Methodsfor Detection of Giardia and Cryptosporidium in Water :

APreliminary Assessment

ReportNo WSAA 25

April 1991

SYNOPSIS

Giardia and Cryptosporidium are significant causes of human gastroenteritisthat can be transmitted by several routes. Water-related outbreaks ofgiardiasis and cryptosporidiosis are known from North America and Britain, andhave involved from fewer than ten to several thousand infections.

Recognitionof infections by Giardia and Cryptosporidium is increasing throughoutAustralia. While there is little evidence yet of transmission by water, thereare strong perceptions among the public and the medical and laboratorycommunity that water is a source of infection, particularly by Giardia. Methods for detecting theseorganisms in water would permit more specific investigation of diseaseoutbreaks as well as prospective studies.

DetectingGiardia and Cryptosporidium in water is essentially a three-stage process,involving primary concentration toreduce a large sample to a volume that can be handled easily in the laboratory,secondary concentration and separation,and microscopic examination, usuallyfacilitated by a stain. Current methods for detecting Giardia and Cryptosporidium inwater 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 inthe field (the “Reference Method”).

Thisstudy concentrated initially on a thorough examination of the secondaryconcentration and microscopic steps. The secondaryconcentration and separation step has been simplified significantly.Centrifugation in a sucrose density gradient permitted recoveries of Giardia and Cryptosporidium exceeding 90%, while segregating mineral materialand much of the organic material into separate fractions. Recovery of theseorganisms in practice will depend on the number and sizes of other organismspresent in a sample. Use of monoclonal antibodies and fluorescent microscopy for microscopic examination, inconjunction with n-propyl gallate to stabilise the fluorochrome fluoresceinisothiocyanate (FITC), improves the recognition of Giardia and Cryptosporidium significantly.

Thesecondary concentration and microscopic steps were used to make a preliminaryassessment of crossflow microfiltration as an alternative method for primary concentration. The importantvariables affecting recovery of Giardiaand Cryptosporidium have beenidentified, but further trials are needed to establish the optimum conditionsfor this process.

Furtherstudy will concentrate on the reproducibility of primary concentration, whichis essential to quantitative analysis, and on field investigations in areaswith potential sources of contamination by Giardiaand Cryptosporidium.

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