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Cryptosporidium/Giardia Detection and Identification Methods

Giardia Detection Kit

Key Points

  • Cryptosporidium parvum and Giardia intestinalis are water- and food-borne intestinal parasites, which pose a threat to human health in the developed world
  • Standard detection methods for clinical and water samples are based on microscopy and require skilled technicians to perform them correctly. They are not capable of identification to species level.
  • Detection methods for the water industry require very large sample volumes and effective concentration procedures to detect the very low numbers of protozoan cysts likely to be present
  • Newer immunology- and PCR-based detection methods are more sensitive, faster and easier to perform than microscopy and offer the possibility of differentiation of species and genotypes

Cryptosporidium

A wide range of intestinal parasites can be transmitted to humans directly, or via contaminated water and foods. Generally, these organisms are much more prevalent in developing countries with poor sanitation, but some are significant threats to public health in the developed world. The most important and widespread group of intestinal parasites are protozoa. Protozoa are microscopic single-celled organisms, some of which are parasites of animals, including humans. A number of genera are capable of causing water- and food-borne illness, but the two most important in the developed world are Cryptosporidium and Giardia. Methods for the detection and identification of these protozoa have been developed for applications in clinical, water and food microbiology laboratories.

Cryptosporidium
Cryptosporidium is a single-celled protozoan parasite belonging to the subclass Coccidia. One species, Cryptosporidium hominis, is specific to humans, but C. parvum, which also infects ruminants, is more important in human disease. Cryptosporidium has a complex life cycle, most of which takes place within the gastrointestinal tract of the host, but the transmissible stage is a resistant, thick-walled spore, known as an oocyst.

Cryptosporidium can cause acute gastrointestinal infection with profuse, watery diarrhoea and abdominal pain being the principal symptoms. A single oocyst may be sufficient to cause illness in very young children. Illness usually lasts for a few days in healthy adults, but can be more severe and longer lasting in vulnerable individuals, especially young children and the elderly. Infected humans and animals excrete large numbers of oocysts during illness and may continue to do so after symptoms have subsided.

Cryptosporidiosis is not especially common, but is probably under-reported in many countries. Large outbreaks are usually associated with contaminated drinking water. For example, in 1993 a water-borne outbreak in Milwaukee in the USA affected over 400,000 people and caused 69 deaths. Outbreaks associated with contaminated food have also been reported, but most cases are thought to be water-borne, or caused by direct contact with an infected person or animal.

Cryptosporidium oocysts are very small (4-6 µm diameter) and resistant to chemicals, including chlorine. These characteristics cause problems for water treatment plants, and where large numbers of oocysts are present it is not always possible to guarantee that public water supplies are uncontaminated. In these situations a temporary 'Boil Water Notice' may be the only way to prevent infection. The availability of sensitive detection methods for Cryptosporidium is very important for the water supply industry.

Giardia
Giardia is a single-celled, flagellate protozoan parasite belonging to the order Diplomonadida. Its cells are unusual in having two nuclei. The important species in relation to human illness is Giardia intestinalis (previously named G. lamblia), which also infects other domestic and wild animals. G. intestinalis exists in two forms during its life cycle. Motile trophozoites live and multiply within the host's gastrointestinal tract and quickly die outside the host. The transmissible stage is a thick-walled resistant cyst.

G. intestinalis can cause an acute gastrointestinal infection in humans, especially children. The main symptoms are diarrhoea and abdominal pain, which may persist for several weeks, or even longer in vulnerable individuals. Infected humans and animals may continue to shed cysts in faeces long after symptoms have subsided, sometimes for years.

G. intestinalis is the most commonly reported human intestinal parasite in the developed world, but is nevertheless thought to be considerably under-reported. Most cases are likely to be caused by contact with contaminated water, infected people and animals, and occasionally by ingestion of contaminated food. Large outbreaks are rare and are mostly water-borne.

The cysts of G. intestinalis are larger and less resistant to chlorine that those of Cryptosporidium and much less likely to pass through water treatment systems into the public water supply. But they survive and remain infective for long periods in surface waters.

