thesitescommonlyinvolved(inorderoffrequency)are:
SystemicBacteriolog
1. TuberculouslymphadenitisistheMCform(35%ofallEPTBcases).Themostcommonsitesare
posteriorcervicalandsupraclavicularlymphnodes.
2. Pleuraltuberculosisaccountsfor20%ofallEPTBcases.Itpresentsaspleuraleffusion.
3. Tuberculosisoftheupperairwaysinvolvinglarynx,pharynx,andepiglottis Tuberculouslymphadenitis
4. Genitourinarytuberculosis: MC form (35%) of all EPTBcases
• Renaltuberculosis MC sites are posterior cervicaland supraclavicular lymphn
• Genitaltuberculosis:Infemalepatients,fallopiantubesandtheendometriumarecomm
onlyinvolvedcausinginfertility.Inmale,epididymisistheMCsite.
5. Skeletaltuberculosis:Weight-bearingjointssuchasspine(Pott’sdiseaseortuberculous
spondylitisismostcommon),hipsandkneesarecommonlyaffected.
6. TuberculosisofCNSoccurscommonlyinchildren.Tuberculousmeningitisandtuberculo
maarethecommonforms
7. Gastrointestinaltuberculosis:TerminalileumandcecumaretheMCsitesinvolved.Transmiss
ionisduetoswallowingofsputum,hematogenousspread,oringestionofcow’smilkcontaminate
dwithM.bovis
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8. Tuberculouspericarditis
9. Tuberculousskinlesions:
• Scrofulodermaisaskinconditioncausedbytuberculousinvolvementoftheskinbydirec
textension,usuallyfromunderlyingtuberculouslymphadenitis
• Lupusvulgaris:Applejellynodulesareformedoverthefaceinfemales.
10. Miliaryordisseminatedtuberculosis:Hematogenousspreadoftuberclebacilliresultsin the
formation of granulomatous lesions resembling millet seeds in various organs. It
ismorecommoninHIVinfectedpeople.
11. HIV-associated tuberculosis: TB is the MC opportunistic diseases among HIV-
infectedpersons. Worldwide, TB affects 70–80% of HIV infected individuals, EPTB being
morecommonthanPTB.
LaboratoryDiagnosisofTuberculosis
DiagnosisofActiveTuberculosis
• Specimencollection:
○ InpulmonaryTB-sputum(2specimens—spotandearlymorning),gastricaspirate
(inchildren)
○ InEPTB—specimensvarydependingonthesiteinvolved.
• Digestion,decontaminationandconcentrationofspecimen:
○ Petroff’smethod(4% NaOH)
○ NALC(N-acetyl-L-cysteine)+2%NaOH.
• Acid-faststainingbyZiehlNeelsen(ZN)technique:
○ Procedure:
▪ Smeariscoveredwithprimarystain,strongcarbolfuchsinfor5minuteswith
intermittentheating
▪ Decolorizationwith25%sulfuricacidfor3minutes
▪ Counterstainingwithmethylenebluefor1min.
○ Advantages:Smearmicroscopyisrapid,easytoperformatperipherallaboratoriesandische
RNTCPgradingofsputumsmearis useful for:
Monitoring the treatment aper.
responseofthepatients ○ Disadvantages:
Assessing the severity ofdisease ▪ Lowsensitivity:Detectionlimitis10,000bacilli/mlofsputum.
Assessing the infectiousnessofthe patient ▪ Viabilityofbacillicannotbedetermined
○ Reporting: M.tuberculosis appears as long slender, beaded, less uniformly stained
redcoloracidfastbacilli.
○ AcidalcoholcanbeusedasdecolorizerinrenaltuberculosistodifferentiateM.tuberculosis(a
cidandalcoholfast)fromM.smegmatis(acidfastbutnotalcoholfast)inurinesample.
○ Sputum microscopyisthemethodofchoicecasefindingtoolfor tuberculosisunderRNTCP
○ RNTCPgrading:Thisgrading isuseful for:
▪ Monitoringthetreatmentresponseofthepatients
▪ Assessingtheseverityofdisease
Section
▪ Assessingtheinfectiousnessofthepatient:Higherthegrademoreistheinfec-
tiousness.Smearnegativepatientsarelessinfectious.
• Othersstainingmethods:
○ Kinyoun’scoldacidfaststaining
○ Auraminephenoltechnique:Itisafluorescentstainingtechnique.
• Conventionalculturemedia:
○ Advantage:Cultureismoresensitivewithdetectionlimitof10-100viablebacilli.
○ Disadvantage:Itistimeconsuming.Minimum8weeksofincubationisneeded.
Examples of media:
○ Solidmedia,e.g.
▪ Eggbased:LowensteinJensen(LJ),Dorseteggmedia,PetragnanimediaLowensteinJe
nsen(LJ):M.tuberculosisproducesrough,toughandbuffcolonies,recommendedbyR
