Direct and sensitive detection of a microsporidian parasite of bumblebees using loop-mediated isothermal amplification (LAMP).

The reduction of bumblebee populations has been reported in the last decades, and the microsporidian parasite Nosema bombi is considered as one of the factors contributing to such reduction. Although the decline of bee populations affects both wild plants and human food supply, the effects of Nosema spp. infections are not known because it is difficult to obtain infective spores from wild bees due to their low prevalence. Microscopical observation of fecal samples or midgut homogenates and/or PCR are generally used for N. bombi detection. However, the germination rate of microsporidian spore declines if they are kept at 4 °C for a long time or frozen. It is therefore crucial to minimize the diagnosis and isolation time of infective spores from field-collected samples. Therefore, we performed a loop-mediated isothermal amplification (LAMP) assay for the direct detection of N. bombi in bumblebee midgut homogenates. Using this method, we could detect N. bombi from individuals from which it was visible under the microscope and directly from wild individuals.

Late-Onset Microsporidial Keratitis in Femtosecond Astigmatic Keratotomy After Laser-Assisted Phacoemulsification.

PURPOSE: To present a case of microsporidial keratitis in a femtosecond laser-created astigmatic keratotomy (AK) incision. METHODS: Case report. RESULTS: A 65-year-old Middle Eastern man presented 2 months after uncomplicated femtosecond laser-assisted cataract surgery (FLACS) and AK with mildly decreased vision and corneal edema in the operative eye. Shortly after treatment with topical corticosteroids, a fulminant corneal infiltrate manifested along the temporal arcuate incision. Multiple corneal scrapings sent for laboratory analysis were inconclusive. Two weeks after the initial presentation, a deep tissue sample was obtained using a 27-gauge cannula passed within the arcuate incision. The gram stain was directly observed, revealing intracellular microsporidial spores. The patient was treated with oral albendazole 400 mg once daily over 2 weeks and topical voriconazole 1% and fumagillin 3 mg/mL eye drops over 10 weeks. During this course, visual function steadily recovered as the infiltrate coalesced and ocular inflammation subsided. CONCLUSIONS: This is the first reported case of microsporidial keratitis presenting as a late-onset infection after femtosecond laser-assisted AK.

Clinical Features, Risk Factors, and Treatments of Microsporidial Epithelial Keratitis.

OBJECTIVE: To report the clinical manifestations, risk factors, and treatments of microsporidial epithelial keratitis in Thailand. METHODS: Twenty eyes of 19 patients were diagnosed and the clinical presentations, risk factors, and management were analyzed. RESULTS: Of 19 patients, six patients (32%) had no apparent risk factors. Predisposing factors included soil exposure (6/19, 32%), water contamination (6/19, 32%), and eye liner (1/19, 4%). Twelve cases (63%) were detected in the rainy season. All cases presented with disseminated, punctated, elevated, epithelial keratitis. Corneal scrapings with Gram-chromotrope staining were positive in all patients. Moxifloxacin 0.5% eye drops were given and all 16 patients experienced complete resolution. Three recurrent cases were resolved with only topical moxifloxacin without corneal scraping or swabbing. CONCLUSIONS: Predisposing factors were not found in some patients; thus, corneal scraping with staining should be considered in cases having a high index of suspicion. The incidence is increased during the rainy season; therefore, clinicians should have more awareness during these times. Debridement with topical moxifloxacin eye drops, without any systemic medication, may be an effective treatment. Corneal scraping or swabbing may not be required in recurrences.

The efficacy of corneal debridement in the treatment of microsporidial keratoconjunctivitis: a prospective randomized clinical trial.

