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.

Unusual fluorescent granulomas and myonecrosis in Danio rerio infected by the microsporidian pathogen Pseudoloma neurophilia.

Abstract Zebrafish are a powerful model organism to study disease. Like other animal models, Danio rerio colonies are at risk of pathogenic infection. Microsporidia, a group of intracellular fungus-like parasites, are one potential threat. Microsporidian spores germinate and spread causing pathological changes in the central nervous system, skeletal muscle, and other anatomic sites. Infection can impair breeding, cause other morbidities, and ultimately be lethal. Previously, detecting microsporidia in zebrafish has required sacrificing animals for histopathologic analysis or microscopic examination of fresh tissues. Here, we show that fish with microsporidial infection often have autofluorescent nodules, and we demonstrate infectious spread from nodule-bearing fish to healthy D. rerio. Histologic analyses revealed that fluorescent nodules are granulomatous lesions composed of spores, degenerating muscle, and inflammatory cells. Additional histologic staining verified that microsporidia were present, specifically, Pseudoloma neurophilia. Polymerase chain reaction (PCR)-based testing confirmed the presence of P. neurophilia. Further PCR testing excluded infection by another common zebrafish microsporidial parasite, Pleistophora hyphessobryconis. Collectively, these studies show that P. neurophilia can induce skeletal muscle granulomas in D. rerio, a previously unknown finding. Moreover, since granulomas autofluoresce, microscopic screening for P. neurophilia infection is feasible in live fish, avoiding the need to sacrifice fish for surveillance for this pathogen.

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.

Humoral immune responses of the grouper Epinephelus akaara against the microsporidium Glugea epinephelusis.

The humoral immune responses of grouper Epinephelus akaara to a natural infection with Glugea epinephelusis was studied by ELISA utilizing intact mature spores as the coated antigen. Results showed that a specific humoral immune response was elicited, but the intensity of infection (in terms of the number of cysts) was not related to the antibody level in naturally infected hosts. The differences in the antigenicity of intact mature spores and soluble spore proteins derived from cracked mature spores were also analyzed. Results suggested that similar antigen epitopes existed between the 2 groups. Additionally, antigen component patterns and the distribution of antigen with immunogenicity were investigated by using the western blot and the immunofluorescent antibody technique (IFAT). The new parasitic microsporidium has specific polypeptide patterns comparable to the reported fish microsporidians. The main antigenic substances are concentrated on the surface of spores, and are mostly located on the anterior and posterior end of the spore bodies. Most surface components of the G. epinephelusis spores are soluble. The potential role of the surface components in initiating infection was also discussed.

Prevalencia de microscoporidios y otros parásitos intestinales en pacientes con infección por VIH, Bogotá, 2001 / Prevalence of microsporidia and other intestinal parasites in HIV infected patients from Bogotá[ a 2001 survey

Los parásitos intestinales oportunistas son protozoos que causan diarrea en pacientes infectados con el virus de inmunodeficiencia humana (VIH). Para determinar la prevalencia de microsporidios y otros par sitos oportunistas intestinales en pacientes infectados con el VIH con síntomas gastrointestinales y sistémicos, se estudiaron 115 pacientes que durante el 2001 consultaron al Hospital Santa Clara (33,0 por ciento), Clínica San Pedro-ISS (20,0 por ciento), Hospital Simón Bolívar (14,8 por ciento), San José (13,9 por ciento), Central de la Policía (6,1 por ciento), Compensar (5,2 por ciento), Liga de Lucha contra el Sida (2,6 por ciento), Hospital San Ignacio (2,6 por ciento) y Hospital Militar (1,7 por ciento). La edad promedio fue de 36 años con un rango de 18 a 71 años; 14 eran mujeres y 101 hombres. La metodología empleada fue la recolección por paciente de dos muestras de materia fecal seriadas para montaje directo, concentración, coloración de Zielh Neelsen modificada para diagnóstico de coccidios intestinales y coloraciones de cromotropo modificada, Gramcromotropo y calcoflúor para diagnóstico de microsporidios. La prevalencia de oportunistas fue de 10,4 por ciento para Cryptosporidium sp. En cuanto a microsporidios, se encontró que de 29 por ciento de positividad con cromotropo modificada como tamizaje, tan sólo 3,5 por ciento de las muestras se confirmaron como positivas con técnicas de calcoflúor y Gram-cromotropo. La prevalencia general de par sitos intestinales fue de 59,1 por ciento, de los cuales los principales patógenos fueron Blastocystis hominis con 25,2 por ciento y Entamoeba histolytica con 13 por ciento. En otros estudios con pacientes inmunosuprimidos por el VIH en Colombia, se han encontrado prevalencias de Cryptosporidium sp. menores que la hallada en esta investigación.

