WORKSHOPS ON OPPORTUNISTIC PROTISTS 23s

Cryptosporidiwn gastrointestinal or

Molecular Tool's Contribution to the Detection of Cryptosporidium KARINE GUYOT,' ANNE FOLLET-DUMOULIN,'.' MARIE-FLORENCE GIREAUDOT-LIEPMA",'

ROSE-IRENE VERDIER: JEAN-CHARLES CAILLIEZ,lJ and EDUARDO DEI.CASb4

'Inrtitut Pasteur de Lille, France ; 'Envuonnement & S a d , Universitk Catholique de Lille. France; 'Centre GHESKIO, Port au Prince, Haifi ; 4Faculti de Midecine el Centre Hospitalier Rigional de Lille, France.

spp. are apicomplexan protozoa that infect the respiratory tract of humans and animals. In

hnmnmpetent individuals, the diarrhea is acute and self-limited whereas in immunocompromised individuals, it can become chronic md may be particularly life threatening, since no specific treatment is available. HIV-infected patients and children in developing countries pe particularly at risk. Oocysts are the infective stage responsible for transmission. Several routes including person-to-person, contamination Dfwater or food and zoonotic infection are possible. In spite of some confusion regarding the speciation within

Cryptosporidiwn, up to 8 named species have been currently proposed I valid on the bases of host specificity, pathogenesis and oocyst morphology [l]. These include C. parvwn in mammals, C. muris in rodents and ruminants, C. felis in domestic cats, C. wrairi in guinea pigs, C. baileyi and C. meleagridis in birds, C. serpentis in reptiles and C. nasorwn in fish. ,Current routine techniques for the diagnosis of cryptosporidiosis rely oh the recognition of specific oocyst morphology by light microscopy, on fecal smears after modified Ziehl-Neelsen or phenol-auramine staining, or immunofluorescence. As the Cryptosporidiwn parasites are hardly morphologically distinguishable, microscopic methods do not allow species identification. Molecular studies have largely contributed to improve our knowledge on human cryptosporidiosis. It has been shown that human infections can be due to isolates of anthroponotic or motic origin. Those have been designed as the human' genotype, which has so far been found exclusively in humans with the exception of a single non-human primate and a dugong. and the tattle' genotype found in domestic livestock and in humans. Multilocus analysis showed that the 2 genotypes are genetically isolated. As no recombination was detected, clonal propagation of C. parvwn can occur. These results imply the existence of two separate transmission cycles for C. parvwn: one that may occur in humans or animals, and other that exclusively passes through a human host. Reports of numerous studies indicate that geographic variation seems to exist in the distribution of these two genotypes in nonoutbreak situations [5]. Indeed, the human genotype is associated with the majority of isolates obtained from individuals in Australia and in the New World whereas the bovine genotype seems to be dominant in Europe. We found that in France, among 11 immunowmpetent, 27 HN-infected, and 9 immunocOmpromised but HIV-uninfected patients, 7/4, 15/12, and 7/2, respectively were infected with the cattlemuman genotypes of C. parvum (submitted). Initial results on Haitian isolates identified the human genotype in most infections among children under 2-years old whereas no significant difference in the distribution of the two genotypes was observed in the HN-infected adults (unpublished).

Recent studies revealed that immunocompromised and immuno- competent humans can be infected with genetically diverse parasite populations (C. parvwn dog genotype, C . meleagridis and C. felis) [5 ] . We identified C. felis (6 cases), C. meleagridis (3 cases) and a new genotype of C. muris (1 case) in immunocompromised humans in France. From such new relevant epidemiological data, it is clear that C. purvwn is not the only species infecting humans.

Numerous KR-based techniques have been described for detecting Crypfosporidiwn parasites. However, most available genotyping tools

Corresponding author: K Guyot - Telephone : +33 3 20 87 7 1 56; Fax : t33 3 20 87 79 08 ; Email : karine.guyot@pasteur-1iIle.fr.

were designed for clinical specimens and analyze single genetic loci shown to be dimorphic for only cattle and human genotypes. Their specificities for other species of Cryptosporidiwn or C . parvwn genotypes were not always investigated and they may fail to amplify DNA from other human pathogenic Cryptosporidiwn parasites. This may lead to an underestimation of potential public health risks. Interpretation of results should be done with caution. For example, one of the first described protocols widely used in the diagnsis of C. parvwn [3] was reevaluated in our laboratory. We showed that a positive PCR did not necessary indicate the presence of C . parvwn oocysts in the sample. We showed DNA heterogeneity at this locus and used this to develop a RFLP method capable of discriminating between C. parvum and C. meleagridis and between C. parvwn of human and cattle origin.

Molecular diagnostic tests for identifying detected oocysts are also of particular interest to the water industry. Currently. Cryptosporidiwn oocysts are identified by immunofluorescence assays on samples concentrated from water. In our laboratory, we performed this concentration step by filtration according to the USEPA 1622 method or by C a C a flocculation. We showed that use of the 1622 method allows the collection of potentially viable oocysts [2]. However, the flocculation process, which affects oocyst viability. appears as an ideal concentration technique to rapidly identify positive sites in ecosystems. Because environmental samples could contain any species or genotypes of Cryptosporidiwn, the best strategy is to use conserved sequences for PCR primers and therefore amplifying the DNA of all potential Cryptosporidiwn parasites present in the analyzed samples. To this aim, we focused on the 18s rDNA locus. This locus was the fust marker of Cryptosporidiwn for which sequences were available in Genbank for all species and genotypes (except C. nasorwn). We tested several primer pairs for DNA amplification and retained those described by Xiao et al. for nested PCR [4]. In this way, we successfully characterized Cryptosporidiwn oocysts from clinical and samples concentrated from surface water.

In recent years, significant advances have been made in developing molecular tools for the detection and genotyping of Cryptosporidiwn. Application of these techniques has largely contributed to our understanding of the genetic diversity among Cryptosporidiwn isolates. However, larger studies focusing on specific mammal populations and the environment are necessary to have a more complete comprehension of the molecular epidemiology of the parasite. Methods capable of sub- genotyping are currently in development, which should permit identificatin of Cryptosporidiwn strains and therefore improve our knowledge of the disease transmission dynamics. Prevention of human cryptosporidiosis would be completely accomplished by a thorough understanding and appreciation of its complex natural history and epidemiology.

m R A T U R E CUED

1 Fayer R, Speer CA & Dubey JP. 1997 concentrated: Fayer, R. (ed). Crypfosporidiwn and Cryptosporidiosis, Boca Raton (FL), CRC Press, p. 1- 41.

2 Guyot K, Gireaudot-Liepnann MF. Cabon A, Riveau-Ricard I, Lange M. Delattre JM & E. Dei-Cns. 2000. Waf. Sci. Tech., 41:189-196.

3 Laxer MA, Timblin BK & Pate1 RJ. 1991. Am. J. Trop. Med. Hyg., 45688- 94.

4 Xiao L. Escalante L. Yang C, Sulaiman I, Escalante AA, Montali RJ. Fayer R & La1 AA. 1999. Appl. Environ. Microbiol., 651578-1583.

5 Xiao L, Morgan UM, Fayer R, Thompson RCA & La1 AA. 2000. Parasifol. Today. 16:287-292.