J Clin Periodontol 2014; 41: 1061–1068 doi: 10.1111/jcpe.12306
Levels of HIV-1 in subgingival biofilm of HIV-infected patients Pavan P, Pereira VT, Souza RC, Souza CO, Torres SR, Colombo APV, da Costa LJ, Sansone C, de Uzeda M, Goncßalves LS. Levels of HIV-1 in subgingival biofilm of HIV-infected patients. J Clin Periodontol 2014; 41: 1061–1068. doi: 10.1111/ jcpe.12306
Abstract Aim: The aims of the current study were to compare the levels of HIV-1 in the subgingival biofilm (SHVL) between detectable and undetectable plasmatic HIV-1 viral load (PHVL) in HIV-infected patients as well as to determine the association of SHVL with PHVL and clinical periodontal parameters. Material and Methods: Forty-one HIV-infected individuals were divided into two groups: detectable (21) and undetectable (20) PHVL. Subgingival biofilm samples were obtained for detection and quantification of HIV-1 by real-time RT-PCR. To estimate the effect of co-variables on the outcome undetectable SHVL, the Generalized Estimation Equation (GEE) was employed. Results: Detectable SHVL was observed only in the detectable PHVL group and the detection of the HIV-1 was observed in 40% of these individuals. In the bivariate analysis between co-variables from the individual level and the outcome SHVL, significant difference was observed only for the CD4+ T lymphocytes levels (p = 0.017). The multiple logistic model demonstrated that only CD4+ T lymphocytes levels had a significant effect on the outcome undetectable SHVL [OR 8.85 (CI 3.6–9.2), p = 0.002]. Conclusion: HIV-1 can be detected and quantified in the subgingival biofilm of HIV-infected individuals, but these findings are not associated with PHVL and periodontal clinical parameters.
Acquired immunodeficiency syndrome (AIDS) is a disease caused by the human immunodeficiency virus type 1 (HIV-1), which has tropism mainly for CD4+ T lymphocytes and macrophages. This disease is characterized by severe immunodeficiency and manifestation of several opportunistic infections (Chermann et al. 1983). HIV-1 can be isolated from many body fluids, excretions and secretions (Hadley 1989). Nevertheless, it has been demonstrated that Conflict of interest and source of funding statement The authors declare that they have no conflict of interests.
only blood, semen, vaginal secretion and maternal milk are considered sources of transmission (Schacker et al. 1996). This virus can also be found in saliva, gingival crevicular fluid, and cerebrospinal fluid (Maticic et al. 2000, Shugars et al. 2001, Cinque et al. 2003, Navazesh et al. 2010). Even though some studies have identified the HIV-1 in saliva (Groopman et al. 1984, Ho et al. 1985, Levy & Greenspan 1988, Barr et al. 1992), the mouth is rarely considered a source of HIV-1 transmission (Maticic et al. 2000). Navazesh et al. (2010) demonstrated an association between plasmatic HIV-1 viral load (PHVL) and HIV-1 viral load in the saliva of
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Priscila Pavan1, Viviane Tiago Pereira1, Rodrigo Carvalho Souza2, Celso Oliveira Souza1, Sandra Regina Torres3, Ana Paula Vieira Colombo4, Luciana Jesus da Costa4, Carmelo Sansone1, Milton de Uzeda2 and Lucio Souza Gonc ß alves2 1
Department of Dental Clinics, Division of Graduate Periodontics, School of Dentistry, Rio de Janeiro, Brazil; 2Dental School, cio de Sa University, Rio de Janeiro, Esta Brazil; 3Department of Oral Pathology and Diagnosis, School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 4Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Key words: chronic periodontitis; HIV infection; viral load; subgingival biofilm; HAART Accepted for publication 3 September 2014
HIV-infected women. Similar results were observed by other authors (Liuzzi et al. 1996, Shugars et al. 1999, 2001), reinforcing the idea that saliva can be a useful and non-invasive way to estimate PHVL (Shugars et al. 1999). This significant association has also been found between PHVL and viral load in gingival crevicular fluid (GCF) (Maticic et al. 2000). In the GCF, viral particles of the HIV-1 are found mainly in advanced HIV infections (Maticic et al. 2000). The infection of periodontal tissues by HIV-1 may be considered a cofactor for local tissue destruction (Parra & Slots 1996, Contreras et al. 1999). Thus, in the subgingival peri-
