Toxins and virulence factors of enterotoxigenic Escherichia coli associated with strains isolated from indigenous children and international visitors to a rural community in Guatemala.

Diarrhoea remains a common cause of illness in Guatemala, with children suffering most frequently from the disease. This study directly compared the frequency, enterotoxin, and colonization factor (CF) profiles of enterotoxigenic Escherichia coli (ETEC) strains isolated from children living in a rural community in Guatemala and from Western visitors to the same location during the same seasons, using similar detection methodologies. We found that ETEC accounted for 26% of severe cases of diarrhoea in children requiring hospitalization, 15% of diarrhoea in the community, and 29% of travellers' diarrhoea in visitors staying ⩾2 weeks. The toxin and CF patterns of the ETEC strains isolated from both groups differed significantly (P < 0·0005) as determined by χ 2 = 60·39 for CFs and χ 2 = 35 for toxins, while ETEC phenotypes found in Guatemalan children were comparable to those found in children from other areas of the world.

Comparative analyses of phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins and colonization factors.

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.

Unstable coronary artery disease in post-menopausal women. Identifying patients with significant coronary artery disease by basic clinical parameters and exercise test. IRIS Study Group.

BACKGROUND: The diagnostic information from an ECG taken while at rest and an exercise test is considered less reliable in women than in men, mostly due to a high percentage of false-positive tests. This can be explained by a lower pre-test likelihood of coronary heart disease. AIMS: To evaluate the diagnostic information that can be gained from basic clinical parameters, an ECG and exercise test in a group of post-menopausal women with symptoms of unstable coronary artery disease in order to identify patients with significant coronary artery stenoses. METHODS AND RESULTS: We prospectively studied 200 post-menopausal women admitted to the coronary care unit with symptoms of unstable coronary artery disease and ECG changes suggestive of ischaemia. The diagnostic value of common risk factors, myocardial enzymes and an early exercise test were assessed. A coronary angiogram was performed within 60 days. Median age was 67 years. On admission, 38% had ST depression on an ECG taken while at rest, 76% had T-wave inversion, and 41% increased enzyme levels. The coronary angiogram revealed that 15% had no atherosclerosis, 14% had atherosclerosis but not lesion > or = 50% of luminal diameter and 71% had at least one significant stenosis. Of patients with known indicators of atherosclerotic disease, all but one had atherosclerosis visualized on the coronary angiogram. A relative ST depression > or = 0.1 mV and a low maximum workload at exercise test were strong predictors of significant coronary artery disease. The positive predictive value of ST depression was 91% and of low maximum workload 84% CONCLUSION: In post-menopausal women with signs of unstable angina and ischaemia on an ECG taken while at rest, the prevalence of coronary atherosclerosis is high, 85%. Contrary to earlier studies, ST T-changes at the early exercise test had a high positive predictive value, especially in combination with a low maximum workload with no false-positive results.

[Initial experience with the use of abciximab in the salvage treatment of acute coronary thrombosis in the Hemodynamics Laboratory]. / Esperienza iniziale sull'uso di abciximab nel trattamento "di salvataggio" della trombosi coronarica acuta nel Laboratorio di Emodinamica.

The optimal treatment of acute thrombotic complications in the Catheterization Laboratory has not been defined yet, due to the limited efficacy shown by various pharmacological regimens, even when associated to coronary angioplasty (PTCA). The aim of our study was therefore to evaluate the effects of abciximab (ReoPro), a new potent inhibitor of the platelet glycoprotein IIb/IIIa, when administered as a "rescue" treatment for acute thrombotic coronary occlusion during diagnostic or interventional procedures. Sixteen patients (12 males, 4 females, mean age 59.3 +/- 9.2 years, range 43-77 years), with unstable angina and consecutively treated with abciximab due to clinical instability attributable to coronary thrombosis angiographically proven during PTCA (9 cases) or diagnostic angiography (7 cases), were identified. The individual angiographic films and medical records were then reviewed in order to evaluate the effects of treatment on coronary flow, thrombus size and occurrence of in-hospital adverse events: death, non-fatal acute myocardial infarction (AMI), need for urgent myocardial revascularization and hemorrhage. The administration of abciximab, in association with PTCA (associated in turn with stent implantation in 8 cases), induced a significant increase of coronary TIMI flow grade (0.3 +/- 0.6 vs 2.4 +/- 0.9; p < 0.05) and a significant decrease of thrombus "score" (size) 2.4 +/- 0.9 vs 1.3 +/- 0.6; p < 0.01). No deaths nor need for urgent myocardial revascularization were observed; in 31% of cases (5 patients) evolution towards AMI occurred, while however 94% of cases (15 patients) had a coronary occlusion before treatment. No major hemorrhagic complications were observed, while in 12% of cases (2 patients) a groin hematoma associated with moderate hemoglobin drop, developed. In conclusion, the administration of abciximab, associated with the common "rescue" interventional procedures, in patients with acute thrombotic coronary occlusion in the Catheterization Laboratory, appears to be effective in restoring adequate coronary flow and reducing the thrombus size (limiting therefore the evolution towards AMI), and safe, not having been associated with significant hemorrhagic complications.

Infection with colonization factor antigen I-expressing enterotoxigenic Escherichia coli boosts antibody responses against heterologous colonization factors in primed subjects.

