Detection of Toxigenic Vibrio cholerae Ol Using Polymerase Chain Reaction for Amplifying the Cholera Enterotoxin Gene

Abstract A real-time polymerase chain reaction (qPCR) assay for the detection of the ctxA gene of toxigenic Vibrio cholerae (Vc) was validated against standard culture techniques. The first experimental phase determined optimal enrichment conditions for detection by culture and qPCR of Vc in shrimp, bottled water, milk, and potato salad. The conditions tested included temperature (35 and 42C), time (6 and 18 h), and effect of shaking (0 and 100 rpm). No definitive trends were found with enrichment temperature or shaking on Vc isolation frequency or detection by qPCR. Generally, Vc was detected by qPCR more frequently than Vc was isolated, but this difference was significant only in the 35C 6 h enrichment without shaking. In the second phase of experiments, shrimp, bottled water, milk, potato salad, and oysters were inoculated with each of 3 toxigenic Vc strains (Latin American O1 strain, an O139 strain, and an O1 strain from the U.S. Gulf Coast) and enriched under static conditions at 42C for 6 and 18 h. Overall, detection frequency of ctx by qPCR was 98 (88/90) and 100 (90/90) after 6 and 18 h enrichments, respectively, while Vc isolation frequency was 87 (78/90) and 83 (75/90) after 6 and 18 h, respectively. Toxigenic Vc can be detected by qPCR within an 8 h work day using the 6 h enrichment procedure, assuming an initial level of at least 12 colony-forming units/g; however, overnight enrichment may be necessary to detect lower levels. These data indicate that the qPCR assay for ctx is a more reliable, sensitive, and rapid alternative to standard Vc culture methods and is applicable to diverse food products.

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Etiology of Diarrhea Requiring Hospitalization in Bangladesh by Quantitative Polymerase Chain Reaction, 2014–2018

Clinical Infectious Diseases ◽

10.1093/cid/ciaa840 ◽

2020 ◽

Author(s):

Mami Taniuchi ◽

Kamrul Islam ◽

Md Abu Sayeed ◽

James A Platts-Mills ◽

Md Taufiqul Islam ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Vibrio Cholerae ◽

Coefficient Of Variation ◽

Quantitative Polymerase Chain Reaction ◽

Age Groups ◽

Public Health Problem ◽

Major Public Health Problem ◽

Surveillance Systems ◽

Chain Reaction ◽

Polymerase Chain

Abstract Background Diarrhea remains a major public health problem and characterization of its etiology is needed to prioritize interventions. However, most data are from single-site studies of children. We tested samples from participants of any age from 11 geographically diverse hospitals in Bangladesh to describe pathogen-specific burdens of diarrhea. Methods We utilized 2 existing diarrhea surveillance systems: a Nationwide network at 10 sentinel hospitals and at the icddr,b hospital. We tested stools from enrolled participants and nondiarrheal controls for enteropathogens using quantitative polymerase chain reaction and calculated pathogen-specific attributable fractions (AFs) of diarrhea. Results We analyzed 5516 patients with diarrhea and 735 controls. Overall, rotavirus had the highest attributable burden of diarrhea (Nationwide AF, 17.7%; 95% confidence interval [CI], 14.3–20.9%; icddr,b AF, 39.9%; 38.0–41.8%), followed by adenovirus 40/41 (Nationwide AF, 17.9%; 95% CI: 13.9–21.9%; icddr,b AF, 16.6%; 95% CI, 14.4–19.4%) and Vibrio cholerae (Nationwide AF, 10.2%; 95% CI, 9.1–11.3%; icddr,b AF, 13.3%; 95% CI: 11.9–15.1%). Rotavirus was the leading pathogen in children <5 years and was consistent across the sites (coefficient of variation = 56.3%). Adenovirus 40/41 was the second leading pathogen in both children and adults. Vibrio cholerae was the leading pathogen in individuals >5 years old, but was more geographically variable (coefficient of variation = 71.5%). Other attributable pathogens included astrovirus, norovirus, Shigella, Salmonella, ETEC, sapovirus, and typical EPEC. Conclusions Rotavirus, adenovirus 40/41, and V. cholerae were the leading etiologies of infectious diarrhea requiring hospitalization in Bangladesh. Other pathogens were important in certain age groups or sites.

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