Natural History of Cryptosporidiosis in a Birth Cohort in Southern India
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
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 . 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 . 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 , 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 .
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 , which tested for multiple bacterial, viral, and parasitic
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
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).
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  or cryptosporidiosis .
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
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
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,
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.
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
Cryptosporidiosis (n = 199)
Noncryptosporidial Diarrhea (n = 1922)
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.
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
Efficacy, % (Range)
Exposure as No. of
Previous Cryptosporidial Person-years of
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. (%).
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
The incidence of 0.86 episodes per child-year is higher
than reported from cohort studies in Peru (0.22 episodes per
child-year)  and Guinea-Bissau (0.33 episodes per
childyear) . 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 . 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%)  and Thailand (64.2%) . However,
2 longitudinal studies from Brazil  and Guatemala 
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  and Guinea-Bissau , 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
No. of children
Any cryptosporidial infection
Moderate or severe symptomatic infection
Any infection in children with nonhomotypicb
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)
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 . 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 .
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 . The predominance of the anthroponotic
C. hominis species may indicate the primary role of person-to-person
transmission of infection in this community . 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 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.
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. The burden of cryptosporidium diarrheal disease among children <24 months of age in moderate/high mortality regions of sub-Saharan Africa and South Asia, utilizing data from the Global Enteric Multicenter Study (GEMS) . PLoS Negl Trop Dis 2016 ; 10 :e0004729.
3. Sarkar R , Tate JE , Ajjampur SS , et al. Burden of diarrhea, hospitalization and mortality due to cryptosporidial infections in Indian children . PLoS Negl Trop Dis 2014 ; 8 : e3042 .
4. Shirley DA , Moonah SN , Kotloff KL . Burden of disease from cryptosporidiosis . Curr Opin Infect Dis 2012 ; 25 : 555 - 63 .
5. Checkley W , White AC Jr, Jaganath D , et al. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium . Lancet Infect Dis 2015 ; 15 : 85 - 94 .
6. Kattula D , Sarkar R , Sivarathinaswamy P , et al. The first 1000 days of life: prenatal and postnatal risk factors for morbidity and growth in a birth cohort in southern India . BMJ Open 2014 ; 4 : e005404 .
7. Ruuska T , Vesikari T. Rotavirus disease in Finnish children: use of numerical scores for clinical severity of diarrhoeal episodes . Scand J Infect Dis 1990 ; 22 : 259 - 67 .
8. Morris SS , Cousens SN , Lanata CF , Kirkwood BR . Diarrhoea-defining the episode . Int J Epidemiol 1994 ; 23 : 617 - 23 .
9. Ajjampur SS , Gladstone BP , Selvapandian D , et al. Molecular and spatial epidemiology of cryptosporidiosis in children in a semiurban community in South India . J Clin Microbiol 2007 ; 45 : 915 - 20 .
10. Xiao L , Escalante L , Yang C , et al. Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus . Appl Environ Microbiol 1999 ; 65 : 1578 - 83 .
11. Xiao L , Morgan UM , Limor J , Escalante A , et al. Genetic diyptosporidium parvum and related Cryptosporidium species . Appl Environ Microbiol 1999 ; 65 : 3386 - 91 .
12. Houpt E , Gratz J , Kosek M , et al. Microbiologic methods utilized in the MAL-ED cohort study . Clin Infect Dis 2014 ; 59 (suppl 4): S225 - 32 .
13. Sarkar R , Ajjampur SS , Prabakaran AD , et al. Cryptosporidiosis among children in an endemic semiurban community in southern India: does a protected drinking water source decrease infection? Clin Infect Dis 2013 ; 57 : 398 - 406 .
14. Lazarus RP , Ajjampur SSR , Geetha JC , et al. Serum anti-cryptosporidial gp15 antibodies in mothers and children less than 2 years of age in India . Am J Trop Med Hyg 2015 ; 93 : 931 - 8 .
15. Priest JW , Kwon JP , Moss DM , et al. Detection by enzyme immunoassay of serum immunoglobulin G antibodies that recognize specific Cryptosporidium parvum antigens . J Clin Microbiol 1999 ; 37 : 1385 - 92 .
16. Sarkar R , Kattula D , Francis MR , et al. Risk factors for cryptosporidiosis among children in a semi urban slum in southern India: a nested case-control study . Am J Trop Med Hyg 2014 ; 91 : 1128 - 37 .
17. Cama VA , Bern C , Roberts J , et al. Cryptosporidium species and subtypes and clinical manifestations in children, Peru . Emerg Infect Dis 2008 ; 14 : 1567 - 74 .
