Microscopio and PCR-based detection of microsporidia spores in human stool samples

Abstract Microsporidia are obligate intracellular fungi with a remarkable ability to infect a wide range of invertebrate and vertebrate hosts. Namely, Enterocytozoon bieneusi is the most frequently microsporidia reported worldwide, and mainly associated with chronic diarrea and wasting syndrome in AIDS patients. Microscopy and PCR-based detection techniques are effective for diagnosis and identification of species and genotypes; however, these methods should be standardized in each laboratory. In this study, we performed microscopy and nested PCR techniques with PCR product sequencing to detect E. bieneusi in human stool samples. These techniques, if applied together, might prove useful for diagnosis and future epidemiological studies of intestinal microsporidiosis in Argentina.

Resumen Los microsporidios son hongos intracelulares obligados con una notable capacidad para infectar una amplia gama de hospedadores invertebrados y vertebrados. Enterocytozoon bieneusi es el microsporidio más frecuentemente reportado en todo el mundo, principalmente tricrómicaasociado con diarrea crónica y síndrome debilitante en pacientes con sida. Las técnicas dedetección basadas en microscopía y PCR son útiles para el diagnóstico y la identificación deespecies y genotipos, pero estos métodos deben estar estandarizados en cada laboratorio.En este estudio evaluamos técnicas de microscopía y PCR anidada, con secuenciación de losproductos, para detectar E. bieneusi en muestras de heces humanas. Estas técnicas, usadas con-juntamente, podrían ser útiles para su aplicación en el diagnóstico de microsporidiosis intestinaly para realizar estudios epidemiológicos de esta afección en Argentina.

Disseminated microsporidiosis: An underdiagnosed and emerging opportunistic disease

@#Disseminated microsporidiosis is a life-threatening disease resulting from the haematogenous spread of microsporidia species. The diagnosis is challenging owing to its subtle nonspecific clinical presentation, which usually reflects the underlying organ involved. Therefore, a high index of suspicion is required for early diagnosis. Besides, tools for confirmatory laboratory diagnosis are limited. Currently, there is no direct diagnostic method that can detect the infection without involving invasive procedures. Clinical confirmation of disseminated microsporidiosis is usually based on light and transmission electron microscopy of infected tissue specimens. These are then followed by species detection using polymerase chain reaction (PCR). Disseminated microsporidiosis shows the potential to be cleared up by albendazole or fumagillin if they are detected and treated early. Based on a series of case reports, this review aims to present a current update on disseminated microsporidiosis with emphasis on the clinical manifestations based on the organ system infected, diagnostic approach and treatment of this devastating condition. Keywords:

Microsporidiosis in Iran: A systematic review and meta-analysis

Objective To examine all evidence about Microsporidia infection in vertebrate/invertebrate hosts and Iranian populations distributed in different regions of the country. Methods All published articles up to December 2015, including descriptive and cross-sectional studies related to the prevalence and genotyping of Microsporidia infection in Iran, was considered in this systematic review. The meta-analysis was done using the random-effects model and Stats Direct statistical software. MEGA 5.05 software and maximum likelihood algorithm with Kimura 2-parameter model were used for phylogenetic analysis. Results Of the 1152 investigated studies, 33 eligible studies reported a prevalence of Microsporidia infection in vertebrate and invertebrate hosts. According to this systematic review, the overall prevalence rate of Microsporidia infection in immunocompromised patients in Iran was 8.18%. Furthermore, the overall prevalence rate of Microsporidia infection in immunocompromised patients with chronic diarrhoea, patients with non-diarrhoea, gastroenteritis, and patients with CD4 (<200 cells/μL) was 15.4%, 4.1%, 0.5%, and 12.9% respectively. The highest prevalence rate of human and animal Microsporidia was estimated in Kerman (29%) and Khuzestan (26.5%). The overall prevalence rate of Microsporidia infection in honeybees using the random-effects model was 40%. Furthermore, the highest prevalence rate of nosemosis was described in East Azerbaijan (48.2%). The most Microsporidia isolates from immunocompromised patients and pigeons in Iran belonged to genotypes D (n = 16; 50%) and E (n = 6; 20.6%) of Enterocytozoon bieneusi. Conclusions This study may be the first systematic review and meta-analysis that provides a broad outlook on the prevalence of microsporidiosis in Iran. It is necessary to investigate Microsporidia infection in vertebrate and invertebrate hosts and environmental resources in Iran.

