Evaluation of photodynamic therapy against Leishmania tropica promastigotes using different photosensitizers.

BACKGROUND: Photodynamic therapy is a two-step procedure, involving the use of photosensitizing agents followed by selective illumination of the target lesion with visible light. Photodynamic therapy has been described recently as a promising strategy for treatment of leishmaniasis. This study aims to evaluate the in vitro phototoxic, morphological, and apoptotic effect of methylene blue, toluidine blue, chloro-aluminum phthalocyanine, and pheophorbide a-mediated photodynamic therapy on the viability of Leishmania tropica promastigotes. METHODS: Parasites were treated with methylene blue, toluidine blue, chloro-aluminum phthalocyanine, and pheophorbide a or/and methylene blue, toluidine blue, chloro-aluminum phthalocyanine, and pheophorbide a-mediated photodynamic therapy, and cell proliferation, morphological changes, and apoptosis were evaluated by XTT, giemsa staining, DAPI staining, and DNA fragmentation, respectively. RESULTS: Parasite viability was significantly different in between the groups treated with methylene blue, toluidine blue, and pheophorbide a, with or without irradiation. chloro-aluminum phthalocyanine treatment did not lead to any alterations in cell viability in Leishmania tropica promastigotes with or without irradiation. DAPI staining results indicated that apoptotic bodies and nucleus fragmentation started to be visible in methylene blue, chloro-aluminum phthalocyanine, and pheophorbide a-mediated photodynamic therapy groups. DNA ladder pattern which is used to define apoptosis was observed in irradiated methylene blue, chloro-aluminum phthalocyanine, and pheophorbide a groups. CONCLUSIONS: The results revealed that apoptosis-induced cell death was observed in Leishmania tropica promastigotes after the application of photosensitizers in combination with light irradiation.

Molecular characterization of Blastocystis in cattle in Turkey.

Blastocystis genus exist in a wide variety of hosts, including humans, birds, insects, annelids, amphibians, fish, and mammals. PCR-based molecular diagnostic methods have been successfully used to detect Blastocystis spp. in feces, and small subunit ribosomal ribonucleic acid (SSU rRNA) gene-based subtyping is the preferred method for diagnosis. There has been discussion about the subtypes of Blastocystis spp. which has been detected so far. To date, 26 different subtypes have been reported. The aim of this study was to determine the existence and diversity of Blastocystis spp. in cattle. In our study, a total of 80 stool samples were collected from cows and calves at 13 different farms in Burdur and one farm in Aydin. Using molecular method, a total of 9 samples out of 80 samples were found to be positive (11.25%) for Blastocystis. As a result of sequence analysis of Blastocystis positive samples, the subtype 14 was detected on seven samples, while in the other two samples, Blastocystis subtype 10 was identified. The ST10 and ST14 subtypes are commonly reported in animals but not isolated from human. Our analyses showed genetic differences among Blastocystis subtypes. Our study is the first Blastocystis subtyping study from cattle in Turkey.

[Subtype Distribution of Blastocystis spp. with DNA Barcoding and Evaluation of Diagnostic Methods]. / DNA Barkotlama Yöntemiyle Blastocystis Alt Tiplerinin Belirlenmesi ve Tani Yöntemlerinin Degerlendirilmesi.

