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    An alternative DNA extraction method for detection of Blastocystis spp. in human fecal samples
    (Academic Press Inc., 2018) Karasartova, Djursun; Güreser, Ayşe Semra; Ruh, Emrah; Türegün Atasoy, Buse; Çalgın, Mustafa Kerem; Taşçı, Leyla; Taylan Özkan, Hikmet Ayşegül
    Polymerase chain reaction (PCR) is an effective technique for diagnosis of Blastocystis infection. Notably, DNA isolation procedure is extremely critical for the PCR step. In the present study, a recently described extraction procedure, named as the “sand method” was modified and adapted for isolation of Blastocystis DNA. To evaluate its efficacy, the current method and QIAamp DNA Stool Mini Kit (Qiagen) were applied to fresh human stool samples. Our results indicated that, the mean DNA concentrations obtained by the sand method and the commercial kit were 48 and 55 ng/?l, respectively. Also, no DNA inhibitors were detected in two methods. The sand method was capable of detecting 16 parasites per 50 mg feces. DNA samples extracted by both methods were subjected to PCR. Blastocystis spp. were detected in 11 (31.4%) of 35 samples, and perfect agreement (?: 1.000) was found between the PCR-sand method and PCR-commercial kit method. The samples that were detected positive by PCR-sand method were successfully sequenced, and Blastocystis subtypes (STs) were identified as ST3, ST2 and ST1. In conclusion, the present study indicates that the sand method provides a simple, rapid and inexpensive procedure for reliable extraction of Blastocystis DNA from stool samples. © 2018 Elsevier Inc.
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    Cystic Echinococcosis in Northern Cyprus: A Literature Review
    (Aves, 2018) Ruh, Emrah; Ozkan, Aysegul Taylan
    Cystic echinococcosis is a zoonotic disease caused by the larval stages of Echinococcus granulosus sensu lato (s.l.). The life cycle of the parasite consists of dogs and other canids as the definitive hosts and ungulates, such as sheep and goats, as the intermediate hosts. Humans are accidental intermediate hosts in the life cycle of the parasite. The island of Cyprus is located in the eastern part of the Mediterranean region where echinococcosis is endemic. The disease was common in the island until the 1970s. In Southern Cyprus, two control programs for echinococcosis were implemented; the first one was initiated in 1971 and continued until 1985, and the second one was introduced in 1993 and implemented for 5 years. The control programs resulted in a decrease of the prevalence in both dogs and livestock. In Northern Cyprus, a control program was implemented between 1997 and 2005 that also resulted in a decrease of the disease rates in definitive and intermediate hosts. However, termination of the program led to an increase in the prevalence. Recent data suggest that sporadic cases of echinococcosis still exist; therefore, control programs should be continued in order to prevent the disease in Northern Cyprus.
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    Epidemiology and prevalence of blastocystis spp. in North Cyprus
    (American Society of Tropical Medicine and Hygiene, 2017) Seyer, Ayşe; Karasartova, Djursun; Ruh, Emrah; Güreser, Ayşe Semra; Turgal, Ebru; Imir, Turgut; Taylan Özkan, Hikmet Ayşegül
    This study was conducted to investigate the prevalence of Blastocystis spp. and its subtypes (STs) in North Cyprus; and to evaluate the presence of this parasite and its STs with respect to demographic, socioeconomic, and epidemiological factors, as well as gastrointestinal symptoms. Stool samples were collected from 230 volunteers. Each participant also filled out a questionnaire. The samples were examined microscopically by native-Lugol and trichrome methods and further tested by polymerase chain reaction (PCR) and sequencing. Prevalence of Blastocystis spp. infection was found to be 10.5%, 10.5%, and 27.8%, by direct microscopy, trichrome method, and PCR, respectively. No other parasites were detected in the specimens except Giardia spp. (n = 2; 0.8%) and Entamoeba coli (n = 1; 0.4%). The most common Blastocystis STs were ST3 (20; 31.2%), ST2 (18; 28.2%), ST1 (8; 12.5%), and ST4 (7; 11%); whereas other STs were identified as ST6 (3; 4.7%), ST7 (2; 3.2%), and non-ST (6; 9.4%). Presence of Blastocystis spp. and its STs was not significantly related to any of the demographic, socioeconomic, and epidemiological factors. Furthermore, no significant association of Blastocystis spp. and its STs with gastrointestinal symptoms was found. This study is the first investigation of the epidemiology of Blastocystis spp. in North Cyprus. Distribution of Blastocystis spp. and its STs among demographic, socioeconomic, and epidemiological factors showed complete homogeneity. Presence of the parasite and its STs was not significantly related with the gastrointestinal symptoms among symptomatic and asymptomatic individuals. These findings suggest that Blastocystis spp. may be part of the intestinal flora in humans. Copyright © 2017 by The American Society of Tropical Medicine and Hygiene.
