Retrospective Determination of the Prevelense of Anti-Echinococcus granulosus Antibodies in Cystic Echinococcosis Pre-diagnosed Patients at Erciyes University Faculty of Medicine

Objective: Cystic echinococcosis (CE) is a parasitic disease that has been known for years in helminth diseases and it is important as human and animal health problem in many parts of the world and in our country due to economic losses. In this study, it was aimed to retrospectively evaluate the distribution of anti-E. granulosus-IgG antibodies in patients with pre-diagnosis of CE that referred to parasitology laboratory between January 2013-December 2018. Methods: Commercial kit was used for indirect hemaglutination (IHA), indirect fluorescent antibody test (IFAT) and Western blot (WB) methods using sera from patient samples was applied according to the kit proposal. In addition, patient materials for CAM, CSF and blood for which polymerase chain reaction (PCR)/QPCR tests were requested were examined. Results: Sera of the patients who were tested with at least one of the IHA, IFAT and WB methods or a combination of these methods, and 443 cases out of 2.283 cases were found to be E. granulosus seropositive. It was determined that 369 (62.03%) of 443 positive patients were female and 330 (37.97%) were male patients. Among these patients, 87 patients whose IFAT and/or IHA tests were negative were found to have positive results with the WB method. IFAT or IHA test results of 13 patients with negative WB tests were found to be positive. Four patients were identified with both tests positive but WB test results negative. In addition, 36 of 72 patients who underwent PCR/QPCR tests were found to be positive. Conclusion: As a result of a six-year retrospective screening, 22% of the cases were found to be positive, and it was concluded that the prevalence of CE is high and the use of a single test may be insufficient in the diagnosis of CE, therefore, test combinations will increase the sensitivity and reliability in reaching the correct diagnosis.

[Investigation of Anti-leishmanial Effects of Bee Products (Honey, Propolis) on Leishmania tropica Promastigotes]. / Ari Ürünlerinin (Bal, Propolis) Leishmania tropica Promastigotlari Üzerine Antileysmanyal Etkilerinin Arastirilmasi.

This study was aimed to investigate the anti-leishmanial effects of bee products (honey and propolis) by using the causative agent of cutaneous leishmaniasis Leishmania tropica promastigotes, in in vitro culture. In vitro anti-leishmanial efficacy of honey (pine, flower and chestnut) and propolis used in the study were evaluated using the microdilution method. Honey, which is a bee product, was dissolved with RPMI medium containing fetal calf serum (FCS) and diluted in the same medium, and serial dilutions were prepared in concentrations between 62.5-1000 mg/ml. Propolis, on the other hand, was dissolved with ethyl alcohol and only 2.5 µl was used from all these concentrations since the alcohol content was more than 50% in these concentrations prepared and we thought that this rate would negatively effect the parasite development. Then, RPMI containing FCS was diluted in the medium and serial dilutions were prepared at concentrations between 50-800 µg/ml. To the dilutions prepared, the promastigot suspension was added so that their final concentrations in the wells were 1 x 106 promastigot/ml and then the medium was incubated for 24 and 48 hours in 26°C. After the incubation, promastigotes were determined microscopically for morphology, mobility and live parasite density, and cell viability was determined by MTS method and 50% inhibitor concentrations (IC50) were compared with control groups. Anti-leishmanial activity of propolis (50, 100, 200, 400 and 800 µg/ml) and honey (62.5, 125, 250, 500 and 1000 mg/ml) on promastigotes was evaluated in vitro. In microscopic examinations, pine honey showed anti-leishmanial activity starting from 62.5 mg/ml, flower honey 250 mg/ml, and chestnut honey 125 mg/ml, and pine honey was more effective on promastigotes (p< 0.05), and propolis was effective from 100 µg/ml concentration. It has been determined that very low concentrations of propolis caused changes in the morphological structure of the parasites and were more effective than the other bee products. The prevention of cell proliferation and decreasing of the IC50 values according with the time of pine honey (IC50= 109.28 mg/ml), flower honey (IC50= 248.07 mg/ml), chestnut honey (IC50= 147.65 mg/ml) and propolis (IC50= 82.98 µg/ml) applied on L.tropica promastigot cell culture was determined by MTS method. In this study, it was found that various concentrations of pine, flower, chestnut honey and propolis showed anti-leishmanial activity on L. tropica promastigotes. It has been observed that pine honey is more effective on promastigotes after 48 hours of incubation period, and propolis is more effective in both morphology and cell inhibition of the parasites even at very low concentrations. It is believed that these data can be used as an alternative treatment method against cutaneous leishmaniasis infections and further studies are required.

