Human Fascioliasis in Bolivia: Serological Surveys in Los Andes Province Ofthe Department of La Paz

HUMAN FASCIOLIASIS IN BOLIVIA: SEROLOGICAL SURVEYS IN LOS ANDES PROVINCE OFTHE DEPARTMENT OF LA PAZ

I 1 2 W. STRAUSS , R. ANGLES , J.G. ESTEBAN & S. MAS-COMA2

t Unidad de Parasitologia, IIISlilLlIONacional de Laboratories de Salud «Nestor Morales Villa~6n» (INLASA), Pasaje Rafaei Subieta No. 1889, Miraflores, La Pat; Bolivia 2DepartamelllO de Parasitologia. Facultad de Farmacia,Universidad de volencia, A v, Vice/lie Andres Estelles sin, 46100 Burjassot - Valencia, Spain

Received 21 ovember 1996: accepted 25 May 1997 REFERENCE:STRAUSS(W.), ANGLES(R.), ESTEBAN(J.G,) & MAS-COMA(S.). 1997.- Human fascioliasis in Bolivia: serological surveys in Los An- des province of the Department of La Paz. Research and Reviews in Parasitology, 57 (2): 109-1 13. ABSTRACTH: uman surveys by different serological techniques and antigen types were undertaken in the human fascioliasis high endemic zone of the orthern Bolivian Altiplano. Five serological studies were carried out in 3 Aymara localities of the Los Andes province in the Department of La Paz. Results show that the orthern Bolivian Altiplano must be considered an endemic region and that human fascioliasis is a real problem of pu- blic health in Bolivia. The large differences detected in prevalences from different localities may be related to the different geographic location within the endemic region and the irregular, patchy distribution of the intermediate Iymnaeid snail hosts. At any rate, these differences, found both between different localities and between different surveys carried out in the same locality. may evidently be related to the sensitivity and specificity of the different test systems and antigen types used for serological analyses. The non-significant differences in prevalences by sex as well as by age groups are worth mentioning: these facts could be explained by the lack of gender role differences and by the immune response of the subjects to Fasciola hepatica, respectively. KEYWORDS:Human fascioliasis, serological surveys, epidemiology, Northern Bolivian Altiplano.

INTRODUCTION ted at 3800-4200 m altitude between Lake Titicaca and the valley of the city of La Paz, are worth mentioning. For the diagnosis of Fasciola hepatica infection, aetio- Nevertheless, an early diagnosis of the acute phase in logical techniques based on the detection of the fluke an F. hepatica infection, when the infected humans have eggs in the faeces are usually used, However, the copro- clinical findings long before eggs could be found in the logical diagnosis of human fascioliasis presents several stools, may be achieved by immunological techniques, disadvantages (ESTEBAN, BARGUES & MAS-COMA, and more concretely by antibody detection. Among the 1998; MAS-COMA, BARGUES & ESTEBA, 1998), The advantages of the indirect methods are the ease in obtai- problems of direct techniques may be avoided simply by ning antigen reagents, the ease of the test systems used, repetitive stool sample collection and respective analy- and the fact that infections can be detected early (by 1-2 ses and by placing the patient on a liver-free diet. More- weeks of infection)(HILLYER, 1993). However, in im- over, coprological examination still remains the main munological techniques there are serious disadvantages method in epidemiological surveys because it allows us or limiting factors because a large number of patients to work both qualitatively and quantitatively: to detect showing parasite eggs in their stools are serologically which and how many subjects are shedding eggs in sto- negative (SAMPAIOSILVA et al., 1996) and serologic ols (prevalence), and how many eggs are shed in faeces cross-reactions with other helminthic infections such as (intensity), The recent paper by BARGUES et al. (1996) paragonimiasis, schistosomiasis, hydatidosis, ascariasis, has demonstrated the viability of humans as definitive trichinellosis and filariasis as well as with hepatitis C vi- host and their epidemiological role in disease transmis- rus infection are common (ESTEBAN, BARGUES & MAS- sion, eggs shed by human subjects being able to develop COMA, 1998; MAS-COMA, BARGUES & ESTEBAN, 1998). normally and continue the parasite life cycle up to the in- Moreover, a consensus concerning the optimal test sys- fection of a subsequent mammal host. Hence the impor- tems for the serological analysis of human fascioliasis tance of knowing how much eggs are being expelled in has been difficult to reach, and even the results obtained human faeces and contaminating the environment in a with the same technique vary according to authors particular geographic zone, In this sense, the world's (STORK et al., 1973; CHEN & MOTT, 1990; HILLYER, highest prevalences (up to 70% in given communities) 1993; ESTEBA, BARGUES & MAS-COMA, 1998; MAS- (HILLYER et al., 1992; MAS-COMA et al., 1995; BJOR- COMA, BARGUES & ESTEBAN, 1998). The major pro- LAND et al., 1995; ESTEBAN et al., 1997 a, b; ANGLES et blem of these serological tests lies in their sensitivity and al., 1997) and intensities (more than 1000 eggs/g of fae- specificity, which are dependent on antigen type and on ces and more than 5000 eggs/g in extreme cases) (ESTE- a lack of consensus concerning the optimal antigen to be BA et al., 1997 a, b) of human fascioliasis infection ob- used in the different tests (HILL YER, 1993; SAMPAIO tained by coprological diagnostic methods in the SILVA et al., 1996; ESTEBAN, BARGUES & MAS-COMA, endemic region of the Northern Bolivian Altiplano, loca- 1998; MAS-COMA, BARGUES & ESTEBAN, 1998)). 110 w. STRAUSS et al.

