Simultaneous Detection Of Rifampicin Resistance Research Articles

English

BACKGROUND In India, everyday more than 6000 people develop tuberculosis (TB) and more than 600 people die of TB (2 death every 5 minutes).1 World Health Organization (WHO) has recently endorsed cartridge based nucleic acid amplification test (CBNAAT) which has the potential to lead a revolution in the diagnosis of tuberculosis. This study intends to assess the performance of CBNAAT for the diagnosis of suspected smear negative pulmonary and extra pulmonary tuberculosis. METHODS The cross-sectional study was carried out in Department of Microbiology, Government Medical College, Thrissur, Kerala, India. CBNAAT was done in district tuberculosis center, Thrissur. The study was done from December 2016 to December 2017. Samples were sent for microscopy, culture and CBNAAT. RESULTS A total of 250 patients were evaluated. Majority of the specimens collected were sputum (61.2 %) followed by bronchial wash (17.6 %). Culture was positive in 48 specimens. CBNAAT was positive in 58 specimens. Both culture and CBNAAT were positive in 47 patients. CBNAAT was negative in 1 specimen but positive in culture test. CBNAAT detected an additional 10 samples. Taking culture as gold standard, culture positives were taken as true positives and culture negatives were taken as true negatives. Accordingly, true positive was 48, true negative was 202, false positive was 10 and false negative was 1. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were respectively 97.95 %, 95.2 %, 82.75 % and 99.5 %. CBNAAT missed out a sputum sample which was culture positive. CONCLUSIONS We found CBNAAT to be an important diagnostic modality especially in smear negative patients for early diagnosis and treatment of TB. Culture of mycobacteria is considered as a gold standard method, but it takes weeks to obtain a positive result and simultaneous detection of rifampicin resistance is not possible with this method. KEY WORDS Tuberculosis, Smear Negative TB, ZN Stain, AFB, Petroff’s Method, CBNAAT, RNTCP, Culture

EFFICACY OF GENE XPERT MTB/RIF ASSAY AND AFB CULTURE IN DIAGNOSIS OF MYCOBACTERIUM TB COMPLEX ON EBUS TBNA

SESSION TITLE: Tuesday Abstract Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: 10/22/2019 01:00 PM - 02:00 PM PURPOSE: Rationale – Tuberculosis and Sarcoidosis are two clinical entity that share same clinical, radiological and pathological features and differentiating between two might be an arduous task. As cultures for M. Tuberculosis in tissue specimens have prolonged turn-around times, rapid molecular methods such as MTB. Rif system has shown great promise in rapid diagnosis of tuberculosis through detection of DNA of M. Tuberculosis and concurrent identification of major mutation that confers rifampicin resistance. We aim to evaluate both efficacy and role of Xpert MTB / RIF Assay and Mycobacterium TB Culture for diagnosis of TB using EBUS TBNA samples in patients with intra-thoracic granulomatous lymphadenopathy. METHODS: This was a retrospective study of randomly selected 100 patients who underwent EBUS TBNA for intra-thoracic granulomatous lymphadenitis with Gene Xpert MTB / RIF Assay and diagnosed with either Tuberculosis or Sarcoidosis RESULTS: EBUS TBNA procedures were performed and a diagnosis of Sarcoidosis (n 17) or Tuberculosis (n 83) was made in 100 patients. Histological results with grades I to II were seen in 74 patients and grade III in 26 patients respectively. Necrotising granulomas were found in 26 / 83 (31.32%) while Non-necrotising granulomas were found in 57 / 83 (68.67%) patients diagnosed with Tuberculosis. Mycobacterium TB Culture was performed in 72 patients diagnosed with Tuberculosis and was positive in 11 patients – 09 / 26 Necrotising Granuloma vs 02 / 57 Non-necrotising Granuloma (34.61% vs 3.5%, P = 0.010). Gene Xpert MTB / RIF Assay were performed in 20 / 83 patients diagnosed with Tuberculosis and was positive in 4 patients – 01 / 07 Necrotising Granuloma vs 03 / 13 Non-necrotising Granuloma (14.30% vs 23.10%, P = 0.547). The Diagnostic Accuracy of Xpert MTB / RIF Assay in diagnosis of Tuberculosis was 70% (95% CI 45.72% to 88.10%), Sensitivity 40%, Specificity 100%, Disease Prevalence 50%, PPV 100%, NPV 62.50%. Of 2 patients finally diagnosed with Sarcoidosis also showed a positive result of Xpert MTB / RIF Assay which possibly suggests casual link between TB and Sarcoidosis CONCLUSIONS: Gene Xpert MTB / RIF Assay can be useful in patients with suspected Pulmonary TB either AFB smear negative or positive due to its rapid and simultaneous detection of Rifampicin resistance and especially beneficial in patients with MDR TB and HIV associated TB. Xpert MTB / RIF Assay has good sensitivity and PPV in context of culture positive intra-thoracic TB lymphadenopathy and can provide immediate diagnosis of likely drug resistance specially in highly endemic area like India CLINICAL IMPLICATIONS: Positive Xpert MTB / RIF Assay with very low MTB detection but culture negative results need to be read cautiously and should be well correlated with clinical and treatment history of patient since Gene Xpert Assay amplifies any DNA, of live or dead bacilli and required to avoid false positive result DISCLOSURES: No relevant relationships by UMANG SHAH, source=Web Response