Blastocystis spp. is one of the most common protozoa in Turkey and throughout the world; laboratory diagnosis, genetic diversity and clinical features are among the most controversial topics related to the parasite. The aims of the present study were to investigate the subtype distribution of Blastocystis spp. Isolates from Aydin, Turkey, to evaluate the efficiency of some diagnostic methods and to evaluate the relationship between Blastocystis spp. infection with demographic factors and clinical findings. According to the direct microscopy results, 100 stool samples with and without Blastocystis spp. were selected by simple random sampling method. All were directly subjected to DNA isolation and cultured in Jones medium. DNA isolation was also carried out in Blastocystis spp. positive cultures with a different kit. Genomic DNA samples were analysed by PCR targeting the Blastocystis spp. small subunit ribosomal RNA (SSU rRNA) gene and subtypes (ST) were determined according to the sequence analyses. Moreover, the samples with undetected ST were further studied with sequence tagged site-PCR (STS-PCR). In addition, the patients with and without Blastocystis spp. were compared in terms of demographic characteristics (gender, age, residence) and clinical findings (itching, diarrhoea, abdominal pain, dyspepsia, nausea, vomiting, constipation and weight loss)., Among 100 stool positive samples diagnosed with direct microscopic examination 81 (81%) and 86 (86%) were found as positive with culture and PCR, retrospectively. Additionally, among 100 Blastocystis spp. negative stool samples five (5%) and seven (7%) samples were found positive with the same methods, respectively. The results of the analysis of Blastocystis spp. with SSU rRNA gene sequencing and STS-PCR methods revealed the subtype distribution of 95 Blastocystis spp. isolates as follows: ST3 (n= 50, 52.6%), ST2 (n= 21, 22.1%), ST1 (n= 17, 17.9%), ST7 (n= 4, 4.2%), ST2 + ST3 (n= 2, 2.1%) and ST1 + ST3 (n= 1, 1.1%). In addition, a complete accordance was observed in subtype distribution between direct DNA isolation from stools and 35 randomly selected isolates from the culture. In our study, the comparison of 107 Blastocystis spp. positive (by any of the methods) cases and 93 negative cases showed that there was no correlation in terms of demographic characteristics and clinical findings. Similarly, there was no significant relationship between symptoms and subtypes. In conclusion, it is recommended that in addition to direct microscopic examination, the use of additional methods such as culture and PCR will be useful in routine laboratory diagnosis of Blastocystis spp. The distribution of Blastocystis subtype in Aydin is mainly in accordance with the global findings. Lack of a relationship between Blastocystis spp. Infection and symptoms in our study was supported the idea that Blastocystis spp. infection is mostly asymptomatic in humans and it may be a member of healthy microbiota.

[Transfection of Leishmania tropica with green fluorescent protein (gfp) gene and investigation of the in vitro drug effect]. / Leishmania tropica'nin yesil floresan protein (gfp) geni ile transfeksiyonu ve in vitro ilaç etkisinin arastirilmasi.

Cutaneous leishmaniasis (CL) is a parasitic disease transmitted by vector sand flies Phlebotomus and Lutzomyia. This disease is characterized by long time non-healing skin lesions, and caused by Leishmania species. CL is the most common infection in Eastern and Southeastern Anatolia in Turkey and L.tropica is known as the main agent of the disease. Number of cases is increasing in our country in time because of malnutrition, migration, travel, low socioeconomic level and ecological changes. For the treatment, the pentavalent antimonials are often used as intralesionally for many years, and it was reported that resistant cases have increased in recent years. New treatment methods and anti-Leishmanial activity of new agents have been investigated because of side effects, resistance development and toxic reactions of the present drugs. These studies are first carried out in vitro and afterwards with in vivo experimental animal models. Reporter gene technology has been used to investigate a variety of purposes like biological events in microorganisms and the efficacy and resistance of drugs in recent years. The major areas that green fluorescent protein (gfp) used are that they can be incorporated into different genes to determine the amount of expression of these genes in different organisms and can be used as markers in living cells. Especially gfp gene, which encodes the green fluorescent protein, is widely used nowadays. Gene-based assays have several advantages like being easy to follow-up, inexpensive and have improved biosecurity. The aim of the present study was to perform the transfection of L.tropica with "enhanced gfp (egfp)" and in vitro usefulness of gfp-transfectants as a drug screening model in comparison to the conventional methods. Promastigotes of L.tropica were transfected with p6.5/egfp by electroporation and selected for tunicamycin-resistance as previously described. L.tropica promastigotes transfected with gfp and in vitro effect of meglumine animoniate was assessed using different methods such as fluorescence microscopy, fluorometer and XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide) assay. The use of gfp-transfected Leishmania strains was found more rapid and more sensitive by fluorescent microscopy and fluorometry than conventional assays for the evaluation of potential anti-leishmanial agents. Consequently, stable gfp-transfected Leishmania species will be used in vitro and in vivo for screening of anti-leishmanial drugs and vaccine development as well as for understanding the biology of the host-parasite interactions at the cellular level. As a result ot this study, gfp transfected model using a Turkish L.tropica isolate was established to be used in further studies.

[Genotyping of Echinococcus granulosus isolates by sequencing of mitochondrial cytochrome C oxidase subunit 1 (cox1) gene in Aydin]. / Aydin'da Echinococcus granulosus izolatlarinin mitokondriyal sitokrom C oksidaz alt ünite 1 geni (cox1) dizi analizi ile genotiplendirilmesi.

