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.

Molecular detection and genetic diversity of Dientamoeba fragilis and Enterocytozoon bieneusi in fecal samples submitted for routine parasitological examination

Objective: To detect Dientamoeba (D.) fragilis and Enterocytozoon (E.) bieneusi and to assess their genetic characteristics in stool samples submitted for routine examination in a clinical laboratory in Southeastern Brazil. Methods: In this survey, 348 stool samples from female and male individuals with age ranging from 0 to ≥55 years were analyzed by PCR amplifying and sequencing based on the small subunit ribosomal RNA (SSU rRNA) gene of D. fragilis and the internal transcribed spacer of E. bieneusi. Results: D. fragilis and E. bieneusi isolates were observed in 2.29% (8/348) and 4.59% (16/348) of the samples, respectively. These parasites were detected in stool samples from individuals of both genders, including young children under nine until adults over 55 years old. No statistically significant differences were found. All D. fragilis isolates were classified as genotype 1 and E. bieneusi isolates included genotypes D (n=15) and A (n=1). Conclusions: The findings provide relevant findings on occurrence and genetic diversity of D. fragilis and E. bieneusi, pointing to the need for the diagnosis of these parasites in routine examinations in clinical laboratories. In addition to sensitive diagnostic methods, it is mandatory that these parasites be considered relevant for physicians and laboratory staff.

Clinical effectiveness of live preparation of lactobacillus in treatment of bacterial vaginosis in pregnancy: A meta-analysis

Objective: To evaluate the clinical effectiveness of live preparation of lactobacillus in treatment of bacterial vaginosis in pregnancy. Methods: Randomized controlled trials of live preparation of lactobacillus in the treatment of bacterial vaginosis in pregnancy were collect by searching PubMed, Web of Science, OVID, CNKI, WanFang, VIP, CBM and Elsevier databases. Quality of the included trials were evaluated by two researchers independently, and data were extracted according to Cochrane systematic evaluation. RevMan 5.3 software was used for meta-analysis. Results: Twenty-one randomized controlled trials involving 2 930 patients were included, which showed that there was significant difference in the clinical effectiveness between vaginal medication of live preparation of lactobacillus and vaginal medication of metronidazole [total effective rate (RR=1.05, 95% CI: 1.02-1.07, P=0.000 4]; significant differences were found in premature delivery rate (RR=0.49, 95% CI: 0.32-0.73, P=0.000 4), premature rupture of membrane rate (RR=0.54, 95% CI: 0.38-0.77, P=0.000 7), infant of low-birth weight rate (RR=0.45, 95% CI: 0.22-0.94, P=0.03), puerperal infection rate (RR=0.60, 95% CI: 0.39-0.94, P=0.03) between the two groups. Conclusions: Vaginal medication of live preparation of lactobacillus was more clinically effective than vaginal medication of metronidazole for bacterial vaginosis in pregnancy. Live preparation of lactobacillus is associated with a lower premature delivery rate, a lower premature rupture of membrane rate, a lower low-birth weight rate and a lower puerperal infection rate.

Occurrence of Cryptosporidium, Enterocytozoon bieneusi and Blastocystis genotypes in Rousettus leschenaultii collected from an urban public park of Hainan Province, China

Objective: To obtain information about the occurrence of Blastocystis spp., Cryptosporidium spp., and Enterocytozoon (E.) bieneusi in fruit bats (Rousettus leschenaultii) collected from an urban public park Hainan Province, China and to analyze the genetic characteristics of the obtained parasites carried by those bats. Methods: On 4th June 2019, ten piles of fresh faecal sample of fruit bats were collected from the Wanlvyuan Gardens in central Haikou, Hainan of China. Blastocystis spp., Cryptosporidium spp., and E. bieneusi were examined by sequencing analysis of the small subunit rRNA gene or internal transcribed spacer (ITS) gene. Results: Among the 10 DNA specimens analyzed, seven (70.0%) were positive for Cryptosporidium spp. and two (20.0%) were positive for E. bieneusi but none of them were positive for Blastocystis. For Cryptosporidium, two novel genotypes were identified which shared 98.2% and 94.4% homology with Cryptosporidium (C.) andersoni Type C, respectively, and were named as C. andersoni Type D (in 6 specimens) and E (in one specimens). The two E. bieneusi-positive isolates were identified as a known zoonotic genotype (PigEbITS7) and a novel genotype (named HNB-I) respectively. Conclusions: The finding of C. andersoni and E. bieneusi genotype PigEbITS7 in fruit bats in Hainan, China suggests that these parasites carried by fruit bats can be transmitted to other animals and humans to cause zoonotic infections.

Prevalence and genotyping of Enterocytoon bieneusi in farmed black goats from Hainan Province, the southernmost part of China

Objective: To investigate the infection rate and genotype distribution of Enterocytoon (E.) bieneusi in farmed black goats from the Hainan Province, China. Methods: A total of 341 fresh fecal samples were collected from black goats farmed in five different locations of the Hainan Province, China. E. bieneusi was examined and genotyped through PCR and sequencing analysis of the internal transcribed spacer (ITS) region of this pathogen. Results: The average prevalence of E. bieneusi in black goats from from the five locations was 24.0% (82/341) ranging from 6.3% (4/63) to 37.2% (32/86) (χ2 =17.252, P<0.01). The detected 82 E. bieneusi isolates belonged to eight ITS genotypes including six known genotypes (AHG1, CHG2, CHG3, CHG5, CM21 and D) and two novel genotypes (HNG-Iand HNG-II). Amongst the genotypes, CHG5 was the the most prevalent with a prevalence of 57.3% (47/82), followed by CHG3 (28.0%, 23/82), CHG2 (4.9%, 4/82), CM21 (3.7%, 3/82), D (2.4%, 2/82), AHG1 (1.2%, 1/82), HNG-I(1.2%, 1/82) and HNG-II(1.2%, 1/82). In those genotypes, only genotype D was found in humans previously. Conclusions: This represents the first report identifying E. bieneusi in black goats from Hainan Province of China. The results indicate that E. bieneusi has a high prevalence and a wide distribution in those animals from Hainan Province, but the risk of zoonotic transmission of E. bieneusi from them to human is low.

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.

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.

Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi in Captive Non-Human Primates in Qinling Mountains

Non-human primates (NHPs) are confirmed as reservoirs of Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi. In this study, 197 fresh fecal samples from 8 NHP species in Qinling Mountains, northwestern China, were collected and examined using multilocus sequence typing (MLST) method. The results showed that 35 (17.8%) samples were positive for tested parasites, including Cryptosporidium spp. (3.0%), G. intestinalis (2.0%), and E. bieneusi (12.7%). Cryptosporidium spp. were detected in 6 fecal samples of Macaca mulatta, and were identified as C. parvum (n=1) and C. andersoni (n=5). Subtyping analysis showed Cryptosporidium spp. belonged to the C. andersoni MLST subtype (A4, A4, A4, and A1) and C. parvum 60 kDa glycoprotein (gp60) subtype IId A15G2R1. G. intestinalis assemblage E was detected in 3 M. mulatta and 1 Saimiri sciureus. Intra-variations were observed at the triose phosphate isomerase (tpi), beta giardin (bg), and glutamate dehydrogenase (gdh) loci, with 3, 1, and 2 new subtypes found in respective locus. E. bieneusi was found in Cercopithecus neglectus (25.0%), Papio hamadrayas (16.7%), M. mulatta (16.3%), S. sciureus (10%), and Rhinopithecus roxellana (9.5%), with 5 ribosomal internal transcribed spacer (ITS) genotypes: 2 known genotypes (D and BEB6) and 3 novel genotypes (MH, XH, and BSH). These findings indicated the presence of zoonotic potential of Cryptosporidium spp. and E. bieneusi in NHPs in Qinling Mountains. This is the first report of C. andersoni in NHPs. The present study provided basic information for control of cryptosporidiosis, giardiasis, and microsporidiosis in human and animals in this area.

