167
Longitudinal Study of Cryptosporidium Infection in Children in Northeastern
Brazil
Robert D. Newman,1,2 Cynthia L. Sears,3
Sean R. Moore,5 James P. Nataro,4 Tadesse Wuhib,3,a
Deborah A. Agnew,1 Richard L. Guerrant,5
and Aldo A. M. Lima6
1
Division of General Pediatrics, Department of Pediatrics, University
of Washington School of Medicine, and 2International Health
Program, University of Washington School of Public Health
and Community Medicine, Seattle; 3Divisions of Infectious Diseases
and Gastroenterology, Johns Hopkins University School of Medicine,
and 4Center for Vaccine Development, University of Maryland,
Baltimore; 5Division of Geographic and International Medicine,
University of Virginia, Charlottesville; 6Unidade Pesquisas Clinicas,
Universidade Federal do Ceará, Fortaleza, Brazil
A prospective, 4-year cohort study of children born in an urban slum in northeastern Brazil
was undertaken to elucidate the epidemiology of Cryptosporidium infection in an endemic
setting, describe factors associated with Cryptosporidium-associated persistent diarrhea, and
clarify the importance of copathogens in symptomatic cryptosporidiosis. A total of 1476
episodes of diarrhea, accounting for 7581 days of illness (5.25 episodes/child-year), were recorded: of these, 102 episodes (6.9%) were persistent. Cryptosporidium oocysts were identified
in 7.4% of all stools, and they were found more frequently in children with persistent diarrhea
(16.5%) than in those with acute (8.4%) or no (4.0%) diarrhea (P ! .001). Low–birth-weight
children and those living in densely crowded subdivisions were at greater risk for symptomatic
infection. Disease course was highly variable and was not associated with the presence of
copathogens. Recurrent Cryptosporidium infection and relapsing diarrhea associated with it
were moderately common. In light of these data, the applicability of the current World Health
Organization diarrheal definitions to Cryptosporidium-associated diarrheal episodes may need
to be reconsidered.
Cryptosporidium parvum is a coccidian protozoan parasite
that is now well recognized as a worldwide cause of diarrhea.
In the United States and other developed nations, this parasite
is a frequent cause of diarrhea among immunocompromised
persons [1, 2], has been implicated in causing epidemic illness
during waterborne and foodborne outbreaks [3–5], and has
been linked to diarrheal outbreaks in day care settings [6]. In
the developing world, the association of Cryptosporidium with
both acute and persistent diarrhea among immunocompetent
children has been striking. Cross-sectional surveys of children
with diarrhea suggest that cryptosporidiosis is endemic in the
Received 4 June 1998; revised 10 February 1999; electronically published
28 May 1999.
Informed consent was obtained from parents or guardians of all children
enrolled in the study. The study was approved by the Johns Hopkins Joint
Committee on Clinical Investigation and by similar committees at the University of Virginia and the Universidade Federal do Ceará.
Grant support: National Institutes of Health (NIH) International Collaboration in Infectious Disease Research ICIDR (AI-26512); and National
Research Service Award (5-T32-PE1002) from NIH to the University of
Washington School of Medicine.
a
Present affiliation: Centers for Disease Control (CDC), Atlanta.
Reprints or correspondence: Dr. Robert D. Newman, Health Alliance
International, 1107 N.E. 45th St., Suite 410, Seattle, WA 98105 (newman@
teledata.mz).
The Journal of Infectious Diseases 1999; 180:167–75
q 1999 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/99/8001-0022$02.00
developing world, with the identification of this parasite in up
to 26.9% of symptomatic children [7]. Community-based longitudinal studies, although far fewer in number, have also consistently found an association between Cryptosporidium infection and diarrheal illness [8–10]. In addition to causing
potentially severe diarrheal illness in young children in the developing world, both symptomatic and possibly asymptomatic
early childhood infections with Cryptosporidium may be associated with a subsequent increase in diarrheal burden or a
decrease in nutritional status (or both) [8, 11, 12].
Since long-term adverse outcomes may be associated with
Cryptosporidium infection [11] and treatment of this parasite
has been elusive [1, 13], it is important to clarify risk factors
not only for disease acquisition but also for illness duration
(i.e., risks for persistent diarrhea). One possibility is that Cryptosporidium species act in conjunction with other pathogens,
particularly to cause persistent diarrhea. Most community studies, however, have not undertaken sufficiently complete microbiologic stool evaluations to clarify this issue [8, 9].
