risk factors for fasciola hepatica infection in children: a case–control study

Transactions of the Royal Society of Tropical Medicine and Hygiene (2006) 100, 158—166

Risk factors for Fasciola hepatica infection inchildren: a case—control study

Luis Marcosa,∗, Vicente Macoa, Frine Samalvidesa, Angelica Terashimaa,Jose R. Espinozab, Eduardo Gotuzzoa

a Institute of Tropical Medicine Alexander von Humboldt (IMTAvH), Universidad Peruana CayetanoHeredia (UPCH), Av. Honorio Delgado 430, Urb. Ingenierıa, San Martın de Porres AP 4314,Lima 100, Perub Unidad de Biotecnologıa Molecular, Laboratorios de Investigacion y Desarrollo de Ciencia y Tecnologıa,Universidad Peruana Cayetano Heredia (UPCH), P.O. Box 4314, Lima 100, Peru

Received 7 December 2004; received in revised form 16 May 2005; accepted 16 May 2005Available online 21 September 2005

KEYWORDSHuman fascioliasis;Fasciola hepatica;Children;Risk factors;Eosinophilia;Peru

Summary We conducted a case—control study to clarify the risk factors for humanfascioliasis in the Northern Peruvian Altiplano. Cases (n = 61) were individuals whowere diagnosed as having chronic fascioliasis by faecal and/or serologic (Fas2-ELISA)studies. Controls (n = 61) had negative serologic and coprological results for Fasciolahepatica. We identified an association between fascioliasis and four variables (40analysed): the habit of drinking alfalfa juice (OR = 4.5; 95% CI 1.8—11.1; P < 0.001);familiarity with aquatic plants (OR = 4.3; 95% CI 1.8—10.6; P < 0.001); dog ownership(OR = 5; 95% CI 1.7—15.1; P = 0.002); and raising more than five sheep (OR = 0.3; 95% CI0.1—0.8; P = 0.01). According to clinical presentation and laboratory studies, dizzyspells (P = 0.01), history of jaundice (P = 0.01), peripheral eosinophilia (P = 0.005)and Ascaris lumbricoides in stools (P = 0.001) were associated with fascioliasis. Theprincipal exposure factor for F. hepatica infection was drinking alfalfa juice. In con-clusion, we suggest that human fascioliasis in Peru should be suspected in patientsfrom livestock-rearing areas, who present with recurrent episodes of jaundice andwho have a history of consumption of alfalfa juice or aquatic plants, or who haveeosinophilia.© 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd.All rights reserved.

∗ Corresponding author. Tel.: +51 1 3821021;fax: +51 1 3821021.

E-mail address: [email protected] (L. Marcos).

1. Introduction

Fascioliasis is a parasitic disease caused by theflukes Fasciola hepatica or F. gigantica. A widerange of herbivorous mammals, such as sheep, cat-tle and goats, are reservoir hosts. The importance

0035-9203/$ — see front matter © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.trstmh.2005.05.016

Risk factors for Fasciola hepatica infection 159

of this disease in livestock-rearing areas is revealedby the fact that it causes large economic lossesin the animal husbandry industry, such that it isa major veterinary public health problem. Up to1990, human fascioliasis was considered a sec-ondary disease (Chen and Mott, 1990); in the lastdecade, however, the number of cases reportedhas increased, reaching 51 countries worldwide(Esteban et al., 1998). Today we know that fas-cioliasis is an important human disease, with upto 17 million people infected (Mas-Coma et al.,1999a).

In Peru, several endemic areas of fascioliasis inanimals and humans have been identified mainly inthe Andean region. The highest prevalence ratesby coprological tests range from 8% in Cajamarca(Knobloch et al., 1985); 34.2% in Santa Ana, Junın(Stork et al., 1973), and 28.3% in the same region31 years later (Marcos et al., 2004); 15.7% in Asillo,Puno (Mas-Coma et al., 1999a; Sanchez et al.,1993), and 35% in the same area 9 years later(Esteban et al., 2002); and by serological tests upto 36.3% in Junın (Marcos et al., 2004). Most casesare school-age children 5—15 years old. Moreover,a high number of human cases have been reportedi21uwrto(w2otip1hftssh

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al., 2001; Vilca, 1982). As human fascioliasis isan important public health problem in the Andeanregion, mainly of Peru and Bolivia, the early detec-tion of most cases ought to be one of the prioritiesof Fasciola control programmes; disclosing the epi-demiology of fascioliasis and awareness of its clin-ical presentations and risks is the first step in thedevelopment of adequate control and preventionprogrammes for endemic areas.

