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.

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.

Survival and gene expression of enterotoxigenic Escherichia coli during long-term incubation in sea water and freshwater.

AIMS: In this study, the main objective was to verify the hypothesis of induction of 'viable but non-culturable' (VBNC) forms of enterotoxigenic Escherichia coli (ETEC) during incubation in water. METHODS AND RESULTS: Six clinically isolated ETEC strains were studied. Viable counts showed culturable ETEC bacteria for up to 3 months in freshwater but only two out of six strains were culturable in seawater at this time point. Although the bacterial cells remained intact, no production or secretion of heat-labile (LT) or heat-stable (ST) enterotoxins was observed using GM1-ELISA methods. However, genes encoding ETEC toxins (STh and LT), colonization factors (CS7 and CS17), gapA and 16S RNA were expressed during 3 months in both sea water and freshwater microcosms as determined by real-time RT-PCR on cDNA derived from the bacteria. CONCLUSIONS: Clinically isolated ETEC strains can survive for long periods in both sea water and freshwater. The bacterial cells remain intact, and the gene expression of virulence genes and genes involved in metabolic pathways are detected after 3 months. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that ETEC bacteria can enter a VBNC state during stressful conditions and suggest that ETEC has the potential to be infectious after long-term incubation in water.

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.

Enterotoxigenic Escherichia coli is detectable in water samples from an endemic area by real-time PCR.

AIMS: We aimed to develop an assay for sensitive detection and quantification of enterotoxigenic Escherichia coli (ETEC) in different types of water samples. METHODS AND RESULTS: Real-time polymerase chain reaction (PCR) assays with primers against ETEC enterotoxin genes estA (STh) estB (STp) and eltB (LT) were designed and the detection levels were determined to be three bacteria per PCR reaction. Gene copy numbers were estimated to be four (LT), two (STh) and one (STp) per bacteria. Twenty-six household and 13 environmental water samples from Bangladesh were filtered through 0.22-microm filters; DNA was extracted from the filters and analysed by real-time PCR. The results were compared with toxin GM1-enzyme-linked immunosorbent assay (ELISA), in which colonies were tested for toxin production after cultivation of the filters. Out of the 39 samples tested, 18 household and 8 environmental samples were positive for ETEC in real-time PCR, but only 6 positive samples were found with GM1-ELISA. CONCLUSIONS: The method allows for highly sensitive detection and quantification of ETEC based on detection of toxin DNA in water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The method facilitates detection and identification of ETEC in water and allows comparison between water contamination and incidence of ETEC diarrhoea in endemic areas.

Induction of B cell responses in the stomach of Helicobacter pylori- infected subjects after oral cholera vaccination.

We have evaluated the possibility of inducing antibody responses locally in the human stomach as a prerequisite for the development of a vaccine against Helicobacter pylori. Both H. pylori-infected and noninfected subjects were immunized with an oral B subunit whole cell (BS-WC) cholera vaccine, and total and vaccine-specific antibody-secreting cells (ASC) were determined by the enzyme-linked immunospot (ELISPOT) technique in cells isolated from the antrum and duodenum, respectively, before and after vaccination. Most of the subjects responded to the vaccination with high frequencies of vaccine-specific ASCs in the duodenum as well as high-serum antibody titers, and no significant differences were seen in the responses between H. pylori- infected and noninfected subjects. When studying the gastric mucosa, on the other hand, there were dramatic differences between the H. pylori-infected and the noninfected subjects. Thus, whereas none of the noninfected subjects responded to the immunization in antrum, most of the H. pylori-infected subjects had high frequencies of vaccine-specific ASCs in this location after vaccination. Furthermore, the H. pylori-infected subjects had strikingly higher (as a mean 80-fold) frequencies of total IgA-secreting cells in antrum than the noninfected subjects, whereas the frequencies of total IgA-secreting cells in the duodenum were comparable between the groups. In conclusion, these results demonstrate the possibility of inducing antibody responses locally in the gastric mucosa of H. pylori-infected individuals, a finding with obvious implications for the future development of a therapeutic vaccine against H. pylori.

Characterization of enterotoxigenic Escherichia coli from diarrhoeal patients in Bangladesh using phenotyping and genetic profiling.

