Blood & CVS

Blood and Cardiovascular system
Essay: Page No
1. Enteric fever
2. HIV/AIDS
3. Hepatitis B infection
4. Malaria
5. Filariasis
6. Dengue fever
7. Brucellosis
Short essay
1. Leptospirosis
2. Visceral leishmaniasis or Kala azar
3. Trypanosomiasis
4. Systemic mycosis
5. Rickettsial infection
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Prepared by: Dr. Deepthy BJ
Department of Microbiology
DM WIMS, WAYANAD
Short notes
1. Ebola
2. Hanta virus
3. Chikungunya
4. Relapsing fever
5. Babesiosis
6. Milk ring test
7. Typhoid vaccine
8. Vi Antigen
9. Weil’s disease
10. Histoplasma capsulatum
11. Epidemic typhus
12. Brill Zinsser disease
13. Japanese B encephalitis
14. Lyme disease
15. HCV or Non A non B hepatitis
16.
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Essay
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Clinical case No 1
A 25 year old male sales representative with history of consumption of food
from outside was admitted with high grade fever, nausea, anorexia and
abdominal discomfort for the past few days. O/E tongue was coated and
hepatosplenomegaly was present. Blood investigation showed TC 8000 cells/ml
with predominant lymphocytes.
1) What is your clinical diagnosis?
The most probable diagnosis is Enteric fever.
2) Enumerate the causative agents

Salmonella typhi, S.Paratyphi A, B and C.
3) Describe the pathogenesis and complications of this disease

 Mode of infection: By ingestion.
 Infective dose: 103- 106 bacilli
Bacterial invasion: On reaching the gut, the bacilli attach themselves to the
microvilli of the ileal mucosa and penetrate to the lamina propria and sub
mucosa.
They are phagocytosed thereby by polymorphs and macrophages.
 Inside the macrophage: They have the ability to resist the intracellular
killing. This is by making an alteration on the LPS, thereby making
themselves resistant to lysozymal enzyme and multiply within these cells.
 Primary bacteraemia: They enter the mesenteric lymph nodes, where
they multiply and, via the thoracic duct, enter the bloodstream. Leads to
Transient bacteraemia.
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Spread: The bacilli are seeded in the liver, gall bladder, spleen, bone marrow,
lymph nodes, lungs and kidneys, where further multiplication takes place.
 Secondary bacteraemia: Towards the end of the incubation period, there
occurs massive bacteremia from these sites of multiplication, heralding
the onset of clinical disease.
Complication:
As bile is a good culture medium for the bacillus, it multiplies abundantly in the
gall bladder and is discharged continuously into the intestine where it involves
Peyer’s patches and the lymphoid follicles of the ileum.
These become inflamed undergo necrosis and slough off leading to typhoid
ulcer, which may lead to intestinal perforation and hemorrhage.
During the3-4 weeks that normally constitute the course of the disease, the
intestinal lesions undergo healing.
4) What are the clinical features?

 Fever: rises gradually to a higher level with spikes which falls down
(Step ladder pattern / Remittent fever).
 Head ache, chill, abdominal discomfort, nausea, vomiting, anorexia,
malaise, myalgia and arthralgia.
About 30 % experience the formation of a maculopapular rash on the trunk and
chest.
 Other signs: Hepatosplenomegaly, epistaxis and bradycardia
5) Mention sample collection and laboratory diagnosis in detail

Samples should be selected based on the duration of the illness.
 First week: Blood, Clot, bone marrow, duodenal aspirate.
 Second week: Serum specimens for WIDAL.
 Third or fourth week: urine and stool culture.
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 Blood culture:
10 ML of blood should be collected at the time of high fever and it should be
directly inoculated into 50-100 ml of glucose broth/ bile broth /brain heart
infusion broth, incubate for 7 days.
Subculture on to solid culture media after 48 hours of incubation or when broth
becomes turbid.
Subcultures are made to solid plating media like Mac conkey agar and NLF
colonies formed are processed further biochemically and serologically to
identify salmonella
Automated blood culture systems BacT/ALERT
 Clot culture
Alternative to blood culture.
Advantages: Serum becomes available for WIDAL test.
Bactericidal activity of serum can be avoided.
 Stool or urine culture –
It is useful for detecting salmonella in the third or fourth week of infection.
They remain positive even after antibiotic treatment.
Stool and urine cultures are also done for the detection of carriers.
Appropriate media should be used to grow salmonella by inhibiting commensals
in the stool.
 Enrichment broth: Selenite F broth, Tetrathionate broth.
 Selective media: DCA, XLD, Wilson and Blair.
 In Wilson and Blair medium salmonella colonies produce jet black
colonies with a metallic sheen due to production of H2S.
 Serology:
WIDAL test
To measure H agglutinins for S.typhi and paratyphi A &B and O agglutinins of
S.typhi.
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Method: Equal volumes of serial dilution of serum and H and O antigens are
mixed in DREYERS and FELIX TUBE respectively and incubate overnight.
H agglutination- loose cottony woolly clumps, significant titre> 200
O agglutination – disc like spreading in bottom, significant titre>100 Positive
Titre depends on the stage of diseases.
Antibodies appear only at the end of first week and increases till fourth
then decline.
Just the presence of agglutinin is not a proof of typhoid fever.
Anamnestic response- prior infection or immunized individuals may have risen
in titer.
6) Add a note on prophylaxis

Enteric fever can be effectively controlled by:
The following methods
 General measures:
Improvement in sanitation, protected water supply.
 Vaccines:
 TAB vaccine: Heat killed typhoid bacillus vaccine contained S.typhi
1000 million and S.paratyphi A and B 750 million each per ml killed.
Dose: 2 doses of 0.5 ml subcutaneously at an interval of 4-6 weeks.
 Typhoral vaccine: live oral vaccine is a stable mutant of S.typhi strain
Ty2 1a, lacking the enzyme UDP-galactose-4-epimerase.
On ingestion it initiates infection but self-destructs after four or five cell
divisions.
Dose: enteric coated capsule and the course consists of one capsule orally taken
an hour before food with a glass of water or milk on days 1,3,5. No antibiotic
should be taken during the course.
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 Vi vaccine: injectable vaccine (typhim-vi) contains purified Vi
polysaccharide antigen from S.typhi strain Ty2.
Dose: single SC or intramuscular injection. Both oral and Vi vaccines are
recommended only for those over five years of age. In both the cases protection
is stated to commence 2-3 weeks after administration and lasts for at least 3
years.
7) Mention about typhoid carriers and the diagnosis of carrier stage in this
condition
There are two types of carrier stage for typhoid, fecal carrier and urinary
carriers
Methods of carrier identification
 Culture isolation: Stool, bile or urine cultures
 Vi antibody detection: By tube agglutination test using S.typhi
suspension carrying Vi antigen
 Isolation of salmonella from sewages:
 Sewer swab technique: Gauze pads left in sewage are cultured on highly
selective media such as Wilson and Blair media.
 Membrane filtration: sewage can be filtered using Millipore membranes
are cultured on highly selective media.
8) Write briefly about treatment and drug resistance

 Currently recommended drugs are
3 rd. generation cephalosporin’s, Azithromycin, Fluroquinolones
 Treatment of carriers
Ampicillin or amoxicillin plus probenecid given for 6 weeks
 Drug resistance:
There are MDR S.typhi: Resistance to chloramphenicol, Ampicillin and
cotrimoxazole
Fluoroquinolone Resistance: Resistance to ciprofloxacin.
Resistance to ceftriaxone.
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Clinical case 2
A 35 year old man presented to the medical OPD with a history of intractable
diarrhea for the past one week. He gave past history of multiple exposures six
months back. On clinical examination he was emaciated and oral thrush was
present.
1) What is the provisional diagnosis?
The patient is suffering from HIV/AIDS infection.
2) What are the morphological characteristics of this organism?

These are Spherical enveloped virus.
Size is about 90-120nm diameter.
Possess two identical copies of single stranded RNA genome.
The genome also contains an enzyme called Reverse transcriptase.
The envelope is lipoprotein in nature. Contain matrix protein, virus coded and
lipid layer which is host cell derived.
 There are three structural genes:

 Envelop gene: gp 160 (cleaved into 2, gp120 &gp 41) the spike protein
Gp 120 is the major antigen and antibodies which are first appearing
against this protein.
 Gag gene: These code for the core and shell protein. The protein is p55
which is again cleaved into p15, p18, p24.
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 Pol gene: This code for the enzymes. The protein is p100 which is
cleaved into p31, p51&p64.
Nonstructural regulatory genes: The transactivation gene (tat)
The regulatory gene (rev)
The negative factor (nef)
The viral infectivity factor (vif)
The long terminal repeat sequences (LTR)
3) Mention the different routes of transmission in this condition?

