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Impact of Vitamin A on Selected Gastrointestinal Pathogen Infections and Associated Diarrheal Episodes among Children in Mexico City, Mexico

  1. Kurt Z. Long1,
  2. Jose Ignacio Santos4,
  3. Jorge L. Rosado6,
  4. Catalina Lopez-Saucedo5,
  5. Rocio Thompson-Bonilla5,
  6. Maricela Abonce5,
  7. Herbert L. DuPont3,
  8. Ellen Hertzmark2 and
  9. Teresa Estrada-Garcia5
  1. 1Department of Nutrition and
  2. 2Department of Epidemiology, Harvard School of Public Health, Boston, Massachusetts, and
  3. 3University of Texas Medical School and School of Public Health, Houston;
  4. 4Hospital Infantil de Mexico Federico Gómez and
  5. 5Centro de Investigación y de Estudios Avanzados, Mexico City, and
  6. 6Universidad Autónoma de Querétaro, Querétaro, Mexico
  1. Reprints or correspondence: Dr. Kurt Z. Long, Dept. of Nutrition, Harvard School of Public Health, 1633 Tremont St., Boston, MA 02115 (klong{at}hsph.harvard.edu)
 
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Abstract

BackgroundThe overall effect of vitamin A supplementation on diarrheal disease in community trials may result from its effect on specific diarrheal pathogens

MethodsWe conducted a placebo-controlled, double-blind trial of the prophylactic effect of vitamin A on gastrointestinal pathogen infections and clinical symptoms among 188 children in Mexico City, Mexico, from January 1998 to May 1999. Children 6–15 months of age were randomly assigned to receive either a vitamin A supplement (for children <12 months of age, 20,000 international units [IU] of retinol; for children ⩾12 months of age, 45,000 IU of retinol) every 2 months or a placebo and were followed for up to 15 months. Stool samples, collected semimonthly, were screened for enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli (ETEC), Shiga toxin–producing E. coli (STEC), enteroinvasive E. coli (EIEC), and Giardia lamblia

ResultsVitamin A supplementation reduced the prevalence of EPEC infections (rate ratio [RR], 0.52 [95% confidence interval {CI}, 0.23–0.86]) and led to shorter durations of both EPEC and ETEC infections. Supplementation also reduced the prevalence of EPEC-associated diarrhea (RR, 0.41 [95% CI, 0.16–1.00]), EPEC-associated fever (RR, 0.15 [95% CI, 0.02–0.98]), and G. lamblia–associated fever (RR, 0.27 [95% CI, 0.13–0.80]). Finally, children who received vitamin A supplementation had shorter durations of EPEC-associated diarrhea than did children who did not receive supplementation but had longer durations of G. lamblia–associated diarrhea

ConclusionsThese results suggest that the effect of vitamin A supplementation on clinical outcomes may be pathogen dependent

Randomized clinical trials concerned with the benefits that vitamin A supplementation produces with respect to childhood health in developing countries have found no consistent effect of supplementation on the incidence of diarrheal disease [1–3]. These inconsistent findings may partly be a result of differences in the baseline vitamin A status of the study populations. However, these results may also be explained by the fact that diarrheal diseases were conceptualized as a single disease outcome in these analyses. Diarrhea is, in fact, caused by an extensive and diverse group of bacterial, viral, and protozoan pathogens that can have very different patterns of transmission and distinct mechanisms of pathogenesis [4, 5]. The impact of vitamin A supplementation on the pathogen-specific immune response also may vary, with supplementation up-regulating the humoral immune response but down-regulating the cell-mediated immune and inflammatory responses [6, 7]. As a result, conceptualizing diarrheal diseases as a single outcome may be a form of misclassification that biases the association between vitamin A supplementation and diarrheal disease. This misclassification may explain why vitamin A supplementation does not have a consistent effect on the incidence of diarrheal disease but does consistently reduce infant mortality and the severity of diarrheal disease episodes [8, 9]

