| ] eee ree Bae IN INFANCY AND CHILDHOOD Philippine Pediatric Society, Inc. Ay Committee on Handbook on ' Childhood Tuberculosis Scanned with CamScannerPHILIPPINE PEDIATRIC SOCIETY, INC. TUBERCULOSIS IN INFANCY AND CHILDHOOD 4™ edition 2016 COMMITTEE ON HANDBOOK ON CHILDHOOD TUBERCULOSIS Scanned with CamScannerGLOSSARY: DEFINITIONS Acquired resistance — refers to th i ; e ba i i q previous treatment cterial resistance in patients with some record of Asymptomatic or latent tuberculosis infecti tc aten ection (LTBI) — i i i tuberculin hypersensitivity as shown by a positive eciciiveniey t ‘sn an striking clinical or roentgenographic manifestations st (TST) with no BCG - stands for Bacillus Calmette-Guerin; is an att ccil * F S tenuated rail 5 used to immunize against tuberculosis yaocing strain of M, bovis Directly observed therapy (DOT) administered directly to the patient by a who observes and documents that the pi — an intervention by which medication is health care professional or trained third person, atient ingests each dose of medication Drug-resistant tuberculosis (DR-TB) — tuberculosis, usually pulmonary, excreting bacilli resistant to one or more antituberculosis drugs Exposed person — a person who has had recent contact with another person with suspected or confirmed contagious pulmonary tuberculosis disease and who has a negative tuberculin (or IGRA) reaction, normal physical examination findings, and chest radiographic findings that are not compatible with tuberculosis Extensively drug-resistant tuberculosis (XDR-TB) - 2 subset of MDR-TB with a strain of Mycobacterium tuberculosis complex that is resistant to_ isoniazid and rifampicin, any quinolone, and at least one of three second-line injectable drugs: kanamycin, capreomycin, or amikacin Extrapulmonary tuberculosis (EPTB) - refers to a case of TB involving oh outside of the lung parenchyma or tracheobronchial tree (e.g., larynx, pleura, nodes, abdomen, genitourinary tract, skin, joints and bones, meninges) Ghon focus - the initial lung lesion of primary TB ium to the Incubation period — time interval from exposure to the mycobacieesitve 1sT development of delayed type hypersensitivity reaction as manifested by (or |GRA) ' x infection i je is comple’ Latent tuberculosis infection (LTBI) Mycobacterium tuberculosis comrstations of in a person who has a positive TST (or IGRA) result, with no clinical mé disease and chest radiograph findings that are normal Scanned with CamScannerGLOSSARY: DEFINITIONS Mantoux test — a skin test for tuberculosis infection, using tuberculin PPD i protein derivative) administered intradermally / intracuianeously at Seeded and positive result read as induration between 48 to 72 hours of injection Miliary tuberculosis— a form of generalized hematogenous TB due to a massive invasion of the blood-stream by the tubercle bacilli seeding in various sites Monoresistance — resistance to one antituberculosis drug Multidrug-resistance (MDR) — resistance to at least isoniazid and rifampicin Mycobacterium tuberculosis (MTB)- the bacterium that causes tuberculosis Poly-resistance — resistance to more than one antituberculosis drug, other than isoniazid and rifampicin Pott’s disease — TB of the spine Primary complex — composed of the primary focus, lymphangitis, localized pleural effusion and regional lymphadenitis, demonstrable by radiographic study Primary resistance — bacterial resistance present in patients who have not received prior treatment with antituberculosis drugs Pulmonary tuberculosis (PTB) - bacteriologically-diagnosed or clinically-diagnosed case of TB involving the lung parenchyma or the tracheobronchial tree. Secondary resistance — bacterial resistance present in patients who have received prior treatment with antituberculosis drugs Subcentimeter — size less than one centimeter in greatest diameter Scrofuloderma — TB of the skin overlying a caseous lymph node that has ruptured to the outside, leaving an ulcer or a sinus Source case — the person, usually with pulmonary TB, who has transmitted infection with Mycobacterium tuberculosis Tuberculin — a protein extracted from Mycobacterium tuberculosis that is used in a skin test to determine if a person has been exposed to tuberculosis and infected with the tuberculoprotein from the organism xvi Scanned with CamScannerGLOSSARY: DEFINITIONS Tuberculin skin testing (TST) - a dia i 1g | gnostic test to i delayed-type hypersensitivity reaction to tuberculoproteins, renee trom nee with the organism or in reaction to the mycobacterium ‘antigen in BCG. fection reaction will manifest as an induration; cut-off size determined by conditions. 3 ane Tuberculosis (TB) — a highly contagious infection and disease caused by the virulent bacterium Mycobacterium tuberculosis, Progressing from inci © disease, when signs and symptoms become manifest, either from pulmona rt extrapulmonary involvement oo Note: Other terms in public health programs such as active case-finding, contact, contact investigation, index case — are found in Chapter 15 (Prevention and Control). Scanned with CamScannerADR AIDS AFB ALT ART AST ATS BCG cDC CDR CMI COz cT CXR DOH-NTP DOT DOTS DOTCh DR DRS GLOSSARY: ABBREVIATIONS adverse drug reaction acquired immunodeficiency syndrome acid-fast bacilli alanine aminotransferase (SGPT) anti-retroviral therapy aspartate aminotransferase (SGOT) American Thoracic Society bacillus Calmette-Guerin Centers for Disease Control and Prevention case detection rate cell-mediated immunity carbon dioxide computerized tomography chest X-ray Department of Health-National Tuberculosis Program directly-observed therapy! treatment directly-observed treatment short-course directly-observed treatment in children drug-resistant drug resistance and susceptibility DTH E, EMB ELISA ELISPOT EPTB FM FDC Gl H, INH HAART HBHA HIV IL-6 IL-12 IFN-y IGRA imc! IRIS ISTC >i delayed-type hypersensitivity ethambutol enzyme-linked immunosorbent assay enzyme-linked immunospot assay extrapulmonary tuberculosis fluorescence microscopy fixed-dose combination growth index isoniazid highly-active antiretroviral therapy heparin-binding hemagglutinin human immunodeficiency virus histocompatibility leukocyte antigen interleukin-6 interleukin-12 interferon gamma interferon gamma release assay Integrated Management Childhood liness = immune reconstitution inflammatory syndrome International Stand: Tuberculosis Care as Scanned with CamScannerIUATLD KNCV LAM LGUs LTBI MDG MDR MRI MGIT MTBC. MPT NAAT NK NGO NNRTI NRTI NO GLOSSARY: ABBREVIATIONS International Union against Tuberculosis and Lung Diseases Royal Netherlands Tuberculosis Foundation lipoarabinomannan local government units Lowenstein-Jensen latent tuberculosis infection Nramp NTP/NTBP NTM NTPS OPV PAS Millennium Development Goal PCR multidrug-resistance magnetic resonance imaging PhilCAT PIDSP mycobacterial growth indicator tube Mycobacterium tuberculosis Mycobacterium tuberculosis complex multiple puncture test nucleic acid amplification test natural killer non-government organization non-nucleoside reverse- transcriptase inhibitor nucleoside reverse- transcriptase inhibitor nitric oxide negative predictive value PLHIV PPD PPM PPS PPV PSMID PTB PTSI R, RIF RR RFLP Negative resista associated ma, Protein "Phage National Tube; ‘ rogram TCulogis, Non-tuberculosis mycob, Nationwide Tuberculog, Prevalence Suey oral polio vaccine Para-aminosalicylic aciq Polymerase chain reaction Philippine Coalition against Tuberculosis Pediatric Infectious Disease Society of the Philippines persons living with HIV purified protein derivative public-private mix Philippine Pediatric Society positive predictive value mrebegy and ects Diseases pulmonary tuberculosis Philippine Tuberculosis Society, Inc. rifampicin, rifampin rifampicin-resistant restriction fragment length polymorphism Scanned with CamScannerTGF-B TNF-a TST USAID WHO XDR Z, PZA ZN GLOSSARY: ABBREVIATIONS streptomycin tuberculosis transforming growth factor-beta tumor necrosis factor-alpha tuberculin skin test tuberculin units United States Agency for International Development World Health Organization extensively drug-resistant pyrazinamide Ziehl-Neelsen Scanned with CamScannerHighlights Estrella Paje-Villar, MD Josefina Carlos, MD Scanned with CamScanner1910 1930 1933 1949 1950 1951 1954 1958 1964 . iippi .ds Anti-Tuberculosis Society n Founding of the Philippine Islan 0 Y Now known Philippine Tuberculosis Society Inc (PTS!) a private agency with govem subsidy as Tuberculosis mortality rate was 487/100,000 population Creation of the Tuberculosis Commission by virtue of Republic Act (RA) 37 under the Philippine Health Service (Ministry of Health), now Dene Health (DOH) Transfer of the TB Commission‘s powers and functions to the Ministry of Health Establishment of Philippine Charity Sweepstakes Office (PCSO) to fund the PTSI through the Philippine Charity Sweepstakes Law (RA 4130) Amiracle drug, streptomycin (S), was first used for TB treatment Creation of the Division of Tuberculosis under the Secretary of Health Establishment of TB Center at