Prospective Cohort Study of Enterotoxigenic Escherichia coli Infections in Argentinean Children

In a follow-up study, enterotoxigenic Escherichia coli (ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children (P = 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872; P = 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea (P = 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors (P = 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81; P = 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6; P = 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea.     

Pathogenicity and Phenotypic Characterization of Enterotoxigenic Escherichia coli Isolates from a Birth Cohort of Children in Rural Egypt

Enterotoxigenic Escherichia coli (ETEC) has consistently been the predominant bacterial cause of diarrhea in many birth cohort- and hospital-based studies conducted in Egypt. We evaluated the pathogenicity of ETEC isolates in a birth cohort of children living in a rural community in Egypt. Between 2004 and 2007, we enrolled and followed 348 children starting at birth until their second year of life. A stool sample and two rectal swabs were collected from children during twice-weekly visits when they presented with diarrhea and were collected every 2 weeks if no diarrhea was reported. From routine stool cultures, five E. coli-like colonies were screened for ETEC enterotoxins using a GM1 enzyme-linked immunosorbent assay (ELISA). The isolates were screened against a panel of 12 colonization factor antigens (CFAs) by a dot blot assay. A nested case-control study evaluated the association between initial or repeat excretion of ETEC and the occurrences of diarrhea. The pathogenicity of ETEC was estimated in symptomatic children compared to that in asymptomatic controls. ETEC was significantly associated with diarrhea (crude odds ratio, 1.37; 95% confidence interval [CI], 1.24 to 1.52). The distribution of ETEC enterotoxins varied between the symptomatic children (44.2% heat-labile toxin [LT], 38.5% heat-stable toxin [ST], and 17.3% LT/ST) and asymptomatic children (55.5% LT, 34.6% ST, and 9.9% LT/ST) (P < 0.001). The CFAs CFA/I (n = 61), CS3 (n = 8), CS1 plus CS3 (n = 24), CS2 plus CS3 (n = 18), CS6 (n = 45), CS5 plus CS6 (n = 11), CS7 (n = 25), and CS14 (n = 32) were frequently detected in symptomatic children, while CS6 (n = 66), CS12 (n = 51), CFA/I (n = 43), and CS14 (n = 20) were detected at higher frequencies among asymptomatic children. While all toxin phenotypes were associated with diarrheal disease after the initial exposure, only ST and LT/ST-expressing ETEC isolates (P < 0.0001) were associated with disease in repeat infections. The role of enterotoxins and pathogenicity during repeat ETEC infections appears to be variable and dependent on the coexpression of specific CFAs.     

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.     

Phenotypic profiles of enterotoxigenic Escherichia coli associated with early childhood diarrhea in rural Egypt

Enterotoxigenic Escherichia coli (ETEC) causes substantial diarrheal morbidity and mortality in young children in countries with limited resources. We determined the phenotypic profiles of 915 ETEC diarrheal isolates derived from Egyptian children under 3 years of age who participated in a 3-year population-based study. For each strain, we ascertained enterotoxin and colonization factor (CF) expression, the O:H serotype, and antimicrobial susceptibility. Sixty-one percent of the strains expressed heat-stable enterotoxin (ST) only, 26% expressed heat-labile enterotoxin (LT) alone, and 12% expressed both toxins. The most common CF phenotypes were colonization factor antigen I (CFA/I) (10%), coli surface antigen 6 (CS6) (9%), CS14 (6%), and CS1 plus CS3 (4%). Fifty-nine percent of the strains did not express any of the 12 CFs included in our test panel. Resistance of ETEC strains to ampicillin (63%), trimethoprim-sulfamethoxazole (52%), and tetracycline (43%) was common, while resistance to quinolone antibiotics was rarely detected. As for the distribution of observed serotypes, there was an unusually wide diversity of O antigens and H types represented among the 915 ETEC strains. The most commonly recognized composite ETEC phenotypes were ST CS14 O78:H18 (4%), ST (or LTST) CFA/I O128:H12 (3%), ST CS1+CS3 O6:H16 (2%), and ST CFA/I O153:H45 (1.5%). Temporal plots of diarrheal episodes associated with ETEC strains bearing common composite phenotypes were consistent with discrete community outbreaks either within a single or over successive warm seasons. These data suggest that a proportion of the disease that is endemic to young children in rural Egypt represents the confluence of small epidemics by clonally related ETEC strains that are transiently introduced or that persist in a community reservoir.     

