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Epidemiology of Enterotoxigenic Escherichia coli Diarrhea in a Pediatric Cohort in a Periurban Area of Lower Egypt
Remon Abu-Elyazeed, Thomas F. Wierzba, Ali S. Mourad,1 Leonard F. Peruski, Bradford A. Kay,2 Malla Rao, Albert M. Churilla,2 A. Louis Bourgeois, Ahmed K. Mortagy,2 Salwa M. Kamal, Stephen J. Savarino, James R. Campbell,2 James R. Murphy,2 Abdollah Nacy, and John D. Clemens
US Naval Medical Research Unit No. 3, Cairo, and University of Alexandria, Alexandria, Egypt; National Institute of Child Health and Human Development, National Institutes of Health, and Naval Medical Research Institute, Bethesda, Maryland

Enterotoxigenic Escherichia coli (ETEC) are diverse pathogens that express heat-labile (LT) and/or heat-stable (ST) enterotoxins, yet little is known about whether epidemiologic patterns of pediatric ETEC diarrhea vary by the expressed ETEC toxin phenotype. In total, 242 Egyptian children aged !3 years were prospectively followed in 19931995. ETEC episodes were detected during twice-weekly home visits, and asymptomatic ETEC excretion was identied from monthly cross-sectional surveys. ETEC episodes were 0.6 per child-year. ST-only ETEC was 2.6 times (P ! .001) more common in warmer than cooler months, while LT-only ETEC showed no seasonal variation. Ownership of a household sanitary latrine, but not breast-feeding, was associated with a lower risk of both enterotoxin phenotypes. Coexpression of a colonization factor by LT- or ST-only ETEC strengthened the association with diarrhea. These ndings indicate that the epidemiologic patterns of LT-only and ST-only ETEC are not identical and that disease interventions should include improved household sanitation.

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It has been estimated that enterotoxigenic Escherichia coli (ETEC) causes nearly 400 million diarrheal episodes and 700,000 deaths annually among children !5 years old [1]. Even

Received 19 May 1998; revised 24 September 1998. Presented in part: 45th annual meeting, American Society of Tropical Medicine and Hygiene, Baltimore, December 1996 (abstract 122). Informed consent was obtained from a parent of each subject, and the protocol was approved by the human use review boards of the US Naval Medical Research Unit No. 3, Cairo, and the Naval Medical Research and Development Command, Bethesda, Maryland. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as ofcial or as reecting the views of the Departments of the Navy or of Defense, the US government, the World Health Organization, or the Egyptian Ministry of Health and Population. Support: US Army Medical Research and Development Command (Fort Detrick, Frederick, MD); US Naval Medical Research and Development Command (Bethesda, MD) work unit No. 9000101PIX3270; Global Programme on Vaccines and Immunization, World Health Organization (Geneva); National Institute of Child Health and Human Development (interagency agreement Y1-HD-0026-01). 1 Deceased. 2 Present afliations: Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta (B.A.K.); Naval Medical Research Institute, Bethesda, Maryland (A.M.C.); Faculty of Medicine, Ain Shams, Cairo (A.K.M.); Naval Research Laboratory, Washington, DC (J.R.C.); and Center for Infectious Diseases and Division of Pediatric Infectious Diseases, Medical School and School of Public Health, University of Texas, Houston (J.M.). Reprints or correspondence: Commanding Ofcer, US Naval Medical Research Unit No. 3 (Attn: Code 101F), PSC 452, Box 5000, FPO AE 09835-0007.
The Journal of Infectious Diseases This article is in the public domain. 0022-1899 1999; 179:3829

these impressive gures may underestimate the disease burden associated with ETEC, since ETEC diarrhea is also associated with chronic nutritional faltering [2]. This disease burden is concentrated primarily in young children in the less-developed countries of Asia, Africa, the Middle East, and Latin America. Among areas in which ETEC diarrhea is endemic, the Middle East is thought to rank high in the toll of morbidity associated with ETEC. A study in lower Egypt revealed a cumulative occurrence of disease of 1.7 episodes for ETEC-expressing heatlabile enterotoxin (LT) and 1.9 episodes for ETEC-expressing heat-stable enterotoxin (ST) per child from birth to age 3 years [3]. The present study was undertaken to better understand the epidemiology of ETEC diarrhea in young Egyptian children, in order to assess the suitability of this population for testing the efcacy of new-generation ETEC vaccines. Our specic objectives were to characterize the descriptive epidemiology of ETEC diarrhea and to evaluate factors associated with the pathogenicity (ability to cause diarrhea) and virulence (ability to cause severe diarrhea) of ETEC in children residing in a eld site near Alexandria, Egypt.

