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Detection of herpes viruses and quantification of its load in periodontitis.

Apart from periodontopathic bacteria there are numerous infectious agents and its interconnected cellular and humoral host response play a important role in periodontitis. The various infectious agents apart from bacteria like viruses, fungi etc may cause the periodontal disease. Majority of studies focus on identification of periodontapathic bacteria, although some recent studies suggest the various viruses like Herpes viruses detection, involvement and in pathogenesis of periodontal disease. (1-3) Herpes viruses are capable of destroying periodontal structural cells and host defense cells thereby reducing its resistance to bacterial colonization. (4) Many studies have shown the presence of HSV viruses like Herpes simplex-1(HSV-1) Human cytomegalo virus(HCMV) and Epstein -Barr virus(EBV) in periodontal sites. Presence of this virus change the periodontal micro flora favoring a severe pathogenesis (3,5).

Many number of studies indicates increased frequency of specific members of the herpes viridae detection, such as EBV-1, HCMV and herpes simplex virus-1 (HSV-1) in various forms of periodontal diseases (1,4,5). In which EBV-1 and HCMV may reside dormant in periodontal cells even after periodontal treatment (6) causing destruction of alveolar bone (7,8) HSV-1 and HCMV synergistically populate porphyromonas gingivalis in periodontitis site (9).

Although prevelance of hsv viruses in periodontal diseases were detected in these various studies (10), definitive role of virus as a unibody causing periodontal disease Is not yet proved. HSV virus may modify microenvironment by reducing host immunity there by supporting periodontopathic bacteria to colonise in diseased site (4). Thus the amount of destruction does not depend only on prevelance but also on virus load in diseased site (10).

Objectives

The current study regards to the detection and quantification of herpes viruses in chronic and aggressive periodontitis and correlate with

the clinical parameters i.e Clinical attachment loss there by showing the role of viral load in diseased sites. Subgingival plaque samples were collected from the diseased sites and quantification of HSV viruses is done with Real time PCR(RT-PCR). Study was done in Thai moogambigai dental college in department of periodontics under the ethical clearance from Dr. MGR University And Research Institute.

MATERIALS AND METHODS

The study included n=60 patients (age range, 23-65 years) who needed a periodontal pocket elimination surgery. Chronic periodontitis patients (n=30) displayed clinical attachment loss of 4-9 mm and gingival inflammation in at least 3 posterior teeth. Aggressive periodontititis patients (n=30 displayed 5-9mm clinical attachment loss in molars and incisors with radiographically seen vertical bone loss. Inclusion criteria were as follows: The patients did not have any systemic condition such as: diabetes, heart disease, asthma. absence of herpetic infection history and didn't have any history of antibiotic medication within the previous 2 months. None of the patients had previous periodontal surgery. They all had at least 20 teeth. They did not report the history of smoking, pregnancy or lactation. The patients were signed with consent form.

Clinical Procedure

Patients screened with periodontal examinations included plaque index (PI), bleeding on probing (BOP), bleeding index, periodontal pocket depth (PPD) and clinical attachment level measurement, from cemento enamel junction (CEJ) to the deepest probable pocket depth, the measurements with Williams probe (Hu-Friedy, USA) in 6 parts: midfacial, midlingual, mesiofacial, mesiolingual, distofacial, distolingual aspects of each tooth. Sub gingival Plaque samples were taken with curettes from Chronic periodontitis patients in deep CAL >6mm and shallow pockets CAL>3mm in same patients and from aggressive periodontitis in CAL>6mm. The plaque samples were immediately immersed in a transport medium 500pl of Phosphate buffered saline (pH 8.0), transported in ice and stored at -20[degrees]C till assay. and given to hi tech diagnostics lab, Chennai.

Laboratory Procedures

The subgingival plaque samples in Phosphate buffer solution (PBS) were microcentrifuged at 8,000rpm for 5minutes, supernatant discarded and the pellet was resuspended in 100pl of lysis broth (10 mmol/L Tris-HCl, 1.0 mmol/L EDTA, 1.0% Triton X-100, pH 8.0). The lysis broth was further vortexed and kept in water bath at 100oC for 5 minutes, cooled and centrifuged at 4000 rpm for 2 minutes and the supernatant was stored at -20[degrees]C. For viruses the last setup of 25 [micro]L Taq Man PCR (including 5 [micro]L of specimen, 12.5 [micro]L Mater Universal mixtures, 5 pmol primer, 4 pmol Taq Man probe) was done. PCR quantification was performed using plasmid dilution (one-copy per mL). The detection process was as follows: 95[degrees]C for 10 minutes, followed by 50 cycles of 94[degrees]C for 10 seconds, 60[degrees]C for 32 seconds, and 72[degrees]C for 25 seconds. The reactions are done in a 7500 RT- PCR system tool.

RESULTS

Pairwise comparisons between plaque samples with CAL< 3 mm in CP and plaque samples with CAL > 6in Both CP AND AP mm were checked using the Wilcoxon-signed rank test. HSV-1 ,EBV and HCMV counts were compared with Friedman test. Differences if were statistically significant, pairwise comparison performed using a Wilcoxon-signed rank test.

The result on the comparison of each virus in plaque samples, cal [less than or equal to] 3 mm and cal > 6 mm is demonstrated in the Table 1 and the results of the comparison between hsv-1, EBV and CMV counts in plaque samples are shown in Table 2.

Herpes simplex virus 1

The HSV-1 count in subgingival plaque was more in sites more than CAL> 6mm and the load in the subgingival plaque samples with CAL <3mm was minimal statistically significant (P<0.05) (table 1) in both chronic periodontitis and aggressive periodontitis

Epstein-Barr Virus

The EBV count in subgingival plaque sample was also more than its amount in the SUBGINGIVAL PLAQUE samples with CAL > 6 mm and the EBV load in CAL d" 3 mm was the least (Table 1). All the differences were statistically significant (P < 0.05) in both chronic periodontitis and aggressive periodontitis.

Human Cytomegalovirus

The HCMV count was similarly higher in subgingival plaque sample CAL> 6 mm. On the other hand, HCMV count was lower in CAL [less than or equal to] 3 mm samples (Table 1) in both chronic and aggressive periodontitis.

Herpes Simplex -1-1, Human Cytomegalovirus and Epstein-Barr Virus Comparison

In comparison of HSV-1, HCMV and EBV counts, the HSV-1 load was statistically higher in the PLAQUE samples with CAL > 6 mm (P = 0.001). In comparison of HCMV and EBV counts, the difference in PLAQUE samples with CAL < 3 mm was not statistically significant (P > 0.05). On the other hand, EBV count was significantly higher than HCMV in tissue samples with PD > 6 mm (P < 0.05) (Table 2). The frequency of HSV-1was 28% in PD > 6 mm in CP AND 32% IN CAL >6MM IN AP and 10.8% in PD d" 3 mm. The frequency of EBV in the same group was 21.6% in PD > 6 mm and 8.1% in PD d" 3 mm. and HCMV was 20.8% in CAL>6MMand 5.1%in CAL <3mm similar in both chronic and aggressive periodontitis table 2

DISCUSSION

The Current Study Was Conducted To Detect HSV-1,HCMV And EBV In Subgingival Plaque Samples Of Chronic Periodontitis Patients And Aggressive Periodontitis Using The RT-PCR. We Observed The Highest Amount Of HSV-1in Samples With CAL> 6 Mm And The Highest Amount Of EBV and HCMV In CAL> 6 Mm Subgingival Plaque Samples Of Patients With Chronic Periodontitis And Aggressive Periodontitis. Many Studies Have Demonstrated The Association Between Herpes Viridae And The Periodontal Disease. Herpes Viruses Like EBV, HCMV, HSV And Human Herpes Virus (HHV 6-8) And Transfusion Transmitted Virus (TTV)(A New One)Causes Periodontal Disease (10,13,14). Latency and reactivation of viruses other than its cytopathic properties effects periodontitis (4). cytokine release during its reactivation produces destruction of periodontal tissues (8). Latency of these viruses may have more damage To The Periodontal Regeneration (10).

EBV and HSV-1 are mostly related to chronic and aggressive periodontitis probing depth and attachment loss were higher in the presence of these viruses (12). Recent studies show the presence of viruses more in GCF sample collected between periodontitis and gingivitis (6,14-17). Few studies evaluated the frequency of viral presence in saliva and gingival tissues. In the study detection of high frequency of viral load was reported in the chronic periodontitis in comparison with healthy sites (6,10,13,15,18-21). Present study confirmed the PRESENCE OF HSV-1 EBV and HCMV counts IN Chronic And Aggressive Periodontitis. As Many Studies Like Contreras et al. could not detect any VIRU S particles in healthy periodontal tissue (1). Sample Collection In The Healthy Sites Less Than 3mm Cal Is Avoided. Most Studies Just End In Detection Of The Viruses Our Study Was Presented With The Viral Counts. Of HSV-1, EBV, HCMV detected.

Viral prevalence have been reported in different populations in different studies Saygun in 2002 (19), Turkish samples. Imbronito (18) and Bilichodmath (13) reported that HSV-1 and EBV-1 were more frequently associated with chronic periodontitis and aggressive periodontitis, respectively this study was done in a south indian population Though the percentages varied greatly for EBV: 78.9% (13), 46.7%, for HSV-1: 100% (13), 40% (18) and for HCMV: 26.31% 13, 50% 18. In other studies, the results were similar in presence of the viral contents in samples.

Though PCR Was The Most Preferred Detection Aid In Many Studies (10) or nested PCR (13,14,18,22) the present study we adopted RT-PCR. Polymerase chain reaction is a gene amplification method, which allows quantification of microorganisms with selective DNA segment manipulation (23). Nested PCR has both advantages (specificity and sensitivity and disadvantages like false positive results due to its contamination susceptibility technique (23). Real-time PCR is a more appropriate quantitative method for detection of viruses. Polymerase chain reaction is a qualitative test that just focuses on the existence of virus, which can be affected by confounding environmental factors. In addition, viruses like EBV and HCMV can remain latent in lymphocytes which are the normal inhabitants of periodontal sulcus and pocket wall; so, mere existence of viruses is not valuable enough (24).

Studies Like Saygun R et al. (17) Which Did Not Compare Their Test Group With A Normal Control Group; We Adopted Similar Study Comparing Samples With Cal < 3 Mm In Chronic Periodontitis And Cal > 6 Mm In Both Chronic And Aggressive Periodontitis (17). Yildirim Et Al. Reported Hcmv And Ebv Detection In Kostmann syndrome with RT- PCR in 2006 (25, Saygun et al. classified chronic and aggressive periodontitis just by age range, which does not seem to be scientific (17). OUR STUDY WE attach radiographic aids to vary the bone loss pattern to differentiate chronic and aggressive periodontitis.. The Aggressive Periodontitis Sites >6mm Showed More Viral Load.

We observed 13.5% HCMV and EBV-1 coinfection, 15% HSV-1 AND EBV COINFECTION in some samples CAL d" 3 mm and PD > 6 mm. This result was in line with Jahangirnezhad (10) and Kubar et al., (21) studies that reported 52% HCMV and EBV-1 coinfection in aggressive periodontitis respectively. Kubar et al. also reported a 27% confection in chronic periodontitis (26). Grenier et al., (14) showed higher copy number of HCMV presence in GCF of deep periodontal pockets which is in agreement with our study on tissue. They also postulated that periodontal therapy can eliminate EBV and HCMV, and reduce HSV in GCF.

Some difficulties of virus DNA extraction from subgingival plaque samples are as follows: 1) It is hard to collect witout contamination of blood and saliva, 2)low amount of plaque samples in aggressive periodontitis sites, and 3) absence of viable proteins in plaque samples to detect viral load accurately we suggest a cohort study for an investigation on the cause and effect relationship of the herpes viruses in the periodontal disease.

Current study demonstrated considerable number of viruses present in this sample population. and increase in load causing severity of disease or vice versa which remained a challenge to find.

REFERENCES

(1.) Contreras A, Umeda M, Chen C, Bakker I, Morrison JL, Slots J. Relationship between herpesviruses and adult periodontitis and periodontopathic bacteria. J Periodontol. 1999; 70(5):478-84. doi: 10.1902/jop.1999.70.5.478. [PubMed] [Cross Ref]

(2.) Contreras A, Slots J. Herpesviruses in human periodontal disease. J Periodontal Res. 2000; 35(1): 3-16. [PubMed]

(3.) Contreras A, Nowzari H, Slots J. Herpesviruses in periodontal pocket and gingival tissue specimens. Oral Microbiol Immunol. 2000; 15(1): 15-8. [PubMed]

(4.) Mardirossian A, Contreras A, Navazesh M, Nowzari H, Slots J. Herpesviruses 6, 7 and 8 in HIV- and non-HIV-associated periodontitis. J Periodontal Res. 2000; 35(5): 278-84. [PubMed]

(5.) Slots J, Contreras A. Herpesviruses: a unifying causative factor in periodontitis? Oral Microbiol Immunol. 2000; 15(5): 277-80. [PubMed]

(6.) Contreras A, Slots J. Mammalian viruses in human periodontitis. Oral Microbiol Immunol. 1996; 11 (6): 381-6. [PubMed]

(7.) Contreras A, Falkler WJ, Enwonwu CO, Idigbe EO, Savage KO, Afolabi MB, et al. Human Herpesviridae in acute necrotizing ulcerative gingivitis in children in Nigeria. Oral Microbiol Immunol. 1997;12(5): 259-65. [PubMed]

(8.) Slots J, Sugar C, Kamma JJ. Cytomegalovirus periodontal presence is associated with subgingival Dialister pneumosintes and alveolar bone loss. Oral Microbiol Immunol. 2002; 17(6): 369-74. [PubMed]

(9.) Slots J, Kamma JJ, Sugar C. The herpesvirus-Porphyromonas gingivalis-periodontitis axis. J Periodontal Res. 2003; 38(3): 318-23. [PubMed]

(10.) Jahangirnezhad M. Subgingival Human Cytomegalovirus and Epstein Barr Virus in Patients with Aggressive Periodontitis in Ahwaz, Iran. J Dent. 2009; 6(2): 63-6.

(11.) Nibali L, Atkinson C, Griffiths P, Darbar U, Rakmanee T, Suvan J, et al. Low prevalence of subgingival viruses in periodontitis patients. J Clin Periodontol. 2009; 36(11): 928-32. [PubMed]

(12.) Das S, Krithiga GS, Gopalakrishnan S. Detection of human herpes viruses in patients with chronic and aggressive periodontitis and relationship between viruses and clinical parameters. J Oral Maxillofac Pathol. 2012; 16(2): 203-9. [PMC free article] [PubMed]

(13.) Bilichodmath S, Mangalekar SB, Sharma DC, Prabhakar AK, Reddy SB, Kalburgi NB, et al. Herpesviruses in chronic and aggressive periodontitis patients in an Indian population. J Oral Sci. 2009; 51(1):79-86. [PubMed]

(14.) Grenier G, Gagnon G, Grenier D. Detection of herpetic viruses in gingival crevicular fluid of patients suffering from periodontal diseases: prevalence and effect of treatment. Oral Microbiol Immunol. 2009;24(6):506-9. [PubMed]

(15.) Parra B, Slots J. Detection of human viruses in periodontal pockets using polymerase chain reaction. Oral Microbiol Immunol. 1996; 11 (5): 289-93. [PubMed]

(16.) Contreras A, Slots J. Active cytomegalovirus infection in human periodontitis. Oral Microbiol Immunol. 1998; 13(4): 225-30. [PubMed]

(17.) Saygun I, Kubar A, Ozdemir A, Slots J. Periodontitis lesions are a source of salivary cytomegalovirus and Epstein-Barr virus. J Periodontal Res. 2005; 40(2): 187-91. [PubMed]

(18.) Imbronito AV, Okuda OS, Maria de Freitas N, Moreira Lotufo RF, Nunes FD. Detection of herpesviruses and periodontal pathogens in subgingival plaque of patients with chronic periodontitis, generalized aggressive periodontitis, or gingivitis. J Periodontol. 2008; 79(12): 2313-21. doi: 10.1902/jop.2008.070388. [PubMed] [Cross Ref]

(19.) Saygun I, Sahin S, Ozdemir A, Kurtis B, Yapar M, Kubar A, et al. Detection of human viruses in patients with chronic periodontitis and the relationship between viruses and clinical parameters. J Periodontol. 2002; 73(12): 1437-43. doi: 10.1902/jop.2002.73.12.1437. PubMed] [Cross Ref]

(20.) Yapar M, Saygun I, Ozdemir A, Kubar A, Sahin S. Prevalence of human herpesviruses in patients with aggressive periodontitis. J Periodontol. 2003;74(11): 1634-40. doi: 10.1902/jop.2003.74.11.1634. [PubMed] [Cross Ref]

(21.) Kubar A, Saygun I, Yapar M, Ozdemir A, Slots J. Real-time PCR quantification of cytomegalovirus in aggressive periodontitis lesions using TaqMan technology. J Periodontal Res. 2004; 39(2): 81-6. [PubMed]

(22.) Contreras A, Zadeh HH, Nowzari H, Slots J. Herpesvirus infection of inflammatory cells in human periodontitis. Oral Microbiol Immunol. 1999; 14(4): 206-12. [PubMed]

(23.) Burkardt HJ. Standardization and quality control of PCR analyses. Clin Chem Lab Med. 2000; 38(2): 87-91. doi: 10.1515/CCLM.2000.014. [PubMed] [Cross Ref]

(24.) Idesawa M, Sugano N, Ikeda K, Oshikawa M, Takane M, Seki K, et al. Detection of Epstein-Barr virus in saliva by real-time PCR. Oral Microbiol Immunol. 2004;19(4):230-2. [PubMed]

(25.) Yildirim S, Yapar M, Kubar A, Slots J. Human cytomegalovirus, Epstein-Barr virus and bone resorption-inducing cytokines in periapical lesions of deciduous teeth. Oral Microbiol Immunol. 2006; 21(2):107-11. [PubMed]

(26.) Kubar A, Saygun I, Ozdemir A, Yapar M, Slots J. Real-time polymerase chain reaction quantification of human cytomegalovirus and Epstein-Barr virus in periodontal pockets and the adjacent gingiva of periodontitis lesions. J Periodontal Res. 2005; 40(2):97-104. [PubMed]

S. Parthiban [1], T. Ramakrishnan [2], V. Balakumar [3], Rathika Rai [1], Umasudhakar [1] and V. Shankarram

[1] Department of Periodontics,Thai Moogambigai Dental College, Golden George Nagar, Chennai 600107, India.

[2] Department of Periodontics, Adhi Parasakthi Dental College, Melmaruvathur, India.

[3] Department of Prosthodontics, Satyabama Dental College, Chennai, India.

(Received: 20 September 2015; accepted: 11 November 2015)

* To whom all correspondence should be addressed. E-mail:
Table 1. Based on the Freidman test. P-values less than
0.05 are considered as statistically significant

Groups                Mean [+ or - ] SD       Median (Mean
                                                  Rank)

HSV-1

  CAL >6mm            5632 [+ or -] 6934
  CAL [less than    103.34 [+ or -] 422.98     0.00 (1.23)
    or equal to]
    3mm

EBV

  CAL >6mm         3170.24 [+ or -] 4877.76   837.00 (2.42)
  CAL [less than    113.22 [+ or -] 416.25     0.00 (1.64)
    or equal to]
    3mm

HCMV

  CAL >6mm          623.27 [+ or -] 1636.25     0.00 (1.99)

  CAL [less than    119.38 [+ or -] 405.67     0.00 (1.80)
    or equal to]
    3mm

Groups             Significant               P Value
                   Pairwaise
                   Comparison

HSV-1                                        0.012

  CAL >6mm
  CAL [less than   0.00 c PD > 6
    or equal to]   mm vs. PD [greater than
    3mm              or equal to] 3mm

EBV
                                             0.00
  CAL >6mm
  CAL [less than   0.00 c PD > 6
    or equal to]   mm vs. PD [less than or
    3mm              equal to] 3mm

HCMV                                         0.013

  CAL >6mm           0.00 c PD > 6

  CAL [less than   mm vs. PD [less than or
    or equal to]     equal to] 3mm
    3mm

Table 2. Based on the Freidman test. P-values less than 0.05 are
considered as statistically significant

Groups                         Mean [+ or -] SD       Median   P value

PD > 16 mm                                                      0.001

  HSV-1                       5632 [+ or -] 6934       0.00
  EBV                      3170.24 [+ or -] 4877.76    0.00
  MCM V                    623.27 [+ or -] 1636.25     0.00

PD [less than or equal                                          0.001
to] 3 mm

  HSV-1                     103.34 [+ or -] 422.98     0.00
  EBV                       113.72 [+ or -] 416.25     0.00
  HCMV                      119.38 [+ or -] 405.67     0.00
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Author:Parthiban, S.; Ramakrishnan, T.; Balakumar, V.; Rai, Rathika; Umasudhakar; Shankarram, V.
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Dec 1, 2015
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