1. DIAGNOSIS OF TUBERCULOSIS
Dr Manie van Rensburg

2. DIAGNOSIS OF TUBERCULOSIS
SPECIMENS
MICROSCOPY
CULTURE
IDENTIFICATION
SENSITIVITY TESTING
DNA DETECTION (PCR)
BIOCHEMICAL MARKERS
IMMUNOLOGICAL

3. SPECIMENS GO WHERE THE GOLD IS QUALITY OF SPECIMEN NUMBER OF SPECIMENS
Nonspecific disease - Which specimen ?
QUALITY OF SPECIMEN
Result as good as the specimen received
Sputum / Spit
Pleural biopsy / Pleural fluid
NUMBER OF SPECIMENS
Sputum 2-3 early morning specimens
Urine – 3 full bladder morning specimens

4. MICROSCOPY DIRECT / CONCENTRATED SPECIMEN ACID-FAST STAIN
ZIEHL- NEELSEN OR FLUOROCHROME
DETECTION ABILITY
ZN stain organisms/ml for smear positivity
Fluorochrome stain – easier to detect organisms
Gram stain – Gram positive or ghost organisms
Sensitivity %

5. Ziehl-Neelsen stain

6. Fluorochrome stain

7. CULTURE LIQUIFIED / [ ] / DECONTAMINATED DECONTAMINATION
Kills normal flora and TB bacilli
SOLID MEDIA / LIQUID MEDIA
Solid media - Egg based (LJ) / Agar based (MB)
Liquid media – Middel brook
TEMPERATURE OF INCUBATION
Lower temp – M. marinum, M. ulcerans, M. chelonae, M fort
AUTOMATED SYSTEMS
Continious monitoring of growth (6 w)

8. Culture media

9. Culture media

10. CULTURE Remains the most sensitive test Insufficient decontamination
Many contaminated specimens
Target level 5-10 %
Over decontamination
No contaminated specimens
Decreased mycobacterial yield

11. IDENTIFICATION BIOCHEMICAL TB protein detection HPLC PROBES (PCR)
Not done – takes weeks to months to do
TB protein detection
Secrete more than 33 proteins
Predominamt protein – MPT64 – rapid immunochromographic detection test
HPLC
Mycolic acids in the cell wall
PROBES (PCR)
DNA probes Commercial / In house
M tuberculosis and Non tuberculous mycobacteria

12. AMPATH statistics 16 % of Specimens are culture positive
12 % M. tuberculosis (75%)
1.5% M. avium – intracellulare (10%)
2.5% Other NTM (15%)
40-50% Culture pos is ZN positive
2-3% Contamination rate

13. SENSITIVITY TESTING Breakpoint method MIC’S GENETIC BASIS (PCR)
Agar dilution
Not done
Broth dilution
Automated method
MIC’S
E test
GENETIC BASIS (PCR)
Line probe assay

16. Representative DNA patterns obtained by the MTBDRplus assay.
Representative DNA patterns obtained by the MTBDRplus assay. Examples of invalid results are also included. Lane 1, example of a pattern of RIFs and INHs; lane 2, example of a pattern of RIFr and INHr; lane 3, example of a pattern of RIFs and INHr; lanes 4 to 9, examples of invalid results. The positions of the oligonucleotides and the control probes are given on the left. The targeted genes and the specific probes lines are shown from top to bottom, as follows: conjugate control (CC); amplification control (AC); M. tuberculosis complex-specific control (TUB); rpoB amplification control; rpoB wild-type probes WT1 to WT8 (505 to 533); four rpoB mutant probes (probes MUT1, MUT2A, MUT2B, and MUT3) in codons D516V, D526Y, H526D, and S531L, respectively; katG amplification control; katG codon 315 wild-type probe; two katG codon 315 mutant probes (probes MUT1 and MUT2) with AGC-ACC (S315T1) and AGC-ACA (S315T2) mutations, respectively; inhA amplification control; inhA wild-type probes WT1 and WT2 covering positions −15 and −16 of the gene regulatory region; four inhA mutant probes (probes MUT1, MUT2, MUT3A, and MUT3B) with mutations C→T at position −15, A→G at position −16, T→C at position −8, and T→A at position −8, respectively. M, colored marker.
A. Lacoma et al. J. Clin. Microbiol. 2008;46:

17. DNA DETECTION DNA is very stable in the environment (lab contam)
Is present in dead bacilli
Cannot be used to monitor treatment
Extraction method of DNA very NB
NB Quality of the specimen
Our laboratory 3 TB DNA tests are done
Hain test
GeneXpert

18. Hain Test
Developed to do sensitivity testing on smear positive samples
70% (1= 40%) sensitivity Smear negative samples
Confirms presence of M. tuberculosis
Sensitivity for INH and Rifampicin
Second line sensitivity testing

19. GeneXpert Presence of MTB and sensitivity to Rif
ZN neg specimens – sens 75%
False rifamicin resistance (few) – major implications
POC testing
Hardware expensive
Reagents expensive
Wastage
Robust system

20. Laboratory flow diagram
Decontamination / ZN / Culture
ZN pos
For molecular ID and Sens
ZN neg
Culture

21. Biochemical markers
ADA
Urinary Lam test

22. Biochemical markers ADA Presently total ADA is determined
Adenosine ---ADA ---> Inosine
Total ADA increased in TB, bacterial inf, RA, lymphoproliferative disorders, empyema
Iso enzymes
ADA1 in all cells
ADA2 in stimulated monocytes and macrophages
ADA2 elevated in TB and RA

23. Biochemical markers Urine Lam test
Cell wall component – lipoarabinomanan
Excreted in urine (lateral flow test)
Used in HIV infected individuals with low CD4 count < 100 cells/ul (WHO 2015)
Sensitivity 28% - 65%
Specificity 90% - 97%
Prognosticator – 2x greater mortality test pos

24. Immunological Mantoux skin test (Tuberculous skin testing)
Nearly 100 years old
Diagnosis latent infection
PPD standarised (RT-23)
PPD 5TU injected intradermally (Wheal)
False negative
Technical, advanced TB, malnutrition, elderly, HIV
False positive
BCG (1yr 40% 10yr 20%), NTM,

25. Immunological Interferon gamma release assay (IGRAS) tests
TB spot test or TB gold test
Lymphocytes stimulated (ESAT6, CFP10, TB7.7)– interferon production pos test
We prefer the TB spot test (less intermediate results)
Not distinguish between active and latent infection
Increased specificity (BCG/NTM not false pos)
Improved sensitivity – TB children with HIV
One patient visit

26. Pearls Go where the gold is Result is as good as the specimen received
Culture remains gold standard
PCR quicker result – less sensitive

27. Questions
Can you do a TB pcr test on blood in a patient with pulmonary TB?
Can you do TB pcr on a post treatment patient?
Does a negative TB microscopy, culture or TB pcr test exclude TB?
Why do you send 3 specimens for TB?

28. REFERENCES
Principles and practices of infectious diseases Mandell, Bennett, Dolin
Manuel of Clinical Microbiology Murry, Baron et. Al
Rapid Molecular Detection of Tuberculosis and Rifampin resistance Boeheme et al. N Eng J Med 361;
Molecular Diagnosis of Mycobacteria
Clinical Chemistry 47: