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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 5  |  Issue : 1  |  Page : 47-53

The coinfection between herpesviruses and periodontopathic microbiota in increasing severity of chronic periodontitis


1 Department of Microbiology, Sinhgad Dental College and Hospital, Pune, Maharashtra, India
2 Department of Microbiology, BJ Government Medical College, Pune, Maharashtra, India

Date of Web Publication12-Mar-2018

Correspondence Address:
Mohammad Mukhit Abdul Gaffar Kazi
Department of Microbiology, Sinhgad Dental College and Hospital, Vadgaon (Bk), Pune - 411 041, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/sjos.SJOralSci_12_17

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  Abstract 


Introduction: Chronic periodontitis is an oral disease and having multiple etiologies. The coinfection between herpesviruses and periodontopathic bacteria might play a crucial role in the increasing severity of the disease. The present study was conducted to find out any specific coinfection which is contributing in increase in the severity of the disease.
Materials and Methods: It was a prospective case–control study. A total of 300 cases with chronic periodontitis (100 each from mild, moderate, and severe chronic periodontitis) and 300 age- and sex-matched controls were included. After fulfilling the inclusion and exclusion criteria, a subgingival plaque specimen was collected and processed for detection of herpesviruses and periodontopathic microbiota.
Results: Herpesviruses were significantly associated with anaerobes and yeasts as compared to aerobes. In severe chronic periodontitis, association of anaerobes and herpesviruses was found to be statistically significant in the present study. All four studied herpesviruses have shown strong association with Porphyromonas gingivalis.
Conclusion: Coinfection helps in the increasing severity of chronic periodontitis when a particular combination of herpesviruses and periodontopathic microbiota is detected from the cases of chronic periodontitis. Herpes simplex virus-2 and P. gingivalis seem to play a crucial role in the increasing severity of chronic periodontitis as compared to other coinfection combinations in the studied populations.

Keywords: Chronic periodontitis, co-infection, herpesviruses, periodontopathic microbiota, severity


How to cite this article:
Gaffar Kazi MM, Bharadwaj R. The coinfection between herpesviruses and periodontopathic microbiota in increasing severity of chronic periodontitis. Saudi J Oral Sci 2018;5:47-53

How to cite this URL:
Gaffar Kazi MM, Bharadwaj R. The coinfection between herpesviruses and periodontopathic microbiota in increasing severity of chronic periodontitis. Saudi J Oral Sci [serial online] 2018 [cited 2018 Aug 15];5:47-53. Available from: http://www.saudijos.org/text.asp?2018/5/1/47/227127




  Introduction Top


Chronic periodontitis is an oral disease and having multiple etiologies.[1] It is initiated by complex subgingival microbiota including Gram-positive and Gram-negative bacteria, facultative and anaerobic organisms, and possibly yeasts.[2] The detection of bacterial pathogens alone cannot explain the pathogenesis of the disease.[3] Additional etiological agents such as herpesviruses were also depicted in the pathogenesis of the disease occurrence and progression.[4]

The coinfection between herpesviruses and periodontopathic bacteria might play a crucial role in the increasing severity of the disease.[5] The herpesvirus infection initiates inflammatory response in the gingival sulcus which leads to decrease in local immune response and increase in the attachment loss and pocket depth. This helps create anaerobic environment which is beneficial for propagation of anaerobic bacteria which ultimately cause more damage during interaction with host immune defense mechanisms leading to more destruction of periodontal tissue.[6] The present study was conducted to find out the coinfection between detected periodontopathic bacteria and herpesviruses in increasing the severity of the disease.