HIV transmitted through the following modes:
Sexual contact:
The most common mode of transmission. But the risk of transmission
through sexual mode is minimal only 0.1 -1%. Anal intercourse has higher risk
of transmission than vaginal intercourse.
Parenteral route:
Blood transfusion: The risk of transmission is more through this mode,
about >90%.
Sharing infected needles or syringes or Accidental inoculation like needle stick
injury. Risk of transmission is 0.5 to 1%.
Perinatal transmission:
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Infection from infected mother to her child either transplacentally or
prenatally, 30%.
The infection can also happen at the time of birth, when the baby comes in
contact with genital secretions.
After birth transmission can occur through breast feeding.
4) Explain the pathogenesis and clinical manifestations of the above

clinical condition?
 After entry into blood stream, HIV surface protein gp 120 attaches to the
host cell principally the CD4 lymphocytes on the host cell surface.
CD4 cells are mainly expressed on the surface of Helper T cells, Monocytes
Macrophages, glial cells etc.
 Inside the host cell the RNA is transcribed into DNA with the help of
enzyme reverse transcriptase. This is called PROVIRUS.
 In the integrated stage HIV establish a latent infection for a variable
period. During this time the virus replicates and spread the infection to
other neighboring cells. This stage is called Latent period.
 The infection causes damages to the T4 lymphocytes and it get depleted
T4:T8 ratio is reversed.
 The viral infection causes functional damage of the cell without making
any damages in the structure.
 Functions of other immunologically active cells like monocytes and
macrophages are affected due to the lack of activating factor by T
Lymphocytes. This Causes Immunodeficiency.
Clinical manifestations:
Incubation period varies from 1-14 days with an average of 6 years.
The clinical features can be classified into:
 Acute infection or Seroconversion illness.
 Asymptomatic infection
 Persistent generalized lymphadenopathy
 Symptomatic HIV infection or Aids related complex
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5) Describe briefly the laboratory investigations.
It is broadly categories into 2 types:

1. Immunological tests or Nonspecific tests : to diagnose
immunological status
2. Specific tests: to diagnose HIV infection.
 Nonspecific tests
 Total and differential leucocyte count:
Total and differential leucocyte count: In AIDS there is leucopenia
with lymphocyte count less than per mm3
 T-lymphocyte subset assay:

CD4: CD8 cell ratio reversed. The count of CD4 lymphocyte falls
below 200 per mm3
 Platelet count: Thrombocytopenia
 IgG and IgA levels: both are raised
 Skin tests for CMI: shows positive reactions because of diminished

CMI
 Specific tests:
The following parameters should be performed before performing the test

(3 Cs)
Take a Consent from the patient; Keep Confidentiality of the testing and
reporting; Do Counselling
 Antibody detection:
 ELISA: This is a screening assay and has high sensitivity and

specificity.
HIV specific antigens like p24, gp120, gp160, gp41 and HIV 2 specific
gp36 are used in the kits.
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NACO recommends that the result of a single test should never be used
as the final interpretation of HIV status as false positive due to technical
errors can occur.
 Other methods:
 Rapid Immuno chromatographic test / Simple test
 DOT Blot assays
 Lateral flow assay
 Particle agglutination assay
 Dip stick comb test
 P 24 Antigen detection
This appears after 12-26 days of infection and last for 3-4 weeks
thereafter.
 Detection of Viral nucleic acid
Viral nucleic acid can be detected by Polymerase chain reaction..

The test is highly specific and sensitive.
There are both DNA and RNA PCR (RT-PCR), helps to monitor
the level of viremia.
The PCR test are costly and are indicated only when other methods
give inconclusive results.
Other methods
Branched DNA assay
NASBA
Virus isolation
Can be isolated from CD4 lymphocytes from peripheral blood &

bone marrow and serum.
Method:
Patients lymphocytes are co cultivated with uninfected lymphocytes in
the presence of interleukin 2.
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Viral replication can be detected by the demonstration of reverse
transcriptase activity and presence of viral antigens p24 in the culture
fluid.
Supplemental tests
These tests are expensive, labor intensive, Need experts to interpret the
result and also gives indeterminate results.
Western Blot:
Detects individual antibodies against various viral antigenic fragments in

serum.
Antibodies to gag gene (p55, p40, p24, p18)
Antibodies to pol gene (p66/65, p55/51, p31)
Antibody to env gene (gp120, gp160, gp41)
The antigen antibody complexes appears as distinct bands on

nitrocellulose strip.
Reactive results are interpreted as Presence of at least two envelop band
with or without gag or pol bands.
6) Name the opportunistic infections seen in HIV/AIDS patients
Bacterial:
Recurrent severe bacterial infections, extra pulmonary tuberculosis

Disseminated non tubercular mycobacterial infection, recurrent
septicaemia.
Viral:
Chronic HSV infections, Progressive multifocal leukoencephalopathy,

CMV infections.
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Fungal:
Pneumocystis jirovecii pneumonia, Oesophageal candidiasis, extra

pulmonary Cryptococcosis, Disseminated mycosis.
Parasitic opportunistic infections
Toxoplasma encephalitis, chronic intestinal cystoisosporiasis, atypical

disseminated Leishmanisis.
7) What are the treatment modalities? What is HAART? What are

the problems pertaining to the use of ART?
Anti-retroviral therapy (ART)
The drug do not kill the virus or cure the disease. The goals of treatment
include:
Clinical, Virological, Immunological and Transmission goals.
 Clinical goals: Prolongation of life and improvement of quality of

life
 Virological goals: Cause reduction of viral load
 Immunological goal: Immune reconstitution both quantitative and
qualitative improvement.
 Transmission goals: Reduction of HIV transmission from infected
person to others.
Anti-retroviral drugs:
The drugs are of different types:

 Nucleoside reverse transcriptase inhibitor
 Non-nucleoside reverse transcriptase inhibitor
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 Protease inhibitor
 Nucleotide reverse transcriptase inhibitors
 Fusion inhibitor
 Highly active antiretroviral therapy (HAART)
HAART is referred to the use of combination of atleast three anti-

retroviral drugs to maximally suppress the HIV and stop the progression
of the disease. Monotherapy with single drug is contra indicated due to
the inefficiency and chance of development of resistance.
Problems pertaining to use of ART:

 Toxicity and adverse effect of ART
 Risk of development of drug resistance
 High cost
 Limited therapeutic options.
8) What are the measures taken on Post exposure prophylaxis?
Post exposure prophylaxis or PEP is required to reduce the risk of

transmission after occupational exposure such as needle stick or sharp
injury or mucocutaneous exposure
Duration: should be started within 2 hours of exposure and continued for

28 days.
Indication: if source is unknown/ positive for HIV
Step of post exposure management:

1. First aid
2. Report to the designated nodal center
3. Take first dose of PEP for HIV
4. Testing for blood borne viral infections:

a. Anti HIV antibody detection
b. HBsAg detection
c. Anti HCV antibody
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d. Anti HBs Antibody
5. Decision on post exposure prophylaxis

6. Informed consent and counselling
7. Documentation and recording of exposure
8. Follow up testing
9. Precautions during the follow-up period
9) What are the confirmatory tests?
 P 24 antigen detection after two weeks of infection.

