Most Cryptosporidium infections in humans are caused by Cryptosporidium hominis (formerly Cryptosporidium parvum human genotype or genotype I 1) and by C. parvum bovine genotype (genotype II). 2 C. hominis is found almost exclusively in humans whereas C. parvum bovine genotype is found in various mammals (cattle, sheep, goats). Other zoonotic cryptosporidia associated with single infections in immunocompetent and/or immunocompromised individuals include Cryptosporidium meleagridis, Cryptosporidium felis, Cryptosporidium muris and several genotypes of C. parvum (dog genotype, pig genotype, cervine genotype). 1, 3
Transmission of infective oocysts occurs by the fecal-oral route, and waterborne outbreaks have been described that affected large numbers of persons in the United States and the United Kingdom. Risk factors of cryptosporidiosis other than water contact include traveling abroad, consumption of contaminated food and contacts to infected persons. 4
Epidemiologic studies from Central Europe have revealed prevalences of Cryptosporidium in symptomatic children from 1.0 to 5.5%. 5, 6 In adults prevalences were as low as 0.2% 7 but could reach >11% in HIV-infected patients with chronic diarrhea. 8 In none of these studies was genetic characterization of the cryptosporidia attempted.
The aim of this study was to determine the prevalence and genotypes of Cryptosporidium spp. occurring in diarrheic immunocompetent children living in Switzerland as well as to identify risk factors for acquiring the infection.
Materials and methods.
Study population and clinical data.
From January 2001 to January 2002, stool samples were collected from diarrheic children presenting to pediatricians or to outpatient clinics of children’s hospitals in the region of Zurich (Switzerland). Samples from children older than 14 years, from immunocompromised children or from children who acquired diarrhea during their hospitalization as well as multiple samples from the same child were excluded from the study. Data including age, sex, nationality, recent travel history (i.e. within a period of 3 months before onset of diarrhea), duration of diarrhea, fever, vomiting, abdominal pain, contact with people with diarrhea and the immune status were collected with the use of a structured questionnaire. Detailed information about the study was provided to and written consent was obtained from all parents or legal representatives.
Stool samples and analyses.
All samples were examined for Cryptosporidium spp. microscopically after direct staining of stool smears (1 per patient) by the modified Ziehl-Neelsen (ZN) technique as well as by the immunologic detection of cryptosporidia-specific coproantigens (CAG) with a commercially available test kit (ProSpecT; Cryptosporidium Microplate Assay, Alexon Trend, Ramsey). DNA from ZN- or CAG-positive samples was isolated as described previously, 9 and a ~420-bp fragment of the 18S ribosomal DNA, which encompasses a variable region, was amplified by PCR using primers CPD-DiagF and PW99R. 9 PCR products were further purified (Qiagen spin columns; Qiagen, Hilden, Germany) and directly sequenced using an ABI PRISM 310 (Applied Biosystems) genetic analyzer by a private company. Sequences were analyzed as described previously. 9
Statistical analyses were performed with software (Instat, StatMate) from GraphPad, Inc. (San Diego, CA).
We analyzed 273 stool samples from the same number of children with diarrhea who met the study criteria. Cryptosporidium oocysts were detected microscopically in 10 of these 273 examined stool samples, yielding a prevalence of 3.7%, whereas Cryptosporidium-specific coproantigens were detected in 17 (prevalence, 6.2%) samples, including all that were positive by microscopy. Results could be confirmed by PCR in 14 of these samples (in 9 of 10 of the ZN-positive and 5 of 7 of the CAG-positive group). ZN-positive and PCR-confirmed CAG-positive samples were classified as true positive, resulting in an overall prevalence of 5.5% (15 of 273). A synopsis of the data of the 15 infected children is given in Table 1. Gender-specific prevalences were 10 of 162 (6.2%) for boys and 5 of 111 (4.5%) for girls, respectively (P > 0.05). Patients were 1 month to 14 years of age (median, 4.3 years; median of study population, 2.1 years). There was a statistically significant trend toward higher infection rates in older children (P = 0.018). Prevalences were 2.3% (age 0 to 2 years) and >10% for children older than 6 years (Table 2).
Traveling abroad within a period of 3 months before the onset of diarrhea was reported for 90 (33.0%) of the children, whereas 160 (58.6%) did not leave Switzerland within this period. Infection rates in children with a travel history were 12.2% (11 of 90) as compared with 1.9% (3 of 160) for children without such a history (P < 0.002). Among the positive cases 10 patients had been traveling to Mediterranean or Balkan States and one to the Philippines (Table 1).
With regard to nationality 6 (4.0%) of 149 Swiss children and 8 (13.3%) of 60 children with other nationalities were Cryptosporidium-positive (P = 0.03).
Among all children 66 had chronic diarrhea (defined as duration of 2 weeks or longer), and 160 had acute diarrhea. No information regarding this was available for 26 children. Prevalence rates were 4.5% (3 of 66) and 6.8% (11 of 161) for those with chronic and acute disease, respectively (not significant).
Among the 14 PCR positive cases, 11 (79%) were genetically characterized as C. hominis and 3 (21%) as C. parvum bovine genotype. Follow-up samples, collected 6 to 8 days after the initial samples, were available for 3 patients and were tested with identical results. Information on travel history and genotype was available for 13 of the infected children. Travel-associated infections were caused by C. hominis in 9 of 10 cases, whereas C. parvum bovine genotype was found in 2 of 3 children without a travel history.