Methods

Detection of the infective cysts of protozoan parasites cannot be accomplished by culture techniques and depends on sufficient cysts being present to detect by microscopy, immunological, or molecular methods. Methods for detection of Cryptosporidium oocysts and Giardia cysts are very similar and can often be carried out simultaneously using the same assay. Most published and commercial assays have been developed for clinical and water applications, but methods for detection of the parasites in certain foods are also available.

1. Clinical methods
Diagnosis of both cryptosporidiosis and giardiasis can be accomplished by microscopic examination of duodenal aspirates and biopsy samples for sporozoites and other stages in the life cycles. However, the presence of large numbers of oocysts in the faeces allows detection by examination of stool samples and most clinical methods are based on this approach.

Collection and storage of samples
Faecal samples for diagnosis of Cryptosporidium and Giardia infection should be tested as soon as possible, ideally within 24 hours. Alternatively, samples can be frozen or preserved with formalin and other fixatives. Since the shedding of cysts may be sporadic, at least three consecutive samples should be collected.

Detection
Large numbers of cysts may be present in the faeces of infected individuals and fresh samples can be examined without further processing. However, for samples preserved in fixatives, a concentration step may be necessary. Typically this involves a formalin-ethyl acetate concentration procedure with centrifugation to separate cysts from debris.

Microscopy
Direct microscopic examination of faecal smears prepared from fresh or concentrated samples is still widely used to detect protozoan cysts in faeces. As the cysts of Cryptosporidium, and to a lesser extent Giardia, are very small, a staining procedure is required to detect them. A number of different staining techniques have been developed, but acid-fast procedures, such as the Ziehl-Neelsen stain and modified Kinyoun's stain are commonly used, along with trichrome stain and fluorescence-based stains, such as auramine phenol. These methods require the skills of an experienced microscopist to be performed and interpreted correctly.

The current preferred method for the detection of protozoan parasite cysts by microscopy is based on immunology. Direct fluorescent-antibody (DFA) testing utilises fluorescent-labelled antibodies specific for cell wall antigens of Giardia cysts and Cryptosporidium oocysts to visualise the parasites and provide a definitive diagnosis. Commercial DFA tests are available, such as the widely used MERIFLUOR® Cryptosporidium/Giardia test from Meridian Biosciences, which are more sensitive than conventional staining techniques and easier to perform.

Immunology
A number of immunology-based methods have also been developed to detect soluble protozoan antigens in faecal samples, thus removing the need for microsopic examination. These methods are now widely used in clinical laboratories for screening large numbers of samples quickly prior to confirmation by microscopy. Enzyme immunoassay (EIA) technologies have been used to develop commercial products for the detection of Cryptosporidium oocysts and Giardia cysts. These assays can be performed in a few hours using a microplate format and the results read using a spectrophotometric plate reader. Examples include Remel's ProSpecT® microplate immunoassays and the RIDASCREEN® range of immunoassays from R-Biopharm.

Immunochromatographic lateral-flow 'dipstick' tests have also been developed for protozoan antigen detection in stool samples. These tests are sensitive and specific, but very easy to use and provide a result in 10 minutes. Examples of commercially available lateral-flow tests include the ImmunoCard STAT!® test from Meridian Biosciences and Remel's Xpect® test for Cryptosporidium.

Molecular biology
In recent years molecular biology-based diagnostic detection methods have been developed for protozoan parasites. These are typically based on PCR, or real-time PCR (RT-PCR) technology and target specific sequences in the protozoan genome. These methods offer highly specific and sensitive assays, but have yet to be developed into widely available commercial clinical diagnostic products. However, the CeeramTOOLS range from bioMérieux includes molecular detection test kits based on RT-PCR technology that are marketed for both Cryptosporidium and Giardia in clinical samples and a few PCR-based tests for screening veterinary samples have become available.

Identification
Identification of Cryptosporidium and Giardia to species level is extremely difficult to accomplish by conventional methods and is a task for specialist parasitology laboratories. It is not usually necessary for routine diagnostic purposes, but may be important in outbreak investigations. Recently, molecular biology techniques have made identification more practical, although specialist skills are still required. PCR followed by restriction fragment length polymorphism (RFLP) analysis or sequencing, targeting different parts of the genome, have been widely employed. Real-time PCR methods have also been described.