One of the singular epidemiological features ofhuman fascioliasis in Peru is how some people areinfected by F. hepatica, as classical watercress isnot as common here as in other countries. In a seriesof 277 patients with fascioliasis diagnosed in Lima,only 45.6% mentioned having eaten watercress;the rest had acquired it from eating other plantssuch as lettuce (31.6%), alfalfa (10.5%) or spinach(5.3%) and drinking water from puquiales (10.5%) oremolientes (5.3%) (the warm beverages made fromvarious plants, chiefly alfalfa and watercress, andsupposed to be good for liver diseases) among oth-ers (Blancas et al., 2004). The emolientes may bea risk factor in the Mantaro Valley, Junın (Marcoset al., 2004). Others mention that the vehicle ofcontamination varies, depending on the region: inFVvoacgta(

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n many Peruvian hospitals since 1981: in Arequipa,20 cases were reported until 1977 (Picoaga et al.,980); in Cuzco, 18 cases were diagnosed in patientsndergoing cholecystectomy (Vilca, 1982); in Lima,here fascioliasis is not endemic, 16 cases were

eported in the 1990s in the Arzobispo Loayza Hospi-al (Jimenez et al., 2001) and 277 cases in a periodf 32 years in Cayetano Heredia National HospitalBlancas et al., 2004); and in Cajamarca, 101 casesere observed from 1996 to 2001 (Alban et al.,002). These results might suggest that Peru is onef the countries with the widest regional distribu-ion of human fascioliasis brought on by F. hepatican the world. A rural population of almost 8 millioneople is estimated at risk in this country (WHO,995). Some regions still have the same, or evenigher, prevalence rates than years before, and thisact is explained by the lack of control and preven-ion measures in those regions. Human fascioliasishould no longer be considered a secondary zoono-is, especially in Peru, but rather, an importantuman parasitic disease.

The clinical picture of human fascioliasis in thecute stage is remarkable, with high eosinophilia,epatomegaly and fever; but in the chronic stage,ost cases have vague or non-specific gastroin-

estinal symptoms (MacLean, 1999). Some of themre complicated and, as a result, are diagnosed inperating rooms, where adult parasites are foundbstructing the bile ducts or causing hepatic dys-unction (Hadden and Pascarelli, 1967; Jimenez et

rance, Taraxacum dens leonis (dandelion leaves),alerianella olitora (lamb’s lettuce) and Menthairidis (spearmint); in the Islamic Republic of Iran,ther green leafy Nasturtium spp. and Mentha spp.;nd in the Bolivian Altiplano, Juncus andicola (Jun-aceae), Juncus ebracteatus (Juncaceae), Mimuluslabratus (Scrophulariaceae), Nostoc sp. (Cianofi-as), among others (Mas-Coma et al., 1999b). Waterlso has been described as a possible risk factorMas-Coma et al., 1995).

According to Esteban et al. (2002), there arewo main vehicles of infection in Peru: water fromtreams and watercress; however, there are ateast 40% of patients who deny exposure to theseBlancas et al., 2004). There is a need to disclosedditional factors involved in fascioliasis trans-ission, to improve control and prevention pro-

rammes in the near future.Other important aspects in human fasciolia-

is are the clinical characteristics and laboratoryndings. These include eosinophil count and hema-ocrit. It is important to detect chronic cases (car-iers of the infection) early on: damage to the livery chronic infection in humans has not yet beenvaluated, while there are many described com-lications, such as anaemia, cholangitis, cholecys-itis and biliary obstruction (Arjona et al., 1995;ilchez et al., 1983). Moreover, in rats there isn increased risk of producing gallstone disease indvanced chronic stage of fascioliasis (Valero et al.,003). The sooner F. hepatica infection is detected,

160 L. Marcos et al.

the smaller the likelihood of severe liver impair-ment.

