Detection of Encephalitozoon spp. from Human Diarrheal Stool and Farm Soil Samples in Korea

Microsporidia are eukaryotic organisms that cause zoonosis and are major opportunistic pathogens in HIV-positive patients. However, there is increasing evidence that these organisms can also cause gastrointestinal and ocular infections in immunocompetent individuals. In Korea, there have been no reports on human infections with microsporidia to date. In the present study, we used real-time PCR and nucleotide sequencing to detect Encephalitozoon intestinalis infection in seven of 139 human diarrheal stool specimens (5%) and Encephalitozoon hellem in three of 34 farm soil samples (8.8%). Genotype analysis of the E. hellem isolates based on the internal transcribed spacer 1 and polar tube protein genes showed that all isolates were genotype 1B. To our knowledge, this is the first report on human E. intestinalis infection in Korea and the first report revealing farm soil samples as a source of E. hellem infection. Because microsporidia are an important public health issue, further large-scale epidemiological studies are warranted.

Prevalencia de Encephalitozoon intestinalis y Enterocytozoon bieneusi en pacientes VIH positivos de Maracaibo, Venezuela / Prevalence of Encephalitozoon intestinalis and Enterocytozoon bieneusi in HIV positive patients to Maracaibo, Venezuela

Los microsporidios pueden provocar infecciones emergentes y oportunistas en individuos inmunocomprometidos de todo el mundo. Se realizó éste estudio para identificar las especies de microsporidios intestinales presentes en pacientes con VIH-SIDA del Servicio Autónomo Hospital Universitario de Maracaibo (SAHUM). Se recolectaron 50 muestras fecales de individuos con diagnóstico confirmado de VIH durante los años 2007-2008; se obtuvieron las cifras de CD4 de solo 42 pacientes. Las muestras se analizaron mediante PCR separadas para la identificación de Encephalitozoon intestinalis y Enterocytozoon bieneusi. Las especies de microsporidios presentaron un 36% de prevalencia, 10 pacientes presentaron Encephalitozoon intestinalis, 4 Enterocytozoon bieneusi y 4 ambas especies. Se determinó una relación inversamente proporcional y estadísticamente significativa entre el contaje de CD4 y la presencia de microsporidios en la muestra fecal. Es destacable la elevada prevalencia de especies de microsporidios observada en los pacientes VIH estudiados, donde predominó E. intestinalis.

Microsporidioses are considered emerging and opportunistic infections in immunocompromised individuals worldwide. The purpose of this study was to identify the species of intestinal microsporidia in patients with HIV-AIDS from the Servicio Autónomo Hospital Universitario de Maracaibo, Venezuela (SAHUM). Fecal samples were collected from 50 patients with confirmed diagnosis of HIV, during the years 2007 and 2008; the CD4 values were obtained from 42 patients. The samples were analyzed by separate PCRs to identify Encephalitozoon intestinalis and Enterocytozoon bieneusi. Microsporidia species showed a 36% prevalence: ten patients had Encephalitozoon intestinalis, four Enterocytozoon bieneusi and four both species. An inverse and statistically significant relationship between the CD4 count and the presence of microsporidia in the fecal sample was also found. It is remarkable the high prevalence of microsporidia species observed in the HIV patients studied, with a predominance of E. intestinalis.

Contribution of multiplex real time PCR for detection and differentiation of Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal samples of immunocompromised patients

