Search

Amaç: Toksoplasma gondii insan ve sıcakkanlı hayvanlarda hastalıklara neden olan bir protozoondur. Bu çalışmanın amacı toksoplazmozis tanısı için T.gondii tekrar bölgesine (RE) özgü primerlerin tasarlanması ile geliştirilen floresans esaslı hızlı LAMP testinin analitik hassasiyetini belirlemektir. Gereç ve Yöntem: PZR ile elde edilen T. gondii RE, pCR2.1-TOPO vektörüne klonlanmıştır. Spesifik primerler kullanılarak geliştirilen LAMP testinin analitik hassasiyeti pCR2.1-RE vektörünün seri seyreltmeleri ile belirlenmiştir. Bulgular: T. gondii RE genine özgü tasarlanan primerler ile geliştirilen LAMP testinin analitik hassasiyetinin 10≤kopya plazmit/reaksiyon olduğu tespit edilmiştir. Sonuç: Geliştirilen test 0,05 takizoit saptama limitine sahip olup oldukça hassas bir testtir. LAMP testinin kolay, ucuz, hızlı ve saha koşullarına uygun olması ve diğer moleküler testlerden daha hassas olması nedeniyle umut verici olduğu öngörülmektedir.

Gastrointestinal parasites of cats can affect animal health and welfare, as well as human health because of some zoonotic parasites including Toxoplasma gondii, Cryptosporidium spp., Isospora spp., Blastocystis sp., and Toxocara spp. Therefore, it is fairly important to investigate the presence of gastrointestinal parasites in stray cats in order to reveal the frequency of parasite diseases and to prevent the spread of parasitic diseases. A total of 465 feces samples were collected from Veterinary Clinics located in 5 different districts of İzmir. For microscopic examination, all feces samples were processed by centrifugation-sucrose flotation. In addition, cat feces with diarrhea were stained by the by Kinyoun acid-fast staining for the diagnosis of Cryptosporidium spp. As a result, 73 of 465 (15.6%) cats were found to be infected with at least one of the following parasites: Blastocystis sp., Isospora spp., Cryptosporidium spp., Toxoplasma gondii-like oocyte, Toxocara spp., Hymenolepis spp. and Dipylidium caninum. Among the studied stray cats, Blastocystis sp. was detected as the most prevalent protozoon parasite (10.5%) in stray cats. Overall, the results show that stray cats are a significant source for distribution of various parasite diseases to humans and animals in İzmir, Turkey.

This study aimed to investigate the Feline immunodeficiency virus (FIV) / Feline leukemia virus (FeLV) infection prevalence among looking healthy stray cats in Western Turkey by serologic and molecular-based tests. A total of 1008 blood samples from the stray cats were used in this study. All samples were tested for FIV antibodies / proviral DNA and FeLV antibodies / antigens / proviral DNA. The genetic characterization and phylogenetic analysis of FeLV and FIV were carried out in this study. These cats also tested for Leishmaniasis and Toxoplasmosis previously. FIV Ab and proviral DNA detected in 25.2 % and 25.5 % of samples, respectively. FeLV Ab, Ag, proviral DNA positivity was in 45.2 %, in 3.3 %, in 69.7 %, respectively. The molecular detection and phylo-genetic analysis of the current FeLV pol gene and FIV gag gene performed. The molecular characterization for the pol gene of FeLV (enFeLV and exFeLV) among Turkey's cat population was reported for the first time. The exFeLV pol sequences closer to the FeLV-A genotype, and the enFeLV pol sequences overlapped with other enFeLV. The current FIV gag sequences were clustered within the subtypes A, B, and C. The findings revealed FeLV subtype A and FIV subtype-A, subtype-B, subtype-C circulate among Turkish stray cats. Single and multiple co-infection positivity was found higher compared to previous reports.

BACKGROUND: Cryptosporidium spp. is a protozoan parasite that infects many vertebrate animals, including humans. Since Cryptosporidium spp. can cause chronic life-threatening diarrhea and severe malabsorption in immunocompromised patients, we investigated the prevalence of this parasite among patients undergoing chemotherapy for malignant solid tumors. OBJECTIVE: Investigate the prevalence of Cryptosporidium spp. in stool samples. DESIGN: Cross-sectional. SETTING: Tertiary care. PATIENTS AND METHODS: Stool samples were collected from adult patients with malignant solid tumors receiving chemotherapy and diarrhea. Cryptosporidium spp. prevalence was determined using Ziehl-Neelsen staining, ELISA, and real-time PCR targeting of the COWP gene. MAIN OUTCOME MEASURE: The prevalence of Cryptosporidium spp. in patients undergoing chemotherapy for malignant solid tumors. SAMPLE SIZE: 94 RESULTS: The prevalence was 2.1% (2/94), 5.3% (5/94), and 5.3% (5/94) as detected by Ziehl-Neelsen staining, real-time PCR and ELISA, respectively. The prevalence reached 8.5% (8/94) using all results obtained from the three methods. Among eight positive stool samples, four were positive by at least two different methods (Ziehl-Neelsen staining-ELISA or ELISA-real-time PCR) whereas the remaining four were positive by either ELISA or real-time PCR. CONCLUSION: These findings show the risk of cryptosporidiosis in cancer patients and the necessity to use at least two diagnostic methods during the diagnosis of cryptosporidiosis to reach more accurate and trustworthy results. LIMITATIONS: Further studies with a larger sample size are recommended. CONFLICT OF INTEREST: None., Izmir Katip Celebi University Scientific Research Projects Coordination Unit [x 2018-TDU-TIPF-0055], Izmir Katip Celebi University Scientific Research Projects Coordination Unit x 2018-TDU-TIPF-0055 to A.A.

Background Diarrhea caused by parasitic agents is common in neonatal calves and causes significant economic losses in cattle farms worldwide. Cryptosporidium spp. is one of the most frequently detected parasitic agents causing diarrhea in neonatal calves. Also, Giardia intestinalis is shown to cause diarrhea in calves. This study aimed to investigate the presence of Cryptosporidium spp. in calves (n:36), cows (n: 11), drinking water and two different artesian water supplies as well as in environmental swap samples (n:32) obtained from the manger, silage, bottle, and doorknob in a dairy farm which has big diarrhea problems. For this purpose, all fecal samples investigated with using direct microscopy for routine parasitological screening. Then, the presence of Cryptosporidium spp. was examined in feces samples and other environmental samples using Kinyoun acid-fast stained slides and Real-Time PCR targeting Cryptosporidium spp.. In addition, Real-Time PCR positive samples were investigated by nested PCR and subsequently by sequencing, BLAST, and phylogenetic analysis for species identification by MEGA7. Results Giardia intestinalis was detected in 10 feces samples (21,27%; 10/47) during routine microscopic examination. Among them, 9 belonged to calves older than two months without diarrhea, and one belonged to an adult cow. Cryptosporidium spp. was found in 11 calves (30.55%; 11/36) by Real-Time PCR, whereas no cows were found positive. Among the PCR positive samples, only five of them were detected as positive by microscopy. Cryptosporidium spp. positivity value was found higher in younger than two month old calves with diarrhea (9/12; 75%). Also, Cryptosporidium spp. was found in one of two water supplies and five of environmental samples by real-time PCR. Of the 18 real time positive samples, 8 were found positive by nested PCR and all positive samples were detected to be Cryptosporidium parvum by sequencing, BLAST, and phylogenetic analysis. Conclusions Our findings showed the importance of C. parvum infection in diarrhea cases occurred in calves. Besides the correct diagnosis and treatment of Cryptosporidium spp. contaminated water resources, and hygiene measures are very important for preventing the cryptosporidiosis in dairy farms.

BackgroundToxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, avian species and humans. Toxoplasmosis is asymptomatic in healthy individuals, whereas it may lead to death in immune suppressed or deficient patients. A vaccine against T. gondii is required to prevent consequences of the infection. the aim of this study is to generate a multivalent recombinant protein vaccine against T. gondii.Methods49 previously discovered antigenic proteins of T gondii were evaluated by their expression level in E. coli and by comprehensive bioinformatics analyses to determine antigenic epitopes. Based on these analyses, six vaccine candidate proteins were selected to generate a hexavalent recombinant protein vaccine adjuvanted with Montanide ISA 50V. Humoral and cellular immune responses were determined by flow cytometry and ELISA. Vaccinated mice were challenged with T. gondii Ankara strain tachyzoites.ResultsIn mice vaccinated with hexavalent vaccine, strong total IgG (P, Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [110S200], This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) grant 110S200 to AYG. the funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Pneumocystis jirovecii is an opportunistic respiratory pathogen causing Pneumocystis pneumonia (PcP) in immunocompromised patients. The aim of this study was to investigate the genetic diversity of P. jirovecii isolates (n: 84) obtained from PcP patients using multilocus sequencing method based on mt26S, SOD, and CYB loci. Among the 84 clinical samples that were positive for P. jirovecii DNA, 31 (36.90%) of them were genotyped using at least one locus. Of the 31 clinical samples, 26 of them were successfully genotyped using all loci whereas three samples were genotyped using either mt26S/CYB loci or mt26S/SOD loci. Additionally, there were two more clinical samples that were genotyped using CYB or SOD locus. Using mt26S locus, genotypes 2, 3, 7, and 8 were detected. Frequencies of genotype 7 and 8 were higher and both of them were found in 11 (n: 29; 37.93%) clinical samples. Using SOD locus, SOD 1, 2, and 4 genotypes were detected. SOD 1 was the predominant genotype (20/28; 71.42%). During the analyses of CYB locus, CYB 1, 2, 5, 6, and 7 as well as a new CYB genotype were detected. CYB 1 (16/29; 55.17%) and 2 (10/29; 34.48%) were the predominant genotypes. Overall, according to the multilocus sequencing results E, F, M, N, P, and V multilocus genotypes were detected among the PcP patients. In addition, SOD 1 was the predominant genotype and CYB had a more polymorphic locus. © 2020, Springer-Verlag GmbH Germany, part of Springer Nature., 2013TIP050, TGA-2019-20253, This study was supported by Ege University Scientific Research Projects Coordination Unit (Project Numbers: TGA-2019-20253 and 2013TIP050).

Introduction: Toxoplasma gondii is an opportunistic protozoan and can be grown using several human cell lines. Breast cancer is the second leading cause of cancer death in women. Her2/Neu-expressing mammary cancer cell lines called TUBO can be grown in vitro. in recent years, protozoan parasites have become popular means of use in cancer therapy research. in this study, we analyzed whether T. gondii tachyzoites can destroy TUBO cells using a novel continuous feed medium approach. Methodology: Two sets of flasks (each containing four groups) containing TUBO cells were inoculated with T. gondii Ankara strain tachyzoites. First set containing 5x10(6) TUBO cells were inoculated with TUBO-tachyzoite ratios of 1:2, 1:1, 2:1, and 4:1 and second set containing 1x10(6) TUBO cells were inoculated with TUBO-tachyzoite ratios of 10:1, 100:1, 1000:1, and 2000:1. Thereafter, culture supernatants were harvested at various days until TUBO cells were destroyed and tachyzoites were counted. Results: in the first and second sets of flasks, TUBO cells were destroyed between days 8 to 12 and 12 to 25, respectively. in addition, the amount of tachyzoites increased 743 and 595 to 112500 times in the first and second set of flasks, respectively. Conclusions: These results show that T. gondii tachyzoites successfully destroy Her2/Neu-expressing mammary cancer cells using a continuous feed medium approach. Although this idea may be too premature for the moment, the approach defined herein may support future researchers investigating the relationship between cancer and parasites which can make important progress toward saving cancer patient lives., Scientific Research Projects Branch Directorate of Ege University, TurkeyEge University [2016TIP082], The authors would like to acknowledge Prof. Federica Cavallo and Irene Fiore Merighi (University of Torino, Torino, Italy) for providing TUBO cells and technical assistance for the maintenance of TUBO cells in our lab. This study was partly supported by the grant given by the Scientific Research Projects Branch Directorate of Ege University, Turkey (Grant No: 2016TIP082) to L.Y.

PubMed ID: 30280697, Toxoplasma gondii is an obligatory intracellular protozoon parasite that causes toxoplasmosis in humans and all warm-blooded animals. In this study, we aimed to investigate the presence of T. gondii DNA in a Eurasian badger (Meles meles) that was found dead in the wildlife area of Izmir. According to the results of real time polymerase chain reaction, T. gondii REP gene was found to be positive in the Eurasian badger brain homogenate. In conclusion, Eurasian badger, a known carnivore, can be a potential source of toxoplasmosis in the natural settings of İzmir, Turkey.

Toxoplasma gondii can infect nearly all warm-blooded animals, including humans. In the laboratory diagnosis of toxoplasmosis, serological tests have importance in detecting antibody response. Traditionally T. gondii tachyzoites grown in vivo are being used as an antigen source in serological assays. Currently, tachyzoites produced in vitro are being tested as an antigen source in order to decrease animal use. Microcarrier technology allowed us to grow anchorage-dependent host cells on microcarrier suspension in short time and approximately 10 times more than traditional flask technique. The ability of T. gondii tachyzoites to grow in host cells adhered to microcarriers has not been analyzed yet. In this study, we aimed to develop a novel in vitro culture method to produce T. gondii tachyzoites abundantly using HeLa cells adhered to Cytodex 1 microcarriers. Initially, the growth of HeLa cells adhered to Cytodex 1 was analyzed using RPMI 1640, DMEM, and EMEM. Next, HeLa cells with a concentration of 1 × 10(5) cells/ml and 2 × 10(5 )cells/ml were adhered to Cytodex 1 and grown in spinner flasks. Then, T. gondii tachyzoites were inoculated with 1:1 and 2:1 cell:tachyzoite ratios to HeLa cells adhered to microcarriers in spinner flaks. During continuous production in spinner flasks, tachyzoites were harvested at the 2nd, 4th, and 7th day of culture and the quality of antigens produced from these tachyzoites were tested in ELISA and Western Blotting using sera of patients with toxoplasmosis. The optimization studies showed that finest HeLa inoculation value was 2 × 10(5 )cells/ml using RPMI 1640, and the cell:tachyzoite ratio to obtain the highest tachyzoite yield (17.1 × 10(7)) was 1:1 at the 4th day of inoculation. According to the results of ELISA comparing HeLa cell and mouse derived antigens, the highest correlation with mouse antigen was achieved at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio (P

Toxoplasma gondii is a protozoan parasite that can infect warm-blooded animals including humans. Vaccination studies mostly use tachyzoite specific proteins however in natural route of infection, toxoplasmosis initiates with tissue cysts (bradyzoites) or oocysts (sporozoites) and thereafter stage conversion takes place where the tachyzoites take action and cause acute infection continues with tachyzoites. Despite this knowledge, challenging models used in the vaccination studies prefer administration of tissue cyst forming strains intraperitoneally or subcutaneously instead of oral administration which is the natural route of infection. In the present study, a multivalent adjuvanted recombinant protein vaccine that contains bradyzoite specific BAG1 and tachyzoite specific GRA1 protein and controls were administered to female Swiss Webster outbred mice. Humoral and cellular immune responses were analyzed by Rec-ELISA, Western blot, and flow cytometry. Mice were infected orally with T. gondii PRU strain tissue cysts using feeding needle to mimic the natural route of infection. 40 days after challenging microscopy and Real Time PCR were performed to determine the protection level. Analysis of sera obtained from vaccinated mice showed strong anti-BAG1 and anti-GRA1 IgG responses. The IgG2a response was significantly higher (P < 0.0001) and the ratio of CD8 + T lymphocytes secreting IFN-? almost doubled compared to PBS control which are indicative of protection against toxoplasmosis. The amount of tissue cysts in vaccinated group was reduced 10.5% compared to control group. To generate a protective vaccine against toxoplasmosis, multistage vaccines and usage of challenging models mimicking natural route of infection are critical cornerstones. In this study, we generated a BAG1 and GRA1 multistage vaccine that induced strong immune response in which the protection was not at anticipated level. In addition, the murine model was orally challenged with tissue cysts to mimic natural route of infection. © 2015 Published by Elsevier Ltd., 110S200, This study was supported by the Grant given by The Scientific and Technological Research Council of Turkey (110S200) to Y.G. T. gondii PRU strain was kindly obtained from Prof. Marie-Laure Dardé. The authors would like to acknowledge AtaFen Inc., Veterinary Vaccine Production Plant and their team for their generous help through the project and Prof. Wolfgang Bohne for providing BAG1 DNA backbone. The topic of the study was presented in 23rd ECCMID (European Congress of Clinical Microbiology and Infectious Diseases) in Berlin, Germany, 2013. The authors state that they have no conflict of interest.

