SPECIFIC PCR ASSAY FOR DIRECT-DETECTION OF INTESTINAL MICROSPORIDIA ENTEROCYTOZOON-BIENEUSI AND ENCEPHALITOZOON INTESTINALIS IN FECAL SPECIMENS FROM HUMAN IMMUNODEFICIENCY VIRUS-INFECTED PATIENTS

A routine assay based on the PCR was developed for the detection of En terocytozoon bieneusi and Encephalitozoon intestinalis in fecal sample s. Two oligonucleotide primer pairs from a conserved region in the sma ll-subunit rRNA genes of E. bieneusi (primer pair V1 and EB450) and E. intestinalis (primer pair V1 and S1500) were used to amplify microspo ridian DNA. We achieved specific amplification of a 382-bp DNA fragmen t in E. intestinalis and a 353-bp DNA fragment in E. bieneusi, Boiling of the samples appeared to be most effective for DNA extraction, Feca l samples containing fewer than 10 microsporidia gave a positive resul t in the PCR assay. Fecal specimens from 30 human immunodeficiency vir us-infected patients with microsporidiosis and fecal specimens from 42 patients suspected of having microsporidiosis were investigated by th e PCR assay. The PCR assay was validated against standard staining met hods (the Uvitex 2B and Chromotrope 2R staining methods) and immunoflu orescence assay specific for E. intestinalis. This comparative study h as shown that PCR improved species determination and can thus be consi dered a fast and reliable method for the detection and identification of each intestinal species.