0095-1137/86/061109-05$02.00/0
CopyrightC 1986, American Society for Microbiology
Cryptosporidium
spp., a
Frequent
Cause of
Diarrhea
in
Liberian Children
NIELS H0JLYNG,* KÂRE M0LBAK, AND S0REN JEPSEN
MalariaResearch Laboratory, Department ofTreponematoses, Statens Seruminstitut, Copenhagen, Denmark Received 21 October1985/Accepted 28 February 1986
Thisreport presentsresults fromasamplesurveydesignedtoinvestigatethepossibleroleof Cryptosporidium
spp. inchildhood diarrhea inadevelopingcountry, Liberia,WestAfrica. Duringthe
four
monthsof January toApril 1983, ahouse-to-house studywascarried outintwogeographicallyandsocially differentcommuni-ties-an urban slumandthreeruralvillages. Stoolsamplesfrom 374children, aged 6to59 months,weretested forCryptosporidiumspp.Amongthe children with diarrhea 8.4%wereCryptosporidiumspp.positivecompared
with a prevalence rate of 5.9% in asymptomatic children. Of the children living in a household with a Cryptosporidium spp.-positive index child, 8.6% hadapositive stool sample.Ofallchildren attendingaclinic because of diarrhea, 14.6% were Cryptosporidium spp. positive. Cryptosporidiosis was more frequent in
youngerchildren; 24 of the total of 29 positivecases(83%)werebelow 2.5yearsold. Actualorpreviousbottle
feeding(formula)was a riskfactor, particularly in children below 18 monthsold. Ofthebottle-fedchildren, 28%wereCryptosporidiumspp.positiveversus9.1%of childrenneverbottlefed. Crowding isanotherpossible risk factor. The prevalence of cryptosporidiosis was 13.5% in big urban households with more than 10
children, whereasthe prevalence in the smallurban households was6.1%. Ethnic andreligious differences were particularly evident in the rural area. No Muslim households had cryptosporidiosis, whereas the prevalence in non-Muslim tribes was9%. Thegeneral belief that cryptosporidiosis is primarily azoonosis is
questionedinthisstudy, partly becausemanycarriers and asymptomatichouseholdcontactswerefound.
Inmany developing countries gastroenteritis is the single biggest causeof early childhood mortality (19). In
industri-alized countries gastroenteritis is rarely fatal, but it is an
important health and socioeconomic burden. Despite mod-erndiagnostic techniques, approximately half of the known
casesofgastroenteritis remainwithoutetiological diagnosis,
making causal therapy difficult.
Enteric cryptosporidiosis, caused by the coccidian para-siteCryptosporidium sp., hasbecomeofincreasing interest since the firstcaseof human cryptosporidiosis wasreported
in 1976 (15). Much of this interest is dueto itsopportunistic behavior in immunocompromized patients, particularly in those withacquired immunedeficiency syndrome (6, 12, 14, 16; Editorial, Lancet, i:492-494, 1984). In these im-munocompromized patients, cryptosporidiosis oftenoccurs
as asevere, persisting waterydiarrhea, withfluid lossesup
to 15 liters per day. A variety of therapies havebeentried unsuccessfully (4), but accordingto recentreportsfrom the Centers for Disease Control, Atlanta, Ga., spiramycin has shown some effect (5). In well-nourished individuals with normal immune functions, the disease often occurs as a
short, self-limiting gastrointestinal illness (14, 16).
Before1982,diagnosis of human cryptosporidiosis usually required intestinal biopsies. Better and faster methods for detecting the infectiveoocystinstoolsamplesnowfacilitate large-scale screening (8,10, 11). At the time of thissurveyDo information had been published on cryptosporidiosis in
developing countries, and the study was therefore
under-taken to elucidate epidemiological and clinical aspects of human cryptosporidiosis in children aged 6 to 59 months froma developingcountry, Liberia, West Africa.
*Corresponding author.
MATERIALS ANDMETHODS
The study was carried out during the dry and late dry
season, JanuarytoApril 1983.