Nueva especie de Parathelohania (Microsporidia) en larvas de Anopheles aquasalis (Diptera: Culicidae) en Venezuela / New species of Parathelohania (Microsporidia) in larva of the Anopheles aquasalis (Diptera: Culicidae) in Venezuela

The symptoms of Anopheles aquasalis larvae naturally infected by a microsporidium, and the ultrastructure of the infecting spores is described. The larvae were maintained under laboratory conditions in salt concentrations of 10 g/l and 20 g/l of water. Daily recordings of the mortality of the larvae were made and the dates of change of instar were noted. Infected larvae were processed for transmission electronic microscopy using conventional methods at pH 7.2 and 260 mOsm/l. The infection by the microsporidia was positively correlated with an increase in the mean duration of the fourth instar of 2.88 to 6.33 days in 10 g/l of salt and of 2.47 to 6.14 days in 20 g/l of salt...

Nosema like organisms found in an immunocompetent human patient: first report in Central América

Stool samples of a immunocompetent hypertense 61 year old woman were stained by the Weber et al. method and studied by electronic microscopy. Mature microsporidia spores were found and according to their diplokaryotic muclei, as well as the typical polar filament with 12 coils, the organisms was classified as Nosema like genus

The human isolate of Brachiola algerae (Phylum Microspora): development in SCID mice and description of its fine structure features.

Ocular, peroral, intraperitoneal, intramuscular, and subcutaneous inoculation of severe combined immunodeficient (SCID) mice with spores of the human isolate (CDC: V404) of Brachiola algerae (syn. Nosema algerae) (Phylum Microspora) revealed that the microsporidium develops in viscera of the immunodeficient mouse host, but only after the ocular administration of spores. It is hypothesized that the physico-chemical milieu of the conjunctiva and cornea helped to adapt the originally 'poikilothermic microsporidian' to the conditions within the homoiothermic organism. Ocular application of spores caused no clinical signs of disease at the application site. However, severe infection in the liver was found 60 days after infection, manifested as hepatosplenomegaly and multifocal miliary necroses and granulomas containing parasites. No microsporidia were found in any other tissues. Transmission electron microscopy revealed characteristic tubulovesicular 'secretory materials' on the plasma membrane of all developmental stages of B. algerae except sporoblasts and spores. These formations increase the parasite surface and allow more efficient metabolic communication of the parasite with the host cell. It is hypothesized that the presence of these structures is a factor helping the parasite to grow in a variety of hosts and tissues. Ultrastructural characters support the likelihood that B. algerae and B. vesicularum are conspecific, and that there exists a relationship between species of the genera Brachiola and Anncaliia.

Diagnóstico laboratorial dos protozoários entéricos oportunistas em Goiânia, GO / Laboratorial diagnosis of intestinal protozoan opportunistic infections in Goiânia, GO

A criptosporidiose, isosporiase, ciclosporiase e as microsporidioses tornaram-se comuns em pacientes imunocomprometidos. O diagnostico destas doencas e importante, pois produzem quadro clinico semelhante, mas possuem manejo terapeutico e prognostico diferente. Visando avaliar a situacao do diagnostico destes parasitas, foram mapeados os laboratorios da rede do SUS e as unidades de saude no municipio de Goiania, GO

Corneal microsporidiosis. Report of case, including electron microscopic observations.

