Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido: 13/08/2025 Aceptado: 09/10/2025 Publicado: 01/11/2025 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 5 Revista Cienﬁca, FCV-LUZ / Vol. XXXV https://doi.org/10.52973/rcfcv-e35770 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Dientamoeba fragilis and Chronic Spontaneous Urcaria: A One Health- Based Zoonoc Perspecve on Human-Parasite Interacon Dientamoeba fragilis y urcaria crónica espontánea: una perspecva zoonóca basada en el principio de “Una Salud” sobre la interacción entre humanos y parásitos Deniz Şentürkoğlu 1 , Özben Özden 2 , Rugıyya Samadzade 1 , Gülcan Saylam Kurtipek 3 , Salih MaÇin 1 * ¹Selcuk University, Faculty of Medicine, Department of Medical Microbiology, Konya, Türkiye ²Acıbadem University, Instute of Health Sciences,Department of Medical Biotechnology, İstanbul, Türkiye ³Selcuk University, Faculty of Medicine, Department of Skin and Venereal Diseases, Konya, Türkiye *Corresponding Author: salihmacin@hotmail.com ABSTRACT Dientamoeba fragilis (D. fragilis) is one of the potenal causes of Chronic Spontaneous Urcaria. The aim of the study is to invesgate the associaon between urcaria and the presence of Dientamoeba fragilis, as well as to compare diagnosc methods. Addionally, this study aims to emphasize the zoonoc potenal of D. fragilis within the One Health approach, which is based on the interacons between human health, animal health, and environmental factors. The study included paents with Chronic Spontaneous Urcaria (n: 90) and healthy individuals (n: 40). Direct microscopic examinaon was performed on stool samples using the nave-Lugol method. DNA was isolated from stool samples, and Real-me Polymerase Chain Reacon was performed for detecon. The incidence of D. fragilis was signiﬁcantly higher in paents with Chronic Spontaneous Urcaria 17/90 (18.9%) compared to healthy controls 1/40 (2.5%) (p = 0.0261). In the direct microscopic examinaon of stool samples from Chronic Spontaneous Urcaria paents, D. fragilis was detected in 3 (2.3%) of the samples. D. fragilis was not detected in the stool samples of healthy volunteers by direct microscopy. However, the Real-me Polymerase Chain Reacon revealed D. fragilis DNA in one healthy sample. The data indicate that D. fragilismay impact on the chronic inﬂammatory skin disorders. Furthermore, the study highlights the value of molecular techniques, such as Real-me Polymerase Chain Reacon, for more accurate detecon of zoonoc parasites. Keywords: Chronic Spontaneous Urcaria; Dientamoeba fragilis; one Health; PCR; zoonozis. RESUMEN Dientamoeba fragilis (D. fragilis), es un parásito zoonóco intesnal, y representa una de las posibles causas de la urcaria crónica espontánea. El objevo de este estudio es invesgar la asociación entre la urcaria y la presencia de D. fragilis, así como comparar métodos diagnóscos. Además, este estudio busca destacar el potencial zoonóco de Dientamoeba fragilis dentro del enfoque “Una Sola Salud”, basado en la interacción entre la salud humana, la salud animal y los factores ambientales. El estudio incluyó a 90 pacientes humanos con urcaria crónica espontánea y 40 individuos sanos. Se realizó un examen microscópico directo de las muestras de heces mediante el método navo con Lugol. Se aisló el ADN de las muestras de heces y se realizó la reacción en cadena de la polimerasa en empo real para su idenﬁcación. La incidencia de D. fragilis fue signiﬁcavamente mayor en pacientes con urcaria crónica espontánea 17/90 (18,9%) en comparación con los controles sanos 1/40 (2,5%) (P= 0,0261). En el examen microscópico directo de muestras de heces de pacientes con urcaria crónica espontánea, se detectó D. fragilis en 3 (2,3%) de las muestras. No se detectó D. fragilis en las muestras de heces de voluntarios sanos mediante microscopía directa. Sin embargo, la reacción en cadena de la polimerasa en empo real reveló ADN de D. fragilis en una muestra sana. Los datos indican que D. fragilis puede tener un impacto en los trastornos inﬂamatorios crónicos de la piel. Además, el estudio destaca el valor de las técnicas moleculares, como la reacción en cadena de la polimerasa en empo real, para una detección más precisa de parásitos zoonócos. Palabras clave: Urcaria crónica espontánea; Dientamoeba fragilis; Una Sola Salud; PCR; zoonosis.

