A Study of Risk Factors Associated With the Prevalence of Cryptosporidium in Villages Around Lake Atitlan, Guatemala

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1 BJID 2004; 8 (August) 319 A Study of Risk Fctors Associted With the Prevlence of Cryptosporidium in Villges Around Lke Atitln, Guteml H. E. Luch 1, C. Z. Bentley 2, E. L. 1 Deprtment of Microiology, College of Medicl Sciences; Ginter 2, J. S. Splter 3 nd L. A. Jensen 2 2 Deprtment of Fmily Medicine nd 3 Infectious Diseses, College of Osteopthic Medicine, Helth Professions Division, Nov Southestern University, Fort Luderdle, Florid USA Cryptosporidium prvum is n endemic, zoonotic coccidin prsitosis tht is highly prevlent in third-world countries where wterorne fecl contmintion of food nd drink or person-toperson contct with oocysts re the most common methods of trnsmission of the enteric protozon. This type of trnsmission of the prsite mde the villges round Lke Atitln, Guteml unique site to compre environmentl risk fctors with the level of Cryptosporidium infections in the locl residents. The study ws crried out in two villges, Sn Antonio Plopo nd Snt Ctrin Plopo, locted in the highlnds ove the shores of the lke. Smers from stool specimens of ptients with gstroenteritis were processed using Kinyoun s modified cid-fst stin nd oserved with light microscopy. Of the 100 residents exmined from the two villges, 32% hd Cryptosporidium infections. Femle children hd the highest prevlence of infection (44% in Sn Antonio Plopo nd 46% in Snt Ctrin Plopo, p<0.05), nd they lso hd significntly higher infection rtes thn mles, 50% vs. 17%, respectively. The prevlence rte ws not influenced y the seson of the yer or y the loction of the residents. We found differences in prevlence rtes due to ge nd gender, nd we suggest tht the high infection rtes of specific groups re ssocited with their exposure to the contminted wter supply from Lke Atitln. Key Words: Cryptosporidium, disese trnsmission, Guteml, risk fctors, lke-wter. Intestinl cryptosporidiosis is cuse of dirrhel disese, worldwide, with 10 species of Cryptosporidium infecting more thn 150 species of mmmls [1,2]. The 1993 outrek of cryptosporidiosis in Milwukee, Wisconsin, ttriuted to contminted city wter, produced n estimted 403,000 infections [3]. This endemic, zoonotic coccidin prsitosis is highly prevlent in third-world countries tht hve wterorne fecl contmintion of food nd drink Received on 21 Jnury 2004; revised 13 June Address for correspondence: Dr. Hrold E. Luch. Deprtment of Microiology, College of Medicl Sciences, Nov Southestern University, Ft. Luderdle, FL 33328, USA. Phone: Fx: E-mil: hrold@nov.edu The Brzilin Journl of Infectious Diseses 2004;8(4): y The Brzilin Journl of Infectious Diseses nd Contexto Pulishing. All rights reserved. with oocysts nd where there is direct infection y person-to-person contct s the common method of trnsmission of this enteric protozon [4]. Dirrhe, following infection with Cryptosporidium, is most common in infnts nd children in developing countries, in immunosuppressed hosts nd in people with deficient nutrition [5-7]. Cryptosporidiosis is serious disese in these countries, ecuse it increses helth prolems ssocited with poverty nd mlnutrition. Epidemiologicl chnges due to iologicl differences nd snittion prctices of individuls in this popultion would hve n overll effect on disese prevlence. In n ttempt to ddress this point, we exmined the risk fctors involved in the prevlence of cryptosporidiosis in villges round Lke Atitln, Guteml.