PURPOSE: To evaluate the efficacy of corneal debridement in the treatment of clinically diagnosed cases of microsporidial keratoconjunctivitis. DESIGN: Prospective, double-masked randomized clinical trial. METHODS: Patients with clinical features such as multifocal, coarse, raised, punctate, round to oval epithelial lesions in the cornea in slit-lamp examination with mild to moderate conjunctival congestion, suggestive of microsporidial superficial keratoconjunctivitis, were included in the prospective study. All patients were randomized into 2 groups. Group 1 patients underwent debridement with the help of a sterile #15 blade on a Bard-Parker handle, whereas only conjunctival swabs were taken from Group 2 patients. All patients were treated with ocular lubricants. RESULTS: One hundred and twenty patients with clinical features suggestive of microsporidial superficial keratoconjunctivitis were included in the study. The mean age was 34.3 ± 13.6 years (Group 1) and 35.8 ± 16.2 years (Group 2) (P = .59). The mean duration of symptoms was 6.8 ± 3.9 days (Group 1) and 7.2 ± 4.6 days (Group 2) (P = .61). Baseline characteristics showed no difference between the 2 groups. The primary outcome was the time from the presentation to complete resolution (ie, absence of corneal lesions) of the clinical signs and symptoms. The secondary outcomes were final visual acuity and residual corneal side effects and/or scarring, if any. The mean resolution time of the corneal lesions was 5.7 ± 4.0 days (Group 1) and 5.9 ± 3.9 days (Group 2) (P = .83). There was no significant difference in final visual outcome in the 2 groups. No serious side effects were observed. CONCLUSION: Debridement does not have any significant advantage in terms of resolution of the corneal lesions and final visual outcome in cases of microsporidial keratoconjunctivitis.

Caenorhabditis elegans as a model for intracellular pathogen infection.

The genetically tractable nematode Caenorhabditis elegans is a convenient host for studies of pathogen infection. With the recent identification of two types of natural intracellular pathogens of C. elegans, this host now provides the opportunity to examine interactions and defence against intracellular pathogens in a whole-animal model for infection. C. elegans is the natural host for a genus of microsporidia, which comprise a phylum of fungal-related pathogens of widespread importance for agriculture and medicine. More recently, C. elegans has been shown to be a natural host for viruses related to the Nodaviridae family. Both microsporidian and viral pathogens infect the C. elegans intestine, which is composed of cells that share striking similarities to human intestinal epithelial cells. Because C. elegans nematodes are transparent, these infections provide a unique opportunity to visualize differentiated intestinal cells in vivo during the course of intracellular infection. Together, these two natural pathogens of C. elegans provide powerful systems in which to study microbial pathogenesis and host responses to intracellular infection.

Microsporidia and coccidia as causes of persistence diarrhea among liver transplant children: incidence rate and species/genotypes.

We determined species/genotype(s) of enteric microsporidia and coccidia causing diarrhea among 44 liver transplant children in Shiraz Nemazee hospital using acid-fast-trichrome staining and polymerase chain reaction-sequencing techniques. Enterocytozoon bieneusi (genotype D), Cryptosporidium (parvum and meleagridis) were detected in 6.81% and 11.36% of the children, respectively.

Zinc PVA versus potassium dichromate for preservation of microsporidian spores of human origin.

Microsporidia are emerging opportunistic parasites. Preservation of the biological properties of microsporidian spores is often required in research work. The present study compared two preservatives; zinc polyvinyl alcohol (zinc PVA) and potassium dichromate solutions for preservation of microsporidian spores separated from human faecal samples. After 0, 1, 2 and 4 months of storage, morphological features and staining characters of the spores were assessed by light microscopy in modified trichrome-stained smears and their viability percentages were calculated using acridine orange/ethidium bromide mixture. Also, spore infectivity was evaluated by faecal spore shedding and intestinal spore load in mice orally inoculated with the preserved spores. Results revealed that morphological features, staining characters and viability of the spores were maintained in both solutions throughout the study period. Spore infectivity was completely preserved in zinc PVA solution but showed significant reduction in potassium dichromate solution at the fourth month of the preservation duration.

Determining potential indicators of Cryptosporidium oocysts throughout the wastewater treatment process.

Most research on wastewater treatment efficiency compliance focuses on physicochemical and microbial indicators; however, very little emphasis has been placed so far on determining suitable indicator organisms to predict the discharge level of pathogens from treatment plants. In this study, raw wastewater, activated sludge, and the resulting final effluents and biosolids in four municipal wastewater treatment plants (WWTPs A, B, C and D) were seasonally investigated for human-virulent water-borne pathogens Cryptosporidium parvum/hominis and Giardia duodenalis, and microsporidia (e.g. Encephalitozoon hellem, E. intestinalis, and Enterocytozoon bieneusi) between 2008 and 2009. A suite of potential microbial indicators for human-virulent protozoa and microsporidia was also determined. A combination of multiple fluorescent in situ hybridization and immunofluorescent antibody assays were applied to detect Cryptosporidium oocysts, Giardia cysts, and microsporidian spores. Escherichia coli, enterococci and Clostridium perfringens spores were cultivated in selective media. Positive correlations were found between the abundance of enterococci and E. coli and abundance of Cryptosporidium oocysts (r(s) > 0.47, p < 0.01) and Giardia cysts (r(s) > 0.44, p < 0.01) at WWTPs A-D. Cryptosporidium perfringens spores were positively correlated to Cryptosporidium oocysts (r(s) = 0.40, p < 0.01) and Giardia cysts (r(s) = 0.46, p < 0.01). There was a strong positive correlation between abundance of Giardia cysts and that of Cryptosporidium oocysts (r(s) > 0.89, p < 0.01). To sum up, a suite of faecal indicator bacteria can be used as indicators for the presence of Cryptosporidium oocysts and Giardia cysts in these activated-sludge systems (WWTPs A, B and C). Overall, Giardia duodenalis was noted to be the best Cryptosporidium indicator for human health in the community-based influent wastewater and throughout the treatment process.

