Application of PCR-based diagnostic tools that target Enterocytozoon hepatopenaei for the molecular detection of a Vittaforma-like microsporidium that infects Penaeus vannamei from Costa Rica.

Several PCR methodologies are available for the detection of Enterocytozoon hepatopenaei (EHP) that target the SSU rRNA gene. However, these methodologies are reported as unsuitable for the detection of EHP due to specificity issues. Here, we report the applicability of two commonly used SSU rRNA methodologies for the detection of additional microsporidia from the genus Vittaforma that is present in cultured Penaeus vannamei from Costa Rica. The molecular detection of DNA of the novel microsporidia can only be achieved using SSU rRNA targeting methodologies and does not cross-react with the highly specific spore wall protein gene PCR detection method.

Structural and Functional Annotation of Hypothetical Proteins from the Microsporidia Species Vittaforma corneae ATCC 50505 Using in silico Approaches.

Microsporidia are spore-forming eukaryotes that are related to fungi but have unique traits that set them apart. They have compact genomes as a result of evolutionary gene loss associated with their complete dependency on hosts for survival. Despite having a relatively small number of genes, a disproportionately high percentage of the genes in microsporidia genomes code for proteins whose functions remain unknown (hypothetical proteins-HPs). Computational annotation of HPs has become a more efficient and cost-effective alternative to experimental investigation. This research developed a robust bioinformatics annotation pipeline of HPs from Vittaforma corneae, a clinically important microsporidian that causes ocular infections in immunocompromised individuals. Here, we describe various steps to retrieve sequences and homologs and to carry out physicochemical characterization, protein family classification, identification of motifs and domains, protein-protein interaction network analysis, and homology modelling using a variety of online resources. Classification of protein families produced consistent findings across platforms, demonstrating the accuracy of annotation utilizing in silico methods. A total of 162 out of 2034 HPs were fully annotated, with the bulk of them categorized as binding proteins, enzymes, or regulatory proteins. The protein functions of several HPs from Vittaforma corneae were accurately inferred. This improved our understanding of microsporidian HPs despite challenges related to the obligate nature of microsporidia, the absence of fully characterized genes, and the lack of homologous genes in other systems.

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.

Encephalitozoon cuniculi and Vittaforma corneae (Phylum Microsporidia) inhibit staurosporine-induced apoptosis in human THP-1 macrophages in vitro.

Obligately intracellular microsporidia regulate their host cell life cycles, including apoptosis, but this has not been evaluated in phagocytic host cells such as macrophages that can facilitate infection but also can be activated to kill microsporidia. We examined two biologically dissimilar human-infecting microsporidia species, Encephalitozoon cuniculi and Vittaforma corneae, for their effects on staurosporine-induced apoptosis in the human macrophage-differentiated cell line, THP1. Apoptosis was measured after exposure of THP-1 cells to live and dead mature organisms via direct fluorometric measurement of Caspase 3, colorimetric and fluorometric TUNEL assays, and mRNA gene expression profiles using Apoptosis RT2 Profiler PCR Array. Both species of microsporidia modulated the intrinsic apoptosis pathway. In particular, live E. cuniculi spores inhibited staurosporine-induced apoptosis as well as suppressed pro-apoptosis genes and upregulated anti-apoptosis genes more broadly than V. corneae. Exposure to dead spores induced an opposite effect. Vittaforma corneae, however, also induced inflammasome activation via Caspases 1 and 4. Of the 84 apoptosis-related genes assayed, 42 (i.e. 23 pro-apoptosis, nine anti-apoptosis, and 10 regulatory) genes were more affected including those encoding members of the Bcl2 family, caspases and their regulators, and members of the tumour necrosis factor (TNF)/TNF receptor R superfamily.

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.

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.

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.

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.

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.

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.

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.

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.

Surveillance of Vittaforma corneae in hot springs by a small-volume procedure.

Vittaforma corneae is an obligate intracellular fungus and can cause human ocular microsporidiosis. Although accumulating reports of V. corneae causing keratoconjunctivitis in both healthy and immunocompromised persons have been published, little is known about the organism's occurrence in aquatic environments. Limitations in detection sensitivity have meant a large sampling volume is required to detect the pathogen up to now, which is problematic. A recent study in Taiwan has shown that some individuals suffering from microsporidial keratitis (MK) were infected after exposure to the pathogen at a hot spring. As a consequence of this, a survey and analysis of environmental V. corneae present in hot springs became an urgent need. In this study, sixty water samples from six hot spring recreation areas around Taiwan were analyzed. One liter of water from each sample site was filtered to harvest the fungi. The positive samples were detected using a modified nested PCR approach followed by sequencing using specific SSU rRNA gene primer pairs for V. corneae. In total fifteen V. corneae-like isolates were identified (25.0% of sites). Among them, six isolates, which were collected from recreational areas B, C and D, were highly similar to known V. corneae keratitis strains from Taiwan and other countries. Furthermore, five isolates, which were collected from recreation areas A, C, E and F, were very similar to Vittaforma-like diarrhea strains isolated in Portugal. Cold spring water tubs and public foot bath pools had the highest detection rate (50%), suggesting that hot springs might be contaminated via untreated water sources. Comparing the detection rate across different regions of Taiwan, Taitung, which is in the east of the island, gave the highest positive rate (37.5%). Statistical analysis showed that outdoor/soil exposure and a high heterotrophic plate count (HPC) were risk factors for the occurrence of V. corneae. Our findings provide empirical evidence supporting the need for proper control and regulations at hot spring recreational waters in order to avoid health risks from this pathogen. Finally, we have developed a small volume procedure for detecting V. corneae in water samples and this has proved to be very useful.

