RESUMO
The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis. The rifampicin resistance determinant region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTC) strains from nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High-resolution melting (HRM) analysis following real-time PCR was used to identify M. tuberculosis strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific single-nucleotide polymorphism (SNP) transversion mutations. This method was used to screen 252 M. tuberculosis clinical isolates, including 154 RIF-resistant strains and 174 INH-resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF-resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in M. tuberculosis strains and offers several advantages over current molecular and culture-based techniques.
Assuntos
DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase/métodos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Catalase/genética , Elementos de DNA Transponíveis , RNA Polimerases Dirigidas por DNA/genética , Genes Bacterianos , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação de Sentido Incorreto , Mycobacterium tuberculosis/isolamento & purificação , Oxirredutases/genética , Sensibilidade e Especificidade , Temperatura de TransiçãoRESUMO
A multiplex real-time PCR assay for the detection of Mycoplasma pneumoniae (MP181), Chlamydia (Chlamydophila) pneumoniae (CP-Arg), Legionella spp. (Pan-Leg), and the human RNase P (RNase P) gene was developed for rapid testing of atypical bacterial respiratory pathogens in clinical specimens. This method uses 4 distinct hydrolysis probes to detect 3 leading causes of community-acquired pneumonia. The assay was evaluated for specificity and sensitivity by testing against 35 related organisms, a dilution series of each specific target and 197 clinical specimens. Specificity testing demonstrated no cross-reactivity. A comparison to previously validated singleplex real-time PCR assays for each agent was also performed. The analytical sensitivity for specific pathogen targets in both the singleplex and multiplex was identical (50 fg), while efficiencies ranged from 82% to 97% for the singleplex assays and from 90% to 100% for the multiplex assay. The clinical sensitivity of the multiplex assay was improved for the Pan-Leg and CP-Arg targets when compared to the singleplex. The MP181 assay displayed equivalent performance. This multiplex assay provides an overall improvement in the diagnostic capability for these agents by demonstrating a sensitive, high-throughput and rapid method. This procedure may allow for a practical and efficient means to test respiratory clinical specimens for atypical pneumonia agents in health care settings and facilitate an appropriate public health response to outbreaks.
Assuntos
Infecções por Chlamydia/diagnóstico , Chlamydophila pneumoniae/isolamento & purificação , Legionella/isolamento & purificação , Legionelose/diagnóstico , Infecções por Mycoplasma/diagnóstico , Mycoplasma pneumoniae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Técnicas Bacteriológicas/métodos , Infecções por Chlamydia/microbiologia , Chlamydophila pneumoniae/genética , Infecções Comunitárias Adquiridas/diagnóstico , Infecções Comunitárias Adquiridas/microbiologia , Humanos , Legionella/genética , Legionelose/microbiologia , Infecções por Mycoplasma/microbiologia , Mycoplasma pneumoniae/genética , Sondas de Oligonucleotídeos/genética , Reação em Cadeia da Polimerase/normas , Ribonuclease P/genética , Sensibilidade e EspecificidadeRESUMO
Four nucleic acid extraction procedures (2 automated and 2 manual) were compared for their efficiency at isolating Mycoplasma pneumoniae DNA. Oropharyngeal swabs from healthy volunteers were spiked with varying amounts of M. pneumoniae, extracted, and tested using real-time polymerase chain reaction. Our data indicate that both automated extraction methods consistently outperform the manual procedures.