Aysu Değirmenci

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.

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 Turkeys 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.

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. Liver Transpl 14:1526–1532, 2008.

Behaviour of Toxoplasma gondii RH Ankara strain tachyzoites during continuous production in various cell lines.

Toxoplasma gondii is an obligate intracellular protozoan parasite. The objective of the present study was to examine the behaviour of Toxoplasma gondii RH Ankara strain tachyzoites in a cell culture environment. The study represents the first step in determining whether T. gondii RH Ankara strain tachyzoites, grown in cell culture, are of sufficient quality to allow cessation of in vivo tachyzoite production for diagnostic assays. In the present study, T. gondii RH Ankara strain tachyzoites were continuously produced in myeloma X63.Ag8.653, HeLa, Hep-2, and Vero cell cultures for 2 months. The average size of the tachyzoites was 3 x 5.7 microm prior to the first inoculation but after continuous production, a marked decrease was noted in average tachyzoite size. The smallest tachyzoite size, was 1 x 2.1 microm after 2 months, in myeloma cell cultures even though the yield of tachyzoites increased. With other cell cultures, tachyzoite yields were not as high as myeloma cell culture although decrease in size was less. The smallest decrease in tachyzoite size, averaging 2 x 3.8 microm after 2 months, was observed in tachyzoites produced in HeLa cell cultures. A virulence assay in small groups of BALB/c mice, using tachyzoites derived from cell cultures, was also conducted. The preliminary results of the virulence assay suggest that as the size of the tachyzoites decreased, the virulence in mice decreased. Future research will focus on the effect of the size of cell culture-derived T. gondii RH Ankara strain tachyzoites on the virulence, protein expression, and the reliability of diagnostic assays. Ultimately, the behaviour of tachyzoites from various T. gondii strains will be observed in cell culture to determine if size is altered.

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.

Cryopreservation of Toxoplasma gondii tachyzoites and tissue cysts

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.

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.

Demonstration of Cryptosporidium parvum in immune suppressed rats using nested PCR

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.

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.