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odontal sites, the presence of the HIV-1 may play a role in the pathogenesis of periodontal diseases in HIV-infected individuals (Ryder 2002). The destructive effect of HIV1 on periodontal tissues occurs due to suppression of CD4+ T lymphocytes and/or due extensive B-cell dysfunction that impairs the quality of the humoral response, leading to an imbalance in the host response (Shirai et al. 1992, Shen & Tomaras 2011). However, the state of B-cell activation found in HIV-infected individuals is comparable to that detected in patients with autoimmune disease (Dar et al. 1988). This polyclonal activation is associated with both the production of autoantibodies and the suppression of immune responses to foreign antigens (Shirai et al. 1992). In the periodontal pocket, the polyclonal B-cell activator may be associated with the production of anti-collagen antibody (Sugawara et al. 1992). The presence of HIV in the subgingival site may play a role in the collagen destruction of the periodontal support tissue, because HIV-1 may be responsible for the polyclonal B-cell activation (Shen & Tomaras 2011), and these cells transform into plasma cell and produce anti-collagen IgG antibody (Sugawara et al. 1992, Nair et al. 2014). Oral lesions are frequently observed in HIV-infected patients, and are considered markers of disease progression and immunosuppression (Coogan et al. 2005). Approximately 40–50% of the HIVinfected patients manifest some fungal, bacterial or viral oral infection (Reznik 2005). Clinical studies have demonstrated a positive correlation between PHVL and some oral infectious diseases, such as periodontal diseases, hairy leukoplakia and candidiasis (Black et al. 2000, Patton 2000, Alpagot et al. 2004, 2007, Ramirez-Amador et al. 2005). In addition, It has been hypothesized that, oral infection, such as infections by periodontal pathogens, can stimulate HIV-1 recrudescence of HIV-latently infected cells, with potential failure of highly active antiretroviral therapy (HAART) and consequent increase in PHVL (Gonzalez et al. 2009, Jordan 2013). Thus, it has been demonstrated that detectable PHVL in HIV-infected
patients is associated with elevated levels of known periodontal pathogens such as Prevotella nigrescens, Tannerella forsythia, and Eikenella corrodens, as well as Campylobacter concisus, Capnocytophaga gingivalis, and Dialister pneumosintes in the subgingival biofilm, (Pereira et al. 2014), and that these associations are not related with CD4+ T lymphocytes levels (John et al. 2012, Pereira et al. 2014). The oral environment may be a favourable site to the pathogenesis of the HIV-1, and also a probable reservoir for this virus, as the viral RNA and proviral DNA have been detected in the saliva and in the GCF (Maticic et al. 2000, Shugars et al. 2001). In addition, it has been demonstrated that oral epithelial cells and Langherans cells may be a reservoir of HIV-1 in the oral mucosa (Herzberg et al. 2011). Nevertheless, the presence of components that inactivate the HIV-1, the reduced salivary HIV-1 viral load, and the presence of specific antibody against HIV-1 may account for the decreased virion infectivity in saliva (Mcneely et al. 1995, Shugars et al. 1999, Kim et al. 2006). However, only one study about HIV-1 in the subgingival environment is available (Maticic et al. 2000). Therefore, the aims of the current study were to compare the levels of HIV-1 in the subgingival biofilm (SHVL) between detectable and undetectable PHVL in HIV-infected patients, as well as to determine the association of SHVL with PHVL and clinical periodontal parameters. Material and Methods Subject population
Forty-one HIV-infected subjects attending the University Hospital Clementino Fraga Filho (HUCFF) at the Federal University of Rio de Janeiro (UFRJ) between 2010 and 2011 were selected. The study protocol was approved by the Review Committee for Human Subjects of HUCFF. All subjects were informed about the aims, risks and benefits of the study and signed a consent form. Patients were >20 years of age and presented at least 15 teeth. Exclusion criteria included the necessity of antibiotic prophylaxis for dental
procedures, pregnancy, diabetes, autoimmune diseases and necrotizing periodontal diseases. The subjects were distributed into two groups: 20 patients with detectable (50 copies/ mL) and 21 patients with undetectable PHVL. Clinical evaluation
