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.

First report of Dientamoeba fragilis infection explaining acute non-specific abdominal pain.

Dientamoeba fragilis is now considered a potentially emerging gastrointestinal pathogen in both developing and developed countries. We first report an autochthonous case of D. fragilis infection in Greece. A 49‑year‑old female with acute non‑specific abdominal pain required emergency surgical admission for active observation and repeated assessment. To the best of our knowledge, this is the first reported case of acute unexplained abdominal pain finally attributed to D. fragilis infection using microscopic and molecular methods.

Detection of Dientamoeba fragilis in patients with HIV/AIDS by using a simplified iron hematoxylin technique / Detecção de Dientamoeba fragilis em pacientes com HIV/AIDS utilizando a técnica de hematoxilina férrica simplificada

INTRODUCTION: Studies strongly indicate Dientamoeba fragilis as one of the causes of diarrhea in human immunodeficiency virus (HIV) patients. METHODS: The objective of the present study was to evaluate the prevalence of D. fragilis associated with the causes of diarrhea in 82 HIV/ AIDS patients hospitalized at the Instituto de Infectologia Emílio Ribas from September 2006 to November 2008. RESULTS: In total, 105 samples were collected from 82 patients. Unprotected sex was the most frequent cause of HIV infection (46.3%), followed by the use of injectable or non-injectable drugs (14.6%). Patients presented with viral loads of 49-750,000 copies/ mL (average: 73,849 ± 124,850 copies/mL) and CD4 counts ranging of 2-1,306 cells/mm³ (average: 159 ± 250 cells/mm³). On an average, the odds of obtaining a positive result by using the other techniques (Hoffman, Pons and Janer or Lutz; Ritchie) were 2.7 times higher than the chance of obtaining a positive result by using the simplified iron hematoxylin method. Significant differences were found between the methods (p = 0.003). CONCLUSIONS: The other techniques can detect a significantly greater amount of parasites than the simplified iron hematoxylin method, especially with respect to Isospora belli, Cryptosporidium sp., Schistosoma mansoni, and Strongyloides stercoralis, which were not detected using hematoxylin. Endolimax nana and D. fragilis were detected more frequently on using hematoxylin, and the only parasite not found by the other methods was D. fragilis.

INTRODUÇÃO: Estudos indicam a Dientamoeba fragilis como uma das causas de diarréia em pacientes com HIV/AIDS. MÉTODOS: Os objetivos deste estudo foram avaliar a prevalência de D. fragilis associadas com as causas de diarréia em pacientes com HIV/AIDS internados no Instituto de Infectologia Emílio Ribas (IIER). Oitenta e dois pacientes internados no IIER fizeram parte deste estudo de setembro de 2006 a novembro de 2008. RESULTADOS: No total, 105 amostras foram coletadas a partir de 82 pacientes neste estudo. Sexo desprotegido foi à causa mais frequente para a aquisição do HIV (46,3%), seguido pelo uso de drogas injetáveis ou não injetáveis (14,6%). Relações heterossexuais foram os mais citados (19,5%). Pacientes apresentaram carga viral entre 49 e 750.000 (média de 7.849 ± 124.850) e CD4 variando de 2 a 1.306 (média de 159 ± 250). Em média, as chances de um resultado ser positivo com outras técnicas foram 2,7 vezes maiores do que a chance de um resultado positivo com hematoxilina férrica simplificada. Foram encontradas diferenças significativas entre os métodos (p=0,003). CONCLUSÕES: As outras técnicas são capazes de detectar uma quantidade significativa maior de parasitas em comparação com a hematoxilina férrica simplificada, especialmente em relação à Isospora belli, Cryptosporidium sp., Schistossoma mansoni e Strongyloides stercoralis que não foram encontrados utilizando a hematoxilina e a Endolimax nana e D. fragilis foram mais detectados pela hematoxilina férrica simplificada, principalmente a D. fragilis que não foi detectada pelos outros métodos.

Manifestaciones clínicas y diagnóstico parasitológico de la infección intestinal causada por dientamoeba Fragilis

Dientamoeba fragilis (Df), un flagelado intestinal humano del orden Trichomonadida, ha sido asociado con síntomas gastrointestinales. El diagnóstico se hace por la observación de trofozoítos binucleados en las heces, en cultivo, o con biología molecular. En este trabajo se integran datos clínicos y parasitológicos de pacientes con Df, para establecer una relación con los síntomas, asociación con otros parásitos intestinales y evaluar los métodos de diagnóstico parasitológico. De 3729 pacientes evaluados entre 1974 y 2005, en el Laboratorio de Amibiasis, Cátedra de Parasitología, Facultad de Medicina de la Universidad Central de Venezuela, se encontró 51 casos (1,4%) con Df, en 33,3% como único agente y en 66,7% asociado con otros protozoarios. La asociación mas frecuente fue con Blastocystis hominis (Bh) (35,3 %), o Bh y otros protozoarios (31,4%). No se observó asociación con nemátodos intestinales. En 16 pacientes con Df sola y en 28 asociada con Bh y comensales, los síntomas más frecuentes fueron respectivamente: diarrea (64,7% y 32,1%), dolor abdominal (11,7% y 25%), vómitos (11,7% y 14,4%). La frecuencia de Df en el grupo etario entre 1-10 años fue 39,2% y resultó estadísticamente significativa (p<0,001). El examen seriado de heces con directo, coloración de Hematoxilina férrica (Hf) y cultivo en medio de Boeck- Drbohlav (modificado) detectó mayor número de casos (58,8%), siendo estadísticamente significativo (p=0,032) al comparar los casos detectados con el directo y Hf (41,2%). Se recomienda considerar a Df como un patógeno intestinal cuando se encuentra en personas con síntomas y en ausencia de otros patógenos conocidos, e investigarla con los métodos recomendados, especialmente con el cultivo que incrementará la posibilidad del hallazgo.

Dientamoeba fragilis (Df), a human intestinal flagellated of the Trichomonadida order, has been associated with gastrointestinal symptoms. The diagnosis is made by observation of binucleated trophozoites in faeces, culture, or by using molecular biology. In this work, clinical and parasitological data of patients with Df are integrated, to establish a relation with the symptoms, the association with other intestinal parasites and to evaluate the methods used for parasitological diagnosis. Of 3729 patients evaluated between 1974 and 2005, in Laboratorio de Amibiasis, Cátedra de Parasitología, Facultad de Medicina, Universidad Central de Venezuela, 51 cases were positive for Df (1.4%); of these, Df was the only agent in 33.3% and in 66.7% it was associated with other protozoa. The most frequent association found was with Blastocystis hominis (Bh) (35.3%), or Bh and other protozoa (31.4%). No intestinal nematodes were found. In 16 patients with Df alone and 28 patients with Df, Bh and comensals, the most frequent symptoms were, diarrhea (64.7% and 32.1%), abdominal pain (11.7% and 25%), vomits (11.7% and 14.4%) respectively. There was statistical significance (p<0.001) on the frequency of Df (39.2%) in the group between 1-10 years. The examination of more than one faecal sample using fresh unpreserved stools samples, ferric hematoxylin stain and culture on Boeck- Drbohlav modified medium detected a greater number of cases (58.8%), as compared when using the two first methods only (41.2%), being statistically significant (p=0.032). It is recommended to consider Df as a pathogen when found in people with intestinal symptoms, in absence of other known pathogens, and to follow the methods mentioned above, specially with the culture which will increase the possibility of recovering this protozoa.