Materials and Methods

Study area and study population. Beginning in November 1993, a cohort of newborns and children !24 months old was assembled in Abees, a periurban agricultural area 10 km southwest of Alexandria, Egypts second largest city. Five Abees villages, each with a population of 1000 persons, were selected for the study. In November 1993, the rst village was enrolled, followed by enroll-

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ment of the second village in January 1994. The third, fourth, and fth villages were subsequently enrolled at 6-month intervals. Enrollment of children in the ve villages was staggered to avoid disruption of the study by too rapid accrual of new subjects. Each village remained under surveillance until the end of the study on 30 September 1995. Enrollment. Just before enrollment, each study village was mapped and its houses were numbered. A house-by-house census was conducted over the ensuing 2 weeks, with collection of demographic and socioeconomic data for all families and individuals. During the next 3 weeks, all children !24 months old were examined by a physician. Newborns (dened as neonates 21 days old) without major congenital anomalies or severe illness were eligible for participation in the study. Older children !24 months old without a clinically important chronic illness were also eligible for enrollment. After the initial enrollment period, eligible newborns in families in the original census continued to be enrolled until the end of the surveillance period. Cohort follow-up. Each child was visited twice weekly until age 36 months or until the end of the study period, whichever occurred rst. At each visit, an interviewer asked the mother about diarrheal symptoms occurring since the previous visit or, in the case of a missed visit, within the last 4 days. If any loose or liquid stools were reported, a fecal specimen was collected (vide infra), mothers were questioned about the severity of the illness, and the child was examined by a study physician. Oral rehydration salts were provided and referral to a treatment facility was made as appropriate. Ongoing monitoring for new births, deaths, and out migrations was done during the follow-up visits. Once each month, all enrolled subjects were asked to give a fecal specimen, which was processed and tested in the same way as specimens collected in association with diarrheal episodes (vide infra). At monthly visits, a history of recent diarrheal symptoms and breast-feeding practice was taken. Collection, transportation, and evaluation of fecal specimens. When any loose or liquid stools were reported during twice-weekly visits, 2 rectal swabs were simultaneously obtained. One was placed in Cary-Blair transport medium for later bacteriologic analysis, and the other was placed in PBS solution for later virologic analysis. In addition, a specimen cup, to be collected the following day, was left with the family to obtain stool for virologic analysis. On the day of collection, all specimens were transported in insulated boxes containing cold packs to the University of Alexandria microbiology laboratory. In the laboratory, rectal swab specimens were evaluated with conventional microbiologic techniques to isolate Shigella, Salmonella, Campylobacter, and Vibrionaceae organisms [4]. In addition, 5 lactose-fermenting colonies morphologically typical of E. coli were picked from MacConkeys agar for later testing by GM1 ELISA for the expression of LT toxin [5] and by inhibition GM1 ELISA for expression of ST enterotoxin [6]. Colonies that were positive for either enterotoxin were tested for expression of colonization factor antigens (CFAs), including CFA/I, CFA/II (CS1, CS2, CS3), CFA/III, and CFA/IV (CS4, CS5, CS6), and for putative colonization factors (PCFs), including PCFO159, PCFO166, CS7, and CS17, using an immunodot blot method that employed monoclonal antibodies against the CFAs and their subtypes [7, 8]. Colonies from diarrhea-associated specimens were tested concurrently

