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Background: Enterotoxigenic Escherichia coli (ETEC) strains cause infectious diarrhea and colonize host intestine epithelia via surface-expressed colonization factors. Colonization factor antigen I (CFA/I), a prevalent ETEC colonization factor, is a vaccine target since antibodies directed to this fimbria can block ETEC adherence and prevent diarrhea., Methods: Two recombinant antigens derived from CFA/I were investigated with a vaccine adjuvant system that displays soluble antigens on the surface of immunogenic liposomes. The first antigen, CfaEB, is a chimeric fusion protein comprising the minor (CfaE) and major (CfaB) subunits of CFA/I. The second, CfaEad, is the adhesin domain of CfaE., Results: Owing to their His-tag, recombinant CfaEB and CfaEad, spontaneously bound upon admixture with nanoliposomes containing cobalt-porphyrin phospholipid (CoPoP), as well as a synthetic monophosphoryl lipid A (PHAD) adjuvant. Intramuscular immunization of mice with sub-microgram doses CfaEB or CfaEad admixed with CoPoP/PHAD liposomes elicited serum IgG and intestinal IgA antibodies. The smaller CfaEad antigen benefitted more from liposome display. Serum and intestine antibodies from mice immunized with liposome-displayed CfaEB or CfaEad recognized native CFA/I fimbria as evidenced by immunofluorescence and hemagglutination inhibition assays using the CFA/I-expressing H10407 ETEC strain., Conclusion: These data show that colonization factor-derived recombinant ETEC antigens exhibit immunogenicity when delivered in immunogenic particle-based formulations., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Wei-Chiao Huang and Jonathan Lovell are named co-inventors on one or more University at Buffalo patent applications describing CoPoP technology and hold equity in POP Biotechnologies, a university startup company licensing the technology., (© 2023 Chang Gung University. Publishing services by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Background: Inflammatory bowel disease (IBD) is a chronic, relapsing disease of the gastrointestinal (GI) tract that is thought to be associated with a complex interplay between the immune system, the GI tract lining, the environment, and the gut microbiome, leading to an abnormal inflammatory response in genetically susceptible individuals. An altered composition of the gut's native microbiota, known as dysbiosis, may have a major role in the pathogenesis of ulcerative colitis (UC) and Crohn disease (CD), two subtypes of IBD. There is growing interest in the correction of this underlying dysbiosis using fecal microbiota transplantation (FMT)., Objectives: To evaluate the benefits and safety profile of FMT for treatment of IBD in adults and children versus autologous FMT, placebo, standard medication, or no intervention., Search Methods: We searched CENTRAL, MEDLINE, Embase, two clinical trial registries, and the reference sections of published trials through 22 December 2022., Selection Criteria: We included randomized controlled trials that studied adults and children with UC or CD. Eligible intervention arms used FMT, defined as the delivery of healthy donor stool containing gut microbiota to a recipient's GI tract, to treat UC or CD., Data Collection and Analysis: Two review authors independently screened studies for inclusion. Our primary outcomes were: 1. induction of clinical remission, 2. maintenance of clinical remission, and 3. serious adverse events. Our secondary outcomes were: 4. any adverse events, 5. endoscopic remission, 6. quality of life, 7. clinical response, 8. endoscopic response, 9. withdrawals, 10. inflammatory markers, and 11. microbiome outcomes. We used the GRADE approach to assess the certainty of evidence., Main Results: We included 12 studies with 550 participants. Three studies were conducted in Australia; two in Canada; and one in each of the following: China, the Czech Republic, France, India, the Netherlands, and the USA. One study was conducted in both Israel and Italy. FMT was administered in the form of capsules or suspensions and delivered by mouth, nasoduodenal tube, enema, or colonoscopy. One study delivered FMT by both oral capsules and colonoscopy. Six studies were at overall low risk of bias, while the others had either unclear or high risk of bias. Ten studies with 468 participants, of which nine studies focused on adults and one focused on children, reported induction of clinical remission in people with UC at longest follow-up (range 6 to 12 weeks) and showed that FMT may increase rates of induction of clinical remission in UC compared to control (risk ratio (RR) 1.79, 95% confidence interval (CI) 1.13 to 2.84; low-certainty evidence). Five studies showed that FMT may increase