SummaryMicrosporidia are recognized as a major aetiological agent in chronic diarrhoea of immunocompromised patients. Their detection by light microscopy is hampered by the small size of the spores. A simple and rapid DNA extraction method has been developed for the detection of microsporidian DNA by PCR directly from stool specimens. It can be performed at room temperature in a I.g-ml microcentrifuge tube format in less than I hour. The subsequent nested polymerase chain reaction permits the detection of 3-100 spores in a 0.1-g stool sample. The amplification products can be verified and the species Enterocytozoon bieneusi, Encephalitozoon cuniculi and Encephalitozoon (Septata) intestinalis distinguished by a simple restriction endonuclease digest.keywords microsporidia, DNA, PCR

Enterocytozoon bieneusi is the most frequently found microsporidium in human infections. In all, 3 distinct genotypes were detected in 12 stool samples from 8 patients with acquired immunodeficiency syndrome (AIDS). A total of 9 polymorphic sites were found in the 243-bp-long internal transcribed spacer (ITS) of the rDNA gene, whereas none was found in 241 bp of adjacent rRNA coding regions. The genotype was stable in samples taken during 11 weeks of infection from one of the patients. The existence of and the ability to discriminate among strains of E. bieneusi are important prerequisites for elucidation of the hitherto unknown reservoirs of this pathogen and the mode of its transmission and may explain its pathogenicity.

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The detection of microsporidial DNA by the polymerase chain reaction (PCR) has been suggested as an alternative or supplement to conventional microscopic methods. However, the relative merits of these techniques remain uncertain. In the present study, clinical specimens of different origin (stool, urine, sputum, nasal discharge, and cerebrospinal fluid) containing four different microsporidial species were blinded after microscopic examination and analyzed by PCR and subsequent restriction fragment length polymorphism (RFLP) to determine the respective species. Thirty-four specimens from 31 patients were evaluated, 16 of which were positive and 18 negative by microscopic examination; PCR detection of microsporidia produced identical results in 82% (28/34) of these specimens. Four samples were microscopically negative, PCR-positive, and two were microscopically positive, PCR-negative. Species determination by RFLP analysis of the amplified product was accurate for all isolates containing Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi compared with microscopic identification of the genus Enterocytozoon or molecular analysis of Encephalitozoon species after in vitro culture. Therefore, PCR-RFLP is useful for the rapid detection and differentiation of microsporidian spores in clinical specimens.

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