Evaluation of methodologies including immunofluorescent assay (IFA) and the polymerase chain reaction (PCR) for detection of human pathogenic microsporidia in water

Microsporidia is a term used to describe a group of emerging protozoan path ogens whose environmental occurrence has only recently been documented due to lack of detection methodologies. This study evaluates and describes curr ent methods for detection of microsporidia in water. Standard methods, for the collection and processing of large volumes of water to detect protozoa, showed only a 4.8% recovery, of microsporidia spores, from 100 I volumes o f tap. Immunofluorescent assay (IFA) analysis was assessed using two differ ent antibodies specific for human pathogenic microsporidia. Results indicat ed that the use of IFA for routine screening of water for microsporidia was not an acceptable approach. The antibodies tested for the IFA resulted in false positives and false negatives and did not react with Enterocytozoon b ieneusi, which is an important human pathogenic microsporidia. Finally, the small sizes of the human pathogenic microsporidia prevent confirmation and species determination by light microscopic methods. Two methods for isolat ing microsporidia DNA from water for use in polymerase chain reaction (PCR) amplification of microsporidia target sequences were assessed. Both of the se DNA isolation methods when combined with the PCR showed the ability to d etect less than ten spores in purified water concentrates. Thus, this study represents the first documentation and evaluation of current methods for t he detection of human pathogenic microsporidia in water. (C) 1999 Elsevier Science B.V. All rights reserved.