Diarrhea burden due to natural infection with enterotoxigenic Escherichia coli in a birth cohort in a rural Egyptian community.

Enterotoxigenic Escherichia coli (ETEC) is commonly associated with diarrhea in Egyptian children. Children less than 3 years old in Abu Homos, Egypt, had approximately five diarrheal episodes per child every year, and at least one of these episodes was due to ETEC. The epidemiology of ETEC diarrhea among children living in a rural Egyptian community was further evaluated in this study. Between January 2004 and April 2007, 348 neonates were enrolled and followed for 2 years. Children were visited twice weekly, and a stool sample was obtained every 2 weeks regardless of symptomatology. A stool sample was obtained whenever a child had diarrhea. From the routine stool culture, five E. coli-like colonies were selected and screened for heat-labile and heat-stable toxins by GM1 enzyme-linked immunosorbent assay (ELISA) and further typed for colonization factor antigens by dot blot assay. Incidence of ETEC infection was estimated among children with diarrhea (symptomatic) and without diarrhea (asymptomatic). Incidence of diarrhea and ETEC-associated diarrhea was 7.8 and 1.48 per child-year, respectively. High risk of repeated ETEC diarrhea was associated with being over 6 months of age, warm season, male gender, and crowded sleeping conditions. Exclusive breast-feeding was protective for repeated ETEC infection. ETEC-associated diarrhea remains common among children living in the Nile Delta. The protective role of breast-feeding demonstrates the importance of promoting exclusive breast-feeding during, at least, the first 6 months of life.

Phenotypic and genotypic analysis of enterotoxigenic Escherichia coli in samples obtained from Egyptian children presenting to referral hospitals.

Hospital surveillance was established in the Nile River Delta to increase the understanding of the epidemiology of diarrheal disease among Egyptian children. Between September 2000 and August 2003, samples obtained from children less than 5 years of age who had diarrhea and who were seeking hospital care were cultured for enteric bacteria. Colonies from each culture with a morphology typical of that of Escherichia coli were tested for the heat-labile (LT) and heat-stable (ST) toxins by a GM-1-specific enzyme-linked immunosorbent assay and colonization factor (CF) antigens by an immunodot blot assay. Enterotoxigenic E. coli (ETEC) isolates were recovered from 320/1,540 (20.7%) children, and ETEC isolates expressing a known CF were identified in 151/320 (47%) samples. ST CFA/I, ST CS6, ST CS14, and LT and ST CS5 plus CS6 represented 75% of the CFs expressed by ETEC isolates expressing a detectable CF. Year-to-year variability in the proportion of ETEC isolates that expressed a detectable CF was observed (e.g., the proportion that expressed CFA/I ranged from 10% in year 1 to 21% in year 3); however, the relative proportions of ETEC isolates expressing a CF were similar over the reporting period. The proportion of CF-positive ETEC isolates was higher among isolates that expressed ST. ETEC isolates expressing CS6 were isolated significantly less often (P < 0.001) than isolates expressing CFA/I in children less than 1 year of age. Macrorestriction profiling of CFA/I-expressing ETEC isolates by using the restriction enzyme XbaI and pulsed-field gel electrophoresis demonstrated a wide genetic diversity among the isolates that did not directly correlate with the virulence of the pathogen. The genome plasticity demonstrated in the ETEC isolates collected in this work suggests an additional challenge to the development of a globally effective vaccine for ETEC.

Detection of fecal and serum antibodies against enterotoxigenic Escherichia coli toxins and colonization factors in deployed U.S. military personnel during Operation Bright Star 2001--Egypt.

Infection caused by enterotoxigenic Escherichia coli (ETEC) poses a significant health problem in children and adults residing in endemic developing countries. Acute and convalescent paired stool and serum samples were obtained from 27 U.S. military personnel with ETEC-induced diarrhea during a military exercise in Egypt. In general, the concentration of total fecal and circulatory anti-LT IgA was significantly increased in convalescent specimens than in the paired acute ones in almost 65 % of the cases. The pattern of specific antibody responses in fecal and serum samples from cases with ETEC expressing the antigens coil surface 1 (CS1), CS2, CS3 and CS6 were, on the other hand, not conclusive due to the small numbers of the study cases. Further research is still required to understand the immunologic responses during the natural course of disease. The data obtained, nevertheless, may help current research efforts on the development of vaccines for humans against ETEC infection.