We therefore undertook a cohort study of children living in
an urban slum (“favela”) in northeastern Brazil to (1) elucidate
the epidemiology of Cryptosporidium infection in an endemic
setting, (2) describe factors associated with Cryptosporidiumassociated persistent diarrhea, and (3) clarify the importance
of copathogens in symptomatic cryptosporidiosis.
168
Newman et al.
Methods
Subjects. We studied an urban slum community (Gonçalves
Dias) of ∼2000 inhabitants near the medical campus of the Federal
University of Ceará in Fortaleza, Brazil. Fortaleza (population of
2 million) is the state capital of Ceará (population of 6 million) in
Brazil’s poor northeastern region. The community was divided into
5 distinct subdivisions created by the presence of major streets, a
swamp, a warehouse, and an adjacent neighborhood of relative
affluence. One of these subdivisions was markedly different from
the others in that houses were extremely small and connected by
narrow alleyways with open sewers.
All pregnant women in the community were identified by study
nurses and were offered enrollment in the project. Following informed consent, women choosing to participate completed a detailed demographic questionnaire with the help of a study nurse.
In general, children were followed from birth, but in 16 cases,
mostly at the beginning of the study, entry was delayed. Sixteen
children entered the study at 12 weeks of age, the latest at 59 days.
Following a study child’s birth, a study nurse visited the house
three times a week (Monday, Wednesday, and Friday) to record
diarrheal illnesses and dietary information. Mothers (or other
guardians) of children with diarrhea were asked to provide detailed
clinical information about the illness, including other symptoms
(fever, vomiting, dehydration), stool consistency and character, and
medication use. Children with diarrhea were visited daily by a study
nurse until 48 h after the illness resolved. The surveillance period
was from August 1989 through April 1993.
Definitions of diarrheal episodes. We used World Health Organization (WHO) criteria to determine diarrheal episodes [14]. A
total of 3 unformed stools in one 24-h period was determined to
be a diarrheal day. Therefore, it was possible for a child to have
a single liquid stool collected and recorded as a nondiarrheal stool
if there was no more than one further liquid stool in that 24-h
period. A minimum of 2 days without diarrhea was required to
delineate distinct episodes of diarrhea. A single day without diarrhea between 2 diarrheal days was therefore counted as 1 day of
a 3-day diarrheal illness. A diarrheal episode !2 weeks in duration
was recorded as acute diarrhea, a 14- to 29-day episode was considered persistent diarrhea, and an episode lasting >30 days was
considered chronic diarrhea [14].
Nutritional evaluation. To assess the association between nutritional status and disease course, we obtained weight and height
information for all children in the cohort at 3-month intervals.
Weight was measured by use of a calibrated sling scale (0.1-kg
scale), and height was measured by use of a supine measuring board
(0.1-cm scale). Weight-for-age, height-for-age, and their weight-forheight Z scores were calculated by use of nutritional software (EPINUT, Epi Info 6.0; CDC, Atlanta, and WHO, Geneva). We defined
children as stunted if their height-for-age Z score was !2 SD below
the mean, as underweight if their weight-for-age Z score was !2
SD below the mean, and as malnourished if weight-for-height Z
score was !2 SD below the mean [15]. To compare nutritional status
between children with Cryptosporidium-associated acute and persistent diarrhea, we used the latest measurements obtained !4
months prior to the diarrheal episode being analyzed.
Stool collection. We attempted to obtain stool samples from
all children with an identified diarrheal episode. We also attempted
JID 1999;180 (July)
to obtain surveillance stool samples (nondiarrheal samples a minimum of 2 weeks from any diarrheal episode) from all children in
the cohort at 4-month intervals. Stools obtained within 3 days
following the conclusion of a diarrheal episode were considered
diarrhea-associated stools. Stool samples obtained 13 days but !2
weeks following a diarrheal episode or within the 2 weeks preceding
a diarrheal episode were considered indeterminate.
Stool samples were collected by parents or guardians and placed
into disposable plastic cups. A portion of each sample was subsequently placed by field staff into a tube containing 10% formalin.
Fresh stools were transported on ice by field staff to the laboratory
for processing and microbiologic evaluation.
Stool evaluation. Stool specimens were concentrated using formalin–ethyl acetate at a centrifuge speed of 800 g in a fecal parasite
concentrator (Evergreen Scientific, Los Angeles), and two slides
were made from the resulting pellet. Slides were stained with modified acid-fast and auramine stains and read at 10003 (oil immersion) and 4003 (fluorescence microscope), respectively. A stool was
considered Cryptosporidium-positive if typical oocysts 4–6 mm in
diameter were visible on both acid-fast and auramine slides.
