Description of microsporidia in simulids: molecular and morphological characterization of microsporidia in the larvae of Simulium pertinax Kollar (Diptera: Simuliidae)

Introduction Microsporidia constitute the most common black fly pathogens, although the species' diversity, seasonal occurrence and transmission mechanisms remain poorly understood. Infections by this agent are often chronic and non-lethal, but they can cause reduced fecundity and decreased longevity. The objective of this study was to identify microsporidia infecting Simulium (Chirostilbia) pertinax (Kollar, 1832) larvae from Caraguatatuba, State of São Paulo, Brazil, by molecular and morphological characterization. Methods Larvae were collected at a single point in a stream in a rural area of the city and were kept under artificial aeration until analysis. Polydispyrenia spp. infection was characterized by the presence of at least 32 mononuclear spores measuring 6.9 ± 1.0 × 5.0 ± 0.7µm in persistent sporophorous vesicles. Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores measuring 4.5 × 3.5µm in sporophorous vesicles. Results The molecular analysis confirmed the presence of microsporidian DNA in the 8 samples (prevalence of 0.51%). Six samples (Brazilian larvae) were related to Polydispyrenia simulii and Caudospora palustris reference sequences but in separate clusters. One sample was clustered with Amblyospora spp. Edhazardia aedis was the positive control taxon. Conclusions Samples identified as Polydispyrenia spp. and Amblyospora spp. were grouped with P. simulii and Amblyospora spp., respectively, corroborating previous results. However, the 16S gene tree showed a considerable distance between the black fly-infecting Amblyospora spp. and the mosquito-infecting spp. This distance suggests that these two groups are not congeneric. Additional genomic region evaluation is necessary to obtain a coherent phylogeny for this group. .

Prevalencia de microsporidios intestinales y otros enteroparásitos en pacientes con VIH positivo de Maracaibo, Venezuela / Prevalence of intestinal microsporidia and other intestinal parasites in hiv positive patients from Maracaibo, Venezuela.

Introducción. Desde 1985, los microsporidios se consideran parásitos causantes de infecciones emergentes y oportunistas en individuos inmunocomprometidos en todo el mundo. Objetivo. Detectar la presencia de microsporidios y otros enteroparásitos en pacientes con VIH/sida del Servicio Autónomo Hospital Universitario de Maracaibo (SAHUM), donde no existían estudios previos en este campo. Materiales y métodos. Las muestras fecales se analizaron mediante examen directo, método de concentración con formol-éter, coloración de Kinyoun y coloración Gram-cromotropo rápida. Se realizaron PCR separadas para diferenciar Entamoeba histolytica o Entamoeba dispar , cuando se observó el complejo E. histolytica/dispar al microscopio. Mediante historia clínica se obtuvo información del paciente. Resultados. De los 56 individuos participantes, 38 (67,86 %) presentaron alguna especie parasitaria comensal o patógena en su muestra fecal. Predominaron los individuos portadores de especies parásitas patógenas (26/38). Fueron diagnosticados protozoos como Isospora belli (17,65 %), Blastocystis spp .(17,65 %), Cryptosporidium spp. (7,84 %), complejo Entamoeba histolytica/dispar (5,88 %) , Entamoeba coli (3,92 %) , Giardia lamblia (3,92 %) , Endolimax nana (3,92 %) , Cyclospora cayetanensis (3,92 %) y Chilomastix mesnili (1,96 %). Entre los helmintos, Ascaris lumbricoides, Trichuris trichiura y Strongyloides stercoralis, presentaron un porcentaje de 27,27 % cada uno, e Hymenolepis nana , de 18,18 %. Solo se detectó E. histolytica en uno de los tres casos que presentaron el complejo al examen microscópico. Mediante Gram-cromotropo, 17 muestras evidenciaron esporas del filo Microsporidia, lo que equivale a un 33,33 % de prevalencia. Conclusión. Los microsporidios pueden ocupar el primer lugar de prevalencia en pacientes con VIH positivo, cuando se utilizan técnicas diagnósticas específicas.