NTCP
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Mycobacteria177
▪ Agarbased:Middlebrook7H11and7H10.Theyarepreferredforisoniazidresist-
antstrainsofM.tuberculosis
○ Liquidmedia:Middlebrook7H9,Dubos,Proskauer,Sula,andSautonmedia. BACTEC MGIT (Mycobacteriagrowth indicator tu
• Automatedculturemethods:Thesesystemsmonitorthegrowthcontinuouslyanddetect Usesanoxygensensitive
growth faster (2-3weeks); however, they areexpensive: fluorescentcompoundtodetect
○ ExampleincludeBACTEC,MGITandBACT/Alert Mycobacterialgrowth
Resistancetofirstlineanti-
○ BACTEC MGIT (Mycobacteria Growth indicator Tube)—Uses an oxygen tuberculardrug
sensitivefluorescentcompoundtodetect:
▪ Mycobacterialgrowthand
▪ Resistancetofirstlineanti-tuberculardrug
• Biochemical identification: Positive for Niacin test, Nitrate reduction test,
PyrazinamidasetestandResistanttoTCH.
• Serology:
○ Antigendetection—LAMandantigen-5detectionbyELISA Lineprobeassay
○ Antibodydetection—notusefulinendemicarea. Detects drug resistance fromsamples (takes one day), buto
Less sensitive for smearnegativecases
• Molecularmethods:
○ PCRdetectingIS6110geneandothergenessuchas65KDaand38KDagenes
○ Line probe assay: Detects drug resistance from samples (takes one day), but
onlyfromsmearpositivecases.Itislesssensitiveforsmearnegativecasesbecauseitisba
sedonhybridizationtechniques.
○ GeneExpert:Itdetectsgrowthandresistancetorifampicin.Ittakesverylesstime(2hou
rs).Itisacartridgebasednucleicacidamplificationtechnique(basedonrealtimePCR).P
leuralbiopsyisbetterspecimenthanpleuralfluidforGeneExpert.
• Animalpathogenicity:UsingGuineapigandrabbit Gene Expert:
It detects growth andresistancetorifampicin
It takes very less time(2hours)
Property M. tuberculosis M. bovis It is a cartridge basedrealtime PCR technique
Acid fast stain Curved,long,beaded,lessuniformlystain Straight, short,
ed stout,uniformlystained
LJmedia Rough,tough,buffcolony White, smooth, moist colony
Growth Eugonic Dysgonic
Glycerol Helpsingrowth Noeffectongrowth
TCH ResistanttoTCH Sensitive toTCH
Niacintest Positive Negative
Nitratetest Positive Negative
Rabbitpathogenicity Notpathogenic Pathogenic
Oxygen Obligateaerobe Microaerophilic
SystemicBacteriolog
Botharepathogenictoguineapigandhumanbeings
DiagnosisofEPTB:EPTBdiffersfromPTBinmanyaspects:
AssuchEPTBspecimensarepaucibacillary,hencesmearmicroscopyislesssensitive.
• Molecularmethodsaremoreuseful.
• PleuralfluidrevealselevatedADA(adenosinedeaminase)and(IFN)-γlevels.
• Renaltuberculosis:Urinaryexcretionofbacilliisintermittent;hence3–
6consecutiveearlymorningurinesamplesarecollected,centrifugedandthesedimentisusedfor
processing.Acidalcoholisusedasdecolorizer.
• CSF examination shows cobweb coagulum on standing, ↑CSF pressure, protein
andchloride;whereas↓glucoselevels.
DiagnosisofLatentTuberculosis
ItisdiagnosedbydemonstrationofdelayedortypeIVhypersensitivityreactionagainstthetuberc
lebacilliantigens.
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A.Tuberculintest
ItisdiscoveredbyVonPirquetin1907.
• Antigensused:
○ Oldtuberculin(OT):Crudepreparationoftuberclebacilli,wasusedbefore.
○ Purifiedproteinderivative(PPD):Purifiedpreparationoftheactivetuberculoprotein,prepar
edbySeibert
○ Dosage: It is expressed in tuberculin unit (TU). One TU is equal to 0.01 ml of OT
or0.00002mgofPPD.
• Procedure:
○ MantouxtestistheMCmethodused.0.1mlofPPDcontaining1TUisinjectedintra-
dermallyintoflexorsurfaceofforearm.
○ HeafandTinemultiplepuncturetests:Boththetechniquesarenotinuse.
• Reading:Indurationsurroundedbyanerythemaisproducedat48–
72hrs.Ifthewidthoftheindurationis:
○ ≥10mm:Positive(tuberculinreactors)
○ 6–9mm:Equivocal/doubtfulreaction
○ <5mm:Negativereaction
• Interpretationofresult:
○ Adults:Positivetestinadultsonlyindicatespresentorpastinfectionwithtuberclebacill