 Co cultivation of virus.
 HIV RNA detection by molecular methods:Gold standard method
for confirmation of HIV.
 HIV DNA detection in pediatric cases.
Clinical case 3
A 35 year old male presented with low grade fever, malaise and fatigue from
the past 2 months. On examination hepatomegaly and right hypochondrial
tenderness present. He reports history of blood transfusion 3 months back.
Laboratory test showed HBsAg positive and HCV antibodies negative
1) What is the probable diagnosis? What are the modes of transmission?
Hepatitis B infection
Modes of transmission
a) Blood transfusion
b) Sexual Transmission
c) Vertical (perinatal) transmission
d) Needle stick injuries
e) Direct skin contact (Infected open skin lesions may spread the virus)
2) Describe in detail the lab diagnosis of this disease

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Confirmed in laboratory using
• Antigen Markers-
 HBsAg: First serological marker to be elevated following infection
 HBeAg: The presence indicates active viral replication and infectivity
 HBcAg: This is a hidden antigen due to surrounding HBsAg coat. This is
non secretory
Can be demonstrated from hepatocytes by Immunofluorescence test
• Antibody markers-
 Anti-HBs: It appears after the clearance of HBsAg and remain elevated
thereafter. This is also a marker of Hepatitis B vaccination.
 Anti-HBe,:Appers after the clearance of HBeAg and remain elevated for
a variable period.
 AntiHBc: Both IgG and IgM antibodies are appeared after the infection.
The first appearing antibody is IgM, it last for another 3 to 6 months and
disappears from circulation. IgG appears late acute stage and remain
positive indefinitely.
Ag detection:
 Rapid Ag detection kits
 Immunochromatographic methods
 ELISA
Ab detection
 ELISA, RIA
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 Molecular markers- HBV DNA
• Non- specific markers- Elevated liver enzymes and serum bilirubin

 Demonstration of viral Markers (HBV antigens and antibodies)
 ELISA, Immuno Chromatographic test
 Molecular diagnosis
 PCR
Non-specific markers:
 Elevated Liver enzymes
 Raised serum bilirubin
3) Add a note on treatment and prophylaxis

Treatment:
Most of the Hep B infections are self-limiting and do not require any specific
treatment.
Tenofovir and telbivudine are the antiviral agents of choice.
Prophylaxis
There are Active, passive and combined immunisation
 Active immunisation:
Hepatitis B vaccines: Recombinant vaccine. Prepared bin baker’s yeast by DNA
recombinant technology by cloning the S gene into the yeast chromosome
Site and route: Intramuscular, to Deltoid (in infant’s anterolateral thigh)
Dosage: 3 doses given at 0,1 and 6 months
Dosage – 10-20 microgram
Revaccination is required if titre remain <mIU/ml
 Passive Immunisation (HBIG)
This is required when immediate protection is needed like acutely exposed to
Hepatitis B positive blood, or sexual contact with acute Hepatitis B patients,
Neonates borne to Hep b positive mother, post liver transplant patient etc
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 Combined immunisation
More effective
Given to neonates born to HBV infected mother,
Dosage: single injection of 0.5 ml of HBIG given to the neonates immediately
after the birth followed by full course of vaccine.
4) What is defective virus?

These are Defective virus which cannot replicate by its own but require Hepatits
B virus for its survival
Transmission is similar to HBV and HCV. Parenteral route is the most common
route followed by sexual and vertical route.
The association of HBV and HDV is of two types:
Co infection and super infection
Co infection: means with the person is exposed to serum containing both HBV
and HDV
Super infection: If a chronic HBV carrier get HDV virus.
Clinical case 4
A 28 year old manual labourer was brought to the causality with high fever,
chill and rigor and profuse sweating. He gives a history of such fever episodes
during the past one weeks. He has recently visited his native place in Madhya
Pradesh on examination pallor and splenomegaly was present.
1) What is your provisional diagnosis?
Malaria
2) Name the species of the causative agent?
Plasmodium vivax,
Plasmodium falciparum,
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Plasmodium malariae,
Plasmodium ovale.
3) Which species cause more severe disease?
Plasmodium falciparum causes the most severe form of infection called

malignant tertian malaria.
4) Name the complications
The complications of falciparum malaria are:
 Cerebral malaria: vascular occlusion and cerebral anoxia due to the

blockage of brain capillaries.
 Black water fever: This is due to an immune reaction towards parasitized
RBC. This leads to fever, haemoglobinuria and dark urine.
 Algid malaria: characterised by cold clammy skin, hypotension,
peripheral circulatory failure and shock.
 Pernicious malaria: Characterised by presence of algid malaria and black
water fever.
 Septicaemia malaria: High grade fever due to the dissemination of the
parasite, leading to multi organ failure.
 Pulmonary edema
5) Explain the life cycle of this parasite
The malarial parasite passes its life cycle in two hosts

IH: Man (Asexual reproduction)
DH: Female anopheles mosquito (Sexual reproduction)
Human acquires the infection by the bite of infected mosquito, which release
sporozoite.
The human cycle shows 4 stages:

1. Pre erythrocytic schizogony
2. Erythrocytic schizogony
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3. Gametogony
4. Exoerythrocytic schizogony
Pre erythrocytic schizogony:
Sporozoite released into the capillaries will reach in the liver parenchymal cell
where they multiply and produce schizont.
The schizont gets matured and releases micro merozoites and macromerozoite.
Micromerozoites enter the circulation.
Macromerozoites re-enter the liver cells.
This cycle last for:
 7-8 days in P.vivax
 5-7 days in P.falciparum
 9 days in P.ovale
 14-16 days P.malariae
Erythrocytic schizogony:
Micromerozoites released in the circulation invade the RBC and forms a
trophozoite. Sometimes it appears like an annular ring, this is called ring stage.
This stage changes into an amoeboid form and then to a schizont.
When schizont matures the nucleus divides and later merozoites are released
into the circulation which again invade the fresh RBC.
During this stage the parasite feed on haemoglobin leaving a residue, haematin
(Malarial pigment).
This cycle last for:

48 hours in P.vivax, P. falciparum and P.ovale.
72 hrs.In P.malariae.
Gametogony:
After repeated cycles of Erythrocytic schizogony, some merozoites develops
into sexually differentiated forms called gametocytes (Gametogony).
This happens inside the internal organs and the mature forms are released in
peripheral circulation.
These are taken up by the mosquitoes.
Exoerythrocytic schizogony:
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It is a local liver cycle seen in P.vivax, P.ovale and P.malariae,but disappears in
P.falciparum.
It act as a source of merozoites and causes relapses.
Mosquito Cycle:
Mosquitoes taken by the gametocyte while feeding on infected person.
Gametocyte reach the midgut where the microgametes and macrogametes
matures and fertilise to form a zygote which transforms into ookinete.
This will penetrate the gut wall and develops into oocyst in the basement
membrane later sporozoitesare developed.
The sporozoite migrate through the body and reaches the salivary gland and the
salivary duct.
This is release into the capillaries during blood meal.
6) Mention the pathogenesis and clinical features of Malaria
Incubation period varies in infection with different species:

 Shortest in P.falciparum – 12 days.
 Longest in P.malariae – 28-30 days.
 In P.vivax and P.ovale – 13 to 17 days.
Nature of fever is different in infection with each species:
Vivax malaria:
It is also called benign tertian malaria, Fever reoccur every third day.
Falciparum malaria:
It is also called malignant tertian malaria. It is called as malignant because of
the severity of infection. Fever may reoccur every 48 hrs.
Malariae malaria:
It is also called quartan malaria caused by plasmodium malariae. Fever reoccur
after 72 hrs or every fourth day.
Ovale tertian malaria:

Caused by Plasmodium ovale. Fever reoccur after interval of 48 hrs.
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Clinical features:
Fever with chill followed by anaemia and splenomegaly.
 Febrile paroxysms: Shows 3 stages

 The cold stage: last for 15-60 minutes.
 The hot stage: last for 2-6 hrs.
 The sweating stage: last for 2-3 hrs.
 Anaemia and splenomegaly.

 Recrudescence: Even after the clinical attack subsides, the parasites are
not completely eliminated from the RBC. These may cause reccurent
attack of malaria. This is known as recrudescence.
 Relapse: This is seen in P.vivax and P.ovale infection. The sporozoite
remain silent in the hepatocytes and activated time to time to form
merozoites which infect RBCs and cause malaria. This is known as
relapse.
7) Discuss the lab diagnosis in detail and describe the finding
Specimen: Capillary blood taken by finger prick
Microscopy-Peripheral blood smear-(Thick and thin smear): Gold standard

method
 Smears are stained using Leishman’s, Giemsa stain, Wrights stain or
fields stain.
Detection of malarial parasite
Thick film allow quick detection and thin film for species identification.
 Amoeboid ring form and schizont: - P. vivax
 Multiple ring forms and banana shaped schizont- P.falciparum
 Band forms- Plasmodium malariae
 Enlarged oval RBC with ring form- P.ovale
 Fluorescent Microscopy: The blood smears are stained with acridine

orange and are examined under fluorescent microscope.
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 QBC: The blood is centrifuged to get a gradient forming discrete layers
as RBC, WBC, Lymphocytes and platelets. This is more faster and
sensitive method. Quantification is also possible.
 Rapid diagnostic Test: Dip stick test: These are simple to perform and
do not require expertise personals.
 Serology: ELISA. IF, CFT, IIF, RIA

 DNA probes
 PCR
8) What are treatment measures?
Choloroquine is the drug of choice.

