Setup, Validation, and Quality Control of a Centralized Whole-Genome-Sequencing Laboratory: Lessons Learned.

Routine use of whole-genome analysis for infectious diseases can be used to enlighten various scenarios pertaining to public health, including identification of microbial pathogens, relating individual cases to an outbreak of infectious disease, establishing an association between an outbreak of food poisoning and a specific food vehicle, inferring drug susceptibility, source tracing of contaminants, and study of variations in the genome that affect pathogenicity/virulence. We describe the setup, validation, and ongoing verification of a centralized whole-genome-sequencing (WGS) laboratory to carry out sequencing for these public health functions for the National Infection Services, Public Health England, in the United Kingdom. The performance characteristics and quality control metrics measured during validation and verification of the entire end-to-end process (accuracy, precision, reproducibility, and repeatability) are described and include information regarding the automated pass and release of data to service users without intervention.

Non-culture detection of Streptococcus agalactiae (Lancefield group B Streptococcus) in clinical samples by real-time PCR.

A real-time PCR assay targeting the cylB gene was developed to detect Streptococcus agalactiae [Lancefield group B Streptococcus (GBS)] from clinical samples. A total of 110 blood culture-negative samples [75 cerebrospinal fluid (CSF) and 35 EDTA blood samples] from neonates with probable GBS sepsis or meningitis were analysed. Among these, 16 of the 75 CSF samples were positive [21.3%, 95% confidence interval (CI) 12.7-32.3%] and two of the 35 EDTA blood samples were positive (5.7%, 95% CI 0.7-19.2%). The proportion testing positive in the CSF samples was significantly higher than in the EDTA blood samples (P=0.05, Fisher's exact test). Overall, this real-time PCR assay was shown to be superior to culture methods for detection of GBS from CSF and EDTA blood samples.

Utility of real-time amplification of selected 16S rRNA gene sequences as a tool for detection and identification of microbial signatures directly from clinical samples.

The potential of incorporating a real-time PCR for amplification and detection of 16S rRNA gene signatures directly from clinical samples was assessed as a tool for diagnostics. Universal PCR primers spanning short variable regions (~500 bp) were optimized for real-time PCR and tested in comparison with a longer fragment (~1400 bp) generated from block-based amplification. Real-time PCR had improved sensitivity of detection (8% increase), decreased amplification time and simplified downstream processing. The real-time PCR primers also offered an improvement in detection of bacteria from samples that demonstrated inhibition with the block-based primers and in the resolution of mixed-sequence traces. In addition to testing primer sensitivity, the effect of amplifying and sequencing two different variable regions of the 16S rRNA gene on organism identification was compared. By amplifying and sequencing a shorter variable region, the number of species that were identified to the species level was reduced by 18%.