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

Diagnosis of Tuberculosis

Clinical Radiological Microbiological Pathological

Biosecurity Level; BSL1

BSL 1 (WHO) WHO, (2004). Laboratory Biosafety Manual Third Edition WHO/CDS/CSR/LYO/2004.11

Laboratory design and infrastructure

Biosecurity Level; BSL2

(WHO) WHO, (2004). Laboratory Biosafety Manual Third Edition WHO/CDS/CSR/LYO/2004.11

Biosecurity Level; BSL3

(WHO) WHO, (2004). Laboratory Biosafety Manual Third Edition WHO/CDS/CSR/LYO/2004.11

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

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

Microscopic methods

Light Microscopy; EZN, Immersion, X100 0,02 mm2

Fluorescence Microscopy; RFKB, dark field, X25;0,34mm2

LED Microscopy; Light emitting diode

Mycobacterium Culture

Advantages Specific

Disadvantages Biosecurity-hazardous

Sensitive Cost Golden Standard Time

Trained Staff

Culture

Microscopy; 5.000-10.000 bacilli/ml Culture;10-100 bacilli/ml %30-50; Diagnosis before becoming

contagious %80; (+) result in all cases

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

Media, general approach

Ingredients Structure Use

- Synthetic - Solid - Isolation

- Semi-synthetic - Liquid - Identification

- Complex - Mixed - Growth

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)

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)

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

Manual; Agar Based Media

Advantages Considerably rapid growth Identifiable colony structure

Disadvantages Expensive CO2 required Sensitive to sun light and heat

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)

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

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

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

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

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

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.