Diagnostic accuracy of the new Xpert MTB/RIF Ultra for tuberculosis disease: A preliminary systematic review and meta-analysis

ObjectivesThe re-engineered Xpert MTB/RIF Ultra (Xpert Ultra) assay was developed due to the poor sensitivity of the Xpert MTB/RIF assay for the detection of tuberculosis (TB) in some conditions. This new assay has been recommended by the World Health Organization since 2017. A systematic review and meta-analysis was performed to assess the accuracy of Xpert Ultra for the detection of TB and rifampicin (RIF) resistance. MethodsThe Medline (via PubMed), Embase (via OvidSP), ISI Web of Science, Cochrane Central Register of Controlled Trials, and Scopus databases were screened for original articles. Summary sensitivity and specificity were calculated with a bivariate mixed-effects model. A Fagan nomogram was used to assess the clinical utility. The sources of heterogeneity were investigated by meta-regression and subgroup analyses. ResultsSixteen studies were identified. The summary diagnostic accuracy of Xpert Ultra for the diagnosis of TB were as follows: sensitivity 87.2% (95% confidence interval (CI) 82.5–90.8%) and specificity 96.5% (95% CI 95.1–97.5%). For the detection of RIF resistance, sensitivity was 95.1% (95% CI 91.6–97.2%) and specificity was 98.9% (95% CI 97.6–99.5%). Meta-regression showed that the category of population, TB prevalence, reference standard, sample state, sample type, and study design attributed to the heterogeneity. Subgroup analyses found good performance of Xpert Ultra in settings with a low TB burden. ConclusionsAs a rapid and highly sensitive test for the detection of TB and simultaneous detection of RIF resistance, Xpert Ultra exhibits a viable alternative in sensitivities in both pulmonary TB (PTB) and extrapulmonary TB (EPTB), which was proved to be higher than Xpert in the comparative analysis, and also shows a good performance in the detection of RIF resistance. Additional studies with comparative consistency tests are needed to precisely describe this finding for more forms of EPTB.

