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Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a speciesspecific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10 4 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh ؉ and trh ؉ strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.Vibrio parahaemolyticus is found worldwide in estuarine waters and is the leading cause of gastroenteritis from seafood in the United States, with most infections resulting from the consumption of raw or mishandled seafood (2, 29). The tlh (thermolabile hemolysin) gene is a species-specific marker for V. parahaemolyticus (42,26), while the tdh (thermostable direct hemolysin) (33, 34) and trh (thermostable-related hemolysin) (35) genes are pathogenicity markers for V. parahaemolyticus. Pathogenic V. parahaemolyticus bacteria in the environment and food samples typically comprise 0.3 to 3% of the total V. parahaemolyticus population (4,8,20,22,43). DNA-based assays targeting these genes were developed for the detection and enumeration of V. parahaemolyticus bacteria (7,10,21,26,27,30,38,44). However, the ability of the PCR assays to detect low levels of pathogenic V. parahaemolyticus bacteria in the presence of a high background of nonpathogenic V. parahaemolyticus bacteria was not reported by the authors (10,30,44). The preferential amplification of nonpathogenic members of the total V. parahaemolyticus population due to the muchhigher copy number of the target could preclude the detection of pathogenic strains pres...

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Abstract. Monkeypox virus (MPXV), a zoonotic orthopoxvirus (OPX), is endemic in the Democratic Republic of Congo (DRC). Currently, diagnostic assays for human monkeypox (MPX) focus on real-time quantitative polymerase chain reaction (PCR) assays, which are typically performed in sophisticated laboratory settings. Herein, we evaluated the accuracy and utility of a multiplex MPX assay using the GeneXpert platform, a portable rapid diagnostic device that may serve as a point-of-care test to diagnose infections in endemic areas. The multiplex MPX/OPX assay includes a MPX-specific PCR test, OPX-generic PCR test, and an internal control PCR test. In total, 164 diagnostic specimens (50 crusts and 114 vesicular swabs) were collected from suspected MPX cases in Tshuapa Province, DRC, under national surveillance guidelines. The specimens were tested with the GeneXpert MPX/OPX assay and an OPX PCR assay at the Institut National de Recherche Biomedicale (INRB) in Kinshasa. Aliquots of each specimen were tested in parallel with a MPX-specific PCR assay at the Centers for Disease Control and Prevention. The results of the MPX PCR were used as the gold standard for all analyses. The GeneXpert MPX/OPX assay performed at INRB had a sensitivity of 98.8% and specificity of 100%. The GeneXpert assay performed well with both crust and vesicle samples. The GeneXpert MPX/OPX test incorporates a simple methodology that performs well in both laboratory and field conditions, suggesting its viability as a diagnostic platform that may expand and expedite current MPX detection capabilities.

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