A total of 99 isolates out of 370 colonization factor (CF)-positive, well-characterized enterotoxigenic Escherichia coli (ETEC) strains belonging to 13 different CF types isolated from diarrhoeal patients admitted to the hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh, were tested. The isolates were selected at random based on expression of the major CFs prevailing in Dhaka, Bangladesh, from 1996 to 1998. These isolates were characterized by O-antigenic serotyping, randomly amplified polymorphic DNA (RAPD) analysis and biochemical fingerprinting using the PhenePlate (PhP) system. The 99 ETEC isolates belonged to 10 O serogroups, the predominant ones being O6 (n=28), O115 (n=20) and O128 (n=20). Most isolates of serogroup O6 (CS1+CS3, 11/14; CS2+CS3, 5/8) belonged to the same PhP/RAPD type (H/f), whereas other isolates of serogroup O6 (n=12) belonged to different PhP/RAPD types (Si/f and F/c). Eleven serogroup O128 (CFA/I) isolates belonged to the same PhP/RAPD type (E/b), whereas the other O128 isolates formed different PhP/RAPD types. Fifteen (75%) serogroup O115 isolates (together with fourteen isolates from serogroups O25, O114, O142 and O159) demonstrated two closely related common groups by PhP typing (A and A1) and belonged to the same PhP/RAPD type (A/a). Three major clonal groups were identified among the ETEC strains in this study, largely based on O-antigenic type, CF expression pattern and toxin profile.

Mucosal immune responses are related to reduction of bacterial colonization in the stomach after therapeutic Helicobacter pylori immunization in mice.

The aim of this study was to investigate the capacity of oral and parenteral therapeutic immunization to reduce the bacterial colonization in the stomach after experimental Helicobacter pylori infection, and to evaluate whether any specific immune responses are related to such reduction. C57BL/6 mice were infected with H. pylori and thereafter immunized with H. pylori lysate either orally together with cholera toxin or intraperitoneally (i.p.) together with alum using immunization protocols that previously have provided prophylactic protection. The effect of the immunizations on H. pylori infection was determined by quantitative culture of H. pylori from the mouse stomach. Mucosal and systemic antibody responses were analyzed by ELISA in saponin extracted gastric tissue and serum, respectively, and mucosal CD4+ T cell responses by an antigen specific proliferation assay. Supernatants from the proliferating CD4+ T cells were analyzed for Th1 and Th2 cytokines. The oral, but not the parenteral therapeutic immunization induced significant decrease in H. pylori colonization compared to control infected mice. The oral immunization resulted in markedly elevated levels of serum IgG+M as well as gastric IgA antibodies against H. pylori antigen and also increased H. pylori specific mucosal CD4+ T cell proliferation with a Th1 cytokine profile. Although the parenteral immunization induced dramatic increases in H. pylori specific serum antibody titers, no increases in mucosal antibody or cellular immune responses were observed after the i.p. immunization compared to control infected mice. These findings suggest that H. pylori specific mucosal immune responses with a Th1 profile may provide therapeutic protection against H. pylori.

