Cryptosporidium spp , a frequent cause of diarrhea in Liberian children

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 different

communi-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 shacks

14

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.

1109

I

)j

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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

sporidium

sp.

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 visited

re-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.

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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

% in

total 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 colonieswere

kept

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 for

production

of heat-labile

en-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).Theplateswereincubatedat

35°C

in an anaerobic

jar

with an activated GasPak and catalyst (BBL

Microbiology Systems,

Cockeysville, Md.)

for48 h.

Suggestive

campylobacterlike

colonieswere

exam-inedby phase-contrast

microscopy

and

categorized

as

Cam-pylobacter

sp. when

spiral

orcurved rods

exhibiting

typical

darting motility

wereobserved.

Swabsweretaken fromall ofthe freshfecal

samples,

kept

in Stuart transportmediumat room

temperature (20

to

25°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 11

months,

theprevalencewas25% (5 of 20). The observed differencesbetween the

prevalences

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 29

positive

children were below 2.5 yearsofage

(X2

= 9.4; P < 0.01) (Table 2).The meanage was 20months.

The

age-specific

prevalences of other enteric

parasites

examined forare shownin Fig. 1. Prevalencesof intestinal parasitoses increased with age and stayed

high

throughout

childhood. In the older

children,

42 to59

months,

approxi-mately 90%hadintestinal

parasites.

Cryptosporidiosis

was

significantly

more

prevalent

among children

being

or

having

been

given

any type offormula

(bottle fed)

than among childrenneverbottlefed

(16

versus

5.7%;

X2

= 7.43; P < 0.01). This pattern was even more

strikingin 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 excreting

Cryptosporidium

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

of

diarrhea,

thusperhaps

having

more

intense 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 children

at-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 a

religious

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

been

questioned

(3). Our

study

demonstratesahuman reservoir with carriers and household contacts; association between crowding,

bottle-feeding,

and

cryptosporidiosis

in regions where domestic animal are

infrequent;

and

peak

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 and

might

very well be themost

important 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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