DOH compound in collaboration with San Lazaro Hospital TB Ward Addition of para-aminosalicylic acid (PAS) to the TB regimen with streplomycin Introduction of BCG vaccination program with assistance from UNICEF Enactment of Tuberculosis Law (RA 1136) in ($) Use of triple anti-TB therapy consisting of isoniazid (H), PAS & streptomyci . jigion Of . Division Establishment of the Bureau of Disease Control which included the TB by virtue of Executive Order no. 288 ce Sune first TB prevalene meat Condu i i ne ict of the Minglanilla Prevalence Survey, .d the prevalen carried out in the province of Cebu, which showe Positive at 4/1,000 population Scanned with CamScannerunder RA 1086 Control Program in all rural health units (RHUs) 3) Use of sputum microscopy as a diagnostic tool for TB Launching of the Domiciliary Care Program Formation of the Philippine College of Chest accredited non-govemment organization (NGO) Medical Association (PMA) Physicians (PCCP) as an society of the Philippine Offering of the Anti -TB treatment using isoniazid, streptomycit ae |, streptomycin and ethambutol Publication of Tuberculosis in Filipino Children by Dr. Mita Pardo de Tavera Establishment of the National Institute of Tuberculosis (NIT) in cooperation with the WHO and UNICEF Rifampicin (R) became available in the Philippines Implementation of the compulsory BCG vaccination in the Philippines Nationwide implementation of the National TB Control Program (NTBP) | of Procedures, which Publication of the First National TB Program Manua d ry diagnostic tool for TB highlighted the use of sputum microscopy as the prima and the introduction of the standard drug regimen for TB treatment Conduct of the First National TB Prevalence Survey by the National instal for Tuberculosis (NIT) Establishment of the Lung Center of the Philippines (LCP) . tality Tuberculosis in all forms as the 3° leading ed oe ala 52.6/100,000 population (Philippine Health Statistics) 3 Scanned with CamScanner1986 1987 1988 1989 1990 1991 1993 i the Office of ice with EO no. 119, under Of Puby; Creation of the TB onto Fath which became the Department of Health) ° ces (Minis Health Servi therapy (SCC) which highlighted the ug ( e Chemothe P Ie Use Launching of Sho postive and cavitary cases by chest X-ray. Pyrazinamige rifampicin for Fg intensive phase of treatment. Fd in the int : : : Gis a = ethambutol used as the 4" drug in the intensive Phase at ‘Streptom: Quezon Institute (Ql). Nationwide implementation of strengthened NTBP as one of the impact programs of DOH with allocation of drugs and supplies Publication of the National TB Program Manual of Procedures (2% edition), which marked the adoption of the Short Course Chemotherapy (SCC) for the management of TB cases; presented the results of the 1981- 1983 First National TB Prevalence Survey (NPS); introduced the itinerant team approach to selected regions / provinces and intensified the training, monitoring / ‘supervision in low performing areas.. First reference to the Public-Private Mix (PPM) for TB Control in the Philippine NTBP manual Issuance of the first consensus Statement by the Tri-Chest organization, led by the Philippine College of Chest Physicians (PCCP) Financial and technical support to the NTBP from the Italian government and World Bank for improving TB control in Regions V, VIII, X and in other cities and provinces Local Government Units (LGUs) became the implementer of TB program — the Local Government Code ' Vil ae of a WHO-assisted external evaluation on NTBP in Regions IV. Ml 1 First NTBP SEAMIC Conference with other Asian Countries Scanned with CamScanner1993 1994 1995 1996 1997 Formation of the National TB Advisory Council Publication of Tuberculosis in Infancy & Childhood (1*edition) by the Philippine Pediatric Society (PPS) Field testing of new NTBP policies and guidelines and establishment of First TB Reference Laboratory in Cebu under the DOH-JICA Project Launching of hospital-based NTBP Founding of the Philippine Coalition Against Tuberculosis (PhilCAT) Tuberculosis, all forms as the 5" leading cause of mortality 39.8/100,000 population (Philippine Health Statistics) Establishment of the TB Reference Laboratory in Regions |, IX, XI Formulation of the revised policies and guidelines on TB management per Administrative Order No. 1-A Launching of Target: Stop TB (slogan: Sigaw ng Bayan, TB ay Labanan) Offering of the Directly Observed Therapy Short Course (DOTS) by the University of Santo Tomas Hospital TB Clinic The 3 Annual Convention of PhilCAT, in collaboration with the PPS and the Pediatric Infectious Disease Society of the Philippines (PIDSP) with the theme: Childhood Tuberculosis, a Concern for All Declaration of August 19 as National TB Day by virtue of Proclamation No. 840 Observance of the first World TB Day on March 24 spearheaded by PhilCAT Launching of Target: StopTB Part Il (slogan: TB ay Labanan, Gamutan ay Tutukan) WHO supported CRUSH TB project in new NTBP policies and guidelines and DOTS Strategy (Tutok Gamutan) Conduct of the Second National Tuberculosis Prevalence Survey in the Philippines Joint publication of National Consensus on Childhood Tuberculosis (NCCT) by the PPS, PIDSP and PhilCAT Scanned with CamScanner1997 1998 1999 2000 2001 : idelines of the New TB Control Program by the Development of Teennica! cont ehilaboration with DOH-JICA Ca Department a Seth Agency), Public Health Development Project and the interment Pate Regional Office (WPRO), in accordance with the WHO ae ce m the external evaluation conducted by WHO in 1993 recomme! Expansion of CRUSH TB using DOTS in seven provinces Development of Technical Guidelines of the New TB Control Program, with emphasis on DOTS or —Tutok Gamutan Establishment of the Center for Tuberculosis in Children, Philippines (CTCP) by Dr. Fe del Mundo Issuance of Memorandum Circular No 98-155 pronouncing the NTBP as the No. 1 priority health program of the LGUs and the prescribing of DOTS Launching of Strategy Project Lusog Baga (—Healthy Lungs) Creation of the Task Force on Childhood Tuberculosis through Department Order No 248-H s. 1998 (amended in 2001): a DOH initiative in cooperation with PPS and other agencies. Development of the first Clinical Practice Guidelines on the Diagnosis, Treatment and Control of TB spearheaded by the Philippine Society of Microbiology and Infectious Diseases (PSMID) with the PCCP and DOH Health Sector Reform Agenda of DOH put NTBP on top priority Formal initiation by the WHO of PPM for TB control during the first global assessment of PPM in TB control Expansion of DOTS Program Publication of the National TB Program Manual of Procedures (3 edition), which served as reference in the orientation/training of the private sector and other government agencies in their implementation of NTP-DOTS. Establishment of the National TB Reference La boratory (NTRL) Strengthening advocacy strategies: TB Healt! i it Slogan: Huwag Mahiya, TB Ipagamot Na in Promotion en) CoH), with Scanned with CamScanner2002 2003 2004 2005 Formulation of the Comprehensive and Integrated Policy for TB Control Creation of TB Diagnostic Committees to review s| putum smear-negative Achievement of a nationwide coverage for DOTS i i sector (from 2002 to 2003) ig Strategy in the public health Development of the Comprehensive and Unified P Sli the DOH and PhilCAT under EO No. 187 oly (CUP) to control TB by Adoption of the Public-Private Mix DOTS (PPMD) Stining of single dose formulation to fixed dose combination (FDC) drugs for adults First National Drug Resistance Survey supported by WHO and JICA became the basis for the policy to address multi-drug resistant (MDR) TB Publication of Tuberculosisin Infancy and Childhood (2"edition) by PPS Issuance of hospital-based NTBP-DOTS policies Issuance of hospital-based NTBP-DOTS policies Lung Center of the Philippines (LCP) became the government's counterpart support to the DOTS-plus initiative Formulation of operational guidelines on PPMD developed by NTBP in cooperation with PhilCAT, WHO and the Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM) Institutionalization of DOTS certification and Philhealth TB OPD Benefit Package Tuberculosis all forms as the 6" leading cause of mortality 31/100,000 Population (Philippine Health Statistics) Publication of the Guidelines for Implementing Tuberculosis Control Program in Children by the DOH Publication of the National TB Program Manual of Procedures (4" edition) which included the use of fixed dose combination anti-TB drugs, external quality assessment, adoption of the Public-Private Mix DOTS, strengthening of the TB Diagnostic Committees, DOTS facility certification and accreditation, and development of the health promotion plan specific to TB. Issuance of Guidelines for the Implementation of Policy and Program on TB Prevention and Control in the Workplace Scanned with CamScannerfl Pa anded to addre 2006 Scaling up of PPMD continued; it expan 88 the Urban POOT nN Manila 0 ulation by PPS of the Evidence-based Clinical p, Formt ie ‘ Tactice Guide); Childhood Tuberculosis 1 edition (published 2008) delines 4 Publication of global guidelines by the WHO and launching Of the Ie met Standards for Tuberculosis Creation of the National TB/HIV Collaborating Committee 2006- Inclusion of the PPMD component in the National Strategic Plan to Stop Tg in 2010 the Philippines 2007 Conduct of the Third National Tuberculosis Prevalence Survey TB- tespiratory as the 6” leading cause of Morbidity in the Philippines 136/100,000 Population; TB - all forms with morbidity rate at 1 52/100,000 Population (Philippine Health Statistics National Epidemiology Center, DOH) 2007- Establishment of 220 PPMD units n; 2008 lationwide Covering 40% of the population and contributing 90% in the national case detection 2008 Publication of the revised G Program in uidelines for Implementing Tuberculosis Contro! Children by th '¢ DOH/NTP 2009 Publication Of the Principle. Control by ¢ cand Practices of PPM for Tuberculosis Care and he DOH & PhilcAT 2010 initia Iitation of the 2010-2016 Philippine Plan of Action to comets (PhIPACT) under the leadershi Program (NTP) Pof the DOH through the National TB Contrl WHO Rapid Advice released: Streptomycin no longer first-line anti-TB dru. 2011 Thex, = Torry eR (Gene Apert) was frst introduced into the country as new SIS Of TB and for determination of rifampicin resistance. Scanned with CamScanner2012 PPS co-sponsored World TB Day (24 March) and the 16” Phil Convention (16-17 August) with (an March) and the 16 PhiCAT Annual 2013 WHO organized external NTP review — Joint Program Revi PhilPACT gram Review (JPR) of the 2014 Reassessment and revision of PhilPACT, led by the National Tuberculosis Control Program, through its midterm evaluation. The updated PhilPACT guides the country in its intensified effort to control TB in 2014-2016. (Updated 2010- 2016 PhilPACT) Institution of PhilHealth Package for TB International Standards for Tuberculosis Care (ISTC), 3" edition The new WHO End TB Strategy was adopted by all WHO Member States at the World Health Assembly in May. It serves as a blueprint for countries to end the global TB epidemic. 2015 WHO released the first Guideline on the management of latent TB infection. 2016 Republic Act No. 10767 —Comprehensive Tuberculosis Elimination Plan Act was signed into law on April 26. 23" PhiICAT Annual Convention co-sponsored by the Pediatric Infectious Disease Society of the Philippines (PIDSP) on 18-19 August. WHO issued the guidance for the use of delamanid in children > 6 years and adolescents on 25 October as a component of a 9-month regimen for MDR- TB. Scanned with CamScanneron DO © &® WD 2 Epidemiology Luis Chan, Jr, MD Ana Liza Duran, MD OUTLINE Overview Tuberculosis Key Facts Global and Regional Burden of TB TB in Children Drug-Resistant TB TB and HIV Co-infection Mode of Transmission of TB Risk Factors for TB Scanned with CamScanneri f death fr ‘ OVERVIEW ide HIV as a leading cause of OM an infer pecs, en unercaos (MTB) cases IN infestous ease ang ima deoase. I 0b ces other sites aS Cea period of transition from i fd dea id involves ot ar h illen fer ne disease ee ee fnew era of Sustainable Development Goals (Spgq)" 7m Forelopment Goals (MDGs) in mortality reduction, a fall in incident cases, ang lives’ gaye have been major eee treatment. Stil there are gaps between esti es a through notification being monitored so that SDGs will be ok Bu alte Set Petiodicaly Thasath anomie co-existing diseases (TB and HIV) are given special mention. p The 12, OsiS KEY FACTS',”: ] / ad ae million people are estimated to have fallen ill with TB: 5.4 million men, illic ili ildren. 3.2 million women and 1.0 million chil 7 0 6 million TB cases were reported to WHO. Of these, 5.7 million were newly diagnosed and another 0.4 million were already on treatment. 0 Among pulmonary TB cases, 58% were bacteriologically confirmed. 0 There were an estimated 700,000 TB deaths among HIV-negative men and 340,000 among HIV-negative women © TB Killed 1.5 million people: 1.1 million HIV-negative and 0.4 million are HIV- Positive. 0 The death toll comprised 890,000 men, 480,000 women and 140,000 children. . ales 2013, the treatment Success rate continued to be high at 86% among all new cases, * Between 2000 and 2014, TI B treatment alone Si illion lives. among HIV-negative people, Saved an estimated 35 millor GLOBAL AND REGIONAL BURDEN OF TB New cases of TB I i ie . eS Ber 109,000 population, oF globally in 2014 is equivalent to 126 cas Region (28%) The absolute nuns ee he half (68%) occurred in Asia and the Arian 28 on ve 20 teas Gey at nae fon CeS2S 1-1 to 1.3 mnt lo with © Proportion of TB -3 million (11-13%) were people living W HIV in ths 2810. Overat gg of rg co-infected with HN ane highest in counts Worldwide. In par ror 'Ccounted for rays Were estimated to be co-infected 7 with Hy, S Of Southern Afr; rot TB cases among people living with Globally, 123 0 “More than 50% of TB equa were infec | 33000 cases Ported in 2014. Inge! MORTB or RR- na oF the total, Pl, the Russian Fea igible for treatment were detected ae « © 123,000 cag, Pn and South Africa accounted for almo: 9 Pulmonary TB pate Presented 41% of the estimated 300,000 ment that were notitied tn ae po of 11 Scanned with CamScannerestimated 480,000 (range 360,001 ay bore :000-610,000) incident MDRTB cases worldwide in The number of incident ‘TB cases relative to population si a . widely among countries. The lowest rates are found pelomieaty i takes countries, where the incidence rate is less than 10 cases/100,000 popilation’s ore Globally, the incidence rate was relatively stable form 1990 uy i then started to fall, achieving the MDG target ahead of the Bots deadine, Betacon 2000 and 2013, the average rate of decline per year was 1.5%. x There were an estimated 13 million prevalent cases (ran illic ili 1B in 2014, equivalent to 174 cases/100,000 populnton By Ie end or 2018, t was estimated that the prevalence rate will have fallen 42% globally since 1990. missing the target. However, two regions met the target before 2015 (the Region of the Americas and the Western Pacific Region) and the Southeast Asia Region reached the target (according to the best estimate) in 2015. TB prevalence is falling in all of the other three regions. The number of TB deaths per 100,000 population averaged 16 globally in 2014, and 21 when TB deaths among HIV positive people are included. There is considerable variation among countries, ranging from under 1 TB death per 100,000 population (Western Europe, Canada, USA, Australia and New Zealand) to more than 40 deaths per 100,000 population in much of the African Region as well as 3 High Burden Countries (HBCs) in Asia (Bangladesh, Cambodia and Myanmar), and the 2 HBCs in the 2 Eastern Mediterranean Region (Afghanistan and Pakistan). Globally, the TB mortality rate (excluding deaths among HIV+ people) fell by 47% between 1990 and 2015, narrowly missing the target of a 50% reduction. WesTERN PaciFic REGION‘?* The Western Pacific Region, where the Philippines belongs, has an estimated population of about 2 bilion. The incidence of TB, including HIV positive patients, e 85 Per 100,000 population. Mortality rate, excluding HIV+TB, is presently at 4. pe 100,000 population while that of HIV+TB patients is 0.27/100,000 Pane - Prevalence rate is 116 per 100,000 population in 2014. The estimate ea a Notified new and relapse cases is close to 4.4 million, while the case detectio , forms, is 85%. (Table 2.1) 12 Scanned with CamScannerBurden 2014, Western Pacific Region imates of TB Table 2.1. Estim: NUMBER (Thousands) RATE (per top Ty Morality (excludes 88 (81-95) 4.8 (4457) HIV+TB) 0.27 023-037 tality (HIV*TB only) 31) 700 (1,900-2,400) 116 TOL reve is (includes 7600 (1500-1600) 85 (80-89) HIV+TB) Incidence (HIV+TB only) Case detection, all forms 31 (28-35) 85 (61-90) NATIONAL PROFILE’ Tuberculosis is the sixth leading cause of morbidity and mortality in the Philippines; the country is ninth out of the 22 highest TB-burden countries in the world and has one of the highest burdens of multi-drug resistant TB®. In 2014, the estimated incidence of A23 TB including HIV positive TB excluding HIV with TB. The WI patients is 290,000 and TB deaths totaled 10,000 HO data of 2014 showed the Philippine TB incidence rate at 288/100,000 Population, while the prevalence rate is at 417/100,000 Population. The percentage of the incidence of TB cases that are HIV positive was at 2.6/100,000 population in 2014, (Table 2.2). i Table 2.2. Estimates of TB Burden 2014, Philippines Incidence (IVETE only Case det (%) tection, all forms 13 Scanned with CamScannerBIN CHILDREN Atleast half a million children become ill with TB each of children with TB have the disease in their lungs (pulm affected by TB disease in other parts of the body (orp ae more children aged <15 years of age accounted for 6.5% of nolified cases. An entingted 550,000 children became ill with TB and 80,000 chit i negative die year. Seventy to eighty percent and relapse cases among children were reported, an increase of about 30% n g e d, % compared with 2013. The largest