Cyst hydatid (CH) is a zoonotic infection that is characterized by the development of metacestode form of Echinococcus granulosus primarily in liver of humans and ruminants. With a worldwide distribution, the infection is still considered as an important parasitic disease that threatens the public health in Turkey as in the other developing countries. Morphological and biological features of parasite fail to discriminate isolates for typing so molecular methods should be used for this purpose. Recently, a total of eleven genotypes of E.granulosus (G1-10 and lion strain) have been identified and these genotypes are highly correlated with host specificity of the parasite. The aim of this study, was to determine the genotypes of E.granulosus isolates from human samples in Aydin. Cyst fluids from CH operated cases in Adnan Menderes University Faculty of Medicine, Training and Research Hospital were used in the present study. Samples were washed with phosphate buffered saline (PBS) and stored in 70% ethanol at -20ºC. Mitochondrial cytochrome C oxidase subunit 1 (cox1) gene was amplified partially by polymerase chain reaction (PCR). The PCR products were sequenced initially, compared to other database in Genbank and evolutionary distances were estimated with references. The genotypes of E.granulosus isolates were determined according to the closest or exact matches to the references. A total of 20 E.granulosus isolates were genotyped in the present study, most of them (15 isolates, 75%) were identified as Genotype 1 (G1), that is defined as sheep genotype and the remaining isolates were defined as pig/camel genotype G6/7 (five isolates, 25%). A possible explanation to our results may be related to the geographical position of Turkey. The identification of G6/7 in addition to sheep genotype G1 indicated that pigs and camels in this area have role in the transmission and distribution of E.granulosus to humans. There is still limited information about the molecular epidemiology of E.granulosus in Turkey. This study reveals the first data about the genotype distribution of E.granulosus in our city, therefore the findings may help to design control program for the disease with a combination of epidemiological data.

[Determination of Leishmania species by PCR-RFLP in the smear samples taken from the lesions of cutaneous leishmaniasis cases]. / Kütanöz leysmanyazli olgularda lezyondan alinan yayma örneginden PCR-RFLP ile Leishmania türünün arastirilmasi.

The forms of the disease caused by Leishmania species in Turkey as well as in Aegean region are cutaneous and visceral leishmaniasis (CL and VL, respectively), and the agent of CL is commonly L.tropica. However, L.infantum was also reported as being CL agent recently. Direct microscopic examination, serological tests and culture are the conventional methods used for the diagnosis of CL. Since the specificities of these methods are high their sensitivities are variable and identification at species level is not possible. Recently, the use of polymerase chain reaction (PCR)-based molecular methods enabled the rapid and reliable diagnosis and species identification. The aim of this study was to investigate the performance of PCR-restriction fragment length polymorphism (RFLP) method both for the detection and identification of Leishmania species simultaneously in CL patients. A total of 30 smear samples that were positive for Leishmania amastigotes with microscopic examination, obtained from CL-suspected cases admitted to Adnan Menderes University Medical School Hospital, Parasitology Laboratory (located at Aydin, in the Aegean region of Turkey) between 2012-2014 period were included in the study. Ten samples taken from the skin lesions caused by Staphylococcus aureus (n= 5) and Candida albicans (n= 5) were also included as negative controls. DNA extractions from the smears were performed by the use of a commercial kit (Macherey-Nagel NucleoSpin Tissue® Kit, Germany). DNA isolation was also performed from L.major, L.infantum and L.tropica promastigotes that were grown in culture as positive controls. In PCR method LITSR and L5.8S primers targeting to ITS (internal transcribed spacer)-1 region were used. In RFLP method, the amplified PCR products were cleaved by BsuRI (HaeIII) restriction enzyme for the species identification. As a result, restriction profiles of all samples (n= 30) were in accordance with L.tropica restriction profile. No band was observed in the control samples (n= 10). The data of this study showed that the most common CL agent in Aydin is L.tropica. In conclusion, ITS-1 PCR-RFLP method may be used directly as a single routine procedure for both the detection and identification of Leishmania species in the clinical samples of CL patients, in laboratories with adequate facilities.

[Investigation of in vitro metronidazole resistance in the clinical isolates of Trichomonas vaginalis]. / Trichomonas vaginalis klinik izolatlarinda in vitro metronidazol direncinin arastirilmasi.

