Evaluation of a Cryptosporidium Internal Standard for Determining Recovery with Environmental Protection Agency Method 1623 Michael W. Ware1, Donna S. Francy2, Emma J. Granger2, Frank W. Schaefer III3, Mark D. Sobsey4, Otto D. Simmons III4 • 10ffice of Research and Development/ National Exposure Research Laboratory • 2U.S. Geological Survey • 3Office of Research and Development/National Homeland Security Research Center • 4University of North Carolina- Chapel Hill Scientific Question A new product, ColorSeed, has been designed as an internal standard for determining the recovery of Cryptosporidium; however, it has not been validated. Are the recoveries achieved with ColorSeed equivalent to those of live oocysts in reagent and stream water samples? Cryptosporidium 1 Parasite with infective oocyst stage 1 Causes human gastroenteritis 1 Caused 1993 Milwaukee waterborne outbreak, affecting over 400,000 people 1 EPA Method 1623 is the baseline detection method for water samples US EPA Method 1623 1 A 10 L water sample is filtered 1 The oocysts are eluted and purified by immunomagnetic separation (IMS) 1 The oocysts are detected by microscopy after fluorescent antibody staining 1 Oocyst recovery is variable and must be evaluated with controls to interpret results 1 Method performance is linked to water quality 1 Commercial costs average $400/ sample Method 1623 QA Program • Determine recovery of spiked oocysts in reagent and raw waters • Two spiking options evaluated Live oocysts ¦ Spiked and natural oocysts appear the same ¦ Requires analysis of 2 samples • 1 spiked sample • 1 reference sample ColorSeed ¦ Gamma irradiated oocysts ¦ Permanently stained with Texas Red ¦ Spiked and natural oocysts look different ¦ Requires analysis of only 1 sample Study Design for Analysis of Samples Three Replicate 10 L Water Samples One 10 L sample for ColorSeed Two 10 L samples for Live Oocysts I ] Spike 10 L with100 ColorSeed Oocysts Spike 10 L with 100 Live Oocysts i 10 L Reference Sample Same field images of mixed live and ColorSeed oocysts when viewed by green (A) and red (B) filters Filtration and Elution Filtration and Elution IMS followed by Fluorescent Staining IMS followed by Fluorescent Staining Microscopic count of sample Analysis of oocysts by GREEN and RED filters Microscopic count of sample Analysis of oocysts by GREEN filters Number of Live and ColorSeed(CS) oocysts Number of Live Oocysts (SP) Filtration and Elution IMS followed by Fluorescent Staining Microscopic count of sample Analysis of oocysts by GREEN filters Number of Live Oocysts (R) Calculate % recovery CS/100x100% Calculate % recovery (SP-R)/100 x100% Results 60 70 Viable oocysts (percent recovery) Summary and Conclusions • No statistical difference in recovery between live and ColorSeed oocysts was observed in reagent or stream waters. • ColorSeed is an appropriate internal standard for Method 1623. • The use of ColorSeed allows spiking studies to be in only one sample, reducing the costs by $400 per control. Disclaimer Although this work has been reviewed by EPA and approved for publication, it may not reflect Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. \ epascienceforum % Collaborative Science for Environmental Solutions kl m fs 45 /r ' PRO"^ sb # \ ------- |