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Subtype Analyses Of 50 Blastocystis spp. Isolates From Symptomatıc Patients

Year 2017, , 6 - 10, 31.01.2017
https://doi.org/10.30934/kusbed.359174

Abstract

Aim: Blastocystis spp. is an enteric protozoon that has grown in importance with recent knowledge of the symptoms, disorders it may cause. Its prevalence varies in different countries, but it is most frequently detected in developing countries. Nine subtypes (ST) of Blastocystis spp. have been detected in humans and it is suggested that these subtypes can be related to its varying clinical symptoms.

Methods: In this study, stool samples were examined with microscopy after performing formol-ethyl acetate sedimentation for the presence of Blastocystis spp. Fifty samples with Blastocystis spp. were cultured in 2 different media and underwent subsequent sequencing to determine the subtypes. Relations between clinical symptoms and the subtypes were examined.

Results: Fourty (80%) of the samples were positive with both culture media; 5 (10%) showed no growth in any media; 5 (10%) grew in only one. 39 (78%) samples had one subtype while 11 (22%) had mixed subtypes. ST3 was determined as the most common subtype followed by ST1, ST5, ST2, and ST4. Abdominal pain was the most common clinical symptom in 23 (46%) patients, followed by 17 (34%) with allergic symptoms. Others had gastrointestinal symptoms and joint paint. No significant relationship detected between symptoms and subtypes.

Conclusion: ST3 was the predominant subtype in patients with gastrointestinal and allergic symptoms. Also, one patient with joint paint suggests that Blastocystis spp. could also be related to arthritis, and even though few cases have been reported, this should be considered by physicians. Although we could not determine a relationship between subtypes and clinical symptoms, further investigations are needed to clarify this subject.