rates of induction of endoscopic remission in UC at longest follow-up (range 8 to 12 weeks); however, the CIs around the summary estimate were wide and included a possible null effect (RR 1.45, 95% CI 0.64 to 3.29; low-certainty evidence). Nine studies with 417 participants showed that FMT may result in little to no difference in rates of any adverse events (RR 0.99, 95% CI 0.85 to 1.16; low-certainty evidence). The evidence was very uncertain about the risk of serious adverse events (RR 1.77, 95% CI 0.88 to 3.55; very low-certainty evidence) and improvement in quality of life (mean difference (MD) 15.34, 95% CI -3.84 to 34.52; very low-certainty evidence) when FMT was used to induce remission in UC. Two studies, of which one also contributed data for induction of remission in active UC, assessed maintenance of remission in people with controlled UC at longest follow-up (range 48 to 56 weeks). The evidence was very uncertain about the use of FMT for maintenance of clinical remission (RR 2.97, 95% CI 0.26 to 34.42; very low-certainty evidence) and endoscopic remission (RR 3.28, 95% CI 0.73 to 14.74; very low-certainty evidence). The evidence was also very uncertain about the risk of serious adverse events, risk of any adverse events, and improvement in quality of life when FMT was used to maintain remission in UC. None of the included studies assessed use of FMT for induction of remission in people with CD. One study with 21 participants reported data on FMT for maintenance of remission in people with CD. The evidence was very uncertain about the use of FMT for maintenance of clinical remission in CD at 24 weeks (RR 1.21, 95% CI 0.36 to 4.14; very low-certainty evidence). The evidence was also very uncertain about the risk of serious or any adverse events when FMT was used to maintain remission in CD. None of the studies reported data on use of FMT for maintenance of endoscopic remission or improvement in quality of life in people with CD., Authors' Conclusions: FMT may increase the proportion of people with active UC who achieve clinical and endoscopic remission. The evidence was very uncertain about whether use of FMT in people with active UC impacted the risk of serious adverse events or improvement in quality of life. The evidence was also very uncertain about the use of FMT for maintenance of remission in people with UC, as well as induction and maintenance of remission in people with CD, and no conclusive statements could be made in this regard. Further studies are needed to address the beneficial effects and safety profile of FMT in adults and children with active UC and CD, as well as its potential to promote longer-term maintenance of remission in UC and CD., (Copyright © 2023 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd.)

Multilocus Sequence Typing has become a useful tool for the study of the genetic diversity and population structure of different organisms. In this study, a MLST approach with seven loci (CP47, MS5, MS9, MSC6-7, TP14, and gp60) was used to analyze the genetic diversity of Cryptosporidium hominis and Cryptosporidium parvum isolated from 28 Colombian patients. Five Cryptosporidium species were identified: C. hominis, C. parvum, Cryptosporidium felis, Cryptosporidium meleagridis, and Cryptosporidium suis. Unilocus gp60 analysis identified four allelic families for C. hominis (Ia, Ib, Id, and Ie) and two for C. parvum (IIa and IIc). There was polymorphic behavior of all markers evaluated for both C. hominis and C. parvum, particularly with the CP47, MS5, and gp60 markers. Phylogenetic analysis with consensus sequences (CS) of the markers showed a taxonomic agreement with the results obtained with the 18S rRNA and gp60 gene. Additionally, two monophyletic clades that clustered the species C. hominis and C. parvum were detected, with a higher number of subclades within the monophyletic groups compared to those with the gp60 gene. Thirteen MLG were identified for C. hominis and eight for C. parvum. Haplotypic and nucleotide diversity were detected, but only the latter was affected by the gp60 exclusion from the CS analysis. The gene fixation index showed an evolutionary closeness between the C. hominis samples and a less evolutionary closeness and greater sequence divergence in the C. parvum samples. Data obtained in this work support the implementation of MLST analysis in the study of the genetic diversity of Cryptosporidium, considering the more detailed information that it provides, which may explain some genetic events that with an unilocus approach could not be established. This is the first multilocus analysis of the intra-specific variability of Cryptosporidium from humans in South America., Competing Interests: The authors have declared that no competing interests exist.