The objective of the present study was toidentify the clinical characteristics of chronic infec-tion by F. hepatica, which may help us to antic-ipate its diagnosis, and to define risk factors offascioliasis, with particular attention to areas ofextreme poverty in the Northern Peruvian Alti-plano. We designed a case—control study with sev-eral variables to find associations; cases of chronicfascioliasis infection were compared with con-trols having the closest demographic characteri-stics.

2. Materials and methods

2.1. Study area

The study took place in March 2001, in the Asillodistrict of the Azangaro Province, located approxi-mately 160 km from the city of Puno, at an altitudeof 3970 m above sea level (14◦47′03′′ LS; 75◦21′13′′LO). The area is cold (annual mean temperature,8.5 ◦C) and has two clearly defined seasons: dry

results for F. hepatica in both coprological and sero-logical tests.

Cases and controls were identified in March 2001from a cross-sectional coprological and serologicalsurvey of human fascioliasis in children 4 to 15 yearsold (Marcos et al., 2005) in Asillo. Controls who metthe inclusion criteria were paired 1:1 by gender andage (±1 year) with cases.

2.5. Clinical-epidemiological interview andphysical examination

The clinical-epidemiological questionnaire wasfilled out by cases and controls in the presenceof their parents or guardians. The questionnairewas used in local terms and included 40 variables:demographic data (first and last name, age, gen-der); alimentary habits (watercress, alfalfa, alfalfajuice, watercress juice, lettuce, ‘totorita’, aquaticplants, spinach, onion and salads); relation to watersupply (drinkable, stream, channel, river or well);housing characteristics (cement or adobe; prox-imity to river, streams, fields; having water andsewage facilities); breeding and number of animals(cows, sheep, swine, guinea pigs, dogs, llamas,ahodatptstorbapppacapstL

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from June to November, and rainy from Decemberto May. Most of the district lacks potable water orelectricity. Most of the population are farmers andraise livestock.

2.2. Study population

The Asillo district has a total population of 2853,including adults and children. There are 1712 (60%)children, and 900 of them are 4 to 15 years old. Theprevalence of human fascioliasis in 1989 was 15.7%(Sanchez et al., 1993 in Mas-Coma et al., 1999a).The sample size (n) for this study was calculatedby using EpiInfo version 6.4, 1996 (CDC Atlanta,GA, USA). We calculated 61 cases and 61 controls(˛ = 0.05, power 80%).

2.3. Case definition

For this study, a case was defined as a resident ofthe Asillo district, whose age was between 4 and 15years old (the highest prevalence is found in this agegroup). Cases had a positive result for F. hepaticaby coprological tests and/or positive serology usingFas2-ELISA (Cordova et al., 1999).

2.4. Control definition

Controls were residents of the Asillo district agedbetween 4 and 15 years old, and who had negative

lpacas, horses, donkeys and rabbits); defecationabits (own bath, silo, open field, river, streamr channel); history of having taken anthelminthicrugs; past coprological diagnosis of intestinal par-sitosis; familiarity with aquatic plants; and his-ory of surgery. An estimated average of animalser family was between five and six animals inhis region (data not shown); we arbitrarily con-idered a cut-off number of five animals to iden-ify people with many animals (more than five),r few animals (less than or equal to five). Withegards to clinical picture, the variables that mighte associated with F. hepatica infection were: hep-tomegaly, right upper quadrant pain, epigastricain, urticaria, dizziness spells, splenomegaly, Mur-hy’s sign, history of jaundice, fat dyspepsia andaleness. To avoid the selection bias between casesnd controls, the interviewer was blinded to theoprological and serological test results. To avoidny confusion with certain signs (dark urine andast history of jaundice), illustrative pictures werehown. To define hepatomegaly we took the ver-ical hepatic diameter stratified by age (Wolf andavine, 2000).