Combined neonicotinoid pesticide and parasite stress alter honeybee queens' physiology and survival.

Honeybee colony survival strongly relies on the queen to overcome worker losses exposed to combined stressors like pesticides and parasites. Queen's capacity to withstand these stressors is however very little known. The effects of the common neonicotinoid pesticide imidacloprid in a chronic and sublethal exposure together with the wide distributed parasite Nosema ceranae have therefore been investigated on queen's physiology and survivorship in laboratory and field conditions. Early physiological changes were observed on queens, particularly the increase of enzyme activities (catalase [CAT] and glutathione-S-transferase [GST] in the heads) related to protective responses to xenobiotics and oxidative stress against pesticide and parasite alone or combined. Stressors also alter the activity of two other enzymes (carboxylesterase alpha [CaE α] and carboxylesterase para [CaE p] in the midguts) involved in metabolic and detoxification functions. Furthermore, single and combined effects of pesticide and parasite decrease survivorship of queens introduced into mating hives for three months. Because colony demographic regulation relies on queen's fertility, the compromise of its physiology and life can seriously menace colony survival under pressure of combined stressors.

Uptake of Encephalitozoon spp. and Vittaforma corneae (Microsporidia) by different cells.

Microsporidia are obligate intracellular parasites infecting a broad range of vertebrates and invertebrates. Various microsporidian species induce different clinical pictures in humans. The reason for this is not clear. It has been speculated that the different microsporidian species are transmitted by various routes, thus causing infections in different organs. Another possibility is that the diverse microsporidia have different tropisms to organ-specific cells, thus causing various diseases. In this study, we investigated the uptake of microsporidian spores by different cells with an immunofluorescence staining technique to investigate whether there is a difference between microsporidian species as well as between different cells. Using this technique, we were able to distinguish between intra- and extracellular microsporidian spores. All examined cell lines were able to internalize microsporidian spores, but the extent of internalization differed significantly between the cells. Although the results showed some patterns that correlate with the distribution of the parasites in humans, the different clinical pictures cannot be sufficiently explained by this phenomenon, so it seems more likely that the various clinical manifestations caused by the different microsporidian species are a consequence of different infection routes rather than of different affinities of the microsporidian species to different cells.

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.

Detection of microsporidia in surface water: a one-year follow-up study.

In order to estimate the rate and seasonal variation of Enterocytozoon bieneusi contamination of surface water, sequential samples of water from the River Seine in France were collected during a 1-year period. Each sample (300-600 l) was submitted to sequential filtrations, and the filters were then examined for microsporidia using light microscopy and nested polymerase chain reaction (PCR) for E. bieneusi. Amplified products were hybridized with a E. bieneusi-specific probe. Twenty-five samples of water were analyzed during 1 year. Microscopic examination of stained filters proved unreliable for the identification of spores. Using nested PCR, 16 of 25 specimens were positive (64%). Unexpectedly, E. bieneusi was identified in only one sample by specific hybridization underlining the lack of specificity of ours primers. Nevertheless, using DNA sequence analysis, unknown microsporidia species were identified in eight cases, which had highest scores of homology with Vittaforma corneae or Pleistophora sp. This study shows a low rate of water contamination by E. bieneusi suggesting that the risk of waterborne transmission to humans is limited.

Characterization of aminopeptidase activity from three species of microsporidia: Encephalitozoon cuniculi, Encephalitozoon hellem, and Vittaforma corneae.

Microsporidia are obligate intracellular parasites of the phylum Microspora. To date, more than 1,200 species within 144 genera have been described, with 14 infecting humans. Currently, no effective treatment exists for human microsporidiosis. In this study, the biochemical properties of the aminopeptidases were investigated within several species of microsporidia. Aminopeptidase activity was detected in 3 species of microsporidia, Encephalitozoon cuniculi, E. hellem, and Vittaforma corneae, using a fluorometric substrate assay. Each species exhibited distinct aminopeptidase properties. The cytosolic neutral aminopeptidase activities of the Encephalitozoon spp. were characterized as preferentially cleaving leucine, whereas those of V. corneae cleaved arginine. Native polyacrylamide gel electrophoresis estimated the molecular mass of E. cuniculi, E. hellem, and V. corneae as 74, 72, and 79 kDa, respectively. Enzymatic activity was inhibited by bestatin and it's analogue, nitrobestatin, indicating that the enzyme was an aminopeptidase for all species. Inhibition with the chelating agents ethylenediaminetetraacetic acid and 1,10phenanthroline characterized the enzymes as metalloaminopeptidases. Subcellular fractionation of the 3 microsporidial species suggested that the enzyme activity was localized in the cytosolic fraction. Optimal enzyme activity was observed at pH 7.2 for all species. This is the first report of enzyme characterization from these 3 species of microsporidia.

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.