WOS: 000430290200079, PubMed ID: 29668747, Toxoplasma gondii is a protozoon parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations, in immune compromised humans. Analyses of the prevalence and genotyping of strains from the definitive host and intermediate hosts will help to understanding the circulation of the different strains and elucidating the role of the genotype (s) in human toxoplasmosis. Turkey has a specific geographic location bridging Africa, Europe, and Asia. We hypothesized that T. gondii strains may have been transferred to Turkey from these continents via migratory birds or vice versa. The present study aimed to assess the prevalence of toxoplasmosis in wild birds of prey of Izmir and Manisa provinces as well as genetically characterize T. gondii strains from these wild birds to show the relation between bird strains and neighboring stray cats as well as human strains previously isolated in Turkey. Tissues obtained from 48 wild birds were investigated for the presence of T. gondii DNA and then bioassayed in mouse. Isolated strains were genotyped using 15 microsatellite markers. The prevalence of T. gondii DNA was found to be 89.6% (n: 43/48) in wild birds. Out of 43 positive samples, a total of 14 strains were genotyped by 15 microsatellite markers. Among them, eight were type II, three were type III and three were mixture of genotypes (two type II/II and one was II/III). These are the first data that showed the presence of T. gondii and types II and III genotypes in wild birds of Turkey. Moreover, Africa 1 was not detected. In addition, cluster analysis showed that T. gondiistrains within type II and III lineage have close relation with strains previously isolated from stray cats in Izmir. Further studies are required to isolate more strains from human cases, other intermediate hosts, and water sources to reveal this relation., Scientific Research Projects Branch Directorate of Ege University, TurkeyEge University [2014-TIP-073], This study was supported by the grant given by the Scientific Research Projects Branch Directorate of Ege University, Turkey (Grant No: 2014-TIP-073) to A.Y.G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Toxoplasma gondii is an obligatory intracellular protozoon parasite that causes toxoplasmosis in humans and all warm-blooded animals. in this study, we aimed to investigate the presence of T. gondii DNA in a Eurasian badger (Meles meles) that was found dead in the wildlife area of Izmir. According to the results of real time polymerase chain reaction, T. gondii REP gene was found to be positive in the Eurasian badger brain homogenate. in conclusion, Eurasian badger, a known carnivore, can be a potential source of toxoplasmosis in the natural settings of İzmir, Turkey., Toxoplasma gondii, insanlarda ve tüm sıcakkanlı hayvanlarda toksoplazmozise neden olan zorunlu hücre içi protozoon bir parazittir. Bu çalışmada, İzmir’in yaban hayatında ölü olarak bulunan bir Avrasya porsuğunda (Meles meles) T. gondii DNA varlığının araştırılması amaçlanmıştır. Real time Polimeraz Zincir Reaksiyonu (PZR) sonucuna göre T. gondii REP geni Avrasya porsuğunun beyin homojenatında pozitif bulunmuştur. Sonuçta önemli bir etobur olan Avrasya porsuğunun İzmir’in doğal çevresinde potansiyel bir toksoplazmoz kaynağı olabileceği gösterilmiştir.

Amac: Kan yoluyla bulasan enfeksiyon etkenleri arasinda hepatit B virusu (HBV) onemini korumaktadir. Serolojik testlerin duyarliliginin arttirilmasina ragmen guvenli kan transfuzyonu icin kanda HBV-DNA varligini saptayan nukleik asit testi (NAT) uygulamalari, ozellikle de tek tek orneklere uygulanan NAT (ID-NAT) uygulamalari gundeme gelmistir. Gerec ve Yontem: Ege Universitesi Tip Fakultesi Kan Merkezi ve Mersin Universitesi Tip Fakultesi Saglik Arastirma Uygulama Hastanesi Kan Merkezi'ne Nisan 2010 ile Ocak 2011 tarihleri arasinda basvuran 18–65 yaslari arasindaki 4352 HBsAg negatif kan vericisinin serum ornekleri incelenmek uzere calismaya alindi. Kan vericilerinin orneklerinde HBV-DNA'yi saptamak amaciyla gercek zamanli polimeraz zincir reaksiyonu (RT-PCR) testi uygulandi. Bulgular: Calisilan orneklerden sadece 2 serumda HBV-DNA supheli pozitif olarak saptandi. Iki pozitif sonuc ayni yontemle iki kez ve farkli alternatif yontemlerle dogrulamak amaciyla tekrarlandi. Tekrarlanan orneklerin negatif bulunmasi sonucunda orneklerin tumunde HBV-DNA negatif olarak degerlendirildi. Iki ayri merkezden alinan HBsAg negatif kan vericilerinin tum orneklerinde HBV-DNA negatif bulundu. Sonuc: Calismaya alinan kan vericilerin sayisi ve merkezlerdeki dusuk seroprevalans gizli HBV gorulmemesinin nedeni olabilir. Turk Kizilayi'nin 2014'de baslayan rutin NAT calismalari bolgemizdeki gizli HBV verilerini daha net ortaya cikaracaktir.

Background Detection of Pneumocystis jiroveci colonization in lungs or oral samples due to high sensitivity of PCR methods results in undue treatment of patients without any symptoms of Pneumocystis pneumonia. The aim of the present study is to demonstrate Pneumocystis carinii in rats, immune suppressed by oral and subcutaneous administration of dexamethasone. Material/Methods Blood, oral, nasal and eye swabs were collected prior to immune suppression and 2, 6, 12 weeks after administration of dexamethasone. Also, samples were collected from lung, heart, liver, kidney, diaphragm, brain, spleen, tongue, muscle, eye, intestine, and feces. Cysts and trophozoites were investigated in stained slides and MSG gene was detected by PCR. Results The results showed that weight loss is significantly higher in rats administered oral dexamethasone (P

The diagnosis of Toxoplasma infection or disease in hematopoietic stem cell transplantation (HSCT) patients is achieved mainly by PCR screening; however screening did not find wide field of use in practice due to costly expenditures of PCR. This study aimed to determine patients at high risk of Toxoplasma infection or disease before transplantation by stem cell originated buffy coat PCR and subsequently to screen them. Buffy coats collected from 12 autologous and 18 allogeneic HSCT patients' donors were investigated by PCR before transplantation. After transplantation, blood and sera collected at fixed time intervals were screened by two PCR methods and serological assays. Screening results first time assessed a toxoplasmosis incidence level as 25% in autologous HSCT patients and increased incidence level in allogeneic HSCT patients to 22%. Importantly, buffy coat PCR was first time performed before transplantation, to determine the risk of toxoplasmosis. Buffy coat PCR results showed that four patients were at high risk of toxoplasmosis before transplantation. After transplantation, these patients experienced toxoplasmosis. In conclusion, for the determination of patients at risk of toxoplasmosis, clinicians should consider buffy coat PCR in combination with serology before transplantation. After transplantation, PCR screening can be initiated in high risk patients upon clinical suspicion.

WOS: 000378184000010, PubMed ID: 27175499, Cystic echinococcosis (CE) and alveolar echinococcosis (AE) caused by Echinococcus granulosus and Echinococcus multilocularis, respectively, are important helminthic diseases worldwide as well as in our country. Epidemiological studies conducted in Turkey showed that the prevalence of CE is 291-585/100.000. It has also been showed that the seroprevalence of AE is 3.5%. For the diagnosis of CE and AE, radiological (ultrasonography, computed tomography, magnetic resonance) and serological methods, in addition to clinical findings, are being used. The definitive diagnosis relies on pathological examination When the hydatid cysts are sterile or does not contain protoscolex, problems may occur during pathological discrimination of E.granulosus and E.multilocularis species. In this study, we aimed to develop a novel multiplex real-time polymerase chain reaction (M-RTPCR) targeting mitochondrial 12S rRNA gene of E.granulosus and E.multilocularis using Echi S (5'-TTTATGAATATTGTGACCCTGAGAT-3') and Echi A (5'-GGTCTTAACTCAACTCATGGAG-3') primers and three different probes;. Anchor Ech (5'-GTTTGCCACCTCGATGTTGACTTAG-fluoroscein-3'), Granulosus (5'-LC640-CTAAGGTTTTGGTGTAGTAATTGATATTTT-phosphate-3') and Multilocularis (5'-LC705-CTGTGATCTIGGTGTAGTAGTTGAGATT-phosphate-3') that will enable the diagnosis of CE and AE in same assay. During M-RTR-PCR, plasmids containing E.granulosus (GenBank: AF297617.1) and E.multilocularis (GenBank: NC_000928.2) mitochondrial 12S rRNA regions were used as positive controls. Cysts samples of patients which were pathologically confirmed to be CE (n: 10) and AE (n: 15) and healthy human DNA samples (n: 25) as negative control as well as DNA samples of 12 different parasites (Taenia saginata, Hymenolepis nana, Trichuris trichiura, Fasciola hepatica, Enterobius vermicularis, Toxoplasma gondii, Pneumocystis jirovecii, Trichomonas vaginalis, Cryptosporidium hominis, Strongyloides stercoralis, Plasmodium falciparum, Plasmodium vivax) were used to develop M-RT-PCR. E.granulosus and E.multilocularis control plasmids were constructed to detect analytic sensitivity of the test using TOPO cloning. Positive control plasmids were diluted to determine analytical sensitivity and specificity by distilled water at 10(6)-10(5)-10(4)-10(3)-10(2)-10(1)-1 plasmid copy of dilution in each reaction. According to the results, analytical sensitivity of the assay for E.granulosus and E.multilocularis was 1 copy plasmid/mu l reaction. The non-existence of cross reactivity with 12 different parasites' DNA samples showed the analytical specificity of the assay. Displaying Echinococcus DNA in cyst samples among 25 patients and species discrimination as well as non-existence of cross reactivity with human DNA samples showed that the clinical sensitivity and specificity of the assay were 100%. As a result, the M-RT-PCR developed in the present study provided a sensitive, specific, rapid, and reliable method in the diagnosis of echinococcosis and the discrimination of E.granulosus and E.multilocularis from cyst samples.