The cross-regulatory effects of vitamin A supplementation on the immune response led us to hypothesize that supplementation with vitamin A would produce distinct health outcomes for different enteric pathogens: vitamin A supplementation would be associated with reductions in the incidence of noninvasive gastrointestinal pathogens, for which a humoral immune response is protective; would have no effect on invasive infections, for which a cell-mediated response is protective; and would be associated with reductions in the onset of clinical symptoms, because of the down-regulation of the inflammatory response. To address these hypotheses, we conducted a randomized, placebo-controlled, double-blind trial of the impact of vitamin A supplementation on diarrheal Escherichia coli and Giardia infections and associated diarrheal episodes among children from marginalized periurban communities of Mexico City, Mexico. These agents were sought because previous work had shown them to be important causes of bacterial and parasite diarrhea in Mexico [10, 11]. Clarification of how vitamin A supplementation may modify pathogen-specific outcomes may allow the development of vitamin A supplementation strategies with increased cost-effectiveness that are designed specifically for such periurban communities. Greater reductions in the prevalence and variety of infection with gastrointestinal pathogens have occurred in such urban communities in Mexico relative to rural communities, as a result of more-widespread implementation of water systems and sanitary facilities

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Subjects, Materials, and Methods

Study populationCommunity health workers conducted a census of all children <2 years of age living in 9 neighborhoods (colonias) within the community of La Magdalena Atlicpac, which is located along the eastern perimeter of Mexico City. These colonias are part of the ring of marginalized communities that surround the periphery of Mexico City and that are made up of families that have migrated to the city from impoverished regions of Mexico within the last 20 years

Mothers of all children from 5–15 months of age in the community were invited to participate in the study, as described elsewhere [12]. Children with diseases that cause immunosuppression or with any congenital or acquired alteration of the digestive tract that could alter the absorption of micronutrients were excluded from the study. In practice, this was primarily chronic diarrhea. Children who were taking vitamin supplements were also excluded from the study. For the overall study, 200 children living in this community were identified and enrolled over a 10-month period, after their parents consented to their participation

Study designEach child, once enrolled, was randomly assigned to receive vitamin A or a placebo, by use of a randomized sequence generated from a random number table by an epidemiologist at the National Institute of Public Health. Children <12 month of age assigned to the vitamin A group were administered a solution containing 20,000 IU of retinol (3.3 IU equals 1 retinol activity equivalent [micrograms]) at baseline and subsequently every 2 months until the end of the study, and children ⩾12 months of age received a solution containing 45,000 IU of retinol. Testing and preparation of the placebo and vitamin A solutions were done by personnel at the National Institute of Nutrition, to assure that the solutions were similar in taste, viscosity, and color and to assure that the field personnel and principal investigator were unaware of the regimens. The field team was in charge of administering the supplements and placebos to children from identical opaque plastic droplet bottles

During the baseline visit, information was collected regarding the number of household members; their ages, education levels, and income; household construction materials; the source and quality of the household water; the type of household sanitation facilities; and household possessions. Information was also collected during the first visit to determine the child’s feeding and weaning patterns as well as morbidity experience during the previous 2 weeks. A previously validated questionnaire was used in the collection of these data by project personnel who had received training in its application

Recruited children were then followed prospectively for up to 15 months, during which time households were visited twice a week. At each visit, mothers or caregivers were interviewed to determine the presence of the following symptoms: diarrhea, the number and consistency of evacuations, the presence of blood and mucus in stool, fever, cough, and respiratory difficulty. A stool sample was collected twice a month for healthy children, and up to 3 stool samples were collected during the week after a diarrheal episode. Project supervisors accompanied ∼5% of all household visits, to assure the quality of data collection. Children were referred to the study physician for diagnosis and treatment when the field-worker or caregiver was concerned about the child’s health status

