Prevalence of herpesviruses in gingivitis and chronic periodontitis: relationship to clinical parameters and effect of treatment.

BACKGROUND: Assess the prevalence of herpesviruses in healthy subjects, gingivitis, and chronic periodontitis patients, to assess the relationship between the prevalence of herpesviruses and periodontal clinical parameters, and to evaluate the effect of phase-I therapy on the level of viral detection.
MATERIALS AND METHODS: Hundred patients consisting of 20 healthy subjects, 40 gingivitis, and 40 chronic periodontitis were included in the study. Clinical parameters recorded included plaque index, gingival index, sulcus bleeding index, probing depth, and clinical attachment level. The gingivitis and chronic periodontitis patients received phase-I periodontal therapy including oral hygiene instructions, full mouth scaling for gingivitis patients and scaling and root planing for chronic periodontitis patients. Gingival crevicular fluid (GCF) was collected, and the presence of herpes simplex virus-1 (HSV-1), HSV-2, cytomegalovirus, and Epstein-Barr virus (EBV) was analyzed using polymerase chain reaction (PCR). Recording of periodontal parameters as well as GCF collection was performed at baseline and 6 weeks postphase-I therapy.
RESULTS: At baseline, the levels of HSV-1 and EBV detection were lower in healthy controls as compared to gingivitis (P < 0.05) and chronic periodontitis cases (P < 0.001). Phase-I therapy led to reduction in the amount of HSV-1 and EBV in gingivitis patients (P < 0.05) and for HSV-1, human cytomegalovirus and EBV in chronic periodontitis patients (P < 0.05) in comparison to baseline. The prevalence of EBV in chronic periodontitis patients was positively associated with increased gingival index, probing depth and loss of clinical attachment (P < 0.05).
CONCLUSIONS: Higher prevalence of HSV-1 and EBV viruses in GCF of gingivitis and chronic periodontitis suggests a strong association between these viruses and periodontal diseases and periodontal therapy can lead to a reduction in herpesviruses at infected sites.

INTRODUCTION

Periodontitis is a multimicrobial and multifactorial inflammatory disease of the periodontal tissues. From the time the search for pathogens causing periodontal diseases began, the organisms in focus and under research have been the bacteria and as such, periodontal microbiology was reduced to periodontal bacteriology. At the turn of the century, the field of periodontal pathophysiology witnessed a revolution when the idea emerged that other oral inhabitants such as viruses could play a role in the transition from periodontal health to periodontal disease. Various viruses which have been investigated for a possible role in periodontal pathogenesis include herpes simplex virus-1 (HSV-1), HSV-2, human cytomegalovirus (CMV), Epstein–Barr virus (EBV), varicella zoster virus, human herpesvirus-6 (HHV-6), HHV-7, HHV-8, human T-lymphotropic virus type 1, torquetenovirus, and hepatitis virus B and C and human papilloma virus.[1] The herpesvirus-bacterial theory of periodontal pathogenesis as put forth by Slots has been around for a decade and during that time ample scientific evidence has accumulated to implicate these viruses in the periodontal disease process.

It has been demonstrated that a single site of active periodontal destruction may harbor more than a million copies of herpesvirus genomes.[2] Herpesviruses have been identified in supragingival and subgingival plaque samples, gingival biopsies, and gingival crevicular fluid (GCF) of healthy and periodontitis patients.[345] The gingival sulcus or periodontal pocket has also been proposed to act as a reservoir between periods of recurrence of herpetic medical infections.[6] Various studies have demonstrated that HSV, CMV, and EBV are significantly associated with chronic periodontitis as compared to healthy controls.[347] It has also been observed that the presence of herpesviruses increases with the increase in disease severity in chronic periodontitis cases.[8] Recently, the effect of phase-I therapy on the levels of herpesviruses in chronic periodontitis cases was assessed, and it was found that such therapy may result in short-term elimination of viruses in the diseased sites.[49] Thus, the present study was conducted with the aim to investigate the prevalence of HSV-1, HSV-2, CMV, and EBV in GCF of healthy controls and gingivitis and chronic periodontitis patients and to evaluate the effect of phase-I periodontal therapy on the GCF virus levels in a population from the Indian subcontinent.

MATERIALS AND METHODS

A total of 100 patients (53 males and 47 females) in the age range of 18–60 years (mean age 34.5 ± 8.3 years) were selected from the out-patient Department of Periodontics. All study subjects were given a detailed verbal and written description of the study and signed a consent form before commencement of the study. The study protocol was approved by the Institutional Review Board.

The study population consisted of 20 clinically healthy, 40 gingivitis, and 40 generalized chronic periodontitis patients who were diagnosed according to the clinical and radiographic criteria proposed by the 1999 International World Workshop for a Classification of Periodontal Diseases and Conditions were included.[10] Patients with any history of a systemic disease, those having taken any antibiotic therapy or underwent any form of periodontal treatment in the past 6 months, pregnant or lactating females, smokers, and those on antiviral or immunosuppressive drugs were excluded from the study. Also, subjects who had any previous history of symptomatic viral (HSV, EBV, and CMV) infection were excluded from the study.

The clinical parameters were assessed at baseline for all the three groups and at 6 weeks after treatment for the gingivitis and chronic periodontitis groups. Clinical parameters recorded for each patient included the plaque index,[11] the gingival index,[12] the sulcus bleeding index,[13] probing depth, and clinical attachment level (CAL) for full mouth. All clinical parameters were evaluated using a UNC-15 probe. Loss of clinical attachment was calculated as the distance from the cementoenamel junction to the base of the periodontal pocket.

After the baseline examination had been done, the patients were recalled 1 week later for GCF collection. GCF was collected at one random site in Group 1 (healthy subjects), at the site showing most severe gingival inflammation in Group 2 (gingivitis group), and at the site showing deepest probing depth in Group 3 (chronic periodontitis group). Before sampling, supragingival plaque was removed using sterile cotton pellets and the sample site was isolated with cotton rolls to avoid salivary contamination. Commercially available filter strips 2 mm × 10 mm (Whatman 3MM, Acerasayan, Bengaluru, India) were gently inserted into the gingival crevice/periodontal pocket until mild resistance was felt carefully avoiding bleeding from the site and kept so for 30 s. The collected sample was immediately transferred to a plastic tube containing 250 µL of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) and placed at −20°C till further processing.

Oral hygiene instructions were given to all the patients. Following the collection of GCF, gingivitis patients received full mouth scaling with ultrasonic instruments, whereas chronic periodontitis patients were subjected to full mouth scaling and root planing. GCF samples were collected at 6 weeks after the periodontal therapy from the same sites used for baseline sampling.

The following morning, GCF samples were centrifuged at 5000 rpm for 2 min. The supernatant was discarded, and the obtained precipitate was washed 3–4 times with TE buffer. Following this, the precipitate was washed with lysis buffer 1 (Tris-HCl and Triton X-100) once, incubated in 100 µL of lysis buffer 2, and 10 µL of proteinase-K was added to the precipitate. The mixture was kept in water bath at 65°C for 2 h and then kept in boiling water bath for 10 min following which it was stored at − 20°C until amplification.

The multiplex polymerase chain reaction (PCR) amplification technique was used to identify viral DNA from HSV-1 HSV-2 human cytomegalovirus (HCMV) EBV. Detection of EBV was carried out using primers sequences (5'-CGTACCTGCGGCTCGTGAAGT-3') as the forward and (5'-AGCAGGGTGCTCGTGTATGGG-3') as the reverse; (5'-TGGTATCGCATGGGAGACAAT-3') as the forward and (5'-CTCCGTCCAGTCGTTTATCTTG-3') as the reverse primers for HSV-2; (5'-ACGTGTTACTGGCGGAGTCG-3') as the forward and (5'-TTGAGTGTGGCCAGACTGAG-3') as the reverse primer for HCMV; and (5'-AGCACTGGCCAGCTCATATC-3') as the forward and (5'-TTGACGTCATGCCAAGGCAA-3') as the reverse primer for EBV. Single PCR was undertaken in a final 25 µL volume. It contained 0.5 ml Taq polymerase, 1X polymerase buffer containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 m M MgCl2, 18 picomoles of each primer, 2 µL of extracted DNA and 200 µM of dNTP. The following number of amplification cycles and annealing temperatures were used: 40 amplification cycles of 30 s at 94°C, 30 s at 54°C and 30 s at 72°C. After the last cycle, the samples were incubated at 72°C for 5 min followed by 4°C for 1 min. Ten micro litters of each amplified product were loaded on a 1% agarose gel with ethidium bromide and detected under an ultraviolet transilluminator.

Statistical analysis

The statistical analysis was performed using SPSS version 16.0 software (IBM, USA). Results are presented as mean ± SD (standard deviation). One-way ANOVA was used for simultaneous multiple group comparisons followed by post hoc Tukey's test for pairwise comparisons. Posttreatment changes within the same group were analyzed by Chi-square statistical tests. A P value of 0.05 or less was considered statistically significant.

RESULTS

Demographic and periodontal conditions

The demographics and periodontal conditions are summarized in Table 1. The percentage of patients carrying, at least, one or two herpesvirus is also presented in Table 1. Among patients suffering from chronic periodontitis 97.5% harbored at least one herpesvirus species as compared to 77.5% in gingivitis and 45% in the periodontally healthy groups. There were no dropouts in the present study.

Table 1

Demographic variables and number of herpesvirus detection at baseline

At baseline, there was significant difference between periodontally healthy, gingivitis, and chronic periodontitis subjects in all evaluated clinical parameters. Periodontal treatment led to improvement in all evaluated clinical parameters after 6 weeks. The posttreatment healing was uneventful in all study patients and no periodontal complications, such as infections or abscesses, were observed throughout the study period [Table 2].

Table 2

Comparative evaluation of clinical parameters at baseline and 6 weeks after periodontal treatment

Prevalence of herpesvirus DNA

At baseline, there was a significant difference between healthy and gingivitis subjects and between healthy and chronic periodontitis patients in the occurrence of HSV-1 and EBV (P < 0.05). Also, the difference between EBV detection in gingivitis and chronic periodontitis patients was statistically significant (P < 0.05) [Table 3].

Table 3

Percentage herpesvirus detection at baseline

Relationship of herpesvirus detection to baseline clinical parameters

The gingivitis group revealed increased herpesvirus detection with an increase in the severity of clinical parameters such as plaque index, gingival index, and sulcus bleeding index [Table 4]. The relationship of EBV detection to increasing plaque index and sulcus bleeding index was statistically significant (P < 0.05). Also, the percentage detection of herpesviruses increased with increased plaque index, gingival index, sulcus bleeding index, probing depth, and clinical attachment loss in the chronic periodontitis group [Figures 1–5]. However, the prevalence of EBV was significantly elevated only in the subgroups of chronic periodontitis group that had higher gingival index, deeper probing depth and increase in CAL values (P < 0.05) [Figures 1–5]. When the overall herpesvirus detection frequency was compared to clinical parameters [Table 5], herpesvirus coinfection with two or more viruses was found to be significantly associated with increased plaque index, gingival index, sulcus bleeding index, and probing depth (P < 0.001).

Table 4

Correlation of clinical parameters to herpesvirus detection at baseline in gingivitis group

Figure 1

Relationship of plaque index to herpesvirus detection at baseline in chronic periodontitis group

Figure 5

Relationship of loss of clinical attachment to percentage herpesvirus detection at baseline in chronic periodontitis group

Table 5

Relationship of number of herpesvirus detection to clinical parameters

Relationship of gingival index to herpesvirus detection at baseline in chronic periodontitis group

Relationship of sulcus bleeding index to herpesvirus detection at baseline in chronic periodontitis group

Relationship of probing depth to percentage herpesvirus detection at baseline in chronic periodontitis group

Effect of phase-I periodontal therapy on herpesvirus detection levels

Phase- I therapy resulted in a significant decrease in the level of HSV-1 detection in gingivitis and chronic periodontitis patients (P < 0.05). The level of CMV detection decreased in chronic periodontitis patients (P < 0.05) whereas the EBV detection level decreased from gingivitis and in chronic periodontitis patients (P < 0.005) [Table 6].

Table 6

Effect of periodontal treatment (phase-I periodontal therapy) on percentage herpesvirus detection at 6 weeks after treatment

DISCUSSION

An etiopathogenic role for herpesviruses has been suggested for chronic periodontitis, aggressive periodontitis, periodontal abscesses, periapical abscesses, and peri-implantitis.[71415] The present study evaluated the presence of herpesviruses in healthy subjects and in gingivitis and chronic periodontitis patients and also assessed the effect of phase-I periodontal therapy on the subgingival occurrence of these viruses. The groups to be assessed were healthy subjects, subjects with gingivitis and subjects with chronic periodontitis. The frequency of herpes virus presence and detection increases with increased probing depth.[5] Hence, even though the herpes viruses can be detected at the sites with shallow pocket depth of periodontitis patients, deepest site of the dentition was chosen as the site to collect GCF. The methodology was based on previous similar studies.[416]

The prevalence of HSV-1 was significantly higher in gingivitis and chronic periodontitis patients as compared to healthy controls. Similar observations were made in the previous studies.[7171819] However, some reports have failed to show a significant relationship between HSV-1 and periodontal disease.[2021] This discrepancy in the frequency detection of HSV-1 may be attributed to sample size, selection criteria, type of sample: (Gingival tissue or GCF), type of PCR used, ethnic differences and tissue tropism of periodontal herpesvirus infection. We observed significant reduction in herpesvirus levels after phase-I therapy. This finding is in agreement with the study by Grenier et al.[4] The reduction of HSV-1 population after phase-I periodontal therapy can be attributed to a reduced influx of HSV-1 infected cells, resolution of gingival inflammation and reduction in pocket depth due to tissue shrinkage.

HSV-2 primarily causes anogenital infections; however, the virus has been recovered from the oral cavity as well.[22] In the present study, the level of HSV-2 detection was very low in all the three study groups and the intergroup difference in detection was not significant. This is in agreement with previous studies.[1617]

CMV has frequently been associated with periodontal disease. The virus infects periodontal monocytes/macrophages and T-lymphocytes, and reactivation of CMV in periodontitis lesions tends to be associated with progressive periodontal disease.[23] Several studies have reported a significantly higher prevalence of CMV in periodontitis patients as compared to healthy controls or gingivitis patients.[4172425] Similarly, in one Indian study, an increased prevalence of HCMV in patients suffering from chronic periodontitis and cervical carcinoma was demonstrated.[26] However, in our study, the detection frequency of CMV was comparatively low, and the difference between the groups was statistically not significant. This finding is consistent with the observations made in some previous studies.[182227] The low frequency of CMV in our study can be explained by two factors, first, that CMV is associated with sites undergoing active periodontal destruction [28] and our study did not identify whether the sample sites were disease-stable or undergoing active destruction. The finding can also be explained by the fact that active CMV infection occurs in human periodontitis in low frequency whereas the latent periodontal CMV infection is a more frequent event.[29] Since CMV may predominantly occur in gingival tissue rather than in GCF, the gingival tissue samples obtained in the other studies might have resulted in increased frequency of CMV than in the present study.

At 6 weeks postphase-I periodontal therapy, the level of CMV in Group 2 was not significantly changed. However, in Group 3, the posttreatment GCF CMV level was significantly different from that of baseline. Similar observation was made in studies which evaluated the effect of surgical and nonsurgical therapies on periodontal abscesses [30] and scaling, root planing of chronic periodontitis patients.[49]

EBV is a herpesvirus, which has been implicated in the pathogenesis of various types of periodontal disease. The virus infects periodontal B-lymphocytes,[23] which may impair the periodontal defense and predispose to overgrowth of periodontal pathogenic bacteria.[3] We found that EBV is associated with a shift from periodontal health to periodontal disease is in agreement with findings of previous studies which reported on a high prevalence of EBV in chronic periodontitis.[202127] However, other studies found an extremely low or nil association between EBV and periodontal disease.[425313233] Again, the large variation in detection frequencies of EBV between different studies may be attributed to the genetic and ethnic background and to geographical and demographic factors of the study groups. Few studies have been conducted assessing the relationship of EBV to chronic periodontitis in Indian population, and our results are in line with the findings of those studies.[163435]

The GCF levels of EBV in Group 2 and 3 were significantly reduced when baseline values were compared to 6 weeks posttreatment. This finding indicating that phase-I periodontal therapy can lead to short-term reduction in EBV frequency from periodontal sites has also been observed after surgical and nonsurgical therapy in periodontal abscesses 30 and after scaling and root planing of chronic periodontitis lesions.[49]

In the gingivitis group, the relationship of EBV detection to increasing plaque index and sulcus bleeding index was statistically significant (P < 0.05). Similarly, the relationship between the prevalence of EBV and the gingival index, probing depth, and CAL in chronic periodontitis group reached statistical significance (P < 0.05). Similar observations were made in previous studies.[836] The positive relationship of herpesvirus frequency and increasing severity of periodontitis emphasizes and provides further evidence for a role of herpesviruses in the pathogenesis of the disease.

We found that number of herpesvirus species increased significantly with an increasing severity of all of the clinical parameters. Patients with coinfection by two or three herpesviruses exhibited significantly more periodontal destruction than patients containing one or no herpesviruses. Previous studies of aggressive periodontitis have also found an association between herpesvirus co-infection and increased gingival bleeding and probing depth.[19] Hence, the present observations reinforce the hypothesis that a simultaneous infection by EBV (B-lymphocytes) and HSV or CMV (T-lymphocytes and macrophages)[23] may exert an additive or synergistic pathogenic effect.

CONCLUSION

The present study showed that the prevalence of HSV-1 and EBV viruses in GCF is higher in patients suffering from gingivitis and chronic periodontitis compared to periodontally healthy subjects. Also, phase-I periodontal therapy results in reduction of herpesvirus level in GCF of gingivitis and chronic periodontitis patients. The current evidence points to a positive relationship between herpesviruses and periodontal disease; however, a cause-effect relationship has yet to be established.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.