with those from nondiarrheal control specimens. Stool specimens or, in lieu of stools, fecal suspensions in PBS from rectal swab samples were tested for rotavirus antigen by a commercial ELISA kit [9]. Denitions. A diarrheal day was dened as the occurrence of 3 unformed stools (or 1, if bloody) in a 24-h period. For breast-fed infants with nonbloody stools, the mother must also have indicated that there had been an increase in frequency or a decrease in consistency of stools in relation to the normal pattern of defecation. An episode of diarrhea began on the rst diarrheal day after 3 consecutive nondiarrheal days and ended on the last diarrheal day followed by 3 consecutive nondiarrheal days. ETEC diarrhea denoted an episode in which ETEC was isolated from any specimen collected during that episode, regardless of whether other diarrheal pathogens were also isolated. A child without any loose or liquid stools on the day of the monthly survey as well as on the 3 days before and 3 days after the survey dened an asymptomatic control. A control was recognized as infected with ETEC when ETEC was isolated from a rectal swab obtained during the monthly survey, regardless of whether other diarrheal pathogens were also detected. A sanitary latrine was a household latrine that drained into a sealed pit or local sewage system. Breast-feeding was dened as receiving any breast milk, regardless of whether other uids or solids were also part of the diet. A child was considered to have stopped breast-feeding on the date that was half way between the monthly survey in which breast-feeding was rst reported to have stopped and the previous monthly survey. A mother or father was considered to be educated if he or she had received any formal schooling at a government or private school. Socioeconomic status of the family was expressed as a score of 028 based on the type of household (apartment vs. detached home) and the number of expensive items found in the household (washing machine, car/ truck, radio, television, etc.). Poor households were those with scores in the lower tertile of the scale. Assessment of crowding was based on the number of residents per sleeping room and was categorized by tertiles into low (0.5 to!3.5 persons per room), medium (3.5 and !4.75), and high (4.75). The presence of blood in stools was based only on a report by the childs caretaker. Analyses. To calculate incidence rates of ETEC diarrhea for a given time interval, the numerator consisted of episodes beginning during the cited interval, and the denominator was the total persontime of actual follow-up less the duration of all ETEC diarrhea episodes during the interval. Days during episodes of ETEC diarrhea were excluded from the denominator, since the denominator was considered to be the period during which subjects were at risk for new ETEC episodes [10]. Poisson regression models, with use of generalized estimating equations [11, 12] to adjust for nonindependence of recurrent outcomes for individual subjects, were tted to assess the independent contributions of multiple predictors of diarrheal incidence. Only predictors found to be statistically signicant in univariate analyses (P ! .05 ) were included in the multivariate Poisson regression models. Relative rates (RR) were calculated from model coefcients. Ninety-ve percent condence intervals (CIs) and P values for RRs, estimated in a two-tailed fashion, were computed using the SEs of the coefcient. A trend test for the association of diarrhea incidence with age and, separately, with crowding was carried out by adding a single

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ordinal variable for age in years (0, 1, and 2 years of age) or household crowding (low, medium, and high) to a Poisson regression model of diarrhea incidence. A P value for the association was obtained using the Wald statistic [13]. A x2 test was used to compare proportions [14]. To assess ETEC pathogenicity, ETEC isolation rates from children during a diarrheal episode were compared to isolation rates for control children without diarrhea as detected by monthly surveys. The association between isolation of ETEC and the occurrence of diarrhea was expressed as an odds ratio (OR). To avoid detection bias from repeat fecal sampling during the same diarrheal episode, ETEC test results from only the rst fecal specimen associated with an episode were included in the analysis. To minimize the effect of natural anti-ETEC immunity in reducing the association between ETEC isolation and diarrhea, only episodes or control results for a subject up to the rst excretion of any ETEC were included in the analysis. All episode or control results for a subject subsequent to the initial excretion were dropped when calculating numerators, denominators, proportions, and ORs. Apart from these exclusions, no constraints were placed on a subject being repeatedly sampled as a case or a control. To control for confounding, ORs were obtained from a multivariate logistic regression model that used generalized estimating equations to adjust for nonindependence of multiple observations for individual subjects. Confounding variables included statistically signicant univariate variables identied as predictors of ETEC incidence from the previous analysis. To ensure that associations reected concurrent comparisons of episodes and controls, an indicator variable for calendar quarter (3-month intervals) was also tted to the model. P values and 95% CIs for ORs were calculated in a two-tailed fashion from coefcients and SEs of these coefcients. SAS 6.12 was used for all analyses [15].

Results Characteristics of the villages and study cohort. The 5 villages were populated by 5318 persons in 581 households. The majority of heads of households were farmers or shermen. Many households had modern features: 94% had electricity, 71% owned a television set, and 94% were connected to the municipal water system, although piped, potable water was sometimes unavailable. Of adults (18 years old), 25% had some formal education. The presence of a sanitary latrine or toilet was reported by 49% of the households. Refuse containers were found in 42% of households either inside or outside the dwelling. The 182 households of children enrolled in the cohort had similar household characteristics when compared with all other village households. Of 260 children !24 months old identied during the study, 242 (93%) were eligible for study participation. The 242 enrolled children were equally divided by gender. At initiation of follow-up, 143 enrollees (59%) were !1 year of age (50 newborns) and 99 (41%) were 12 years old. Prolonged breast-feeding was common; 50% of children were still receiving breast milk at age 24 months. Over the course of the study, historical information was successfully obtained during 91% of the twice-weekly visits. In