Mantoux test Interpretation: ibutdoesnotconfirmpresenceofactivestageofthedisease.Itisonlyusedasepidemiolo
gicalmarker:
Adults: Positive test indicatespresent or past infection withtuberclebacilli
Butdoesnotconfirm ▪ Prevalenceiscalculatedbycountingalltuberculinreactorsinacommunity
activedisease ▪ Incidence–Bycountingnewconverterstotuberculintestinacommunity.
Used as epidemiologicalmarker.
○ Children:Positivetestindicatesactiveinfectionandusedasdiagnosticmarker.
Children: Positive testindicatesactiveinfectionandusedasdiagnosticmarker.
• Falsepositive:Thetestbecomesfalsepositiveafter:
○ BCGvaccination(after8–14weeks),
○ NTMinfection
• Falsenegative:Thetestmaybecomefalsenegativeinconditionssuchas:
○ Early oradvanced TB,
○ MiliaryTBandpostmeasles
○ Lowimmunity,HIVinfectedpeople.
• Twosteptesting:Inadults,tuberculinreactivityslowlywaneswithtimeanditmaybecomenega
tiveaftersomeyears.Repeattest1–
2weeksafterthefirsttestexertsaboostereffectandgivesastrongpositivereaction(>20mm).
B. InterferonGammaReleaseAssay(IGRA)
• Procedure:SensitizedTlymphocytesofsuspectedindividuals,areexposedtohighlyspecific
M.tuberculosis antigen such as CFP10 (culture filtrate protein) and ESAT6
(earlysecretedantigenictarget-6),whichleadstoreleaseofhighlevelofIFNγ.
• Itisaninvitrotest.ELISAformatsareavailable(calledasQuantiFERON-TBGoldassay)
• Advantage:Itishighlyspecific;therearenofalsepositiveconditions.
Section
Treatment
Anti-tuberculardrugscanbeclassifiedinto:
• Firstline drugs: Isoniazid (H),Rifampin (R),Pyrazinamide (Z),Ethambutol (E),
Streptomycin(S)
• Secondlinedrugs:
○ Ethionamide,Cycloserine,Macrolides(Clarithromycin)
○ Quinolones(Ofloxacin,ciprofloxacin)
○ Aminoglycosides:Kanamycin,capreomycinandamikacin
Thefollowingstrategiesarefollowed:
1. MultidrugtherapyforShortcourselastingfor6months(or8monthsinpreviously
treatedcases)
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Mycobacteria179
2. Twophasechemotherapy:Intensivephase(2–3months)followedbyContinuationphase
(4–5 months)
3. DOTSstrategy(DirectlyObservedTreatment,Shortcourse)isrecommendbyRNTCP
andWHO
4. Treatmentregimens:CategoryIandIIregimensarefollowedasperRNTCP.
DrugSusceptibilityTesting(DST)
SeveralDSTmethodsareavailablefortuberclebacilli.
Phenotypicmethods:Commonlyusedmethodsare:
• Proportionmethodisthegoldstandardmethod
• AutomatedsystemssuchasBACTECMGITarewidelyusedthesedays.
• Others:
○ Absoluteconcentrationmethod
○ Resistanceratiomethod.
Molecularmethods:TodetectdrugresistantgenesbymethodssuchasPCRbasedassays,DNAProbe
based(Lineprobeassay),DNAmicroarrayandGeneExpert
DrugresistancegenespresentinM.tuberculosis(acquiredbymutation)
MultidrugResistantTuberculosis(MDR-TB)
Drugs Drug-resistantgenes
Isoniazid
MDR- Enoyl ACP reductase (inhA), Catalase-peroxidase
(katG)Alkylhydroperoxidereductase(AhpC)
Rifampicin RNApolymerasesubunitB(rpoB)
Pyrazinamide Pyrazinamidase (pncA)
MDR-TB
Ethambutol Ribosomalproteinsubunit12(rpsL)
Resistance to isoniazid andrifampicin with or withoutresis
Streptomycin Ribosomalproteinsubunit12(rpsL),16sribosomalRNA(rrs)Aminoglycosidephosphot
ransferasegene(strA)
TBisdefinedasresistancetoisoniazidandrifampicinwithorwithoutresistancetootherfirstlined
Fluoroquinolones DNAgyrase(gyrAandB)
rugs.
Epidemiology:
• AsperWHO,60%oftotalMDR-
TBcasesresidesinBRICScountries:Brazil,Russia,India,China,SouthAfrica,withIndiaaccou
ntingforthemaximumcases.
• InIndia,MDR-TBaccountsfor2.8%ofallnewTBcasesand12–17%ofre-treatmentcases.
TreatmentofMDR-TBisdoneunderDOTS-
Plusprogrammebyusingcomplexregimenof2ndlinedrugs:intensivephase(6drugs)for6–
SystemicBacteriolog
9monthsfollowedbycontinuationphase(4drugs)for18months.
ExtensivelyDrugResistantTuberculosis(XDR-TB) Extensively Drug-ResistantTuberculosis (XDR-TB