18. Valentiner-Branth P , Steinsland H , Fischer TK , et al. Cohort study of Guinean children: incidence, pathogenicity, conferred protection, and attributable risk for enteropathogens during the first 2 years of life . J Clin Microbiol 2003 ; 41 : 4238 - 45 .
19. Newman RD , Sears CL , Moore SR , et al. Longitudinal study of Cryptosporidium infection in children in northeastern Brazil . J Infect Dis 1999 ; 180 : 167 - 75 .
20. Fraser D , Dagan R , Naggan L , et al. Natural history of Giardia lamblia and Cryptosporidium infections in a cohort of Israeli Bedouin infants: a study of a population in transition . Am J Trop Med Hyg 1997 ; 57 : 544 - 9 .
21. Korpe PS , Haque R , Gilchrist C , et al. Natural history of cryptosporidiosis in a longitudinal study of slum-dwelling Bangladeshi children: association with severe malnutrition . PLoS Negl Trop Dis 2016 ; 10 :e0004564.
22. Ajjampur SS , Rajendran P , Ramani S , et al. Closing the diarrhoea diagnostic gap in Indian children by the application of molecular techniques . J Med Microbiol 2008 ; 57 : 1364 - 8 .
23. Bern C , Ortega Y , Checkley W , et al. Epidemiologic differences between cyclosporiasis and cryptosporidiosis in Peruvian children . Emerg Infect Dis 2002 ; 8 : 581 - 5 .
24. Chacín-Bonilla L , Barrios F , Sanchez Y. Environmental risk factors for Cryptosporidium infection in an island from western Venezuela . Mem Inst Oswaldo Cruz 2008 ; 103 : 45 - 9 .
25. Janoff EN , Mead PS , Mead JR , et al. Endemic Cryptosporidium and Giardia lamblia infections in a Thai orphanage . Am J Trop Med Hyg 1990 ; 43 : 248 - 56 .
26. Cruz JR , Cano F , Caceres P , et al. Infection and diarrhea caused by Cryptosporidium sp. among Guatemalan infants . J Clin Microbiol 1988 ; 26 : 88 - 91 .
27. Newman RD , Zu SX , Wuhib T , et al. Household epidemiology of Cryptosporidium parvum infection in an urban community in northeast Brazil . Ann Intern Med 1994 ; 120 : 500 - 5 .
28. Mølbak K , Andersen M , Aaby P , et al. Cryptosporidium infection in infancy as a cause of malnutrition: a community study from Guinea-Bissau , West Africa . Am J Clin Nutr 1997 ; 65 : 149 - 52 .
29. Kirkpatrick BD , Haque R , Duggal P , et al. Association between Cryptosporidium infection and human leukocyte antigen class I and class II alleles . J Infect Dis 2008 ; 197 : 474 - 8 .
30. Kirkpatrick BD , Huston CD , Wagner D , et al. Serum mannose-binding lectin deficiency is associated with cryptosporidiosis in young Haitian children . Clin Infect Dis 2006 ; 43 : 289 - 94 .
31. Borad A , Ward H. Human immune responses in cryptosporidiosis . Future Microbiol 2010 ; 5 : 507 - 19 .
32. Ajjampur SS , Sarkar R , Sankaran P , et al. Symptomatic and asymptomatic Cryptosporidium infections in children in a semi-urban slum community in southern India . Am J Trop Med Hyg 2010 ; 83 : 1110 - 5 .
33. Ajjampur SS , Liakath FB , Kannan A , et al. Multisite study of cryptosporidiosis in children with diarrhea in India . J Clin Microbiol 2010 ; 48 : 2075 - 81 .
34. Gatei W , Das P , Dutta P , et al. Multilocus sequence typing and genetic structure of Cryptosporidium hominis from children in Kolkata , India. Infect Genet Evol 2007 ; 7 : 197 - 205 .
35. Nagamani K , Pavuluri PR , Gyaneshwari M , et al. Molecular characterisation of Cryptosporidium: an emerging parasite . Indian J Med Microbiol 2007 ; 25 : 133 - 6 .
36. Okhuysen PC , Chappell CL , Sterling CR , et al. Susceptibility and serologic response of healthy adults to reinfection with Cryptosporidium parvum . Infect Immun 1998 ; 66 : 441 - 3 .
37. Chappell CL , Okhuysen PC , Sterling CR , et al. Infectivity of Cryptosporidium parvum in healthy adults with pre-existing anti-C. parvum serum immunoglobulin G . Am J Trop Med Hyg 1999 ; 60 : 157 - 64 .