Specimens were examined for the presence of ova and parasites
by use of Lugol’s iodine stain of both fresh and concentrated stool
at 4003. Stools were examined for the presence of fecal leukocytes,
using both microscopy (Gram’s stain) and a latex agglutination
assay (LEUKO-TEST; Techlab, Blacksburg, VA) for the detection
of lactoferrin [16]. Stools from children with ongoing breast-feeding
were not tested for fecal lactoferrin because of cross-reactivity with
breast milk. A stool guaiac test (Hemoccult; SmithKline Diagnostics, Santa Cruz, CA) was used to detect the presence of occult
blood.
Monoclonal ELISAs were used on fresh-frozen stool samples to
detect rotavirus (Rotaclone; Cambridge Biotech, Worcester MA)
and adenovirus types 40 and 41 (Adenoclone, Cambridge Biotech).
Fresh stool was plated onto appropriate agar medium (consisting
of MacConkey, xylose-lysine-deoxycholate, thiosulfate-citrate-bilesucrose, trypticase soy with 5% defibrinated sheep blood and ampicillin, or trypticase soy with 5% defibrinated sheep blood with
Campylobacter jejuni supplement), to isolate Shigella species, Salmonella species, Vibrio species, Yersinia enterocolitica, and C. jejuni.
Three lactose-fermenting colonies identified as Escherichia coli by
the API biochemical system (bioMérieux, Hazelwood, MO) were
isolated from each stool and subcultured to agar slants for storage
at 47C and subsequent characterization.
One E. coli isolate from each patient was examined, using established methods, for HEp-2 cell adherence and enterotoxigenicity
[17]. Adherence to HEp-2 cells was scored as aggregate, localized,
or diffuse [18]. Aggregate adherence was interpreted as the presence
of enteroaggregative E. coli, and localized adherence was interpreted as the presence of enteropathogenic E. coli (EPEC), both
of which have been implicated as pathogens in childhood diarrhea
[19]. The role of diffusely adherent E. coli in the etiology of diarrheal disease remains unclear [19].
One E. coli strain per stool was also evaluated, using established
methods, by oligonucleotide probe hybridization for the heat-labile
and heat-stable toxins at the University of Virginia or the University of Maryland [17] (Dupont NEN Research Products, Boston) and for eae (E. coli attaching and effacing trait) [20], EAF
(EPEC adherence factor) [21], DA (diffuse adherence) [22], IEC
JID 1999;180 (July)
Cryptosporidium Infection in Brazil
(enteroinvasive E. coli) [23], and EHEC (enterohemorrhagic E. coli)
[24] at the University of Maryland, Baltimore.
Strains were classified into virulence categories of E. coli on the
basis of one or more tests being positive. Therefore, E. coli strains
were classified as EPEC if the eae probe, EAF probe, or HEp-2
assay for localized adherence was positive; as enterotoxigenic if
either the heat-stable or heat-labile probe was positive; as diffusely
adherent if either the HEp-2 assay or diffuse adherence probe was
positive; as enteroinvasive if the IEC probe was positive; and as
enterohemorrhagic if the EHEC probe was positive. Because no
IEC or EHEC strains were identified in the first 47 stools, the
remaining 24 stools were not tested.
Statistical analysis. Univariate analyses were performed using
x2 or Fisher’s exact tests (for cross-tabulations with an expected
value in any cell <5) to compare proportions for categorical variables; t or Wilcoxon rank sum tests (for variables with nonnormal
distributions) were used to compare continuous variables. Tests
were considered significant when the two-sided P value was ! .05.
To determine whether there were identifiable risk factors for
symptomatic infection with Cryptosporidium species, we used Cox
regression [25]. We used age in days as the time variable and assessed risks for a first symptomatic episode of cryptosporidiosis.
Children without symptomatic infection were censored as they left
the study or on 30 April 1993, the end of the surveillance period.
Because this was an open cohort, multiple entry and exit was permitted for each child prior to failure.
To account for possible secular trends, we stratified the analysis
by year of birth. We included the following factors as potential
covariates in regression models: birth weight (!2500 vs. >2500 g),
sex, breast-feeding practices (divided a priori into tertiles), family
size, house type and location, water sources and usage, hygiene
practices, presence of pets, and maternal education. Variables were
considered independent predictors if the 95% confidence interval
associated with the point estimate did not include 1. The initial
model suggested that only low birth weight and subdivision of
residence were independent predictors. The proportional hazards
assumption was met for the independent predictors. Additional
variables were included as confounders if they changed the hazard
ratio of either independent predictor by 110%.
STATA software (release 5.0; STATA, College Station, TX) was
used for all statistical analyses.
Results
We enrolled 189 children during the study period. Children
had a mean of 543 days (range, 9–1350) of follow-up, and the
Table 1.
