Download to read offline
![Xpert MTB/RIF Ultra assay
• The xpert MTB/RIF ultra assay (hereafter called xpert ultra) uses the same genexpert platform as
the xpert MTB/RIF test,
• Developed to improve the sensitivity and reliability of detection of MTBC and RIF resistance.
• To address sensitivity, xpert ultra uses two multicopy amplification targets
• (IS6110 and IS1081) and a larger PCR chamber; thus, xpert ultra has a lower LOD than xpert
MTB/RIF (16 colony forming units [cfu]/ml and 131 cfu/ml, respectively).
Ref : WHO operational handbook on tuberculosis Module 3](https://image.slidesharecdn.com/newerdiagnosticmodalityintuberculosis-240330160804-20c1ac03/75/Newer-Diagnostic-Modality-in-Tuberculosis-pptx-21-2048.jpg)
![Other Automated NAAT
• Moderate complexity automated NAATs
• The moderate complexity automated NAATs class of tests includes rapid and
accurate tests for the detection of pulmonary TB from respiratory samples.
• Overall pooled sensitivity for TB detection was 93.0% (95% confidence interval
[CI]: 90.9–94.7%) and specificity 97.7% (95% CI: 95.6–98.8%) .
• Moderate complexity automated NAATs are also able to simultaneously detect
resistance to both RIF and INH, and are less complex to perform than phenotypic
DST and LPAs.
Ref : WHO operational handbook on tuberculosis Module 3](https://image.slidesharecdn.com/newerdiagnosticmodalityintuberculosis-240330160804-20c1ac03/75/Newer-Diagnostic-Modality-in-Tuberculosis-pptx-25-2048.jpg)
More Related Content
Similar to Newer Diagnostic Modality in Tuberculosis.pptx
![HIGH ALTITUDE PULMONORY EDEMA (HAPE)[2305843009213699724].pptx](https://cdn.slidesharecdn.com/ss_thumbnails/highaltitudepulmonoryedemahape2305843009213699724-230505035742-4455e82f-thumbnail.jpg?width=640&height=640&fit=bounds)
![Rheumatic Fever CASE PRESENTATION [Autosaved].pptx](https://cdn.slidesharecdn.com/ss_thumbnails/casepresentationautosaved-251123182512-9d9b0da4-thumbnail.jpg?width=640&height=640&fit=bounds)
Newer Diagnostic Modality in Tuberculosis.pptx
- 1. Introduction : Burdenand Need for Newer Modalities • Significant public health problem, • 10 million TB cases ( 2019 ) • 7.1 million reported to have been diagnosed and notified • The relative gap was even larger for MDR/RR-TB in 2019, • 0.5 million new cases of MDR/RR-TB • 206030 were detected and notified • Drug-resistant TB (DR-TB) is another area of concern, • The effective management of TB relies on • the rapid diagnosis of TB, • rapid detection of drug resistance and • prompt initiation of an effective treatment regimen. Ref : WHO operational handbook on tuberculosis Module 3 , 2021
- 2. Introduction : Burdenand Need for Newer Modalities.. • WHO new definition of XDR TB to improve treatment of drug resistance TB needs advanced methods to detect additional drug resistance : • MDR TB : No change • Pre XDR TB : MDR and RR plus Fluoroquinolones • XDR TB : MDR and RR plus Fluoroquinolone plus atleast one of group A drugs (levofloxacin, moxifloxacin, bedaquiline and linezolid) • Thus, there is a need for access to fast and accurate detection tests and rapid and accurate drug- susceptibility testing (DST) for all TB patients Ref : WHO operational handbook on tuberculosis Module 3 , 2021
- 3. An overview ofConventional methods : Microscopy Ziehl – Neelsen stained light microscopy • Rapid and cost effective • Need 10,000 bacilli per ml for detection • Sensitivity : 45-80% and depends on organism burden and no of samples examined , HIV status • Specificity : > 90% • Positive predictive value : 50 – 80% Ref : Department of Laboratory Medicine, University of California, San Francisco. https://microbeonline.com/auramine-rhodamine-fluorochrome-staining-principle-procedure-results-limitations/
- 4. Microscopy… • Auramine Rhodainestaining with fluorescent microscopy • Primary stain: auramine rhodamine solution binds to mycolic acid of MTB • Decolorizer: 0.5% acid alcohol • Counter stain: 0.5% pottassium permanganate • Advantages… • For large no of samples need to be examined • 10X more sensitive than ZN staining • Disadvantage… • Costly
- 6. Overview of conventionalmethod : Detection of TB infection • Tuberculin skin test • For the diagnosis of active TB • Sensitivity : 94 %, • Specificity : 88% • Designed to support diagnosis of TB infection but not to establish active disease • A negative result doesnot rule out the disease • Ref : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100264/
- 7. An overview ofConventional methods : culture • Conventional culture methods • Highly sensitive tool for detection of tuberculosis • Detects upto 10 bacteria/ml • Sensitivity : 80 % • Specificity : 98% • 3 types of conventional culture media • Lowenstein-Jensen • Egg based medium • Middlebrook 7H10 or 7H11 • Agar based medium • Middlebrook 7H12 • Liquid medium • Growth on Liquid media is faster (1-3 week vs 3-8 weeks ) • Comparative study of a biphasic culture system (Roche MB Check system) with a conventional egg medium for recovery of mycobacteria. Aichi Mycobacteriosis Research Group.