Fluoroquinolones (ofloxacin/
1. Definition:TheseareMDR-TBcasesthatarealsoresistantto:
levofloxacin)
• Fluoroquinolones(ofloxacin/levofloxacin)and At least one injectableaminoglycosides (kanamycin,amika
• Atleastoneinjectableaminoglycosides(kanamycin,amikacinorcapreomycin).
2. Epidemiology:InUSA,3%ofMDR-TBcaseshavebeenfoundtobeXDR.Theexactincidence of
XDR-TB in India is not known. The MDR-TB treatment failure cases (2–
6%)maybepresumedtobeXDR-TBcases.
3. Treatment of XDR-TB is extremely difficult. XDR-TB has a very rapidly
progressingclinicalcoursewithhighmortality.
ProphylaxisbyBCGVaccine(BacillusCalmetteGuerin)
BCGvaccinewasdevelopedbyCalmetteandGuerin(1921).Theyattenuatedthestrainbyserialsubcul
turinginglycerolbilepotatomediumfor230timesoveraperiodof13years.
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• BCGStrain:LiveattenuatedM.boviswasthestrainoriginallyused:
○ Thoughthesamestrainisusedcurrently,duetodifferentmethodsofmaintenanceinvar
iousvaccinelaboratories,manysubstrainshaveevolvedinpastfewdecades.
○ InIndia,WHOrecommendedDanish1331strainofBCGisused.Itispreparedin
CentralBCGlaboratory,Guindy,Chennai.
AdministrationofBCG:
• Reconstitution: BCG is available in lyophilized form which should to be reconstituted
Doseandstrength:0.1mlcontaining0.1mgTU innormal saline before administration. Distilled water is never used as it is irritant.
Alcohol should not used towipethe skin Oncereconstituted;itisadministeredwithin1hour.
Site:Itisgivenaboveinsertionofleftdeltoid • AdministrationofBCG:
Route:Byintradermalroute
○ Doseandstrength:0.1mlcontaining0.1mgTU
○ Alcoholshouldnotusedtowipetheskin
○ Site:Itisgivenaboveinsertionofleftdeltoid
○ Route:Byintradermalroutebyusinga26gaugetuberculinsyringe.
• PhenomenaafterBCG:Ifproperlyinjectedintradermally,thenatinoculationsite:
○ 6–12weeks:Permanenttinyroundscar(4–8mmdiameter)isformed
○ 8–14weeks:Mantouxtestbecomespositive.
ProtectionofBCG:
Ifoverdoseisgiven:Thelesionorscarbecomeslargerandirregularsize.
Variableefficacyof0–80%.
Durationofimmunitylastsfor • Protection:
15–20years ○ Efficacy:ManytrialshaveshownthatBCGhasavariableefficacyof0–80%.
○ Durationofimmunitylastsfor15–20years
○ Though BCG may not protect from the risk of tuberculosis infection, but it
surelygivesprotectiontoinfantsandyoungchildrenagainstthemoreserioustypesofth
edisease,suchasmeningitisanddisseminatedtuberculosis.
IndicationsofBCG:
• ComplicationsfollowingBCG:
Direct BCG: BCG is giventonewbornsoonafterbirth
directly,followedindevelopingcountries. ○ MC complications include ulceration at the vaccination site and regional lymphad-
enitis
Indirect BCG: BCG is givenafterperformingtuberculintest,followed in less prevalencecountry
○ Rarely,keloidorlupuslesion, andosteomyelitismaydevelop
○ Very rarely, non-fatal meningitis and progressive tuberculosis, disseminated
BCGinfection(‘BCGitis’)arereportedinpeoplewithlowimmunity.
• IndicationofBCG:
○ DirectBCG:BCGisgiventonewbornsoonafterbirthdirectly.Thisstrategyisfol-lowed
by most of the developing countries including India. If not given at birth
itcanbegivenlater,maximumupto2years.
○ IndirectBCG:BCGisgivenafterperformingtuberculintest.
OtherusesofBCG:
• ContraindicationstoBCG:HIVpositivechild,ChildbornetoAFBpositivemother,Childwithlow
Stimulates the immunesystem, providing someprotectionagainstleprosyandleukemia
immunity,GeneralizedeczemaandPregnancy
In malignancies such asbladdercarcinoma(OncoTICEstrainofBCG)
BCG may be superior to PPDfor tuberculin test Other usesofBCG:
•
○ BCGprovidessomeprotectionagainstleprosyandleukemia
○ BCG has been tried as adjunctive therapy in malignancies such as bladder carci-
noma(OncoTICEstrainofBCG)
○ BCGmaybesuperiortoPPDfortuberculintest,asreportedbysomeworkers.
Section
NONTUBERCULOUSMYCOBACTERIA
NonTuberculousMycobacteria(NTM)or atypicalmycobacteriaorMycobacteriaotherthan
tubercle bacilli (MOTT) are isolated from birds, animals, soil and water. They
areopportunisticpathogensinhumans.
Table3.6.1:Runyon’sclassificationofnon-Tuberculousmycobacteria(NTM)
Runyongroup Property Species
I.Photochromogens Producepigmentsonlyinlight MASK-M.marinum,M.asiaticum,M.simiae,M.kansasii
II.Scotochromogens Producepigmentsevenindark,butintensityofcolorm M.scrofulaceum,M.szulgai,M.gordonae,M.celatum,
ayincreaseonexposuretolight M. flavescens
Contd...
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Mycobacteria181
Contd...
Runyongroup Property Species