Intestinal microsporidiosis is among the most frequent opportunistic diseases in immunocompromised patients. Routine diagnosis is generally performed by light microscopy of stained fecal samples. While unequivocal non-molecular species identification, important for cases management, is achievable only through electron microscopy. This study aimed to evaluate the contribution of multiplex real time PCR for simultaneous detection and differentiation of Enterocytozoon bieneusi and Encephalitozoon intestinalis in stool specimens of patients with immunosuppressive conditions. Stool samples were obtained from 78 immunocompromised patients suffering from diarrhea. The samples were screened for intestinal microsporidiosis by light microscopy using Weber's modified trichrome stain. The samples were subjected to multiplex real time PCR using Enterocytozoon bieneusi [E. bieneusi] primers and a probe specific on the internal transcribed spacer [ITS] sequence. Encephalitozoon intestinalis [E. intestinalis] primers and probe were specific for the small ribosomal subunit RNA gene sequence. Of 78 samples, 20 [25.6%] were detected positive by multiplex real time PCR. E. intestinalis was identified in 8 cases [40%], E. bieneusi in 7 [35%], and both species in 5 [25%]. Light microscopy detected a total of 22 samples [28.2%], 7 of which did not show the belt-like structure characteristic for microsporidial spores [empty-looking spores]. Compared to real time PCR, light microscopy had 75% sensitivity, 87.9% specificity, 68.2% PPV, 91.1% NPV and 84.6% accuracy in detection of microsporidia. No significant difference was found regarding the detection of E. intestinalis, E. bieneusi or both species by microscopy. Multiplex real time PCR proved to be more effective than classical trichrome stain for simultaneous identification and differentiation between E. bieneusi and E. intestinalis

Ceratoconjuntivite por Encephalitozoon hellem em periquitos agapornis (Agapornis spp) no Brasil: relato de caso / Keratoconjunctivitis by Encephalitozoon hellem in lovebirds (Agapornis spp) in Brazil: case report

Relata-se um caso de ceratoconjuntivite causada por Encephalitozoon hellem em agapornis (Agapornis spp.) adultos, provenientes de um criatório comercial. Cinco animais apresentaram sinais clínicos de ceratoconjuntivite, blefaroespasmo e blefaroedema bilateral, com presença de secreção seropurulenta. Amostras fecais foram colhidas e foi realizado exame coproparasitológico, com resultado negativo. Dois animais foram necropsiados, sendo detectados, em impressões de raspado de conjuntiva ocular, esporos e outros estádios evolutivos de Microsporidium. A confirmação do diagnóstico foi feita pela reação em cadeia de polimerase e sequenciamento de fragmentos amplificados, com utilização de primers específicos para o gene da subunidade 18S do rRNA de E. hellem. A análise dos fragmentos amplificados demonstrou 100 por cento de similaridade com outras sequências de E. hellem publicadas no GenBank. Este é primeiro relato de infecção por E. hellem em aves no Brasil.

A clinical case of keratoconjunctivitis by Encephalitozoon hellem in adult lovebirds (Agapornis spp.) from a commercial flock is reported. Five animals presented clinical symptoms of keratoconjunctivitis, blepharospasm, and bilateral blepharoedema, with seropurulent secretion. Coproparasitological diagnosis was carried out in fecal samples, with negative results. Two animals were necropsied, with detection of spores and other developmental stages of Microsporidium in conjunctival smears. The confirmation of the diagnosis was accomplished by the polimerase chain reaction with specific primers for 18S subunit of the rRNA of E. hellem, followed by sequencing of amplified fragments, which revealed 100 percent of genetic similarity to E. hellem. This study is the first report of E. hellem infection in birds in Brazil.

In vitro culture of various species of microsporidia causing keratitis: evaluation of three immortalized cell lines.

Being intracellular parasites, microsporidia can only be propagated in cell culture systems. This study evaluated three cell lines to determine the most suitable host-parasite In vitro system. Confluent monolayers of vero, SIRC, and HeLa cell lines, grown in 24-well tissue culture plates, were inoculated with varying concentrations (1 x 10(4) to 1 x 10(8) spores/mL) of Vittaforma corneae, Encephalitozoon hellem, Encephalitozoon cuniculi, and Encephalitozoon intestinalis spores. Growth was compared quantitatively at weekly intervals. Encephalitozoon species showed the highest amount of growth when cultured in vero cell line, while there was no significant difference in their growth in SIRC and HeLa cell lines. In comparison, V. corneae showed the highest growth in SIRC cells, followed by vero cells. The analytical sensitivity was found to be 1 x 10(4) spores/mL for vero cell line compared to 1 x 10(5) spores/mL for SIRC cell line and 1 x 10(7) spores/mL for HeLa cell line. HeLa cells also showed rapid disruption of cells, and the spores could not be easily distinguished from cell debris. This is the first report of the comparison of vero, SIRC, and HeLa for the propagation of microsporidial spores. Vero cell line was found to be more sensitive than SIRC and HeLa cells, and we believe that the inclusion of vero cell line in the routine culture protocols of ocular parasitology laboratories would result in a significant increase in the diagnostic yield.