total, 95% of scheduled monthly visits were completed. Rectal swabs, which were used to detect ETEC, were collected for 76% of the 628 reported diarrheal episodes and obtained at 81% of 1898 surveillance visits. Specimens were not collected from other subjects because of either parental refusal to give a specimen or subject absenteeism. Overall incidence of diarrhea. During 216.2 child-years at risk of diarrhea, 628 episodes of diarrhea were detected in the study cohort (table 1). The incidence of diarrheal episodes (per child-year) decreased with increasing age: 4.9 during year 1; 2.5 during year 2; and 1.0 during year 3 of life (P ! .001 for trend). Only 5 (1%) episodes lasted 14 days, and 36 (6%) were described as bloody. Incidence of ETEC diarrhea. ETEC were detected in 125 (20%) fecal specimens associated with diarrheal episodes, for an incidence of 0.6 episodes per child-year at risk (table 1). Sixty-six (53%) ETEC episodes were associated with ST-only strains, 43 (34%) were associated with LT-only strains, and 16 (13%) were associated with strains that produced both LT and ST (LT/ST) enterotoxins. The incidence (episodes per child-year at risk) of ETEC diarrhea exhibited a marked inverse relationship with age: 1.0 in year 1, 0.6 in year 2, and 0.1 in year 3 of life (P ! .001 for trend). Recognized CFAs were identied in 30 (24%) ETEC episodes. CFA/I, CFA/II, or CFA/IV was expressed by 28 (94%) of these isolates; PCFO166 and CS7 were expressed in 1 isolate each. Among ETEC isolates associated with diarrheal episodes, CFA/I, CFA/II, or CFA/IV was expressed by 7 (44%) LT/ST ETEC, 15 (23%) ST-only ETEC, and 6 (14%) LT-only ETEC (P .05, for differences among the 3 enterotoxin phenotypes). One or more non-ETEC enteric copathogens (rotavirus, Shigella, or Campylobacter species) were isolated in only 9 (7%) ETEC-associated diarrheal episodes. Table 2 shows the incidence of ETEC diarrhea and the crude and adjusted RRs for the associations between selected sociodemographic and other features and the rates of ETEC diarrhea. The overall incidence of ETEC diarrhea increased during the warmer months (adjusted RR [RRa] 1.85 ; 95% CI, 1.26 2.72) and was suggestively elevated when a refuse container was located in the home (RR a 1.49; 95% CI, 0.992.25). The presence of a sanitary latrine was associated with a lower inTable 1. Incidence of diarrhea in a cohort of 242 infants and children from Abees, Egypt, November 1993 to September 1995.
Age (months) at follow-up 011 1223 2435 Total
a b c d

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All a diarrhea 4.9 2.5 1.0 2.9 (331) (245) (052) (628)

ETEC toxin phenotypes LT 0.3 0.2 0.0 0.2

b

ST 0.5 0.3 0.1 0.3

LT/ST 0.1 0.1 0.0 0.1

All 1.0 0.6 0.1 0.6 (64) (55) (06) (125)

(21) (21) (01) (43)

(34) (28) (04) (66)

(09) (06) (01) (16)

Incidence/child year (episodes). Includes strains expressing heat-labile toxin (LT) only. Includes strains expressing heat-stable toxin (ST) only. Includes strains expressing LT and ST toxins.

Table 2. Incidence and relative rate (RR) of ETEC diarrhea in Egyptian children, aged 035 months, in Abees, Egypt, by selected features and toxin phenotype, November 1993 to September 1995.
ETEC toxin phenotype All ETEC
a

LT only
b a

ST only
b a

Feature 1.59 (1.11, 2.35) 1 0.97 (0.65, 1.47) 1 1.07 (0.68, 1.69) 1 1.13 (0.72, 1.75) 1 1.41 (0.56, 2.10) 1 0.51 (0.35, 0.77) 1 1.31 (0.80, 1.06) 1.08 (0.62, 1.83) 1 0.77 (0.48, 1.20) 1 2.49 (1.63, 3.83) 1
f e d

Incidence 1.85 (1.26, 2.72) 1 1.10 (0.74, 1.62) 1 1.26 (0.81, 1.96) 1 0.86 (0.55, 1.35) 1 1.49 (0.99, 2.25) 1 0.49 (0.32, 0.74) 1 1.53 (0.96, 2.45) 1.10 (0.63, 1.92) 1 0.83 (0.51, 1.34) 1 1.27 (0.65, 2.45) 1 0.26 (28) 0.14 (15) 0.12 (07) 0.23 (33) 0.18 (13) 0.23 (17) 0.19 (13) 0.95 (0.47, 1.98) 1.21 (0.58, 2.56) 1 0.52 (0.25, 1.16) 1 1.86 (0.95, 3.49) 1
f e

