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Newer Diagnostic test in Tuberculosis ..
- 1. ADVANCES IN TBDIAGNOSIS Dr. Rasika Deshmukh
- 2. TB burden • In2019, an estimated 10 million people fell ill with tuberculosis (TB); 3 million were not reported to have been diagnosed and notified • 465 000 patients with multidrug-resistant TB or rifampicin-resistant TB (MDR/RR-TB), only 206 030 (44%) were diagnosed and notified • India >>the total number of incident TB patients (new and relapse) notified during 2021 were 19,33,381
- 3. WHO-endorsed and emergingmolecular tests for TB and drug resistance
- 4. Next-generation Xpert testing •development of the Xpert MTB/RIF assay was a significant step forward for improving the diagnosis of TB and the detection of rifampicin resistance globally. • Nucleic Acid Amplification Test, or NAAT>>qPCR • TAT 2hours • Pulmonary and extrapulmonary TB
- 5. MTB/RIF MTB/RIF Ultra DiagnosisMTB complex MTB complex Resistance Detects rifampicin resistance Detects rifampicin resistance Amplification for TB detection Single target : rpoB core region Multi-copy target : rpoB core region Insertion elements : IS6110+ IS1081 (short pieces of DNA occurs multiple times in genome, conserved region) Resistance detection Real time PCR 5 probes binds to rpoB gene Melting curve 4 probes binds to rpoB gene Sputum input 2ml 2ml PCR reaction 25ul 50ul Chamber for DNA amplification smaller larger Assay TAT 112 min 65-87 min Limit of detection 131 cfu/ml 16 cfu/ml Cost App. Rs. 1000 App. Rs. 1000 Sensitivity 85 88 Specificity 98 96
- 8. Next-generation Xpert testing Newcategory : MTB trace • semiquantitative “trace” category = bacilli at the lowest limits of detection>>one of the two multicopy amplification targets, but not the rpoB sequences, are detected. Consideration for interpretation of trace report • HIV (PLHIV) positives, children and ETPB = positives >>paucibacillary. • other cases with no risk or previous history of TB >> a fresh specimen retested r/o false positives • Trace positives >> previously treated for TB • Repeat trace = TB positives • minimally trained technicians, • continuous power supply and computer which limits its use as a true point-of-care (POC) test.
- 9. Diagnostic accuracy ofXpert MTB/RIF Pulmonary TB Rifampicin resistance
- 10. Diagnostic accuracy ofXpert MTB/RIF
- 11. Recommendations on XpertMTB/RIF and Xpert Ultra as initial tests (WHO) 1. In adults and children with signs and symptoms of pulmonary TB 2. In adults with signs and symptoms of pulmonary TB and without a prior history of TB (≤5 years) or with a remote history of TB treatment (>5 years since end of treatment) 3. In adults with signs and symptoms of pulmonary TB and with a prior history of TB and an end of treatment within the last 5 years 4. In adults and children with signs and symptoms of TB meningitis 5. In adults and children with signs and symptoms of extrapulmonary TB 6. In HIV-positive adults and children with signs and symptoms of disseminated TB 7. In adults with signs and symptoms of pulmonary TB who have an Xpert Ultra trace positive result on the initial test 8. In adults in the general population who had either signs or symptoms of TB or chest radiograph with lung abnormalities or both 9. In adults in the general population who had either a positive TB symptom screen or chest radiograph with lung abnormalities or both,
- 12. Made in India:Truelab by Molbio • Truenat MTB, Truenat MTB Plus, and Truenat MTB-Rif Dx (Molbio Diagnostics, Goa, India) • chip-based, micro real-time PCR-based assays for TB detection • 1 hour on the portable Truelab platform (Molbio Diagnostics). • more affordable alternative to Xpert • Truenat MTB and Truenat MTB Plus = M. tuberculosis bacilli in sputum • Truenat MTB-Rif Dx >>optional add-on chip = RIF resistance detection • Truelab, which comes in Uno-, Duo-, and Quattro-throughput formats and POC friendly, as it has a dust filter and runs in temperatures up to 30°C, but multiple micropipetting steps necessitate a trained technician for its operation.
- 13. Recommendations on TruenatMTB or MTB Plus as initial tests (WHO) • In adults and children with signs and symptoms of pulmonary TB • In adults and children with signs and symptoms of pulmonary TB and a Truenat MTB or MTB Plus positive result>>Truenat MTB-RIF Dx
- 15. Diagnostic accuracy ofMolbio Truenat MTB MTB-RIF Dx Molbio Truenat MTB Plus Molbio Truenat MTB
- 16. Abbott Molecular 1. Mycobacteriumtuberculosis detection (Mtb) (RealTime MTB test) 2. both rifampicin and isoniazid resistance (RealTime MTB RIF/INH). • TB detection targets = both the IS6110 genetic element and the pab gene. • The rifampicin and isoniazid resistance test uses eight dye-labelled probes to detect variants in the rifampicin resistance determining region (RRDR) of the rpoB gene and four probes to detect isoniazid resistance, with two probes each for the katG and inhA genes. • limit of detection (LoD) of 17 cfu/mL for the RealTime MTB assay and of 60 colony forming units (cfu)/mL for the RealTime RIF/INH assay • test is performed on the m2000 platform, m2000sp for automated DNA extraction and m2000rt for the real-time PCR
- 18. Becton Dickinson (BD) •multiplexed real-time PCR (BD MAX™ MDR-TB) NAAT • MTB = IS6110 and IS1081 • Rifampicin resistance = RRDR codons rpoB gene; • Isoniazid resistance = both the inhA promoter region and katG gene • LoD reported by the company is 0.5 cfu/mL for Mtb detection and 6 cfu/mL for resistance detection • Runs 24 samples at a time • BD MAX platform, with the DNA automatically extracted and real-time PCR performed.
