Intestinal Protozoan Parasitic Infections in Immunocompromised Child Patients with Diarrhea.

Intestinal protozoan parasites are common causes of infectious diarrhea in children receiving anticancer therapy or undergoing transplantation. Additionally, immunosuppression therapy in such patients may exacerbate the symptoms related to these parasitic infections. The aim of this study was to evaluate the prevalence and diagnostic importance of parasitic protozoan infections in children treated for malignancies or undergoing transplantation, and to highlight the control of intestinal parasitic infections for immunosuppressed patients at a hospital in Izmir, Turkey. In total, 82 stool samples from 62 patients were analyzed by microscopic examination and polymerase chain reaction (PCR) for the presence of coccidian parasites. Our results showed that Cryptosporidium, Cyclospora, and Cystoisospora were present in 22.5% (14/62), 9.6% (6/62), and 3.2% (2/62) of the cases using either method, respectively. The prevalence of these coccidian parasites identified with both methods was 35.4% (20/62). Other intestinal parasites (Blastocystis, Giardia, and Entamoeba coli) were detected in 10 patients. PCR analysis showed the presence of all coccidian parasites in the same stool sample for one patient. Finally, both PCR and microscopic examination of the stools revealed that there is a higher prevalence of Cryptosporidium, Cyclospora, and Cystoisospora in immunocompromised children. These examinations allowed an early start of appropriate antibiotic treatments and led to an increased percentage of correctly treated patients.

Investigation of Anti-Toxoplasma gondii Antibodies in Cats Using Sabin-Feldman Dye Test in Ankara in 2016

Objective: Toxoplasmosis, in which obligate intracellular protozoa Toxoplasma gondii (T.gondii) is the causative organism, is a multisystemic disease that can be seen all over the world and can impair all vertebrates. The only hosts known for T.gondii are members of Felidae family. Our study aimed to determine anti-Toxoplasma gondii antibodies with Sabin-Feldman Dye Test (SFDT) in cats in Ankara. It's aimed to evaluate the current situation in terms of Toxoplasmosis spread by comparing our findings with previous studies in the same region. Methods: Rh strain of Toxoplasma used in our study is maintained in our laboratory. SFDT is still accepted as the gold standard. Material of the study was obtained by taking blood samples from cats who were admitted to the clinics between March 2016 and October 2016 in Ankara. Blood samples were inactivated and measurements were done with SFDT 1/4, 1/16, 1/64, 1/256, 1/1024 titers. Results: SFDT resulted positive in 56 (43.4%) cats at a dilution of 1/16, in 7 (5.4%) cats at a dilution of 1/64, in 23 (17.8%) cats at a dilution of 1/256 and negative in 43 (33.3%) cats. Comparison of demographic data with SFDT results showed that positive test results did not differ according to gender and age (P=0.803 and P=0.991, respectively). Seropositivity was higher in stray cats than house cats (P<0.001). Test results were negative in the cats that fed only by commercial dry food (P<0.001). Positivity in hunter cats was more than in non-hunters (P<0.001). Conclusion: Seropositivity was detected in 66.6% of the cats, which was quite a high rate. As a result, taking precautions in terms of Toxoplasma for stray cats that are hunting and feeding naturally is a necessity for public health.

The use of Toxoplasma gondii tachyzoites produced in HeLa cells adhered to Cytodex 1 microcarriers as antigen in serological assays: an application of microcarrier technology.

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 × 105 cells/ml and 2 × 105 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 × 105 cells/ml using RPMI 1640, and the cell:tachyzoite ratio to obtain the highest tachyzoite yield (17.1 × 107) 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 < 0.0001). The sensitivity and specificity ratios of ELISA were 100%. In addition, Western blotting banding patterns of the antigen derived at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio was comparable with mouse derived antigen. Overall, this novel methodology can be an alternative source of antigen in diagnostic assays, decrease animal use for antigen production, and contribute to the solution of ethical and economic problems.

The association between Cytomegalovirus co-infection with Pneumocystis pneumonia and mortality in immunocompromised non-HIV patients.

