Blastocystis: A Mysterious Member of the Gut Microbiome.

Blastocystis is the most common gastrointestinal protist found in humans and animals. Although the clinical significance of Blastocystis remains unclear, the organism is increasingly being viewed as a commensal member of the gut microbiome. However, its impact on the microbiome is still being debated. It is unclear whether Blastocystis promotes a healthy gut and microbiome directly or whether it is more likely to colonize and persist in a healthy gut environment. In healthy people, Blastocystis is frequently associated with increased bacterial diversity and significant differences in the gut microbiome. Based on current knowledge, it is not possible to determine whether differences in the gut microbiome are the cause or result of Blastocystis colonization. Although it is possible that some aspects of this eukaryote's role in the intestinal microbiome remain unknown and that its effects vary, possibly due to subtype and intra-subtype variations and immune modulation, more research is needed to characterize these mechanisms in greater detail. This review covers recent findings on the effects of Blastocystis in the gut microbiome and immune modulation, its impact on the microbiome in autoimmune diseases, whether Blastocystis has a role like bacteria in the gut-brain axis, and its relationship with probiotics.

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.

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.

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 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.

Wound Myiasis in Diabetic Foot Ulcer: Calliphoridae and Sarcophagidae Family.

BACKGROUND: Myiasis is a parasitic infestation of tissues or body cavities of mammals with dipterous larvae. The patients with diabetic foot ulcers are more vulnerable to acquiring infestation; however, the infestation may be neglected and mistreated in some cases. METHODS: Data were collected of twelve myiasis cases with diabetic foot ulcers in Nazli-Selim Eren Chronic Wound and Infections Care Unit, Aydin, Turkey between 2017 and 2019. Demographic, clinical characteristics of the patients and clinical examination of the wound were recorded. To morphology-based identification method of the agents, the developmental stages of the maggots were examined. RESULTS: The cases aged between 46 and 81 years (10 males, two females). Eight of the larvae collected from wounds had Calliphoridae and four had Sarcophagidae family. The larvae were infested right/left foot sole, thumb, ankle, and mostly left toes. The number of larvae collected from the cases ranged from 2 to 48. Third-stage larvae (L3) were mostly detected. Mixed (L1-L2, L2-L3) larvae were detected in a patient. The infestations were more common in July and August. According to the score of Infectious Diseases Society of America (IDSA), ten (83%) cases had moderate and two (17%) cases were mild diabetic foot infections (DFIs). CONCLUSION: Diabetic foot ulcers should be evaluated in terms of myiasis. This was the first study in our province indicating that myiasis should not be neglected and different species of flies were responsible for myiasis cases.

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.

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.

Retrospective Analysis of Cystic Echinococcosis Results in Aydin Adnan Menderes University Training and Research Hospital Parasitology Laboratory Between 2005 and 2017

Objective: Echinococcus granulosus is the causative helminth of cystic echinococcosis (CE). The parasite is known to form fluid-filled cysts that grow slowly in the internal organs, particularly the liver and/or lungs. This disease is still important in terms of public health and economically in Turkey and other countries where animal husbandry is widespread. The aim of our study was to retrospectively evaluate the cases that were admitted to the Adnan Menderes University, Training and Research Hospital Parasitology laboratory on suspicion of CE between January 2005 and January 2017. Methods: Totally, 3446 sera (from 2019 female and 1427 male) were tested with an in-house ELISA for the presence of E. granulosus specific IgG antibodies at the timeswhen they were sent. Socio-demographic characteristics (age, gender, residence, and dog ownership), positivity titers, and cyst locations of pathologically confirmed CE patients were analyzed retrospectively. Results: The ages of patients varied between 4-87 years. It was found that 1104 (32%) of the 3446 sera were positive, and of them, 642 (58.1%) were female and 462 (41.9%) were male. Patients who had pathologically confirmed CE diagnosis constituted 247 (22.3%) of the total seropositive sera. Liver was the most commonly affected organ (81.8%), followed by lungs (6.1%). Conclusion: CE remains an important public health problem in our city; therefore, it is once again emphasized that preventive studies should be planned.

[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.

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.

Investigation of the Applicability of a Rapid Diagnosis Test in the Diagnosis of Cystic Echinococcosis

OBJECTIVE: Echinococcus granulosus, the etiological agent of cystic echinococcosis (CE) in humans and livestock, is a widely distributed zoonotic pathogen tapeworm. The infection is transmitted to humans by the ingestion of E. granulosus eggs released in the feces of definitive hosts such as dogs. The larval stage of the parasite develops a slowly enlarging cyst in the visceral organs, particularly in the liver and/or lung. The aim of the present study was to evaluate the diagnostic value of an immunochromatographic test (ICT) for CE. METHODS: A total of 50 sera from surgically and/or pathologically confirmed patients with CE were included in the study as the study group; the control group comprised patients who tested negative for enzyme-linked immunosorbent assay (ELISA). Sera were selected from the collection at Adnan Menderes University, Faculty of Medicine, Parasitology Laboratory, by simple random sampling. The collection included sera obtained between 2010 and 2014; antibody titers of each serum sample were determined using in-house ELISA, before storage at -20°C. The presence of E. granulosus antibody in the sera was determined using a commercially available ICT (VIRAPID® HYDATIDOSIS) kit method. RESULTS: In the study group (E. granulosus-confirmed cases), two (4%) of the 50 sera were negative and 48 (96%) were positive with ICT. In the control group (ELISA-negative), all were negative with ICT. CONCLUSION: The rapid diagnostic test has been evaluated as a practical, easy-to-use method for detecting CE, and it can be used as a screening test in routine diagnosis and research.