Identification and characterization of diarrheal pathogensStool samples were plated onto Salmonella-Shigella MacConkey, and MacConkey-tellurite agars for the identification of Salmonella species, Shigella species, and E. coli [13]. The presence of ova and parasites in stool were determined by the Kato-Katz technique, to identify gastrointestinal infections with Entamoeba histolytica and G. lamblia [14]. Five lactose-fermenting colonies with a morphological appearance resembling that of E. coli (when present) were selected from the MacConkey agar plates and speciated biochemically. Diarrheagenic E. coli were characterized by a single multiplex technique (described elsewhere [15]) that detects the following pathogenic genes: heat-stable and heat-labile enterotoxins (st and lt) for enterotoxigenic E. coli (ETEC); intimin (eaeA) and bundle-forming pilus (bfp) for enteropathogenic E. coli (EPEC); Shiga toxin 1 and 2 (stx1 and stx2) and intimin (eaeA) for Shiga toxin–producing E. coli (STEC); and invasive-associated loci (ia1) for enteroinvasive E. coli (EIEC). STEC isolated from children were further characterized by expression of the O157 lipopolysaccaride antigen and enterohemolysine gene (hlyA), by use of a latex particle agglutination kit (Oxoid Limited)

Outcome definitionsThe primary end points for the study were the prevalence and duration of infections with G. lamblia, E. histolytica and diarrheagenic E. coli among children and the incidence and duration of the diarrhea and fever episodes associated with these infections. An infection with G. lamblia or E. histolytica was defined as a stool sample that was positive for the given parasite. Infection with any of the specific diarrheagenic E. coli was defined as any lactose-positive bacteria isolated from stool that had the pathogenic genes for the respective pathogens. Durations of pathogen infections were defined as encompassing the time between the first and last sequentially collected stool samples that were positive for the pathogen. A single positive stool sample was assigned a duration of 1 day. Episodes of fever or diarrhea were defined as pathogen associated if the episode occurred within 7 days before or after a positive stool sample. A diarrheal episode was defined as the mother’s reporting of symptoms in the child and was confirmed by the passage of 3 or more liquid stools in 1 day. The duration of a diarrheal episode was defined as encompassing the sequential days of reported diarrheal symptoms in the child followed by 3 or more symptom-free days

Statistical analysisRate ratios (RRs) for pathogen prevalence among all children randomized to the 2 groups were first estimated by fitting Poisson regression models to counts of stool samples that were positive for pathogens. The duration (in weeks) of pathogen infections was then compared between the 2 groups by use of Weibull parametric regression survival-time models estimated with maximum likelihood procedures. Infection duration was defined as the time from first positive stool sample to the first negative stool sample among sequentially collected samples. RRs for the incidence of pathogen-associated diarrhea and fever were estimated by fitting Poisson regression models to counts of diarrheal and fever episodes among children with pathogen-positive stool samples [16]. Finally, the duration (in days) of pathogen-specific diarrheal episodes was compared between the 2 groups by use of Weibull parametric regression survival-time models. For each outcome analyzed, separate variables for coinfections with other specific pathogens was included in the model, to control for the modifying effect of these coinfections on the association between vitamin A supplementation and the analyzed outcome. Coinfections were defined as the presence of another pathogen in the analyzed stool sample or in any stool sample collected 2 weeks before that positive stool sample. The age and breast-feeding status of each child were also included in these analyses. Statistical significance was set at P<.05, and P<.1 was used for interactions. The GENMOD and LIFEREG procedures from SAS software (version 8.2; SAS Institute) were used in the Poisson regression and hazard analyses, respectively

Sample sizes were calculated on the basis of the assumptions that the study population has a diarrheal disease rate of 3 episodes/child/year and that the vitamin A supplement would reduce this rate by ∼20%. This effect approximated the reduction in diarrhea incidence reported by Barreto et al. in their vitamin A trial in Brazil [17]. It was calculated that a sample size of 100 persons/group was required to detect a 20% reduction between the control and treatment groups with a power of 80%, a 95% significance level, and an expected loss to follow-up of 20%. This calculation allowed for repeated measurements of the outcome and a correlation between measurements at different time points of 0.7 [18]