- 20. Line probe assays assaysinclude • GenoType MTBDRplus (Hain Lifesciences-Bruker, Nehren, Germany) and Nipro NTM+MDRTB II (Osaka, Japan). New-generation LPAs (higher sensitivity): • GenoType MTBDRsl version 2.0; Hain Lifesciences-Bruker) can detect mutations associated with fluoroquinolones (FLQs) and second-line injectables, kanamycin, amikacin, and capreomycin • recommended to guide MDR-TB treatment initiation
- 21. LPA Procedures: KeySteps DNA extraction: a. Clinical pulmonary specimens (decontaminated) b. Cultured isolates (solid or liquid cultures) Amplification Reverse Hybridization Analysis
- 22. MTBDRplus Assay: Testsfor Mutations in INH and Rifampin rpoB: If mutation present, this may correlate with resistance to Rifampin inhA: If mutation present, this may correlate with resistance to INH (low level resistance) katG: If mutation present, this may correlate with resistance to INH (high level resistance)
- 23. Attributes and advantages •Highly sensitive and specific (98.9%, 100%) • Detects both Rifampicin and Isoniazid • Meant for low and high throughput labs • Short Turn around time of 5hrs • 48 samples can be processed simultaneously • Affordable – ~$10 per test Disadvantages • Cannot be used as point of care test • Requires biosafety facilities • Does not eliminate need of culture and microscopy • Only for smear positive sample • Sensitivity to detect INH resistance is low Line probe assays
- 25. Loop-mediated isothermal amplification •A commercial molecular assay, the Loopamp™ Mycobacterium tuberculosis complex (MTBC) detection kit (Eiken Chemical Company, Tokyo, Japan), • Loop-mediated isothermal amplification (LAMP) reaction. • manual assay that requires less than 1 hour to perform and can be read with the naked eye under UV light. • little infrastructure and is relatively easy to use, rapid diagnostic test, high throughput, no sophisticated instruments, and has biosafety requirements similar to those of sputum-smear microscopy
- 26. Recommendations on LAMP(WHO) • in adults with signs and symptoms consistent with TB • in adults with signs and symptoms consistent with pulmonary TB, especially when further testing of sputum smear-negative specimens is necessary.
- 29. Alere Determine TBLAM Ag tests (AlereLAM) • immunocapture assay • detects LAM antigen in urine, • lipopolysaccharide present in mycobacterial cell walls released from metabolically active or degenerating bacterial cells during TB disease • Recommendations : diagnosis of active TB in HIV- positive adults, adolescents and children • 60 µL of urine to the test strip and incubating at room temperature for 25 minutes
- 30.
- 31. GeneXpert Omni andother point-of-care devices. • POC GeneXpert Omni platform is a long-awaited development, as it will permit the use of Xpert MTB/RIF and Ultra assays in decentralized locations (e.g., primary care centers) • 2-day battery life and no tablet or computer requirement • Q-POC from QuantuMDx (Newcastle-upon-Tyne, United Kingdom) >>less than 30 min>> combination with oral swabs as a sample
- 32. Next-generation sequencing • promisingoption for comprehensive DST for TB • Faster results • provide detailed and accurate sequence information for whole genomes, as with whole-genome sequencing (WGS), or multiple gene regions of interest, as with targeted NGS
- 33. Target sequencing • DeeplexMyc-TB (Genoscreen, Lille, France) ultradeep sequencing of 24-plex amplicon mixes for mycobacterial species identification, genotyping, and DST>>can detect heteroresistance • DR-TB is DeepChek-TB (Translational Genomics Research Institute, Flagstaff, USA)
- 34. Next-generation sequencing Strengths andlimitations of WGS versus targeted sequencing via next-generation sequencing
- 35. TECHNOLOGY METHOD INTENDEDUSE SENSITIVTY % SPECIFICITY % TARGET TAT (hrs) Xpert MTB/RIF 2010 qPCR MTB diagnosis and RIF resistance detection 85 (pooled), 96 (RIF resistance) 99 (MTB detection) 98 (RIF resistance) DL/SDL <2 Xpert MTB/RIF ultra 2017 qPCR/melting temperature analysis (RIF resistance) MTB diagnosis and RIF resistance detection 90 (pooled), 94 (RIF resistance) 96 (MTB detection), 98 (RIF resistance) DL/SDL <2 First-line probe assays (e.g., GenoType MTBDRplus and NIPRO) 2008 PCR, hybridization Diagnosis of RIF and INH resistance 98 (RIF resistance), 84 (INH resistance) 99 (RIF resistance), >99 (INH resistance) RL 5 Second-line probe assays (e.g., GenoType MTBDRsl) 2016 PCR, hybridization Diagnosis of FLQ and SLID resistance 86 (FLQ resistance), 87 (SLID resistance) 99 (FLQ resistance), 99 (SLID resistance) RL 5
- 36. TECHNOLOGY METHOD INTENDEDUSE SENSITIVTY % SPECIFICITY % TARGET TAT (hrs) Loop amp MTBC assay 2016 Loop-mediated isothermal amplification MTB diagnosis 78 (pooled) 98 (MTB detection) PL <2 Truenat MTB plus 2020 Micro RT-PCR MTB diagnosis 80 (pooled) 96 (RIF resistance) PL <2 Truenat MTB-RIF Dx 2020 Micro RT-PCR Diagnosis of RIF resistance 84 (RIF resistance) 97 (RIF resistance) PL <2
- 37.
Editor's Notes
- #5 Ultra test cartridges have a larger chamber for DNA amplification than Xpert MTB/RIF and two multicopy amplification targets for TB, namely, IS6110 and IS1081