Diagnosis of disseminated microsporidian Encephalitozoon hellem infection by PCR-Southern analysis and successful treatment with albendazole and fumagillin

Abstract

A 37-year-old AIDS patient presented with foreign body sensation. Microsporidia were detected in smears from a conjunctival swab and urine sediment stained with calcofluor and a modified trichrome blue stain and by indirect fluorescent-antibody staining with murine polyclonal antiserum raised against Encephalitozoon hellem. This antiserum cross-reacted with other Encephalitozoon species, so PCR was performed to amplify the microsporidian ribosomal DNA (rDNA) with pan-Encephalitozoon primers. The PCR DNA products from the urine and conjunctival clinical specimens, along with the tissue culture-derived microsporidian controls, were assayed by Southern analysis with oligonucleotide probes specific for Encephalitozoon cuniculi, E. hellem, and Encephalitozoon (Septata) intestinalis. The PCR product amplified from the urine specimen hybridized with the E. hellem probe only, while insufficient DNA was amplified from the conjunctiva specimen for detection by Southern analysis. For corroboration of the PCR-Southern analysis results, aliquots of the urine and conjunctiva specimens were seeded onto RK-13 cell monolayers. The rDNA extracts of the cultured microsporidia were amplified by PCR with pan-Encephalitozoon primers, and the PCR DNA products were subjected to digestion with restriction endonuclease FokI. The amplified rDNA of both the urine and conjunctiva isolates generated digestion patterns that were identical to the E. hellem PCR rDNA digestion pattern. In addition, double-stranded heteroduplex mobility shift analysis with these PCR products indicated that the urine and conjunctiva isolates 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 study demonstrate that PCR-Southern analysis provides a basis for distinguishing E. cuniculi, E. hellem, and E. intestinalis in clinical specimens.

Phacoemulsification for the management ofEncephalitozoon cuniculi‐induced phacoclastic uveitis in a rabbit

“…If other primers are used, Southern analysis, PCR‐restriction fragment length polymorphism, or double‐stranded DNA heteroduplex mobility shift analysis would be necessary to distinguish closely related species of Encephalitozoon. 5,34,35 Serologic tests available for assaying serum of rabbits suspected of E. cuniculi infection include indirect immunofluorescence (IFAT), carbon immunoassay, ELISA, complement fixation, immunoperoxidase and microagglutination tests 34,36,37 . High IFAT titers of similar magnitude to the present case have been documented in laboratory rabbits infected with E. cuniculi.…”

Encephalitozoon helleminfection as the cause of a unilateral chronic keratoconjunctivitis in an umbrella cockatoo (Cacatua alba)

“…There are no broadly effective antimicrosporidial drugs that are labeled for human or veterinary use. Oral albendazole therapy has been used in humans with varying success to treat ocular, respiratory, enteric, and systemic infections of Encephalitozoon spp 44,45 . In humans, patients often show clinical improvement with treatment, but have a recrudescence or relapse of the microsporidial infection at a later time.…”

Identification of Enterocytozoon bieneusi spores in respiratory samples from an AIDS patient with a 2-year history of intestinal microsporidiosis

“…Until recently, the "gold standard" method for demonstrating the presence of these parasites was electron microscopy (18,19). However, for diagnostic purposes, electron microscopy is time- consuming and relatively insensitive and requires considerable expertise (8). The development of reliable differential staining techniques, such as that with Weber's chromotrope-based stain, has facilitated the widespread diagnosis of microsporidia at the light-microscope level, and electron microscopy is mainly applied to species differentiation.…”

“…Although the 607-bp band could be clearly detected with the undiluted sample, the stronger bands obtained after QIAquick concentration only corroborated this result. Species-specific identification of microsporidia in clinical samples is becoming increasingly important due to previously described variable responses to some drugs (8).…”