The prevalence of cryptosporidiosis of 5.5% found in our study in immunocompetent children with diarrhea is comparable with those from previous reports from Switzerland. 5, 6 Infection rates were highest in children older than 6 years, which is in contrast to a study from Ireland reporting highest rates in children of up to 3 years of age. 10 Given the low prevalence of cryptosporidiosis in Switzerland, infection is thought to be acquired mainly abroad during traveling. 7, 11 In our patient group a travel history was reported only for 23 to 32% of the children younger than 6 years, whereas for older children traveling frequencies were between 53 and 63% (data not shown). This observation could at least partially explain the differences found in the age-related infection rates between regions of low and high endemicity.
Our study design did not allow us to draw conclusions concerning the association of cryptosporidial infection and clinical symptoms, and we cannot exclude that children of the same area are asymptomatically infected. In an Australian study, however, it was shown that the vast majority (89%) of children who tested positive for Cryptosporidium on fecal microscopy indeed suffered from diarrhea. 12
Genetic characterization of Cryptosporidium isolates of human origin in several countries revealed different proportions of C. hominis and C. parvum bovine genotype. In the United Kingdom McLauchlin et al. 13 found C. hominis in 37.8% of 1705 human samples and C. parvum bovine genotype in 61.5%. Interestingly the relation was reversed in travel-associated cases, with C. hominis being significantly more common (64%) than C. parvum bovine genotype (36%). This was also observed in our study, where the majority of infected children had a travel history, and 90% (9 of 10) of the travel-associated isolates were characterized as C. hominis. Despite the high infection pressure with C. parvum bovine genotype in parts of Europe (e.g. prevalence in Switzerland in calves up to 40%), symptomatic cases in immunocompetent individuals are rarely found. However, in our study area AIDS patients with chronic diarrhea were mostly infected with the zoonotic C. parvum bovine genotype. 14
C. hominis has been responsible for most water-borne outbreaks in North America and in the United Kingdom, causing symptomatic cryptosporidiosis in immunocompetent adults and children. 4 Until now no such outbreaks have been reported in Central Europe, presumably because of the use of different drinking water sources or technical aspects of drinking water purification. Several studies in Central Europe confirm the ubiquitous presence of oocysts of Cryptosporidium spp., including genotypes known to be pathogenic to humans, in surface waters. 9
However, our study clearly supports earlier risk analyses 5 that showed that transmission of Cryptosporidium spp. among immunocompetent children is primarily of anthroponotic nature in our study area.
The strong commitment of all pediatricians and the physicians at the children’s hospitals of Zürich and Winterthur involved in this study is greatly acknowledged. This work represents the medical dissertation of CG.
1. Morgan-Ryan UM, Fall A, Ward LA, et al. Cryptosporidium hominis
n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens.
J Euk Microbiol 2002;49:433–40.
2. McLauchlin J, Pedraza-Diaz S, Amar-Hoetzeneder C, Nichols GL. Genetic characterization of Cryptosporidium
strains from 218 patients with diarrhea
diagnosed as having sporadic cryptosporidiosis. J Clin Microbiol 1999;37:3153–8.
3. Xiao L, Bern C, Limor J, et al. Identification of 5 types of Cryptosporidium
parasites in children
in Lima, Peru. J Infect Dis 2001;183:492–7.
4. Fayer R, Morgan U, Upton SJ. Epidemiology of Cryptosporidium
: transmission, detection and identification. Int J Parasitol 2000;30:1305–22.
5. Mausezahl D, Egger M, Odermatt P, Tanner M. Clinical aspects and epidemiology of cryptosporidiosis in immunocompetent children
. Schweiz Rundsch Med Prax 1991;80:936–40.
6. Essers B, Burnens AP, Lanfranchini FM, et al. Acute community-acquired diarrhea
requiring hospital admission in Swiss children
. Clin Infect Dis 2000;31:192–6.
7. Baumgartner A, Marder HP, Muniziger J, Siegrist HH. Frequency of Cryptosporidium
spp. as cause of human gastrointestinal disease in Switzerland
and possible source of infection. Schweiz Med Wochenschr 2000;36:1252–8.
8. Weber R, Ledergerber B, Zbinden R, et al. Enteric infections and diarrhea
in human immunodeficiency virus-infected persons: prospective community-based cohort study. Arch Intern Med 1999;159:1473–80.
9. Ward PI, Deplazes P, Regli W, Rinder H, Mathis A. Detection of eight Cryptosporidium
genotypes in surface and waste waters in Europe. Parasitology 2002;124:359–68.
10. Corbett-Feeney G. Cryptosporidium
with acute diarrhoea in the west of Ireland. J Infect 1987;14:79–84.
11. Egger M, Mausezahl D, Odermatt P, Marti HP, Tanner M. Symptoms and transmission of intestinal cryptosporidiosis. Arch Dis Child 1990;65:445–7.
12. Assadamongkol K, Gracey M, Forbes D, Varavithya W. Cryptosporidium
in 100 Australian children
. Southeast Asian J Trop Med Public Health 1992;23:132–7.
13. McLauchlin J, Amar C, Pedraza-Diaz S, Nichols GL. Molecular epidemiological analysis of Cryptosporidium
spp. in the United Kingdom: results of genotyping Cryptosporidium
spp. in 1,705 fecal samples from humans and 105 fecal samples from livestock animals. J Clin Microbiol 2000;38:3984–90.
14. Morgan U, Weber R, Xiao LH, et al. Molecular characterization
isolates obtained from human immunodeficiency virus-infected individuals living in Switzerland
, Kenya and the United States. J Clin Microbiol 2000;38:1180–3.
Keywords:© 2004 Lippincott Williams & Wilkins, Inc.
Cryptosporidium spp; diagnosis; coproantigen; molecular characterization; children; diarrhea; Switzerland