Crude RR (95% CI) Adjusted RR (95% CI) Incidence 0.18 (21) 0.22 (22) 0.21 (23) 0.19 (20) 0.23 (16) 0.18 (27) 0.16 (09) 0.21 (33) 0.27 (28) 0.13 (15) 0.12 (15) 0.31 (28) 0.39 (0.21, 0.72) 1
e

Crude RR (95% CI) Adjusted RR (95% CI) Incidence 0.82 (0.46, 1.52) 1 1.11 (0.61, 2.13) 1 1.28 (0.65, 2.42) 1 0.76 (0.36, 1.52) 1 2.08 (1.11, 3.85) 1
d

Crude RR (95% CI) Adjusted RR (95% CI) 2.16 (1.30, 3.62) 1

e

0.70 (81) 0.44 (44) 1.27 (0.71, 2.25) 0.29 (31) 0.33 (35) 0.32 (22) 0.30 (44) 0.38 (22) 0.28 (43)
d

0.91 ( 0.48, 1.70) 1

0.41 (47) 0.19 (19)

2.58 (1.56, 4.26) 1

0.57 (62) 0.59 (63) 1.55 (0.82, 2.92) 1 0.62 (0.29, 1.30) 1 2.12 (1.15, 3.89) 1

0.88 (0.51, 1.49) 1 1.07 (0.60, 1.89) 1 1.36 (0.79, 2.45) 1

1.01 (0.60, 1.70) 1 1.24 (0.70, 2.17) 1 1.04 (0.58, 1.87) 1

0.61 (42) 0.57 (83)

0.63 (36) 0.56 (87)

0.69 (70) 0.49 (55)

0.30 (31) 0.31 (35)

e

0.97 (0.58, 1.67) 1 0.23 (29) 0.41 (37 ) 0.56 (0.34, 0.95) 1
d

1.04 (0.58, 1.86) 1 0.54 (0.32, 0.93) 1

d

0.41 (52) 0.81 (73)

0.32 (0.19, 0.66) 1

0.67 (48) 0.55 (41) 0.51 (36)

1.24 (0.59, 2.60) 1.40 (0.64, 3.08) 1 0.53 (0.24, 1.16) 1 0.82 (0.28, 2.35) 1

0.38 (27) 0.30 (22) 0.24 (17) 0.22 (13) 0.34 (50) 0.47 (51) 0.14 (15)

1.58 (0.79, 2.97) 1.25 (0.60, 2.42) 1 0.65 (0.36, 1.21) 1 3.36 (1.74, 6.27) 1
f

1.75 (0.90, 3.40) 1.22 (0.61, 2.43) 1 0.76 (0.40, 1.42) 1 1.77 (0.63, 4.97) 1

Season at follow-up Warmer Colder Sex Female Male Socioeconomic status Poor Nonpoor Mothers education Some None Refuse container in home Yes No Sanitary latrine Yes No No. of persons per bedroom 4.75 3.5 to !4.75 0.5 to !3.5 Household heads education Some None Breast-feeding Yes No

0.47 (27) 0.61 (90)

0.82 (90) 0.33 (35)

NOTE. All features except breast-feeding status and season of follow-up were ascertained at baseline. CI, condence interval; LT, heat-labile toxin; ST, heat-stable toxin. a Incidence/child year (episodes). b Adjusted for statistically signicant univariate predictors including age, season of follow-up, presence of sanitary latrine, breast-feeding, refuse container in home, and socioeconomic status (dened in text) using multivariate Poisson regression. c Warmer months are MayOctober; colder months are NovemberApril. d P ! .05. e P ! .01. f P ! .001.

