Received: 17/01/2025 Accepted: 26/02/2025 Published: 21/04/2025 1 of 5 https://doi.org/10.52973/rcfcv-e35617 Revista Científica, FCV-LUZ / Vol. XXXV ABSTRACT Blastocystis species are zoonotic protist commonly found in animals and humans. To date, 17 subtypes of Blastocystis have been identified, nine of which have been isolated from humans. This study aimed to determine the frequency of Blastocystis subtypes in patients diagnosed with urticaria and to explore the relationship between patient symptoms and Blastocystis subtypes. Stool samples from 100 urticaria patients and 100 healthy volunteers were analyzed for the presence of Blastocystis spp. using direct microscopic examination with the native–Lugol method and the subtypes were identified through PCR and sequencing techniques. A questionnaire was administered to the patient group to gather information on symptoms, socio–economic status, and hygiene practices. Blastocystis spp. was detected in 9% (9/100) of the urticaria patients and 5% (5/100) of the control group. The distribution of Blastocystis subtypes in the patient group was as follows: ST2 (n = 4, 44.4%), ST3 (n = 3, 33.3%), ST1 (n = 1, 11.1%), and ST4 (n = 1, 11.1%). In the control group, the distribution was ST3 (n = 2, 40%), ST1 (n = 2, 40%), and ST2 (n = 1, 20%). Regarding the relationship between symptoms and Blastocystis subtypes, 8 of 9 (88.9%) Blastocystis–positive patients reported rash, 7 (77.8%) experienced itching, 6 (66.7%) had fever, 3 (33.3%) experienced swelling, and 1 (11.1%) reported abdominal pain. Notably, bloating and abdominal pain were observed exclusively in patients with ST2. It is crucial to highlight the elevated prevalence of Blastocystis in areas where livestock farming is prevalent and the zoonotic cycle in the transmission of the parasite. While limited studies have suggested a correlation between Blastocystis subtypes and urticaria, the high prevalence of ST2 in urticaria patients may indicate its significant role in pathogenicity. The data derived from the patient questionnaire highlight a notable association between ST2 and symptoms such as bloating and abdominal pain, warranting further investigation. Key words: Blastocystis spp., urticaria, sequencing, subtypes, zoonosis RESUMEN Las especies de Blastocystis son protistas zoonóticos comúnmente encontrados en animales y humanos. Hasta la fecha, se han identificado 17 subtipos de Blastocystis, de los cuales nueve han sido aislados de humanos. Este estudio tuvo como objetivo determinar la frecuencia de los subtipos de Blastocystis en pacientes diagnosticados con urticaria y explorar la relación entre los síntomas de los pacientes y los subtipos de Blastocystis. Se analizaron muestras fecales de 100 pacientes con urticaria y 100 voluntarios sanos en busca de la presencia de Blastocystis spp. mediante examen microscópico directo con el método nativo–Lugol, y los subtipos fueron identificados a través de técnicas de PCR y secuenciación. Se administró un cuestionario al grupo de pacientes para recopilar información sobre síntomas, estado socioeconómico y prácticas de higiene. Blastocystis spp. se detectó en el 9 % (9/100) de los pacientes con urticaria y en el 5 % (5/100) del grupo de control. La distribución de los subtipos de Blastocystis en el grupo de pacientes fue la siguiente: ST2 (n = 4, 44,4 %), ST3 (n = 3, 33,3 %), ST1 (n = 1, 11,1 %) y ST4 (n = 1, 11,1 %). En el grupo de control, la distribución fue ST3 (n = 2, 40 %), ST1 (n = 2, 40 %) y ST2 (n = 1, 20 %). En cuanto a la relación entre los síntomas y los subtipos de Blastocystis, 8 de 9 (88,9 %) pacientes positivos para Blastocystis informaron erupciones cutáneas, 7 (77,8 %) experimentaron picazón, 6 (66,7 %) tuvieron fiebre, 3 (33,3 %) presentaron hinchazón y 1 (11,1 %) reportó dolor abdominal. Es notable que la distensión abdominal y el dolor abdominal se observaron exclusivamente en pacientes con ST2. Es crucial destacar la elevada prevalencia de Blastocystis en áreas donde la cría de ganado es prevalente y el ciclo zoonótico en la transmisión del parásito. Aunque estudios limitados han sugerido una correlación entre los subtipos de Blastocystis y la urticaria, la alta prevalencia de ST2 en pacientes con urticaria puede indicar su papel significativo en la patogenicidad. Los datos derivados del cuestionario de los pacientes resaltan una notable asociación entre ST2 y síntomas como distensión abdominal y dolor abdominal, lo que justifica una investigación adicional. Palabras clave: Blastocystis spp. , urticaria, secuenciación, subtipos, zoonosis Detection of Blastocystis spp. in patients with urticaria and identification of subtypes using sequencing techniques Detección de Blastocystis spp. en pacientes con urticaria e identificación de subtipos mediante técnicas de secuenciación Laman Musayeva 1 , Gülcan Saylam Kurtipek 2 , Özben Özden 3 , Salih Macin 1 * 1 Selcuk University, Faculty of Medicine, Department of Medical Microbiology. Konya, Türkiye. 2 Selcuk University, Faculty of Medicine, Department of Skin and Venereal Diseases. Konya, Türkiye. 3 Acıbadem Mehmet Ali Aydınlar University, Faculty of Medicine, Department of Medical Biotechnology. İstanbul, Türkiye. *Corresponding author: salihmacin@hotmail.com

