Optimised immunofluorescence procedure for enumeration of Cryptosporidium parvum oocyst suspensions

The aim of this study was to evaluate an optimised immunofluorescence assay in terms of the variability of sets of counts for Cryptosporidium parvum oocyst suspensions and data recovery and the reliability of the procedure. A coefficient of variation (CV) of 10% was determined to be the maximum value acceptable for count variability. It was found that the optimised IFA tested provided a high precision for the sets of enumerations for suspensions containing 800-20,000 oocysts/mL. The procedure was found to be robust and providing high recovery level (96.3%). In terms of counting precision, the technique described here approaches the performance of flow cytometry and surpasses other manual techniques with a CV of 10% for a concentration close to 800 oocysts/mL. The procedure described is particularly suitable for the production of seed doses and for other applications requiring the titration of oocyst suspensions with a high degree of precision and accuracy.