2. Water and environmental methods
Monitoring of treated drinking water, raw water and waste water samples for Cryptosporidium and Giardia cysts is hampered by the low numbers usually present. This means that large volumes of water need to be examined, especially when testing treated water. A concentration step is therefore essential and current methods incorporate a filtration procedure before the detection method. Standard methods have been published for the water treatment industry. One example widely used around the world is USEPA method 1623 as summarised below.

Collection and storage of samples In water treatment plants sampling points can be built in so that large volumes (up to 1,000 litres for treated water) of water at different stages of the process can be diverted directly through a suitable filter. The filter is then sent to the laboratory for further analysis under refrigeration and processed as soon as possible. However, where remote testing is required, water can be collected in large carboys (10-50 litres) and transported to the laboratory chilled to a temperature below 10oC. Sample processing should begin within 96 hours of sampling.

Detection
The water sample is first passed through a filter system able to capture the small Cryptosporidium oocysts and Giardia cysts. Filter capsules designed specifically for the purpose are available commercially and examples include the Envirochek™ capsule from Pall Life Sciences and the Filta-Max® system from IDEXX. The filter is then eluted and the eluate centrifuged to concentrate the cysts, which are then separated from other material using an immunomagnetic separation (IMS) procedure. The IMS procedure can be semi-automated using the TCS Biosciences Isolate® system. The oocysts and cysts are then stained on well slides using labelled monoclonal antibodies and examined by immunofluorescence assay (FA) microscopy. Confirmation can be accomplished by staining with 4',6-diamidino-2-phenylindole (DAPI) and by differential interference contrast (DIC) microscopy. Staining systems for FA microscopy are available commercially and include the Meridian Biosciences MERIFLUOR system and BTF EasyStain™ system. The EasyStain has also been found to be suitable for use with PCR analyis. (Download report - Di Giovanni, G. D., R. M. Hoffman, and G. D. Sturbaum. 2010. Cryptosporidium Genotyping Method for Regulatory Microscope Slides, Project 4099 Report. Denver, CO: Water Research Foundation)

Cytometry has been investigated as a detection method for protozoa in water samples. A validated protocol has been developed for the ChemScan RDI® Solid-Phase Cytometry instrument marketed by bioMérieux. This is able to detect Cryptosporidium oocysts retained on a filter membrane using fluorescent cell labelling and laser scanning technology. The instrument is highly automated and capable of producing results within four hours of sampling. The method has gained approval from the UK Drinking Water Inspectorate (DWI).

Molecular biology and PCR-based detection methods have also been developed for Cryptosporidium oocysts and Giardia cysts in water. As with clinical methods, these target specific sequences on the protozoan genome and can be used as alternatives to FA microscopy. Some commercial products are available, for example CeeramTOOLS range from bioMérieux and Norgen Biotek market Cryptosporidium RT-PCR Detection Kits for water and environmental samples.

Identification
Unfortunately the standard methods used in the water industry do not identify protozoan species or genotypes. As in the clinical sector, RT-PCR based methods have been developed for identification and some commercial applications have been marketed. For example, the Cryptosporidium Genotyping Kit from Invitrogen can distinguish between infective and non-infective species and genotypes.

Quality Control and Reference Material
EasySeed™ is the global reference standard for Cryptosporidium and Giardia testing and is approved by the UK Drinking Water Inspectorate and the US Environmental Protection Agency. ColorSeed™ is an easy to use as an internal standard that can be added to every sample for Cryptosporidium and Giardia testing. The inactivated Cryptosporidium oocysts and Giardia cysts of ColorSeed fluoresce red whilst those that were present in the sample fluoresce green.

3. Food methods
Food samples are rarely tested for Cryptosporidium oocysts or Giardia cysts. The low numbers likely to be present and the difficulty of extracting them from most food matrices preclude practical routine analysis. However, methods have been developed for certain 'high risk' foods, notably ready-to-eat salads and fresh produce. The FDA Bacteriological Analytical Manual (BAM) includes such a method, designed to detect Cryptosporidium oocysts in fresh produce washes and in filterable samples such as juices. The methods described are adapted from those used in water analysis and include both microscopy and PCR-based detection.

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Water Filters and IMS

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Fluorescent Assay Staining

Molecular Biology

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This guide has been prepared by Food Safety Info, scientific and technical information providers for the food industry. For more information, visit our web site at www.foodsafetywatch.com

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