Enterotoxigenic Escherichia coli (ETEC) adhere to the intestinal mucosa by a number of fimbrial colonization factors (CFs) that have been claimed to induce only type-specific immunity. However, adult Bangladeshi patients infected with CFA/I-expressing bacteria, developed significant plasma IgA antibody responses, as determined by enzyme-linked immunosorbent assay, not only against the homologous fimbriae but also against several heterologous CFs, i.e. CS1, CS2, CS4 and PCFO166 fimbriae. In contrast, North American volunteers, who had probably not been infected by ETEC previously, responded with serum IgA against CFA/I fimbriae but not against any other CFs after symptomatic infection with CFA/I-expressing ETEC. Thus, infection with CFA/I-expressing bacteria may boost immune responses against CFs with a related amino acid sequence in previously primed subjects.

Optimization of the intestinal lavage procedure for determination of intestinal immune responses.

Optimal conditions to process, concentrate and store intestinal lavage fluid were studied in samples collected from volunteers before and after oral immunization with a prototype vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhoea. Total IgA and specific IgA antibody titres against enterotoxin and colonization factor antigen were determined in 22 lavage samples which were either enzyme-inhibited or heat-inactivated and then subjected to different long-term storage conditions. Samples were analysed within 1 month of collection and also after 3, 6 and 24 months of storage. Total IgA concentrations and specific IgA antibody levels were higher in lavage samples treated with enzyme inhibitors (soybean trypsin inhibitor and phenylmethylsulfonyl fluoride) than in those heat-inactivated. Similarily, concentration of the lavage fluid by freeze-drying was superior to concentration against polyethylene glycol. Specific antibody titres remained elevated after storage for at least 6 months but declined after 2 years in frozen compared with freeze-dried samples.

Epidemiological aspects of enterotoxigenic Escherichia coli diarrhoea in infants in the Jerusalem area.

BACKGROUND: As most of the clinical laboratories in Israel do not use toxigenicity testing as a routine procedure in cases of Escherichia coli gastroenteritis, current information regarding clinical and epidemiological aspects of infection by enterotoxigenic E. coli (ETEC) is lacking. This survey aimed at retrieving that information by conducting routine enterotoxigenicity tests in a clinical laboratory for patients suspected of E. coli gastroenteritis. METHODS: The survey population included 415 children < or = 2.5 years old with gastroenteritis, during the second half of 1992. Fresh first stool cultures were checked daily for toxigenicity by ELISA. Thereafter the serotypes, and the colonization factor antigens (CFAs) were evaluated for the ETEC strains. RESULTS: in 86 of the 415 children (20.7%) we found ETEC. Half these strains would have not been reported as pathogenic without this routine toxigenicity test. CFA were defined in 48 (55.8%) of the ETEC strains. The most common serogroups were O-153, O-6, O-126, and O-128.

Roles of different putative colonization factor antigens in colonization of human enterotoxigenic Escherichia coli in rabbits.

The role of some well-characterized putative colonization factors (PCFs) in enterotoxigenic Escherichia coli (ETEC), i.e. PCFO159, PCFO166, CS7, CS17 and CFA/III, for colonization of the bacteria in the intestine was studied in a non-ligated rabbit intestine model (RITARD). Intestinal administration of 10(11) organisms of the various strains only resulted in very mild symptoms with loose stools during a few days in most of the animals. Strains expressing PCFO159, CS7, CS17 and CFA/III were shed in the stool for a significantly longer period than PCF/CS-negative ETEC. However, the mean time of shedding PCFO166 positive organisms did not significantly exceed that of non-fimbriated E. coli. All strains that colonized rabbit intestine, as assessed by prolonged fecal excretion, also gave rise to high serum antibody responses against the homologous fimbriae whereas non-colonizing strains failed to induce such responses. This study strongly suggests that several of the recently described PCFs, e.g. PCFO159, CS7, CS17 and CFA/III are colonizing factors and strong immunogens.

Detection of colonization factor antigen I-positive enterotoxigenic Escherichia coli with a cloned polynucleotide probe.

We compared a new colony hybridization assay with an established enzyme-linked immunosorbent assay for detection of enterotoxigenic Escherichia coli (ETEC) expressing colonization factor antigen I (CFA/I). The tests were applied to 135 human ETEC strains. Of these isolates, 30 had previously been characterized for CFAs. A strain harboring the plasmid vector of the polynucleotide gene probe, nine non-ETEC strains from healthy infants, and eight ETEC strains of animal origin were included for further evaluation of probe specificity. The two assays showed a high level of concordance in the specific detection of ETEC strains expressing CFA/I. A total of 24 strains tested positive in the CFA/I hybridization assay, while 23 of those strains were positive in the CFA/I enzyme-linked immunosorbent assay. The single discrepant result could be explained by the loss of a regulatory gene. The strain harboring the plasmid vector of the probe, the non-ETEC E. coli strains, and the ETEC strains of animal origin were all negative in the CFA/I probe assay.

Rapid GM1-enzyme-linked immunosorbent assay with visual reading for identification of Escherichia coli heat-labile enterotoxin.

A modified, quicker and simpler GM1-enzyme-linked immunosorbent assay procedure for detection of Escherichia coli heat-labile enterotoxin (LT) has been developed with the intent that it should be useful in less well-equipped laboratories. The method, which makes use of stable reagents including commercially available horseradish peroxidase immunoglobulin conjugate, can detect LT in overnight cultures within 1 working day (8 h), and the tests can be read with the naked eye. This GM1-horseradish peroxidase-enzyme-linked immunosorbent assay shows excellent quantitative and qualitative correlation with the conventional GM1-enzyme-linked immunosorbent assay. When 100 human E. coli strains were analyzed blindly and in parallel by the two methods, LT production was identified in 50 out of 50 LT-positive strains and in 0 out of 50 LT-negative strains by either method.