Background. To design an efficacy trial of a killed oral vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhea in Egyptian children, we derived for ETEC diarrhea an empirical definition that increased the probability that diarrhea associated with excretion of ETEC was caused by the detected ETEC. Methods. We conducted a cohort study of 397 Egyptian children <24 months old and monitored them until they were 3 years old. Vaccine-preventable (VP) ETEC was defined as ETEC expressing ⩾1 of the toxin- (heat-labile [LT] toxin) and colonization-factor antigens (CFA I, II, and IV) in the vaccine. Results. Although fecal excretion of VP-ETEC was highly associated with diarrhea, excretion of LT-ETEC per se was not related to diarrhea (adjusted odds ratio [ORJ, 1.16 [95% confidence interval {CI}, 0.90-1.49]). The fecal excretion of antigenic types of VP-ETEC other than LT-ETEC (non-LT VP-ETEC) was highly associated with diarrheal symptoms (OR A, 3.91 [95% CI, 2.78-5.49]; P<.001), and this association was greater for nonbloody than for bloody diarrhea. Conclusions. Because the vaccine had been anticipated to protect primarily against symptomatic ETEC diarrhea, these results indicate that the primary-outcome definition of ETEC diarrhea for the trial should be restricted to nonbloody diarrheal episodes associated with fecal excretion of non-LT VP-ETEC. Enteric pathogens are frequently ingested and excreted by persons in developing countries, and ingestion frequently is insufficient to cause diarrheal symptoms; therefore, in such settings, fecal excretion of a particular enteropathogen in a patient with diarrhea can reflect the chance coincidence of 2 common events: diarrhea on the one hand and ingestion and excretion of the enteric pathogen on the other hand [1]. As a result, the detection, in these settings, of the fecal excretion of a pathogen in a patient with diarrhea does not establish per se that the detected pathogen has played a pathogenic role in causing the patient's symptoms. This diagnostic ambiguity creates potential problems in the evaluation of vaccines against enteric infections in developing countries. Most such vaccines are designed to prevent symptomatic infections. However, the evaluation of a vaccine's preventive effect on all diarrheal episodes associated with the fecal excretion of the target pathogen will include an appreciable fraction of episodes in which symptoms are associated with, but not caused by, the excreted target pathogen. If a vaccine has a greater protective effect against disease-causing infection than against intestinal colonization per se, an analysis of all episodes of diarrhea associated with the fecal excretion of the target pathogen will yield underestimates of vaccine efficacy against symptomatic infections. A recent efficacy trial of a new vaccine against pediatric enterotoxigenic Escherichia coli (ETEC) diarrhea in Egypt presented us with the challenge of defining diarrheal outcomes that were etiologically related to excreted ETEC. We report the application of an evidence-based, pathogenicity-driven method used to define these outcomes for the trial. Killed oral ETEC vaccine tested in Egypt. The agent under study was an orally administered, formalinized whole-cell ETEC plus recombinant cholera toxin B-subunit (ETEC/rCTB) vaccine that was developed at the University of Goteborg [2, 3]. The vaccine consists of cells that express colonization-factor antigen (CFA)/I, CFA/II, and CFA/IV, the most epidemiologically prevalent CFAs of ETEC, together with rCTB, which is antigen-ically related to the subunit of the heat-labile toxin (LT) of ETEC [4]. On the basis of earlier encouraging results regarding the safety and immunogenicity of this vaccine [5-7], a field trial of the efficacy of a 3-dose regimen of this oral vaccine was planned for Egyptian infants and children 6–18 months old, an age group with a high risk of ETEC diarrhea [8]. Pretrial cohort study of pediatric diarrhea in Abu Homos. To estimate the incidence rates of ETEC diarrhea at the site and the population to be used for the vaccine efficacy trial, we conducted a cohort study. This study also provided data necessary for evaluation of alternative outcome definitions for the trial. The cohort study was conducted in 2 rural villages ∼40 km from the city of Alexandria, in the Abu Homos district of the Beheira Governorate in lower Egypt. The enrollment of children <24 months old began in February 1995, after a census was done. Thereafter, all newborns in the 2 villages were enrolled until September 1997. Eligibility criteria