Prevalence of toxin types and colonization factors in enterotoxigenic Escherichia coli isolated during a 2-year period from diarrheal patients in Bangladesh

The prevalence of toxin types and colonization factors (CFs) of enterotoxigenic Escherichia coli (ETEC) was prospectively studied with fresh samples (n = 4,662) obtained from a 2% routine surveillance of diarrheal stool samples over 2 years, from September 1996 to August 1998. Stool samples were tested by enzyme-linked immunoassay techniques and with specific monoclonal antibodies for the toxins and CFs. The prevalence of ETEC was 14% (n = 662), with over 70% of the strains isolated from children 0 to 5 years of age, of whom 93% were in the 0- to 3-year-old age range. Of the total ETEC isolates, 49.4% were positive for the heat-stable toxin (ST), 25.4% were positive for the heat-labile toxin (LT) only, and 25.2% were positive for both LT and ST. The rate of ETEC isolation peaked in the hot summer months of May to September and decreased in winter. About 56% of the samples were positive for 1 or more of the 12 CFs that were screened for. The coli surface antigens CS4, CS5, and/or CS6 of the colonization factor antigen (CFA)/IV complex were most prevalent (incidence, 31%), followed by CFA/I (23.5%) and coli surface antigens CS1, CS2, and CS3 of CFA/II (21%). In addition, other CFs detected in decreasing order were CS7 (8%), CS14 (PCFO166) (7%), CS12 (PCFO159) (4%), CS17 (3%), and CS8 (CFA/III) (2.7%). The ST- or LT- and ST-positive ETEC isolates expressed the CFs known to be the most prevalent (i.e., CFA/I, CFA/II, and CFA/IV), while the strains positive for LT only did not. Among children who were infected with ETEC as the single pathogen, a trend of relatively more severe disease in children infected with ST-positive (P < 0.001) or LT- and ST-positive (P < 0.001) ETEC isolates compared to the severity of the disease in children infected with LT only-positive ETEC isolates was seen. This study supports the fact that ETEC is still a major cause of childhood diarrhea in Bangladesh, especially in children up to 3 years of age, and that measures to prevent such infections are needed in developing countries.     

Molecular Characterization of Enterotoxigenic Escherichia coli Isolates Recovered from Children with Diarrhea during a 4-Year Period (2007 to 2010) in Bolivia

Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood diarrhea. This study aimed to characterize ETEC strains isolated from Bolivian children aged <5 years according to enterotoxin profile, colonization factors (CFs), suggested virulence genes, and severity of disease. A total of 299 ETEC isolates recovered from children with diarrhea and 55 ETEC isolates from children without diarrhea (controls) were isolated over a period of 4 years. Strains expressing heat-labile toxin (LT) or heat-stable toxin (ST) alone were about equally common and twice as common as ETEC producing both toxins (20%). ETEC strains expressing human ST (STh) were more common in children aged <2 years, while ETEC strains expressing LT plus STh (LT/STh) were more frequent in 2- to 5-year-old children. Severity of disease was not related to the toxin profile of the strains. CF-positive isolates were more frequently identified in diarrheal samples than in control samples (P = 0.02). The most common CFs were CFA/I and CS14. CFA/I ETEC strains were more frequent in children aged <2 years than CS1+CS3 isolates and CS14 isolates, which were more prevalent in 2- to 5-year-old children. The presence of suggested ETEC virulence genes (clyA, eatA, tia, tibC, leoA, and east-1) was not associated with disease. However, east-1 was associated with LT/STh strains (P < 0.001), eatA with STh strains (P < 0.001), and tia with LT/STh strains (P < 0.001). A minor seasonal peak of ETEC infections was identified in May during the cold-dry season and coincided with the peak of rotavirus infections; this pattern is unusual for ETEC and may be important for vaccination strategies in Bolivia.     

