Rapid Microarray-Based Detection of Rifampin, Isoniazid, and Fluoroquinolone Resistance in Mycobacterium tuberculosis by Use of a Single Cartridge.

The rapid and robust identification of mutations in Mycobacterium tuberculosis complex (MTBC) strains mediating multidrug-resistant (MDR) and extensively drug-resistant (XDR) phenotypes is crucial to combating the MDR tuberculosis (TB) epidemic. Currently available molecular anti-TB drug susceptibility tests either are restricted to a single target or drug (i.e., the Xpert MTB/RIF test) or present a risk of cross-contamination due to the design limitations of the open platform (i.e., line probe assays). With a good understanding of the technical and commercial boundaries, we designed a test cartridge based on an oligonucleotide array into which dried reagents are introduced and which has the ability to identify MTBC strains resistant to isoniazid, rifampin, and the fluoroquinolones. The melting curve assay interrogates 43 different mutations in the rifampin resistance-determining region (RRDR) of rpoB, rpoB codon 572, katG codon 315, the inhA promoter region, and the quinolone resistance-determining region (QRDR) of gyrA in a closed cartridge system within 90 min. Assay performance was evaluated with 265 clinical MTBC isolates, including MDR/XDR, non-MDR, and fully susceptible isolates, from a drug resistance survey performed in Swaziland in 2009 and 2010. In 99.5% of the cases, the results were consistent with data previously acquired utilizing Sanger sequencing. The assay, which uses a closed cartridge system in combination with a battery-powered Alere q analyzer and which has the potential to extend the current gene target panel, could serve as a rapid and robust point-of-care test in settings lacking a comprehensive molecular laboratory infrastructure to differentiate TB patients infected with MDR and non-MDR strains and to assist clinicians with their early treatment decisions.

Rapid microarray-based assay for detection of pyrazinamide resistant Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a key antibiotic for the treatment of drug susceptible tuberculosis. PZA-resistance is mainly mediated by mutations in the pncA gene; however the current gold standard is a phenotypic drug susceptibility test requiring a well-adjusted pH-value for reliable results. Our melting curve assay detects a non-wild type genotype in selected pncA regions in at least 3750 gene copies/mL within 2.5 hours. The prototype assay was further evaluated by analyzing 271 Mycobacterium tuberculosis complex isolates from Swaziland originating from a previously published drug resistance survey and including 118 isolates with pncA mutations. Sensitivity was 83% (95% CI 75-89%) and specificity was 100% (95% CI 98-100%). Under consideration of further improvements with regard to the target range our melting curve assay has the potential as a rapid rule-in test for PZA susceptibility (wild type pncA), however false resistant results (mutant pncA, but PZA susceptible) cannot be ruled out completely.

An application of competitive reporter monitored amplification (CMA) for rapid detection of single nucleotide polymorphisms (SNPs).

Single nucleotide polymorphisms (SNPs) are essential parameters in molecular diagnostics and can be used for the early detection and clinical prognosis in various diseases. Available methods for SNP detection are still labor-intensive and require a complex laboratory infrastructure, which are not suitable for the usage in resource-limited settings. Thus, there is an urgent need for a simple, reliable and rapid approach. In this paper we modified the previously developed competitive reporter monitored amplification (CMA) technique for the detection of resistance mediating SNPs in Mycobacterium tuberculosis complex (MTBC) strains. As a proof-of-principle for the application of the CMA-based SNP assay in routine molecular tuberculosis diagnostic, we show that the assay recognizes resistance mediating SNPs for rifampicin, isoniazid and ethambutol from either isolated DNA or heat inactivated M. tuberculosis cell cultures. The CMA-based SNP assay can identify the most prevalent resistance mediating mutations in the genes rpoB, katG, embB, and the promotor region of inhA within one hour.