Detection of EBV, CMV and HSV-1 in subgingival samples of HIV positive and negative patients with chronic periodontitis

Cite as: Escalona L, Veitía D & Correnti M. Detection of EBV, CMV and HSV-1 in subgingival samples of HIV positive and negative patients with chronic periodontitis. J Oral Res 2016; 5(4): 168-174. Abstract: Objective: To detect the presence of infection by EBV (Epstein-Barr Virus), CMV (Cytomegalovirus) and HSV-1 (Herpes Simplex Virus type 1) in subgingival samples from HIVpositive patients under HAART (High Activity Antiretroviral Therapy), HIVpositive patients without HAART, HIV-negative patients with chronic periodontitis and healthy controls. Methodology: Crevicular fluid samples of 11 HIV+ patients on therapy were evaluated, 6 without antiretroviral therapy, 7 HIVnegative subjects with chronic periodontitis and 7 periodontally-healthy controls. PI (Plaque index), GI (Gingival Index), PD (probing depth) and CAL (Clinical Attachment Loss) were registered at six sites per each tooth in all teeth and subgingival plaque samples of a tooth were collected per quadrant. Nested PCR was used to detect EBV and endpoint PCR to detect infection by CMV and HSV-1. Results: Clinical parameters showed statistically significant differences between HIV-positive patients and subjects with chronic periodontitis compared with the control group (p<0.05). DNA of EBV was detected mainly in HIV-positive patients under HAART, 91% (10/11). DNA of CMV was detected mainly in patients without HAART, 67% (4/6), while HSV-1 was observed in 27% (3/11) of patients under HAART. In the control group no virus was detected. Coinfection was observed in 50% of HIV patients without HAART, 36% of HIV patients with HAART and 14% of HIV-negative with chronic periodontitis. Conclusion: Viral infection was prevalent in HIV patients under HAART and EBV was the primary viral infection detected in HIV-positive patients with chronic periodontitis.


INTRODUCTION.
Periodontal disease is a multifactorial condition caused by a complex community of microorganisms that interact with host cells and tissues, causing the release of a wide range of inflammatory cytokines, chemokines, and mediators that lead to the destruction of periodontal tissues 1,2 . The coexistence of herpesvirus and periodontopathic bacteria, together with the host's local immune response, would be involved in the disease progression 3 . Different Herpesviri-dae detected in gingival tissue, such as Epstein-Barr virus (EBV) and cytomegalovirus (CMV), have been detected.
They seem to play a pathogenic role in periodontitis 4-6 . EBV is transmitted by contact with saliva and remains throughout life in the form of latent infection, usually asymptomatic 7 . EBV or Human Herpesvirus 4 (HH-4) belongs to the genus Lymphocryptovirus. Its main feature is the ability to establish itself as a latent infection within the host cell and the ability to induce proliferation of these 8 .
The role of herpesviruses in the etiology of periodontal disease has been suggested by their presence in gingival tissue, crevicular fluid and subgingival plaque in teeth with periodontal disease. However, the role of CMV and EBV in the pathogenesis of periodontal disease has not yet been ted at 65°C overnight in ATL buffer, then AL buffer was added, and samples were incubated at 72°C for 10 minutes. Absolute ethanol was added and transferred to a column; washes were performed with buffers W1 and W2.
Elution was performed with 200µl of elution solution.

PCR for viral detection
EBV was detected using a nested PCR, following the specifications described by Arreaza et al. 15

Statistic Analysis
To determine the existence of statistically significant differences among all the groups studied with statistical variables the Kruskal-Wallis and Mann-Whitney tests were used. Statistical significance was considered for values p<0.05.

