Targeted Next Generation Sequencing (tNGS) for detection of drug-resistant tuberculous meningitis: Is this sequencing technology ready for prime time?

Purpose: Tuberculous meningitis (TBM) is the most severe form of tuberculosis (TB). Difficulty in diagnosing the condition along with other factors, increases its potential for high morbidity and mortality. Targeted Next Generation Sequencing (tNGS) generates high quality sequence read depths, enabling the identification of low-frequency alleles linked to Drug resistance (DR).

Morbidity and Mortality of the Transmetatarsal Amputation: A Comparative NSQIP Analysis.

The objective of this investigation was to compare the morbidity and mortality of transmetatarsal amputation to other frequently performed surgical procedures utilizing a large US database. The American College of Surgeons National Surgical Quality Improvement Program (NSQIP) database was interrogated for the purposes of this investigation. We initially extracted data related to the Current Procedural Terminology (CPT) code 28805 (amputation, foot; transmetatarsal) and the variable labels "estimated probability of morbidity" and "estimated probability of mortality.

Targeted next generation sequencing directly from sputum for comprehensive genetic information on drug resistant Mycobacterium tuberculosis.

Background: Timely drug resistance detection is essential to global tuberculosis management. Unfortunately, rapid molecular tests assess resistance to only a few drugs, with culture required for comprehensive susceptibility test results.

Methods: We evaluated targeted next generation sequencing (tNGS) for tuberculosis on 40 uncultured sputum samples.

Evaluation of CSF pyrosequencing to diagnose tuberculous meningitis: A retrospective diagnostic accuracy study.

Background: We evaluated the performance of pyrosequencing, a genotypic test which detects TB and XDR-defining mutations within 6 h, directly on CSF samples for diagnosing TB meningitis(TBM).

Methods: This retrospective, diagnostic accuracy study was conducted in Hinduja hospital, Mumbai from May-2017 to May-2019. 107 consecutive patients with physician-suspected TBM for whom CSF pyrosequencing was requested were screened.

Diagnosis and Management of Osteoarticular Tuberculosis: A Drastic Change in Mind Set Needed-It is Not Enough to Simply Diagnose TB.

Background: In the era of increasing drug resistance in pulmonary tuberculosis (TB), it is prudent to assess causes of poor response to anti tubercular therapy (ATT) and drug sensitivity pattern (DSP) in osteoarticular TB.

Materials And Methods: As a part of Bombay Orthopaedic society's research project, members were asked to refer non responders to ATT to our institute. Cases were enrolled from October 2010 to March 2014.

Interpreting very low detected on Xpert /rifampicin.

The development and rollout of the Xpert Mycobacterium tuberculosis/rifampicin assay for the GeneXpert platform is considered an important breakthrough in the fight against tuberculosis. Xpert though robust is known to have issues that occur with very low load of tuberculosis detection, wherein it is recommended to confirm resistance if resistance is not suspected using another genotypic test.

Multicentre study to establish interpretive criteria for clofazimine drug susceptibility testing.

To conduct a multicentre study to establish the critical concentration (CC) for clofazimine (CFZ) for drug susceptibility testing (DST) of on the MGIT™960™ system using the distribution of minimum inhibitory concentrations (MIC) and genotypic analyses of mutations. In phase I of the study, the MIC distribution of laboratory strains (H37Rv and in vitro-selected mutants) and clinical pan-susceptible isolates were determined ( 70). In phase II, a tentative CC for CFZ ( 55) was proposed.

Multiplex PCR to detect pAmpC β-lactamases among enterobacteriaceae at a tertiary care laboratory in Mumbai, India.

Drug-resistance due to AmpC β-lactamases represents a growing problem worldwide. In this study, a previously collected sample of 108 cefoxitin-resistant clinical isolates was assessed for AmpC β-lactamase production through routine phenotypic testing and double-disc cefoxitin/cloxcallin (DD-CC), cefoxitin/phenylboronic acid (CDT-PBA) and AmpC disc tests. The same isolates were characterized by a novel multiplex polymerase chain reaction molecular assay to detect the presence of blaACT, blaDHA, blaCIT, blaFOX, blaMIR and blaMOX.

