BackgroundThe 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.Methodology/Principal FindingsThe assay showed a diverse range of sensitivity and specificity, 91.26% [95% CI, 84–96] and 95.5% [95% CI, 87–99] for FQ (PPV ∼97% & NPV ∼ 87.67%), 56.19% [95%CI, 46–66] and 81% [95%CI, 66–91] for EMB (PPV ∼ 88.06% & NPV ∼ 43.21%) and 100% for SLD. Diagnostic accuracy for FQ, SLD and EMB was 94%, 100% and 63.51%, respectively. 1.17% (2/170) were heteroresistance strains, where the heteroresistance was linked to rrs gene. A varying rate of validity was observed 100% (170/170) for FQ, 94.11% (160/170) for EMB, 88.23% (150/170) for SLD.Conclusions/SignificanceGenotype MTBDRsl is simple, rapid, economical assay that can be used to detect commonly known resistance associated with Fluoroquinolone, second line injectable drugs and ethambutol. The assay detects the targeted resistance in less time as compared to phenotypic DST. But due to low NPV to FQ (88%) and EMB (43.21%), the assay results must be interpreted in coordination with the phenotypic DST.

BackgroundControl of the global Tuberculosis (TB) burden is hindered by the lack of a simple and effective diagnostic test that can be utilized in resource-limited settings.MethodsWe evaluated the performance of Truenat MTB™, a chip-based nucleic acid amplification test in the detection of Mycobacterium tuberculosis (MTB) in clinical sputum specimens from 226 patients with suspected pulmonary tuberculosis (TB). The test involved sputum processing using Trueprep-MAG™ (nanoparticle-based protocol run on a battery-operated device) and real-time PCR performed on the Truelab Uno™ analyzer (handheld, battery-operated thermal cycler). Specimens were also examined for presence of MTB using smear microscopy, liquid culture and an in-house nested PCR protocol. Results were assessed in comparison to a composite reference standard (CRS) consisting of smear and culture results, clinical treatment and follow-up, and radiology findings.ResultsBased on the CRS, 191 patients had “Clinical-TB” (Definite and Probable-TB). Of which 154 patients are already on treatment, and 37 were treatment naïve cases. Remaining 35 were confirmed “Non-TB” cases which are treatment naïve cases. The Truenat MTB test was found to have sensitivity and specificity of 91.1% (CI: 86.1–94.7) and 100% (CI: 90.0–100) respectively, in comparison to 90.58% (CI: 85.5–94.3) and 91.43% (CI: 76.9–98.2) respectively for the in-house nested PCR protocol.ConclusionThis preliminary study shows that the Truenat MTB test allows detection of TB in approximately one hour and can be utilized in near-care settings to provide quick and accurate diagnosis.

Objective To correlate rrs and eis promoter mutations, found in Mycobacterium tuberculosis (MTB) isolates,with corresponding Minimum Inhibitory Concentrations (MICs) of amikacin (AMK), kanamycin (KAN), and capreomycin (CAP). Methods Ninety MTB clinical isolates were analyzed in this study. MICs were determined by MGIT 960 for 59 isolates with resistance-associated mutations in the rrs and eis promoter gene regions and 31 isolates with wild-type sequences, as determined by the GenoTypeMTBDRsl (version 1) assay. Results The rrs A1401G mutation was identified in 48 isolates resistant to the second line injectables. The eis promoter mutations C-14T (n=3), G-10C (n=3), G-10A (n=3), and C-12T (n=2) were found within 11 isolates with various resistance profiles to the second line injectables. Thirty-one isolates had wild-type sequences for the rrs and eis promoter gene regions of interest, one of which was AMK, KAN and CAP-resistant. Isolates with the A1401G rrs mutation had AMK, KAN, and CAP MICs of >40, >20, and 5–15mg/L, respectively. Isolates with eis promoter mutations had AMK, KAN, and CAP MICs of 0. 25–1. 0, 0. 625–10, and 0. 625–2. 5mg/L, respectively. Conclusions This study provides a preliminary basis for the prediction of phenotypic resistance levels to the second line injectables based upon the presence of genetic mutations associated with AMK, KAN and CAP-resistance. Results suggest that isolates with eis promoter mutations have consistently lower resistance levels to AMK, KAN, and CAP than isolates with the rrs A1401G mutation.

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. A total of 113 consecutive, non-duplicate carbapenem-resistant clinical isolates were collected from July to December 2012. These isolates were subjected to the modified Hodge test (MHT) for phenotypic detection of carbapenemases, an inhibitor-based test employing EDTA for the detection of metallo-β-lactamase (MBL), and phenylboronic acid for the detection of Klebsiella pneumoniae carbapenemase (KPC). A multiplex polymerase chain reaction (PCR) assay that characterized the five most predominant carbapenemases (bla NDM, bla OXA, bla VIM, bla IMP, bla KPC) was designed. The 113 isolates consisted of Klebsiella spp. (46), Enterobacter spp. (32), Escherichia coli (31), Citrobacter spp. (2), Proteus spp. (1), and Morganella spp. (1). bla NDM-1 was the most prevalent carbapenemase and accounted for 75.22 % (85/113) of the isolates. This was followed by bla OXA [4.42 % (n = 5)]. 18.5 % (21/113) of the isolates possessed dual carbapenemase genes. 98.9 % concordance was observed between the phenotypic tests and the molecular tests for the detection of MBL. In conclusion, patients infected with resistant bacteria require early appropriate antimicrobial treatment for good clinical outcome. Thus, identifying the resistant mechanisms of suspected pathogens becomes crucial. Also, the high incidence of plasmid-mediated bla NDM-1 calls for the implementation of strict infection control and contact isolation precautions in order to prevent the spread of these organisms.

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