[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.

Use of Internal Transcribed Spacer Sequence Polymorphisms as a Method for Trichomonas vaginalis Genotyping.

OBJECTIVE: Trichomonas vaginalis is the most common non-viral, sexually transmitted pathogen with a worldwide distribution. The aim of the present study was to design a new genotyping tool for T. vaginalis isolates using internal transcribed spacer (ITS) sequences. METHODS: First, a total of 20 cryopreserved T. vaginalis isolates were thawed and genomic DNA was isolated from fresh cultures. A polymerase chain reaction (PCR) was performed to amplify the ITS regions and the amplicons were sequenced. These sequences were aligned with others from Genbank and polymorphisms were detected. At last, each ITS sequence was given a different sequence type. RESULTS: More than 99% homology was observed among sequences. Of 20 isolates, five had identical ITS sequence to reference (L29561) defined as ITST1. Moreover, 13 had A58 deletion (ITST10), one had C203T mutation (ITST2), and one had both A58 deletion and C203T mutation (ITST11). ITS typing of T. vaginalis sequences on Genbank revealed a total of 11 ITS types with the predominance of ITST1 (44.4%) globally. CONCLUSIONS: ITS typing seems to be an applicable and useful tool for a better understanding of molecular epidemiology as well as for the dissemination of T. vaginalis clones.

The Distribution of Blastocystis Subtypes among School-aged Children in Mugla, Turkey.

BACKGROUND: Blastocystis is a common protozoon that inhabits human intestinal tract and has a worldwide distribution. This study aimed to determine subtype (ST) distribution of Blastocystis among school-aged children in a western city of Turkey between Mar and Jun 2014. METHODS: This cross-sectional study was conducted among primary school children in Mugla between Mar and Jun 2014. Overall, 468 stool samples from children were examined by direct microscopy and inoculated into Jones medium. Blastocystis partial small subunit ribosomal RNA gene (SSU-rDNA) was amplified and sequenced from culture positive isolates. Subtypes were determined according to closest or exact match at GenBank and Blastocystis ST (18S) database. RESULTS: The positive rate of Blastocystis was 7.4% (n=35) with xenic in-vitro culture (XIVC). The subtypes could be identified for 33 (94.2%) isolates; 12 (34.2%) were ST3, 11 (31.4%) were ST1, 9 (25.7%) were ST2, one was (2.8%) ST7. No relationship was found between Blastocystis infected and non-infected cases in terms of gastrointestinal symptoms. Additionally, none of the possible risk factors was related to Blastocystis infection. CONCLUSION: Subtypes in children was similar to those reported in most of the studies that found ST3 as the most common subtype.

Determination of Trichomonas vaginalis Genotypes Using PCR-Restriction Fragment Length Polymorphism (RFLP).

OBJECTIVE: Trichomonas vaginalis is an anaerobic protozoon and the most common non-viral sexually transmitted pathogen. The present study was designed to determine the genotypes of T. vaginalis using polymerase chain reaction (PCR)-restriction fragment length polymorphism of actin gene. METHODS: A total of 20 isolates from symptomatic females isolated and cryopreserved at Adnan Menderes University, Research and Training Hospital Parasitology Laboratory were included. The isolates from liquid nitrogen were thawed and grown in trypticase-yeast extract-maltose medium prior to the study. Following nucleic acid extraction, the actin gene of T. vaginalis was amplified using nested PCR and amplicons were concentrated with phenol-chloroform-isoamyl alcohol precipitation. The final products were digested with HindII, MseI, and RsaI and were visualized using agarose gel electrophoresis. RESULTS: Most isolates were actin genotype E (n=9, 45%). The remaining isolates were genotype G (n=7, 35%), genotype N (n=1, 5%), and genotype H (n=1, 5%); two were mixed genotypes of E and H (10%). CONCLUSION: To the best of our knowledge, this study is the first to provide data on T. vaginalis genotypes in Turkey. However, further studies should be conducted to understand the molecular epidemiology of T. vaginalis at the national and global levels.

Blastocystis in ulcerative colitis patients: Genetic diversity and analysis of laboratory findings.

OBJECTIVE: To determine Blastocystis frequency and subtypes (ST) in ulcerative colitis (UC) patients and analyse some laboratory findings between Blastocystis positive and negative cases. METHODS: Faecal samples from 150 UC patients in Adnan Menderes University, Training and Research Hospital were examined by direct microscopy and cultivated in Jones medium. Blastocystis positive cultures were subjected to DNA isolation and subtypes were identified by sequencing of barcode region. A retrospective analysis was conducted on C reactive protein (CRP), leucocyte counts (WBC), neutrophil counts, and sedimentation rates. RESULTS: The overall positive rate of Blastocystis was 8% (12 patients) and the most abundant subtype was ST3 (eight isolates, 66.7%), followed by ST1, ST2 and ST7. Laboratory findings between Blastocystis infected and non-infected UC patients were not significantly different. Blastocystis frequency was 3.8% among the patients in active stage, while it was 11.8% among the patients in remission stage. CONCLUSIONS: The present study confirms previous findings that have indicated the predominance of Blastocystis ST3 in humans and contributes additional evidence that suggests the low colonisation of Blastocystis infection in ulcerative colitis patients during active stage.