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cidence of ETEC diarrhea (RR a 0.49; 95% CI, 0.320.74). The incidence of ETEC diarrhea increased with each tertile increase in household crowding (!3.5 persons, RR a 1 ; 3.5 to !4.75, RR a 1.10; 4.75, RR a 1.53), but this was not statistically signicant (P .08 for trend, adjusted for covariates). Breast-feeding, gender, lower socioeconomic status, and parental education were not associated with incidence of ETEC diarrhea. ST-only and LT-only ETEC episodes were the only enterotoxin phenotypes separately analyzed, since there were too few LT/ST diarrheal episodes to permit tting of Poisson regression models. The presence of a sanitary latrine was associated with a marked decrease in the incidence of both LT-only (RR a 0.32; 95% CI, 0.190.66) and ST-only ETEC diarrhea (RR a 0.54; 95% CI, 0.320.93). As shown in table 2, the rate of ST-ETEC diarrhea increased notably during warmer months (RR a 2.58; 95% CI, 1.564.26) compared with the rate during cool months. In contrast, no seasonal variations were apparent for the rate of LT-only ETEC diarrhea. Moreover, a refuse container in the home was associated with an increased incidence of LT-only ETEC (RR a 2.12; 95% CI, 1.153.89) but not an increase of ST-only ETEC diarrhea. Pathogenicity of ETEC infections. In table 3, diarrheal cases and nondiarrheal controls are compared for isolation of ETEC by enterotoxin phenotype and by expression of a CFA. Overall, fecal isolation of any ETEC was associated with the presence of diarrheal symptoms (adjusted OR [ORa] 2.02; 95% CI, 1.382.96). When analyzed by enterotoxin phenotype, isolation of ST-only ETEC (OR a 2.26; 95% CI, 1.383.70) was associated with diarrheal symptoms, whereas the associ-

ation for LT-only ETEC was suggestive but not statistically signicant (OR a 1.52; 95% CI, 0.982.37). Further, ST-only ETEC was associated with diarrhea both when a CFA was detected (OR a 3.45; 95% CI, 1.527.84) or not detected (OR a 1.71; 95% CI, 1.032.83), whereas LT-only ETEC was not associated with diarrhea with or without detection of CFA. Still, for both ST-only and LT-only ETEC, the risk of diarrhea increased when a CFA was identied, compared with no identication of a CFA. LT/ST ETEC isolation was not associated with diarrhea, but the small number of LT/ST ETEC isolates limited the interpretability of this analysis. Since age was inversely associated with the incidence of ETEC diarrhea, we also examined whether ETEC pathogenicity (the association of ETEC with diarrhea symptoms) decreased with age. Table 4 shows that the overall association between ETEC isolation and diarrheal symptoms did not change with age for either the crude OR or, when the distribution of outcomes or covariates allowed computation, the adjusted OR. Virulence of ETEC infections. We tested whether ETEC phenotype or CFA expression was associated with disease severity as manifested by a history of vomiting or the presence of 1 objective sign of dehydration. Signs of dehydration included sunken eyes, tented skin, dry mucous membranes, or feeble radial pulse and were recorded after a physical examination by a physician. No associations were detected. For 115 episodes (clinical data were incomplete for 10 episodes), 23% had a history of vomiting and 16% showed 1 sign of dehydration. For 41 LT-, 59 ST-, and 15 LT/ST-ETEC, 17%, 14%, and 20%, respectively, had 1 sign of dehydration (P .79). For

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Table 3. Comparison of diarrhea episodes with controls for frequency of ETEC isolation by toxin and colonization factor antigen (CFA) phenotype, Abees, Egypt, November 1993 to September 1995.
ETEC phenotype LT only CFA CFA ST only CFA CFA LT/ST only CFA CFA All ETEC CFA CFA % episodes 10.7 1.6 8.0 14.5 3.5 10.5 2.6 1.4 1.4 30.1 7.2 21.0 (338) (430) (363) (331) (425) (351) (417) (435) (438) (239) (376) (272)
a

% controls 8.5 0.6 8.0 6.9 1.5 5.6 3.9 2.0 1.7 19.4 4.5 16.7

Crude odds ratio 1.29 2.57 1.00 2.31 2.38 2.00 0.67 0.70 0.79 1.79 1.63 1.32 (0.85, (0.95, (0.64, (1.55, (1.21, (1.29, (0.34, (0.29, (0.32, (1.27, (1.01, (0.93, 1.94) 6.93) 1.54) d 3.43) e 4.70) d 3.08) 1.29) 1.70) 1.96) d 2.52) e 2.63) 1.88)

Adjusted odds ratio 1.52 2.31 1.24 2.26 3.45 1.71 0.63 0.61 0.76 2.02 1.91 1.39 (0.98, (0.83, (0.77, (1.38, (1.52, (1.03, (0.31, (0.25, (0.28, (1.38, (1.09, (0.94, 2.37) 6.46) 1.98) d 3.70) d 7.84) e 2.83) 1.30) 1.53) 2.08) f 2.96) e 3.35) 2.07)

(1037) (1404) (1085) (919) (1322) (1005) (1251) (1375) (1335) (597) (1167) (676)

NOTE. LT, heat-labile toxin; ST, heat-stable toxin; , positive; , negative. a % of total episodes; see text for calculation of total episodes. b % of total controls; see text for calculation of total controls. c Adjusted for study quarter (18 quarters), age (011, 1223, and 2435 months), socioeconomic status (poor/nonpoor), presence of sanitary latrine (yes/no), and breast-feeding (yes/no) using multivariate logistic regression. d P ! .01. e P ! .05. f P ! .001.