Dientamoeba fragilis and Chronic Spontaneous Urcaria / Şentürkoğlu et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Urcaria is a skin condion that aﬀects both the skin and mucous membranes, causing symptoms such as congeson, redness (erythema), swelling (edema), and itchy plaques in the capillaries. This condion can signiﬁcantly impact quality of life [1]. Urcaria is generally divided into two types: Acute Urcaria (AU) and Chronic Urcaria (CU). When the condion lasts for less than six weeks, it is classiﬁed as AU , if it persists more than six weeks, it is considered CU [2 ,[3]. CU due to a known trigger is called Inducible Urcaria. However, when it occurs without an idenﬁable cause, it is referred to as CSU, which aﬀects approximately 0.5 % to 5 % of the populaon [4 , 5 , 6]. Dientamoeba fragilis is a single-celled zoonoc organism and a common intesnal protozoan worldwide [7]. Known as a “ﬂagellate-less” protozoan, it was ﬁrst described approximately 100 years ago [8]. D. fragilis exhibits a pleomorphic trophozoite form, ranging in size from 4 μm to 20 μm [9]. The life cycle of D. fragilis has not been understood yet. It was previously thought that transmission occurred through trophozoites that were preserved within the eggs of helminths, such as Enterobius vermicularis and Ascaris lumbricoides [10]. Incidence rates are underesmated as a result of diﬃcules in accurate diagnosis [11]. A deﬁnive diagnosis is performed by observaon the parasite in stool samples that ﬁxed, stained, and examined under a microscope [12]. Also, culture methods for D. fragilis include xenic culture might perform from stool sample [13]. The RT-PCR method conﬁrmed speciﬁc detecon of D. fragilis without interference from other similar Trichomonad organisms in human samples [14]. Treatments for D. fragilis infecons include hydroxyquinoline, difetarsone, paromomycin, metronidazole, secnidazole, tetracycline, iodoquinol, and erythromycin [15]. Although the pathogenicity of D. fragilis is controversial, it can cause disnct symptoms, including abdominal pain and diarrhea [16]. Recent studies have also suggested an associaon between D. fragilis and urcaria [17 ,[18]. Advances in molecular techniques have enhanced the understanding of its pathogenicity, and research has indicated a rise in posive cases among suspected infecons [19]. The One Health approach, which considers environmental, animal, and human health, has become increasingly important in understanding infecous diseases, including parasic infecons. Many pathogens, especially zoonoc ones, are impacted by complex ecological and host-related factors and do not exist only within a single species [20]. Intesnal protozoa are implicated in non-gastrointesnal diseases such as CSU, a common inﬂammatory skin disorder with unknown eology. Evidence suggests that chronic infecons, immune dysfuncon, and microbial interacons contribute to its development [21]. This study aims to invesgate the presence of D. fragilis in paents with urcaria and examine its potenal associaon with the disease. Addionally, it aims to compare the eﬀecveness of direct microscopic examinaon (DM) and PCR as diagnosc methods for D. fragilis. Furthermore, by exploring the associaon between D. fragilis and CSU, this study aims to emphasized a clinical associaon that has received limited aenon parasic infecons within the One Health approach. MATERIALS AND METHODS Ethical statement This study was approved as a research project by the Selcuk University Faculty of Medicine Local Ethics Commiee with the decision dated April 1, 2020 (No: 2020/149). Selecon of study groups, collecon, and storage of samples This study included 90 paents diagnosed with CSU applied the outpaent clinic of the Department of Dermatology and Venereal Diseases at Selçuk University Faculty of Medicine between June 2021 and December 2021. Paents were excluded from