PubMed ID: 27905280, OBJECTIVE: Malaria is an important tropical disease that is detected in 198 million people and causes 367-755 thousand deaths annually. Recently, the real-time Polymerase Chain Reaction (PCR) technique has enabled quick determination of Plasmodium spp. and species identification in the same assay with a low contamination risk. In the present study, we aimed to use real-time PCR targeting the 18S rRNA gene to diagnose Plasmodium spp. and perform species identification.METHODS: DNA samples of 15 patients with malaria (14 caused by P. vivax, 1 caused by P. falciparum) confirmed by microscopy as well as positive control plasmids were used. As the negative control, DNA samples of 15 individuals without malaria were used.RESULTS: According to the results of real-time PCR, samples of 15 patients with malaria were found to be positive for Plasmodium spp. Melting curve analysis showed that 14 of them were P. vivax and the remaining was P. falciparum. In addition, mixed infection with P. falciparum and P. vivax was successfully detected by real-time PCR when DNA of P. falciparum- and P. vivax-positive samples was experimentally mixed.CONCLUSION: The present study showed that real-time PCR can be useful in the diagnosis and species identification of Plasmodium spp. as well as the detection of mixed infections in addition to microscopy in Turkey.

Dünyada ve ülkemizde Echinococcus granulosus ve Echinococcus multilocularis'in neden olduğu sırasıyla kistik ekinokokkoz (KE) ve alveolar ekinokokkoz (AE) önemli helmint hastalıkları arasındadır. Türkiye'de yapılan epidemiyolojik çalışmalar KE prevalansının 291-585/100.000 arasında olduğunu göstermiştir. AE seroprevalansının da %3.5 oranında olduğu bildirilmiştir. KE ve AE tanısında klinik bulgular yanında genelde radyoloji (ultrason, bilgisayarlı tomografi , manyetik rezonans) ve serolojik yöntemler kullanılmaktadır. Kesin tanı patolojik incelemeye dayalıdır. Hidatik kistlerin steril olması ya da protoskoleks içermemesi durumunda ise, patolojik tekniklerle E.granulosus ve E.multilocularis tür ayrımında zorluklar yaşanmaktadır. Bu çalışmada, aynı test içerisinde AE ve KE tanısının yapılmasına imkan verebilecek Echi S (5'-TTTATGAATATTGTGACCCTGAGAT-3') ve Echi A (5'-GGTCTTAACTCAACTCATGGAG-3') primerleri ve üç farklı prob; Anchor Ech (5'-GTTTGCCACCTCGATGTTGACTTAG-fl oresan-3'), Granulosus (5'-LC640-CTAAGGTTTTGGTGTAGTAATTGATATTTT-fosfat-3') ve Multilocularis (5'-LC705CTGTGATCTTGGTGTAGTAGTTGAGATT-fosfat-3') kullanılarak, E.granulosus ve E.multilocularis mitokondriyal 12S rRNA genini hedefl eyen yeni bir multipleks gerçek zamanlı polimeraz zincir reaksiyonu (M-RTPCR) yönteminin geliştirilmesi amaçlanmıştır. M-RT-PCR sırasında, E.granulosus (GenBank: AF297617.1) ve E.multilocularis (GenBank: NC_000928.2) mitokondriyal 12S rRNA gen bölgelerini içeren plazmidler pozitif kontrol olarak kullanılmıştır. M-RT-PCR yönteminin geliştirilmesinde ayrıca, patolojik olarak KE (n: 10) ve AE (n: 15) olduğu doğrulanmış hastalara ait kist örneklerinin yanı sıra, negatif kontrol olarak sağlıklı insan DNA örnekleri (n: 25) ve 12 farklı parazite (Taenia saginata, Hymenolepis nana, Trichuris trichiura, Fasciola hepatica, Enterobius vermicularis, Toxoplasma gondii, Pneumocystis jirovecii, Trichomonas vaginalis, Cryptosporidium hominis, Strongyloides stercoralis, Plasmodium falciparum, Plasmodium vivax) ait DNA örnekleri de çalışılmıştır. Testin analitik duyarlılığının saptanması için TOPO klonlama ile E.granulosus ve E.multilocularis pozitif kontrol plazmidleri oluşturulmuştur. Pozitif kontrol plazmidi, analitik duyarlılık ve özgüllüğün belirlenmesi amacıyla her reaksiyonda 106-105-104-103-102-101-1 plazmid kopya olacak şekilde distile su ile sulandırılmıştır. Çalışmamızın sonuçları, testin pozitif kontrol plazmidi kullanılarak elde edilen analitik duyarlılığının, E.granulosus ve E.multilocularis için 1 plazmid kopya/?l örnek olduğunu göstermiştir. Testin 12 farklı parazite ait DNA örneği ile çapraz reaksiyon vermemesi, analitik özgüllüğünü ortaya koymuştur. Yirmibeş hastaya ait kist örneğinde Echinococcus DNA varlığının gösterilmesi ve tür ayrımının yapılabilmesinin yanında, sağlıklı (negatif kontrol) insan DNA örnekleri ile çapraz reaksiyon vermemesi, testin klinik duyarlılık ve özgüllüğünün %100 olduğunu göstermiştir. Sonuç olarak, bu çalışmada geliştirilen M-RT-PCR yönteminin, kist örneklerinde ekinokokkoz tanısının konulmasına ek olarak, E.granulosus ve E.multilocularis'in ayırt edilmesinde duyarlı, özgül, hızlı ve güvenilir bir yöntem olduğu tespit edilmiştir, Cystic echinococcosis (CE) and alveolar echinococcosis (AE) caused by Echinococcus granulosus and Echinococcus multilocularis, respectively, are important helminthic diseases worldwide as well as in our country. Epidemiological studies conducted in Turkey showed that the prevalence of CE is 291-585/100.000. It has also been showed that the seroprevalence of AE is 3.5%. For the diagnosis of CE and AE, radiological (ultrasonography, computed tomography, magnetic resonance) and serological methods, in addition to clinical fi ndings, are being used. The defi nitive diagnosis relies on pathological examination When the hydatid cysts are sterile or does not contain protoscolex, problems may occur during pathological discrimination of E.granulosus and E.multilocularis species. In this study, we aimed to develop a novel multiplex real-time polymerase chain reaction (M-RTPCR) targeting mitochondrial 12S rRNA gene of E.granulosus and E.multilocularis using Echi S (5'-TTTATGAATATTGTGACCCTGAGAT-3') and Echi A (5'-GGTCTTAACTCAACTCATGGAG-3') primers and three different probes; Anchor Ech (5'-GTTTGCCACCTCGATGTTGACTTAG-fl uoroscein-3'), Granulosus (5'-LC640-CTAAGGTTTTGGTGTAGTAATTGATATTTT-phosphate-3') and Multilocularis (5'-LC705-CTGTGATCTTGGTGTAGTAGTTGAGATT-phosphate-3') that will enable the diagnosis of CE and AE in same assay. During M-RTR-PCR, plasmids containing E.granulosus (GenBank: AF297617.1) and E.multilocularis (GenBank: NC_000928.2) mitochondrial 12S rRNA regions were used as positive controls. Cysts samples of patients which were pathologically confi rmed to be CE (n: 10) and AE (n: 15) and healthy human DNA samples (n: 25) as negative control as well as DNA samples of 12 different parasites (Taenia saginata, Hymenolepis nana, Trichuris trichiura, Fasciola hepatica, Enterobius vermicularis, Toxoplasma gondii, Pneumocystis jirovecii, Trichomonas vaginalis, Cryptosporidium hominis, Strongyloides stercoralis, Plasmodium falciparum, Plasmodium vivax) were used to develop M-RT-PCR. E.granulosus and E.multilocularis control plasmids were constructed to detect analytic sensitivity of the test using TOPO cloning. Positive control plasmids were diluted to determine analytical sensitivity and specifi city by distilled water at 106-105-104-103-102-101-1 plasmid copy of dilution in each reaction. According to the results, analytical sensitivity of the assay for E.granulosus and E.multilocularis was 1 copy plasmid/?l reaction. The non-existence of cross reactivity with 12 different parasites' DNA samples showed the analytical specifi city of the assay. Displaying Echinococcus DNA in cyst samples among 25 patients and species discrimination as well as non-existence of cross reactivity with human DNA samples showed that the clinical sensitivity and specifi city of the assay were 100%. As a result, the M-RT-PCR developed in the present study provided a sensitive, specifi c, rapid, and reliable method in the diagnosis of echinococcosis and the discrimination of E.granulosus and E.multilocularis from cyst samples

Malaria was expected to be a major problem during blood donation in Turkey due to existence of malaria cases in southeastern region of Turkey. The present study aimed for the first time, to investigate malaria in "donors deferred for malaria risk" and to determine the regional rates of malaria deferral in Turkey. Blood samples were collected from several Blood Banks of southeastern provinces where local malaria cases still exist and from Blood Bank of Ege University Medical School (EUMS) located in western Turkey where malaria is eradicated decades ago. Plasmodium spp. and specific antibodies were investigated by stained smears, antigen detection, PCR and ELISA. Among the donors deferred for malaria risk, Plasmodium spp. were not detected by microscopy, PCR or antigen detection. Seroprevalances were 2% and 3.92% in western and southeastern regions, respectively. Rate of donor deferral for malaria risk was 0.9% in EUMS and deferrals were exclusively because of travel to southeastern Turkey. In southeastern provinces, deferrals were mainly due to malaria like fever history. The present study first time assessed regional rates of donor deferral due to malaria risk in Turkey. Previously, malaria was expected to be a major problem during blood donation in Turkey due to existence of malaria cases in southeastern region of Turkey. The results of the study showed that 97% of the deferrals were unnecessary. In conclusion, to reduce unnecessary donor deferrals in Turkey, in addition to comprehensive questioning for malaria history, the usage of a malaria antibody screening method should be initiated prior to deferral decision.