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    Investigation of pregnancy-associated malaria by microscopy, rapid diagnostic test and PCR in Bandundu, the Democratic Republic of Congo
    (Oxford University Press, 2018) Ruh, Emrah; Bateko, Jean Paul; Imir, Turgut; Taylan Özkan, Hikmet Ayşegül
    Background: The study was conducted to investigate malaria prevalence among a group of women in the Democratic Republic of Congo (DRC) who received intermittent preventive treatment in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP). Methods: A total of 250 women from Bandundu city who received two doses of IPTp-SP were enrolled in the survey. Blood samples were collected at the time of delivery and malaria prevalence was determined using microscopy, rapid diagnostic test (RDT), and nested polymerase chain reaction (PCR). Results: Malaria infection was detected in 81 (32.4%), 93 (37.2%), and 92 (36.8%) samples by microscopy, RDT, and PCR, respectively. Among 92 samples, P. falciparum mono-infection (n=87; 94.5%), P. falciparum+P. vivax (n=2; 2.2%) and P. falciparum+P. malariae (n=1; 1.1%) mixed infections, and P. vivax mono-infection (n=2; 2.2%) were detected. Prevalence of malaria was not affected by age and number of pregnancies (p<0.05). Microscopy and RDT, either alone ( ?=0.29; p<0.001) or in combination (?=0.33; p<0.001) showed a fair agreement with PCR. Conclusions: Our findings indicate that two doses of IPTp-SP did not protect the women against malaria in the DRC, and support the World Health Organization (WHO) guidelines that ensure a minimum of three doses of SP in pregnancy. © The Author(s) 2018. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved.
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    Is “dried stool spots on filter paper method (DSSFP)” more sensitive and effective for detecting Blastocystis spp. and their subtypes by PCR and sequencing?
    (Springer Verlag, 2016) Seyer, Ayşe; Karasartova, Djursun; Ruh, Emrah; Güreser, Ayşe Semra; Imir, Turgut; Taylan Özkan, Hikmet Ayşegül
    PCR and DNA sequencing are currently the diagnostic methods of choice for detection of Blastocystis spp. and their suptypes. Fresh or frozen stool samples have disadvantages in terms of several aspects such as transportation, storage, and existence of PCR inhibitors. Filter paper technology may provide a solution to these issues. The aim of the present study was to detect Blastocystis spp. and their subtypes by employing two different preservation methods: conventional frozen stool (FS) and dried stool spots on filter paper (DSSFP). Concentration and purity of DNA, sensitivity of PCR, and DNA sequencing results obtained from the two methods were also compared. A total of 230 fecal samples were included and separated into two parts: one part of the fecal samples were directly frozen and stored at ?20 °C. The remaining portion of the specimens were homogenized with saline and spread onto the filter papers as thin layer with a diameter of approximately 3 cm. After air-dried, the filter papers were stored at room temperature. DSSFP samples were collected by scraping from the filter papers. DNA were extracted by EURx Stool DNA Extraction Kit from both samples. Concentration and purity were measured with Nano-Drop, then PCR and sequencing were conducted for detection of Blastocystis spp. and its genotypes. Pure DNA was obtained with a A260/A280 ratio of 1.7–2.2 in both methods. DNA yield from FS was 25–405 ng/?l and average DNA concentration was 151 ng/?l, while these were 7–339 and 122 ng/?l for DSSFP, respectively. No PCR inhibition was observed in two methods. DNA from DSSFP were found to be stable and PCR were reproducible for at least 1 year. FS-PCR- and DSSFP-PCR-positive samples were 49 (21.3 %) and 58 (25.3 %), respectively (p = 0.078). The 43 specimens were concordantly positive by both FS-PCR and DSSFP-PCR. When the microscopy was taken as the gold standard, sensitivity of DSSFP-PCR and FS-PCR was 95.5 and 86.4 %, while specificity of both tests was 99.4 and 98.3 %, respectively. DNA sequencing results of 19 microscopically confirmed cases were strictly identical (concordance 100 %) in both methods, and ST2:6, ST3:8, ST4:3, and ST6:2 were the detected subtypes. Among the 230 fecal samples, the most predominant subtypes were ST3, ST2, ST4, and ST1 by both FS and DSSFP methods. Concordance of DNA sequencing results obtained from the two methods was noted to be 90.7 %. To our knowledge, this is the first study that demonstrates DNA extraction from DSSFP is more sensitive and effective than the FS method for diagnosis of Blastocystis spp. and their subtypes by PCR and DNA sequencing. © 2016, Springer-Verlag Berlin Heidelberg.
