J Oral Pathol Med (2017) 46: 644–648
© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
wileyonlinelibrary.com/journal/jop
Effect of HIV infection in the micronuclei frequency on the
oral mucosa
Celina Faig Lima1, Monica Ghislaine Oliveira Alves1, Juvencio Jose Duailibe Furtado2, Marcelo Marcucci3,
Ivan Balducci4, Janete Dias Almeida5
1
University Braz Cubas, Mogi das Cruzes, S~ao Paulo, Brazil; 2Department of Infectology, Heliopolis Hospital, S~
3
Department of Stomatology, Heliopolis Hospital, S~ao Paulo, Brazil; 4Department of Social and Pediatric Dentistry, S~
University (Unesp), Institute of Science and Technology, S~
ao Jose dos Campos, S~
Diagnosis, S~ao Paulo State University (Unesp), Institute of Science and Technology, S~
BACKGROUND: The genotoxic impact of HIV infection
on the oral cavity malignancies is unknown. The aim of
this study was to evaluate the effect of HIV infection in
micronucleus (MN) frequency on the oral mucosa of
HIV+ patients and establish a relationship with early
cytogenetic changes in oral carcinogenesis.
METHODS: Thirty HIV+ individuals who are under
highly active antiretroviral therapy (HAART) and 30
non-HIV patients were evaluated. Two smears were
taken from the lateral border of the tongue and mouth
floor and stained by Feulgen. The frequency of MN was
examined in 3000 cells per subject under common
microscopy.
RESULTS: MN analysis showed no significant difference
between groups by Mann–Whitney U-test for total MNs
(P = 0.178). The presence of single MN was greater in
control group with statistical significance (P = 0.009),
while in HIV group, multiple MNs were exhibited in
higher mean.
CONCLUSIONS: HIV patients under HAART therapy
and low viral load values showed higher frequency of
multiple MNs, which, although not statistically significant,
may be caused by the action of the Vpr gene, an
accessory gene of HIV. These results corroborate the
theory of HIV infection cytogenetic damage.
J Oral Pathol Med (2017) 46: 644–648
Keywords: AIDS cytodiagnosis; exfoliative cytology; HIV; HIV
infection; micronuclei; oral mucosa
Correspondence: Celina Faig Lima, Universidade Braz Cubas - Faculdade
de Odontologia. Av. Francisco Rodrigues Filho, 1233. Mogilar, Mogi das
Cruzes. Cep: 08773-380, S~ao Jose dos Campos, S~ao Paulo, Brazil. Tel:
055114791 8000, Fax: 114790 3844, E-mail: celinafaig@yahoo.com.br
Accepted for publication November 17, 2016
doi: 10.1111/jop.12527
ao Paulo, Brazil;
ao Paulo State
ao Paulo, Brazil; 5Department of Bioscience and Oral
ao Jose dos Campos, S~
ao Paulo, Brazil
Introduction
The acquired immunodeficiency syndrome (AIDS) is an
infectious disease caused by the HIV, leading to immuno-
suppression, and with disease progression, these individuals
were usually affected by opportunistic systemic infections
which lead to death (1). With antiretroviral therapy (2), there
was a change in this scenario and now HIV+ individuals
have higher survival rate. In accordance with Cobucci et al.
(3), after active antiretroviral therapy (HAART), there were
changes in the incidence cancers among HIV/AIDS patients,
with their overall risk increased (4–6). There is no an exact
number of the frequency of oral squamous cell carcinoma
(OSCC), which can be variable due the geography and
social conditions. The fact is there is an increase in the
number of oral and oropharyngeal cancer in HIV population
of approximately 50% in USA (7). The incidences of oral
cavity infections are also reduced, but HIV+ individuals are
more susceptible to exposure to carcinogens and the
development of oral malignancies.
The literature reveals that the most aggressive oral
mucosa genotoxic agents are alcohol and tobacco (8–11).
The exclusion of these chemical carcinogens raises ques-
tions about the action of biological aggressors such as virus.
Nevertheless, few studies have been carried out about
genotoxic effects of virus on oral mucosa. Even among the
most studied category, human papilloma virus (HPV), only
one case assessing the frequency of MN in cervical mucosa
was found in the literature. Authors concluded that HPV
infection increased MN frequency (12). The action of 16E6
and E7 oncogene from HPV reduces telomerase activity and
increases chromosomal instability with consequent MN
formation (13).
Within in this context, the genotoxic effect of HIV
infection on the oral carcinogenesis is still unknown. The
HIV infection in neoplastic processes could be explained by
the action of the accessory gene Vpr, from HIV, which leads
to abnormalities in the cell cycle and accumulates in the G2/
M cell division phase culminating in ploidy alterations (14,
15).

