A total of 73 subjects enrolled and were confirmed eligible in the UNC HIV
Demonstration Project from 2008 to 2011; 73 were followed from baseline to 12 months, and 45 participants were followed from baseline to 24 months. All underwent consecutive periodontal measurements every six months. There were 54 men and 19 women (Table 1).
graphics, see Table 1. Of individuals, 45% were smokers. At baseline, the overall mean CD4 and HIV viral load (VL) were 517 cells/ml and 72 043 copies/ml, respectively. At baseline,
demographic values were collected and comprehensive oral examination with treatment planning was performed. Prophylaxis/debridement was carried out at baseline, 6, 12, 18, and 24 months (Table 2.) At baseline, 12 and 24 months, CD4 and HIV VL were obtained and whole un- stimulated saliva was collected for the measurement of IL-6. For the 73 participants over the course of the study, there were 293 dental prophylaxis/periodontal maintenance procedures, 19 scaling and root planing procedures, 33 periodontal debridements, 127 dental extractions, and 325 dental restorations placed.
Dental intervention in HIV-positive subjects resulted in periodontal disease
resolution Seventy-three HIV-positive subjects were classified according to AAP, CDC/AAP, and BGI periodontal disease classification systems (Table 3. and Figure 1).
AAP Classification- AAP measures periodontal disease based on clinical attachment levels, reflecting past disease status. The AAP classification at both baseline and post-
intervention categorized 87.6% of subjects as having mild periodontitis at both baseline and post- intervention at 12 months. Pre- and post-intervention differences were not statistically significant for the AAP classification (P = 0.38, extended McNemar test). Of subjects, 4% improved by 1 category and 6.8% decreased by 1 category. The perceived worsened disease status was likely due to gingival recession associated with disease resolution.
AAP/CDC classification- AAP/CDC measures periodontal disease based on clinical attachment levels and probing depth based on a subset of sites, and this metric estimates disease. Using the AAP/CDC classification, 51% were classified in the moderate periodontitis group at baseline. At 12 months post-treatment, 13.7% of subjects improved by one category and 20.5% of subjects improved by 2 categories. The inclusion of periodontal pocket depths provides a measure of current disease status. However, pre- and post-intervention differences in classification were not statistically significant for the AAP/CDC classification (P = 0.255, extended McNemar test).
BGI Classification- BGI measures periodontal disease based on bleeding on probing (BOP) values and probing depths, and this metric considers inflammation (BOP). The
biologically based classification, BGI, determined that 63% of the subjects had severe disease at baseline. Post- treatment at 12 months, only 1 subject remained in the severe disease category, with increasing number of subjects categorized less severe categories. At 12 months post- treatment, 56.2% of subjects improved by one category, 9.6% of subjects improved by two categories, and 9.6% of subjects improved by three categories. Thus, 54 patients showed
significant for the BGI classification (P < 0.0001, extended McNemar test). The improvement was likely due to the resolution of gingival inflammation. Compared to the AAP classification, the CDC/AAP and BGI classifications reflected current active disease and periodontal disease resolution over time (Figure 1).
In HIV-infected subjects, differences in age, smoking, and income were not associated with more severe periodontal disease BGI case classifications were used to assess the frequency of periodontal health, gingivitis, and disease in the context of demographic variables. High prevalence of severe periodontal disease in the HIV cohort was detected across all income levels and across all ages (Figure 2). An equivalent number of smokers and non-smokers had severe periodontal disease. Severe periodontal disease was detected in 70% of HIV-positive NH Blacks and 50% NH Whites. At baseline, severe periodontal disease was detected in 63% of men and in 63% of women with HIV infection. Similar percentages of men and women were detected in the moderate disease category.
Binary logistic regression analysis determined the likelihood of moderate/severe
periodontal disease based on CDC/AAP classification (Figure 2). Moderate/severe periodontitis was positively associated with White and non-White races, smoking and non-smoking, male and female gender, all income levels, and all age groups. The probability of disease development was over 50% for both women and men and for both smokers and non-smokers. The probability of disease development was over 60% across all age groups and across all income levels. The probability of disease development was at least 50% for Whites and over 60% for Blacks and Hispanics.
ART and HIV status were associated with severe periodontal disease Of the 73
individuals followed longitudinally for one year, at baseline 63 were on ART and 10 were not. Of those subjects on ART, 51 were on ART for at least 12 months at baseline. Of these
individuals, 28 demonstrated undetectable viral load (Figure 3a). There were 45 individuals followed for 24 months, of whom 39 were on ART for at least 12 months at baseline. Of these individuals, 25 demonstrated viral suppression with undetectable viral load at baseline (Figure 3b).
Regardless of ART status, over 80% of subjects had moderate/severe disease as determined by BGI classification (n = 73). BGI classification detected severe periodontal disease in 55% of those with undetectable viral loads (n = 28) and 75% of subjects on short-term ART (n = 12) Table 4. Interestingly, McNemar analysis did not detect statistically significant improvement in BGI classification using tests in the absence of ART (n = 10, P = 0.63), on short-term ART (n = 12, P = 0.53), or on long-term ART who were not suppressed (n = 21, P = 0.09). This lack of statistical association may be related to the small size of the groups. However, in those on long- term ART who were suppressed, a statistically significant improvement in BGI was detected (n = 28, P = 0.026) Table 5b. At least fifty percent of subjects in each of the four groups demonstrated at least one category of periodontal improvement (Table 5a).
At baseline, the log median CD4 counts were similar across the groups ranged from 437 to 469 cells/ml in those subjects who were on ART and 568 cells/ml in those who were not on ART. In the group of 45 participants at 24 months, all ART groups demonstrated statistically significant increases in CD4 count (n = 45, P = 0.007 to P = 0.01). At 24 months, median CD4 counts in the group not on ART were 792 cells/ml, on short-term ART were 655 cells/ml, in the long-term ART-not-suppressed group were 783 cells/ml, and in the long-term ART-sup- pressed group were 647 cells/ml (Figure 4a). In those individuals on no ART, short-term ART, or on long-term ART who were not suppressed, differences in BGI were not significantly associated with changes in CD4 count at 24 months. However, in those who were in the long-term ART- suppressed group, improved BGI/ periodontal status was associated with improved CD4 (P = 0.0436). In the long-term ART-suppressed group, univariate analysis did not reach a statistical significance at 12 months (P = 0.058); however, by 24 months, a statistically significant association was detected between improved BGI and increased CD4 counts (P = 0.01). Of interest, in the group of subjects that were on long-term ART and not suppressed, univariate analysis detected highly statistically significant drops in viral load at both 12 months (P < 0.0002) and 24 months (P = 0.0052) (Figure 4b).
Salivary pro-inflammatory cytokine levels were associated with an increased risk of developing moderate/severe periodontal disease The pro-inflammatory cytokine IL-6 was measured in the oral fluids from HIV-positive individuals (n = 26) (Figure 5). Age-adjusted predicted probabilities (95% CI) of moderate/severe periodontitis were determined. Those individuals with the highest IL-6 levels had a 60% probability of moderate/severe periodontal disease development at baseline (Figure 5a). Importantly, dental intervention resulted in overall decreased salivary IL-6 levels for all diseased groups, with the moderate group, P2, showing a
significant decrease from baseline to 24 months (P = 0.043) as determined by the Wilcoxon signed rank test (Figure 5b). At 12 months, IL-6 levels were assessed across ART groups. Mean IL-6 levels were highest at baseline in the long-term virologically suppressed group and
demonstrated a significant decrease from baseline to 12 months (P = 0.031, Wilcoxon signed rank test) (Figure 5c).
No ART Short
Term LongTermSuppressed Long TermNot Suppressed
0 12 24 0 12 24 0 12 24 0 12 24 Months A. B. 0.0 20,000 40,000 60,000 80,000 100,000 120,000 No ART Short
Term LongTermSuppressed
Long Term Not Suppressed 0 12 24 0 12 24 0 12 24 0 12 24 Months p=ns p=ns p=0.005 p=0.0002 p=0.039 p=0.015 0 200 400 600 800 1000 1200 CD 4 c el ls /m l HI V V L (c opi es /m l) p=ns p=0.007 p=0.01 p=0.01 p=0.087
No ART Short Term 20 140 40 p=0.031 Healthy Gingivitis Moderate Severe 0 20 60 p=0.043 0.0 0.2 0.4 0.6 0.8 1.0
Low Moderate High
40 o A. B. C. Baseline 12 Months 24 Months LongTerm Suppressed Long Term Not Suppressed 0 12 0 12 0 12 0 12 Months 60 80 100 120 IL 6 pg/ m l IL 6 pg/ m l IL6 pg/ml A ge A dju ste d Pr oba bl ilt y of M ode ra te /S ev er e P er io do n ti ti s (9 5 %CI )
Discussion
Oral microbial load and associated inflammation may influence both local and systemic disease outcomes. In a group of HIV-positive subjects who were virologically suppressed at baseline, dental intervention was associated with decreased periodontitis, increased CD4 counts (P = 0.023) and decreased IL-6 (P = 0.03). While the data presented in this study do not remove the possibility that effective ART was responsible for viral load and CD4 count change in the other ART groups, it does suggest that decreased oral infection and inflammation were associated with improved HIV metrics.
Inflammation is an important driver of both periodontal disease and HIV progression. Our observation of the utility of classification systems that include a measure of inflammation contributes to this literature. Increasing evidence suggests that the chronic periodontal infection is implicated in the generation of a systemic inflammatory response, which represents a potential risk factor for worsening systemic conditions87,88. Use of a periodontal classification system that reflects the biology of disease is an important metric in those with systemic inflammation- associated disorders. This was well illustrated in this study of HIV infected subjects with gingival inflammation.
Of 73 individuals in our population, 63 were on ART and significant inflammation was detected that resolved with dental intervention and aggressive oral hygiene. Distinct profiles of periodontal disease and disease resolution were detected comparing different periodontal
classification systems. While AAP provided an important glimpse of historical disease based on attachment levels, inclusion of probing depths in the CDC/AAP classification facilitated
oral and periodontal inflammation. With BGI, the majority of subjects were classified as severe at baseline. In previous studies, bleeding on probing was twice as high in an antiretroviral- untreated group compared to those on ART98. In our study regardless of ART status, 80% of subjects had moderate/severe disease at baseline using a classification system that includes current inflammation. Our findings were similar to the findings of John et al in a South African population where HIV stage and ART were not associated with higher levels of periodontal disease in HIV-positive subjects. In the South African group inclusion of a gingival index of inflammation reversed the significant association between antiretroviral therapy, probing depth and clinical attachment loss99. Of all classification systems, BGI, the inflammation-based classification system, demonstrated the most significant shifts in category associated with the dental intervention, moving virtually all individuals of the severe category into a moderate disease group by 12 months. These findings demonstrate the importance of including
inflammation as a disease indicator. The periodontal field has now recognized the importance of this indicator, and a recent task force was convened to address the addition of bleeding on probing to the AAP classification to begin 2017100.
In a Nationally representative sample, NHANES 2009- 2012, moderate/severe
periodontitis was positively associated with non-white race, aging, smoking, male gender, and low income86. In our study of HIV positive individuals, frequency of periodontitis was high across all demographic variables, including gender, age and income. Interestingly, in our study there was not a significant difference with regard to smoking status. HIV may be a more important driver of the oral inflammatory process than the traditional demographic risk factors typically associated with periodontal disease in a nationally representative sample.
group who was not suppressed at baseline. The presence of detectable viral loads, however, may signify ineffective ART. Given that there were individuals with detectable viral load on ART, the improvement in HIV status could have been related to more effective ART use. At baseline, 50% of individuals on long-term ART in the severe disease group were undetectable, and by 24 months, two-thirds of subjects in this group were undetectable. The dental intervention decreased oral microbial load and was associated with decreased low-level HIV viremia. Detectable HIV viral load has been associated with the presence of oral pathogens. A recent Brazilian study determined that detectable HIV VL was associated with elevated levels of known periodontal pathogens, such as P. nigrescens, T. forsythia, and E. corrodens101. There is also the potential for a direct pathogen–pathogen relationship, as others and we have shown that periodontal bacterial end products can increase HIV replication102.
Periodontal disease is associated with circulating microbial products. Bacterial translocation into systemic circulation from the periodontal pocket is a common event, as supported by the detection of bacteremia subsequent to relatively minor periodontal events and procedures. The massive bacterial load of the gut is thought to drive microbial translocation, causing HIV-related systemic immune activation93. Importantly, oral antigens have been shown to facilitate trafficking of activated oral antigen-specific intestinal T-cell responses through CD18103. Hence, periodontal antigens may facilitate intestinal immune activation. We posit that the mouth contributes to microbial translocation in HIV- associated systemic immune activation. While periodontitis does not cause atherosclerotic vascular disease, periodontal interventions do result in reduced systemic inflammation104. Statistically significant improvement in BGI
classification was detected in individuals who were virologically suppressed, suggesting
to the study size of this convenience population, particularly the small sample size of the
virologically suppressed group. This does limit generalizability. However, this population and its distribution are similar to the demographic distribution of the HIV epidemic in the southeastern US. Despite the limitations, statistically significant changes were detected in the virologically suppressed group. Another limitation was retention rate at 24 months; baseline to 12 months there were 73 subjects followed longitudinally, at 24 months there were only 45 subjects seen at all 5 visits. Importantly, despite these limitations, in every group with detectable HIV VL at baseline, HIV VL was reduced with the intervention.
It has previously been shown that the achievement of undetectable HIV VL was
associated with decreased risk of comorbid events and strongly associated with increased CD4 cells105. Low CD4 counts have previously been associated with chronic periodontitis in cross- sectional studies106. In this study, a median sustained increase in CD4 count of over 100 cells/ml
was detected with the dental intervention in a group of subjects who were suppressed at baseline. This suggests that over and above ART, dental interventions that diminish the oral microbial reservoir may provide a significant benefit to the immune system.
Periodontal interventions in HIV significantly reduced periodontal inflammation that may be associated with systemic inflammation. Additional studies of systemic immune activation markers and periodontal disease resolution are needed. Here, we describe a simple and relatively inexpensive dental intervention that achieved decreased oral IL-6 and increased CD4 counts in a subset of individuals on effective ART.