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EPIDEMIOLOGY OF SHIGELLA-ASSOCIATED DIARRHEA IN RURAL EGYPTIAN CHILDREN

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  • 1 U.S. Naval Medical Research Unit No. 3, Cairo, Egypt; U.S. Naval Research Unit No. 2, Jakarta, Indonesia; National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland; Abu Homos Field Research and Training Center, Egyptian Ministry of Health and Population, Beheira, Egypt; International Vaccine Institute, Seoul, Korea

During the period from February 1995 to February 1998, the epidemiology of Shigella diarrhea was studied among children less than three years of age residing in Egypt’s Nile Delta. Children were visited twice a week and a stool sample was collected from any of them with diarrhea. The incidence of Shigella-associated diarrhea was 0.2 episodes/child-year, with S. flexneri being the most common serogroup isolated (55% of Shigella episodes). Younger age and the warm months increased the risk of developing Shigella-associated diarrhea, while breastfeeding was protective. Children with Shigella were ill for a mean of four days and passed a mean of six stools per day. Common symptoms included fever (35%), vomiting (19%), and dehydration (16%). Dysentery, however, was unusual, occurring in only 11% of the cases. In conclusion, Shigella-associated diarrhea remains relatively common in Egyptian children and supports the need for additional control measures including vaccine development.

INTRODUCTION

Shigella infections remain a global public health concern, causing diarrhea in both the developing and developed regions.1 Shigellosis is common among children less than five years of age in developing countries and in persons who travel from industrialized to less developed countries.2,3 In the developing world, it is estimated that 113 million episodes of shigellosis occur annually, resulting in more than 400,000 deaths.4 Industrialized countries also report outbreaks of Shigella infections among high-risk populations such as children attending day care,5–7 persons with human immunodeficiency virus/acquired immunodeficiency syndrome,8–11 and inmates of custodial institutions.12 Prevention of Shigella infections has proven difficult, mainly due to the low inoculum needed to produce disease13,14 and inadequate empirical therapy options secondary to antimicrobial resistance.15–18 The World Health Organization has called for the development of candidate vaccines against Shigella.19 In considering the efficacy of a Shigella vaccine, it is necessary to have reliable estimates of the burden of disease in targeted endemic areas, including age-specific incidence data, risk factors, and the distribution of the various serogroups and subtypes of Shigella-causing infection.

In Egypt, limited data exists regarding Shigella-associated diarrhea, with the last population-based study20 of Shigella conducted more than 15 years ago. Additionally, Zaki and others20 did not fully characterize the Shigella isolates with respect to serogroup. Therefore, this study was performed and the results of a three-year longitudinal study of diarrheal diseases in children living in the Nile River Delta in Egypt are reported.

METHODS

Study population and surveillance.

Details about the study population, surveillance methodologies, specimen collection, and transport and laboratory processing have been previously reported by Naficy and others.21,22 Briefly, a prospective cohort study was conducted in two villages in Abu Homos, Egypt, a rural agricultural district located in the Nile Delta, 40 km southeast of Alexandria. Children less than 24 months of age and all newborns, without congenital abnormalities or a history of hospitalization, were identified and recruited for study enrollment. Children remained in the cohort until they reached 36 months of age or February 1998, whichever came first. Each enrolled child was visited at home twice a week by a trained field worker, and a standardized questionnaire was administered to the caregiver. In addition, a determination was made regarding any diarrheal symptoms since the previous visit. Those children reported to have loose or liquid stools had a rectal swab and fresh stool specimen collected. The rectal swab was immediately placed in Cary-Blair (CB) transport media and the stool specimen placed in a plastic capped container and both were refrigerated and subsequently transport to the Naval Medical Research Unit No. 3. For the purpose of case-control comparison, cross-sectional surveys of all enrolled children were conducted at two-month intervals over the three-year period of the study. In these surveys, rectal swabs and stool specimens were collected from all the children regardless of their diarrheal symptoms.

A diarrheal day was defined as the occurrence of ≥3 unformed stools (or ≥1, if bloody) in a 24-hour period. For breastfed infants with non-bloody 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 first diarrheal day after three or more consecutive non-diarrheal days and ended on the last diarrheal day, followed by three consecutive non-diarrheal days. Shigella diarrhea denoted an episode in which Shigella was isolated from any specimen collected during that episode of diarrhea, regardless of whether other pathogens were also isolated from the stool. Shigella infection was considered to be asymptomatic whenever diarrhea did not occur within seven days before or after the isolation of Shigella from feces. The presence of blood in stools was based on the report of the child’s care-giver.