III.Non- NoPigmentation M.avium-intracellularecomplex(MAC),M.ulcerans,
photochromogens M.xenopi,M.paratuberculosis,M.malmoense
IV.Rapidgrowers Growwithinoneweek M.chelonae,M.fortuitum,M.smegmatis,M.abscessus
NTMdifferentiatedfromMTBcomplexby:
• Resistance to paranitrobenzoic acid (PNB), but sensitive tothiophen-2-carboxylic
acidhydrazide(TCH)
• Arylsulfatasetestpositive
• StrongCatalasepositive
• Nonpathogenicforguineapig;butpathogenicformouse
CommonNTMinfections:
• Resistanttoantituberculardrugs. M.marinum:Swimmingpool granuloma or fish tankgranu
M.ulcerans:Buruliulcer
DiseasesCausedbyNontuberculousMycobacterium M.fortuitum and M.chelonaecause post-trauma injection
MAC: Causes opportunisticinfectioninHIV
Photochromogens(ProducePigmentsOnlyinLight)
• M.marinum:Itcausesthefollowinginfections
○ Swimmingpoolgranulomaorfishtankgranuloma.
○ Tendonitisandtendernodules–spreadinasporotrichoidpattern.
• M.asiaticumisrarelyassociatedwithpulmonarydiseaseandbursitis.
• M.simiae:Itgivesapositiveniacintest.Itproducespulmonarylesions.
• M.kansasii:Causeschronicpulmonarydiseaseresemblingtuberculosis.
Scotochromogens(ProducePigmentsinLightandDark)
• M.scrofulaceumcausesscrofula(cervicallymphadenitis)inchildren.
• M.gordonaeisoftenfoundascommensalintapwater.
• M.szulgai:Itmayoccasionallycausepulmonarydiseaseandbursitis.
• M.celatumisararecauseofpulmonaryinfection.
Nonphotochromogens(DonotProducePigments)
• M.avium-intracellularecomplex(MAC):
○ They comprise of two related organisms: M.avium (Battey bacillus) and
M.intracellulare.
○ They are opportunistic pathogens in HIV with low CD4 Tcell count (< 50/μl).
○ Manifestations-lymphadenitis,respiratoryinfectionanddisseminateddisease.
• M.xenopihasbeenisolatedfromhospitalwatersupplies,andnosocomialoutbreaks.
• M.ulceransisawaterborneskinpathogen,foundmainlyinthetropicsofAfrica,AmericaandSout
heastAsia:
○ ItistheagentofBuruliulcer.
SystemicBacteriolog
○ Itcanalsocauseosteomyelitisandlimbdeformities.
○ Exotoxin:Itproducesmycolactonetoxin.
• M.malmoensecancausepulmonarydiseaseandrarelylymphadenitis.
• M.paratuberculosis(Johne’sbacillus)isassociatedwiththepathogenesisofCrohn’sdisease,butt
hislinkhasnotbeenprovedyet.
RapidGrowers(Growwithin1WeekofIncubation)
• M.fortuitumandM.chelonaecausepost-traumainjectionabscessandcatheterinfections
• M.abscessuscancausepulmonaryinfection Skin Lesions of LepromatousLeprosy:
• Testforrapidgrowers:Arylsulfatasetest—Positiveforrapidgrowers. Many,symmetrical
Marginisirregular
Appear as: Multiple nodules(lepromata), PlaquesandXant
MYCOBACTERIUMLEPRAE Leonine facies and eyebrowalopecia
MycobacteriumlepraeorHansen’sbacillusisthecausativeagentofleprosy,anancientdisease that
remained as a social stigma over many years due to the superstitious beliefs
andcharacteristicdeformitiesproducedinthepatients.
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ClinicalManifestationsandClassification
Leprosyprimarilyinvolvescoolerpartsbutiscapableofaffectinganytissueororganscausingbon
ydeformitiesanddisfigurementsinuntreatedcases.
Incubationperiod:Leprosyhasalongincubationperiod,anaverageof3–5years.
• Thiscanbeexplainedduetothelongergenerationtimeofleprabacilli(12–13days)
• Lepromatousleprosy(LL)caseshavelongerincubationperiodthantuberculoid(TT).
Table3.6.2:Clinicalclassificationofleprosy
RidleyJoplingclassification Madridclassification Indianclassification
Lepromatousleprosy(LL) Lepromatoustype Lepromatoustype
BorderlineLepromatousleprosy(BL) Borderline Borderline
Borderlineleprosy(BB) Indeterminate type Indeterminate type
SlikLesionsofTuberculoidLeprosy: BorderlineTuberculoidleprosy(BT) Tuberculoidtype Pureneurotictype
Oneorfew,asymmetrical
Tuberculoidleprosy(TT) - Tuberculoidtype
Marginissharp
Appear as: Hypopigmented,annularmaculeswithelevatedborders
Leprosy is a bipolar disease. Under any classification scheme, lepromatous and
Tendencytowardscentralclearing tuberculoidcasesarethetwoextremepolesofthedisease(describedintable).Othervarietiesare:
• Borderline type: It is seen in patients possessing characteristics in between
tuberculoidandlepromatoustypes.Theymayshifttoeithertypedependingonchemotherapyor
alterationsinthehostresistance.
• The indeterminate type: This denotes those early unstable cases with one or
twohypopigmentedmaculesanddefinitesensoryimpairment.Bacteriologicallynegative.
• Pure neuritic type: Shows neural involvement without any skin lesion. Cases
arebacteriologicallynegative.
Characters Lepromatous leprosy (LL) Tuberculoidleprosy(TT)

