Abstract. 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.

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/μl and average DNA concentration was 151 ng/μl, while these were 7-339 and 122 ng/μl 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.

* bu çalışmanın bir bölümü 6. ulusal Tanısal ve Moleküler Mikrobiyoloji Kongresi (15-19 Haziran 2010, Ankara)'nde poster olarak sunulmuştur. ÖZET Giriş: Aspergillus cinsi küf mantarlarının klinik örneklerde kültürde üretilme başarısı %50'yi geçmemektedir. Nötropenik ateş dönemindeki hastaların bir çoğunda ampirik antifungal tedavi başlanması nedeniyle, kültürde herhangi bir mantarın üretilebilme olasılığı azalmaktadır. Moleküler yöntemlerde hedeflenen mikroorganizmanın DNA'sı gösterildiği için tedavi başlanmış olması tanıyı olumsuz yönde etkilememektedir. Bu çalışmada, Aspergillus cinsi mantarların DNA'sının gösterilmesi için geliştirilen kalitatif gerçek zamanlı polimeraz zincir reaksiyonu yöntemi sunulmuştur. Materyal ve Metod: Örneklerden DNA izolasyonu hazır sistem kullanılmadan yapılmıştır. Aspergillus cinsi mantarların rDNA gen bölgesinden 28S alt ünitesi hedeflenmiş, TaqMan amplifikasyon gerçekleştirilmiştir. Yöntemin aspergillozdaki tanısal değeri farklı örnekler kullanılarak incelenmiştir. Aspergillus kolonisi ile kontamine edilen "simüle" kan örnekleri, aspergilloz oluşturulan deney hayvanı kan ve dokuları ve aspergilloz şüphesiyle takip edilen hasta örnekleri ile toplam 60 örnek çalışılmıştır. Bulgular: En çok yalancı negatiflik deneysel olarak aspergilloz oluşturulmuş hayvanların doku örneklerinde elde edilmiştir. Hayvan doku örneklerinin 4 (%40)'ünde, hayvan kan örneklerinin 3 (%30)'ünde, simüle kan örneklerinin 2 (%20)'sinde, hasta örneklerinin 1 (%10)'inde yalancı negatif sonuç alınmıştır. Koloni süspansiyonlarının tamamı pozitif bulunmuştur. Sonuç: Geliştirdiğimiz yöntemin kan örneklerinde Aspergillus DNA'sının gösterilmesinde başarılı olduğu ancak, doku örnekleri için geliştirilmesi gerektiğine karar verilmiştir. Yöntemin tanısal değerinin geniş gruplarda yapılacak çalışmalar ile desteklenmesi gerekmektedir.