Development and Accuracy of Quantitative Real-Time Polymerase Chain Reaction Assays for Detection and Quantification of Enterotoxigenic Escherichia coli (ETEC) Heat Labile and Heat Stable Toxin Genes in Travelers' Diarrhea Samples

Abstract

Abstract. Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

In Situ Analyses Directly in Diarrheal Stool Reveal Large Variations in Bacterial Load and Active Toxin Expression of Enterotoxigenic Escherichia coli and Vibrio cholerae

“…By use of quantitative real-time PCR, we could determine the gene copy number of the genes encoding the disease-causing V. cholerae and ETEC toxins, to up to 10 8 copies per ml ( Table 2 ). These numbers were corroborated by the results of the quantitative culture and other studies reporting between 10 7 and 10 8 CFU of diarrheal pathogens per milliliter or gram of stool ( 31 , 32 ) and up to 10 8 to 10 9 pathogen gene copies per gram of stool ( 30 ). Toxin gene copy numbers in ETEC have been determined to be between 1 and 16 copies per cell ( 32 , 33 ), indicating that quantification of gene copies might overestimate bacterial load up to 10-fold.…”

“…These numbers were corroborated by the results of the quantitative culture and other studies reporting between 10 7 and 10 8 CFU of diarrheal pathogens per milliliter or gram of stool ( 31 , 32 ) and up to 10 8 to 10 9 pathogen gene copies per gram of stool ( 30 ). Toxin gene copy numbers in ETEC have been determined to be between 1 and 16 copies per cell ( 32 , 33 ), indicating that quantification of gene copies might overestimate bacterial load up to 10-fold. Regardless, shedding of 10 7 to 10 8 bacteria per ml of stool in patients, who can lose several liters of fluid per day, is probably one of the main factors contributing to the large epidemic outbursts of diarrhea caused by these pathogens.…”

“…The development of quantitative molecular diagnostics, including real-time PCR and other PCR methods for identification of the pathogens, may aid in finding the major disease-causing pathogen(s) in an infection ( 30 , 36 ). With the use of these techniques, ETEC has been shown to be frequently underestimated ( 32 ). It might also turn out that a high proportion of the cholera cases are, in fact, mixed infections, since we detected E. coli or other species in all V. cholerae -positive stool samples.…”

Promotion of Early Gut Colonization by Probiotic Intervention on Microbiota Diversity in Pregnant Sows

“…To detect possible presence of the most common intestinal pathogens in piglets’, PCR reactions were performed using as a template 100 ng of DNA extracted from fecal samples [as described in Section “Denaturing Gradient Gel Electrophoresis (DGGE) Analysis”]. Primers used in the study were complementary to eltA gene and invA gene, specific for enterotoxigenic Escherichia coli ( Youmans et al, 2014 ) and Salmonella sp. ( Li and Chen, 2013 ), respectively.…”

Diarrheagenic Escherichia coli

“…The diagnosis of ETEC strains should include, in addition to LT and ST detection, complementary PCR assays for the detection of virulence genes such as clyA , eatA , tia , tibC , leoA , and east-1 340 . A sensitive and specific PCR assay with primers targeting the genes lt and st was reported by Stacy-Phipps et al, 365 and later by Youmans et al, 366 using quantitative real-time PCR. Moreover, several multiplex PCR assays were also developed using these two genes 367, 368, 369…”