Ethics approvalThe present study was approved by the ethical review committees of the National Center for the Health of Infants and Adolescents of Mexico and of the Harvard School of Public Health

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Results

Twelve of the 200 children who were initially recruited at baseline were lost to follow-up or were excluded; 188 remained in the study and were followed for up to 15 months, 93 in vitamin A group and 95 in the placebo group (figure 1). The sociodemographic characteristics of the study children and households were similar between the children who received vitamin A and those who received placebo (table 1). Approximately 47%–50% of households had no access to piped water, and ∼65%–70% had no indoor toilets; 34%–40% of children were stunted at baseline. The duration of follow-up for the children in the 2 groups was not significantly different (for the vitamin A group, a mean±SE of 11.5±5.5 months; for the placebo group, a mean±SE of 11.9±5.6 months; P=.56). Approximately 1035 of the planned 1120 doses of vitamin A or placebo were administered by the project personnel to the study children, for a compliance rate of 92%

Figure 1
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Figure 1

Trial profile

Figure 2
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Figure 2

Cumulative hazards for duration of pathogen infections and pathogen-associated diarrheal episodes in the vitamin A supplementation (dashed lines) and placebo (solid lines) groups. A Enteropathogenic Escherichia coli (EPEC) infections. B Enterotoxigenic E. coli (ETEC) infections. C EPEC-associated diarrhea. D, Giardia lamblia–associated diarrhea. Shorter infections and episodes are associated with higher cumulative hazards

Table 1
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Table 1

Sociodemographic characteristics of the study children and households

EPEC, G. lamblia and ETEC were the pathogens most frequently isolated from stool samples (table 2). STEC, EIEC, and E. histolytica were isolated far less frequently. In the Poisson regression analysis, vitamin A supplementation was found to be associated with reductions in EPEC infections (RR, 0.52 [95% confidence interval {CI}, 0.23–0.86]) (table 2). There was no association between supplementation and the prevalence of infections with ETEC and G. lamblia. No analysis was done for the remaining pathogens, because of the small number of isolates. The effect of vitamin A supplementation on the duration of pathogen infections was determined by fitting Weibull parametric regression models to these durations. In this analysis, the children who received vitamin A supplementation had significantly reduced durations of EPEC infections, compared with the children in the placebo group (mean±SE, 1.56±0.05 and 2.96±0.11 weeks, respectively), and significantly reduced durations of ETEC infections (mean±SE, 1.76±0.16 and 2.75±0.22 weeks, respectively) (table 3). The greater increase in the cumulative hazard among the children who received vitamin A supplementation relative to the children in the placebo group for EPEC and ETEC infections indicated that there is a greater probability that the infection will end in the former group than in the latter group (figure 2)

Table 2
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Table 2

Impact of vitamin A supplementation on the prevalence of gastrointestinal pathogen infections

Table 3
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Table 3

Impact of vitamin A supplementation on duration of pathogen infections

Approximately 26%, 23%, and 15% of infections with EPEC, ETEC, and G. lamblia were associated with diarrheal episodes, respectively. Diarrhea was also associated with more than half of the 9 EIEC isolates. Children who received vitamin A supplementation had a significantly reduced prevalence of EPEC-associated diarrheal disease (RR, 0.41 [95% CI, 0.11–1.00]) (table 4) and a marginally significant reduced prevalence of G. lamblia–associated diarrhea (RR, 0.25 [95% CI, 0.05–1.17]). Vitamin A supplementation had no effect on ETEC-associated diarrhea. There were too few episodes of diarrhea associated with the remaining pathogens to perform the analysis

Table 4
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Table 4

Impact of vitamin A supplementation on the prevalence of pathogen-associated diarrhea and fever

Approximately 10%–12% of infections with EPEC, ETEC, STEC, EIEC, and G. lamblia were associated with fever. Approximately 31%, 40%, and 54% of these fever episodes co-occurred with diarrheal symptoms among EPEC-, ETEC-, and G. lamblia–infected children, respectively. Vitamin A supplementation was associated with important reductions in the prevalence of fever among EPEC-infected (RR, 0.15 [95% CI, 0.02–0.98]) and G. lamblia–infected (RR, 0.27 [95% CI 0.13–0.08]) children (table 4). Vitamin A supplementation had no effect on ETEC-associated fever

Mean durations of diarrheal episodes were 3.40, 3.38, and 3.27 days for EPEC-, ETEC-, and G. lamblia–associated diarrhea, respectively. When Weibull parametric regression models were fit to durations of EPEC-associated diarrhea, the mean duration was found to be significantly shorter among the children who received vitamin A supplementation than among the children who receive placebo (mean±SE, 1.74±0.19 and 3.41±0.30 days, respectively) (table 5 and figure 2). The mean duration of G. lamblia–associated diarrhea was significantly longer among the children who received vitamin A supplementation (mean±SE, 6.17±1.35 and 2.93±0.34 days, respectively). Supplementation was not associated with any alterations in the duration of ETEC-associated diarrheal episodes

Table 5
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Table 5

Impact of vitamin A supplementation on the duration of pathogen-specific diarrheal episodes

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Discussion

We have found that the effect of vitamin A supplementation on clinical outcomes is pathogen specific. Supplementation was associated with reductions in the prevalence of EPEC infections and reductions in the durations of EPEC and ETEC infections. Children infected with EPEC and G. lamblia who received vitamin A supplementation had fewer diarrheal and fever episodes than did the children in the placebo group. They also had reduced durations of EPEC-associated diarrhea but increased durations of G. lamblia–associated diarrhea. To our knowledge, these results are the first to indicate that vitamin A supplementation has such differential effects on specific gastrointestinal pathogens, given that a majority of vitamin A trials have focused on general diarrhea as an outcome

A number of studies concerned with the impact of vitamin A on overall diarrhea have reported that vitamin A supplementation is associated with reductions in the incidence of more-severe episodes [17] and reductions in clinical visits [9]. However, other studies have reported that supplementation has no effect [2, 3] or even a negative effect on diarrhea [19]. A recently published meta-analysis has reported that, when the results of these and other trials are pooled together, vitamin A supplementation has no effect on diarrhea [20]. We have argued previously that these inconsistent findings may reflect a differential effect of vitamin A supplementation on diverse groups of diarrheal pathogens, the prevalence and makeup of which may vary in different community settings [21]. It is difficult to compare these results with our present findings, because we have analyzed pathogen-specific outcomes as end points and not general diarrhea

The regulatory effects of vitamin A on pathogen-specific immune responses may also contribute to these previous inconsistent findings and, thus, may provide insight into our findings here. Vitamin A and other retinoids can induce important changes in the differentiation and activation of monocyte populations that are involved in innate immunity and the expression of cytokines by these cell populations [22, 23]. Vitamin A deficiency is associated with reduced cytotoxic activity of natural killer spleen cells [24], reduced T lymphocyte proliferation and functionality [25], a reduced response against specific antigens [26, 27], and reduced homing of T cells to the gut [28]. Deficiency can also lead to a polarized Th1 response [29], whereas retinoid treatment can up-regulate a Th2 response [6]

The reduction in the prevalence and duration of EPEC-associated diarrheal and fever symptoms we observed among the children who received vitamin A supplementation is consistent with the known anti-inflammatory effects of vitamin A. EPEC colonization and attachment to epithelial cells in the host leads to inflammation and pathological disease as a result of the induction of the inflammatory cytokine response [30, 31]. We have previously reported that vitamin A supplementation may reduce fecal concentrations of the proinflammatory cytokines interleukin-6 and interferon (IFN)–γ after EPEC infections and diarrheal episodes [12]. This anti-inflammatory effect of retinol may reduce the onset and duration of diarrhea and fever associated with EPEC and G. lamblia infections