References

  • Popruk P, Pintong A, Radomyos P. Diversity of Blastocystis Subtypes in Humans. J. Trop. Med. Parasitol. 2013; 36:88-97.
  • Wawrzyniak I, Poirier P, Viscogliosi E, ve diğ. Blastocystis,an unrecognized parasite:an overview of pathogenesis and diagnosis. Ther Adv Infect Dis. 2013;1:167-78.
  • Scanlan PD, Marchesi JR Micro-eukaryotic diversity of the human distal gut microbiota : quantitative assessment using culture-dependent and independent analysis of faeces. ISME J. 2008;2:1183-93.
  • Tan TC, Ong SC, Suresh KG. Genetic variability of Blastocystis sp. isolated obtained from cancer and HIV/AIDS patients. Parasitol Res. 2009; 105: 1283-6.
  • Katsarou-Katsari A, Vassalos CM, Tzanetou K, ve diğ. Acute urticaria associated with amoeboid forms of Blastocystis sp. Subtype 3. Acta Derm Venereol. 2008; 88 :80-1.
  • Ozyurt M, Kurt O, Molbak K, ve diğ. Molecular epidemiology of Blastocystis infections in Turkey. Parasitol Int. 2008; 57:300-6.
  • Dogruman-Al F, Yoshikawa H, Kustimur S, ve diğ. PCR- based subtyping of Blastocystis isolates from symptomatic and asymptomatic individuals in a major hospital in Ankara,Turkey. Parasitol Res. 2009; 106:263-8.
  • Souppart L, Sanciu G, Cian A, ve diğ. Molecular epidemiology of human Blastocystis isolates in France. Parasitol Res. 2009;105:413-21.
  • Dominguez-Marquez M, Guna R, Munoz C, ve diğ. High prevalence of subtip 4 among isolates of Blastocystis hominis from symptomatic patients of health district of Valencia (Spain). Parasitol Res. 2009; 105:949-55.
  • Stendsvolt C, Christiansen D, Olsen K, ve diğ. Blastocystis sp. Subtype 4 is common in Danish Blastocystis-positive patients presenting with acute diarrhea. Am J Trop Med Hyg. 2011; 84:883-5.
  • Forsell J, Granlund M, Stendsvold C, ve diğ. Subtype analysis of Blastocystis isolates in Swedish patients. Eur J Clin Microbiol Infect Dis. 2012; 31:1689-96.
  • Jones WR. The experimental infection of rats with Entamoeba histolytica; with a method for evaluating the anti-amoebic properties of new compounds. Ann Trop Med Parasitol. 1946;40: 130-140.
  • Zaman V, Ng GC, Suresh K, ve diğ. Isolation of Blastocystis from the cockroach (Dictyoptera, Blattidae). Parasitol Res. 1993; 79 : 73-4.
  • Grecu (Mătiuț) DS, Neagu AN, Hărmănescu EA, ve diğ. In vitro division modalities developed by Blastocystis hominis examined with the acridine orange stain. Analele Științifice ale Universității, „Alexandru Ioan Cuza” dinIași, s. Biologie animală, Tom LIX,2013. 8 Ocak 2017’de http://www.bio.uaic.ro/publicatii/anale_zoologie/issue/2013/2013fa02Grecu&al.pdf adresinden indirildi.
  • Stensvold CR. Comparison of Sequencing (Barcode Region) and Sequence- Tagged-Site PCR for Blastocystis Subtyping. J Clin Microbiol. 2013; 51: 190–4.
  • Santín M, Gómez-Muñoz MT, Solano-Aguilar G, ve diğ. Development of a new PCR protocol to detect and subtype Blastocystis spp. from humans and animals. Parasitol Res. 2011; 109(1):205-12.
  • Scanlan PD, Stensvold CR, Cotter PD. Development and Application of a Blastocystis Subtype-Specific PCR Assay Reveals that Mixed-Subtype Infections Are Common in a Healthy Human Population. Appl Environ Microbiol. 2015; 81 (12): 4071-6.
  • Zhang X, Qiao J, Wu X, ve diğ. In vitro culture of blastocystis hominis in three liquid media and its usefulness in the diagnosis of Blastocystosis. Int J Infect Dis. 2012; 16:23-8.
  • Roberts T, Barratt J, Harkness J,ve diğ. Comparison of Microscopy, Culture, and Conventional Polymerase Chain Reaction for Detection of Blastocystis sp. in Clinical Stool Samples. Am J Trop Med Hyg. 2011; 84: 308–12.
  • Leelayoova S, Taamasri P, Rangsin R, ve diğ. In-vitro cultivation: a sensitive method for detecting Blastocystis hominis. Ann Trop Med Parasitol. 2002; 96:803-7.
  • Stensvold CR, Arendrup MC, Jespersgaard C, ve diğ. Detecting Blastocystis using parasitologic and DNA-based methods: a comparative study. Diagn Microbiol Infect Dis. 2007; 59: 303-7.
  • Meloni D, Sanciu G, Poirier P, ve diğ. Molecular subtyping of Blastocystis sp. isolates from symptomatic patients in Italy. Parasitol Res. 2011; 109: 613-9.
  • Menounos PG, Spanakos G, Tegos N, ve diğ. Direct detection of Blastocystis sp. in human faecal samples and subtype assignment using single strand conformational polymorphism and sequencing. Mol Cell Probes. 2008; 22: 24-9.
  • Forsell J, Granlund M, Stendsvold C. Subtype analysis of Blastocystis isolates in Swedish patients. Eur J Clin Microbiol Infect Dis. 2012; 31:1689-96.
  • Alfellani MA, Mulla DT, Jacob AS, ve diğ. Genetic Diversity of Blastocystis in Livestock and Zoo Animals. Protist. 2013;164:497-509.
  • Souppart L, Moussa H, Cian A, ve diğ. Subtype analysis of Blastocystis isolates from symptomatic patients in Egypt. Parasitol Res. 2010;106: 505-11.
  • Moosavi A, Haghighi A, Mojarad E, ve diğ. Genetic variability of Blastocystis sp. isolated from symptomatic and asymptomatic individuals in Iran. Parasitol Res. 2012; 111:2311-5.
  • Noël C, Dufernez F, Gerbod D, ve diğ. Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis. J Clin Microbiol. 2005; 43: 348-55.
  • Tan KS .New insights on classification, identification, and clinical relevance of Blastocystis spp. Clin Microbiol Rev. 2008;21:639-65.
  • Ertuğ S, Malatyalı E, Ertabaklar H, ve diğ. Subtype distribution of Blastocystis isolates and evaluation of clinical symptoms detected in Aydin province, Turkey. Mikrobiyol Bul. 2015;49:98-104.
  • Sakalar C, Kandemir İ, Uyar Y, ve diğ. Polymerase Chain Reaction Based Subtyping of Blastocystis spp. Isolates from Symptomatic Patients in Turkey. Turkiye Klinikleri J Med Sci. 2013;33:1064-8.
  • Pasqui AL, Savini E, Saletti M, ve diğ. Chronic urticaria and Blastocystis hominis infection: a case report. Eur Rev Med Pharmacol Sci. 2004; 8:117-20.
  • Kick G, Rueff F, Przbilla B. Palmoplantar pruritus subsiding after Blastocystis hominis eradication. Acta Derm Venereol. 2002;82: 60.
  • Valsecchi R, Leghissa P, Greco V. Cutaneous lesions in Blastocystis hominis infection. Acta Derm. Venereol. 2004;84:322-3.
  • Safadi DE, Meloni D, Poirier P, ve diğ. Short Report: Molecular Epidemiology of Blastocystis in Lebanon and Correlation between Subtype 1 and Gastrointestinal Semptoms. Am J Trop Med Hyg. 2003;88:1203-6.
  • Dogruman-Al F, Dagci H, Yoshikawa H, ve diğ. A possible link between subtype 2 and asymptomatic infections of Blastocystis hominis. Parasitol Res 2008;103:685-9.
  • Vogelberg C, Stendsvolt C, Monecke S, ve diğ. Blastocystis sp. Subtip 2 detection during recurrence of gastrointestinal and urticarial symptoms. Parasitol Int. 2010;59:469-71.
  • Zuel-Fakkar N, Abdel Hameed DM, Hassanin OM. Study of Blastocystis hominis isolates in urticaria: a case control study. Clin Exp Dermatol. 2011;36:908-10.
  • Lee MG, Rawlins SC, Didier M, ve diğ. Infective arthritis due to Blastocystsis hominis. Ann Rheum Dis. 1990; 49: 192 – 3.
  • Krüger K, Kamilli I, Schattenkirchner M. Blastocystis hominis as a rare arthritogenic pathogen. A case report. Z Rheumatol. 1994; 53:83-5.