A child was classified as breast-fed if breast milk constituted any portion of the child’s diet. A mother was considered to be educated if she received any formal schooling. Using our previously described methods,23 socioeconomic status of the family was expressed as a score of 0–28, based on the type of housing (apartment versus detached home) and the number of amenities belonging to the family (washing machine, car, television, etc.). Poor households were classified as those with scores in the lower third of the scale. Assessment of crowding was based on the number of residents per sleeping room.

Laboratory evaluation.

Standard microbiology laboratory techniques were used to isolate and identify Shigella, Salmonella, Campylobacter, Escherichia coli, and Vibrio sp. from the rectal swab in CB media, while the stool sample in buffered-glycerol-saline was cultured only on Salmonella-Shigella plates.24–27 Shigella serogroups and serotypes were determined using standard commercially available antisera (Difco Laboratories, Detroit, MI),28 and S. flexneri were ascertained using a panel of monoclonal antibody reagents that were kindly provided by Nils Carlin (Swedish Biological Laboratories, Vaccin, Stockholm, Sweden). Rotavirus testing was performed on the frozen stool specimens as previously described.21

Statistical analyses.

Incidence rates were calculated by dividing the number of diarrhea episodes or the number of Shigella-associated episodes by child-years at risk of follow-up. To identify variables predictive of Shigella-associated diarrhea, the strength of association between the dependent and independent variables was computed using relative rates. Crude relative rates were obtained by adding a single independent variable to a Poisson regression model. To adjust for confounding among variables, a Poisson regression model was fit by simultaneously adding all independent variables to a single multivariate model. Since repeated measures on the same child are likely correlated, generalized estimating equations (GEEs) were used in the multivariate model. Both crude and adjusted relative risks (aRRs) were obtained from these models by exponentiating the model coefficients. Ninety-five percent confidence intervals (CIs) were calculated using model coefficients and empirical standard errors and exponentiating those results.

The virulence of Shigella by species was assessed by comparing the frequency distribution of each sign or symptom (i.e., vomiting, bloody stools, fever, etc.) between S. flexneri, S. sonnei, and S. dysenteriae using a chi-square test. Too few episodes of S. boydii or episodes with more than one Shigella sp. were detected to include these groups in the analysis.

To evaluate pathogenicity, a case-control analysis was performed to examine the association between the occurrence of diarrheal symptoms and excretion of Shigella. To minimize the effect of prior infections with Shigella and potential immunity to subsequent infections with the organism, analysis was restricted to the first Shigella infection, as either a case or control experienced by the subject since beginning enrollment in the study. Cases were defined as study subjects with Shigella diarrhea detected during the twice a week home visits. Controls were defined as children without diarrhea who had cultures performed on fecal samples obtained during the every-other-month routine surveys. Since the controls had a single fecal specimen collected at each visit, only the microbiologic result of the first fecal specimen from each episode was included in the analysis to avoid detection bias.

To identify signs or symptoms related to Shigella-associated diarrhea, a second case-control analysis was conducted. Cases were children with Shigella-associated diarrhea without co-pathogens and controls were children with diarrhea not associated with Shigella. The strength of the association between Shigella infection and independent variables was measured using crude and adjusted odds ratios (aORs). Crude ratios were obtained by fitting a single independent variable into the logistic regression model. To control for confounding between variables, multivariate models were fit by entering all independent variables to the regression model. As with the previous multivariate models, GEEs were also used to adjust for the correlation between repeated measures on the same individual. Parameter estimates from both the crude and multivariate models were used to calculate odds ratios and 95% CIs and obtain P values. All statistical tests were two-tailed. P values <0.05 were considered statistically significant. All statistical tests were completed using SAS version 6.12 (SAS Institute, Cary, NC).

The study was reviewed and approved by the Naval Medical Research Unit No. 3 Institutional Review Board. Before initiation of the study, the project was explained in detail to the parents or guardians of all the children participating in the study and informed consent was obtained before a child was enrolled in the study.

RESULTS

Incidence and disease burden.

