Enterotoxigenic Escherichia coli (ETEC), a major cause of infectious diarrhea, produce heat-stable and/or heat-labile enterotoxins and at least 25 different colonization factors that target the intestinal mucosa. The genes encoding the enterotoxins and most of the colonization factors are located on plasmids found across diverse E. coli serogroups. Whole-genome sequencing of a representative collection of ETEC isolated between 1980 and 2011 identified globally distributed lineages characterized by distinct colonization factor and enterotoxin profiles. Contrary to current notions, these relatively recently emerged lineages might harbor chromosome and plasmid combinations that optimize fitness and transmissibility. These data have implications for understanding, tracking and possibly preventing ETEC disease.
Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of childhood diarrhea in developing countries and in travelers. However, this pathogen has often not been reported in surveys of diarrheal pathogens, due to lack of simple standardized methods to detect ETEC in many laboratories. ETEC expresses one or both of two different enterotoxin subtypes: heat-stable toxins, a heat-labile toxin (LT), and more than 22 different colonization factors (CFs) that mediate adherence to the intestinal cell wall. Here we compare established phenotypic and genotypic detection methods and newly developed PCR detection methods with respect to sensitivity, specificity, positive predictive value, and ease of performance. The methods include GM1-enzyme-linked immunosorbent assay and dot blot techniques using specific monoclonal antibodies (MAbs) for phenotypic detection of the toxins and CFs, respectively, as well as different PCR and DNA/DNA hybridization techniques, including new PCR assays, for genotypic identification of the toxin and CF genes, respectively. We found very good general agreement in results derived from genotypic and phenotypic methods. In a few strains, LT and CFs were identified genetically but not phenotypically. Based on our analyses, we recommend initial screening for ETEC in clinical samples by multiplex toxin gene PCR. Toxin-positive strains may then be analyzed by dot blot tests for detection of the CFs expressed on the bacterial surface and by PCR for determination of additional CFs for which MAbs are currently lacking as well as for strains that harbor silent CF genes.Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of morbidity and mortality in children under the age of five in developing countries and in travelers and soldiers positioned in these areas (3,18,21,25). ETEC has, however, been grossly underestimated as the cause of diarrhea in many studies, mainly because few laboratories have suitable methods in place for ETEC diagnostics (18). Hence, it is of great importance to introduce simple and reliable methods for diagnosis of ETEC in all laboratories that are involved in identifying microbes associated with diarrhea. This study was undertaken to identify ETEC diagnostic methods that are suitable for more widespread introduction into research and clinical laboratories.ETEC expresses one or both of two types of toxin: heatstable toxin (ST) and heat-labile toxin (LT), both of which induce diarrhea. The ST toxin is encoded by two different genes, estA and st1, both of which code for small peptides (19 and 18 amino acids) with similar functions and gene sequences. The translated toxins are called STh (estA), originally found in ETEC isolated from humans, and STp (st1), found in ETEC isolated from pigs. The ST genes may be expressed alone or in combination with the LT genes eltA and eltB. Since eltAB may also be expressed alone, there are seven possible toxin combinations that can be expressed in individual ETEC strains: STh, STp, STh/LT, STp/LT, LT, and less commonly, STh/STp...
Background Enterotoxigenic Escherichia coli causes diarrhoea, leading to substantial mortality and morbidity in children, but no specific vaccine exists. This trial tested an oral, inactivated, enterotoxigenic E coli vaccine (ETVAX), which has been previously shown to be safe and highly immuongenic in Swedish and Bangladeshi adults. We tested the safety and immunogenicity of ETVAX, consisting of four E coli strains overexpressing the most prevalent colonisation factors (CFA/I, CS3, CS5, and CS6) and a toxoid (LCTBA) administered with or without a double-mutant heat-labile enterotoxin (dmLT) as an adjuvant, in Bangladeshi children. Methods We did a randomised, double-blind, placebo-controlled, dose-escalation, age-descending, phase 1/2 trial in Dhaka, Bangladesh. Healthy children in one of three age groups (24-59 months, 12-23 months, and 6-11 months) were eligible. Children were randomly assigned with block randomisation to receive either ETVAX, with or without dmLT, or placebo. ETVAX (half [5•5 × 10¹⁰ cells], quarter [2•5 × 10¹⁰ cells], or eighth [1•25 × 10¹⁰ cells] adult dose), with or without dmLT adjuvant (2•5 µg, 5•0 µg, or 10•0 µg), or placebo were administered orally in two doses 2 weeks apart. Investigators and participants were masked to treatment allocation. The primary endpoint was safety and tolerability, assessed in all children who received at least one dose of vaccine. Antibody responses to vaccine antigens, defined as at least a two-times increase in antibody levels between baseline and post-immunisation, were assessed as secondary endpoints. This trial is registered with ClinicalTrials.gov, NCT02531802.