- 8. An overview ofConventional methods : Measurement of ADA level • ADA is an essential enzyme of purine catabolic pathway catabolising Adenosine to Inosine • Increased in TB as stimulation of T cell by tubercular antigen • Serum ADA is not very useful as high false positive rate as in : • Infectious mononucleosis • Enteric fever • Liver disease • Sarcoidosis , RA • Leukemia and other malignancies • Very low ADA level seen in SCID • ADA in body fluids other than serum has high sensitivity • Pleural fluid : at 0.71 mu kat/L sensitivity of 100% ; specificity of 92% • CSF : at level 0.15 mu cat/L sensitivity of 100 % and specificity of 99% • Ref : https://pubmed.ncbi.nlm.nih.gov/2720966/
- 9. Recent Advances : Microscopy •Light emitting diode fluorescence microscopy Method for detection of TB infection • IGRA Culture • BACTEC radiometric system (BACTEC TB- 460) • Mycobacteria growth indicator tube (MGIT) system (Manual) • Fully automated MGIT system (BACTEC MGIT 960) • FAST Plaque
- 10. Recent Advances : Moleculartest • Xpert MTB/RIF assay • Xpert MTB/RIF Ultra assay • Truenat MTB, MTB Plus and MTB-RIF Dx assays • Moderate complexity automated NAATs • Low complexity automated • Line probe Assay • TB LAMP • High complexity reverse hybridization NAAT • Whole Genome Sequencing Other • Lateral flow – Lipoarabinomannan Assay (LF - LAM)
- 11. Recent advances :Light emitting diode fluorescence microscopy • Meta analysis of 12 studies involving LED-FM showed • Pooled sensitivity of 66.9% and • Pooled specificity of 96.8% • Less expensive and atleast equal or more sesnsitive than conventional microscopy • WHO recommended conventional fluorescent microscopy to be replaced by LED-FM • Advantages: • Increased performance • Increased lamp life time • Reduced initial , operating and maintainance costs • No need for dark room • European respiratory journal : https://erj.ersjournals.com/content/26/2/339
- 12. Recent advances :Method for detection of TB infection • IGRA • Sensitivity 93%, and the specificity 95% • Similar to TST, IGRA is used to support the diagnosis of TB infection but not to establish the active disease ; • Have advantage : • higher specificity, • better correlation with previous exposure to M. Tuberculosis, • low cross-reaction due to BCG vaccination or previous exposure to NTM • Review article on NCBI website : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100264/
- 14. Recent Advances inCulture methods : • Its introduction in 1980s was a breakthrough since it allowed the detection of M. tuberculosis in a few days compared with weeks in the conventional culture media • Radometric method • When bactec vial is inoculated , mycobacterium if present utilises 14C laballed palmitic acid and release 14CO2 • Reports in term of growth index and >10 is significant • Recovery rate for BACTEC TB 460 was 39% vs 29 % for LJ media and average recovery time 13.3 days to 31.2days • https://pubmed.ncbi.nlm.nih.gov/17377350/ BACTEC radiometric system (BACTEC TB-460)
- 15. Mycobacteria growth indicatortube (MGIT) system • Principle : A fluoroscent compound which is sensitive to O2 is embedded in silicone on the bottom of the tube . the actively respiring microorganisms consumes the 02 and allow the fluorescence to be observed using UV trans illuminator lamp • In one study • Bactec 460 system : 201 isolates • BACTEC MGIT 960 system : 190 isolates • Lowenstein-jensen medium : 168 isolates • Mean time to isolate : the shortest times to detection were obtained with the BACTEC MGIT 960 system (13.3 days); 1.5 days earlier than that with the BACTEC 460 system (14.8 days) and 12 days earlier than that with lowenstein-jensen medium (25.6 days). • IT is a nonradiometric, noninvasive system with the tubes incubated in a compact system that reads them automatically • https://journals.asm.org/doi/full/10.1128/JCM.37.11.3578-3582.1999