Of 2,566 persons distributed among 272 houses across the two villages, 397 children, 205 of whom were boys (52%), were enrolled. Of the total study population, 186 children (47%) from birth to 24 months of age were enrolled at study initiation with the other 211 (53%) enrolled as newborns over the course of the study. Twenty-three children were lost to follow-up during the observation period. Thirteen children died, but none of these deaths were related to Shigella-associated illness, and 10 subjects out-migrated.

During the study period, 73,507 twice a week household surveillance visits were scheduled and 69,893 (95%) were successfully completed. A rectal swab was collected from every child reported to have loose or liquid stools at the time of the household visit. From the every two-month cross-sectional survey, a study questionnaire was completed and rectal swabs were collected from 99% of the scheduled visits. From the twice a week visits, 3,477 episodes of diarrhea were reported during the study period, with an incidence of 5.5 episodes of diarrhea per child per year.

Shigella was the third most common pathogen identified (0.2 episodes per child-year), behind enterotoxigenic E. coli (ETEC) (1.4 episode per child-year) and Campylobacter (0.6 episode per child-year) and ahead of rotavirus (0.17 episodes per child-year). Shigella was isolated from 134 episodes of diarrhea that occurred in 101 children (61% boys), giving an incidence of 0.2 cases per child-year. Twenty children had two separate episodes of Shigella-associated diarrhea, five children had three episodes, and one child had four episodes. A co-pathogen was isolated in 15 of the 134 episodes of Shigella. Campylobacter was the sole co-pathogen in nine cases, while enterotoxigenic Escherichia coli or rotavirus were isolated in addition to Shigella and Campylobacter in five cases and one case, respectively. The incidence of Shigella infection varied significantly from year to year of the study. Incidence per child-year of follow up was highest during the first year (0.5 episodes per child-year), compared with 0.05 episodes per child-year and 0.1 episodes per child-year during the second and third years, respectively.

Shigella was less frequently isolated from the stool of children less than six months old with diarrhea. The incidence of Shigella-associated diarrhea progressively increased after six months of age, peaking during the second year of life, followed by a steady decrease in incidence in children more than two years of age (Table 1).

Shigella flexneri was the most common serogroup identified, found in 55% of the 134 episodes, followed by S. sonnei (22%), S. dysenteriae (19%), and S. boydii (2%) (Table 1). In one episode, both S. flexneri and S. dysenteriae were isolated from the stool and were classified as a mixed infection (Table 1). Further characterization of the 134 episodes found that S. flexneri type 2a was the most common overall isolate of Shigella, as well as the most common sub-type of S. flexneri, while all S. dysenteriae were type 2.

Risk factors.

Table 2 lists the associations between several risk factors and symptomatic Shigella infection, with only two variables being statistically associated with disease: age and season. Compared with children 24–35 months old (reference group), the highest risk was noted among children 12–23 months old (aRR = 7.0, 95% CI = 1.7–29.1), followed by infants (aRR = 4.7, 95% CI = 1.1–21.3) (Table 2). Also, Shigella-associated diarrhea was more likely during warmer (May through October) (aRR = 2.9, 95% = CI 1.5–5.6) than cooler weather. The risk of Shigella-associated diarrhea was not related to village of residence, maternal education, household crowding, socioeconomic status, or hygiene factors such as source of water or presence of a household latrine. However, breastfeeding in the first year of life significantly decreased the incidence of Shigella-associated diarrhea among study children (aRR = 0.4, 95% CI = 0.2–0.8) (Table 2).

Clinical presentation of Shigella infection.

Analysis of Shigella virulence (Table 3) was limited to those 119 cases where Shigella was the sole pathogen isolated from the specimen of diarrhea. Fever was the symptom most commonly reported in association with Shigella (35% of cases), followed by vomiting (19%). Bloody diarrhea, a classic symptom of shigellosis, was reported by only 11% of the mothers whose children were found to have Shigella-associated diarrhea. Sixteen percent of children were diagnosed with severe dehydration. The mean duration of illness was four days and 23% of the children were ill for five days. Children averaged six stools per day at the peak of their illness, but 24% of the children had eight or more stools per day. Of the five symptoms examined, only two variables, visible blood (aOR = 2.3, 95% CI = 1.3–3.8) and ill greater than three days (aOR = 1.4, 95% CI = 1.0–2.0), were predictive of symptomatic infection with Shigella (Table 4).

Pathogenicity of Shigella infection.