Characterization of an enterotoxigenic Escherichia coli strain from Africa expressing a putative colonization factor.

An enterotoxigenic Escherichia coli (ETEC) strain of serotype O114:H- that expressed both heat-labile and heat-stable enterotoxins and tested negative for colonization factors (CF) was isolated from a child with diarrhea in Egypt. This strain, WS0115A, induced hemagglutination of bovine erythrocytes and adhered to the enterocyte-like cell line Caco-2, suggesting that it may elaborate novel fimbriae. Surface-expressed antigen purified by differential ammonium sulfate precipitation and column chromatography yielded a single protein band with M(r) 14,800 when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (16% polyacrylamide). A monoclonal antibody against this putative fimbrial antigen was generated and reacted with strain WS0115A and also with CS1-, CS17-, and CS19-positive strains in a dot blot assay. Reactivity was temperature dependent, with cells displaying reactivity when grown at 37 degrees C but not when grown at 22 degrees C. Immunoblot analysis of a fimbrial preparation from strain WS0115A showed that the monoclonal antibody reacted with a single protein band. Electron microscopy and immunoelectron microscopy revealed fimbria-like structures on the surface of strain WS0115A. These structures were rigid and measured 6.8 to 7.4 nm in diameter. Electrospray mass-spectrometric analysis showed that the mass of the purified fimbria was 14,965 Da. The N-terminal sequence of the fimbria established that it was a member of the CFA/I family, with sequence identity to the amino terminus of CS19, a new CF recently identified in India. Cumulatively, our results suggest that this fimbria is CS19. Screening of a collection of ETEC strains isolated from children with diarrhea in Egypt found that 4.2% of strains originally reported as CF negative were positive for this CF, suggesting that it is biologically relevant in the pathogenesis of ETEC.

Evaluation of the response of human humoral antibodies to Salmonella typhi lipopolysaccharide in an area of endemic typhoid fever.

Because of the limited value of Widal's test in the diagnosis of typhoid fever in areas of endemicity, individual serum levels of IgM, IgA, IgG, and IgG subclass antibodies to Salmonella typhi lipopolysaccharide were evaluated in samples collected in Egypt. The study involved 106 febrile patients, including 40 patients for whom cultures were positive for S. typhi and 66 patients for whom diseases other than typhoid were diagnosed. Multivariate regression modeling revealed that detection of the combination of IgA, IgG, and IgG2 correlated best, although not perfectly (adjusted r(2) = 68), with a positive culture; the sensitivity and specificity of testing for IgA, IgG, and IgG2 (i.e., all three tests positive vs. all three tests negative) were 91.7% and 98.1%, respectively. These results suggested that testing for IgA, IgG, and IgG2 in combination is of diagnostic value for S. typhi infection.

Human resistance to reinfection with schistosomes. II. Specific IgA titres before & 3 months after praziquantel treatment.

Specific IgA assays by ELISA technique for schistosomal cases of school age children (12-14 years), were performed before and 3 months after praziquantel treatment. Sera of the resistant cases (non-reinfected after chemotherapeutic cure) showed a significant higher titres of anti-soluble egg antigen (anti-SEA) and anti-soluble worm antigen preparation (anti-SWAP) IgA antibodies than that of reinfected schistosomal cases before and 3 months after treatment. Praziquantel therapy insignificantly (P > 0.05) increased anti-SEA and anti-SWAP IgA antibodies in the sera of schistosomal cases and 3 months after treatment. Anti-SEA IgA & IgE anti-SWAP IgA & IgE were correlated positively with each other among schistosomal cases either resistant or reinfected while anti-SEA and anti-SWAP IgA antibody levels of schistosomal cases with eosinophilia were significantly higher than that with normal eosinophilic counts. Anti-SEA IgA antibody titres of schistosomal cases were correlated negatively, before treatment, with intensity of infection but anti-SWAP IgA titres were not, which might indicates that anti-SEA IgA antibodies inhibit schistosomes for egg laying and subsequently pathological complications due to granuloma formation. The higher titres of specific IgA antibody of resistant cases, its positive correlation with IgE and its negative correlation with intensity of infection might support the role of IgA as an essential component of acquired (resistance) immunity to share with IgE in protection of cured schistosomal cases against reinfection.

Applicability of ELISA on buffer-eluates of capillary blood spotted on filter papers for the diagnosis and clinical staging of human schistosomiasis.