Subjects were submitted to anamnesis questionnaire, and data about gender, age, ethnicity, education, income, smoking, alcohol consumption, drug use and means of HIV transmission were obtained. The history of AIDS-defining opportunistic infections, CD4+ T lymphocyte counts, HIV viral load and antiretroviral therapy were obtained from patients’ medical records. Oral examination included visual inspection of the oral mucosa and periodontal evaluation. Periodontal measurements were recorded at 6 sites per tooth (distobuccal, buccal, mesiobuccal, distolingual, lingual, mesiolingual) in all teeth, excluding third molars, and included probing depth (PD), clinical attachment level (CAL), visible supragingival biofilm (VSB) (presence/absence) and bleeding on probing (BOP). These measurements were performed by one calibrated examiner (Intraclass Correlation Coefficient of 0.96 and 0.97 for PD and CAL respectively), using conventional manual periodontal probe (PCP UNC 15; Hu-Friedy, Chicago, IL, USA). Chronic periodontitis (CP) was defined as the presence of >10% of teeth with PD and/or CAL ≥5 mm and BOP. Periodontal health (PH) was considered when ≤10% of sites with BOP, no PD or CAL >3 mm, although PD or CAL = 4 mm in up to 5% of the sites without BOP was allowed. After clinical examination, patients with evidence of destructive periodontal disease received full-mouth scaling and root planing under local anaesthesia and oral hygiene instruction. Immunologic assessment
In all HIV-infected subjects, laboratory analyses of CD4+ T lymphocyte, CD8+ T lymphocyte, and neutrophil levels in peripheral blood were routinely performed every 4 months for all patients at the
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
HIV-1 in subgingival biofilm HUCFF/UFRJ. For comparison purposes, the laboratory data of each patient were obtained within the same week that the periodontal clinical examination was performed. Detection and quantification of HIV-1 in the subgingival biofilm
Sample collection Subgingival biofilm samples were sampled from 12 sites of each subject with CP (6 sites with the deepest PD and 6 with PD 0–3 mm), and 6 randomly selected healthy sites from subjects with periodontal health, using sterile Gracey curettes (HuFriedy, Chicago, IL, USA). The samples were placed in individual 1.5 mL tubes containing TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.6) and stored at 80°C. These samples were used for detection and quantification of HIV-1 by real-time RT-PCR. Nucleic acid extraction and cDNA synthesis The RNA was extracted from a pool of subgingival biofilm samples (samples from two different periodontal sites were combined in 140 lL of TE buffer to generate a pool) using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions. The synthesis of the cDNA was performed with 10 lM of random oligomers using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s instructions. Real-time quantitative RT-PCR cDNAs were amplified using the TaqManâ Universal Master Mix amplification kit (Roche Molecular System, Branchburg, NJ, USA). Primers and probe were designed to a conserved region within the HIV-1 LTR: sense SSF1 (50 -GCT AAC TAG GGA ACC CAC TGC TT-30 ), anti-sense SSR1 (50 -CAA CAG ACG GGC ACA CAC TAC T-30 ), probe SS (50 FAM-AGC CTC AAT AAA GCT TGC CTT GAG TGC TTC-BHQ1-30 ) (Aguiar et al. 2007). Briefly, 5 lL of RNA was added into 12.5 lL amplification mix containing 2.5 lM of each primer, 5 lM of probe and RNAse-free water to a 25 lL final volume.
Thermal cycling programme was as follows: 50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min. Cycling was performed in ABI 7500 sequence detection system (Applied Biosystems). RNAse-free water was used as negative template control (NTC). Serial dilutions of the pNL4-3 plasmid bearing the full-length HIV-1 subtype B genome were used as positive external quantitation controls. An aliquot of 5 lL of 101–1010 copies of the pNL4-3 DNA was added to 20 lL of the qPCR mixture prepared as described above and submitted to the same cycling conditions as the test samples. The standard curve was generated each time a qPCR reaction was performed. An obtained Cycle threshold (Ct)-value was valid if the NTC was undetectable and if Ct values of the positive controls were in the range of mean two times the standard deviation (STD). Data analysis
All statistical tests were performed using Statistical Package for the Social Sciences (SPSS) software, version 19.0 (IBM, S~ ao Paulo, SP, Brazil). Descriptive analysis included socio-demographic characteristics, HAART use, HIV-related diseases, specific antiretroviral drugs, smoking, alcohol consumption and immunological data. Full-mouth periodontal clinical measurements were averaged for each patient and then within each group, and presented as mean of PD and CAL and the percentage of sites with BOP and VSB. Continuous variables were compared using Mann– Whitney test, and categorical variables were analysed using Fisher’s exact test. In order to estimate the effect of co-variables on the outcome undetectable subgingival HIV-1 viral load (SHVL), the multiple logistic method Generalized Estimation Equation (GEE) was employed. This model was used to consider the clustered nature of the probing depth’s data. For this analysis, only co-variables with p < 0.05 in the comparison between detectable and undetectable PHVL were included. The level of significance for all analyses was 5%.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
1063
Results Sociodemographic, clinical, health-related behaviour and immunological features of the studied sample
Of the 41 subjects who participated in this study, 51.2% were males, 39% were white and 50% were over 41 years of age. The majority of the participants reported no use of drugs (85.4%), while a few were current smokers (14.6%) and alcohol consumers (12.2%). The time of exposure to HIV was greater than 10 years in 68.3% of the subjects, and 51.2% had CP. Diarrhoea (63.6%), recurrent oral ulcers (70%), oral herpes simplex (31.7%), and pneumocystis pneumonia (31.7%) were the most frequent clinical conditions reported by these subjects (data not shown). Regarding antiretroviral therapy, 65.9% of the subjects were undergoing HAART, and the most frequently used antiretrovirals were the nucleoside reverse transcriptase inhibitors (NRTIs), such as lamivudine (70.7%), tenofovir (57.5%) and zidovudine (37.5%). In addition to the antiretroviral therapy, 15% were taking sulfamethoxazole plus trimethoprim as prophylaxis for Pneumocystis jirovecii pneumonitis (data not shown). There were no statistical differences regarding socio-demographic characteristics (gender, age, ethnicity, education, income) and healthrelated behaviour (smoking, alcohol consumption, drug use) between the two groups (detectable and undetectable PHVL) (data not shown). When HIV-infection related aspects were compared between the undetectable and detectable PHVL groups, only the use of HAART (p < 0.001) and SHVL detection (p = 0.001) were significantly different between them (Fisher’s exact test) (Table 1). Regarding laboratory data, CD4+ T lymphocytes levels were significantly higher in the undetectable PHVL group than in the detectable PHVL group (p = 0.038, Mann–Whitney test) (Table 2). Table 3 summarizes the periodontal clinical parameters of the sampled population. No significant differences were found between detectable and undetectable PHVL groups regarding the frequency of
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Table 1. Comparison of HIV-infection related aspects between undetectable and detectable PHVL groups Characteristics
Undetectable PHVL (N = 21)
N (%) Time of exposure to HIV 1–10 years 11–20 years ≥21 years HAART* Yes No SHVL Undetectable Detectable
Detectable PHVL (N = 20)
p value Microbiological data
5 (23.8) 16 (76.2) 0 (0.0)
8 (40.0) 9 (45.0) 3 (15.0)
0.060
20 (95.2) 1 (4.8)
7 (36.8) 12 (63.2)
0.05, Mann– Whitney test) (Table 4).
odontal health showed statistically significant differences for PD, CAL and BOL (p < 0.05), while no significant differences were observed for VSB, PHVL (undetectable and detectable) and SHVL (undetectable
HIV-1 viral load was detected and quantified in the subgingival biofilm samples by RT-PCR. The sensibility of the assay was first evaluated and was in the range of 10-100 copies of HIV-1 RNA. In order to confirm that the virus was detected in the subgingival biofilm instead of the provirus in the mononuclear cells from the blood, a RT-PCR was performed on positive samples, without c-DNA reaction. All these samples were negative, suggesting that the detection of the HIV-1 was from the subgingival biofilm. Detectable SHVL was observed only in the detectable PHVL group (20 patients) and the detection of the HIV-1 was found in 8 (40%) of these patients (Table 1). A bivariate analysis was performed between variables that showed significant differences between the undetectable and detectable PHVL groups (CD4+ T lymphocytes levels, HAART, and mean PD), PHVL (stratified into two levels: 0–3 log and > 3 log) and the outcome SHVL (Table 5). A statistically significant difference was observed only for the co-variable TCD4+ lymphocytes levels (p = 0.017, Fisher’s exact test). The results of the multiple logistic model employed to estimate the effect of the previously used co-variables in the bivariate analysis (Table 5) on the outcome undetectable SHVL are presented in Table 6. In this model, all measurements of PD from the 372 sampled sites were included. The only co-variable that demonstrated a significant effect on the outcome was CD4+ T lymphocytes levels. The interpretation of this finding is that patients with CD4+ T lymphocytes levels ≥500 cells/mm3 have odds eight times higher to present undetectable SHVL than those with CD4+ T lymphocytes levels
