Recovery and viability of Cryptosporidium parvum oocysts and Giardia intestinalis cysts using the membrane
R M McCuin1, Z Bukhari, J L Clancy
1Environmental Consultants, Inc., St. Albans, VT 05478, USA.
Abstract:
Previously, the cellulose acetate membrane filter dissolution method was reported to yield Cryptosporidium parvum oocyst recoveries of 70.5%, with recovered oocysts retaining their infectivity. In contrast, high spike doses (approximately 1 x 10(5) Cryptosporidium parvum oocysts and Giardia intestinalis cysts) yielded recoveries ranging from 0.4% to 83.9%, and 3.2% to 90.3%, respectively, in this study. Recoveries with low spike doses (approximately 100 (oo)cysts) continued to demonstrate high variability also. Efforts to optimize the method included increased centrifugation speeds, suspension of the final concentrate in deionized water for organism detection on well slides, and analysis of the entire concentrate. A comparison of two monoclonal antibodies was also conducted to identify potential differences between antibodies in detection of organisms. Archived source and finished water samples were spiked, yielding variable recoveries of C. parvum oocysts (11.8% to 71.4%) and G. intestinalis cysts (7.4% to 42.3%). Effects of organic solvents used in the membrane dissolution procedure on the viability of recovered (oo)cysts was determined using a fluorogenic vital dyes assay in conjunction with (oo)cyst morphology, which indicated > 99% inactivation. These data indicate that the membrane dissolution procedure yields poor and highly variable (oo)cyst recoveries, and also renders the majority of recovered organisms non-viable.
Insights
The cellulose acetate membrane filter dissolution method shows poor and inconsistent recovery rates for Cryptosporidium parvum oocysts and Giardia intestinalis cysts. Furthermore, the method inactivates over 99% of recovered organisms, rendering them non-viable.
Area of Science:
- Environmental microbiology
- Water quality analysis
- Parasitology
Background:
- Accurate detection of waterborne parasites like Cryptosporidium parvum and Giardia intestinalis is crucial for public health.
- Previous studies suggested high recovery rates for Cryptosporidium parvum using the cellulose acetate membrane filter dissolution method.
- Variability in recovery and viability of oocysts and cysts is a significant challenge in water quality monitoring.
Purpose of the Study:
- To evaluate the efficacy and reliability of the cellulose acetate membrane filter dissolution method for recovering Cryptosporidium parvum oocysts and Giardia intestinalis cysts from water samples.
- To assess the viability of recovered organisms after the membrane dissolution procedure.
- To optimize the method for improved recovery and detection.
Main Methods:
- Spiking archived source and finished water samples with known quantities of Cryptosporidium parvum oocysts and Giardia intestinalis cysts.
- Applying the cellulose acetate membrane filter dissolution method with variations in centrifugation speed and sample analysis.
- Utilizing monoclonal antibodies for organism detection.
- Assessing organism viability using fluorogenic vital dyes and morphological examination.
Main Results:
- High spike doses yielded variable recoveries (0.4%–83.9% for oocysts, 3.2%–90.3% for cysts).
- Low spike doses also showed high variability in recoveries.
- Recoveries from spiked water samples ranged from 11.8% to 71.4% for oocysts and 7.4% to 42.3% for cysts.
- >99% inactivation of recovered organisms was observed, confirmed by vital dyes and morphology.
Conclusions:
- The membrane dissolution method demonstrates poor and highly variable recovery rates for Cryptosporidium and Giardia.
- The procedure significantly impacts organism viability, leading to >99% inactivation.
- This method is not reliable for accurate quantification or detection of viable Cryptosporidium parvum oocysts and Giardia intestinalis cysts in water samples.