For treating Choloroquine resistant P.falciparum, a combination of
sulphadoxine and pyrimethamine is used.
9) Write briefly on the measures to prevent this disease
Personal and community prophylaxis

Personal prophylaxis:
Protection against mosquito bite.
Chemoprophylaxis to the travellers visiting malaria endemic areas.
(Choloroquine and pyrimethamine)
Community prophylaxis:
Treatment of carriers.
Destruction mosquitoes and larvae.
Clinical case: 5
A 39 year old man from Alappey was presented to the emergency department
with fever, lymphadenitis and lymphangitis. The man complained of swelling in
her right leg below the knee.
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1) What is the probable diagnosis?
The patient is suffering from filariasis.
2) Describe the life cycle

The life cycle complete in 2 hosts.
DH: Human
IH: Mosquitoes (Culex, aedes and anopheles)
Infective form: Third stage larvae
Human cycle:
Human get the infection by the bite of infected mosquito, the third stage larvae
get deposited in the bite wound.
The larvae penetrate through the puncture wound and reaches the lymphatics
and to draining lymph node and develop into adult worm.
The male fertilise the female and it give birth to microfilariae. It matures and
liberate into the circulation. During blood meal it is taken up by mosquito.
Mosquito cycle:
Microfilariae lose their sheath and penetrate the gut wall and reaches thorax
where they moults thrice, which is infective to man.
The infective form, the 3 rd stage larvae enter the proboscis and released.
3) What are the features of microfilariae?

It is the embryo of microfilariae. Size is about 275 to 300µ long and 8-10
micron breadth.
It is the first stage larvae.
It is found in peripheral blood, hydrocele fluid and chylous urine.
They are actively motile.
They have a hyaline sheath which is seen around the larval body.
The larvae move forward and backward in the hyaline sheath.
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Head end is blunt and tail end is pointed.
In unstained preparation they appear colourless.
It is stained by Romanowsky stain.
The microfilariae do not undergo further development in human body unless
they are taken up by mosquito.
4) Mention the pathogenesis and clinical features

The infection with this parasite is called Wuchereriasis or bancroftian filariasis
or lymphatic filariasis.
Clinically it occurs as:
 lymphatic filariasis or classical filariasis (caused by adult worm)
 Occult filariasis (Caused by embryos)
Classical filariasis:
The typical manifestation is caused by the adult worm blocking lymph nodes
and vessels.
The blocking may be due to mechanical or allergic inflammatory reaction. This
will lead to functional damage of the vessel and it will become incompetent.
Increased permeability of vessel walls leads to the leakage of protein rich lymph
into the surrounding tissue. This causes lymphoedema and elephantiasis.
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The parts mostly affected are Limbs, Testes, scrotum, Penis, breast and vulva.
Clinical features:
Acute filariasis:
Fever, chill, malaise, lymphoedema, lymph adenitis, lymphangitis
Chronic filariasis:
Lymph in urine: Chyluria
Hydrocele
Orchitis
Funiculitis and
Epididymitis
Occult filariasis
It is due to hypersensitivity reaction to microfilarial antigens.
Microfilaria are not found in peripheral circulation. Other features of filariasis
are absent.
Manifestation:
 Tropical pulmonary eosinophilia:
Present as low grade fever, loss of weight and pulmonary symptoms

Dry cough, dyspnoea and wheezing.
Raised eosinophil count.
Chest x-ray shows mottled shadow.
Raised filarial antibodies.
5) Explain the lab diagnosis of filariasis?
 Specimen: Blood is collected during night time or

Day time after DEC provocation test.
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The most favorable site of collection is ear lobes because the number of
microfilariae are more there.
Species identification:
W.bancrofti: pointed tail with cephalic space free of nuclei.
B.malayi: Tail tip blunt, nuclei extend upto the tip.
 Demonstration of microfilariae
 Wet mount preparation for demonstrating motile larvae
 Peripheral blood smear examination
 Giemsa stained thin and thick smear.
 QBC method: If the direct smear is negative, confirmation can be
done by QBC test.
 Demonstration of:
 microfilariae from:
o Chylous urine
o Exudate of lymph varix
o Hydrocele fluid
 Adult worm:
o Lymph node biopsy
 Calcified worm
o X-ray
 Serology
o ELISA
o RIA
o CR
o FAT
 Molecular methods
o PCR
 Xenodiagnosis
o Demonstration of microfilariae from the stomach blood of
mosquito vector.
6) What are the treatment and prophylactic measures?
 Vector control
 Protection against mosquito bite
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Treatment:
 Diethycarbamazine: long term low dose therapy is required

6mg/kg daily for 12 days.
 Albendazole: 400mg twice daily for 21 days(both adult worm and
microfilariae)
 Ivermectin: 0.4 mg/kg single dose (to kill microfilariae)
Clinical case 6
19 years old female patient complaints of fever biphasic in nature with severe
bone pains in the back and limbs. She had maculo-papular rash and developed
hemorrhagic manifestations. Answer the following:
1) What is the probable diagnosis?

The patient is showing the symptoms of viral hemorrhagic fever- Dengue fever.
2) What is the transmitting vector for this disease?

Aedes aegypti is the principal vector followed by Aedes albopticus.
3) What are the signs and symptoms of dengue fever?
 Abrupt onset of high fever,

 Maculopapular rashes,
 Headache,
 Muscle and joint pain,
 Retro bulbar pain,
 lymph adenopathy,
 Nausea and vomiting.
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4) Discuss the laboratory diagnosis of this disease
Specimen: blood, CSF, autopsy tissues
NS1 Ag detection:
By Immunochromatographic assay and ELISA test.
This can be detected in the early stage of the disease and remain positive upto 2
weeks.
Detection of Antibody:
By ELISA and Immunochromatographic assays.
 IgM: Detection of IgM antibody for the early diagnosis.
 IgG: This is appearing in serum only after 10 days of infection.
Isolation of Virus:
By tissue culture, inoculating clinical specimens into mosquito cell line
Animal inoculation using suckling mice.
Identification of growth using fluorescent antibody technique.
Molecular method:
PCR
Genotyping.
5) What are the two complications of this disease?

• Mention the preventive measures to prevent this disease
The two complications are:
 Dengue hemorrhagic fever (DHF)
 Dengue shock syndrome (DSS)
Dengue hemorrhagic fever is characterised by-

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 High grade continuous fever
 Hepatosplenomegaly
 Thrombocytopenia
 Raised hematocrit
Dengue shock syndrome is characterised by-
 Rapid and weak pulse
 Hypotension
 Presence of cold and clammy skin
 Restlessness
Clinical case: 7
A 29 year old women presents with intermittent fever since 45 days along with
fatigue, arthralgia and night sweats. On examination she has a foul smelling
perspiration and hepatosplenomegaly. She also gives history of consumption of
unpasteurized milk.
1) What are the tests for diagnosing Brucellosis?
1. Blood culturing
2. Serology: for detecting IgM and IgG antibodies
3. Agglutination test: STAT, standard tube agglutination test.
4. Brucellin skin test
Diagnosis of Animal Brucellosis

1. Rapid plate agglutination test
2. Rose Bengal card test
3. Milk ring test
4. Whey agglutination test
2) Explain prozone phenomenon
Precipitation occurs clearly and abundantly when Ag and Ab are

present in optimum proportion. When serial dilutions of serums are tested
for antibodies equal quantities of Ag are added in all tubes, precipitation
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occurs most rapidly and abundantly in one of the middle tube where Ag
and Ab are present in optimum proportion.
If the amount of precipitation formed are plotted the resulting curve
shows three phases:
1. A zone of Ab excess (Prozone)
2. A zone of equivalence
3. A zone of Ag excess (Post zone)
This is called as zone phenomenon.
3) Pathogenesis of Brucellosis?
The three major species causing Human infections are:

B.melitensis
B. suis
B.abortus
These are transmitted to human by:
 Drinking contaminated raw milk or by ingestion of milk
products.
 Direct contact with infected animal tissues.
 Accidental Ingestion or inhalation.
 Mucosal contamination.
The bacilli from the site of entry travels through the lymphatic channels to
regional lymph nodes to thorasic duct after entering into the blood stream.
They can grow intracellularly. So mostly seen inside the phagocytic cells.
The incubation period is 2 to 3 weeks and may extend to 6 months.
The onset of symptoms may be acute or insidious.
Human infections are of 3 types

1. Subclinical or latent infection
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2. Acute Brucellosis or Undulant fever or Malta fever: Associated
with prolonged bacteraemia.
3. Chronic brucellosis: If the disease persist for more than 6
months. This is mostly due to hypersensitivity.
Presence of which antibody indicates intrauterine infection in newborns

Presence of IgG antibodies indicates intrauterine infection.
4) What is Castaneda’s method of Blood culture?

