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REPUBLIC OF TURKEY MINISTRY OF HEALTH Tuberculosis Control Deaprtment

REPUBLIC OF TURKEY MINISTRY OF HEALTH Tuberculosis Control Deaprtment. Kurs Programı. Feyzullah GÜMÜŞLÜ, MD, Specialist Head of TB Control Department. Diagnosis and Treatment of Tuberculosis New Methods in TB bacteriological Diagnosis 23 April 2008 – Hall 2. Diagnosis of Tuberculosis.

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REPUBLIC OF TURKEY MINISTRY OF HEALTH Tuberculosis Control Deaprtment

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  1. REPUBLIC OF TURKEYMINISTRY OF HEALTHTuberculosis Control Deaprtment Kurs Programı Feyzullah GÜMÜŞLÜ, MD, Specialist Head of TB Control Department Diagnosis and Treatment of Tuberculosis New Methods in TB bacteriological Diagnosis 23 April 2008 – Hall 2

  2. Diagnosis of Tuberculosis • Clinical • Radiological • Microbiological • Pathological

  3. Biosecurity Level; BSL1 Laboratory design and infrastructure BSL 1 (WHO) WHO, (2004). Laboratory Biosafety Manual Third Edition WHO/CDS/CSR/LYO/2004.11

  4. Biosecurity Level; BSL2 (WHO) WHO, (2004). Laboratory Biosafety Manual Third Edition WHO/CDS/CSR/LYO/2004.11

  5. Biosecurity Level; BSL3 (WHO) WHO, (2004). Laboratory Biosafety Manual Third Edition WHO/CDS/CSR/LYO/2004.11

  6. Immune system Amount of bacilli Exposure Disease Very high Very long Very poor + + + + + + + + + + + + + + Certain High long Poor Quite possible + + + + + + ++ Low short Good + + + + + + + ++++ Probable Not possible Very low Very short Very good Biosecurity and Development of Disease

  7. Microscopy • %43; Smear (direct), all cases • %55; Smear (centrifuged sputum), all cases • %90; Smear (direct), contagious cases • (1.) %80-83, (2.) %10-14, (3.) %5-8

  8. Microscopic methods • Light Microscopy; EZN, Immersion, X100 0,02 mm2 • Fluorescence Microscopy; RFKB, dark field, X25;0,34mm2 • LED Microscopy; Light emitting diode

  9. Mycobacterium Culture

  10. Culture • Microscopy; 5.000-10.000 bacilli/ml • Culture;10-100 bacilli/ml • %30-50; Diagnosis before becoming contagious • %80; (+) result in all cases

  11. Ideal Media • Should permit growth even in case of low number (0-100) of bacilli. • Should be affordable • Should support identification • Should inhibit contaminants • Should be compatible with drug sensitivity testing

  12. Media, general approach

  13. Diagnostic media • Manual Methods • Egg based media(L J, Ogawa) • Agar based media (MB7H10-11) • Liquid media • Herman – Kirschner liquid media • Dubos OAA liquid media • Automatic (Rapid) Methods • Solid media • TK Media (Salubris) • Liquid media • Middlebrook 7H12 • BACTEC 460 (Becton Dickinson) • MGIT 960 (Becton Dickinson) • MB / Bact (BioMerieux) • Septicheck – AFBTB (Becton Dickinson) • ESP Culture II System (Trek Diagnostic)

  14. Manual; egg based media • Advantages • Easy, affordable, • Identifiable colony structure • Storage for long periods of time, • Resistant to contamination, • Coagulation • Malachite green • Culture without the need for centrifugation and neutralisation (Ogawa)

  15. Manual; egg based media • Disadvantages • 6 - 8 weeks for growth • Especially in case of low number of bacilli • In case of heavy decontamination procedures • Sample loss during contamination • Misapplication • Bubbles and nodes

  16. Manual; Agar Based Media • Advantages • Considerably rapid growth • Identifiable colony structure • Disadvantages • Expensive • CO2 required • Sensitive to sun light and heat

  17. Automatised Media(Radiometric – With Fluorescense Indicator) • Adavantages • Rapid growth • 5-7 days ; BACTEC, • 11-12 days; MGIT • Rapid identification of species • Specific-sensitive • Low work load • Disadvantages • Both media and equipment are expensive • Requires certain infrastructure • Equipment and maintanance services • Radiometric waste disposal (BACTEC)

  18. Sensitivity of Smear and Culture

  19. Biomolecular Methods • Nucleic-Acid Amplification Tests(NAA) • PCR, TMA, SDA %100 Specivity Sensitivity; Microscopy < NAA < Culture ss (+) cases; %95 Sensitive ss (+) and c(-) cases; %60-70 sensitive

  20. Serology Disadvantages Sensitivity Low in Ss(-) cases Distinction of LTB and active TB is difficult Distinction of TB/MOTT bacilli is difficult Adavantages • Material supply • Easy • Results in 1 hour • Simple technology • Considerably inexpensive

  21. Phage method Disadvantages Low sensitivity in Ss(-), c(+); Requires expert staff Limited to sputum Advantages • Results in 48-72 hours • No need for complex equipment • High sensitivity in Ss(+) and semi-quantitative results • 70% sensitivity and 99% specivity in new cases

  22. Cytotoxin Assay Disadvantages Only just obtained blood samples are eligable Difficult to distinguish infection and disease Expensive Advantages • Single sample • More objective as compared to Ppd • TB/MOTT bacilli distinction

  23. FINAL REMARKS • Numerous methods have been used and recommended both for smear and culture. However none of them has been granted as the universal best option. Because the applicability of methods depend on technical capacity, trained laboratory staff, appropriate equipment and calibration.

  24. thank you

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