Methods for Detection of Giardia and Cryptosporidium in Water: A Preliminary Assessment
This report was produced for the Urban Water Research Association of Australia, a now discontinued research program.
Report No UWRAA 25
Giardia and Cryptosporidium are significant causes of human gastroenteritis that can be transmitted by several routes. Water-related outbreaks of giardiasis and cryptosporidiosis are known from North America and Britain, and have involved from fewer than ten to several thousand infections.
Recognition of infections by Giardia and Cryptosporidium is increasing throughout Australia. While there is little evidence yet of transmission by water, there are strong perceptions among the public and the medical and laboratory community that water is a source of infection, particularly by Giardia. Methods for detecting these organisms in water would permit more specific investigation of disease outbreaks as well as prospective studies.
Detecting Giardia and Cryptosporidium in water is essentially a three-stage process, involving primary concentration to reduce a large sample to a volume that can be handled easily in the laboratory, secondary concentration and separation, and microscopic examination, usually facilitated by a stain. Current methods for detecting Giardia and Cryptosporidium in water are time-consuming and give poor recovery or are poorly reproducible. There appears to be scope for simplifying the initial concentration step, particularly in the most widely-used method for concentrating large volumes in the field (the “Reference Method”).
This study concentrated initially on a thorough examination of the secondary concentration and microscopic steps. The secondary concentration and separation step has been simplified significantly. Centrifugation in a sucrose density gradient permitted recoveries of Giardia and Cryptosporidium exceeding 90%, while segregating mineral material and much of the organic material into separate fractions. Recovery of these organisms in practice will depend on the number and sizes of other organisms present in a sample. Use of monoclonal antibodies and fluorescent microscopy for microscopic examination, in conjunction with n-propyl gallate to stabilise the fluorochrome fluoresceinisothiocyanate (FITC), improves the recognition of Giardia and Cryptosporidium significantly.
The secondary concentration and microscopic steps were used to make a preliminary assessment of crossflow microfiltration as an alternative method for primary concentration. The important variables affecting recovery of Giardia and Cryptosporidium have been identified, but further trials are needed to establish the optimum conditions for this process.
Further study will concentrate on the reproducibility of primary concentration, which is essential to quantitative analysis, and on field investigations in areas with potential sources of contamination by Giardia and Cryptosporidium.