A case-control analysis was performed to determine whether there was an association between isolation of Shigella from the stool and the presence of diarrheal symptoms. The isolation of Shigella from the stool was significantly associated with symptomatic infection (aOR = 1.8, 95% CI = 1.2–2.7). Even after adjustments for age, sex, seasonality and breastfeeding, the significant association with symptomatic infection remained although lower (aOR = 1.2, 95% CI = 1.0–1.6). Additionally, the association with symptomatic disease varied by serogroup, particularly pronounced for S. dysenteriae (aOR = 1.8, 95% CI = 1.1–3.1) (Table 5). While isolation of Shigella from the stool on the whole was associated with symptomatic disease, it was not universal since 33 of the 116 isolates of Shigella (28%) were cultured from control children. All serogroups were isolated from control children, but 73% of the isolates from control children were S. flexneri.

DISCUSSION

In this prospective, community-based study, we were able to demonstrate that Shigella was an important cause of diarrhea among children in the Nile River Delta of Egypt. With an incidence of 0.2 episodes per child-year, Shigella, behind ETEC and Campylobacter, was the third most common cause of bacterial diarrhea and more common than rotavirus-associated diarrhea in the current cohort of children. While numerous studies have demonstrated Shigella to be a common cause of diarrhea among young children living in developing nations, most of the studies have been hospital-based, which potentially detect only the more severe cases of diarrhea, and thus may underestimate the true incidence of Shigella-associated diarrhea.29–39 Compared with the only other community-based study of Shigella in Egyptian children,20 Shigella was isolated nearly three times more frequently in the present study. Unlike the report by Zaki and others,20 we demonstrated significant yearly variations in the annual incidence of shigellosis during the three years of our study. The most dramatic variation occurred during the first year of our study, with isolation rates of Shigella being 10 and 4 times the rates they were during the second and third years, respectively. If the data from the first year of our study was excluded, the isolation rate of Shigella is comparable to that reported by Zaki and others,20 suggesting the first year isolation rate in our study may have been due to an outbreak. Further evidence that an epidemic of Shigella occurred during the first year of the study is that neither the total rate of diarrhea nor the specific rate of ETEC- and Campylobacter-associated diarrhea varied significantly over the study period.

We found that S. flexneri was the most common serogroup and S. flexneri 2a the most common subtype. The next most common serogroup was S. sonnei, the most commonly reported Shigella serogroup from industrialized countries.4 Thus, development of a polyvalent vaccine that covers strains of S. flexneri and S. sonnei would be predicted to provide protection against 78% of Shigella infections occurring in Egypt, assuming 100% efficacy.

An interesting finding of the present study was that 28% of the episodes of Shigella were asymptomatic, isolated from children with no recent history of diarrhea. This is in contrast to studies showing that isolation of Shigella from asymptomatic individuals occurred but was unusual.20,40,41 Possible reasons for the discrepancy from previous studies include children in this study not reporting a recent of diarrhea or having partial immunity from prior infections with Shigellla. In support of the later possibility is a community-based study of diarrhea in Mexico, where 55% of the Shigella infections were recovered from asymptomatic children.42

In contrast to the classic picture of dysentery, we found it common for subjects with symptomatic Shigella infections to have a relatively mild to moderate illness, with the majority of children having no fever or blood in stools. This finding underscores the fact that while the presence of bloody diarrhea may be indicative of shigellosis, the absence of this symptom cannot preclude the diagnosis. If similar observations are made in other studies, it may necessitate altering clinical guidelines for the diagnosis and treatment of diarrheal illness in settings such as rural Egypt, where primary care physicians are unlikely to have access to a laboratory capable of performing cultures.

The observation of Shigella being commonly associated with mild disease is in stark contrast to the majority of children from hospital-based studies where disease is typically severe and frequently associated with dysentery.43 The mild/moderate nature of disease in the current study is further demonstrated by the absence of Shigella-associated diarrhea deaths during the study period compared with case fatality rates of up to 30% among children hospitalized for treatment of Shigella-associated diarrhea.29 The findings in our study thus expand the spectrum of clinical illness associated with Shigella, and demonstrate the importance of performing active surveillance in the community to detect illness that may otherwise go unrecognized.

