title: Evaluation in Cameroon of a Novel, Simplified Methodology to Assist Molecular Microbiological Analysis of V. cholerae in Resource-Limited Settings.
abstract: BACKGROUND
  Vibrio 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.
  
  
  RESULTS
  Forty-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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, United States of America.
authors.forename: Amanda K
authors.initials: AK
authors.lastname: Debes
authors.affiliation: Department of Biomedical Sciences, University of Dschang, Dschang, Cameroon.|M.A. SANTE (Meilleur Accès aux Soins de Santé), Yaoundé, Cameroon.|Division of Health Operations Research, Ministry of Public Health, Yaoundé, Cameroon.
authors.forename: Jerome
authors.initials: J
authors.lastname: Ateudjieu
authors.forename: Etienne
authors.initials: E
authors.lastname: Guenou
authors.affiliation: M.A. SANTE (Meilleur Accès aux Soins de Santé), Yaoundé, Cameroon.
authors.forename: Etiene
authors.initials: E
authors.lastname: Guenou
authors.affiliation: University of the Philippines Manila-National Institutes of Health, Manila, Philippines.
authors.forename: Anna Lena
authors.initials: AL
authors.lastname: Lopez
authors.affiliation: University of the Philippines Manila-National Institutes of Health, Manila, Philippines.|Research Institute for Tropical Medicine, Muntinlupa, Philippines.
authors.forename: Mark Philip
authors.initials: MP
authors.lastname: Bugayong
authors.affiliation: University of the Philippines Manila-National Institutes of Health, Manila, Philippines.|Research Institute for Tropical Medicine, Muntinlupa, Philippines.
authors.forename: Pearl Joy
authors.initials: PJ
authors.lastname: Retiban
authors.affiliation: Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique.
authors.forename: Marcelino
authors.initials: M
authors.lastname: Garrine
authors.affiliation: Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique.|Instituto Nacional de Saúde (INS), Ministério da Saúde, Maputo, Mozambique.
authors.forename: Inacio
authors.initials: I
authors.lastname: Mandomando
authors.affiliation: University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Shan
authors.initials: S
authors.lastname: Li
authors.affiliation: University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: O Colin
authors.initials: OC
authors.lastname: Stine
authors.affiliation: Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, United States of America.
authors.forename: David A
authors.initials: DA
authors.lastname: Sack
mesh_terms: D002163:Cameroon / Q000453:epidemiology; D002675:Child, Preschool; D002771:Cholera / Q000453:epidemiology / Q000382:microbiology*; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D018598:Minisatellite Repeats; D017720:Molecular Epidemiology / Q000379:methods; D058889:Molecular Typing / Q000379:methods*; D009073:Mozambique / Q000453:epidemiology; D010209:Paper; D010679:Philippines / Q000453:epidemiology; D013048:Specimen Handling / Q000379:methods*; D014734:Vibrio cholerae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification