Abstract
BackgroundVibrio cholerae is endemic in South Asia and Africa where outbreaks of cholera occur widely and are particularly associated with poverty and poor sanitation. Knowledge of the genetic diversity of toxigenic V. cholerae isolates, particularly in Africa, remains scarce. The constraints in improving this understanding is not only the lack of regular cholera disease surveillance, but also the lack of laboratory capabilities in endemic countries to preserve, store and ship isolates in a timely manner. We evaluated the use of simplified sample preservation methods for molecular characterization using multi-locus variable-number tandem-repeat analysis (MLVA) for differentiation of Vibrio cholerae genotypes.Methods and FindingsForty-seven V. cholerae isolates and 18 enriched clinical specimens (e.g. stool specimens after enrichment in broth) from cholera outbreaks in Cameroon were preserved on Whatman filter paper for DNA extraction. The samples were collected from two geographically distinct outbreaks in the Far North of Cameroon (FNC) in June 2014 and October 2014. In addition, a convenience sample of 14 isolates from the Philippines and 8 from Mozambique were analyzed. All 87 DNAs were successfully analyzed including 16 paired samples, one a cultured isolate and the other the enriched specimen from which the isolate was collected. Genotypic results were identical between 15 enriched specimens and their culture isolates and the other pair differed at single locus. Two closely related, but distinct clonal complexes were identified among the Cameroonian specimens from 2014.ConclusionsCollecting V. cholerae using simplified laboratory methods in remote and low-resource settings allows for subsequent advanced molecular characterization of V. cholerae O1. These simplified DNA preservation methods identify V. cholerae and make possible timely information regarding the genetic diversity of V. cholerae; our results set the stage for continued molecular epidemiological research to better understand the transmission and dissemination of V. cholerae in Africa and elsewhere worldwide.
Highlights
Cholera remains a major public health problem in developing countries, in Africa and Asia, where endemic and epidemic disease continues to devastate vulnerable populations
Collecting V. cholerae using simplified laboratory methods in remote and low-resource settings allows for subsequent advanced molecular characterization of V. cholerae O1
These simplified DNA preservation methods identify V. cholerae and make possible timely information regarding the genetic diversity of V. cholerae; our results set the stage for continued molecular epidemiological research to better understand the transmission and dissemination of V. cholerae in Africa and elsewhere worldwide
Summary
Cholera remains a major public health problem in developing countries, in Africa and Asia, where endemic and epidemic disease continues to devastate vulnerable populations. The etiologic agent of cholera, V. cholerae, has more than 200 serogroups, differentiated by the O-antigen on the lipopolysaccharide (LPS) of the bacteria’s outer membrane [1]. Of these 200 serogroups, only those that produce cholera toxin (CT) are known to cause epidemic and pandemic disease, primarily serogroups O1 and O139 [2]. The method uses the number of repeats at each specific locus to differentiate between isolates [4] and has shown substantial variation between isolates in a single outbreak [5]. We evaluated the use of simplified sample preservation methods for molecular characterization using multi-locus variable-number tandemrepeat analysis (MLVA) for differentiation of Vibrio cholerae genotypes
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