Confirming previous reports, we found the risk of Shigella-associated diarrhea to be low in infancy and steadily increased through the second year of life.44 The low rate in infancy may be due to breastfeeding because a child who was reported to be breastfed was significantly less likely to develop Shigella-associated diarrhea, a finding noted in several past reports.21,44–46 We found boys to be more likely to acquire Shigella-associated diarrhea, possibly due to cultural or behavioral norms in our study area. However, the virulence of Shigella infections were not related to the sex of the child, as shown by equivalent rates of bloody diarrhea and severe dehydration in boys and girls. This is in contrast to a study from Bangladesh, where girls were twice as likely as boys to die of Shigella-associated diarrhea, but this may be due to a cultural practice of preferentially seeking medical care for boys.47 As with other reports, the risk of acquiring Shigella-associated diarrhea was significantly more likely to occur during the summer months. Although many previous studies have reported poor household sanitation and high population density to be risk factors for developing Shigella-associated diarrhea, we were unable to reproduce these findings, possibly due to the homogeneity of our study population.

In summary, through a community-based, prospective cohort study, we have shown Shigella to be a common cause of diarrhea among Egyptian children living in the Nile Delta. The most common Shigella serogroup identified was S. flexneri in which age and season were significant risk factors for Shigella-associated diarrhea, while breastfeeding was protective. The results of this study demonstrate that a significant burden of disease associated with Shigella, which lends itself to a viable field site to conduct clinical trials testing candidate Shigella vaccines.

Table 1

Incidence rates of Shigella sp. among rural Egyptian children*

Age (months)All ShigellaS. flexneriS. sonneiS. dysenteriaeS. boydiiMixed Shigella sp.
* Incidence per child-year of follow-up at risk (episodes/person years of follow-up). The number of children are 101 distributed as follows: 75 with 1 episode, 20 with 2 episodes, 5 with 3 episodes, and 1 with 4 episodes. Shigella co-pathogens are included.
1–110.14 (32/221.5)0.09 (19/221.6)0.02 (5/221.7)0.03 (7/221.8)0.005 (1/221.8)0
12–230.31 (77/251.8)0.15 (39/252.0)0.09 (23/252.1)0.05 (12/252.1)0.008 (2/252.2)0.004 (1/252.2)
24–350.11 (25/221.9)0.07 (16/222.0)0.01 (2/222.0)0.03 (7/222.0)00
    Total0.19 (134/695.2)0.11 (74/695.5)0.04 (30/695.9)0.04 (26/695.9)0.004 (3/696.0)0.001 (1/696.0)
Table 2

Crude and adjusted relative risks for the association between selected sociodemographic and environmental factors and the incidence of Shigella diarrhea*

No. of episodes (n = 119)†Incidence rate (episodes per child-year)Adjusted RR‡ (95% CI)
* RR = relative risk; CI = confidence interval.
† 15 cases with co-pathogens were excluded.
‡ Adjusted for all other variables in the model and for repeated measures on the same child using generalized estimating equations.
Age (months)
    0–11290.14.7 (1.1–21.3)
    12–23680.37.0 (1.7–29.1)
    24–35220.11
Sex
    Female450.10.8 (0.5–1.3)
    Male740.21
Breastfeeding
    Yes200.10.4 (0.2–0.8)
    No90.31
Village
    820520.20.7 (0.4–1.2)
    830670.21
Season
    Warm830.22.9 (1.5–5.6)
    Cold360.11
Maternal education
    Any70.10.9 (0.4–2.0)
    None1120.21
Crowded (persons per room)
    <370.21.6 (0.8–3.2)
    ≥31120.171
Latrine
    Yes510.20.6 (0.3–1.0)
    No680.021
Socioeconomic status
    Medium/High820.21.2 (0.6–2.4)
    Low370.21
Main water source
    Other90.10.7 (0.3–1.5)
    Municipal1100.21
Table 3

Clinical characteristics associated with Shigella episodes

S. flexneri no. (%)S. sonnei no. (%)S. dysenteriae no. (%)S. boydii no. (%)Mixed no. (%)All no. (%)
* 15 episodes with co-pathogens were excluded.
Vomiting11 (17)7 (24)4 (17)0 (0)0 (0)22 (19)
Fever22 (34)13 (45)7 (30)0 (0)0 (0)42 (35)
Severe dehydration11 (17)6 (21)1 (4)0 (0)1 (100)19 (16)
Blood in stool9 (14)2 (7)2 (9)0 (0)0 (0)13 (11)
Diarrhea ≥8 stools/day19 (30)5 (17)4 (17)0 (0)1 (100)29 (24)
Illness ≥3 days34 (53)16 (55)14 (61)1 (50)1 (100)66 (56)
Total64292321119*
Table 4

