REPUBLIC OF TURKEY MINISTRY OF HEALTH Tuberculosis Control Deaprtment
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REPUBLIC OF TURKEYMINISTRY OF HEALTH
Tuberculosis Control Deaprtment
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
Kurs Programı
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Diagnosis of Tuberculosis
Clinical Radiological Microbiological Pathological
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Biosecurity Level; BSL1
BSL 1 (WHO) WHO, (2004). Laboratory Biosafety Manual Third Edition WHO/CDS/CSR/LYO/2004.11
Laboratory design and infrastructure
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Biosecurity Level; BSL2
(WHO) WHO, (2004). Laboratory Biosafety Manual Third Edition WHO/CDS/CSR/LYO/2004.11
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Biosecurity Level; BSL3
(WHO) WHO, (2004). Laboratory Biosafety Manual Third Edition WHO/CDS/CSR/LYO/2004.11
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Biosecurity and Development of Disease
Amount of bacilli
ExposureImmune system Disease
Very high Very long Very poor+ + + + + + + + + + + + + + Certain
High + + + + + +long Poor ++
+ +Low short + + Good + + +
++++
Quite possible
Very low Very short Very good
Probable
Not possible
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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
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Microscopic methods
Light Microscopy; EZN, Immersion, X100 0,02 mm2
Fluorescence Microscopy; RFKB, dark field, X25;0,34mm2
LED Microscopy; Light emitting diode
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Mycobacterium Culture
Advantages Specific
Disadvantages Biosecurity-hazardous
Sensitive Cost Golden Standard Time
Trained Staff
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Culture
Microscopy; 5.000-10.000 bacilli/ml Culture;10-100 bacilli/ml %30-50; Diagnosis before becoming
contagious %80; (+) result in all cases
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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
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Media, general approach
Ingredients Structure Use
- Synthetic - Solid - Isolation
- Semi-synthetic - Liquid - Identification
- Complex - Mixed - Growth
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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)
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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)
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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
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Manual; Agar Based Media
Advantages Considerably rapid growth Identifiable colony structure
Disadvantages Expensive CO2 required Sensitive to sun light and heat
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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)
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Sensitivity of Smear and Culture
Observed number of
bacilli
Estimated number of
bacilli in 1 ml of sputum
Probability of positive result
In 300 fields; 0 Less than 1000
Less than %10
In 300 fields 1-2 5000-10.000 %50
In 100 fields 1-9 Approximately 30.000
%80
In 10 fields 1-9 Approximately 50.000
%90
For each field 1-9
Approximately 100.000
%96.2
For each field 10 or more
500.000 %99.95
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Biomolecular Methods
%100 Specivity Sensitivity; Microscopy < NAA < Culture
ss (+) cases; %95 Sensitive
ss (+) and c(-) cases; %60-70 sensitive
Nucleic-Acid Amplification Tests(NAA) PCR, TMA, SDA
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Serology
Adavantages Material supply Easy Results in 1 hour Simple technology Considerably
inexpensive
DisadvantagesSensitivityLow in Ss(-) casesDistinction of LTB and active TB is difficultDistinction of TB/MOTT bacilli is difficult
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Phage method
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
Disadvantages Low sensitivity in Ss(-), c(+); Requires expert staffLimited to sputum
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Cytotoxin Assay
Advantages Single sample More objective as compared to
Ppd TB/MOTT bacilli distinction
DisadvantagesOnly just obtained blood samples are eligableDifficult to distinguish infection and diseaseExpensive
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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.
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