This is a biphasic blood culturing system. Blood culture bottle containing
solid and liquid phases in a single bottle. Frequent sub culturing can be avoided
by this method.
The blood is directly inoculated into the liquid phase and the bottles are
incubated in the upright position. For sub culturing, bottles are tilted so broth
runs over solid media and incubated in the upright position.
In case of positive blood culture colonies can be seen on the agar slant.
Advantages:
 Reduces the chances of contamination.
 Eliminate the risk of infection to laboratory workers.
5) What is SAT test? (Standard agglutination test)

Serum agglutination test or Standard agglutination test.
Is the most widely used test for diagnosis of Brucellosis.
This is a tube agglutination test.
Equal volume of serial two fold dilution of patient serum is treated with
Brucella antigen.
Blocking effect of antibodies if present can be removed by keeping the serum at
55C for 30 minutes.
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After properly mixing the serum and the reagent the tubes are kept at 37C
overnight.
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Short Essay
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Leptospirosis
It is a zoonotic disease caused by L.interogans.
It is transmitted to man by direct or indirect contact with water contaminated
with urine and faeces of carrier animals.
The bacteria enters through the minute cut or aberrations on skin or through
intact mucus membrane of mouth, nose and eyes.
After an incubation period of about 6-8 days, there is onset of febrile illness
with leptospires in blood. This phase is called septicaemic phase.
The organism from the blood settled down into other organs like liver, Kidney,
spleen, brain, lungs etc.
Clinical features:
Severe leptospirosis associated with fever, conjunctivitis, albuminuria, jaundice.
Complication:
 Disseminated intravascular coagulation (DIC)
 Acute renal failure.
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Lab diagnosis:
Specimen: Blood, Urine, CSF
 Collection:
The Leptospira will die off in acidic urine so it should be made in alkaline.
 Microscopic examination: Demonstration of leptospires from
samples:
 Fluorescent antibody technique

 Silver impregnation test
 Dark ground microscopy
 Culture
Difficult to grow. Media used are
 EMJH medium
 Modified Korthoffs medium
 Fletchers semi solid medium
Plates are incubated at 30 C, examined every alternate days up to 6 weeks.
 Serology:
It is the usual method of lab diagnosis.
IgM Antibodies begin to appear at the end of first week and continue to rise till
the 4 th week and then declines.
IgG appear after at the end of third week and persist for months and years.
Two types of serological tests
1. Screening test: Rapid dip stick assay, ELISA, Immunochromatographic
test.
2. Serovar specific test: MAT test or Microscopic agglutination test:
This is a Gold standard test and the reference test for diagnosing
leptospirosis.
Formalised or living suspension of leptospires is allowed to agglutinate
with the antibodies and observed under microscope.
 Molecular methods:
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PCR is a useful tool to diagnose leptospirosis.
Visceral leishmaniosis/ Kala azar
This is a parasitic disease caused by Leishmania donovani.

This is a protozoan parasite.
It has two morphologic forms Amastigote form and Promastigote form.
The parasite cause a disease called visceral leishmaniasis or KALA AZAR
(Black disease).
Infection transmit by the bite of sand-fly named phlebotomus argentipes.
From the site of inoculation the parasite multiplies in the RES especially in the
spleen, liver, bone marrow and lymph nodes.
Clinical features:
Pyrexia, splenomegaly, hepatomegaly, lymphadenopathy and general features
of anaemia.
Lab diagnosis
Direct and Indirect evidences
 Direct evidences:
Donovan bodies (LD bodies): This is the Amastigote forms which can be seen
in peripheral blood
 Peripheral blood smear
 culture
Bone marrow aspiration, Splenic puncture
 Antigen detection
 Indirect Evidences
 Reversed albumin- globulin ratio

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 Specific serological test: IHA, IFAT, ELISA
 Rapid Immunochromatographic strip test
Trypanosomiasis
This is a parasitic disease caused by haemoflagellated protozoan trypanosomes.
Human infections are produced by
 T.brucei causes Sleeping sickness.
 T.cruzi causes Chagas disease.
 T.rangeli -Does not cause any disease
Morphology
The parasite occurs in different forms
T.brucei: They occurs as trypomastigote form which has 3 forms long slender,
short stumpy and intermediate form.
T.cruzi: There are 2 morphological forms:
Amastigote form
Trypomastigote form
Sleeping sickness:
Human gets the infection by the bite of tse tse fly.
A chancre develops at the site of bite.
The chancre heals but the organisms enters the blood stream and causes
parasitaemia which leads to high fever.
Clinical features:
Fever, Weakness, Rapid loss of weight
Characteristic signs of meningitis. Inability to concentrate and day time sleepy
stage (Sleeping sickness)
In the final stage it it ends in coma and death.
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Chagas disease:
The manifestations are due to the growing Amastigote stage in tissues.
The myocardium, skeletal muscle, neurological cells and skeletal muscles are
involved.
Lab diagnosis of Trypanosomiasis:

Microscopy: Giemsa stained blood film
Culture: NNN Medium
Ag detection: ELISA
Ab detection: IFAT, IHA, ELISA
Xenodiagnosis
Intradermal test: Cruzin test: Delayed type of hypersensitivity.
Systemic mycosis
These are fungal infections of deeper part of the body or internal organs.
There are 4 types
1. Histoplasmosis
2. Blastomycosis
3. Coccidioidomycosis
4. Paracoccidioidomycosis
 Histoplasmosis
This is primarily a disease of reticuloendothelial system caused by Histoplasma
capsulatum.
It is a dimorphic fungus.
The disease is also known as Darlings disease because it was first reported by
Samuel Darling in 1905.
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This is a soil saprophyte and the source of human infection is inhalation spores
from the soil.
There are two varieties:
H.capsulatum causing Classical histoplasmosis
H.duboisii causing African histoplasmosis.
Clinical features:
 Pulmonary Histoplasmosis
 Disseminated Histoplasmosis: Involvement of RES results in
lymphadenopathy, Hepatosplenomegaly, fever, anemia and high rate of
fatality.
 Mucocutaneous Histoplasmosis.
Lab diagnosis:
Specimens: Sputum, bone marrow aspirate, blood, scraping from
mucosal lesions, Lymph node aspirate.
Microscopy:
Direct examination: smears made from the specimens are stained with
Giemsa and wrights stains.
H.capsulatum appears as small, oval yeast cell packed with in the
macrophages.
Culture:
This is the Gold standard method of diagnosis.
Cultures are made on antibiotics added SDA or BHI. Since this is a
dimorphic fungi 2 tubes should be kept at different temperatures.
One at 37 C and the other at 25C.
The yeast phase will be grown in tube kept at 37C.
Mycelial growth shows finger like projection with spores.
Serology:
Latex agglutination, Precipitation and Complement fixation.
Histoplasmin skin test:
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This is a delayed hypersensitivity test. The antigen used is
histoplasmin. A positive histoplasmin test indicates present or past
infection.
Molecular method: PCR
 Blastomycosis
This is a mycotic infection caused by Blastomycetes dermatitidis.