INTRODUCTION: Impact of Cytomegalovirus (CMV) co-infection pneumonia in non-HIV patients with Pneumocystis jirovecii pneumonia (PCP) is unclear. OBJECTIVES: The aim of our study was to determine whether CMV co-infection is associated with an increased risk of mortality. METHODS: Our study was conducted at Ege University Hospital, Turkey. We used molecular assays to diagnose Pneumocystis jirovecii in respiratory samples, and CMV in both respiratory and blood samples. We compared morbidity and mortality stratified by CMV co-infection status. RESULTS: Between 2009 and 2015, 43 patients (mean age: 56.7 ± 15.3 years) were diagnosed with PCP. Only 3 of 43 patients had received PCP prophylaxis. We microbiologically confirmed CMV co-infection in 28 of 43 (65.1%) patients. Acute respiratory distress syndrome (ARDS) and requirement of mechanical ventilation were more common in the CMV co-infection group (P = .019 and P = .031 respectively), and duration of intensive care unit was also longer (P = .006). In univariate analyses, mortality at 30 days was higher in the CMV co-infection group as compared to the group with PCP alone (78.6% and 46.7% respectively; P = .046). In multivariate analyses, mortality was independently associated only with the presence of ARDS [OR: 6.22 95% CI 1.3-29.32] and the association with CMV co-infection was no longer significant [OR: 2.6 95% CI 0.49-13.72, P = .257]. CONCLUSION: The risk of mortality appears to be increased in the setting of CMV and PCP co-infection in HIV-uninfected immunocompromised patients. PCP prophylaxis use was lower than expected, suggesting low physician awareness of the risks of PCP in this population.

Diagnosis and Species Discrimination of Positive Malaria Samples by Real-Time Polymerase Chain Reaction.

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.

Seroprevalence of Leishmania infection and molecular detection of Leishmania tropica and Leishmania infantum in stray cats of Izmir, Turkey.

Leishmaniasis caused by more than 20 species of genus Leishmania is transmitted by the bite of infected phlebotomine sand flies. The studies on Leishmania infection in cats is very few in Turkey and therefore we aimed to screen stray cats living in city of Izmir located in western Turkey using nested PCR targeting kinetoplast DNA and serological techniques (ELISA and IFA). Leishmania DNA positive samples were also studied by ITS1 real time PCR. Whole blood and serum samples were obtained from stray cats (n: 1101) living in different counties of Izmir. In serological assays, a serum sample was considered positive in 1:40 dilution in IFA and for ELISA a serum sample was accepted positive when the absorbance value (AV) exceeded the mean AV + Standard Deviation (SD) of the negative control serum samples. According to the results, the seropositivity rates were 10.8% (119/1101) and 15.2% (167/1101) by in house ELISA and IFA, respectively. Among serology coherent samples, the seropositivity rate was 11.1% (116/1047) as detected by both assays after discordant samples (n: 54) were discarded. Of the 1101 stray cats, six (0.54%) were positive by nested PCR while only one of these six samples was positive by ITS1 real time PCR. During PCR, three controls designated as Leishmania infantum, Leishmania tropica, and Leishmania major were used for species identification. According to nested PCR results, L. tropica was identified in two cats (no.76 and 95). In another cat (no. 269), there were two bands in which one of them was well-matched with L. infantum and the other band had ∼850 bp size which does not match with any controls. Remaining three cats (no. 86, 514, and 622) also had the ∼850 bp atypical band size. ITS1 real time PCR detected L. tropica in only one cat (no. 622) which showed an atypical band size in nested PCR. These results indicated that three cats with only one atypical band (no. 86, 514, and 622) and the cat with mixed infection (no. 269) were infected with L. tropica. Altogether, L. tropica was detected in all six DNA positive cats and L. infantum was detected in one cat with mixed infection. In conclusion, although the reservoir role of cats in nature is still unclear the high seroprevalence rate against Leishmania parasites and detecting parasite DNA in stray cats in Izmir indicates that the stray cats are frequently bitten by infected sand flies. Further research activities are required to reveal the frequency of leishmaniasis in cats in different regions of Turkey where Leishmania species are endemic.

