Cryptosporidium is a protozoan parasite which causes acute gastrointestinal illness in humans and animals. Numerous species and genotypes have been identified (Feng et al., 2018), and although Cryptosporidium parvum and Cryptosporidium hominis cause most human infections, other mainly animal-adapted species have also caused infections and outbreaks (Ryan et al., 2021). The oocyst stage shed in faeces confers resistance to environmental conditions, can break through water treatment barriers and is resistant to chlorine-based disinfectants at the concentration used to treat drinking water (Betancourt and Rose, 2004). Water monitoring for Cryptosporidium oocysts continues to rely on microscopy-based detection and enumeration (ISO 15553; USEPA 1623; Anon 2010) but does not identify the contaminating species or genotypes, which requires molecular testing. This level of characterisation can help reveal the source of contamination, and is an important part of the investigation of water quality incidents and outbreaks (Chalmers, 2012) but appropriate methods and training need to be in place (Deksne et al., 2020).

Where molecular characterisation of Cryptosporidium spp. from water samples is carried out, this is by downstream DNA extraction and testing of material retrieved from Cryptosporidium-positive microscope slides (Li et al., 2015; DiGiovanni et al., 2010). The benchmark method is amplicon sequencing from nested PCR of the SSU rRNA gene (DiGiovanni et al., 2015; Jiang et al., 2005), which is present in multiple copies in all species and genotypes, and has variable and conserved regions. The whole process is known as “slide genotyping”. This has been applied during operational management and in outbreak investigations in the UK, but not all slides are successfully genotyped and poor typeability is often, but not always, related to low numbers of oocysts (Puleston et al., 2014; Chalmers et al., 2010; Nichols et al., 2006).

Regulatory monitoring and risk assessments for Cryptosporidium oocysts in drinking water in the UK contributed to improvements in water quality and a reduction in cryptosporidiosis cases and outbreaks in the first decade of the 21st century (Lake et al., 2007; Drury and Lloyd 2003). The preventive management for the whole supply chain, encapsulated in the Water Safety plan approach since 2004, also aims to improve drinking water quality (WHO/IWA, 2017). A net effect appears to be continued reduction in the number of oocysts detected in drinking water samples that poses a challenge to slide genotyping; for example, the proportion of slides received in our laboratory for genotyping that had single oocysts seen increased annually from 18% in 2015 to 57% in 2018 (CRU data).

The small amount of target DNA in the sample is not the only challenge posed by slide genotyping. The multiple hosts and sources of Cryptosporidium oocysts that contaminate water mean that different species and genotypes may be captured on a single slide. Strategies to resolve these mixtures have included increasing the number of technical replicates by repetitive nested PCR and limited template dilution (Ruecker et al., 2012; Xiao et al., 2006). Although next generation sequencing technologies have been applied to single Cryptosporidium oocysts these were highly purified from larger populations and free from sample matrix (Troell et al., 2016; Guo et al., 2015).

The aim of the work reported here was to increase the typeability of Cryptosporidium slide genotyping, and to improve the laboratory workflow and turnaround times using real-time PCR. Such an assay needed to incorporate primer and probe sequences homologous with all known Cryptosporidium spp. and genotypes, and generate a suitably short sequence for efficient amplification (Debode et al., 2017), while incorporating the polymorphic regions necessary for species discrimination. Sequence differentiation between species and genotypes needed to be possible from the amplicon generated by the assay. Typeability was required at least equal to the benchmark method (DiGiovanni et al., 2015). We favoured using a real-time PCR incorporating a hydrolysis probe because such assays potentially offer greater sensitivity than conventional PCR (Debode et al., 2017), improved biocontainment and as a single standalone assay they are quicker, offering reduced turnaround time from receipt to results.

Real-time PCR assays have been published previously for the detection and discrimination of individual, important Cryptosporidium spp., for example: C. hominis or C. parvum (Yang et al., 2013) and C. hominis and C. parvum together, C. andersoni individually and C. bovis individually (Burnet et al., 2013). Some assays were developed for specific applications, for example to detect those species important in cattle: C. parvum, C. andersoni, C. bovis and C. ryanae (Thomson et al., 2016), or targeting particular species and sample types e.g. human pathogenic species in water (Li et al., 2015) or clinical samples (Hadfield et al., 2011). Other assays provided for generic detection of Cryptosporidium (Burnet et al., 2013) or provided only limited species discrimination e.g. C. hominis and C. parvum (Samie et al., 2006), or did not differentiate between C. bovis and C. xiaoi (Zahedi et al., 2018). Our challenge was to detect and differentiate all species and genotypes of Cryptosporidium, for which the SSU rRNA is the gene of choice (Xiao et al., 1999).

Here we present the development and validation of real-time PCR amplification and sequencing of the SSU rRNA gene, for improved slide genotyping. Other applications include sensitive and specific detection and genotyping of Cryptosporidium spp. in stools or other sample matrices such as food (Mayer-Scholl et al., 2022).