Microsporidiosis in Iran: A systematic review and meta-analysis

OBJECTIVE@#To examine all evidence about Microsporidia infection in vertebrate/invertebrate hosts and Iranian populations distributed in different regions of the country.@*METHODS@#All published articles up to December 2015, including descriptive and cross-sectional studies related to the prevalence and genotyping of Microsporidia infection in Iran, was considered in this systematic review. The meta-analysis was done using the random-effects model and Stats Direct statistical software. MEGA 5.05 software and maximum likelihood algorithm with Kimura 2-parameter model were used for phylogenetic analysis.@*RESULTS@#Of the 1152 investigated studies, 33 eligible studies reported a prevalence of Microsporidia infection in vertebrate and invertebrate hosts. According to this systematic review, the overall prevalence rate of Microsporidia infection in immunocompromised patients in Iran was 8.18%. Furthermore, the overall prevalence rate of Microsporidia infection in immunocompromised patients with chronic diarrhoea, patients with non-diarrhoea, gastroenteritis, and patients with CD4 (<200 cells/μL) was 15.4%, 4.1%, 0.5%, and 12.9% respectively. The highest prevalence rate of human and animal Microsporidia was estimated in Kerman (29%) and Khuzestan (26.5%). The overall prevalence rate of Microsporidia infection in honeybees using the random-effects model was 40%. Furthermore, the highest prevalence rate of nosemosis was described in East Azerbaijan (48.2%). The most Microsporidia isolates from immunocompromised patients and pigeons in Iran belonged to genotypes D (n = 16; 50%) and E (n = 6; 20.6%) of Enterocytozoon bieneusi.@*CONCLUSIONS@#This study may be the first systematic review and meta-analysis that provides a broad outlook on the prevalence of microsporidiosis in Iran. It is necessary to investigate Microsporidia infection in vertebrate and invertebrate hosts and environmental resources in Iran.

Human Microsporidiosis In Malaysia: Review Of Literatures

The purpose of this study is to review the literature on microsporidiosis in various high-risk groups among the Malaysian population, i.e., HIV/AIDS, cancer, hospitalised patients and Orang Asli, and to update information with regards to microsporidia prevalence, diagnosis and association of the disease with gastrointestinal symptoms in Malaysia. Hospitalised patients showed the highest prevalence (28.3%) of microsporidiosis compared to other risk groups. This review did not find any direct correlations between gastrointestinal symptoms and microsporidiosis. Since microsporidiosis is an emerging threat to the high-risk groups, greater awareness should be instilled among clinicians to consider microsporidiosis in their differential diagnosis if no other causes can be defined.

Prevalencia de microsporidiosis intestinal en niños con desnutrición severa de un hospital de la ciudad de Maracaibo / Intestinal Microsporidiosis Prevalence in Children With Severe Malnutrition at a Hospital in the City of Maracaibo

Con la finalidad de comparar la prevalencia de Microsporidiosis intestinal en niños con desnutrición severa y niños eutróficos de la Unidad de Recuperación Nutricional del Hospital Chiquinquirá de Maracaibo, Estado Zulia, se realizó la técnica de reacción en cadena de la polimerasa (PCR) para la identificación de Encephalitozoon intestinalis y Enterocytozoon bieneusi en las muestras de heces de 50 niños desnutridos graves y 50 niños eutróficos, cuyas edades fueron estratificadas de la siguiente manera: lactantes menores (0-11 meses), lactantes mayores (12-23 meses), preescolares (2-6 años), y escolares (7 a 12 años). De las especies de microsporidios investigadas, se evidenció Enterocytozoon bieneusi en el 14% de los niños desnutridos graves y 8% en los niños eutróficos, no se obtuvieron amplificaciones para Encephalitozoon intestinalis en los grupos estudiados; al aplicar la prueba estadística X², resultó no significativa para las variables microsporidiosis intestinal y desnutrición. Se concluye que la medida en la que se relacionan la desnutrición, y la microsporidiosis es difícil de esclarecer, ya que la desnutrición es una condición multifactorial y aún no está bien establecido cuál de estos dos factores es la causa y cual la consecuencia.