Therefore, HIV+ patients should be biomonitored for the
development of early changes in OSCC, for which the
micronucleus test (MN) can be used. The MN investigation
has been widely used for biomonitoring and risk assessment
of groups exposed to different genotoxic agents, because
this test helps to identify the initial changes of the
carcinogenesis process (16, 17). Micronucleus are formed
by fragments of delayed chromatin or chromosomes with
abnormalities that separate from nucleus during anaphase of
mitosis. They apparently reflect chromosomal aberrations
that occurred during the proliferation of the basal layer (17,
18).
The aim of this study was to evaluate the effect of HIV
infection in MN frequency on the oral mucosa of HIV+
patients and establish a relationship with early cytogenetic
changes in oral carcinogenesis.
Material and methods
This study was approved by the Ethical Committee of the
Heliopolis Hospital, S~ao Paulo, Brazil (819/2011), and the
Institute of Science and Technology, S~ao Jose dos Campos
(ICT-UNESP), Brazil, (073/2011-PH/CEP). Thirty consec-
utive HIV+ patients, whose diagnosis was confirmed by
ELISA test, undergoing HAART were selected from the
Department of Infectious Diseases, Heli opolis Hospital, S~ao
Paulo, in the period of 1 year, along with 30 non-HIV
patients of the Department of Biosciences and Oral
Diagnosis, ICT-UNESP.
The inclusion criteria followed those described by Lima
et al. (11): both male and female subjects, no history of oral
malignant neoplasia, no visible clinical signs at the local
evaluation, no illicit drugs users, non-smokers, drinkers of
no more than three doses of alcoholic beverages per week.
Gender and age (3 years) groups were matched. The
exclusion criterion was the presence of any clinical
alteration in the oral site of collection including oral mucosa
infectious diseases (e.g. candidiasis and HSV infection).
Patients from outpatient clinic of oral diagnosis submitted
to treatment of non-neoplastic lesions (e.g. hyperplasia)
were invited to participate in the control group.
Patients who agreed to participate signed the informed
consent. They were subjected to extra- and intra-oral clinical
examination and answered a questionnaire about general
data, habits and health condition. CD4+/mm3 lymphocytes
and viral load values were registered.
The smears were taken using a cytobrush, from the left
lateral border of the tongue and mouth floor, without the
prior use of mouthwash (19, 20). Three smears were
performed and immediately fixed by alcoholic spray (21),
and the samples were taken to ICT-UNESP. The samples
were transferred to hydrochloric acid (HCl) 5N at room
temperature for 5 min, and then, the material was incubated
in Schiff reagent (Merck, Darmstadt, Germany) for 90 min
at 4°C. After this, three consecutive washings in distilled
water were carried out before the samples were immersed
into the Fast Green FCF (Sigma, St.Louis, MO, USA) 0.5%
for 15 s. After dipping, the samples were rinsed three times
with absolute ethanol and subsequently clarified with
xylene.
Changes in oral mucosa of HIV patient
Lima et al.
645
The slides were examined under a Zeiss Axioplan 2 light
microscope equipped with the Axiophot 2 Photo System.
The slides were first examined at 9400 magnification and
then at 91000 magnification for the confirmation of MN
presence. A total of at least 3000 cells per subject were
screened. Only the cells with intact nuclei with smooth and
distinct nuclear perimeter, which provides cytoplasm defi-
nition, were included in the count. The criteria used to count
the MNs were the presence of a surrounding halo which is
representative of a homogeneous membrane, to have a
diameter less than one-third of the associated nucleus, the
intensity of staining should be similar to that in the same
focal plane (22) and to have no connection to the core. The
MNs were separated into two categories: single MN and
multiples MNs (23).
The data analysis was performed using the Mann–
Whitney U-test. The association between variables was
carried out using the Spearman correlation test. Significance
level for each test was a < 5%.
Results
Sample profile
Thirty HIV+ patients and control subjects were evaluated,
each group consisting of 22 men and 8 women. In the HIV+
group, 25 individuals were Caucasians and five were Black
with a mean age (mean  SD) of 49.51  8.2 years for the
HIV+ group (range 32–75 years). In the control group, there
were 26 Caucasians and four Blacks with a mean age of
49.3  8.06 years (range 35–75 years). Both groups con-
sisted of non-smokers who did not consume more than three
units of alcoholic beverages per week and did not use illicit
drugs.
The viral load of the HIV+ group was ˂50 copies/ml,
with only only case with viral load of 99 copies/ml. The
mean CD4+ T cell was 551.33  272.39 mm3.
The use of mouthwash was reported by four subjects from
HIV+ group, one case of cetylpyridinium chloride use and
three cases of mouthwash containing essential oils use. In
the control group, five subjects reported the use of
mouthwash containing essential oils.
MN investigation
The values obtained in the MN test for overall frequency of
MN and micronucleated cells (3000 cells) were investigated
and were categorized into four conditions. A comparison
was made in each category by Mann–Whitney U-test
(a = 5%). The result is shown in Table 1.
Of the 30 cases evaluated for each study group, three
were excluded due to low cellularity for both groups. The
presence of MN was observed in only 40.74% of cases of
HIV+ group, with a mean of 1.11  1.63, while in the
control group, the incidence of MN occurred in 66.67% of
cases, with mean of 1.40  1.45. However, there was no
statistically significant difference between the frequencies of
total MNs according to the Mann–Whitney U-test
(P = 0.178) as well as between the total of micronucleated
cells (P = 0.087). The comparison between groups showed
statistical significance in the case of cells with the presence
of a single MN (P = 0.009), but this difference cannot be
J Oral Pathol Med
 Changes in oral mucosa of HIV patient
Lima et al.