Levels of HIV-1 in subgingival biofilm of HIV-infected patients Pavan P, Pereira VT, Souza RC, Souza CO, Torres SR, Colombo APV, da Costa LJ, Sansone C, de Uzeda M, Goncßalves LS. Levels of HIV-1 in subgingival biofilm of HIV-infected patients. J Clin Periodontol 2014; 41: 1061–1068. doi: 10.1111/ jcpe.12306
Abstract Aim: The aims of the current study were to compare the levels of HIV-1 in the subgingival biofilm (SHVL) between detectable and undetectable plasmatic HIV-1 viral load (PHVL) in HIV-infected patients as well as to determine the association of SHVL with PHVL and clinical periodontal parameters. Material and Methods: Forty-one HIV-infected individuals were divided into two groups: detectable (21) and undetectable (20) PHVL. Subgingival biofilm samples were obtained for detection and quantification of HIV-1 by real-time RT-PCR. To estimate the effect of co-variables on the outcome undetectable SHVL, the Generalized Estimation Equation (GEE) was employed. Results: Detectable SHVL was observed only in the detectable PHVL group and the detection of the HIV-1 was observed in 40% of these individuals. In the bivariate analysis between co-variables from the individual level and the outcome SHVL, significant difference was observed only for the CD4+ T lymphocytes levels (p = 0.017). The multiple logistic model demonstrated that only CD4+ T lymphocytes levels had a significant effect on the outcome undetectable SHVL [OR 8.85 (CI 3.6–9.2), p = 0.002]. Conclusion: HIV-1 can be detected and quantified in the subgingival biofilm of HIV-infected individuals, but these findings are not associated with PHVL and periodontal clinical parameters.
Acquired immunodeficiency syndrome (AIDS) is a disease caused by the human immunodeficiency virus type 1 (HIV-1), which has tropism mainly for CD4+ T lymphocytes and macrophages. This disease is characterized by severe immunodeficiency and manifestation of several opportunistic infections (Chermann et al. 1983). HIV-1 can be isolated from many body fluids, excretions and secretions (Hadley 1989). Nevertheless, it has been demonstrated that Conflict of interest and source of funding statement The authors declare that they have no conflict of interests.
only blood, semen, vaginal secretion and maternal milk are considered sources of transmission (Schacker et al. 1996). This virus can also be found in saliva, gingival crevicular fluid, and cerebrospinal fluid (Maticic et al. 2000, Shugars et al. 2001, Cinque et al. 2003, Navazesh et al. 2010). Even though some studies have identified the HIV-1 in saliva (Groopman et al. 1984, Ho et al. 1985, Levy & Greenspan 1988, Barr et al. 1992), the mouth is rarely considered a source of HIV-1 transmission (Maticic et al. 2000). Navazesh et al. (2010) demonstrated an association between plasmatic HIV-1 viral load (PHVL) and HIV-1 viral load in the saliva of
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Priscila Pavan1, Viviane Tiago Pereira1, Rodrigo Carvalho Souza2, Celso Oliveira Souza1, Sandra Regina Torres3, Ana Paula Vieira Colombo4, Luciana Jesus da Costa4, Carmelo Sansone1, Milton de Uzeda2 and Lucio Souza Gonc ß alves2 1
Department of Dental Clinics, Division of Graduate Periodontics, School of Dentistry, Rio de Janeiro, Brazil; 2Dental School, cio de Sa University, Rio de Janeiro, Esta Brazil; 3Department of Oral Pathology and Diagnosis, School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 4Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Key words: chronic periodontitis; HIV infection; viral load; subgingival biofilm; HAART Accepted for publication 3 September 2014
HIV-infected women. Similar results were observed by other authors (Liuzzi et al. 1996, Shugars et al. 1999, 2001), reinforcing the idea that saliva can be a useful and non-invasive way to estimate PHVL (Shugars et al. 1999). This significant association has also been found between PHVL and viral load in gingival crevicular fluid (GCF) (Maticic et al. 2000). In the GCF, viral particles of the HIV-1 are found mainly in advanced HIV infections (Maticic et al. 2000). The infection of periodontal tissues by HIV-1 may be considered a cofactor for local tissue destruction (Parra & Slots 1996, Contreras et al. 1999). Thus, in the subgingival peri-