2 320 Risk Fctors of Cryptosporidium BJID 2004; 8 (August) Mterils nd Methods Study popultion The study ws crried out in Sn Antonio Plopo nd Snt Ctrin Plopo in the highlnds of centrl Guteml. The two villges re locted 12 miles from ech other on the shores of Lke Atitln, lrge, freshwter reservoir with strems running into it ut without wter leving it, mking it confined wter source for the locl inhitnts. Dt were otined during two Jnury nd June visits in 2001 nd in Children etween the ges of 2 nd 13, with symptoms of dominl pin nd cute, wtery, dirrhe, were selected for the study. The history of infection with Cryptosporidium, the immune sttus, prior or current pthogen infections, nd the degree of mlnutrition of the ptients in our study were not known. Therefore, other unidentified pthogens nd conditions could hve hd n ffect on our results of risk fctors ssocited with children hving dominl pin nd dirrhe nd hroring Cryptosporidium. Of the 100 children in the study, 58 were mle nd 42 were femle. Sn Antonio Plopo hd 21 mles nd 25 femles nd Snt Ctrin Plopo hd 37 mles nd 17 femle prticipnts in the study. The Institutionl Review Bord of Nov Southestern University pproved the study nd informed consent ws otined from sujects, prents or gurdins. Smple collection nd lortory nlysis Children with symptoms of dirrhe nd dominl pin were identified in the towns of Sn Antonio Plopo nd Snt Ctrin Plopo. Fecl smples were collected from ptients, nd smers were prepred on glss slides y mixing with PVA, followed y drying t room temperture. Dried specimens were trnsported to lortory t NSU nd Cryptosporidium oocysts were identified fter stining with Kinyoun s modified cid-fst stin [8]. A smple ws considered negtive for Cryptosporidium if oocysts were not detected fter 20 minutes of scnning using oil immersion (1000x) light microscopy. Sttisticl nlysis Infected nd non-infected ptients were identified, nd the numer of people infected within ge groups, gender nd villges ws used to determine the prevlence of Cryptosporidium. Comprisons of the prevlence rtes of infection etween ge groups, gender nd villges were mde using McNemr s test [9]. Differences etween groups were considered significnt if p <0.05. Results All three groups of children in the study hd high infection rtes (16-44%) for cryptosporidiosis, nd there were no significnt differences in the prevlence rtes etween younger nd older ge groups (Tle 1). In Snt Ctrin Plopo, femle children in the 2 to 5 yer ge group hd higher prevlence of infection nd hd significntly overll higher infection rtes thn did mles. Mles in the 2 to 5 yer ge group in Snt Ctrin Plopo hd lower prevlence rtes for Cryptosporidium thn the older mles. However, significnt differences (p<0.05) in the presence of Cryptosporidium were not found etween ge groups or genders in Sn Antonio Plopo. Totl prevlence rtes for Cryptosporidium infections were high for oth Sn Antonio Plopo nd Snt Ctrin Plopo, with n overll infection rte for oth towns of 32% (Tle 2). Prevlence rtes for mles were lower thn those for femles when the dt from oth villges were comined. Overll, mles in the 2 to 5 yer ge group hd lower prevlence rte for cryptosporidiosis thn did older mle children. The prevlence of infection recorded for Jnury, in the dry seson, ws 29% nd it ws35% for June, during the wet seson (Tle 3). Mles, in the 2 to 5 yer ge group, exhiited significntly lower prevlence rtes thn did femles, for oth Jnury nd June. Mles, etween the ges of 2 nd 10, hd lower prevlence rtes thn did femles in Jnury. There ws no significnt difference (p<0.05) in the overll numers of infected children due to the wether conditions.