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    Leishmaniasis in Northern Cyprus
    (2019) Ruh, Emrah; Özkan, Hikmet Ayşegül Taylan
    Leishmaniasis is a vector-borne disease that is caused by Leishmania parasites. Sandflies are the vectors, and dogs are the primary reservoir of Leishmania spp. Cutaneous leishmaniasis (CL) is the most common form of the disease, whereas visceral leishmaniasis (VL) is the most severe form and is generally fatal if left untreated. The disease is seen in 98 countries and distributed through three regions on five continents. The island of Cyprus is located in the eastern part of the Mediterranean region where leishmaniasis is endemic. The presence of sandflies, canine leishmaniasis (CanL), and human VL and CL cases has been documented in Northern and Southern Cyprus. CanL cases were found at various rates between 1.9% and 13.2% in Northern Cyprus. In 1990, Leishmanin skin test positivity was detected in Northern Cyprus, and Leishmania infantum was found to be the infecting agent. In 2016, three pediatric VL cases caused by L. infantum were reported in Northern Cyprus. More recently, in a study conducted in Kyrenia District, three seropositive individuals have been detected. In the study, seven individuals, including the seropositive persons, were found to have a history of CL. Consequently, these studies indicate the presence of leishmaniasis in Northern Cyprus. Therefore, vector and reservoir control programs should be implemented for prevention of the disease.
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    Leishmaniasis in Northern Cyprus: Human cases and their association with risk factors
    (Malaria Research Center, 2017) Ruh, Emrah; Bostancı, Ayşegül; Kunter, Vasfiye; Tosun, Özgür; Imir, Turgut; Schallig, Henk; Taylan Özkan, Hikmet Ayşegül
    Background & objectives: Cyprus is located in the eastern part of the Mediterranean Region where leishmaniasis is endemic. The primary objective of this study was to investigate human visceral leishmaniasis (VL) in the northern region of Cyprus where presence of canine leishmaniasis (CanL) and sandflies has been documented in earlier studies. The secondary objective was to assess the association of leishmaniasis with demographic and epidemiological variables. Methods: Intravenous blood samples were collected from 249 volunteers in Kyrenia district (located in the northern coastal region of Cyprus). Whole blood samples were tested for DNA of Leishmania spp by polymerase chain reaction (PCR), while serum samples were analyzed using direct agglutination test (DAT) and rK39 test. For evaluation of possible risk factors, a questionnaire was applied to the participants. Results: Only three (1.2%) of 249 participants were found seropositive by DAT (n = 2) or rK39 test (n = 1). The remaining samples were negative in serology, and no PCR positivity was detected in any of the 249 participants. Seven individuals, including the seropositive cases, had a history of cutaneous leishmaniasis (CL). Seropositivity and CL were not significantly related with gender (M/F: 40.2/59.8%), age [Mean: 42.85 ± 17.45, Median: 40 (7–86)], occupation (Indoor/Outdoor: 84.7/12.9%), dog ownership (52.6%), and CanL history (5.3%). However, a statistical association was found between seropositivity and past CL infection. Also, a significant relation was observed between participants living in peripheral area (63.1%) and CL infection. Furthermore, leishmaniasis awareness (28.1%) among the study population was statistically correlated with past CL infection and dog ownership. Interpretation & conclusion: This study demonstrates the presence of leishmaniasis and highlight the need for implementation of efficient control measures on the northern coast of Cyprus. © 2017, Malaria Research Center. All rights reserved.