required that the enrolled child have no significant congenital defects or chronic illnesses. Before enrollment, written informed consent was obtained from the parent or guardian of each child. Human-subject guidelines of the US Departments of Defense and Health and Human Services were followed throughout the study. Children were followed from the time of enrollment until age 36 months or February 1998, whichever came first. Surveillance was conducted by twice-weekly visits by trained field-workers to the homes of the enrolled children. Children reported to have had loose or liquid stools had a rectal swab and a fresh stool specimen collected by the field-worker. When a mother or caretaker reported that the child had loose or liquid stools, a physician examined the child to assess the degree of dehydration and other measures of disease severity, and arranged for appropriate therapy. For visits during which, because of the absence of the child or the caretaker, histories could not be obtained, another visit was attempted the next day. In addition, cross-sectional surveys of all children in the cohort were conducted at 2-month intervals over the 3-year period of the study. In these surveys, rectal swabs were collected and stool specimens were obtained and processed from all children, regardless of the occurrence of diarrheal symptoms, in a manner identical to that for the twice-weekly longitudinal surveillance. Demographic events such as deaths and out-migrations, which were censoring events for follow-up, were ascertained during these visits to the homes. A total of 180 children <24 months old were enrolled at the start of surveillance, and an additional 217 newborns were added to the cohort thereafter; 95% of the scheduled twice-weekly visits were successfully completed. Rectal-swab specimens collected in the field were immediately placed in Cary-Blair medium and, along with the stool specimens, were transported on ice packs to the Abu Homos field laboratory, where they were refrigerated. Twice each week, the specimens were transported on ice packs for microbiological evaluation at the laboratories of US Naval Medical Research Unit-3 in Cairo. Rectal swabs were plated on MacConkey's media, and 5 lactose-fermenting colonies were evaluated for both LT and heat-stable toxin (ST) by GM1-ganglioside ELISAs [9, 10]. Colonies that were positive for either LT or ST were further evaluated for CFAs by dot-blot tests with monoclonal antibodies specific for CFA/I, CFA/II (CS1, CS2, and CS3) and for CFA/IV (CS4, CS5, and CS6) [11, 12]. Conventional methods were used for isolation of Campylobacter, Salmonella, Shigella, and Vibrionaceae from rectal-swab specimens and for detection of rotavirus antigen in stool specimens [13-15]. Definitions.A diarrheal day was defined as the passage of ⩾3 loose or liquid stools during any 24-h period (in addition, for breast-fed infants, the mother had to state that the stools were less formed or more frequent than usual) or ⩾ 1 loose or liquid stool with the presence of visible blood. A diarrheal episode was considered to have begun on the first day of loose or liquid stools after ⩾3 consecutive nondiarrheal days and to have ended on the first diarrheal day that was followed by ⩾3 consecutive nondiarrheal days. Pathogens, including ETEC, were considered to be related to a diarrheal episode if they were detected on any day of the episode. An ETEC diarrheal episode denoted an episode in which ⩾ 1 tested lactose-fermenting colony from a fecal specimen expressed either LT or ST. A vaccine-preventable (VP) ETEC episode (an episode associated with ETEC expressing CFA or toxin antigens contained in the vaccine) was one in which every detected ETEC colony expressed either LT or CFA/I, CFA/II, or CFA/IV. A pure ETEC episode was one in which all detected ETEC had the same Toxin and CFA phenotypes. The intensity of ETEC excretion was measured in terms of the number of the 5 tested lactose-fermenting colonies that were ETEC. Diarrheal episodes were considered to be severe if ⩾1 of the following features was present by history or examination: (1) a history of ⩾1 generalized convulsions, (2) a history of drowsiness to the point that the subject no longer could take fluids, (3) a history of vomiting ⩾3 times within a 24-h period since the onset of diarrhea, (4) inability to awaken on examination, or (5) evidence of ⩾1 objective sign of dehydration on examination (e.g., sunken fontanelle, sunken eyes, absence of tears during crying, tenting of skin, parched oral mucous membranes, or weak-to-absent radial pulse); all