Trichomonas vaginalis, a flagellated, urogenital anaerobic protozoon is reported as an important cause of vaginitis with a global distribution. Although metronidazole is the primary choice of drug for the treatment of trichomoniasis, the presence of resistant isolates from many different countries highlights the need of novel drugs for the treatment. Many studies from Turkey mostly dealing with the in vitro effects of compounds and natural products against T.vaginalis have been reported, however, only one study has been encountered searching the metronidazole resistance in a single T.vaginalis isolate. The aim of this study was to determine the in vitro metronidazole resistance and minimum lethal concentrations (MLCs) of the isolates from symptomatic cases. T.vaginalis strains isolated from vaginal discharge samples of symptomatic women that were sent to Adnan Menderes University Faculty of Medicine, Research and Training Hospital Parasitology Laboratory, between 2009-2014 period, were included in the study. The strains were isolated by the inoculation of samples into trypticase-yeast-maltose medium supplemented with 10% fetal calf serum. A total of 40 T.vaginalis isolates stored by cryopreservation were revived before the experiments. T.vaginalis trophozoites were incubated with different concentrations of metronidazole (200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56 µg/ml) and the viability of cells were examined in both aerobic and anaerobic conditions under phase contrast microscope. Additionally, non-motile isolates were further inoculated into fresh media and viability was checked. The wells containing motile trophozoites after 48 hours of incubation with 15 µg/ml and/or higher metronidazole concentration in anaerobic condition and 75 µg/ml and/or higher metronidazole concentration in aerobic conditions were determined as resistant isolates. Of the 40 T.vaginalis isolates three (7.5%) were resistant to metronidazole. MLC mean values of metronidazole-sensitive isolates were 27.17 µg/ml in aerobic and 7.75 µg/ml in anaerobic conditions. The rate of metronidazole resistance detected in this study was higher than most of reports from different countries. Despite being limited to the isolates from Aydin province (located at Agean region of Turkey), the present study has a value as it presented the existence of metronidazole-resistant isolates in Turkey for the first time. More research from other parts of Turkey is needed to better understand the metronidazole resistance at a national scale and to investigate novel strategies for the treatment. Moreover, further studies need to be carried out in order to clarify the relationship between clinical treatment response and in vitro metronidazole resistance in trichomoniasis.

[Genotyping of Giardia intestinalis strains isolated from humans in Aydin, Turkey]. / Aydin'da insanlardan izole edilen Giardia intestinalis suslarinin genotiplendirilmesi.

Giardia intestinalis which is a flagellate, intestinal protozoon of humans and a variety of mammalian species, shows worldwide distribution. To date, eight genotypes of the parasite have been identified. Among these genotypes, assemblage A and B have zoonotic characteristics with low host specificity, thus they are responsible for the human infections. The aim of this study was to identify G.intestinalis genotypes in Aydin, located in Aegean region of Turkey. A total of 40 stool samples that were found positive for G.intestinalis by direct microscopic examination, from Adnan Menderes University, Research and Training Hospital, Parasitology Laboratory from January 2011 to December 2014 were included in the study. DNA isolation from stool samples performed with commercial kit (QIAamp DNA Stool Mini Kit, Qiagen, Germany) followed by polymerase chain reaction (PCR) for G.intestinalis 16S rRNA and beta-giardin genes and then the amplicons were sequenced. Out of 40 isolates 11 (27.5%) were positive with 16S rRNA PCR and 10 (25%) were positive with beta-giardin PCR. Of 21 sequenced amplicons, 10 (47.6%) of them showed 98%-100% similarity with reference sequences and their genotypes could be identified. The distribution of genotypes were as follows: cluster A1 (n: 3), cluster A2 (n: 3), cluster A3 (n: 2) and assemblage B (n: 2). In the light of our results the isolates detected in humans might be zoonotic origin. In accordance with the previous reports in Turkey, assemblage A (8/10) was more common than assemblage B (2/10). In the present study, 10 (25%) out of 40 isolates could be genotyped and sequencing of beta-giardin gene yielded more effective results than sequencing of 16S rRNA for the determination of assemblages. The present study indicated that, there is a need for prospective studies with extended number of cases allowing the comparison of the two genes used for G.intestinalis genotyping.