According to HILLYER et al. (1992) and HILL YER & serial sections from each block were affixed to clean microscope APT (1997), serological techniques demonstrate that the slides with formalin 4% for 10 min and stored at -20° C until coprological studies underestimate real prevalences. In used. -Soluble stabilized total antigen: Live, intact adult F. hepatica the human fascioliasis endemic region of the Northern worms were obtained from ovine livers at the abbatoir of Bata- Bolivian Altiplano the serological surveys carried out lIas and washed 3-4 times at room temperature during I hr with with different techniques showed human prevalences cold 0,0 IM phosphate buffered saline (PBS), pH 7,2, to remove between 39% and 100% (see review of MAS-COMA et all traces of blood and bile. The worms were homogenized in a al., 1995). tissue grinder with PBS containing 0,5 ml of a calcicrein solution The aim of this paper is to present the results obtained (500 inactivations units/rnl). The homogenized product was soni- in different serological surveys carried out in three Ay- cated in a Fisher sonic dismembrator model 300 at 20 KhzlI mA for 60 seconds four times; then the product was stored for 2 hr at mara localities from the Northern Bolivian Altiplano in 4° C stirring it slowly every 10 minutes. The antigen preparation the 1986-1991 period. The purpose of these studies was was centrifuged at 5,000 X gat 4° C and the supernatant collec- to contribute to the knowledge of the geographical distri- ted. Protein concentration was determined by the LOWRY et at. bution and prevalence of human fascioliasis in this ende- (1951) technique and the antigens were stored at _70° C. mic region, and simultaneously to adapt and optimize se- -Excretory-secretory antigen: Live, intact adult F. hepatica worms veral serological diagnostic techniques for human were obtained from ovine livers at the abbatoir of Batallas and washed 3-4 times at room temperature during I hr with 0,01 M fascioliasis in the INLASA Institute of La Paz, the Boli- phosphate buffered saline (PBS), pH 7,2, to remove all traces of vian national disease reference center. blood and bile. The worms were then incubated for 3 hr (20 worms/ I00 ml) in PBS at 37" C containing 0,5 ml of a calcicrein solution (500 inactivations units/ml). The antigen preparation MATERIAL A D METHODS was centrifuged at 5,000 X g at 4° C and the supernatant collected. Protein concentration was determined by the LOWRY et al. Study area:The geographic area studied is located in the Depart- (1951) technique and the antigens were stored at -70° C. ment of La Paz, where more than 2400000 natives live in approximately 133985 km2 The surveys were conducted in the Los Andes Serological techniques: The serological techniques employed in province, in the orthern Bolivian Altiplano, in the so-called «cor- this study (see Table I) were performed essentially as described by ridor of Batallas», a zone located at an altitude of 3800-4000 m FRAGA DE AZEVEDO & COELHO ROMBERT(1965) for the Indirect between Lake Titicaca and the valley of the city of La Paz. immunofluorescent antibody assay (IFA) and SANTIAGODE WElL, HILLYER & PACHECO (1984) and CEPANZO (1989) for the Enzyme- Serosurveys: Samples of sera were collected between 1986 and linked immunosorbent assay (ELISA): 1991 from three localities of the Los Andes province located near -IFA: Fifty ml of the inactivated sera (56° C for 30 min) diluted in to the shore of Lake Titicaca. The localities of Batallas and Cala- phosphate-buffered saline (PBS), pH 7,2, at serial dilutions star- saya were studied only once, while Cutusuma was surveyed on ting at 1:4 were placed on each antigen slide and incubated in a two different occasions in 1986 and 