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Gene Xpert & Advances
The document presents an overview of tuberculosis (TB) cases and advancements in diagnostics, with a focus on the Gene Xpert technology. In 2017, global TB incidence was reported at 10 million cases, with significant drug resistance issues highlighted, particularly in India and other countries. The document details the sensitivity and specificity of various TB detection methods, including the Gene Xpert and its Ultra version, emphasizing the need for improved testing in vulnerable populations.
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Gene Xpert & Advances
- 1. Gene Xpert & Advances Dr.Sayan Chakraborty, MD Senior Resident, DM Infectious diseases AIIMS, New Delhi Email: [email protected]
- 2. Breaking NEWS • 10million new cases in 2017 • India (27%), China (9%), Indonesia (8%), Pakistan (5%), Bangladesh (4%) • 9% in PLHIV (72% in Africa) • 1.3 million deaths in HIV – • 300,000 deaths in HIV +
- 3. Global Drug resistantTB in 2017 • 558,000 developed Rifampicin resistant (RR) TB • 82% were multidrug-resistant (MDR-TB) • India (24%), China (13%), Russian Federation (10%) • 3.5% of new cases & 18% of previously treated cases had MDR/RR-TB • 8.5% (95% confidence interval, 6.2–11%) had extensively drug-resistant TB (XDR-TB) Global Tuberculosis report WHO 2018
- 4. Indian Scenario Estimates NumberRate (per 100,000) Incidence (includes HIV+TB) 2.7 million 204 (140–281) Incidence (HIV+TB only) 86,000 6.4 (4.3–9) Incidence (MDR/RR-TB) 135,000 10 (5.8–16) Mortality (excludes HIV+TB) 410,000 31 (28–33) Mortality (HIV+TB only) 11,000 0.79 (0.48–1.2) Global Tuberculosis report WHO 2018
- 5. Indian scenario -Treatment • TB treatment coverage in 2017: 65% (47–96) Treatment success Rate New and relapse cases in 2016 69% Previously treated cases, excluding relapse in 2016 70% HIV-positive TB cases in 2016 75% MDR/RR-TB cases started on second-line treatment in 2015 46% XDR-TB cases started on second-line treatment in 2015 28% Global Tuberculosis report WHO 2018
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- 9. WHO endorsements inTB diagnostics Timeline YEAR TECHNOLOGY TURNAROUND TIME Before 2007 ZN stain Solid culture 2 days 30-60 days 2007 Liquid culture Upto 42 days 2008 Line probe assay (Genotype MTBDR plus) 24 – 48 hours 2011 LED based fluorescence microscopy 1 day 2011 Xpert MTB/RIF 112 mins 2013 Line probe assay SL (Genotype MTBDR SL) 24 – 48 hours 2017 Xpert MTB/RIF Ultra 65 – 87 mins
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- 11. Principle of XpertMTB/RIF • Semi- quantitative hemi-nested Real time PCR • Segment Amplified – 192 bp region of M.tuberculosis rpoB gene • Set of three primers amplify the target region • Detection is by five overlapping molecular beacon probes Lawn et al. Future Microbiology, 2011, 6(9), 1067–1082
- 12. RIF RESISTANCE iseither NOT DETECTED or DETECTED There is no high or low detection for RIF RESISTANCE Interpretation
- 13. Interpretation MTB DETECTED Any NegativeProbe (Ct = 0) or Δ Ctmax > 3.5 between any two probes All the 5 probes Positive & Δ Ctmax ≤ 3.5 between them MTB DETECTED RIF Resistance DETECTED MTB DETECTED RIF Resistance NOT DETECTED ∆ Ctmin ≤ 2.0
- 14. Min = 22.4Max = 23.9 Ct <3.5 : 23.9-22.4 = 1.5 : Resistance NOT DETECTED RIF Resistance NOT DETECTED
- 15. Ct 9.6 RIF ResistanceDETECTED Max. = 36.5 Min. = 26.9 Ct >3.5 Rif Resistance DETECTED
- 16. Interpretation • RIF ResistanceINDETERMINATE : 1. If Ct of any probe exceeds the Valid Ctmax ( 39 for A, B & C & 36 for D & E ) 2. Earliest Ct value is greater than (Valid Ctmax of probe in condition 1) - (delta Ct max cut-off of 3.5 ) • Invalid: Sample Processing Control (SPC) not detected in a negative test • Error: Probe check fails