Background: Hepatitis C virus infection is a leading cause of blood-borne hepatitis disease worldwide. Hepatitis C is a silent liver disease that, without treatment, leads to late-onset complications, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, in 10-40% of patients., Objective: This study aimed to review the epidemiology, clinical features, diagnosis, treatment, and prevention of hepatitis C among perinatally exposed children., Methods: Public databases, including MEDLINE and PubMed, and websites from the Centers for Disease Control and Prevention, the Food and Drug Administration, the World Health Organization, and the National Institutes of Health were searched for relevant articles published between 2006 and 2021., Results: The prevalence of hepatitis C has increased among women of childbearing age in the United States and is associated with risk factors, such as intravenous drug use, health inequities, and low socioeconomic background. Infants born to hepatitis C virus-infected mothers have a 6% risk of vertical transmission, and among those infected, 75% will develop chronic hepatitis C and late complications. However, hepatitis C-exposed infants are frequently lost to follow-up, and those infected have delayed diagnosis and treatment and are at high risk for late-onset complications. Direct- acting antivirals and the establishment of effective treatment guidelines cure hepatitis C virus infections., Conclusion: Hepatitis C predominantly affects underserved communities. Early screening of mothers and infants is critical for the diagnosis, treatment, and prevention of chronic infections and lateonset complications. New policies are needed to address hepatitis C health care inequities affecting mothers and infants in the United States., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Coronavirus disease 2019 (COVID-19) is a viral respiratory infection caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). While SARS-CoV-2 is a leading cause of morbidity and mortality in older adults, COVID-19 also affects newborn infants in nurseries and the Neonatal Intensive Care Units (NICUs). The majority of infected neonates are believed to acquire SARS-CoV-2 by horizontal transmission, and most of them have asymptomatic or mild symptomatic infections. In rare cases, infants with COVID-19 may have severe complications resulting in death. We report a case of COVID-19 in a premature neonate born at 34 weeks gestational age who presented with hypothermia and respiratory distress and subsequently developed clinical and radiological signs of necrotizing enterocolitis (NEC). The neonate received medical management, including antibiotics, suspension of gastric feeds, and intensive NICU support. The neonate's clinical condition improved without surgical intervention, and after 10 days of antibiotics and gradual reestablishment of gastric feeds, patient health condition returned to normal, and weeks later, he was discharged home. COVID-19 in infants is frequently asymptomatic or associated with mild disease, and in rare cases, it may be associated with severe gastrointestinal complications including NEC., Competing Interests: The authors state that they have no conflicts of interest to declare., (Copyright © 2021 Mary K. Mannix et al.)

Background: Haemolytic uraemic syndrome (HUS) is a common cause of acquired kidney failure in children and rarely in adults. The most important risk factor for development of HUS is a gastrointestinal infection by Shiga toxin-producing Escherichia coli (STEC). This review addressed the interventions aimed at secondary prevention of HUS in patients with diarrhoea who were infected with a bacteria that increase the risk of HUS., Objectives: Our objective was to evaluate evidence regarding secondary preventative strategies for HUS associated with STEC infections. In doing so, we sought to assess the effectiveness and safety of interventions as well as their potential to impact the morbidity and death associated with this condition., Search Methods: We searched the Cochrane Kidney and Transplant Register of Studies up to 12 November 2020 through contact with the Information Specialist using search terms relevant to this review. Studies in the Register are identified through searches of CENTRAL, MEDLINE, and EMBASE, conference proceedings, the International Clinical Trials Register (ICTRP) Search Portal and ClinicalTrials.gov., Selection Criteria: Studies were considered based on the methods, participants, and research goals. Only randomised controlled trials were considered eligible for inclusion. The participants of the studies were paediatric and adult patients with diarrhoeal illnesses due to STEC. The primary outcome of interest was incidence of HUS., Data Collection and Analysis: We used