Adjusted odds ratios for the associations between selected clinical characteristics and Shigella diarrhea*

Shigella no. (%)Non-Shigella no. (%)Adjusted OR† (95% CI)
* Co-pathogens are excluded from two groups. OR = odds ratio; CI = confidence interval.
† Adjusted for all clinical characteristics in the model and repeated measures using generalized estimating equations.
Fever
    Yes42 (35)1,264 (38)1.1 (0.8–1.5)
    No77 (65)2,079 (62)Reference
Vomiting
    Yes22 (18)721 (22)1.2 (0.8–1.9)
    No97 (82)2,622 (78)Reference
Severe dehydration
    Yes19 (16)360 (11)1.4 (0.8–2.4)
    No100 (84)2,983 (89)Reference
Bloody stool
    Yes13 (11)144 (4)2.3 (1.3–3.8)
    No106 (89)3,199 (96)Reference
Illness duration
    ≥3 days66 (55)1,511 (45)1.4 (1.0–2.0)
    <3 days53 (45)1,832 (55)Reference
Total episodes1193,343
Table 5

Comparison of diarrhea episodes with controls for frequency of Shigella isolation by species*

Cases† no. (%)Controls no. (%)Adjusted OR‡ (95% CI)
* OR = odds ratio; CI = confidence interval.
† Where Shigella was isolated in the first episodes and in the first sample collected.
‡ Adjusted for age, sex, season, and breastfeeding using logistic regression.
§ P < 0.05.
¶ Mixed Shigella = S. flexneri plus S. dysenteriae.
S. flexneri41 (1.2)24 (1.0)1.6 (1.1–2.3)§
S. sonnei2 (0.7)7 (0.3)1.1 (0.8–1.7)
S. dysenteriae17 (0.5)1 (0.04)1.8 (1.1–3.1)§
S. boydii1 (0.03)1 (0.04)2.4 (0.6–9.6)
Mixed Shigella1 (0.03)0 (0.0)
All Shigella83 (2.4)33 (1.3)1.2 (1.0–1.6)
Total children sampled3,4772,493

Authors’ addresses: Remon R. Abu-Elyazeed, Thomas F. Wierzba, Robert W. Frenck, Karim A. Kamal, and Ibrahim A. Abd-El Messih, U.S. Naval Medical Research Unit No. 3, Cairo, Egypt, Telephone: 20-2-342-1381, Fax: 20-2-342-1382, E-mail: frenckr@namru3.org. Shannon D. Putnam, U.S. Naval Medical Research Unit No. 2, Jakarta, Indonesia. Malla R. Rao and Abdollah B. Naficy, National Institute of Child Health and Human Development, Building 31, Roonm 2A32, MSC 2425, National Institutes of Health, 31 Center Drive, Bethesda, MD 20892-2425. Stephen J. Saravino, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910. Leonard F. Peruski Jr., Indiana University School of Medicine, 3400 Broadway, Gary, IN 46408. Sahar A. El-Alkamy, Abu Homos Field Research and Training Center, Egyptian Ministry of Health and Population, Beheira, Egypt. John D. Clemens, International Vaccine Institute, Seoul, Korea.

Acknowledgments: We thank the children who participated in the study and their families; the staff of the Abu-Homos Field Research and Training Center for their contributions to field and laboratory work; and Drs. Badria Z. Morsy (Director General) and Mahmoud Abu El Nasr (First Undersecretary, Ministry of Health and Population of Egypt) for their support. We also thank Khaled Hassan and Dr. Atef El-Gendy (U.S. Naval Medical Research Unit No. 3) for their technical support.

Financial support: This work is supported by the U.S. Army Medical Research and Development Command (Fort Detrick, Frederick, MD), the Naval Medical Research Center (Bethesda, MD) work units M00101.HIX.3421 and M00101.PIX.3270, the Global Program on Vaccines and Immunization of the World Health Organization (Geneva, Switzerland), and the National Institute of Child Health and Human Development, National Institutes of Health (Bethesda, MD).

Disclaimer: The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the U.S. Department of the Navy, the U.S. Department of Defense, the U.S. Government, the World Health Organization, or the Egyptian Ministry of Health.

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