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.

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.

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.

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.

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.

Aplicaçäo do método de coloraçäo tricrômica, em fezes diarréicas de infectados pelo HIV, para pesquisa de microsporídios / Trichrome staining method applied in stools from HIV-infected patients with diarrhorea, for microsporidia investigation

Os microsporídios säo implicados em várias manifestaçöes clínicas em pacientes com AIDS; os quadros diarréicos säo os mais comuns. O diagnóstico das microsporidioses dependia da microscopia eletrônica para exame de materiais obtidos por procedimentos invasivos. A técnica de coloraçäo tricrômica modificada permite o diagnóstico sem necessidade deste procedimento, através da microscopia óptica. No presente estudo foi aplicado o método de coloraçäo tricromica em fezes de 62 pacientes com diarréia, infectados pelo HIV ou com AIDS. Das 62 amostras, identificou-se esporos de microsporídios em uma. O trabalho corrobora a presença destes protozoários em nosso meio, associada a quadros de diarréia crônica em pacientes com AIDS e grave comprometimento imunológico, constata que este método de coloraçäo promove satisfatória visualizaçäo de esporos de microsporídios em fezes e aponta caminhos para novos estudos

Microsporidiasis en pacientes con SIDA y diarrea crónica. Experiencia en el Instituto Nacional de la Nutrición "Salvador Zubirán" / Microsporidiasis in a patient with AIDS and chronic diarrhea. Experience in the Instituto Nacional de la Nutricion Salvador Zubiran

Antecedentes. Microsporidium sp. se ha considerado una causa poco frecuente de diarrea en pacientes con SIDA. Sin embargo, el uso de algunas tinciones histoquímicas en la evaluación de las biopsias de intestino delgado, ha evidenciado incremento en su prevalencia. En México no existe información sobre la prevalencia y las características anatomoclínicas de este patógeno. Diseño. Se revisaron 98 biopsias de intestino delgado teñidas con HE y Giemsa de pacientes con SIDA y diarrea crónica (enero 1987-diciembre 1994). La información clínica y de laboratorio se obtuvo de los expedientes clínicos. Resultados. En 50 pacientes se identificaron patógenos oportunistas en las biopsias de intestino delgado. Microsporidium sp. se identificó en 30 pacientes (prevalencia de 31 por ciento). En 24 de 30 se obtuvo información del expediente clínico. Todos los pacientes (17/24 hombres, 7/24 mujeres) se encontraba en estadio C3 de SIDA con edad promedio de 33 años. Los factores de riesgo para SIDA fueron homosexualidad en hombre y transfusiones en mujeres. Se identificó nivel socioeconómico bajo en 75 por ciento de los casos. La manifestación inicial de SIDA fue diarrea en 67 por ciento. La cuenta de CD4 fue < 200/mm3 en 13/24 y > 200/mm3 en 2/24. Los exámenes de heces y la interpretación inicial de la biopsia fue negativa para Microsporidium sp. En la revisión de las biopsias, se identificó inflamación linfoplasmocitaria con eosinófilos y atrofia intestinal en un alto porcentaje de casos. Las esporas se tiñeron de color rojo pálido. Conclusión. Microsporidium sp. fue frecuente en pacientes de nivel socioeconómico bajo, en estadio C3 de SIDA con cifras de CD4<200 mm3. La tinción de Giemsa es un método útil y barato para la identificación de esporas y merontes de Microsporidium sp

A powerful DNA extraction method and PCR for detection of microsporidia in clinical stool specimens.

The diagnosis of intestinal microsporidiosis has traditionally depended on direct visualization of the parasite in stool specimens or intestinal biopsy samples by light and/or electron microscopy. Limited information about the specificity and sensitivity of PCR for the detection microsporidia in clinical stool specimens is available. To establish a sensitive and specific method for the detection of microsporidia in clinical samples, we studied clinical stool specimens of 104 randomly selected human immunodeficiency virus-infected patients with diarrhea to compare light microscopy and PCR. Fluorochrome Uvitex 2B staining was used for light microscopy. To raise the sensitivity of PCR, we used a powerful and fast DNA extraction method including stool sedimentation, glass bead disruption, and proteinase K and chitinase digestion. PCR was performed with primer pairs V1-PMP2, V1-EB450, and V1-SI500, and the nature of the PCR products was confirmed by Southern blot hybridization. Microsporidiosis was diagnosed by light microscopy in eight patients. Ten patients tested positive for microsporidiosis by PCR. Enterocytozoon bieneusi was found in seven cases, and Encephalitozoon intestinalis was found in four cases. In one case a double infection with E. bieneusi and E. intestinalis was diagnosed by PCR, whereas light microscopy showed only E. bieneusi infection. PCR testing of stool specimens is useful for diagnosis and species differentiation of intestinal microsporidiosis in HIV patients.

Cytologic detection of microsporidia spores in bile. A comparison of stains.

OBJECTIVE: To compare stains in preparations of bile in a patient with AIDS and microsporidial cholangitis. STUDY DESIGN: Bile was obtained from a 30-year-old male with AIDS and symptoms of cholangitis. Comparative staining of the specimen was performed using a formalin-fixed preparation stained with Chromotrope 2R stain and with alcohol-fixed preparations stained with Gram and Giemsa stain and Diff-Quik. An alcohol-fixed ThinPrep slide was stained with Papanicolaou stain. RESULTS: Diagnostic microsporidia spores were detected under oil immersion using Papanicolaou, Chromotrope 2R, Giemsa and Gram stain. The Diff-Quik-stained preparation also revealed microsporidia but with suboptimal morphology. CONCLUSION: Detection of microsporidia in bile can be achieved using several different stains routinely available to cytologists, most optimally with alcohol-fixed Papanicolaou- or Giemsa-stained preparations or with Chromotrope 2R stain, which is available in parasitology laboratories. These findings should be applicable to fluids from other body sites with this emerging pathogen in AIDS.

