Detection of Microsporidia and Identification of Enterocytozoon bieneusi in Surface Water by Filtration Followed by Specific PCR.

Microsporidiosis due to species from five different genera, Enterocytozoon, Eiiceplialitozoori, Nosema. Vittafoma and Trachipleistophora, has emerged as an opportunistic infection in immunocompromised patients. Up to now, the source of contamination remains unknown, especially for E. bieneusi, the most common species found in humans. As for other parasitic diseases with fecal excretion of infective stages, the presence of parasites in the environment is likely and surface water may represent a possible source of contamination or a vehicle of transmission. We report on the development of a filtration and PCR-based method for detection of microsporidial spores and we demonstrated the presence of E. bieneusi in one sample of surface water from the river Seine. Experimental studies using water spiked with spores obtained from infected stools (E. bieneusi), or tissue cultures inikted with E. cunicrrli, E. hellem and E. intestinalis (kindly provided by E. Canning and T. Van Gool) were performed to assess for efficiency and sensitivity of the filtration and PCR methods. The filtration comprised two sequential steps. The first step consisted in a concentration of the sample. Water was flushed at 4 litershin. through a filtration cartridge wluch retains all particles of more than 1 micron size (Fulflo. M39R10A, Parker Filtration, France) ; then the collected material was eluted from the cartridge into 2 liters of distilled water. In the second step, this eluate was submitted to three sequential filtrations at 200 d m i n . on 180, 60, 11 microns pore-size nylon filters (47 mm of diameter) (Millipore, France) then finally filtered on a 0.4 micron pore-size polycarbonate filter (Millipore). Each filter was examined by PCR and one part of the 0.4 micron filters was examined by light microscopy using Uvitex 2B staining. DNA extraction of the material colIected from the filters was pertormed using guanidine thiocyanate, according to Boom et al. [l], then identification of parasitic DNA was performed by hvo sequential PCR directed against different DNA fragments of the small sirbunit rRNA gene of microsporidia. The first PCR used generic primers designed for detecting human infecting microsporidia [2] for a 35 cycles amplification, then the resulting products were submitted to a second amplification (35 cycles), using primers specific for E. bieneusi [3], E. intestinalis [ 3 ] , E. cuniculi and E. hellem (kindly provided by ARJ Schuitema). The 1265 bp DNA fragments obtained with E. bieneusi specific PCR primers from 4 filters were sequenced (E.S.G.S., Montigny le Bretonneux, France) and analyzed comparatively to previously published sequences of microsporidia (Genbank). For field studies, five 200400 liters samples were collected : one sample of surface water from the river Seine, 15 km down-stream to Paris, three Goni the river Loire, 25 km up-stream to Nantes, and one sample of ground water collected near Paris. Two liters of each sample were directly filtered (without concentration) and the remaining was concentrated then filtered as described above. Experimental studies using spiked distilled water showed that the spores of the different species of microsporidia were retained by the 0.4 micron filter and could be detected by light microscopy and by PCR. When increasing dilutions of spores of E. cuniculi, E. liellem or E. intestinalis were tested, they were no longer detectable by microscopy but generic and specific PCR were positive, allowing the detection of approximatively 1-3 spores in 2 liters of distilled MATERIAL, AND METHODS.