INTRODUCTION

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The most common medical disorder among travelers from developed countries to developing countries is diarrheal illness (28, 29), which also is the most common reason that Peace Corps volunteers (PCVs) seek medical care (4, 7). Various bacterial enteropathogens, such as enterotoxigenic Escherichia coli, are the most commonly identified etiologic agents of travelers' diarrhea (5, 18, 21). However, surveillance data from 1990 suggested that intestinal parasitism was prevalent among PCVs and that infection with Entamoeba histolytica was particularly common among PCVs in Guatemala (7, 10). In that context, we conducted studies among PCVs in Guatemala to identify risk factors for diarrheal illness in general and to determine how common various parasitic infections are in this setting. We first conducted a clinic-based, case-control study, which included 48 case (diarrheal) episodes, 26 control episodes, and 115 stool specimens obtained during these episodes (10). Six (12.5%) of the case episodes could be accounted for by protozoal pathogens, specifically, Cyclospora cayetanensis (three episodes), Cryptosporidium parvum (one), Giardia lamblia (one), and E. histolytica-Entamoeba dispar (one). Infection with Blastocystis hominis was equally prevalent among case episodes (31%) and control episodes (32%).

Next, we conducted a prospective, longitudinal study in which a cohort of 36 newly arrived PCVs recorded daily dietary and symptom data and provided at least monthly stool specimens throughout their >2-year stay in Guatemala, even when they were asymptomatic (11). Data for 23,689 person-days and for 1,168 stool specimens were collected. Our findings concerning risk factors for diarrheal illness have already been published (11). Here we present our analyses of the stool data concerning intestinal parasitism. Although we predicted that parasites would account for a minority of the PCVs' diarrheal episodes, we were interested in studying the incidence and natural history of infection with both pathogenic and nonpathogenic parasites.

	MATERIALS AND METHODS

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General. In October 1991, we recruited participants among PCVs en route to Guatemala. The study was approved by the institutional review board of the Centers for Disease Control and Prevention (CDC), and participants provided informed consent. Participants contributed person-days to the study from their arrival in Guatemala until they completed Peace Corps service or withdrew from the study. PCVs were asked to provide daily exposure and symptom data, irrespective of health status, on a structured log, which had one row per day of the month and columns for placing check marks by specific symptoms and exposures (11). PCVs also recorded their medications.

Stool and serum specimens. We asked PCVs to provide a baseline stool specimen in October 1991, before they left for Guatemala; at least one stool per month thereafter; a median of three at midservice; and a median of three at close of service. We encouraged PCVs to collect additional specimens when they had gastrointestinal (GI) symptoms, irrespective of whether the PCV was evaluated in the Peace Corps clinic in Guatemala City. The monthly specimens and those collected when PCVs were symptomatic are not distinguished in the analyses because stools could be of both types and PCVs varied in their thresholds for collecting nonroutine specimens and for being evaluated by medical staff. According to usual practice and separate from the study protocol, specimens collected from PCVs evaluated by medical staff because of GI symptoms also were examined by local Guatemalan laboratories. Whenever possible, the results of this testing were obtained, but details about testing methods were not.

Definitions and statistical methods. An episode of infection with a specific parasite was defined as the occurrence of at least one stool positive for that parasite, irrespective of the findings for other parasites. Successive episodes with the same parasite had to be separated by at least four consecutive stools that were negative for that parasite and at least 30 days between the last stool documented to be positive in one episode and the first stool in the next episode. Because of these criteria, we assumed that successive episodes were statistically independent. Although the true dates of acquisition and clearance of infection were unknown, the duration of an episode was defined as the number of days from the first through the last positive stool within the episode.

	RESULTS

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General information about the PCVs. As previously described (11), 36 (55.4%) of the 65 eligible PCVs participated in the study. The participants had a median age of 24 years (range, 22 to 70), and 18 (50.0%) were women. Participants and nonparticipants were similar with respect to age, sex, and the ultimate duration of Peace Corps service (11). Most participants stayed in the study throughout their Peace Corps service, which began in late October 1991 and usually ended in late 1993 or early 1994. Eight left the Peace Corps after a median of 10 months, and two provided stool specimens somewhat erratically for >1 year and ultimately dropped out of the study before their Peace Corps service ended.