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Table 4. Comparison of diarrhea cases with controls for frequency of ETEC isolation by age and season, Abees, Egypt, November 1993 to September 1996.
Age (months) and ETEC strain 011 LT only ST only LT/ST All ETEC 1223 LT only ST only LT/ST All ETEC 2435 LT only ST only LT/ST All ETEC % a episodes 8.9 12.5 2.5 25.2 14.3 17.3 2.7 36.3 4.8 19.1 3.3 57.1 % b controls (337) (302) (388) (226) (453) (383) (542) (253) (247) (234) (321) (118) 1.33 2.40 0.61 1.66 1.68 2.14 0.68 2.39 0.47 4.35 0.89 4.49 Odds ratio Crude (0.69, (1.26, (0.23, (1.01, (0.93, (1.17, (0.23, (1.38, (0.06, (1.27, (0.11, (0.95, 2.56) d 4.56) 1.57) e 2.75) 3.03) e 3.93) 2.00) d 4.16) Adjusted 1.77 2.44 0.84 2.19 1.87 1.85 0.58 2.18 (0.89, (1.01, (0.29, (1.18, (1.01, (0.91, (0.19, (1.28,
c

(191) 6.8 (200) 5.6 (236) 4.1 (155) 16.8 (126) 9.1 (110) 8.9 (151) 3.9 (77) 20.2 (21) (21) (30) (4) 9.7 5.1 3.7 22.9

3.54) e 5.92) 2.48) e 4.06) 3.45) 3.76) 1.73) d 3.71)

e

3.62) 0.43 (0.04, 4.77) e ND 14.96) 7.07) 0.68 (0.07, 6.60) 21.33) ND

NOTE. LT, heat-labile; ST, heat-stable; ND, not done because distribution of outcomes or covariates did not allow computation. a % of total episodes; see text for calculation of total episodes. b % of total controls; see text for calculation of total controls. c Adjusted for age, calender quarter of study (18 quarters), socioeconomic status (poor/nonpoor), presence of sanitary latrine (yes/no), refuse container in home (yes/no), and breast-feeding status (yes/no) using multivariate logistic regression. d P ! .01. e P ! .05.

25 CFA-expressing episodes and 90 episodes without a detected CFA, the proportions with 1 sign of dehydration were 20% and 22%, respectively (P .50). The proportions with a history of vomiting were 32%, 20%, and 13% for LT-, ST-, and LT/ ST-ETEC (P .26), respectively, and emesis was 28% and 22% for CFA-positive and -negative episodes, respectively (P .81). Discussion Our results demonstrate that ETEC infections imposed a major burden of diarrheal morbidity upon this pediatric study population in lower Egypt. The incidence in this cohort is consistent with those reported previously from Egypt [3] and is equal to or higher than those reported in other areas of the developing world [1618]. Potential limitations. Before discussing the implications of these data, it is important to address the potential limitations of our study. As an observational study, our analyses of predictors of ETEC diarrhea can only be considered as suggestive, although the credibility of these analyses was strengthened by our analytical control for relevant confounding variables. Because our case-control comparisons measured excretion rather than exposure to ETEC, the ORs relating ETEC isolation to diarrheal symptoms cannot be strictly interpreted as reecting the relationship between pathogen ingestion and the risk of