Resistance Pattern and Molecular Characterization of Enterotoxigenic Escherichia coli (ETEC) Strains Isolated in Bangladesh.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection leading to acute watery diarrhea in infants and young children as well as in travellers to ETEC endemic countries. Ciprofloxacin is a broad-spectrum antimicrobial agent nowadays used for the treatment of diarrhea. This study aimed to characterize ciprofloxacin resistant ETEC strains isolated from diarrheal patients in Bangladesh. METHODS: A total of 8580 stool specimens from diarrheal patients attending the icddr,b Dhaka hospital was screened for ETEC between 2005 and 2009. PCR and Ganglioside GM1- Enzyme Linked Immuno sorbent Assay (ELISA) was used for detection of Heat labile (LT) and Heat stable (ST) toxins of ETEC. Antimicrobial susceptibilities for commonly used antibiotics and the minimum inhibitory concentration (MIC) of nalidixic acid, ciprofloxacin and azithromycin were examined. DNA sequencing of representative ciprofloxacin resistant strains was performed to analyze mutations of the quinolone resistance-determining region of gyrA, gyrB, parC and parE. PCR was used for the detection of qnr, a plasmid mediated ciprofloxacin resistance gene. Clonal variations among ciprofloxacin resistant (CipR) and ciprofloxacin susceptible (CipS) strains were determined by Pulsed-field gel electrophoresis (PFGE). RESULTS: Among 1067 (12%) ETEC isolates identified, 42% produced LT/ST, 28% ST and 30% LT alone. Forty nine percent (n = 523) of the ETEC strains expressed one or more of the 13 tested colonization factors (CFs) as determined by dot blot immunoassay. Antibiotic resistance of the ETEC strains was observed as follows: ampicillin 66%, azithromycin 27%, ciprofloxacin 27%, ceftriazone 13%, cotrimaxazole 46%, doxycycline 44%, erythromycin 96%, nalidixic acid 83%, norfloxacin 27%, streptomycin 48% and tetracycline 42%. Resistance to ciprofloxacin increased from 13% in 2005 to 34% in 2009. None of the strains was resistant to mecillinam. The MIC of the nalidixic acid and ciprofloxacin of representative CipR strains were 256 µg/ml and 32µg/ml respectively. A single mutation (Ser83-Leu) in gyrA was observed in the nalidixic acid resistant ETEC strains. In contrast, double mutation in gyrA (Ser83-Leu, Asp87-Asn) and a single mutation in parC (Glu84-Ly) were found in ciprofloxacin resistant strains. Mutation of gyrB was not found in either the nalidixic acid or ciprofloxacin resistant strains. None of the ciprofloxacin resistant strains was found to be positive for the qnr gene. Diverse clones were identified from all ciprofloxacin resistant strains by PFGE analysis in both CF positive and CF negative ETEC strains. CONCLUSION: Emergence of ciprofloxacin resistant ETEC strains results in a major challenge in current treatment strategies of ETEC diarrhea.

Colonization factors of human enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of childhood and travellers' diarrhoea. The ability of ETEC to adhere to the intestinal epithelium of the host is an important virulence determinant, and adhesion is mediated by proteinaceous surface appendages called colonization factors.

T and B cell responses to chimeric proteins containing heterologous T helper epitopes inserted at different positions.

The ability of a T helper (Th) epitope to induce help for B cells recognizing different determinants within a multideterminant antigen was investigated. Chimeric fusion proteins, containing inserts of single or multiple copies of the Th epitope ovalbumin 323-339 (ova) at two different positions, were compared with respect to their ability to induce specific antibody production and ova-specific T cell activation. The antibody responses against B cell determinants at the amino and carboxy terminus, respectively was differently influenced by the molecular positioning of the inserted Th determinant. All ova-containing fusion proteins induced antibody production against the B cell determinant at the amino terminal end irrespective of the positioning of ova. In addition, multiple copies of ova in any position led to increased levels of antibody production against this epitope. In contrast, T cell help for antibody production against the determinant at the carboxy terminus was more effective after insertion of multiple copies of ova in a distal than in an adjacent position. Furthermore a fusion protein, containing four copies of ova effectively elicited T cell help for high levels of antibody production against both examined B cell determinants, showing that activated Th cells recognizing a single epitope could simultaneously provide help for distinct sets of B cells specific for widely separated epitopes within a protein. Immunodominant T cell recognition of ova in all chimeric peptides, independently of its position, was demonstrated by lymph node cell (LNC) proliferation of primed BALB/c mice. The level of ova-specific T cell proliferation was similar, irrespective of which chimeric peptide that had been used for priming, and thus did not reveal any differences in T cell priming efficiencies related to the number of ova copies in the fusion proteins. However, when the peptides were presented to a ova-specific T cell line by A20 B lymphoma cells, a close correlation between IL-2 production by the clonal T cells and the number of ova epitopes in the chimeric peptides was observed. Thus, increased cytokine production by ova-specific T cells may be important for the increased level of in vivo antibody production observed in response to multiple copies of ova in the chimeric antigens.

Genetic fusion of a non-toxic heat-stable enterotoxin-related decapeptide antigen to cholera toxin B-subunit.

A decapeptide highly homologous to the STa Escherichia coli heat-stable enterotoxin and to several other heat-stable enterotoxins was fused genetically to the amino-end of the B-subunit of cholera toxin (CTB) and the hybrid protein gene expressed from a tacP overexpression system. The STa-related decapeptide used, which was encoded by a synthetic oligodeoxynucleotide, contained a single mutation which substituted a disulfide-linked cysteine by alanine. After its fusion to CTB the decapeptide was able to both react with and to give rise to anti-STa antibodies. Expression of the decapeptide-CTB hybrid by non-toxigenic Vibrio cholerae resulted in its full secretion into the extracellular milieu from where it could then be readily purified by single-step affinity chromatography using immobilized GM1 ganglioside. Bacteria producing this non-toxic, immunogenic decapeptide-CTB toxoid might be useful for the development of oral vaccines against diarrhea caused by E. coli and other bacteria producing immunologically related heat-stable enterotoxins, and as a source of immunoreagents for methods used to diagnose disease caused by these bacteria.

Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination.

The B subunit portion of cholera toxin (CTB) is a safe and effective oral immunizing agent in humans, affording protection against both cholera and diarrhoea caused by enterotoxigenic Escherichia coli producing heat-labile toxin (LT) (Clemens et al., 1986; 1988). CTB may also be used as a carrier of various "foreign" antigens suitable for oral administration. To facilitate large-scale production of CTB for vaccine development purposes, we have constructed recombinant overexpression systems for CTB proteins in which the CTB gene is under the control of strong foreign (non-cholera) promoters and in which it is also possible to fuse oligonucleotides to the CTB gene and thereby achieve overexpression of hybrid proteins (Sanchez and Holmgren, 1989; Sanchez et al., 1988). We here expand these findings by describing overexpression of CTB by a constitutive tacP promoter as well as by the T7 RNA-polymerase promoter, and also by describing gene fusions leading to overexpression of several hybrid proteins between heat-stable E. coli enterotoxin (STa)-related peptides to either the amino or carboxy ends of CTB. Each of the hybrid proteins, when tested as immunogens in rabbits, stimulated significant anti-STa as well as anti-CTB antibody formation, although the anti-STa antibody levels attained (c.a. 1-15 micrograms/ml specific anti-STa immunoglobulin) were too low to give more than partial neutralization of STa intestinal challenge in baby mice. The hybrid proteins also had a near-native conformation, as apparent from their oligomeric nature and their strong reactivity with both a neutralizing antibody against the B subunit and a neutralizing monoclonal antibody (mAb) against STa.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucosal immunity: implications for vaccine development.

The mucosal surfaces in e.g. the gastrointestinal, respiratory and urogenital tracts represent a very large exposure area to exogenous agents including microorganisms. Not surprising, therefore, those mucosal tissues are defended by a local immune system with properties and functions that in many respects are separate from the systemic immune system. The intestine is the largest immunological organ in the body. It comprises 70-80% of all immunoglobulin-producing cells and produces more secretory IgA (SIgA) (50-100 mg/kg body weight/day) than the total production of IgG in the body (ca. 30 mg/kg/day). The local immune system of the gut has two main functions: to protect against enteric infections, and to protect against uptake of and/or harmful immune response to undergraded food antigens. The best known entity providing specific immune protection for the gut is the SIgA system. The resistance of SIgA against normal intestinal proteases makes antibodies of this isotype uniquely well suited to protect the intestinal mucosal surface. The main protective function of SIgA antibodies is the "immune exclusion" of bacterial and viral pathogens, bacterial toxins and other potentially harmful molecules. SIgA has also been described to mediate antibody-dependent T cell-mediated cytotoxicity (ADCC), and to interfere with the utilization of necessary growth factors for bacterial pathogens in the intestinal environment, such as iron. It is now almost axiomatic that in order to be efficacious, vaccines against enteric infection must be able to stimulate the local gut mucosal immune system, and that this goal is usually better achieved by administering the vaccines by the oral route rather than parenterally. Based on the concept of a common mucosal immune system through which activated lymphocytes from the gut can disseminate immunity also to other mucosal and glandular tissues there is currently also much interest in the possibility to develop oral vaccines against e.g. infections in the respiratory and urogenital tracts. It has previously been widely assumed that only live vaccines would efficiently stimulate a gut mucosal immune response. However, an oral cholera vaccine, composed of the nontoxic B subunit of cholera toxin in combination with killed whole cell (WC) cholera vibrios has been shown to stimulate a strong intestinal SIgA antibody response associated with long-lasting protection against cholera. We have used this new cholera subunit vaccine and developed ELISPOT methods for examining at the clonal B and T cell level the dynamics of intestinal and extra-intestinal immune responses in humans after enteric immunizations.(ABSTRACT TRUNCATED AT 400 WORDS)

