Point-of-Care System for Detection of Mycobacterium tuberculosis and Rifampin Resistance in Sputum Samples

Abstract

Early detection of Mycobacterium tuberculosis complex (MTBC) and markers conveying drug resistance can have a beneficial impact on preventive public health actions. We describe here a new molecular point-of-care (POC) system, the Genedrive, which is based on simple sample preparation combined with PCR to detect MTBC and simultaneously detect mutation markers in the rpoB gene directly from raw sputum sample. Hybridization probes were used to detect the presence of the key mutations in codons 516, 526, and 531 of the rpoB gene. The sensitivities for MTBC and rpoB detection from sputum samples were assessed using model samples spiked with known numbers of bacteria prepared from liquid cultures of M. tuberculosis. The overall sensitivities were 90.8% (95% confidence interval [CI], 81, 96.5) for MTBC detection and 72.3% (95% CI, 59.8, 82.7) for rpoB detection. For samples containing >1,000 CFU/ml, the sensitivities were 100% for MTBC and 85.7% for rpoB detection, while for samples containing <100 CFU/ml, the sensitivities were 86.4% and 65.9% for MTBC and rpoB detection, respectively. The specificity was shown to be 100% (95% CI, 83.2, 100) for MTBC and rpoB. The clinical sputum samples were processed using the same protocol and showed good concordance with the data generated from the model. Tuberculosis-infected subjects with smear samples assessed as scanty or negative were detectable by the Genedrive system. In these paucibacillary patients, the performance of the Genedrive system was comparable to that of the GeneXpert assay. The characteristics of the Genedrive platform make it particularly useful for detecting MTBC and rifampin resistance in low-resource settings and for reducing the burden of tuberculosis disease.

Analytical and Clinical Evaluation of the Epistem Genedrive Assay for Detection of Mycobacterium tuberculosis

“…Insufficient cell lysis or insufficient removal of PCR inhibitors from sputum by the cassette may also adversely affect sensitivity, although we did not specifically study these parameters. Our results differ markedly from the only other paper published on this assay to date (6).…”

“…This test is CE marked for sale in European Economic Area (EEA) member states and is also licensed for distribution in India (7)(8)(9). However, this test's performance has not been extensively evaluated or reported in peer-reviewed journals apart from a single study published by the manufacturer and collaborators (6).…”

“…This test uses a simple paper-based DNA extraction method coupled with PCR amplification and detection on Epistem's Genedrive instrument, a lightweight, portable, benchtop PCR platform with real-time PCR and melting temperature analysis capabilities. Epistem's Genedrive MTB/RIF (Genedrive) assay detects the Mycobacterium tuberculosis complex by targeting two different regions of the M. tuberculosis complex genome, a short repetitive region, rep13E12, and a segment of the rpoB gene (4)(5)(6). This test is CE marked for sale in European Economic Area (EEA) member states and is also licensed for distribution in India (7)(8)(9).…”

Detection of multidrug-resistant Mycobacterium tuberculosis strains isolated in Brazil using a multimarker genetic assay for katG and rpoB genes

“…Also, the frequency of 62.8% for S531L (rpoB) found in this work was consistent with other studies, considering that published rates range between 40.1% and 82.4%. [31][32][33][34] In the same way, H526D (rpoB) polymorphism, with a found frequency of 7% in this work amply oscillates worldwide, with values ranging from 5.9% to 40%. 31,34 Therefore, great frequency variations are evident between countries, making it necessary to study locally the prevalence of the canonical mutations for each geographical region in order to figure out which polymorphisms have the best predictive accuracy.…”

“…[31][32][33][34] In the same way, H526D (rpoB) polymorphism, with a found frequency of 7% in this work amply oscillates worldwide, with values ranging from 5.9% to 40%. 31,34 Therefore, great frequency variations are evident between countries, making it necessary to study locally the prevalence of the canonical mutations for each geographical region in order to figure out which polymorphisms have the best predictive accuracy. As early diagnosis is the best strategy to control MDRTB, several molecular methods to detect drug resistance in M. tuberculosis are currently widely employed.…”