[Comparison of direct microscopy, culture and polymerase chain reaction methods for the diagnosis of cutaneous leishmaniasis]. / Kutanöz leysmanyazis tanisinda direkt mikroskopi, kültür ve polimeraz zincir reaksiyonu yöntemlerinin karsilastirilmasi

Cutaneous leishmaniasis (CL) is an endemic disease especially in Southeastern Anatolia of Turkey and recently shows a trend for spread to other regions of the country including the Aegean region. The diagnosis of CL is based on combined evaluation of epidemiological data with the clinical symptoms and laboratory findings. Direct microscopic examination and culture methods are mainly used in the routine diagnosis of CL, while molecular methods are mainly used for research. The aim of this study was to detect the presence of Leishmania spp. in samples obtained from CL-suspected patients by using direct microscopy, culture and polymerase chain reaction (PCR) methods and to compare the results. A total of 55 patients who were admitted to Parasitology Laboratory of Adnan Menderes University Hospital, Aydin (located at Aegean region in Turkey), between 2012-2014 were included in the study. Smear preparations from the skin lesions of cases were fixed and stained with Giemsa, and the presence of amastigote forms were evaluated by direct microscopy. NNN medium was used for the cultivation of samples. Total genomic DNA of Leishmania from the samples were extracted with a commercial kit (NucleoSpin Tissue(®) Kit, Macherey-Nagel, Germany) and PCR was performed by using 13A and 13B primers to amplify the 116 base pair fragment of Leishmania spp. specific kinetoplast DNA. Amastigotes were observed in 29 (53%) of the 55 samples by direct microscopy, promastigotes were detected among 34 (62%) samples in culture, and parasite-specific amplicons were revealed in 30 (55%) samples by PCR. All assays were positive in 24 patients while in 18 patients all of the tests yielded negative results. Thirty-seven (67%) out of 55 cases were diagnosed as CL when reactivity in at least one of these three methods were considered as positive. Accordingly the positivity rates of the methods were 78.4% (29/37) for direct microscopy, 92% (34/37) for culture and 81.1% (30/37) for PCR in CL-diagnosed patients, indicating culture as the most sensitive method. Regarding the culture method as gold standard, the sensitivity and specificity of direct microscopy were calculated as 76.4% and 86%, respectively, while PCR presented with 85.3% sensitivity and 95% specificity. In conclusion, it was thought that the usage of more than one method for CL diagnosis leads to increase the sensitivity and specificity which enables the diagnosis of a wide range of patients.

[Subtype distribution of Blastocystis isolates and evaluation of clinical symptoms detected in Aydin province, Turkey]. / Aydin ilinde elde edilen Blastocystis izolatlarinin alt tip dagilimi ve klinik semptomlarin degerlendirilmesi.

The pathogenic potential and genetic diversity of Blastocystis are poorly understood despite being one of the most frequent intestinal parasites in routine fecal examination all around the world as well as Turkey. There are numerous defined subtypes (ST) of Blastocystis which infect animals and nine of them were isolated from human fecal samples. Blastocystis is an anaerobic parasite and generally recognized as nonpathogenic microorganism that colonizes the colon. However recent studies have indicated that the genotypes may be related with the pathogenicity and clinical symptoms of the infection. The aims of this study were to investigate the subtypes of Blastocystis isolates obtained from stool samples submitted to the parasitology laboratory of Adnan Menderes University, Faculty of Medicine, and to evaluate the clinical symptoms of infected cases. A total of 61 cases (40 male, 21 female; age range: 5-69 years, mean age: 35 ± 19.1 years) were included in the study. Stool samples that were positive for Blastocystis cysts in direct microscopic examination, were inoculated in Jones medium and incubated at 37°C for 72 hours for the growth of parasite. Genomic DNAs were isolated from Jones medium directly or frozen samples with a commercial kit (DNAzol, Invitrogen, USA). The subtypes of Blastocystis were detected by polymerase chain reaction (PCR) using ST-specific primers and the symptoms of patients were evaluated retrospectively. Forty-four (72.1%) out of 61 isolates were subtyped by PCR, while 17 (27.9%) could not be typed. The distribution of Blastocystis subtypes were found as follows; ST3 in 17 (38.6%), ST2 in 13 (29.5%), ST1 in 9 (20.5%), ST1 + ST3 in 4 (9.1%), and ST1 + ST2 in one (2.3%) of the samples. The most common symptoms among Blastocystis infected cases were abdominal pain (n= 24, 39.4%), pruritus (n= 22, 36.1%), diarrhea (n= 4, 6.6%) and constipation (n= 2, 3.3%), respectively. This is the first study investigating the genotypes of Blastocystis in Aydin province (located at Aegean region of Turkey), and the findings were consistent with those reported from other regions. The predominant subtype was found as ST3, like other studies in our country and this data supports that ST3 is a human originated genotype of Blastocystis. Additionally, the higher rate of pruritus detected among our patients infected with Blastocystis compared with the other studies was considered remarkable. In conclusion, multicenter and large-scaled molecular and clinical studies are needed to elucidate the pathogenicity and the epidemiology of Blastocystis infections.