3 BJID 2004; 8 (August) Risk Fctors of Cryptosporidium 321 Tle 1. Comprison of prevlence rtes of Cryptosporidium etween mle nd femle children in villges round Lke Atitln, Sn Antonio Plopo Snt Ctrin Plopo Age group Mles Femles Totl Mles Femles Totl 2-5 2/10 (20) 4/9 (44) 6/19 (32) 3/19 (16) c 5/9 (56) d 8/28 (29) 6-9 2/7 (29) 4/9 (44) 6/16 (38) 4/11 (36) 1/3 (33) 5/14 (36) /4 (25) 2/7 (29) 3/11 (27) 2/7 (29) 2/5 (40) 4/12 (33) All ges 5/21 (24) 10/25 (40) 15/46 (33) 9/37 (24) 8/17 (47) d 17/54 (31) Prevlence rtes of infected mles nd femles did not differ etween villges. c Mles 2 to 5 yers old hd significntly lower prevlence rtes (p<0.05) thn mles in other ge groups in the sme villge. d Femles hd significntly higher prevlence rtes thn mles (p<0.05) in the sme villge. Tle 2. Prevlence rtes of Cryptosporidium in mles nd femles within ge groups for oth Sn Antonio Plopo nd Snt Ctrin Plopo Age group Mles Femles Totl 2-5 5/29 (17),c 9/18 (50) 14/47 (30) 6-9 6/18 (33) 5/12 (42) 11/30 (37) /11 (27) 4/12 (30) 7/23 (30) All ges 14/58 (24) 18/42 (40) 32/100 (32) Prevlence rtes of mles were significntly less thn those of femles (p<0.05) within ge groups. c Mles in the 2-5 ge group hd lower prevlence rte (p<0.05) thn mles in the other ge groups. Discussion The prevlence of cryptosporidiosis in children from Ltin Americn countries vries from 2% to 31% [10], suggesting high level of infection in Gutemln villges nd ws the sis for this study. The demonstrtion of Cryptosporidium in children in villges round Lke Atitln provided n opportunity to compre infection rtes etween geogrphiclly isolted villges tht shred the sme sources of infection y hving the sme flor, fun, wter source nd life style risk fctors. It ws uncertin how often Cryptosporidium infections were cquired in these villges. The locl environmentl distriution of Cryptosporidium ws dependent upon humn, griculturl nd wildlife sources nd the significnce of ech individul source with regrd to the presence of oocysts in the environment ws unknown. Bovine reservoirs were considered s sources of humn cryptosporidiosis, s previously demonstrted y Awd-El-Kriem [11,12]. There were few niml reservoirs of Cryptosporidium round the lke nd they did not seem to hve n effect on the prevlence rtes of humn infection, or if they did, the effect ws hidden y the shred use of wter. Pst studies of the effect of the wether on prevlence rtes for cryptosporidiosis hve hd vrying

4 322 Risk Fctors of Cryptosporidium BJID 2004; 8 (August) Tle 3. Cryptosporidium infections in children: comprison of prevlence rtes of mles nd femles within ge groups y seson for Sn Antonio Plopo nd Snt Ctrin Plopo Jnury June Age group Mles Femles Totl Mles Femles Totl 2-5 3/17 (18) 4/7 (57) 7/24 (29) 2/12 (17) 5/11 (45) 7/23 (30) 6-9 2/8 (25) 2/5 (40) 4/13 (31) 4/10 (40) 3/7 (43) 7/17 (41) /5 (20) 2/7 (29) 3/12 (25) 2/6 (33) 2/5 (40) 4/11 (36) All ges 6/30 (20) 8/19 (42) 14/49 (29) 8/28 (29) 10/23 (43) 18/51 (35) Mles in Jnury nd June hd significntly lower prevlence rtes (p<0.05) thn did femles. results. High levels of infection re found in the dry seson in Lim, Peru, 33% [13], the United Sttes, 22-27% [14] nd in Guteml City, 8.3% [5]. Other investigtions found high prevlence rtes during the wet sesons in the United Sttes, % [14], Mexico City, 11% [6], in Englnd nd Wles, 2-22% [15], nd round Guteml City, 1% [16]. In contrst, we found no significnt differences in the levels of Cryptosporidium infections etween sesons. The one consistent source of infection ws the deep volcnic lke. The wter level ws constnt nd it ws the min source for drinking, thing nd wshing of household goods. It consisted of run-off wter from the mountins round it, where the villges were locted nd where the villgers prcticed very poor snittion. In our study, lke wter ws the most importnt method of Cryptosporidium trnsmission. Children hd tendency to e less wre of hygiene prctices nd plyed in dirt yrds contminted with fecl mteril. Chickens nd dogs were commonly found roming freely in ech of the towns, resulting in widespred contmintion of the soil with their feces. Cttle nd other domestic nimls were rrely seen, however, humn feces were frequently found on the ground close to humn dwellings. A previous study of children with Cryptosporidium found tht there were no significnt differences in infection due to ge or gender [17]. Our dt, however, showed tht mles in the ge group 2-5 yers hd fewer infections thn did femles (17% vs. 50%, respectively, p<0.05), suggesting tht the mle s exposure to Cryptosporidium ws ffected y life style risk fctors tht incresed the exposure of the femles to the untreted wter. Mle nd femle children of ges 2-5 were siclly treted the sme y their prents, mking their risk fctors very similr, nd the resons for the high level of infection in this ge group of femle