Primer aislamiento de Encephalitozoon intestinalis a partir de muestra de materia fecal de un paciente colombiano con sida / First isolate of Encephalitozoon intestinalis from stools of a Colombian patient with AIDS

Introducción. Los microsporidios son agentes de infecciones oportunistas en pacientes con sida y con trasplantes, principalmente. Enterocytozoon bieneusi y Encephalitozoon intestinalis son los más frecuentes, asociados con infecciones entéricas. Los cultivos celulares han contribuido al conocimiento de los microsporidios. En Colombia no se han obtenido aislamientos provenientes de pacientes con microsporidiosis y, por consiguiente, no existen cepas autóctonas de los mismos. Objetivo. Establecer el cultivo celular de microsporidios intestinales a partir de materia fecal de pacientes parasitados. Materiales y métodos. Se realizó concentración agua-éter de la materia fecal positiva para microsporidios y el sedimento resultante se trató con una mezcla de antibióticos y antimicóticos durante 18 horas a 37 oC. Se inocularon células Vero previamente cultivadas en placas de 24 pozos y en medio RPMI con suplemento de suero bovino fetal al 10 por ciento y antibióticos, con las esporas concentradas. Los cultivos se mantuvieron a 37 oC al 5 por ciento de CO2. Se cambió de medio cada dos días y se evaluó la presencia de esporas en los sobrenadantes mediante Gram-cromótropo rápido en caliente. Resultados. En la segunda semana después de la infección, se encontraron esporas de microsporidios con morfología y coloración características. Mediante PCR se determinó que el microsporidio encontrado correspondía a la especie E. intestinalis. Conclusión. Se estableció el cultivo in vitro de microsporidios de materia fecal. Este protocolo es importante para la obtención y el mantenimiento de cepas autóctonas en Colombia, y contribuirá a las investigaciones de aspectos bioquímicos, inmunológicos y epidemiológicos de dichas cepas.

Immune responsiveness associated with experimental Encephalitozoon intestinalis infection in immunocompetent rats.

PURPOSE: Microsporidial infections have been recognized as an increasingly important infection in immunocompromized patients, particularly those infected with HIV/AIDS. This study was designed to study immune responses associated with experimental Encephalitozoon intestinalis infection in immunecompetent rats. MATERIALS AND METHODS: Thirty-four rats in 3 groups, A (Control), B (Intraperitoneal) and C (Oral) were given injections of 0.5 ml of 2 x 10(6) of purified spores of Encephalitotozoon intestinalis spores and were observed for serum specific IgG for 21 days using both Direct and Indirect ELISA. RESULTS: In indirect ELISA, specific lgG were detected on days 7, 14 and 21 for the group B rats and on day 21 for group C and in direct ELISA method, specific lgG were detected in-group B rats on days 7 and 21, for group C rats on day 21 only, while in the control rats, specific lgG were not detected. There was no significant difference between the direct and indirect methods (df=1, X(2), P>0.05). E. intestinalis was observed in stool samples of rats in 1/12 (08.33%) on days 14 and 21 in group B and in 4/10 (33.33%), 3/10 (25.00%) and 2/10 (16.67%) on days 7, 14 and 21 respectively in group C. In-group, A which is the control rats, no microsporidia were observed on days 0, 7, 14 and 21. CONCLUSIONS: There were no changes in the T-lymphocyte counts of rats prior to and after inoculation with spores. Extensive lesions were observed along the intestinal walls especially on the middle and lower sections of group C rats only.