Utility of pyrosequencing for rapid detection of tubercular meningitis (TBM) and associated susceptibility directly from CSF specimens.

Tubercular meningitis (TBM) is a serious form of tuberculosis (TB). The diagnosis of TBM & susceptibility/resistance is difficult as TB MGIT culture lacks sensitivity & timeliness. Timely and accurate diagnosis of the TBM is the need of the hour for initiation of appropriate therapy.

Evaluation of pyrosequencing for extensive drug resistance-defining anti-tuberculosis drugs for use in public healthcare.

MGIT 960 drug susceptibility testing (DST) for Mycobacterium tuberculosis was compared for performance and speed with pyrosequencing (PSQ). Pulmonary samples (n = 100), from GeneXpert/MTB/Rifampicin-resistant patients receiving second-line treatment for 1-3 months, were subjected to DST and PSQ for seven drugs (isoniazid, rifampicin, kanamycin, amikacin, capreomycin, moxifloxacin, and ofloxacin). The mean time-to-result was 35 and two days for DST and PSQ, respectively.

Pyrosequencing to resolve discrepant Xpert MTB/RIF and Mycobacterial Growth Indicator Tube 960.

Delayed diagnosis of drug resistance has been a major obstacle to proper management and control of drug-resistant tuberculosis (TB). Expanded access to rapid molecular diagnostics such as Xpert MTB/RIF has been helpful, but has generated confusion about how to interpret genotype-phenotype discordance. Optimal management is not clearly defined for patients with rifampin resistance by Xpert MTB/RIF but rifampin susceptibility by phenotypic testing.

A performance evaluation of MTBDRplus version 2 for the diagnosis of multidrug-resistant tuberculosis.

Objective: To evaluate the performance of a recently updated rapid molecular diagnostic test, GenoType® MTBDRplus version 2, designed to detect drug resistance in both acid-fast bacilli (AFB) smear-negative and -positive specimens.

Design: Sputum samples from 1128 patients at risk for multidrug-resistant tuberculosis (MDR-TB) were tested using MTBDRplus v2 and compared with reference standard MGIT™ 960™ drug susceptibility testing. The relationship of participant human immunodeficiency virus (HIV) status, diabetic status, previous treatment, and smear gradation to the likelihood of obtaining an interpretable result was assessed using logistic regression.

Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables.

Objective/background: The in vitro drug-susceptibility testing of Mycobacterium tuberculosis reports isolates as resistant or susceptible on the basis of single critical concentrations. It is evident that drug resistance in M. tuberculosis is quite heterogeneous, and involves low level, moderate level, and high level of drug-resistant phenotypes.

Performance of a pyrosequencing platform in diagnosing drug-resistant extra-pulmonary tuberculosis in India.

Setting: Pyrosequencing diagnostic assays have shown great utility in identifying and characterizing pulmonary drug-resistant tuberculosis (TB) infections. However, the method has yet to be evaluated for the diagnosis of drug-resistant extra-pulmonary TB (EPTB).

Objective: To evaluate the performance of a pyrosequencing platform in establishing molecular drug resistance profiles for 79 clinical EPTB specimens at a referral center for drug-resistant TB in India.

Redefining MTBDRplus test results: what do indeterminate results actually mean?

Background: Although line-probe assays (LPAs) are promising, little research has been conducted to elucidate the true nature of indeterminate LPA results or assess the ability of these assays to perform on a wide range of clinical samples.

Objective: To evaluate the performance of the commercially available GenoType(®) MTBDRplus LPA against conventional BACTEC™ MGIT™ 960 culture and drug susceptibility testing (DST) among 308 pulmonary tuberculosis (PTB) and 32 extra-pulmonary TB samples.

