Association of Herpesvirus and Periodontitis: A Clinical and Laboratorial Case–Control Study

Objectives  A significant influence of the Herpesviridae family in the progression of periodontal disease has been suggested. The aim of this study was to investigate the potential association of four Herpesviruses (HSV-1, HSV-2, cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) with periodontal disease using a qualitative test for evaluating the presence or absence of viral DNA in crevicular fluid samples of both healthy periodontal patients and periodontal compromised patients. Materials and Methods  A case–control study was conducted in 100 participants at a university clinic. A qualitative test was used for evaluating the presence/absence of viral DNA in crevicular fluid samples of both healthy periodontal patients and periodontal compromised patients, and considering the periodontitis staging (stage II, stage III, and stage IV) and grading (grade A, grade B, and grade C). Statistical Analysis  The distribution of the same exposure variables to the periodontitis staging and grading was compared using Chi-square, Fisher's exact, and Gamma tests depending on the variable characteristics. The significance level was set at 5%. The association of the variables: age, sex, diabetes, smoking, alcohol, and oral hygiene was also considered. Results  The prevalence of Herpesviridae family virus DNA was 6% for the periodontal healthy group and 60% for the periodontitis group (roughly 60% on periodontitis stages II, III, and IV, p <0.001; and twofold increase in moderate and rapid progression grades compared with the slow progression grade, p <0.001). HSV1 DNA was prevalent in all periodontitis stages and grades. HSV 2, EBV, and CMV DNA had increasing prevalence rates in more severe stages (stages III and IV, p <0.001); while considering periodontitis grade, HSV2 ( p  = 0.001), CMV ( p  = 0.019) and EBV ( p <0.001) DNA were prevalent only in grades B and C, with EBV DNA registering a marked prevalence in grade C. Conclusion  A significant different distribution of Herpesviridae virus DNA per each stage of disease was registered.


Sample collection
For the extraction of the crevicular fluid samples, two numbered 35 paper points were used per participant.The paper points were previously sterilized in an autoclave under 134 °C and consequently placed in sterilized Eppendorfs of 1.5 mL capacity. 2 The Eppendorf were numbered from 1 to 100 for the purpose of later matching the sample number to the survey number, allowing future identification.For in-stance, the patient filling in the survey number 37 got his crevicular fluid collected with the paper points addressed to the Eppendorf number 37.
For the crevicular fluid sample collection the two deepest periodontal pockets (in mouth) were chosen.The sample collection was done on an illuminated dental chair.Using a periodontal probe, the selected pockets were remeasured to confirm their depth.Following, the collection site was dried with an air jet (sani-tip) so local saliva could be removed.Subsequently, the paper point was inserted in the periodontal socket and let to absorb the crevicular fluid for 30 seconds. 3 This process was repeated and done on other location of the mouth, in a periodontal socket, that was similarly, large in depth.In total two sterilized paper points were used per participant.
After the sample collection the paper points were placed in the respective sterile Eppendorf for future laboratory analysis with PCR technique.
In the healthy group the process was similar, however, the paper points were placed in the gingival sulcus of molars or premolars for the collection of crevicular fluid.

Sample analysis DNA extraction
The analysis of the crevicular fluid samples took place at the Egas Moniz Applied Microbiology Laboratory.
First, DNA extraction was executed.The procedure for the DNA extraction was carried for each one of the 100 samples individually, following the same protocol.Nitril gloves were used throughout the whole process.
To each sample Eppendorf, 1 mL of ultrapure water (Sigma, Portugal) was added with a micropipette and let to rest at room temperature for 30 minutes.Subsequently, the Eppendorfs were vortex for 10 seconds at low rpm.Following, the samples were placed in a centrifuge for 3 minutes at 14,000 rpm.After being centrifugated the supernatant was removed, not damaging the pellet with a micropipette.The tip of the micropipette was discarded and a sterile one was used for every single one of the samples.Consecutively, to each sample 200 µL of Chelex (Bio-Rad, Portugal) 5% was added and then placed in bain-marie (56°C) for 15 minutes.The samples were vortexed for 10 seconds at high rpm and the tips from the caps pierced with a sterile needle.Following, the samples were placed in floating supports on boiling water for 8 minutes, not letting surpass the protocoled time.Posteriorly, the samples were vortexed (10 seconds) and centrifuged (14,000 rpm for 3 minutes), one final time.Succeeding the DNA extraction procedure, the samples were stored in a freezer at À20°C for future use.
European Journal of Dentistry © 2023.The Author(s).
Presence of Herpesviridae Family Virus Associated with Periodontitis Picolo et al.

