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Tuberculosis: From Basic Research to Patient Care

Tuberculosis: From Basic Research to Patient Care. Moses L. Joloba. Mycobacterium tuberculosis. Mycobacterium tuberculosis (MTB), is the causative agent of tuberculosis (TB) 1.7 billion people infected with MTB worldwide The majority have latent infection

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Tuberculosis: From Basic Research to Patient Care

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  1. Tuberculosis: From Basic Research to Patient Care Moses L. Joloba

  2. Mycobacterium tuberculosis • Mycobacterium tuberculosis (MTB), is the causative agent of tuberculosis (TB) • 1.7 billion people infected with MTB worldwide • The majority have latent infection • Each year, 9 million develop disease • 2 million die of TB annually

  3. 3.0 2.0 Millions of deaths in 2002 1.0 0.0 HIV/AIDS Tuberculosis Malaria Measles TB is the single largest cause of death from a curable and preventable infectious disease

  4. TB in Uganda • Uganda is one of the 22 countries most affected by TB in the world. • In Uganda we detect 40,000 cases per year • This represents 50% of the cases to be detected. • About 70% of those detected are cured • WHO recommends 70% detection rate and 85% cure rates

  5. Advances in TB diagnostics or Uganda • Presented under the following areas: • A – Diagnosis • B – Identification • C – Drug susceptibility testing (DST) • D – Epidemiology

  6. Diagnosis Previous (< 2006) Current • Light microscopy • Solid culture (LJ and Middlebrook) • Semi automated Liquid culture Bactec 460 in JCRC • Florescent Microscope • Automated Liquid culture system (MGIT) • Portable Liquid culture system (Manual MGIT) • Direct phenotypic DST methods • HAIN Test for MDR detection • HAIN Test for speciation • PCR for Identification • Capillia for Identification • RFLP, Spoligotyping, RD analysis and MIRU for epidemiology

  7. Microscopy

  8. TB diagnostic testing in endemic countries Fundamental diagnostic: 2007 Fundamental diagnostic: 1882

  9. TB diagnostic testing in endemic countries Fundamental diagnostic: 2008 Fundamental diagnostic: 1882

  10. CULTURE

  11. Recent advances in microscopy in E. Africa • Binocular microscopes – covers 80% of the country in Uganda • Florescent microscopes (LEDs) for very busy centers • More sensitive than light X-scopes • Decrease workload – scan fewer fields • LEDs do not need dark rooms and have long life bulbs • Introduction of External Quality Assurance (EQA), covers 90% of the country (Uganda) – ensures quality (2005) • Change in diagnostic policy (2008) • 2 smears instead of 3 • One positive smear instead of 2 enough to start therapy

  12. Current TB culture labs in Uganda • NTRL – Wandegeya • FIND – Wandegeya • JCRC - Mengo • MRC- Entebbe • AERAS – Medical School • MSF – Epicentre - Mbarara

  13. Culture methods • Solid – LJ… • Liquid culture • Automated MGIT 960 • Manual • MGIT • 3. Bactec 9000 series for fluids • Solid – LJ… • Liquid Bactec 460

  14. Identification Before 2006 Current • Presumptive (accurate for solids) • Colony morphology • Growth rate • ZN • NAP for Bactec 460 • Presumptive for solid –phasing out • PCR for IS6110 • Capillia – key for liquid system

  15. Drug susceptibility testing (DST) Before 2006 • Indirect proportion by LJ and Bactec 460 TAT 3 weeks TAT 3 month

  16. PCR Hybridization Detection Rapid DST (Current) 1. Genotype MTBDR – Detects MDR TB in one day 3. Nitrate reductase assay – 14d 4. MODS assay – 10d 2. Drect MGIT – 5d

  17. www.modsperu.org

  18. TOTAL HANDS-ON TIME = 2 MINUTES

  19. GeneXpert The system is the first to fully automate and integrate all the steps required for PCR-based DNA testing: sample preparation, DNA amplification and detection. Designed to simplify hands-on preparation, the system provides PCR test results from a raw sample in about an hour, enabling time-critical DNA tests at the point of need.

