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.

Occurrence of zoonotic Enterocytozoon bieneusi in cats in Brazil / Ocorrência de Enterocytozoon bieneusi zoonótico em gatos no Brasil

Abstract Enterocytozoon bieneusi is an opportunistic intestinal pathogen that infects humans and a wide variety of animals worldwide. Our aim in this study was to investigate the occurrence of E. bieneusi in a domestic cat population in Campo Grande, Mato Grosso do Sul, Brazil. Sixty fecal samples from diarrheic cats were subjected to polymerase chain reaction (PCR) and the amplicons were sequenced for identification. E. bieneusi was detected in two samples (3.3%), both identified as genotype D. This genotype has already been reported in animals and humans and is considered a zoonotic genotype. Our findings represent the first report of E. bieneusi in domestic cats in Brazil, reinforcing the importance of identifying this agent as a source of infection in animals and humans.

Resumo Enterocytozoon bieneusi é um patógeno intestinal oportunista que infecta humanos e uma variedade de animais em todo o mundo. O objetivo no presente estudo foi investigar a ocorrência de E. bieneusi em uma população de gatos domésticos em Campo Grande, Mato Grosso do Sul, Brasil. Sessenta amostras fecais de gatos diarréicos foram submetidas a reação em cadeia da polimerase (PCR) e os produtos de amplificação foram sequenciados para identificação molecular. E. bieneusi foi detectado em duas amostras (3,3%), ambos identificados como genótipo D. Esse genótipo tem sido relatado em animais e humanos e é considerado um genótipo zoonótico. Nossos resultados representam a primeira descrição de E. bieneusi em gatos domésticos no Brasil, reforçando a importância desse agente como fonte de infecção para animais e humanos.

Enterocytozoon bieneusi detected by molecular methods in raw sewage and treated effluent from a combined system in Brazil

BACKGROUND Enterocytozoon bieneusi are the most common microsporidia associated with different clinical manifestations such as diarrhoea, respiratory tract inflammation and acalculous cholecystitis, especially in immunocompromised patients. Infection usually occurs by ingestion of food and water contaminated with spores, but can also result from direct contact with spores through broken skin, eye lesions, and sexual transmission, depending on the microsporidian species. Although there are reports of E. bieneusi found in humans and animals in Brazil, there are no published studies of environmental samples examined by molecular methods. OBJECTIVES The purpose of this study was to verify the presence of E. bieneusi in raw sewage and treated effluent from a combined system by molecular methods. METHODS Raw sewage and treated effluent samples collected from a combined system were analysed for the presence of E. bieneusi using the internal transcriber spacer (ITS) region of E. bieneusi by nested polymerase chain reaction. FINDINGS The analysis revealed E. bieneusi presence and a novel genotype (EbRB) in one raw sewage sample and one treated effluent. MAIN CONCLUSIONS The presence of E. bieneusi in final effluent indicates that the combined system may not remove microsporidian spores. This study is the first report of E. bieneusi in environmental samples in Brazil.

Molecular Characterization of Enterocytozoon bieneusi in Domestic Rabbits (Oryctolagus cuniculus) in Northeastern China

A study of 426 rabbits from 3 cities in Jilin province (Changchun City and Jilin City) and Liaoning province (Shenyang City) was conducted between May and June 2015. The overall prevalence of E. bieneusi in rabbits was 0.94% (4/426), with 0% (0/116), 1.72% (3/174), and 0.74% (1/136) in Jilin, Changchun, and Shenyang City, respectively. Only 3 farms (farm 1 and farm 3 in Changchun City, farm 8 in Shenyang City) were PCR-positive for E. bieneusi. Moreover, rabbits of more than 6 months (1.72%) had the highest E. bieneusi prevalence, followed by rabbits of 4-6 months (1.26%), 2-3 months (0.58%), and less than 1 month (0%). Analysis of ITS gene of E. bieneusi suggested that all 4 E. bieneusi isolates were genotype D, and were classified as group 1a. The present results first demonstrated the existence of zoonotic E. bieneusi in domestic rabbits in China. Effective control measures should be implemented to prevent E. bieneusi infection in domestic rabbits, other animals, and humans.

Detection of Enterocytozoon bieneusi (Microsporidia) by polymerase chain reaction (PCR) using species-specific primer in stool samples of HIV patients.

BACKGROUND & OBJECTIVE: Microsporidia are obligate intracellular parasites causing infections predominantly in immunocompromised patients. Enterocytozoon bieneusi is the most important microsporidian causing chronic diarrhoea in AIDS patients. The current method used for diagnosing the microsporidia spores is based on light microscopy using stained smears, which do not differentiate spores at species level. The present study was undertaken to detect microsporidia and confirm at species level (E. bieneusi) by PCR from stool samples of HIV positive patients. METHODS: During September 2002 to April 2003, stool samples from 153 HIV-positive patients (with chronic diarrhoea n = 105; without diarrhoea n=48) were collected and examined microscopically for microsporidia spores using modified Weber's chromotrope stain. Stool samples were subjected to PCR assay using species-specific primer EBIEFI/EBIER1, which amplifies small subunit ribosomal RNA (SSU rRNA) of this microsporidian RESULTS: A total of 10 HIV positive patients with chronic diarrhoea were positive for microsporidia by microscopic analysis and confirmed as Enterocytozoon bieneusi by PCR. No false positive results were observed. A diagnostic DNA fragment of 607 bp of the unique SSU rRNA was amplified from all samples infected with E. bieneusi. INTERPRETATION & CONCLUSION: The study revealed that polymerase chain reaction is a useful tool for accurate species identification of microsporidia in stool samples, which serves the benefit of treatment to the patients.