standard methodological procedures as recommended by Cochrane. Summary estimates of effect were obtained using a random-effects model, and results were expressed as risk ratios (RR) and their 95% confidence intervals (CI) for dichotomous outcomes. Confidence in the evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach., Main Results: We identified four studies (536 participants) for inclusion that investigated four different interventions including antibiotics (trimethoprim-sulfamethoxazole), anti-Shiga toxin antibody-containing bovine colostrum, Shiga toxin binding agent (Synsorb Pk: a silicon dioxide-based agent), and a monoclonal antibody against Shiga toxin (urtoxazumab). The overall risk of bias was unclear for selection, performance and detection bias and low for attrition, reporting and other sources of bias. It was uncertain if trimethoprim-sulfamethoxazole reduced the incidence of HUS compared to no treatment (47 participants: RR 0.57, 95% CI 0.11-2.81, very low certainty evidence). Adverse events relative to this review, need for acute dialysis, neurological complication and death were not reported. There were no incidences of HUS in either the bovine colostrum group or the placebo group. It was uncertain if bovine colostrum caused more adverse events (27 participants: RR 0.92, 95% CI 0.42 to 2.03; very low certainty evidence). The need for acute dialysis, neurological complications or death were not reported. It is uncertain whether Synsorb Pk reduces the incidence of HUS compared to placebo (353 participants: RR 0.93, 95% CI 0.39 to 2.22; very low certainty evidence). Adverse events relevant to this review, need for acute dialysis, neurological complications or death were not reported. One study compared two doses of urtoxazumab (3.0 mg/kg and 1.0 mg/kg) to placebo. It is uncertain if either 3.0 mg/kg urtoxazumab (71 participants: RR 0.34, 95% CI 0.01 to 8.14) or 1.0 mg/kg urtoxazumab (74 participants: RR 0.95, 95% CI 0.79 to 1.13) reduced the incidence of HUS compared to placebo (very low certainty evidence). Low certainty evidence showed there may be little or no difference in the number of treatment-emergent adverse events with either 3.0 mg/kg urtoxazumab (71 participants: RR 1.00, 95% CI 0.84 to 1.18) or 1.0 mg/kg urtoxazumab (74 participants: RR 0.95, 95% CI 0.79 to 1.13) compared to placebo. There were 25 serious adverse events reported in 18 patients: 10 in the placebo group, and 9 and 6 serious adverse events in the 1.0 mg/kg and 3.0 mg/kg urtoxazumab groups, respectively. It is unclear how many patients experienced these adverse events in each group, and how many patients experienced more than one event. It is uncertain if either dose of urtoxazumab increased the risk of neurological complications or death (very low certainty evidence). Need for acute dialysis was not reported., Authors' Conclusions: The included studies assessed antibiotics, bovine milk, and Shiga toxin inhibitor (Synsorb Pk) and monoclonal antibodies (Urtoxazumab) against Shiga toxin for secondary prevention of HUS in patients with diarrhoea due to STEC. However, no firm conclusions about the efficacy of these interventions can be drawn given the small number of included studies and the small sample sizes of those included studies. Additional studies, including larger multicentre studies, are needed to assess the efficacy of interventions to prevent development of HUS in patients with diarrhoea due to STEC infection., (Copyright © 2021 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd.)

Background: Inflammatory bowel disease (IBD) is a chronic, relapsing disease of the gastrointestinal tract that is thought to be associated with a complex interplay between microbes and the immune system, leading to an abnormal inflammatory response in genetically susceptible individuals. Dysbiosis, characterized by the alteration of the composition of the resident commensal bacteria in a host compared to healthy individuals, is thought to play a major role in the pathogenesis of ulcerative colitis (UC) and Crohn's disease (CD), two subtypes of IBD. There is growing interest to correct the underlying dysbiosis through the use of fecal microbiota transplantation (FMT) for the treatment of IBD., Objectives: The objective of this systematic review was to assess the efficacy and safety of FMT for the treatment of IBD., Search Methods: We searched the MEDLINE, Embase, Cochrane Library, and Cochrane IBD Group Specialized Register databases from inception to 19 March 2018. We also searched ClinicalTrials.gov, ISRCTN metaRegister of Controlled Trials, and the Conference Proceedings Citation Index., Selection Criteria: Only randomized trials or non-randomized studies with a control arm were considered for inclusion. Adults or pediatric participants with