Gene-Xpert: Diagnosis of Pulmonary Tuberculosis in a Sputum Smear Negative Patient

This case report has tried to highlight the ease and benefit of Gene-Xpert testing in difficult to diagnose patient with sputum smear negative pulmonary tuberculosis. Early treatment of tuberculosis is usually delayed by lack of rapid and accurate diagnostic modalities, especially in resource-limited settings like ours. Gene-Xpert is a rapid test based on real time PCR assay and molecular technology for the detection of Mycobacterium tuberculosis. It is highly sensitive tool and enables simultaneous detection of rifampicin resistance within short period of time i,e. <2hrs. It has distinct advantage of providing same-day diagnosis which could potentially limit loss to follow up during diagnostic evaluation of smear negative tuberculosis patients.Keywords: Gene-Xpert; pulmonary tuberculosis; sputum microscopy.

Evaluation of GeneXpert MTB/RIF assay in diagnosis of extrapulmonary tuberculosis and rifampicin resistance

Background: The prevalence of tuberculosis is still on the rise particularly extrapulmonary cases. There is an urgent need for rapid diagnosis of these cases for prompt initiation of treatment. Aim: To evaluate the role of GeneXpert MTB/RIF Assay in the diagnosis of clinically suspected extrapulmonary tuberculosis (EPTB) and to detect rifampicin resistance in these cases. Materials and methods: A total of 241 samples from April 2016 to July 2018, from all clinically suspected EPTB patients with support of either laboratory or radiological evidence were included in study. 132 pleural fluid, 59 lymph node aspirate, 21 CSF, 17 pus, 9 ascitic fluid and 3 synovial fluid samples were screened for presence of acid fast bacilli (AFB) by conventional Zeihl-Neelsen (ZN) technique. The same samples were also used for testing by GeneXpert MTB/RIF Assay. Results: Out of 241 EPTB samples tested, none were positive for AFB by ZN staining. Overall 9.54 % (23 out of 241) samples were positive for MTB by GeneXpert with a positivity rate of 23.80%, 13.55%, 11.76% & 6.06% respectively for CSF, lymph node aspirate, pus and pleural fluid. MTB was not detected in any of ascitic fluid & synovial fluid samples. Of the 23 MTB positive samples detected by GeneXpert, all were sensitive to rifampicin. Conclusions: GeneXpert was found to be simple, effective and useful diagnostic method for detection of EPTB due to its rapidity & simultaneous detection of rifampicin resistance, thus reducing the time taken for initiation of treatment.

Diagnosis of Pulmonary Tuberculosis in Resource Limited Setting of Rawalpindi

Introduction:Tuberculosis is an infectious disease with a high prevalence of about 9 million cases occurring annually. Ziehl Neelsen microscopy is the most widely used technique to detect Acid Fast Bacilli, but it is less sensitive. However, fluorescent microscopy is more helpful with simple diagnostic criteria. Gene Xpert®MTB/RIF assay is a rapid molecular assay that enables diagnosis of Tuberculosis with simultaneous detection of rifampicin resistance. Owing to this fact, we aimed at evaluating the diagnostic accuracy of Ziehl Neelsen microscopy, fluorescent microscopy and Xpert MTB/RIF keeping MTB culture (Mycobacterial Growth Indicator Tube) as a gold standard for the diagnosis of tuberculosis.Methodology:This study was carried out at a tertiary care hospital of Rawalpindi in the year 2016. Patients aged 18 to 70 years irrespective of gender with suspected TB based on history, clinical and radiological examination were included in the study. Respiratory clinical specimens including sputum, Broncho-Alveolar Lavage (BAL), and endobronchial washings were collected. Specimens were processed by MGIT (MTB culture), ZN microscopy, fluorescent microscopy and Gene Xpert MTB/RIF assay.Results:A total of 352 respiratory specimens were tested among which 160 (45%) samples were positive by culture. Out of culture positive samples, 158 samples (98.7%) were GeneXpert TB positive while 2 were negative. While only 49 (30.6%) were positive on ZN microscopy and 89 (55%) were positive on fluorescent microscopy. Out of the culture negative samples, 2 were positive with ZN microscopy, one was positive with fluorescent microscopy and 3 were positive on Gene Xpert. Sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and diagnostic accuracy of ZN Smear microscopy was 39%, 99.5%, 96%, 63% and 14.5% respectively. Sensitivity, specificity, PPV, NPV and diagnostic accuracy of fluorescent smear microscopy was 55% and 99.5%, 98%, 72% and 79% respectively. Sensitivity, specificity, PPV, NPV and diagnostic accuracy of Gene XPERT was 98% and 99%, 98%, 99% and 98% respectively.Conclusion:In countries like Pakistan where Tuberculosis is endemic, the diagnostic accuracy with highest sensitivity and specificity was Gene Xpert Polymerase Chain Reaction (PCR) MTB/RIF assay which can help in well-timed diagnosis of the disease.

