Dataset Information

Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use.

ABSTRACT: Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrs genes and the promoters of inhA and eis genes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of Mycobacterium tuberculosis in 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.

SUBMITTER: Chakravorty S

PROVIDER: S-EPMC5228229 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use.

Chakravorty Soumitesh S Roh Sandy S SS Glass Jennifer J Smith Laura E LE Simmons Ann Marie AM Lund Kevin K Lokhov Sergey S Liu Xin X Xu Peng P Zhang Guolong G Via Laura E LE Shen Qingyu Q Ruan Xianglin X Yuan Xing X Zhu Hong Zhu HZ Viazovkina Ekaterina E Shenai Shubhada S Rowneki Mazhgan M Lee Jong Seok JS Barry Clifton E CE Gao Qian Q Persing David D Kwiatkawoski Robert R Jones Martin M Gall Alexander A Alland David D

Journal of clinical microbiology 20161228 1

Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four St ...[more]

PMID: 27807153

Similar Datasets

Project description:BACKGROUND: The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) makes the treatment and control of tuberculosis difficult. Rapid detection of drug-resistant strains is important for the successful treatment of drug-resistant tuberculosis; however, not all resistance mechanisms to the injectable second-line drugs such as amikacin (AK), kanamycin (KM), and capreomycin (CAP) are well understood. This study aims to validate the mechanisms associated with AK, KM, and CAP resistance in M. tuberculosis clinical strains isolated in Thailand. RESULTS: A total of 15,124?M. tuberculosis clinical strains were isolated from 23,693 smear-positive sputum samples sent from 288 hospitals in 46 of 77 provinces of Thailand. Phenotypic analysis identified 1,294 strains as MDR-TB and second-line drugs susceptibility was performed in all MDR-TB strains and revealed 58 XDR-TB strains. Twenty-nine KM-resistant strains (26 XDR-TB and 3 MDR-TB) could be retrieved and their genes associated with AK, KM, and CAP resistance were investigated compared with 27 KM-susceptible strains. Mutation of the rrs (A1401G) was found in 21 out of 29 KM-resistant strains whereas mutations of eis either at C-14 T or at G-37 T were found in 5 strains. Three remaining KM-resistant strains did not contain any known mutations. Capreomycin resistance was determined in 28 of 29 KM-resistant strains. Analysis of tlyA revealed that the A33G mutation was found in all CAP-resistant strains and also in susceptible strains. In contrast, the recently identified tlyA mutation T539G and the novel Ins49GC were found in two and one CAP-resistant strains, respectively. In addition, our finding demonstrated the insertion of cytosine at position 581 of the tap, a putative drug efflux encoding gene, in both KM-resistant and KM-susceptible strains. CONCLUSIONS: Our finding demonstrated that the majority of KM resistance mechanism in Thai M. tuberculosis clinical strains was rrs mutation at A1401G. Mutations of the eis promoter region either at C-14 T or G-37 T was found in 5 of 29 strains whereas three strains did not contain any known mutations. For CAP resistance, 3 of 28 CAP-resistant strains contained either T539G or Ins49GC mutations at tlyA that might be associated with the resistant phenotype.

Project description:Objectives This is a protocol for a Cochrane Review (diagnostic). The objectives are as follows: To estimate the diagnostic accuracy of Xpert MTB/XDR for the detection of pulmonary tuberculosis in people with signs and symptoms of pulmonary tuberculosis. To estimate the diagnostic accuracy of Xpert MTB/XDR for resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin in people irrespective of rifampicin resistance and people with detected rifampicin resistance. In these populations, pulmonary tuberculosis will have been detected by Xpert MTB/XDR (as it is a reflex test). Such populations typically will have received prior testing verifying tuberculosis with another WHO‐approved test. Secondary objectives To compare the diagnostic accuracy of Xpert MTB/XDR by direct testing versus indirect testing (whereby Xpert MTB/XDR is performed on a Mycobacterium tuberculosis isolate grown from culture). To investigate the effects of potential sources of heterogeneity on test accuracy. For pulmonary tuberculosis, potential sources include HIV status, smear status, history of tuberculosis, treatment status (no treatment or currently on treatment), and treatment response status (culture conversion, yes or no). For drug resistance, potential sources include the type of reference standard and history of tuberculosis treatment. In addition, for fluoroquinolone resistance, a potential source of heterogeneity is the specific drug (e.g. ofloxacin or moxifloxacin) used in the phenotypic culture‐based DST (pDST) reference standard. We will also consider whether the WHO‐recommended critical drug concentration was used for the pDST reference standard (WHO Critical Concentrations 2018; WHO Critical Concentrations 2021). Regarding previously treated people, these investigations are important questions for clinical practice. For tuberculosis detection, studies have highlighted the challenges in interpreting Xpert MTB/RIF‐positive and Xpert Ultra‐positive results in previously treated people (Mishra 2020; Theron 2016a). As mentioned, for detection of drug resistance, previous treatment may increase the likelihood of having drug resistance.

Dataset Information

Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use.

Publications

Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets