Natural History of Cryptosporidiosis in a Birth Cohort in Southern India

Clinical Infectious Diseases, Jan 2017

Background. Cryptosporidium is a leading cause of moderate to severe childhood diarrhea in resource-poor settings. Understanding the natural history of cryptosporidiosis and the correlates of protection are essential to develop effective and sustainable approaches to disease control and prevention.

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Natural History of Cryptosporidiosis in a Birth Cohort in Southern India

CID Natural History of Cryptosporidiosis in a Birth Cohort in Southern India Deepthi Kattula 1 3 Nithya Jeyavelu 1 3 Ashok D. Prabhakaran 1 3 Prasanna S. Premkumar 1 2 Vasanthakumar Velusamy 1 3 Srinivasan Venugopal 1 3 Jayanthi C. Geetha 1 3 Robin P. Lazarus 1 3 Princey Das 1 3 Karthick Nithyanandhan 1 3 Chandrabose Gunasekaran 1 3 Jayaprakash Muliyil 1 3 Rajiv Sarkar 1 3 Christine Wanke 0 1 5 Sitara Swarna Rao Ajjampur 1 3 Sudhir Babji 1 3 Elena N. Naumova 1 4 5 Honorine D. Ward 0 1 5 Gagandeep Kang 1 3 0 Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center 1 Received 17 May 2016; editorial decision 12 October 2016; accepted 1 November 2016; published online December 24, 2016. lege, Vellore 632 004 , India 2 Biostatistics, Christian Medical College , Vellore, Tamil Nadu , India 3 Gastrointestinal Sciences 4 Friedman School of Nutrition Science and Policy, Tufts University , Boston, Massachusetts 5 Department of Public Health and Community Medicine, Tufts University School of Medicine Background. Cryptosporidium is a leading cause of moderate to severe childhood diarrhea in resource-poor settings. Understanding the natural history of cryptosporidiosis and the correlates of protection are essential to develop effective and sustainable approaches to disease control and prevention. Methods. Children (N = 497) were recruited at birth in semiurban slums in Vellore, India, and followed for 3 years with twiceweekly home visits. Stool samples were collected every 2 weeks and during diarrheal episodes were tested for Cryptosporidium species by polymerase chain reaction (PCR). Serum samples obtained every 6 months were evaluated for seroconversion, defined as a 4-fold increase in immunoglobulin G directed against Cryptosporidium gp15 and/or Cp23 antigens between consecutive sera. Results. Of 410 children completing follow-up, 397 (97%) acquired cryptosporidiosis by 3 years of age. PCR identified 1053 episodes of cryptosporidiosis, with an overall incidence of 0.86 infections per child-year by stool and serology. The median age for the first infection was 9 (interquartile range, 4-17) months, indicating early exposure. Although infections were mainly asymptomatic (693 [66%]), Cryptosporidium was identified in 9.4% of diarrheal episodes. The proportion of reinfected children was high (81%) and there was clustering of asymptomatic and symptomatic infections (P < .0001 for both). Protection against infection increased with the order of infection but was only 69% after 4 infections. Cryptosporidium hominis (73.3%) was the predominant Cryptosporidium species, and there was no species-specific protection. Conclusions. There is a high burden of endemic cryptosporidiosis in southern India. Clustering of infection is suggestive of host susceptibility. Multiple reinfections conferred some protection against subsequent infection. - cryptosporidiosis is not clearly understood. There is a dearth of longitudinal data on the course of infection in the absence of overt diarrheal disease. An understanding of the natural history of cryptosporidiosis and correlates of protection are essential in developing effective disease control and preventive measures. We conducted intensive active surveillance of children from birth till 3 years of age in a semiurban area in southern India by harnessing the synergistic benefits of a birth cohort design in a community setting and efficient molecular approaches to detect cryptosporidial infections. MATERIALS AND METHODS Site Recruitment, Follow-up, and Definitions A birth cohort of 497 newborns was recruited between March 2009 and May 2010 in periurban Vellore, Tamil Nadu, India. In this population, human immunodeficiency virus (HIV) prevalence in antenatal women is <0.3% and there is an effective prevention of transmission program; hence, we did not screen for HIV. Children with very low birth weight (<1500 g) or congenital malformations were excluded. Enrollment, twice-weekly follow-up, and a description of illness in the cohort have been published [6]. Surveillance stool samples were collected every 2 weeks. For diarrhea, 3 stool samples were collected on separate days from day 1 to day 7 from the start of the episode. Severity of diarrheal episodes was assessed using the Vesikari scoring system [7]. Cord blood, where possible, or peripheral blood within 45 days after birth was collected from the infant. Every 6 months, 3- to 5-mL blood samples were collected from the study children. Diarrhea was defined as ≥3 loose, watery stools in a 24-hour period [8], with an episode defined as at least 1  day of diarrhea, preceded and followed by at least 2 days without diarrhea. Cryptosporidiosis/cryptosporidial infection was identified by polymerase chain reaction (PCR) detection of Cryptosporidium species in stool or a 4-fold increase in immunoglobulin G (IgG) levels to gp15 and/or Cp23 between 2 serum samples. A diarrheal episode was termed symptomatic cryptosporidiosis if a stool sample collected within ±7 days was PCR positive. Cryptosporidiosis identified by stool PCR was asymptomatic if the stool was PCR positive but there was no diarrhea ±14 days. A new asymptomatic episode was considered when there was at least a 2-week gap with an intervening stool sample negative for Cryptosporidium species. Asymptomatic cryptosporidiosis identified by serology was a 4-fold increase in IgG to gp15 and/or Cp23 between 2 sera with no diarrhea or stool PCR positivity during that period. When a child was positive for asymptomatic infection by both stool PCR and serology during a defined interval, only stool PCR was considered and the child was not double-counted. Symptomatically undifferentiated cryptosporidiosis occurred when there was a 4-fold increase in IgG levels between 2 sera with a history of diarrhea during the period, but all stools were PCR negative. Cryptosporidial infection includes asymptomatic and symptomatic infections, while disease refers to symptomatic infection. Testing for Cryptosporidium in Stool DNA extracted from stool using a QIAamp DNA stool mini kit (Qiagen, Valencia, California) was screened by conventional 18S ribosomal RNA (rRNA) nested PCR for Cryptosporidium species using published protocols [9, 10]. Appropriate negative (no DNA template) and positive (known C.  hominis or C.  parvum PCR-positive stool) controls were included in every extraction and PCR run. For PCR-positive samples, species was determined by restriction fragment-length polymorphism and confirmed by 18S rRNA amplicon sequencing [11]. Identification of Enteric Pathogens in Diarrhea Other than testing for Cryptosporidium species, which was by PCR, all diarrheal stool samples were tested for the presence of bacterial, viral, and parasitic pathogens using the Interactions of Malnutrition and Enteric Infections (MAL-ED) study protocols [12], which tested for multiple bacterial, viral, and parasitic pathogens. ELISA for IgG to Gp15 and Cp23 in Serum Serum IgG was measured by enzyme-linked immunosorbent assay (ELISA) using recombinant gp15 and Cp23 [13–15] as antigens, with results expressed as arbitrary ELISA units. Samples with positive ELISA unit values were considered seropositive. MHC Class II Typing Major histocompatibility complex (MHC) class  II typing was performed in a subset of 74 children, of whom 41 had only asymptomatic infection and 33 had ≥2 infections with at least 1 symptomatic infection. We chose children from whom sufficient genomic DNA was available, so a power calculation was not performed to determine the ideal sample size. There is no information of human leukocyte antigen (HLA) alleles in this population, which consists of a mix of religions and ethnic backgrounds in southern India. DNA was extracted from blood using a DNeasy Blood and Tissue kit (Qiagen). HLA class  II (DR and DQ) alleles expressed by the MHC genes were identified by PCR with sequence-specific oligonucleotide probes using a Luminex-based method with a Rapid Lifecodes kit (Tepnel Lifecodes Corporation, Stamford, Connecticut). Statistical Analysis Data were analyzed using Stata software, version 12.1 for Windows (StataCorp, College Station, Texas) and R version 2.12.1 (http://www.r-project.org/), with analyses confined to the 410 children who completed 3 years of follow-up. The baseline demographic characteristics of 87 children who did not complete the study did not differ significantly from those included in the analysis (Supplementary Table 1). Comparisons used the χ2 or Fisher exact test for categorical variables and 2-tailed t test or Wilcoxon rank-sum test for continuous variables. The cumulative incidence of cryptosporidial infections was calculated using survival analysis, adjusted for the duration of follow-up of each child and expressed as number of episodes per child-year. Based on the assumption that cryptosporidiosis would follow a Poisson distribution, the number of children expected to have 0, 1, 2, 3, 4, 5, and 6 episodes of cryptosporidiosis was calculated and compared with the observed frequency to verify if there was clustering of cryptosporidiosis in children. For protective effects of prior cryptosporidial infection, parametric Poisson regression survival models were used to obtain relative risks and confidence intervals adjusted for repeated infection in the same child. The adjusted relative risks and protective efficacy of prior cryptosporidial infections were adjusted for factors previously reported to be associated either with overall morbidity [6] or cryptosporidiosis [16]. The role of antibody-mediated protection was studied using the effect of presence of serum IgG to gp15 and Cp23 on subsequent infection rates. When there were multiple cryptosporidial infections between 2 sera, analysis was restricted to the first episode. Parametric Poisson regression survival models were used to model the risk of cryptosporidial infection or disease as a function of preexisting antibody levels. Serum IgG levels to gp15 and Cp23 were categorized into quartiles to examine a dose–response relationship between antibody levels and protection. The IgG level was included as a categorical variable with absence of antibody as the reference category. The model was adjusted for age and number of previous infections. Allele frequencies were counted for analysis of HLA data, with an allele frequency of >10% used for genetic association analysis. The association between HLA markers and occurrence of symptomatic and asymptomatic cryptosporidial infection was measured by calculating the odds ratio, using logistic regression. Ethics Approval Written informed consent was obtained from the parents or guardians of children in the study. The study was approved by the institutional review boards of the Christian Medical College, Vellore, India, and the Tufts University Health Sciences campus, Boston, Massachusetts. Burden of Cryptosporidiosis Of 497 recruited children, 410 completed 3 years of follow-up contributing 1218 child-years. The most common reason for children not completing the study was permanent migration out of the study area. Of the 410, 397 (97%) had cryptosporidiosis; 237 (59.7%) had only asymptomatic infections and 119 (30%) developed both symptomatic and asymptomatic cryptosporidial infections (Figures 1 and 2). Among the remaining children, 24 (6.0%) children had 1 or more episodes of symptomatic infection but no asymptomatic infection and 17 Figure 1. Number of children in a birth cohort in Vellore, India, who completed 3 years of follow-up (n = 410) who had cryptosporidiosis detected by polymerase chain reaction (PCR) or serology. (4.3%) children had undifferentiated cryptosporidial infection detected only by serology. There was no difference by sex for asymptomatic (P  =  .10) or symptomatic infections (P = .17). Infection occurred by 6 months of age in 165 (40.2%) children, by 2 years in 379 (92.4%), and by 3 years in 397 (97%). Symptomatic cryptosporidiosis was seen in 24 (5.8%), 55 (13.4%), 116 (28.3%), and 143 (35%) children by 6  months and 1, 2, and 3  years of age, respectively. The median age at first cryptosporidial infection was 9 (interquartile range [IQR], 4–17) months, with no difference (P =  .75) between asymptomatic (8 [IQR, 4–14]) and symptomatic (9 [IQR, 3–16]) infection (Supplementary Figure 1). Overall, the incidence of cryptosporidiosis was 0.86 episodes (symptomatic 0.16, asymptomatic 0.57) per child-year, with the highest incidence (1.01 episodes per child-year) in the first year of life (Table 1). Figure 2. Number of symptomatic and asymptomatic cryptosporidial infections detected in a birth cohort in Vellore, India, who completed 3 years of follow-up. Abbreviation: PCR, polymerase chain reaction. Cryptosporidial Infection Episodes Incidence rates are shown as incidence rate (95% confidence interval) per child-year. Clustering of Cryptosporidial Infection Among 410 children, 63 (15.4%) had only 1 infection, 132 (32.2%) had 2, 113 (27.6%) had 3, 64 (15.6%) had 4, and 25 (6.1%) had ≥5 infections. There was clustering of infection (χ2 test for goodness of fit = 1755.21; degrees of freedom = 6; P < .0001). Similarly, among 143 children with symptomatic infection, 99 (69.2%) had only 1 episode, 36 (25.2%) had 2 episodes, and 8 (5.6%) had ≥3 episodes of symptomatic cryptosporidiosis, with evidence of clustering (χ2 test for goodness of fit = 25.04; degrees of freedom = 5; P < .0001). Clinical Features of Symptomatic Cryptosporidiosis Of 2134 diarrheal episodes, at least 1 stool sample was obtained for 2121 (99.4%) episodes, and 199 (9.4%) were Cryptosporidium positive by PCR. Most (197 [99%]) were acute