increases were in India (about 30,000) an Philippi trout 10,000)" ( 000) and the Philippines DRUG RESISTANT TB‘? Resistance to drugs Poses a grave threat to the prevention, treatment and control of TB. Drug-resistant TB is extremely difficult and costly to treat. Persons with drug- resistant TB are more likely to die of TB than those who have drug-susceptible TB. The problem of drug resistance is man-made, which arises from noncompliance and improper use of anti-tuberculous drugs particularly in areas with poor TB control programs. Locally, there is limited data on drug resistance of MTB in children. Most of the culture and sensitivity studies on MTB are done on adult patients. Globally, there is an estimated 3.3% of new TB cases and 20% of previously-treated cases have MDR-TB. The number of cases detected (123,000) worldwide represented 41% of the global estimate. Detection gaps were worst in the Western Pacific Region where the number of cases detected was only 19% of the number of notified cases estimated to have MDR-TB. In the Philippines, MDRTB among all new TB cases was 2%, with 21% for retreatment cases. (Table 2.3). The estimate total of MDR-TB cases among notified pulmonary TB cases, both new and retreatment, is at 11,100 in 2014. There was also a noted increase in the total cases of Rifampicin Resistant/Multidrug Resistant Tuberculosis (RR MDRTB) from 17,241 cases in 2013 to 27,287 cases in 2014, There were 3,000 laboratory-confirmed RR-/MDR-TB cases and 2680 patients who were started on MDR -TB treatment in 2014 (Table 2.4). Table 2.3. Estimates of MDR-TB burden 2014, Philippines RETREATMENT 21 (16-29) % of TB cases with MDR-TB [ 2 (142.7) 4,600 (3,300-6,300) MDR-TB- cases among notified Pulmonary TB cases 14 Scanned with CamScannerag notified | 4,600 (3,300-6,30 =| D HIV CO-INFECTION*® i is a fatal combination. HIV infection contributes : on Mint of TB cases. With its ability to destroy the immu - is the most significant risk factor for the progression of dormant TB infe disease. HIV positive individuals are 20 to 30 times more likely to de those who do not have HIV. TB is the leading cause of death in adults every four people with HIV dying from TB. Globally, 51% of notified TB patients had a documented HIV result in estimated 12% of the 9.6 million people who developed TB in 2014 were figure highest in the African Tegion at 79%." Of the 1.5 million people killed, 0 are HIV+ with an estimated 190,000 TB deaths among HIV+ men, and 140, 5,000 (range: 50,000-60,000) TB of the total number of HIV+ death ional 8.4 million lives among HIV+ peo HIV+ women. There wel TB cases in 2014, an estimate of 53 %), were TB patients with known HIV ta 10 times (5,034) in 2013° tus, thus it ir a : (Table 2.5), 'S increasing the estimate to Scanned with CamScannerdroplet nuclei are generated when Persons who hi i i > lave pulmonary or lai | TB disease cough, sneeze, shout, sing or spit. De 7 ary ryngeal i it cs Pending o1 ir particles can paras Suspended in the air for severat , i“ nl ak Sionment, these tiny a person inhales the droplet nuclei Containing MTB, and the droplet el teva net mouth or nasal passages, upper respiratory tract, To rosa traverse the the lungs. and bronchi to reach the alveoli of The other modes of transmission are through ingestion of i i dairy products from infected cattle resulting in ‘boving Fe (cause te unpasteurized bovis); skin inoculation from contaminati 'y Mycobacterium k (on of an abrasion, which usuall pathologists and laboratory personnel (prosector's wart),? Fomites are hot important its transmission, thus special handling of utensils and bed linens are not necessary.° INFECTIOUSNESS OF TB PATIENTS* The infectiousness of a person with TB disease is directly relate: tubercle bacilli expelled into the air. A Person is considered relies where tubercle bacilli are expelled. Infectiousness of TB Patients also depends on a variety of factors: susceptibility (immune status) of the exposed individual, environmental factors that affect the concentration of M. tuberculosis organisms and the proximity, frequency, and duration of exposure to an infectious source. In general patients who have suspected or confirmed TB disease should be considered infectious if: 1. They are coughing, undergoing cough-inducing procedures, or have positive sputum ‘smear results for acid-fast bacilli (AFB), presence of pulmonary cavity; and 2. They are not receiving adequate anti-tuberculosis therapy, have just started therapy, or have a poor clinical or bacteriologic response to therapy. The risk of TB infection among child contacts is associated with the proximity and duration of contact and the degree of lung involvement and/or sputum smear positivity of the index case, which is usually from an infectious adolescent or adult.’ Childhood TB arises most often as a result of the inhalation of droplet nuclei approximately 1 to 5 microns in size containing M. tuberculosis bacilli expectorated by a sputum smear- Positive individual with pulmonary TB. TB infected infants and postpubertal adolescents are at increased risk for progression to TB disease, and children aged less than five years are at increased risk for disseminated disease. * RISK FACTORS FOR TB” TB disease may develop within weeks in some individuals infected with TB and who lave weak immune eystems. Other TB infected individuals may cevelor TB disease. years after exposure once their immune system is compromised. (vers , Lee 10% of infected persons who do not receive treatment for LTBI will deve lop es at some time in their lives. For persons whose immune systems are weal a Socal those with HIV infection and AIDS, the risk of developing TB disease is sigi ly higher than for persons with normal immune systems. 16 Scanned with CamScanneref 10N URE AN NE a | tion is dependent on «i. Risk OF EXPOS of acquiring infection is dependent on Sigtiticany The risk of exposure on extent of disease of nfectiousnons ot a su closeness OF, Cor ily or household member a Seti it oF UNFecogn usually an older farm 1B. Family and community c tituti ies 3S Overeroyg: untreated pan ae A similar risk holds for institutionalizeq Children and im Se rare prisons, homes for the elderly and shelters fo Street chy th in hospitals, , RISK OF PROGRESSION FROM INFECTION TO ACTIVE DISEASE ISK is i ie infection to active disease is determined b 5 ee Caen intial infection, nutritional status, intercurrent cae immunosuppression and other intrinsic host factors. Generally 3 to 4’ mt individuals progress to active TB during the first year after infection (tubereuin conversion); it occurs in 5 015%, thereafter, Persons at high risk for developing TB disease fall into two who have been recently infected with TB bacteria and t main categories ~ those conditions that weaken the immune system. hose who have Medical Persons who have been recently infected with TB bacteria * Close contacts of a person with infectious TB disease * Persons who have Migrated from areas of the world with high rates of TB * Children less than 5 years of age who have a Positive Tuberculin Skin Test (TST) * Groups with high rates of TB transmission, such as homeless persons, injection drug users, and Persons with HIV infection * Persons who work or reside with people who are at high risk for TB in facilities or institutions Such as hospitals, homeless shelters, correctional facilities, nursing lomes, and residential homes for those with HIV : ; Conditions that weaken the immune system B young children often have weak immune systems. Other people ca? Hiviaties cee Systems, too, specially people with any of these conditions: Silicosis Diabetes Mellitus Severe kidney qi y disea: Undemutrition . Organ transplants alignancies ™Munosuppregg; Pression, e.g, by Corticosteroids, anti-neoplastics, etc. 17 Scanned with CamScanneriousness appears to decline rapidly Patients with drug-resistant TB diseace fremain infectious for weeks or even months. may remain infectious until they receive adequate teams initial drug regimen and Addendum: For the 2016 Global TB Report just released 1 obe 13 October 2016, the full report can b obtained from this link: htto:wwwwho.intitb/pblications/global_eportien! : REFERENCES 1. World Health Organization. Global Tuberculosis Report 2015 z% http://www. who .int/tb/publication/2015/factsheet_tb_2015 update Ne 2015.pdf lovember 3. World Health Organization_HQ_Reports TB Country Profile 4. Centers for Disease Control and Prevention, National Center for HIV, STD and TB Prevention Division of Tuberculosis Elimination. Core Curriculum on Tuberculosis, What the Clinician Should Know. 6" ed, 2013 5. Frampton MW. An outbreak of tuberculosis among hospital personnel caring for a Patient with a skin ulcer. Ann Intern Med. 1992;117:312. 6. Haas DW. Mycobacterial diseases. Philadelphia, Churchill Livingstone. 2000. p. 2576-2607. (GL Mandell, Douglas, JE Bennett, editors. Principles and Practice of Infectious Diseases. 5" ed.). 7. Grzybowski S$, Barnett GD, Styblo K. Contacts of cases of active pulmonary tuberculosis. Bull Int Union Tuberc. 