OBJECTIVE: To report a case of corneal stromal infection caused by a protozoon of the genus MICROSPORIDIA:, including clinical, histopathologic, and electron microscopic observations. DESIGN: Case report. METHODS: Light and electron microscopy studies were performed on keratectomy specimens from a 67-year-old immunocompetent man who had a unilateral chronic stromal keratitis that was refractory to medical treatment. Initial corneal biopsy followed by lamellar and penetrating keratoplasty were performed on the patient. All the specimens were studied histopathologically. RESULTS: Light microscopy of the corneal biopsy and the subsequent keratectomy specimens demonstrated myriad small, round to oval microsporidial organisms measuring 3.5 to 5.0 micrometer in length that stained positively with the periodic acid-Schiff, Grocott-methenamine silver, and acid-fast methods and were gram positive. Electron microscopic observations demonstrated viable blastospores that had a thin osmiophilic outer cell wall and contained 11 to 13 coils of the filament. The light and electron microscopic features, the tinctorial characteristics, and the selective corneal stromal involvement are consistent with microsporidial keratitis. CONCLUSIONS: Microsporidiosis should be considered in the differential diagnosis of a culture-negative stromal keratitis refractory to medical treatment. The diagnosis can be easily established based on the morphologic features of the protozoa in the keratectomy specimens. No effective medical treatment for the stromal disease is available. Full-thickness keratoplasty is suggested because, in our patient, lamellar keratoplasty did not preclude recurrence of the disease.

Molecular detection and phylogenetic placement of a microsporidian from English sole (Pleuronectes vetulus) affected by X-cell pseudotumors.

Flatfish tissue samples exhibiting X-cell pseudotumors were tested with a number of ribosomal DNA (rDNA) general primers in polymerase chain reactions (PCRs). Microsporidian primers resulted in the amplification of an rDNA fragment and molecular phylogenetic analysis indicated that although the organism did not relate closely with any current microsporidian genera, it was most similar to Nucleospora salmonis and branched within the Enterocytozoonidae. Re-examination of the original tissues used for DNA extractions revealed the presence of putative microsporidian spores in PCR-positive samples. These observations reiterate the highly sensitive diagnostic feature of PCR, allowing detection of organisms overlooked by conventional methods and demonstrate the occurrence of rare, coinfecting organisms.

Effect of antiretroviral therapy on cryptosporidiosis and microsporidiosis in patients infected with human immunodeficiency virus type 1.

To better understand whether potent antiretroviral therapies can modify the natural history of HIV-1-associated microsporidiosis and cryptosporidiosis, the response to antimicrobial treatment of these opportunistic infections was evaluated in patients with or without antiretroviral treatment. Fifty patients with diarrhoea, all positive for Cryptosporidium parvum or Enterocytozoon bieneusi, were included in the study. Retrospective data were collected concerning demographics, clinical and microbiological characteristics of the parasitic infection, antiretroviral therapy and prophylaxis against opportunistic infections. Faecal samples were prepared using the Richie formalin-ethyl acetate method and stained using the modified Ziehl-Neelsen method for detection of Cryptosporidium parvum and Isospora belli, the modified trichrome and calcofluor white technique for detection of Enterocytozoon spp., and iodine for detection of ova, cysts or vegetative forms. Diarrhoea was defined as an abnormal increase in stool liquidity, an abnormal increase in stool frequency and a daily stool weight of more than 250 g for a period of at least 4 days. Patients treated with double antiretroviral therapy or protease inhibitors demonstrated an excellent response and a sustained therapeutic effect after follow-up (range, 5-36 months). The relapse of cryptosporidiosis in two patients who discontinued antiretroviral therapy suggests that the infection might remain in a latent stage. The resolution of the diarrhoea seems to be related to an increased CD4+ cell count rather than to the viral load. In conclusion, these data strongly support the hypothesis that combination antiretroviral therapy is able to greatly modify the course of cryptosporidiosis and microsporidiosis in patients infected with HIV-1.

