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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.

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.

MGIT 960 system with PNBA assay for identification of M. tuberculosis complex is a rapid and useful method in laboratories processing a large number of specimens.

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.

We screened and spoligotyped 150 consecutive phenotypically confirmed extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) isolates (January 2008 to March 2009) for rifampin, isoniazid, fluoroquinolone, and aminoglycoside resistance targeting rpoB, inhA, katG, gyrA, gyrB, and rrs. Mutations predominant among XDR-TB were S315T (katG) (100% of isolates), S531L (rpoB) (97% of isolates), D94G (gyrA) (53% of isolates), and A1401G (rrs) (71% of isolates). Spoligotyping revealed 62% of the isolates to be Beijing.The worldwide emergence of extensively drug resistant Mycobacterium tuberculosis (XDR-TB) is a major setback to tuberculosis (TB) control (10,11,12,15). XDR-TB, defined as multidrug resistant (MDR) M. tuberculosis (i.e., resistant to rifampin and isoniazid) with additional resistance to any fluoroquinolone (FQ) and to any one of three s-line injectables, i.e., capreomycin, kanamycin, or amikacin (20).The mycobacteriology laboratory of P. D. Hinduja National Hospital (PDHNH), a tertiary care center in central Mumbai, received 7,482 clinical specimens for TB culture using a mycobacterial growth indicator tube (MGIT) and Lowenstein-Jensen medium (LJ) from January 2008 to March 2009. Our laboratory has a referral bias toward nonresponders, as we mainly receive treatment failures and complicated TB cases and drug susceptibility testing (DST) is performed only on request. A total of 3,899 samples were positive for the Mycobacterium tuberculosis complex, and 2,522 patients requested DST, of which 1,640 (65%) were MDR and 150 (9.1%) were XDR-TB by the MGIT (19). We studied mutations targeting the genes conferring resistance to drugs included in the definition of XDR-TB (inhA, katG, rpoB, and gyrA [1, 5, 8, 10, 14, 22, 27] and gyrB and rrs [2,16,28]) and fingerprinted these patients' isolates by spoligotyping (6). DNA extraction from pure culture was performed by the cetyl trimethyl ammonium bromide (CTAB-NaCl) method (23). A single tube touchdown multiplex PCR for three target genes associated with resistance to isoniazid (inhA and katG) and rifampin (rpoB) was done and screened for mutations using an in-house reverse line blot hybridization (RLBH) assay (21). The screening of katG resulted in the identification of G315C (100%), the S315T mutation in katG (Table 1) conferring high-level resistance to isoniazid with an altered catalase-peroxidase activity (24). Twenty-one (14%) of these isolates showed the C15T mutation in the promoter region of the inhA gene in addition to the katG mutation. The promoter mutation was also seen among XDR-TB isolates in Portugal (18) and China (25). The S531L mutation in rpoB was more prevalent, observed in 146 (97%) of these isolates, whereas the H526Y mutation was observed only in 4 (3%) of the isolates. A previous study from this center (21) on MDR isolates revealed an array of point mutations from position 511 to position 531 in rpoB; however, the S531L mutation in rpoB dominated among XDR-TB isolates. This is similar to what was found in a study from Portugal (18), where...

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. Results gyrA mutations at Ala90Val (n=25), Ser91Pro (n=6), Asp94Ala (n=4), Asp94Asn/Tyr (n=13), Asp94Gly (n=42) and Asp94His (n=3) were observed. Isolates with mutations at Ala90Val or Ser91Pro had MIC90 of 4.0µg/ml and 1.0µg/ml for OFX and MXF, respectively, and isolates with mutations at Asp 94Ala, Asn/Tyr, Gly and His had MIC90 of 8.0µg/ml, and 2.5µg/ml for OFX and MXF, respectively. Conclusions MTB MICs were found to be consistently lower for MXF than for OFX among isolates with the same gyrA mutation (e.g. Ala90Val). The majority of MTB isolates containing mutations at Asp94Ala, Asn/Tyr, Gly and His in gyrA were associated with a moderate level of resistance to MXF (MIC=2.5µg/ml), although 3 isolates with the mutations Asp94Asn/Tyr/Gly were associated with a high level of resistance to both fluoroquinolones (MXF MICs = 5.0–8.0µg/ml, OFX MICs = ≥10.0µg/ml).

The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first-and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.