Routine laboratory diagnosis of Pneumocystis jirovecii is currently achieved by PCR in almost all laboratories with sufficient equipment due to its high sensitivity and specificity compared to staining methods. A current issue that limits the reliability and sensitivity of PCR is the degree of inhibition caused by inhibitory substances in respiratory samples. The present study aimed to analyse the degree and frequency of inhibition in real-time PCR detecting P. jirovecii in respiratory specimens submitted to a Pneumocystis pneumonia (PcP) diagnosis laboratory in Ege University Medical School, Turkey. Between July 2009 and December 2010, 76 respiratory specimens [63 bronchoalveolar lavage (BAL) fluid, 10 sputum samples, two tracheal aspiration fluid and one thoracentesis fluid] obtained from 69 PcP-suspected patients were investigated for the presence of P. jirovecii using real-time PCR targeting the cdc2 gene. Of these samples, 42 of the specimens were stained and examined by microscopy according to the request of the clinicians. PCR was positive in 15 specimens in the initial run. Of the remaining 61 samples, 41 of them were negative with positive internal inhibition controls (i.e. true-negative group). The frequency of inhibition in the initial run was 26.31 % (20/76) as determined by spiked negative controls. All of the inhibited samples were resolved after 1 : 2, 1 : 5, 1 : 10 and 1 : 20 dilutions. P. jirovecii was detected by PCR in two inhibited specimens after retesting with diluted samples which were also positive by microscopy. The incidence of P. jirovecii in respiratory specimens was 22.36 % (17/76) as determined by real-time PCR and 7.14 % (3/42) by microscopy. Overall, the incidence of P. jirovecii in respiratory samples was 23.68 % (18/76) as detected by both methods. In conclusion, inclusion of spiked positive controls in each sample and retesting with diluted samples to resolve inhibition increased the reliability of the real-time PCR assay in terms of determining false-negative results and influencing the treatment of the patient. Furthermore, results of the present study determined for the first time the frequency and degree of inhibition in a real-time PCR detecting P. jirovecii in respiratory specimens during routine diagnosis of PcP.

Toxoplasma gondii is one of the most researched parasite due to its easy growth both in vitro and in vivo. Tachyzoites, derived from mouse or rat peritoneum encounters ethical and economical problems when used for research or diagnostic purposes. Currently, research has focused on determining the most suitable cell culture environment to reach highest amount of viable tachyzoites with least host cell contamination. However, gene expression changes that take place throughout the adaptation of evolving T. gondii strains to continuous cell cultures appear as a problem. The present study aimed to determine a novel cell culture strategy for T. gondii RH Ankara strain tachyzoites to harvest abundant tachyzoites with least host cell contamination and minimal antigenic variation at predetermined dates to use as an antigen source in serological assays that will facilitate reduction in animal use. To achieve this purpose, T. gondii RH Ankara strain tachyzoites were incubated with HeLa cell at different ratios for two or three days. In all flasks incubated for two days, viability rate reached to 100% and HeLa cell contamination decreased to levels between 0.12–0.5 × 106/ml. In the flasks with HeLa-tachyzoite ratio 1/8, the tachyzoite yield and viability ratio were 3 × 106/ml and 100%, respectively, with accompanying 10 fold decrease (0.12 × 106/ml) in HeLa contamination. During continuous production, highest tachyzoite yield was obtained from the first passage (3.55 × 106/ml) and until the end of third subculture viability rates and HeLa cell contaminations were between 98.2–99.4% and 0.31–0.37 × 106/ml, respectively. ELISA, IFA and Western blot analyses showed that the quality, specificity and sensitivity of the antigen harvested from the first passage of cell culture performed at two days intervals were comparable to the antigen harvested from mice and decreased in the following subcultures. Overall, these results demonstrated that T. gondii RH Ankara strain is still evolving to adapt to cell culture environment and therefore such strains continuously produced in cell cultures should be avoided for serological assays. However, the two day short interval cell culture method described herein offers a chance to reduce the animal use intended for the preparation of serological assays’ antigen from local evolving strains.

The successful folding of a recombinant protein after expression and purification is essential for structural, biochemical and vaccination studies. Toxoplasma gondii recombinant GRA1 protein is a promising vaccine candidate against toxoplasmosis. In the present study, the folding of recombinant GRA1 protein has been evaluated by web based bioinformatics tools that predict protein folding. Subsequently, trypsin digestion, which is a simple indication of proper protein folding, has been used to determine whether recombinant GRA1 protein is likely to be folded. The results indicate that the recombinant GRA1 protein is predicted to be folded by most of the web based bioinformatics predictors. Moreover, in protease digestion experiments, the recombinant GRA1, which was purified to homogeneity without the use of denaturants, gives rise to a discrete band pattern that is indicative of a folded protein. Together, the results suggest that recombinant GRA1 protein is in a folded conformation, suitable for structural, biochemical and vaccination studies.

Toxoplasmosis is a serious and potentially life-threatening disease in liver transplant recipients while they are immunosuppressed. We report the clinical and laboratory findings related to active toxoplasma infection associated with 40 immunosuppressed liver transplant procedures that took place over a 12-month period at a major transplant unit in Izmir, Turkey. Twenty-seven (67.5%) of the 40 transplant recipients were found to be seropositive for toxoplasma infection and therefore at risk of reactivated infection. From the serological status of the donors, which was ascertained in 38 of 40 cases, we identified 3 (7.9%) of 38 transplants to be from a seropositive donor to a seronegative recipient. In 10 (26.3%) of 38 transplants, both the donor and recipient were seronegative, and this excluded toxoplasma as a risk. A comparison of real-time polymerase chain reaction (PCR) and nested PCR was undertaken in combination with a range of serological assays (the Sabin-Feldman dye test, enzyme immunoassay immunoglobulin M, and immunosorbent agglutination assay immunoglobulin M). Ethylene diamine tetraacetic acid blood samples from 3 of the 30 recipients at risk from toxoplasma were found positive by PCR, but only 1 of these was found positive in both assays. Among the 3 PCR-positive patients, immunoglobulin M and immunoglobulin G antibody levels increased in only 1 patient. Correlations between symptoms, laboratory findings, and clinical management (use of anti-toxoplasma therapy) are presented. Our findings suggest that toxoplasma presents a significant risk to our liver transplant population and that PCR is a helpful addition in identifying active infections and hence in informing clinical management decisions.

WOS: 000347840000013, PubMed ID: 25790534, Background/aim: The West Nile virus (WNV) is a mosquito-borne flavivirus causing different forms of infection among humans, varying from asymptomatic illness to fetal central nervous system infection. Turkey lies within an endemic region for WNV. Transfusion of infected blood products is another well-documented major route of transmission. The aim of our study was to investigate the presence of WNV viremia among a healthy donor population from the western part of the country. Materials and methods: A total of 438 healthy volunteer blood donors were included in the study. The presence of WNV RNA was investigated by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) and anti-WNV IgG was detected by a commercial ELISA test. Results: Ages of volunteer donors were 18-62 years (mean: 34.7) and 34 (7.76%) were women. All samples were negative for WNV RNA by qRT-PCR. Eleven (2.51%) samples, 1 of which was borderline, were positive for anti-WNV IgG. All positive samples were from the western part of the country and 9 of them were from Izmir. Conclusion: Although all donor samples were negative for WNV RNA by qRT-PCR, the risk of WNV transmission via blood products should not be ignored in endemic regions.