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.     

Controlled study of Escherichia coli diarrheal infections in Bangladeshi children.

Diarrheal diseases are highly prevalent in Bangladesh. However, the relative contribution of diarrheagenic Escherichia coli organisms--those that are enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroinvasive, enterohemorrhagic, enteroaggregative, and diffuse adherent--to diarrhea in Bangladeshi populations is not known. With DNA probes specific for these diarrheagenic E. coli strains, we analyzed fecal E. coli from 451 children up to 5 years of age with acute diarrhea seeking treatment at a Dhaka hospital and from 602 matched control children without diarrhea from July 1991 to May 1992. Enteroinvasive E. coli was not isolated from any children; enterohemorrhagic E. coli was not isolated from any diarrheal children but was isolated from five control children; enteroaggregative and diffuse adherent E. coli strains were isolated with similar frequencies from children with and without diarrhea, thereby showing no association with diarrhea; ETEC was significantly associated with diarrhea in the diarrheal children as a whole and especially in the age groups of 0 to 24 months and 37 to 48 months (further analysis suggests an association with diarrhea for the heat-stable toxin only and for both heat-labile- and heat-stable-toxin-producing ETEC only); and EPEC was significantly associated with diarrhea in the diarrhea group as a whole and particularly in infants up to 1 year of age. Further analysis suggested that EPEC strains of only the traditional serogroups were significantly associated with diarrhea. ETEC and EPEC infections peaked during warm months. Our data thus suggest that EPEC and ETEC are important causes of acute diarrhea in children in this setting.     

Campylobacter jejuni antibodies in Nigerian children.

Titers of complement-fixing (CF) antibody to Campylobacter jejuni were demonstrated in 87 (36.7%) of 237 infants 6 to 15 months old in Jos, Nigeria. Of the total number of children examined, 81 had acute diarrhea and 27 of them (33.3%) were found to have CF antibodies in their serum. The remaining 156 children were asymptomatic, and 60 (38.4%) of them had CF antibodies. In the diarrheal group, 27 of 75 children 6 to 8 months old were CF antibody positive. There was no significant difference in the incidence of CF C. jejuni antibodies in the diarrheal and nondiarrheal infants (P greater than 0.05). Also, infants 9 to 15 months old had a higher incidence of CF antibodies (46.5%) than those 6 to 8 months of age (25%). The data suggest that the infants whose sera were CF antibody positive had had an exposure to C. jejuni. All 33 infants 6 to 8 months of age who had no diarrhea were CF antibody negative.     

Distribution of colonization factor antigens among enterotoxigenic Escherichia coli strains isolated from patients with diarrhea in Nepal, Indonesia, Peru, and Thailand.

Samples (1,318) of enterotoxigenic Escherichia coli (ETEC) isolated in 1994-1995 from children with diarrhea from Nepal, Indonesia, Peru, and Thailand were examined for colonization factor antigen (CFA) and coli surface (CS) antigens. Fifty-five percent of 361 heat-labile and heat-stable (LT-ST), 14% of 620 LT-only, and 48% of 337 ST-only ETEC had CFA/CS antigens. LT-ST ETEC strains were predominantly in the CFA II group, and ST only strains were in the CFA IV group. Additional studies are needed to identify ETEC strains that do not have CFA/CS antigens.     

Comparative safety and immunogenicity of two attenuated enterotoxigenic Escherichia coli vaccine strains in healthy adults