UC or CD were eligible for inclusion. Eligible interventions were FMT defined as the administration of fecal material containing distal gut microbiota from a healthy donor to the gastrointestinal tract of a someone with UC or CD. The comparison group included participants who did not receive FMT and were given placebo, autologous FMT, or no intervention., Data Collection and Analysis: Two authors independently screened the titles and extracted data from the included studies. We used the Cochrane risk of bias tool to assess study bias. The primary outcomes were induction of clinical remission, clinical relapse, and serious adverse events. Secondary outcomes included clinical response, endoscopic remission and endoscopic response, quality of life scores, laboratory measures of inflammation, withdrawals, and microbiome outcomes. We calculated the risk ratio (RR) and corresponding 95% confidence interval (95% CI) for dichotomous outcomes and the mean difference and 95% CI for continuous outcomes. Random-effects meta-analysis models were used to synthesize effect sizes across trials. The overall certainty of the evidence supporting the primary and selected secondary outcomes was rated using the GRADE criteria., Main Results: Four studies with a total of 277 participants were included. These studies assessed the efficacy of FMT for treatment of UC in adults; no eligible trials were found for the treatment of CD. Most participants had mild to moderate UC. Two studies were conducted in Australia, one study was conducted in Canada, and another in the Netherlands. Three of the included studies administered FMT via the rectal route and one study administered FMT via the nasoduodenal route. Three studies were rated as low risk of bias. One study (abstract publication) was rated as unclear risk of bias. Combined results from four studies (277 participants) suggest that FMT increases rates of clinical remission by two-fold in patients with UC compared to controls. At 8 weeks, 37% (52/140) of FMT participants achieved remission compared to 18% (24/137) of control participants (RR 2.03, 95 % CI, 1.07 to 3.86; I² = 50%; low certainty evidence). One study reported data on relapse at 12 weeks among participants who achieved remission. None of the FMT participants (0/7) relapsed at 12 weeks compared to 20% of control participants (RR 0.28, 95% CI 0.02 to 4.98, 17 participants, very low certainty evidence). It is unclear whether there is a difference in serious adverse event rates between the intervention and control groups. Seven per cent (10/140) of FMT participants had a serious adverse event compared to 5% (7/137) of control participants (RR 1.40, 95% CI 0.55 to 3.58; 4 studies; I² = 0%; low certainty evidence). Serious adverse events included worsening of UC necessitating intravenous steroids or surgery; infection such as Clostridium difficile and cytomegalovirus, small bowel perforation and pneumonia. Adverse events were reported by two studies and the pooled data did not show any difference between the study groups. Seventy-eight per cent (50/64) of FMT participants had an adverse event compared to 75% (49/65) of control participants (RR 1.03, 95% CI 0.81 to 1.31; I² = 31%; moderate certainty evidence). Common adverse events included abdominal pain, nausea, flatulence, bloating, upper respiratory tract infection, headaches, dizziness, and fever. Four studies reported on clinical response at 8 weeks. Forty-nine per cent (68/140) of FMT participants had a clinical response compared to 28% (38/137) of control participants (RR 1.70, 95% CI 0.98 to 2.95, I² = 50%, low certainty evidence). Endoscopic remission at 8 weeks was reported by three studies and the combined results favored FMT over the control group. Thirty per cent (35/117) of FMT participants achieved endoscopic remission compared to 10% (11/112) of control participants (RR 2.96, 95 % CI 1.60 to 5.48, I² = 0%; low certainty evidence)., Authors' Conclusions: Fecal microbiota transplantation may increase the proportion of participants achieving clinical remission in UC. However, the number of identified studies was small and the quality of evidence was low. There is uncertainty about the rate of serious adverse events. As a result, no solid conclusions can be drawn at this time. Additional high-quality studies are needed to further define the optimal parameters of FMT in terms of route, frequency, volume, preparation, type of donor and the type and disease severity. No studies assessed efficacy of FMT for induction of remission in CD or in pediatric participants. In addition, no studies assessed long-term maintenance of remission in UC or CD. Future studies are needed to address the therapeutic benefit of FMT in CD and the long-term FMT-mediated maintenance of remission in UC or CD.