  20. MTB Speciation • Previously none • Now using PCR RD analysis we can speciate MTB complex into • M. tuberculosis • M. africanun • M. bovis • M. bovis BCG • M. canneti • M. microti • M. pimpinedi • …… • Asiimwe BB, Koivula T,Kallenius G, Huard RC, Ghebremichael S, Asiimwe J, Joloba ML. Mycobacterium tuberculosis Uganda Genotype is the predominant cause of TB in Kampala, Uganda. Int J Tuberc Lung Dis2008; 12(4): 386 - 389.

  21. MOTT speciation Through use of PCR based GenoType Mycobacterium CM from HAIN we can speciate MOTT into 14 species (2008) • M. avium spp. • M. chelonae • M. abscessus • M. fortiutum • M. gordonae • M. intracellulare • M. scrofulaceum • M. interjectum • M. Kansasii • M. malmoense • M. peregrinum • M. marinum/M. ulcerans • M. tuberculosiscomplex • M. xenopi.

  22. Molecular epidemiology of TB • RFLP • Spoligotyping – PCR Based • MIRU – PCR based

  23. RFLP with 50% clustering Benon B.Asiimwe,1,2Moses L. Joloba2,Solomon Ghebremichael1,3, Tuija Koivula4,David P. Kateete2, Fred K. Ashaba2, Alexander Pennhag3, Ramona Petersson3, and GunillaKallenius*,1,3­. DNA Restriction Fragment Length Polymorphism Analysis of Mycobacterium tuberculosis Isolates from HIV-Seropositive and HIV-seronegative Patients in Kampala, Uganda. (Submitted to Clinical Microbiology and Infection)

  24. Fig. 1. Description of 33 clustered spoligotypes (2 to 49 cases) in the study Order STa Nb (%) Spoligotype Patternc Labeld Beijing T2 T2-Uganda T2 ND T2 CAS1_KILI T2 T2 LAM9 CAS1_DELHI T2 T1-T2 T2 T2 T2 T2 T2 T2 LAM3/S CAS1 T2 LAM11-ZWE T2 ND ND ND ND ND ND LAM6 CAS2 ND • 1 04 (1.2) • 128 49 (14.2) • 135 38 (11.0) • 52 26 (7.6) • UGA1 17 (4.5) • 590 15 (4.4) • 21 12 (3.5) • 125 11 (3.2) • UGA2 10 (2.9) • 42 09 (2.6) • 26 09 (2.6) • UGA3 09 (2.6) • 78 09 (2.6) • UGA4 09 (2.6) • UGA5 09 (2.6) • UGA6 09 (2.6) • UGA7 08 (2.3) • UGA8 07 (2.0) • UGA9 07 (2.0) • 4 06 (1.7) • 142 06 (1.7) • UGA10 06 (1.7) • 59 05 (1.5) • UGA11 05 (1.5) • UGA12 04 (1.2) • UGA13 04 (1.2) • UGA14 03 (0.9) • UGA15 03 (0.9) • UGA16 03 (0.9) • UGA17 02 (0.6) • 64 02 (0.6) • 288 02 (0.6) • UGA18 02 (0.6) aAs identified in SpolDB4.0;ST, Shared Type; Nb, Number of isolates (as a percentage of total M. tuberculosis strains in the study); cFilled boxes represent positive hybridization while empty boxes represent absence of spacers; dLabel defining the lineage/sub lineage; ND, Not yet determined in SpolDB4.0. Asiimwe BB, Ghebremichael S, Kallenius G, Koivula T, Joloba ML.Mycobacterium tuberculosis spoligotypes and drug susceptibility pattern of isolates from tuberculosis patients in peri-urban Kampala, Uganda. BMC Infect Dis. 2008 Jul 28;8(1):101. [Epub ahead of print] PMID: 18662405 [PubMed - as supplied by publisher]

  25. MIRU to detect multiple infections

  26. Way Foward • Need to strengthen systems to utilize existing and new technology • Develop good tools to monitor lab performance • Train TB teams at point of care

  27. Searching for Point of care test

  28. Thank you

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