[Silencing the target rhoptry neck protein (RON) gene responsible for the invasion of Toxoplasma gondii with siRNA transfection]. / Toxoplasma gondii'nin invazyonundan sorumlu hedef Rhoptry Neck Protein (RON) geninin siRNA transfeksiyonu ile susturulmasi.

Toxoplasma gondii is a coccidian protozoan that causes toxoplasmosis is a common disease in Turkey as well as all over the world. It causes various clinical symptoms depending on the immune system status, age, or location of the disease. There is an organelle called the apical complex at the anterior end of the parasite. Rhoptry Neck Proteins (RONs), a component of this organelle, play a critical role in the formation of "moving junction" and parasitophorous vacuoles during host cell invasion. On the other hand, interfering RNA (iRNA) treatment options developed in recent years have emerged. With small iRNAs (siRNA) it is also possible to treat and control parasitic diseases, too. From here it is thought to use this method against toxoplasmosis. Within the scope of the project, it is aimed to silence RON1 gene the target invasion molecules of T.gondii with siRNA transfection. In the study, the negative control group constitute HeLa cells, the positive control group constitute HeLa cells infected with T.gondii tachyzoites and the experimental group constitute HeLa cells infected with T.gondii tachyzoites after siRNA transfection were used. Samples were collected in each study group at 30 seconds, 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 36 hours and 48 hours. In order to calculate the proportional change of the parasite loads between control groups and experimental groups, RNA and protein isolations were performed from these samples for real time polymerase chain reaction (qRt-PCR) and WB analyzes, respectively. The statistical difference between control groups and experimental groups was calculated. Significant difference in gene expression in experiments with siRNA1 (p< 0.0055), siRNA2 (p< 0.0003), siRNA3 (p<0.0001), siRNA4 (p< 0.0001), siRNA5 (p< 0.0001), siRNA6 (p< 0.0001), siRNA7 (p< 0.0182), siRNA9 (p< 0.0011) and siRNA10 (p< 0.0004) but there was no significant difference statistically in the experiment with siRNA8 (p< 0.4049) was detected. Thus, it has been detected that invasion was inhibited due to the lack of production of parasite antigen in the cell lysate belongs to experimental groups at WB assays with anti-T.gondii RON1 and total anti-T.gondii antibodies resulting in silencing of the RON1 gene. The suppression of the TgRON1 gene expression by this method is a promising step in the development of anti-toxoplasmosis vaccines and therapeutic agents.

[Plasmodium ovale malaria and molecular diagnosis: Could it be a relapse?] / Plasmodium ovale sitmasi ve moleküler tanisi: relaps olabilir mi?

Malaria caused by Plasmodium species continues to affect the half of the world population. According to the World Health Organization 2017 data, 445.000 cases of malaria and 219 million cases of new clinical malaria cases were reported during the year. African continent is the geographical region where the disease is most frequent. In recent years there has been an increase in the number of imported cases after travels to this continent. In this case report, relaps caused by Plasmodium ovale in a male Republic of Turkey citizen patient who has travelled to Uganda only and no other place a year and half ago was presented. Thin blood smear was prepared from the peripheral blood of the patient who admitted to our hospital with complaints of fever and chills. The smear was stained with Giemsa and examined with a x100 objective microscope and trophozoites belonging to Plasmodium genus were detected. Considering the size and locality of the trophozoites in the erythrocytes, it is thought that the parasite may be Plasmodium vivax. Nested PCR method was used for the species identification. Nested PCR studies were performed using Plasmodium genus and specific primers for P.vivax, Plasmodium falciparum, P.ovale and Plasmodium malariae. Nested PCR products were run on gel and P.ovale was visualized in 787 bp region. P.vivax, P.malariae, P.falciparum, P.ovale and Plasmodium knowlesi species specific primers and probe-based quantitative real-time PCR (qRt-PCR) study revealed that the patient was infected with P.ovale. The patient had no history of chronic illness but had a history of recovered malaria 7-8 years ago. The patient did not have any complaints other than these complaints. CMV IgM and IgG and Brucella aglutinisation tests were negative. It is clear that relapse cases can also be seen when P.ovale species are in hypnozoite stage in the liver. Although there are 18 reported cases of relapse in the last century, these phenomena do not provide sufficient evidence for the theory of relapse. A true relapse is thought to be mild symptoms and even subclinical disease. It is also known that it is difficult to distinguish a true recurrence in cases of relapses that can occur after a long time from primer infection. The best way to overcome this difficulty is to assume being in a malaria endemic area or not between primary infection and recurrence. We think that the applications that are carried out together with the microscope and molecular studies, especially in cases where there is relapses in which low parasitemia or travel story are insufficient, are extremely important both in terms of diagnosis and accurate identification of species and in the selection of treatment.