the study if they had suspected drug use, signiﬁcant infecons (viral, bacterial, or fungal) that could trigger urcaria within approximately one week, or had experienced intense stress. The control group consisted of 40 healthy adults who did not have any known acute or chronic diseases and had no history of CSU. Examinaon of stool samples by direct microscopy method As part of the study, the presence of Dientamoeba fragilis was invesgated in 130 stool samples using the DM method during roune parasitological examinaon. The stool samples were collected in ﬁxave-free plasc containers and examined immediately upon arrival at the laboratory. Stool samples were carefully mixed with 0.9 % NaCl (saline), and Lugol’s iodine soluon was applied to both edges of the slide. The samples were then examined under a light microscope at 20X and 40X magniﬁcaon (Zeiss, Germany). Lugol staining facilitated the idenﬁcaon of D. fragilis by enhancing the visibility of its internal structures, allowing for detailed diﬀerenaon. Aﬅer processing, the stool samples were stored at -80 °C for further analysis. Genomic DNA isolaon DNA isolaon was performed using the ZymoBIOMICS™ DNA Miniprep Kit (Merck KGaA, Darmstadt, Germany). The extracted DNA samples were transferred into numbered Eppendorf tubes, labeled according to the corresponding volunteer, and stored at -20 °C. Real-me Polymerase Chain Reacon In the RT-PCR analysis, DNA isolates obtained from the extracon process were used. The reacon mixture included the abm BlasTaq 2X qPCR MasterMix (Thermo Fisher Scienﬁc, Germany) and Fluorogenic SYBR Green (Thermo Fisher Scienﬁc, Germany). The ampliﬁcaon was performed in a 96-well plate using Roche LightCycler 96 (Basel, Switzerland) with appropriate primers. 2 of 5

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Stascal analysis R version 3.6.0 (The R Foundaon for Stascal Compung, Vienna, Austria; [hps://www.r-project.org] analysis was used. The Shapiro-Wilk test and Q-Q plot were used to assess the normality of the data, while the Levene test was used to evaluate the homogeneity of variances. Descripve stascs for numerical data were presented as mean ± standard deviaon (minimum – maximum), whereas categorical variables were summarized as numbers (n) and percentages (%). Stascal diagnosc measures were reported with a 95% conﬁdence interval (CI). A 5 % signiﬁcance level (P < 0.05) was considered for hypothesis tesng. RESULTS AND DISCUSSION The study parcipants ranged in age from 19 to 85 years (41.1 ± 16.85). The group included 57 males (43.8 %) and 73 females (56.2 %), with 90 CSU paents and 40 healthy individuals. Age and gender distribuons were similar between the groups (P= 0.753 and P= 0.713, respecvely). In the direct microscopic examinaon from 90 CSU paents, D. fragilis was detected in 3 cases (2.3 %). However, D. fragilis was not found in any of the stool samples from 40 healthy volunteers. According to RT-PCR results, D. fragilis DNA was detected in 17 (18.9 %) of the stool samples from the paent group. In contrast, among the stool samples of healthy volunteers, D. fragilis DNA was idenﬁed in only one case (2.5 %) (P= 0.0261). The diagnosc performance of direct microscopy, using RT- PCR as the gold standard, is presented in TABLE I. The consistency between PCR and direct microscopy results was found to be 25.6 % (κ = 0.256), indicang a low level of concordance between the two methods. TABLE I Comparison of the performance of PCR and Direct Microscopy methods in the diagnosis of D.fragilis PCR results Negave Posive Total Direct Microscopy results Negave 112 (TN) 15 (FN) 127 (97.7%) Posive 0 (FP) 3 (TP) 3 (2.3%) Total (86.2 %) (13.8 %) Stascal Diagnosc Measures Sensivity (95 % CI) 16.7 (3.6 – 41.4) Speciﬁcity (95 % CI) 100 (96.8 – 100) PPV (95 % CI) 100 (29.2 – 100) NPV (95 % CI) 88.2 (81.3 – 93.2) Accuracy (95 % CI) 97.7 (93.4 – 99.5) Κ 0.256 