1990; moreover, the sera ob- moist chamber at 37° C for 30 min. The slides were rinsed once tained in the latter year were tested twice by the same serological with PBS, pH 7,2, and then washed with PBS for 20 min. Fifty 111 technique but with a different type of antigen. of I :200 dilution in Evans blue of fluorescein isothiocyanate- Because of their ethnic and religious customs, Aymara natives conjugated rabbit anti-human immunoglobulin (IgG/TgM kappa usually offer great difficulties in allowing the collection of blood and lambda) antiserum (Pasteur Institut, France) were then pla- samples. Therefore, several meetings for previous information and ced on each slide and incubated in a moist chamber at 37° C for sensitization of local inhabitants were organized. Despite these ef- 30 min. The slides were rinsed once and washed with PBS for 20 forts, inhabitants participated to a different degree according to lo- min, then air-dried, mounted with buffered glycerol, covered calities. However, the sample size in each total population studied with a cover glass, and examined with an Olympus fluorescence was always at least 20% of the subject number of each locality, microscope. Positive and negative reference sera were also inclu- and at least 75% of the number of subjects present the day of the ded in every batch tested. To reduce nonspecific reactivity, a di- survey. lution ~ I: 16, instead of the value of I: J 0 that is currently used Survey participants gave informed consent and a simple ques- (FRAGA DE AZEVEDO & COELHO ROMBERT, 1965), was conside- tionnaire including name, age, sex, site of residence and an identi- red antibody positive. The endpoint titer was the highest serum fication number was recorded for each one. Approximately 5 ml of dilution giving typical and visible fluorescent reading. venous blood were drawn from each subject. The blood was main- -ELISA: The optimum concentration of soluble stabilized total and tained at ambient temperature until serum was obtained.All sera excretory-secretory antigens, as determined by block titration, were transported in thermic boxes at-4° C and were kept frozen at were 25 pg/rnl and 20 mg/ml, respectively. Both antigens were _20° C in the laboratory until screened for F. hepaiica antibodies diluted in carbonate buffer, pH 9,6, incubated for I hr at 37° C in the INLASA Institute. and overnight at 4° C. Sera and horseradish peroxidase-conjuga- Characteristics of the human population studied in the localities ted goat rabbit anti-human IgA, IgG, IgM, Kappa, Lambda (Da- surveyed are shown in Table I. kopatts of Dako Corp.) were used at I :200 and I: 1000 dilutions, respectively. Incubations were done at 37° C for 30 min.ABTS Antigen preparations: In the ILASA Institute, the following an- 92,2, azino-di(ethil-thiazolin) sulfonic acid was used as substrate tigens were prepared: and was incubated for 5 min at 37° C. A I M HF solution was ad- -Figured antigen: Live, intact adult F. hepatica worms were obtai- ded to terminate the reaction (100 Ill/well) before the plates were ned from bovine and ovine livers at the abbatoirs of La Paz and read spectrophotometrically in a Wellcozyme Plus Mk II ELISA Batallas, respectively. The worms were transported in a penici- reader. Final volume of all reactants was 200 111.CUll-off value: llin-streptomycin solution to the laboratory and, after washing Normal values were determined with different individual normal several times at room temperature with physiologic solution, human serum from 50 subjects from the Altiplano but not from they were mounted in carboxymethylcellulose blocks. Four mm the endemic region; these were run in triplicate in 96-well plates. Serological surveys on human fascioliasis in Bolivia III