.6. Stool samples

wo stools were collected on two consecutive dayser subject. One gram of faeces was placed in aeparate container for performing the Kato—Katz


technique, before the bulk of each sample wasplaced in 10% formalin. Faeces were transportedto the Laboratory of Parasitology at the Insti-tuto de Medicina Tropical Alexander von Humboldt(IMTAvH), Universidad Peruana Cayetano Heredia(UPCH), for processing on the same day they werepassed. Stools were examined by the rapid sed-imentation technique described by Lumbreras etal. (1962) for Fasciola eggs (Maco et al., 2002),the spontaneous sedimentation technique for allintestinal parasites, and the Kato—Katz techniquefor Fasciola egg count (Katz et al., 1972), accord-ing to the national guidelines (Beltran et al., 2003).However, there are some helminth and protozoanspecies that might be absent or might alter theprevalence rates of each group in the final com-parative analysis, as in the cases of: Enterobiousvermicularis (anal swabs would be the adequatetechnique for the detection of the eggs of thisnematode); ancylostomids, Strongyloides sterco-ralis and Trichostrongylus (the agar plate methodwould be better for the detection of the larvaeof these geohelminths); and coccidia (the modifiedZiehl—Neelsen technique would be an adequatemethod to see these protozoa).

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tinuous variables by Student’s t test. A value ofP < 0.05 was considered as the critical level of sig-nificance. Odds ratio (OR), 95% confidence inter-vals and P-values were calculated. Subsequently,stepwise conditional logistic regression was used todetermine independent potential risk factors asso-ciated with F. hepatica infection.

2.10. Ethical considerations

Informed consent was obtained from parents orguardians and assent from children; all wereinformed on the nature of the study, benefits andrisks. All cases were treated with triclabendazole.The use of triclabendazole was approved by theInstitutional Ethics Committee (CEI) of the UPCHand by the Directive Committee of the IMTAvH; itis currently the treatment of choice for patientswith fascioliasis seen in the institute. For the otherintestinal parasite species, the patients receiveddonated anthelminthic and antiprotozoal treat-ments.

3. Results

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.7. Blood samples

even millilitres of venous blood was obtained fromll consenting subjects, and 2 ml were separatedor complete blood counts. Sera from the other 5 mlere centrifuged the same day, transported to Liman ice and stored at —20 ◦C; the sera were assayedy Fas2-ELISA as originally described (Cordova etl., 1999).

.8. Analysis of livestock faeces

aeces from a total of 44 animals from the Asilloistrict, in the place in which cases and controlsriginated, were analysed. They belonged to 20 cat-le, 13 sheep, four swine, five dogs and two rabbits.ecently excreted animal faeces were gatheredrom the ground and assayed for parasites using theapid sedimentation technique.

.9. Statistical analysis

he statistical analyses were done using SPSS 9.0or Windows (SPSS Inc., Chicago, IL, USA). This con-isted of a univariate descriptive analysis, whichalculated frequencies and percentages for cat-gorical variables. For continuous variables, theeans and standard deviations were calculated.ivariate analysis was applied to categorical vari-bles by �2 or Fisher’s exact test and to con-

f 61 cases, 54 subjects had positive results by botherological and coprological tests, and seven fromhe coprological test only. Thus, all 61 cases had. hepatica chronic infections. The 61 controls allesulted negative in both coprological and serolog-cal tests. The distribution of cases and controls byge and gender is shown in Table 1. It is knownhat the prevalence of human fascioliasis varies asge increases (Esteban et al., 1999), and the lab-ratories findings, including eosinophil counts andematocrit levels might also vary. Because of this,e tried to separate cases in groups, each withlarge enough number of members, to arrive at

tatistically significant conclusions. Thus, the casesnd matched controls were divided into three ageroups (4—7, 8—11 and 12—15 years) of roughly sim-lar size, but with some more cases in the secondroup.

Among the 40 variables that we examined bynivariate analysis, eight of them were signifi-antly associated with F. hepatica infection, sevenith increased risk: water supply from chan-els (OR = 2.4, 95% CI 1.1—5.3, P = 0.03); eatingquatic plants (OR = 2.5, 95% CI 1.1—5.6, P = 0.028);rinking alfalfa juice (OR = 2.8, 95% CI 1.3—5.8,= 0.006); breeding five or more cattle (OR = 2.5,5% CI 1.1—5.6, P = 0.028); owning dogs (OR = 3.2,5% CI 1.3—8.1, P = 0.01); defecating in fieldsOR = 2.6, 95% CI 1.3—5.6, P = 0.01); familiarity withquatic plants (OR = 3.9, 95% CI 1.8—8.3, P = 0.000).