Subtype Distribution of Blastocystis in Türkiye.

Blastocystis is an anaerobic protozoan with global importance because of infecting a variety of hosts and having high prevalence in many countries. Blastocystis isolates display remarkable genetic differences, and many subtypes (STs) have currently been defined based on polymorphism in SSU rRNA coding gene. Each 25 subtype may have different characteristics such as pathogenicity, host specificity, and structural variations. Most current research on Blastocystis has focused on these differences and molecular epidemiology. This review aimed to provide a summary of Blastocystis subtype distribution in Türkiye. Regarding human samples, 16 manuscripts were found in the literature, which presented 783 Blastocystis isolates from 9 cities in Türkiye. The most common subtype was ST3 (47.9%), the others were ST1 30 (17.5%), ST2 (14.7%), ST4 (4%), and ST5-ST7 (15.9%). There were few studies on animal hosts and environmental samples. The faecal samples from rats, farm, and pet animals were examined for Blastocystis subtypes and ST1, ST3, ST4-ST7, ST10, and ST12-ST14 were reported. In addition, two studies reported Blastocystis ST1 and ST3 subtypes in environmental water samples. In conclusion, the review of available literature showed that a systematic understanding of the subtype distribution of 35 Blastocystis in Türkiye is still lacking. Most of the studies were performed in a limited number of cities, animal hosts, and environmental samples, therefore, more studies from different provinces are needed in forthcoming research. The majority studies were performed in a limited number of provinces, animal species and very few environmental samples, so in the future; there is a need of novel studies that evaluate more samples from different provinces.

Antiprotozoal activity of different Xenorhabdus and Photorhabdus bacterial secondary metabolites and identification of bioactive compounds using the easyPACId approach.

Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce antimicrobial compounds as secondary metabolites to compete with other organisms. Our study is the first comprehensive study screening the anti-protozoal activity of supernatants containing secondary metabolites produced by 5 Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, Acanthamoeba castellanii, Entamoeba histolytica, Trichomonas vaginalis, Leishmania tropica and Trypanosoma cruzi, and the identification of novel bioactive antiprotozoal compounds using the easyPACId approach (easy Promoter Activated Compound Identification) method. Though not in all species, both bacterial genera produce antiprotozoal compounds effective on human pathogenic protozoa. The promoter exchange mutants revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides. Among the bacteria assessed, only P. namnaoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. These discovered antiprotozoal compounds might serve as starting points for the development of alternative and novel pharmaceutical agents against human parasitic protozoa in the future.

First Molecular Characterisation of Blastocystis from Experimental Rats in Turkey and Comparison of the Frequencies Between Obese and Non-obese Groups

Objective: Blastocystis is a zoonotic protozoan that infects a wide range of animals, including humans and rodents. This study aimed to determine the frequency and subtype distribution of Blastocystis in laboratory rats at a laboratory animal facility in Turkey. Methods: This study included 54 male Sprague-Dawley rats from Aydin Adnan Menderes University Laboratory Animal Center. Among these rats, 30 were fed with high-fat diet (obese group) and the remaining 24 received standard chow (non-obese group). Blastocystis positivity was determined with amplification of small subunit 18S rRNA gene following their nucleic acid extraction from faecal samples. Subtypes were detected by submitting the partial 18S rRNA gene sequences to the database (pubmlst. Results: Blastocystis infection was detected in 33 (61.1%) of 54 laboratory rats. The frequency of Blastocystis was significantly different between obese and non-obese rats (p<0.05), with 43.3% and 83.3%, respectively. When referred to the database, exact matches were identified with Blastocystis subtype 4 (ST4) for all isolates. In the phylogenetic analysis of the partial 18S rDNA sequence, the sequence was closely clustered with reference ST4 subtypes from other countries, including China, Japan, United Kingdom and Czech Republic. Conclusion: This study revealed the high rate of Blastocystis colonisation in laboratory rats, posing a risk for human transmission. The comparison of obese and non-obese groups supported the idea that Blastocystis might be an indicator of healthy gut flora. The detection of ST4 in all rats agreed with previous reports of the predominance of this subtype in rodents.

Investigation of the relationship between obesity and Blastocystis infection in an adult population in Aydin, Turkey.