[Detection of Echinococcus granulosus and Echinococcus multilocularis in cyst samples using a novel single tube multiplex real-time polymerase chain reaction]. / Kist örneklerinde yeni bir tek tüp multipleks gerçek zamanli polimeraz zincir reaksiyonu ile Echinococcus granulosus ve Echinococcus multilocularis'in saptanmasi.

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-RT-PCR) 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-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 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/µl 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.

Preliminary analysis of Plasmodium vivax genotypes isolated in southeastern Turkey.

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.

Identification of Toxoplasma gondii antigens associated with different types of infection by serum antibody profiling.

Acquisition of acute toxoplasmosis during the first trimester of pregnancy can have catastrophic consequences for the foetus. Diagnosis is routinely based on the detection of maternal Toxoplasma gondii--antibodies using whole parasite extracts as detection antigen. While such assays are sensitive, they show no specificity for the stage of infection. We hypothesized diagnosis might be improved using recombinant antigens for detection, particularly if antibodies to certain antigen(s) were associated with early or late stages of infection. To address this, protein microarrays comprising 1513 T. gondii exon products were probed with well-characterized sera from seronegative ('N') controls, and acute ('A'), chronic/IgM-persisting ('C/M') and chronic ('C') toxoplasmosis cases from Turkey. Three reactive exon products recognized preferentially in A infections, and three recognized preferentially in C/M infections, were expressed in Escherichia coli and tested for discrimination in IgG ELISAs. The best discriminators were exon 1 of TGME49_086450 (GRA5) which detected C/M infections with 70.6% sensitivity and 81.8% specificity, and exon 6 of TGME49_095700 (ubiquitin transferase domain-containing protein) which detected A infections with 84.8% sensitivity and 82.4% specificity. Overall, the data support a recombinant protein approach for the development of improved serodiagnostic tests for toxoplasmosis.

Diagnostic value of a Rec-ELISA using Toxoplasma gondii recombinant SporoSAG, BAG1, and GRA1 proteins in murine models infected orally with tissue cysts and oocysts.

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<0.01). The anti-BAG1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly at days 15, 40, and 120 (P<0.05). The anti-GRA1 IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 2, 10, and 40 (P<0.01). The anti-GRA1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly only at day 40 (P<0.05). The anti-SporoSAG, anti-BAG1, and anti-GRA1 IgG titers of mice showed significant increases at day 40 (P<0.05) and decrement started for only anti-GRA1 IgG at day 120. The presence of anti-SporoSAG IgM and IgG antibodies can be interpreted as recently acute infection between days 10-40 because IgM decreases at day 40. Similarly, presence of anti-BAG1 IgM and absence of IgG can be evaluated as a recently acute infection that occurred 40 days before because IgG peaks at day 40. A peak in anti-GRA1 antibody level at first testing and reduction in consecutive sample can be considered as an infection approximately around day 40 or prior. Overall, recombinant SporoSAG, BAG1 and GRA1 proteins can be accepted as valuable diagnostic markers of recently acute toxoplasmosis.

Genetic characterization of Toxoplasma gondii isolates and toxoplasmosis seroprevalence in stray cats of Izmir, Turkey.

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.

[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.

Co-expression of the Bcl-xL antiapoptotic protein enhances the induction of Th1-like immune responses in mice immunized with DNA vaccines encoding FMDV B and T cell epitopes.