Table 1 General characteristics of cases and controls

Variable Cases (n = 61) Controls (n = 61) Total (n = 122)

Mean age ± SD 9.46 ± 2.8a 9.39 ± 2.84 9.43 ± 2.8

Age group (years)4—7 [no. (%)] 17 (27.9)b 18 (29.5) 35 (28.7)8—11 [no. (%)] 29 (47.5)c 28 (45.9) 57 (46.7)12—15 [no. (%)] 15 (24.6)d 15 (24.6) 30 (24.6)

GenderMale [no. (%)] 30 (49.2) 31 (50.8) 61 (50)Female [no. (%)] 31 (50.8) 30 (49.2) 61 (50)e

a P = 0.898.b P = 0.841.c P = 0.856.d P = 1.000.e P = 0.856.

Breeding more than five sheep appeared as a pro-tection factor (OR = 0.3, 95% CI 0.1—0.7, P = 0.003).

In stepwise conditional logistic regression anal-ysis, three variables remained independently andsignificantly associated with increased risk of F.hepatica infection: drinking alfalfa juice, familiar-ity with aquatic plants and owning dogs. Breed-ing more than five sheep was associated with adecreased risk (Table 2).

Cases were more likely to have dizziness(OR = 2.5; P = 0.016) and a history of jaundice(OR = 3.5; P = 0.011) than were controls. No othersymptoms or signs had a significant association withF. hepatica infection.

In laboratory analysis, we were unable to cal-culate the eosinophil count in seven cases andnine controls because the anticoagulated bloodwas lost in transit. There was a significant differ-ence between cases and controls, when we com-pared the absolute eosinophil count (mean ± SD =680.5 ± 850.5 cases versus 297.4 ± 392.9 controls;t = 2.87; P = 0.005) and percentage with eosinophilia(43.5% cases versus 17.6% controls; �2 = 7.7;P = 0.006). The mean eosinophil count for eachage group was: group I, 931 ± 1280 in cases and

differences in eosinophil counts in each agegroup between cases and controls, the mean ofeosinophils are much higher in cases, particularly inthe first years. There was no significant differencebetween the hematocrit mean between both groups(mean ± SD = 45.9% ± 3.9 cases versus 45.4% ± 3.9controls; t = 0.66; P = 0.51).

Fifty-four percent of cases and 57% of controlshad other pathogenic intestinal parasites, such as,in descending order of prevalence (controls versuscases): Giardia lamblia (18.3 versus 33.3%), Ascarislumbricoides (20 versus 1.7%), Entamoeba histolyt-ica/Entamoeba dispar (10 versus 5%), Hymenolepisnana (3.3 versus 5%), Trichuris trichiura (1.7 ver-sus 5%), Enterobius vermicularis (1.7 versus 3.3%),Strongyloides stercoralis (1.7 versus 0%). One childhad seven species of intestinal parasites. Amongthe intestinal parasites found, only A. lumbricoidesinfection was associated with F. hepatica (�2 = 10.7;P = 0.001).

The results of the intensity of infection measuredby the Kato—Katz technique and its relationshipwith laboratory data and clinical findings are shownin Table 3.

Fasciola eggs were detected in 20 of the 44 ani-mas0

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403 ± 558 in controls; group II, 675 ± 630 in casesand 260 ± 268 in controls; group III, 392 ± 293in cases and 248 ± 237 in controls. Despite thefact that there were no statistically significant

Table 2 Logistic regression analysis for risk factors fo

Variable Cases (n = 61) Co

Drinking alfalfa juice 42 (68.9) 27Familiarity with aquatic plants 39 (63.9) 19Dog ownership 53 (86.9) 41Breeding >5 sheep 8 (13.1) 22

Paired analysis (conditional logistic regression).

als studied (45.5%). The proportion of infectednimals was: cattle 13/20 (65%); swine 2/4 (50%);heep 4/13 (30.8%); dogs 1/5 (20%); and rabbits/2.

