Molecular detection and genotyping of Dientamoeba fragilis and Blastocystis sp. in housefly Musca domestica (Diptera: Muscidae): first report for Dientamoeba fragilis.

Dientamoeba fragilis and Blastocystis sp. are single-celled protozoan parasites of humans and animals. Although they are found in the intestines of healthy hosts, the pathogenicity of them is still unclear. To date, there is no report on D. fragilis and only two studies (without subtyping) on the occurrence of Blastocystis sp. in Musca domestica. In this study, fly samples were collected from livestock farms and their surroundings in the Kirsehir province (Central Anatolia Region) of Türkiye from May to August 2023. A total of 150 microscopically identified M. domestica samples were analyzed for the detection of D. fragilis and Blastocystis sp. molecularly. The overall prevalence of Blastocystis sp. and D. fragilis in M. domestica was determined to be 3.3% (5/150) and 8.0% (12/150), respectively. The SSU rRNA gene sequences of the isolates indicated genotype 1 of D. fragilis. Eleven isolates were identical and represented a single isolate (KAU-Dfrag1). BLAST analysis of KAU-Dfrag1 indicated identity with the isolates reported from humans, cattle, sheep, and budgerigars. The other isolate (KAU-Dfrag2) was polymorphic at two nucleotides from KAU-Dfrag1 and three nucleotides from known genotypes from GenBank and represented a variant of genotype 1. The Blastocystis sp. isolates were found to be identical and represent a single genotype (KAU-Blast1). BLAST analysis revealed that the KAU-Blast1 genotype belonged to the potentially zoonotic subtype 5 (ST5) and exhibited the highest genetic identity (ranging from 99.4 to 99.6%) with pigs, cattle, and sheep from different countries. Our study provides the first data on the molecular prevalence, epidemiology, and genotypic characterization of D. fragilis and Blastocystis sp. in M. domestica.

Observations on the transmission of Dientamoeba fragilis and the cyst life cycle stage.

Little is known about the life cycle and mode of transmission of Dientamoeba fragilis. Recently it was suggested that fecal­oral transmission of cysts may play a role in the transmission of D. fragilis. In order to establish an infection, D. fragilis is required to remain viable when exposed to the pH of the stomach. In this study, we investigated the ability of cultured trophozoites to withstand the extremes of pH. We provide evidence that trophozoites of D. fragilis are vulnerable to highly acidic conditions. We also investigated further the ultrastructure of D. fragilis cysts obtained from mice and rats by transmission electron microscopy. These studies of cysts showed a clear cyst wall surrounding an encysted parasite. The cyst wall was double layered with an outer fibrillar layer and an inner layer enclosing the parasite. Hydrogenosomes, endoplasmic reticulum and nuclei were present in the cysts. Pelta-axostyle structures, costa and axonemes were identifiable and internal flagellar axonemes were present. This study therefore provides additional novel details and knowledge of the ultrastructure of the cyst stage of D. fragilis.

The clinical significance of Dientamoeba fragilis and Blastocystis in human stool-retrospective cohort study.

OBJECTIVES: The aim of this study was to assess the clinical significance of Dientamoeba fragilis (DF) and Blastocystis species (Bs) in human stool. METHODS: Observational study of patients ≥18 years, who were tested by stool multiplex PCR for bacteria and parasites between April 2019 and March 2022. Although DF and Bs are part of the PCR kit, these results are not routinely reported to the patient or the ordering physician. The main outcomes were the incidence of symptoms during 14 days before the referral to stool PCR test, and the incidence of several clinical outcomes during 60 days after the PCR test (symptoms, referrals to further evaluation, prescription of symptomatic, or antibiotic treatment). RESULTS: A total of 27 918 patients were tested by stool PCR during the 3 study years. A total of 6215 (22.3%) and 5337 (19.2%) were positive for DF and Bs, respectively. The incidence of symptoms before the test was similar in those positive for Bs or DF and those with all-negative PCR (adjusted OR and 95% CI of 0.87 [0.80-0.95] and 0.82 [0.76-0.88] for Bs and DF, respectively), whereas significantly higher (2.47 [2.23-2.73]) in those positive for the other multiplex PCR assay components. During the 60 days after the test, the prevalence of any of the outcomes was similar in those positive for Bs or DF and those with negative PCR (adjusted OR and 95% CI of 0.92 [0.83-1.02] and 0.89 [0.81-0.97] for symptoms, 0.84 [0.75-0.94] and 0.93 [0.85-1.01] for referrals, 0.88 [0.75-1.03] and 0.82 [0.71-0.94] for symptomatic treatment, and 0.88 [0.75-1.02] and 0.86 [0.75-0.98] for antibiotic treatment in the Bs and DF positive individuals, respectively). The PCR cycle threshold was not associated with any of the outcomes. DISCUSSION: Positive stool PCR for DF or Bs was not associated with any of the measured clinical outcomes.