diarrhea. Finally, we failed to obtain fecal specimens for nearly one-quarter of the reported diarrhea episodes and one-fth of controls. This missing microbiologic data could have led to underestimates of the true incidence of ETEC diarrhea. It is unlikely, though, that the missing data led to biased estimates of the RRs or ORs, since the proportion of missing fecal specimens was similarly distributed between different levels of categorized variables (e.g., age, seasons, presence of latrines) for episodes and for controls. Similarities and distinctions of ETEC toxin phenotypes. From these data, we identied both similarities and differences in clinical, epidemiologic, and microbiologic features of LT-only versus ST-only ETEC diarrheal episodes. Episodes associated with these 2 toxin types were of similar clinical severity and had similar preventive associations with the presence of a household latrine and a lack of any association with breastfeeding. Moreover, each toxin phenotype exhibited stronger associations with the presence of diarrhea symptoms when the isolate also expressed a CFA. For all ETEC phenotypes combined, the disease incidence was higher in warmer months, as reported in northeastern Brazil [19]. However, the 2 enterotoxin phenotypes in this study were distinguished by a clear difference in seasonality. ST-only ETEC was more common in the warm summer months, and LT-only ETEC exhibited no seasonal variation. The last observation underscores the fact that ETEC represents a heterogenous collection of enteropathogens, and that future epidemiologic studies of ETEC should be designed to consider the distinctive patterns of different toxin phenotypes. Implications for the prevention of ETEC diarrhea. The striking age-related decline in the incidence of ETEC diarrhea observed in our cohort supports the notion that ETEC is naturally immunizing and that age-related acquisition of natural immunity confers important levels of protection against diarrhea due to this infection. These ndings are in accord with studies from Bangladesh and Latin America and with experiments in North American volunteers that demonstrated the protective effects of natural and vaccine-induced immunity to ETEC [1722]. This observation is further supported by our case-control results showing that, in the absence of a previous episode associated with a homologous phenotype, there was no clear association between symptoms of ETEC diarrhea, ETEC isolation, and age. That asymptomatic excretion of ETEC among controls did not decrease with age (table 4) implies that exposure to ETEC is considerable throughout the rst 3 years of life and that natural immunity may not be equally protective against ETEC colonization per se. If immunization with new ETEC vaccines can simulate natural immunity, these vaccines could have a major impact on symptomatic ETEC disease during childhood. Our results also point to the importance of environmental factors in modifying the risk of ETEC diarrhea during childhood. The presence of a refuse container in the home was sug-

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gestively associated with an overall increase in risk of ETEC diarrhea and was statistically related to the risk of LT-only ETEC diarrhea. This association with refuse containers in the home was expected, since refuse containers were frequently uncovered in the study households, providing children with easy access to garbage and serving as an attractant to ies. Conversely, the presence of a sanitary latrine was protectively related to the risk of ETEC diarrhea. Although none of these associations with environmental variables identied specic vehicles or routes of transmission of ETEC to children, the associations strongly suggest that behaviors related to hygiene and cleanliness in the household affect disease incidence. Further studies of specic hygienic behaviors are needed to formulate rational behavioral interventions to reduce risk of disease. It is noteworthy that breast-feeding was not associated with a reduced risk of ETEC diarrhea, since breast-feeding has been associated with a reduced overall risk of diarrhea and of diarrheal mortality [2325]. That breast-feeding might lead to a reduction of the incidence of ETEC diarrhea is supported by observations that secretory IgA antibodies against CFAs and LT ETEC can be identied in colostrum and human milk samples from mothers in developing countries [2629] and that bovine colostrum that is hyperimmune to ETEC virulence factors can protect adult volunteers against an experimental challenge with an ETEC strain expressing homologous virulence factors [30]. Moreover, in a small cohort study in Mexico, breast-feeding was associated with a reduction in the risk of diarrhea after ETEC infection, but not with a reduction in the risk of ETEC infection per se [31]. However, a larger study from Bangladesh found no overall reduction of the incidence of severe ETEC diarrhea during the rst 3 years of life among breast-fed infants and children [32]. That breast-feeding was not associated with a reduction in the risk of ETEC diarrhea in our cohort does not exclude the possibility that breast-feeding, prior to the onset of weaning, may be protective. However, our data suggest that promotion of breast-feeding cannot be relied upon to effect major reductions in the incidence of pediatric ETEC diarrhea in less-developed settings like Egypt. Conclusions. The high incidence of ETEC diarrhea and clear-cut indication of ETEC pathogenicity in our study support the notion that ETEC is a signicant pathogen in this population. Despite the contrasting epidemiologic proles of LT-only and ST-only ETEC illnesses and despite the apparent complex epidemiology of ETEC transmission in this study, our data support the notion that hygienic interventions, such as installation of sanitary latrines and education about proper personal and household hygiene, may exert a salutary preventive effect on ETEC disease in this setting. Moreover, our data suggest that natural immunity may be highly protective against ETEC diarrhea, lending optimism to current attempts to develop an effective ETEC vaccine [3335]. Substantial progress has been made in the development of new-generation oral vac-

cines against ETEC [36], and trials of the clinical efcacy of these vaccines in infants and young children are planned for lower Egypt and for other ETEC-endemic areas.

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