The relative concentrations of IgM and IgG antibodies (Ab) to Schistosoma mansoni soluble egg antigen (SEA) were evaluated in paired samples of venous blood sera and buffer-eluates of capillary blood drops dried on filter papers. The samples were obtained from school children at early and chronic stages of schistosomiasis diagnosed on the basis of history, clinical symptomatology and parasitological criteria. Enzyme-linked immunosorbent assay (ELISA), simultaneously performed, revealed paired samples to display comparable Ab levels in all cases. Samples from children with early schistosomiasis had specific IgM:IgG ratios greater than 1 [optical densities (O.D.) in sera and blood eluates of 0.77 +/- 0.32 and 0.68 +/- 0.30, respectively for IgM and 0.52 +/- 0.25 and 0.50 +/- 0.25 for IgG]. This ratio, however, was less than 1 in samples from chronically infected children (O.D. of 0.20 +/- 0.11 and 0.20 +/- 0.11 for IgM and 0.69 +/- 0.33 and 0.73 +/- 0.32 for IgG). The specific advantages of this simplified technique are the use of anti-SEA Abs in fingerstick blood eluates, rather than sera of venous blood to serologically diagnose schistosomiasis and to differentiate early from chronic infections particularly when used for mass screening, such as epidemiologic surveys.

Prevalence of morbidity of schistosomiasis among preschool children in Bani-Suef governorate.

The present study was carried out on 429 children below 6 years of age from a village in Bani-Suef governorate to show the schistosomiasis prevalence rate among them. Direct sedimentation techniques of the urine and stool beside an indirect serological test (Dot-ELISA) were used for the diagnosis. The results indicate 14.5% and 26.3% positive cases as detected parasitologically and serologically, respectively. Other parasitic infections were also diagnosed. The serologic test enabled us to differentiate acute from chronic schistosomiasis cases in the studied sample. The serologically positive infants may suggested the congenital transmission of immunologic information rather than active infection. On the other hand, there were no correlations between the schistosomiasis incidence and both the anthropometric measurement and organ involvements.

Dot-enzyme-linked immunosorbent assay (dot-ELISA) for the rapid diagnosis of human fascioliasis.

A dot-enzyme-linked immunosorbent assay (dot-ELISA) was developed as a fast and field applicable antibody detection tool for the diagnosis of human fascioliasis. The assay is performed using partially purified antigens from a species of Fasciola at 180 ng protein/dot (2 microliters) and serum samples at 1:20 dilution (1 microliter). Dot-ELISA results completely agreed with those of micro-ELISA. Antigen-coated nitrocellulose sheets stored for 3 mo at -20 C showed results identical to fresh sheets. Sera from patients with fascioliasis (n = 30) and other parasitic or viral infections (n = 120) were compared with sera from healthy controls (n = 14). Ninety samples can be tested within 90 min. The sensitivity, specificity, and speed of the assay may justify its use in laboratory and field studies.

Immunoaffinity fractionation of Schistosoma mansoni worm antigens using human antibodies and its application for serodiagnosis.

A crude Schistosoma mansoni soluble worm antigen preparation (SWAP) was fractionated using an immunoaffinity column consisting of specific human anti-SWAP antibodies obtained from chronic S. mansoni-infected human sera and bound to CNBr-activated Sepharose 4B. The chromatographic separation resulted in three fractions: the unbound material (FW), and the eluted antigens with glycine-HCl (F1) and glycine-HCl-NaCl (F2). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the purified antigens F1 and F2 consisted of several bands when stained with Coomassie blue and silver stain, with molecular weights between 20 X 10(3) and 200 X 10(3). The F1 and F2 fractions in addition to FW and SWAP were used in an enzyme-linked immunosorbent assay (ELISA) to measure antibody levels in sera from schistosomiasis patients. Each individual serum assessed with the purified F2 antigen gave 100% positivity and three to four times higher optical density in comparison to SWAP with only 88% positivity. No detectable cross-reactive antibodies against F2 were found when a limited number of sera from filariasis, fascioliasis and trichinellosis patients were screened. Furthermore, F2 was also used and found to be more sensitive generally in detecting anti-adult worm antibodies than SWAP in recently schistosomiasis-infected persons. Thus, F2 appears to be a highly sensitive and specific reagent for the serodiagnosis of schistosomiasis infection.