A vaccine against enterotoxigenic Escherichia coli (ETEC) is needed to prevent diarrheal illness among children in developing countries and at-risk travelers. Two live attenuated ETEC strains, PTL002 and PTL003, which express the ETEC colonization factor CFA/II, were evaluated for safety and immunogenicity. In a randomized, double-blind, placebo-controlled trial, 19 subjects ingested one dose, and 21 subjects ingested two doses (days 0 and 10) of PTL-002 or PTL-003 at 2 x 10(9) CFU/dose. Anti-CFA/II mucosal immune responses were determined from the number of antibody-secreting cells (ASC) in blood measured by enzyme-linked immunospot assay, the antibody in lymphocyte supernatants (ALS) measured by enzyme-linked immunosorbent assay (ELISA), and fecal immunoglobulin A (IgA) levels determined by ELISA. Time-resolved fluorescence (TRF) ELISA was more sensitive than standard colorimetric ELISA for measuring serum antibody responses to CFA/II and its components, CS1 and CS3. Both constructs were well tolerated. Mild diarrhea occurred after 2 of 31 doses (6%) of PTL-003. PTL-003 produced more sustained intestinal colonization than PTL-002 and better IgA response rates: 90% versus 55% (P = 0.01) for anti-CFA/II IgA-ASCs, 55% versus 30% (P = 0.11) for serum anti-CS1 IgA by TRF, and 65% versus 25% (P = 0.03) for serum anti-CS3 IgA by TRF. Serum IgG response rates to CS1 or CS3 were 55% in PTL-003 recipients and 15% in PTL-002 recipients (P = 0.02). Two doses of either strain were not significantly more immunogenic than one. Based on its superior immunogenicity, which was comparable to that of a virulent ETEC strain and other ETEC vaccine candidates, PTL-003 will be developed further as a component of a live, oral attenuated ETEC vaccine.     

Survey in Vanuatu on enterotoxigenic Escherichia coli in children and infants with and without acute diarrhea.

We have studied the incidence of enterotoxigenic Escherichia coli (ETEC) strains isolated from infants with and without diarrheal diseases in Vanuatu, South Pacific. Over a period of 5 months we have isolated enterotoxigenic E. coli strains from 29 (26.6%) of 109 children with acute diarrhea and from 13 (21.6%) of 60 children of the control group. In the group with diarrhea, 7 (6.4%) strains released heat-labile toxin, 7 (6.4%) released heat-stable toxin, and 15 (13.7%) produced both heat-labile and heat-stable toxin. In the control group, only one strain (1.6%) produced heat-stable toxin, 12 (20%) produced heat-labile toxin, and none produced both. Association of strains releasing heat-stable toxin or both heat-labile and heat-stable toxin with diarrhea was highly significant as shown by statistical analysis. The O serogroups and colonization factors CFA/I and CFA/II are presented.     

Analysis of incidence of infection with enterotoxigenic Escherichia coli in a prospective cohort study of infant diarrhea in Nicaragua.

Diarrheal episodes with enterotoxigenic Escherichia coli (ETEC) were prospectively monitored during the first 2 years of life in a cohort of 235 infants from Leon, Nicaragua. ETEC was an etiological finding in 38% (310 of 808) of diarrheal episodes and in 19% (277 of 1,472) of samples taken as asymptomatic controls at defined age intervals (P = <0.0001). The majority of diarrheal episodes (80%) occurred before 12 months of age. The major ETEC type was characterized by colonization factor CFA I and elaboration of both heat-labile enterotoxin and heat-stable enterotoxin (ST). The proportion of E. coli strains with CFA I was significantly higher in cases with diarrhea (P = 0.002). The second most prevalent type showed putative colonization factor PCFO166 and production of ST. The prevalence of PCFO166 was approximately 20%, higher than reported before. Children with a first CFA I episode contracted a second ETEC CFA I infection 24% of the time, compared with 46% for ETEC strains of any subtype. Most of the ETEC episodes were of moderate severity, and only 5% (15 of 310) were characterized as severe. In conclusion, our results give valuable information for the planning of intervention studies using ETEC vaccines.     

Enterotoxigenic Escherichia coli in central Canada.