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odontal sites, the presence of the HIV-1 may play a role in the pathogenesis of periodontal diseases in HIV-infected individuals (Ryder 2002). The destructive effect of HIV1 on periodontal tissues occurs due to suppression of CD4+ T lymphocytes and/or due extensive B-cell dysfunction that impairs the quality of the humoral response, leading to an imbalance in the host response (Shirai et al. 1992, Shen & Tomaras 2011). However, the state of B-cell activation found in HIV-infected individuals is comparable to that detected in patients with autoimmune disease (Dar et al. 1988). This polyclonal activation is associated with both the production of autoantibodies and the suppression of immune responses to foreign antigens (Shirai et al. 1992). In the periodontal pocket, the polyclonal B-cell activator may be associated with the production of anti-collagen antibody (Sugawara et al. 1992). The presence of HIV in the subgingival site may play a role in the collagen destruction of the periodontal support tissue, because HIV-1 may be responsible for the polyclonal B-cell activation (Shen & Tomaras 2011), and these cells transform into plasma cell and produce anti-collagen IgG antibody (Sugawara et al. 1992, Nair et al. 2014). Oral lesions are frequently observed in HIV-infected patients, and are considered markers of disease progression and immunosuppression (Coogan et al. 2005). Approximately 40–50% of the HIVinfected patients manifest some fungal, bacterial or viral oral infection (Reznik 2005). Clinical studies have demonstrated a positive correlation between PHVL and some oral infectious diseases, such as periodontal diseases, hairy leukoplakia and candidiasis (Black et al. 2000, Patton 2000, Alpagot et al. 2004, 2007, Ramirez-Amador et al. 2005). In addition, It has been hypothesized that, oral infection, such as infections by periodontal pathogens, can stimulate HIV-1 recrudescence of HIV-latently infected cells, with potential failure of highly active antiretroviral therapy (HAART) and consequent increase in PHVL (Gonzalez et al. 2009, Jordan 2013). Thus, it has been demonstrated that detectable PHVL in HIV-infected
patients is associated with elevated levels of known periodontal pathogens such as Prevotella nigrescens, Tannerella forsythia, and Eikenella corrodens, as well as Campylobacter concisus, Capnocytophaga gingivalis, and Dialister pneumosintes in the subgingival biofilm, (Pereira et al. 2014), and that these associations are not related with CD4+ T lymphocytes levels (John et al. 2012, Pereira et al. 2014). The oral environment may be a favourable site to the pathogenesis of the HIV-1, and also a probable reservoir for this virus, as the viral RNA and proviral DNA have been detected in the saliva and in the GCF (Maticic et al. 2000, Shugars et al. 2001). In addition, it has been demonstrated that oral epithelial cells and Langherans cells may be a reservoir of HIV-1 in the oral mucosa (Herzberg et al. 2011). Nevertheless, the presence of components that inactivate the HIV-1, the reduced salivary HIV-1 viral load, and the presence of specific antibody against HIV-1 may account for the decreased virion infectivity in saliva (Mcneely et al. 1995, Shugars et al. 1999, Kim et al. 2006). However, only one study about HIV-1 in the subgingival environment is available (Maticic et al. 2000). Therefore, the aims of the current study were to compare the levels of HIV-1 in the subgingival biofilm (SHVL) between detectable and undetectable PHVL in HIV-infected patients, as well as to determine the association of SHVL with PHVL and clinical periodontal parameters. Material and Methods Subject population
Forty-one HIV-infected subjects attending the University Hospital Clementino Fraga Filho (HUCFF) at the Federal University of Rio de Janeiro (UFRJ) between 2010 and 2011 were selected. The study protocol was approved by the Review Committee for Human Subjects of HUCFF. All subjects were informed about the aims, risks and benefits of the study and signed a consent form. Patients were >20 years of age and presented at least 15 teeth. Exclusion criteria included the necessity of antibiotic prophylaxis for dental
procedures, pregnancy, diabetes, autoimmune diseases and necrotizing periodontal diseases. The subjects were distributed into two groups: 20 patients with detectable (50 copies/ mL) and 21 patients with undetectable PHVL. Clinical evaluation
