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

• Published in: Journal of microbiological methods 1999-02, Vol.35 (1), p.43-52
• Main Authors: Dowd, Scot E, Gerba, Charles P, Kamper, Matthew, Pepper, Ian L
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Detection; DNA, Protozoan - analysis; Enterocytozoon bieneusi; False Negative Reactions; False Positive Reactions; Fluorescent Antibody Technique, Indirect; Fundamental and applied biological sciences. Psychology; General aspects and techniques; Humans; IFA; Immunomagnetic Separation - methods; Microsporida - genetics; Microsporida - isolation & purification; Microsporida - physiology; Microsporidia; PCR; Polymerase Chain Reaction - methods; Protozoa; Species Specificity; Spores - isolation & purification; Water; Water Microbiology; Waterborne pathogen
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/S0167-7012(98)00101-8

Microsporidia is a term used to describe a group of emerging protozoan pathogens whose environmental occurrence has only recently been documented due to lack of detection methodologies. This study evaluates and describes current 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 l volumes of tap. Immunofluorescent assay (IFA) analysis was assessed using two different antibodies specific for human pathogenic microsporidia. Results indicated 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 bieneusi, 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 isolating microsporidia DNA from water for use in polymerase chain reaction (PCR) amplification of microsporidia target sequences were assessed. Both of these DNA isolation methods when combined with the PCR showed the ability to detect less than ten spores in purified water concentrates. Thus, this study represents the first documentation and evaluation of current methods for the detection of human pathogenic microsporidia in water.