International multi-laboratory evaluation of a method for genotyping Cryptosporidium recovered from water regulatory slides

This webinar presentation is based on a research presentation by Dr. George Di Giovanni of the University of Texas School of Public Health, Rebecca Hoffman of the Wisconsin State Laboratory of Hygiene, and Dr. Gregory D. Sturbaum of CH Diagnostic and Consulting. The speaker is Dr. Di Giovanni.

Abstract: USEPA Method 1622/23 and the UK DWI method allow the microscopic enumeration of Cryptosporidium oocysts in water, but they do not determine the species or genotypes of the Cryptosporidium detected. This research group previously developed a method for recovering and genotyping the Cryptosporidium oocysts present on regulatory water microscope slides. Human infectious and animal-associated Cryptosporidium genotypes were distinguished using single-round multiplex 18S and hsp70 PCR compatible with conventional or real-time formats. Real-time High Resolution Melting Analysis using the QIAGEN Rotor-Gene Q instrument allowed further discrimination of human- and animal-associated Cryptosporidium spp. An international method evaluation was performed with the participation of 12 laboratories located in 6 countries. Diverse surface-water field slides that were found negative for Cryptosporidium using microscopy were seeded with single flow-sorted C. parvum or C. muris oocysts. Single oocyst-seeded matrix-free slides and unseeded slides were also included. Based on a total of 990 seeded slides, the overall detection rate was 50%, but the detection rates varied significantly by laboratory, with a range of 28% to 88%. Detection of C. parvum was slightly higher than for C. muris (57% and 43%, respectively). The presence of a field matrix did not affect detection, indicating that the method was robust for environmental samples. Further evaluation of the method using slides with naturally occurring Cryptosporidium are planned. The Cryptosporidium slide genotyping method may be used by water utilities as a resesarch tool to gain added value from regulatory monitoring by providing additional information on human health risk and as an aid for the development of effective watershed management plans.

George Di Giovanni

George Di Giovanni is Professor of Environmental Microbiology at the University of Texas School of Public Health. Dr. Di Giovanni received his PhD from the University of Arizona and did postdoctoral work as a National Research Council Associate with the US Environmental Protection Agency. Prior to joining the University of Texas School of Public Health, he was a Professor and Faculty Fellow with the Texas A&M System, and a Senior Environmental Scientist for the American Water Works Company. He has served as the Chair of the AWWA Microbiological Contaminants Research Committee. His current resesarch program focuses on the detection and molecular analysis of waterborne pathogens, including Cryptosporidium, Giardia, and viruses, and on microbial source tracking to determine the human and animal sources of fecal pollution of water supplies. He and his research team have been honored with a Texas Environmental Excellence Award.