RESULTS.
The   The presence of EBV, CMV and HSV-1 among all the groups in study are shown in Table 2

DISCUSSION.
Microbiological research in human periodontal disease has typically focused on bacteria and to a lesser extent on parasites and yeasts. However, in the last decade the presence of herpesviruses has been shown, including HSV1 and 2, EBV, and CMV in periodontal pockets of patients with periodontitis [16][17][18][19][20][21][22] .
A systematic review of microbiological studies in patients with periodontal disease in Central and South America by Contreras et al. 23 indicates that the genomes of HSV-1, CMV and EBV have been detected in the periodontal pocket, saliva and gingival immune cells. The three viruses have been associated with chronic periodontitis, aggressive periodontitis, acute ulcerative necrotizing gingivitis and periodontal abscess. Zhu et al. 19 suggest that EBV and CMV are significantly associated with chronic periodontitis.
In the present study the increased frequency of infection was observed in HIV-positive patients, EBV infection being the most common, followed by CMV and HSV-1; while in the control group no infection was detected. When comparing HIV-positive patients to each other, it was noted that for patients on HAART, EBV and HSV-1 infections were the most frequent, with 91% and 27%, respectively. However, for patients without HAART, a frequency of infection of equal magnitude for EBV and CMV (67% each) was observed, which was not the case for HSV-1.
There are few studies evaluating the frequency of infection by virus in GCF in HIV positive and negative patients with and without HAART. Some studies report a significant relationship between herpesviruses and the risk of chronic periodontitis, however, their findings are inconsistent 19 . Only two similar studies evaluating some of the variables included in this research were found. Large et al. 24 evaluated the frequency of infection by EBV, CMV and HSV-1 in two groups of patients, HIV-positive and HIVnegative, both diagnosed with chronic periodontitis, without distinction between the application or non-application of antiretroviral therapy.
Their results indicated a higher frequency of detection of EBV in saliva and subgingival plaque in HIV-positive patients compared to HIV-negative patients. Consequently, the authors suggest an association between the presence of EBV-1 and coinfection by EBV-1 and CVM with the diagnosis of periodontitis in HIV-positive patients 25 . The results of these two studies are similar to those of the present study regarding the type and frequency of the virus detected, although samples are larger. Some studies report that HAART has been shown to reduce the number of opportunistic infections, however these infections may occur and damage the immune system of patients under HAART 9 .
A larger number of studies have been conducted on patients with periodontitis. Grenier et al. 17    presence of herpesvirus in patients with periodontitis and periodontally healthy patients, finding that the prevalence of CMV and HSV in GCF was higher in patients with periodontitis, and that the prevalence of CMV increased in proportion to the depth of the pocket. Sharma et al. 18 compared the presence of CMV and EBV in patients with chronic and aggressive periodontitis with healthy individuals, reporting higher prevalence of EBV in patients with chronic periodontitis, while CMV infection was higher in patients with aggressive periodontitis. The highest frequency for both viral agents was observed in deep pockets. Rones et al. 26 demonstrated in vitro positivity for HSV-1 in epithelial cells and fibroblasts in the area of the gingival sulcus cells, suggesting that these cells could be reservoir of latent virus. Furthermore, Petrovic et al. 27 reported the presence of HSV-1 in GCF by PCR, indicating that the presence of this virus coincides with a high degree of tissue destruction in patients with chronic periodontitis. These observations were confirmed by Das et al. 28 . In the present study detection frequency for EBV and CMV observed in patients with periodontitis is consistent with previous reports, confirming their presence in patients with periodontal disease and deep periodontal pockets.
The present study evaluated coinfection between these herpesviruses, finding that the highest rate occurred in HIV positive patients without HAART (36%) and in patients with chronic periodontitis (14%). Coinfection with these viruses may increase the complexity of the clinical picture. Botero et al. 29 found a correlation between periodontal pathogen detection, CMV and deep pockets. These authors suggest that viral infections are acquired at an early age and that the prevalence in the population increases between 30-35 years old.
The results obtained in this research reinforce the hypothesis that herpesviruses may be involved in increased periodontal destruction, supporting the evidence that herpesviruses have a pathogenic role in the etiology of periodontal disease.
In conclusion, there is a higher frequency of herpesvirus infection in HIV-positive patients, the most prevalent being EBV in the HIV-positive group with HAART, and CMV in the HIV-positive group without HAART.