During epidemiological studies carried out in urban and rural areas of the midwestern Canadian province of Manitoba, we cultured enterotoxigenic Escherichia coli (ETEC) from 16 (1.7%) of 945 diarrheal stools and 4 (0.3%) of 1,282 normal stools. ETEC was found in not more than 2.3% of diarrheal stools obtained from any population during any season. Diarrhea associated with ETEC persisted for a mean of 9 days. Two children were dehydrated and required intravenous fluid therapy, and one adult suffered a cholera-like syndrome. Half of the children required hospitalization for management of their diarrhea. Two adults and two children who harbored ETEC were completely asymptomatic. The pattern of toxin production correlated with serotype and the serotypes encountered were (with a few exceptions) similar to those found in other areas. We conclude that ETEC is an uncommon cause of diarrhea, both in rural and urban areas of central Canada. However, the possibility that ETEC might cause severe sporadic cases or epidemics of gastroenteritis remains.     

Genotypic and Phenotypic Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Peruvian Children

Enterotoxigenic Escherichia coli (ETEC) is a major cause of childhood diarrhea. The present study sought to determine the prevalence and distribution of toxin types, colonization factors (CFs), and antimicrobial susceptibility of ETEC strains isolated from Peruvian children. We analyzed ETEC strains isolated from Peruvian children between 2 and 24 months of age in a passive surveillance study. Five E. coli colonies per patient were studied by multiplex real-time PCR to identify ETEC virulence factors. ETEC-associated toxins were confirmed using a GM1-based enzyme-linked immunosorbent assay. Confirmed strains were tested for CFs by dot blot assay using 21 monoclonal antibodies. We analyzed 1,129 samples from children with diarrhea and 744 control children and found ETEC in 5.3% and 4.3%, respectively. ETEC was more frequently isolated from children >12 months of age than from children <12 months of age (P < 0.001). Fifty-two percent of ETEC isolates from children with diarrhea and 72% of isolates from controls were heat-labile enterotoxin (LT) positive and heat-stable enterotoxin (ST) negative; 25% and 19%, respectively, were LT negative and ST positive; and 23% and 9%, respectively, were LT positive and ST positive. CFs were identified in 64% of diarrheal samples and 37% of control samples (P < 0.05). The most common CFs were CS6 (14% and 7%, respectively), CS12 (12% and 4%, respectively), and CS1 (9% and 4%, respectively). ST-producing ETEC strains caused more severe diarrhea than non-ST-producing ETEC strains. The strains were most frequently resistant to ampicillin (71%) and co-trimoxazole (61%). ETEC was thus found to be more prevalent in older infants. LT was the most common toxin type; 64% of strains had an identified CF. These data are relevant in estimating the burden of disease due to ETEC and the potential coverage of children in Peru by investigational vaccines.Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children from developing countries and in adult travelers from industrialized countries to the developing world (16, 21). According to the World Health Organization (WHO), ETEC is the second most common cause of diarrhea after rotavirus in children less than 5 years of age and is therefore an important target for vaccine development (11). Diarrhea due to ETEC develops between 8 and 72 h after initial infection, usually due to the ingestion of contaminated food and water (21). The disease varies from a mild illness to one of great severity, usually without leukocytes or fecal blood but often with vomiting and, potentially, dehydration (10).The ability of ETEC to adhere to and colonize the human intestinal mucosa has been correlated with the presence of specific antigenic fimbriae called colonization factors (CFs), which have been designated colonization factor antigens (CFAs), coli surface antigens (CSs), or putative colonization factors (PCFs), followed by a numeric designation. The CFs are mainly fimbrial or fibrillar proteins, although some are not fimbrial in structure (21). To date, over 25 human ETEC CFs have been described. In turn, these CFs have been divided into different families: (i) a CFA/I-like group including CFA/I, CS1, CS2, CS4, CS14, and CS17; (ii) a CS5-like group including CS5, CS7, CS18, and CS20; and (iii) a unique group including CS3, CS6, and CS10 to CS12 (8, 21, 33).Following CF-mediated mucosal adhesion, ETEC elaborates one or both of two enterotoxins: heat-labile toxin (LT), a protein multimer which shares many features with cholera toxin and which binds to intracellular adenylylcyclase, leading to increased cyclic AMP levels, and/or heat-stable toxin (ST), a small-peptide molecule that similarly activates guanylylcyclase and which produces increased intracellular cyclic GMP. For both toxins, the increased chloride secretion resulting from these toxins produces a watery diarrhea (10, 16). Both of these virulence factors are plasmid encoded. ST is encoded by two different genes: estA and st1, which produce STh (originally isolated from ETEC in humans) and STp (originally from a pig isolate), respectively. LT toxin is encoded by the eltA and eltB genes (12). The diagnosis of ETEC infection relies upon the detection of either the genes themselves or their gene products in clinical specimens.Currently, derivatives of LT and the CFs are targets for the development of vaccines against ETEC. However, the great variability of ETEC CFs requires determination of the CF types prevalent in different geographic locations (21, 33). The aims of this study were (i) to determine the clinical and epidemiological characteristics of ETEC diarrhea in Peruvian children, (ii) to determine the presence of ST and LT, (iii) to determine the presence and distribution of colonization factors in these strains, and (iv) to determine the antibiotic susceptibilities of these strains.     