1975;50:90-106. en f 8. Marks SM, Taylor Z, Qualls NL, et al. Outcomes of contact investigations of infectious tuberculosis patients. Am J Respir Crit Care Med. 2000;162:2033-8. i for transmission of 9. Kenyon TA, Creek T, Laserson K, et al. Risk factors fc Mycobacterium tuberculosis from HIV-infected tuberculosis patients, Botswana. Int J Tuberc Lung Dis. 2002;6:843-50. 10. Lienhardt C, Sillah J, Fielding K, et al. Risk factors for tuberculosis infection in 18 Scanned with CamScannera children in contact with infectious tuberculosis cases in The Gambia, West Ai Pediatrics. 2003;111:e608-14. cg, 11. American Academy of Pediatrics, Committee on Infectious Diseases, Tubercuy Elk Grove Village, IL: American Academy of Pediatrics. 2003, . 642-5 (Pickering LK, editor. 2003 Redbook: report of the Committee oj a Diseases. 26" ed.). Infectious 12. National Tuberculosis Controllers Association, Center for Diseas: ane ar : Contro}, Guidelines for the Investigation of Contacts of Persons with Infectious Ti Ig MMWR Weekly. 2005 December 16;54:RR-15. Ubereulosis, Scanned with CamScannero 3 Microbiology Jaime Santos, M.D Mary Antonette Madrid, M.D OUTLINE Overview Morphology and Taxonomy Physiology and Growth Characteristics Antigenic Structure and Determinants of Virulence Laboratory Diagnosis M. tuberculosis Genome Sequence Scanned with CamScannerOVERVIEW Mycobacterium tuberculosis (MTB), classified under the M. tuberculosis i agent that causes tuberculosis in humans. Tuberculosis continues to ieee the interest in the medical field as there are still a lot to be investigated on the dses g agent's virulence and new information derived from the Mycobacterium tuberculoss, genome can contribute to the development of therapeutic and preventive strategiog The availability of Xpert MTB/RIF assay, a real-time DNA-based test, as revolutionized TB case detection by providing early results. This chapter discusses the morphology, taxonomy, growth characteristics, virulence factors, laboratory diagnosis, and the MTB genome sequence. i MORPHOLOGY AND TAXONOMY Mycobacteria are aerobic, nonmotile, and slightly curved or straight bacilli. They retain carbolfuchsin dye when decolorized with acid-ethanol by the Ziehl-Neelsen method (acid-fastness). The organisms contain high-molecular-weight (60 to 90 carbons) mycolic acids in their cell walls.’ They are classified into: (a) M. tuberculosis complex; and (b) non-tuberculous mycobacteria previously referred to as atypical mycobacteria or mycobacteria other than tuberculosis (MOTT), which includes M. avium- intracellulare, M. kansasii and all other species. Mycobacterium tuberculosis complex (MTBC) includes species that cause tuberculosis in humans and other mammals. Though member species are very closely related genetically, not all have been given species status. The following are the named species of this complex: Mycobacterium tuberculosis (M. tuberculosis), M. bovis, M. caprae, M. africanum, M. microti, M. canetti, and M. pinnipedii.'°* Bacille Calmette-Guerin (BCG), which is used for vaccination, is presumed to be derived from M. bovis. BCG however, is now seen to have features that differentiate it from other strains of M. bovis.’ M.bovis causes disease in cattle, humans and other mammals, while M. tuberculosis causes disease only in humans. In addition to M. tuberculosis, humans are also the principal host of M.africanum and M, canetti, Goal, voles, and seals are the principal hosts of M. caprae, MV. microti and M. piniipedi, respectively.* The primary mode of transmission of M. bovis is ingestion of contaminated milk = meat. While M. bovis requires oxygen for growth, it is inhibited by the higher Lae required by M. tuberculosis. It is also intrinsically resistant to pyrazinamide. Over ae pasteurization, tuberculin testing of cattle, and killing of infected animals, considerably decreased the incidence of M. bovis.”” . sed The Runyon classification scheme groups non-tuberculous mycobacteria (NTM) be 0 i : i F t rate and pigmentation. This ‘scheme is becoming less important al a oe jrailability of new methods of rapid detection of mycobactent A eon growing or pigmented isolate of mycobacteria is not to be mistaken as M. tubercu! . sii ‘Among the important members of NTM are (1) M. avium complex (ac), M. Kans 21 Scanned with CamScanneri that can cause pulmonary disease; (2) 1, avium and M. Scrofulaceu and M. ert aphadenits Particularly in children, and, (3) MAC and iu, kansasii as can cane sigseminated disease among HIV patients. oad of the MTBC, NTMs have a wi Uni ide range of Pathogenicity and are n issible from one human to another. The NTMs are also more difficult to treat vans they are relatively drug resistant compared to ise becal M. tuberculosi raphically-relevant clonal distri Hla the SpolDB4database. °° Amore recent characterization of Mycobacterium tuberculosis diversity and molecular epidemiology utilizing the mycobacterial interspersed repetitive units—variable number of DNA tandem repeats (MIRU-VNTRs) method described database, the SITVITWEB. This databa another international c ise has genotyping information on more than 62,582 isolates from 153 Countries compared to only spoligotyping information on 39,295, isolates from 122 countries Contained in the previous version of the SpolDB4 data-base,'” bution of the MM. tuberculosis complex can PHYSIOLOGY AND GROWTH CHARACTERISTICS Mycobacteria are Obligate aerobes; generally, they have simple growth requirements. Many strains have surface growths and clumping characteristics in liquid, synthetic media. Tweens (nonionic detergents) are added to Promote dispersed growth. The growth rate of mycobacteria is slow but is enhanced by 6% to 8% carbon dioxide. Optimal temperature for the growth of M. tuberculosis is 35°C to 40°C. ANTIGENIC STRUCTURE AND DETERMINANTS OF VIRULENCE The specific structures, antigens, and mechanisms responsible for the virulence of M. tuberculosis are not known with certainty; but trehalose dimycolate (cord factor) and Sulfatides have been associated with the ability of virulent strains to produce disease. ig Wilto, cord factor is fesponsible for the morphologic appearance of M. tuberculosis ce > Setpentine Cords of bacilli close, parallel arrangements. However, its role a nan rele S enknown. Studies in mice demonstrated iio bes strong inducers of cytokine acai 28, Well as neutrophil recruitment eae are perphoraly Ibeatod pestis that inhibit fusion of secondary lysosomes Sih teat ne Osomes withi i oting intracellu organisme within a macrophage, possibly promoting Upoarabinoman i he mycobacterial cell walll, is a ; Wan (LAM), an important molecule in the my‘ LAM). variant Ominant mycobacteri figen.* Man-LAM (mannan-capped bresent in Myeobactetial_ antigen." The a fival and has] one shown to be responsible for enhanced bacilli survivs entry into Macrophages. 22 Scanned with CamScannerLABORATORY DIAGNOSIS TB bacilli have a slow doubling time of 18 to 24 hours.”Although the newly develop, techniques like PCR give earlier results, demonstration of the organism by Cute still considered the gold standard for the diagnosis of TB. SPECIMEN Specimens include pulmonary secretions, urine, genital discharge, CSF, blood, peur fluid, and other biologic fluids. Demonstration of the bacilli in children is limited by the fact that childhood TB (primary tuberculosis) is often paucibacillary and that very young children cannot raise any sputum. For these reasons, a gastric aspirate specimen assumes greater importance for children compared to adults. (For collection and handling of specimens see chapter 8 on diagnostic tests) The quality of specimens submitted for microbial investigation affects the performance of the laboratory test. Specimens should reflect the site of infection and preferably collected aseptically.’ Microscopy Acid-fast staining techniques: a. The ZN technique (or the hot method) uses carbol fuchsin, which is taken up by the cell even after treatment with acid alcohol. Mycobacteria will appear as red rods against a blue background. Heat is applied in this method. b. Kinyoun or cold stain is a modification of the ZN method and does not involve application of heat. It uses a higher concentration of phenol with carbol fuchsin. Mycobacteria appear as above. c. Auramine-O stain makes use of auramine instead of carbol fuchsin. TB bacilli are seen fluorescing under the fluorescence microscope. The use of fluorescent stain increases sensitivity of microscopy."* CULTURE Non-sterile specimens contaminated with commensal organisms _underg0 decontamination prior to inoculation onto culture media. This is achieved through the use of either NaOH method or Nalc-NaOH method. '® Culture Media’® The following are the suggested media for cultivation of Mycobacteria fro specimens: i 1. Solid Media is either egg-based or agar-based. Among the egg-based medi, the most popular 1 + Agar-based: Middlebrook 7H10, Middlebrook 7H11, Mitchison's selective 71". 