Systemic microsporidiosis in inland bearded dragons (Pogona vitticeps).

One laboratory-hatched and -reared inland bearded dragon (Pogona vitticeps) (No. 1) and two privately owned inland bearded dragons (Nos. 2 and 3) died, showing nonspecific signs of illness. Light microscopic examination of hematoxylin and eosin-stained tissue sections from lizard No. 1 revealed severe hepatic necrosis with clusters of light basophilic intracytoplasmic microorganisms packing and distending hepatocytes and free in areas of necrosis. Similar microorganisms were within cytoplasmic vacuoles in distended renal epithelial cells, pulmonary epithelial cells, gastric mucosal epithelial cells, enterocytes, and capillary endothelial cells and ventricular ependymal cells in the brain. In lizard Nos. 2 and 3, microorganisms of similar appearance were in macrophages in granulomatous inflammation in the colon, adrenal glands, and ovaries. The microorganism was gram positive and acid fast and had a small polar granule that stained using the periodic acid-Schiff reaction. Electron microscopic examination of deparaffinized liver of lizard No. 1 revealed merogonic and sporogonic stages of a protozoan compatible with members of the phylum Microspora. This report provides the first description of microsporidiosis in bearded dragons and is only the second report of this infection in a lizard.

Enterocytozoon bieneusi multiorgan microsporidiosis in a HIV-infected patient.

Multiorgan microsporidiosis due to Enterocytozoon bieneusi was diagnosed in an HIV-infected patient. The parasite was found and identified as E. bieneusi by transmission electron microscopy in stools, duodenal biopsy, nasal discharge and sputum. No clinical improvement or parasite eradication was obtained after albendazole therapy, but the patient remained alive 9 months after diagnosis.

Detection of Enterocytozoon bieneusi in two human immunodeficiency virus-negative patients with chronic diarrhea by polymerase chain reaction in duodenal biopsy specimens and review.

Intestinal microsporidiosis has been associated traditionally with severely immunocompromised patients with AIDS. We describe two new cases of intestinal microsporidiosis due to Enterocytozoon bieneusi in human immunodeficiency virus-negative adults. Both patients presented with chronic nonbloody diarrhea, and one had intestinal lymphangiectasia as well. Intestinal microsporidiosis was diagnosed by evaluation of stool samples, and the specific species was determined by use of polymerase chain reaction (PCR) in duodenal biopsy specimens. To our knowledge, this is the first report of confirmation of E. bieneusi in the intestinal epithelium of HIV-negative individuals by use of PCR in duodenal biopsy specimens. Cases of intestinal microsporidiosis in HIV-negative individuals reported in the English-language literature are reviewed. These two new cases along with those described previously corroborate the need to evaluate for microsporidia in HIV-negative individuals with unexplained diarrhea.

Ultrastructural morphology of Enterocytozoon bieneusi in biliary epithelium of rhesus macaques (Macaca mulatta).

Enterocytozoon bieneusi is the most common microsporidian parasite found in humans with acquired immunodeficiency syndrome. A nearly identical organism was recently recognized in rhesus macaques (Macaca mulatta). Ultrastructural examination of this microsporidian parasite in biliary epithelium of rhesus macaques reveals characteristics unique to E. bieneusi, including 1) a lack of sporophorus vesicles or pansporoblastic membranes, 2) direct contact of all stages with the host-cell cytoplasm, 3) elongated nuclei present within proliferative and sporogonial stages, 4) late thickening of the sporogonial plasmodium plasmalemma, 5) electron-lucent inclusions present throughout the life cycle, 6) precocious development of electron dense discs before plasmodial division to sporoblasts, and 7) the presence of polar tube doublets within spores and sporoblasts visualized as 5-7 coils in section.

Microsporidia in duodenal biopsies from 72 HIV-infected patients with abdominal complaints.

In order to evaluate the capacity of routine histological examination to detect microsporidiosis, a retrospective study was performed on 72 duodenal biopsies from 72 HIV-infected patients with upper abdominal symptoms of unknown cause. Two light microscopic cytological staining techniques, modified trichrome stain and the fluorochrome Calcofluor, were used. Two cases of microsporidiosis were detected among the 20 patients with prolonged diarrhoea of unknown origin in whom no etiological agent had been demonstrated by stool examination, mycobacterial and cytomegalovirus culture of biopsies, and histological routine staining of duodenal biopsies. The calculated confidence interval of 3-30% corresponds to the prevalence of intestinal microsporidiosis in HIV patients with prolonged diarrhoea in various parts of the world. The findings motivate attempts to identify microsporidia using special cytological staining methods. Improved methods of species identification are needed to aid in the choice of chemotherapy.

Tetramicra brevifilum (Matthews & Matthews, 1980) (Microsporida: Tetramicriidae) in a new fish host, Lophius budegassa (Spinola, 1807) in Spain.

Tetramicra brevifilum, a microsporidian parasite of Scophthalmus maximus, was found in Lophius budegassa for the first time. This parasite was detected in 5 of 199 hosts captured in the coastal waters of Barcelona (Northwest Mediterranean), which enlarges the geographic distribution of this microsporidian. Affected fish did not show any external sign of disease, and cysts of T. brevifilum were found associated with the body musculature but were easily differentiated from those of Spraguea lophii, another microsporidian present in this host. A case of simultaneous infection by both T. brevifilum and S. lophii was found.