1. TB – LABORATORY INVESTIGATIONS by Dr. Zubaidah Abdul Wahab Datin Dr. Ganeswrie Raj 1

2. LEARNING OBJECTIVES To update current laboratory investigations & techniques for diagnosing TB To strengthen laboratory facilities for TB management 2

3. INTRODUCTION Diagnosis of TB is based on the detection of acid fast bacilli (AFB) on smears & culture of Mycobacterium tuberculosis from clinical specimens. 3

4. INTRODUCTION Specimen collection Lab investigations – Microscopy – Culture – Identification – AntiTB sensitivity – Molecular 4

5. SPUTUM COLLECTION Sputum 1 At least 2 specimens At least one early morning specimen For patients who are unable to spontaneously expectorate adequate sputum specimens Sputum induction with nebulised hypertonic saline Fiberoptic bronchoscopy with bronchoalveolar lavage Gastric lavage especially in paediatric group (neutralise with sodium bicarbonate) 1 WHO, 2010 5

6. OTHER SAMPLES Sterile body fluids include blood, CSF, pleural fluid, peritoneal fluid should be collected aseptically transported immediately to laboratory in sterile container - 6

7. MICROSCOPY Microscopy Presumptive diagnosis Sputum Ziehl-Neelsen staining for AFB Conventional microscope low sensitivity (20 - 60%) 1 Light emitting diode-based fluorescence microscopy (LED FM) 2 10% more sensitive shorter time spent quicker turnaround time 1 Steingart KR et al., Lancet Infect Dis, 2006 2 Shenai S et al., Int J Tuberc Lung Dis, 2011 7

8. Acid fast bailli CONVENTIONAL LIGHT MICROSCOPY

9. AFB bacilli – IF stain (low mycobacterial load) IMMUNOFLUORESCENCE MICROSCOPY

10. CULTURE & SENSITIVITY Culture conventional method using LJ or Ogawa 6 - 8 weeks Drug-susceptibility testing 1 - 2 months 10

11. Without drugWith drug - INH M. tuberculosis AB Susceptible to INH PHENOTYPIC METHOD – Proportion method

12. Conventional culture medium (solid) – TB. Rough, buff, cream-coloured colonies, compact, corded colonies

13. CULTURE & SENSITIVITY Liquid culture media a. Bactec MGIT - detection by 2 weeks - identification within 3 weeks - sensitivity testing by 4 weeks b. Bactec MycoF only for blood sample 13

14. NUCLEIC ACID AMPLIFICATION TEST (NAAT) NAAT molecular technique provide rapid results within 24 - 48 hours ability to confirm presence of Mycobacterium in 50 - 80% AFB smear negative & culture positive 1,2 detect Mycobacterium in specimens weeks earlier than culture for 80 - 90% patients 2 1 CDC, MMWR Morb Mortal Wkly Rep, 2009 2 Sarmiento OL et al., J Clin Microbiol, 2003 14

15. LINE PROBE ASSAY (LPA) Line Probe Assay (LPA) detect rifampicin & isoniazid resistance in smear positive sputum or culture isolates carried out in a TB risk level 2 laboratory 15

16. GENE XPERT MTB Gene Xpert MTB for detection of rifampicin resistance Mycobacterium fast & accurate can be used in peripheral laboratories fully automated, near the patient, uses a robust system & technically simple 16

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18. COMMERCIAL SEROLOGICAL ASSAY Recommendation 5 Commercial serological assay should not be used to diagnose pulmonary & extrapulmonary TB. 18

19. WHO published a policy statement in 2011 stating that commercial serological tests for TB provides inconsistent & imprecise estimates of sensitivity & specificity which can adversely impact patient safety. 1 1 WHO, Commercial serodiagnostic tests for diagnosis of tuberculosis, 2011 19 COMMERCIAL SEROLOGICAL ASSAY

20. ROLE OF ADENOSINE DEAMINASE (ADA) IN TB DIAGNOSIS ADA is an enzyme involved in purine metabolism of the cells. Measurement of ADA level in pleural effusion & CSF is useful additional test in pleural & meningeal TB. In pleural TB, sensitivity = 92% & specificity = 90% 1 In CSF-ADA in TB meningitis, sensitivity = 93% & specificity of 97% if cut-off level of >10 U/L used 2 1 Liang QL et al., Respir Med, 2008 2 Rana SV et al., Clin Neurol Neurosurg., 2010

21. TAKE HOME MESSAGES LED FM has more advantages than conventional microscopy NAAT e.g. LPA is able to shorten the turn- around time for identification & antiTB testing Measurement of Adenosine Deaminase level in pleural or cerebrospinal fluid may be considered as an adjunct in diagnosing pleural TB & tuberculous meningitis respectively. 21

22. 22 THANK YOU  dr_zubaidah@sel.moh.gov.my dr_zubaidah@sel.moh.gov.my  ganiraj_balan@yahoo.com ganiraj_balan@yahoo.com