Bacillaryload Multibacillary Paucibacillary
Bacteriologicalindex 4–6+ 0–1+
Skinlesions Many, symmetrical, Margin is irregular, Lesions Oneorfew,asymmetrical,Marginissharp,Lesionsappearas,Hyp
appearasMultiplenodules(lepromata),PlaquesandXanthoma- opigmented,annularmaculeswithelevatedborders,Tendencyto
likepapules,Leoninefaciesandeyebrowalopecia wardscentralclearing
Nervelesion Appearlate Early anesthetic skin lesion, Enlarged thickened nerves(MC
Hypoesthesiaisalatesign nerves involved are ulnar nerve followed by
postauricularnerve),Nerveabscessseen(commoninBT)
Cellmediatedimmunity CMIlow CMInormal
Lepromintest Negative Positive
Lymphocytetransformat Negative Positive
iontest
CD4/CD8Tcellratio 1:2 2:1(normal)
Humoralimmunity Exaggerated Normal
Section
AutoAntibodies Elevated Notseen

VDRLtest Biologicalfalsepositive VDRLtestnegative
Antibodies to PGL-1 Elevatedin95%ofcases Elevatedin60%ofcases
Macrophages Foamytype(lipidladen) Epithelioid type
Langhansgiantcells Notseen Found
Deformities
About25%ofuntreatedcasesdevelopdeformitiesinduecoursewhichmayarisedueto:
• Nerveinjuryleadingtomuscleweaknessorparalysisor
• Diseaseprocess(facialdeformitiesorlossofeyebrow)or
• Infectionorinjury(ulcers).
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Mycobacteria183
Commondeformitiesinclude:
• Face:Leoninefacies,saggingface,lossofeyebrow/eyelashesandcornealulcers.
• Hands:Clawhandandwristdrop
LepraReactionTypeI:
• Feet:Footdrop,clawingoftoes,inversionoffoot,andplantarulcers. If occurs before treatment:ProgressestowardsLL(downgra
If occurs after treatment:ProgresstowardsTT(reversalreact
LepraReactions
Thoughleprosyrunsasachronicdisease,severalallergictypeacuteexacerbationsoccurthroughou
titscourse,calledasleprareactionswhichareoftwotypes:IandII.
Characters LepraReactionTypeI LepraReactionTypeII

