Detection and genotyping of Dientamoeba fragilis from symptomatic patients: New insights from Italy into a little-known gastrointestinal protozoan.

Dientamoeba fragilis (D. fragilis) represents a common protozoan in both high and low income countries. Despite this, epidemiological data on dientamoebiasis are still limited, and it is possible that the actual prevalence rates of D. fragilis have been underestimated due to the challenges in its detection and identification. In the present study, symptomatic patients from Rome (Central Italy) were surveyed for two years to determine D. fragilis percentage of infection and genotypes. Stool samples collection was performed over 864 patients, DNA extracted, and RT-PCR performed by the SeeGene Allplex™ Gastrointestinal Parasite Panel Assays. Seventy-nine resulted positive for D. fragilis (9.1%). Co-infections were detected in 22 isolates: 21 displayed Blastocystis sp. + D. fragilis (27.8%). Based on the sequence of a central fragment of the SSU rRNA gene, only genotype 1 was identified. These findings are among the few available data regarding genetic diversity of D. fragilis in Italy. Large-scale human and animal research are required to enhance our knowledge of prevalence, host range, genetic variability and zoonotic transmission of this little-known intestinal protozoan.

No evidence of pathogenicity of Dientamoeba fragilis following detection in stools: A case-control study.

Dientamoeba fragilis is a ubiquitous intestinal parasite with detection in the stools that has become increasingly frequent following the advent of PCR as a routine screening tool. However, the pathogenicity of this parasite is still much debated. In order to assess the potentially pathogenic nature of this protozoan, a retrospective case-control study was carried out between January and December 2020 on patients from Toulouse University Hospital, with the aim of evaluating the potential clinical effects and changes in laboratory parameters linked to the presence and load of D. fragilis in stools. After matching age, sex and mode of care (consultation or hospitalisation), no significant difference was observed in the frequency of clinical signs between the 36 patients who tested positive for Dientamoeba fragilis PCR in their stools and the 72 control patients who were PCR negative for this protozoan. The presence of D. fragilis in the faeces was not associated with changes in laboratory parameters. Furthermore, a high digestive load of D. fragilis had no identifiable impact on clinical and laboratory parameters. Only the concomitant presence of Blastocystis sp. in stools was significantly more frequent in the D. fragilis group (uni- and multivariate analysis). Finally, this study showed no significant difference in clinical or laboratory signs between patients carrying Dientamoeba fragilis and the control group, regardless of the intestinal parasite load, suggesting that D. fragilis could be considered a commensal of the digestive tract.

Title: Aucune preuve de la pathogénicité de Dientamoeba fragilis détecté dans les selles : une étude cas-témoins. Abstract: Dientamoeba fragilis est un parasite digestif ubiquitaire dont la détection dans les selles est devenue de plus en plus fréquente avec l'avènement de la PCR comme outil de détection de routine. Cependant, la pathogénicité de ce parasite est encore très discutée. Afin d'évaluer le caractère potentiellement pathogène de ce protozoaire, une étude rétrospective cas-témoins a été réalisée entre janvier et décembre 2020 sur des patients du CHU de Toulouse, dans le but d'évaluer les effets cliniques et biologiques potentiels associés à la présence et à la charge de D. fragilis dans les selles. Après appariement sur l'âge, le sexe et le mode de prise en charge (consultation ou hospitalisation), aucune différence significative n'a été observée dans la fréquence des signes cliniques entre les 36 patients testés positifs pour la PCR de Dientamoeba fragilis dans les selles et les 72 patients témoins avec une PCR négative pour ce protozoaire. La présence de D. fragilis dans les selles n'était pas associée à des modifications des paramètres biologiques. De plus, une charge digestive élevée de D. fragilis n'avait pas d'impact identifiable sur les paramètres cliniques et biologiques. Seule la présence concomitante de Blastocystis sp. dans les selles était significativement plus fréquente dans le groupe D. fragilis (analyse uni- et multivariée). En conclusion, cette étude n'a pas montré de différence significative concernant les signes cliniques ou biologiques entre les patients porteurs de Dientamoeba fragilis et le groupe témoin, quelle que soit la charge parasitaire digestive, indiquant que D. fragilis pourrait être considéré comme un commensal du tube digestif.

Detection of Blastocystis sp. and Dientamoeba fragilis using conventional and molecular methods in patients with celiac disease.