Study population. Two communities, anurban slum and three rural villages, were selected and geographically and socially stratifiedtoenablecomparison. Social stratification
wasbasedontheincome andproperty level ofahousehold
and the educational level and socialstatusinthecommunity of allmembers ofa household.
The urbanslum, WestPoint, wasa shanty town situated
on a 0.25-km2 peninsula extending from the capital city,
Monrovia, into the Atlantic Ocean. The
estimated
popula-tion was 30,000, living primarily in one-storey tin shacks14
12-
10-
6-
4-prevalence of Cryptosporidium % prevalence of:
1001
G.lambliaor
Ehistolytica helminths enteric parasites 60
00
20
2 3 4 Ae inyears
FIG. 1. Age-specific prevalences of Cryptosporidium sp. and
other intestinal parasites. Enteric parasites were G. lamblia, E.
histolytica, T. trichiura, A. lumbricoides, hookworm, and S. stercoralis.
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TABLE 1. Data on29Cryptosporidium sp.-positive children
Days Campy- Follow-up
Case Age Sex Diar- of Vomit- Abdominal Fever Bottle Other intestinal lobacter Pathogenic
Crypto-no. (mo) Region rhea diar- ing pains fed parasites bacterium Diar- YPO
rhea
p.rhea
sporidiumsp.
1 8 F WPa + 7 - + + - +T.T.b - EPECé
2 9 ?" WP + 4 - + + + - +
3 9 F WP + 3 - - + - +T.T.
-4 10 M WP + 7 + + - +
-5 7 M WP + 2 + - + +
-6 24 M WP + 5 - - - -
-7 29 M WP - - -
-8 43 F WP + 2 - - - - + T.T. ? Shigellaboydii
9 48 M WP + 5 + + - + + G.L.,eT.T.,
H.W!
10 10 M WP + 5 - - - -
-il 15 F WP - - - + - + + +
12 22 M WP + 6 - - - - + T.T., H.W.
-13 20 M WP + 2 - ? - - + T.T. - Shigellasonneé
14 24 F WP + 30 - + - + +T.T. +
15 25 M WP + 30 + + - + +G.L., T.T., +
H.W.
16 52 M WP + 2 ? ? - + +G.L., A.L.,g +
T.T.
17 6 F Bong + ? ? ? +
18 il F Bong + 2 ? ? ? - + H.W. - EPEC -
-19 14 M Bong - - - + - + Plesiomonas
shigelloides
20 12 F Bong - - - - + G.L.
21 12 F Bong + 3 - - - + +H.W. - - +
22 20 M Bong - - ? ?
23 27 M Bong - - ? ?
24 31 F Bong - - - - + A.L. - +
25 9 M Bong + 6 + + +
26 16 M Bong - - + +
27 38 M Bong - - - + + T.T. +
28 6 M Bong + 3
29 22 ? Bong + 7 - + +
a
WP,
WestPoint.bT.T., T. trichiura.
' EPEC,EnteropathogenicE.coli. d
?,
Noinformation.eG.L., G.lamblia. fH.W., Hookworm. g A.L., A.lumbricoides.
with few or nofacilities. Thecommunitywas served by one public health clinic without a permanent doctor and one school. In the rural area, Bong county, situated 200 km inland in a forest area, three typical villages with a total population of approximately 3,500 were selected. People lived inadobe houses with no facilities, but a small public health clinic and a school were functional in two of the villages.
A household was defined as an extended family living together and sharing one cooking pot. Only children aged 6
to 59
months
were included. Households were visitedre-peatedlyuntil allchildren living for more than 4 weeks in the household were seen. The refusal and dropout rates were less than4%in bothareas.
In all households the medical history was recorded, and the children were physically examined by a physician. A freshly voided stool and a blood sample were collected from each child. To consider a child as having diarrhea, two criteriahad to be fulfilled. The child should have diarrhea, accordingtoitsparents, and thefecalsampleshould take the shape ofthe stoolcontainer used.
