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Determining the frequency of Epstein-Barr virus (EBV) in two inflammatory lesions of the periapical cyst and dental granuloma
Gene Reports 18 (2020) 100565
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Determining the frequency of Epstein-Barr virus (EBV) in two inflammatory lesions of the periapical cyst and dental granuloma
T
⁎
Parisa Zeraatia, Laleh Hoveidaa, , Atousa Aminzadehb a b
Department Of Microbiology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran Department Of Oral Pathology, School of Dentistry, Islamic Azad University, Isfahan (Khorasgan) Branch, Isfahan, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Epstein-Barr virus (EBV) Periapical cyst Dental granuloma PCR
Background: One of the significant problems of Partial root canal therapy is periapical cyst and granuloma. Microorganisms contribute to the creation of periapical lesions, causing chronic illness and dental granuloma. This study aims to determine the effect of Epstein-Barr virus (EBV) on the exacerbation of the disease using the PCR technique. Methods: Paraffin tissue samples containing 25 periapical cysts, and 25 dental granulomas underwent DNA extraction. The extracted DNA quality was confirmed by the β-globin component replication using polymerase chain reaction (PCR). Moreover, the samples were evaluated using the nested-PCR technique in terms of the frequency of EBV using dedicated primers. The data analysis was performed using chi-squared, Fischer, and independent t-tests. Results: EBV was detected in 4% (1/25) of the periapical sample and 4% (1/25) of the 25 dental granuloma samples. Despite the above observations, there was no statistically significant difference in the frequency of EBV between the periapical cyst and dental granuloma samples. Conclusion: Based on the results of the study, EBV is likely to affect the pathogenesis or the growth of periapical lesions. Given that there is not a lot of research in this area and the results of the studies are conflicting, the findings attract more research.
1. Introduction Dental granuloma is a relatively common lesion consisting of the proliferating mass of granulation tissue with bacterial nature, which is generated in response to the dead tissue in the tooth canal section. This inflammatory process is a reaction to chronic stimulation caused by the damaged root canal system (Deyhimi and Aminzadeh, 2008). However, not all dental granulomas lead necessarily to the creation of a periapical cyst (Cohen, 2009). The periapical (radicular) cyst is the most prevalent type of mandibular odontogenic cyst in the world, accounting for about 50–75% of oral cysts (Lin et al., 2017; Shetty et al., 2010). Due to inflammatory pathogenesis, this cyst can be found in all age groups; however, it is usually observed between the third and fifth decade (30–49 years), and in many cases in the fourth and fifth and even the sixth decade (40–59 years) (Lin et al., 2017). The periapical cyst is reportedly more common in males than females (Deyhimi, 2006). Inflammatory cells may have latent herpes viruses (Popovic et al., 2015). The interaction between herpes viruses and acute inflammation is
mutual. Active viral infection causes acute periapical inflammation and inflammatory intermediates, which can activate periapical herpes virus (Sabeti et al., 2003). The Epstein-Barr virus (EBV) was detected in 1964 by Epstein and Barr (Tayebi et al., 2006). EBV belongs to the herpesviruses family, and one of the eight known human viruses belonging to the herpes family that infects a significant percentage (> 90%) of the population worldwide and is the causative agent of infectious mononucleosis (Rostamzadeh et al., 2007; Küppers, 2003). It is the most prevalent and stable cancer-related virus, which was first discovered in the cells separated from African Burkitt's lymphoma (Cohen, 2009). It is detected frequently in Burkitt's lymphoma, Hodgkin's disease, and Tcell lymphoma (Sin and Dittmer, 2012). The difficulties in the infection of primary epithelial cells indicate that EBV is essential in natural complex multi-dimensional epithelial malignancies (Tayebi et al., 2006; Makino et al., 2015). Epstein-Bar virus can be hidden secreted in epithelial cells and oral tissue. Studying periapical lesions is essential for EBV, as the virus can be important in immune-response reactions (Chen et al., 2009).
Abbreviations: EBV, Epstein-Barr virus; PCR, polymerase chain reaction ⁎ Corresponding author. E-mail address: [email protected] (L. Hoveida). https://doi.org/10.1016/j.genrep.2019.100565 Received 15 September 2019; Received in revised form 10 November 2019; Accepted 12 November 2019 Available online 13 November 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
Gene Reports 18 (2020) 100565
P. Zeraati, et al.
Fig. 1. PCR gel electrophoresis images. A) PCR analysis of DNA samples extracted from periapical and dental granuloma cystic tissue with β-globin primers. B) PCR analysis of DNA samples extracted from the periapical cyst is using dedicated primers. C) PCR analysis of DNA samples extracted from dental granuloma cyst using dedicated primers.
globin) (Table 2) to confirm the quality of the extracted DNA (Mahmoudvand et al., 2015). PCR reactions were carried out for each isolate in the final volume of 25 μl containing about 50 ng (5 mL) of the extracted DNA with 2.5 μl of Buffer 1× (CinnaGene, Iran), 1 μl of MgCl2 (CinnaGen, Iran), 0.5 μl of dNTP (CinnaGene, Iran), 0.2 μl of Taq DNA Polymerase (Taq DNA Polymerase), and 1 μl of each dedicated primer (PCO3 and PCO4) (Mahmoudvand et al., 2015). PCR experiments for β-globin were performed as follows: The initial denaturation was carried out for 10 min at 94 °C, followed by 35 cycles at 94 °C for 45 s of denaturation. Annealing was done at 72 °C for 45 s. Then, the extension was performed for 1 min at 72 °C, and the final extension was performed for 10 min at 72 °C. Nested-PCR experiments were performed to identify EBV from the DNA extracted from the paraffin blocks in two stages: The first stage consists of the DNA combination extracted from the paraffin blocks and Blend Master Mix A (Kianmed, Iran). The second stage includes of the output of the early stage together with Blend Master Mix B with dedicated primers and the initial denaturation temperature program for 5 min at 95 °C, 40 cycles of denaturation for 30 s at 95 °C, cooling for 30 s at 72 °C, extension for 30 s at 72 °C. The final format was performed for 5 min at 72 °C (Table 2). The PCR results were then loaded on the 2% agarose gel and were observed using the UV device (BIO-RAD, the USA) (Fig. 1).
Immunohistochemical and immunohistological diagnostic methods can be used to identify the EBV-related tumors (Hess, 2004; Mowry et al., 2008). Polymerase chain reaction (PCR) is an appropriate method for a small number of EBV variants. PCR technique is often used for the identification of EBV in random samples collected from transplant patients and blood samples taken from patients with infectious mononucleosis (Telenti et al., 1990). Since microorganisms may have contribution to the pathogenesis or progression of periapical lesion and, to the best knowledge of the researchers, very few studies have been conducted in this field so far, this study aimed at evaluating the presence of EBV applying the PCR technique. 2. Materials and methods 2.1. Sample collection Fifty paraffin blocks with clinical and histopathological diagnosis of periapical cyst (n = 25) and dental granuloma (n = 25) were gathered from archive of oral pathology laboratory, dental school, Islamic Azad University, Isfahan, Iran, which belongs to the 2010–2017 period were collected and investigated in this study. 2.2. DNA extraction Ten sections (10 μm) of the paraffin blocks were cut and placed in a 5 mL Eppendorf tube. The first phase of deparaffinization was carried out by initially adding 800 mL of Xylene into the above tubes. After turning the tubes on the shaker and incubation for 10 min at room temperature, the tubes were centrifuged at a 14,000 rpm speed for 10 min at 18 °C. The steps 2 and 4 mentioned above were again performed with new Xylene, then incubated for 10 min at 37 °C. In the next step, the surface materials were removed, and 800 mL of ethanol was added to the tubes and incubated for 3 min at room temperature. Finally, the centrifuge and the surface materials were discarded and processed for 15 min at room temperature for the evaporation of alcohols. In this stage, DNA extraction was performed by Gene All kit concerning the manufacturer's instruction. The extracted DNA was stored at −20 °C until it was used.
3. Results Out of the 50 samples studied, 25 were male, and 25 were female. This study aimed to determine the presence of EBV in periapical and dental granuloma cysts. The age range of the subjects in the group of periapical cyst was between 12 and 50 years with the mean age of 34.1 ± 10.1 years, and the age range of dental granuloma was between 13 and 55 years with the mean age of 36.3 ± 10.6 years. The independent t-test showed that there was no significant difference between the two groups (p = 0.46). In the periapical cyst group, 13 (52%) were male, and 12 (48%) were female. In the dental granuloma group, 12 (48%) were male 13 (52%) were female. Chi-square test showed that there was no significant difference in the frequency distribution of gender of individuals investigated between the two groups (p = 0.78). One sample (4%) of periapical cysts and one sample (4%) of periapical granuloma were reported positive in terms of the presence of EBV (Table 1 and Fig. 1). Fisher's exact test showed that there was no significant correlation between the two groups in terms of the frequency of EBV presence (p= 1). Given the positive sample was
2.3. Polymerase chain reaction (PCR) First, all extracted DNA samples were analyzed by spectrophotometer and NanoDrop in terms of concentration and DNA purity. Then, PCR was carried out with dedicated PCO4/PCO3 primers (β2
Gene Reports 18 (2020) 100565
P. Zeraati, et al.
was observed in none of the periapical periodontitis samples. This difference stems from host factors, environmental factors such as genetic background, ethnicity, economic status, DNA extraction method, as well as PCR application methods. In their study, Yildirim et al. (2006) proposed that the frequency of EBV is related to the amount of bone damage and the resulting cytokines, so there will be more viruses in larger lesions. In another study, Hernádi et al. (2010) reported the presence of EBV in large-scale periapical lesions (5 mm) compared to the small lesions with the rate of difference (i.e., 50%). In addition to detecting the presence of EBV and HCMV in periapical granulomatous tissues by RT-PCR, Sabeti et al. (2003) suggested that the prevalence of herpes simplex viruses was higher in symptomatic periapical lesions compared to asymptomatic periapical lesions. Therefore, the result confirms the role of the size of lesions in virus positivity and multiplicity. In their study, Andric et al. (2007) also reported that in some studies, EBV could be false-positive responses due to tissue infection examined to the patient's saliva. However, the present study showed that the presence of the virus was not significantly different between the two groups (P > 0.05). The results of this study are consistent with the studies of Saboia-Dantas et al. (2007), Li et al. (2009), Jakovljevic et al. (2015). In their research, Kabak et al. (2005) proposed that despite the presence of HSV virus in periapical cyst and not in dental granuloma, the findings of the study are consistent with the present study. The limitations of our study include the following: Firstly,
Table 1 Frequency of EBV genome among different groups. Tissue type
Positive for EBV
Mean age
Periapical cyst (n = 25) Dental granuloma (n = 25)
1/25 1/25
12–50 13–55
associated with a 35-year-old male in the periapical group, and a 33year-old female in the periapical granuloma group, it was not possible to study its relevance to age and gender considering the low frequency of EBV.
4. Discussion The periapical lesion is an inflammatory immune disease affecting periapical dental tissue and its surrounding bones. These processes are initially established by multi-microbial bacterial infection from the root canal (Yildirim et al., 2006). Microbial infection in the root canal of the tooth is necessary to form a periapical lesion. However, no signs of periapical inflammatory lesions have been observed in Germ-free (GF) animals (Andric et al., 2007). EBV infection is often associated with different types of oral diseases (Satija and Rai, 2015). Researchers stated that EBV plays a key role in the inception of the periapical inflammatory process and possibly in its development through cell damage (Makino et al., 2015). In the present study, 4% of EBV periapical lesions were observed, which is much lower compared with similar studies, such as Makino et al. (2015) (75%). In addition, the results of this study are consistent with the findings of Yazdi et al.'s (2008) study in which the presence of EBV in 6% of apical periodontitis was explored using the PCR technique. In addition, in their research on the presence of EBV in 6% of apical periodontitis using PCR technique, Yazdi et al. (2008) reported that they are consistent with the findings of the present study. Researchers believe that the possibility of diagnosing EBV using the PCR technique in the acute phase of the disease (i.e., symptomatic phase of the disease) is more applicable (Jakovljevic et al., 2015). In their study, Sabeti et al. (2003) specified that it is impossible to diagnose EBV in periapical lesions without symptoms (e.g. pain, swelling, and secretion) while the presence of EBV was diagnosed in 88% of symptomatic periapical lesions. Moreover, in the study of Hernádi et al. (2013), EBV was reported as the predominant pathogen. They reported the simultaneous presence of HCMV infection and EBV infection in periapical lesions while the presence of the EBV genome was not reported in any asymptomatic lesions. Based on a study conducted via the PCR technique, Slots et al. (2004) stated that HCMV was observed in 37% of symptomatic and asymptomatic specimens, whereas EBV was present only in HCMV-infected lesions. Thus, the result showed the role of other viruses in the activation of EBV in periapical lesions. Besides, given the low amount of EBV and HCMV in the studies of Dawson III et al. (2009) and Nibali et al. (2009) performed using the RT-PCR method, they stated that the absence of an active viral infection might cause a decrease in the amount of virus in the samples studied. Sunde et al. (2008) also reported that the presence of EBV was 45% via the RT-PCR technique; however, HCMV presence
5. Conclusion According to the results of the present study, it was not precisely revealed whether or not EBV has a role in pathogenesis or progression of periapical lesions. Hence, further studies with large sample sizes are required. Ethical approval This manuscript does not cover any studies with human participants or animals performed by the authors. Financial disclosure The authors announce that they had no financial interests related to the material in the manuscript. Funding/support Self-funding. Author's contributions LH, AA conceived, designed and supervised the study and revised the manuscript; PZ collected and analyzed the data; PZ, AA and LH drafted the manuscript. Declaration of competing interest No conflict of interest was announced by the authors.
Table 2 Sequences and other primer profiles used in this study. Locus
Primer
5′ to 3′ sequence
Size, bp
Reference
β-Globin
PCO3 PCO4 EBV-1
5′-ACACAACTGTGTTCACTAGC-3′ 5′-CAACTTCATCCACGTTCACC-3′ 5′-TATGCTATCCAATCTATATGC-3′ 5′-ATTGTAACCTCGCTAACC-3′ 5′-TATGCTATCCAATCTATATGC-3′ 5′-ATTGTAACCTCGCTAACC-3′
110 bp
(Mahmoudvand et al., 2015)
450 bp
This study
360 bp
This study
EBV
EBV-2
3
Gene Reports 18 (2020) 100565
P. Zeraati, et al.
References
e0121548. Mowry, S.E., Strocker, A.M., Chan, J., Takehana, C., Kalantar, N., Bhuta, S., et al., 2008. Immunohistochemical analysis and Epstein-Barr virus in the tonsils of transplant recipients and healthy controls. Archives of Otolaryngology–Head & Neck Surgery 134 (9), 936–939. Nibali, L., Atkinson, C., Griffiths, P., Darbar, U., Rakmanee, T., Suvan, J., et al., 2009. Low prevalence of subgingival viruses in periodontitis patients. J. Clin. Periodontol. 36 (11), 928–932. Popovic, J., Gasic, J., Zivkovic, S., Kesic, L., Mitic, A., Nikolic, M., et al., 2015. Prevalence of human cytomegalovirus and Epstein-Barr virus in chronic periapical lesions. Intervirology 58 (5), 271–277. Rostamzadeh, K.Z., Makhdoumi, K., Salarilak, S., 2007. Prevalence of Epstein Barr Virus Infection and Effecting Factors in Renal Allograft Recipients for Controlling Ptld in Imam Khomeini Hospital From 2001 to 2004. Sabeti, M., Valles, Y., Nowzari, H., Simon, J., Kermani-Arab, V., Slots, J., 2003. Cytomegalovirus and Epstein–Barr virus DNA transcription in endodontic symptomatic lesions. Oral Microbiol. Immunol. 18 (2), 104–108. Saboia-Dantas, C., Coutrin de Toledo, L., Sampaio-Filho, H., Siqueira Jr., J., 2007. Herpesviruses in asymptomatic apical periodontitis lesions: an immunohistochemical approach. Oral Microbiol. Immunol. 22 (5), 320–325. Satija, A., Rai, J.J., 2015. Viruses: a paradox in etiopathogenesis of periodontal diseases. Journal of Dental and Medical Sciences 14 (12), 58–64. Shetty, S., Angadi, P.V., Rekha, K., 2010. Radicular cyst in deciduous maxillary molars: a rarity. Head and neck pathology 4 (1), 27–30. Sin, S.-H., Dittmer, D.P., 2012. Cytokine homologs of human gammaherpesviruses. J. Interf. Cytokine Res. 32 (2), 53–59. Slots, J., Nowzari, H., Sabeti, M., 2004. Cytomegalovirus infection in symptomatic periapical pathosis. Int. Endod. J. 37 (8), 519–524. Sunde, P.T., Olsen, I., Enersen, M., Beiske, K., Grinde, B., 2008. Human cytomegalovirus and Epstein–Barr virus in apical and marginal periodontitis: a role in pathology? J. Med. Virol. 80 (6), 1007–1011. Tayebi, D., Mokhtari, M., Salehi, S., Mohammadi, B., Ebrahimpour, M., 2006. Seropidemiology of Epstein–Barr Virus Infection Among Asymptomatic Students of Islamic Azad University of Kazeroon, Southwest of Iran. Telenti, A., Marshall, W., Smith, T., 1990. Detection of Epstein-Barr virus by polymerase chain reaction. J. Clin. Microbiol. 28 (10), 2187–2190. Yazdi, K., Sabeti, M., Jabalameli, F., Eman eini, M., Kolahdouzan, S., Slots, J., 2008. Relationship between human cytomegalovirus transcription and symptomatic apical periodontitis in Iran. Oral Microbiol. Immunol. 23 (6), 510–514. Yildirim, S., Yapar, M., Kubar, A., Slots, J., 2006. Human cytomegalovirus, Epstein-Barr virus and bone resorption-inducing cytokines in periapical lesions of deciduous teeth. Oral Microbiol. Immunol. 21 (2), 107–111.
Andric, M., Milasin, J., Jovanovic, T., Todorovic, L., 2007. Human cytomegalovirus is present in odontogenic cysts. Oral Microbiol. Immunol. 22 (5), 347–351. Chen, V., Chen, Y., Li, H., Kent, K., Baumgartner, J.C., Machida, C.A., 2009. Herpesviruses in abscesses and cellulitis of endodontic origin. J Endod 35 (2), 182–188. Cohen, J.I., 2009. Optimal treatment for chronic active Epstein-Barr virus disease. Pediatr. Transplant. 13 (4), 393. Dawson III, D.R., Wang, C., Danaher, R.J., Lin, Y., Kryscio, R.J., Jacob, R.J., et al., 2009. Real-time polymerase chain reaction to determine the prevalence and copy number of Epstein-Barr virus and cytomegalovirus DNA in subgingival plaque at individual healthy and periodontal disease sites. J. Periodontol. 80 (7), 1133–1140. Deyhimi, P., 2006. Pathology of Tooth and Odontogenic Lesions. Isfahan University Medical Scinces, Isfahan, pp. 224–230. Deyhimi, P., Aminzadeh, A., 2008. A case report of an unusual radicular cyst associated with deciduous toot. J Isfahan Dent Sch 89~ 93. Hernádi, K., Szalmás, A., Mogyorósi, R., Czompa, L., Veress, G., Csoma, E., et al., 2010. Prevalence and activity of Epstein-Barr virus and human cytomegalovirus in symptomatic and asymptomatic apical periodontitis lesions. J. Endod. 36 (9), 1485–1489. Hernádi, K., Gyöngyösi, E., Mészáros, B., Szakács, L., Szalmás, A., Csoma, E., et al., 2013. Elevated tumor necrosis factor-alpha expression in periapical lesions infected by Epstein-Barr virus. J. Endod. 39 (4), 456–460. Hess, R.D., 2004. Routine Epstein-Barr virus diagnostics from the laboratory perspective: still challenging after 35 years. J. Clin. Microbiol. 42 (8), 3381–3387. Jakovljevic, A., Andric, M., Knezevic, A., Soldatovic, I., Nikolic, N., Karalic, D., et al., 2015. Human cytomegalovirus and Epstein-Barr virus genotypes in apical periodontitis lesions. J. Endod. 41 (11), 1847–1851. Kabak, S., Kabak, Y., Anischenko, S., 2005. Light microscopic study of periapical lesions associated with asymptomatic apical periodontitis. Annals of Anatomy-Anatomischer Anzeiger 187 (2), 185–194. Küppers, R., 2003. B cells under influence: transformation of B cells by Epstein–Barr virus. Nat. Rev. Immunol. 3 (10), 801. Li, H., Chen, V., Chen, Y., Baumgartner, J.C., Machida, C.A., 2009. Herpesviruses in endodontic pathoses: association of Epstein-Barr virus with irreversible pulpitis and apical periodontitis. J. Endod. 35 (1), 23–29. Lin, L.M., Ricucci, D., Kahler, B., 2017. Radicular cysts review. JSM Dental Surgery 2 (2), 1017.1–3. Mahmoudvand, S., Safaei, A., Erfani, N., Sarvari, J., 2015. Presence of human papillomavirus DNA in colorectal cancer tissues in Shiraz, Southwest Iran. Asian Pac. J. Cancer Prev. 16 (17), 7883–7887. Makino, K., Takeichi, O., Hatori, K., Imai, K., Ochiai, K., Ogiso, B., 2015. Epstein-Barr virus infection in chronically inflamed periapical granulomas. PLoS One 10 (4),
4
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Determining the frequency of Epstein-Barr virus (EBV) in two inflammatory lesions of the periapical cyst and dental granuloma
T
⁎
Parisa Zeraatia, Laleh Hoveidaa, , Atousa Aminzadehb a b
Department Of Microbiology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran Department Of Oral Pathology, School of Dentistry, Islamic Azad University, Isfahan (Khorasgan) Branch, Isfahan, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Epstein-Barr virus (EBV) Periapical cyst Dental granuloma PCR
Background: One of the significant problems of Partial root canal therapy is periapical cyst and granuloma. Microorganisms contribute to the creation of periapical lesions, causing chronic illness and dental granuloma. This study aims to determine the effect of Epstein-Barr virus (EBV) on the exacerbation of the disease using the PCR technique. Methods: Paraffin tissue samples containing 25 periapical cysts, and 25 dental granulomas underwent DNA extraction. The extracted DNA quality was confirmed by the β-globin component replication using polymerase chain reaction (PCR). Moreover, the samples were evaluated using the nested-PCR technique in terms of the frequency of EBV using dedicated primers. The data analysis was performed using chi-squared, Fischer, and independent t-tests. Results: EBV was detected in 4% (1/25) of the periapical sample and 4% (1/25) of the 25 dental granuloma samples. Despite the above observations, there was no statistically significant difference in the frequency of EBV between the periapical cyst and dental granuloma samples. Conclusion: Based on the results of the study, EBV is likely to affect the pathogenesis or the growth of periapical lesions. Given that there is not a lot of research in this area and the results of the studies are conflicting, the findings attract more research.
1. Introduction Dental granuloma is a relatively common lesion consisting of the proliferating mass of granulation tissue with bacterial nature, which is generated in response to the dead tissue in the tooth canal section. This inflammatory process is a reaction to chronic stimulation caused by the damaged root canal system (Deyhimi and Aminzadeh, 2008). However, not all dental granulomas lead necessarily to the creation of a periapical cyst (Cohen, 2009). The periapical (radicular) cyst is the most prevalent type of mandibular odontogenic cyst in the world, accounting for about 50–75% of oral cysts (Lin et al., 2017; Shetty et al., 2010). Due to inflammatory pathogenesis, this cyst can be found in all age groups; however, it is usually observed between the third and fifth decade (30–49 years), and in many cases in the fourth and fifth and even the sixth decade (40–59 years) (Lin et al., 2017). The periapical cyst is reportedly more common in males than females (Deyhimi, 2006). Inflammatory cells may have latent herpes viruses (Popovic et al., 2015). The interaction between herpes viruses and acute inflammation is
mutual. Active viral infection causes acute periapical inflammation and inflammatory intermediates, which can activate periapical herpes virus (Sabeti et al., 2003). The Epstein-Barr virus (EBV) was detected in 1964 by Epstein and Barr (Tayebi et al., 2006). EBV belongs to the herpesviruses family, and one of the eight known human viruses belonging to the herpes family that infects a significant percentage (> 90%) of the population worldwide and is the causative agent of infectious mononucleosis (Rostamzadeh et al., 2007; Küppers, 2003). It is the most prevalent and stable cancer-related virus, which was first discovered in the cells separated from African Burkitt's lymphoma (Cohen, 2009). It is detected frequently in Burkitt's lymphoma, Hodgkin's disease, and Tcell lymphoma (Sin and Dittmer, 2012). The difficulties in the infection of primary epithelial cells indicate that EBV is essential in natural complex multi-dimensional epithelial malignancies (Tayebi et al., 2006; Makino et al., 2015). Epstein-Bar virus can be hidden secreted in epithelial cells and oral tissue. Studying periapical lesions is essential for EBV, as the virus can be important in immune-response reactions (Chen et al., 2009).
Abbreviations: EBV, Epstein-Barr virus; PCR, polymerase chain reaction ⁎ Corresponding author. E-mail address: [email protected] (L. Hoveida). https://doi.org/10.1016/j.genrep.2019.100565 Received 15 September 2019; Received in revised form 10 November 2019; Accepted 12 November 2019 Available online 13 November 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
Gene Reports 18 (2020) 100565
P. Zeraati, et al.
Fig. 1. PCR gel electrophoresis images. A) PCR analysis of DNA samples extracted from periapical and dental granuloma cystic tissue with β-globin primers. B) PCR analysis of DNA samples extracted from the periapical cyst is using dedicated primers. C) PCR analysis of DNA samples extracted from dental granuloma cyst using dedicated primers.
globin) (Table 2) to confirm the quality of the extracted DNA (Mahmoudvand et al., 2015). PCR reactions were carried out for each isolate in the final volume of 25 μl containing about 50 ng (5 mL) of the extracted DNA with 2.5 μl of Buffer 1× (CinnaGene, Iran), 1 μl of MgCl2 (CinnaGen, Iran), 0.5 μl of dNTP (CinnaGene, Iran), 0.2 μl of Taq DNA Polymerase (Taq DNA Polymerase), and 1 μl of each dedicated primer (PCO3 and PCO4) (Mahmoudvand et al., 2015). PCR experiments for β-globin were performed as follows: The initial denaturation was carried out for 10 min at 94 °C, followed by 35 cycles at 94 °C for 45 s of denaturation. Annealing was done at 72 °C for 45 s. Then, the extension was performed for 1 min at 72 °C, and the final extension was performed for 10 min at 72 °C. Nested-PCR experiments were performed to identify EBV from the DNA extracted from the paraffin blocks in two stages: The first stage consists of the DNA combination extracted from the paraffin blocks and Blend Master Mix A (Kianmed, Iran). The second stage includes of the output of the early stage together with Blend Master Mix B with dedicated primers and the initial denaturation temperature program for 5 min at 95 °C, 40 cycles of denaturation for 30 s at 95 °C, cooling for 30 s at 72 °C, extension for 30 s at 72 °C. The final format was performed for 5 min at 72 °C (Table 2). The PCR results were then loaded on the 2% agarose gel and were observed using the UV device (BIO-RAD, the USA) (Fig. 1).
Immunohistochemical and immunohistological diagnostic methods can be used to identify the EBV-related tumors (Hess, 2004; Mowry et al., 2008). Polymerase chain reaction (PCR) is an appropriate method for a small number of EBV variants. PCR technique is often used for the identification of EBV in random samples collected from transplant patients and blood samples taken from patients with infectious mononucleosis (Telenti et al., 1990). Since microorganisms may have contribution to the pathogenesis or progression of periapical lesion and, to the best knowledge of the researchers, very few studies have been conducted in this field so far, this study aimed at evaluating the presence of EBV applying the PCR technique. 2. Materials and methods 2.1. Sample collection Fifty paraffin blocks with clinical and histopathological diagnosis of periapical cyst (n = 25) and dental granuloma (n = 25) were gathered from archive of oral pathology laboratory, dental school, Islamic Azad University, Isfahan, Iran, which belongs to the 2010–2017 period were collected and investigated in this study. 2.2. DNA extraction Ten sections (10 μm) of the paraffin blocks were cut and placed in a 5 mL Eppendorf tube. The first phase of deparaffinization was carried out by initially adding 800 mL of Xylene into the above tubes. After turning the tubes on the shaker and incubation for 10 min at room temperature, the tubes were centrifuged at a 14,000 rpm speed for 10 min at 18 °C. The steps 2 and 4 mentioned above were again performed with new Xylene, then incubated for 10 min at 37 °C. In the next step, the surface materials were removed, and 800 mL of ethanol was added to the tubes and incubated for 3 min at room temperature. Finally, the centrifuge and the surface materials were discarded and processed for 15 min at room temperature for the evaporation of alcohols. In this stage, DNA extraction was performed by Gene All kit concerning the manufacturer's instruction. The extracted DNA was stored at −20 °C until it was used.
3. Results Out of the 50 samples studied, 25 were male, and 25 were female. This study aimed to determine the presence of EBV in periapical and dental granuloma cysts. The age range of the subjects in the group of periapical cyst was between 12 and 50 years with the mean age of 34.1 ± 10.1 years, and the age range of dental granuloma was between 13 and 55 years with the mean age of 36.3 ± 10.6 years. The independent t-test showed that there was no significant difference between the two groups (p = 0.46). In the periapical cyst group, 13 (52%) were male, and 12 (48%) were female. In the dental granuloma group, 12 (48%) were male 13 (52%) were female. Chi-square test showed that there was no significant difference in the frequency distribution of gender of individuals investigated between the two groups (p = 0.78). One sample (4%) of periapical cysts and one sample (4%) of periapical granuloma were reported positive in terms of the presence of EBV (Table 1 and Fig. 1). Fisher's exact test showed that there was no significant correlation between the two groups in terms of the frequency of EBV presence (p= 1). Given the positive sample was
2.3. Polymerase chain reaction (PCR) First, all extracted DNA samples were analyzed by spectrophotometer and NanoDrop in terms of concentration and DNA purity. Then, PCR was carried out with dedicated PCO4/PCO3 primers (β2
Gene Reports 18 (2020) 100565
P. Zeraati, et al.
was observed in none of the periapical periodontitis samples. This difference stems from host factors, environmental factors such as genetic background, ethnicity, economic status, DNA extraction method, as well as PCR application methods. In their study, Yildirim et al. (2006) proposed that the frequency of EBV is related to the amount of bone damage and the resulting cytokines, so there will be more viruses in larger lesions. In another study, Hernádi et al. (2010) reported the presence of EBV in large-scale periapical lesions (5 mm) compared to the small lesions with the rate of difference (i.e., 50%). In addition to detecting the presence of EBV and HCMV in periapical granulomatous tissues by RT-PCR, Sabeti et al. (2003) suggested that the prevalence of herpes simplex viruses was higher in symptomatic periapical lesions compared to asymptomatic periapical lesions. Therefore, the result confirms the role of the size of lesions in virus positivity and multiplicity. In their study, Andric et al. (2007) also reported that in some studies, EBV could be false-positive responses due to tissue infection examined to the patient's saliva. However, the present study showed that the presence of the virus was not significantly different between the two groups (P > 0.05). The results of this study are consistent with the studies of Saboia-Dantas et al. (2007), Li et al. (2009), Jakovljevic et al. (2015). In their research, Kabak et al. (2005) proposed that despite the presence of HSV virus in periapical cyst and not in dental granuloma, the findings of the study are consistent with the present study. The limitations of our study include the following: Firstly,
Table 1 Frequency of EBV genome among different groups. Tissue type
Positive for EBV
Mean age
Periapical cyst (n = 25) Dental granuloma (n = 25)
1/25 1/25
12–50 13–55
associated with a 35-year-old male in the periapical group, and a 33year-old female in the periapical granuloma group, it was not possible to study its relevance to age and gender considering the low frequency of EBV.
4. Discussion The periapical lesion is an inflammatory immune disease affecting periapical dental tissue and its surrounding bones. These processes are initially established by multi-microbial bacterial infection from the root canal (Yildirim et al., 2006). Microbial infection in the root canal of the tooth is necessary to form a periapical lesion. However, no signs of periapical inflammatory lesions have been observed in Germ-free (GF) animals (Andric et al., 2007). EBV infection is often associated with different types of oral diseases (Satija and Rai, 2015). Researchers stated that EBV plays a key role in the inception of the periapical inflammatory process and possibly in its development through cell damage (Makino et al., 2015). In the present study, 4% of EBV periapical lesions were observed, which is much lower compared with similar studies, such as Makino et al. (2015) (75%). In addition, the results of this study are consistent with the findings of Yazdi et al.'s (2008) study in which the presence of EBV in 6% of apical periodontitis was explored using the PCR technique. In addition, in their research on the presence of EBV in 6% of apical periodontitis using PCR technique, Yazdi et al. (2008) reported that they are consistent with the findings of the present study. Researchers believe that the possibility of diagnosing EBV using the PCR technique in the acute phase of the disease (i.e., symptomatic phase of the disease) is more applicable (Jakovljevic et al., 2015). In their study, Sabeti et al. (2003) specified that it is impossible to diagnose EBV in periapical lesions without symptoms (e.g. pain, swelling, and secretion) while the presence of EBV was diagnosed in 88% of symptomatic periapical lesions. Moreover, in the study of Hernádi et al. (2013), EBV was reported as the predominant pathogen. They reported the simultaneous presence of HCMV infection and EBV infection in periapical lesions while the presence of the EBV genome was not reported in any asymptomatic lesions. Based on a study conducted via the PCR technique, Slots et al. (2004) stated that HCMV was observed in 37% of symptomatic and asymptomatic specimens, whereas EBV was present only in HCMV-infected lesions. Thus, the result showed the role of other viruses in the activation of EBV in periapical lesions. Besides, given the low amount of EBV and HCMV in the studies of Dawson III et al. (2009) and Nibali et al. (2009) performed using the RT-PCR method, they stated that the absence of an active viral infection might cause a decrease in the amount of virus in the samples studied. Sunde et al. (2008) also reported that the presence of EBV was 45% via the RT-PCR technique; however, HCMV presence
5. Conclusion According to the results of the present study, it was not precisely revealed whether or not EBV has a role in pathogenesis or progression of periapical lesions. Hence, further studies with large sample sizes are required. Ethical approval This manuscript does not cover any studies with human participants or animals performed by the authors. Financial disclosure The authors announce that they had no financial interests related to the material in the manuscript. Funding/support Self-funding. Author's contributions LH, AA conceived, designed and supervised the study and revised the manuscript; PZ collected and analyzed the data; PZ, AA and LH drafted the manuscript. Declaration of competing interest No conflict of interest was announced by the authors.
Table 2 Sequences and other primer profiles used in this study. Locus
Primer
5′ to 3′ sequence
Size, bp
Reference
β-Globin
PCO3 PCO4 EBV-1
5′-ACACAACTGTGTTCACTAGC-3′ 5′-CAACTTCATCCACGTTCACC-3′ 5′-TATGCTATCCAATCTATATGC-3′ 5′-ATTGTAACCTCGCTAACC-3′ 5′-TATGCTATCCAATCTATATGC-3′ 5′-ATTGTAACCTCGCTAACC-3′
110 bp
(Mahmoudvand et al., 2015)
450 bp
This study
360 bp
This study
EBV
EBV-2
3
Gene Reports 18 (2020) 100565
P. Zeraati, et al.
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