Background: The epidemiology of antibiotic-resistant Enterobacteriaceae intestinal carriage in healthy US children has not been well characterized., Methods: Children between 14 days and 14 years of age were enrolled during well-child visits in Oakland, California, Kansas City, Kansas, and Nashville, Tennessee, between December 2013 and March 2015. Data on recent antibiotic use by the child and travel and hospitalization history of all members of each child's household were obtained with a risk-factor survey. Stool specimens collected from the subjects were screened for extended-spectrum β-lactamase-producing (ESBL-P) bacteria using CHROMagar ESBL medium. Putative ESBL-P Escherichia coli and Klebsiella colonies underwent phenotypic confirmation by double-disk synergy testing; confirmed third-generation cephalosporin-resistant (3GCR) isolates underwent additional antibiotic-susceptibility testing., Results: In 519 subjects, the overall 3GCR Enterobacteriaceae carriage rate was 4.4% (n = 23) and ranged from 3.4% to 5.1% among the study sites. The ESBL-P Enterobacteriaceae carriage rate was 3.5% (n = 18). The rates of 3GCR Enterobacteriaceae carriage was highest in 1 to <2 year olds at 6.5%, and was 5.2% in <5 year-olds vs 1.7% in ≥5-year-olds (P = .11). 3GCR and ESBL-P Enterobacteriaceae carriage was associated with international travel within the previous year; 11.1% of ESBL-P Enterobacteriaceae carriers reported this history compared with 1.6% of noncarriers (P = .004). No other queried factor was found to increase risk. Of the 24 analyzed 3GCR isolates, 58% were multidrug resistant., Conclusions: The 3GCR Enterobacteriaceae carriage rate exceeds 5% in healthy US children <5 years of age. International travel within the previous year increased the risk of 3GCR and ESBL-P Enterobacteriaceae carriage. In contrast, we found no differences in the rates of hospitalization or recent antibiotic exposure between carriers and noncarriers. Young children, who have the highest prevalence of colonization, might be a sentinel population to study to gain a better understanding of community sources of antibiotic-resistant Enterobacteriaceae.

Background: Enteropathogenic Escherichia coli (EPEC) is one of the leading causes of watery diarrhea among children., Methods: In this study EPEC isolates from the pediatric population of Pakistan (2010-2012) were subjected to phylotyping, antibiotic susceptibility, extended-spectrum beta-lactamase (ESBL) profiling and evaluation of one representative strain from each panel of phylotypesin Galleria mellonella, infection model., Results: A total of 46/225 (20.4%) stool samples were positive for EPEC. Isolates mainly belong to D phylogroup (18, 39%) followed by nontypeable (10, 22%), B1 (9, 20%), B2 (8, 17%) and A (1, 2%). High resistance was observed for ampicillin (42, 91%), erythromycin (41, 89%), cefaclor (37, 80%), trimethoprim/sulfamethoxazole (36, 78%), tetracycline (36, 78%). Among nalidixic acid resistant isolates 13 (28%) showed presence of single nucleotide polymorphism (SNP) in parC (C330-T330) whereas 1 (2%) isolate showed gyrB (A660-T660) SNP. Furthermore, 27 (59%) isolates were ESBL producers. Representative isolates of phlyotypes A and B2 showed enhance killing of G. mellonella compared to ones belonging to phylotypes B1 and D., Conclusions: Non-typeable EPEC strains were frequently observed. ESBL production in ESBL producers was found to be plasmid mediated. No significant association of antibiotic resistance profile with specific phylogroup of EPEC was found, however G. mellonella infection model differentiated representative phylotypes., (© The Author 2016. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Intestinal Escherichia coli pathogens are major causes of diarrhoeal disease in children under five years of age worldwide. The aim of this study is to evaluate the association of E. coli pathotypes with childhood diarrhoea in Colombia., Methodology: A case-control study was conducted in 815 samples from children younger than five years of age in Cartagena, Colombia (466 cases and 349 controls). Controls were randomly selected 1:1 to cases, to obtain 349 cases and 349 controls., Results: This study revealed that 27 (7.44%) cases and 12 (3.43%) controls were positives for any of the E. coli pathotypes. The difference observed was statistically significant indicating that E. coli pathotypes were associated with cases of childhood diarrhoea. Enterotoxigenic E. coli (ETEC) was the most common pathotype associated with childhood diarrhoea. Additional E. coli pathotypes were also identified., Conclusions: We conclude that after the adjustment by age, sex and socioeconomic stratum, the odds ratio obtained by logistic regression shows an association between infection with ETEC and childhood diarrhoea.

