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. Methods and Findings 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.
References
[1]
Chatterjee SN, Chaudhuri K (2003) Lipopolysaccharides of Vibrio cholerae. I. Physical and chemical characterization. Biochim Biophys Acta 1639: 65–79. pmid:14559113 doi: 10.1016/j.bbadis.2003.08.004
[2]
Chen Y, Johnson JA, Pusch GD, Morris JG Jr., Stine OC (2007) The genome of non-O1 Vibrio cholerae NRT36S demonstrates the presence of pathogenic mechanisms that are distinct from those of O1 Vibrio cholerae. Infect Immun 75: 2645–2647. pmid:17283087 doi: 10.1128/iai.01317-06
[3]
Kendall EA, Chowdhury F, Begum Y, Khan AI, Li S, et al. (2010) Relatedness of Vibrio cholerae O1/O139 isolates from patients and their household contacts, determined by multilocus variable-number tandem-repeat analysis. J Bacteriol 192: 4367–4376. doi: 10.1128/JB.00698-10. pmid:20585059
[4]
Danin-Poleg Y, Cohen LA, Gancz H, Broza YY, Goldshmidt H, et al. (2007) Vibrio cholerae strain typing and phylogeny study based on simple sequence repeats. J Clin Microbiol 45: 736–746. pmid:17182751 doi: 10.1128/jcm.01895-06
[5]
Rebaudet S, Mengel MA, Koivogui L, Moore S, Mutreja A, et al. (2014) Deciphering the origin of the 2012 cholera epidemic in Guinea by integrating epidemiological and molecular analyses. PLoS Negl Trop Dis 8: e2898. doi: 10.1371/journal.pntd.0002898. pmid:24901522
[6]
Kiiru J, Mutreja A, Mohamed AA, Kimani RW, Mwituria J, et al. (2013) A study on the geophylogeny of clinical and environmental Vibrio cholerae in Kenya. PLoS One 8: e74829. doi: 10.1371/journal.pone.0074829. pmid:24066154
[7]
Azarian T, Ali A, Johnson JA, Mohr D, Prosperi M, et al. (2014) Phylodynamic analysis of clinical and environmental Vibrio cholerae isolates from Haiti reveals diversification driven by positive selection. MBio 5. doi: 10.1128/mbio.01824-14
[8]
Ghosh R, Nair GB, Tang L, Morris JG, Sharma NC, et al. (2008) Epidemiological study of Vibrio cholerae using variable number of tandem repeats. FEMS Microbiol Lett 288: 196–201. doi: 10.1111/j.1574-6968.2008.01352.x. pmid:18811655
[9]
Alam M, Hasan NA, Sadique A, Bhuiyan NA, Ahmed KU, et al. (2006) Seasonal cholera caused by Vibrio cholerae serogroups O1 and O139 in the coastal aquatic environment of Bangladesh. Appl Environ Microbiol 72: 4096–4104. pmid:16751520 doi: 10.1128/aem.00066-06
[10]
Stine OC, Alam M, Tang L, Nair GB, Siddique AK, et al. (2008) Seasonal cholera from multiple small outbreaks, rural Bangladesh. Emerg Infect Dis 14: 831–833. doi: 10.3201/eid1405.071116. pmid:18439375
[11]
Bhuiyan NA, Nusrin S, Ansaruzzaman M, Islam A, Sultana M, et al. (2012) Genetic characterization of Vibrio cholerae O1 strains isolated in Zambia during 1996–2004 possessing the unique VSP-II region of El Tor variant. Epidemiol Infect 140: 510–518. doi: 10.1017/S0950268811000926. pmid:21676349
[12]
Mohamed AA, Oundo J, Kariuki SM, Boga HI, Sharif SK, et al. (2012) Molecular epidemiology of geographically dispersed Vibrio cholerae, Kenya, January 2009-May 2010. Emerg Infect Dis 18: 925–931. doi: 10.3201/eid1806.111774. pmid:22607971
[13]
Lam C, Octavia S, Reeves PR, Lan R (2012) Multi-locus variable number tandem repeat analysis of 7th pandemic Vibrio cholerae. BMC Microbiol 12: 82. doi: 10.1186/1471-2180-12-82. pmid:22624829
[14]
Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, et al. (2013) Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382: 209–222. doi: 10.1016/S0140-6736(13)60844-2. pmid:23680352
[15]
Nhampossa T, Mandomando I, Acacio S, Quinto L, Vubil D, et al. (2015) Diarrheal Disease in Rural Mozambique: Burden, Risk Factors and Etiology of Diarrheal Disease among Children Aged 0–59 Months Seeking Care at Health Facilities. PLoS One 10: e0119824. doi: 10.1371/journal.pone.0119824. pmid:25973880
[16]
Debes AK, Ateudjieu J., Guenou E., Ebile W., Sonkoua I.T., Njimbia A.C., Steinwald P., Ram M., Sack D.A. (In Press) Clinical and Environmental Surveillance for Vibrio cholerae in Resource Contstrained Areas: Application during a one year surveillance in the Far North Region of Cameroon. Am J Trop Med Hyg.
[17]
Hijmans RJ, Guarino, L., Bussink, C., Mathur, P., Cruz, M., Barrentes, I (2015) DIVA-GIS 7.5.
Debes AC, S; Sack, D.A. (2015) Manual for Detecting Vibrio cholerae O1 from Fecal Samples using an Enriched Dipstick Assay—A Low-Cost, Simplified Method of Confirming Cholera. Baltimore, MD.
[20]
Panchalingam S, Antonio M, Hossain A, Mandomando I, Ochieng B, et al. (2012) Diagnostic microbiologic methods in the GEMS-1 case/control study. Clin Infect Dis 55 Suppl 4: S294–302. doi: 10.1093/cid/cis754. pmid:23169941
[21]
Kain KC, Lanar DE (1991) Determination of genetic variation within Plasmodium falciparum by using enzymatically amplified DNA from filter paper disks impregnated with whole blood. J Clin Microbiol 29: 1171–1174. pmid:1864936
[22]
Nandi B, Nandy RK, Mukhopadhyay S, Nair GB, Shimada T, et al. (2000) Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW. J Clin Microbiol 38: 4145–4151. pmid:11060082
[23]
Hoshino K, Yamasaki S, Mukhopadhyay AK, Chakraborty S, Basu A, et al. (1998) Development and evaluation of a multiplex PCR assay for rapid detection of toxigenic Vibrio cholerae O1 and O139. FEMS Immunol Med Microbiol 20: 201–207. pmid:9566491 doi: 10.1111/j.1574-695x.1998.tb01128.x
[24]
Bauer A, Rorvik LM (2007) A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. Lett Appl Microbiol 45: 371–375. pmid:17897378 doi: 10.1111/j.1472-765x.2007.02195.x
[25]
Rashed SM, Azman AS, Alam M, Li S, Sack DA, et al. (2014) Genetic variation of Vibrio cholerae during outbreaks, Bangladesh, 2010–2011. Emerg Infect Dis 20: 54–60. doi: 10.3201/eid2001.130796. pmid:24377372