2 Egg-based: Wallenstein, Lowenstein-Jensen (LJ) with RNA, LJ with py acid; Ogawa’ m clinical Luis 23 Scanned with CamScannererr 415 iquid Media i 2, Ligue rook 7H9 brott . vi een Albumin broth ' : Commercial liquid-based mycobacterial culture systems: ol * 5 BACTEC 460 TB BACTEC Mycobacterial Growth Indicator Tube (MGIT) 960 system 9 VersaTREK (ESP I!) culture system MB/BacT system oo Of these culture media, the currently available ones in some local laboratories are Lowenstein-Jensen, Middlebrook, Ogawa and BACTEC. Culture Method 1. Conventional Conventional culture of mycobacteria involves inoculation on solid media. If this is the only method used, inoculation of both the egg-based and agar-based media is recommended. For specimens such as sputum that are contaminated with normal bacterial flora, a selective medium containing antimicrobial agents, such as Mitchison's selective 7H11 agar, should also be inoculated.’ Conventional culture media should be examined for growth twice a week, for the first four weeks, starting from day 3 to 5 postinoculation, and thereafter, once a week until the eighth week. Positive cultures should be identified to the level of species using either biochemical or molecular methods. ® The major advantage of conventional culture is that it allows visualization of colony Morphology and pigmentation. Disadvantages of conventional solid media are Prolonged time of growth of mycobacteria (3 to 4weeks) and low sensitivity."°"""* This 's because the doubling time (every 12-24 hours) of M. tuberculosis is extremely slow Compared to most bacteria. © While solid media are still being used by most centers, the use of liquid media such as Middlebrook 7H of 9 and Dubos Tween broths offers the advantage of more rapid growth mycobacteria as well as a higher isolation rate, than on solid media. 2 BACTEC TB System and other commercial iquid-based culture systems _ The Most widely used system is the semi-automated BACTEC TB system ee labelen 2 (one for blood and another for all other specimen types) that conta labeled palmitic acid substrate. Each specimen (5.0 mL of blood or 0.5 Mio acy 4 Seeen t¥Pes) is inoculated into one vial and an antibiotic, mixture fs BTR. og supe eRs other than blood. Bacilli multiply in the broth and utllze tne ee gt COr bstrate, releasing CO, info the head space above the broth. «aa, greater thocasured by the BACTEC 460, which calculates the growth index than ten suge : acteria are Sub-cultured i ining mycobs jests that mycobacteria are present. Vials containing qe eTEC ‘identification are done. to a solid medium, and direct tests for identification 24 Scanned with CamScanner460 (Beckton Dickinson) was the first commercial liquid-based mycobacteria) ria system used. It has been considered a standard against which m Cut were compared.’ ost other Syste There are advantages to the BACTEC TB system: rapid detection of growth (5 fi by days); increased sensitivity compared with solid media; the ability to distingy from other mycobacterial species; and rapid susceptibility testing of fee. MTB tuberculosis. The major disadvantage of the BACTEC system is its radioactiy Ad disposal of which is expensive and may be problematic for some institut a an However, there are now non-radiometric BACTEC systems, which provide a i alternative to the original radiometric BACTEC. ® @ Viable Other available commercial liquid-based mycobacterial culture systems. ing BACTEC MGIT 960 culture system, Versa TREK culture system, and MB/BacT ca BACTEC MGIT is a system that uses a modified 7H11 broth containing an indica that fluoresces under UV light. As oxygen in the medium is consumed, the indicator wit fluoresce. Fluorescence is detected either manually using a Wood's lamp, or automatically and continuously if BACTEC 960 instrument is available. MB/BacT uses a colorimetric sensor that changes color as increased amounts of COz produced by the growing organism is detected. VersaTREK system monitors gas-related pressure changes that occurs during bacterial multiplication. This was previously marketed as ESP Il culture system. * DNA FINGERPRINTING Analysis of the M. tuberculosis genome is made throu: uses the restriction fragment-length polymorphism (RFLP) \ differentiates M. tuberculosis from other members of the MTBC and other atypical isolates; it also permits the identification of patterns of spread of a particular M. tuberculosis strain among individuals, which makes it possible to obtain the source infection, track down changes in antimicrobial susceptibility and allows _ differentiation between human and bovine isolates.°"*'° This technique utilizes restriction enzymes or endonucleases that cut DNA at specific "recognition" sites = which, in tum, results in short segments or restriction fragments of DNA with leng! > that vary according to the particular enzyme used. The DNA fingerprints with are lengths and specific nucleotide sequences can be compared, in a map-like br te with the DNA pattems of M. tuberculosis organisms from other, individuals. patterns are the same, they suggest a common source of exposure." gh DNA fingerprinting, which ) method. DNA fingerprinting NucLeic Acip AMPLIFICATION TEST (NAAT) Jeic acid probe, M. tuberculosis-specific nucleic acid s De direct differences between mycobacterial DNA are amplified, allowing TBC organisms in clinical specimens. NAATs can also distinguish Tray itis identify NTM, and has a role in rapid detection of drug resi — the conventional laboratory approached its. Limitations to its use includ os eee Scanned with CamScanner equences located OF Using a nucl regions of detection of organisms, can regarded as a complementary tool to tl diagnosis, giving accurate and rapid resul 25nsitvity in smear-negative and non-respiratory samples, and cost2! se : ‘Acommonly used NAAT is PCR. XperT MTBIRIF ASSAY This is a new rapid diagnostic tool that 0 toc t simultaneously detects the tuberculosis complex and tifampicin resistance, giving fesults in a: from test run. It is a nucleic acid amplification test (developed by Cepheid, Sunnyvale CA, US.). §; amplification and detection of target se Presence of M, e iS early as 2 hours using the Gene Xpert platform ample Purification, nucleic acid quence are combined in a single Xpert MTB/RIF b rtridge and loaded in the instrument. Following sample loading, all Succeeding steps are c e fully automated. There is thus very minimal operator training needed. In addition to this, safety concerns on handling i ized because the assay's reagent is tuberculocidal. lake the test suitable for use outside a central or reference laboratory. The instrument, however, will Need an uninterrupted, stable electrical supply, temperature control and yearly calibration,22 2% Rifampicin resistance can been used to represent multidrug resistance since rifampicin Monoresistance is uncommon,* and most isolates that are resistant to rifampicin are also resistant to INH. The use of molecular methods for rapid detection of rifampicin is feasible since >95% of resistance is associated with mutations in a limited region of the M. tuberculosis "poB gene (the gene that encodes the beta Subunit of RNA Polymeraseto which rifampicin binds under wild-type conditions), while INH resistance involves at least four different genes and would be more difficult to detect." Xpert MTB/ IF makes use of molecular beacon technology to identify rifampicin are Molecular beacons are nucleic acid hybridization probes that bind to oe Sequences in regions (such as the rpoB gene) where there are resistance an can '€ probes will fluoresce when bound to a target. Presence of mutation 2 ion ; fluorescence." Xpert MTB/RIF Sesay amplifies MIBC: specific eequenes af ‘recion Gere. then mutations are searched for in the rifampicin registance determining (RROR) using 5 molecular beacon probes (Probes A to E). ee The test will determine if MTBC was detected or not detected in ne owes detected, it Ui eS, 2180 possible for which the test should be repeated. If MTEC was oerempicin Will ‘also indicate the following: 1) Rifampicin resistance oan te. Detailed steps fesistance not detected, or, 3) Rifampicin resistance indeterminate 'MvVolved in the test are discussed in a number of web sources. MTBRIF_is being . rt L Based on WHO's Current Policies and Guidance, ee of MOR-TB of ite fecommended as an initial diagnostic test in suspect -negative specimens. ons: &ssociated TB, as well as a follow-on test for ant to the following ie ‘de and peommendation on the use of Xpert MTBIRIF apply ts CSF, lymp! V pirate, tool, blood Processed or unprocessed sputum, gastric lavage er utlly ee gine’ tissues, There are stil inadequate data on t and urine specimens? 26 Scanned with CamScannerConventional culture methods for TB detection take around 2-6 weeks before Growth ig detected and an additional 3 weeks waiting time is needed for drug resistance results to become available. The availability of Xpert MTBIRIF assay sets an important milestone in TB diagnosis and control as it offers a rapid means of detecting MTBC ang drug resistance. Unnecessary regimens are avoided, appropriate drugs are Provided, and infection control is timely implemented. For more information on the use of this test for TB diagnosis, please refer to Chapter 7, M. TUBERCULOSIS GENOME SEQUENCE Two strains of M. tuberculosis have been fully sequenced to date: the CDC-1551 (the so-called "Oshkosh" strain) isolated from a male working ina children's clothing factory, which is highly virulent in mice but has not caused widespread epidemics in man;”’ and H37Ry, first isolated in 1905 but which has retained its virulence over the years in animal models, is drug-susceptible, and can be genetically manipulated.” The genome is a circular chromosome with 4.4 million base pairs (surpassed in size only by E. coli among the bacterial genomes completed to date), 4000 genes, and a high G+ C content (65%). A large part of its coding capacity is devoted to enzymes involved in lipogenesis and lipolysis (explaining the large amount of lipids in the mycobacterial cell wall) and to two new families of glycine-rich proteins. Updated gene-based information on the H37RV strain is presented in TubecuList, a relational database which can be accessed at http://tuberculist,epfl.ch/. In their 2013 release, over 700 protein coding sequences have been updated.” Comparing isolates from clinical specimens shows remarkable genome sequence conservation but these novel proteins may be a significant source of antigenic variation among strains. There are four copies of the previously known mce gene which encodes a macrophage-colonizing factor. New mobile genetic elements (insertion sequences) have also been found. Sequencing of whole genomes of the other members of the M.tuberculosis complex, including that for M. avium subspecies paratuberculosis” M. smegmatis and M. bovis, has been described. The members of the M. tuberculosis complex has increased the numbers of insertion sequence elements, which might explain higher intra-species variability in M, tuberculosis compared to other species of mycobacteria.” Although aerobic, M. tuberculosis als0 has pathways associated with anaerobic metabolism which may be responsible for long-term persistence in tissues. Knowledge gained from the M. tuberculosis genome completion will elucidate pathways of cell wall synthesis and bacterial metabolism—these can become potential drug targets in the future. It can also add to our understanding of how drug resistance occurs and can contribute to both future vaccine design and investigational attempts at immunomodulation. 27 Scanned with CamScannerREFERENCES , Gail. Mycobacteria. In: Henry's clinical diagnosis and Management 1. Wontay methods. 22nd ed._/ [edited be} Richard’. McPherson, Matthew 2 Pincus. Saunders Elsevier Ltd. PA. 2011, p. 1145, : n Baron S. Mycobacteria and Nocardia. In Medical Microbiology. 4 ed. UTMC; 1996. . Grange JM, Yates MD, de Kantor IN. ¢ Guidelines for Specification within the Mycobacterium tuberculosis complex. WHO/EMC/ZO00/96 4. . Grange, GM. The genus Mycobacterium and Mycobacterium tuberculosis complex. \n: Tuberculosis A Comprehensive Clinical Reference. Zumla A.|. and Schaaf HS. eds. Saunders Elsevier Ltd., Oxford, UK, 200: 9, pp. 44-59. . Grange JM. Pathogenesis of tuberculosis: pathway to apical localization. Tubercle and Lung Disease. 1995;76:276-277, . Baron EJ, et. al. Tuberculosis. In: Bailey and Scott's Diagnostic Microbiology. 9" ed. p. 590-633, a 2 7. Mycobacterium. In: Medical Microbiology 7" ed. Murray PR, Rosenthal KS, Pfaller MA. eds. Saunders, Elsevier Inc. Philadelphia, PA. 2013, Pp. 235-247 2 Daley CL, Heifets L. Other mycobacteria causing human disease. In: Tuberculosis A Comprehensive Clinical Refere: nce. Zumla A.I. and Schaaf H.S. eds. Saunders Elsevier Ltd., Oxford, UK, 2009, pp.60-74. SpolDB4database Web link: http:/Awww.pasteur-quadeloupe fr/tb/bd_myco.html or hitp:/iwww.pasteur-guadeloupe fr:B081/SITVITDeme 10.Brudley et al. Mycobacterium tuberculosis complex genetic diversity: mining the fourth intermationat spoligotyping database (SpolDB4) for classification, population Genetics and epidemiology. BMC Microbiol 2006 Mar 6:6 (1): 23. 11. Demay et al. A Publicly available international multimarker database for studying Mycobacteriu ft t m tuberculosis genetic diversity and molecular epidemiology. Infect ‘enet Evol. vol 12, issue 4, 2012 June. 2 12, hitp:iwn.pasteur-quadeloupe f1:8081/SITVIT_ONLINE _ ei II lipids. 18. Geisel RE, Sakamoto K, Russell DG, etal. In ive sci of (eae Or yohakise ot Mycobacterium bovis bacillus Calmette-Gue'tin is due princip ycolates. J Immunol 2005;174:5007-5015. 14. Kan se receptor 9 PB, Azad AK, Torrelles JB, et al. The human macrophage mannose recsrt ltects “Mycobacterium tuberculosis lipoarabinomannan- biogenesis. J Exptl Med 2008:202:087-999. 28 Scanned with CamScanner18.Whitelaw AC and Sturm WA. Microbiological testing for Mycobac. tuberculosis. In: Tuberculosis A Comprehensive Clinical Reference, Zumig Are Schaaf H.S. eds. Saunders Elsevier Ltd., Oxford, UK, 2009, pp. 169-17, any 16. Palomino JC, Ledo SC, Ritacco V. Tuberculosis 2007: From basic sch patient care. sed. Available from: www. TuberculosisTextbook com” SW" 17. Luquin M, et. al. Comparison of a biphasic non-radiometric system with Lowens; Jensen and Bactec-460 system for recovery of mycobacteria from ct specimens. Tubercle and Lung Disease. 1996;77:449-453, 18. Behr H, Small PM. Molecular fingerprinting of Mycobacterium tuberculosis. can it help the clinician? Clinical Infectious Diseases. 1997;25:806-810, How 19. Nogueira JM, et. al. Correspondence: Molecular approach to identifying Toute of, tuberculosis transmission in a village. The Int. Journal of Tuberculosis and Lung Diseases. 2000;4(1):91-93. 20. Rigouts L, et. al., Use of DNA restriction fragment typing in the differentiation of M, tuberculosis complex isolates from animals and humans in Burundi. Tubercle and Lung Diseases. 1996;77:264-268. 21. Daniel Brodie and Neil W Schluger. Nucleic acid amplification for detection of Mycobacterium tuberculosis. In: Tuberculosis A Comprehensive Clinical Reference, Schaaf and Zumla (Eds). pp 197-204 22.CDC. A new diagnostic tool to diagnose TB. The Xpert MTBIRIF Assay. Downloaded from: http:/ww.cde.gov/tb/publications/factsheets/pdf/xpertmtb- rifassayfactsheet_final.pdf 23.World Health Organization. Automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF assay for the diagnosis of pulmonary and extrapulmonary TB in adults and children, Policy update. 24. Cruz AT, Starke J. Tuberculosis. In: Feign and Cherry's Textbook of Pedic Infectious Diseases. Feign RD, Cherry JD, Demmler-Harrison GJ and Kaplan SL (eds). Philadelphia USA Saunders Elsevier, 7" ed, 2014, 1335-1380 25. Xpert MTBIRIF” package insert. Cephaid, Sunnyvale CA, USA. Revised Febru) 2015. Downloaded from: _ http://www. cepheid.com/administrator/componen! com_productcatalog/library-files/2cc1 15cd6ba267207c04661a3d0fefc8-Xper MIE: RIF-ENGLISH-Package-Insert-301-1404-Rev-B-February-2015.pdf 26. http://www. finddiagnostics.ora/programs/tb/find_activities/automated_naat.html 27.Genome sequence and analysis. The Institute for Genomic Research. Available from: www.tigr.com 29 Scanned with CamScanner98, Cole ST, Brosch R, et. al. Deciphering the biology of i lycobacterium tub is from the complete genome sequence. Nature, 1998;393;537-544, so JM, Kapopoulou A, Jones LM, Cole ST, TubercuList—10 ears afi 29. Hpeculosis (Edinb). 2011 Jan:4( 11-7. | 30. TubercuList - Mycobacterium tuberculosis Database: tuberculist.epfl.ch/ 31.Li L, Bannantine JP, Zhang Q, et. al. The complete genome Sequence of Mycobacterium avium subspecies paratuberculosis. Proc Natl Acad Sci (USA). 2005;102:12344-9. 32. Maari PR, Bannantine JP, Golding GB. Comparative genomics of metabolic pathways in Mycobacterium species: gene duplication, gene decay and lateral gene transfer. FEMS Microbiol Rev. 2006;30:906-2! 33. Young DB. Blueprint for the white plague. Nature, 1998;393:515-516. 30 Scanned with CamScanner4 Immune Responses Jaime Santos, M.D Mary Antonette Madrid, M.D OUTLINE Overview The Role of Macrophages The Role of T-lymphocytes Role of Genetic Determinants Immune Response and Course of TB Infection Scanned with CamScannerOVERVIEW / lin reactor and a patient with severe miliary tube, loi matic tubercul of the spectrum of TB infection and disease—oy (Tay represen Ge eee poresel to M. tuberculosis. A relatively goss inn efiecive aind po patient with TB pleuritis (as evidenced by few baci in pun’ response is eat disease ‘without treatment and preserved tuberculin skin pl fluid, Ce a relatively poor immune response leads to advanced, Progress personales While immune responses do not entirely account for the clinica, einer TB infection as it does in leprosy, an enhanced understanding Of the immunology of TB coupled with the knowledge Gained of the i tuberculosis ganar? cou sghficany lead to breakthroughs in vaccine development ang B immunotherapy." there are differences between murine models of TB and human TB disease, teoeennae agreement and experimental support for the following: Macrophages and T-lymphocytes are the Primary defenses against TB; antibodies and B lymph 8 have little role, if any, in Protecting one against TB; and the role of neutrophils and natural killer (NK) cells is unclear. An asympto THE ROLE OF MACROPHAGES*5 Puacocytosis AND KILLING The initial encounter of TB bacilli with macrophages in the lungs may be the most important contributor to subsequent events, While intracellular killing follows phagocytosis, M. tuberculosis can evsus Such Killing mechanisms. Among their survival Strategies a tr : (1) prevention of acidification of phagosomes (where TB bacilli are found within Macrophages); (2) Neutralization of the effects of reactive oxygen intarmedieies by mycobacterial cell wall components; (3) inhibition of plasma membrane repair,” ant () probable inhiotion cf Phagosome-lysosomal fusion through secretion of SapM (edd p osphatase) » PknG® and glycosylated Phosphotadyl-inositol Loreen ir hat can aan, Man-LAM) Mant at 2 cell wall component of M. tubereloss ee ae as a mit tic of mammalian Phosphatidylinositols and can block the the mater atovROStO! 