General information about the stool specimens. The 36 PCVs submitted 1,168 stool specimens, including 36 baseline stools, 74 midservice stools, 88 close-of-service stools, and 970 other stools (83.0%) (Table 1). The median number of specimens per PCV was 35 (range, 6 to 63). The compliance rate for submitting at least one specimen per month while enrolled in the study was 96%. Of the 1,168 specimens, 453 (38.8%) were positive for at least one parasite and 48 (4.1%) were positive for a pathogen (includes E. histolytica-E. dispar) (Table 1). Infections with 11 different parasites were documented, and up to five parasites were found per specimen. The proportions of specimens positive for the various protozoal and helminthic pathogens ranged from 0.2 to 1.5%, and those for the various nonpathogens ranged from 0.2 to 7.1% (Table 1); the proportion positive for B. hominis, which was placed in its own category because of uncertainty about its pathogenicity, was 29.9%.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Distributions (shown in box plots) of the number of days from arrival in Guatemala until infection with sequential parasites (e.g., the first parasite to infect the PCV, irrespective of which parasite it was) was documented. For each box plot, the number of PCVs is shown on the left and the number of PCVs (percentage of total on left) infected with pathogens is shown on the right. E. histolytica-E. dispar is classified as a pathogen, and B. hominis is not. On the x axis, both the number of days and the corresponding year are shown.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Distributions (shown in box plots) of the number of days from arrival in Guatemala until infection with particular parasites was documented. The data are for each PCV's first episode of infection with the parasite. For each box plot, the number of PCVs is shown on the left. On the x axis, both the number of days and the corresponding year are shown. The box plots are ordered by ascending medians.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Month of onset of episodes of infection with various parasites. (A) Data for episodes of infection with C. parvum (black bars) and G. lamblia (white bars). (B) Data for nonpathogens. (C) Data for B. hominis. The stool specimens were collected from 22 October 1991 through 26 April 1994 (median, 1 December 1992). Thus, each month includes data for multiple years.

Episodes of infection. The PCVs had 116 episodes of parasitic infection, some of which overlapped because of coinfection with multiple parasites (Tables 2 and 3). The median number of episodes per PCV for the 32 infected PCVs was two (range, 1 to 12). The 18 women (50.0% of 36 PCVs) contributed 12,757 person-days to the study (53.9% of 23,689) and 69 episodes (59.5% of 116), and the 23 persons who were <30 years of age (63.9% of 36) contributed 15,976 person-days (67.4%) and 76 episodes (65.5%); the proportions were not significantly different. The incidence, in episodes per 100 person-years, was highest for B. hominis (65), followed by E. coli (31), C. parvum (17), and E. hartmanni (17) (Table 2). Although no PCV had more than one documented episode of infection with the same pathogen, individual PCVs had up to four episodes with specific nonpathogens and B. hominis. On average, successive episodes were separated by >130 days and by >6 negative stools, which suggests that the episodes represented reinfections rather than relapses (Table 3).

Testing of stool specimens by local laboratories. Separate from the study protocol, 129 stool specimens were tested by local Guatemalan laboratories, typically during evaluations of GI illness. Portions of 126 specimens from 28 PCVs were tested for parasites, and portions of 35 specimens from 22 PCVs were tested for bacteria. Leukocytes were noted in 20 specimens (15.5%), some of which also had blood or mucus. The testing for bacteria yielded one specimen positive for Shigella sonnei, one positive for a Shigella sp., and one reportedly positive for "pathogenic" Escherichia coli; all three specimens had many leukocytes.

Serologic testing. Two or three serum specimens from each of 26 PCVs were available for serologic testing. All specimens were negative for antibody to E. histolytica, including those from the two PCVs with stools positive for E. histolytica-E. dispar.

	DISCUSSION

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We conducted a multiyear study of the incidence and natural history of intestinal parasitic infections in a cohort of 36 PCVs in Guatemala. The study, which included 65 person-years of data, 1,168 stool specimens, and 116 episodes of infection with 11 parasites, was unusual in that it was prospective and longitudinal rather than cross-sectional or clinic based. We are not aware of any other study of intestinal parasitism in which a group of initially asymptomatic adults was monitored so closely, for so long, and with such a high compliance rate.

Our main conclusions are as follows. Intestinal parasitism, considering the parasites as a group, was common. New parasitic infections were gradually acquired throughout the PCVs' >2-year stay in Guatemala and were not markedly seasonal. As expected, infections with pathogenic parasites could account for a minority of the PCVs' diarrheal episodes. C. parvum was the most commonly identified pathogen, E. histolytica-E. dispar was rarely found, and A. lumbricoides was the only identified helminth. B. hominis was by far the most commonly identified parasite, and in several respects, such as prolonged shedding, it behaved more like a nonpathogen than like a pathogen.

The continued acquisition of parasitic infections throughout the PCVs' >2-year overseas stay indicates that the PCVs repeatedly had fecal exposures. Although many of the infections were with nonpathogens, the fecal exposures also placed the PCVs at risk for infections with pathogens, both parasitic and nonparasitic. Our previously published data about the PCVs' risk factors for diarrheal illness in general also led us to conclude that the PCVs repeatedly had potentially risky dietary exposures and that the risk for diarrheal episodes persisted, although it decreased, as the length of stay in Guatemala increased (11). We did not attempt to identify risk factors for infection with specific parasites because we identified insufficient numbers of evaluable episodes for meaningful multivariate analyses. In addition, given that routine specimens were collected monthly, we often were uncertain when the parasitic infections actually were acquired and therefore what the exposure period of interest was. We decided to focus instead on qualitative comparisons of the patterns of infection with individual parasites and with the pathogens versus the nonpathogens.