ciola hepatica infection

ls (n = 61) Odds ratio 95% CI P

3) 4.46 1.796—11.076 0.00061) 4.34 1.785—10.546 0.00062) 5.00 1.663—15.074 0.00221) 0.30 0.107—0.847 0.0189


Table 3 Relationship between the intensity [eggs per gram of faeces (epg)] of Fasciola hepatica infection and thesymptoms, signs and laboratory data among infected children

Variable Range AM/GM Intensity (epg) (% positive) P

<100 101—400 401—1000

Total no. cases (n = 60) 72—528 238/224 95.0 —Male (n = 30) 72—384 228/215 1.7 96.7 — 0.366Female (n = 30)a 120—528 248/234 — 46.7 3.3

Laboratory dataEosinophilia

Positive (n = 19) 144—528 260/245 — 39.1 2.2 0.152Negative (n = 27) 72—360 224/212

Symptoms and signsHepatomegaly

Yes (n = 2) 144—240 192/185 — 3.3 — 0.44No (n = 58) 72—528 240/225

RUQ painYes (n = 12) 72—480 231/215 — 20 — 0.13No (n = 48) 120—528 242/229

Epigastric painYes (n = 22) 72—480 231/215 1.7 20 1.7 0.656No (n = 38) 120—580 241/229

UrticariaYes (n = 31) 72—528 243/224 1.7 46.7 3.3 0.64No (n = 29) 144—360 243/224

History of jaundiceYes (n = 16) 120—528 239/220 — 25 1.7 0.98No (n = 44) 72—480 238/225

Fat dyspepsiaYes (n = 23) 72—528 242/221 1.7 35 1.7 0.77No (n = 37) 144—480 236/226

Dark urineYes (n = 14) 120—360 204/194 — 23.3 — 0.089No (n = 46) 72—528 248/234

DiarrhoeaYes (n = 16) 72—528 252/224 1.7 21.7 3.3 0.45No (n = 44) 144—384 233/224

DizzinessYes (n = 29) 72—528 240/221 1.7 43.3 3.3 0.85No (n = 31) 144—360 236/227

AM/GM = arithmetic mean/geometric mean. <100 to >1000: distribution of the intensity ranges in epg in cases, expressed as apercentage of the total number of cases. There were no cases with more than 1001 epg.a One female case was excluded from the analysis because her stool was insufficient for performing the Kato—Katz technique.

4. Discussion

In this study, we examined Fasciola cases and con-trols to determine epidemiologic and clinical riskfactors in the Northern Peruvian Altiplano, whereone of the highest prevalence rates of human fas-cioliasis in Peru has been reported (Esteban et al.,2002).

The present study shows that eating aquaticplants, drinking alfalfa juice and having a watersupply from irrigation channels increases the riskof F. hepatica infection by at least twice. Waterhas been described as a risk factor; in Bolivia 13%of experimentally obtained metacercariae werefloating (Mas-Coma et al., 1999b). Water supplyfrom streams (small lateral channels) was not


associated with fascioliasis (P = 0.346). Water fromstreams that comes from irrigation channels wasnot consumed as frequently as water from largerirrigation channels, which permanently conductwater; the small lateral channels are flooded onlywhen needed; this was noted also by Esteban etal. (2002). Thus, infection with F. hepatica fromcontaminated water may require large or fre-quent innocula from where freshwater lymnaeidsdevelop.

One of the alimentary habits associated with fas-cioliasis in this region was consumption of aquaticplants. Interestingly, inhabitants from this areahave the habit of eating aquatic plants found grow-ing along the edge of streams. The constructionof the Asillo-Azangaro irrigation project involvedthe creation of a channel system, which might bethe cause of the appearance of such endemicity,as others have mentioned (Esteban et al., 2002).Consequently the environmental factors have beenfavourable for the development of a high burden ofaquatic plants, which are likely to play an impor-tant role in the dissemination of the disease, bymaking contact with the metacercariae floating inthe water.

