LEVELS OF HIV-1 IN SUBGINGIVALBIOFILM OF HIV-INFECTED PATIENTS.Pavan P et al, JCP 2014.

Shilpa ShivanandII MDS

Introduction • 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.

• HIV-1 can be isolated from many body fluids, excretions and secretions

Hadley 1989• It has been demonstrated that 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• Even though some studies have

identified HIV-1 in saliva…..Groopman et al

1984……The mouth is rarely considered a

source of HIV-1 transmissionMaticic 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 HIV-infected subjects.

• This reinforced 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 GCF

Maticic et al 2000

Aim • 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• Forty-one HIV-infected subjects were

selected. • The subjects were distributed into two

groups: 20 patients with detectable (50 copies/mL) and 21 patients with undetectable PHVL.

INCLUSION CRITERIA• Patients were >20 years of age and

presented at least 15 teeth.EXCLUSION CRITERIA• Included the necessity of antibiotic

prophylaxis for dental procedures, pregnancy, DM, autoimmune diseases and necrotizing periodontal diseases.

Clinical evaluation• Subjects were submitted to a

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• 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).

• After clinical examination, patients with evidence of destructive periodontal disease received full-mouth SRP under LA and OHI.

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 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 biofilmSample 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.

• 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 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 140μL of TE buffer to generate a pool) using the QIAamp Viral RNA Mini Kit.

• The synthesis of the cDNA was performed with 10µM of random oligomers using the High Capacity cDNA Reverse Transcription kit.

Real-time quantitative RT-PCR

• cDNAs were amplified.• Primers and probe were designed to a

conserved region within the HIV-1 LTR• 5 µL of RNA was added into 12.5 µL

amplification mix containing 2.5 µM of each primer, 5 µM of probe and RNAse-free water to a 25 µL final volume.

• Thermal cycling programme was performed.

• The standard curve was generated each time a qPCR reaction was performed.

Data analysis• All statistical tests were performed using

Statistical Package for the Social Sciences (SPSS) software.

• 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.

Results• 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 time of exposure to HIV was greater than 10 years in 68.3% of the subjects, and 51.2% had CP.

• Regarding antiretroviral therapy, 65.9% of the subjects were undergoing HAART, and the most frequently used were NRTIs (lamivudin 70.7%, tenofovir 57.5%, zidovudine 37.5%).

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

Regarding laboratory data, CD4+ T lymphocytes levels were significantly higher in the undetectable PHVL group than in the detectable PHVL group (p =

0.038)

Regarding periodontal parameters no significant differences were found between detectable and

undetectable PHVL groups regarding the frequency of CP (p = 0.563) and only PD showed significant differences (p

= 0.016)

• Comparisons between patients with chronic periodontitis and individuals with periodontal health showed statistically significant differences for PD, CAL and BOP (p < 0.05), while no significant differences were observed for VSB, PHVL (undetectable and detectable) and SHVL (undetectable and detectable) (p > 0.05)

Microbiological data• HIV-1 viral load was detected and

quantified in the subgingival biofilm samples by RT-PCR.

• 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.

• A statistically significant difference was observed only for the co-variable TCD4+ lymphocytes levels (p = 0.017).

• 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 <200 cells/mm3 (p = 0.002).

Discussion• The current study assessed the

association among SHVL, PHVL and periodontal clinical parameters.

• No association among those parameters was found in this HIV-1 infected sample population.

• Goncalves et al. (2007) reported that long term HAART, which usually decreases PHVL and increases CD4+ T lymphocytes levels was related to lower severity of clinical periodontal parameters in a HIV-infected Brazilian population.

• In the present study, CD4+ T lymphocytes levels were significantly lower in the detectable PHVL group than in the undetectable group.

• This finding suggests that the patients with detectable PHVL can be at higher risk for oral infection, such as periodontal disease.

• In contrast, Goncalves et al. (2005) compared HIV-infected patients with and without CP and they did not observe any association between CD4+ T lymphocytes levels and clinical periodontal parameters.

• In the current study, the detection and quantification of HIV-1 in subgingival sites were performed by real-time PCR.

• Of the 20 individuals from the detectable PHVL group, 40% showed detectable SHVL.

• On the other hand, all patients with undetectable PHVL demonstrated also undetectable SHVL.

• This can be influenced by the CD4+ T lymphocytes levels which were significant higher in the undetectable group, when compared to the detectable PHVL group.

• These findings are in agreement with the study of Maticic et al. (2000), who detected HIV-1 in the GCF of 49% of the patients, with a significant correlation with PHVL.

• Moreover, these authors demonstrated that the HIV-1 was not detected in the gingival crevicular fluid of any patients with CD4+ T lymphocytes > 500 cells/mm3.

• Regarding the use of HAART, no significant association was observed for the SHVL, and this result is in agreement with Maticic et al. (2000) who did not observe an association between HAART and the detection of HIV-1 in the crevicular gingival fluid either.

• Only CD4+ T lymphocytes levels demonstrated a significant effect on the outcome undetectable SHVL, suggesting that patients with CD4+ T lymphocytes levels ≥ 500 cells/mm3 present undetectable SHVL than patients with lymphocytes levels <200 cells/mm3.

Conclusion• The current study can contribute for a

better understanding of the presence of HIV-1 in the subgingival biofilm.

• Nowadays, HIV-infected individuals present a longer life expectancy, and consequently a long exposure to the HIV.

• Thus, further studies are necessary to improve knowledge about the behaviour of the HIV-1 in the subgingival biofilm, and to investigate the possible local (periodontal tissue) and systemic effects of this virus over time.

Cross References

I. Association of T CD4 lymphocyte levels and subgingival microbiota of chronic periodontitis in HIV infected Brazilians

under HAARTOral Surg Oral Med Oral Pathol Oral Radiol Endod 2004,

Gonçalves LS et al.• The aim of this study was to determine the subgingival

microbiota of HIV-infected patients with chronic periodontitis and different T CD4 lymphocyte levels under HAART.

STUDY DESIGN:• 64 HIV+ patients were distributed into Group I: chronic

periodontitis and Group II: periodontal health. All subjects received conventional periodontal therapy. Periodontal clinical parameters were evaluated at 6 sites/tooth in all teeth at baseline and 4 months after therapy. The levels of T CD4 were obtained from the patient's medical record. Subgingival plaque samples were taken from the 6 sites with the largest pocket depth in each subject of Group I, and 6 randomly selected sites in subjects of Group II. The presence of 22 subgingival species was determined using the checkerboard DNA-DNA hybridization method.

RESULTS:• Sixty-one percent of the HIV-infected patients

represented AIDS cases, although 69% of them were periodontally healthy. The T CD4 lymphocyte mean level was 333 cells/mm3 and viral load was 12,815 +/- 24,607 copies/mm3. Yet, the prevalence of chronic periodontitis was relatively low (36%). Several periodontal pathogens, in particular T. forsythensis (P < .05), were more prevalent in HIV-positive patients with periodontitis than in HIV-positive subjects with periodontal health. Most of the species decreased in frequency after therapy, particularly P. gingivalis (P < .05). E. faecalis and F. nucleatum were significantly more prevalent in the subgingival microbiota of patients with chronic periodontitis and lower levels of T CD4 (P < .05), while beneficial species tended to be more frequently detected in individuals with T CD4 counts over 500 cells/mm3.

CONCLUSION:• The subgingival microbiota of HIV-infected patients with

chronic periodontitis include a high prevalence of classical periodontal pathogens observed in non-infected individuals. Furthermore, the severe immunosuppression seems to favor the colonization by these species, as well as by species not commonly found in the subgingival microbiota.

II. The Association Between Detectable Plasmatic Human Immunodeficiency Virus (HIV) Viral Load and Different Subgingival

Microorganisms in Brazilian Adults With HIV: A Multilevel Analysis

Viviane Tiago Pereira etal, JOP 2014.Background: This study investigates the association between detectable plasmatic human immunodeficiency virus (HIV) viral load (HVL) and high levels of periodontal- and non-periodontal-related microorganisms in the subgingival microbiota of individuals with HIV.Methods: Thirty-seven individuals with HIV were divided into two groups: 1) detectable HVL (n = 15); and 2) undetectable HVL (n = 22). Subgingival biofilm samples were obtained, and the levels of 35 microbial species were determined by the checkerboard DNA–DNA hybridization method. Periodontal clinical measures and laboratory and sociodemographic data were also registered. χ2 test, Fisher exact test, and Mann-Whitney U test were used to compare groups. Multilevel ordinal regression models were used to test the association between HVL and the levels of 35 microbial species in subgingival biofilm, adjusted for confounders.

Results: Of the 35 species studied, 11 (31.4%) showed higher mean levels in the detectable HVL group than undetectable HVL group (P <0.001). These species included Actinomyces naeslundii II, Actinomyces israelii, Actinomyces odontolyticus, Veillonella parvula, Capnocytophaga gingivalis, Eikenella corrodens, Campylobacter concisus, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Candida albicans. Significant associations between detectable HVL and high levels of microorganisms, adjusted for confounders, were observed for A. naeslundii I, Actinomyces gerencseriae, C. gingivalis, E. corrodens, C. concisus, Prevotella nigrescens, T. forsythia, and Dialister pneumosintes.Conclusion: Detectable plasmatic HVL in individuals with HIV was associated with elevated levels of known periodontal pathogens, such as P. nigrescens, T. forsythia, and E. corrodens, as well as C. concisus, C. gingivalis, and D. pneumosintes in the subgingival biofilm.