C. parvum was the most commonly identified pathogen, in part because we examined stools with an immunofluorescence technique that is more sensitive than acid-fast staining (1). Had we not done immunofluorescence testing for either C. parvum or G. lamblia, the numbers of infected PCVs would have fallen from 11 (30.6%) to 2 (5.6%) for C. parvum and from 6 (16.7%) to 5 (13.9%) for G. lamblia. Thus, G. lamblia would have been the most commonly identified pathogen. If the results of the serologic testing for antibody to C. parvum, which was done with an investigational enzyme immunoassay for epidemiologic rather than clinical purposes, are added to the results of the stool testing, the number of infected PCVs rises from 11 (30.6%) to 17 (47.2%). Even more seropositive persons might have been identified had serum specimens been collected more frequently and therefore closer to episodes of infection. The C. parvum cases diagnosed by stool examination were not markedly seasonal, which is consistent with what was found in 1997 and 1998 in a study among Guatemalans in outpatient facilities (3).

Although E. histolytica infection purportedly was common among PCVs in Guatemala before we began our study (10), we documented only two episodes of infection with E. histolytica-E. dispar. None of the PCVs who were tested had developed detectable antiamebic antibody, which suggests that invasive amebiasis was uncommon. Although local Guatemalan laboratories identified E. histolytica-E. dispar more often than study staff did, this could reflect misidentification of other parasites, cells (e.g., leukocytes), or debris as E. histolytica, which is a notoriously common problem (16).

We did not identify any cases of cyclosporiasis in this study, although we identified three in our case-control study (10). We could have missed some infections with this and other parasites by testing insufficient numbers of specimens during some illness episodes (13); by collecting routine stool specimens monthly rather than more often, which would have been impractical; and by using techniques that by present standards are suboptimally sensitive for detection of particular parasites. For example, UV fluorescence microscopy is now known to be more sensitive than examination of acid-fast-stained slides for detection of Cyclospora cayetanensis (12). In addition, we did not use special stains for microsporidia or any molecular techniques for detection of parasites. Unfortunately, the present repertoire of well-evaluated assays for detection of serum antibodies to GI parasites is very limited.

Although we did not design our study to address the controversial question of whether B. hominis is a pathogen (9, 14, 15, 17, 20, 26, 27, 31, 32), we took advantage of the high incidence of B. hominis infection by comparing the patterns of acquisition and excretion of B. hominis with those of the known pathogens and nonpathogens. We were intrigued to note that, in several respects, B. hominis behaved more like a nonpathogen than a pathogen. First, infection was more common with B. hominis and with the nonpathogens as a group than with the pathogens as a group. Second, some PCVs had several episodes of infection with B. hominis and individual nonpathogens but not with any pathogen. Third, PCVs more commonly excreted B. hominis and nonpathogens for prolonged periods, sometimes hundreds of days. Lastly, PCVs were somewhat more apt, although not significantly so, to be symptomatic if infected with pathogens rather than B. hominis or nonpathogens. In our case-control study, B. hominis was equally prevalent during case and control episodes (31 to 32%) (10).

The above comparisons should be considered only qualitative and suggestive, in part because the factors that we could address in the context of our study do not reliably differentiate pathogens and nonpathogens. For example, although commensals probably are more apt than pathogens to be shed for prolonged periods (6, 23-25, 33), pathogens can be excreted for weeks and sometimes months, and they sometimes cause asymptomatic infection and reinfection. Clearly, the durations of excretion we observed were partly dependent on our definition of an episode, which might not have always distinguished accurately between intermittent shedding and reinfection. Our symptom analyses were also complicated by the high background rate of GI symptoms, the need to exclude many infection episodes from the analyses because of documented or possible coinfection with other microbes, and the possibility that what is now called B. hominis includes both pathogenic and nonpathogenic species or strains (9, 14). Although we could not resolve the long-standing controversy about the pathogenicity of B. hominis, we urge caution in attributing symptomatology to B. hominis infection. In situations such as ours, in which the incidence and prevalence of B. hominis infection are high, so is the probability of coincidentally finding B. hominis when symptomatic persons are evaluated.

Although we purposefully focused on parasitic infections, we had hoped to test some stool specimens for other enteropathogens as well. Unfortunately, having PCVs freeze or refrigerate fresh stool specimens in their homes for later testing for bacteria and viruses was impractical, and the aliquots of the specimens provided in clinic that we froze were ruined when the freezer malfunctioned. Therefore, the only testing for bacteria was done by local laboratories, and no specimens were tested for viruses. However, even in studies that include testing for all known enteropathogens, the etiologic agents for at least a substantial minority of episodes of travelers' diarrhea remain unidentified (5, 21, 30). Clearly, our understanding of the epidemiology and natural history of intestinal infection among travelers and expatriates will increase as the diagnostic tools improve.