Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis

BACKGROUND: Rapid molecular diagnostics, with their ability to quickly identify genetic mutations associated with drug resistance in Mycobacterium tuberculosis clinical specimens, have great potential as tools to control multi- and extensively drug-resistant tuberculosis (M/XDR-TB). The Qiagen PyroMark Q96 ID system is a commercially available pyrosequencing (PSQ) platform that has been validated for rapid M/XDR-TB diagnosis. However, the details of the assay’s diagnostic and technical performance have yet to be thoroughly investigated in diverse clinical environments. METHODS: This study evaluates the diagnostic performance of the PSQ assay for 1128 clinical specimens from patients from three areas of high TB burden. We report on the diagnostic performance of the PSQ assay between the three sites and identify variables associated with poor PSQ technical performance. RESULTS: In India, the sensitivity of the PSQ assay ranged from 89 to 98 % for the detection of phenotypic resistance to isoniazid, rifampicin, fluoroquinolones, and the injectables. In Moldova, assay sensitivity ranged from 7 to 94 %, and in South Africa, assay sensitivity ranged from 71 to 92 %. Specificity was high (94–100 %) across all sites. The addition of eis promoter sequencing information greatly improved the sensitivity of kanamycin resistance detection in Moldova (7 % to 79 %). Nearly all (89.4 %) sequencing reactions conducted on smear-positive, culture-positive specimens and most (70.8 %) reactions conducted on smear-negative, culture-positive specimens yielded valid PSQ reads. An investigation into the variables influencing sequencing failures indicated smear negativity, culture negativity, site (Moldova), and sequencing of the rpoB, gyrA, and rrs genes were highly associated with poor PSQ technical performance (adj. OR > 2.0). CONCLUSIONS: This study has important implications for the global implementation of PSQ as a molecular TB diagnostic, as it demonstrates how regional factors may impact PSQ diagnostic performance, while underscoring potential gene targets for optimization to improve overall PSQ assay technical performance. TRIAL REGISTRATION: ClinicalTrials.gov (#NCT02170441). Registered 12 June 2014. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12879-016-1781-y) contains supplementary material, which is available to authorized users

Mycobacterium africanum Cases, California

Five Mycobacterium tuberculosis complex isolates in California were identified as M. africanum by spoligotyping, single nucleotide polymorphisms, a deletion mutation, and phenotypic traits, confirming it as a cause of tuberculosis in the United States. Three of the five patients from whom M. africanum was isolated had lived in Africa

Rifabutin and rifampin resistance levels and associated rpoB mutations in clinical isolates of Mycobacterium tuberculosis complex

Cross-resistance in rifamycins has been observed in rifampin (RIF)-resistant Mycobacterium tuberculosis complex isolates; some rpoB mutations do not confer broad in vitro rifamycin resistance. We examined 164 isolates, of which 102 were RIF-resistant, for differential resistance between RIF and rifabutin (RFB). A total of 42 unique single mutations or combinations of mutations were detected. The number of unique mutations identified exceeded that reported in any previous study. RFB and RIF MICs up to 8 μg/mL by MGIT 960 were studied; the cut-off values for susceptibility to RIF and RFB were 1 μg/mL and 0.5 μg/mL, respectively. We identified 31 isolates resistant to RIF but susceptible to RFB with the mutations D516V, D516F, 518 deletion, S522L, H526A, H526C, H526G, H526L, and two dual mutations (S522L + K527R and H526S + K527R). Clinical investigations using RFB to treat multidrug-resistant tuberculosis cases harboring those mutations are recommended

Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis.

BackgroundRapid molecular diagnostics, with their ability to quickly identify genetic mutations associated with drug resistance in Mycobacterium tuberculosis clinical specimens, have great potential as tools to control multi- and extensively drug-resistant tuberculosis (M/XDR-TB). The Qiagen PyroMark Q96 ID system is a commercially available pyrosequencing (PSQ) platform that has been validated for rapid M/XDR-TB diagnosis. However, the details of the assay's diagnostic and technical performance have yet to be thoroughly investigated in diverse clinical environments.MethodsThis study evaluates the diagnostic performance of the PSQ assay for 1128 clinical specimens from patients from three areas of high TB burden. We report on the diagnostic performance of the PSQ assay between the three sites and identify variables associated with poor PSQ technical performance.ResultsIn India, the sensitivity of the PSQ assay ranged from 89 to 98 % for the detection of phenotypic resistance to isoniazid, rifampicin, fluoroquinolones, and the injectables. In Moldova, assay sensitivity ranged from 7 to 94 %, and in South Africa, assay sensitivity ranged from 71 to 92 %. Specificity was high (94-100 %) across all sites. The addition of eis promoter sequencing information greatly improved the sensitivity of kanamycin resistance detection in Moldova (7 % to 79 %). Nearly all (89.4 %) sequencing reactions conducted on smear-positive, culture-positive specimens and most (70.8 %) reactions conducted on smear-negative, culture-positive specimens yielded valid PSQ reads. An investigation into the variables influencing sequencing failures indicated smear negativity, culture negativity, site (Moldova), and sequencing of the rpoB, gyrA, and rrs genes were highly associated with poor PSQ technical performance (adj. OR > 2.0).ConclusionsThis study has important implications for the global implementation of PSQ as a molecular TB diagnostic, as it demonstrates how regional factors may impact PSQ diagnostic performance, while underscoring potential gene targets for optimization to improve overall PSQ assay technical performance.Trial registrationClinicalTrials.gov ( #NCT02170441 ). Registered 12 June 2014

Additional file 1: Table S1. of Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis

Pyrosequencing (PSQ) Success of Each Gene Target by Smear- and Culture-Status. Table S2. Proportion of Successful Pyrosequencing (PSQ) Reactions by Smear- and Culture-Status. (DOCX 18 kb

Additional file 2: of Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis

Pyrosequencing (PSQ) Performance Dataset. (TXT 400 kb

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

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