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.

Prevalence des infections opportunistes digestives parasitaires a Kinshasa (Republique Democratique du Congo). Resultats d'une enquete preliminaire chez 50 patients au stade SIDA

En Republique Democratique du Congo (RDC); comme dans de nombreux pays africains; le sida et son cortege d'infections opportunistes sont une cause majeure de morbidite et de mortalite. A Kinshasa; on estime entre 4 et 5le taux de prevalence de sujets infectes par le VIH; soit plus de 200 000 personnes (chiffres du Programme National de Lutte contre le Sida; PNLS 2005). A ce jour; faute de personnels formes et de moyens diagnostiques adaptes; aucune enquete n'a encore ete menee sur la prevalence des parasites opportunistes digestifs dans la population des patients infectes par leVIH; prealable indispensable a la mise en place d'une politique de soin adaptee. Une enquete preliminaire a ete realisee a Paris sur 50 echantillons de selles de 50 patients malades du sida; hospitalises dans 3 hopitaux de references de Kinshasa. Onze patients (22) avaient une symptomatologie digestive avec un syndrome diarrheique. La realisation des examens specialises a mis en evidence 2 cas d'infection digestive par des parasitoses opportunistes (4); une a Cryptosporidium sp. et une a Enterocytozoon bieneusi; premier cas decrit dans la litterature en RDC

Prevalence des infections opportunistes digestives parasitaires a Kinshasa (Republique Democratique du Congo); resultats d'une enquete preliminaire chez 50 patients au stade sida

En Republique Democratique du Congo (RDC); comme dans de nombreux pays africains; le sida et son cortege d'infections opportunistes sont une cause majeure de morbidite et de mortalite. A Kinshasa; on estime entre 4 et 5le taux de prevalence de sujets infectes par le VIH; soit plus de 200 000 personnes (chiffres du Programme National de Lutte contre le Sida; PNLS 2005).A ce jour; faute de personnels formes et de moyens diagnostiques adaptes; aucune enquete n'a encore ete menee sur la prevalence des parasites opportunistes digestifs dans la population des patients infectes par leVIH; prealable indispensable a la mise en place d'une politique de soin adaptee. Une enquete preliminaire a ete realisee a Paris sur 50 echantillons de selles de 50 patients malades du sida; hospitalises dans 3 hopitaux de references de Kinshasa. Onze patients (22) avaient une symptomatologie digestive avec un syndrome diarrheique. La realisation des examens specialises a mis en evidence 2 cas d'infection digestive par des parasitoses opportunistes (4); une a Cryptosporidium sp. et une a Enterocytozoon bieneusi; premier cas decrit dans la litterature en RDC

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

Using pig biliary system, in vivo propagation of Enterocytozoon bieneusi, an AIDS-related zoonotic pathogen

A microsporidian parasite Enterocytozoon bieneusi is the most common microorganism recognized in AIDS patients, and slow scientific progress is attributed to our inability to propagate the parasite. We report upon the development of a system of propagation using the pig biliary system. The parasite spores were continuously detected in the bile samples post onset of spore shedding in the gall bladder, which suggests that this organism maintain persistent infection in the biliary system and that the hepatobiliary tree may represent a reservoir of infection. In conclusion the biliary tree is an adequate niche for the propagation of E. bieneusi. This work has also resulted in the development of a procedure of ultrasound-guided cholecystocentesis for aspirating biles. This is a simple and non-surgical procedure, and creates no signs of clinical complications in the livers and the gall bladders after dozens of separate attempts. Thus, this is a very useful and safe technique for the aspiration of bile from live animals.