They are soil saprophytes.
Human gets the infection through inhalation of conidia.
Clinical forms:
 Pulmonary form
 Cutaneous form
 Disseminated form
 Miscellaneous form
Lab diagnosis
Specimens: Sputum, pus, biopsy
Processing of specimen:
KOH mount: Thick, double walled yeast cells.
Culture: Specimens are to be inoculated in two tubes of SDA, BHI.
Skin test: It is a delayed hypersensitivity reaction.
Serology: CFT, ELISA, RIA.
 Coccidioidomycosis:
This causes a systemic fungal infection by C.immitis
This is a dimorphic fungi seen in soil.
Human gets the infection by inhaling arthrospores.
The spores swells inside the body and develops spherules.
This contains endospores which comes out through the rupture of the cell and
disseminate.
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Clinical forms:
 Pulmonary form
 Chronic meningitis
 Disseminated form
Lab diagnosis
Specimens: sputum, pus, CSF
Microscopy:
 KOH mount shows thick double walled spores with spherules.
 Histopathological examination by H& E staining
 Immunofluorescence test
Culture:
Specimens are to be inoculated in two tubes of SDA, BHI.
Skin test: Coccidioidin skin test. It is a delayed hypersensitivity reaction.
Positive test indicates past infection.
Serology: CFT, ELISA, RIA.
Treatment: Itraconazole, Amphoterecin B
 Paracoccidioido mycosis:
Caused by a dimorphic fungus called Paracoccidioides brasiliensis.
It produce chronic granulomatous disease involving lung, mucosa, skin and
lymphatic system.
It is also called South American Blastomycosis because the disease is more
confined to South America.
Human gets the infection by inhaling the spores.
Clinical features:
 Pulmonary form
 Mucocutaneous form
 Lymphatic form
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Lab diagnosis
Specimens: Sputum, pus, biopsies
Microscopy:
 KOH mount shows numerous yeast cells with multiple buds.
 Histopathological examination by H& E and PAS staining
Culture:
Culture: Specimens are to be inoculated in two tubes of SDA, BHI.
Skin test: It is a delayed hypersensitivity reaction against paracoccidioidin
antigen.
Serology: ELISA, RIA.
Treatment: Itraconazole, Amphoterecin B
Rickettsial infection
Based on the clinical manifestations, Rickettsial infections are classified into:
 Typhus fever group
 Spotted fever group
 Scrub typhus
The disease coming under typhus fever group are:
 Epidemic typhus
 Endemic typhus
 Recrudescent typhus or Brill Zinsser disease
The disease coming under spotter fever are
 Indian tick typhus
 African tick typhus
 Rickettsial pox
Another type of Rickettsial infection is Scrub typhus produced by Orientia
tsutsugamushi.
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Pathogenesis
All the Rickettsial infections are transmitted to human through Arthropod
vectors.
Tick, mite, louse and flea are the common vectors involved.
After entering through the bite wound the bacteria enters the lymphatic channels
to the lymph nodes and spills into blood stream.
Attaches to the endothelial membrane.
Phagocytosed by macrophages.
Inside the host cell they multiply slowly by binary fission.
 Epidemic typhus:
The causative organism is R.prowazekii, the vector is human body louse
Pediculous humanis corporis.
The infected lice defecate while feeding on human and the organisms present
will be enter into the body through the scratches and abrasions.
The incubation period is 5 to 21 days.
Patient shows severe head ache, myalgia, fever and a rash appears on 4 to 7
days.
The patient develops cloudy state of consciousness.
 Brill Zinsser disease

This is a kind of latent infection produced by Rickettsia prowazekii.
This is a milder form and the duration is less.
 Endemic typhus:
The disease is caused by R.typhi. Vector is Rat flea.
Man gets the infection through the bite of infected flea.
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There is no man to man transmission. Human infection is a dead end.
 Spotted fever Group:

Rocky mountain spotted fever:
Causative agent is R.rickettsii. It is transmitted by ticks.
The incubation period is one week.
 Indian tick typhus:
It is caused by R.conori. It is transmitted by ixodid tick. An eschar with a
necrotic centre is produced at the site of tick bite.
 Rickettsial pox
This is a febrile disease carried from mouse to man by a mite and produce a
vesicular rash.
The disease is self-limiting, non-fatal and resembles chicken pox.
 Scrub typhus
It is the most common Rickettsial disease in India.
It is caused by Orientia isutsugamushi.
It is transmitted through the larval form of mite.
Human acquires the infection by the bite of infected mite larvae (Chigger).
So scrub typhus is also called chigerrosis.
Incubation period is 7 to 10 days.
The patient develops a necrotic lesion at the site of bite and also shows fever,
chill, headache, conjunctivitis and maculopapular rash.
It is a fatal disease and the rates ranges from 10-60%
Laboratory diagnosis
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1. Serology
2. Isolation of Rickettsia
3. Molecular methods
 Weil felix reaction:

It is heterophile agglutination test that detect antibodies in patients with
Rickettsial fever.
These Ab cross react with some strains of non-motile strains of proteus.
This is due to the sharing of an alkali stable carbohydrate antigen of Rickettsia
and O antigens of Proteus (Proteus vulgaris, OX-19,OX-2,OX-K)
 Indirect Immunofluorescence
 ELISA
 Latex agglutination test
Isolation:
Rickettsiae are highly infectious organisms. So it is not done for a routine
diagnosis.
It can also be grown in Chick embryo and by tissue culture.
Molecular diagnosis
PCR
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Short notes
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Ebola virus
These are a group of RNA viruses which causes haemorrhagic fever.
This was first identified in 1976 and WHO had announced this as a public
health emergency after the outbreak happened on 2014.
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They are pleomorphic, long, filamentous structures, size ranging from 80-
1000nm.
Ebola virus has 6 subtypes or species, all differs from each other by their
nucleotide sequence.
The first disease was identified in 1976 in Sudan.
An outbreak was first happened in a village near the EBOLA River, so the virus
got the name Ebola.
Pathogenesis:
The natural reservoir host is Fruit bat.
It is transmitted from one person to other through close contact.
Incubation period is about 2-12 days.
The disease start with fever, head ache, sore throat, muscle pain which
progresses to abdominal discomfort, vomiting and diarrhoea.
The mortality rate is 25-90%.
Diffuse erythematous rash appears later leading to shock and death.
Lab diagnosis:
Specimen: Throat swab, nasopharyngeal swab
Electron Microscopy: To demonstrate the filamentous forms
Virus isolation: Tissue culture. Highly infectious so should be done in Level 4
biosafety cabinets.
Serology
ELISA, Antibody phage indicator assay
IFT
Molecular method
RTPCR
Treatment
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Supportive care to manage dehydration and symptomatic treatment.
Hanta virus
This belongs to Bunyaviridae.
These are the major rodents borne virus transmitted to human through rodent
excreta or contaminated fomites.
Hanta viruses are spherical enveloped virus contain segmented single stranded
RNA.
They cause haemorrhagic fever and renal syndrome (HFRS).
Diagnosis:
Is made by detection of viral RNA by RT-PCR
Treatment:
There is no specific antiviral therapy. Supportive Symptomatic care is given.
Chikungunya
It is an arbovirus causing febrile illness and severe arthralgia.
It belongs to the family Togaviridae.
It is a single stranded RNA virus.
The vector is Aedes aegypti.
The disease is characterised by fever, severe joint pain, lymphadenopathy,
conjunctivitis, arthritis and rash (Rare).
Arthritis with swellings in the small joints of wrist and ankles.
Biphasic fever reoccurring every 1 – 6 days.
It has similar manifestations like dengue. Both can be differentiated only with
lab diagnosis.
Chikungunya is less severe than dengue. Haemorrhagic manifestations are
rarely seen.
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Lab diagnosis
 Specimen: serum
 Serology:
 ELISA: IgM antibody appear after 4 days of infection and persist
for about 2 months. IgG antibodies appears late but persist for a
longer period.
 ICT: Rapid method to detect both IgM and IgG antibodies.
 Isolation of virus: Virus isolation using mosquito cell line.
 Molecular method: PCR
Relapsing fever/ Borreliosis

Borreliosis or relapsing fever is a disease caused by spirochete Borrelia.
Important species are:
 B.recurrentis Causes epidemic replapsing fever.
 B.burgdorferi causes lyme disease.
 B.vincentii causes ulcerative gingivostomatitis or vincent’s angina.
Relapsing fever
These are of two types:
1. Louse borne relapsing fever or epidemic relapsing fever caused by
B.recurrentis.
This is a vector borne disease transmitted by human body louse.
Borrelia enters the host’s body through minute aberrations or through
mucous membrane.
2. Tick borne relapsing fever or endemic relapsing fever is caused by
B.duttoni transmitted by the bite of an infected tick.
This lead to bacteraemia and fever.
Clinical manifestations:
Incubation period varies between 2 to 10 days.
The disease is characterised by febrile and afebrile period with 3 to 5
days of gap.
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Louse borne and tick-borne relapsing fevers are clinically
indistinguishable.
Lab diagnosis:
 Specimen: Blood collected at the time of fever.
 Dark ground microscopy: Borrelia can be detected by their

motility.
 Giemsa or Leishman stained smears to detect Borrelia.
 Direct fluorescent antibody test: Can be done using monoclonal

antibodies.
 Blood culture: To isolate Borrelia from blood.
 Serology: ELISA and IFAT to detect antibodies from serum
Babesiosis
This is a malaria like disease seen in animals.
The most common species is B.microti, others: B.bovis, B.divergens.
It is transmitted by the bite of hard tick.
There are 3 morphological forms:
 Sporozoite
 Trophozoite
 Merozoites
The disease is characterised by fever, chill, sweating muscle pain and fatigue.
Mild hepatosplenomegaly and hemolytic anemia can be seen in some cases.
Lab diagnosis:
Thin and thick smear of blood stained using Giemsa stain.
The parasite closely resembling Pfalciparum
Differentiating feature:
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Absence of pigmet
Absence of schizont and gametocyte.
The characteristic feature of attaching the daughter cells to the parent parasitic
cell is an identifying feature.
Milk ring test