  Materials and Methods Top


It was a prospective case–control study. Ethical approval and informed consent were obtained before enrolling patients in the study. The total study period was from June 2011 to December 2014. A total of 300 cases with chronic periodontitis (100 each from mild, moderate and severe chronic periodontitis) and 300 age- and sex-matched controls were included. After fulfilling the inclusion and exclusion criteria, a subgingival plaque specimen was collected and transferred to Tris-EDTA (TE) buffer for the detection of herpesviruses by multiplex polymerase chain reaction (MPCR), brain–heart infusion (BHI) broth for aerobic and yeast detection, and Robertson's cooked-meat medium (RCM) for the detection of periodontopathic anaerobes. DNA extraction and MPCR were done as described earlier.[7] DNA extraction was done as follows: samples were vortexed to dislodge the plaque into the TE buffer medium (HiMedia) and then centrifuged at 5000 rpm for 5 min and supernatant was discarded. Then, 500 μl fresh TE buffer was added and centrifuged for 3–4 min. The procedure was repeated for 2–3 times using fresh buffer each time. Supernatant was discarded. Fifty microliters lysis buffer I (HiMedia) was added and vortexes for 5 min. Fifty microliters lysis buffer II (HiMedia) and 10 μl proteinase K (100 μg/ml) (Chromous Biotech, India) was added, and the mixture was vortexes vigorously. The tubes kept in water bath (60°C) for 2 h and then kept in boiling water bath for 10 min to deactivate the enzyme. Finally, all extracted samples were stored at −20°C. PCR master mix was prepared containing dNTPs (10 mM), PCR Taq polymerase buffer (10×), and Taq DNA polymerase (1.5 U/reaction) (Chromous Biotech, India). The primers (BioServe India Pvt. Ltd., India) used in the study were as follows:[3] Herpes simplex virus-1 (HSV-1) (Forward: 5'-CGTACCTGCGGCTCGTGAAGT-3' and Reverse: 5'-AGCAGGGTGCTCGTGTATGGGC-3'), HSV-2: Forward: 5'-TGGTATCGCATGGGAGACAAT-3' and Reverse: 5'CTCCGTCCAGTCGTTTATCTTG-3') cytomegalovirus (CMV): Forward: 5'-ACGTGTTACTGGCGGAGTCG-3' and Reverse:5'-TTGAGTGTGGCCAGACTGAG-3'), Epstein–Barr virus (EBV): Forward: 5'-AGCACTGGCCAGCTCATATC-3' and Reverse: 5'-TTGACGTCATGCCAAGGCAA-3'). The oligonucleotide primers were prepared at a concentration of 2.5 pm each in DEPC water. A premix was prepared and aliquoted. The premix contains the following components in a final volume of 50 μl/aliquot (25 μl PCR Master Mix (Chromous Biotech Ltd.), 1 μl of forward and reverse oligonucleotide primers (BioServe India Pvt. Ltd.) of each herpesviruses (HSV-1, HSV-2, CMV, and EBV), DEPC water adjusted to the final volume of 50 μl, 3 μl of sample DNA added to each tube and subjected to MPCR. MPCR conditions were as follows: initial denaturation at 95°C for 5 min, 45 cycles of denaturation at 95°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 30 s, and final extension was done at 72°C for 5 min. PCR products were detected by gel electrophoresis (2%).

A subgingival plaque specimen collected in BHI broth was used for cultivation of aerobes by platting on sheep blood agar and MacConkey agar as per standard method. RCM was used to cultivate anaerobes as per standard method.[8] The data were entered in an Excel sheet with results and were analyzed using SPSS software version 17.0 (USA) and Chi-square test with P < 0.001 being used to find out any significant associations between periodontopathic bacteria and herpesviruses.


  Results Top


Herpesviruses were significantly associated with anaerobes and yeasts as compared to aerobes [Table 1]. In severe chronic periodontitis, association of anaerobes and herpesviruses was found to be statistically significant in the present study [Table 2]. HSV-1 and Porphyromonas gingivalis coinfection was seen with an odds ratio (OR) 6.56 as compared to Bacteroides fragilis (3.99) and Veillonella parvula (2.31) [Table 3]. HSV-2 and P. gingivalis coinfection was seen with an OR 13.28 as compared to B. fragilis (4.98), V. parvula (3.03), Candida spp. (0.49), Staphylococcus aureus (0.25), and Enterococcus faecalis (1.88) [Table 4]. EBV and P. gingivalis coinfection was seen with an OR 5.23 as compared to B. fragilis (2.71) and V. parvula (2.16) [Table 5]. Similarly, human CMV (HCMV) and P. gingivalis coinfection was seen with an OR 2.91 [Table 6].
Table 1: Association between other organisms and herpesviruses in chronic periodontitis

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Table 2: Association between periodontopathic microbiota and herpesviruses in severity of chronic periodontitis

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Table 3: Association between herpes simplex virus-1 and periodontopathic microbiota

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Table 4: Association between herpes simplex virus-2 and periodontopathic microbiota

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Table 5: Association between Epstein-Barr virus and periodontopathic microbiota

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Table 6: Association between cytomegalovirus and periodontopathic microbiota

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  Discussion Top


Chronic periodontitis is known to be a polymicrobial disease. The coinfection of herpesviruses along with other periodontopathic microbiota could aggravate the disease. The coinfection between herpesviruses and anaerobes was found in 82.7% of patients with chronic periodontitis in the present study [Table 1]. Overall, herpesviruses and anaerobes were detected more significantly from severe type of chronic periodontitis than the mild and moderate [Table 2] suggesting their role in the increasing severity of the disease.