Results: Invalid LPA results (defined as those with a missing Mycobacterium tuberculosis identification band) were obtained for 18 PTB samples, which were excluded from further analysis.

Determination of MICs of levofloxacin for Mycobacterium tuberculosis with gyrA mutations.

The purpose of the present study was to correlate gyrA mutations found in Mycobacterium tuberculosis isolates using the GenoType(®) MTBDRsl assay with minimum inhibitory concentrations of the fluoroquinolone levofloxacin (LVX). Of 123 archived clinical M. tuberculosis isolates evaluated, 93 isolates had an Ala90Val, Ser91Pro, Asp94Ala, Asn/Tyr, Gly or His mutation and 30 were wild-type.

Defining multidrug-resistant tuberculosis: correlating GenoType MTBDRplus assay results with minimum inhibitory concentrations.

This study correlates MICs of rifampicin (RIF) and isoniazid (INH) with GenoType MTBDRplus assay results for drug-resistant Mycobacterium tuberculosis (MTB) clinical isolates. MICs of RIF and INH were established for 84 and 90 isolates, respectively, testing 7 concentrations of each drug. Genotypic resistance to each drug was determined by GenoType MTBDRplus assay with 50 representative mutations confirmed by pyrosequencing, with mutations in the rpoB gene associated with RIF resistance and mutations in the katG and/or inhA genes associated with INH resistance.

Correlating Minimum Inhibitory Concentrations of ofloxacin and moxifloxacin with gyrA mutations using the genotype MTBDRsl assay.

Objective: To correlate gyrA mutations found on the Genotype MTBDRsl assay in Mycobacterium tuberculosis (MTB) isolates with Minimum Inhibitory Concentrations (MICs) to the fluoroquinolones compounds ofloxacin (OFX) and moxifloxacin (MXF).

Methods: MICs for OFX and MXF were ascertained for 93 archived clinical MTB isolates that showed gyrA mutations at Ala90Val, Ser91Pro, Asp94Ala, Asn/Tyr, Gly and His. Thirty fluoroquinolones susceptible isolates as determined by presence of all wild-type gyrA bands on the Genotype MTBDRsl assay were also included.

Evaluation of pyrosequencing for detecting extensively drug-resistant Mycobacterium tuberculosis among clinical isolates from four high-burden countries.

Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance in Mycobacterium tuberculosis isolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines.

Molecular characterization of carbapenem-resistant Enterobacteriaceae at a tertiary care laboratory in Mumbai.

Carbapenem-hydrolyzing β-lactamases are increasingly reported worldwide, leading to therapeutic failure. In an era where the drug development pipeline is stagnant, it is crucial to preserve current classes of antibiotics to help fight against infection caused by multidrug-resistant organisms (MDROs), by practicing a rational approach for the use of antibiotics. Identifying the mechanisms of resistance gives us much needed insights in this field.

Second-line drug susceptibility breakpoints for Mycobacterium tuberculosis using the MODS assay.

Objective: To establish breakpoint concentrations for the fluoroquinolones (moxifloxacin [MFX] and ofloxacin [OFX]) and injectable second-line drugs (amikacin [AMK], kanamycin [KM] and capreomycin [CPM]) using the microscopic observation drug susceptibility (MODS) assay.

Setting: A multinational study conducted between February 2011 and August 2012 in Peru, India, Moldova and South Africa.

Design: In the first phase, breakpoints for the fluoroquinolones and injectable second-line drugs (n = 58) were determined.

Evaluation of genotype MTBDRsl assay to detect drug resistance associated with fluoroquinolones, aminoglycosides and ethambutol on clinical sediments.

Background: The emergence of resistant tuberculosis (TB) is a major setback to the global control of the disease as the treatment of such resistance is complex and expensive. Use of direct detection of mutations by molecular methods could facilitate rapid diagnosis of resistance to offset diagnostic delays. We evaluated the performance of the Genotype MTBDRsl (Hain Life Sciences) for the detection of second line resistant TB directly from stored smear positive sputum sediments.