Objective: To detect the presence of microsporidia and other enteric parasites in patients with HIVAIDS of the Autonomous Services University Hospital of Maracaibo (SAHUM), where there are no previous studies in this field. Materials and methods: Fecal samples were analyzed by means of direct exam, concetration method with formal-ether, Kinyoun coloration and fast Gram-Chromotrope coloration. Separate PCR were perfomed to differentiate Entamoeba histolytica and Entamoeba dispar , when the E. histolytica/E. dispar complex was observed in the microscope. Information on the patient was obtained trough clinical history. Results: Of 56 individuals that participated, 38 (67.86%) presented some commensal parasite and/ or pathogenic species in their fecal sample. Carriers of pathogenic species were predominat (26/38). Protozoa such as Isospora belli protozoa (17.65%), Blastocystis spp. (17.65%), Cryptosporidium spp. (7.84%), E. histolytica/E. dispar (5.88%), Entamoeba coli (3.92%), Giardia lamblia (3.92%), Endolimax nana (3.92%), Cyclospora cayetanensis (3.92%), and Chilomastix mesnilli (1.96%) were diagnosed. Among the helminths, Ascaris lumbricoides, Trichuris trichiura and Strongyloides stercoralis , had a percentage of 27.27% each, and Hymenolepis nana , 18.18%. Entamoeba histolytica was only detected in one of three cases presenting complex microscopic examination. By Gram-chromotrope, 17 samples showed spores of the Microsporidia phylum, equivalent to 33.33% prevalence. Conclusion: Microsporidia may be first prevalente in HIV positive patients when specific diagnostic techniques are used.

Microsporidial keratoconjunctivitis in an immunocompetent patient with a past history of laser in situ keratomilieusis surgery.

Ocular infection with microsporidia has been documented in both immunocompetent and immunocompromised patients. Sources and mode of human infection with microsporidia have been difficult to ascertain although exposure to water may be an important risk factor. Of four genera that have been reported in human disease, only the genera Nosema, Encephalitozoon and Septata are documented to cause ocular infection. Here, in our case a healthy 30-year-old man who had undergone bilateral laser in situ keratomilieusis surgery two and half years back presented with a 10-day history of redness and 4-day history of blurring of vision in the right eye. On presentation, his best-corrected visual acuity was 20/20 partial in both eyes. Slit lamp examination revealed multiple pin head shaped infiltrates in the right cornea. Examination of the left eye was unremarkable. Based on microscopic demonstration of numerous microsporidial spores in the corneal scrapings, a diagnosis of microsporidial keratitoconjunctivitis was made. On treatment with oral albendazole, the cornea became clear with complete resolution of symptoms and signs within two weeks.

Multiplex PCR Detection of Waterborne Intestinal Protozoa; Microsporidia, Cyclospora, and Cryptosporidium

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 101 to 102 oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.

Ocorrência de microsporídios em pequenos mamíferos silvestres no Estado de São Paulo / Occurrence of Microsporidia in small wildlife mammals in the state of São Paulo, Brazil

This study describes infection of microsporidia in the natural fauna of small wildlife animals in an area of deforestation for a water reservoir construction in the state of São Paulo, Brazil. It was focused on marsupials, small rodents, frogs, primates, bats, and others mammals taken from this area to access whether they may represent environmental sources of these zoonoses. From all captured animals, microsporidia spores were observed in the feces of bats, small rodents, and marsupials. This study emphasises the importance of wild animals, particularly small mammals as potential sources of microsporidia to human and animal populations from deforested areas.

Microsporidiosis in a Brazilian University Hospital: case report

This is the report on a patient with chronic diarrhea caused by microsporidia. He is married, infected with HIV and has low CD4 cell count. The diagnosis was established through stool parasite search using concentration methods and Gram - chromotrope staining technique. Ileum biopsy was also performed in this case. The etiological diagnosis may be established in a clinical laboratory, by chromotrope staining technique in routine microscopic examination of stool specimens.

Este é o relato de caso de doente com diarréia crônica causada por Microsporidia. O doente era homem, casado, infectado com HIV e tinha baixa taxa de linfócitos CD4+. O diagnóstico foi feito em exame de fezes utilizando métodos de concentração e técnica de coloração de Gram-Chromotrope. Biópsia de íleo também foi realizada neste caso. O diagnóstico etiológico pode ser feito em laboratório clínico, por técnicas de coloração baseada em cromotrope na rotina da observação microscópica direta.

