Aetiology and epidemiology of human cryptosporidiosis cases in Galicia (NW Spain), 2000-2008.

Cryptosporidium infects millions of people worldwide causing acute gastroenteritis, but despite its remarkable epidemiological and economic impact, information on the epidemiological trends of human cryptosporidiosis is still scarce in most countries. Here we investigate a panel of 486 cases collected in Galicia (NW Iberian Peninsula) between 2000 and 2008, which sheds new light on the epidemiology in this region of the South Atlantic European façade. Incidence rates in Galicia are one order of magnitude higher than those reported in other regions of Spain, suggesting that this parasite remains largely underdiagnosed in this country, and are also larger than those typical of other European countries with available data. Two species dominate our dataset, Cryptosporidium hominis (65%) and C. parvum (34%). The sex ratio of patients infected by either species was 0·5, but C. hominis was significantly more common in younger males. C. parvum infections were more acute and required more specialized medical attention, which suggests a differential adaptation of each species to human hosts. The parasites display strong seasonal and geographical variation. C. parvum incidence peaked during summer and was mainly detected in rural areas while C. hominis infections were more frequent in autumn and exhibited a more even geographical distribution. Such differences probably reflect their distinct sources of infection - C. parvum is mainly zoonotic and C. hominis anthroponotic - and the effects of climatic variables, like temperature and rainfall.

High-throughput genotyping assay for the large-scale genetic characterization of Cryptosporidium parasites from human and bovine samples.

The epidemiological study of human cryptosporidiosis requires the characterization of species and subtypes involved in human disease in large sample collections. Molecular genotyping is costly and time-consuming, making the implementation of low-cost, highly efficient technologies increasingly necessary. Here, we designed a protocol based on MALDI-TOF mass spectrometry for the high-throughput genotyping of a panel of 55 single nucleotide variants (SNVs) selected as markers for the identification of common gp60 subtypes of four Cryptosporidium species that infect humans. The method was applied to a panel of 608 human and 63 bovine isolates and the results were compared with control samples typed by Sanger sequencing. The method allowed the identification of species in 610 specimens (90·9%) and gp60 subtype in 605 (90·2%). It displayed excellent performance, with sensitivity and specificity values of 87·3 and 98·0%, respectively. Up to nine genotypes from four different Cryptosporidium species (C. hominis, C. parvum, C. meleagridis and C. felis) were detected in humans; the most common ones were C. hominis subtype Ib, and C. parvum IIa (61·3 and 28·3%, respectively). 96·5% of the bovine samples were typed as IIa. The method performs as well as the widely used Sanger sequencing and is more cost-effective and less time consuming.

Multilocus patterns of genetic variation across Cryptosporidium species suggest balancing selection at the gp60 locus.

Cryptosporidium is an apicomplexan protozoan that lives in most vertebrates, including humans. Its gp60 gene is functionally involved in its attachment to host cells, and its high level of genetic variation has made it the reference marker for sample typing in epidemiological studies. To understand the origin of such high diversity and to determine the extent to which this classification applies to the rest of the genome, we analysed the patterns of variation at gp60 and nine other nuclear loci in isolates of three Cryptosporidium species. Most loci showed low genetic polymorphism (πS <1%) and similar levels of between-species divergence. Contrastingly, gp60 exhibited very different characteristics: (i) it was nearly ten times more variable than the other loci; (ii) it displayed a significant excess of polymorphisms relative to between-species differences in a maximum-likelihood Hudson-Kreitman-Aguadé test; (iii) gp60 subtypes turned out to be much older than the species they were found in; and (iv) showed a significant excess of polymorphic variants shared across species from random expectations. These observations suggest that this locus evolves under balancing selection and specifically under negative frequency-dependent selection (FDS). Interestingly, genetic variation at the other loci clusters very well within the groups of isolates defined by gp60 subtypes, which may provide new tools to understand the genome-wide patterns of genetic variation of the parasite in the wild. These results suggest that gp60 plays an active and essential role in the life cycle of the parasite and that genetic variation at this locus might be essential for the parasite's long-term success.