Group Acomprised 41 children with diarrhea (mean age,
20.5months; range, 6 to 58 months) seen at the public health clinic, West Point, and 20 household
contacts
(11 with diarrhea) ofCryptosporidium sp.-positivechildren. All stool samples were examined forcryptosporidiae.Group Bincluded 284 West Point children with or without diarrhea living in 144 households randomly sampled from a house-to-house map. Stools were examined for cryp-tosporidiae in 80 children with diarrhea (mean age, 33.2 months; range, 6 to 59 months) and in a further 65 randomly picked children without diarrhea with similar age distribu-tion asgroups A and B. Another 13 çhildren without diarrhea were examined as household contacts of Cryptosporidium sp.-positive children.
Group Ccomprisedall of thechildren in the three villages in Bong county. From 260 households, 518 children were examined. Stools were examined forcryptosporidiaein 105 children with diarrhea (mean age, 30.6 months; range, 6 to 59 months) and in a further 37 randomly picked children with-out diarrhea with a similar age distribution. Another 13 children without diarrhea were household contacts of posi-tive children. Children (n = 191) fromgroups A,
B,
and C were reexamined after 1 month.on April 11, 2020 by guest
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TABLE 2. Age-specific prevalenceforCryptosporidium sp.
Cryptosporidium-positive children in":
Age Group A Group B Group C GroupsA, B, andC
(mo) Nositive/. % Avg% in Nositive/ % Avg% in
Npositive!
% Avg% in positive! %Avg
% intotal age groups total age groups total age groups total age groups
6-11 5/22 22.7 18.4 1/21 4.8 7.6 4/23 17.4 13.6 10/66 15.2 12.1
12-17 0/5 1/25 4.0 5/19 26.3 5/49 10.2
18-23 0/5 2/13 15.4 1/17 5.9 4/35 11.4
24-29 2/6 33.3 2/20 10.0 1/22 4.5 5/48 10.4
30-35 0/4 8.7 0/18 1.4 1/20 5.0 2.7 1/42 2.4 2.9
36-41 0/4 0/8 1/16 6.3 1/28 3.6
42-47 1/4 25.0 0/22 0/12 1/38 2.6
48-53 1/3 33.3 1/13 7.7 0/8 2/24 8.3
54-59 0/8 0/13 0/18 0/39 0
Unknown 0/5 0/5 0
a The percentages of positive children in groups A, B, and C were14.8,4.4, and8.4%,respectively,and theoverall percentage was 7.8%.
Microbiological methods. Stools were collected in sterile containers and kept in an insulated box with ice packs (temperature,5 to 10°C) until processingwithin 24 h.
Two slides withunconcentrated stool, one unstainedand oneiodine stained (Lugols),wereexaminedfor identification of Entamoeba histolytica and Giardia lamblia cysts; Trichuris trichiura, Ascaris lumbricoides, and hookworm ovae;and Strongyloides stercoralislarvae and ovae. Speci-mens were preserved in 10% Formalin and later concen-tratedby the method of Ritchie as improved by Allen and Ridley (1). A thick smear was made from the sediment, which was then air dried and fixed in a flame and with methanol. The smears were stained by a modified Ziehl-Neehlsen technique,asdescribed by Henriksen and Pohlenz (10). Because a large amount of material took up car-bolfuchsin,along staining and decolorizing periodwasused. Known Cryptosporidium sp.-positive slides were stained with each batch as a control. Stained preparations were examined under oil immersion at amagnification ofx 1,000. A
preparation
wasconsiderednegative onlyafterscreening ofmorethan 200fields.Thebacteriological examination included culturing within 24haftersampling for Shigella sp.,Salmonellasp., Yersinia enterocolitica, and Escherichia coliby routine methods and media from the Department of Diagnostic Bacteriology, Statens
Seruminstitut,
Copenhagen, Denmark.Ail
isolated colonieswerekept
inbeefextractagar, and within1month theyweresent to thelaboratoryattheStatens Seruminstitut forverification andfurtheridentification.Strains isolatedfrom diarrheic children
aged
lessthan 30 months were, if identified biochemically as E. coli, serotyped and tested forproduction
of heat-labileen-terotoxin by the Y-1 adrenal cell assay and heat-stable enterotoxin by thesucklingmouse assay.