Microsporidia in household dogs and cats in Iran; a zoonotic concern.

Microsporidia in dogs and cats is primarily caused by the obligate, intracellular parasite Encephalitozoon cuniculi, which is a member of the phylum Microsporidia. The aim of the current study is the detection of this parasite in stool samples of small animals of Iran, by polymerase chain reaction. Microsporidia spp. was found in 31% (31/100) of dogs (E. cuniculi (18/100), Encephalitozoon bieneusi (8/100) and Encephalitozoon intestinalis (5/100)), and 7.5% (3/40) of the specimens obtained from cats were infected with E. bieneusi. Sequencing of PCR products confirmed these results. In conclusion, Microsporidia infection seems to be fairly common in pet animals of Iran, especially in dogs. This finding could indicate the importance of pet animals as zoonotic reservoirs of microsporidial human infections.

Clinical trial of 0.02% polyhexamethylene biguanide versus placebo in the treatment of microsporidial keratoconjunctivitis.

PURPOSE: To evaluate the efficacy of 0.02% polyhexamethylene biguanide (PHMB) in the treatment of keratoconjunctivitis caused by microsporidia. DESIGN: Prospective, double-masked, randomized, placebo-controlled clinical trial. METHODS: One hundred forty-five patients in a single-center, institutional setting were recruited. Patients with superficial keratoconjunctivitis and corneal scrapings with positive results for microsporidial spores were included. Patients with any known allergy to PHMB, and clinically suspected bacterial, viral, or fungal infection were excluded from the study. One hundred forty-five patients were treated at 4-hour intervals with either topical 0.02% PHMB (n = 72) or placebo (n = 73). The patients were followed-up on day 3 +/- 1, day 7 +/- 1, and weekly thereafter, until complete resolution of the corneal lesions. Patients with deterioration of clinical symptoms and signs were removed from the study and were treated with PHMB. Main outcome measures included resolution time, cure time, and final visual outcome. RESULTS: Resolution time was defined as the amount of time until disappearance of corneal epithelial infiltrates. Cure time was defined as the interval until absence of conjunctival congestion, corneal epithelial lesion, and superficial punctate keratitis. Baseline characteristics showed no relevant difference between the groups. The mean resolution time was 4.9 +/- 2.2 days and 4.6 +/- 2.3 days in the PHMB and placebo groups, respectively (P = .49). The mean time for cure was 13.5 +/- 6.6 days and 9.4 +/- 5.1 days in PHMB and placebo groups, respectively (P = .004). There was no significant difference in the final visual outcome between the groups (P = .10). No serious adverse effects were noted. CONCLUSIONS: Treatment of microsporidial keratoconjunctivitis with PHMB does not offer any significant advantage over placebo, suggesting self-limiting nature of the disease.

Emerging prevalence of microsporidial keratitis in Singapore: epidemiology, clinical features, and management.

