Detection and identification of bacterial pathogens directly from sputum samples by pyrosequencing

Purpose. The standard culture findings for detecting and identifying bacterial pathogens in patients with lower respiratory tract infections (LRTIs) are usually not available for two to three days, which delays the initiation of appropriate antibiotic therapies. We aimed to develop a faster method of identification of bacterial pathogens in LRTIs which would offer a timelier guide to initial antibiotic choices. Methodology. The developed PCR-pyrosequencing-based method was defined as mask PCR-pyrosequencing (MPP). This method uses primer pairs deliberately designed to mask the interference of colonised bacteria in sputum to detect and identify bacterial pathogens directly from LRTI patient sputum samples within 5 h. Accordingly, the standard PCR-pyrosequencing method was defined as normal PCR-pyrosequencing (NPP) here. The clinical performance of the novel system was evaluated by comparing with traditional semi-quantitative culture and identification results. Results. The coincidence for culture and MPP was 91.3%. Compared with the semi-quantitative culture results, NPP identified 89.9% strains of grade 3+ (corresponding to 1.0x 10(6)c.f.u ml(-1)) and 100% of grade 4+ (corresponding to 1.0x 10(7)c.f.u ml(-1)), both of which were considered to be the presumptive pathogens in the clinics. MPP identified 98.9 % strains of grade 3+ and 100 % of grade 4+. Additionally, PCR-pyrosequencing could detect a minimum concentration of 1.0x 10(6)c.f.u ml(-1) of bacteria in sputum, with no significant difference between NPP and MPP. Conclusion. The PCR-pyrosequencing technique developed in this study is an accurate, fast, and high throughput method for the direct detection and identification of bacterial pathogens from sputum.