Debes Amanda K, Ateudjieu Jerome, Guenou Etienne, Lopez Anna Lena, Bugayong Mark Philip, Retiban Pearl Joy, Garrine Marcelino, Mandomando Inacio, Li Shan, Stine O Colin, Sack David A
Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, United States of America.
Department of Biomedical Sciences, University of Dschang, Dschang, Cameroon.
PLoS Negl Trop Dis. 2016 Jan 6;10(1):e0004307. doi: 10.1371/journal.pntd.0004307. eCollection 2016 Jan.
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.
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.
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.
霍乱弧菌在南亚和非洲为地方病，在这些地区霍乱广泛暴发，尤其与贫困和卫生条件差有关。关于产毒霍乱弧菌分离株的遗传多样性的知识，特别是在非洲，仍然匮乏。改善这种认识的制约因素不仅在于缺乏常规霍乱疾病监测，还在于流行国家缺乏及时保存、储存和运送分离株的实验室能力。我们评估了使用简化样本保存方法进行分子特征分析，采用多位点可变数目串联重复分析（MLVA）来区分霍乱弧菌基因型。
从喀麦隆霍乱暴发中采集的47株霍乱弧菌分离株和18份富集临床标本（如肉汤富集后的粪便标本）保存在Whatman滤纸上用于DNA提取。样本取自2014年6月和2014年10月喀麦隆极北地区（FNC）两个地理位置不同的暴发。此外，还分析了来自菲律宾的14株分离株和来自莫桑比克的8株分离株的便利样本。所有87份DNA均成功分析，包括16对样本，一对是培养的分离株，另一对是从中分离出该分离株的富集标本。15份富集标本与其培养的分离株之间的基因型结果相同，另一对在单个位点存在差异。在2014年喀麦隆的标本中鉴定出两个密切相关但不同的克隆复合体。
在偏远和资源匮乏地区使用简化实验室方法收集霍乱弧菌，便于随后对霍乱弧菌O1进行先进的分子特征分析。这些简化的DNA保存方法可识别霍乱弧菌，并使及时获取有关霍乱弧菌遗传多样性的信息成为可能；我们的结果为继续开展分子流行病学研究奠定了基础，以便更好地了解霍乱弧菌在非洲和世界其他地方的传播和扩散情况。
