ABSTRACT
Objective:
This study aimed to detect the presence of Echinococcus spp. in formalin-fixed paraffin-embedded (FFPG) samples of hydatid cyst cases and to discuss the DNA isolation problems in FFPG samples.
Methods:
FFPG samples of 47 cases diagnosed with hydatid cyst were included in this study. Demographic characteristics of the cases were investigated. Microtome sections were taken from the samples and deparaffinization, DNA extraction, polymerase chain reaction (PCR), and gel agarose electrophoresis procedures were performed.
Results:
Of the cases, 55.3% were female, whereas 45.7% were male. Average age was 45.47 and 68.1% of the cases were located in the liver, 17.0% in the lung, 12.8% in the abdomen, and 2.1% in the brain. DNA was obtained in only 11 (23.4%) of the FFPG cyst samples and no proliferation was detected in the PCR products of any of the sample.
Conclusion:
The scolex/germinal membrane’ absence in the FFPG sections, intense inflammatory cell reaction, presence of fibrosis and stromal/parenchymal tissue, DNA damage due to formaldehyde action, long-term archiving, and insufficient amount of DNA obtained were considered as factors preventing DNA replication in PCR.
Keywords: Hydatid cyst, Echinococcus, human, PCR
References
1
Schneider R, Gollackner B, Edel B, Schmid K, Wrba F, Tucek G, et al. Development of a new PCR protocol for the detection of species and genotypes (strains) of Echinococcus in formalin-fixed, paraffin-embedded tissues. Int J Parasitol 2008; 38: 1065-71.
2
Beyhan YE, Çobanoğlu U, Çelik S, Yılmaz H, Halidi AG. Molecular characterization of human lung and liver cystic echinococcosis isolates in Van Province, Turkey. Acta Trop 2020; 206: 105451.
3
Eckert J, Schantz PM, Gasser RB, Torgerson PR, Bessonov AS, Movsessian SO, et al. Geographic Distribution and Prevalence. In: Eckert J, Gemmell MA, Meslin F. -X, Pawlowski ZS (Eds.). WHO/OIE Manual on Echinococcosis in Humans and Animals: a Public Health Problem of Global Concern. Paris: World Organisation for Animal Health; 2001; 101-43.
4
Kurt A, Avcioglu H, Guven E, Balkaya I, Oral A, Kirman R, et al. Molecular characterization of Echinococcus multilocularis and Echinococcus granulosus from cysts and formalin-fixed paraffin-embedded tissue samples of human isolates in Northeastern Turkey. Vector Borne Zoonotic Dis 2020; 20: 593-602.
5
Örsten S, Çiftçi T, Azizova A, Yüce G, Uysal A, İmamoğlu Ç, et al. Investigation of the relationship between CE cyst characteristics and genetic diversity of Echinococcus granulosus sensu lato in humans from Turkey. Parasitology 2020; 147: 1712-7.
6
Alvarez Rojas CA, Romig T, Lightowlers MW. Echinococcus granulosus sensu lato genotypes infecting humans--review of current knowledge. Int J Parasitol 2014; 44: 9-18.
7
Nakao M, Lavikainen A, Yanagida T, Ito A. Phylogenetic systematics of the genus Echinococcus (Cestoda: Taeniidae). Int J Parasitol 2013; 43: 1017-29.
8
Thompson RCA, McManus DP. Aetiology: Parasites and Life-Cycles. In: Eckert J, Gemmell MA, Meslin F. -X, Pawlowski ZS (Eds.). WHO/OIE Manual on Echinococcosis in Humans and Animals: a Public Health Problem of Global Concern. Paris: World Organisation for Animal Health; 2001; 1-19.
9
Utük AE, Sımşek S, Köroğlu E. Echinococcus cinsinin moleküler genetik karakterizasyonu [Molecular genetic characterization of genus Echinococcus.]. Turkiye Parazitol Derg 2005; 29: 171-6.
10
Eroğlu F, Genç A, Koltaş İS. Adana ilindeki Echinoccus granulosus izolatlarının PCR-RFLP yöntemi ile genotiplendirilmesi. Zirve Tıp Derg 2016; 1: 22-5.
11
Mansourian S, Sadjjadi SM, Hosseini SV, Barzin Z, Mehrabani D, Tanideh N, et al. Evaluation of different chemical agents on the germinative layer of sheep hydatid cyst after implantation to peritoneal cavity of BALB/c. J Invest Surg 2009; 22: 183-7.
12
Ergin S, Saribas S, Yuksel P, Zengin K, Midilli K, Adas G, et al. Genotypic characterisation of Echinococcus granulosus isolated from human in Turkey. Afr J Microbiol Res 2010; 4: 551-5.
13
Akyol ÇV. Echinococcus Türlerinin Epidemiyolojisi. “Echinococcosis” N Altıntaş, R Tınar ve A Çoker (Editörler). Ege Üniversitesi Matbaası. Bornova, İzmir: 2004; 259-83.
14
Eryıldız C. Echinococcus granulosus İzolatlarının Genotiplendirilmesi. Doktora Tezi. T.C. Trakya Üniversitesi Tıp Fakültesi Tıbbi Mikrobiyoloji Anabilim Dalı. Edirne: 2010.
15
Snábel V, Altintas N, D’Amelio S, Nakao M, Romig T, Yolasigmaz A, et al. Cystic echinococcosis in Turkey: genetic variability and first record of the pig strain (G7) in the country. Parasitol Res 2009; 105: 145-54.
16
Moro P, Schantz PM. Echinococcosis: a review. Int J Infect Dis. 2009; 13: 125-33.
17
Eryıldız C, Sakru N. Molecular Characterization of Human and Animal Isolates of Echinococcus granulosus in the Thrace Region, Turkey. Balkan Med J 2012; 29: 261-7.
18
Tamarozzi F, Akhan O, Cretu CM, Vutova K, Akinci D, Chipeva R, et al. Prevalence of abdominal cystic echinococcosis in rural Bulgaria, Romania, and Turkey: a cross-sectional, ultrasound-based, population study from the HERACLES project. Lancet Infect Dis 2018; 18: 769-78.
19
Güreser AS, Özcan O, Özünel L, Boyacıoğlu Zİ, Taylan Özkan A. Çorum’da kistik Ekinokokkoz ön tanısı ile başvuran hastaların radyolojik, biyokimyasal ve serolojik analizlerinin değerlendirilmesi. Mikrobiyoloji Bülteni 2015; 49: 231-9.
20
Yazar S, Ozkan AT, Hökelek M, Polat E, Yilmaz H, Ozbilge H, et al. Türkiye’de 2001-2005 Yillari Arasinda Kistik Ekinokokkozis [Cystic echinococcosis in Turkey from 2001-2005]. Turkiye Parazitol Derg 2008; 32: 208-20.
21
Yılmaz GR, Babür C. Ekinokokkosis tanısı. Türk Hij Den Biyol Derg 2007; 64: 35-44.
22
Do H, Dobrovic A. Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin Chem 2015; 61: 64-71.
23
Simsek S, Kaplan M, Ozercan IH. A comprehensive molecular survey of Echinococcus granulosus in formalin-fixed paraffin-embedded tissues in human isolates in Turkey. Parasitol Res 2011; 109: 411-6.
24
Quach N, Goodman MF, Shibata D. In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR. BMC Clin Pathol 2004; 4: 1.
25
Al-Jawabreh A, Ereqat S, Dumaidi K, Nasereddin A, Al-Jawabreh H, Azmi K, et al. The clinical burden of human cystic echinococcosis in Palestine, 2010-2015. PLoS Negl Trop Dis 2017; 11: e0005717.
26
Siles-Lucas M, Casulli A, Conraths FJ, Müller N. Laboratory Diagnosis of Echinococcus spp. in Human Patients and Infected Animals. Adv Parasitol 2017; 96: 159-257.
27
Ludyga N, Grünwald B, Azimzadeh O, Englert S, Höfler H, Tapio S, et al. Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses. Virchows Arch 2012; 460: 131-40.
28
Erdoğan E, Özkan B, Mutlu F, Karaca S, Şahin İ. Farklı konaklardan elde edilen Echinococcus granulosus izolatlarının moleküler karakterizasyonu [Molecular characterization of Echinococcus granulosus isolates obtained from different hosts]. Mikrobiyoloji Bülteni 2017; 51: 79-86.
29
Hamamcı B, Açıkgöz G, Kılıç E, Karaaslan K, Çetinkaya Ü, Durgun Yetim T, et al. Biochemical Analysis of Germinal Membrane and Cyst Fluid by Raman Spectroscopy in Echinococcosis. Turkiye Parazitol Derg 2019; 43: 175-81.
30
Gökpınar S, Değirmenci R, Yıldız K. Genotyping of Echinococcus granulosus obtained from cattle slaughtered in Kırıkkale Province. Ankara Univ Vet Fak Derg 2017; 64:51-4.
