Investigation of West Nile virus among healthy blood donors in the western part of Turkey.

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. Turkeylies 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.

[Demonstration of Cryptosporidium parvum in immune suppressed rats using nested PCR]. / Immun Sistemi Baskilanmis Siçanlarda Cryptosporidium parvum'un Nested PZR Yöntemi ile Gösterilmesi.

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 < sup > th < /sup > 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.

Isolation of Toxoplasma gondii strains similar to Africa 1 genotype in Turkey.

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.

[Cryopreservation of Toxoplasma gondii tachyzoites and tissue cysts]. / Toxoplasma gondii Takizoit ve Doku Kistlerinin Kriyoprezervasyonu.

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.

[Comparison of Immune Responses Elicited by Adjuvanted Tachyzoite Lysate Vaccines Developed from Two Different Toxoplasma gondii Strains Isolated in Turkey]. / Türkiye'de Izole Edilen Iki Farkli Toxoplasma gondii Susundan Üretilen Adjuvanli Takizoit Eriyik Protein Asilarinin Uyardigi Immün Yanitlarin Karsilastirilmasi

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-g 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-g 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-g 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.

Detection of Pneumocystis in the nasal swabs of immune-suppressed rats by use of PCR and microscopy.

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<0.05). Microscopy was positive only in lungs of rats orally administered dexamethasone. PCR was positive in lungs and oral swabs of rats prior to the administration of dexamethasone. After the administration of dexamethasone, the MSG gene was detected in oral swabs, lungs, spleen, kidney and (for the first time) in nasal swabs. PCR was positive in nasal swabs during the second and sixth weeks of oral and subcutaneous administration of dexamethasone, respectively. CONCLUSIONS: Presence of P. jiroveci in nasopharyngeal aspirate, oropharyngeal wash, oral swab, induced sputum or BAL, and absence in nasal swab in a patient without symptoms of PCP may support clinician's decision regarding colonization. Overall, detection of P. carinii in nasal swabs of rats by PCR demonstrated that nasal sampling can be used for the diagnosis of Pneumocystis pneumonia.

Determining Toxoplasma high-risk autologous and allogeneic hematopoietic stem cell transplantation patients by systematic pre-transplant PCR screening of stem cell originated buffy coat.

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.

Action plan to regain unnecessary deferred blood donors due to malaria risk in Turkey.

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.

Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey.

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 RH Ankara: production of evolving tachyzoites using a novel cell culture method.

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 0.12-0.5×10(6)/ml. In the flasks with HeLa-tachyzoite ratio 1/8, the tachyzoite yield and viability ratio were 3×10(6)/ml and 100%, respectively, with accompanying 10 fold decrease (0.12×10(6)/ml) in HeLa contamination. During continuous production, highest tachyzoite yield was obtained from the first passage (3.55×10(6)/ml) and until the end of third subculture viability rates and HeLa cell contaminations were between 98.2-99.4% and 0.31-0.37×10(6)/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.

[Investigation of anti-HTLV I/II seroprevalence in healthy blood donors in Izmir region, Turkey]. / Izmir bölgesinde saglikli kan vericilerinde anti-HTLV-I/II seroprevalansinin arastirilmasi

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.

Investigation of folding of purified recombinant GRA1 protein using web based protein disorder servers and trypsin digestion.

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.

Incidence and diagnosis of active toxoplasma infection among liver transplant recipients in Western 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.

Comparison of the effects of Artemisia vulgaris and Artemisia absinthium growing in western Anatolia against trichinellosis (Trichinella spiralis) in rats.

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<0.05) during the enteral and parenteral phases. Thus, the results of the present study revealed that A. vulgaris could be an alternative drug against trichinellosis.

[Distribution of intestinal parasites detected in the parasitology laboratory of the Ege University Medical School Hospital, in 2005]. / Ege Universitesi Tip Fakültesi Hastanesi parazitoloji laboratuvarinda 2005 yili boyunca saptanan bagirsak parazitlerinin dagilimi.

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.