The coinfection between individual herpesviruses and periodontopathic microbiota was evaluated in the present study. HSV-1 was strongly associated with P. gingivalis with an OR 6.56 as compared to B. fragilis (3.99) and V. parvula (2.31) in the present study [Table 3]. Kamma et al.[9] have reported a strong association between HSV-1 and P. gingivalis in their study. HSV-2 was strongly associated with P. gingivalis with an OR 13.8 as compared to B. fragilis (4.98) and V. parvula (3.03) in the present study [Table 4]. The HSV-2 was seen to be involved in increasing the severity of disease in association with P. gingivalis. This is the first study as per our knowledge which strongly suggesting association between HSV-2 and P. gingivalis (OR 13.8).

EBV was strongly associated with P. gingivalis with an OR 5.23, B. fragilis (2.71) and V. parvula (2.16) in the present study [Table 5]. Kato et al.[10] have reported that coexistence of EBV and P. gingivalis was significantly higher in the deeper pocket depths. Contreras and Slots[11] have also reported association between EBV and P. gingivalis which increases the severity of the periodontitis in their study. Kamma et al.[9] have reported that periodontitis sites demonstrating any combination of HCMV, EBV-1, and HSV revealed significantly higher occurrence of P. gingivalis and Dialister pneumosintes coinfection than sites showing no viruses (P = 0.002). Saygun et al.[12] have reported correlations between counts of EBV and P. gingivalis and Tannerella forsythia in severe chronic periodontitis. Sugano et al.[13] findings suggested that the interaction between EBV and P. gingivalis is bidirectional, with EBV reactivation suppressing host defenses and permitting overgrowth of P. gingivalis, and P. gingivalis has the potential to induce EBV reactivation. However, Slots et al.[14] have reported no association between EBV and P. gingivalis in their study. They have reported that EBV may not be predictive of the presence of subgingival P. gingivalis.

HCMV was strongly associated with P. gingivalis (OR 2.91) in the present study [Table 6]. Botero et al.[15] have reported that the frequency of detection of P. gingivalis, Prevotella intermedia/Prevotella nigrescens, and Eikenella corrodens was increased in HCMV-positive periodontally diseased subjects compared to healthy subjects. Saygun et al.[12] have reported positive correlations between counts of HCMV and P. gingivalis, T. forsythia, and C. rectus in their study. Contreras and Slots[11] have reported association between HCMV and P. gingivalis which leads to increasing severity of the periodontitis. Slots et al.[14] have reported that association between HCMV and P. gingivalis leads to progression of periodontitis.

The coexistence of herpesvirus and periodontopathic microbiota seemingly leads to synergistic effects and aggravates the progress of periodontal disease. The mixed herpesvirus infections encourage subgingival colonization of periodontopathic microbiota by various mechanisms. Herpesvirus activation leads to suppression of periodontal immune mechanism resulting in overgrowth of periodontopathic microbiota that releases proinflammatory cytokines and chemokines. These interactions subsequently could result in breakdown of periodontal tissue, thereby increasing the severity of the disease. In addition, herpesvirus infection can lyse oral epithelial cells which can facilitate access for periodontopathic microbiota to deeper tissues and also create more binding sites.[5]

There was no significant association found between aerobes detected and herpesviruses and also in severity of the disease in the present study [Table 2]. However, when compared with individual herpesviruses and aerobic flora, it has revealed association with S. aureus and E. faecalis.

No association between HSV-1 and any of the isolated aerobes was found in the present study [Table 3]. HSV-2 was found to be associated with S. aureus with an OR 0.25 and E. faecalis (1.88) in the present study [Table 4]. These findings have been reported first time in the literature as per our knowledge. No association between EBV and any of the detected aerobes was found in the present study [Table 5]. No association between HCMV and any of the detected aerobes was found in the present study [Table 6].