Brazil.
169
cumulative number of days of observation was 102,681. A total
of 157 children (83%) were followed for at least 3 months. There
were 1476 episodes of diarrhea identified in 153 children, accounting for 7581 days of illness; the rate of diarrhea was 5.25
episodes/child-year. The mean number of days per diarrheal
episode was 5.1 (SD, 6.3); the median number of days was 3.
Of these episodes, 371 (25.1%) represented single-day diarrheal
illnesses, 987 (66.9%) represented acute diarrhea longer than 1
day in duration, 102 (6.9%) represented persistent diarrhea, and
16 (1.1%) represented chronic diarrhea. We obtained samples
from 611 (41.4%) of diarrheal episodes. Overall, there were 1091
stool samples tested during the study period. Only 36 children
(19%) experienced no episodes of diarrhea. Among the 157
children with 13 months of surveillance, 9 had no recorded
episodes of diarrhea.
We identified Cryptosporidium oocysts in 58 (31.2%) of 189
cohort children and in 58 (36.9%) of 157 children followed for
at least 3 months. Of 1054 specimens from unique diarrheal
episodes or from nondiarrheal or indeterminate stools, we identified Cryptosporidium oocysts in 73 (6.9%; table 1). Cryptosporidium oocysts were found more frequently in stools from
episodes of persistent (16.5%) and acute (8.4%) diarrhea than
in nondiarrheal stools (4.0%; P ! .0001 , x2, and test for trend).
No symptomatic infections with Cryptosporidium were identified in children !3 or 130 months of age. The mean age at
diagnosis of the first symptomatic Cryptosporidium episode was
11.9 months. Cryptosporidium infection in this community was
highly seasonal (figure 1). The percentage of laboratory specimens positive for Cryptosporidium oocysts ranged from 16.4%
during the rainiest month of the year (April) to 0% during 4
of the 5 driest months of the year (August–December).
Among the 12 asymptomatic children with a stool specimen
positive for Cryptosporidium oocysts, 3 had short (1 or 2 days)
episodes of diarrhea between 16 and 22 days prior to the positive nondiarrheal stool, and 3 had previous episodes of symptomatic cryptosporidiosis between 2 and 22 months earlier. The
other 6 children had no diarrhea within a month of the Cryptosporidium-positive stool. In addition, there were 2 children
with positive stool samples that were deemed indeterminate.
One child had a short episode of diarrhea 6 days following the
positive stool; the other had 2 days of diarrhea 9 days before
the positive stool and then a 5-day episode 11 days later.
Identification of Cryptosporidium oocysts in laboratory samples from children in northeastern
Group
Nondiarrheal (>14 days from any diarrheal episode)
Acute diarrhea (!14 days’ duration)
Persistent diarrhea (>14 days’ duration)
Indeterminate (collected 13 but !14 days from diarrheal episode)
Total
a
No. total
No. positive
%
P
299
514
97
144
1054
12
43
16
2
73
4.0
8.4
16.5
1.4
6.9
)
.02
b
!.001
.24
NOTE. Data do not include 37 samples obtained as follow-up within diarrheal episodes.
a 2
x test, vs. nondiarrheal stools.
b
x2 test, persistent vs. acute, P 5 .003 ; x2 test, 2 3 3 table (excluding indeterminate), P ! .001 ; x2 test for trend (excluding
indeterminate), P ! .001.
170
Newman et al.
JID 1999;180 (July)
Figure 1. Seasonal distribution of Cryptosporidium-positive stools compared with monthly rainfall totals (mm), Fortaleza, Brazil, August
1989–April 1993.
We found a total of 15 cases of recurrent Cryptosporidium
infection among 13 children, which includes the 3 cases of secondary asymptomatic infection (table 2). In 6 cases (5 children),
the 2 positive stool specimens were in close succession (!14day gap between diarrheal episodes); in 4 cases (3 children),
there was a moderate gap of 1–2.5 months; and among the
remaining 5 children, there were gaps of 13–22 months. Two
children who had persistent diarrhea at the time of presumptive
primary infection had secondary episodes of acute diarrhea,
whereas 2 children had a converse experience: acute diarrhea
followed by persistent diarrhea during secondary infection.
Twelve children who were not diagnosed with recurrent Cryptosporidium infection nevertheless had a relapse of diarrhea
within 7 days of the completion of the primary episode. These
relapses occurred a median of 5 days later and lasted a median
of 3 days. Two children had a second relapse within 1 week of
the first relapse. In 2 of these 14 total relapses, a stool sample
was obtained and was negative for Cryptosporidium oocysts;
stool samples were not submitted for the other episodes.