Blastocystis is one of the most frequent protozoa in human faecal samples, however, little is known about its relation with obesity. The present study aimed to analyse Blastocystis infection and subtypes in three adult populations classified according to body mass index (BMI). Faecal samples from 346 individuals were classified according to BMI: control (124 cases), overweight (110 cases), and obese (112 cases). Nucleic acid extraction from the samples was followed by amplification of partial 18S ribosomal RNA (18S rRNA) gene of Blastocystis. The neighbourjoining method was used to construct a phylogenetic tree from evolutionary distance data. Clinical findings were compared between Blastocystis infected and non-infected cases. Blastocystis was detected in 52 (15%) of 346 individuals with PCR assay. Blastocystis was less frequent in obese group (8%) than both control group (18.2%) and overweight group (18.5%). Subtype distribution was as follows: ST3 (n=21; 43.8%), ST2 (n=15; 31.3%), ST1 (n=10; 20.8%) and ST7 (n=2; 4.2%). The overall nucleotide diversity of 18S ribosomal RNA gene was 0.049. None of the gastrointestinal symptoms and gender was not significantly related with the infection. Despite the cross sectional nature of the study including a specific population, it suggests a negative association between Blastocystis infection and obesity. In addition, the lack of significant relation further supports asymptomatic colonization of Blastocystis.

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.

Microsatellite-Based Genotyping, Analysis of Population Structure, Presence of Trichomonas vaginalis Virus (TVV) and Mycoplasma hominis in T. vaginalis Isolates from Southwest of Turkey.

BACKGROUND: The present study aimed to determine genetic diversity of Trichomonas vaginalis (T. vaginalis) isolates with microsatellite markers in Turkey (Nov 2015 to 2016) and to create a web-based microsatellite typing (MT) approach for the global interpretation of the data. In addition, the endosymbiosis of Mycoplasma hominis (M. hominis) and T. vaginalis virus (TVV) in the isolates was also examined. METHODS: The allele sizes for each locus were calculated and microsatellite types were determined according to the allele profiles. The population structure was examined with Bayesian clustering method. A website (http://mttype.adu.edu.tr) was created for collection and sharing of microsatellite data. Presence of TVV and M. hominis in T. vaginalis isolates were investigated with electrophoresis and PCR. RESULTS: Of 630 vaginal samples T. vaginalis was detected in 30 (4.7%) and those were used for further analysis. The structure produced by a clustering algorithm revealed eight genetic groups. The typing of isolates according to microsatellites revealed 23 different microsatellite types. Three clones were determined among isolates (MT10 16.7%; MT18 10% and MT3 6.7%). The frequency of TVV and M. hominis was 16.6% (n=5) and 20% (n=6), respectively. CONCLUSION: Presence of three clones among 30 T. vaginalis isolates indicated that microsatellite-based genotyping was efficient to determine the clonal distribution of T. vaginalis isolates. Therefore, a promising tool might be developed further and adapted to the studies dealing with molecular epidemiology of T. vaginalis. Microsatellite data from forthcoming studies will be deposited and presented on the website. In addition, we also presented the frequency of two endosymbionts in T. vaginalis isolates for the first time in Turkey.

The Retrospective Analysis of Demodex spp. Results in Aydin Adnan Menderes University Faculty of Medicine Hospital Parasitology Laboratory.

OBJECTIVE: Demodex spp. is one of the most common ectoparasites in humans. The aim of the present study is to evaluate the positivity of Demodex spp. in our Parasitology Laboratory, retrospectively. METHODS: The study included Demodex spp. suspected cases from different departments between 2008 and 2017. The link between Demodex spp. and demographics and symptoms was investigated. In addition, Demodex spp. was evaluated regarding symptoms and distribution pattern (U, T and diffuse region). RESULTS: Demodex spp. was detected in 576 (78%) of 738 cases. There was no relationship between sex and parasite positivity, but frequency was lower in cases below 19 years. There was a relationship between presence of parasite and redness, itching, burning and rash. The parasite density was higher in U region (n=335, 58.2%). When clinical findings and parasite number were statistically compared; itching, burning and rash were significantly higher in patients with parasite density ≥5 parasites/cm2, while a similar result was not observed in patients with redness. CONCLUSION: Given its prevalence and its relationship with the clinical findings; we believe that Demodex is an important parasitic disease for our province and should be evaluated in cases with various dermatological complaints in the face.

Subtype Distribution of Blastocystis in Pregnant Women and Analysis of Possible Risk Factors.