This is the most commonly used test to diagnose Brucellosis in animals.
It is carried out to detect infected animals in farms by using pooled milk
samples.
Method:
Whole milk is mixed with a drop of stained Brucella antigen and incubated in
water bath at 70C for 40-50 minutes.
If antibodies are present it will agglutinate the bacilli and rise with cream to
form a blue ring at the top leaving the milk unstained.
Ring is not formed if antibodies are absent in milk and the complete milk will
appear blue coloured.
Typhoid vaccine
a) TAB vaccine: Heat killed typhoid bacillus vaccine contained S.typhi 1000
million and S.paratyphi A and B 750 million each per ml killed.
Dose: 2 doses of 0.5 ml subcutaneously at an interval of 4-6 weeks.
b) Typhoral vaccine: live oral vaccine is a stable mutant of S.typhi strain Ty2
1a, lacking the enzyme UDP-galactose-4-epimerase.
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On ingestion it initiates infection but self-destructs after four or five cell
divisions.
Dose: enteric coated capsule and the course consists of one capsule orally taken
an hour before food with a glass of water or milk on days 1,3,5. No antibiotic
should be taken during the course.
c) Vi vaccine: injectable vaccine (typhim-vi) contains purified Vi

polysaccharide antigen from S.typhi strain Ty2.
Dose: single SC or intramuscular injection. Both oral and Vi vaccines are

recommended only for those over five years of age. In both the cases protection
is stated to commence 2-3 weeks after administration and lasts for at least 3
years.
Vi Antigen
It is the surface polysaccharide antigen covering the O antigen.
The name Vi represents the virulence of the organism. It is heat labile.
The vi polysaccharide act as a virulence factor by inhibiting phagocytosis,
resisting compliment activation and bacterial lysis.
The vi antigen is poorly immunogenic and only low titres of antibodies are
produced following infection.
The antibody formed are disappeard during the convalescent period so it is not
useful for the diagnosis.
Its presence indicates the carrier state.
This is present only in few Serovar but tend to lose Vi antigen on serial
subculture.
It covers the O antigen and make the strain inagglutinable with O antisera, but
this is reverted by heating for 1 hour.
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There are no diagnostic utility for Vi antigens but are useful for epidemiological
studies.
There are Vi vaccines, contain purified Vi polysaccharide antigen which is
prepared from s.typhi strain Ty2.
Weil’s disease
It is a severe form of leptospirosis where hepato renal damage occurs in 10% of
patients.
Leptospirosis is a zoonotic disease caused by Leptospira iterogans.
The disease is transmitted by direct or indirect contact with water contaminated
with urine of carrier animals.
Leptospires enters the body through cut or aberrations on the skin or directly
through mucous membranes.
Incubation period is about 6-8 days.
The disease is biphasic. There are septicaemic phase and immune phase.
The disease start with febrile illness with leptospires in blood. This phase is
called septicaemic phase.
From the blood the organism will be seeded into different organs such as liver,
kidney, spleen and meninges.
The disease can cause liver and kidney damage, meningitis, respiratory distress
and even death in untreated cases.
Laboratory diagnosis
 Specimen: CSF, Blood and urine
 Microscopy:
Demonstration of Leptospires in Blood and in urine by Dark ground
microscopy.
Silver impregnation technique: For demonstrating the tightly coiled bacteria

with hooked ends.
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 Culture:
Blood and urine can be cultured in
 EMJH medium
 Modified Korthof’s medium
 Fletchers semisolid medium
The Inoculated medias are incubated at 28-32 C aerobically up to 6 weeks
before declaring as negative.
 Animal Inoculation:
Intraperitoneal inoculation into guinea pigs and the peritoneal fluids are
examined daily for leptospires by dark ground illumination.
 Serological test:
ELISA, ICT, Lepto dip stick assay.
 IgM antibodies appear early within one week of illness and reaches a
peak level in 10-18 days.
 IgG antibodies appears after 3 rd week and persist for a longer period.
 Confirmatory test or Gold standard test:

 Microscopic agglutination test or MAT test
It detect antibodies against specific serovars of Leptospira.
Patient serum is mixed in a microtitre plate with a specific leptospiral serovar to
be tested.
The tubes are then incubated for 2 to 4 hrs at 37 C and examined under dark
ground microscope for the presence of agglutination.
Molecular method: PCR
Histoplasma capsulatum
It is a dimorphic fungus.
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This is primarily a disease of reticuloendothelial system caused by Histoplasma
capsulatum.
The disease is also known as Darlings disease because it was first reported by
Samuel Darling in 1905.
This is a soil saprophyte and the source of human infection is inhalation spores
from the soil.
There are two varieties:
H.capsulatum causing Classical histoplasmosis
H.duboisii causing African histoplasmosis.
Clinical features:
 Pulmonary Histoplasmosis
 Disseminated Histoplasmosis: Involvement of RES results in
lymphadenopathy, Hepatosplenomegaly, fever, anemia and high rate of
fatality.
 Mucocutaneous Histoplasmosis.
Lab diagnosis:
 Specimens: Sputum, bone marrow aspirate, blood, scraping from

mucosal lesions, Lymph node aspirate.
 Microscopy:
Direct examination: smears made from the specimens are stained with
Giemsa and wrights stains.
H.capsulatum appears as small, oval yeast cell packed with in the
macrophages.
 Culture:
This is the Gold standard method of diagnosis.
Cultures are made on antibiotics added SDA or BHI. Since this is a
dimorphic fungi 2 tubes should be kept at different temperatures.
One at 37 C and the other at 25C.
The yeast phase will be grown in tube kept at 37C.
Mycelial growth shows finger like projection with spores.
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 Serology:
Latex agglutination, Precipitation and Complement fixation.
 Histoplasmin skin test:

This is a delayed hypersensitivity test. The antigen used is
histoplasmin. A positive histoplasmin test indicates present or past
infection.
Laboratory diagnosis of undulant fever
Acute Brucellosis is called Undulant fever or Malta fever.

The disease is called undulant fever because the fever is typically undulant,
rising and falling like a wave.
Laboratory diagnosis include:
 Culture of Brucellae
 Serology
 Hypersensitivity test
Specimens: Blood culture is the most definitive test
Other samples: bone marrow, liver, lymph node, CSF, urine, vaginal discharge,
sputum and seminal fluids.
Blood culture:
Blood is collected during the peak of fever because the chances of isolation of
Brucellae is more at this phase.
5-10 ml of blood is inoculated on to 50 to 100 ml of broth and incubate at 37 C
in capnophilic condition. Subcultures are made on solid media in every alternate
day’s up to 8 weeks. Because Bruclla is a slow grower and also a fastidious
organism.
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Epidemic typhus
This is also called Classical typhus. The causative organism is Rickettsia
prowazekii.
The disease got the name typhus because of the cloudy state of conscious
developed by the patient in the acute stage of the illness typhus meaning smoke
or cloud).
It is a vector born disease and the vector is human body louse (Pediculous
humanis corporis)
The lice become infected by ingesting the blood from infected patients.
The organism multiply in the gut of the vector and discharges through faeces in
3 to 5 days.
Human gets the infection after the lice faces being rubbed onto the aberrations
produced during scratching.
The incubation period ranges from 5-21 days.
Clinical features:
Head ache, chill, myalgia, high fever and vomiting.
A maculopapular rash appears 4 to 7 days after the onset of illness which first
appears on the trunk and then spread to the limbs.
On the second week of illness the patient develop a cloudy state of
consciousness.
Lab diagnosis
 Serology
 Isolation of Rickettsiae
 Molecular methods
Serology
Nonspecific test and Specific test
 Nonspecific test: Weil felix reaction
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 Specific test: Using Rickettsial antigens.
Weil Felix reaction

It is a heterophile agglutination test that detect antibodies in the patients serum
with Rickettsial fever that cross react with some strains of Proteus ,due to
sharing of alkali stable carbohydrate antigen of Rickettsia and O group antigens
present in certain non-motile strains of Proteus. E.g.P.vulgaris OX 19, OX-2
and Proteus mirabilis OXK.
 Neil-Mooser reaction
It is also called Tunica reaction.
When male guinea pigs are injected intraperitoneally
 With blood from a case of endemic typhus or
 Blood from a case of endemic typhus
 With a culture of R.typhi
The animal develop fever and characteristic scrotal inflammation. The
scrotum become enlarged and the testes cannot be pushed back into the
abdomen because of the inflammatory reaction between the layers of
Tunica vaginalis. This is called Neil Mooser or Tunica reaction.
This is negative in infection with R.prowazekii (Epidemic typhus)
The test gives positive reaction in R.conori and R akari.
Brill Zinsser disease
It is a typhus fever disease also called Recrudescent typhus.

