The impact of Toxocara-seropositivity on attention and motor skills in children with attention-deficit hyperactivity disorder.

BACKGROUND: The study aims to compare neurological soft signs and executive functions between Toxocara-seropositive and seronegative groups in children with attention-deficit/hyperactivity disorder. METHODS: The study included 60 boys with ADHD, aged 7-12. After blood samples were taken, the Stroop Color Word Test and Judgment of Line Orientation test (JLOT) were implemented to measure executive functions. Neurological soft signs were evaluated with Physical and Neurological Examination for Subtle Signs (PANESS). RESULTS: Serological tests were positive for Toxocara antibodies in 20 cases. There was no significant difference between Toxocara seropositive and seronegative regarding age, socioeconomic status, developmental stages, and ADHD severity. However, Toxocara-seropositive children had higher Stroop time and Stroop interference scores and lower JLOT scores than Toxocara-seronegative children. Furthermore, Toxocara-seropositive children exhibited more neurological soft signs, such as gait and station abnormalities, dysrhythmia, and a longer total time in timed movements compared to Toxocara-seronegative children. CONCLUSION: Our study indicates a link between Toxocara-seropositivity and impaired neurological soft signs and executive functions in ADHD. Further research is needed to understand ADHD mechanisms, develop practical treatments considering immunological factors, and thoroughly evaluate how Toxocara seropositivity affects executive functions and motor skills in children with ADHD.

[Genetic Diversity of Blastocystis in Diarrheal Cases: Identification of Subtypes and Alleles]. / Ishalli Olgularda Blastocystis'in Genetik Çesitliligi: Alt tipler ve Alellerin Belirlenmesi.

Blastocystis spp. are the most common intestinal protozoan parasites detected in human stool samples. While identified long before today, its pathogenicity remains controversial. It is generally asymptomatic but in symptomatic cases, many gastrointestinal symptoms, especially diarrhea, have been associated with Blastocystis infection. In recent years, the relationship between the symptoms observed in cases and Blastocystis subtypes (ST) has been reported. The aim of this study was to detect Blastocystis in diarrheal cases admitted to the Aydin Adnan Menderes University Faculty of Medicine, Department of Parasitology Laboratory, to determine subtypes and allele diversity and to investigate its relationship with clinical symptoms. For this purpose, diarrheal stool samples of 200 cases were included in the study and their demographic characteristics (age, gender, residence) and clinical findings (abdominal pain, dyspepsia, nausea-vomiting, weakness, weight loss, anal itching, rash, urticaria) were recorded. Blastocystis was detected by direct microscope method (DM) and by molecular analyses which were performed with polymerase chain reaction (PCR). Subtype diversity was determined based on DNA sequence analysis by PCR targeting the Blastocystis ribosomal ribonucleic acid small subunit (SSU rRNA) gene. In addition, alleles related to Blastocystis subtypes were determined and statistically compared between all data and clinical findings. In the current study, Blastocystis was detected in 31 (15.5%) samples by DM and in 35 (17.5%) samples by PCR specific to the Blastocystis SSU rRNA gene among 200 diarrheal stool samples. No statistical difference was detected between Blastocystis and demographic characteristics. Dyspepsia and nausea-vomiting symptoms differed significantly in cases with Blastocystis compared to negative ones (p= 0.0025, p= 0.0498). Blastocystis subtype was detected in 33 samples by SSU rRNA sequence analysis, and the subtype distribution was ST1 (n= 10, 30.3%), ST2 (n= 4, 12.1%) and ST3 (n= 19, 57.6%). In the statistical evaluation between clinical findings and Blastocystis subtypes, a relationship was found between dyspepsia and Blastocystis ST3 (p= 0.0039). The allele diversity of Blastocystis subtypes was determined as allele 4 (10/10) in all ST1, allele 11 (2/4) and 12 (2/4) in ST2, allele 34 (14/19), 36 (4/19), and 38 (1/19) in ST3. In conclusion, our study provides important data on the molecular epidemiological characteristics of the Blastocystis by determining positivity, subtypes and alleles in diarrheal cases. Therefore, within the scope of the one health approach, comprehensive molecular epidemiological studies are required to determine the presence and genotypes of Blastocystis in human, animal and environmental samples.

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.

Arthropod vectors of disease agents: Their role in public and veterinary health in Turkiye and their control measures.

Mosquitoes, sandflies, and ticks are hematophagous arthropods that pose a huge threat to public and veterinary health. They are capable of serving as vectors of disease agents that can and have caused explosive epidemics affecting millions of people and animals. Several factors like climate change, urbanization, and international travel contribute substantially to the persistence and dispersal of these vectors from their established areas to newly invaded areas. Once established in their new home, they can serve as vectors for disease transmission or increase the risk of disease emergence. Turkiye (formerly Turkey) is vulnerable to climate change and has experienced upward trends in annual temperatures and rising sea levels, and greater fluctuations in precipitation rates. It is a potential hotspot for important vector species because the climate in various regions is conducive for several insect and acari species and serves as a conduit for refugees and immigrants fleeing areas troubled with armed conflicts and natural disasters, which have increased substantially in recent years. These people may serve as carriers of the vectors or be infected by disease agents that require arthropod vectors for transmission. Although it cannot be supposed that every arthropod species is a competent vector, this review aims to (1) illustrate the factors that contribute to the persistence and dispersal of arthropod vectors, (2) determine the status of the established arthropod vector species in Turkiye and their capability of serving as vectors of disease agents, and (3) assess the role of newly-introduced arthropod vectors into Turkiye and how they were introduced into the country. We also provide information on important disease incidence (if there's any) and control measures applied by public health officials from different provinces.

[An In Vitro Study on Sonodynamic Therapy of Leishmania tropica Using Curcumin]. / Kurkumin Kullanilarak Leishmania tropica'nin Sonodinamik Tedavisi Üzerine Bir In Vitro Çalisma.

Leishmaniasis is an infectious disease that is transmitted by Phlebotomus, 400 thousand new cases appearing every year, and approximately 350 million people are at risk, and accepted by the World Health Organization as one of the six important tropical diseases. Cutaneous leishmaniasis is a disease that occurs on exposed areas of the body and is characterized by long-term non-healing skin lesions. Although the treatment methods applied today vary according to the clinical picture of the patient, the immune system of the person and the causative agent Leishmania species, there is still no standard treatment scheme that has few side effects and can be used in the treatment of leishmaniasis. Therefore, alternative treatment methods with less side effects are being tried. Sonodynamic therapy (SDT) has also emerged as an active antimicrobial research area in recent years. SDT, a new modality for antibacterial therapy, aims to increase antibacterial effects with the simultaneous combination of low-intensity ultrasound and sonosensitizer. There is no information in the literature about the effect of SDT on parasites. In this study, it was aimed to demontrate the anti-leishmanial effect and possible mechanisms of curcumin mediated SDT on L.tropica promastigotes in vitro. Parasites were incubated with 0.25, 1.0, 4.0 and 15.6 micromolar (µM) of curcumin for one hour and subjected to 1 MHz frequency, 50% duty cycle and 3 W/cm2 intensity ultrasound irradiation. XTT assay was used to evaluate the viability of the cells and morphological changes were analyzed by Giemsa staining. Flow cytometry was used to quantify the fluorescence emitted by intracellular reactive oxygen species (ROS) signal, JC-1, cell cycle, Annexin V/PI staining reagents. With the combination of curcumin (15.6 µM) and ultrasound (3 W/cm2 intensity, seven minutes), L.tropica promastigote viability was found to be significantly decreased compared to the control group. Giemsa staining results showed that 15.6 µM curcumin mediated SDT induced several morphological alterations in L.tropica promastigotes typical for apoptosis. Late apoptosis was observed in 15.6 µM curcumin combined SDT treated parasites according to Annexin/PI staining. Besides, curcumin mediated SDT caused mitochondrial membrane potential (∆á´ªm) loss. Cell cycle analysis data indicated that curcumin based SDT caused an subG1 arrest in the cell cycle of L.tropica promastigotes. The generation of intracellular ROS detected by flow cytometry was increased in L.tropica promastigotes treated with curcumin mediated SDT. This study provided new data elucidating the molecular mechanism underlying the anti-leishmanial effect of curcumin mediated SDT. Curcumin mediated SDT has the potential to inactivate L.tropica promastigotes. However, further testing with amastigote or animal models is needed.

