Full Molecular Typing of Neisseria meningitidis Directly from Clinical Specimens for Outbreak Investigation.
Mark ItskoAdam C RetchlessSandeep J JosephAbigail Norris TurnerJose A BazanAdodo Yao SadjiRasmata Ouédraogo-TraoréXin WangPublished in: Journal of clinical microbiology (2020)
Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis worldwide and an occasional cause of meningococcal urethritis. When isolates are unavailable for surveillance or outbreak investigations, molecular characterization of pathogens needs to be performed directly from clinical specimens, such as cerebrospinal fluid (CSF), blood, or urine. However, genome sequencing of specimens is challenging because of low bacterial and high human DNA abundances. We developed selective whole-genome amplification (SWGA), an isothermal multiple-displacement amplification-based method, to efficiently enrich, sequence, and de novo assemble N. meningitidis DNA from clinical specimens with low bacterial loads. SWGA was validated with 12 CSF specimens from invasive meningococcal disease cases and 12 urine specimens from meningococcal urethritis cases. SWGA increased the mean proportion of N. meningitidis reads by 2 to 3 orders of magnitude, enabling identification of at least 90% of the 1,605 N. meningitidis core genome loci for 50% of the specimens. The validated method was used to investigate two meningitis outbreaks recently reported in Togo and Burkina Faso. Twenty-seven specimens with low bacterial loads were processed by SWGA before sequencing, and 12 of 27 were successfully assembled to obtain the full molecular typing and vaccine antigen profile of the N. meningitidis pathogen, thus enabling thorough characterization of outbreaks. This method is particularly important for enhancing molecular surveillance in regions with low culture rates. SWGA produces enough reads for phylogenetic and allelic analysis at a low cost. More importantly, the procedure can be extended to enrich other important human bacterial pathogens.
Keyphrases
- cerebrospinal fluid
- fine needle aspiration
- endothelial cells
- nucleic acid
- single molecule
- public health
- low cost
- single cell
- intensive care unit
- acute kidney injury
- circulating tumor
- gene expression
- genetic diversity
- cell free
- ultrasound guided
- candida albicans
- circulating tumor cells
- data analysis
- bioinformatics analysis
- amino acid