other episodes were classified as nonsevere. Diarrhea was classified as bloody if the caretaker reported visible blood in stool and as nonbloody if no blood was described. An episode of ETEC was considered to have a copathogen if Shigella species, Campylobacter jejuni, or rotavirus was isolated in a fecal specimen collected during the episode. Seasons of the year were divided into the cool (November-April) and warm (May-October) months. Strategies for evaluation of the pathogenic roles of ETEC in diarrheal episodes. The field trial of the efficacy of ETEC/ rCTB began in 1998, after several phase 2 trials in descending age groups of Egyptian adults and children had found the vaccine to be safe and immunogenic [5-7, 16]. The trial targeted healthy infants and children 6-18 months old who were randomized to receive, at 2-week intervals, 3 oral doses of either the ETEC/rCTB vaccine or a placebo that consisted of a killed K12 strain of E.coli whole cells. After they had received an assigned regimen, participants were monitored for 1 year, by twice-weekly home visits, for the detection of ETEC diarrhea, by surveillance methods identical to those in the preceding cohort study. The goal of the trial was to assess the protective efficacy, over a 1-year period of follow-up, of a complete course of vaccine against VP-ETEC diarrheal episodes. The potential age range of follow-up for the trial was 6-30 months. Because the goal of the trial was to determine whether the ETEC/rCTB vaccine prevented symptomatic episodes that were etiologically attributable to VP-ETEC, we investigated various strategies for refining the definition of VP-ETEC diarrhea in ways that increased the strength of the pathogenic relationship between detected VP-ETEC organisms and the occurrence of diarrheal symptoms. To discern features for refining this definition, we conducted nested case-control analyses of the pretrial cohort study. Case patients had episodes of diarrhea detected during the cohort study. Control subjects were children selected during the bimonthly surveys who did not have diarrheal episodes within 7 days of the survey visit. The pathogenicity of VP-ETEC was assessed by comparing the prevalence of fecal excretion of VP-ETEC in the case patients and in the control subjects. These associations were expressed as odds ratios (ORs), with ORs >1 denoting a pathogenic relationship and progressively larger ORs corresponding to progressively stronger pathogenic relationships. Because a single diarrheal episode could have been evaluated on the basis of multiple rectal-swab specimens if the episode spanned several visits, only the results of the first rectal swab of each case were considered in the case-control comparisons, to maintain comparability with the control subjects, from whom only 1 such specimen was obtained. In these analyses, we considered several features for refining the definition of VP-ETEC diarrhea; these included characteristics of the diarrheal episode (clinical severity and diarrhea character) and characteristics of the isolated VP-ETEC (toxin phe-notype, expression of a CFA, and intensity of excretion). We also evaluated whether the presence or absence of a fecal copathogen, in addition to VP-ETEC, modified the pathogenic relationship between VP-ETEC and diarrheal symptoms, because, a priori, we reasoned that episodes in which such copathogens were present should be excluded from the trial outcomes. To simulate the conditions of the field trial, the case-control analyses were restricted to children 6–30 months old, the age range of follow-up for the field trial. We excluded case patients and control subjects who had had <5 lactose-fermenting colonies tested for toxin and CFA expression, because we wanted to use uniform criteria to judge the intensity of excretion, and we calculated this intensity on the basis of the number of colonies that were ETEC. Of the 2624 age-eligible diarrheal episodes in children who were potentially eligible as case patients, 423 were excluded because the specimens had <5 tested colonies; this left 2201 eligible diarrheal episodes for analysis. Of the 2295 age-eligible visits to control subjects during the cross-sectional surveys, 414 were excluded because the specimens had <5 tested colonies; this left 1881 eligible visits to control subjects for analysis. Because children could be selected more than once as either a case patient or a control subject, we used, for this repeated sampling of cases and controls, logistic-regression models that used a robust variance estimator to adjust the variance of