A CRITICAL REVIEW OF MOLECULAR BIOLOGY TECHNIQUES FOR DETECTION AND ENUMERATION OF CRYPTOSPORIDIUM OOCYSTS.
Report No DWI0375/16

Feb 1994

SUMMARY:

This report reviews the recent literature on techniques for the detection, identification and enumeration of Cryptosporidium. Although background information was presented on the present state of research leading to the adoption of molecular techniques for the study of Cryptosporidium, emphasis was placed on the requirements of the water industry.

The importance of cryptosporidiosis as a waterborne disease is now well recognised as well as the attendant difficulty of detecting precisely the offending pathogen in water.

Low oocyst counts, preponderance in water of extraneous materials that could interfere with tests and the many ways through which water contamination could arise are some of the factors that preclude the use of conventional diagnostic techniques for testing water samples.

The sensitivity and specific nature of molecular techniques, with some of them being able to detect fractions of the parasite, make them an obvious choice in the quest for identifying techniques suitable for use by the water industry.

The electrophoretic mobility pattern of specified enzymes (Isoenzyme analysis) has been used to distinguish between Cryptosporidium species. However, the large number of oocysts required for the assay precludes its use for screening water samples.

Flow cytometry following concentration and fluorescent staining has been found reliable for species differentiation and oocyst concentration. The technique relies on the separation of Cryptosporidium oocysts based on size, light scattering and the intensity of fluorescence. While flow cytometry has promise for use by the water industry as it is time and labour saving, problems of cost effectiveness and the non-availability of dedicated software as well as a lack of appropriate species-specific monoclonal antibodies number among the factors that hinder its routine use for screening water samples.

Magnetizable particle technology is another novel method that has been investigated for use. The technique "captures" oocysts exposed to specific antibodies coated on magnetic beads in a magnetic field. It is time and labour saving and not capital intensive. The snag with the use of the technique is that, at the moment, it is not automated and has only been developed for small scale use. Furthermore, the fact that visual confirmation of results is still necessary is a drawback.

Dielectrophoresis, a technique that characterises particles based on their migration pattern when exposed to a non-uniform electric field has been evaluated for the detection of Cryptosporidium, concentration and the assessment of the viability of cryptosporidal oocysts. Preliminary findings indicate that the technique is potentially sensitive, labour saving and easily adaptable to computer technology. The high cost of the equipment and particularly its low limit of sensitivity have to be further improved upon before the technique can be adopted for use by the water industry.

Electrorotation assay detects oocysts exposed to specific antibodies or DNA "probes" based on their rotation rate in an electromagnetic field. Although the stage of development of the technique for use in the study of cryptosporidiosis is still rudimentary, it appears to be a technique that will be useful to the water industry when fully developed. The fact that it is potentially highly sensitive, has prospects for not only concentrating oocysts but also distinguishing viable from non-viable oocysts are some of its major attractions.

Polymerase chain reaction (PCR) has been found highly sensitive for distinguishing between different species of Cryptosporidium. Unlike chromosomal DNA analysis, PCR requires small amounts of parasite material for its application. This point makes it well suited for use by the water industry. However, the ease with which the test system can be contaminated and the elaborate and well organised laboratory facilities needed for its application, are some of the crucial factors that possibly would affect its use for the routine monitoring of water.

Cooled charged coupled device (CCD) and enhanced chemiluminescence (ECL), methods that detect light emissions from fluorescent-labelled oocysts have also been investigated for use in the study of cryptosporidiosis. The major drawback of using CCD is the high equipment cost while low resolution, detection of false positives, the need for concentrating the oocysts before use and the confirmation of observations by microscopy are some of the problems associated with the use of ECL.

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