Detection of major diarrheagenic bacterial pathogens by multiplex PCR panels.

Diarrheal diseases remain a major threat to the youngest population in low- and middle-income countries. The main bacterial pathogens causing diarrhea are diarrheagenic Escherichia coli (DEC) that consists of enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorrhagic EHEC and enteroinvasive E. coli (EIEC), Salmonella, Shigella spp. (S. dysenteria, S. sonnei, S. flexneri) Campylobacter (C. coli, C. jejuni), Vibrio (V. vulnificus, V. parahaemolyticusm, V. cholerae), Yersinia enterocolitica and Aeromonas hydrophila. The aim of this study was to set up rapid multiplex PCR (mPCR) panels to identify these diarrheagenic pathogens based on their specific virulence genes. Primers against specific target genes were combined into three mPCR panels: one for diarrheal E. coli, one for pathogens causing mainly bloody diarrhea, and the third for the remaining pathogens. The panels were tested against a set of stool samples from Swedish children with diarrhea and controls and the analysis identified bacterial pathogens in 14/54 (26%) of the samples. These results show that our three developed mPCR panels can detect main bacterial diarrheagenic pathogens in clinical samples.

Sjöling Å ，Sadeghipoorjahromi L ，Novak D ，Tobias J ... -  《-》

A quantitative polymerase chain reaction assay for rapid detection of 9 pathogens directly from stools of travelers with diarrhea.

Every year, 80 million tourists traveling to tropical and subtropical areas contract traveler's diarrhea (TD). Forty percent to 80% of cases are caused by bacteria, yet clinical diagnostic tests are available to identify only a few of the strains that cause TD. We aimed to develop a quantitative polymerase chain reaction (qPCR) assay to identify all major pathogens in stool samples. We developed a low-cost, high-throughput, multiplex qPCR assay for simultaneous detection of 9 bacterial pathogens in stool samples: Salmonella, Yersinia, Campylobacter, and Vibrio cholerae, as well as Shigella or enteroinvasive Escherichia coli, enterohemorrhagic E coli, enterotoxigenic E coli (ETEC), enteroaggregative E coli (EAEC), and enteropathogenic E coli (EPEC). The assay was validated using positive (n = 245) and negative (n = 243) control strains, as well as preselected positive and negative stool samples. In addition, stool samples were collected from 96 returning travelers with TD. The findings were compared with those from routine diagnostic tests. The assay detected the bacterial strains with 100% sensitivity and specificity, compared with results from the reference tests. Of all stool samples collected from travelers with TD, EPEC was found in 47%, EAEC in 46%, ETEC in 22%, enterohemorrhagic E coli in 7%, Campylobacter in 6%, Shigella or enteroinvasive E coli in 2%, and Salmonella in 2%. Multiple pathogens were found in 37% of all samples. We developed a low-cost, high-throughput qPCR assay for use in routine diagnostic analysis and research. It detects the pathogenic bacteria most commonly associated with TD in stool samples with 100% sensitivity and specificity, compared with reference methods. The assay requires 4 hours, whereas current detection methods require 1 to 7 days. At least 1 TD pathogen was identified in stool samples from 76% of returning travelers, whereas conventional methods found a pathogen in only 17%. The most commonly detected bacteria were EPEC, EAEC, and ETEC.

Antikainen J ，Kantele A ，Pakkanen SH ，Lääveri T ，Riutta J ，Vaara M ，Kirveskari J ... -  《-》

Real-time multiplex polymerase chain reaction with high-resolution melting-curve analysis for the diagnosis of enteric infections associated with diarrheagenic Escherichia coli.

Devi TS ，Durairaj E ，Lyngdoh WV ，Duwarah SG ，Khyriem AB ，Lyngdoh CJ ... -  《-》

A novel single-step multiplex polymerase chain reaction assay for the detection of diarrheagenic Escherichia coli.

Escherichia coli that causes diarrhea in humans is referred to as diarrheagenic E. coli (DEC), and has been categorized into the following 5 groups: shigatoxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), and enterotoxigenic E. coli (ETEC). In this study, we developed a novel one-step multiplex polymerase chain reaction (mPCR) for the rapid detection of 10 pathogenic genes (stx1, stx2, eae, bfpA, invE, aggR, esth, estp, elt, and astA) of DEC. Five categorized strains were used as positive controls for DEC harboring each pathogenic gene, and 828 DEC-like strains, isolated from diarrheal stool samples and assumed to be DEC on the basis of serotyping, were used in the mPCR-based detection of the pathogenic genes. To demonstrate the utility of mPCR, the 828 strains were subjected to our optimized protocol, and the results obtained were compared with those obtained by monoplex PCR. The results showed agreement for all strains. Using mPCR, we also detected 65 DEC and 41 astA-positive E. coli, and 7 of these DEC strains were "O antigen untypable" (OUT). This novel mPCR protocol allowed for rapid, convenient, and economical pathogenicity-based identification of the DEC.

Fujioka M ，Otomo Y ，Ahsan CR 《-》

Detection of diarrheagenic Escherichia coli by multiplex PCR.

Hegde A ，Ballal M ，Shenoy S 《-》