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.

Prevalence of Blastocystis and Dientamoeba fragilis in diarrheal patients in Corum, Türkiye.

To investigate the prevalence of Blastocystis and Dientamoeba fragilis in diarrhea patients and healthy individuals in Corum, Türkiye, fecal samples from 92 diarrhea patients and 50 healthy individuals were collected and evaluated using direct microscopy and molecular methods to screen for bacteria, protozoa, and viruses. The prevalence of Blastocystis was 24.6% in total and more frequent in the healthy group (30.0%). The commonly detected STs (subtypes) were ST3 (40.0%) and ST2 (34.2%). The distribution of Blastocystis STs in the healthy and diarrheal groups did not show any difference in sex and age, but ST3 was detected more frequently in patients aged from 40 to 59 years (p < 0.05). Alleles 4 (8/12) and 2 (4/12) were present in ST1; 9 (3/5) and 12 (2/5) in ST2; 34 (9/14), 36 (3/14), and 38 (2/14) in ST3; and only allele 42 (2/2) in ST4. D. fragilis was present in 8.4% of the population. However, there was no statistically significant difference between the healthy and diarrheic groups (12.0% and 6.5%, respectively), neither with respect to age nor sex. Co-infection was 58.3% and was more frequent in healthy individuals (33.3%) than in diarrhea patients (25.0%). Blastocystis ST3 was the most common subtype detected, with D. fragilis at 33.3%. Salmonella, Shigella, or helminth eggs were not observed in all groups, but Entamoeba histolytica, Giardia intestinalis, Cryptosporidium, Rotavirus, Adenovirus, and Clostridium difficile toxin were found only in diarrhea patients. These findings support the hypothesis that Blastocystis and D. fragilis may be part of the healthy human gut microbiome.

Dientamoeba fragilis associated with microbiome diversity changes in acute gastroenteritis patients.

This study examined the correlation between intestinal protozoans and the bacterial microbiome in faecal samples collected from 463 patients in New Zealand who were diagnosed with gastroenteritis. In comparison to traditional microscopic diagnosis methods, Multiplexed-tandem PCR proved to be more effective in detecting intestinal parasites. Among the identified protozoans, Blastocystis sp. and Dientamoeba fragilis were the most prevalent. Notably, D. fragilis was significantly associated with an increase in the alpha-diversity of host prokaryotic microbes. Although the exact role of Blastocystis sp. and D. fragilis as the primary cause of gastroenteritis remains debatable, our data indicates a substantial correlation between these protozoans and the prokaryote microbiome of their hosts, particularly when compared to other protists or patients with gastroenteritis but no detectable parasitic cause. These findings underscore the significance of comprehending the contributions of intestinal protozoans, specifically D. fragilis, to the development of gastroenteritis and their potential implications for disease management.

A Cross-Sectional Study on the Occurrence of the Intestinal Protist, Dientamoeba fragilis, in the Gut-Healthy Volunteers and Their Animals.

Dientamoeba fragilis is a cosmopolitan intestinal protist colonizing the human gut with varying prevalence depending on the cohort studied and the diagnostic methods used. Its role in human health remains unclear mainly due to the very sporadic number of cross-sectional studies in gut-healthy populations. The main objective of this study was to expand knowledge of the epidemiology of D. fragilis in gut-healthy humans and their animals. A total of 296 stool samples from humans and 135 samples from 18 animal species were analyzed. Using qPCR, a prevalence of 24% was found in humans in contrast to conventional PCR (7%). In humans, several factors were found to influence the prevalence of D. fragilis. A more frequent occurrence of D. fragilis was associated with living in a village, traveling outside Europe and contact with farm animals. In addition, co-infection with Blastocystis spp. was observed in nearly half of the colonized humans. In animals, D. fragilis was detected in 13% of samples from eight species using qPCR. Our molecular phylogenies demonstrate a more frequent occurrence of Genotype 1 in gut-healthy humans and also revealed a likely a new protist species/lineage in rabbits related to D. fragilis and other related organisms.

