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TCS Water Sciences for Cryptosporidium Equipment, Reagents and Controls
Cryptosporidium spp Detection and Identification Methods

Cryptosporidium oocysts
Fluorescent Cryptosporidium oocysts
Key Characteristics
  • a protozoan parasite known to cause disease in mammals, including humans
  • Capable of completing life-cycle within host
  • Produces acute long lasting gastrointestinal disease (self-limiting, 3-4 weeks), non-resolving in immuno-compromized individuals
  • No effective treatment
  • Resistant to standard disinfection procedures, e.g. chlorine

Introduction
The above list of attributes relate to Cryptosporidium spp, a protozoan parasite that is known to cause disease in mammals, including humans. It is therefore surprising to learn that there is such a diverse range of regulatory control surrounding Cryptosporidium.

At one extreme, laboratory regulation and data that must be admissible as evidence in a court of law (DWI, UK), through enhanced surface water treatment regulations (EPA, USA) to little or no regulation (Republic of Ireland). All three of these jurisdictions have had recent outbreaks impacting not only human health but also tourism and commerce.

There are other protozoans that are also implicated in human disease, e.g. Giardia, Plasmodium and Toxoplasma. However it appears that Cryptosporidium is the ‘emerging’ protozoan pathogen that is receiving the most attention. Indeed, within the last five years it has been reported that the number of published research articles on the subject of Cryptosporidium was equivalent to that for Campylobacter, itself the biggest cause diarrhoeal disease in humans.

Aetiology and epidemiology
Of the known species of Cryptosporidium, it is the zoonotic C. parvum and anthroponotic C. hominis that are most often associated with cryptosporidiosis in humans. The life cycle of the organism is completed within the human gut and transmission of the disease occurs upon the excretion of stable oocysts, each containing four infectious sporozoites, from the host into the environment. Human disease consists of watery diarrhoea, abdominal pain, vomiting and consequent weight loss.

Most commonly associated with waterborne infections, Cryptosporidium is also implicated in foodborne outbreaks involving fresh produce and processed meats. The significance of Cryptosporidium as a risk factor within food production has seen the development of risk assessment models within HACCP frameworks in those sectors of the industry where heat processing is minimal. As chemical control is largely ineffective, environmental control employs physical techniques, e.g. heat, sedimentation, flocculation and filtration. It is reported that ozonation and UV treatments are also effective against Cryptosporidium oocysts.

It is now accepted that Cryptosporidium is ubiquitous in the environment and thus will find its way into the food chain from time-to-time. New molecular techniques are improving the understanding of the epidemiology of outbreaks and are also reporting in real-time. Analysis at the subgenotype level allows sources of infection to be tracked, pathogenicity to be compared and may lead to the development of novel therapeutics.

Isolation Techniques
Supplier reference
for these items:

acid fast staining

automation

concentration/capture

immunoassay

fiiltration

lateral flow assay

immunofluorescence

molecular methods

QC organisms

slide controls

Until relatively recently (pre-1980) diagnosis of human disease caused by Cryptosporidium was by histological preparations from biopsy samples. Moving forward, the difficulty in environmental control and lack of effective treatment for the disease condition, have lead to developments in both laboratory methodology and management of the organism in the environment.

Stool samples in human disease provide for high numbers of oocysts and relatively simple preparation and processing. Non-invasive, acid-fast staining techniques are used widely in clinical laboratories. These techniques also provide data for other parasitic infections. Antigen produced by the parasites in the gut can be detected from faecal specimens quickly and easily using lateral flow assays.

Environmental samples containing very few oocysts require increased levels of pre-treatment to collect sufficient oocysts for analysis.

Large volumes of water are filtered to concentrate the oocysts together in a small volume or pellet. A secondary concentration step is performed using immuno-magnetic separation (IMS), where magnetic beads are coated with anti-cryptosporidium antibodies that capture the oocysts from the particulate matter of the pellet. After washing, the oocysts are disassociated from the magnetic beads and placed on special slides for subsequent examination.

This takes the form of a combination of treatments all used in conjunction with an epifluorescence microscope. By selecting different filters, the microscope will reveal characteristic staining patterns for the oocysts. One immunofluorescent technique utilizes monoclonal antibodies (Mab) conjugated to a fluorescent dye (FITC). Another observation is performed using the viability stain 4, 6-Diamidino-2-phenylindole (DAPI) highlighting the nuclei within the sporozites. Finally, the presence of internal contents and morphology is performed by differential interference contrast (DIC) microscopy.

Automation eliminates the initial time consuming examination of the prepared slides by using automatic laser scanning devices, leaving the operator to microscopically validate only the detected events.

The development of molecular protocols has allowed the typing of Cryptosporidium species to the sub-genotype level. Techniques such as real-time polymerase chain reaction (RT-PCR) offer speed, specificity and sensitivity in the future analysis of many human pathogens including Cryptosporidium.



Comments
Human cryptosporidiosis was historically associated with waterborne disease, more recently implicated in foodborne outbreaks.

Cryptosporidium presents significant public health challenges that need to be addressed through quantitative risk assessment, leading to better environmental control and reduced incidence win the food chain.

Laboratory methodology is developing to allow faster, more specific data to be generated allowing characterization and tracking of disease causing strains.

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