diarrhea (≤14  days’ duration), with a median duration of 3 (IQR, 2–4) Table 2. Clinical Characteristics of Diarrheal Episodes in Which Cryptosporidium Species Was Identified by Polymerase Chain Reaction and Other Episodes in a Birth Cohort in Vellore, India, Followed up to 3 Years of Age Symptomatic Cryptosporidiosis (n = 199) Noncryptosporidial Diarrhea (n = 1922) Clinical Characteristics Median (IQR) age, mo 16 (9–24) Median (IQR) duration, d 3 (2–4) Accompanying symptoms, No. (%) Vomiting 45 (22.6) Fever 40 (20.1) Treatment required, No. (%) Clinic visits 117 (58.8) Hospitalization 7 (3.5) Intravenous fluids 1 (0.5) Severity of diarrheal episodesa, No. (%) Mild 95 (47.7) Moderate 80 (40.2) Severe 24 (12.0) Abbreviation: IQR, interquartile range. aData not available for 6 episodes. 455 (23.6) 450 (23.4) 955 (49.8) 758 (39.5) 203 (10.6) days, similar to that of noncryptosporidial diarrhea (Table  2). Of the 199 symptomatic cryptosporidial episodes, clinical features and severity did not differ from noncryptosporidial diarrhea (Table  2). There were 48 (24%) episodes of symptomatic cryptosporidiosis where co-pathogens, predominantly Giardia (27 [13%]) and Shigella (12 [6%]), were found. Protection Conferred by Natural Infection The incidence of cryptosporidial infection, but not disease, decreased as the number of infections increased (Table 3). The adjusted efficacy after 4 prior infections was 69% against infection, 70% against asymptomatic infection, and 66% against undifferentiated cryptosporidial infection, although the efficacy against symptomatic infection was lower and insignificant (19%). To explore the reason for the lower protection against symptomatic infection, a subgroup analysis was performed excluding 8 children who had >2 symptomatic infections. It showed improved protective efficacy against symptomatic infection, indicating that certain children pushed the protective effect toward the null (Supplementary Table 2). In children with multiple infections, the severity of diarrhea did not significantly decrease between the first and second infections (P = .93) or between the second and third infections (P  =  .32). Similarly, in children with multiple symptomatic infections, the severity of diarrhea between the first and second (P = .19) or between the second and third (P = .39) episodes did not significantly decrease. Association of Serum Antibodies With Protection Against Cryptosporidial Infections and Diarrheal Disease Serum gp15 and Cp23 IgG levels increased with age till 24  months, after which levels started to decline (Supplementary Figure  2). Geometric mean IgG levels increased with an increasing number of previous infections (Supplementary Figure  3). There was no association Relative Risk of Subsequent Event (95% CI) Adjusted for Age Adjusted for Other Factorsa Adjusted Protective Efficacy, % (Range) Exposure as No. of Previous Cryptosporidial Person-years of Infections Follow-up Incidence Rate per Child-year (95% CI) Abbreviation: CI, confidence interval. aAdjusted for age, sex, presence of sibling, maternal age <23 years, use of firewood for cooking. between preexisting gp15 or Cp23 IgG levels and subsequent cryptosporidial infection (Supplementary Table  3) or diarrheal disease after adjusting for age and previous infections (Supplementary Table 4). Distribution of Cryptosporidium Species in the Cohort Species could be determined for 473 of 733 (64.5%) episodes of cryptosporidiosis detected by stool PCR (77% and 67% of cryptosporidial diarrheal and nondiarrheal stool, respectively). Cryptosporidium hominis predominated and was identified in 347 (73.3%) infections in which species could be identified, followed by C.  parvum (81 [17.1%]) (Table  4). Other species were C.  meleagridis (5.2%) and C.  felis (1%). Mixed infection with both C. parvum and C. hominis was identified in 14 (2.9%) episodes and C.  andersoni and C.  muris in 1 episode (0.2%). Species could not be identified in 260 (35.5%) episodes, of which 199 (76.5%) episodes were asymptomatic. On the gels, untyped samples generally had weaker bands, which may indicate lower amounts of parasite in samples. To assess species-specific repeated infection and protection against subsequent infection, analysis was restricted to 161 children for whom complete species data were available for all infections. For protection against subsequent infection, we evaluated the risk of primary and subsequent infections with C. hominis or non–C. hominis species (Table 5). There was no decrease in the risk of overall or species-specific asymptomatic or symptomatic cryptosporidiosis in later infections, indicating a lack of species-specific protection. There was no significant association