Should Dientamoeba fragillis be looked for in pediatric digestive pathology of an unknown cause? A proposed pilot case-control study.

INTRODUCTION: Dientamoeba (D.) fragilis is a common intestinal protozoan with an unresolved clinical significance. The association between D. fragilis and the etiology of gastrointestinal symptoms in children is unclear. Metronidazole is often used for treatment. The aims of this study are to clarify the clinical relevance of D. fragilis in children with gastrointestinal symptoms, and to determine the clinical and microbiological efficacy of metronidazole in D. fragilis-infected children with gastrointestinal complaints. METHODS: A prospective case-control study was performed from October 2017 to February 2019. A total of 106 individuals aged 1-17 were included. Out of the 106; 59 showed gastrointestinal symptoms (case group), and 47 were without gastrointestinal symptoms (control group). We excluded 2 patients from the case group. D. fragilis was diagnosed by real-time PCR in stool samples. A 10-day course of oral Metronidazole was prescribed in D. fragilis positive children with GI symptoms. Clinical data before and after the treatment as well as peripheral eosinophilia in previous blood samples, were recorded. RESULTS: Of the 104 participants, D. fragilis was found in 17 (29.8%) children from the case group, whereas in the control group the parasite was detected in 11 patients (23.4%) with an odds ratio (OR) of 1.39 (IC 95% 0.53-3.75, p=0.46). The most prevalent clinical manifestation was abdominal pain (46/57, 80.7%). Seventeen cases with a positive PCR received anti-parasitic treatment according to the established protocol, although during the collection period we received only 11 stool samples to perform the post-treatment follow-up. The PCR of the D. fragilis remained positive in 3 patients (3/11, 27.27%). Despite achieving the eradication of the parasite, 4/8 patients (50%) continued with digestive symptoms. CONCLUSIONS: According to our study there were no differences between the D. fragilis infection in children with or without gastrointestinal symptoms. No relation was found between the clinical and microbiological responses after said D. fragilis treatment. Therefore, we conclude that it is not justified to look specifically for D fragilis in pediatric patients with abdominal symptoms.

Prevalencia de Dientamoeba fragilisy otros protozoários intestinales en porcinos de una granja en la región Andina de Colombia / Prevalence of Dientamoeba fragilis and other intestinal protozoa from a swine farm at the Andean region of Colombia

RESUMEN Dientamoeba fragilis es un protozoário que parasita el intestino grueso del hombre y animales domésticos. Hasta el momento, aún no son claros aspectos de su ciclo de vida, como el rango de hospedadores, reservorios, mecanismo de infección, entre otros. Se postula que el cerdo es un hospedador natural para este protozoario y que, debido a su cercanía con el humano, podría facilitar una transmisión zoonótica. En Colombia no existen a la fecha estudios sobre la presencia de D. fragilis en hospedadores animales, incluidos los cerdos, y los datos sobre la prevalencia de otros protozoarios intestinales en estos animales son escasos. El objetivo fue determinar la frecuencia de protozoarios intestinales, incluyendo D. fragilis, en cerdos de una granja tecnificada de una zona rural de Medellín (región Andina de Colombia). Se recolectaron muestras de materia fecal de 70 cerdos en etapa de preceba, que fueron evaluadas mediante examen directo, tinción tricrómica y Ziehl-Neelsen modificado. Se realizaron análisis univariados con medidas de frecuencia relativa y tendencia central y análisis bivariados para la exploración de factores de riesgo. Se detectó la presencia de D. fragilis en un 13% de las muestras y de otros parásitos como Entamoeba spp. (66%), Blastocystis spp. (64%), Balantioides coli (36%), Cryptosporidium spp. (36%), Iodamoeba butschlii (17%), coccidias (4%) y Giardia spp. (1,4%). En este primer reporte de D. fragilis en cerdos en Colombia se observó la alta prevalencia de otros protozoarios patógenos, lo que corrobora el papel de los cerdos como importantes reservorios de infecciones humanas. Estudios sobre la presencia de este protozoario tanto en cerdos como en el personal encargado de su manejo contribuirían al conocimiento sobre su dinámica de transmisión.