[Retrospective evaluation of serologic and molecular test results of toxoplasmosis suspected patients]. / Toxoplasmosis süpheli hastalara ait serolojik ve moleküler test sonuçlarinin retrospektif dlarak degerlendirilmesi.

Toxoplasma gondii is a compulsory intracellular protozoan parasite with a wide range of host in warm-blooded vertebrates and has importance in terms of health and economy. Toxoplasmosis is very common because it can infect people with a variety of ways; ingestion of contaminated water and nutrients; raw or undercooked meats containing tissue cysts, blood transfusions, organ transplantantation and transplacental transfer. The aim of this study was to evaluate serologic and molecular test results of toxoplasmosis pre-diagnosed patients. Anti-T.gondii-IgG, anti-T.gondii-IgM ELISA, anti-T.gondii-IgM IFAT and anti-T.gondii-IgG avidity serological tests and PCR tests were applied by using blood, cerebrospinal fluid, amniotic fluid, pericardial fluid and abscess samples from patients who have admitted to Erciyes University Faculty of Medicine Department of Parasitology routine serology and molecular diagnosis laboratories with a pre-diagnosis of toxoplasmosis. Among 6547 patients 3.3% (n= 220) were only IgM positive, 9.2% were both IgG and IgM positive (n= 598). Among male patients, the positivity rates were lower and only IgM seropositive patients were 0.6% (n= 45) while the frequency of both IgG and IgM positive patients was 0.8% (n= 47). The number of both IgG and IgM seropositive cases among new borns, constituting 6.4% (n= 425) of the total number of patients, was 20 (0.3%) and the number of IgM seropositive samples was 25 (0.4%). Only 290 patients positive for IgM antibodies were studied for IFAT and 22 of these patients were positive for anti-T.gondii-IFAT IgM. Anti-T.gondii IgG avidity test was performed in all IgG positive patients regardless of their IgM seropositivity; low avidity was found in 0.7% (n= 18) of IgM-negative patients' sera and equivocal avidity was detected in 6.5% (n= 179). Low avidity was detected in 2.6% of IgM positive patients. Nine of the patients evaluated as anti-T.gondii IgM negative and IgG positive were detected as positive by PCR and two of them were negative. One of these PCR-positive patient's amniotic fluid was sent after the serological test results and detected as positive. Twenty CSF samples were studied by PCR and 7 samples were positive. Also, 8 blood samples which were anti-T.gondii IgM negative and IgG positive were found to bepositive in 7 and negative in one sample with PCR results, subsequently. PCR tests with pericardial fluid and abscess materials were found to be negative. In the case of suspicious or risky situations such as false negatives or false positives resulting from cross-reaction that can occur in ELISA tests, unnecessary medication or interventional approaches can be avoided by applying molecular-based testing at laboratories with appropriate infrastructure. For this reason, we believe that the application of molecular tests in addition to serological tests in risky situations may give more reliable results.

[Dientamoeba fragilis infection in patients with gastrointestinal system complaints]. / Gastrointestinal sikayeti olan hastalarda dientamoeba fragilis enfeksiyonu.