Efficacy of malachite green mediated photodynamic therapy on treatment of Cutaneous Leishmaniasis: In vitro study.

BACKGROUND: Leishmaniasis is a common zoonotic disease that is transmitted by phlebotomus and causes several clinical conditions, from self healing lesion to deadly internal organ involvement. Photodynamic therapy (PDT) is a treatment method that leads to the generation of cytotoxic species and consequently to cell death and tissue destruction by visible light in the presence of a photosensitizer and oxygen. The aim of this study was to investigate effect of malachite green (MG)-mediated PDT in Leishmania tropica (L. tropica) promastigotes. MATERIAL AND METHODS: Parasites were incubated with 0.19, 0.39, 1.56, 3.25 and 6.25 µM of MG for one hour and subjected to 46.4 J/cm2 light irradiation. Trypan blue assay was used to evaluate the viability of the cells and mitochondirial activity alteration was determined by MTT. Morphological changes were analyzed by Giemsa staining and Scanning electron microscopy (SEM) analyses. Flow cytometry was used to quantify the fluorescence emitted by cell volume, JC-1, Cell Cycle and Annexin V/PI staining reagents. RESULTS: Malachite green mediated photodynamic therapy at 1.56 and 3.125 µM decreased the viability of the L. tropica promastigotes and induced changes in the mitochondrial membrane potential. L.tropica promastigotes was bloked in G0/G1 phase. The morphology of the parasite was affected at the 1.56 and 3.125 µM MG+PDT, resulting in rounded cells with loss of flagellum and irregular shape. CONCLUSIONS: This study demonstrated that antileishmanial effects through mitochondrial dysfunction, cell cycle arrest, and apoptosis-like cell death to parasites. This work showed PDT with MG effectedparasites. Therefore, MG-mediated PDT may provide a promising approach for L. tropica promastigotes.

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.

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.

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.

[Determination of Antimony Resistance Mechanism of Leishmania tropica Causing Cutaneous Leishmaniasis in Turkey]. / Türkiye'de Kutanöz Leysmanyazis Etkeni Leishmania tropica'da Antimon Direnç Mekanizmasinin Belirlenmesi.

World Health Organization reported that approximately one billion people are at risk in endemic areas, one million cases of cutaneous leishmaniasis (CL) and approximately 300,000 cases of visceral leishmaniasis (VL) were reported per year in the last five years. The number of deaths due to VL is reported to be approximately 20,000 per year. Approximately 2500 cases/year have been reported as CL, caused by Leishmania tropica and Leishmania infantum, in Turkey. The significant increase observed in many cities mainly in the provinces of Mediterranean and Aegean regions in cases and foci in recent years, suggests that there may be an increase in this infections in the following years as well. In Turkey, the causative agent of CL is L.tropica and meglumine antimoniate is used in the treatment of CL. We aimed to determine antimony resistance genes specific for L.tropica by comparing the gene and protein expressions of antimony-resistant and non-resistant L.tropica strains. L.tropica isolates obtained from 3 CL patients without antimonate resistance from Aegean, Mediterranean and Southeastern regions of Turkey were provided to transform into 3 resistant isolates against meglumine antimony in the laboratory conditions. Gene expression alterations by microarray method; protein profiles by two-dimensional gel electrophoresis (2D-PAGE) and relevant proteins by MALDI-TOF/TOF MS of these isolates were accomplished and compared. L.tropica isolates from 10 CL patients who did not respond to antimony therapy were analyzed for resistance to antimonial compounds and quantitative real-time polymerase chain reaction was performed to detect the expression of genes responsible for resistance development. Moreover, differences in protein expression levels in isolates with and without antimony resistance were determined by comparing protein profiles and identification of proteins with different expression levels was carried out. Enolase, elongation factor-2, heat shock protein 70, tripanthione reductase, protein kinase C and metallo-peptidase proteins have been shown to play roles in L.tropica isolates developing resistance to antimonial compounds and similar expression changes have also been demonstrated in naturally resistant isolates from patients. In conclusion, it was revealed that L.tropica strains in our country may gain resistance to meglumine antimoniate in a short time. It is foreseen that if the patients living in our country or entering the country are treated inadequately and incompletely, there may be new, resistant leishmaniasis foci that may increase the number of resistant strains and cases rapidly.

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.