Efecto de la ciclosporina A en ratones C57BL/6 infectados con encephalitozoon intestinalis / Cyclosporine A effect in mice C57BL/6 infected with Encephalitozoon intestinalis

Introducción. Encephalitozoon intestinalis es un microsporidio parásito del intestino, que puedediseminarse en pacientes inmunocomprometidos. Existen referencias de modelos animales inmunosuprimidos para el estudio de la microsporidiosis utilizando fármacos que producen supresión total de la respuesta inmune; sin embargo, no se han estudiado los efectos deinmunosupresores con acción selectiva sobre los componentes de esta respuesta. Objetivo. Evaluar el efecto de la inmunosupresión con ciclosporina A (CsA) en ratones C57BL/ 6 infectados con E. intestinalis.Materiales y métodos. Se utilizaron 80 ratones C57BL/6 distribuidos en cuatro grupos: infectados, inmunosuprimidos e infectados, inmunosuprimidos no infectados y controles. La inmunosupresión con CsA (50 mg/kg) se realizó vía intraperitoneal durante todo el estudio. En la semanas 2, 3, 4 y 6 posteriores a la infección se obtuvo sangre para determinar los anticuerpos, y materia fecal para evaluar la cinética de excreción de esporas. Además, se extrajeron varios órganos para estudiar la histopatología y observar la posible diseminación del parásito. Resultados. La producción de anticuerpos IgG fue mayor en los ratones inmunocompetentes infectados que en los inmunosuprimidos infectados con E. intestinalis. No se encontró elparásito en órganos diferentes al intestino delgado en los dos grupos infectados. Sin embargo, la excreción de esporas, tanto en heces como en líquido duodenal, fue mayor en el grupo inmunosuprimido infectado. Conclusión. La CsA en el modelo en ratón no indujo la diseminación de E. intestinalis ni la exacerbación de la enfermedad, pero contribuyó al aumento en la cinética de excreción deesporas y la disminución de la producción de anticuerpos IgG en los ratones inmunosuprimidos infectados.

Microsporidiosis intestinal: una visión integral / Intestinal microsporidiasis: an integral vision

La microsporidiosis intestinal es la infección del tracto digestivo alto, Enterocytozoon bieneusi y Encephalolytozoon intestinalis, pertenecientes al phylum Microspora, que en el hospedero inmunocomprometido especialmente con el síndrome de inmunodeficiencia adquirida (SIDA), produce cuadros de diarrea prolongada y malabsorción. Se revisarán sus aspectos históricos, biológicos, fisiopatológicos, inmunológicos, epidemiológicos, clínicos y de tratamiento. Método: para la elaboración de esta revisión se emplearon las bases de datos Medline y Pubmed a partir de 1981 hasta la fecha

Microsporidiosis generalizada por encephalitozoon sp en un paciente pediátrico con enfermedad de Bruton / Generalized microsporidiosis caused by encephalitozoon sp in a pediatric patient with Brutonïs disease

We present the case of a four year old boy with a history of repeated upper respiratory tract infections and pyoderma. He presented fever, seizures, inhability to talk, loss of swallowing, fine tremor in the upper extremities; positive bilateral Babinski reflex and quadriparesis. The diagnosis of Brutonïs disease and generalized microporidiosis was based on immunologic analysis, smear tests with chromotrope R2 stain and indirect immunofluorescense with monoclonal 3B6 antibody for Encephalitozoon species in samples of spinal fluid, bronchial and paranasal sinus aspirates and stool, which were all positive. The patient was treated with albendazol during 72 days; he left the hospital in a good condition, walking, talking and able to swallow. His laboratory test controls were negative; he is followed up in the outpatient department

Mantenimiento en cultivo y caracterización de un microsporidio encephalitozoon hellem aislado en un paciente con SIDA y neumonía / Culture maintanance and characterization of a microsporidian encephalitozoon hellen isolated from AIDS patient with neumonia