Demographic and nutritional characteristics of the 59 children with symptomatic Cryptosporidium infection are shown in
table 3. The mean age of all children was 13.7 months (range,
2.7–42.5); age did not differ between children with acute or
persistent diarrhea. Mild malnutrition was common, and moderate or severe malnutrition (stunting, underweight, or wasting)
was rare, regardless of the clinical course of Cryptosporidium
infection. Exclusive breast-feeding averaged 6 weeks, and any
breast-feeding averaged 48 weeks. Just over 50% of children
had pets in the home, and just under 50% had running water.
No demographic or nutritional characteristics were significantly
different between those with acute or persistent diarrhea.
Cryptosporidium-associated diarrhea ranged from 1–31 days,
and the median duration was lengthy (6 days). The range in
the peak number of stools in a single 24-h period was also great
(3–20); the associated means (in peak stool output) between
those with acute diarrhea (5 stools) and persistent diarrhea (9
stools) differed significantly (table 4). The most common symptoms associated with cryptosporidial diarrhea were vomiting
JID 1999;180 (July)
Cryptosporidium Infection in Brazil
Table 2. Recurrent Cryptosporidium infections in children in northeastern Brazil.
Child
no.
1
2
b
2
3
4
5
6
7
8
b
8
9
10
11
12
13
Gap between
a
diarrheal episodes
1st episode type
2d episode type
2 days
2 days
6 days
3 days
9 days
12 days
37 days
37 days
2.5 months
2.5 months
13 months
19 months
22 months
22 months
22 months
Acute
Acute
Acute
Acute
Acute
Acute
Persistent
Acute
Acute
Acute
Persistent
Acute
Acute
Acute
Acute
Acute
Acute
Acute
Acute
Acute
Acute
Acute
Acute
Acute
Asymptomatic
Acute
Persistent
Persistent
Asymptomatic
Asymptomatic
NOTE. Acute diarrhea, episode !2 weeks in duration; persistent diarrhea,
episode lasting 14–29 days.
a
If one stool sample was asymptomatic, time was measured from end of
episode to time of stool sample.
b
For children with 12 episodes, the second row contains 2d and 3d episode
types.
and tactile fever, although even these were found in !50% of
cases. These symptoms did not differ between those with acute
or persistent diarrhea. Mild dehydration was rare, and moderate to severe dehydration was not observed. Concomitant
diarrhea in other family members was common. A significantly
higher percentage of children with persistent diarrhea received
antibiotics during their illness than did those with acute diarrhea (67% vs. 34%, P 5 .05).
Laboratory characteristics of 72 Cryptosporidium-positive
stool samples are also shown in table 4. One-quarter of nondiarrheal stools had a liquid consistency upon laboratory examination. Although visible blood was observed in none of the
171
specimens, occult blood was found in ∼18%. Fecal fat was
common in the stools of symptomatic individuals but nonexistent in nondiarrheal stools; reducing substances were rare.
Fecal leukocytes were seen by direct microscopy in approximately one-third of stools from symptomatic children but in
no nondiarrheal stools. Fecal lactoferrin, a more sensitive
marker for the presence of fecal leukocytes, was also detected
only in symptomatic children. There was no significant association between fecal leukocytes or fecal lactoferrin and the
presence of any copathogen (data not shown). No stool characteristics were significantly more common in persistent diarrheal stools than in acute diarrheal stools.
Potential copathogens detected in stools of cohort children
with Cryptosporidium infection are shown in table 5. Enteroaggregative E. coli, enterotoxigenic, diffusely adherent E. coli,
Giardia lamblia, and helminths were the most commonly identified potential copathogens. Entamoeba histolytica, rotavirus,
adenovirus, IEC, and EHEC were not identified in any stool
specimens. In addition, we found no Vibrio species, Y. enterocolitica, or C. jejuni in any Cryptosporidium-positive stool
(data not shown). We found no difference in the prevalence of
copathogens between acute and persistent diarrheal stools. No
test for trends for stool copathogens among the 3 groups was
statistically significant.
In a Cox proportional hazards model, we found that low
birth weight and residence in the most crowded subdivision
within the slum were associated with an increased risk of symptomatic Cryptosporidium infection, after adjusting for weeks of
exclusive breast-feeding, sex, type of house, and previous childhood deaths in the home (table 6). Children weighing !2500 g
at birth were at a 15-fold increased risk compared with all other
children of having a symptomatic episode of Cryptosporidium
infection detected.
Table 3. Demographic and nutritional characteristics of 59 children in northeastern Brazil with symptomatic Cryptosporidium infection.