ABSTRACT Dientamoeba fragilis is a protozoan that parasitizes the large intestine of humans and domestic animals. To date, some aspects regarding D. fragilis life cycle, including hosts, reservoirs, infection mechanism, among others, are not yet clear. Swine are considered natural hosts for this protozoan, therefore their close contact with humans promotes its zoonotic transmission. In Colombia there are no studies on the presence of D. fragilis in animal hosts, including pigs, and data about other intestinal protozoa are scarce. The objective was to determine the prevalence of intestinal protozoan, including D. fragilis, in pigs raised in a farm from a rural area of Medellin (Andean region of Colombia). Fecal samples from 70 pigs in prefattening stage were collected. Direct fecal smear examination, trichrome and modified Ziehl-Neelsen stain were used in the study. Univariate analysis (frequency distribution and central tendency measures) and bivariate analysis were used to explore risk factors. Dientamoeba fragilis was found in 13% of the evaluated fecal samples. Other parasites detected included: Entamoeba spp. (66%), Blastocystis spp. (64%), Balantioides coli (36%), Cryptosporidium spp. (36%), Iodamoeba butschlii (17%), coccidias (4%), and Giardia spp. (1,4%). This is the first report of D. fragilis in swine in Colombia, and the high prevalence of other pathogen protozoa was also observed, which corroborates the role of pigs as important reservoirs for human infections. Studies focused on the evaluation of both swine and swine-exposed farm workers should be done in order to know the dynamics of transmission of this parasite.

First detection and molecular characterization of Dientamoeba fragilis in cattle.

Dientamoeba fragilis is a flagellated protozoan with amoeba-like morphology that inhabits the human gastrointestinal tract. It is endemic in a vast geography around the world, including developed countries. There are limited studies on non-human hosts of the parasite, and suitable hosts have not been clarified. The parasite has been detected in non-human primates, pigs, cats, dogs and rats. There is no study in the literature investigating and detecting the presence of this parasite in cattle. In this study, stool samples taken from 163 different cattle and calves from 11 different farms between March 2017 and May 2022 were examined for the detection of D. fragilis via PCR. Trichrome staining was performed on all PCR-positive samples. The isolates with the expected amplicon size were sequenced using the 18S ribosomal RNA region, and their genotypes were determined by BLAST analysis. Sequences were analysed with the most similar and reference sequences in the literature, forming a phylogenetic tree. We detected D. fragilis in 31 (19.01%) of the 163 stool samples. D. fragilis cysts/trophozoites were detected by trichrome staining method in six of 31 samples. All PCR products selected for molecular analysis from positive samples had the same nucleotide sequence. As a result of BLAST analysis, all sequences were determined to belong to D. fragilis genotype 1. This study determined for the first time that cattle are suitable hosts for D. fragilis. Furthermore, the parasite subtype we detected belongs to genotype 1, which is the most common type in humans, suggesting that the parasite may have a zoonotic character. Our result is important in terms of the epidemiology of the parasite, as the mode of transmission is controversial, and available data on its suitable hosts are limited.

Retrospective Results of Hacettepe University Faculty of Medicine Parasitology Laboratory Between 2014-2019

Objective: Parasitic infections emerge as a significant health problem, especially in underdeveloped and developing countries. Epidemiological data play an important role in taking effective measures against parasitic diseases. Methods: Clinical samples (stool, blood, bone marrow and tissue samples, etc.) that were sent to Hacettepe University Hospitals Parasitology Laboratory between 2014 and 2019 were analyzed retrospectively. Results: The positivity rates of the parasites detected in this study are as follows; Blastocystis sp. (71.6%), Dientamoeba fragilis (13.3%), Giardia lamblia (4.7%), Echinococcus spp. (1.9%), Enterobius vermicularis (1.8%) and Taenia spp. (0.3%). In this study, four of the patients were found to be positive for Leishmania spp. and two patients for Plasmodium falciparum and four patients for Plasmodium spp. E. histolytica/E. dispar cysts and/or trophozoites examined by Trichrome staining in our study were not detected within six years. Conclusion: According to this data and in the light of the results obtained from different regions of our country, it will be possible to properly direct the necessary strategies for the diagnosis, treatment of parasitic infections and the implementation of preventive measures.

First report and genotyping of Dientamoeba fragilis in pet budgerigars (Melopsittacus undulatus), with zoonotic importance.