Comunicamos la identificación, a nivel de especie, de un microsporidio aislado en cultivo celular a partir de un lavado broncoalveolar de un paciente con Sida y neumonía. La caracterización del aislado se realizó mediante: 1) estudio morfológico de microscopía óptica y electrónica, 2) estudio inmunológico con antisuero específicos, inmunofluorescencia indirecta (IFI) e inmunoblot (WB) y 3) estudio molecular tras reacción en cadena de la polimerasa (PCR) con iniciadores especie-específicos diseñados a partir de la región que codifica la subunidad menor del ARN ribosomal. Las características ultraestructurales del aislado permitieron su identificación en el género Encephalitozoon. La identificación específica del microsporidio como Encephalitozoon hellem se realizó mediante IFI y WB, empleando suero policlonal de conejo anti-E. hellem (CDC:0291:V213), y mediante la amplificación por PCR del fragmento diagnóstico utilizando el par de iniciadores EHELF/EHELR específicos para esta especie. El aislado ha sido denominado EHVS-96 y se mantiene en cultivo continuo en células Vero-E6. Este es el primer aislamiento en cultivo y caracterización de E. hellem en España

Chitinolytic activity in viable spores of encephalitozoon species

By employing 4-methylumbelliferyl-beta-D-NN',N"-triacetylchitotriose substrate in a semi quantitative assay, chitinolytic activity in viable spores of Encephalitozoon cuniculi and E. intestinalis was detected and dependence on reaction time, spore concentration, concentration of substrate and temperature were demonstrated. It was possible to block the chitinolytic activity by chitin hydrolysate. By incubation at 80§C for 10 min or at 55§C for 20 min the spores were loosing the chitinolytic activity. Incubation of the spores in trypsin reduced the chitinolytic activity. Cellulase activity could not be detected.

Infecçöes oportunistas por protozoários parasitos - uma revisäo sobre Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli e microsporídios / Opportunistic infections caused by protozoan parasites - a review of cryptosporidium parvum, cyclospora cayetanensis, isospora belli e microsporidios

Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli e várias espécies de microsporídios säo reconhecidos atualmente como protozoários parasitos emergentes e oportunistas para o homem. Os organismos membros dos grupos de coccídios e microsporídios, com ampla distribuiçäo geográfica, säo reportados também com aumento de freqüência nas populaçöes de imunocompetentes e imunocompromissados. Esses parasitos säo transmitidos, principalmente, pela água, mas säo descritas infecçöes através dos alimentos. Os organismos säo responsáveis por surtos agudos de diarréia com várias outras seqüelas relacionadas com a doença. Os coccídios incluem três gêneros - Cryptosporidium, Cyclospora e Isospora. Os dois últimos säo de menor importância em termos de mortalidade e morbidade. Os microsporídios incluem gêneros (Enterocytozoon, Encephalitozoon) que somente agora estäo sendo reconhecidos como importantes agentes de doenças. As mais importantes rotas de transmissäo dos microsporídios no homem é a ingestäo ou inalaçäo de esporos. Diferentemente dos coccídios, esses organismos estäo mais restritos às populaçöes de imunocomprometidos. O aumento da incidência e o número de pacientes com diarréia prolongada devida a esses parasitos indicam a necessidade de aumentar a vigilância clínica relacionada com prevençäo, diagnóstico e tratamento.

Actualizaciones sobre microsporidiosis en el hombre / Update on microsporidiosis in man

The importance of microsporidium as an opportunistic agent in immunocompromised and AIDS patients is reviewed. Five strains of the agent have been described: Encephalitozoon, Enterocytozoon, Nosema, Pleistophora and Septata. The clinical presentation may be as 1) Generalized infections with multisystemic involvement, specially of the central nervous system; 2) Intestinal, that is the most important and frequent localization in man, and that may cause death in AIDS patients; 3) Ocular, that affects cornea, conjunctiva and may extend to paranasal sinuses; 4) Liver and biliary tract infection with granulomatous lesions, hepatic necrosis or sclerosing colangitis and 5) Muscular, affecting skeletal muscle. The diagnosis is difficult and is established finding spores in the affected tissues with light or electron microscopy. Lately, the diagnosis of intestinal microsporidiosis is made looking for faecal spores. The resistant wall of spores hampers treatment. However, good results are obtained with albendazole in intestinal microsporidiosis