- 17. FASTPlaque • Bacteriophage basedsystem • in a comparative study with auramine smear microscopy and LJ medium in 1,692 sputum specimens found that : • FASTPlaque TB test detected TB in 75% of culture-confirmed cases and 70% of cases with a clinical diagnosis of TB, with a specificity of 98.7% and 99.0% , respectively. • In contrast, the concentrated auramine smear microscopy had a sensitivity of 63.4% and 61.3% and a specificity of 97.4% and 97.3% in culture- confirmed and all cases, respectively
- 18. PCR : aquick review
- 19. Molecular test :Xpert MTB/RIF assay (CBNAAT) • WHO endorsed Cadridge based closed real time PCR for • Detection of MTB and • RIF Resistance • Amplifies a 192 bp region of MTB rpoB gene • can detect upto 131 bacteria / ml of sample • Sensitivity : 99% • Specificity: 99% • Time to detect : 2 hr • Diagnosis of smear negative tuberculosis is greatly improved by Gene Xpert • For extrapulmonary tuberculosis Xpert MTB/Rif is the best test to diagnose in many study but is suboptimal in pauci bacillary Tb infection
- 21. Xpert MTB/RIF Ultraassay • The xpert MTB/RIF ultra assay (hereafter called xpert ultra) uses the same genexpert platform as the xpert MTB/RIF test, • Developed to improve the sensitivity and reliability of detection of MTBC and RIF resistance. • To address sensitivity, xpert ultra uses two multicopy amplification targets • (IS6110 and IS1081) and a larger PCR chamber; thus, xpert ultra has a lower LOD than xpert MTB/RIF (16 colony forming units [cfu]/ml and 131 cfu/ml, respectively). Ref : WHO operational handbook on tuberculosis Module 3
- 22. MTB/RIF MTB/RIF Ultra ResistanceDetects Rifampicin Resistance Detects Rifampicin Resistance Amplification for TB detection Single target : rpoB core region Multicopy target : rpoB core region ; Insertion elements Resistance deterction Real time PCR 5 probes binds to RpoB gene Melting curve 4 probe binds to RPOB genes Sputum input 2 ml 2 ml Assay time 112 min 65-87 min Limit of detection 131 cfu/ml 16cfu/ml Cost 9.98 usd 9.98 usd
- 23.
- 24. Truenat MTB, MTBPlus and MTB-RIF Dx assays • The Truenat MTB and MTB Plus assays use chip-based real-time micro PCR for the semiquantitative detection of MTBC directly from sputum specimens • can report results in under an hour. • The assays use automated, battery operated devices to extract, amplify and detect specific genomic DNA loci. • Truenat MTB-RIF Dx may be used as an initial test for RIF resistance in cases with Truenat MTB , MTB plus positive cases Ref : WHO operational handbook on tuberculosis Module 3
- 25. Other Automated NAAT •Moderate complexity automated NAATs • The moderate complexity automated NAATs class of tests includes rapid and accurate tests for the detection of pulmonary TB from respiratory samples. • Overall pooled sensitivity for TB detection was 93.0% (95% confidence interval [CI]: 90.9–94.7%) and specificity 97.7% (95% CI: 95.6–98.8%) . • Moderate complexity automated NAATs are also able to simultaneously detect resistance to both RIF and INH, and are less complex to perform than phenotypic DST and LPAs. Ref : WHO operational handbook on tuberculosis Module 3
- 26. Other Automated NAAT… •Low complexity automated • This test uses a cartridge designed for the GeneXpert instrument to detect resistance to INH, FQs, ETO and second-line injectable drugs (AMK, kanamycin and capreomycin) • Follow on test to determine resistance once MTB is confirmed • The overall pooled sensitivity for detection of INH resistance was 94% and specificity was 98% Overall pooled sensitivity for detection of FQ resistance was 93 % and specificity was 98% Ref : WHO operational handbook on tuberculosis Module 3
- 27. Other Automated NAAT… •High complexity reverse hybridization NAAT • Useful for the detection of resistance to PZA. • Based on the same principle as the FL-LPA and SL-LPA but requires the use of a large number of hybridization probes to cover the full pncA gene (>700 base pairs ). • The overall pooled sensitivity for the detection of PZA resistance was 81.2% and the pooled specificity was 97.8% • WHO recommendation • Used on M. tuberculosis culture isolates for detection of PZA resistance, rather than culture-based phenotypic DST Ref : WHO operational handbook on tuberculosis Module 3