of homozygous and heterozygous HLA class  II alleles in children with only asymptomatic or at least 1 symptomatic cryptosporidial infection (Supplementary Tables 5 and 6). Table  4. Frequency of Infections Caused by Different Species of Cryptosporidium in a Birth Cohort of Children (n  =  410) Followed up to 3 Years of Age in Vellore, India Data are presented as No. (%). DISCUSSION All Infections Symptomatic Infection 199 138 Asymptomatic Infection 534 335 This intensive birth cohort study in southern India revealed a high burden of cryptosporidiosis with 97% of children infected by 3  years of age. Both infection and cryptosporidial diarrhea clustered in some children, but limited testing of HLA class II alleles did not find an association with susceptibility or protection. In this cohort, protection conferred by prior infection was slow to develop, required multiple infections for partial protection, and was not associated with the presence of serum antibodies. The incidence of 0.86 episodes per child-year is higher than reported from cohort studies in Peru (0.22 episodes per child-year) [17] and Guinea-Bissau (0.33 episodes per childyear) [18]. This is because of the inclusion of serology; based on PCR alone, incidence would have been 0.60 episodes per child-year. Among urban Brazilian children followed for 4  years, Israeli Bedouin children followed for 2  years, and an urban Bangladeshi cohort followed for 2  years, approximately 31%, 49%, and 77%, respectively, developed cryptosporidial infection [19–21]. The high rates of infection in this study are likely due to intensive surveillance and use of sensitive diagnostic methods and complementation by serology, as reported for other enteric pathogens [5, 22]. A recent report building on the case-control analysis from the Global Enteric Multicenter Study (GEMS) study also demonstrated the importance of cryptosporidial infection, although the focus was on severe disease and mortality [2]. Similar to this study, the GEMS reanalysis showed that cryptosporidial diarrhea was more common in toddlers (Table 2). The majority (60%) of children had only asymptomatic cryptosporidial infections, similar to cohorts in Peru (67%) and Bangladesh (72%) [17, 23] and cross-sectional studies in Venezuela (86.3%) [24] and Thailand (64.2%) [25]. However, 2 longitudinal studies from Brazil [19] and Guatemala [26] reported 79% and 65% cryptosporidial diarrhea, respectively. The high proportion of asymptomatic infections may be due to the highly infectious nature of the parasite, with constant exposure resulting in subclinical infections, especially in endemic areas where transmission is through multiple routes [13, 27]. Only 24 (1.1%) episodes of persistent diarrhea were observed in this cohort, of which 2 episodes were associated with Cryptosporidium, differing from longitudinal studies from Brazil [27] and Guinea-Bissau [28], which reported greater persistent diarrhea in children, but are older studies. During the 3-year follow up, 81% of children had >1 episode of cryptosporidiosis. Although the majority of children (97%) were infected, some children were more at risk for repeated infections and Infection No. of children Follow-up, child-months Any cryptosporidial infection Asymptomatic infection Symptomatic infection Moderate or severe symptomatic infection Homotypica infection Any infection in children with nonhomotypicb primary infection Homotypic symptomatic infection Any symptomatic infection in children with nonhomotypic primary infection Data are shown as percentage (95% CI) unless otherwise indicated. Abbreviation: CI, confidence interval; NA, not applicable. aInfection with the same species as the primary infection. bInfection with a different species than the primary infection. Rate Ratio (95% CI) 1.02 (.7–1.4) 0.80 (.5–1.2) 1.6 (.7–3.5) 2.6 (.6–12.1) disease, with children with symptomatic cryptosporidiosis tending high rate of transmission in the community. Children with sympto have a higher risk of diarrhea when reinfected. This suggests tomatic infection tended to have a higher probability of repeated host susceptibility for cryptosporidiosis. Studies by Kirkpatrick diarrhea, suggestive of genetic susceptibility. Further understandin Bangladesh and Haiti reported associations with HLA class II ing of susceptibility to or protection from disease will require DQB1*0301 and class I B*15 I [29, 30]. In our study there was no larger studies with more detailed analysis of genetic associations significant association of any class II allele with asymptomatic or and cell-mediated immunity in conjunction with epidemiological symptomatic infection, in contrast to the study in Bangladesh in which the class  II