Novel real-time PCR assays for the specific detection of human infective Cryptosporidium species

Cryptosporidium is a parasite that causes the majority of waterborne protozoan outbreaks of gastrointestinal disease worldwide. Cryptosporidiosis usually presents as self-limiting, although often prolonged, diarrhea with abdominal pain, nausea, vomiting and low-grade fever. Infection can be life threatening in some severely immunocompromised groups. Post infection sequelae have also been reported. On a global level, Cryptosporidium hominis and Cryptosporidium parvum cause the majority of human infections and drinking waterborne outbreaks. Recently, Cryptosporidium cuniculus has also caused a sizeable waterborne outbreak. Molecular detection of human infective Cryptosporidium species is usually based on conserved housekeeping genes, as is the case for other microorganisms. However, novel detection targets have been investigated for Cryptosporidium, but previous investigations aiming to identify C. hominis and C. parvum specific genes had limited success because the majority of putative species-specific genes were subsequently found to be present in the other species. This was attributed to the limited numbers of genome sequences available and their low quality that hindered the accurate prediction of genes’ presence. Nevertheless, evidence was found for one C. hominis specific gene (Chos1), which was further validated independently using comparative genomics of newly sequenced clinical isolates. Chos-1 has a telomeric location and the predicted secreted protein of 50 kDa has interesting features, (highly glycosylated, serine rich and several internal repeats), suggestive of a role in host pathogen interaction. It is plausible that other Cryptosporidium speciesspecific genes and virulence factors are also located at the telomeres. Subtelomeres are hotspots for genetic recombination in other parasitic protozoa, resulting in significant chromosome length and sequence composition polymorphisms and an over-representation of highly-diverged loci which have been utilized for discriminatory diagnostic purposes. Thanks to decreased costs and technological advances, an increasing number of full genome sequences are becoming available for Cryptosporidium spp. and are contributing to improved comparative genomics analyses. While many investigators used these genomes to identify genes and proteins based on their presence in other parasites and bacteria, we have continued to look for species-specific genes that are likely to be involved in host parasite interaction and virulence. In addition, these genes provide a source of highly specific and stable detection markers. In this study, we used an increased number of genome sequences to identify species-specific genes, focusing on genes close to the chromosome ends. Subsequently, primers and probes targeting these loci were designed and evaluated as novel real-time PCR assays for the specific detection of the major human infective waterborne parasites C. hominis and C. parvum. Previous investigation of putative species-specific genes led to the characterization of the telomeric Chos-1 gene. This finding suggested that further genes are likely to be analogously positioned and warranted further investigation of subtelomeric regions of C. hominis and C. parvum genome sequences. In the first instance, the reference genome sequences of C. hominis TU502 (gp60 subtype IaA25R3) 13 and C. parvum Iowa (gp60 subtype IIaA15G2R1) 14 were retrieved from CryptoDB (http:// cryptodb.org) and used for analysis. Subsequently, genomic data of newly sequenced genomes from C. hominis and C. parvum clinical isolates (UKH4 gp60 subtype IaA14R3 and UKP6 gp60 IIaA15G2R1, respectively) were used to verify our findings. Comparative analysis of sequence data was achieved by aligning subtelomeric regions spanning 30000 bp from the 50and 30-ends of