tions. By contrast, breeding sheep was a protec-tive factor against F. hepatica infection (univariateanalysis and logistical regression analysis). This pro-tective effect could be explained by the fact thatfewer sheep are infected and the duration of infec-tion is short (1—5 months). Alternatively, owningmany sheep may be associated with anthelminthictreatment of the sheep or living in an area in whichthe metacercariae burden is low while the presenceof freshwater lymnaeids is low or absent. The dis-tribution of fascioliasis by foci as a consequence oflymnaeid presence in the water bodies and of snailpopulation size and extent shows that people whoare living in places with low lymnaeid burden havea lower possibility of becoming infected, and thus,the sheep have less mortality. These results suggestthat the type of livestock in a region might be takeninto account as an epidemiological marker to iden-tify endemic areas for fascioliasis. These findingsmay be supported by the well-known patchy distri-bution of fascioliasis, with foci being related to thelocal distribution of intermediate snail host popu-lations in freshwater bodies, as well as to generalphysiographic and climatic conditions (Mas-Coma etal., 1999b). Therefore, these results, which indi-rbti

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The other alimentary habit associated with fas-cioliasis, drinking alfalfa juice, was also foundto be associated with fascioliasis in the logisti-cal regression analysis. Alfalfa juice is widely con-sumed throughout Peru as a popular medicinalplant. In Arequipa, located near Puno, metacer-cariae were found in ‘emolientes’ (warm bever-ages made from the tender leaves of watercressor alfalfa, among others) (Leguıa, 1991). We sug-gest that when the fields are watered from con-taminated channels, the metacercariaea encyst onalfalfa stems or leaves and may be the route bywhich infection is acquired. Our results suggestthat alfalfa juice should be taken into account forfuture epidemiological surveys, especially wherethis habit is present. The magnitude of this prob-lem has not yet been measured, but there arereports of children and adults outside endemicareas with chronic liver diseases, possibly caused byF. hepatica infection (Almendras-Jaramillo et al.,1997; Heredia et al., 1984; Sanchez-Sosa et al.,2000).

In the breeding of animals, we found that breed-ing more than five head of cattle raised the risk offascioliasis infection 2.5 times. Having more thanfive sheep reduced the risk. In this area, cattleare the predominant livestock. Fascioliasis is morelethal in sheep than in cows in this area (C. Sanchez,unpublished data). Thus, exposure to cattle is likelyto be associated with an increased risk, due to botha higher prevalence and longer duration of infec-

ectly show that fascioliasis distribution may occury foci, might be applied in other regions or beaken into account for future epidemiological stud-es.

We found that owning dogs increased the risk of. hepatica infection, 3.2 times by univariate anal-sis, and five times by logistic regression analysisone in five dogs studied had fascioliasis by copro-ogical tests). However, the eggs may have beenxcreted after ingestion of raw liver, as no sero-ogical tests were done in dogs. Close contact ofhe dogs with cows and sheep in the transmissionoci might explain why owning dogs may be a pos-ible risk factor, but the participation of the dogsn fascioliasis transmission is unlikely. This findinghould be further studied, to determine its validity.

Defecation in open fields was associated with a.6 times risk of fascioliasis. Perhaps subjects withoor hygienic habits have more contact with theontaminated environment, as was described bythers in this same region (Esteban et al., 2002).

As well as eating aquatic plants, familiarity withquatic plants was also associated with fasciolia-is. Although watercress is not very common in thisegion, aquatic plants similar to watercress coulde an important source of fascioliasis. This is proba-ly the most important risk factor, along with drink-ng alfalfa juice.

The only significant clinical difference betweenhe case and control groups was a history ofaundice and dizziness. The history of jaundice


associated with chronic fascioliasis suggests thatthese patients could have had episodes of transientbiliary obstruction, caused by the parasite. A highproportion of intestinal parasitoses found in bothgroups may obscure the clinical picture of fasciolia-sis. In order to avoid this bias, a study of the clinicalcharacteristics of chronic human fascioliasis in anendemic area could consider the prior eliminationof other intestinal parasites beforehand; this wouldbe hard to do because of difficult patient follow-up, the lack of necessary infrastructure, the limitedaccessibility of these places and low compliance ofpatients because of frequent migration, etc.

