Cryptosporidium hominis gp60 subtype IbA10G2 is a common cause of cryptosporidiosis. This subtype is responsible for many waterborne outbreaks as well as sporadic cases and is considered virulent and highly important in the epidemiology of cryptosporidiosis. Due to low heterogeneity within the genome of C. hominis it has been difficult to identify epidemiological markers with higher resolution than gp60. However, new markers are required in order to improve outbreak investigations and studies of the transmission dynamics of this clinically important subtype. Based on the whole genome sequences of 17 C. hominis isolates, we have identified several differential loci and developed a new sequence based typing panel with higher resolution than gp60. An amplicon sequencing method was also developed which is based on a one-step PCR which can be sequenced using a Next Generation Sequencing (NGS) platform. Such a system provides a rapid and high-throughput workflow. A panel of nine loci with 10 single nucleotide variants (SNV) was selected and evaluated using clinical IbA10G2 isolates from sporadic, cluster and outbreak associated cases. The specimens were separated into 10 different genetic profiles named sequence types (STs). All isolates within an outbreak or cluster belonged to the same ST, including several samples from the two large waterborne outbreaks which occurred in Sweden between 2010 and 2011 indicating that these outbreaks might be linked. The results demonstrate the methods suitability for improved genotyping of C. hominis IbA10G2.

ABSTRACT In order to improve genotyping and epidemiological analysis of Cryptosporidium spp., genomic data need to be generated directly from a broad range of clinical specimens. Utilizing a robust method that we developed for the purification and generation of amplified target DNA, we present its application for the successful isolation and whole-genome sequencing of 14 different Cryptosporidium hominis patient specimens. Six isolates of subtype IbA10G2 were analyzed together with a single representative each of 8 other subtypes: IaA20R3, IaA23R3, IbA9G3, IbA13G3, IdA14, IeA11G3T3, IfA12G1, and IkA18G1. Parasite burden was measured over a range of more than 2 orders of magnitude for all samples, while the genomes were sequenced to mean depths of between 17× and 490× coverage. Sequence homology-based functional annotation identified several genes of interest, including the gene encoding Cryptosporidium oocyst wall protein 9 (COWP9), which presented a predicted loss-of-function mutation in all the sequence subtypes, except for that seen with IbA10G2, which has a sequence identical to the Cryptosporidium parvum reference Iowa II sequence. Furthermore, phylogenetic analysis showed that all the IbA10G2 genomes form a monophyletic clade in the C. hominis tree as expected and yet display some heterogeneity within the IbA10G2 subtype. The current report validates the aforementioned method for isolating and sequencing Cryptosporidium directly from clinical stool samples. In addition, the analysis demonstrates the potential in mining data generated from sequencing multiple whole genomes of Cryptosporidium from human fecal samples, while alluding to the potential for a higher degree of genotyping within Cryptosporidium epidemiology.

We have developed a novel strategy for the purification of Cryptosporidium oocysts from clinical samples using IMS and PCR amplification of target DNA to facilitate uniform coverage genome sequencing and de novo assembly. Our procedure could also be used for other microbial pathogens from clinical specimens.