OBJECTIVE: To investigate the incidence and epidemiologic factors involved in the development of microsporidial keratitis. The association of host immune status and clinical pattern, clinical features, and the role of fluoroquinolone monotherapy in treatment are also examined. DESIGN: Retrospective, noncomparative case series. PARTICIPANTS: All cases (124 patients, 134 eyes) of microsporidial keratitis confirmed with modified trichrome stain positive of corneal scrape over a 4-year period. METHODS: Epidemiologic factors were observed. Host immune status with human immunodeficiency virus (HIV) serology and CD4/CD8 analysis was performed when consent was obtained. Visual acuity (VA) and slit-lamp examination throughout the course of keratitis was recorded. Treatment used included topical fluoroquinolones (ciprofloxacin 0.3%, moxifloxacin 0.5%, gatifloxacin 0.5%, levofloxacin 0.5%, or norfloxacin 0.3%) as monotherapy or in combination with topical fumagillin and/or systemic albendazole. Where corneal edema developed, ultrasound corneal pachymetry was recorded. MAIN OUTCOME MEASURES: Demographic features and epidemiologic factors, including host immune status. Clinical features and disease course, including the response to different therapeutic regimes. RESULTS: Patients ranged in age from 11 to 68 years (mean, 31.9; median, 30) with a male:female ratio of 8:1 (females n = 17 [13.7%]). We performed HIV serology and CD4/CD8 in 45.9% of cases (n = 57); all the cases tested were negative with normal T-cell indices. Epidemiologic factors included soil exposure (50%), contact lens wear (21.1%), and topical steroid treatment (17.1%). The VA on presentation ranged from 20/20 to 20/100 (median, 20/30) with no loss in lines of VA on resolution. Common features were follicular papillary conjunctivitis and coarse punctate epithelial lesions in 3 patterns--diffuse, peripheral, and paracentral--evolving into nummular keratitis before resolution. Resolution occurred in 99% of cases on topical fluoroquinolone monotherapy. Four patients had recurrent disease that resolved with repeat fluoroquinolone or fluoroquinolone/oral albendazole combination. Two new clinical features were identified--diffuse endotheliitis (19.4%) with corneal edema and limbitis. CONCLUSIONS: This study identifies an increasing incidence of microsporidial keratitis in Singapore with a strong correlation with prior soil exposure. Diffuse endotheliitis and limbitis have not been described and resolves with topical steroid therapy. Topical fluoroquinolone monotherapy is a valid treatment option.

Examination of naturally exposed bottlenose dolphins (Tursiops truncatus) for Microsporidia, Cryptosporidium, and Giardia.

Bottlenose dolphins (Tursiops truncatus) captured in the estuarine waters off the coasts of South Carolina and Florida were examined for the presence of Microsporidia, Cryptosporidium sp., and Giardia sp. DNA extracted from feces or rectal swabs was amplified by polymerase chain reaction using parasite-specific small subunit ribosomal RNA gene primers. All positive specimens were subjected to gene sequence analysis. Of 83 dolphins, 17 were positive for Microsporidia. None was positive for Cryptosporidium or Giardia. Gene sequence data for each of the positive specimens were compared with data in GenBank. Fourteen specimens were found similar to, but not identical to, the microsporidian species Kabatana takedai, Tetramicra brevifilum, and Microgemma tinca, reported from fish, and possibly represent parasites of fish eaten by dolphins. Gene sequence data from 3 other specimens had approximately 87% similarity to Enterocytozoon bieneusi, a species known primarily to infect humans and a variety of terrestrial mammals, including livestock, companion animals, and wildlife. It is not clear if these specimens represent a species from a terrestrial source or a closely related species unique to dolphins. There were neither clinical signs nor age- or gender-related patterns apparent with the presence of these organisms.

Bilateral microsporidial keratoconjunctivitis in an immunocompetent non-contact lens wearer.

PURPOSE: To describe an immunocompetent male with bilateral microsporidial keratoconjunctivitis who responded to treatment with albendazole, propamidine, and fumagillin. METHODS: Corneal and conjunctival epithelial scrapings from a man with bilateral keratoconjunctivitis previously treated with topical corticosteroids were evaluated by Gram stain and by fluorescence microscopy. RESULTS: Gram stain and fluorescence microscopy of corneal epithelial scraping revealed organisms characteristic of microsporidia. Results of human immunodeficiency virus antibody testing were reported as nonreactive. Symptoms of ocular discomfort and clinical signs of keratoconjunctivitis resolved after five weeks of treatment that included systemic albendazole and topical propamidine isethionate 0.1% and fumagillin bicyclohexylammonium salt. A follow-up conjunctival scraping failed to detect any residual organisms 2 weeks after cessation of all treatment. CONCLUSION: Microsporidial ocular infection occurred in an immunocompetent non-contact lens wearer. Microsporidial keratoconjunctivitis should be considered in any individual with atypical multifocal diffuse epithelial keratitis, regardless of immune status or recent history of contact lens wear.

Diagnostic pathology of microsporidiosis.

