Microsporidial stromal keratitis: characterisation of clinical features, ultrastructural study by electron microscopy and efficacy of different surgical modalities.

AIMS: To report the clinical manifestations, ultrastructure and evaluate the efficacy of therapeutic lamellar keratectomy (TLK) and penetrating keratoplasty (PK) for microsporidial stromal keratitis (MSK). METHODS: Fourteen MSK cases between 2009 and 2018 were recruited. Each patient's clinical presentation, light microscopy, histopathology, PCR and electron microscopy (EM) of corneal samples were reviewed. RESULTS: The patients were 70.0±4.7 years old (average follow-up, 4.5 years). Time from symptoms to presentation was 10.6±13.0 weeks. The corneal manifestations were highly variable. Corneal scrapings revealed Gram stain positivity in 12 cases (85.7%) and modified Ziehl-Neelsen stain positivity in 9 (64.3%). Histopathology revealed spores in all specimens, while sequencing of small subunit rRNA-based PCR products identified Vittaforma corneae in 82% of patients. EM demonstrated various forms of microsporidial sporoplasm in corneal keratocytes. All patients were treated with topical antimicrobial agents or combined with oral antiparasitic medications for >3 weeks. As all patients were refractory to medical therapy, they ultimately underwent surgical intervention (TLK in 7, PK in 6 and 1 received TLK first, followed by PK). Postoperatively, the infection was resolved in 78.6% of the patients. Nevertheless, a high recurrence rate (21.4%) was noted during 3-year follow-up, with only two patients retained a final visual acuity ≥20/100. CONCLUSION: MSK usually presents with a non-specific corneal infiltration refractory to antimicrobial therapy. The diagnosis relies on light microscopic examinations on corneal scrapings and histopathological analyses. Surgical intervention is warranted by limiting the infection; however, it was associated with an overall poor outcome.

Molecular identification of microsporidian species in patients with epithelial keratitis.

Introduction. Ocular microsporidiosis is a significant emerging infectious disease reported in immunocompromised patients and immunocompetent persons throughout the world.Aim. To identify the pathogens responsible for human keratitis, via corneal scrapings.Methodology. Thirty-three hospitalized patients with epithelial keratitis were examined using staining and DNA sequencing. DNA was extracted from corneal samples and the small-subunit ribosomal RNA gene was amplified by polymerase chain reaction (PCR) and sequenced.Results. Twenty-one samples were positive by staining while PCR generated amplicons in 18 cases. Of the 18 sequences, 16 were identical with, or very similar to, those of Vittaforma corneae (99-100 % similarity) and the remaining two sequences were similar to that of unidentified Microsporidium species deposited in the GenBank.Conclusion. This study has reconfirmed that V. corneae causes epithelial keratitis in humans and that a newly detected Microsporidium species is also involved in microsporidial keratitis as one of the emerging pathogens in Thailand. Ophthalomologists should be aware of microsporidial keratitis in people from Thailand and those from neighbouring countries.

Swimming Pool-Associated Vittaforma-Like Microsporidia Linked to Microsporidial Keratoconjunctivitis Outbreak, Taiwan.

We analyzed 2 batches of environmental samples after a microsporidial keratoconjunctivitis outbreak in Taiwan. Results indicated a transmission route from a parking lot to a foot washing pool to a swimming pool and suggested that accumulation of mud in the foot washing pool during the rainy season might be a risk factor.

Microsporidial keratitis retrospectively diagnosed by ultrastructural study of formalin-fixed paraffin-embedded corneal tissue: a case report.