A case of Enterocytozoon bieneusi infection in an HIV-negative renal transplant recipient.

Reported here is a case of microsporidiosis that occurred in an HIV-negative renal transplant recipient. The patient developed protracted diarrhea 18 months following transplant surgery. Many spores of Enterocytozoon bieneusi were detected in stool smears using a modified trichrome staining method. Identification was confirmed using the polymerase chain reaction. Histological examination of duodenal biopsies revealed numerous spores in the cytoplasm of enterocytes. Tacrolimus and steroid regimens were decreased, treatment with mycophenolate mofetil was discontinued, and the patient was given albendazole and metronidazole for 2 weeks. The diarrhea resolved after 15 days of treatment; 2 months later the patient had recovered completely. A more systematic search for microsporidia using specific staining procedures should be performed in transplant recipients who develop severe diarrhea.

Identificación de Enterocytozoon bieneusi en un paciente con colangitis esclerosante y SIDA / Identification of Enterocytozoon bieneusi in a patient with sclerosing cholangitis and AIDS

Enterocytozoon bieneusi es el microsporidio que más comúnmente ha sido identificado en pacientes con SIDA. En este trabajo, se describen las manifestaciones clínicas de un paciente con diarrea crónica, pancreatitis y colangitis esclerosante asociada con SIDA. Los estudios por imágenes, con ultrasonografía y colangiopancreatografia retrógrada endoscópica, revelaron alteraciones en la vía biliar intra-y extrahepática, idénticas a las observadas en colangitis esclerosante. Se detectó Enterocytozoon bieneusi en duodeno y duodeno peripapilar por microscopia óptica y se confirmó por la reación en cadena de la polimerasa (PCR) utilizando primers específicos en muestras incluidas en parafina. La infección con microsporidios se debería sospechar en nuestro país en pacientes con inmunodeficiencia severa y colangitis esclerosante asociada con SIDA.

Microsporidia and acquired immunodeficiency syndrome

Microsporidia is a common term that has been used to refer to a group of eukaryotic, obligate intracellular protozoan parasites belonging to the phylum Microspora. They are important agricultural parasites, contaminating commercial insects; they are also important by infecting laboratory rodents, rabbits and primates. Ever since the early cases found by Magarino Torres, who reported the presence of Encephalitozoon in a patient suffering of a meningoencephalomyelitis, some human pathology caused by microsporidia has been described. However, only after the acquired immunodeficiency syndrome outbreak have these organisms appeared as significant etiological agents in different pathologies. Even so, they remain underestimated. In the present article, the importance of microsporidia for the human pathology in immunocompromised host has been stressed

Microsporidiosis ocular: primer caso en un preescolar inmunocompetente reportado en Venezuela / Ocular microsporidiosis: first case to immunocompetent preschool in Venezuela

Este informe presenta el primer caso de queratitis producida por microspora reportado en Venezuela, en un paciente immunocomprometido de 5 años y 10 meses de edad, sin antecedentes de traumatismo ocular, quien consultó por presentar, desde hacía 8 días, molestias en el ojo izquierdo. El examen biomicroscópico revela la presencia de queratitis punctata. Se practicó estudio parasitológico y microbiológico de secreción conjuntival, así como raspado de epitelio corneal, confeccionándose extendidos y coloreándose mediante las técnicas de Kinyoun, Gram, PAS y Giemsa. Se observaron al microscopio de luz con objetivo de inmersión, pudiendo constatar la presencia de estructuras compatibles morfológica y tintorialmente con esporas de microspora, algunas de las cuales estaban libres y otras eran intracelulares, de forma esférica u ovales, con tamaños comprendidos entre 1,5 y 2,5 um de diámetro X 2,5 a 3,0 um. El paciente recibió tratamiento local utilizando colirio a base de corticoesteroides y sulfisoxazol seis veces al día, y por vía oral 200 mg diarios de albendazol. Todo el tratamiento duró catorce días, lográndose desaparición de los síntomas, restitución ad integrum del epitelio corneal y desaparición de las esporas en los extendidos confeccionados con la secreción ocular

Production of monoclonal antibodies directed against the microsporidium Enterocytozoon bieneusi.