This is a latent infection seen in epidemic typhus infected persons.
In some cases who recovered from epidemic typhus, the rickettsiae may remain
latent in lymphatic tissues or organs for years. Such latent infection may be
reactivated leading to recrudescent typhus or Brill Zinsser disease.
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It is a milder illness and the duration of the disease is shorter. Case fatality is
lower.
Japanese B encephalitis
This is an arthropod born disease, and was first recognised in Japan.
The vector is culex mosquito.
In India it was first identified in 1955 in Tamilnadu.
No man to man transmission. Humans are considered as the dead end.
Clinical features:
The incubation period is 5 to 15 days.
The large majority of infections are asymptomatic.
The disease has an abrupt onset with fever, headache and vomiting.
After 1-6 days signs of encephalitis starts with neck rigidity, convulsions,
convulsions altered sensorium and coma.
Lab diagnosis
Specimens: Serum, CSF
These are tested for JE specific IgM antibodies.
Molecular methods
Reverse transcriptase PCR.
Treatment
No specific treatment. Only supportive measures.
Prophylaxis
 A live attenuated vaccine prepared from JE strains SA 14-14-2. It is a cell
line derived vaccine.
Two doses are given, first dose at the age of 9 to 12 months and 16-24 months.
0.5 ml administered subcutaneously at left upper arm
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 Inactivated Vero cell vaccine.
 Formalin inactivated mouse brain vaccine.
Kala azar
This a parasitic disease caused by Leishmania donovani. The disease is called
visceral leishmaniasis or Kala azar (Black Disease).
The disease spread to human by the bite of Sand-fly.
The parasite spread from the site of inoculation and multiply in
reticuloendothelial system especially in spleen, bone marrow and lymph node.
This leads to enlargement of these organs.
There are two morphological forms for this parasite, Amastigote form and
Promastigote form.
Amastigote forms are the aflagellar form which are seen inside the RES, in
human.
Promastigote forms are flagellar forms which is present inside the insect vector.
Visceral leishmaniasis is one of the most important opportunistic infection in
HIV patient.
Diagnosis
There are Direct and Indirect evidences.
Direct evidences:
 Demonstration of Amastigote form or LD bodies from
 Peripheral blood
 Bone marrow aspirate
 Splenic aspirate
 Lymph node aspirate
 Culture
Blood bone marrow aspiration
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Lymph node aspirate
 Antigen detection
 Animal Inoculation
 PCR
Indirect Evidences:
 Blood examination to see leukopenia an neutropenia
 Elevated serum globulin
 Reversal of albumin- globulin ratio
Non-specific serological test:
 Napier aldehyde test
 Chopra’s antimony test
 Complement fixation test with WKK antigens.
Specific serological test

 Direct agglutination test
 Indirect hemagglutination test
 Indirect fluorescent antibody test
 ELISA
 Immunochromatographic strip test
Skin test:
The Leishmanin skin test. There is no diagnostic value for this test.This test is
negative in active cases of kala-azar as these patient have impaired cell
mediated immunity.
Differential diagnosis:
Malaria, Trypanosomiasis, brucellosis, tuberculosis, relapsing fever.
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WIDAL test
The most widely used serological method to diagnose enteric fever.
Principle:
It is a tube agglutination test, which detect antibodies produced against
Salmonella typhi O and H antigens and H antigens of both Paratyphi A and B.
To measure H agglutinins for S.typhi and paratyphi A &B and O agglutinins of
S.typhi.
Antigen used are:
H and O antigens of Salmonella typhi
H antigens of S.paratyphi A and B
Method:
 WIDAL rack with 4 rows of test tubes are taken for the test.
 H agglutination will be carried out in DREYERS tubes (Conical bottom)
O agglutination is tested in FELIX TUBE (Round bottom)
 Equal volumes of serial dilution of patients serum and H and O antigens
are mixed in respective tubes and incubate overnight.
 H agglutination shows loose cottony woolly clumps, significant titre>
200
O agglutination shows disc like spreading in bottom, significant
titre>100 Positive
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Lyme disease:
It is caused by Borrelia burdogferi and is transmitted through tick bite.
There are 3 stages for the disease:
 Localised infection: Maculo popular rash appears at the site of tick bite
called erythema migrans.
 Disseminated Infection: Borrelia spread through blood to different sites
resulting in secondary skin lesions, arthralgia, malaise and neurological
abnormalities.
 Persistent infection: about 60% of the patients develop arthritis which last
for months.
Lab diagnosis:
 Serology: Antibody detection
 Isolation: BSK medium (Barbour-stoenner-kelly)
 Molecular method: PCR
HCV or non-A non-B hepatitis
This is an RNA virus belonging to the family Flaviviridae which causes

transfusion associated hepatitis.
It is an enveloped virus carrying glycoprotein spikes.
There are 6 genotypes and many subtypes.
Pathogenesis:
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Source of infection: Carriers
Mode of transmission:
 Blood transfusion
 Contact with infected blood or blood products
 Vertical transmission
High risk persons include:
 Transplant recipients
 Injectable drug abusers
 Immuno-compromised persons
Clinical features:
Incubation period: 5-12 weeks
The disease starts with milder symptoms like fever, nausea, Jaundice
(absent or milder forms)
Few of them progresses to chronic hepatitis which leads to liver damage.
Complications: cirrhosis or hepatocellular carcinoma.
Laboratory diagnosis:
Serological diagnosis is the most widely used method.
Ag detection from hepatocytes by Immunofluorescence test.
ELISA
Recombinant Immunoblot test
RIA
HCV RNA detection by RT PCR
Prophylaxis:
No vaccine is available.
General prophylaxis:
Screening of HCV before transfusing blood.
Avoid using unused or unsterile needle.
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Blood and Cardiovascular system

6. Milk ring test

10. Histoplasma capsulatum

11. Epidemic typhus

12. Brill Zinsser disease

13. Japanese B encephalitis

14. Lyme disease

15. HCV or Non A non B hepatitis

2) Enumerate the causative agents

3) Describe the pathogenesis and complications of this disease

4) What are the clinical features?

5) Mention sample collection and laboratory diagnosis in detail

6) Add a note on prophylaxis

8) Write briefly about treatment and drug resistance

2) What are the morphological characteristics of this organism?

 There are three structural genes:

3) Mention the different routes of transmission in this condition?

4) Explain the pathogenesis and clinical manifestations of the above

It is broadly categories into 2 types:

 T-lymphocyte subset assay:

 Platelet count: Thrombocytopenia

 IgG and IgA levels: both are raised

 Skin tests for CMI: shows positive reactions because of diminished

The following parameters should be performed before performing the test

 ELISA: This is a screening assay and has high sensitivity and

 Detection of Viral nucleic acid

Viral nucleic acid can be detected by Polymerase chain reaction..

Can be isolated from CD4 lymphocytes from peripheral blood &

Detects individual antibodies against various viral antigenic fragments in

The antigen antibody complexes appears as distinct bands on

6) Name the opportunistic infections seen in HIV/AIDS patients

Recurrent severe bacterial infections, extra pulmonary tuberculosis

Chronic HSV infections, Progressive multifocal leukoencephalopathy,

Pneumocystis jirovecii pneumonia, Oesophageal candidiasis, extra

Parasitic opportunistic infections

Toxoplasma encephalitis, chronic intestinal cystoisosporiasis, atypical

7) What are the treatment modalities? What is HAART? What are

Anti-retroviral therapy (ART)

Clinical, Virological, Immunological and Transmission goals.

 Clinical goals: Prolongation of life and improvement of quality of

The drugs are of different types:

HAART is referred to the use of combination of atleast three anti-

Problems pertaining to use of ART:

8) What are the measures taken on Post exposure prophylaxis?

Post exposure prophylaxis or PEP is required to reduce the risk of

Duration: should be started within 2 hours of exposure and continued for

Indication: if source is unknown/ positive for HIV

Step of post exposure management:

4. Testing for blood borne viral infections:

5. Decision on post exposure prophylaxis

9) What are the confirmatory tests?

 P 24 antigen detection after two weeks of infection.

2) Describe in detail the lab diagnosis of this disease

• Non- specific markers- Elevated liver enzymes and serum bilirubin

3) Add a note on treatment and prophylaxis

4) What is defective virus?

2) Name the species of the causative agent?

3) Which species cause more severe disease?

Plasmodium falciparum causes the most severe form of infection called

4) Name the complications

The complications of falciparum malaria are:

 Cerebral malaria: vascular occlusion and cerebral anoxia due to the

5) Explain the life cycle of this parasite

The malarial parasite passes its life cycle in two hosts

Blood & CVS

Blood & CVS