Blastocystis sp. and Dientamoeba fragilis are intestinal protists, which are common worldwide, but the pathogenic role of these organisms in gastrointestinal diseases is still controversial. This study aimed to investigate the frequency of Blastocystis sp. and D. fragilis in stool samples from adult patients with celiac disease (CD) by using conventional and molecular methods. A total of 75 patients with CD and 75 healthy individuals were included in this study. Fresh stool specimens collected from each individual were analyzed by conventional and molecular methods. The overall prevalence of Blastocystis sp. and D. fragilis was 41.3% (31/75) and 24% (18/75) in patients with CD, and 46.7% (35/75) and 13.3% (10/75) in healthy controls, respectively. There was no statistically significant difference in the prevalence of Blastocystis sp. and D. fragilis between CD patients and healthy individuals. Blastocystis sp. subtypes were identified in 20 CD and 16 control patients and the overall subtype distribution was observed as ST1 13.9%, ST2 30.6%, and ST3 55.6%. The prevalence of Blastocystis sp. and D. fragilis in adults with CD is similar to the prevalence of protozoa in healthy adults. In this study, the most prevalent Blastocystis subtype was ST3 and the most frequent allele was a34 in both CD patients and healthy individuals. No significant difference was found between the two groups in terms of the detection rates of Blastocystis sp. and D. fragilis, and it is thought that both protists may be colonisers of the intestinal microbiome.

The epidemiology of intestinal protozoa in the Israeli population based on molecular stool test: a nationwide study.

Stool examination using microscopy was the traditional method for the diagnosis of intestinal parasites. Recently, the use of molecular tests to identify stool protozoa has become the main tool used in most clinical laboratories in Israel. This study aimed to evaluate the prevalence of intestinal parasites in Israel and to compare this prevalence in laboratories that use molecular tests vs a laboratory that uses microscopy. Samples collected from January to October 2021 at seven laboratories were analyzed by real-time PCR (RT-PCR) or by microscopy. The multiplex panel included the following pathogens: Giardia lamblia, Entamoeba histolytica, Cryptosporidium spp., Cyclospora, Dientamoeba fragilis, and Blastocystis spp. Overall, 138,415 stool samples were tested by RT-PCR and 6,444 by microscopy. At least one protozoa species was identified in 28.4% of the PCR-tested samples compared to 4.6% of the microscopy-tested samples. D. fragilis was the most common PCR-identified species (29%). D. fragilis, G. lamblia, and Cryptosporidium spp. were mainly found in pediatric population, while Blastocystis spp. was most prevalent among adults (P < 0.001). In a sub-cohort of 21,480 samples, co-infection was found in 4,113 (19.15%) samples, with Blastocystis spp. and D. fragilis being the most common (14.9%) pair. Molecular stool testing proved more sensitive compared to microscopy. D. fragilis was the most commonly detected pathogen. The above profile was identified during the COVID pandemic when traveling was highly restricted and most likely represents the locally circulating protozoa. IMPORTANCE: This study sheds light on the prevalence of stool parasites in Israel. Additionally, this study indicates that the shift from microscope analysis to molecular tests improved protozoa diagnosis.

Detección molecular de Dientamoeba fragilis en heces: eliminación de los inhibidores de la DNA polimerasa / Molecular detection of Dientamoeba fragilis in faeces: removal of DNA polymerase inhibitors

Se realizó un estudio de purificación del DNA de los trofozoitos de Dientamoeba fragilis a partir de muestras fecales humanas congeladas, no fijadas en formol, y con elevadas concentraciones de inhibidores de la DNA polimerasa. Para tal fin, se utilizaron columnas ("spin-columns") disponibles en el comercio. Esta metodología se comparó con una técnica tradicional de purificación con fenol/cloroformo. Ambos métodos fueron seguidos de la amplificación del DNA de D. fragilis por una "nested" PCR y posterior electroforesis en un gel de agarosa de los amplicones obtenidos. La extracción del DNA a partir de trofozoitos de D. fragilis - mediante el procedimiento de las columnas - produjo DNA de elevada calidad, libre de impurezas e inhibidores de la polimerasa. Por el contrario, con la técnica de fenol/cloroformo se observó la inhibición de la enzima, cuando se analizaron los productos de amplificación. Además, se confirmó que el agregado de SAF (solución de acetato de sodio - ácido acético - formol) a las muestras fecales afecta la integridad del DNA y como consecuencia impide su posterior amplificación.