DIAGNOSIS OF DISSEMINATED MICROSPORIDIAN ENCEPHALITOZOON HELLEM INFECTION BY PCR-SOUTHERN ANALYSIS AND SUCCESSFUL TREATMENT WITH ALBENDAZOLE AND FUMAGILLIN

A 37-year-old AIDS patient presented with foreign body sensation. Micr osporidia were detected in smears from a conjunctival swab and urine s ediment stained with calcofluor and a modified trichrome blue stain an d by indirect fluorescent-antibody staining with murine polyclonal ant iserum raised against Encephalitozoon hellem. This antiserum cross-rea cted with other Encephalitozoon species, so PCR was performed to ampli fy the microsporidian ribosomal DNA (rDNA) with pan-Encephalitozoon pr imers. The PCR DNA products from the urine and conjunctival clinical s pecimens, along with the tissue culture-derived microsporidian control s, were assayed by Southern analysis viith oligonucleotide probes spec ific for Encephalitozoon cuniculi, E. hellem, and Encephalitozoon (Sep tata) intestinalis. The PCR product amplified from the urine specimen hybridized with the E. hellem probe only, while insufficient DNA was a mplified from the conjunctiva specimen for detection by Southern analy sis. For corroboration of the PCR-Southern analysis results, aliquots of the urine and conjunctiva specimens were seeded onto RK-13 cell mon olayers. The rDNA extracts of the cultured microsporidia were amplifie d by PCR with pan-Encephalitozoon primers, and the PCR DNA products we re subjected to digestion with restriction endonuclease FokI. The ampl ified rDNA of both the urine and conjunctiva isolates generated digest ion patterns that were identical to the E. hellem PCR rDNA digestion p attern. In addition, double-stranded heteroduplex mobility shift analy sis with these PCR products indicated that the urine and conjunctiva i solates were identical to each other and to E. hellem. The patient was treated with albendazole and topical fumagillin and responded rapidly , with no recurrence of ophthalmologic signs. The results of this stud y demonstrate that PCR-Southern analysis provides a basis for distingu ishing E. cuniculi, E. hellem, and E. intestinalis in clinical specime ns.