RESUMO
Detection of bacterial gene transcripts in low density mucosal samples is challenging. We evaluated the NanoString nCounter system for transcript detection in Neisseria meningitidis (Nm) cultures. The method was sensitive, reproducible (R2â¯=â¯0.99) and demonstrated changes in gene expression. Studying Nm transcripts from pharyngeal samples may be feasible using this approach.
Assuntos
Perfilação da Expressão Gênica/métodos , Neisseria meningitidis/genética , Testes Diagnósticos de Rotina/métodos , Perfilação da Expressão Gênica/instrumentação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estresse FisiológicoRESUMO
Improved methods for the detection and characterization of carried Neisseria meningitidis isolates are needed. We evaluated a multiplex PCR algorithm for the detection of a variety of carriage strains in the meningitis belt. To further improve the sensitivity and specificity of the existing PCR assays, primers for gel-based PCR assays (sodC, H, Z) and primers/probe for real-time quantitative PCR (qPCR) assays (porA, cnl, sodC, H, E, Z) were modified or created using Primer Express software. Optimized multiplex PCR assays were tested on 247 well-characterised carriage isolates from six countries of the African meningitis belt. The PCR algorithm developed enabled the detection of N. meningitidis species using gel-based and real-time multiplex PCR targeting porA, sodC, cnl and characterization of capsule genes through sequential multiplex PCR assays for genogroups (A, W, X, then B, C, Y and finally H, E and Z). Targeting both porA and sodC genes together allowed the detection of meningococci with a sensitivity of 96% and 89% and a specificity of 78% and 67%, for qPCR and gel-based PCR respectively. The sensitivity and specificity ranges for capsular genogrouping of N. meningitidis are 67% - 100% and 98%-100% respectively for gel-based PCR and 90%-100% and 99%-100% for qPCR. We developed a PCR algorithm that allows simple, rapid and systematic detection and characterisation of most major and minor N. meningitidis capsular groups, including uncommon capsular groups (H, E, Z).
Assuntos
Algoritmos , Meningite Meningocócica , Reação em Cadeia da Polimerase Multiplex/métodos , Neisseria meningitidis/genética , Porinas/genética , Superóxido Dismutase/genética , Feminino , Humanos , Masculino , Mali/epidemiologia , Meningite Meningocócica/diagnóstico , Meningite Meningocócica/epidemiologia , Meningite Meningocócica/genética , Neisseria meningitidis/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: Detection of meningococcal carriers is key to understanding the epidemiology of Neisseria meningitidis, yet no gold standard has been established. Here, we directly compare two methods for collecting pharyngeal swabs to identify meningococcal carriers. METHODS: We conducted cross-sectional surveys of schoolchildren at multiple sites in Africa to compare swabbing the posterior pharynx behind the uvula (U) to swabbing the posterior pharynx behind the uvula plus one tonsil (T). Swabs were cultured immediately and analyzed using molecular methods. RESULTS: One thousand and six paired swab samples collected from schoolchildren in four countries were analyzed. Prevalence of meningococcal carriage was 6.9% (95% CI: 5.4-8.6%) based on the results from both swabs, but the observed prevalence was lower based on one swab type alone. Prevalence based on the T swab or the U swab alone was similar (5.2% (95% CI: 3.8-6.7%) versus 4.9% (95% CI: 3.6-6.4%) respectively (p=0.6)). The concordance between the two methods was 96.3% and the kappa was 0.61 (95% CI: 0.50-0.73), indicating good agreement. CONCLUSIONS: These two commonly used methods for collecting pharyngeal swabs provide consistent estimates of the prevalence of carriage, but both methods misclassified carriers to some degree, leading to underestimates of the prevalence.