The protozoan Dientamoeba fragilis is one of the most common parasites in the digestive system of humans worldwide. The host range and transmission routes of D. fragilis, including the role of animals, are still ambiguous with few reports from non-human primates, sheep, rodents, pigs, a cat and a dog. In this study, we used microscopic and TaqMan qPCR analyses to investigate D. fragilisin 150 faecal samples from pet budgerigars (Melopsittacus undulatus) in the Central Anatolia Region of Turkey. Dientamoeba fragilis DNA was detected in 32 samples, resulting in a mean prevalence of 21.3%. In microscopic examination, trophozoites/cysts of D. fragilis were detected in 13 of 32 qPCR-positive samples. SSU rRNA sequence analyses of the qPCR-positive isolates identified genotype 1 of D. fragilis as predominant in budgerigars. Phylogenetic analyses of the SSU rRNA gene region clustered D. fragilis genotypes, as well as other trichomonads, in separate monophyletic clusters with bootstrap values ≥79.0. Our study provides the first evidence for the natural host status of pet budgerigars for D. fragilisand contributes to the knowledge of the epidemiology of this parasite. The high prevalence of genotype 1 of D. fragilis suggests that pet budgerigars are suitable reservoirs for zoonotic transmission. Our findings contribute to an increased awareness and knowledge of D. fragilis infections in the context of a one-health approach.

Investigation of Dientamoeba fragilis and Blastocystis in patients from Turkey with ulcerative colitis and irritable bowel syndrome: Any relation with genotypes?

Blastocystis sp. and Dientamoeba fragilis are two most common protists worldwide, whose pathogenic potentials are a matter of debate since their discovery. This study aims to investigate the relationship between the activation of ulcerative colitis (UC) and irritable bowel syndrome (IBS) with these protists. A total of 100 patients (35 IBS, 35 active UC, and 30 remittent UC), diagnosed at Hacettepe University Adult Hospital (Ankara, Turkey), were screened for D. fragilis and Blastocystis sp. with microscopic examination using the methods of wet mount, trichrome staining, conventional PCR, nested PCR, real-time PCR and genotyping. Eight patients (4 IBS, 2 active, and 2 remittent UC patients) were found to be D. fragilis positive. 18S rRNA region of the parasite was amplified in four of the patients, whereas cathepsin L-like cysteine peptidase; clan Sc, family S9, serine peptidase; and clan MH, family M20 metallopeptidase in six different patients. All isolates were Genotype 1. Sequence results showed very limited diversity. A total of nine patients (3 IBS, 5 active UC, 1 remittent UC) were found to be positive for Blastocystis sp., all of which were Subtype 3. One active UC and one IBS patient were found to be positive for both parasites. No statistically significant difference was detected between the patient groups in means of parasite detection. D. fragilis was found to be related to older age (p=0,045). In our study, no significant correlation was identified between D. fragilis and Blastocystis sp., and the activation of UC and IBS. More studies are needed on the host-parasite relationship, including the role of gut microbiota, together with transcriptomic and metabolomic assessments to unveil the pathogenicity of both protists.

Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods.

INTRODUCTION: The clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing. MATERIAL AND METHODS: Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes. RESULTS: The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients. CONCLUSION AND RECOMMENDATION: Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

Diagnostic and Therapeutic Considerations Towards Dientamoeba fragilis in Children: A Survey Amongst General Practitioners and Pediatricians in the Netherlands.

ABSTRACT: This survey was undertaken to obtain insight in the attitude of Dutch physicians towards pathogenicity, diagnostic- and therapeutic approach towards Dientamoeba fragilis in children. Physicians were invited by e-mail for a questionnaire. A total of 211 of 450 physicians (46.9%) completed the questionnaire, including 67 general practitioners (GPs) and 144 pediatricians. Of all respondents, 175 of 211 (82.9%) considered D fragilis a "potential pathogen", when other causes of gastro-intestinal complaints are ruled out. Only 16 of 211 (7.6%) performed diagnostic tests regularly. Diagnostic tests were performed by 162 of 211 (77%) of respondents in children with diarrhea and abdominal pain in consideration of duration of symptoms. Fecal polymerase chain reaction (PCR) was diagnostic modality of preference. Eighty-nine of 142 (62.7%) prescribed metronidazole as antibiotic of first choice. This study shows heterogeneity in clinical practice amongst Dutch physicians regarding diagnostic- and therapeutic approach of D fragilis in children. Different attitude towards pathogenicity and inconsistent guidelines could be causative factors.

