Clinical Evaluation of the XDR-LFC Assay for the Molecular Detection of Isoniazid, Rifampin, Fluoroquinolone, Kanamycin, Capreomycin, and Amikacin Drug Resistance in a Prospective Cohort.

While the goal of universal drug susceptibility testing has been a key component of the WHO End TB Strategy, in practice, this remains inaccessible to many. Rapid molecular tests for tuberculosis (TB) and antituberculosis drug resistance could significantly improve access to testing. In this study, we evaluated the accuracy of the Akonni Biosystems XDR-TB (extensively drug-resistant TB) TruArray and lateral-flow-cell (XDR-LFC) assay (Akonni Biosystems, Inc., Frederick, MD, USA), a novel assay that detects mutations in seven genes associated with resistance to antituberculosis drugs: katG, the inhA promoter, and the ahpC promoter for isoniazid; rpoB for rifampin; gyrA for fluoroquinolones; rrs and the eis promoter for kanamycin; and rrs for capreomycin and amikacin. We evaluated assay performance using direct sputum samples from 566 participants recruited in a prospective cohort in Moldova over 2 years. The sensitivity and specificity against the phenotypic reference were both 100% for isoniazid, 99.2% and 97.9% for rifampin, 84.8% and 99.1% for fluoroquinolones, 87.0% and 84.1% for kanamycin, 54.3% and 100% for capreomycin, and 79.2% and 100% for amikacin, respectively. Whole-genome sequencing data for a subsample of 272 isolates showed 95 to 99% concordance with the XDR-LFC-reported suspected mutations. The XDR-LFC assay demonstrated a high level of accuracy for multiple drugs and met the WHO's minimum target product profile criteria for isoniazid and rifampin, while the sensitivity for fluoroquinolones and amikacin fell below target thresholds, likely due to the absence of a gyrB target in the assay. With optimization, the XDR-LFC shows promise as a novel near-patient technology to rapidly diagnose drug-resistant tuberculosis.

Laboratory Evaluation of a Lateral-Flow Cell for Molecular Detection of First-Line and Second-Line Antituberculosis Drug Resistance.

Despite the WHO's call for universal drug susceptibility testing for all patients being evaluated for tuberculosis (TB), a lack of rapid diagnostic tests which can fully describe TB resistance patterns is a major challenge in ensuring that all persons diagnosed with drug-resistant TB are started on an appropriate treatment regime. We evaluated the accuracy of the Akonni Biosystems XDR-TB TruArray and lateral-flow cell (XDR-LFC), a novel multiplex assay to simultaneously detect mutations across seven genes that confer resistance to both first- and second-line anti-TB drugs. The XDR-LFC includes 271 discrete three-dimensional gel elements with target-specific probes for identifying mutations in katG, inhA promoter, and ahpC promoter (isoniazid), rpoB (rifampin), gyrA (fluoroquinolones), rrs and eis promoter (kanamycin), and rrs (capreomycin and amikacin). We evaluated XDR-LFC performance with 87 phenotypically and genotypically characterized clinical Mycobacterium tuberculosis isolates. The overall assay levels of accuracy for mutation detection in specific genes were 98.6% for eis promoter and 100.0% for the genes katG, inhA promoter, ahpC promoter, rpoB, gyrA, and rrs The sensitivity and specificity against phenotypic reference were 100% and 100% for isoniazid, 98.4% and 50% for rifampin (specificity increased to 100% once the strains with documented low-level resistance mutations in rpoB were excluded), 96.2% and 100% for fluoroquinolones, 92.6% and 100% for kanamycin, 93.9% and 97.4% for capreomycin, and 80% and 100% for amikacin. The XDR-LFC solution appears to be a promising new tool for accurate detection of resistance to both first- and second-line anti-TB drugs.

Integrated amplification microarray system in a lateral flow cell for warfarin genotyping from saliva.

BACKGROUND: Genetic polymorphisms in the CYP2C9 and VKORC1 genes have been linked to sensitivity of the anticoagulant drug warfarin. The aim of this study is to demonstrate a method for warfarin sensitivity genotyping using gel element microarray technology in a simplified workflow from sample collection to analysis and detection. METHODS: We developed an integrated amplification microarray system combining PCR amplification, target labeling, and microarray hybridization within a single, closed-amplicon "lateral flow cell" for genotyping three single nucleotide polymorphisms (SNPs) that influence warfarin response. We combined nucleic acid extraction of saliva using the TruTip technology together with the lateral flow cell assay and with fully automated array detection and analysis. RESULTS: The analytical performance of the assay was tested using 20 genotyped human genomic DNA samples and found to be sensitive down to 330 input genomic copies (1 ng). A follow-up pre-clinical evaluation was performed with 65 blinded saliva samples and the genotyping results were in agreement with those determined by bidirectional sequencing. CONCLUSIONS: Combined with the use of non-invasive saliva samples, rapid DNA extraction, the lateral flow cell, automatic imaging and data analysis provides a simple and fast sample-to-answer microarray test for warfarin sensitivity genotyping.