Several hybridomas producing antibodies detected by indirect immunofluorescence antibody test (IFAT) were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized against whole spores (protocol 1) or chitinase-treated spores (protocol 2) of Enterocytozoon bieneusi and were cloned twice by limiting dilutions. Two monoclonal antibodies (MAbs), 3B82H2 from protocol 1, isotyped as immunoglobulin M (IgM), and 6E52D9 from protocol 2, isotyped as IgG, were expanded in both ascites and culture. IFAT with the MAbs showed that both MAbs reacted exclusively with the walls of the spores of E. bieneusi, strongly staining the surface of mature spores, and produced titers of greater than 4,096. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. No cross-reaction, either with the spores of the other intestinal microsporidium species Encephalitozoon intestinalis or with yeast cells, bacteria, or any other intestinal parasites, was observed. The MAbs were used to identify E. bieneusi spores in fecal specimens from patients suspected of having intestinal microsporidiosis. The IFAT was validated against standard staining methods (Chromotrope 2R and Uvitex 2B) and PCR. We report here the first description and characterization of two MAbs specific for the spore wall of E. bieneusi. These MAbs have great potential for the demonstration and species determination of E. bieneusi, and their application in immunofluorescence identification of E. bieneusi in stool samples could offer a new diagnostic tool for clinical laboratories.

[Microsporidiasis in AIDS patients with chronic diarrhea. Expieriences at the National Institute of Nutrition "Salvador Zubrirán"]. / Microsporidiasis en pacientes con SIDA y diarrea crónica. Experiencia en el Instituto Nacional de la Nutrición "Salvador Zubirán".

BACKGROUND: Microsporidium sp. has been considered as a rare cause of diarrhea in AIDS patients. However, the improvement of some histochemical stains in the analysis of small bowel biopsies has shown an increase in its prevalence. In Mexico there are no series reporting intestinal microsporidiasis. DESIGN: Small bowel biopsies of 98 patients with AIDS and chronic diarrhea stained with HE and Giemsa were reviewed (January 1987-December 1994). The clinical, demographic and laboratory information was obtained from the clinical charts. RESULTS: In 50 patients an opportunistic microorganism was identified in the small bowel biopsy (51%). Microsporidium sp. was identified in 30 patients (31%). The clinical charts were reviewed in all but six cases. Of the 24 patients with microsporidiasis as the cause of diarrhea, 17 were male and seven female with a median age, of 33 years, old. Homosexuality was the main risk factor in males (11/17), and blood transfusion in females (4/7). A low socioeconomical classification was found in 75% cases. The initial manifestation of AIDS was diarrhea in 16/24 (67%), CD4 count cell below 200 mm3 was identified in 13/24 patients and more than 200 mm3 in 2/24. The stool examination and the original histologic interpretations were negative for Microsporidium sp. Lymphoplasmocytic inflammatory infiltrate with eosinophils in the lamina propia and atrophy was frequently seen. A pale red and gray color was observed in spore and merogonial phases of Microsporidium stained with Giemsa. CONCLUSION: Microsporidium sp. was present as the only pathogen in 31% of the small bowel biopsies reviewed by light microscopy. Diarrhea due to Microsporidium sp. is frequently seen in advanced stages of AIDS with CD4 count cell below 200 mm3 Giemsa stain in the evaluation of small biopsies is a cheap and useful method to, identify Microsporidium sp.