TN: true negave, FN: false negave, FP: false posive, TP: true posive, 95 % CI: 95 % conﬁdence interval, Sensivity: sensivity, Speciﬁcity: speciﬁcity, PPV: posive predicve value, NPV: negave predicve value, Accuracy: accuracy, κ: Value of Kappa test stasc. Stool samples were also examined microscopically for intesnal parasites other than D. fragilis. Blastocyss spp. was the most frequently detected intesnal parasite in both groups, idenﬁed in 8 cases (8.8 %) among CSU paents and 1 cases (2.5 %) in the healthy control group. Addionally, Entamoeba spp. was detected in 2 CSU paents (2.2 %) and 1 healthy individual (2.5 %) (TABLE II). TABLE II Intesnal parasites detected by direct microscopy Direct Microscopy D. fragilis n / % Blastocyss spp. n / % Entamoeba spp. n / % Control Group (n:40) 0/ 0,0 1 / 2.5 1 / 2.5 Paent Group (n:90) 3 / 3.3 8/ 8.8 2 / 2.2 Dientamoeba fragilis can cause disnct disease manifestaons, such as abdominal pain and diarrhea [22 ,[23]. Advancements in molecular diagnosc methods have allowed for a more precise examinaon of its pathogenicity. Studies have shown that the detecon rate of D. fragilis has increased in suspected cases, further supporng its potenal role as a pathogen. D. fragilis is among the parasites suspected to contribute to urcaria [24]. A meta-analysis conducted in Germany examined the possible relaonship between D. fragilis and CSU. The analysis found that the incidence of intesnal parasites, including D. fragilis, in CSU paents can reach 75 % [25]. D. fragilis DNA was detected in 17 (18.9 %) of the stool samples from CSU paents in this study. Addionally, parasites such as Blastocyss spp. and Entamoeba spp. were detected from CSU paents. Dientamoeba fragilis and Enterobius vermicularis were detected in 49 paents presenng with nausea, voming, diarrhea, abdominal pain, urcaria, anal itching, and weight loss in Spain. These paents were treated with metronidazole, and symptoms completely resolved in 43 cases. However, in 6 paents, symptoms persisted, and follow-up stool examinaons aﬅer four weeks sll tested posive for D. fragilis. A second treatment with paromomycin was administered to these paents, aﬅer which all recovered, and no D. fragilis was detected in their stool samples [26]. In this study, the success rate of direct stool microscopy was found to be lower than that of molecular methods. Direct stool microscopy may not be suﬃcient for diagnosis in clinically symptomac paents. Vezir et al. [1] invesgated the prevalence of intesnal parasites in CSU paents, including 76 children and 38 adults in Turkey. In the pediatric group, stool sample analysis revealed Blastocyss spp. in 18.4 % (n=14), D. fragilis in 2.6 % (n=2), and Giardia lamblia in 1.3 % (n=1). In the adult group, Blastocyss spp. was the most frequently detected parasite, found in 18.4 % (n=7) of samples. Following an-parasic treatment, urcaria symptoms signiﬁcantly improved in 57.1 % of pediatric paents and 60% of adult paents. In this study, a total of 8 Blastocyss spp. were detected in 90 adult CSU paents, which is consistent with the ﬁndings. These ﬁndings suggest that Blastocyss spp. and D. fragilis may contribute to chronic urcaria, signiﬁcantly aﬀecng paents’ quality of life [1]. 3 of 5

Dientamoeba fragilis and Chronic Spontaneous Urcaria / Şentürkoğlu et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico D. fragilis was detected in 59 (12.04 %) of 490 samples using RT-PCR analysis. Among paents with D. fragilis infecon, diarrhea was signiﬁcantly more common (16.3 %; P = 0.001). The diarrhea rate was higher in D. fragilis posive paents (84.09 %) compared to D. fragilis negave paents (P = 0.0005), showing a stascally signiﬁcant associaon. According to the study results, the incidence of D. fragilis was signiﬁcantly higher in paents with CSU (18.9 %) compared to controls (2.5 %) (P = 0.0261) [27]. D. fragilis DNA was