- 28. Line Probe Assay •LPAs are a family of DNA strip-based tests that detect mutations associated with drug resistance. • They do this either directly, through binding DNA amplification products (amplicons) to probes targeting the most commonly occurring mutations (MUT probes), or indirectly, inferred by the lack of binding the amplicons to the corresponding wild-type probes. Ref : WHO operational handbook on tuberculosis Module 3
- 29. LPA… • First-line LPAs •Allow the detection of resistance to RIF and INH. • WHO recommendation • For people with a smear-positive sputum specimen or a cultured isolate of MTBC, commercial LPAsmay be used as the initial test instead of phenotypic DST to detect resistance to RIF and INH. • FL-LPAs are not recommended for the direct testing of sputum-smear-negative specimens for the detection of MTBC. Ref : WHO operational handbook on tuberculosis Module 3
- 30. SL- LPA • Second-lineLPAs (SL-LPAs) • Allow the detection of resistance to FQS and AMK. • Who recommendation : • For patients with confirmed MDR/RR-TB, an SL-LPAs may be used as the initial test, instead of phenotypic dst, to detect resistance to fqs and amk. Ref : WHO operational handbook on tuberculosis Module 3
- 32. Loop Mediated IsothermalAmplification (LAMP) • Directly from sputum specimens. • Manual assay that provides results in less than 1 hour, • Advantages : • Does not require sophisticated instrumentation and • Can be used at the peripheral health centre level, given biosafety requirements similar to those for sputum-smear microscopy. • Disadvantages • TB-LAMP does not detect resistance to anti-tb drugs. Ref : WHO operational handbook on tuberculosis Module 3
- 33. LAMP… • For thedetection of TB in adults with signs and symptoms consistent with pulmonary TB , sensitivity of 0.78 specificity of 0.98 ( ref :microbiological reference standard.) • Who recommendation • May be used as a replacement test for sputum-smear microscopy for diagnosing pulmonary TB in adults with signs and symptoms consistent with TB. • May be used as a follow-on test to smear microscopy in adults with signs and symptoms consistent with pulmonary tb, especially when further testing of sputum-smear negative specimens is necessary • Ref : WHO operational handbook on tuberculosis Module 3
- 34.
- 35. Whole Genome Sequencing •Culture Isolates of M tuberculosis is used • Genome read in small sections • Sections compared with database Study shown high Concordance in Results with Xpert MTB RIF with Benefits of additional information like PZA - DST
- 36. Lateral flow –Lipoarabinomannan Assay (LF - LAM) • Immunocapture assay based on the detection of the mycobacterial LAM antigen in urine; • A potential point-of care test for certain populations being evaluated for TB. • Although the assay lacks sensitivity, it can be used as a fast, bedside, rule-in test for HIV-positive individuals, especially in urgent cases where a rapid TB diagnosis is critical for the patient’s survival. • . The detection of mycobacterial LAM antigen in urine does not provide any information on drug resistance. Ref : WHO operational handbook on tuberculosis Module 3
- 37. LF –LAM… • WHOrecommendation in Inpatient Setting to assist in diagnosis of active TB in HIV-positive adults, adolescents and children: – • With signs and symptoms of TB (pulmonary or extrapulmonary); – • With advanced HIV disease; – • Who are seriously ill; or – • Who have a CD4 cell count of less than 200 cells/mm3 , irrespective of signs and symptoms of TB. • WHO recommendation in Outpatient Setting to assist in diagnosis of active TB in HIV-positive adults, adolescents and children : – • Have signs and symptoms of TB (pulmonary or extrapulmonary); – • Are seriously ill; or – • Have a CD4 cell count of less than 100 cells/mm3 , irrespective of signs and symptoms of TB. Ref : WHO operational handbook on tuberculosis Module 3
- 38.
- 39. Newer Modalities practicedin Nepal • Xpert MTB/Rif • LPA • Cultures • LAMP
- 40. Diagnostic algorithm oftuberculosis in NEPAL : quick review
- 41.
- 42.