RESUMO
There is a growing awareness that molecular diagnostics for detect-to-treat applications will soon need a highly multiplexed mutation detection and identification capability. In this study, we converted an open-amplicon microarray hybridization test for multidrug-resistant (MDR) Mycobacterium tuberculosis into an entirely closed-amplicon consumable (an amplification microarray) and evaluated its performance with matched sputum and sediment extracts. Reproducible genotyping (the limit of detection) was achieved with â¼25 M. tuberculosis genomes (100 fg of M. tuberculosis DNA) per reaction; the estimated shelf life of the test was at least 18 months when it was stored at 4°C. The test detected M. tuberculosis in 99.1% of sputum extracts and 100% of sediment extracts and showed 100% concordance with the results of real-time PCR. The levels of concordance between M. tuberculosis and resistance-associated gene detection were 99.1% and 98.4% for sputum and sediment extracts, respectively. Genotyping results were 100% concordant between sputum and sediment extracts. Relative to the results of culture-based drug susceptibility testing, the test was 97.1% specific and 75.0% sensitive for the detection of rifampin resistance in both sputum and sediment extracts. The specificity for the detection of isoniazid (INH) resistance was 98.4% and 96.8% for sputum and sediment extracts, respectively, and the sensitivity for the detection of INH resistance was 63.6%. The amplification microarray reported the correct genotype for all discordant phenotype/genotype results. On the basis of these data, primary sputum may be considered a preferred specimen for the test. The amplification microarray design, shelf life, and analytical performance metrics are well aligned with consensus product profiles for next-generation drug-resistant M. tuberculosis diagnostics and represent a significant ease-of-use advantage over other hybridization-based tests for diagnosing MDR tuberculosis.
Assuntos
Técnicas de Genotipagem/métodos , Sedimentos Geológicos/microbiologia , Mycobacterium tuberculosis/genética , Análise de Sequência com Séries de Oligonucleotídeos , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Genes MDR/genética , Genótipo , Humanos , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana , Técnicas Analíticas Microfluídicas , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Rifampina/farmacologia , Sensibilidade e EspecificidadeRESUMO
Clinical laboratories are recognizing the importance of implementing sensitive and specific molecular diagnostic tests. However, widespread adoption of these tests requires simplified workflows without requiring expensive supporting instrumentation. To enable microarray-based analysis to meet these requirements, we describe a valveless flow cell for disposable use that supports PCR coupled with microarray hybridization in the same chamber. The flow cell assembly consists simply of double-faced tape, a plastic microarray substrate, an absorbent, and a commercially-available hydrophilic thin film. The simple construction lends itself to low-cost and ease of manufacturing, yet several features reduce the complexity of the standard microarray workflow. First, there is no requirement for custom instrumentation. Second, the hydrophilic thin film allows uniform filling of a microfluidic chamber. Third, a geometric capillary stop design confines liquid to the microarray chamber during PCR, and thus eliminates the need for a valve or hydrophobic surface treatment. And fourth, imbibition drives the uniform removal of liquid reagents from the array chamber. Three hundred genomic copies of methicillin-resistant Staphylococcus aureus (MRSA) are detected in a flow cell with gel drop microarrays printed on an unmodified plastic substrate. This sensitivity is shown to be comparable to conventional methods (i.e., PCR in a tube, with separate hybridization in a microarray chamber, where amplicon is exposed to the workspace before and after hybridization). However, the flow cell combines these multiple steps into a simple, compact workflow without the need for complex valves or custom instrumentation and is less susceptible to contamination of the workspace than conventional methods because the amplicon is confined to the device.