Evaluation of Pyrosequencing for Detecting Extensively Drug-Resistant Mycobacterium tuberculosis among Clinical Isolates from Four High-Burden Countries

Ajbani, Kanchan; Lin, Shou-Yean Grace; Rodrigues, Camilla; Nguyen, Duylinh; Arroyo, Francine; Kaping, J; Jackson, Lynn; Garfein, Richard S.; Catanzaro, Donald G.; Eisenach, Kathleen D.; Victor, Thomas C.; Crudu, Valeru; Gler, Maria Tarcela; Ismail, Nazir; Desmond, Edward; Catanzaro, Antonino; Rodwell, Timothy C.

doi:10.1128/aac.03614-14

Cited by 37 publications

(26 citation statements)

References 38 publications

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“…The inclusion of the quinolone resistancedetermining region of the gyrA gene spanning codons 88 to 95 was adequate to detect the majority (64 to 96%) of FQ resistance in each of the three sites, and observed variations in the sensitivity of the assay for FQ resistance detection between the sites were similar to those reported previously (41)(42)(43).…”

Section: Rifsupporting

confidence: 80%

Frequency and Distribution of Tuberculosis Resistance-Associated Mutations between Mumbai, Moldova, and Eastern Cape

Georghiou

Seifert

Catanzaro

et al. 2016

Antimicrob Agents Chemother

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f Molecular diagnostic assays, with their ability to rapidly detect resistance-associated mutations in bacterial genes, are promising technologies to control the spread of drug-resistant tuberculosis (DR-TB). Sequencing assays provide detailed information for specific gene regions and can help diagnostic assay developers prioritize mutations for inclusion in their assays. We performed pyrosequencing of seven Mycobacterium tuberculosis gene regions (katG, inhA, ahpC, rpoB, gyrA, rrs, and eis) for 1,128 clinical specimens from India, Moldova, and South Africa. We determined the frequencies of each mutation among drug-resistant and -susceptible specimens based on phenotypic drug susceptibility testing results and examined mutation distributions by country. The most common mutation among isoniazid-resistant (INH r ) specimens was the katG 315ACC mutation (87%). However, in the Eastern Cape, INH r specimens had a lower frequency of katG mutations (44%) and higher frequencies of inhA (47%) and ahpC (10%) promoter mutations. The most common mutation among rifampin-resistant (RIF r ) specimens was the rpoB 531TTG mutation (80%). The mutation was common in RIF r specimens in Mumbai (83%) and Moldova (84%) but not the Eastern Cape (17%), where the 516GTC mutation appeared more frequently (57%). The most common mutation among fluoroquinolone-resistant specimens was the gyrA 94GGC mutation (44%). The rrs 1401G mutation was found in 84%, 84%, and 50% of amikacin-resistant, capreomycin-resistant, and kanamycin (KAN)-resistant (KAN r ) specimens, respectively. The eis promoter mutation ؊12T was found in 26% of KAN r and 4% of KAN-susceptible (KAN s ) specimens. Inclusion of the ahpC and eis promoter gene regions was critical for optimal test sensitivity for the detection of INH resistance in the Eastern Cape and KAN resistance in Moldova. (This study has been registered at ClinicalTrials.gov under registration number NCT02170441.) I n 2014, an estimated 9.6 million people developed tuberculosis (TB), and 1.5 million people died of their infection (1). Although global TB incidence rates have fallen an average of 1.5% per year since 2000, the rise of drug-resistant TB (DR-TB) globally has complicated TB control efforts (1). The World Health Organization (WHO) estimates that as many as 1 in every 20 new, active TB infections is now drug resistant (1). One of the major roadblocks in combating this growing problem has been the lack of diagnostic technology for DR-TB. Current growth-based culture and drug susceptibility testing (DST) methods can take several weeks to months to yield results (2). While waiting on culture results, physicians are forced to treat their patients empirically, adjusting treatment regimens only once DST results become available. As a result, many undiagnosed DR-TB patients are being given medications that are ineffective, which amplifies resistance, increases the risk of mortality, and increases the risk of transmitting DR-TB infections in the community.Rapid molecular diagnostic assays for DR-TB have the p...

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Section: Rifsupporting

confidence: 80%

Frequency and Distribution of Tuberculosis Resistance-Associated Mutations between Mumbai, Moldova, and Eastern Cape

Georghiou

Seifert

Catanzaro

et al. 2016

Antimicrob Agents Chemother

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“…Pyrosequencing not only shows the presence or absence of these mutations, but also displays detailed sequence data, which enables users to distinguish mutations conferring resistance from silent mutations as well as from those conferring different levels of resistance. 267 Commercial kits are available but have not yet received regulatory approval for diagnostic use. For example, the Ion AmpliSeq TB Research Panel examines eight genes involved in resistance to first-line and second-line drugs (embB, eis, gyrA, inhA, katG, pncA, rpoB, and rpsL).…”

Section: Sequencing Approachesmentioning

confidence: 99%

The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis

Dheda

Gumbo

Maartens

et al. 2017

The Lancet Respiratory Medicine

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506

474

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“…55 False positive results may stem from the detection of silent rpoB mutations that do not affect phenotypic susceptibility. 61,62 Therefore, in countries with low prevalence of rifampin resistance, confirmation of a positive result is recommended (Box 2). 63 The Xpert roll-out has encountered several implementation issues, including inconsistent power supply and high cost.…”

Section: Xpert Mtb/rifmentioning

confidence: 99%

“…Compared with conventional DST, PSQ has demonstrated 89% to 94% sensitivity and 96% to 100% specificity for isoniazid; 95% to 96% sensitivity and 100% specificity for rifampin; 61% sensitivity and 85% specificity for ethambutol; 87% to 93% sensitivity and 100% specificity for fluoroquinolones; and 68% to 88% sensitivity and 97% to 100% specificity for second-line injectable agents. 62,[117][118][119] Of note, Sanger sequencing, which uses a different technology, offers comparable sensitivities and specificities, but PSQ, an easier and shorter procedure, is more suitable for high-throughput processing. 117,118 Whole genome sequencing (WGS) has emerged as another strategy for DST.…”

Section: Dna Sequencingmentioning

confidence: 99%

New Diagnostics for Childhood Tuberculosis

Chiang

Swanson

Starke

2015

Infectious Disease Clinics of North America

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Evaluation of Pyrosequencing for Detecting Extensively Drug-Resistant Mycobacterium tuberculosis among Clinical Isolates from Four High-Burden Countries

Cited by 37 publications

References 38 publications

Frequency and Distribution of Tuberculosis Resistance-Associated Mutations between Mumbai, Moldova, and Eastern Cape

Frequency and Distribution of Tuberculosis Resistance-Associated Mutations between Mumbai, Moldova, and Eastern Cape

The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis

New Diagnostics for Childhood Tuberculosis

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