Detection of heteroresistant Mycobacterium tuberculosis by pyrosequencing.
Ontology highlight
ABSTRACT:
SUBMITTER: Engstrom A
PROVIDER: S-EPMC3838067 | biostudies-literature | 2013 Dec
REPOSITORIES: biostudies-literature
Publications
Detection of heteroresistant Mycobacterium tuberculosis by pyrosequencing.
Engström Anna A Hoffner Sven S Juréen Pontus P
Journal of clinical microbiology 20130918 12
The ability of pyrosequencing to detect a resistant minority population of a heteroresistant Mycobacterium tuberculosis strain was investigated by performing a titration study. A mutant signal was noted only when the proportion of mutant DNA in the DNA target was 35 to 50%, showing that the sensitivity is significantly lower than that of phenotypic drug susceptibility test methods. ...[more]
Similar Datasets
Project description:BackgroundThe worldwide expanding Mycobacterium tuberculosis W-Beijing family is associated with treatment failure and relapse. Its identification currently relies on spoligotyping and conventional sequencing. We developed pyrosequencing as an alternative method for its identification.FindingsPyrosequencing found a G/A substitution in the Rv0927c-pstS3 intergenic spacer and a RD105 deletion, identifying 8/104 M. tuberculosis isolates as W-Beijing isolates. In addition, pyrosequencing found a previously unreported TGC deletion in the Rv0927c gene of W-Beijing isolates. Total concordance was found between the pyrosequencing data and conventional sequencing, as well as reference molecular identification. Multispacer Sequence Typing assigned the W-Beijing isolates to the Asian lineage and the 96 non-W-Beijing isolates to the Euro-American lineage (P < 10-5). The W-Beijing isolates were all susceptible to streptomycin, rifampin, isoniazid, ethambutol, and pyrazinamide; no resistance-associated mutations were detected in these eight W-Beijing isolates. There were no statistically significant differences in the antibiotic susceptibility of W-Beijing and non-W-Beijing isolates (p = 0.2, X2 test). Pyrosequencing correctly identified M. tuberculosis organisms in 26/26 sputum specimens exhibiting acid-fast bacilli. Pyrosequencing results were obtained within four hours, incurring an estimated cost of 1.86 euro/test.ConclusionPyrosequencing of the Rv0927c gene and adjacent intergenic spacer is an efficient, low-cost technique for the rapid identification of W-Beijing isolates.
| S-EPMC2799434 | biostudies-literature
Project description:Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci: rpoB, katG, embB, rrs, gyrA, and the promoter regions of inhA and eis. The molecular method was evaluated on a panel of 290 clinical isolates of M. tuberculosis. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant M. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant M. tuberculosis. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in M. tuberculosis.
| S-EPMC3372151 | biostudies-literature
Project description:Treating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization.
| S-EPMC3911348 | biostudies-literature
Project description:BackgroundIdentification of the Mycobacterium tuberculosis complex organisms to the species level is important for diagnostic, therapeutic and epidemiologic perspectives. Indeed, isolates are routinely identified as belonging to the M. tuberculosis complex without further discrimination in agreement with the high genomic similarity of the M. tuberculosis complex members and the resulting complex available identification tools.FindingsWe herein develop a pyrosequencing assay analyzing polymorphisms within glpK, pykA and gyrB genes to identify members of the M. tuberculosis complex at the species level. The assay was evaluated with 22 M. tuberculosis, 21 M. bovis, 3 M. caprae, 3 M. microti, 2 M. bovis BCG, 2 M. pinnipedii, 1 M. canettii and 1 M. africanum type I isolates. The resulted pyrograms were consistent with conventional DNA sequencing data and successfully identified all isolates. Additionally, 127 clinical M. tuberculosis complex isolates were analyzed and were unambiguously identified as M. tuberculosis.ConclusionWe proposed a pyrosequencing-based scheme for the rapid identification of M. tuberculosis complex isolates at the species level. The assay is robust, specific, rapid and can be easily introduced in the routine activity.
