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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?

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Abstract

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.

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Acknowledgments

We thank to BM Laboratory Company and Dr. Humen Cebbari for the technical support and Dr. Selma Usluca from the National Parasitology Laboratory, Turkish Public Health Agency, for providing the negative control samples.

Financial support

This study was funded by the Hitit University Scientific Research Projects with the grant number TIP19002.15.003. We would like to thank the Hitit University for providing the financial support.

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Authors and Affiliations

  1. Department of Medical Microbiology and Clinical Microbiology, Faculty of Medicine, Near East University, Nicosia, Cyprus

    Ayse Seyer, Emrah Ruh, Ayse Semra Güreser, Turgut Imir & Aysegul Taylan-Ozkan

  2. Faculty of Health Sciences, Girne American University, Kyrenia, Cyprus

    Ayse Seyer

  3. Department of Medical Microbiology, Faculty of Medicine, Hitit University, Çorum, Turkey

    Djursun Karasartova, Ayse Semra Güreser & Aysegul Taylan-Ozkan

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  1. Ayse Seyer
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  2. Djursun Karasartova
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  5. Turgut Imir
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  6. Aysegul Taylan-Ozkan
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Correspondence to Aysegul Taylan-Ozkan.

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The study was approved by the Clinical Research Ethics Board of Ankara Numune Training and Research Hospital, Turkey, No/Year: E.Kurul-E-15-446/06.04.2015.

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Written informed consent was obtained from each patient who participated in this study.

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Seyer, A., Karasartova, D., Ruh, E. et al. Is “dried stool spots on filter paper method (DSSFP)” more sensitive and effective for detecting Blastocystis spp. and their subtypes by PCR and sequencing?. Parasitol Res 115, 4449–4455 (2016). https://doi.org/10.1007/s00436-016-5231-y

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