Search

Acute viral gastroenteritis is an intestinal infection that can be caused by several different viruses. Here we describe the evaluation and verification of Seeplex Diarrhea-V ACE (Seeplex DV), a novel commercial multiplex reverse transcription-PCR (RT-PCR) assay that detects 5 diarrheal pathogens, including adenovirus, rotavirus, norovirus genogroup I (GI) and GII, and astrovirus. We describe a retrospective study of 200 clinical specimens of which 177 were stool specimens previously tested for the presence of gastrointestinal viruses by electron microscopy (EM) and/or real-time RT-PCR (rRT-PCR). The remaining 23 specimens comprised other human pathogens of viral or bacterial origin. Discordant norovirus GI and GII results were resolved using a commercial kit; discordant adenovirus and rotavirus results were resolved using a home brew multiplex rRT-PCR assay. Diagnostic sensitivities and specificities were calculated before and after discordant analysis. After discordant analysis, estimated diagnostic sensitivities were 100% for adenovirus, rotavirus, and norovirus GI and 97% for norovirus GII. Diagnostic specificities after discordant analysis were 100% for adenovirus, rotavirus, and norovirus GI and 99.4% for norovirus GII. The 95% limits of detection were 31, 10, 2, and 1 genome equivalent per reaction for adenovirus, rotavirus, and norovirus GI and GII, respectively. The results demonstrate that the Seeplex DV assay is sensitive, specific, convenient, and reliable for the simultaneous detection of several viral pathogens directly in specimens from patients with gastroenteritis. Importantly, this novel multiplex PCR assay enabled the identification of viral coinfections in 12 (6.8%) stool specimens.

Background In early 2017 an outbreak of Mumps virus affected over 100 individuals in the province of Ontario, concurrent with multiple mumps virus outbreaks across North America. Traditional genotyping of mumps outbreaks relies on sequencing a portion of the small hydrophobic (SH) gene, but has limited capability to distinguish between strains of the same genotype. Most mumps cases in Ontario in recent years are of genotype G. We used a novel whole genome sequencing (WGS) protocol to perform a molecular epidemiological investigation of the outbreak. Methods Throat (n = 5) and buccal (n = 15) swabs positive by RT-PCR for SH or Fusion (F) gene targets were cultured in primary Rhesus monkey kidney cells. Cell free viral extract underwent RT-PCR and subsequent PCR amplification using overlapping primer pairs to cover the entire 15 kilobase (kb) genome. The first 8 samples were amplified with 18 pairs of overlapping primers, which was reduced to 9 sets (average fragment size 1.9 kb, range 1.6–2.8 kb) for the final 12 samples. Mumps cDNA libraries were prepared with Nextera XT kit and WGS of the indexed fragments was performed with V2 reagent kits on the Illumina MiSeq instrument. Reference based genome assembly was performed using samtools version 1.4. Phylogenetic analysis was performed by maximum likelihood method in MEGA7. Results We identified two distinct genotype G lineages comprised of 9 patients each and closely related to a 2009–2010 outbreak in Ontario and New York (Figure 1). Inter-lineage single nucleotide polymorphism (SNP) differences ranged from 25 to 31, whereas intra-lineage SNPs ranged from 0 to 8 SNPs. Two outlying sequences, of genotype C and G respectively, may represent sporadic introduction of virus from other areas. Time from virus isolation to SNP based analysis was approximately 4 days. Conclusion WGS of Mumps virus culture isolates using the PCR fragment method identified two distinct genotype G lineages in a large provincial outbreak. This method may aid public health authorities identify separate transmission chains in the case of large outbreaks. Disclosures All authors: No reported disclosures.