| S-EPMC3214197 | biostudies-literature
Project description:We developed a novel method, PyroTyping, for discrimination of Mycobacterium tuberculosis isolates combining pyrosequencing and IS6110 polymorphism. A total of 100 isolates were analysed with IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, mycobacterial interspersed repetitive units - variable number tandem repeats (MIRU-VNTR), and PyroTyping. PyroTyping results regarding clustering or discrimination of the isolates were highly concordant with the other typing methods performed. PyroTyping is more rapid than RFLP and presents the same discriminatory power, thus, it may be useful for taking timely decisions for tuberculosis control.
| S-EPMC5533701 | biostudies-literature
Project description:Fluoroquinolones (FQ) are important second-line drugs to treat tuberculosis; however, FQ resistance is an emerging problem. Resistance has been mainly attributed to mutations in a 21-bp region of the Mycobacterium tuberculosis gyrA gene, often called the quinolone resistance-determining region (QRDR). We have developed a simple, rapid, and specific assay to detect FQ resistance-determining QRDR mutations. The assay amplifies the M. tuberculosis gyrA QRDR in an asymmetrical PCR followed by probing with two sloppy molecular beacons (SMBs) spanning the entire QRDR. Mutations are detected by melting temperature (T(m)) shifts that occur when the SMBs bind to mismatched sequences. By testing DNA targets corresponding to all known QRDR mutations, we found that one or both of the SMBs produced a T(m) shift of at least 3.6°C for each mutation, making mutation detection very robust. The assay was also able to identify mixtures of wild-type and mutant DNA, with QRDR mutants identified in samples containing as little as 5 to 10% mutant DNA. The assay was blindly validated for its ability to identify the QRDR mutations on DNA extracted from clinical M. tuberculosis strains. Fifty QRDR wild-type samples, 34 QRDR mutant samples, and 8 heteroresistant samples containing mixtures of wild-type and mutant DNA were analyzed. The results showed 100% concordance to conventional DNA sequencing, including a complete identification of all of the mixtures. This SMB T(m) shift assay will be a valuable molecular tool to rapidly detect FQ resistance and to detect the emergence of FQ heteroresistance in clinical samples from tuberculosis patients.
| S-EPMC3067712 | biostudies-literature
Project description:An estimated one-third of tuberculosis (TB) cases go undiagnosed or unreported. Sputum samples, widely used for TB diagnosis, are inefficient at detecting infection in children and paucibacillary patients. Indeed, developing point-of-care biomarker-based diagnostics that are not sputum-based is a major priority for the WHO. Here, in a proof-of-concept study, we tested whether pulmonary TB can be detected by analyzing patient exhaled breath condensate (EBC) samples. We find that the presence of Mycobacterium tuberculosis (Mtb)-specific lipids, lipoarabinomannan lipoglycan, and proteins in EBCs can efficiently differentiate baseline TB patients from controls. We used EBCs to track the longitudinal effects of antibiotic treatment in pediatric TB patients. In addition, Mtb lipoarabinomannan and lipids were structurally distinct in EBCs compared to ex vivo cultured bacteria, revealing specific metabolic and biochemical states of Mtb in the human lung. This provides essential information for the rational development or improvement of diagnostic antibodies, vaccines and therapeutic drugs. Our data collectively indicate that EBC analysis can potentially facilitate clinical diagnosis of TB across patient populations and monitor treatment efficacy. This affordable, rapid and non-invasive approach seems superior to sputum assays and has the potential to be implemented at point-of-care.
| S-EPMC9751131 | biostudies-literature
Project description:Heteroresistance is defined as the coexistence of both susceptible and resistant bacteria in a bacterial population. Previously published data show that it may occur in 9 to 57% of Mycobacterium tuberculosis isolates for various drugs. Pyrazinamide (PZA) is an important first-line drug used for treatment of both drug-susceptible and PZA-susceptible multidrug-resistant TB. Clinical PZA resistance is defined as a proportion of resistant bacteria in the isolate exceeding 10%, when the drug is no longer considered clinically effective. The ability of traditional drug susceptibility testing techniques to detect PZA heteroresistance has not yet been evaluated. The aim of this study was to compare the capacity of Bactec MGIT 960, Wayne's test, and whole-genome sequencing (WGS) to detect PZA-resistant subpopulations in bacterial suspensions prepared with different proportions of mutant strains. Both Bactec MGIT 960 and WGS were able to detect the critical level of 10% PZA heteroresistance, whereas Wayne's test failed to do so, with the latter falsely reporting highly resistant samples as PZA susceptible. Failure to detect drug-resistant subpopulations may lead to inadvertently weak treatment regimens if ineffective drugs are included, with the risk of treatment failure with the selective growth of resistant subpopulations. We need clinical awareness of heteroresistance as well as evaluation of new diagnostic tools for their capacity to detect heteroresistance in TB.
| S-EPMC8370246 | biostudies-literature
Project description:In this study, our objectives was to analyze the molecular characteristics of mutation at embB codon306 in Mycobacterium tuberculosis in Qingdao by pyrosequencing technology, and to assess the value of embB codon306 used as a molecular marker to diagnose multidrug resistant (MDR) TB strains. Pyrosequencing was used to detect mutations at embB codon306 among M. tuberculosis isolates from tuberculosis (TB) patients in Qingdao. The correlation between embB306 mutation and MDR phenotype was evaluated by comparing with conventional drug susceptibility testing results. 60.9% of MDR strains and 15.2% of non-MDR strains carried embB306 mutation, respectively. The percentage of MDR-TB harboring embB306 mutation was significantly higher than that of non- MDR-TB (?(2)=15.09, P < 0.01). EmbB306 mutation serving as a marker to diagnose MDR-TB comparing with the traditional susceptibility test, the specificity, sensitivity and accuracy were 85%, 61%, and 77%, respectively. EmbB306 mutation is the main mechanism of TB resistance to multidrug in Qingdao, showing an association with the MDR. Pyrosequencing should be a good diagnostic tool for MDR-TB in Qingdao.
| S-EPMC4565318 | biostudies-literature
Project description:ObjectivesThe light-emitting diode (LED) fluorescence microscopy has made acid-fast bacilli (AFB) detection faster and efficient although its optimal performance in resource-limited settings is still being studied. We assessed the optimal performances of light and fluorescence microscopy in routine conditions of a resource-limited setting and evaluated the digestion time for sputum samples for maximum yield of positive cultures.DesignCross-sectional study.SettingFacility-based involving samples of routine patients receiving tuberculosis treatment and care from the main tuberculosis case referral centre in northern Nigeria.ParticipantsThe study included 450 sputum samples from 150 new patients with clinical diagnosis of pulmonary tuberculosis.MethodsThe 450 samples were pooled into 150 specimens, examined independently with mercury vapour lamp (FM), LED CysCope (CY) and Primo Star iLED (PiLED) fluorescence microscopies, and with the Ziehl-Neelsen (ZN) microscopy to assess the performance of each technique compared with liquid culture. The cultured specimens were decontaminated with BD Mycoprep (4% NaOH-1% NLAC and 2.9% sodium citrate) for 10, 15 and 20 min before incubation in Mycobacterium growth incubator tube (MGIT) system and growth examined for acid-fast bacilli (AFB).ResultsOf the 150 specimens examined by direct microscopy: 44 (29%), 60 (40%), 49 (33%) and 64 (43%) were AFB positive by ZN, FM, CY and iLED microscopy, respectively. Digestion of sputum samples for 10, 15 and 20 min yielded mycobacterial growth in 72 (48%), 81 (54%) and 68 (45%) of the digested samples, respectively, after incubation in the MGIT system.ConclusionsIn routine laboratory conditions of a resource-limited setting, our study has demonstrated the superiority of fluorescence microscopy over the conventional ZN technique. Digestion of sputum samples for 15 min yielded more positive cultures.
| S-EPMC3948456 | biostudies-literature