coefficients [17]. The method is the same as the generalized estimating equations method under the identity working correlation. To obtain crude ORs for associations in these models, case-control status was taken as the dependent variable, and a variable for ETEC excretion was fitted as the independent variable. The coefficient for the ETEC-excretion variable in these models was exponentiated to estimate ORs, and 2-tailed Pvalues and 95% confidence intervals (CIs) for the ORs were estimated using SEs of the coefficients. To obtain estimates of ORs adjusted for important confounding variables (OR A), we refitted the models after forcing 2 additional independent variables in the models: age (in months) and season at the time of selection. All analyses were performed by the SAS macro GLIMMIX (version 6.12; SAS Institute). Of the 2201 eligible episodes of diarrhea observed among children 6–30 months old, 610 (28%) were associated with the fecal excretion of ETEC, and 321 (15%) were associated with the fecal excretion of VP-ETEC. Most episodes of ETEC diarrhea were associated with the excretion of pure ST-ETEC (61%); lesser proportions of ETEC episodes were associated with the excretion of pure LT-ETEC (24%), pure LT/ST ETEC (7%), or mixtures of ETEC-toxin phenotypes (8%). Among episodes associated with the excretion of a single ETEC-toxin phenotype, the expression of CFA/I, CFA/II, or CFA/IV was most prevalent among those with either pure LT/ST-ETEC (49%) or pure ST-ETEC (29%) and was uncommon among those with pure LT-ETEC (6%). Bloody diarrhea and severe diarrhea were in and of VP-ETEC As in the association between the fecal excretion of ETEC and diarrheal symptoms by the toxin of the excreted ETEC. associations were significant for pure ST-ETEC (OR A, [95% CI, and pure LT/ST-ETEC (OR A, [95% CI, to be no relationship between the excretion of pure LT-ETEC and the occurrence of diarrheal symptoms (OR A, 1.16 [95% CI, was a significant association between the excretion of VP-ETEC and diarrheal symptoms (OR A, [95% CI, P<.001), of pure ETEC from the definition of VP-ETEC that left only non-LT VP-ETEC) the strength of this association (OR A, 3.91 [95% CI, between fecal isolation of enterotoxigenic Escherichia coli (ETEC) and the occurrence of diarrheal symptoms, in a cohort of Egyptian children. that an analysis of either the of a CFA or the intensity of ETEC excretion of pure LT-ETEC episodes for which were pathogenic the excretion of pure LT-ETEC with diarrheal symptoms, regardless of the of a vaccine CFA and regardless of the intensity of excretion the other of VP-ETEC and each a association with diarrheal symptoms Although was a that the association between the fecal excretion of non-LT VP-ETEC and diarrheal symptoms increased with an intensity of excretion a significant association was observed only 1 positive colony was detected (OR A, [95% CI, between the fecal isolation of enterotoxigenic Escherichia coli (ETEC) and the occurrence of diarrheal symptoms, in a cohort of Egyptian children, by toxin and colonization-factor antigen phenotypes of vaccine-preventable (VP) ETEC. between fecal isolation of enterotoxigenic Escherichia coli (ETEC) and the occurrence of diarrhea, in a cohort of Egyptian children, by intensity of ETEC excretion As in pure LT-ETEC was not associated with the occurrence of diarrheal symptoms, if cases considered were restricted to episodes of nonbloody diarrhea (OR A, 1.16 [95% CI, or to patients with severe symptoms (OR A, [95% CI, The association between the excretion of non-LT VP-ETEC and the occurrence of diarrheal symptoms was in severe (OR A, [95% CI, and (OR A, [95% CI, diarrhea, but to be more associated with nonbloody (OR A, [95% CI, than with bloody (OR A, [95% CI, diarrhea. between fecal isolation of enterotoxigenic Escherichia coli (ETEC) and the occurrence of diarrheal symptoms, in a cohort of Egyptian children, by features of the diarrheal episode as in the fecal excretion of VP-ETEC, the excretion of was associated with the occurrence of diarrheal symptoms (OR A, [95% CI, the same for the excretion of non-LT VP-ETEC (OR A, [95% CI, The isolation of pure LT-ETEC from the isolation of was not associated with the occurrence of diarrheal symptoms (OR A, [95% CI, between fecal isolation of enterotoxigenic Escherichia coli (ETEC) and the occurrence of diarrheal symptoms, in a cohort of Egyptian children, by excretion of enteric copathogens The results of the present study an empirical basis for the of criteria for pathogenicity-driven definitions of in a trial of the efficacy of an ETEC vaccine in infants and children in Egypt. We have that the detection of pure LT-ETEC in a diarrheal episode in target population did not a pathogenic role for the detected that this regardless of (1) additional on the definition of pure in terms of the of a CFA or the intensity of fecal excretion, and (2) whether diarrhea was only to nonbloody episodes or those which were As the definition of pure LT-ETEC to in which copathogens were excreted no pathogenic Because the isolation of pure LT-ETEC in was not associated with diarrheal symptoms in the and population of the trial and because the vaccine to be tested in the trial was to against only symptomatic analyses indicate that was no for including diarrhea associated with the excretion of pure LT-ETEC in the primary-outcome definition for the trial. analyses also that the excretion of VP-ETEC, was to pure was associated with diarrhea regardless of either the intensity of fecal excretion of ETEC or the severity or of diarrhea; because the association was for bloody diarrhea, which is not the to be caused by ETEC, and because bloody diarrhea was in only 5 of non-LT VP-ETEC we to the case definition to include only nonbloody episodes in the outcome definition of the trial. because the vaccine was not to protect against diarrhea caused by was that the excretion of non-LT VP-ETEC was associated with diarrheal symptoms such enteric copathogens were not also potential of study should be that methods only to in which is to study outcome events that are related to the isolation of the targeted pathogen. be in which this goal is not such as trials of vaccines that are designed to the of a pathogen by of the definition derived by the method used in analysis have been to the target population for trial and not be to other trials of the same vaccine in target for in the present was found that the isolation of pure LT-ETEC was not associated with diarrhea, and was that this should be excluded from the primary-outcome definition for the trial. Although a of pathogenicity of LT-ETEC has been reported for other in the developing we the that performed in other LT-ETEC to be associated with diarrhea. earlier vaccine trials that the oral ingestion of cholera toxin subunit against diarrhea associated with an that that LT-ETEC was a cause of diarrhea in the in those trials in the pathogenicity of LT-ETEC in could be the of several features that can between such as the to which persons are and the of to the target pathogen. In addition, because LT-ETEC organisms be with to a of other the pathogenicity of LT-ETEC from to evidence that at of LT-ETEC are pathogenic from both common and in in which LT-ETEC is be important to include LT-ETEC diarrhea as a primary-outcome for trials of vaccines against ETEC, such as the vaccine that we tested in Egypt. the results of analysis to the of surveillance used in for we the that LT-ETEC have been associated with diarrhea had surveillance been an that have detected cases that were more severe than those detected in surveillance. these are several of the method that we the method the definition of outcome events in a is the method definitions of outcomes in a manner that is on empirical data than on the data required for these empirical definitions derived from the method are collected as of the of a site for a vaccine trial and, not a of additional A of the method that we used is potential could to case definitions for other infections in which the isolation of a pathogen does per se, the pathogen as the cause of a patient's symptoms; for an has been used for the definition of episodes of among persons in and additional include other enteric infections and infections. the is not to trials of preventive such as The method is also potentially to the definition of infections in of as as in In data the use of an evidence-based, pathogenicity-driven method for the definition of outcomes in trials of vaccine The application of the method to a trial of the efficacy of a killed oral vaccine against ETEC in Egyptian infants and children in the of episodes of pure LT-ETEC diarrhea from the outcome definition, a that had not been anticipated These that definitions for outcomes in phase 3 vaccine trials not be and in in which the vaccine is targeted to symptomatic they can be on analyses of pathogenicity that use data that are obtained as of the of a field site for a vaccine trial. definitions that to the pathogenicity of organisms detection does not an role for a patient's symptoms can the vaccine measured by a trial.
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