LEVELS OF HIV-1 IN SUBGINGIVAL BIOFILM OF HIV-INFECTED PATIENTS.

2. LEVELS OF HIV-1 IN
SUBGINGIVAL
BIOFILM OF HIV-INFECTED
PATIENTS.
Pavan P et al, JCP 2014.
Shilpa Shivanand
II MDS

3. 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

4. • 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 transmission
Maticic et al 2000

5. • 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

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.
• 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.

12. 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.

13. 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.

14. 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.

15. 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%).

16. 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

17. Regarding laboratory data, CD4+ T lymphocytes levels were
significantly higher in the undetectable PHVL group than in
the detectable PHVL group (p = 0.038)

18. 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)

19. • 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)

21. 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.

22. • 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).

23. 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.

24. • 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.

25. • 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.

26. • 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.

27. • 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.

28. 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.

29. Cross References

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

31. 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

32. 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.

33. 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.