Chronic microsporidial stromal keratitis in an immunocompetent, non-contact lens wearer.

An 82-year-old healthy man with unilateral chronic stromal keratitis, initially diagnosed to have viral keratitis and refractory to medical therapy, showed numerous oval, microsporidial organisms, measuring 4-5 m in length in the corneal biopsy. Penetrating keratoplasty, followed by treatment with systemic albendazole and topical propamidine isethionate resulted in resolution of the infection. Electron microscopy of the keratoplasty specimen demonstrated sporoblasts with diplokaryotic nuclei and multiple coils of the filament. The light and electron microscopic features were consistent with microsporidial keratitis.

Clinical and diagnostic aspects of intestinal microsporidiosis in HIV-infected patients with chronic diarrhea in Rio de Janeiro, Brazil

The objectives of this study were to determine both the prevalence of microsporidial intestinal infection and the clinical outcome of the disease in a cohort of 40 HIV-infected patients presenting with chronic diarrhea in Rio de Janeiro, Brazil. Each patient, after clinical evaluation, had stools and intestinal fragments examined for viral, bacterial and parasitic pathogens. Microsporidia were found in 11 patients (27.5 percent) either in stools or in duodenal or ileal biopsies. Microsporidial spores were found more frequently in stools than in biopsy fragments. Samples examined using transmission electron microscopy (n=3) or polymerase chain reaction (n=6) confirmed Enterocytozoon bieneusi as the causative agent. Microsporidia were the only potential enteric pathogens found in 5 of the 11 patients. Other pathogens were also detected in the intestinal tract of 21 patients, but diarrhea remained unexplained in 8. We concluded that microsporidial infection is frequently found in HIV infected persons in Rio de Janeiro, and it seems to be a marker of advanced stage of AIDS

Esporos de microsporidios en heces de dos niños HIV seronegativos de Tucumán, Argentina / Microsporidian spores in the stool specimens of two HIV-negative children from Tucuman, Argentina

La incidencia mundial de microsporidiosis intestinal en pacientes infectados por HIV es relevante. En contraste, son muy pocos los casos de infección del parásito en pacientes HIV negativos. Aquí se informa que en diciembre de 1994 fueron detectadas infecciones microsporidianas intestinales en dos niños, hermanos, de 16 y 33 meses de edad, HIV-negativos, desnutridos, nativos y residentes en la provincia de Tucumán, Argentina. El diagnóstico se logró empleando microscopía óptica para la detección de esporos microsporidianos en frotis de especímenes fecales coloreados con una nueva modificación del método de Ziehl-Neelsen modificado. El reconocimiento de los esporos estuvo basado en sus características morfológicas y propiedades tintoriales, y en su comparación con frotis testigo positivo para Enterocytozoon bieneusi coloreados con el método de Weber. Ambos niños mostraron definidos signos de desnutrición y otras patologías asociadas, lo que sumado a la falta de colaboración de sus progenitores para un adecuado estudio clínico, hicieron dificultoso poner en claro el rol desempeñado por los parásitos en estos casos. No obstante, las infecciones microsporidianas de los dos niños tuvieron evolución autolimitada

Microsporidiosis humana / Human microsporidiosis

La microsporidiosis humana ha cobrado relevancia clínica con la aparición del virus de la inmunodeficiencia humana. La diarrea crónica constituye la expresión clínica más frecuente es estos pacientes aunque se han descrito infecciones localizadas en los distintos parénquimas del organismo e infecciones generalizadas. Las especies más relevantes son enterocytozoon bieneusi y encephalitozoon intestinalis. Se plantean como desafíos definir claramente los métodos diagnósticos y terapia adecuadapara ambas especies, una eventual terapia profiláctica primaria y/o secundaria y actualizar además las cifras de prevalencia en los distintos países

Enterocytozoon bieneusi (orden microsporidia, familia entrerocytozoonidae) in Costa Rica: report of the first human case in central america

