Evaluation of Potential Modifications to EPA Method 1623 for Rapid Accurate Low Level Enumeration of Cryptosporidium oocysts

摘要

In response to proposed federal regulations calling for increased monitoring ofprotozoans in raw source waters, several different systems are under evaluation todetermine their accuracy and precision for determining protozoan concentrations. Theapproved method for the detection of Cryptosporidium (EPA Method 1623) under EPA'sLT2ESWTR (proposed) has wide method performance acceptance criteria and isextremely labor intensive. Our objective is to evaluate the accuracy of oocyst recoveryand detection methods using a variety of immunocapture methods (IMS) and labelingsystems in conjunction with a new automated rapid detection technology (RBD2100). Incontrast to traditional flow cytometry, this new technology provides an easy to usemicrobial counting platform that would eliminate the need for microscope-basedenumeration by a technician, saving time and potentially improving the accuracy of themeasurement process. Working with water samples spiked with formaldehyde preservedoocysts of a known concentration (Waterborne), commercially availableCryptosporidium oocyst specific immunomagnetic beads (Dynal and Aureon) were usedaccording to the manufacturers recommended procedures. Following the dissociation ofbeads/oocyst complex (13.3.3 from 1623), the oocysts were labeled with a small volumeof the Cy5-conjugated antibody (Waterborne) and analyzed on the RBD2100 forenumeration. Parallel samples were treated only with the Cy5-conjugated antibody2followed by enumeration on the RBD2100 (no IMS performed) for use as a total countused in IMS recovery calculations. The Dynal and Aureon beads respectively providedrecoveries of 72 % and 75 % respectively based upon RBD2100 enumeration.Alternatively, Protein G bound paramagnetic beads (50 nm) were coated with oocystspecific antibody from Biogenesis, mixed with the Cy5 Cryptosporidium antibody(Waterborne) and added to water samples spiked with a wide range of oocystsconcentrations (176 to 2832 per ml). The mixture was incubated for 1 hr at 37°C prior toIMS and the captured sample was then analyzed on the RBD2100 (without the beaddissociation and removal step). The flow cytometer counts correlated very well(R~2=0.966) with the expected counts within this concentration range. The next phase ofthis work will involve using matrix-containing water from the 1623 method (through step13.2) in conjunction with this alternative enumeration technology. The immunocaptureprocedure described in combination with the RBD2100 technology represents a potentialmodification to the currently available procedures for the assessment of oocystcontamination in drinking water.