Production and characterization of monoclonal antibodies to Vibrio cholerae soluble haemagglutinin.

Monoclonal antibodies (MAbs) against a protease of Vibrio cholerae, the soluble haemagglutinin (sHA), have been prepared and characterized with regard to their ability to inhibit different biological properties of sHA and to protect against experimental V. cholerae infection. Four fusion experiments yielded two specific immunoglobulin G1 MAbs that reacted with sHA produced by different V. cholerae O1 and non-O1 strains but that differed in their antigen-binding capacity. Both MAbs were capable of inhibiting the haemagglutinating and protease activities of sHA as well as its ability to nick the enterotoxin A subunit, but neither of them had any effect on the mucinase activity of sHA. One of the MAbs significantly inhibited experimental cholera infection induced by sHA-producing V. cholerae O1 in rabbit small intestine.

Enzyme-linked immunosorbent assay for determination of antibodies to Vibrio cholerae toxin-coregulated pili.

An ELISA for determination of antibodies to V. cholerae TCP was developed. Since purified TCP preparations contained detectable amounts of LPS (as shown by ELISA and immunoelectron microscopy with anti-LPS polyclonal serum), a capture ELISA was used. In this test the plate was coated with anti-TCP monoclonal antibody followed by incubation with TCP fimbriae. By this procedure no LPS bound to the solid phase as shown by the loss of reactivity with anti-LPS serum. The capture ELISA allowed sensitive and specific determination of TCP antibodies in sera of rabbits immunized with classical but not El Tor V. cholerae strains. There was good agreement between results in the TCP ELISA and reactivity with the TcpA band in immunoblot analyses when antisera raised against classical and El Tor vibrios were studied.

Mechanism of spontaneous loss of heat-stable toxin (STa) production in enterotoxigenic Escherichia coli.

Five strains of enterotoxigenic Escherichia coli (ETEC) showing spontaneous loss of heat-stable enterotoxin (STa) production were studied to elucidate the underlying genetic mechanisms. Southern blot analysis revealed that loss of STa production, and the corresponding lack of hybridization with the STa gene probes, were associated with deletions of DNA fragments harboring the relevant toxin genes rather than with loss of plasmids.

Effects of undernutrition on infection with Vibrio cholerae O1 and on response to oral cholera vaccine.

The association between undernutrition and the risk of colonization and disease with Vibrio cholerae O1, concentrations of salivary IgA and the serologic response to infection and to orally administered cholera B subunit were examined prospectively in a family study in Bangladesh. Children ages 1 to 8 years who were family contacts of patients hospitalized with culture-confirmed cholera were visited within 24 hours of the hospitalization and daily for 10 days, queried for the presence of diarrhea and cultured for V. cholerae O1. On Day 1 each child was weighed and saliva was collected to measure total IgA. On Days 1 and 21 blood was taken to assess vibriocidal and antitoxin titers, and on Days 1 and 2 B subunit or placebo was given orally as part of a trial to look for a toxin-blocking effect. Of 412 children enrolled in the study 35% (143) became infected with V. cholerae O1 and 49% (70) of these developed diarrhea. Undernutrition, defined in a child as weight less than 70% of the Harvard reference weight-for-age, was not associated with colonization, disease or the duration or severity of cholera. Moreover well-nourished children did not differ from undernourished children in their concentrations of salivary total IgA, initial serum antitoxin or vibriocidal antibodies or in their serologic response to colonization, disease or B subunit. The immune system in its response to cholera appears to be quite resistant to nutritional insults. The good antitoxin response to B subunit among undernourished children is of particular importance in considering the use of future oral cholera vaccines in areas where such undernutrition is common.