- 17. Diagnostic value inPulmonary TB • Smear +VE/ Culture +VE TB: Pooled sensitivity 98% (95% CI, 97-99%) (23 studies, 1952 participants) • Smear –VE/ Culture +VE TB: Pooled sensitivity 68% (95% CI, 61-74%) (23 studies, 7151 participants) Pooled specificity of 99% (95% CI, 98-99%) (23 studies, 7151 participants) WHO Xpert MTB/RIF implementation manual 2014
- 18. Diagnostic value inPulmonary TB • PLHIV: Pooled sensitivity 79% (95% CI, 70-86%) (7 studies, 1789 participants) • Detecting rifampicin resistance: Pooled sensitivity 95% (95% CI, 90-97%) (17 studies, 555/2624 total specimens) Pooled specificity of 98% (95% CI, 97-99%) (24 studies, 2414 specimens). WHO Xpert MTB/RIF implementation manual 2014
- 19. M.tuberculosis vs NTM •Xpert MTB/RIF highly accurate in distinguishing TB from NTM in clinical specimens • Among 180 specimens with NTM, Xpert MTB/ RIF had a positive result in only 1 specimen that grew NTM (14 studies, 2626 participants) WHO Xpert MTB/RIF implementation manual 2014
- 21. Sharma SK, etal. PLoS ONE 2015; 10(10): e0141011
- 22. Sharma SK, etal. PLoS ONE 2015; 10(10): e0141011
- 25. Diagnose extra-pulmonary TB inadults & children Review: 15 published studies, 7 unpublished studies (5922 samples) WHO Xpert MTB/RIF implementation manual 2014 Pooled sensitivity Studies/Samples Lymph node aspirate 84.9% 14 studies, 849 samples Pleural fluid 43.7% 17 studies, 1385 specimen CSF 79.5% 16 studies, 709 specimens Gastric fluid 83.8% 12 studies, 1258 samples Other tissue specimens 81.2% 12 studies, 699 specimens
- 26. EPTB in India Pooledsensitivity Studies/Samples Lymph node aspirate 83.1% 13 studies, 955 samples Pleural fluid 46.4% 14 studies, 841 specimen CSF 80.5% 13 studies, 839 specimens Index TB guidelines 2017
- 27. Sample Sensitivity SpecificitySamples Pleural fluid 40% 97% 364 Lymph node 88% 91% 273 CSF 68% 98% 230 Cold abscess 95% 71% 153 Ascitic fluid 18% 99% 102 Endometrial aspirate 33% 100% 95 Urine 33% 100% 55 Pericardial fluid 25% 94% 20 Overall 71% 95% 1292 EPTB from AIIMS, New Delhi SK Sharma et al. Eur Respir J 2014, 44(4), 1090- 1093
- 28. • 60 patients,52 (88.3%) had a final diagnosis of PTB and 16 (26.7%) were culture confirmed. • Xpert® MTB/RIF: In culture confirmed cases, sensitivity 81% (54%–96%) and specificity of 73% (56%–85%) • In culture negative cases, Sensitivity 32% (17%–51%) and Specificity 100% (54%–100%) • Culture had a sensitivity of 32% (20%–47%) for the final diagnosis. Lung India 2018;35:295-300
- 29. • The sensitivityof stool Xpert was 11.54% and specificity 98.65% as compared to culture • Conclusion: Stool sample for Xpert cannot replace gastric aspirate and induced sputum for diagnosis, and hence should not be used as a first line test
- 30. Need for XpertMTB/RIF Ultra • Limitations of Xpert MTB/RIF: Sputum negative Pulmonary TB: Pooled sensitivity 68% (95% CI, 61-74%) CSF: Pooled sensitivity 79.5% (95% CI, 62.0-90.2% PLHIV: Pooled sensitivity 79% (95% CI, 70-86%) WHO Xpert MTB/RIF implementation manual 2014
- 32. MTB/RIF MTB/RIF Ultra DiagnosisMTB complex MTB complex Resistance Detects rifampicin resistance asa surrogate for MDR- TB Detects rifampicin resistanceas a surrogate for MDR-TB Amplification forTB detection Single target: rpoB core region Multi-copy target: rpoBcore region Insertion elements: IS6110+ IS1081 Resistance detection Real-time PCR 5 probes bind to RpoBgene Nested PCR, Melting curve; 4 probes bind to RpoBgene Sputum input 2ml 2ml PCRreaction 25ul 50ul AssayTAT 112 min 65-87min Limit of detection 114 cfu/ml 16 cfu/ml Short pieces of DNA, occurs multiple times in genome, conserved in MTBC