For identification of
Campylobacter
sp.,culturing
was done onSkirrowmedium (18).Theplateswereincubatedat35°C
in an anaerobicjar
with an activated GasPak and catalyst (BBLMicrobiology Systems,
Cockeysville, Md.)
for48 h.
Suggestive
campylobacterlike
colonieswereexam-inedby phase-contrast
microscopy
andcategorized
asCam-pylobacter
sp. whenspiral
orcurved rodsexhibiting
typical
darting motility
wereobserved.Swabsweretaken fromall ofthe freshfecal
samples,
kept
in Stuart transportmediumat room
temperature (20
to25°C),
and cultured inand seededfrom seleniteenrichmentmedium within 1 month for identification of Salmonella sp. and Y. enterocolitica. A suspension from the swabs was used to examine for rotavirus by enzyme-linked immunosorbent assay(Dakopatts, Copenhagen, Denmark).
Thechi-squaretest with Yatescorrection and the Mantel-Haenszel test were used when appropriate for statistical comparison. A Pvalue less than 0.05 wasconsidered signif-icant.
RESULTS
Cryptosporidium oocysts were isolated from 29 of 374 children examined.Data on thesechildrenaregiven in Table 1.
Of children with diarrhea, 8.4% (20 of 237) were
Cryptosporidium
sp.positive
compared with 5.9% (6 of 102) ofchildren without diarrhea and 8.6% (3 of35) ofchildren from a Cryptosporidium sp.-positive household. The differ-ences were not significant. Of the nine Cryptosporidium sp.-positive children without diarrheaon the dayof exami-nation, six had a history ofdiarrhea during the preceding 2-weekperiod.Childrenattending WestPoint clinic(groupA) complain-ing of diarrheahad ahigher prevalenceofcryptosporidiosis
(14.6%;
6of41) thandidchildrenwith diarrheain groups B(7.5%;
6of80)andC(5.7%;
6of105). Amongtheyoungest clinicchildren, aged6 to 11months,
theprevalencewas25% (5 of 20). The observed differencesbetween theprevalences
in the urban groups, A versus B, and between the sample groups, B versusC, arenot
significant.
Thepeakprevalencewasin the age group 6 to 11 months
(14.9%),
and24 of the 29positive
children were below 2.5 yearsofage(X2
= 9.4; P < 0.01) (Table 2).The meanage was 20months.The
age-specific
prevalences of other entericparasites
examined forare shownin Fig. 1. Prevalencesof intestinal parasitoses increased with age and stayed
high
throughout
childhood. In the older
children,
42 to59months,
approxi-mately 90%hadintestinal
parasites.
Cryptosporidiosis
wassignificantly
moreprevalent
among childrenbeing
orhaving
beengiven
any type offormula(bottle fed)
than among childrenneverbottlefed(16
versus5.7%;
X2
= 7.43; P < 0.01). This pattern was even morestrikingin the younger age groups below 18 months
(Table
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TABLE 3. PrevalenceofCryptosporidiumexcretorsin relationtohistory of bottle feeding
Bottlefed Neverbottlefed
Frequency of
Location and age range Total No. % Total No. % bottlefeeding
no.
Cryptosporidium
Positive no.Cryptosporidium
Positive (%)sp. positive sp.positive
WestPoint
Allages 68 8 11.8 136 7 5.1 33.3
Below 18months 21 4 19.1 48 3 6.3 30.4
Bong
Allages 7 4 57.1 147 9 6.1 4.5
Below18 months 4 3 75.0 40 5 12.5 9.1
West Point and Bong
Allages 75 12 16.0 283 16 5.7 20.9
Below 18 months 25 7 28.0 88 8 9.1 22.1
3). Forall children below 18 months ofage, the difference was significant (X2 = 4.5;P <0.05). Ofthe total number of Cryptosporidium sp.-positive children, 43% (12 of 28) had been bottle fed, whereas only 19% (63 of 330) of the
Cryptosporidium sp.-negative children had been bottle fed (X2 = 7.4; P<0.01). Noinfantsexclusively breast fedwere foundto excrete Cryptosporidiumoocysts.
