Effective Concentration and Detection of Cryptosporidium, Giardia, and the Microsporidia from Environmental Matrices

Cryptosporidium spp., Giardia spp., and members of Microsporidia are enteropathogenic parasites of humans and animals, producing asymptomatic to severe intestinal infections. To circumvent various impediments associated with current detection methods, we tested a method providing multistage purification and separation in a single, confined step. Standard real-time PCR was used as a detection method. Samples spiked with C. parvum and G. intestinalis were split for comparison to standard Method 1623. Results were equivalent to immunomagnetic procedures for Cryptosporidium, and Giardia. Overall percent recovery for Cryptosporidium with Method 1623 averaged 26.89% (std 21.44%; min = 0%; max = 73%) and was similar but less variable for qPCR method at an estimated average of 27.67 (std 17.65%; min = 5%; max = 63%). For Giardia, Method 1623 had an overall average recovery of 27.11% (std 17.98%; min = 1%; max = 58%), while multistage purification and qPCR had an estimated lower overall recovery at 18.58% (std 13.95%; min = 0%; max = 35%). Microsporidia were also readily detected with an estimated recovery of 46.81% overall (std 17.66%; min = 18%; max = 70%) for E. intestinalis and 38.90% (std 14.36%; min = 13%; max = 62%) for E. bieneusi. 1. Introduction Cryptosporidium, Giardia, and Microsporidia are enteropathogenic parasites of humans and animals, producing asymptomatic to severe intestinal infections [1, 2]. Detection of these pathogens continues to be of great interest for public health, and direct detection monitoring is warranted given a poor correlation with standard fecal pollution indicators [3]. Currently, the U.S. and several European nations mandate the use of combined immunomagnetic and microscopy-based (IMS) procedures (i.e., IT rule; Method 1623) for monitoring surface and drinking waters for Cryptosporidium and Giardia. These methods are hampered by cost and their time consuming nature [4] and require specific expertise to distinguish between human and animal pathogenic Cryptosporidium and Giardia species [5]. Problems also arise with false positive and negative results [6] and poor dissociation of oocysts from magnetic beads in the purification step [7]. These methods are specific only to Cryptosporidium and Giardia, and though Microsporidia and other waterborne pathogens are listed in various contaminate lists, no cost and time-effective method for their detection exists. It is well known that molecular techniques have been developed that are more effective than immunofluorescence microscopy in detecting specific pathogens [8]. Notably,