Microsporidia are ubiquitous spore-forming parasites that are important worldwide pathogens in the HIV/AIDS pandemic. They are also increasingly being seen in HIV(-) individuals. Infection has been documented in almost every tissue and organ in the body and in a broad spectrum of cell types, including epithelial, mesenchymal, and neural. Microsporidia elicit a wide range of pathology, e.g., inflammation and cell death, and symptoms, e.g., shortness of breath, sinusitis, and diarrhea with wasting. Untreated, microsporidiosis has been documented as a cause of death.

Diagnostic use of 3 techniques for identification of microsporidian spores among AIDS patients in Portugal.

The calcofluor stain (CF), the monoclonal antibody (MAb) 3B6 indirect immunofluorescence assay (IFA) and the modified trichrome blue stain (MT) were compared in terms of their reproducibility in a routine laboratory and in order to evaluate the percentage of cases of microsporidiosis in Portuguese HIV patients. A total of 166 faeces samples, 71 pulmonary specimens and 43 urine samples were studied using the 3 techniques. CF had a high sensitivity and a moderate specificity when applied to faeces samples. The sensitivity was lower with pulmonary specimens. The method is easy and quick to perform but readings take a long time to obtain. The MAb 3B6 IFA had a good to excellent sensitivity when applied to faeces and urine samples, but moderate sensitivity in pulmonary specimens. Readings were quick and easy to obtain, but the assay took longer to perform than the other 2 techniques. There was a greater correlation between the results obtained with the MT and MAb 3B6 IFA techniques than between those obtained with the MT and CF techniques. In conclusion, the MT performed better than the MAb 3B6 IFA and CF and continues to have an important place in a routine laboratory for the diagnosis of microsporidiosis. This work also confirms the existence of a relatively high proportion (30%) of cases of infection with Microsporidia, especially intestinal microsporidiosis, in HIV patients in Portugal.

Brachiola algerae spore membrane systems, their activity during extrusion, and a new structural entity, the multilayered interlaced network, associated with the polar tube and the sporoplasm.

The microsporidial genus, Brachiola, contains three species: the type species Brachiola vesicularum (identified from an AIDS patient) and two species transferred from the genus Nosema, becoming Brachiola connori and Brachiola algerae. A developmental feature of the genus Brachiola is the "thickened" plasmalemma from sporoplasm through sporoblast stage. The sporoplasm has been reported to have a thick plasmalemma at 1-h postextrusion. The purpose of this investigation was to observe B. algerae spores before, during and after germination to determine if the plasmalemma is thick at the point of extrusion and if not, when and how it forms. New understandings regarding the polar filament position inside the spore, places it outside the sporoplasm proper with the sporoplasm limiting membrane invaginations surrounding it. These invaginations, present a possible location for aquaporins. The multilayered interlaced network (MIN), a new organelle (possibly of Golgi origin from the sporoblast), was observed inside the spore and sporoplasm; it formed an attachment to the end of the extruded polar tube and contributed to the thickening of the sporoplasm plasmalemma. A thin "unit limiting membrane", present on the sporoplasm at the time of extrusion, is connected to the MIN by many cross-connections forming the "thick blistered" surface by 30 min-postextrusion.

PCR detection of Tetramicra brevifilum (Microspora) infection in turbot (Scophthalmus maximus L.) musculature.

This study investigated the spatial distribution of Tetramicra brevifilum spores in the musculature of infected turbot Scophthalmus maximus, with the aim of identifying the most appropriate body locations for diagnostic assays. A PCR protocol optimized for the detection of T. brevifilum spores in turbot muscle is also described. In fish showing low- and moderate-intensity infection, the spatial distribution of spores was best fitted by a negative binomial distribution, indicating a clumped spatial pattern; the negative binomial coefficient k was lower for fish with low-intensity infection, indicating a more markedly clumped pattern in these fish. In fish with high-intensity infection, the spatial distribution of spores was best fitted by the Poisson distribution, indicating a random pattern. In both low- and moderate-intensity infection, spores were present at highest density in the musculature adjoining the dorsal fins. Samples for PCR were therefore obtained from this location. PCR amplification was of the small subunit ribosomal DNA (SSUrDNA), using a pair of species-specific primers that amplify the 1250 bp product. The PCR protocol developed showed better sensitivity than microscopical techniques (detection rate by microscopy 25%, versus 42% by PCR), suggesting that it may be useful for routine screening for Tetramicra brevifilum infection in cultured turbot.