BACKGROUND: The utility of formalin-fixed paraffin-embedded (FFPE) corneal tissue specimens for retrospective diagnosis of microsporidial keratitis was evaluated by transmission electron microscopy (TEM) analysis and the possible second case of microsporidial keratitis after Descemet stripping automated endothelial keratoplasty (DSAEK) was described. CASE PRESENTATION: A 68-year-old man presented with multiple crystalline opacities in the corneal stroma that progressed extremely slowly after DSAEK. Fungiflora Y staining of corneal scrapings from the affected regions revealed an oval microorganism. Topical voriconazole administration was ineffective and penetrating keratoplasty was performed. Histological and molecular analyses were carried out on the excised cornea. Ziehl-Neelsen staining revealed an acid-fast, oval organism that was visible by ultraviolet illumination after Fungiflora Y and Uvitex 2B staining, whereas periodic acid-Schiff and Grocott's staining did not yield any significant findings. Microsporidium was detected by TEM of FFPE tissue. Nosema or Vittaforma sp. was suspected as the causative microorganism by PCR of FFPE tissue and by the fact that those species are known to cause eye infection. The corneal graft has maintained transparency at 1 year and half postoperatively. CONCLUSIONS: This is the first known case of microsporidial keratitis diagnosed retrospectively by molecular and ultrastructural study of FFPE tissue, and the possible second case of microsporidial keratitis after DSAEK. Microsporidial keratitis should be considered when corneal opacity refractory to conventionally known therapy would occur after DSAEK. Our findings suggest that more microsporidial keratitis cases than have been reported to date can be identified by TEM or PCR examination of FFPE corneal specimens.

Stromal Keratitis with Endophthalmitis Caused by Vittaforma Corneae in an Immunocompetent Patient: A Case Report.

Purpose: To describe a case of microsporidial stromal keratitis with endophthalmitis in an immunocompetent patient.Methods: Case reportResults: A 58-year-old HIV-negative man presented with stromal keratitis in his right eye. The patient demonstrated subsequent vitritis, multifocal retinitis and arteritis, and macular edema with recurrent vitreous hemorrhage after therapeutic keratoplasty. Numerous microsporidial spores were detected in corneal tissues by modified trichrome stain. Both corneal tissues and vitreous sample of the affected eye showed positive results by polymerase chain reaction targeting the microsporidial small subunit rRNA gene whose sequences belonged to Vittaforma corneae. Post-keratoplasty and vitrectomy, his best-corrected visual acuity was hand motion due to pale optic disc.Conclusion: Endophthalmitis can be a consequence of microsporidial stromal keratitis in an immunocompetent host. Early recognition and prompt treatment should be considered in patients diagnosed with microsporidial keratitis presenting with mild vitritis, retinitis, and recurrent vitreous hemorrhage.

Molecular surveillance of Vittaforma-like microsporidia by a small-volume procedure in drinking water source in Taiwan: evidence for diverse and emergent pathogens.

Vittaforma corneae belongs to microsporidia, which include over 1500 species of opportunistic obligate intracellular fungi infecting almost all known animal taxa. Although outbreaks of ocular infections caused by waterborne V. corneae have been reported in recent years, little is known about the occurrence of this pathogen in aquatic environments. In this study, 50 water samples from rivers and reservoirs around Taiwan in two seasons were analyzed to explore the presence of this pathogen in natural aquatic environments. A high detection rate of Vittaforma-like amplicons (94%; 47/50) was observed in the water samples when examined by nested PCR with primer pairs specific to the small ribosomal subunit (SSU) rRNA gene. After electrophoresis, many lanes showed multiband patterns with expected molecular weights. After confirmation by DNA sequencing and by sequence alignment in the NCBI database, we identified a variety of Vittaforma-like microsporidia with weak sequence similarity, with approximately 85% identity to V. corneae, thus indicating high diversity of microsporidia in aquatic environments. Phylogenetic analysis showed clear-cut microsporidian clade classification and indicated that the most Vittaforma-like microsporidia in this study belong to clade IV and cluster into four major groups. The first group is similar to the microsporidia associated with ocular microsporidiosis. The second group is associated with the diarrheal pathogens, whereas the third and fourth groups are a novel group and a zoonotic group, respectively. This study provides abundant sequencing information, which will be useful for future molecular biological studies on microsporidia. Because microsporidia are important pathogens of animals and humans, it is urgently necessary to determine via a survey whether there are species with potential threats that have not yet been revealed.