The first case of microspotidiosis in Central America is describes in en AIDS patient from Costa Rica. Electronic microscopy studies indicate that the spores were not included in a parasitophorous vacuole, but they are in direct contact with the cell cytoptasm. Sporogonic proliferative plasmodial forms presence and localization of the polar tubes in the anterior region of the spore, confirmed the specie Enterocytozoon bieneusi as the cause of this microsporidian infection

Microsporidioses humanas: desenvolvimento de métodos para o diagnóstico morfológico das microsporidioses intestinais em pacientes HIV-Positivos no município do Rio de Janeiro

As infecções intestinais por Enterocytozoon bieneuse e Septata intestinalis têm sido frequêntemente diagnosticadas em pacientes com SIDA/AIDS. Entretanto a detecção de esporos de microsporídios nas fezes é particularmente difícil quando o número dessas estruturas de resistência é pequeno. A partir de um método específico para a detecção de oocistos de Cryptosporidium, desenvolveu-se neste trabalho uma modificação que permitiu a concentração de esporos de microsporídios nas fezes. Foram estudados 140 pacientes do Hospital Evandro Chagas/FIOCRUZ e 95 do Hospital Universitário Pedro Ernesto/UERJ, todos soropositivos para o vírus da imunodeficiência humana(HIV). As amostras fecais desses pacientes foram investigadas pelo método direto e pelo método de concentração, desenvolvido neste trabalho. Todas as lâminas foram coradas pelas técnicas do Chromotrope e do Gram-Chromotrope, sendo essa última coloração desenvolvida e testada durante os trabalhos experimentais. Foram obtidas prevalências++. para microsporídios de 17,86 por cento (método direto) e 25 por cento (método de concentração) no grupo de indivíduos do HEC/FIOCRUZ e de 11,58 por cento no grupo investigado do HUPE/UERJ. A microscopia eletrônica de transmissão, realizada em fragmentos de biópsia duodenal de um paciente com diagnóstico positivo pela microscopia óptica, permitiu a identificação de esporos que apresentaram ultra-estrutura caracter161stica, compatível com a espécie E. bieneusi. O número de pacientes que foi diagnosticado com microsporidiose intestinal no presente estudo mostrou que os microsporídios são importantes agentes de infecção em indivíduos portadores do vírus HIV no Município do Rio de Janeiro

Microsporidioses humanas: desenvolvimento de métodos para o diagnóstico morfológico das microsporidioses intestinais em pacientes HIV-Positivos no município do Rio de Janeiro / Microsporídiosis human: development of methods for the morphological diagnosis of intestinal microsporídiosis in HIV-positives in the municipality of Rio de Janeiro

As infecções intestinais por Enterocytozoon bieneuse e Septata intestinalis têm sido frequêntemente diagnosticadas em pacientes com SIDA/AIDS. Entretanto a detecção de esporos de microsporídios nas fezes é particularmente difícil quando o número dessas estruturas de resistência é pequeno. A partir de um método específico para a detecção de oocistos de Cryptosporidium, desenvolveu-se neste trabalho uma modificação que permitiu a concentração de esporos de microsporídios nas fezes. Foram estudados 140 pacientes do Hospital Evandro Chagas/FIOCRUZ e 95 do Hospital Universitário Pedro Ernesto/UERJ, todos soropositivos para o vírus da imunodeficiência humana(HIV). As amostras fecais desses pacientes foram investigadas pelo método direto e pelo método de concentração, desenvolvido neste trabalho. Todas as lâminas foram coradas pelas técnicas do Chromotrope e do Gram-Chromotrope, sendo essa última coloração desenvolvida e testada durante os trabalhos experimentais. Foram obtidas prevalências++. para microsporídios de 17,86 por cento (método direto) e 25 por cento (método de concentração) no grupo de indivíduos do HEC/FIOCRUZ e de 11,58 por cento no grupo investigado do HUPE/UERJ. A microscopia eletrônica de transmissão, realizada em fragmentos de biópsia duodenal de um paciente com diagnóstico positivo pela microscopia óptica, permitiu a identificação de esporos que apresentaram ultra-estrutura caracter161stica, compatível com a espécie E. bieneusi. O número de pacientes que foi diagnosticado com microsporidiose intestinal no presente estudo mostrou que os microsporídios são importantes agentes de infecção em indivíduos portadores do vírus HIV no Município do Rio de Janeiro