detected in 17 (18.9 %) of the stool samples from the paent group. D. fragilis DNA was idenﬁed in only one case of healthy volunteers (2.5 %) which is consistent with the ﬁndings. Thirty-one stool samples taken from paents with GIS disease in Australia and previously determined to be D. fragilis posive by microscopy were studied with the “Convenonal PCR” method, targeng the SSU rRNA gene region. According to the results, D. fragilis was found posive in 29 of 31 samples. In Italy, researchers developed a new RT-PCR method for D. fragilis by targeng the SSU rRNA gene region. With this new method, false posives by cross-reacng with other Trichomonads other than D. fragilis, seen in previous RT-PCR methods, are eliminated. It has also been determined that this new method is more sensive than “Microscopy” and “Convenonal PCR” methods [6]. In Australia, 31 stool samples from paents with gastrointesnal system (GIS) disease, previously idenﬁed as D. fragilis posive through microscopy, were analyzed using the Convenonal PCR method, targeng the SSU rRNA gene region. The results showed that D. fragilis was detected in 29 out of 31 samples [28]. A total of 472 stool samples were analyzed to compare the eﬀecveness of Mulplex PCR (MT-PCR), RT-PCR, and Microscopy methods. The study found that RT-PCR and MT- PCR demonstrated 100% sensivity and speciﬁcity. In contrast, the microscopy method showed a speciﬁcity of 90 % but signiﬁcantly lower sensivity (40-50 %) [29]. These ﬁndings conﬁrm that molecular methods provide higher sensivity and speciﬁcity compared to microscopy, making them more reliable for detecng D. fragilis. Stool samples from paents suspected to be D. fragilis posive in Netherlands were analyzed using the RT-PCR method. The results showed that 43 % of the samples tested posive for D. fragilis DNA. In comparison, posivity rates detected by microscopy ranged between 8 % and 19.8 %. These ﬁndings demonstrate that RT-PCR provides more accurate and reliable detecon of D. fragilis compared to microscopy [30]. In this study, D. fragilis DNA was detected in 17 out of 90 CSU paent samples using PCR, whereas only 3 cases were idenﬁed through direct microscopy. Based on these results, the agreement between PCR and direct microscopy was found to be 25.6 % (κ = 0.256). In conclusion, RT-PCR exhibited higher sensivity and speciﬁcity in detecng D. fragilis compared to direct microscopy, conﬁrming its superiority as a diagnosc tool [31]. CONCLUSION The results of the study suggest a potenal associaon between D. fragilis infecon and Chronic Spontaneous Urcaria, emphasizing the importance of considering intesnal parasic infecons in the diﬀerenal diagnosis of chronic inﬂammatory skin condions. Furthermore, the results highlight the importance of using molecular diagnosc techniques, such as Real-me Polymerase Chain Reacon, together with convenonal microscopic methods for detecon. ACKNOWLEDGEMENTS This study was supported by Selcuk University Coordinator- ship of Research Projects (Project No: 20202019). Conﬂict of Interest The authors declare no conﬂicts of interest. BIBLIOGRAPHIC REFERENCES [1] Vezir S, Kaya F, Vezir E, Karaosmanoglu N, Adiloglu AK. Evaluaon of intesnal parasites in paents with chronic spontaneous urcaria in a territory hospital in Turkey. J. Infect. Dev. Ctries. [Internet]. 2019; 13(10):927-932. doi: hps://doi.org/p9m9 [2] Kolkhir P, Muñoz M, Asero R, Ferrer M, Kocatürk E, Metz M, Xiang YK, Maurer M. Autoimmune chronic spontaneous urcaria. J. Allergy Clin. Immunol. [Internet]. 2022; 149(6):1819-1831. doi: hps://doi.org/gr96qc [3] Zuberbier T, Bernstein JA, Maurer M. Chronic spontaneous urcaria guidelines: What is new? J. Allergy Clin. Immunol. [Internet]. 2022; 150(6):1249-1255. doi: hps://doi.org/gs6bnz [4] Kaplan A, Lebwohl M, Giménez-Arnau AM, Hide M, Armstrong AW, Maurer M. 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