Plasmodium vivax is the most common cause of malaria worldwide as well as southeastern Turkey. After the implementation of a successful national elimination program that the local malaria cases were not reported in 2011, malaria returned to county of Savur located in southeastern Turkey in summer of 2012. The present study aimed to determine the prevalent P. vivax genotypes isolated from southeastern Turkey. Genetic polymorphism in P. vivax CSP gene was analyzed by PCR-RFLP to assess the ratio of VK210 and VK247 types. Blood samples were obtained from 15 patients who lived in southeastern between 2005-2006. According to the results, VK210 type was detected in 10 samples (66.6%), VK247 type was observed in three samples (20%). Remaining two samples showed mixed infection (13.3%). The results of the present study first time showed the ratio of P. vivax genotypes in southeastern Turkey before the elimination in 2011. The results of the present study will be enable researchers to compare the new isolates with the previously detected ones and design new treatment and/elimination strategies.

Toxoplasma gondii tachyzoites and tissue cysts are largely used for developing diagnostic assays, vaccines and in drug research as well as biochemical and molecular structure studies. Continuous passaging of tachyzoites or tissue cysts in animal models encounter ethical and economical problems and it is a time consuming procedure. Cryopreservation of tachyzoites and tissue cysts and revitalization of cryopreserved samples whenever needed, can decrease the economical loss, ethical problems and labour. In the present article, production of tachyzoites and tissue cysts in mice, preparation of samples for cryopreservation, cryopreservation of tachyzoites and tissue cysts, defrosting of cryopreserved samples and reinoculation to mice have been described in detail.

WOS: 000343671700080, PubMed ID: 25268351, Toxoplasma gondii causes congenital toxoplasmosis in newborns resulting with fetal anomalies. Determining the initiation time of infection is very important for pregnant women and current serological assays have drawbacks in distinguishing the recently acute toxoplasmosis. Diagnosis of recently acute infection may be improved by using stage specific antigens in serological assays. In the present study, the diagnostic value of sporozoite specific SporoSAG, bradyzoite specific BAG1 proteins and GRA1 protein expressed by all forms of the parasite have been evaluated ELISA using sera systematically collected from mice administered orally with tissue cyst and oocysts. The anti-SporoSAG IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 1, 10, and 15 (P, Scientific and Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [107S359]; Scientific Research Projects Branch Directorate of Ege UniversityEge University [2007TIP015], This study was supported partly by the grants given by The Scientific and Technological Research Council of Turkey (107S359) and the Scientific Research Projects Branch Directorate of Ege University (2007TIP015) to Yuksel Guruz. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

WOS: 000340879300061, PubMed ID: 25127360, Currently, some Toxoplasma gondii genotypes are being associated with serious clinical presentations. A recent report showing the Africa 1 genotype in two local congenital toxoplasmosis cases acquired in Turkey formed the basis of this study because atypical Africa 1 genotype is most frequently detected in animals and patients from sub-Saharan Africa. Since stray cats are considered as the linkage between wild life and urban life in T. gondii transmission, the present study aimed to isolate and characterize T. gondii strains circulating in stray cats of Izmir (Western Turkey). A secondary objective was to determine toxoplasmosis seroprevalence in this cat population. Tissues obtained from 100 deceased stray cats were bioassayed and isolated strains were genotyped using 15 microsatellite markers. In addition, toxoplasmosis seroprevalence was analyzed in 1121 cat sera collected from several large veterinary clinics in Izmir. Among the 22 isolates, 19 were Type II (86.3%), two were Type III (9%) and one was Africa 1 genotype (4.5%). The overall seropositivity rates in cats were 42-48% and 33.4-34.4% according to IFA and ELISA, respectively. Seroprevalence in deceased cats was significantly higher than in healthy cats (P = 0.0033). Finding both the major clonal Type II lineage together with the Type III lineage also found in Middle East, and an atypical genotype, Africa 1 appears consistent with the specific geographic location of Turkey between three continents and raises the possibility of transportation of these strains between continents through trade routes or long distance migratory birds. In addition, the first large study of toxoplasma seroprevalence in a stray cat population was also reported. The relatively high seropositivity rates and the variety of T. gondii genotypes confirm the local stray cat population as a risk factor for human toxoplasmosis in Izmir., Scientific Research Projects Branch Directorate of Ege University, TurkeyEge University [2010-TIP-091, 2011-TIP-034], This study was supported by the grants given by the Scientific Research Projects Branch Directorate of Ege University, Turkey (Grant No: 2010-TIP-091 and 2011-TIP-034) to Y.G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

OBJECTIVE In the present study, the aim is to demonstrate Cryptosporidium parvum 18S small-subunit rRNA gene, in lung and stool samples of immune suppressed rats. This gene region is specific for Cryptosporidium spp. and thus can be used in humans for routine diagnostic procedures. METHODS Three groups (n=4) of Rattus norvegicus rats were used. The first and second groups were administered dexamethasone, subcutaneously and orally, respectively, for 12 weeks. Rats in the control group were not immune suppressed. Lung and stool specimens were obtained from rats at the end of 12 th week and examined for the presence of C. parvum DNA using Nested PCR. RESULTS C. parvum DNA was demonstrated in lung and stool samples of rats which were immune suppressed by oral dexamethasone. On the other hand, C. parvum DNA was demonstrated only in stool specimens of the rats which were immune suppressed by subcutaneous dexamethasone. No band pattern was observed in the specimens of the control group. CONCLUSION The results of the study showed that oral dexamethasone administration was more efficient in generating disseminated cryptosporidiosis in rats compared to subcutaneous dexamethasone administration. In addition, Nested PCR targeting 18S small-subunit rRNA gene can be used to detect Cryptosporidium spp. in respiratory and stool specimens of animals and humans.

International audience; INTRODUCTION: Toxoplasma gondii is a protozoon parasite that has a worldwide dissemination. It can cause serious clinical problems such as congenital toxoplasmosis, retinochoroiditis, and encephalitis. Currently, T. gondii genotypes are being associated with these clinical presentations which may help clinicians design their treatment strategy. CASE REPORTS: Two T. gondii strains named Ankara and Ege-1 were isolated from newborns with congenital toxoplasmosis in Central and Western Anatolia, respectively. Ankara and Ege-1 strains were isolated from the cerebrospinal fluid of newborns. According to microsatellite analysis, Ankara and Ege-1 strains were sorted as Africa 1 genotype. CONCLUSION: T. gondii strains isolated in Turkey were first time genotyped in this study. Africa 1 genotype has previously been isolated in immunosuppressed patients originating from sub-Saharan Africa. The reason of detecting a strain mainly detected in Africa can be associated with Turkey's specific geographical location. Turkey is like a bridge between Asia, Europe and Africa. Historically, Anatolia was on the Silk Road and other trading routes that ended in Europe. Thus, detecting Africa 1 strain in Anatolia can be anticipated. Consequently, strains detected mainly in Europe and Asia may also be detected in Anatolia and vice versa. Therefore, further studies are required to isolate more strains from Turkey.

Toxoplasma gondii the causative agent of toxoplasmosis is an obligate intracellular parasite with a wide host range including all warm-blooded animals and birds. T.gondii infection causes congenital toxoplasmosis in newborns and this may lead to fetal anomalies, retinochoroiditis leading to blindness, lethal toxoplasmic encephalitis in immune compromised patients, and organ failure in transplantation patients. The pathogenesis of toxoplasmosis change due to differences in the specific immune response elicited by diverse T.gondii strains. The protective immunity against toxoplasmosis is conferred by cellular immune responses. In the present study, two different strains isolated from Turkey named T.gondii Ankara and Ege were used to evaluate the types of humoral and cellular immune responses elicited by adjuvanted tachyzoite protein vaccines in an animal model. In the study, 6-8 weeks old female BALB/c mice were used and six study groups (each contains three mice) were composed for vaccination. The first and second groups were vaccinated with T.gondii Ankara and Ege (TAnkPE and TEgePE, respectively) tacyhzoite lysates, the third and fourth groups were vaccinated by tacyhzoite lysates adjuvanted with Freund's adjuvant (TAnkPE-Freund; TEgePE-Freund, respectively). The fifth and sixth groups were vaccinated with PBS and Freund's adjuvant as controls. Immunization of the animals was performed two times at three weeks intervals. The serum samples were collected before vaccination and after each vaccination to determine the IgG response by Western blotting, and IgG1 and IgG2a responses by ELISA. To determine the cellular immune response, CD8/CD4 cell ratio, intracellular IFN-gamma and IL-4 levels were determined in stimulated spleen cells grown in cell culture systems by flow cytometry. Toxoplasma IgG antibodies were only detected in TAnkPE-Freund group. IgG1 and IgG2a responses did not increase in any vaccination groups and there was not any polarization towards IgG1 or IgG2a. There was no significant increase in CD8/CD4 ratio of stimulated spleen cells. IFN-gamma level was increased in only TAnkPE-Freund vaccination group, however IL-4 levels were increased in TAnkPE-Freund, TEgePE-Freund and TEgePE groups. Our data showed that TAnkPE-Freund vaccine led to increase in IgG and IFN-gamma responses in BALB/c mice, however, tachyzoite lysate vaccines developed in this study did not induce sufficient protective immune response against toxoplasmosis. Thus, use of specific immunogenic proteins must be taken into consideration in the future vaccine development studies against toxoplasmosis.