Enterotoxigenic Escherichia coli (ETEC) strains produce a type IV pilus named Longus. We identified a 16-gene cluster involved in the biosynthesis of Longus that has 57 to 95% identity at the protein level to CFA/III, another type IV pilus of ETEC. Alleles of the Longus structural subunit gene lngA demonstrate a diversity of 12 to 19% at the protein level with strong positive selection for point replacements and horizontal transfer.

Two Salmonella enterica serovar Typhi strains that express and export a truncated version of Plasmodium falciparum circumsporozoite surface protein (tCSP) fused to Salmonella serovar Typhi cytolysin A (ClyA) were constructed as a first step in the development of a preerythrocytic malaria vaccine. Synthetic codon-optimized genes (t-csp1 and t-csp2), containing immunodominant B- and T-cell epitopes present in native P. falciparum circumsporozoite surface protein (PfCSP), were fused in frame to the carboxyl terminus of the ClyA gene (clyA::t-csp) in genetically stabilized expression plasmids. Expression and export of ClyA-tCSP1 and ClyA-tCSP2 by Salmonella serovar Typhi vaccine strain CVD 908-htrA were demonstrated by immunoblotting of whole-cell lysates and culture supernatants. The immunogenicity of these constructs was evaluated using a "heterologous prime-boost" approach consisting of mucosal priming with Salmonella serovar Typhi expressing ClyA-tCSP1 and ClyA-tCSP2, followed by parenteral boosting with PfCSP DNA vaccines pVR2510 and pVR2571. Mice primed intranasally on days 0 and 28 with CVD 908-htrA(pSEC10tcsp2) and boosted intradermally on day 56 with PfCSP DNA vaccine pVR2571 induced high titers of serum NANP immunoglobulin G (IgG) (predominantly IgG2a); no serological responses to DNA vaccination were observed in the absence of Salmonella serovar Typhi-PfCSP priming. Mice primed with Salmonella serovar Typhi expressing tCSP2 and boosted with PfCSP DNA also developed high frequencies of gamma interferon-secreting cells, which surpassed those produced by PfCSP DNA in the absence of priming. A prime-boost regimen consisting of mucosal delivery of PfCSP exported from a Salmonella-based live-vector vaccine followed by a parenteral PfCSP DNA boosting is a promising strategy for the development of a live-vector-based malaria vaccine.

The lack of a practical small animal model to study the immunogenicity of Salmonella typhi-based live vector vaccines expressing foreign antigens has seriously impeded the vaccine development process. For some foreign antigens, stimulation of serum IgG antibody is the desired, protective immune response. We administered to mice, by orogastric or intranasal (i.n.) routes, attenuated delta aroC delta aroD S. typhi CVD 908 carrying a plasmid encoding fragment C (fragC) of tetanus toxin fused to the eukaryotic cell receptor binding domain of diphtheria toxin (fragC-bDt), and monitored serum antibody. While orogastric inoculation of three doses was not immunogenic, i.n. immunization elicited high titers of serum IgG tetanus antitoxin, generating peak ELISA geometric mean titers (GMT) of 27024 and 35658 with 10(8) and 10(9) c.f.u. dosages, respectively; 10(9) c.f.u. i.n. of an delta aroA S. typhimurium live vector stimulated a peak antitoxin GMT of 376 405. Mice immunized with the S. typhi live vector were 100% protected against challenge with 100 50% lethal doses of tetanus toxin that rapidly killed all control mice. Intranasal immunization with two doses of S. typhi expressing unfused fragment C under control of an anaerobically-activated promoter derived from nirB stimulated significantly higher titers of serum neutralizing antitoxin than fused fragC-bDt controlled by the same promoter (GMT 0.10 AU ml-1 vs 0.01 AU ml-1, P = 0.0095). Two i.n. doses of S typhi encoding fragC under control of powerful constitutive promoter 1pp led to significantly higher peak serum neutralizing antitoxin titers than the otherwise identical construct utilizing the nirB promoter (peak GMT 0.72 AU ml-1 vs 0.10 AU ml-1, P = 0.022). The i.n. route of inoculation of mice may constitute a practical breakthrough that could expedite the development of some S. typhi-based live vector vaccines by allowing, for the first time, quantitative measurement of serum antibody responses to candidate constructs following i.n. mucosal immunization.