Tuberculosis Recommendations for Solid Organ Transplant Recipients and Donors.

Tuberculosis Recommendations for Solid Organ Transplant Recipients and Donors.

Extra pulmonary tuberculosis: rapid identification of mycobacterium tuberculosis and simultaneous detection of rifampicin resistance by genexpert assay

Extra pulmonary tuberculosis: rapid identification of mycobacterium tuberculosis and simultaneous detection of rifampicin resistance by genexpert assay

Rapid implementation of new TB diagnostic tests: is it too soon for a global roll-out of Xpert MTB/RIF?

In 2011 the World Health Organization approved Xpert MTB/RIF for tuberculosis diagnosis and recommended its rapid implementation. Xpert MTB/RIF is accurate: sensitivity is 72.5 -98.2% (smear-negative and -positive cases, respectively) and specificity 99.2%. Benefits include same-day diagnosis and simultaneous detection of rifampicin resistance. However, the test has some shortcomings and has not had time for thorough evaluation. Cost-effectiveness studies are difficult to perform and few have been completed. Existing data suggest cost-effectiveness in some, but not all, settings. The urgent need for better diagnostics is evident. Yet, serial implementation of new technologies causes ineffective spending and fragmentation of services. How new tests are incorporated into existing diagnostic algorithms affects both outcomes and costs. More detailed data on performance, effect on patient-important outcomes, and costs when used with adjunct tests are needed for each setting before implementation. While awaiting further clarification it seems prudent to slow its implementation among resource-constrained tuberculosis control programs.

Simultaneous species identification and detection of rifampicin resistance in staphylococci by sequencing of the rpoB gene

In recent years, coagulase-negative staphylococci (CoNS) have been increasingly recognised as causative agents of various infections, especially in immunocompromised patients and related to implanted foreign body materials. In this study, rpoB sequencing was used for simultaneous species identification and detection of rifampicin resistance in clinical staphylococci isolates. Forty-nine (96%) out of 51 isolates, representing 17 different Staphylococcus species according to the initial phenotypic species identification, were identified to the species level using rpoB sequencing. Furthermore, the two remaining isolates were Kocuria sp. and Corynebacterium sp. respectively, according to 16S rRNA sequencing. Comparison with the phenotypic diagnostics also revealed that 8 (16%) of the 49 isolates differed regarding identified species. Discrepant analysis confirmed the result of the rpoB sequencing for all except 2 of these isolates, which could not be distinguished as single species using 16S rRNA sequencing. Regarding detection of rifampicin resistance, isolates obtained pre- and post-treatment with rifampicin were examined. These isolates comprised S. aureus (7 patients) and S. lugdunensis (1 patient). Rifampicin resistance was mainly detected following short-term treatment with rifampicin in combination with isoxazolyl-penicillin, or long-term treatment with rifampicin and ciprofloxacin. Each rifampicin-resistant isolate displayed an identical rpoB sequence as their corresponding rifampicin-susceptible isolates except for one (n = 6) or two (n = 1) nonsynonymous single nucleotide polymorphisms, or insertion of one codon (n = 1). In conclusion, rpoB sequencing is a rapid, objective and accurate method of species identification and simultaneous detection of rifampicin resistance in staphylococci.