Detection of Blastocystis spp. subtypes using sequencing techniques / Musayeva et al._____________________________________________ 2 of 5 INTRODUCTION Blastocystis is a zoonotic protist that is commonly found in gastrointestinal tract of humans and other mammals, as well as birds, reptiles, fish and insects. Transmission occurs via the fecal– oral route or through direct contact with the microorganism’s reservoir [1]. Although Blastocystis was identified long ago, its pathogenicity remains a subject of debate [2]. The distribution of Blastocystis varies not only between countries but also across different regions within the same country. The incidence and prevalence of this parasite is increasing due to factors such as low socio–economic status, inadequente infrastructure, consumption of contaminated water and food, and poor hygiene. This parasite has been found both in the human intestine and in vertebrate animals such as mice, rats, chickens, cattle, and pigs [3]. The subtypes of Blastocystis differ based on their reservoirs, geographical distribution. To date, nine subtypes (ST1-ST9) have been identified in humans with ST3 being the most common. Forty two different subtypes have been identified in total, and they may have varying effects on the host, ranging from harmful to potentially beneficial [4]. Studies have shown that different subtypes are dominant in different regions [5, 6]. Researches indicate that ST1–ST4 are more prevalent in humans compared to other hosts. Blastocystis spp. isolated from various hosts are currently classified into 18S rRNA gene subtypes (STs) . ST1, ST2, ST3, and ST4 are frequently detected among humans. [7, 8, 9]. ST1, ST2, and ST4 have been implicated in gastrointestinal symptoms, and associated with Irritable Bowel Syndrome (IBS) [10]. Additionally, ST1 has been reported to have increasing pathogenicity [11]. This study aimed to detect Blastocystis spp. isolates in patients diagnosed with urticaria, determine the subtype distribution of these isolates through sequencing, evaluate the relationship between Blastocystis spp. and urticaria symptoms, and compare findings with those of a healthy control group. Urticaria is a condition of unknown etiology, with protozoa and helminths recognized as potential causes [12]. The itchy lesions characteristic of urticaria result from histamine release in the skin, triggered by immune mechanisms. It has been reported that parasites can stimulate the secretion of immunological mediators such as IL-3, IL-4, IL-5, and IL-13 from Th2 cells in the intestinal lumen, leading to histamine release through the IgE response. Intestinal parasites also influence the development of tolerance to allergens by modulating the immune system [13]. MATERIALS AND METHODS Research group and method: This study included 100 patients who applied the Selcuk University hospital, Dermatology outpatient clinic between February 2019 and February 2020 with a diagnosis of urticaria, as well as 100 healthy volunteers. A questionnaire was administered to the patient group to gather information on complaints, socio– economic status, and hygiene practices. Blastocystis spp. was considered positive if at least one microorganism was observed in microscope field at 400× magnification. Genomic DNA isolation was performed using a commercial kit (Norgen Biotek, Canada). Blastocystis isolates were analyzed using a PCR thermal cycler (Sensoquest Labcycler, Germany). PCR products were examined through 1.5% agarose gel electrophoresis followed by staining with ethidium bromide and imaging with the Gel Logic 200 Imaging System (Kodak, USA). During the purification stage, single–band PCR products were purified using the HighPrep™ PCR Clean–up System (AC-60005) according to the manufacturer’s instructions. Stool samples were processed using the ABI 3730XL Sanger sequencer and the BigDye Terminator v3.1 Cycle Sequencing Kit in the Macrogen laboratory (Applied Biosystems, Foster City, CA). Ethical Consideration Patients meeting the diagnostic criteria for urticaria were informed about the study’s purpose, details, and procedures. Each participant signed an informed consent form. Ethical approval was obtained from the Local Ethics Committee of Selcuk University Faculty of Medicine (Date: 06.02.2019; Approval Number: 2019/04). RESULTS AND DISCUSSION A total of 200 stool specimens were included in the study. The patient group consisted of 36 males and 64 females, while the