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. A total of 187 archived isolates were run through a PSQ assay in order to identify M. tuberculosis (via the IS6110 marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets included katG, the inhA promoter and the ahpC-oxyR intergenic region for isoniazid (INH) resistance; the rpoB core region for rifampin (RIF) resistance; gyrA for fluoroquinolone (FQ) resistance; and rrs for amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). 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.     

First evaluation of an improved assay for molecular genetic detection of tuberculosis as well as rifampin and isoniazid resistances

The commercially available line probe assay MTBDRplus 2.0 (Hain Lifescience, Nehren, Germany) was evaluated for its ability to detect Mycobacterium tuberculosis complex (MTBC) and mutations conferring resistance to rifampin (RMP) and isoniazid (INH) directly in smear-negative and smear-positive pulmonary clinical specimens under routine laboratory conditions. A total of 348 samples originating from Moldova, a high-incidence country for tuberculosis (TB), were investigated. Two hundred fifty-seven (73.9%) were smear negative, 12 samples were excluded, and 81 (23.3%) were smear positive. Two DNA extraction methods were applied. Compared to culture and clinical data as the reference standard (adapted from Vadwai V et al., J. Clin. Microbiol. 49:2540-2545, 2011), overall sensitivity and specificity were 87.6 and 99.2%, respectively. One hundred four of the 257 smear-negative samples turned out to be culture positive, and 20 were MTBC culture negative but were positive based on clinical symptoms. The combined sensitivity and specificity in the subgroup of smear-negative samples were calculated to be 79.8 and 99.2%, respectively. MTBDRplus 2.0 detected RMP and INH resistance with sensitivity and specificity of 94.3 and 96.0%, respectively. In conclusion, the MTBDRplus 2.0 assay is a rapid and highly sensitive test for the detection of M. tuberculosis strains from smear-positive and -negative clinical specimens and provides additional information on RMP and INH resistance status, which can easily be included in routine laboratory work flow.     

Increasing tuberculosis case detection: lessons from the Republic of Moldova

The Republic of Moldova undertook reforms in tuberculosis (TB) control and health care consistent with international recommendations and advanced towards the global target for case detection. The number of TB cases notified increased overall by 50% during 2001-2005. Expansion of the DOTS strategy and full coverage coincided with a greater role for primary health care (PHC) in TB control and the advent of national insurance for TB diagnosis and treatment. These developments and improvements in laboratories, surveillance, medical personnel skills, and public awareness contributed to increased case detection. The Republic of Moldova addressed both demand and supply sides in these efforts. It increased effective demand for TB services by dispersing diagnostic capability, instituting financing mechanisms and saturating the public with information on symptoms, transmission and treatment. It increased the supply of TB services by upgrading the laboratory network, revamping surveillance and training practitioners. The Republic of Moldova's experience offers lessons for other countries: TB-PHC integration allowed more suspect cases to be diagnosed at nearby PHC clinics, contributing to more cases being notified. Innovative TB communications reached the general public, vulnerable groups, practitioners and the media. TB control projects built on each other and national coordination mechanisms served to identify funding for the most pressing needs. There are challenges remaining for TB control in the Republic of Moldova, not least the stable treatment success rate, but the country can list valuable lessons and achievements.     

Complications related to access site after percutaneous coronary interventions

Objectives : The primary objective was to compare the NCDR rate of four access site related complications (arteriovenous fistula, pseudoaneurysm, retroperitoneal bleed, and blood transfusion) with that obtained independently within a Quality Improvement (QI) Database. The secondary objective was to determine the in‐hospital mortality related to these complications. Background : NCDR is the most comprehensive database of post‐PCI patients and a method by which hospitals are compared for quality of care and outcomes. The collected data include in‐hospital events only, therefore reporting for same day and/or early discharges may fail to capture adverse events. We hypothesized that the actual rate of complications post‐PCI may be higher than the rate reported in NCDR. Methods : Adverse events following PCIs were collected in a QI database. We compared the rate of four abovementioned complications between NCDR and our QI database for the time period between January 1, 2005 and December 31, 2008. Results : A total of 3,940 PCIs were performed on 3,430 patients in the four‐year interval. The incidence of the combined endpoint of the four adverse events was 4.1% (161 events) in NCDR, vs. 4.7% (186 events) in QI database, a 13% higher yield. There was significantly higher in‐hospital mortality in the complication group with regards to both all cause (4.7% vs. 1.1%, P < 0.0001) and cardiovascular mortality (2.7% vs. 1%, P = 0.046). Conclusions : Access site related complications occurred 13% more than what was reported in NCDR and were associated with a greater than fourfold increase in in‐hospital mortality. © 2011 Wiley‐Liss, Inc.     