The design and analysis of cholera vaccine trials: recent lessons from Bangladesh.

The recent spread of cholera to Latin America, together with the persistent burden of this disease in Asia and Africa, have stimulated efforts to evaluate new cholera vaccines in field settings. Although the standard experimental paradigm for vaccine field trials is well established, the success of these trials will also depend on suitable consideration of the epidemiology of cholera and of cholera vaccination in the setting under study. Epidemiological studies done in Bangladesh emphasize the importance of appreciating the poorly predictable, multifocal occurrence of cholera in estimating a probable incidence of cholera for a field trial. They also underscore how the filtering effect of enrolling subjects into a prospective trial can dramatically reduce the available population for study, and can yield a study sample whose expected risk of cholera differs markedly from that for the source population. Finally, the data highlight the subtle effects that the mode of surveillance and the choice of an outcome definition can have upon protective efficacy, and emphasize the need for subgroup analyses that address the distinctive variations in vaccine protection that may occur in subjects differing in age and in ABO blood groups, and in subjects exposed to classical versus El Tor cholera.

PIP: It is important to predict cholera incidence for clinical trials, but it is hard to do so, even in areas where there is a regular endemic pattern. In Matlab, Bangladesh, cholera tends to occur seasonally, right before and after the summer monsoon. Various host facts affect cholera incidence, e.g., insufficient natural immunity and exposure to infected persons. Passive surveillance data in Matlab demonstrate that cholera occurs in a temporo-spatially clustered, multifocal pattern within a community. Ineligibility, refusal to participate, and absenteeism have a strong filtering effect on the population in a field trial. For example, in a field trial of killed oral cholera vaccines in Bangladesh, only 62.285 of the original 124,035 people completed the trial. As a result of nonparticipation, the expected incidence for nonvaccinees was overestimated. The definition of cholera used also determines incidence. For example, if a trial in endemic areas uses the classical definition (watery diarrhea with excretion of cholera vibrios but no copathogens), it would miss those cases with atypical symptoms (e.g., 21% of treated cholera episodes in the oral vaccine trial were clinically atypical or microbiologically mixed). Age related distinctions, ABO blood group, and biotype of the infecting organisms all influence vaccine efficacy. The above issues were considered when designing field trails of killed oral cholera vaccines in Colombia and Peru, but most people in these countries lack preexisting natural immunity, these areas have only E1 for cholera, and it will be very difficult to predict incidence among nonvaccines. This review of epidemiological studies of cholera in Bangladesh, including a large-scale field trial of killed oral cholera vaccines, describes the significance of these considerations.

Exclusion of clinically atypical or microbiologically mixed diarrhoeal episodes from outcome events in a field trial of oral cholera vaccines.

We investigated whether alternative clinical and microbiological criteria for outcome events affected estimates of vaccine efficacy in a randomized, double-blind field trial of B subunit-killed whole cell (BS-WC) and killed whole cell-only (WC) oral cholera vaccines among 62,285 rural Bangladeshi participants. At one year of follow-up estimates of vaccine protective efficacy (PE = 60%, P less than 0.0001 for BS-WC; PE = 54%, P less than 0.0001 for WC) against all treated diarrhoeal episodes associated with V. cholerae 01 were similar to estimates of efficacy against only those episodes which were clinically typical and unassociated with additional enteric pathogens (PE = 62%, P less than 0.0001 for BS-WC; PE = 52%, P less than 0.0001 for WC). In contrast, estimates of vaccine cross-protection against episodes associated with each of several agents antigenically related to V. cholerae 01 (LT-ETEC, non-cholera Vibrio sp, Aeromonas sp) were substantially reduced when mixed infections with V. cholerae 01 were excluded. We conclude that restrictive criteria intended to improve the specificity of the definition of cholera did not increase the detectability of vaccine efficacy against V. cholerae 01, but that exclusion of mixed infections with V. cholerae 01 was necessary to avoid false-positive conclusions about vaccine cross-protection against other potential target pathogens.