Case Report: Ocular Microsporidiosis: Case in a Patient Returning from India and Review of the Literature.

Microsporidia are protists close to the kingdom of fungi that may cause eye infections. Most cases are reported in Asia and affect both immunocompromised and immunocompetent patients. Here, we report a rare case of microsporidial keratoconjunctivitis in an immunocompetent French patient 3 weeks after returning from India. In our patient, Weber trichrome staining of conjunctival scrapings revealed rounded elements approximately 1-3 µm in size. Conventional polymerase chain reaction analysis by ribosomal RNA subunit sequencing showed 100% identity with Vittaforma corneae. Treatment by corneal debridement combined with fluoroquinolone eye drops allowed complete resolution of the lesions. Although rare, ocular microsporidiosis should be investigated in a patient who is native to Asia or has returned from an endemic area and presents with keratoconjunctivitis of undetermined etiology.

In vitro culture of various species of microsporidia causing keratitis: evaluation of three immortalized cell lines.

Being intracellular parasites, microsporidia can only be propagated in cell culture systems. This study evaluated three cell lines to determine the most suitable host-parasite In vitro system. Confluent monolayers of vero, SIRC, and HeLa cell lines, grown in 24-well tissue culture plates, were inoculated with varying concentrations (1 x 10(4) to 1 x 10(8) spores/mL) of Vittaforma corneae, Encephalitozoon hellem, Encephalitozoon cuniculi, and Encephalitozoon intestinalis spores. Growth was compared quantitatively at weekly intervals. Encephalitozoon species showed the highest amount of growth when cultured in vero cell line, while there was no significant difference in their growth in SIRC and HeLa cell lines. In comparison, V. corneae showed the highest growth in SIRC cells, followed by vero cells. The analytical sensitivity was found to be 1 x 10(4) spores/mL for vero cell line compared to 1 x 10(5) spores/mL for SIRC cell line and 1 x 10(7) spores/mL for HeLa cell line. HeLa cells also showed rapid disruption of cells, and the spores could not be easily distinguished from cell debris. This is the first report of the comparison of vero, SIRC, and HeLa for the propagation of microsporidial spores. Vero cell line was found to be more sensitive than SIRC and HeLa cells, and we believe that the inclusion of vero cell line in the routine culture protocols of ocular parasitology laboratories would result in a significant increase in the diagnostic yield.

Antimicrosporidial activities of fumagillin, TNP-470, ovalicin, and ovalicin derivatives in vitro and in vivo.

Therapies for microsporidiosis in humans are limited, and fumagillin, which appears to be the most broadly effective antimicrosporidial drug, is considered to be moderately toxic. The purpose of this study was to apply an in vitro drug screening assay for Encephalitozoon intestinalis and Vittaforma corneae and an in vivo athymic mouse model of V. corneae infection to assess the efficacy of TNP-470 (a semisynthetic analogue of fumagillin), ovalicin, and eight ovalicin derivatives. TNP-470, ovalicin, and three of the ovalicin derivatives inhibited both E. intestinalis and V. corneae replication by more than 70% in vitro. Another three of the ovalicin derivatives inhibited one of the two microsporidian species by more than 70%. None of the treated athymic mice survived the V. corneae infection, but they did survive statistically significantly longer than the untreated controls after daily treatment with fumagillin administered at 5, 10, and 20 mg/kg of body weight subcutaneously (s.c.), TNP-470 administered at 20 mg/kg intraperitoneally (i.p.), or ovalicin administered at 5 mg/kg s.c. Of two ovalicin derivatives that were assessed in vivo, NSC 9665 given at 10 mg/kg i.p. daily also statistically significantly prolonged survival of the mice. No lesions associated with drug toxicity were observed in the kidneys or livers of uninfected mice treated with these drugs at the highest dose of 20 mg/kg daily. These results thus support continued studies to identify more effective fumagillin-related drugs for treating microsporidiosis.

Sequence survey of the genome of the opportunistic microsporidian pathogen, Vittaforma corneae.

The microsporidian Vittaforma corneae has been reported as a pathogen of the human stratum corneum, where it can cause keratitis, and is associated with systemic infections. In addition to this direct role as an infectious, etiologic agent of human disease, V. corneae has been used as a model organism for another microsporidian, Enterocytozoon bieneusi, a frequent and problematic pathogen of HIV-infected patients that, unlike V. corneae, is difficult to maintain and to study in vitro. Unfortunately, few molecular sequences are available for V. corneae. In this study, seventy-four genome survey sequences (GSS) were obtained from genomic DNA of spores of laboratory-cultured V. corneae. Approximately, 41 discontinuous kilobases of V. corneae were cloned and sequenced to generate these GSS. Putative identities were assigned to 44 of the V. corneae GSS based on BLASTX searches, representing 21 discrete proteins. Of these 21 deduced V. corneae proteins, only two had been reported previously from other microsporidia (until the recent report of the Encephalitozoon cuniculi genome). Two of the V. corneae proteins were of particular interest, reverse transcriptase and topoisomerase IV (parC). Since the existence of transposable elements in microsporidia is controversial, the presence of reverse transcriptase in V. corneae will contribute to resolution of this debate. The presence of topoisomerase IV was remarkable because this enzyme previously had been identified only from prokaryotes. The 74 GSS included 26.7 kilobases of unique sequences from which two statistics were generated: GC content and codon usage. The GC content of the unique GSS was 42%, lower than that of another microsporidian, E. cuniculi (48% for protein-encoding regions), and substantially higher than that predicted for a third microsporidian, Spraguea lophii (28%). A comparison using the Pearson correlation coefficient showed that codon usage in V. corneae was similar to that in the yeasts, Saccharomyces cerevisiae (r = 0.79) and Shizosaccharomyces pombe (r = 0.70), but was markedly dissimilar to E. cuniculi (r = 0.19).

In vitro cultivation of microsporidia of clinical importance.

Although attempts to develop methods for the in vitro cultivation of microsporidia began as early as 1937, the interest in the culture of these organisms was confined mostly to microsporidia that infect insects. The successful cultivation in 1969 of Encephalitozoon cuniculi, a microsporidium of mammalian origin, and the subsequent identification of these organisms as agents of human disease heightened interest in the cultivation of microsporidia. I describe the methodology as well as the cell lines, the culture media, and culture conditions used in the in vitro culture of microsporidia such as Brachiola (Nosema) algerae, Encephalitozoon cuniculi, E. hellem, E. intestinalis, Enterocytozoon bieneusi, Trachipleistophora hominis, and Vittaforma corneae that cause human disease.