The decreased prevalence and shorter duration of EPEC infections observed among the children who received vitamin A supplementation was unexpected. A protective Th1 cellular mucosal immune response is induced by EPEC infections in rabbits and by the homologous mouse pathogen Citrobacter rodentium [32, 33]. Vitamin A down-regulates this response and up-regulates the Th2 response, which would suggest that supplementation should have either no effect or a negative effect on EPEC infections [6]. However, we recently reported elsewhere that vitamin A supplementation is associated with down-regulation of the IFN-γ response among children who receive supplementation after EPEC infections [12]. Additionally, Geissmann et al. have reported that the activation of immature dendritic cells or the induction of apoptosis by retinoids depends on costimulation with TNF-α [22]. These findings suggest that the presence or absence of inflammatory signals may induce vitamin A to produce opposite effects on the immune response and on pathogen-specific clinical outcomes

It is also unclear why vitamin A supplementation reduces the durations of ETEC infections and G. lamblia–associated fever but increases the duration of G. lamblia–associated diarrhea. ETEC infections induce an early Th1 response but late Th2 response [34]. The noninvasive character of ETEC infections and the protection provided by anti-enterotoxin intestinal IgA suggest that the late Th2 response is more important in providing protection. A number of studies have reported that IgA antibodies and Th2 cytokines are important in eradicating Giardia muris or G. lamblia infection [35]. However, it is not clear whether a Th2 response is more protective against Giardia infection, because a strong Th1 response is also important in reducing G. lamblia intestinal trophozoite numbers and fecal cyst counts [36, 37]. The increase in the duration of G. lamblia–associated diarrhea among the children who received vitamin A supplementation may be due to a decreased ability to reduce the number of trophozoites and cysts as a result of vitamin A down-regulating this response. Overall, there is need for further clarifying the role that these different immune-response components play in outcomes associated with ETEC and G. lamblia infections and for clarifying how the regulatory effect of vitamin A on these responses modifies these outcomes

One limitation of the present study is that we have no indicators of the children’s vitamin A status, because blood samples were not collected from children. Differences in the initial status of study children could determine the effectiveness of supplementation, with deficient children benefiting more and nondeficient children benefiting less. A probabilistic national survey conducted in Mexico has shown that overt vitamin A deficiency (<10 μg/dL) and subclinical deficiency (10–20 μg/dL) among children living in Mexico City is minimal but that low levels of serum retinol are prevalent [38]. A second limitation of the study was our nonrandom selection of children for the study, which may have introduced biases into our results. Finally, we have not addressed what effect vitamin A supplementation has on infections with viruses such as rotavirus, which are important causes of diarrheal disease among young children worldwide. Efforts to clarify the effect of vitamin A on these infections could clarify the overall effect of vitamin A supplementation on the burden of diarrheal disease in pediatric populations

If these findings can be confirmed and extended to include other pathogens, they may explain why vitamin A has not been found to have a consistent effect on diarrheal disease in studies conducted in different parts of the world [2, 3, 17, 20, 39]. The contrasting pathogen-specific effects of vitamin A supplementation may produce diverse effects in different community settings, where there is considerable variation in the prevalence of different types of diarrheal pathogens. The greatest differences in effects may be produced among communities that differ with respect to such factors as water and sanitation facilities and maternal education, each of which can modify pathogen transmission [40]. In the future, one obvious strategy to enhance the effectiveness of supplementation programs would be to use such community hygiene measures as markers of pathogen prevalence to target subgroups of children

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Acknowledgment

We thank Matilde Juarez of the Clinical Microbiological Laboratory, Hospital La Perla, Secretary of Health, Cda Netzahuaycoytl, for her assistance in the laboratory

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Footnotes

  • ↵Presented in part: annual meeting of Experimental Biology, San Diego, 2–6 April, 2005 (abstract 848.8)

    Potential conflicts of interest: none reported

    Financial support: Instituto de Nutricion Danone, CONACYT (National Council of Science and Technology of Mexico); National Institutes of Health (grant K01 DK06142-02)

  • Received April 11, 2006.
  • Accepted July 8, 2006.
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  1. J Infect Dis. (2006) 194 (9): 1217-1225. doi: 10.1086/508292
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