DQB1*0301 allele was significantly associated with asymptomatic infection. The number of children who were HLA typed was small (41 asymptomatic and 33 symptomatic) but similar to those in Bangladesh (32 asymptomatic and 34 symptomatic). Impaired cell-mediated immunity may be another reason for susceptibility, since CD4 T cells have a crucial role in protection against and resolution of cryptosporidiosis [31]. Previous analysis of data from this and another cohort in the same location that did not include serology or HLA typing showed that presence of older important risk factors for childhood cryptosporidiosis [16]. The majority of cryptosporidial infections were associated with C. hominis (73%). Cryptosporidium hominis has been identified as the most common (ranging from 79% to 88%) species in children from the same region [9, 32], India [33–35], and elsewhere [5]. The predominance of the anthroponotic C. hominis species may indicate the primary role of person-to-person transmission of infection in this community [16]. We could not demonstrate species-specific protection in children with primary C. hominis infection, and this may reflect the diversity of C. hominis, with a need for subtyping to determine whether there is subtype-specific protection. Our study found limited protection (69%) against subsequent infection after 4 prior infections, without significant protection against symptomatic cryptosporidiosis after adjustment for potential confounders, reflecting partial immunity developed over time. Studies on US healthy adult volunteers have demonstrated that prior exposure results in reduction in disease severity and intensity of infection but not rate and duration of illness, indicating that protection is not conferred by a single exposure. However, volunteers with preexisting serum antibodies required higher challenge doses to acquire subsequent infections compared with seronegative adults [36, 37]. Although geometric mean antibody levels increased with number of infections in this cohort (Supplementary Figure 3), indicating a serological response to cryptosporidial infections, preexisting antibodies did not demonstrate any association with protection from subsequent infection or diarrheal disease. This highlights that humoral immunity may not play a major role in protection against cryptosporidial infections and disease, and the role of cell-mediated immunity needs more detailed consideration. This study provides important insights into the natural history of cryptosporidiosis in an endemic semiurban slum community in southern India. The fact that almost all children in the study acquired cryptosporidial infection by 3  years of age indicates a Supplementary Data Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. Acknowledgments. We are grateful to the parents and children from the urban slums of Vellore for their enthusiastic participation and support. We also thank the field workers for their tireless work monitoring the cohort, and all the support staff of the Division of Gastrointestinal Sciences, Christian Medical College, Vellore, who made the study possible. Author contributions. G.  K., H.  W., C.  W., E.  N., J.  P. M., and S.  S. R. conceived the study. D. K., R. S., V. V., and S. V. managed the cohort and data. S. S. R., S. B., G. K,. N. J., A. D. P., J. C. G., R. P. L., P. D., K. N., and C. G. led the laboratory work. D. K. and P. S. P. led the statistical analysis. All authors contributed to the interpretation of the data and writing of the report, and approved the final manuscript. Financial support. This work was supported by the National Institute of Allergy and Infectious Diseases (R01 A1072222 to H. W.). D. K. was supported by the Fogarty International Center (training grants D43 TW007392 to G. K. and D43 TW009377 to G. K., C. W., and H. W.). Potential conflicts of interest. All Authors certifies no potential conflicts of interest. The authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Kotloff KL , Nataro JP , Blackwelder WC , et  al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study . Lancet 2013 ; 382 : 209 - 22 . 2. Sow SO , Muhsen K , Nasrin D , et  al. 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Deepthi Kattula, Nithya Jeyavelu, Ashok D. Prabhakaran, Prasanna S. Premkumar, Vasanthakumar Velusamy, Srinivasan Venugopal, Jayanthi C. Geetha, Robin P. Lazarus, Princey Das, Karthick Nithyanandhan, Chandrabose Gunasekaran, Jayaprakash Muliyil, Rajiv Sarkar, Christine Wanke, Sitara Swarna Rao Ajjampur, Sudhir Babji, Elena N. Naumova, Honorine D. Ward, Gagandeep Kang. Natural History of Cryptosporidiosis in a Birth Cohort in Southern India, Clinical Infectious Diseases, 2017, 347-354, DOI: 10.1093/cid/ciw730