control group included 47 males and 53 females (TABLE I). Various forms of Blastocystis were observed, with the vacuolar form being the most common. Overall, Blastocystis spp. was detected in 14 (7%) of the 200 stool samples. Among the patient group, Blastocystis spp. was positive in 9 (9%) of 100 stool samples. Of these, 2 (2%) samples contained both Entamoeba spp. and Blastocystis spp., while 7 (7%) contained only Blastocystis spp.. No other parasites were found in the Blastocystis spp. negative stool samples. Blastocystis spp. was positive in 5 (5%) of 100 stool samples in the control group. Among these, 1 (1%) sample contained both Entamoeba spp. and Blastocystis spp., while 4 (4%) contained only Blastocystis spp. (TABLE II). The ameboid form of Blastocystis spp. has been suggested as a potential contributor to pathogenicity [14]. A study conducted in Egypt found the ameboid form in 60.6% of Blastocystis–positive urticaria patients, whereas it was absent in healthy controls TABLE I Age and gender distribution of the patient and control groups Age Patient Group Control Group Total Female Male Female Male N % 0-18 4 4 11 7 26 13 18-51 46 26 28 26 126 63 >50 14 6 16 12 48 24 Total 64 36 55 45 200

_________________________________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol.XXXV 3 of 5 by including 54 urticaria patients and 50 healthy controls with stool samples examined microscopically and evaluated via PCR. A significantly higher number of parasites were detected in the patient group compared to the control group (P<0.001), though no significant difference was observed between acute and chronic urticaria patients (P=0.2). Among the urticaria patients, 33 (61.1%) were Blastocystis–positive, compared to only 4 (8%) of the controls. The vacuolar and cyst forms were the predominant forms observed [15]. After direct microscopic examination, genomic DNA was isolated from the 14 Blastocystis–positive stool samples, and PCR targeting the SSU rRNA gene of Blastocystis was performed. Amplification was observed at the expected size (~119 bp). DNA sequencing was conducted bidirectionally, and the sequences were aligned with reference sequences. Chromatogram images were reviewed to correct any false nucleotide readings. Four distinct Blastocystis subtypes were identified: ST1, ST2, ST3, and ST4. In the patient group, the subtype distribution was as follows: ST2 (n = 4, 44.4%), ST3 (n = 3, 33.3%), ST1 (n = 1, 11.1%), and ST4 (n = 1, 11.1%). In the control group, the subtype distribution was: ST3 (n = 2, 40%), ST1 (n = 2, 40%), and ST2 (n = 1, 20%) (TABLE III). In this study, the five most commonly reported symptoms in the patient group, itching, rash, swelling, fever, and bloating were analyzed. It is hypothesized that the symptoms of itching and rash may be associated with the subtype distribution of Blastocystis. Additionally, a notable relationship between ST2 and symptoms such as bloating and abdominal pain was observed. Several studies have investigated the clinical significance of Blastocystis spp. in relation to symptoms. In a study, 554 stool samples were examined to assess the prevalence of Blastocystis in symptomatic and asymptomatic groups. The prevalence rates were 16.08% (64/398) in asymptomatic patients and 18.58% (29/156) in symptomatic patients, with no significant association between symptoms and Blastocystis positivity (P=0.528). However, a statistically significant relationship was observed between urticaria and Blastocystis positivity (P<0.05). Among specific symptoms, the association rates were diarrhea (16.37%), constipation (16.94%), bloating (15.57%), nausea (14.28%), and urticaria (71.42%) [16]. The host specificity and pathogenic potential of Blastocystis isolates are influenced by sequence variations in the SSU rRNA gene [17]. The genetic heterogeneity among Blastocystis isolates suggests that different subtypes may have varying pathogenicity. It remains unclear whether Blastocystis directly contributes to allergic manifestations or is merely a common component of the gut microbiota. A study conducted in Brazil in 2019 analyzed the molecular diversity of Blastocystis spp. in urticaria patients. Using conventional methods and PCR, six subtypes (ST1, ST2, ST3, ST4, ST6, and mixed ST1+ST3) were identified. The most common subtypes were ST1 (a4), ST3 (a34 and a36), and ST4 (a42) [18]. The relationship between symptoms and subtypes of Blastocystis spp. positive patients was evaluated (TABLE V). According to the survey results, 87% (87/100) of urticaria patients reported living in the city center, while 13% (13/100) lived in rural areas. In terms of water usage, 11% (11/100) used bottled water, 28% (28/100) used tap water, and 61% (61/100) used both bottled and tap water. Antibiotic use was reported by 11% (11/100) of the patients, and 4% (4/100) mentioned