Subjects were submitted to anamnesis questionnaire, and data about gender, age, ethnicity, education, income, smoking, alcohol consumption, drug use and means of HIV transmission were obtained. The history of AIDS-defining opportunistic infections, CD4+ T lymphocyte counts, HIV viral load and antiretroviral therapy were obtained from patients’ medical records. Oral examination included visual inspection of the oral mucosa and periodontal evaluation. Periodontal measurements were recorded at 6 sites per tooth (distobuccal, buccal, mesiobuccal, distolingual, lingual, mesiolingual) in all teeth, excluding third molars, and included probing depth (PD), clinical attachment level (CAL), visible supragingival biofilm (VSB) (presence/absence) and bleeding on probing (BOP). These measurements were performed by one calibrated examiner (Intraclass Correlation Coefficient of 0.96 and 0.97 for PD and CAL respectively), using conventional manual periodontal probe (PCP UNC 15; Hu-Friedy, Chicago, IL, USA). Chronic periodontitis (CP) was defined as the presence of >10% of teeth with PD and/or CAL ≥5 mm and BOP. Periodontal health (PH) was considered when ≤10% of sites with BOP, no PD or CAL >3 mm, although PD or CAL = 4 mm in up to 5% of the sites without BOP was allowed. After clinical examination, patients with evidence of destructive periodontal disease received full-mouth scaling and root planing under local anaesthesia and oral hygiene instruction. Immunologic assessment
In all HIV-infected subjects, laboratory analyses of CD4+ T lymphocyte, CD8+ T lymphocyte, and neutrophil levels in peripheral blood were routinely performed every 4 months for all patients at the
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
HIV-1 in subgingival biofilm HUCFF/UFRJ. For comparison purposes, the laboratory data of each patient were obtained within the same week that the periodontal clinical examination was performed. Detection and quantification of HIV-1 in the subgingival biofilm
Sample collection Subgingival biofilm samples were sampled from 12 sites of each subject with CP (6 sites with the deepest PD and 6 with PD 0–3 mm), and 6 randomly selected healthy sites from subjects with periodontal health, using sterile Gracey curettes (HuFriedy, Chicago, IL, USA). The samples were placed in individual 1.5 mL tubes containing TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.6) and stored at 80°C. These samples were used for detection and quantification of HIV-1 by real-time RT-PCR. Nucleic acid extraction and cDNA synthesis The RNA was extracted from a pool of subgingival biofilm samples (samples from two different periodontal sites were combined in 140 lL of TE buffer to generate a pool) using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions. The synthesis of the cDNA was performed with 10 lM of random oligomers using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s instructions. Real-time quantitative RT-PCR cDNAs were amplified using the TaqManâ Universal Master Mix amplification kit (Roche Molecular System, Branchburg, NJ, USA). Primers and probe were designed to a conserved region within the HIV-1 LTR: sense SSF1 (50 -GCT AAC TAG GGA ACC CAC TGC TT-30 ), anti-sense SSR1 (50 -CAA CAG ACG GGC ACA CAC TAC T-30 ), probe SS (50 FAM-AGC CTC AAT AAA GCT TGC CTT GAG TGC TTC-BHQ1-30 ) (Aguiar et al. 2007). Briefly, 5 lL of RNA was added into 12.5 lL amplification mix containing 2.5 lM of each primer, 5 lM of probe and RNAse-free water to a 25 lL final volume.
Thermal cycling programme was as follows: 50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min. Cycling was performed in ABI 7500 sequence detection system (Applied Biosystems). RNAse-free water was used as negative template control (NTC). Serial dilutions of the pNL4-3 plasmid bearing the full-length HIV-1 subtype B genome were used as positive external quantitation controls. An aliquot of 5 lL of 101–1010 copies of the pNL4-3 DNA was added to 20 lL of the qPCR mixture prepared as described above and submitted to the same cycling conditions as the test samples. The standard curve was generated each time a qPCR reaction was performed. An obtained Cycle threshold (Ct)-value was valid if the NTC was undetectable and if Ct values of the positive controls were in the range of mean two times the standard deviation (STD). Data analysis
All statistical tests were performed using Statistical Package for the Social Sciences (SPSS) software, version 19.0 (IBM, S~ ao Paulo, SP, Brazil). Descriptive analysis included socio-demographic characteristics, HAART use, HIV-related diseases, specific antiretroviral drugs, smoking, alcohol consumption and immunological data. Full-mouth periodontal clinical measurements were averaged for each patient and then within each group, and presented as mean of PD and CAL and the percentage of sites with BOP and VSB. Continuous variables were compared using Mann– Whitney test, and categorical variables were analysed using Fisher’s exact test. In order to estimate the effect of co-variables on the outcome undetectable subgingival HIV-1 viral load (SHVL), the multiple logistic method Generalized Estimation Equation (GEE) was employed. This model was used to consider the clustered nature of the probing depth’s data. For this analysis, only co-variables with p < 0.05 in the comparison between detectable and undetectable PHVL were included. The level of significance for all analyses was 5%.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Results Sociodemographic, clinical, health-related behaviour and immunological features of the studied sample
