DIAGNOSIS OF TUBERCULOSIS Dr Manie van Rensburg

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

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

MICROSCOPY DIRECT / CONCENTRATED SPECIMEN ACID-FAST STAIN ZIEHL- NEELSEN OR FLUOROCHROME DETECTION ABILITY ZN stain 10 000 organisms/ml for smear positivity Fluorochrome stain – easier to detect organisms Gram stain – Gram positive or ghost organisms Sensitivity 22-80 %

Ziehl-Neelsen stain

Fluorochrome stain

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)

Culture media

Culture media

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

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

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

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

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;46:3660-3667

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

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

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

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

Biochemical markers ADA Urinary Lam test

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

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

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,

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

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

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?

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;11 1005-1015 Molecular Diagnosis of Mycobacteria Clinical Chemistry 47:5 809-814