Blastocystis spp. Saptanan 50 Semptomatik Hastada Subtip Dağılımın Saptanması

Year 2017, , 6 - 10, 31.01.2017
https://doi.org/10.30934/kusbed.359174

Abstract

Amaç: Blastocystis spp. neden olduğu klinik semptomlar nedeniyle günümüzde önem kazanan enterik bir protozoondur. Prevalansı dünyanın farklı bölgelerinde değişmekle birlikte gelişmekte olan ülkelerde daha sık görüldüğü bildirilmektedir. Bugüne kadar insanlarda 9 Blastocystis spp. alt tipi saptanmıştır ve bu örneklerin farklı klinik semptomlar ile ilişkili olabileceği öne sürülmüştür.

Yöntem: Bu çalışmada formol-etil asetat yöntemi ile Blastocystis spp. varlığı saptanan 50 semptomatik hastadan elde edilen izolatlarının iki farklı besiyerinde kültürleri yapılmış, bu izolatların alt tip dağılımı sekanslama yöntemi ile belirlenmiş, alt tipler ile klinik bulgular arasındaki ilişkiler incelenmiştir.

Bulgular: Örneklerden 40 (%80)’ı her iki besiyerinde de üremiş, 5 (%10)’i iki besiyerinde de ürememiş, 5 (%10) örnek ise iki besiyerinden sadece birinde üremiştir. 39 (%78) örnekte tek bir alt tip saptanırken, 11 (%22) örnekte birden fazla alt tip varlığı saptanmıştır. Örnekler arasında en sık ST3 saptanırken bunu sırasıyla ST1, ST5, ST2 ve ST4 takip etmiştir. Karın ağrısı 23 (%46) hastada olmak üzere en sık rastlanan klinik semptom olup, allerjik semptomlar ise 17 (%34) hastada gözlenmiştir. Bunun dışında hastalarda çeşitli gastrointestinal semptomlar ve eklem ağrısı varlığı da gözlenmiştir. Klinik semptomlar ile alt tipler arasında anlamlı bir ilişki saptanamamıştır.