Of the 41 subjects who participated in this study, 51.2% were males, 39% were white and 50% were over 41 years of age. The majority of the participants reported no use of drugs (85.4%), while a few were current smokers (14.6%) and alcohol consumers (12.2%). The time of exposure to HIV was greater than 10 years in 68.3% of the subjects, and 51.2% had CP. Diarrhoea (63.6%), recurrent oral ulcers (70%), oral herpes simplex (31.7%), and pneumocystis pneumonia (31.7%) were the most frequent clinical conditions reported by these subjects (data not shown). Regarding antiretroviral therapy, 65.9% of the subjects were undergoing HAART, and the most frequently used antiretrovirals were the nucleoside reverse transcriptase inhibitors (NRTIs), such as lamivudine (70.7%), tenofovir (57.5%) and zidovudine (37.5%). In addition to the antiretroviral therapy, 15% were taking sulfamethoxazole plus trimethoprim as prophylaxis for Pneumocystis jirovecii pneumonitis (data not shown). There were no statistical differences regarding socio-demographic characteristics (gender, age, ethnicity, education, income) and healthrelated behaviour (smoking, alcohol consumption, drug use) between the two groups (detectable and undetectable PHVL) (data not shown). When HIV-infection related aspects were compared between the undetectable and detectable PHVL groups, only the use of HAART (p < 0.001) and SHVL detection (p = 0.001) were significantly different between them (Fisher’s exact test) (Table 1). Regarding laboratory data, CD4+ T lymphocytes levels were significantly higher in the undetectable PHVL group than in the detectable PHVL group (p = 0.038, Mann–Whitney test) (Table 2). Table 3 summarizes the periodontal clinical parameters of the sampled population. No significant differences were found between detectable and undetectable PHVL groups regarding the frequency of
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Table 1. Comparison of HIV-infection related aspects between undetectable and detectable PHVL groups Characteristics
Undetectable PHVL (N = 21)
N (%) Time of exposure to HIV 1–10 years 11–20 years ≥21 years HAART* Yes No SHVL Undetectable Detectable
Detectable PHVL (N = 20)
p value Microbiological data
5 (23.8) 16 (76.2) 0 (0.0)
8 (40.0) 9 (45.0) 3 (15.0)
0.060
20 (95.2) 1 (4.8)
7 (36.8) 12 (63.2)
0.05, Mann– Whitney test) (Table 4).
odontal health showed statistically significant differences for PD, CAL and BOL (p < 0.05), while no significant differences were observed for VSB, PHVL (undetectable and detectable) and SHVL (undetectable
HIV-1 viral load was detected and quantified in the subgingival biofilm samples by RT-PCR. The sensibility of the assay was first evaluated and was in the range of 10-100 copies of HIV-1 RNA. In order to confirm that the virus was detected in the subgingival biofilm instead of the provirus in the mononuclear cells from the blood, a RT-PCR was performed on positive samples, without c-DNA reaction. All these samples were negative, suggesting that the detection of the HIV-1 was from the subgingival biofilm. Detectable SHVL was observed only in the detectable PHVL group (20 patients) and the detection of the HIV-1 was found in 8 (40%) of these patients (Table 1). A bivariate analysis was performed between variables that showed significant differences between the undetectable and detectable PHVL groups (CD4+ T lymphocytes levels, HAART, and mean PD), PHVL (stratified into two levels: 0–3 log and > 3 log) and the outcome SHVL (Table 5). A statistically significant difference was observed only for the co-variable TCD4+ lymphocytes levels (p = 0.017, Fisher’s exact test). The results of the multiple logistic model employed to estimate the effect of the previously used co-variables in the bivariate analysis (Table 5) on the outcome undetectable SHVL are presented in Table 6. In this model, all measurements of PD from the 372 sampled sites were included. The only co-variable that demonstrated a significant effect on the outcome was CD4+ T lymphocytes levels. The interpretation of this finding is that patients with CD4+ T lymphocytes levels ≥500 cells/mm3 have odds eight times higher to present undetectable SHVL than those with CD4+ T lymphocytes levels