children were not ovious. Most mles over 10 yers of ge were up on the mountin sides working in the fields during the dy while the femle children helped their mother wshing clothes in the lke, clening nd cooking, thus hving more exposure to untreted wter, incresing their risk of infection. There re quite likely severl unidentified routes of disese spred in these villges nd further studies re required to determine trnsmission ptterns. In conclusion, we oserved lrge numer of Cryptosporidium infections in children living in two villges round Lke Atitln, Guteml, nd we postulte tht the lke wter ws responsile for trnsmission of the disese. The finding tht children etween the ges of 2 nd 10 were highly susceptile (30-37%) to infection with Cryptosporidium, nd the high prevlence rte in femles (50%), suggested difference in exposure to infections from tht of mle (17%) children. There ws n pprent consistency of infection during oth the dry nd wet sesons (significntly different infection rtes were not

5 BJID 2004; 8 (August) Risk Fctors of Cryptosporidium 323 oserved) corresponding to constnt exposure to the untreted wter supply, coming minly from the lke. This study extends the knowledge of the chrcteristics of Cryptosporidium infections y demonstrting tht constnt source of contminted wter, regrdless of the time of seson, results in high prevlence rtes in children, nd snittion prctices determine the cquisition of infections. Further studies of risk fctors in the villges re needed to identify the trnsmission ptterns of cryptosporidiosis. Acknowledgements This work received finncil support from n inhouse grnt (HEL) nd President s Scholrship Awrd (CZB, JSP, ELG, HEL) from NSU. The ssistnce of numerous DOCARE Interntionl volunteers is pprecited. References 1. Duey J.P., Speer C.A., Fyer R. Cryptosporidiosis in Mn nd Animls. Boc Rton, Florid: CRC Press, Fyer R., Morgn U., Upton S.J. Epidemiology of Cryptosporidium: trnsmission, detection nd identifiction. Int J Prsitol 2000;30: Mc Kenzie W.R., Hoxie N.J., Proctor M.E., et l. A mssive outrek in Milwukee of Cryptosporidium infection trnsmitted through the pulic wter supply. N Eng J Med 1994;331: Alles A.J., Wldron M.A., Sierr L.S., et l. Prospective comprison of direct immunofluorescence nd conventionl stining methods for detection of Girdi nd Cryptosporidium spp. in humn fecl specimens. J Clin Microiol 1995;33: Cruz J.R., Cno F., Cceres P., et l. Infection nd dirrhe cused y Cryptosporidium sp. mong Gutemln infnts. J Clin Micro 1988;26: Enriquez F.J., Avil C.R., Tnk-Kido J.I., et l. Cryptosporidium infections in Mexicn children: Clinicl, nutritionl, enteropthogenic nd dignostic evlutions. Am J Trop Med Hyg 1997;56: Holley H.P., Jr, Dover C. Cryptosporidium spp., common cuse of prsitic dirrhe in otherwise helthy individuls. J Infect Dis 1986;153: Henriksen S.A., Pohlenz J.F.L. Stining cryptosporidi y modified Ziehl-Neelsen technique. Act Vet Scnd 1981;22: Pgno M., Guvreu K. Principles of Biosttistics, 2nd Edition, Duxury, Pcific Grove, Cliforni: Duxury Pulishers, Ungr B.L., Gilmn R.H., Lnt C.F., et l. Seroepidemiology of Cryptosporidium infection in two Ltin Americn popultions. J Infect Dis 1988;157: Awd-El-Kriem F.M., Roinson H.A., Dyson D.A., et l. Differentition etween humn nd niml isoltes of Cryptosporidium prvum using moleculr nd iologicl mrkers. Prsitol 1995;110: Awd-El-Kriem F.M., Roinson H.A., Dyson D.A., et l. Differentition etween humn nd niml isoltes of Cryptosporidium prvum using moleculr nd iologicl mrkers. Prsitol Res 1998;84: Bern C., Orteg Y., Checkley W., et l. Epidemiologic differences etween cyclosporisis nd cryptosporidiosis in Peruvin children. Emerg Inf Dis 2002;8: Amin O.M. Sesonl prevlence of intestinl prsites in the United Sttes during Am J Trop Med Hyg 2002;66: Smerdon W.J., Nichols T., Chlmers R.M., et l. Foot nd Mouth Disese in livestock nd reduced cryptosporidiosis in humns, Englnd nd Wles. Emerg Inf Dis 2003;9: Bern C., Hernndez B., Lopez B., et l. The contrsting epidemiology of Cyclospor nd Cryptosporidium mong outptients in Guteml. Am J Trop Med Hyg 2000;63: Xio L., Bern C., Limor J., et l. Identifiction of 5 types of Cryptosporidium prsites in children in Lim, Peru. J Infect Dis 2001;183:492-7.