In order to compare the prevalence of intestinal microsporidiosis in children with severe malnutrition and eutrophic children at the Nutritional Recovery Unit, Chiquinquirá Hospital in Maracaibo, State of Zulia, a chain reaction technique was performed on the polymerase (PCR) to identify Encephalitozoon intestinalis and Enterocytozoon bieneusi in stool samples from 50 healthy and 50 severely malnourished children, whose ages were stratified as follows: younger infants (0-11 months), older infants (12-23 months), preschool (2-6 years) and school children (7-12 years). Of the microsporidia species investigated, Enterocytozoon bieneusi was evident in 14% of severely malnourished children and 8% of the eutrophic children. No amplifications for Encephalitozoon intestinalis were obtained in the groups studied. On applying the chi-square statistical test, the result was not significant for the variables intestinal microsporidiosis and malnutrition. Conclusions are that the extent to which malnutrition and microsporidiosis relate is difficult to clarify, because malnutrition is a multifactorial condition, and it is not yet well established which of these two factors is the cause and which is the consequence.

Microsporidiosis gastrointestinal: una actualización / Gastrointestinal microsporidiosis: an update

La microsporidiosis es una infección emergente y oportunista producida por microorganismos intracelulares obligados formadores de esporas que han sido taxonómicamente reclasificados como hongos. La eficacia y seguridad de diferentes productos farmacéuticos ha sido evaluada a lo largo de los años, sin embargo, es limitado el arsenal terapéutico del que se dispone a la hora de tratar individuos infectados por esos microorganismos. La actualización necesaria en relación con esta temática, así como la necesidad de un documento de consulta para los estudiantes de pre y post graduación determinó la realización de este trabajo, con el que se pretende ofrecer información actual, pertinente y de calidad que permita una mejor atención a los usuarios del sector salud y mantener el proceso de educación continuada de los profesionales en todos los niveles de atención.

The microsporidiosis is an emergent and opportunist infection produced by spore-forming intracellular microorganisms, recently reclassified by taxonomists as fungi. The efficacy and safety of different pharmaceutical products has been evaluated along the years, however, the therapeutic arsenal is limited when treating infected people. The necessary update on this theme, and the necessity of a consultation document for pre and post graduate students determined the accomplishing of this review, pretending to offer the updated, pertinent and qualitative information that may allows a better medical care to the users of the health sector and maintaining the continued educational process of the professionals at all the public health care levels.

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.

Experiencia terapéutica con nitazoxanida en diarrea crónica por microsporidios y criptosporidios en pacientes inmunocomprometidos oncológicos / Therapeutic experience with nitazoxanide in chronic diarrhea for microsporidia and cryptosporidia in immunocompromised cancer patients

Informar la experiencia de erradicación de Cryptosporidium y Microsporidium spp., en pacientes inmunocomprometidos con diarrea crónica utilizando nitazoxanida como terapia. Se evaluaron 50 pacientes con diarrea crónica (38 mujeres y 12 hombres), edad promedio de 45 y 56 años respectivamente; mayores de edad, dieron su consentimiento informado por escrito; con diagnósticos de cáncer de vías digestivas (veintidos), mama (diez y seis), cuello uterino (siete) y próstata (cinco); además 6 presentaban infección por VIH. Se hizo diagnóstico parasitológico empleando la coloración de Kinyoun. Recibieron nitazoxanida 500 mg/VO/BID por períodos de 3,6,9 días dependiendo del resultado de los controles microbiologicos. Se identificó como agente único a Cryptosporidium spp. en 2 pacientes y Microsporidium sp., en 6 pacientes, y combinados en 42 pacientes. Al tercer día de tratamiento, en 30 pacientes se logró erradicación de la criptosporidiosis, persistiendo con clínica diarreica en 28 de ellos; al sexto día se evidenció cura microbiológica (erradicación parasitaria de ambos microorganismos) y clínica (cese diarrea), en un total de 40 pacientes; al noveno día de tratamiento, se logró cura clínica y microbiológica en todos los pacientes incluyéndose en este último grupo todos los pacientes con infección VIH. Se evidenció epigastralgia en 4 pacientes que recibieron 9 días de terapia. Nitazoxanida parece ser un tratamieto eficaz y seguro en diarrea crónica micro y/o criptosporidiana en pacientes inmunocomprometidos oncológicos y es necesario realizar pesquisa microbiológica de parasitosis emergente y prolongación del tratamiento más allá de losa 3 días convencionalmente recomendados.