Predicting Extensively Drug-Resistant Mycobacterium tuberculosis Phenotypes with Genetic Mutations

Molecular diagnostic methods based on the detection of mutations conferring drug resistance are promising technologies for rapidly detecting multidrug-/extensively drug-resistant tuberculosis (M/XDR TB), but large studies of mutations as markers of resistance are rare. The Global Consortium for Drug-Resistant TB Diagnostics analyzed 417 Mycobacterium tuberculosis isolates from multinational sites with a high prevalence of drug resistance to determine the sensitivities and specificities of mutations associated with M/XDR TB to inform the development of rapid diagnostic methods. We collected M/XDR TB isolates from regions of high TB burden in India, Moldova, the Philippines, and South Africa. The isolates underwent standardized phenotypic drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), ofloxacin (OFX), amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) using MGIT 960 and WHO-recommended critical concentrations. Eight genes (katG, inhA, rpoB, gyrA, gyrB, rrs, eis, and tlyA) were sequenced using Sanger sequencing. Three hundred seventy isolates were INH^r, 356 were RIF^r, 292 were MOX^r/OFX^r, 230 were AMK^r, 219 were CAP^r, and 286 were KAN^r. Four single nucleotide polymorphisms (SNPs) in katG/inhA had a combined sensitivity of 96% and specificities of 97 to 100% for the detection of INH^r. Eleven SNPs in rpoB had a combined sensitivity of 98% for RIF^r. Eight SNPs in gyrA codons 88 to 94 had sensitivities of 90% for MOX^r/OFX^r. The rrs 1401/1484 SNPs had 89 to 90% sensitivity for detecting AMK^r/CAP^r but 71% sensitivity for KAN^r. Adding eis promoter SNPs increased the sensitivity to 93% for detecting AMK^r and to 91% for detecting KAN^r. Approximately 30 SNPs in six genes predicted clinically relevant XDR-TB phenotypes with 90 to 98% sensitivity and almost 100% specificity.     

Synthesis, evaluation of antimicrobial activity, and molecular modeling of novel 2-((4-(2H-benzo[d] [1,2,3] triazol-2-yl)piperidin-1-yl)methyl)-5-substituted phenyl-1,3,4-oxadiazoles

A new series of 2-((4-(2H-benzo[d][1,2,3]triazol-2-yl)piperidin-1-yl)methyl)-5-substituted-1-yl)-acetic acid hydrazide (3) with various substituted aromatic carboxylic acids phenyl-1,3,4-oxadiazoles (4a–j) were synthesized by cyclization of 4-benzotriazol-2-yl-piperidin in the presence of POCl₃. The structures were characterized by spectral data. The representative examples were screened invitro antibacterial activity and antifungal activity. The compounds showed significant antibacterial activity and showed moderate antifungal activity as that of standards, respectively. The data were further compared with structure-based investigations using docking studies with the crystal structure of oxidoreductase (1XDQ) protein organism: Escherichia coli for antibacterial activity and oxidoreductase (3QLS) protein, organism: Candida albicans for antifungal activity. The score values estimated by genetic algorithm were found to have a good correlation with the experimental inhibitory potencies.     

MTBDRplus and MTBDRsl Assays: Absence of Wild-Type Probe Hybridization and Implications for Detection of Drug-Resistant Tuberculosis

Accurate identification of drug-resistant Mycobacterium tuberculosis is imperative for effective treatment and subsequent reduction in disease transmission. Line probe assays rapidly detect mutations associated with resistance and wild-type sequences associated with susceptibility. Examination of molecular-level performance is necessary for improved assay result interpretation and for continued diagnostic development. Using data collected from a large, multisite diagnostic study, probe hybridization results from line probe assays, MTBDRplus and MTBDRsl, were compared to those of sequencing, and the diagnostic performance of each individual mutation and wild-type probe was assessed. Line probe assay results classified as resistant due to the absence of wild-type probe hybridization were compared to those of sequencing to determine if novel mutations were inhibiting wild-type probe hybridization. The contribution of absent wild-type probe hybridization to the detection of drug resistance was assessed via comparison to a phenotypic reference standard. In our study, mutation probes demonstrated significantly higher specificities than wild-type probes and wild-type probes demonstrated marginally higher sensitivities than mutation probes, an ideal combination for detecting the presence of resistance conferring mutations while yielding the fewest number of false-positive results. The absence of wild-type probe hybridization without mutation probe hybridization was determined to be primarily the result of failure of mutation probe hybridization and not the result of novel or rare mutations. Compared to phenotypic culture-based drug susceptibility testing, the absence of wild-type probe hybridization without mutation probe hybridization significantly contributed to the detection of phenotypic rifampin and fluoroquinolone resistance with negligible increases in false-positive results.