In the present study, coinfection of Candida spp. with HSV-1 was seen [Table 3]. No other herpesviruses have shown any coinfection with Candida spp. in the present study. This is the first study reporting a strong association between Candida spp. and HSV-1 in patients with chronic periodontitis. HSV-2 and P. gingivalis seems to be one of the major coinfections found in the present study as compared to other herpesviruses and periodontopathic microbiota. This needs to be evaluated further for any specific characteristics sharing between coinfections.


  Conclusion Top


Coinfection helps in the increasing severity of chronic periodontitis when a particular combination of herpesviruses and periodontopathic microbiota is detected from the cases of chronic periodontitis. HSV-2 and P. gingivalis confection was seen predominantly as compared to other coinfection combinations in the studied populations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Newman MG, Socransky SS, Savitt ED, Propas DA, Crawford A. Studies of the microbiology of periodontosis. J Periodontol 1976;47:373-9.  Back to cited text no. 1
    
2.
Daniluk T, Tokajuk G, Cylwik-Rokicka D, Rozkiewicz D, Zaremba ML, Stokowska W. Aerobic and anaerobic bacteria in subgingival and supragingival plaques of adult patients with periodontal disease. Adv Med Sci 2006;51 Suppl 1:81-5.  Back to cited text no. 2
    
3.
Slots J. Herpesvirus periodontitis: Infection beyond biofilm. J Calif Dent Assoc 2011;39:393-9.  Back to cited text no. 3
    
4.
Slots J. Human viruses in periodontitis. Periodontol 2000 2010;53:89-110.  Back to cited text no. 4
    
5.
Sanghavi A, Dave D, Nadig P, Sanghavi T, Khanpara N. Human herpes virus: Bacteria and periodontium. J Oral Dis Markers 2016;1:1-5.  Back to cited text no. 5
    
6.
Ling LJ, Ho CC, Wu CY, Chen YT, Hung SL. Association between human herpesviruses and the severity of periodontitis. J Periodontol 2004;75:1479-85.  Back to cited text no. 6
    
7.
Shin CH, Park GS, Hong KM, Paik MK. Detection and typing of HSV-1, HSV-2, CMV and EBV by quadruplex PCR. Yonsei Med J 2003;44:1001-7.  Back to cited text no. 7
    
8.
Summanen P, Baron EJ, Citron DM, Strong C, Wexler HM, Finegold SM. Wadsworth Anaerobic Bacteriology Manual. 5th ed. Belmont, California: Star Publishing Company; 1993.  Back to cited text no. 8
    
9.
Kamma JJ, Contreras A, Slots J. Herpes viruses and periodontopathic bacteria in early-onset periodontitis. J Clin Periodontol 2001;28:879-85.  Back to cited text no. 9
    
10.
Kato A, Imai K, Ochiai K, Ogata Y. Higher prevalence of Epstein-Barr virus DNA in deeper periodontal pockets of chronic periodontitis in Japanese patients. PLoS One 2013;8:e71990.  Back to cited text no. 10
    
11.
Contreras A, Slots J. Mammalian viruses in human periodontitis. Oral Microbiol Immunol 1996;11:381-6.  Back to cited text no. 11
    
12.
Saygun I, Kubar A, Sahin S, Sener K, Slots J. Quantitative analysis of association between herpesviruses and bacterial pathogens in periodontitis. J Periodontal Res 2008;43:352-9.  Back to cited text no. 12
    
13.
Sugano N, Ikeda K, Oshikawa M, Idesawa M, Tanaka H, Sato S, et al. Relationship between Porphyromonas gingivalis, Epstein-Barr virus infection and reactivation in periodontitis. J Oral Sci 2004;46:203-6.  Back to cited text no. 13
    
14.
Slots J, Kamma JJ, Sugar C. The herpesvirus-Porphyromonas gingivalis-periodontitis axis. J Periodontal Res 2003;38:318-23.  Back to cited text no. 14
    
15.
Botero JE, Parra B, Jaramillo A, Contreras A. Subgingival human cytomegalovirus correlates with increased clinical periodontal parameters and bacterial coinfection in periodontitis. J Periodontol 2007;78:2303-10.  Back to cited text no. 15
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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