Clinical and pathological characteristics of microsporidian encephalitis / 中华神经科杂志

Objective To study the clinical and pathological manifestations of microsporidian encephalitis.Methods The clinical findings and the brain pathological features of a patient with microsporidian encephalitis hospitalized in 2004 were studied.Results The onset was subacute or chronic. The body temperature was usually normal or below 37.5℃,but it rose when patient's condition deteriorated and coma appeaxed.The patient had hypoimmunity but without human immunodeficiency virus infection. Multifocal lesions in the whole brain,signs of meningeal irritation and infective myelogram were observed. Rheumatoid factor increased in the early stage and indirect bilirubin,proteins in cerebrospinal fluid(CSF), and immunoglobulin IgG,IgA increased in the middle stage.Cytological examination of CSF showed lymphocyte reaction.Blood routine test showed normal eosinophil granulocyte count.The patient was found to have pleurisy,peritonitis and cystitis.Brain magnetic resonance image(MRI)manifested plaque-like isometric T1 weight image and long T2 weight image signal in white matter of bilateral cerebral hemisphere and cerebella where FLAIR sequence showed hyperintensity.No apparent mass was identified.Contrast- enhanced MRI scan showed patchy and ring-like intensification.The neural system impairments were permanent and not improved after treatment.The pathology of brain tissue showed neuronal degeneration, karyopycnosis and Derivasculitis.The infectious agents were observed in the cytoplasm of neurons.Wister rats had muhiple organ inflammatory reaction 2 weeks after intraperitoneal inoculation of the patient's CSF and a large quantity of pathogens were found in the peritoneal lavage fluid.Conclusions The patient was PAS staining method is useful for detecting the pathogen in neurons and the rate can be raised by animal intraperitoneal cultivation

Prevalence of intestinal microsporidiosis in Human Immunodeficiency Virus-infected patients with diarrhea in major United States cities

To determine the prevalence of intestinal microsporidiosis in HIV-infected patients, we performed a prospective study of HIV-infected patients with diarrheal illnesses in three US hospitals and examined an observational database of HIV-infected patients in 10 US cities. Among 737 specimens from the three hospitals, results were positive for 11 (prevalence 1.5 percent); seven (64 percent) acquired HIV through male-to-male sexual contact, two (18 percent) through male-to-male sexual contact and injection drug use, and one (9 percent) through heterosexual contact; one (9 percent) had an undetermined mode of transmission. Median CD4 count within six months of diagnosis of microsporidiosis was 33 cells/µL (range 3 to 319 cells/µL). For the national observational database (n = 24,098), the overall prevalence of microsporidiosis was 0.16 percent. Prevalence of microsporidiosis among HIV-infected patients with diarrheal disease is low, and microsporidiosis is most often diagnosed in patients with very low CD4+ cell counts. Testing for microsporidia appears to be indicated, especially for patients with very low CD4+ cell counts.

Para determinar a prevalência de microsporidiose intestinal em pacientes infectados pelo HIV foi realizado um estudo prospectivo em três hospitais dos Estados Unidos da América do Norte (EUA) e analizada uma base de dados nacional composta de dados coletados de pacientes infectados pelo HIV em 10 cidades dos EUA. De um total de 737 amostras de fezes de pacientes infectados pelo HIV que apresentavam diarréia, amostras de 11 pacientes (prevalência de 1,5 por cento) foram positivas para microsporídios. Todos os positivos eram do sexo masculino e, entre eles, sete (64 por cento) pacientes adquiriram a infecção pelo HIV através de relação homossexual, dois (18 por cento) através de relação sexual e drogas injetáveis e um (9 por cento) através de contato heterosexual, enquanto que em um paciente o modo de transmissão do HIV não foi determinado. A contagem média de linfócitos CD4 realizada até seis meses do diagnóstico de microsporidiose foi de 33 células/microlitro (3 a 319 células/microlitro). A análise da base de dados nacional (n = 24.098) mostrou uma prevalência de microsporidiose de 0,16 por cento. A prevalência de microsporidiose em pacientes HIV-positivos com diarréia é baixa. Entretando, como a microsporidiose é mais frequentemente diagnosticada em pacientes com contagens de CD4 muito baixas, a indicação de pesquisa de microsporídios é justificada, especialmente para estes pacientes.