Crowding was found to be associated with cryp-tosporidiosis. In the condensed urban slum with many big households, the prevalence of cryptosporidiosis in big households withmore than 10children below5yearsofage was20% ingroup A (2of10)and11.1% ingroup B(3of 27). The prevalence in households with fewer than 10 children below5 yearsofage was14.3% (7 of 49) ingroup Aand3.1% (4of 131) ingroup B. In therural
villages,
thecritical size of a household was found to be smaller. The prevalence of cryptosporidiosis in all of the villages was11.5% (3 of26)in householdswith fiveor morechildren and 7.8%(10of 129)in smaller households.In Bong county, 21 children who belonged to a Muslim tribewereexamined(mean age, 33.1months).Noneofthese children hadcryptosporidiosis, whereastheprevalencewas 9% inchildren from other tribes (mean age, 30.3 months).
No socioeconomic parameters other than crowding and bottle-feeding practices were found to correlate with cryptosporidiosis.
A total of 45 children had their stools reexamined for cryptosporidiae 1 month after the first examination, and 3
(6.7%)
werepositive. Of the45children, 6 werepositiveat the first examination, and 2 ofthese were still excretingCryptosporidium
oocysts 1 month later.DISCUSSION
Although human cryptosporidiosis was first reported in 1976 (15), it is only in the last few years, following the epidemic outbreak of acquired immune deficiency syn-drome, that an increase in studies on cryptosporidiosis has beenreported.
Very little information is available on the prevalence, significance, andprognosis ofCryptosporidium infection in children (9, 21), particularly in developing countries, where childhood diarrheaconstitutes a major problem (2, 13).
Inthissamplesurvey there was no significantdifferencein the prevalences of cryptosporidiosis among children with diarrhea, asymptomaticchildren, and household contacts of positive cases(8.4, 5.9, and 8.6%, respectively). However, two-thirds of the asymptomatic excretors (carriers) had a
history of diarrheain the 2 weekspriortoexamination. High carrierrates werealso found with other well-known patho-gens likeShigella spp. (3.7% in the group of children with diarrhea; 1.6% in the controlgroup)andCampylobactersp. (39.6%in thegroup of children withdiarrhea; 31.0% in the controlgroup). It is well documented in other studies from developingcountries, thoughnotwellexplained, thatmany asymptomatic infections occur when diarrhea is very pre-valentand fecal transmission andpoint prevalence of known pathogens arehigh (7).
Among allofthechildrenattending the clinic in the slum areaand
complaining
ofdiarrhea,
thusperhapshaving
moreintense diarrhea, the prevalence of cryptosporidiosis was
muchhigher
(14.6%)
than among the samplegroupchildren with diarrhea(6.5%). This pattern was not seen for any of the other intestinalparasites lookedfor and could indicate thatCryptosporidium infectioncauses a more severetypeof diarrheabutmightalsosimply be explained bythefactthat younger children are overrepresented among childrenat-tending the clinic. Larger studies would be neededto con-firm this.