Characteristic
Age, months
Male sex
Breast-feeding, weeks (37, 12)
Exclusive breast-feeding, weeks (40, 14)
Not weaned
Height-for-age Z score, mean (42, 15)
!2 SD height-for-age Z score (42, 15)
Weight-for-age Z score, mean (43, 15)
!2 SD weight-for-age Z score (43, 15)
Weight-for-height Z score, mean (42, 15)
!2 SD weight-for-height Z score (42, 15)
No. residents/no. rooms, mean (41, 16)
Running water in home (37, 16)
Pet in home (42, 16)
Acute diarrhea
(n 5 43)
Persistent diarrhea
(n 5 16)
P
12.3 (6.5)
26 (60)
46.2 (38.9)
6.2 (6.4)
18 (42)
20.87 (1.2)
9 (21)
20.45 (1.1)
2 (5)
0.15 (0.9)
0 (0)
3.58 (1.4)
17 (45)
15 (36)
14.3 (7.2)
9 (56)
54.1 (37.6)
6.0 (7.5)
10 (63)
21.20 (0.8)
4 (27)
20.49 (1.4)
2 (13)
0.44 (67)
0 (0)
3.53 (2.3)
10 (63)
7 (44)
.30
.77
.75
.75
.16
.33
.73
.93
.27
.36
1.00
.92
.31
.62
NOTE. For continuous variables, data are mean (SD), P value from t test or Wilcoxon
rank sum test. For dichotomous variables, data are no. (%), P value from x2 test or Fisher’s
exact test. Acute diarrhea, episode !2 weeks in duration; persistent diarrhea, episode lasting
14–29 days. If information was incomplete, n for acute and persistent diarrhea, respectively,
is listed beside characteristic.
172
Newman et al.
JID 1999;180 (July)
Table 4. Clinical and laboratory characteristics of 59 children in northeastern Brazil with
symptomatic Cryptosporidium infection.
Characteristic
Acute diarrhea
(n 5 43)
Persistent diarrhea
(n 5 16)
5.1 (2.3)
4.1 (1.1)
19 (44)
15 (35)
4 (9)
0 (0)
10 (32)
12 (34)
5 (14)
14 (35)
9 (4.2)
5.1 (2.0)
8 (50)
10 (63)
3 (19)
2 (13)
2 (20)
8 (67)
4 (33)
6 (43)
Clinical
Peak no. of stools/day, mean
Mean no. of stools/day, mean
a
Vomiting
a
Tactile fever
a,b
Mild dehydration
Hospitalized
Antibiotic use in previous 30 days (n 5 31, 10)
Antibiotic use during illness (n 5 35, 12)
Antiperistaltic during illness (n 5 34, 12)
Family member with diarrhea (n 5 40, 14)
Laboratory
c
Occult blood
Visible mucous
Fecal fat (n 5 27, 12)
Reducing substances
Fecal leukocytes
d
Fecal lactoferrin (n 5 21, 4)
6
9
4
2
12
14
(14)
(21)
(15)
(5)
(28)
(67)
5
3
5
0
6
3
P
!.0001
.03
.69
.06
.38
.07
.69
.05
.21
.60
(31)
(19)
(42)
(0)
(38)
(75)
.15
1.00
.10
1.00
.53
1.00
NOTE. For continuous variables, data are mean no. (SD), P from t test or Wilcoxon rank sum
test. For dichotomous variables, data are no. (%), P from x2 test or Fisher’s exact test. Acute diarrhea,
episode !2 weeks in duration; persistent diarrhea, episode lasting 14–29 days. If information was incomplete, n is listed beside characteristic for acute and persistent diarrhea, respectively.
a
Recorded as positive if it occurred any time during illness.
b
No moderate or severe dehydration was recorded for any Cryptosporidium-positive children.
c
No visible blood was found in any laboratory specimens.
d
Stools from children with ongoing breast-feeding were not tested because of cross-reactivity with
breast milk.
Discussion
These data demonstrate that Cryptosporidium infection is
common among children !4 years of age in a Brazilian slum
community, affecting nearly one-third of all children followed.
Cryptosporidium was isolated significantly more frequently
from children with diarrhea than from asymptomatic children;
children with persistent diarrhea were also more likely to have
Cryptosporidium oocysts identified in their stools than those
with acute diarrhea. This association with persistent diarrhea
is remarkably similar in magnitude to that found in Guinea
Bissau, West Africa [8]. In children with more than one documented episode of Cryptosporidium infection, persistent diarrhea was equally likely to be associated with either episode.