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    Molecular identification of sulfadoxine-pyrimethamine resistance in malaria infected women who received intermittent preventive treatment in the Democratic Republic of Congo
    (BioMed Central Ltd., 2018) Ruh, Emrah; Bateko, Jean Paul; İmir, Turgut; Taylan Özkan, Hikmet Ayşegül
    Background: Point mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes which confer resistance to sulfadoxine-pyrimethamine (SP) occur at increasing rates. The present study aimed to identify Pfdhfr and Pfdhps mutations in P. falciparum isolates recovered from women who received two doses of SP during pregnancy in Bandundu, the Democratic Republic of Congo (DRC). Methods: A total of 48 women with confirmed P. falciparum infection were enrolled in the study. Finger-prick blood samples that were collected on filter paper at the time of delivery were used for DNA isolation. Pfdhfr and Pfdhps genes were amplified by a nested PCR protocol. DNA sequencing was performed on both strands, and the point mutations were analysed. Results: All of the 48 (100.0%) P. falciparum isolates carried at least one polymorphism in both genes. The wild-type haplotypes of Pfdhfr (CNCSI [C50, N51, C59, S108, I164]) and Pfdhps (SAKAA [S436, A437, K540, A581, A613]) were not observed in the study. In Pfdhfr, N51I (85.4%), C59R (60.4%), and S108N (100.0%) polymorphisms were detected. Triple mutation (CIRNI) (mutant amino acids are underlined) was the most prevalent (47.9%) Pfdhfr haplotype. In the study, all P. falciparum isolates (100.0%) harboured the A437G allele in Pfdhps gene. Also, K540E and A581G polymorphisms were observed in one (2.1%) isolate. Single mutant haplotype (SGKAA) was detected in 97.9% of the isolates. Mutant Pfdhfr and Pfdhps allele combinations revealed quintuple (CICNI-SGEGA; 2.1%), quadruple (CIRNI-SGKAA; 47.9%), triple (CICNI-SGKAA; 35.4%, CNRNI-SGKAA; 12.5%), and double (CNCNI-SGKAA; 2.1%) haplotypes. Conclusions: In the study, the rate of SGEGA haplotype was low (2.1%). Although K540E and A581G alleles are more common in Eastern Africa, a distinct lineage of SGEGA is also present in the DRC, which is located in Central Africa. This haplotype is associated with decreased efficacy of SP in pregnant women and infants, therefore, it should be carefully considered in the DRC and SP resistance should be routinely monitored. © 2018 The Author(s).
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    Serological screening of West Nile virus among blood donors in northern Cyprus
    (Wiley, 2020) Balaman, Nagat; Gazi, Umut; Imir, Turgut; Şanlıdağ, Tamer; Ruh, Emrah; Tosun, Özgür; Özgül, Aykut; Taylan Özkan, Hikmet Ayşegül
    Background: West Nile virus (WNV) is a neurotropic arbovirus that can also be transmitted through blood transfusion. Even though its geographic distribution has been expanding, there has not yet been any epidemiological data on WNV in northern Cyprus. The aim of our study is to fill this gap by using donated blood samples. Methods: Samples collected from the main government hospital blood bank in Nicosia were analyzed by anti?WNV enzyme?linked immunosorbent assay (ELISA) (immunoglobulin M [IgM] and immunoglobulin G [IgG]). Seropositive samples were subjected to plaque reduction neutralization test (PRNT) for confirmation and analyzed by ELISA IgG avidity test and reverse transcription real?time polymerase chain reaction (rRT?PCR). Results: Of the 760 sera samples, 2 (0.3%) were IgM+ and 31 (4.1%) were IgG+. Neutralization activity was detected in none (0.0%) of the IgM+ and 26 (83.9%) of IgG+ donor specimens. ELISA IgG avidity test reported high avidity in 21 (67.7%) and low avidity in one (3.2%) IgG+ sample. PRNT?confirmed anti?WNV IgG+ samples exhibited only borderline (19.2%) or high avidity (80.8%) values. rRT?PCR results were negative for both IgM+ and IgG+ samples. Conclusion: Anti?WNV antibodies were detected in northern Cyprus among blood donors. The establishment of preventive measures and evaluation of the geographic extent of the WNV in northern Cyprus are highly recommended.