In this study, we aimed to investigate the incidence of Dientamoeba fragilis with different diagnostic methods in patients with gastrointestinal symptoms and determine the sensitivity and specificity of existing diagnostic methods. Fecal samples collected from 101 patients with gastrointestinal complaints (especially upper abdominal pain, abdominal and pelvic pain, nausea and vomiting, gastroenteritis and colitis, unexplained fever and diarrhea) and 20 control cases from various clinics were included in the study. Samples were first examined with native-Lugol (N-L) method and cultured in Robinson medium. All 121 stool and culture samples were stained with iron hematoxylin stain (IHS) and trichrome stain (TS) methods and examined by PCR and QPCR for D.fragilis. Among 121 stool samples 13 (10.7%), 2 (1.7%), 7 (5.7%) 13 (10.7%), and 7 (5.8%), 4 (3.3%), 2 (1.7%), 3 (2.5%) of cultured samples were determined positive with IHS, TS, PCR, QPCR respectively. Fifteen of the 121 stool samples were determined as diarrheal. All diarrheal stool samples were negative with IHS and TS. One of the diarrheal stools and 6 (4.9%) of the non-diarrheal stools were positive by PCR. All of the diarrheal stools were negative. Thirteen of the non-diarrheal stool samples (10.7%) were positive by QPCR. When the QPCR method was considered as gold standard, sensitivity and specificity values were determined as 46% and 93% in IHS, 0% and 99% in TS, 54% and 100% by PCR and sensitivity and specificity values were 67% and 96% in IHS, 33% and 98% in TS, 67% and 100% by PCR among cultured stool samples. As a result, it was determined that there was a statistically significant difference between the samples of the patients and the control groups and the sensitivity and specificity of the conventional and molecular methods (IHS, TS, PCR and QPCR) determined in this study supported the results of other compared studies. It has been determined that staining methods used for the diagnosis of D.fragilis gave false positivite or negativite results. In addition, the QPCR method is more advantageous in terms of time saving for the diagnosis and initiation of the treatment and in cases where QPCR is not available, IHS and conventional PCR methods should be used together. In our opinion, this study will contribute to the results of epidemiological and scientific studies on D.fragilis in Turkey.

Evaluation of four commercial DNA extraction kits for the detection of Microsporidia and the importance of pretreatments in DNA isolation.

Microsporidia are obligate intracellular parasitic protozoa infecting the wide variety of hosts and are commonly known as a cause of chronic diarrhea particularly in immunocompromised individuals. Molecular-based tests have high sensitivity and specificity in disease diagnosis. However, these tests' performance relies on the isolation of DNA in a good concentration. The standard procedures of commercial DNA extraction kits are usually insufficient for this purpose due to the tough walls of spores. This study aimed to test the significance of pretreatments by glass beads and freeze-thawing processes in DNA isolation from microsporidia spores. The parasite was cultured in growing Vero cells and seven serial dilutions were prepared from the collected spores. DNA purification was performed according to different tissue kits and stool kit procedures with and without any pretreatment. Concentration of isolated DNA samples were evaluated by real-time PCR. As a result of this study, the detectable amount of spores is minimum 10 spores in each 100 µ! sample according to the different tissue kits' standard protocols. However, according to the DNA stool mini kit, the detectable amount of spores was found to be 1,000 spores/100 µl of stool sample when pretreated with both the freeze-thawing and glass beads methods.In conclusion, the current study demonstrated that further pretreatments are an essential process for DNA extraction from the stool specimens in order to avoid possible false negativity in the diagnosis of microsporidiosis.

The high prevalence of Encephalitozoon intestinalis in patients receiving chemotherapy and children with growth retardation and the validity of real-time PCR in its diagnosis.

BACKGROUND/AIM: The aim of this study was to investigate the presence of Encephalitozoon intestinalis in different patient groups consisting of immunocompromised and immunocompetent individuals. MATERIALS AND METHODS: The stool samples of 100 patients consisting of 25 patients receiving chemotherapy and with acute gastrointestinal complaints, 25 with bone marrow transplant and acute gastrointestinal complaints, 25 with urticaria, and 25 with growth retardation were included in the study. As control groups, 25 subjects without any chronic disease but with acute gastrointestinal complaints and 25 healthy volunteers, making a total of 50 subjects, were included in the study. E. intestinalis was investigated by IFA-MAbs and molecular methods. RESULTS: Forty percent of patients receiving chemotherapy and with acute gastrointestinal complaints, 24% of patients with bone marrow transplant and acute gastrointestinal complaints, 20% of patients with urticaria, 40% of children with growth retardation, and 28% of patients without any chronic disease but with acute gastrointestinal complaints were determined as positive. CONCLUSION: To the best of our knowledge, this is the first report to assess the relationship between E. intestinalis and growth retardation. We think that the reliability of the use of molecular methods, especially real-time PCR, should be improved for the diagnosis of E. intestinalis.