DNA pyrosequencing-based bacterial pathogen identification in a pediatric hospital setting.

Sole reliance on biochemical methods can limit the clinical microbiology laboratory's ability to identify bacterial pathogens. This study describes the incorporation of DNA pyrosequencing-based identification for routine pathogen identification of atypical clinical isolates in a large children's hospital. The assay capitalized on the highly conserved nature of 16S rRNA genes by positioning amplification and sequencing primers in conserved target sequences flanking the variable V1 and V3 regions. A total of 414 isolates of 312 pediatric patients were tested by DNA pyrosequencing during the time period from December 2003 to July 2006. Seventy-eight different genera were specified by DNA pyrosequencing, and isolates were derived from diverse specimen types. By integrating DNA sequencing of bacterial pathogens with conventional microbiologic methods, isolates that lacked a definitive identification by biochemical testing yielded genus- or species-level identifications in approximately 90% of cases by pyrosequencing. Improvements incorporated into the assay process during the period of clinical testing included software enhancements, improvements in sequencing reagents, and refinements in database search strategies. Coupled with isolation by bacteriologic culture and biochemical testing, DNA pyrosequencing-based bacterial identification was a valuable tool that markedly improved bacterial pathogen identification in a pediatric hospital setting.