Abstract
The dynamic nature of Vibrio parahaemolyticus epidemiology has presented a unique challenge for disease intervention strategies. Despite the continued rise of disease incidence and outbreaks of vibriosis, as well as the global emergence of pandemic clones and serovariants with enhanced virulence, there is a paucity of molecular methods for the serotyping of V. parahaemolyticus strains to improve disease surveillance and outbreak investigations. We describe the development of a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the simultaneous identification of 11 clinically most common V. parahaemolyticus serotypes spanning a 10-year period. Through extensive sequence analyses using 418 genomes, specific primers and probes were designed for a total of 22 antigen gene targets for the O- and K- serogroups. Additionally, the toxR gene was incorporated into the assay for the confirmation of V. parahaemolyticus. All gene targets were detected by the assay and gave expected Tm values, without any cross reactions between the 11 clinically common serotypes or with 38 other serotypes. The limit of identification for all gene targets ranged from 0.1 to 1 ng/μL. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 1°C and <1% respectively, indicating a highly reproducible assay. A multicenter double-blind clinical study was conducted using the traditional V. parahaemolyticus identification workflow and the MLMA assay workflow in parallel. From consecutive diarrheal stool specimens (n = 6118) collected over a year at 10 sentinel hospitals, a total of 153 V. parahaemolyticus isolates (2.5%) were identified by both workflows. A total agreement (kappa = 1.0) between the serotypes identified by the MLMA assay and conventional serological method was demonstrated. This is the first molecular assay to simultaneously identify multiple clinically important V. parahaemolyticus serotypes, which satisfies the acute need for a practical, rapid and robust identification of V. parahaemolyticus serotypes to facilitate the timely detection of vibriosis outbreaks and surveillance.
Highlights
Vibrio parahaemolyticus is a halophilic bacterium that thrives in estuarine, marine, and coastal waters and is recognized as the primary cause of gastroenteritis associated with the consumption of seafood and marine products worldwide (Cabrera-Garcia et al, 2004; Mclaughlin et al, 2005; Letchumanan et al, 2014)
We have identified an additional gene locus, orf9, to be specific for serogroup O10 (Accession no: CNA0002415) and was included in the assay as a target in addition to wvcP for serogroup O10 identification
Since early 1970s, the tracking of disease spread has relied on conventional serological typing, which is considered the international gold standard pertaining to V. parahaemolyticus epidemiology
Summary
Vibrio parahaemolyticus is a halophilic bacterium that thrives in estuarine, marine, and coastal waters and is recognized as the primary cause of gastroenteritis associated with the consumption of seafood and marine products worldwide (Cabrera-Garcia et al, 2004; Mclaughlin et al, 2005; Letchumanan et al, 2014). In China, V. parahaemolyticus has been the leading cause of foodborne outbreaks and infectious diarrhea among adults, in coastal cities (Li Y. et al, 2014; Liu et al, 2018). V. parahaemolyticus infections are recognized as a multiserogroup affliction caused by diverse serotypes (Bhuiyan et al, 2002), leading primarily to acute gastroenteritis, and occasionally, wound infection and septicemia (Daniels et al, 2000). In 1996, the emergence of a more virulent serotype O3:K6 was subsequently being recognized as a pandemic clone owing to its rapid dissemination and the cause of numerous gastroenteritis outbreaks globally (Nair et al, 2007). The pandemic clone and its serovariants are known to represent 49 serotypes, which are widely distributed among 22 countries in Asia, Europe, America and Africa (Han et al, 2016). The serotyping of V. parahaemolyticus has played a pivotal role in significantly advancing our understanding of its epidemiology
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