Phenotypic Diversity of Enterotoxigenic Escherichia coli Strains from a Community-Based Study of Pediatric Diarrhea in Periurban Egypt

No past studies of diarrhea in children of the Middle East have examined in detail the phenotypes of enterotoxigenic Escherichia coli (ETEC) strains, which are important pathogens in this setting. During a prospective study conducted from November 1993 to September 1995 with 242 children under 3 years of age with diarrhea living near Alexandria, Egypt, 125 episodes of diarrhea were positive for ETEC. ETEC strains were available for 98 of these episodes, from which 100 ETEC strains were selected and characterized on the basis of enterotoxins, colonization factors (CFs), and O:H serotypes. Of these representative isolates, 57 produced heat-stable toxin (ST) only, 34 produced heat-labile toxin (LT) only, and 9 produced both LT and ST. Twenty-three ETEC strains expressed a CF, with the specific factors being CF antigen IV (CFA/IV; 10 of 23; 43%), CFA/II (5 of 23; 22%), CFA/I (3 of 23; 13%), PCFO166 (3 of 23; 13%), and CS7 (2 of 23; 9%). No ETEC strains appeared to express CFA/III, CS17, or PCFO159. Among the 100 ETEC strains, 47 O groups and 20 H groups were represented, with 59 O:H serotypes. The most common O serogroups were O159 (13 strains) and O43 (10 strains). O148 and O21 were each detected in five individual strains, O7 and O56 were each detected in four individual strains, O73, O20, O86, and O114 were each detected in three individual strains, and O23, O78, O91, O103, O128, and O132 were each detected in two individual strains. The most common H serogroups were H4 (16 strains), 12 of which were of serogroup O159; H2 (9 strains), all of which were O43; H18 (6 strains); H30 (6 strains); and H28 (5 strains); strains of the last three H serogroups were all O148. Cumulatively, our results suggest a high degree of clonal diversity of disease-associated ETEC strains in this region. As a low percentage of these strains expressed a CF, it remains possible that other adhesins for which we either did not assay or that are as yet undiscovered are prevalent in this region. Our findings point out some potential barriers to effective immunization against ETEC diarrhea in this population and emphasize the need to identify additional protective antigens commonly expressed by ETEC for inclusion in future vaccine candidates.     

Colonization factors of enterotoxigenic Escherichia coli isolated from children with diarrhea in Argentina.

A prospective study was performed to evaluate the presence of colonization factor antigens (CFAs) in enterotoxigenic Escherichia coli (ETEC) strains isolated from 1,211 children with diarrhea in Argentina. One hundred nine ETEC strains that were isolated from seven different laboratories in various regions of the country were tested for CFAs by using monoclonal antibodies against CFA/I and E. coli surface antigens CS1, CS2, and CS3 of CFA/II and CS4 and CS5 of CFA/IV; a polyclonal antiserum against CS6 was used. The CFAs searched for were found in 52% of the ETEC strains: 23% of the strains carried CFA/I, 17% carried CFA/IV, and 12% carried CFA/II. All of the CFA/I strains produced heat-stable enterotoxin, and several of them were of the prevalent serotypes O153:H45 and O78:H12. Among the 19 strains expressing CFA/IV, 16 expressed CS5 and CS6 and produced the heat-stable enterotoxin and most were of serotype O128:H21; the remaining 3 strains produced CS6 only. No ETEC strains expressing CS4 were found. Most (11 of 13) of the CFA/II-carrying ETEC strains expressed CS1 and CS3, and 10 of them were of the O6:K15:H16 serotype and produced both heat-labile and heat-stable toxins. As many as 24 of the 109 CFA-negative ETEC strains gave mannose-resistant hemagglutination with erythrocytes from different species; 4 strains had high surface hydrophobicity, suggesting the presence of additional, as yet undefined, colonization factors in up to 25% of the ETEC isolates.     

Fecal leukocytes in children infected with diarrheagenic Escherichia coli

The purpose of this study was to determine the presence and quantity of fecal leukocytes in children infected with diarrheagenic Escherichia coli and to compare these levels between diarrhea and control cases. We analyzed 1,474 stool samples from 935 diarrhea episodes and 539 from healthy controls of a cohort study of children younger than 2 years of age in Lima, Peru. Stools were analyzed for common enteric pathogens, and diarrheagenic E. coli isolates were studied by a multiplex real-time PCR. Stool smears were stained with methylene blue and read by a blinded observer to determine the number of polymorphonuclear leukocytes per high-power field (L/hpf). Fecal leukocytes at >10 L/hpf were present in 11.8% (110/935) of all diarrheal episodes versus 1.1% (6/539) in controls (P < 0.001). Among stool samples with diarrheagenic E. coli as the only pathogen isolated (excluding coinfection), fecal leukocytes at >10 L/hpf were present in 8.5% (18/212) of diarrhea versus 1.3% (2/157) of control samples (P < 0.01). Ninety-five percent of 99 diarrheagenic E. coli diarrhea samples were positive for fecal lactoferrin. Adjusting for the presence of blood in stools, age, sex, undernutrition, and breastfeeding, enterotoxigenic E. coli (ETEC) isolation as a single pathogen, excluding coinfections, was highly associated with the presence of fecal leukocytes (>10 L/hpf) with an odds ratio (OR) of 4.1 (95% confidence interval [CI], 1.08 to 15.51; P < 0.05). Although diarrheagenic E. coli was isolated with similar frequencies in diarrhea and control samples, clearly it was associated with a more inflammatory response during symptomatic infection; however, in general, these pathogens elicited a mild inflammatory response.     

Entamoeba histolytica infection in children and protection from subsequent amebiasis

The contribution of amebiasis to the burden of diarrheal disease in children and the degree to which immunity is acquired from natural infection were assessed in a 4-year prospective observational study of 289 preschool children in an urban slum in Dhaka, Bangladesh. Entamoeba histolytica infection was detected at least once in 80%, and repeat infection in 53%, of the children who completed 4 years of observation. Annually there were 0.09 episodes/child of E. histolytica-associated diarrhea and 0.03 episodes/child of E. histolytica-associated dysentery. Fecal immunoglobulin A (IgA) anti-parasite Gal/GalNAc lectin carbohydrate recognition domain (anti-CRD) was detected in 91% (183/202) of the children at least once and was associated with a lower incidence of infection and disease. We concluded that amebiasis was a substantial burden on the overall health of the cohort children. Protection from amebiasis was associated with a stool anti-CRD IgA response. The challenge of producing an effective vaccine will be to improve upon naturally acquired immunity, which does not provide absolute protection from reinfection.     

Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II,and CFA/IV

Diarrhea is the second leading cause of death in children younger than 5 years and continues to be a major threat to global health. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacteria causing diarrhea in developing countries. ETEC strains are able to attach to host small intestinal epithelial cells by using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines that induce antiadhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, and CS3), and CFA/IV (CS4, CS5, and CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an antiadhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects and developed immune responses to CFA/I, CFA/II, and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC or E. coli strains expressing CFA/I, CFA/II, or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross-reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence of E. coli strains expressing these seven adhesins and suggested that this CFA MEFA could be a candidate to induce broad-spectrum antiadhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating an MEFA) may be generally used in vaccine development against heterogenic pathogens.