The age distribution of cryptosporidiosis was very dif-ferent from the age distribution of the other intestinal parasitoses examined forinthis study. Whereas these other intestinal parasitoses were characterized by prevalences increasing with age to a
high
and stable level in older children, Cryptosporidium prevalence peaked in the age group 6 to 12 months and declined to a stable low level in older children(Fig. 1).These results agreewiththefew other reports from developing countries where similarage distri-butions ofcryptosporidiosisandrelationstootherintestinal parasiteshave beenreported. Thus,in ahospital study from Rwanda (2) the mean age of Cryptosporidium sp.-positive children was 13.3 months,andonly4of 20positivechildren had other entericparasites. InCostaRica(13) the mean age varied between rural (22.7 months) and urban (9.7 months) children, and only 3 of 12 Cryptosporidium sp.-positive children had other entericparasites. This reverse pattern in age-specific prevalence might reflect a different way of transmission or more likely the induction of protective immunity againsttheCryptosporidiumparasite. If the latter, this would facilitate the development of a vaccine for risk groups. More knowledge, particularly on the immune re-sponse in cryptosporidiosis, is needed to elucidate this.Oneaimof thisstudywastopinpointpossible risk factors of acquiring cryptosporidiosis. The prevalence of Cryp-tosporidiumexcretors wassignificantly higher in those
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dren beingorhaving been bottle fed for some period (P < 0.01). This was particularly true for children less than 18 months old. Interestingly, the two children who shed oocysts at thereexamination 4 weeks later were bothbottle fed and below 18 months of age. These results support a study from CostaRica, where a strong negativecorrelation was found between cryptosporidiosis and breast feeding (13). In the present study no infants exclusively breast fed were found to excrete Cryptosporidium oocysts.
Inthechildren beingbottle fed at thetime of the survey, the higherrate of infection was probably best explained by theuse of often contaminated food and bottles.
The reason why this association persisted even in older children notbeing further bottle fed is notclear. Apossible explanation is the earlier acquisition of infections added to theoftenvery poordietary bottle food, leadingto malnutri-tion and possibly impaired immune funcmalnutri-tion (17, 20).
Crowdingwasdifficultto define intheurban slum, where conditions were sobad that virtually everyone lived under similar crowded, unhygienic conditions. Crowding, ex-pressed as the number of residents (or children) per the number ofrooms, was morethan twiceashighasinthe rural area. Moreover, the average room was smallerin theurban slum. In this area the prevalence of cryptosporidiosis was highest in verylarge families withmorethan 10children. In rural villages with better-spaced housing conditions, the criticalfamily size seemedtobe smaller.Webelievethatthis association with crowding is indicative oftheimportance of person-to-persontransmission in these areas.
Theobservation that oocysts were not found in a single child from the Muslim tribeliving in Bongcountyisprobably explained by
differences
in hygienic practices based on areligious
background. Many Muslim households (47.1 com-pared with 17.7% in the non-Muslim tribes) hadprivate pit latrines. Better access to water, no domestic pigs, and traditions of hand washing may also play a part in the explanation.Inconclusion, this studysuggestswidespread existence of
Cryptosporidium
sp. in children in Liberia.Cryp-tosporidiosis was more prevalent in younger children and particularly in infants with severediarrhea.
The concept ofcryptosporidiosis as a zoonosis has
re-cently
beenquestioned
(3). Ourstudy
demonstratesahuman reservoir with carriers and household contacts; association between crowding,bottle-feeding,
andcryptosporidiosis
in regions where domestic animal areinfrequent;
andpeak
prevalence among infants still carried on their mothers' backs and thus not yetin intimate contact with theoutside world and animals. These
findings
indicate that person-to-persontransmission exists andmight
very well be themostimportant way of transmitting
Cryptosporidium
oocysts.ACKNOWLEDGMENTS
We thankAloysiusP.Hanson, Director of the Liberian Institute for BiomedicalResearch,under whoseauspicesthe survey in Liberia was carried out. Nils Axelsen, Department of Treponematoses, Knud Gaarslev, Department of Diagnostic Bacteriology, Fritz 0rskov and Ida 0rskov, Centre for E. coli, and Severin Olesen-Larsen, Department ofBiostatistics,areallacknowledgedfor their contributions. Grethe Wegener and Gunild Frederiksen are thanked for their valuable technical assistance. S. Â. Henriksen,
StatensVeterinæreSerumlaboratorium,Copenhagen,is thankedfor isassistance inconfirmingCryptosporidium-positive slides.
The studywas supported byDanish International Development
Agency grant104.Dan.8/341.
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