Sonuç: Çalışmamızda, gastrointestinal ve allerjik semptomları olan hastalarda ST3 alt tipi en sık saptanmıştır. Ayrıca her ne kadar az sayıda olgu bildirilmiş olsa da bir hastada eklem ağrısı saptanmış olması Blastocystis spp.’nin artrit ile ilişkisi olabileceğini ve klinisyenler tarafından göz önünde bulundurulması gerektiğini düşündürmektedir. Her ne kadar çalışmamızda klinik bulgular ile alt tipler arasında anlamlı bir ilişki bulunamamış olsa da Blastocystis spp. alt tiplerinin, klinik bulgularla ilişkilerinin anlaşılması için daha fazla araştırmaya ihtiyaç vardır.

References

  • Popruk P, Pintong A, Radomyos P. Diversity of Blastocystis Subtypes in Humans. J. Trop. Med. Parasitol. 2013; 36:88-97.
  • Wawrzyniak I, Poirier P, Viscogliosi E, ve diğ. Blastocystis,an unrecognized parasite:an overview of pathogenesis and diagnosis. Ther Adv Infect Dis. 2013;1:167-78.
  • Scanlan PD, Marchesi JR Micro-eukaryotic diversity of the human distal gut microbiota : quantitative assessment using culture-dependent and independent analysis of faeces. ISME J. 2008;2:1183-93.
  • Tan TC, Ong SC, Suresh KG. Genetic variability of Blastocystis sp. isolated obtained from cancer and HIV/AIDS patients. Parasitol Res. 2009; 105: 1283-6.
  • Katsarou-Katsari A, Vassalos CM, Tzanetou K, ve diğ. Acute urticaria associated with amoeboid forms of Blastocystis sp. Subtype 3. Acta Derm Venereol. 2008; 88 :80-1.
  • Ozyurt M, Kurt O, Molbak K, ve diğ. Molecular epidemiology of Blastocystis infections in Turkey. Parasitol Int. 2008; 57:300-6.
  • Dogruman-Al F, Yoshikawa H, Kustimur S, ve diğ. PCR- based subtyping of Blastocystis isolates from symptomatic and asymptomatic individuals in a major hospital in Ankara,Turkey. Parasitol Res. 2009; 106:263-8.
  • Souppart L, Sanciu G, Cian A, ve diğ. Molecular epidemiology of human Blastocystis isolates in France. Parasitol Res. 2009;105:413-21.
  • Dominguez-Marquez M, Guna R, Munoz C, ve diğ. High prevalence of subtip 4 among isolates of Blastocystis hominis from symptomatic patients of health district of Valencia (Spain). Parasitol Res. 2009; 105:949-55.
  • Stendsvolt C, Christiansen D, Olsen K, ve diğ. Blastocystis sp. Subtype 4 is common in Danish Blastocystis-positive patients presenting with acute diarrhea. Am J Trop Med Hyg. 2011; 84:883-5.
  • Forsell J, Granlund M, Stendsvold C, ve diğ. Subtype analysis of Blastocystis isolates in Swedish patients. Eur J Clin Microbiol Infect Dis. 2012; 31:1689-96.
  • Jones WR. The experimental infection of rats with Entamoeba histolytica; with a method for evaluating the anti-amoebic properties of new compounds. Ann Trop Med Parasitol. 1946;40: 130-140.
  • Zaman V, Ng GC, Suresh K, ve diğ. Isolation of Blastocystis from the cockroach (Dictyoptera, Blattidae). Parasitol Res. 1993; 79 : 73-4.
  • Grecu (Mătiuț) DS, Neagu AN, Hărmănescu EA, ve diğ. In vitro division modalities developed by Blastocystis hominis examined with the acridine orange stain. Analele Științifice ale Universității, „Alexandru Ioan Cuza” dinIași, s. Biologie animală, Tom LIX,2013. 8 Ocak 2017’de http://www.bio.uaic.ro/publicatii/anale_zoologie/issue/2013/2013fa02Grecu&al.pdf adresinden indirildi.
  • Stensvold CR. Comparison of Sequencing (Barcode Region) and Sequence- Tagged-Site PCR for Blastocystis Subtyping. J Clin Microbiol. 2013; 51: 190–4.
  • Santín M, Gómez-Muñoz MT, Solano-Aguilar G, ve diğ. Development of a new PCR protocol to detect and subtype Blastocystis spp. from humans and animals. Parasitol Res. 2011; 109(1):205-12.
  • Scanlan PD, Stensvold CR, Cotter PD. Development and Application of a Blastocystis Subtype-Specific PCR Assay Reveals that Mixed-Subtype Infections Are Common in a Healthy Human Population. Appl Environ Microbiol. 2015; 81 (12): 4071-6.
  • Zhang X, Qiao J, Wu X, ve diğ. In vitro culture of blastocystis hominis in three liquid media and its usefulness in the diagnosis of Blastocystosis. Int J Infect Dis. 2012; 16:23-8.
  • Roberts T, Barratt J, Harkness J,ve diğ. Comparison of Microscopy, Culture, and Conventional Polymerase Chain Reaction for Detection of Blastocystis sp. in Clinical Stool Samples. Am J Trop Med Hyg. 2011; 84: 308–12.
  • Leelayoova S, Taamasri P, Rangsin R, ve diğ. In-vitro cultivation: a sensitive method for detecting Blastocystis hominis. Ann Trop Med Parasitol. 2002; 96:803-7.
  • Stensvold CR, Arendrup MC, Jespersgaard C, ve diğ. Detecting Blastocystis using parasitologic and DNA-based methods: a comparative study. Diagn Microbiol Infect Dis. 2007; 59: 303-7.
  • Meloni D, Sanciu G, Poirier P, ve diğ. Molecular subtyping of Blastocystis sp. isolates from symptomatic patients in Italy. Parasitol Res. 2011; 109: 613-9.
  • Menounos PG, Spanakos G, Tegos N, ve diğ. Direct detection of Blastocystis sp. in human faecal samples and subtype assignment using single strand conformational polymorphism and sequencing. Mol Cell Probes. 2008; 22: 24-9.
  • Forsell J, Granlund M, Stendsvold C. Subtype analysis of Blastocystis isolates in Swedish patients. Eur J Clin Microbiol Infect Dis. 2012; 31:1689-96.
  • Alfellani MA, Mulla DT, Jacob AS, ve diğ. Genetic Diversity of Blastocystis in Livestock and Zoo Animals. Protist. 2013;164:497-509.
  • Souppart L, Moussa H, Cian A, ve diğ. Subtype analysis of Blastocystis isolates from symptomatic patients in Egypt. Parasitol Res. 2010;106: 505-11.
  • Moosavi A, Haghighi A, Mojarad E, ve diğ. Genetic variability of Blastocystis sp. isolated from symptomatic and asymptomatic individuals in Iran. Parasitol Res. 2012; 111:2311-5.
  • Noël C, Dufernez F, Gerbod D, ve diğ. Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis. J Clin Microbiol. 2005; 43: 348-55.
  • Tan KS .New insights on classification, identification, and clinical relevance of Blastocystis spp. Clin Microbiol Rev. 2008;21:639-65.
  • Ertuğ S, Malatyalı E, Ertabaklar H, ve diğ. Subtype distribution of Blastocystis isolates and evaluation of clinical symptoms detected in Aydin province, Turkey. Mikrobiyol Bul. 2015;49:98-104.
  • Sakalar C, Kandemir İ, Uyar Y, ve diğ. Polymerase Chain Reaction Based Subtyping of Blastocystis spp. Isolates from Symptomatic Patients in Turkey. Turkiye Klinikleri J Med Sci. 2013;33:1064-8.
  • Pasqui AL, Savini E, Saletti M, ve diğ. Chronic urticaria and Blastocystis hominis infection: a case report. Eur Rev Med Pharmacol Sci. 2004; 8:117-20.
  • Kick G, Rueff F, Przbilla B. Palmoplantar pruritus subsiding after Blastocystis hominis eradication. Acta Derm Venereol. 2002;82: 60.
  • Valsecchi R, Leghissa P, Greco V. Cutaneous lesions in Blastocystis hominis infection. Acta Derm. Venereol. 2004;84:322-3.
  • Safadi DE, Meloni D, Poirier P, ve diğ. Short Report: Molecular Epidemiology of Blastocystis in Lebanon and Correlation between Subtype 1 and Gastrointestinal Semptoms. Am J Trop Med Hyg. 2003;88:1203-6.
  • Dogruman-Al F, Dagci H, Yoshikawa H, ve diğ. A possible link between subtype 2 and asymptomatic infections of Blastocystis hominis. Parasitol Res 2008;103:685-9.
  • Vogelberg C, Stendsvolt C, Monecke S, ve diğ. Blastocystis sp. Subtip 2 detection during recurrence of gastrointestinal and urticarial symptoms. Parasitol Int. 2010;59:469-71.
  • Zuel-Fakkar N, Abdel Hameed DM, Hassanin OM. Study of Blastocystis hominis isolates in urticaria: a case control study. Clin Exp Dermatol. 2011;36:908-10.
  • Lee MG, Rawlins SC, Didier M, ve diğ. Infective arthritis due to Blastocystsis hominis. Ann Rheum Dis. 1990; 49: 192 – 3.
  • Krüger K, Kamilli I, Schattenkirchner M. Blastocystis hominis as a rare arthritogenic pathogen. A case report. Z Rheumatol. 1994; 53:83-5.
There are 40 citations in total.

