CryptoNet: Molecular-based Tracking to Better Understand U.S. Cryptosporidiosis Transmission

As recently as the late 1990s, cryptosporidiosis in humans and other animals was thought to be caused by only one Cryptosporidium species, C. parvum. Molecular testing has since rewritten Cryptosporidium’s taxonomy. We now know that what we thought of as a single species is really a collection of at least 30 species, many with multiple subtypes causing varying severities of illness. All forms are morphologically indistinguishable by traditional clinical lab tests. Only a few of the species cause human infection. National molecular surveillance data in the United Kingdom has demonstrated that >95% of human Cryptosporidium infections are caused by multiple subtypes of C. hominis and C. parvum. C. hominis primarily exists in a human-to-human transmission cycle in more urban settings, while C. parvum is more rural transmission impacting both humans and ruminants (e.g., pre-weaned calves). Other rarer species can also infect humans. Only molecular methods can distinguish these species, genotypes, and subtypes to elucidate the complex transmission of Cryptosporidium in the U.S.

In response to the inability of traditional clinical diagnostics to distinguish Cryptosporidium species, genotypes, and subtypes and increased national reporting of cryptosporidiosis, CDC is developing CryptoNet, the first molecular tracking system for a parasitic infection. CryptoNet will be a multidisciplinary, molecular-based surveillance system built on the common BioNumerics platform successfully used by PulseNet and CaliciNet. CryptoNet planning is aimed at the efficient use of existing infrastructure to facilitate the systematic collection and molecular characterization of Cryptosporidium isolates to further understand cryptosporidiosis epidemiology.

By facilitating real-time sharing of molecular epidemiology data among U.S. national, state, and local public health departments, CryptoNet will further elucidate the epidemiology of cryptosporidiosis and Cryptosporidium species, genotype, and subtype transmission by:

• Improving detection, investigation, and interpretation of waterborne, zoonotic, person-to-person, and foodborne cryptosporidiosis outbreak data
• Identifying geographic and temporal changes in the distribution of Cryptosporidium
• Increasing capacity to identify traditional and novel epidemiological links and risk factors, outbreak sources, and sources of contamination
• Identifying Cryptosporidium species, genotypes, and subtypes not previously known to infect humans

Examples of how molecular typing has improved our epidemiologic understanding of cryptosporidiosis include:

• Oklahoma, 2007: Determined that two distinct outbreaks of cryptosporidiosis had occurred in adjacent counties during the same month.
• Colorado, Idaho, New Mexico, and Iowa, 2007: Identified a previously rare C. hominis subtype in 40 cases (70%) from all four states.
• New Mexico, 2007: Identified Cryptosporidium horse genotype in a pet shop employee with severe clinical symptoms.
• North Carolina, 2009: Identified zoonotic transmission of C. parvum that had occurred from pre-weaned calves to camp attendees and staff, an epidemiologic link that the retrospective cohort study failed to identify.

Cryptosporidium Genotyping Techniques. CryptoNet has been built for comparison of the 18S rRNA genes and 90 kDa glycoprotein gene (gp60) and has established a DNA sequence database using:

• A nested PCR-RFLP method on the 18S rRNA gene extracted from cryptosporidiosis diagnostic samples. The 18S rRNA gene has high copy numbers and the presence of conserved regions interspersed with highly polymorphic regions. The 800 bp nested PCR amplicon is used in RFLP reactions with different sets of restriction enzymes to differentiate Cryptosporidium species and genotypes.
• A nested PCR method on the gp60 gene extracted from clinical samples submitted for subtyping of Cryptosporidium species. GP60 is the most commonly used target for subtyping C. parvum, C. hominis, and C. meleagridis. Sequencing of the 800 bp nested PCR product identifies subtype family and subtype.

Laboratory Resource Requirements and Costs. Laboratories that regularly perform molecular analysis will need little to no additional equipment or resources to complete the necessary methodologies for Cryptosporidium genotyping. Specific equipment needs include a thermal cycler, microcentrifuge, electrophoresis system, water bath, PCR workstation, ABI 3130xl Genetic Analyzer or similar sequencing machine, and BioNumerics software. The estimated cost per sample beginning with DNA isolation and ending with DNA sequencing is approximately $9.50 ². CDC will cover expenses to train and certify partnering laboratories across the U.S.