owning pets. The symptoms of patients were distributed between acute and chronic urticaria groups, as detailed in TABLE IV. TABLE II Parasite distribution according to patient and control groups Parasite Distribution Patient Group Control Group Total N=100 N=100 N=200 N % N % N % Blastocystis spp. 7 7 4 4 11 5,5 Blastocystis spp. + Entamoeba spp. 2 2 1 1 3 1,5 Entamoeba spp. – – 2 2 2 1 TABLE III Distribution of Blastocystis subtypes Subtype Patient Group Control Group N % N % ST1 1 1 2 2 ST2 4 4 1 1 ST3 3 3 2 2 ST4 1 1 – – Total 9 9 5 5 TABLE IV Distribution of patient complaints according to acute and chronic urticaria patient groups Symptoms Acute Urticaria Chronic Urticaria Total N=36 N=64 N=100 N % N % N % Itching 34 94,4 59 92,1 93 93 Redness 31 86,1 58 90,6 89 89 Blister 11 30,5 35 54,6 46 46 Fever 8 22,2 36 56,2 44 44 Swelling 9 11,1 31 48,4 40 40 Skin Rash 3 8,3 19 29,6 22 22 Abdominal Pain 1 2,7 8 12,5 9 9 Diarrhea 3 8,3 1 1,5 4 4 Nausea 1 2,7 3 4,6 4 4 Constipation – – 3 4,6 3 3 Indigestion – – 3 4,6 3 3 Womiting 1 2,7 1 1,5 2 2 Weight loss – – – – – –

Detection of Blastocystis spp. subtypes using sequencing techniques / Musayeva et al._____________________________________________ 4 of 5 Although most individuals infected with Blastocystis are asymptomatic, recent research has proposed that Blastocystis may even serve as a marker of healthy gut flora [19, 20]. Clinical findings related to Blastocystis infection are thought to depend on various factors, including genotype, host immune response, parasite density, gut microbiota, and co–infections [21, 22]. Several studies have highlighted a strong correlation between Blastocystis positivity and urticaria, with some suggesting that the parasite may induce urticaria by activating specific immune mediators [23, 24, 25]. Recent studies investigating the zoonotic transmission potential of Blastocystis have revealed significant findings. For instance, a study conducted in Egypt reported Blastocystis positivity rates of 38% in 136 human fecal samples and 19% in 190 cattle fecal samples, highlighting cattle as a potential reservoir in the zoonotic cycle [26]. Similarly, research conducted in Iran detected Blastocystis positivity in 29.1% of fecal samples collected from 395 animals, including poultry, sheep, and cattle. Among the positive samples, cattle exhibited the highest prevalence (50.6%), followed by sheep (32.0%) and poultry (20.4%) [27]. These findings emphasize the importance of further exploring animal– to–human transmission pathways and the role of livestock in the epidemiology of Blastocystis. It was observed that Blastocystis positivity was significantly higher among individuals living in rural areas (59.3%) compared to those in urban areas (40.7%). This discrepancy was attributed to factors such as direct contact with animals and inadequate sanitation in rural settings. In this study, the majority of patients reported living in the city center, while 13% resided in rural areas, potentially influencing the transmission dynamics of Blastocystis. CONCLUSION In this study, Blastocystis spp. isolates were detected in 9% (9/100) of the urticaria patient group and 5% (5/100) of the control group The relatively low prevalence compared to other studies may be attributed to the high socio–economic status and better hygiene practices in the study population. Among the nine subtypes identified in the urticaria patient group, ST2 was the most dominant (4/9), followed by ST3 (3/9), ST1 (1/9), and ST4 (1/9). In the control group, the subtypes detected were ST3 (2/5), ST1 (2/5), and ST2 (1/5). ST2 was notably more prevalent in the patient group. Regarding the relationship between symptoms and subtypes, 8 of the 9 Blastocystis–positive patients exhibited rash, 7 experienced itching, 6 had fever, 3 reported swelling, and 1 had abdominal pain. Bloating and abdominal pain were observed exclusively in patients with ST2. Based on the analysis of the questionnaire data, it is concluded that there may be a significant association between ST2 and specific symptoms in urticaria patients. ACKNOWLEDGEMENT This study was supported by Selcuk University Coordinatorship of Research Projects (Project No: 19202027). Conflict of interest statement The authors have no conflict of interests. BIBLIOGRAPHIC REFERENCES [1] Rudzinska M, Sikorska K. Epidemiology of Blastocystis infection: a review of data from Poland in relation to other reports. Pathogens [Internet]. 2023; 12(8):1050. doi: https:// doi.org/pfv7 [2] Tileklioğlu E, Ertabaklar H. Genetic Diversity of Blastocystis in Diarrheal Cases: Identification of Subtypes and Alleles. Bull. Microbiol. [Internet]. 2024; 58(2):196-208. doi: https://doi. org/pfv8 [3] Mei X, Su C, Wang W, Zhang B, Wei L, Zhang Z, Tian X, Yang Z, Li X, Duan A, Wang S. 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