Hypersensitivityreaction TypeIV(delayedhypersensitivity) TypeIII(immunecomplexmediated)
Seenwith Borderlineleprosy Lepromatousvariety(BL,LL)
Manifestsas Inflammationofpreviouslesions,newskin Cropsofpainfulerythematouspapuleswhichbecomenodular
lesionsandneuritis
Progressesas If occurs before treatment— Itusuallyoccursfollowingthestartofchemotherapy.
ProgressestowardsLL(downgradingreacti
on)
Ifoccursaftertreatment—
ProgresstowardsTT(reversal reaction)
Thelperresponse TH1predominates TH2predominates
Otherorgans Usuallynotaffected Eyes,testesandkidneyareaffected
Treatment Glucocorticoid Glucocorticoid, thalidomide, clofazimine
Epidemiology
• Sourceofinfection:Multibacillary(LLandBL)casesarethemostimportantsource.
• Modeoftransmission:Nasaldropletinfection(MC)>Contacttransmission>breast
milk,verticalmode,tattooing Leprosyeliminationlevel:
• Communicability:Leprosyisnothighlycommunicable.Intimateandprolongedcontactisnece Leprosyeliminationlevel:
ssary. <1/10,000population
PrevalenceinIndia:0.68
GeographicalDistribution casesper10,000population
ChhattisgarhandDadraandNagar Haveli are above theleve
CurrentlyleprosyisalmostexclusivelyconfinedtothedevelopingnationsofAsia,Africa,LatinAm
erica,andPacific.
Indiaaccountsformaximumcasesglobally.However,thediseaseburdenisdeclining:
• TheprevalencerateinIndiaisabout0.68casesper10,000populationin2012.
• About32states/
UnionTerritory(UT)havealreadyachievedthelevelofleprosyeliminationi.e.prevalencera
SystemicBacteriolog
teoflessthan1caseper10,000population.
• ChhattisgarhandDadraandNagarHaveliaretheonlystate/UTinwhichitremainsabove
thelevel ofleprosyelimination. Biharliesat theborderline.
LaboratoryDiagnosis
SmearMicroscopy
Smearmicroscopyisdonetodemonstrateacidfastbacilliinthelesions: Smearmicroscopy:
Site:Edgeofthelesion
• Totalsixsamplesarecollected;fourfromskin(forehead,cheek,chinandbuttock),ear
Method: Slit skin smear
lobeandnasalmucosa Appearance:Cigarbundlesof AFB arranged in globi,which
• Theedgeofthelesionisthepreferredsite. macrophagescalledVirchow's'lepra cells'
• Slitskinsmearisthetechniquefollowedtocollecttheskinandearlobespecimens.
• Nasalspecimensarecollectedbynasalblow,ii)nasalscraping
• Biopsyfromthethickenednervesandnodularlesionsmaybenecessaryinsomecases.
• AcidfaststainingisdonebyZiehl-Neelsentechniquebyusing5%sulfuricacid.
• Appearance:Underoilimmersionobjective,redacidfastbacilliareseen,arranged
singlyoringroups,boundtogetherbylipid-likesubstance,thegliatoformglobi(called
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ascigarbundleappearance).Theglobiarepresentinsidethefoamymacrophagescalled
asVirchow’s‘lepracells’or‘foamycells’:
○ Livebacilliwillbeuniformlystainedwithparallelsidesandroundendsandlengthisfivet
imesthewidth.
○ Deadbacilliarelessuniformlystainedandhavefragmentedandgranular appearance.
• Gradingofthesmearisdoneby:
○ Bacteriologicalindex(BI)isbasedonthetotalnumberofbacilli(liveanddead)seenperoilim
mersionfield.
○ Morphologicalindex(MI):Percentageofuniformlystainedbacillioutofthetotalnumber
ofbacillicounted.MIisabettermarkertomonitorthetreatmentresponse.
○ SFGpercentage(solid,fragmentedgranularrodpercentage).
MouseFootPadCultivation
M.lepraeisnotcultivableeitherinartificialculturemediaorintissueculture.Theonlycertain
waytocultivateM.lepraeisbyinoculatingthespecimensinto:
• Ninebandedarmadilloand
AntibodyDetectioninLeprosy:
FLA-ABS • Footpadofmice.
ELISAdetectingAbtoPGL-1
AntibodyDetection
• FLA-ABS(FluorescentLeprosyAntibodyAbsorptiontest)
• ELISAdetectingIgMantibodiestoPGL-1(phenolicglycolipid-1)antigenofM.leprae
TestforDetectingCMI(LeprominTest)
LepromintestdemonstratesthedelayedhypersensitivityreactionandanintactCMIagainst
thelepraantigen.
• Procedure:0.1mlofleprominantigenisgivenintradermallytoforearmandreadingistaken
UsesofLeprominTest: twice;at48hrsand21days.
Classifyinglesionsofleprosy • Early reading or Fernandez reaction at 48 hrs: Induration and erythema (red area) of
Assessingprognosis
Assessing resistance toleprosyinindividuals >10 mm diameter indicates delayed hypersensitivity reaction and hence past exposure
toleprabacilli.However,itdoesnotindicateactiveinfection.
• Late reading or Mitsuda reaction at 21 days: Nodule of > 5 mm size formed at the site
ofinoculationwhichulcerateslateron.Itindicatesthatthepatient’sCMIis intact.
• UsesofLepromintest:ThelatereactionmeasurestheCMI;hencecanbeusedfor:
○ Classifyinglesionsofleprosy:InTTpatientswithintactCMI,thetestisstronglyposi-
tive.InLLpatients;thetestisnegativeindicatingalowCMI.
○ Assessingprognosis:IntactCMI(asinTTpatients)indicatesgoodprognosis.
○ Assessingresistancetoleprosyinindividuals:Leprominnegativepersonsareathigher
riskofdevelopingmultibacillaryleprosythanleprominpositivepersons.
Treatment
Section
Becauseofriskofdevelopingresistance,WHOrecommendsmultidrugtherapy(MDT)fortreatmentof
leprosy:
• Recommendeddrugs:Dapsone,rifampicinandclofazimine
• Alternatedrugs:Ethionamide,quinolones(ofloxacin),minocyclineandclarithromycin
• WHOtreatmentregimensareadministeredbasedontheclinicaltypeofleprosy.
Table3.6.3:ClinicalclassificationofleprosyandWHOtreatmentregimens
Criteria Paucibacillary leprosy Multibacillary leprosy
Skinlesions 1–5 6ormore
Nerveinvolvement 1 2ormore
Microscopy Smearnegative Smearpositive
Contd...
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Sandhya BhatKMDDNBMNAMS PDCR