Details

Primary Language Turkish
Subjects Health Care Administration
Journal Section Original Article
Authors

Özden Büyükbaba Boral

Deniz Gözde Çelik Payçu This is me

Akın Akgül This is me

Halim İşsever This is me

Publication Date January 31, 2017
Submission Date June 27, 2016
Acceptance Date September 25, 2016
Published in Issue Year 2017

Cite

APA Büyükbaba Boral, Ö., Çelik Payçu, D. G., Akgül, A., İşsever, H. (2017). Blastocystis spp. Saptanan 50 Semptomatik Hastada Subtip Dağılımın Saptanması. Kocaeli Üniversitesi Sağlık Bilimleri Dergisi, 3(1), 6-10. https://doi.org/10.30934/kusbed.359174
AMA Büyükbaba Boral Ö, Çelik Payçu DG, Akgül A, İşsever H. Blastocystis spp. Saptanan 50 Semptomatik Hastada Subtip Dağılımın Saptanması. KOU Sag Bil Derg. January 2017;3(1):6-10. doi:10.30934/kusbed.359174
Chicago Büyükbaba Boral, Özden, Deniz Gözde Çelik Payçu, Akın Akgül, and Halim İşsever. “Blastocystis Spp. Saptanan 50 Semptomatik Hastada Subtip Dağılımın Saptanması”. Kocaeli Üniversitesi Sağlık Bilimleri Dergisi 3, no. 1 (January 2017): 6-10. https://doi.org/10.30934/kusbed.359174.
EndNote Büyükbaba Boral Ö, Çelik Payçu DG, Akgül A, İşsever H (January 1, 2017) Blastocystis spp. Saptanan 50 Semptomatik Hastada Subtip Dağılımın Saptanması. Kocaeli Üniversitesi Sağlık Bilimleri Dergisi 3 1 6–10.
IEEE Ö. Büyükbaba Boral, D. G. Çelik Payçu, A. Akgül, and H. İşsever, “Blastocystis spp. Saptanan 50 Semptomatik Hastada Subtip Dağılımın Saptanması”, KOU Sag Bil Derg, vol. 3, no. 1, pp. 6–10, 2017, doi: 10.30934/kusbed.359174.
ISNAD Büyükbaba Boral, Özden et al. “Blastocystis Spp. Saptanan 50 Semptomatik Hastada Subtip Dağılımın Saptanması”. Kocaeli Üniversitesi Sağlık Bilimleri Dergisi 3/1 (January 2017), 6-10. https://doi.org/10.30934/kusbed.359174.
JAMA Büyükbaba Boral Ö, Çelik Payçu DG, Akgül A, İşsever H. Blastocystis spp. Saptanan 50 Semptomatik Hastada Subtip Dağılımın Saptanması. KOU Sag Bil Derg. 2017;3:6–10.
MLA Büyükbaba Boral, Özden et al. “Blastocystis Spp. Saptanan 50 Semptomatik Hastada Subtip Dağılımın Saptanması”. Kocaeli Üniversitesi Sağlık Bilimleri Dergisi, vol. 3, no. 1, 2017, pp. 6-10, doi:10.30934/kusbed.359174.
Vancouver Büyükbaba Boral Ö, Çelik Payçu DG, Akgül A, İşsever H. Blastocystis spp. Saptanan 50 Semptomatik Hastada Subtip Dağılımın Saptanması. KOU Sag Bil Derg. 2017;3(1):6-10.