Vittaforma corneae keratitis in southern India: role of a novel duplex PCR.

Microsporidia are obligate intracellular parasites that infect eukaryotic cells and have emerged as major opportunistic human pathogens. Due to the difficulties in definitive laboratory diagnosis and insufficient knowledge, ocular microsporidiosis is infrequently reported in India. To improve diagnostic facilities, we have developed a novel duplex PCR (dPCR) for the simultaneous identification of both genera and species of isolates with microsporidian aetiology that cause keratitis. The material scraped from the corneas of 12 clinically diagnosed microsporidial keratitis patients was subjected to routine microbiological examinations and molecular diagnosis using a novel dPCR that targeted the small-subunit rRNA gene (SSU-rRNA) of microsporidia and Vittaforma corneae using genus- and species-specific primers. Of the 12 corneal scrapes, 6 showed positive results in smears, while dPCR provided positive amplification with both pan-microsporidial and V. corneae species-specific primers for 9 corneal scrapes. The results were validated by sequencing and blast analysis. The sensitivity of this novel dPCR method was higher than that of conventional microscopy in the diagnosis of corneal microsporidial infection. dPCR with specific primers is potentially more sensitive, specific and depends less on more complicated methods for exact identification of the aetiology of microsporidial keratitis.

Low natural levels of Nosema ceranae in Apis mellifera queens.

Queens are the primary female reproductive individuals in honey bee colonies and, while they are generally free from Nosema ceranae infection, they are nevertheless susceptible. We sought to determine whether queens are naturally infected by N. ceranae, as these infections could be a factor in the rapid spread of this parasite. Queens were analyzed using real-time PCR and included larval queens, newly emerged, and older mated queens. Overall, we found that all tissues we examined were infected with N. ceranae at low levels but no samples were infected with Nosema apis. The infection of the ovaries and spermatheca suggests the possibility of vertical transmission of N. ceranae.

Microsporidial keratitis in patients with hot springs exposure.

This retrospective study included 10 eyes of 9 patients diagnosed with microsporidial keratitis. All of them were known to contract this disease after taking baths in hot springs. The disease was diagnosed based on detecting microsporidia in corneal scrapings using Gram stain and the modified Kinyoun's acid-fast stain. The specimens from the last six patients were subjected to PCR and then sequencing. All of them revealed that the microorganism identified has a high similarity to Vittaforma corneae. Repeated debridement of the epithelial lesions successfully eradicated the microsporidial infection in all nine patients.

Ultrastructural examination of two cases of stromal microsporidial keratitis.

Two cases with chronic stromal keratitis are described in immunocompetent hosts where the diagnosis was originally thought to be herpetic or adenoviral disease. Light microscopy and ultrastructural examination of corneal tissue by electron microscopy were performed following penetrating keratoplasty (case 1) and corneal biopsy (case 2). Specimens from both cases were analysed for viral identification by PCR. Two different species of Microsporidia were identified. Case 1 represents the fourth reported case of corneal stromal Vittaforma corneae where the spores measured 3.3 x 1.4 microm, arranged in characteristic linear groups of about four to eight. Each spore contained a diplokaryotic nucleus and a single row of ten polar tube coils. By contrast, case 2 is the first reported case of stromal keratitis caused by Trachipleistophora hominis. In this case, spores measured 4 x 2.4 microm, located typically within packets. In this species, the polar tube was arranged as a single row of about 10-13 profiles. Viral DNA could not be amplified by PCR. In conclusion, microsporidial stromal keratitis should be considered in culture-negative cases refractory to medical therapy. As microbiological culture techniques are unsuccessful, diagnosis may only be established following histopathological and ultrastructural examination of corneal tissue.

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.