The absorbances obtained with all of these sera were analyzed The distribution of prevalences according to sex is statistically. The mean +3 SD absorbance at 405 nm was 0,248. shown in Table 1. No significant differences were found Using this value of the normal/negative group, positive samples between sexes. were defined as those with absorbance values greather than Table 2 shows the distribution of prevalences accor- 0,250, which was considered the endpoint of the negative sera. ding to age groups. Although differences detected Institutional ethical review procedure: The surveys were carried among the various age groups are not significant, when out after informed consent was obtained from the local authorities analysing the type of human population studied in each of the community (among Aymaras, community authorities are locality several important data appear: the highest preva- responsible for transmitting parental consent after previous mee- lences are detected among the 1-10 year age group and tings), as well as from the school Director and teachers. These stu- the prevalences are maintained at a high rate in all age dies were approved by the Secretarfa Nacional de Salud del Minis- terio de Desarrollo Humano (La Paz, Bolivia) and were performed groups, including adult subjects. by the INLASA Institute in La Paz, the official disease reference center for Bolivia. DISCUSSIO

RESULTS The results obtained are on the same line as those obtained in previous studies carried out in the Northern Bo- The results obtained in Table I show high fascioliasis livian Altiplano. Human F. hepatica infection detected prevalence rates in the three localities surveyed from the up to the present in this region by parasitological met- province of Los Andes. The large differences detected in hods, serological tests, parasitological and/or serological prevalences from different localities and overall in the methods and clinical symptoms (see MAS-COMAet al., same locality are worth mentioning. 1995; BJORLA D et al., 1995; ESTEBANet al., 1997 a, b;

Prevalence

Total Males Females

Localities Year Human Age range Techniques o. positive / % o. positive / % o. positive / % survey population (years) applied No. tested o. tested o. tested

Cutusuma 1986 total 2-76 IFA 67/74 90,5 35/38 92,1 32/36 88,9 Cutusuma 1990 total 3-76 ELlSA* 84/148 56,8 43179 54,4 41/69 59,4 Cutusuma 1990 total 3-76 ELlSA** 73/148 49,3 40179 50,6 33/69 47,8 Batallas 1990 school 10-21 ELlSA* 1061234 45,3 82/177 46,3 24/57 42,1 Calasaya 1991 total 1-81 ELlSA* 20/81 24,7 8/41 19,5 12/40 30,0

Table 1.- Characteristics of the human population, techniques used, prevalence of Fasciola hepatica infection in the localities surveyed, and the relationship of the latter to sex, in the Northern Bolivian Altiplano. [FA", immunofluorescence assay; ELlSA* '" enzyme-linked imrnu- nosorbent assay with soluble stabilized total antigen; ELlSA** '" enzyme-linked immunosorbent assay with excretory/secretory antigen.

Cutusuma" Cutusuma" Cutusuma" Batallas" Calasaya'' ( 1986) ( 1990) ( 1990) ( 1990) (1991)

Age group No. positive/ No. positive/ No. positive/ o. positive/ No. positive/ (years) No. tested (%) No. tested (%) No. tested (%) No. tested (%) No. tested (%)

1-10 36/39 (92,3) 34/45 (75,6) 30/45 (66,7) 104/223 (46,6) 4/9 (44,4) 11 - 20 27/31 (87, I) 31/59 (52,5) 26/59 (44, I) 2/11 (18,2) 5/14 (35,7) 21 - 30 212 (100) 8/13 (61,5) 7/13 (53,8) 0/0 (-) 6/15 (40) 31 - 40 0/0 (-) 5/15 (33,3) 5/15 (33,3) 0/0 (-) 3/19 (15,9) 41 - 50 1/1 (100) 4/8 (50) 3/8 (37,5) 0/0 (-) 1/11(9,1) > 50 1/1 (100) 2/8 (25) 2/8 (25) 0/0 (-) 1/13 (7,7)

Total 67/7 4 (90,5) 84/148 (56,8) 73/148 (49,3) 106/234 (45,3) 20/81 (24,7)

Table 2.- Prevalence of Fasciola hepatica infection by age groups in the localities surveyed with different techniques and antigen types. a = immunofluorescence assay; b", enzyme-linked immunosorbent assay with soluble stabilized total antigen; C '" enzyme-linked immunosorbent assay with excretory/secretory antigen. 112 W. STRAUSS et al.

ANGLESet al., 1997) have allowed us to consider this differences (ESTEBA et al., 1997 a, b). This fact is pro- zone between the city of La Paz and Lake Titicaca as a bably related with the immune response of the subjects high endemic zone of human fascioliasis including seve- to F. hepatica. It is known that antibody-mediated res- ral hyperendemic subzones (ESTEBANet al., 1997 b). ponse varies from host to host, and in the same host, ac- The large differences detected in prevalences from dif- cording to the phase of the infection (OLDHAM, 1985). ferent localities may be related to the different geograp- Although studies on immunity in man are limited, anti- hic location within the endemic region and the irregular, body levels to F. hepatica remain uniformly high in in- patchy distribution of the intermediate Iymnaeid snail fected individuals for many years (HILLYERet al., 1992). hosts. At any rate, these differences, found between dif- Moreover, there is another question that must not be for- ferent localities as well as between different surveys ca- gotten: nothing is today known about whether in a hu- rried out in the same locality, may evidently be related to man endemic area the adult subjects are able to maintain the sensitivity and specificity of the different test sys- the parasite acquired when young or not, or can be newly tems and antigen types used for the serological analyses. infected as the consequence of inhabiting a zone of high Unfortunately, moreover, data known on human fascio- infection risk. Some data suggest that liver fluke adults liasis prevalences detected by serological methods in se- may survive in the human host up to 13,5 years (CHAT- veral localities of the same Altiplanic endemic zone can- TERJEE, 1975; KAR AUKHOV,1978; DA et al., 1981). not be easily compared because different immunological techniques (intradermal tests, haemolysis, fluorescence, and enzymatic tests), and different antigen types (from ACKNOWLEDGEMENTS crude to excretory-secretory antigens) (see MAS-COMA et al., 1995; BJORLA D et al., 1995) were used. Data This work was partially supported by funding from the Regional Office of the Pan American Health Organization in La Paz, the Di- concerning the localities of Cutusuma and Calasaya are reccion Nacional de Epidemiologfa of the Ministerio de Prevision a good example, as seen when comparing the results of Social y Salud Publica in La Paz, the STD Programme of the Com- the present paper (see Table I) with those obtained in the mission of the European Communities (DGXII: Science, Research same localities by other authors using other techniques and Development) (Contract No. TS3-CT94-0294), Brussels, EU, (80,5% prevalence with the intradermoreaction test in and the Programme of Scientific Cooperation with Latin America, Cutusuma and 49% prevalence with Falcon assay scree- Instituto de Cooperacion Iberoarnericana, Agencia Espafiola de Cooperacion Internacional (1.C.I.-A.E.C.I.), Madrid, Spain. ning test-enzyme-Iinked immunosorbent assay -FAST- The authors would like to thank Lic. S. Ramirez and Miss M. ELISA- in Calasaya)(see MAS-COMA et aI., 1995; Magarifio (lNLASA, La Paz, Bolivia) for technical assistance in BJORLA D et al., 1995). the surveys. Other problems may be added to the above-mentioned The authors also acknowledge the facilities provided and the co- ones, such as those related to differences in the antibody llaboration received from the following Bolivian institutions and responses to F. hepatica reported by different investiga- centres, as well as their respective representatives or directors: Di- tors due to different antigen preparation methods and reccion Nacional de Epidemiologfa of the Ministerio de Prevision Social y Salud Publica in La Paz; Cornite Regional de Zoonosis storage conditions, or varying times of blood collection and Centro Piloto La Paz of the Unidad Sanitaria La Paz; and Of- during the course of infection (HILLYER, 1993; SAMPAIO fice of the Pan American Health Organization in La Paz. SILVAet al., 1996). 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