Amaç: Bu çalışmada immun sistemi baskılanmış sıçanların dışkı ve akciğer örneklerinde Nested PZR ile Cryptosporidium parvum 18S small- subunit rRNA geninin gösterilmesi amaçlanmıştır. Bu gen bölgesi Cryptosporidium spp. için özgün olup, insanlardaki rutin enfeksiyonun tanısında kullanılabilmektedir. Yöntemler: Bu çalışmada üç grup (n=4) Rattus norvegicus türü sıçan kullanılmıştır. Birinci ve ikinci grup sıçanların immun sistemlerinin baskılanması için sırasıyla deri altından ve ağızdan 12 hafta boyunca deksametazon uygulanmıştır. Kontrol grubuna herhangi bir ilaç uygulanmamıştır. İmmun sistemi baskılanmış ve kontrol grubu sıçanlardan 12. hafta sonunda alınan akciğer ve dışkı örneklerinde Nested PZR ile C. parvum varlığı araştırılmıştır. Bulgular: Ağızdan deksametazon uygulanan grubun dışkı ve akciğer örneklerinde C. parvum DNA’sı saptanmıştır. Buna karşın deri altından deksametazon uygulanan sıçan grubunun yalnızca dışkı örneklerinde C. parvum DNA’sı saptanmıştır. Kontrol grubu sıçanlarda herhangi bir bant paterni saptanmamıştır. Sonuç: Bu çalışma sıçanlarda ağızdan deksametazon uygulamasının, deri altından uygulamaya göre daha yaygın cryptosporidiosis oluşumuna sebep olduğunu göstermiştir. Ayrıca immun sistemi baskılanmış hayvan veya insanlara ait dışkı ve solunum örneklerinde 18S small-subunit rRNA genine özgü Nested PZR testinin Cryptosporidium spp. tanısındaki etkinliği gösterilmiştir. (Turkiye Parazitol Derg 2013; 37: 165-8), Objective: In the present study, the aim is to demonstrate Cryptosporidium parvum 18S small-subunit rRNA gene, in lung and stool samples of immune suppressed rats. This gene region is specific for Cryptosporidium spp. and thus can be used in humans for routine diagnostic procedures. Methods: Three groups (n=4) of Rattus norvegicus rats were used. The first and second groups were administered dexamethasone, subcutaneously and orally, respectively, for 12 weeks. Rats in the control group were not immune suppressed. Lung and stool specimens were obtained from rats at the end of 12th week and examined for the presence of C. parvum DNA using Nested PCR. Results: C. parvum DNA was demonstrated in lung and stool samples of rats which were immune suppressed by oral dexamethasone. On the other hand, C. parvum DNA was demonstrated only in stool specimens of the rats which were immune suppressed by subcutaneous dexamethasone. No band pattern was observed in the specimens of the control group. Conclusion: The results of the study showed that oral dexamethasone administration was more efficient in generating disseminated cryptosporidiosis in rats compared to subcutaneous dexamethasone administration. In addition, Nested PCR targeting 18S small-subunit rRNA gene can be used to detect Cryptosporidium spp. in respiratory and stool specimens of animals and humans. (Turkiye Parazitol Derg 2013; 37: 165-8)

Toxoplasma gondii, çok geniş konak aralığında, sıcak kanlı hayvanlarda ve kuşlarda enfeksiyon oluşturabilen zorunlu hücre içi bir parazittir. İnsanlarda T.gondii enfeksiyonu, yeni doğanlarda fetal anomalilere yol açan konjenital toksoplazmoz, körlüğe sebep olan retinokoroidit, immün sistem yetmezliği olan kişilerde fatal seyreden toksoplazmik ensefalit ve organ nakli yapılanlarda organ reddi ve ölüme sebep olmaktadır. Farklı T.gondii suşlarının hayvan modellerinde uyardıkları immün yanıta bağlı olarak, oluşturdukları patogenez de değişiklik göstermektedir. Toksoplazmoza karşı korunmada hücresel immün yanıtın daha önemli olduğu belirtilmektedir. Bu çalışmada, ülkemizde izole edilen T.gondii Ankara ve Ege suşlarından üretilen adjuvanlı takizoit protein aşılarının hayvan modellerinde uyardığı hümoral ve hücresel immün yanıt tiplerinin araştırılması amaçlanmıştır. Çalışmada 6-8 haftalık dişi BALB/c fareler kullanılmış ve aşılama için her biri üç adet fare içeren altı grup oluşturulmuştur. Birinci ve ikinci grup T.gondii Ankara ve Ege (TAnkPE; TEgePE) takizoit eriyiği ile, üçüncü ve dördüncü grup Freund adjuvanı ile birleştirilmiş TAnkPE (TAnkPE-Freund) ve TEgePE (TEgePE-Freund) takizoit eriyiği ile, kontrol grupları olan beşinci ve altıncı gruplar ise PBS ve Freund adjuvanı ile aşılanmıştır. Hayvanların immünizasyonu üç hafta aralıklarla iki kez yapılmıştır. Aşılama öncesi ve her aşılama sonrası alınan serum örneklerinde Western blot yöntemiyle IgG yanıtı, ELISA testi ile IgG1 ve IgG2a yanıtları araştırılmıştır. Hücresel immün yanıtları belirlemek için, hücre kültürü ortamında uyarılan dalak hücrelerinin CD8/CD4 oranı, hücre içi IFN-? ve IL-4 sitokinleri akış sitometrisi ile ölçülmüştür. Çalışmamızda, Toxoplasma IgG antikorları sadece TAnkPE-Freund aşısı uygulanan grupta saptanmış; IgG1 ve IgG2a antikor yanıtlarının hiçbir aşılama grubunda artmadığı tespit edilmiş, IgG1 veya IgG2a yönünde belirgin bir polarizasyon olmadığı belirlenmiştir. Aşılanan grupların hiçbirisinde uyarılmış dalak hücrelerinde CD8/CD4 oranında değişiklik saptanmamıştır. IFN-? üretimi sadece TAnkPE-Freund ile aşılanan grupta artarken; IL-4 üretimi TAnkPE-Freund, TEgePE-Freund ve TEgePE ile aşılanan gruplarda artmıştır. Çalışmamızın verileri, TAnkPE-Freund aşısının BALB/c farelerinde IgG ve IFN-? yanıtlarını artırdığını göstermiş, ancak toksoplazmoza karşı geliştirdiğimiz bu eriyik protein aşılarının koruyucu immün yanıtı yeterince uyarmadıkları saptanmıştır. Bu nedenle ileride yapılacak olan aşı çalışmalarında daha özgül proteinlerin kullanılması yönünde kanaat oluşmuştur., Toxoplasma gondii the causative agent of toxoplasmosis is an obligate intracellular parasite with a wide host range including all warm-blooded animals and birds. T.gondii infection causes congenital toxoplasmosis in newborns and this may lead to fetal anomalies, retinochoroiditis leading to blindness, lethal toxoplasmic encephalitis in immune compromised patients, and organ failure in transplantation patients. The pathogenesis of toxoplasmosis change due to differences in the specific immune response elicited by diverse T.gondii strains. The protective immunity against toxoplasmosis is conferred by cellular immune responses. In the present study, two different strains isolated from Turkey named T.gondii Ankara and Ege were used to evaluate the types of humoral and cellular immune responses elicited by adjuvanted tachyzoite protein vaccines in an animal model. In the study, 6-8 weeks old female BALB/c mice were used and six study groups (each contains three mice) were composed for vaccination. The first and second groups were vaccinated with T.gondii Ankara and Ege (TAnkPE and TEgePE, respectively) tacyhzoite lysates, the third and fourth groups were vaccinated by tacyhzoite lysates adjuvanted with Freund’s adjuvant (TAnkPE-Freund; TEgePE-Freund, respectively). The fifth and sixth groups were vaccinated with PBS and Freund’s adjuvant as controls. Immunization of the animals was performed two times at three weeks intervals. The serum samples were collected before vaccination and after each vaccination to determine the IgG response by Western blotting, and IgG1 and IgG2a responses by ELISA. To determine the cellular immune response, CD8/CD4 cell ratio, intracellular IFN-γ and IL-4 levels were determined in stimulated spleen cells grown in cell culture systems by flow cytometry. Toxoplasma IgG antibodies were only detected in TAnkPE-Freund group. IgG1 and IgG2a responses did not increase in any vaccination groups and there was not any polarization towards IgG1 or IgG2a. There was no significant increase in CD8/CD4 ratio of stimulated spleen cells. IFN-γ level was increased in only TAnkPE-Freund vaccination group, however IL-4 levels were increased in TAnkPE-Freund, TEgePE-Freund and TEge- PE groups. Our data showed that TAnkPE-Freund vaccine led to increase in IgG and IFN-γ responses in BALB/c mice, however, tachyzoite lysate vaccines developed in this study did not induce sufficient protective immune response against toxoplasmosis. Thus, use of specific immunogenic proteins must be taken into consideration in the future vaccine development studies against toxoplasmosis.

