Evaluation of the immunofluorescence procedure for detection of Giardia cysts and Cryptosporidium oocysts in water

Abstract

The accurate determination of the presence of Giardia cysts and Cryptosporidium oocysts in surface waters requires a reliable method for the detection and enumeration of these pathogenic organisms. Published methods have usually reported recovery efficiencies of less than 50% for both cysts and oocysts. Typically, the losses are greater for Cryptosporidium oocysts than they are for Giardia cysts. The purpose of this study was to examine procedures used for sample collection, elution, concentration, and clarification to determine when losses of cysts and oocysts occurred during processing. The results showed that major losses of cysts and oocysts occurred during centrifugation and clarification. Depending on the centrifugation force, oocyst losses of as high as 30% occurred for each centrifugation step. A 1.15-specific-gravity Percoll-sucrose gradient was needed to optimize recovery of oocysts from natural water samples. Minor improvements in the procedure could be accomplished by selecting a filter other than the recommended 1-m-pore-size (nominal-porosity) polypropylene filter. Giardia cysts and Cryptosporidium oocysts are environmentally resistant intestinal parasites that can cause gastroenteritis in humans when they are ingested. These organisms can be transported by water and have caused documented waterborne outbreaks of giardiasis and cryptosporidiosis (6). The method of detection for Giardia cysts and Cryptosporidium oocysts in water samples relies primarily on microscopic observation of water samples by an immunofluorescence technique (1). This method involves filtration of a large volume of water through a 1-m-pore-size (nominal-porosity) cartridge filter, elution of the microorganisms from the filter using a detergent-based elution medium, concentration by centrifugation, clarification using a 1.10-specific-gravity Percoll-sucrose density gradient, indirect fluorescent-antibody labeling, and microscopic examination of the sample. The immunofluorescence assay (IFA) for Giardia cysts and Cryptosporidium oocysts is time-consuming and labor intensive and requires a large degree of analytical expertise. Moreover, the recovery efficiency of the procedure, especially for Cryptosporidium oocysts, is relatively low. LeChevallier et al. (5) reported an average method efficiency of 68.6% for Giardia cysts in seeded tap water; however, Cryptosporidium oocysts were recovered at an average level of only 25.3%. A recent study of 12 commercial laboratories that processed spiked proficiency samples showed an average of 9% recovery of Giardia cysts and 3% recovery of Cryptosporidium oocysts (2). Moreover, 36% failed to detect Giardia cysts and 55% failed to find Cryptosporidium oocysts, even though the samples contained 99 cysts and 80 oocysts. Large variations in recovery efficiencies have been reported between different laboratories and even within a single laboratory (2, 3). The objective of the present study was to analyze the IFA technique, to determine when the losses of cysts and oocysts occurred during sample pr...

Application of Monitoring Data forGiardia andCryptosporidium to Boil Water Advisories

“…The mean recovery efficiency of the filter analysis procedure was 44.0 Ϯ 4.4% for Giardia cysts and 14.1 Ϯ 4.7% for Cryptosporidium oocysts ( n = 78 control filters). These recoveries compare favorably to the recoveries of 8.8% and 10.8%, respectively, reported by Clancy et al (11) in an interlaboratory comparison, and fit the pattern of recoveries of less than 50% with Cryptosporidium being the lowest that was identified by LeChevallier et al (12) The analytical results from Temagami North and South and from Thunder Bay, Loch Lomond, and Bare Point, are summarized in Table I. The raw and treated Temagami results are divided into "outbreak" and "postoutbreak" periods to show the marked increase in the number of cysts and the very high concentrations of Giardia cysts in both treated and raw water samples from Temagami South during the outbreak.…”

An evaluation of the Gelman Envirochek® capsule for the simultaneous concentration ofCryptosporidiumandGiardiafrom water

“…Giardia recoveries averaged 12% 2 18 using the USEPA method and 49% 2 91 using the Ongerth membrane method; Cryptosporidium recoveries averaged 8% 2 13 with the USEPA method and 9% 2 4 using the Ongerth membrane method. Other studies have reported higher recoveries (LeChevallier et al 1991(LeChevallier et al , 1995 but these recoveries are from seeded packed pellets which do not include the sampling and elution steps. The ICR method recoveries in this study were higher than previously reported (Clancy 1996) and may be due to sampling only a 10 l sample volume rather than the 100 l normally sampled.…”

“…Previous seeding and recovery studies frequently used (oo)cyst doses at concentrations of 10 4 -10 6 in order to achieve some detection of the target organisms (LeChevallier et al 1995;Clancy 1996). These levels are far in excess of the concentrations expected in natural samples.…”

Assessing the risk posed by oocysts in drinking water

“…However, Cryptosporidium detection levels currently used vary considerably and may either underestimate or overestimate actual viable and infective organism levels. [40][41][42] This analysis relies on extrapolation of a doseresponse relationship in healthy subjects to a broader population. A single, laboratory-stored strain of C. parvum was used in the dose-response experiments.…”