Whole genome sequence of Vibrio cholerae directly from dried spotted filter paper

Abstract

Background Global estimates for cholera annually approximate 4 million cases worldwide with 95,000 deaths. Recent outbreaks, including Haiti and Yemen, are reminders that cholera is still a global health concern. Cholera outbreaks can rapidly induce high death tolls by overwhelming the capacity of health facilities, especially in remote areas or areas of civil unrest. Recent studies demonstrated that stool specimens preserved on filter paper facilitate molecular analysis of Vibrio cholerae in resource limited settings. Specimens preserved in a rapid, low-cost, safe and sustainable manner for sequencing provides previously unavailable data about circulating cholera strains. This may ultimately contribute new information to shape public policy response on cholera control and elimination. Methodology/Principal findings Whole genome sequencing (WGS) recovered close to a complete sequence of the V . cholerae O1 genome with satisfactory genome coverage from stool specimens enriched in alkaline peptone water (APW) and V . cholerae culture isolates, both spotted on filter paper. The minimum concentration of V . cholerae DNA sufficient to produce quality genomic information was 0.02 ng/μL. The genomic data confirmed the presence or absence of genes of epidemiological interest, including cholera toxin and pilus loci. WGS identified a variety of diarrheal pathogens from APW-enriched specimen spotted filter paper, highlighting the potential for this technique to explore the gut microbiome, potentially identifying co-infections, which may impact the severity of disease. WGS demonstrated that these specimens fit within the current global cholera phylogenetic tree, identifying the strains as the 7 th pandemic El Tor. Conclusions WGS results allowed for mapping of short reads from APW-enriched specimen and culture isolate spotted filter papers. This provided valuable molecular epidemiological sequence information on V . cholerae strains from remote, low-resource settings. These results identified the presence of co-infecting pathogens while providing rare insight into the specific V . cholerae strains causing outbreaks in cholera-endemic areas.

On the transformation of MinHash-based uncorrected distances into proper evolutionary distances for phylogenetic inference

“…It enables phylogenetic trees to be quickly reconstructed from a large number of genomes without the burden of multiple sequence alignments (see e.g. 2,29, [44][45][46][47][48][49][50][51][52]. This report confirms this view by showing that proper evolutionary distances can be easily derived from MH p-distance estimates, therefore enabling accurate phylogenetic inferences.…”

NewVibrio choleraesequences from Eastern and Southern Africa alter our understanding of regional cholera transmission

“…Other than sequencing costs, one of the barriers to generating V. cholerae whole genome sequences is heterogeneity in the sample types available; sequencing typically requires relatively large amounts of purified nucleic acid, and is therefore often not attempted when only crude specimens (as opposed to cultured isolates) are available. Previous studies have shown that V. cholerae sequencing from Alkaline Peptone enriched (APW) stool or isolates on filter paper can be successful 26 , so we attempted to take this one step further, comparing the success of various specimen types preserved on filter paper across samples from several countries and using different nucleic acid extraction techniques. Specifically, we compared sequencing from bacterial cultures, bacterial isolates preserved on filter paper, and whole stool directly spotted onto filter paper.…”

“…In this study, we aimed to fill some of the gaps in our understanding of 7PET V. cholerae O1 circulation in Southeast Africa using isolates collected from 2007–2018 in Uganda, Kenya, Tanzania, Malawi, and Zambia. In addition, we explored the potential of performing whole genome sequencing from low-cost sample preservation methods (e.g., stool or isolates on filter paper), which facilitates specimen collection in resource-constrained areas where isolates would otherwise not be captured 18,26 . We found that sequencing from these samples can produce whole genome sequences, which we used to identify multiple co-circulating V. cholerae lineages in Southeast Africa.…”

Putting rapid tests to work in surveillance and control of cholera

“…Ideally, the new generation of tests should help clinicians to decide on appropriate antibiotic use in a context of growing concern of antimicrobial resistance. Furthermore, as we rely more on genomics for the classification of circulating V. cholerae strains, more work is needed to understand whether the RDTs used could serve as an appropriate medium to temporarily preserve and transport the genetic material needed for subsequent molecular analyses, as has been done with filter paper [ 14 ].…”