Toxoplasma gondii takizoit ve doku kistleri çalışmaları aşı, tanı testleri ve ilaç araştırmaları, biyokimyasal ve moleküler yapı çalışmaları gibi birçok bilimsel çalışmada kullanılmaktadır. Doku kisti ve takizoitlerin sürekli in vivo pasajlanması zahmetli bir işlem olduğu kadar diğer yandan sürekli hayvan kullanılmasından kaynaklanılan yüksek maliyetli ve etik sorunlar çıkaran bir işlemdir. Takizoitlerin ve doku kistlerinin kriyoprezervasyonunun gerektiğinde in vivo canlandırılması ekonomik kayıpları, iş gücü ve hayvan etik sorunlarını azaltılabilecektir. Bu makalede takizoit ve doku kistlerinin farelerde, intraperitoneal üretimi, kriyoprezervasyon için hazırlanması ve kriyolanmış örneklerin çözdürülerek farelere tekrar uygulanması detaylı olarak tarif edilmiştir. (Turkiye Parazitol Derg 2013; 37: 44-6), Toxoplasma gondii tachyzoites and tissue cysts are largely used for developing diagnostic assays, vaccines and in drug research as well as biochemical and molecular structure studies. Continuous passaging of tachyzoites or tissue cysts in animal models encounter ethical and economical problems and it is a time consuming procedure. Cryopreservation of tachyzoites and tissue cysts and revitalization of cryopreserved samples whenever needed, can decrease the economical loss, ethical problems and labour. In the present article, production of tachyzoites and tissue cysts in mice, preparation of samples for cryopreservation, cryopreservation of tachyzoites and tissue cysts, defrosting of cryopreserved samples and reinoculation to mice have been described in detail. (Turkiye Parazitol Derg 2013; 37: 44-6)

Almost 10-20 million people in the world are thought to be infected by human deltaretroviruses, namely human T-cell lymphotropic virus (HTLV) type I and II, recently. HTLV-I is endemic in southwestern Japan, the Caribbean and sub-Saharan Africa, whereas HTLV-II is more prevalent in intravenous drug addicts, and in American indian populations, endemically. HTLV-I is mainly responsible for adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), however, HTLVII is not clearly associated with a known clinical disease. Both viruses may be transmitted by sexual contact, parenteral route, whole blood transfusion and breast-feeding. In most of the countries [USA, Canada, South America, Caribbean, Japan, Taiwan and some Europe countries (France, UK, Ireland, Sweden, Denmark, The Netherlands, Portugal, Romania, Greece)] routine screening of anti-HTLV-I/II in blood donors is mandatory, however, there is no such practice in Turkey since seroepidemiologic data on HTLVI/II infections is insufficient. In this study, the seroprevalence of HTLV-I/II in healthy blood donors admitted to the blood bank of Ege University Medical Faculty Hospital, Izmir (located at Aegean region), was investigated to support data on the decision making process on routine screening of anti-HTLV-I/II in blood centers. Serum samples from 10.000 healthy blood donors (mean age: 32.6 years; 87.8% were male), who succeeded the donor history questionnaire, were included to the study, and HTLV-I/II antibodies were screened by a commercial enzyme immunoassay (ELISA) (Murex HTLVI-II, Murex Diagnostics, UK) method. Serum samples which were yielded reactive and borderline results were retested by ELISA, and repeated reactive/borderline results were then confirmed by HTLV-I/II confirmation test (INNO-LIA HTLV-I/II, Innogenetics, Belgium). Seven samples yielded reactive/borderline reactive results by both ELISA lots, however, all of them were found negative by confirmatory test. According to our data HTLV-I/II infections are not endemic in Izmir region, and anti-HTLV-I/II screening of blood donors is not required in our blood center currently. Nevertheless, screening HIV which is very rare in prevalence among the donor population, is mandatory for blood donors in our country. Thus, even its prevalence is very low, much more comprehensive and multi-centered studies are necessary for making the decision of integrating HTLV-I/II in routine blood bank screening tests in Turkey.

Toxoplasmosis is a serious and potentially life-threatening disease in liver transplant recipients while they are immunosuppressed. We report the clinical and laboratory findings related to active toxoplasma infection associated with 40 immunosuppressed liver transplant procedures that took place over a 12-month period at a major transplant unit in Izmir, Turkey. Twenty-seven (67.5%) of the 40 transplant recipients were found to be seropositive for toxoplasma infection and therefore at risk of reactivated infection. From the serological status of the donors, which was ascertained in 38 of 40 cases, we identified 3 (7.9%) of 38 transplants to be from a seropositive donor to a seronegative recipient. In 10 (26.3%) of 38 transplants, both the donor and recipient were seronegative, and this excluded toxoplasma as a risk. A comparison of real-time polymerase chain reaction (PCR) and nested PCR was undertaken in combination with a range of serological assays (the Sabin-Feldman dye test, enzyme immunoassay immunoglobulin M, and immunosorbent agglutination assay immunoglobulin M). Ethylene diamine tetraacetic acid blood samples from 3 of the 30 recipients at risk from toxoplasma were found positive by PCR, but only 1 of these was found positive in both assays. Among the 3 PCR-positive patients, immunoglobulin M and immunoglobulin G antibody levels increased in only 1 patient. Correlations between symptoms, laboratory findings, and clinical management (use of anti-toxoplasma therapy) are presented. Our findings suggest that toxoplasma presents a significant risk to our liver transplant population and that PCR is a helpful addition in identifying active infections and hence in informing clinical management decisions.

Trichinellosis often causing diarrhea and more rarely fever, periorbital edema and myositis in human, is commonly treated with benzimidazole derivatives. The Artemisia genus has been found to be effective against a variety of parasites. In the present study, the efficacy against trichinellosis (Trichinella spiralis) of Artemisia vulgaris and Artemisia absinthium was examined for the first time in rats. The results of trichinoscopy and artificial digestion, during the enteral (adult) phase of the illness show that 300 mg/kg doses of methanol extracts of the aerial parts of A. vulgaris and A. absinthium reduced the larval rate by 75.6% and 63.5% in tongue, 53.4% and 37.7% in diaphragm, 67.8% and 46.2% in quadriceps, and 66.7% and 60.5% in biceps-triceps muscles of rats, respectively. Furthermore, during the parenteral (encapsulated larvae) phase, 600 mg/kg doses of A. vulgaris and A. absinthium extracts decreased the larval rate by 66.4% and 59.9% in tongue, 57.4% and 50.0% in diaphragm, 47.6% and 43.7% in quadriceps, 60.2% and 46.4% in biceps-triceps muscles of rats, respectively. Analysis of antibody also showed that A. vulgaris significantly reduced the antibody response (P

The aim of this study was to determine the parasite frequency in 3925 patients during 2005 from January 1- December 31 in the parasitology laboratory of the Ege University Medicine School. During the laboratory investigation, 3925 fecal specimens and cellophane tapes from the patients were examined. After the microscope examination of 3925 feces samples, it was found that 590 (15.03%) of these samples contained one or more intestinal parasites. Blastocystis hominis (4.96%), Cyclospora spp. (1.91%), Enterobius vermicularis (1.86%), Entamoeba coli (1.78%) and Giardia intestinalis (1.78%) were the five most common parasites obtained during the examination.

Bu çalışmada, 1 Ocak - 31 Aralık 2005 tarihleri arasında, Ege Üniversitesi Tıp Fakültesi Hastanesi Parazitoloji Laboratuvarı’na başvuran 3925 hastada bağırsak parazitlerinin incelenmesi amaçlanmıştır. 3925 hastadan alınan gaita ve selofan bant preparatlarının mikroskobik incelenmesinde toplam 590 (%15,03) örnekte bir veya birden fazla bağırsak paraziti saptanmıştır. En sık saptanan 5 parazitin, Blastocystis hominis (%4,96), Cyclospora spp. (%1,91), Enterobius vermicularis (%1,86), Entamoeba coli (%1,78) ve Giardia intestinalis (%1,78) olduğu görülmüştür., The aim of this study was to determine the parasite frequency in 3925 patients during 2005 from January 1– December 31 in the parasitology laboratory of the Ege University Medicine School. During the laboratory investigation, 3925 fecal specimens and cellophane tapes from the patients were examined. After the microscope examination of 3925 feces samples, it was found that 590 (15.03%) of these samples contained one or more intestinal parasites. Blastocystis hominis (4.96%), Cyclospora spp. (1.91%), Enterobius vermicularis (1.86%), Entamoeba coli (1.78%) and Giardia intestinalis (1.78%) were the five most common parasites obtained during the examination.