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Laboratory Diagnosis
Tuberculosis
Dr. Kiarash Ghazvini
Department for bacteriology and virology,
Mashhad University of medical Sciences
Mycobacterium tuberculosis
long, slender, straight or curved, acid fast bacilli
slow growers, obligate aerobes, intracellular bacterium structure composed of high molecular weight acidic waxes, mycolic acid, cord factor
Diagnosis
E p i d e m i o l o g y 2
M. tuberculosis appearing as bright red bacilli (rods) in a sputum smear stained with the Ziehl-Neelsen stain
AFB MICROSCOPY
Advantages
- High specificity (AFB in sputum = TB)
-Rapid
- Accurate diagnoses
All mycobacterium are acid fast, no exception ; > 98% for AFB in high burden countries - Using simple and available equipment
Disadvantage
Low sensitivity; Reported sensitivity ranging 25 to 65% when compared to culture Species differentiation impossible. False positive; Saprophytic mycobacteria.
Cumulative Positivity
50%
0%
First
Second
Third
Quality of sputum, morning sputum 10-100% more positives Number of samples Quality of smear, staining, quality of microscopes, Effort in examining number of fields
Smear-positive
Volume
Pleural, peritoneal fluids Cerebrospinal fluid
E p i d e m i o l o g y 3
Culture
M. tuberculosis grows in Lowenstein Jensen medium, which contains inhibitors to keep contaminants from outgrowing the organism. Because of its slow growth, it takes 4-6 weeks before small buffcoloured colonies are visible on the medium.
Culture
BACTEC TB System
based
on the principal that the organisms multiply in the broth and metabolize C 14containing palmaticacid, producing radioactively labeled 14CO2.
Middle Brook 7H12 containing palmatic acid PANTA (Polymyxin B , Amphotericin B , Nalidic acid , trimethoprim ,Azlocillin ) OADC enrichment
*.Containing
Draw blood from patient, expose white cells to TB antigens, look for signs of immune response
Two most common methods are ELISA and ELISPOT
TST
Blood tests
More expensive
Reliability fair
Cross-reacts with BCG, NTM
Blood Tests
67-96% 95-100%
Specificity
Summary
Likely more sensitive for active TB, but clinical utility in this setting less clear
Logistical and cost barriers to implementation Ideally will replace TST in many settings
Adenosine deaminase (ADA) and interferon gamma studied for dx of extrapulmonary TB Both are markers of immune response to TB Not that specific
Immune dx of TB
In low-incidence areas, specificity and sensitivity are not good enough for routine use
More specific markers needed
PCR
2) Determine rapidly whether acid-fast organisms identified by microscopic examination in clinical specimens are M.tuberculosis
3) Identify the presence of genetic modifications known to be associated with resistance to some anti mycobacterial agents. 4) Determine whether or not isolates of M.tuberculosis from different patients have a common origin in the context of epidemiological studies.
Molecular method
Incase of smear and culture positive the sensitivity is ranging 80% to 90% with specificity of 97%-99%.
Incase of smear negative and culture positive the sensitivity is ranging 60% to 80% with specificity of 97%-99%.
Identification of the target sequence of DNA doesn't imply organism viability. Contamination of samples by product from previous PCR experiments
Disadvantages:
NAA Summary
NAA is useful to distinguish TB from NTM in smear + specimens Less sensitive in smear specimens Clinical judgement must always take priority Relatively expensive tests; need data to support costeffectiveness
TB/HIV
MDR/XDR-TB