In laboratory analysis, absolute eosinophilia wasmore frequent in cases than in controls. Despitethe high prevalence of other intestinal parasites,both groups have similar prevalences of helminths,except for A. lumbricoides infection. While it isnot possible to rule out acute stages of the otherhelminths, in particular those that cause higheosinophil counts, such as T. trichiura, A. lum-bricoides, S. stercoralis or E. vermicularis, bothgroups were equally exposed (in time and space)to the environment throughout the study; the like-lihood of acquisition of these infections is the samefelottobaaadttPf

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attention: women show more complication ratesthan do men in adulthood, as has been reported in atotal of approximately 622 cases in Peruvian hospi-tals since 1970; around 55% are women, in whichthere is an increase of abdominal surgeries. Thepercentage is more evident as age increases (Albanet al., 2002; Blancas et al., 2004; Jimenez et al.,2001; Picoaga et al., 1980; Vilca, 1982).

The possible causes of these findings requiremore studies and explanations, given that hor-monal, immunological and environmental factorsmight be involved. Among the signs and symp-toms analysed and their relationship with the inten-sity of the infection, only fat dyspepsia, diarrhoeaand dizziness had arithmetic and geometric meansmildly higher than asymptomatic patients for thesame symptoms; however, they were not statisti-cally associated (P > 0.05).

In conclusion, the consumption of alfalfa juiceor raw aquatic plants is a risk factor for Fasciolainfection in endemic areas; as the clinical pictureof chronic infection is non-specific, any patientfrom these areas with a history of jaundice andeosinophilia should be tested for F. hepatica infec-tion.

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or both exposed groups. The highest levels ofosinophilia was observed in younger children, mostikely caused by recently acquired acute infection,bserved in the last period of the acute phase andhe beginning of the chronic stage. By contrast,hese results might coincide with the acute stagesf other helminths, as we mentioned. Ascaris lum-ricoides is a common cause of high eosinophilia,nd we found an association between ascaridiasisnd fascioliasis. To avoid a possible bias, ascaridi-sis cases and their corresponding controls wereropped from analysis, and the result showed thathe eosinophil counts remained different betweenhe two groups (711 versus 300 eosinophils/mm3;= 0.028). Thus, eosinophilia was probable due to

ascioliasis, rather than to A. lumbricoides.The significant association between F. hepatica

nd A. lumbricoides probably reflects increasedaecal—oral exposure to faecal contamination inhis human group. These individuals might alsorink contaminated water or eat raw vegetablesith metacercariae, increasing their risk of both

nfections.Finally, the intensity of the infection among

ases was mild to moderate (101 and 400 epg); twoases had more than 401 epg (up to 528 epg); andnly one had less than 100 epg. The intensity wasigher in girls than boys, as was reported in a previ-us study in the same region (Esteban et al., 2002)nd in the Northern Bolivian Altiplano (Estebant al., 1999). This latter finding deserves special

onflicts of interest statementhe authors have no conflicts of interest concerninghe work reported in this paper.

cknowledgements

e would like to kindly thank: Dr Vicente Macoardenas, Department of Pathology, Police Hospi-al, Lima, for his assistance obtaining the bloodamples; Vıctor Neyra and Elizabeth Chavarry,nidad de Biotecnologıa Molecular, Facultad deiencias y Filosofıa, Universidad Peruana Cayetanoeredia for their help in the processing of sera;r Raul Tello, Marco Canales and Carmen Quijano,aboratorio de Parasitologıa, Instituto de Medicinaropical Alexander von Humboldt for their help inhe processing of stool samples; Dr Cesar Wegener,irector of Hospital Regional of Azangaro, Puno,eru, for storing blood samples; Dr Abdias Hur-ado, President of CTAR — Puno, Peru, for providingransport to work in the study area; Dr A. Clin-on White, Jr, Infectious Diseases Section, Depart-ent of Medicine, Baylor College of Medicine,ouston, Texas, USA, and Dr Humberto Guerra,.D., Ph.D., Dr. Med., Professor of Medicine, Insti-

uto de Medicina Tropical Alexander von Hum-oldt, Universidad Peruana Cayetano Heredia,ima, Peru for their help editing and reviewing thisanuscript.


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