Consistent with findings in other longitudinal studies in the
developing world [26, 27], we found that cryptosporidiosis was
a highly seasonal disease: throughout the 4-year study period,
recovery of Cryptosporidium oocysts occurred almost exclusively in months in which there was significant rainfall. It is
Table 5. Potential copathogens associated with 72 Cryptosporidium-positive stool samples from children in
northeastern Brazil.
Pathogen
Enteropathogenic Escherichia coli
c
Enterotoxigenic E. coli
Enteroaggregative E. coli
Diffusely adherent E. coli
Salmonella or Shigella species
Giardia lamblia
Any helminth
Nondiarrheal
(n 5 12)
1
2
5
0
0
0
3
(8.3)
(16.7)
(41.7)
(0)
(0)
(0)
(25.0)
a
Acute diarrhea
(n 5 44)
2
6
17
6
2
3
6
(4.5)
d
(14.0)
(38.6)
(13.6)
(4.5)
(6.8)
(13.6)
a
Persistent diarrhea
(n 5 16)
0
1
8
2
0
2
1
(0)
(6.3)
(50.0)
(12.5)
(0)
(12.5)
(6.3)
a
P, diarrheal
vs.
b
nondiarrheal
P, acute
vs.
b
persistent
.45
.64
1.00
.33
1.00
.58
.35
1.00
.66
.56
1.00
1.00
.60
.66
NOTE. Indeterminate stools (n 5 2 ) were excluded from table. Where microbiology was incomplete, n is listed beside pathogen.
Forty-seven stools tested for enteroinvasive E. coli and enterohemorrhagic E. coli were all negative. Sixty-one stools tested for
rotavirus and adenovirus were all negative. Acute diarrhea, episode !2 weeks in duration; persistent diarrhea, episode lasting 14–29
days.
a
Values are no. (%).
b
Fisher’s exact test.
c
Heat-stable toxin only, n 5 4; heat-labile toxin only, n 5 4; both, n 5 1.
d
n 5 43 for this group.
JID 1999;180 (July)
Cryptosporidium Infection in Brazil
173
Table 6. Cox proportional hazards model for first symptomatic Cryptosporidium
episode (Stratified by year of birth) in children in northeastern Brazil.
Characteristic
Low birth weight (!2500 g)
Residence in most crowded subdivision
Exclusive breast-feeding
!14 days
14–41 days
>42 days
Unknown no. of days
Nonbrick house (adobe or mixed)
Any previous childhood deaths in household
Male sex
notable that no children 13 years of age were diagnosed with
symptomatic Cryptosporidium infection in this cohort, although
previous work in this community has demonstrated sporadic
symptomatic secondary cases among older children and adults
in households with an index case [28].
We found that both fecal leukocytes and fecal lactoferrin
were common in the stools of children with symptomatic Cryptosporidium infection. This unexpected finding may be explained by recent data that indicate that Cryptosporidium infection of intestinal epithelial cells stimulates cellular
production of interleukin-8. Production of interleukin-8 may
lead to the mucosal recruitment of leukocytes and contribute
to stimulation of secretion in Cryptosporidium infection [29].
In multivariate analysis, we found that children with low
birth weight were significantly more likely to be diagnosed with
cryptosporidiosis. Whether this is a result of ongoing immunologic immaturity or is related to persistently lower weightfor-age and height-for-age Z scores for these children prior to
diagnosis, when compared with other children (data not
shown), remains unclear. There did not appear to be any risk
associated with self-reported water usage patterns, perhaps because scattered water sources in the community as well as the
city water supply have been shown to be contaminated [30]. In
addition, we found that children in the most densely crowded
subdivision (with open sewers) within the slum were indeed at
greater risk for cryptosporidiosis. It is likely that the use of
data from the full cohort and the use of multivariate analysis
(Cox regression) unmasked geographic location as a risk factor
in this analysis, compared with our previous univariate analysis
of this same population [11].
These findings suggest that in developing world settings, there
may be a blurring between the typical pattern of epidemic Cryptosporidium disease with a single-source exposure and endemic
disease, in which person-to-person transmission has been presumed to play a dominant role. Both the current finding that
crowding appears to be a risk factor and previous evidence that
there is a high incidence of secondary infection among household members of index cases in this community [28] suggest
endemic disease. However, we have previously demonstrated
that various water sources in this community, including the city
water supply, are contaminated with Cryptosporidium oocysts
Hazard ratio
P
95% confidence interval
5.34
3.18
.007
.001
1.57–18.2
1.55–6.48
1
0.48
0.79
0.09
1.57
1.62
1.59
)
.07
.57
.002
.30
.24
.15
Reference group
0.22–1.07
0.36–1.75
0.18–0.41
0.67–3.67
0.73–3.62
0.84–2.99
[30], suggesting that epidemic infection may also play a role.