OBJECTIVE: Since the identification of Blastocystis subtypes (ST) in the last decade, much has been learned about the genetic diversity of Blastocystis isolates in different populations, except pregnant women. The objective of this study is to investigate the genetic diversity of Blastocystis in pregnant women and analyse some demographic factors. METHODS: The faecal samples from 100 pregnant women were collected at an Obstetrics and Gynecology Department in Mugla, Turkey. Thereafter, Blastocystis positivity was detected by direct microscopy and culture. The positive cultures were subjected to DNA isolation, and the Blastocystis barcode region was amplified with polymerase chain reaction. Next, the sequences were queried against GenBank nucleotide and Blastocystis STs (18S) databases. RESULTS: Blastocystis was detected in 14% (14 out of 100) of the faecal samples by culture and 10% (10 out of 100) of the samples by direct microscopy. Nine of Blastocystis isolates (64.4%) were ST3, three (21.4%) were ST1 and two (14.2%) were ST2. Neither the demographic features nor the gastrointestinal symptoms were statistically related to Blastocystis infection. CONCLUSION: The findings in this study agreed with the most of the previous human studies that found ST3 as the most abundant genotype. This study reported the frequency of Blastocystis in pregnant women and highlighted the importance of comprehensive studies with more cases of Blastocystis during pregnancy.

Molecular characterisation of Trichomonas vaginalis isolates in Southwest Turkey with multilocus sequence typing and genetic structure analysis in relation to different countries.

Trichomonas vaginalis, a flagellated protozoan parasite, is among the most common sexually transmitted pathogens in the world. The present study aimed to identify the genetic profiles of T. vaginalis in the southwest of Turkey with multilocus sequence typing (MLST) and to analyse the genetic structure of the parasite in a collection of isolates from different countries. The study included 27 T. vaginalis isolates from symptomatic females in Aydin, Turkey. Seven housekeeping genes of T. vaginalis were partially amplified and sequenced after genomic DNA extraction from in vitro cultures. The allele profiles and sequence types (STs) of the isolates were determined by using the MLST database (https://pubmlst.org/tvaginalis). The genetic structure and differentiation of the parasite were analysed in relation to findings from other countries by assembling the available MLST sequences. When referred to the database, a total of 22 STs, including 18 new STs were found; besides, there were two new allele types. The genetic analysis of MLST data demonstrated the presence of two main genetic structures: Type I and Type II. In addition, the neighbor-joining method also revealed that the isolates were clustered into two groups. The genetic types distributed almost equally in the Netherlands and the USA, however, the predominance of Type I was noted in Turkey and the UK. The genetic differentiation among four countries was significant (p < .05), the gene flow was relatively high between the Netherlands and the USA, in contrast to Turkey. Finally, genetic variations were originated within populations (93.8%) rather than among populations (6.2%). In conclusion, we studied the genetic diversity of T. vaginalis isolates with MLST in the southwest of Turkey and showed the origin of genetic differentiation of the parasite among different countries. The presentation of MLST profiles and genetic variance of T. vaginalis isolates will contribute to the development of new diagnostic and treatment options for the parasite.

Retrospective Analysis of Toxoplasma gondii Serology Results from Adnan Menderes University Faculty of Medicine Parasitology Laboratory from 2007 to 2017

Objective: Toxoplasma gondii is a common apicomplexan parasite of humans and can cause significant morbidity and mortality due to congenital transmission and in patients with immune deficiency. The aim of this study was to evaluate T. gondii serology results of 11 years and to determine compatibility of serologic diagnosis methods. Methods: The study was conducted between 2007 and 2017, and anti-T. gondii IgG antibodies were investigated by an in-house Enzyme Linked Immunosorbent Assay (ELISA) and Indirect Fluorescence Antibody (IFA) methods. Moreover, T. gondii-specific IgM antibodies were also studied by ELISA and a commercial kit. In our study, compatibility of ELISA and IFA methods was also investigated statistically. Results: Serology of T. gondii was studied in 8095 individuals including 1123 (13.9%) males and 6972 (86.1%) females. The overall rate of anti-T. gondii IgG positivity was 31.5% (n=2550) and anti-T. gondii IgM positivity was 1.6% (n=127). There was no significant relationship between sex and seropositivity. A high degree of correlation was found between ELISA and IFA. Conclusion: The current findings reveal that toxoplasmosis is still an important public health disease and that the seropositivity rate is consistent with the region in general. Moreover, using IFA and ELISA methods together in the laboratory seems to be effective.