Primer diagnóstico de microsporidiosis humana en Uruguay / First diagnosis of microsporidiosis human in Uruguay

Los microsporidios son protozoarios conocidos desde hace mucho tiempo como causantes de plagas en invertebrados y vertebrados. En la década de 1980 se les reconoce como agentes etiológicos de diarrea en VIH-sida y posteriormente en trasplantados renales así como en personas inmunocompetentes (diarrea del viajero). El objetivo de este trabajo es comunicar el primer hallazgo de estos agentes en un paciente VIH-sida y contribuir al conocimiento de las características patogénicas y diagnósticas más importantes de los microsporidios. En 1998 en el Departamento de Parasitología se implementaron las técnicas de coloración para microsporidios, gram-cromotrope y tricrómica. El primer hallazgo se observó en un paciente VIH-sida, con diarrea de nueve meses de evolución; no se realizó tratamiento específico ya que concomitantemente presentaba una meningoencefalitis criptococócica, la que precipitó su fallecimiento. El reconocimiento de estos agentes oportunistas como causa de diarrea en Uruguay, contribuye al diagnóstico etiológico de la diarrea crónica en pacientes VIH-sida. En adelante, las coloraciones específicas de microsporidios deberían ser incluidas en el algoritmo diagnóstico para enteroparasitosis en grupos de riesgo.

Summary Microsporidia are protozoan parasites found in vertebrates and invertebrates. In the 80's they were recognized as a cause of diarrhea in HIV-AIDS patients and lately in patients with renal transplantation and immunocompetent patients (traveler's diarrhea). The objective of this paper is to communicate the first founding of this agent in a patient with HIV-AIDS and to contribute to determine the most important pathogenic and diagnostic characteristics of microsporidia. In 1998 the Parasitoloy Departament implemented colouring techniques for microsporidia, gram-chromotrope and trichrome. The first founding was observed in a HIV-AIDS patient with a 9 month-course of diarrhea; specific treatment was not performed since the patient carried a concomitant cryptococcal meningoencephalitis, that provoked his death. Recognition of theses oportunist agents as a cause of diarrhea in Uruguay contribute to determine the aetiologic diagnosis of chronic diarrhea in patients with HIV-AIDS. Specific colouring techniques should be incorporated in diagnosis algorithm for enteroparasitosis in risk groups.

Résumé Les microsporidiens ce sont des protozoaires connus depuis longtemps en tant que responsables de certains fléaux chez les vertébrés et les invertébrés. Aux années 80, on les reconnaît comme agents étiologiques de diarrhée en VIH-sida et quelque temps après, chez des greffés rénaux ainsi que chez des personnes immunocompétentes (diarrhée du voyageur). Le but de ce travail est de communiquer la première trouvaille de ces agents chez un patient VIH-sida ainsi que de contribuer à la connaissance des caractéristiques pathogéniques et diagnostiques les plus importantes de ces protozoaires. En 1998, au Département de Parasitologie, on a utilisé les techniques de coloration pour microsporidiens, gram-chromotrope et thrichomona. La première observation a été faite chez un patient VIH-sida, avec diarrhée de neuf mois d'évolution; aucun traitement spécifique n'a été fait puisqu'il présentait une méningo-encéphalite cryptococcose qui a accéléré sa mort. Le repérage de ces agents opportunistes responsables de diarrhée en Uruguay, contribue au diagnostique étiolo-gique de la diarrhée chronique chez des patients VIH-sida. Désormais, les colorations spécifiques de miscrospori-diens devarient être inclues parmi les techniques diagnostiques pour entéro-parasitoses dans des groupes de risque.

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

Diagnosis and Treatment of Major Protozoal Infections among Acquired Immune Deficiency Syndrome Patients / 中国寄生虫学与寄生虫病杂志

Protozoal infection is one of the most important opportunistic infections among patients with acquired immune deficiency syndrome(AIDS). In order to enhance the knowledge of protozoal infections in AIDS, the current status of diagnosis and treatment of toxoplasmic encephalitis, cryptosporidiosis, microsporidiosis and isosporiasis was reviewed in this paper.