Foot-and-mouth disease (FMD) is one of the most devastating animal diseases, affecting all cloven-hoofed domestic and wild animal species. Previous studies from our group using DNA vaccines encoding FMD virus (FMDV) B and T cell epitopes targeted to antigen presenting cells, allowed demonstrating total protection from FMDV homologous challenge in those animals efficiently primed for both humoral and cellular specific responses (Borrego et al. Antivir Res 92:359-363, 2011). In this study, a new DNA vaccine prototype expected to induce stronger and cross-reactive immune responses against FMDV which was designed by making two main modifications: i) adding a new B-cell epitope from the O-serotype to the B and T-cell epitopes from the C-serotype and ii) using a dual promoter plasmid that allowed inserting a new cistron encoding the anti-apoptotic Bcl-xL gene under the control of the internal ribosomal entry site (IRES) of encephalomyocarditis virus aiming to increase and optimize the antigen presentation of the encoded FMDV epitopes after in vivo immunization. In vitro studies showed that Bcl-xL significantly prolonged the survival of DNA transfected cells (p < 0.001). Accordingly, vaccination of Swiss out-bred mice with the dual promoter plasmid increased the total IgG responses induced against each of the FMDV epitopes however no significant differences observed between groups. The humoral immune response was polarized through IgG2a in all vaccination groups (p < 0.05); except peptide T3A; in correspondence with the Th1-like response observed, a clear bias towards the induction of specific IFN-γ secreting CD4⁺ and CD8⁺ T cell responses was also observed, being significantly higher (p < 0.05) in the group of mice immunized with the plasmid co-expressing Bcl-xL and the FMDV B and T cell epitopes.

[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.

[Comparison of polymerase chain reaction with wet mount and culture methods for the diagnosis of trichomoniasis]. / Trichomoniasis Tanisinda Polimeraz Zincir Reaksiyonu ile Mikroskobi ve Kültür Yöntemlerinin Karsilastirilmasi

OBJECTIVE: Wet mount, culture and staining methods are generally used in the diagnosis of trichomoniasis. Polymerase chain reaction (PCR) methods with different primer pairs have been tested and used for research in recent years. METHODS: In this study, T. vaginalis was tested for in the vaginal samples of 102 patients referred to obstetrics and gynecology polyclinic of Aydin Obstetrics and Children Hospital for various reasons, with direct microscopy, culture and PCR with primers targeting Tv-E650. In addition, PCR was applied to 20 T. vaginalis strains that were isolated from patients who were previously diagnosed with vaginitis. RESULTS: Of 102 samples, T. vaginalis was found to be positive in 2.94, 4.90 and 4.90% with wet mount, TYM medium and PCR respectively. The positivity rate reached 5.88% using the 3 methods together. All 20 strains isolated from patients with vaginitis were reported as positive by the PCR method. CONCLUSION: The wet mount had 60% sensitivity and 100% specificity, while PCR showed 80% sensitivity and 97.95% specificity when compared with the culture method, which is accepted as the "gold standard". The PCR method was performed for the first time as a diagnostic assay for trichomoniasis in this study and it is concluded that it can be used routinely for its diagnosis.

Identification of potential serodiagnostic and subunit vaccine antigens by antibody profiling of toxoplasmosis cases in Turkey.

Toxoplasmosis, caused by infection of the protozoan parasite Toxoplasma gondii, is associated with mild disease in healthy individuals, whereas individuals with depressed immunity may develop encephalitis, neurologic disorders, and other organ diseases. Women who develop acute toxoplasmosis during pregnancy are at risk of transmitting the infection to the fetus, which may lead to fetal damage. A diagnosis is usually confirmed by measuring IgG, or IgM where it is important to determine the onset of infection. A negative IgM result essentially excludes acute infection, whereas a positive IgM test is largely uninterpretable because IgM can persist for up to 18 months after infection. To identify antigens for improved diagnosis of acute infection, we probed protein microarrays displaying the polypeptide products of 1357 Toxoplasma exons with well-characterized sera from Turkey. The sera were classified according to conventional assays into (1) seronegative individuals with no history of T. gondii infection; (2) acute infections defined by clinical symptoms, high IgM titers, and low avidity IgG; (3) chronic/convalescent cases with high avidity IgG but persisting IgM; (iv) true chronic infections, defined by high avidity IgG and no IgM. We have identified 38 IgG target antigens and 108 IgM target antigens that can discriminate infected patients from healthy controls, one or more of which could form the basis of a 'tier-1' test to determine current or previous exposure. Of these, three IgG antigens and five IgM antigens have the potential to discriminate chronic/IgM persisting or true chronics from recent acutely infected patients (a 'tier-2' test). Our analysis of the antigens revealed several enriched features relative to the whole proteome, which include transmembrane domains, signal peptides, or predicted localization at the outer membrane. This is the first protein microarray survey of the antibody response to T. gondii, and will help in the development of improved serodiagnostics and vaccines.