Laboratory diagnosis of Tuberculosis (Pulmonary and extra pulmonary Tuberculosis)

Microbiological laboratory diagnosis of Tuberculosis

1. Laboratory Diagnosis TUBERCULOSIS 1 Dr Saumya Singh Prof. & Head Microbiology UIMS, Prayagraj

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3. 3 Sputum 2 specimens—spot and early morning Gastric aspirate /lavage *Early morning lavage should be collected and processed early (<4 hours) Recommended for children (tend to swallow sputum) or ICU patients (aspiration) Bronchial secretions 2–5 mL Bronchoalveolar lavage 20–50 mL Samples

4. 4 CSF, pericardial fluid, synovial fluid and ascitic fluid Pleural fluid Blood Indicated only for disseminated TB and co- infected with HIV Transbronchial and other biopsies collected in sterile normal saline Swabs The only recommended swabs are: Laryngeal swabs: Collected early morning in empty stomach or Swab from discharging sinus Urine 3 early morning specimens collected (500 mL/ specimen) Stool For disseminated TB in HIV infected patients and infants Suboptimal specimens (organism load is less)

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6. Laboratory diagnosis of Tuberculosis (Cont..) 6 Direct microscopy A. ZN staining of sputum smear showing long, slender and beaded red colored acid–fast bacilli; B . Auramine phenol staining of sputum smear —Tubercle bacilli appear bright brilliant green against the dark background. (Fluorescence Microscopy)

7. NTEP grading is useful for - ZN stained sputum smears: *Monitoring the treatment response of the patients *Assessing the severity of disease /infectiousness of the patient ZN: Smear sensitivity ≥ 10,000 bacilli /mL of sputum FM : Smear sensitivity ≥ 1000 bacilli /mL of sputum sputum smears 7 No. of AFB seen OIF to be screened Grading Result No AFB in 100 OIF 100 Negative 1–9/ 100 OIF 100 Scanty* (no.) Positive 10–99/100 OIF 100 1+ Positive 1–10/ OIF 50 2+ Positive >10/ OIF 20 3+ Positive

8. 8 Sensitivity: 10-100viable bacilli/ml Sensitivity >PCR Speciation possible Drug sussceptibility

9. Culture (Cont..) 9 A. Lowenstein-Jensen medium (arrow showing rough, tough and buff-colored colonies); B. BACTEC MGIT; C. MGIT liquid culture medium Conventional culture media 6–8 weeks Automated culture methods 3–4 weeks (- ve 6weeks) MGIT system: Detects growth as well as resistance to antitubercular drugs (ATDs) Prelim: 21 days Final report: 42 days BacT /Alert

10. Laboratory diagnosis of Tuberculosis (Cont..) 10 Culture identification Acid fast stain Automated identification—by MALDI-TOF MPT 64 antigen Biochemicals

11. NAAT (Nucleic acid amplification tests) GeneXpert - CBNAAT GeneXpert MTB/RIF GeneXpert Ultra MTB/RIF GeneXpert P-XDR (Rif R) TrueNAT 11 Molecular methods

12. CBNAAT (Cartridge based nucleic acid amplification test) (Cont..) 12 Qualitative: For identification of MTB complex (DNA) and detection of resistance to rifampicin Limit of detection-(LOD) - 16-131 bacilli /ml Fast ,Automated ,Turn around time - 2 hours GeneXpert system / GeneXpert ultra MTB/RIF

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14. Genexpert P-XDR (Rif R) MTB DNA Kanamycin,Isoniazide,Fluroquiolone,Amikacin,Capreomycin,Ethionamide 14 MTB /RIF MTB/RIF Ultra MTB complex Rifampicin Resistance 2ml sputum TAT-112min 65-87 min LOD 131 bacilli /ml 16 bacilli/ml Single target rpoB core Multiple target rpoB core /IS6110+ IS1081 Real time PCR Nested PCR

15. XPERT MTB/RIF: WHO recommendation Prefer over Microscopy and Culture in : CSF -suspected TB meningitis Use replacement in: Lymph nodes and Extrapulmonary Tissues in presumed EP TB Do not use for Diagnostic Sampling in: Stool ,Urine , Blood ( lack of data on the utility of genexpert ) 15

16. TrueNAT - Chip based NAAT 16 Identification of MTB complex (DNA) Detection of resistance to rifampicin Limit of detection (LOD): 100 bacilli/ml Turnaround time – Appx.2.15 hours Separately

17. 17 Indeterminate : MTB concentration very low Repeat sampling Still – Culture and drug sensitivity testing /LPA

18. 18 Line probe assay (e.g. Genotype MTB DR Plus)-Hain’s test Probe based detection of amplified DNA in the specimen for identification and detection of resistance to 1st and 2nd line ATDs; Speciation of MTB complex and NTM Turnaround time -3-4 days. *Positive Smear and culture

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20. (Cont..) 20 Diagnosis of latent tuberculosis Tuberculin skin test (e.g. Mantoux test), 48-72hrs Adults: epidemiological marker: does not confirm presence of active infection Children ;diagnostic marker -active infection False positive :Atypical mycobacterial infection False negative : Early /Advance tuberculosis ,Miliary tuberculosis Immunosuppressive indivisuals

21. 21 Interferon gamma release assay (IGRA)- TB gold or Platinum /TB sure

22. Diagnostic algorithm for pulmonary tuberculosis 22

23. Tests Recommended ZN Stain /Modified ZN stain TB culture / BACTEC MGIT / BacT /Alert TrueNAT CBNAAT (GeneXpert) Xpert MTB/RIF GeneXpert Ultra MTB/RIF GeneXpert P-XDR (Rif R) LPA Mantoux test TB Platinum/gold or TB Sure 23

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