Improved diagnosis of gastrointestinal infections using a semi-automated multiplex real-time PCR for detection of enteropathogens.

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis.Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination.Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures.Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013-2018) included stool culture, microscopy and immunochromatography.Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013-2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis.Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

Investigation of Dientamoeba fragilis Frequency in Faecal Samples of Patients with Enterobius vermicularis Infection by Polymerase Chain Reaction

Objective: Dientamoeba fragilis (D. fragilis) is a flagellated protozoan with an amoeba-like morphology, located in the gastrointestinal tract. The hypothesis was that the parasite was transported by Enterobius vermicularis (E. vermicularis) eggs. This study aimed to determine the association of D. fragilis and E. vermicularis with the genotypes of the identified strain of D. fragilis. Results of trichrome staining were compared with those of polymerase chain reaction (PCR), which is widely used in the diagnosis of D. fragilis. Methods: A total of 391 samples were obtained. The stool and cellophane slide samples were sent together to the Parasitology Department Laboratory, Faculty of Medicine, Aydin Adnan Menderes University, between 1 October 2017 and 1 October 2018. Stool samples of all patients with E. vermicularis (n=74) and without E. vermicularis (n=74) infection were used. All samples were examined for the presence of D. fragilis by trichrome staining and PCR. The 18S ribosomal RNA region of D. fragilis isolates was sequenced. Demographic characteristics and clinical findings of the patients were evaluated. Results: D. fragilis was detected in 42 (28.37%) of 148 samples; 28 (66.6%) of them were detected in patients with E. vermicularis infection. The coexistence of two parasites was significant (p<0.05). All isolates sequenced were genotype 1. No significant relationship was found between the presence of parasites and clinical findings, living area and gender (p>0.05). Conclusion: D. fragilis is frequently associated with E. vermicularis, so the presence of D. fragilis should be also considered in affected patients. The use of high-sensitivity molecular methods such as PCR is important in preventing false results. Amaç: Dientamoeba fragilis (D. fragilis), amip benzeri morfolojiye sahip, gastrointestinal yerlesimli, kamçili bir protozoondur. Parazitin Enterobius vermicularis (E. vermicularis) yumurtalariyla tasindigi hipotezi kabul görmektedir. Çalismamizda D. fragilis ve E. vermicularis birlikteligini incelemek, bulunan D. fragilis'lerin genotiplerini belirlemek ve D. fragilis tanisinda yaygin olarak kullanilan trikrom boyama ile polimeraz zincir reaksiyon (PZR) yöntemlerini karsilastirmak amaçlanmistir. Yöntemler: Çalismamizda Aydin Adnan Menderes Üniversitesi Tip Fakültesi, Parazitoloji Laboratuvari'na 1 Ekim 2017-1 Ekim 2018 tarihleri arasinda diski ve selofan lam örnegi birlikte gönderilmis toplam 391 olgu örnegi incelenmistir. Selofanli lam örneklerinde E. vermicularis saptanan tüm gönüllü olgularin (74 olgu) diski örnegi ile E. vermicularis negatif 74 olgunun diski örnegi çalisilmistir. Tüm diskilar trikrom boyama ve PZR yöntemleri ile D. fragilis varligi açisindan incelenmistir. Saptanan D. fragilis izolatlarinin 18S ribozomal RNA bölgesi sekanslanmistir. Olgularin demografik özellikleri ve klinigi degerlendirilmistir. Bulgular: Toplam 148 olgunun 42'sinde (%28,37) D. fragilis saptanmistir. D. fragilis pozitif olan 42 olgunun %66,6'sini E. vermicularis pozitif olgular olusturmus ve iki parazitin birlikteligi anlamli bulunmustur (p<0,05). Sekanslanan tüm izolatlar genotip 1 olarak saptanmistir. Klinik bulgular, yasanilan bölge ve cinsiyet ile parazit varligi arasinda anlamli bir iliski saptanamamistir (p>0,05). Sonuç: Arastirmamizda D. fragilis'in siklikla E. vermicularis ile birliktelik gösterdigi ve bu olgularda D. fragilis varligina ayrica dikkat edilmesi gerektigi vurgulanmistir. Yanlis sonuçlari engellemede, yüksek duyarliliga sahip PZR gibi yöntemlerin önemi bir kez daha görülmüstür.