Jaypee Brothers Medical Publishers (P)

Methods toImproveYour Memory

Ouradvice as timeis precious:

MMR+2Pox Hepatitisviruses Malaria STD

KFD 3–8d HIV 10 Pertussis 7–14d Cl.novyi 5–6d

SoftTick Endemic relapsing fever (B. duttoni),Q-

KochWeekbacillus Haemophilusaegyptius Eaton’sagent Mycoplasmapneumoniae

2. Adansonian (orphonetic)classification:To avoidtheuse ofweighted

Gram-negative bacteria-peptidogly can layer

Characters Gram-Positivecellwall Gram-Negativecellwall

Characters Gram-Positivecellwall Gram-Negativecellwall

▪ Hemagglutination: Many fimbriated bacteria (e.g. Escherichia coli,

Lag phase Log phase Stationary phase Declinephase

Heat labile toxin of ETEC Activationofadenylatecyclase→↑cAMPintargetcell→Secretorydiarrhea

HISTORY 10. Swarmingisseenin: (NEETPatternBased)

20. Acidfastbacilli–stainedby: (TNPG2015)

(PGIDec2007) 35. Allthefollowingstatementsaretrueregardingendotoxi

PHYSICALMETHODSOFSTERILIZATION/DISINFECTION Mechanism of action of heatsterilization:

• Mercury salts such as mercuric chloride, thiomersal and mercurochrome were

18. Surgicalbladeissterilizedby? (DNBJune2009)

• More sensitive:Double roundofamplification yieldshighquantity ofDNA.

• Proteus(proticin typing),Pseudomonas (pyocin typing) 5. Pulsefieldgelelectrophoresis

CULTUREMEDIA/METHODS 9. Bloodagarisanexample: (DNBJune2012)

• Foursubtypes: IgG1-4(Decreasing orderfor DHSisIgG1>IgG 2> IgG3> IgG4)

2. Waldenstrom’s Macroglobulinemia: It is lymphoma affecting B-cells producing

C. Precipitationin Gelin PresenceofElectricCurrent

Hemagglutination inhibition(HAI) test

Abbreviation Immunoassaymethod Molecules used for labeling Typeofvisibleeffect

ANTIGEN 9. Antibodydiversityisdueto: (PGIDec2008)

a. IgG b. IgA c. IgM d. IgE

ANTIGENANTIBODYREACTION 50. Heterophileagglutinationis/areusedinalltestexcept:

Macrophages differ frommonocytesby: Macrophage

Antigen-PresentingCells(APCs) Antigen-presenting cells(APCs)

Immunity PartofInnate immunity,Ag nonspecific Acquired immunity,Agspecific

3. Effector functions: Production of secreted antibodies by plasma cells which in

6. Cellmediatedimmunityisbyvirtueof: c. IL-7 d. IFN-Y

15. MHCmoleculesarecodedby: (NEETPatternBased)

RAST: Detects allergenspecific serum IgE

• Brucellaabortus • Cryptococcusneoformans • Hashimoto’sthyroiditis

Idiopathic Platelet membrane Auto-antibodies against platelet membrane antigens leads to

tosus antigenssuch as DNA, nuclear • Immunecomplexes(selfAg-

Pathogen T-celldefect B-celldefect Granulocyte defect Complementdefect

OPV1,2and3 At6weeks,10weeksand14weeks 2drops Oral Oral

DPTBooster 5–6years 0.5ml Intramuscular Upperarm

Rabiesimmuneglobulin(RIG) Equine,Human TreatmentofrabiesandPEPinpeoplenotpreviouslyimmunizedwithrabiesvaccine

Immunoglobulin preparations Source Indications

HepatitisA Human PostexposureprophylaxisforFamilycontactsandTravelers

Rhisoimmunization(RhIG) Human Treatment–

TRANSPLANTATIONIMMUNOLOGY 18. Allograftrejectionisanexampleof: (JIPMER2014)

• Botryomycosis (mycetoma-like condition)

8. Staphaureuscauses: (NEETPatternBased) rtheoutbreak: (AI2011)

Streptolysin-O (SL-O) Streptolysin-S (SL-S)

forretrospective diagnosis of streptococcal infections (except streptococcalinfections

• For10yrs (with carditis)

Characteristics Early-onset disease Late-onset disease

Characters S. pyogenes S.agalactiae

Pathogenic Viridansstreptococci: copeptides.

15. Which of the following organisms, when isolated

Itcan be differentiatedfrom other commensal insputum, i.e. Strept Viridans:

Properties Gonococcal urethritis (GU) Nongonococcalurethritis(NGU)

OthercomplicationsarecommontobothGUandNGUsuchas: Selective media are useful inchronicgonococcalc