Four multiplex PCR assays for detection of 19 enterotoxigenic Escherichia coli (ETEC) colonization factorsand an improved ETEC toxin multiplex PCR were developed and tested on Bangladeshi and Bolivian ETEC strain collections. The assays will be useful for surveillance of ETEC infections in diagnostic laboratories that have access to PCR.Infection with enterotoxigenic Escherichia coli (ETEC) is one of the main causes of childhood diarrhea in developing countries and in travelers to these areas (7). ETEC strains express one or both of two different enterotoxins, the heatstable toxins (STh and STp) and a heat-labile toxin (LT), and more than 23 different colonization factors (CFs) that are named CFA/I and CS1 to CS22 (2). The LT enterotoxin and the CF antigens are immunogenic, and one or both of these components are included in most vaccines that are being developed against ETEC diarrhea (14,15). To evaluate the putative impact of different vaccine compositions on ETEC diarrhea, it is important to characterize clinical ETEC isolates from different parts of the world with regard to toxin and CF profiles, since they have been observed to vary from one geographic region to another (4,7,8,10,12).In order to simplify and accelerate the detection of the different CFs and toxins by PCR, we have established a set of multiplex PCR assays for detection of the most-prevalent CFs and the toxins using previously established primers (11), as well as new primers when appropriate. The CF primers were assembled into four panels designed to amplify 19 of the most common CFs, and a new set of STh primers was included in the previously established toxin multiplex PCR (Table 1).To establish the multiplex assays for the CFs and toxins, 10-to 100-ng amounts of DNA from ETEC reference strains boiled in water (Table 1) were mixed with master mix as described previously (11), except that 400 nM deoxynucleoside triphosphates and 8 to 10 forward and reverse primers at a final concentration of 200 nM each were added to the mix. The PCRs were amplified by an initial denaturation at 94°C (1 min), followed by 35 cycles of amplification (94°C for 30 s, 52°C for 30 s, and 72°C for 1 min), and finally, 5 min at 72°C. The products were run on 3% agarose gels or 3% MetaPhor gels (MetaPhor agarose; Cambrex Bio Science Rockland, Inc.) for better resolution and visualized under UV light (Fig. 1). Only the correct CF and toxin PCR products were amplified, and by mixing several strains and performing multiplex PCR on the mixed DNA, we verified that the primers in each panel could detect the presence of several CFs in one reaction.To evaluate the new multiplex PCR methods for ETEC toxins and CFs, 106 clinical ETEC isolates were analyzed by multiplex PCR and the results were compared with results obtained by using the previously established ETEC toxin multiplex PCR and with the dot blot method using available monoclonal antibodies (11). We used two different sets of ETEC strains that were isolated in two geographically different developing countries: 65 Boli...
Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea among children in developing countries and in travelers to areas of ETEC endemicity. ETEC strains isolated from humans may produce a heat-labile enterotoxin (LT) and two types of the heat-stable enterotoxin STa, called STh and STp, encoded by the estA gene. Two commonly used assay methods for the detection of STa, the infant mouse assay or different enzyme-linked immunosorbent assays, are unable to distinguish between the two subtypes of ST. Different genotypic methods, such as DNA probes or PCR assays, may, however, allow such discrimination. Using gene probes, it has recently been reported that ETEC strains producing STp as the only enterotoxin are not associated with diarrhea. In this study, we have used highly specific PCR methods, including newly designed primers for STh together with previously described STp primers, to compare the relative distribution of STh and STp in ETEC isolated from children with diarrhea in three different geographically distinct areas, i.e., Bangladesh, Egypt, and Guatemala, and from travelers to Mexico and Guatemala. It was found that ETEC strains producing STp were as commonly isolated from cases of diarrhea as strains producing STh both in Egypt and Guatemala, whereas STp strains were considerably less common in Bangladesh. No difference was found in the relative distribution of STh and STp in ETEC strains isolated from travelers with diarrhea and from asymptomatic carriers. Irrespective of ST genotype, the disease symptoms were also similar in both children and travelers.
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