Polymerase Chain Reaction
The technique used in the study was qualitative multiplex PCR.A real time thermal cycler was not available to enable the quantification of viral load.Multiplex PCR enables to simultaneously detect multiple targets in a single reaction.For the preparation of PCR solutions, reverse and forward primers were used for HSV1, HSV2, CMV, and EBV (►Appendix Table 1), 4 with 25 µL of MaterMix Taq Polymerase (Nzytech, Lisbon, Portugal), 14 µL of ultrapure water and 1 µL of each primer were added to numbered Eppendorfs.Subsequently, 3 µL of the previously extracted DNA sample was pipetted. 4A control Eppendorf was prepared, containing all the aforementioned solutions, however, the 3 µL of genetic material (DNA) was replaced by ultrapure water.
Primer solutions were prepared with a concentration of 10 µM and stored at À20°C.This procedure as well as the following, was executed with proper refrigeration and nitril gloves, as per protocol.
To prepare the PCR solutions: 25 µL of Taq Polymerase MasterMix (Nzytech), 1 µL of each primer and 14 µL of ultrapure water (Sigma), and 3 µL of DNA were added to each Eppendorf.Alongside the preparation of each 10 PCR solutions, a control solution was prepared to ensure that no contamination occurred during the experience.The control solution was similar to the PCR ones, however, 3 µL of DNA was replaced by 3 µL of ultrapure water.The prepared PCR solutions, alongside the control solution, were placed in a thermocycler.The program used in the thermocycler consisted of 95°C for 5 minutes for initial denaturation; 45 cycles, where each cycle consists of 30 seconds at 95°C (denaturation), 30 seconds at 54°C (annealing) and 30 seconds at 72°C (extension), and the final extension at 72°C for 5 minutes. 4The entire process in the thermal cycler lasted approximately two and a half hours.
For the preparation of the agarose gel (2%), 4 g of agarose and 200 mL of TAE (Tris-acetate-EDTA) were used.Furthermore, 10 µL of RedSafe nucleic acid staining solution dye (Merck, Algés, Portugal) was added.The agarose solution was then poured into a 22-well comb bed.
On a disinfected stand the Eppendorfs containing the PCR solutions were placed on ice and pipetted into the respective wells with a micropipette.The tips of the micropipette were always discarded between wells: moreover, the procedure was done using nitril gloves.Two DNA band markers, V and VI (Nzytech) were applied, which by having guide DNA bands on the order of 200 nanometers (nm), 300 nm and 400 nm, allowed better identification of the viruses.The base pairs of the respective virus' bands are listed in ►Appendix Table 2. 4 Positive control tests for the investigated viruses were not used, due to unavailable resources.
Each PCR solution was applied in the respective well with a 25 µL micropipette.The micropipettes were discarded and changed after every PCR solution placement.First, the V marker was applied; an interval well was left, and 25 µL of the control solution was poured.Subsequently, 25 µL of the samples were added in 10 sequent wells; Finally, leaving one well gap, the marker VI was applied.
A power of 50 volts for 3 hours was used for the electrophoresis procedure and the results were posteriorly observed under ultraviolet light.After the observation of the agarose gel under the UV light a copy of the shown image was printed for future analysis.Consecutively, the presence or absence of viruses was recorded on an excel table for statistical analysis.