3-kinase "ang a seeYmalic, Product PI3-P required for the B baci caf SgCSOMES ino phagaiyscns AME, Itis also likely, but not proven a mechanisms C2202. the, hopes from the phagosome. Among the Ki S of macrophages, the role of nitric oxide is very likely, ANTIGEN PRESENTATION AND CYTOKINE Propuction TB bacilli can also induc ‘ ind transforming growth factor bese Phages to produce a vari se include Tesponses, and int iety of cytokines; these inet lerleukin-12 (IL-9) vy interieukin-6 (IL-6), which can suppress T (IL-12), which can enhance T-helper responses- 32 Scanned with CamScannerProf mycobacteria throu ss OBEN? Hougardy and co nisms : IFN-gamma responses to pro ether aes mechanism of M. tubercul ANTIGENS il i ji i by live, i munity against TB can be induced y In 02 nile espe hypersensitivity (En) a be ind by anyoob organi by cell wall components. is_ phenomenot s scion tervereatn reaction are dissociable; ees secreted aad a ive 4 structural antigens, and that the way the antigen i ee a4 pete cudal for en Among the prime candidates of pI San aa ESAT-6,"° and the 30 kDa a or 85 B antigen.* Another protective anti hat has come to prominence is heparin-binding hemagglutinin (HBHA), mycobacterial antigen that can induce mycobacterial aggregation. Presence antibodies against HBHA (anti-HBHA antibodies) inhibits hemagglutina mycobacterial aggregation, as well as attachment to epithelial cells. "° HBHA has been shown to stimulate secretion of high levels of IFN-y by peripheral bl lymphocytes among subjects with latent TB infection. '7"® THE ROLE OF T-LYMPHOCYTES T-CELL-DERIVED CYTOKINES" In animal Studies, CD4 T-cell( T-helper cell at transfer resulted in ths e oe tion resulted in severe TB and higher levels of mycobacteremia. These dé the key role of CD4 T-cells in antimycobacterial immunity. CD4 T-cells show varied cytokine production with a subset (termed Th1) pr “ntegengamma (IFN-y), tumor necrosis factor-alpha (TNF-a), and IL~ many nen IFN-y is known to enhance macrophage intracel tuberculosis oa Pathogens. Its role in macrophage intracellular Cells—calleg demonstrated in mice. At the other extreme, a Ve a ce cytokines important in humoral immi Protective role in TB. Diminished Tht respon: Mononuclear cells in pulmonary TB patients ; high levels of Th1 cytokines have }) depletion resulted in severe TB, while le protection of recipients. In humans, HIV infection Scanned with CamScannerit ts which is important in-vj, ah || pattern of cytokine effect va likely that it ii feetions € a measles, immunosuppressants and others) which pe ty agents (eid significantly affect protection against M. tuberculosis or Could even in alal g clinical presentation. iat reulin skin test (delayed-type hypersensitivity) are Cells LEE ately Th1 cytokines. DTH response is associated with, but nat Peal i cell-mediated immunity. The reasons may be: (1) DTH responses are a function of cD4T cells, whereas protective immunity involves CD8 T-cells (cytotoxic 7. lymphocytes) and unconventional T-cell subsets as well; (2) PPD does not Contain ay the antigens derived from M. tuberculosis; and (3) in TB patients who have granuloma formation but have negative tuberculin test, T-cells may be trapped in granulomas reflecting their accumulation at the site of defense. Arecognized subset of T-helper cells, called Th17 cells, were found to be an important modulator of inflammation and CD4+ T-cell recall or memory response. Thi7 cells secrete IL-17, can recruit neutrophils, monocytes and CD4+ T-cells producing IFN- y, and stimulate chemokine expression. “2 Anergy, indicated by a negative tuberculin test, reflects suppression of DTH from Processes which interfere with Th1 response /.e. malnutrition, HIV infection, steroid therapy and severe tuberculosis itself as a result of production of inhibitory cytokines like TGF-B, Anergy is often transient and reverts to normal when underlying causes are resolved.*3 The possibility of modify Tesponse to a direction ing deleterious immune responses or shunting the immune Tesearch on cytokines ani favorable to the patient is among the driving forces behind dd tuberculosis, ‘ Cytotytic or Cytotoxic T- Cytolytic oF cytotoxic T-cells kil ing i including ee Ues These cals ciher exhiit were ge neacellular pathogens including LYMPHOCYTES likewise been shown to bi b 2 Cy The relative Contribution of -ytotoxic ostly through apoptosis.?"° CD8 T-cellshave ic ag 4 is. a is unknown but is exper jainst M. bovis-infected macrophages in-vitro. cytolytic T-celisto the t i ive ii ine response tO cled to be signing? (tal Protective immu ROLE OF GENETIC DETERMINANTS A BCG resistanc: ) is associated marsspGene (BCS) linked with the molecule Nramp (natural resistance Protection against S. 19 protein) has been Characterized in mice associated with mice against virulent Muu and L. conc a However, evidence that it protects . tuberculosis ig equivocal at best.” Likewise, a human 34 Scanned with CamScannere to Nramp has been found on chromosome 2. There is evidence that variant oo he Nramp1 gene are associated with increased replication of mycobacterial oer than susceptibility to TB. « amiologic data from twin studies, however, indicate that Susceptibility has ti epider and may be related to the largely unexplored relation between pistocompatibility leukocyte antigen (HLA) complex and TB. Si c xX a tudies on the completed human genome sequence could give leads in predicting human susceptibility genes. IMMUNE RESPONSE AND COURSE OF TB INFECTION® if not removed by mucociliary defenses, TB bacilli enter the alveoli where they encounter alveolar macrophages; there may be unknown genetic determinants making the macrophages more permissive to the growth of these bacilli. TB bacilli can also evade intracellular killing mechanisms and get carried to lymph nodes and other organs. In the lungs, macrophages may introduce antigen to T-cells resulting in its expansion, including the development of memory T-cells of which DTH cells may be a subset. The result may be tuberculin-positive persistence and/or long-term protection. The inflammatory Th1 responses elicited may result to local pulmonary infiltrates and hilar adenopathy-the picture of primary complex—resulting in containment. Other children may get on such a response but sometimes fail to contain the bacilli, thus resulting to a Progressive disease: this could be a consequence of the imbalance between proinflammatory and immunosuppressive effects. Still, other children may fail to mount an effective Th1 response, intrinsically or through immunosuppression (even a heavy bacterial load may cause immunosuppressive cytokines to predominate), and develop miliary TB, which is characterized by poor inflammatory and DTH responses and which Often occurs among the very young who may have inadequate immune responses. The development of progressive disease occurs in about 10% of affected individuals. Even after adequate treatment, some bacilli persist in a dormant state, which is known as latent TB infection—a state where the individual is. clinically aaymplomate Reactivation of dormant bacilli leads to active disease.”* Factors that promote development and maintenance of latency include: low concentrations of oO) futrients in chronic granulomas; and local production of TNF-a and nitric oxic ee - Animal studies show TNF-a, interferon-y, and NO significantly maintain infot the latent state.312 : In case: HIV inf 15% ated immunity (e.g. with S where infection is contained, depression of cell-mediated Immunity (8. Hi ection, malnutrition, disease or drugs) may lead to reactivarer ss Of cases occurs at extrapulmonary sites without apparent lung IMMUNE RESPONSE IN THE VERY YOUNG: There is relati one year are Of developing a serious, Of young children are comparatively weaker allowing for more i se under age ive immaturity of the immune system of young children. tnereased risk Particularly susceptible to develop tuberculosis, faptive responses disseminated dicease, particularly TBM. Innate and adapte fr 35 Scanned with CamScanner