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PLoS Negl Trop Dis. 2019 May 30;13(5):e0007330. doi: 10.1371/journal.pntd.0007330. eCollection 2019 May.

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

Author information

1
Wellcome Trust Sanger Institute, Genome campus, Hinxton United Kingdom.
2
M.A. SANTE (Meilleur Accès aux Soins de Santé), Yaoundé, Cameroon.
3
Department of Microbiology and Parasitology, Faculty of Science, University of Buea, Buea, Cameroon.
4
Department of Public Health, Faculty of Medicine and Pharmaceutical Sciences, University of Dschang, Cameroon Dschang Cameroon.
5
Clinical Research Unit, Division of Health Operations Research, Ministry of Public Health, N°8, quartier du Lac (Yaoundé III), Cameroon.
6
Ministry of Health, Lilongwe, Malawi.
7
John Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America.
8
Assistance Publique-Hôpitaux de Marseille (APHM), Marseille, France.
9
Hôpital Européen, Marseille, France.
10
National Laboratory of Public Health in Haiti (LNSP), Ministry of Public Health and Population, Haiti.
11
Directorate for Epidemiology, Laboratory and Research, Ministry of Public Health and Population, Haiti.
12
Sorbonne Université, INSERM, Institut Pierre-Louis d'Epidémiologie et de Santé Publique, APHP, Hôpital Pitié-Salpêtrière, Paris, France.
13
London School of Hygiene and Tropical Medicine, Keppel St, Bloomsbury, London WC1E 7HT, United Kingdom.

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 7th 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.

Conflict of interest statement

The authors have declared that no competing interests exist.

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