Experimental infections in healthy volunteers have demonstrated that the mean infective dose for Cryptosporidium is low
(132 oocysts) [31] and that the quantity of oocysts excreted in
the stools of infected individuals can be enormous (up to a
billion oocysts per infection) [32], indicating the ease with which
this infection might be amplified in crowded communities. In
poor urban areas such as this, it may be that seasonal rains
amplify ongoing low-level water source contamination to produce increased numbers of infections and that person-to-person
transmission then further sustains endemic disease in the community until the return of the dry season, when the number of
cases again becomes negligible.
We found that a pattern of intermittent diarrhea associated
with Cryptosporidium infection was moderately common, consistent with data from the large waterborne outbreak in Milwaukee in 1993 [3, 33]. In that study, 39% of subjects reported
the recurrence of diarrhea after >2 days of normal stools. This
pattern of recurrent diarrhea brings into question the WHO
criteria for diarrheal episodes, in which the recurrence of diarrhea after 2 symptom-free days marks a new episode [14].
This definition may need to be revised in areas with endemic
cryptosporidiosis. We propose that the term “persistent relapsing diarrhea” may be a better description of disease pattern
in children with diarrheal episodes separated by a week or less,
particularly for children in whom Cryptosporidium infection has
been diagnosed.
Our conclusions about risk associations with first Cryptosporidium episodes are limited by the fact that Cryptosporidium
infection, unlike measles, for example, does not have a pathognomonic clinical presentation. Because the causes of diarrhea are multiple, stool examination is necessary for diagnosis
of cryptosporidiosis. Although our sampling rate was quite
good (41%), we are obviously missing many cases of symptomatic Cryptosporidium infection and many more asymptomatic
infections. This fact is underscored by data from this same
community showing that antibodies to Cryptosporidium are
present in 95% of tested individuals and that infection, whether
it is detected or not, is nearly ubiquitous [28]. In addition, our
sensitivity in the detection of pathogenic E. coli was reduced
by our inability to test 11 strain per sample. Finally, our model
174
Newman et al.
of risks associated with symptomatic Cryptosporidium infection
must be interpreted with caution. The small numbers of failures
(symptomatic Cryptosporidium infections) are reflected in the
wide confidence intervals for all covariates, even those found
to be statistically significant.
Consistent with previous studies, we found that even in an
endemic setting, infection with Cryptosporidium results in heterogeneous disease. Short-term adverse outcomes (e.g., dehydration, hospitalization) were rare, possibly because of our clinical interventions (e.g., oral rehydration solution, maternal
education) or a muted clinical course in this highly endemic
setting, compared with adverse outcomes in naive children exposed under outbreak conditions [34]. The fact that we were
unable to find relationships between illness duration and either
infection with copathogens or demographic and nutritional
characteristics suggests the existence of a yet-to-be-defined hostparasite interaction, even among children who are clinically
immunocompetent. A recent case report of a child with chronic
cryptosporidiosis and interferon-g deficiency [35], as well as
experimental evidence from animal models regarding interferon-g and other cytokines [36–38], suggests that these mediators may play a role in the course of human disease.
In addition, there is recent evidence of genetic heterogeneity
among C. parvum isolates, although the clinical significance of
these differences in endemic disease has yet to be elucidated
[39]. Further work in characterizing the immunologic status of
individuals with cryptosporidiosis and clarifying subtypes of C.
parvum, and in attempting to correlate this information with
clinical course, might begin to explain the relatively protean
presentations of this disease.
In summary, we found that in this Brazilian slum, early childhood infection with Cryptosporidium oocysts was very common, highly seasonal, and strongly associated with persistent
diarrhea. Low–birth-weight children and those living in densely
crowded subdivisions appear to be at greater risk of symptomatic infection. Disease course was highly variable and was
not associated with the presence of copathogens. Recurrent
Cryptosporidium infection and relapsing diarrhea associated
with it were common. In light of these data, the applicability
of the current WHO diarrheal definitions to Cryptosporidiumassociated diarrheal episodes may need to be reconsidered.
Acknowledgments
We thank Sayonara Alencar and Luzia Sousa de Melo for invaluable
field assistance; Isabel McAuliffe, Jania Teixeira, Maria do Carmo
Nunes de Pinho, Leah Barrett, and Conceição Nogueria Raulino for
laboratory assistance; the people of Gonçalves Dias for their cooperation; and David K. Shay, Stephen Gloyd, and Robert L. Davis for
thoughtful reviews of the manuscript.
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