- 33. Temperature Fluorescence Melting curve analysis:If amutation is present, dsDNA(probe + TBDNA) dissociates sooner thanif ‘normal’ DNApresent Therange of Tmis known for mutant and normal Mutant ’Normal’ Mixed population Tm mutant TB Tm normal TB Principle ofdetection WHO meeting report of a technical expert consultation 2017 (WHO/HTM/TB/2017.04)
- 34. Resultsfor Ultra MTBcategories CategoryMTB/RIF Xpert Ultra Interpretation Not detected X X No TBdetected High X X TBdetected Med X X TBdetected Low X X TBdetected Verylow X X TBdetected Trace X Traceamounts MTB detected MTBnot detected =neither of multi-copy target probes are positive but SPC(Samplie Processing Control) is positive (valid) MTBdetected =one or both probes for multi-copy target are positive and at least two rpoB probes positive WHO meeting report of a technical expert consultation 2017 (WHO/HTM/TB/2017.04)
- 35. Newcategory: MTBtrace • Anewresultcategory: •Trace=Improvedsensitivity =Lowestbacillaryburden detected •Oneor both probes for multi-copy targetsarepositive withCt <37 andno more than onerpoBprobes haveaCt<40 Considerationsfor interpretation of “Trace"results: •In HIV-positives, children andEPTB = TBpositive •If in apatient with no-risk of HIVor previoushistory ofTB = repeat test on newspecimen •if trace detected on repeat =TB positive WHO meeting report of a technical expert consultation 2017 (WHO/HTM/TB/2017.04)
- 37. Performanceof Ultra vsMTB/RIF •1,520 persons with suspected TB enrolled. • Overall, sensitivity of the Ultra was 5% higher than that of Xpert MTB/RIF (95%CI +2.7, +7.8) • Sensitivity increases were highest among smear- negative culture-positive patients (+17%, 95%CI +10, +25) and among PLHIV (+12%, 95%CI +4.9, +21). • But specificity was 3.2% lower (95%CI -2.1, -4.7). • Specificity-decrease were higher in patients with a history of TB (-5.4%, 95%CI -9.1, -3.1) than in patients with no history of TB (-2.4%, 95%CI -4.0, - 1.3) WHO meeting report of a technical expert consultation 2017 (WHO/HTM/TB/2017.04)
- 38. • In CSF:Sensitivity raised to 95% by Ultra for detection of TB meningitis • Respiratory samples from children: Sensitivity 71% for Ultra versus 47% for Xpert MTB/RIF • Primarily due to the ‘trace call’ • In low TB burden settings: Specificity of Ultra is very high (99.3%, 95%CI 96-99) Performanceof Ultra vsMTB/RIF WHO meeting report of a technical expert consultation 2017 (WHO/HTM/TB/2017.04)
- 39. Lancet Infect Dis2018; 18: 76–84
- 40. Lancet Infect Dis2018; 18: 68–75
- 41. Lancet Infect Dis2018; 18: 68–75
- 42. • Detection ofhetero-resistance: rpoB S531L, L511P and H526N RRDR in mixtures containing as little as 10% mutant DNA (even 5%) Chakravorty et al. 2017; mBio 8:e00812-17.
- 43. GeneXpert Omni • Single-cartridge,point-of-care platform • Low power consumption • Integrated battery (4 hrs) + supplemental battery (12 hrs) • Automatic connectivity Rapid, onsite molecular testing at primary care clinics in high-burden countries http://www.cepheid.com/en/genexpert-omni/
- 44. GeneXpert Xtend • Identifiesresistance to INH, the FQs, amikacin and kanamycin • Same testing procedures and platform as the Xpert, • Largely restricted to patients who are Xpert +ve & RIF resistant • Funding Agency: National Institute of Health (NIH) • Project Start: 2014-04-01 • Project End: 2019-03-31 http://grantome.com/grant/NIH/R01-AI111397-01
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Editor's Notes
- #47 The TrueNat machines costs less (Rs 4.5 lakh) to the department as it is Indian. The cost for the diagnosis of a single person will be around Rs 300-400 whereas the cost of cartridges of CB-NAAT machines is between Rs 2,500 and Rs 3,000. One cartridge is used to test six people