646
Table 1 Frequency of MN and micronucleated cells in HIV and control groups

Single MN ˃01 MN Micronucleated cells MN Total

Statistics HIV Control HIV Control HIV Control HIV Control
Mean 0.33 0.93 0.78 0.48 0.67 1.11 1.11 1.40
StDev 0.55 0.87 1.55 1.09 0.83 0.97 1.63 1.45
Median 0 1 0 0 0 1 0 1
IQI 1 2 0 0 1 2 2 2
CV (%) 166.41 94.37 166.41 225.83 124.81 87.66 146.26 102.89
P-value 0.009 0.614 0.087 0.178

StDev, Standard deviation; IQI, Interquartile range; CV, Coefficient of variation.

Table 2 Relationship between the percentage of cases and the frequency

of MN according to the count of CD4+/mm3

Count (%) of CD4 + T cell/mm3

Total number of MN ≤200 201–550 ˃550
1 0 3.7 14.81
2 0 3.7 3.7
4 11.11 0 3.7
5 0 0 3.7
Total of cases evaluated 4 11 12

Figure 1 Single MN from control group indicated by the arrow.
P = 0.904), cells with multiple MNs (rs = 0.130,
P = 0.515) and for the total number of micronucleated
cells (rs = 0.026, P = 0.895). However, these correlations
were not significant for any of these groups.
Only a small number of individuals used mouthwashes
and using the Mann–Whitney U-test (a = 5%) showed no
difference between groups which was observed for cells
with single MN (P = 0.217), micronucleated cells
(P = 0.088) and total MNs (P = 0.06). The average MN
for HIV+ group was 0.25  0.5 and for the control group
was 1.2  0.49. The results of the descriptive analysis are
shown in Table 3.

Figure 2 Graphic of the distribution of all micronucleus in the cells of
HIV and control groups.
observed in the presence of cells with multiple MNs
(P = 0.614). The single MN is showed in Fig. 1.
The frequency and distribution of MN in both groups
evaluated can be observed in Fig. 2. Although observed in
the control group the number of cases with total frequency
of MN was higher than in HIV+ group, the highest
frequency of multiple MNs was observed in cases where
the count of CD4+ T cells was less than 200/mm3 (Table 2).
The Spearman correlation test (a = 5%) showed a weak
negative correlation between MN frequency and count of
CD4+ lymphocytes from the HIV+ group for total MNs
(rs = 0.003, P = 0.987), cells with single MN (rs = 0.024
Discussion
The aim of this study was to evaluate the effect of HIV
infection in the cytogenetic damage in oral mucosa consid-
ering that DNA damage may lead to oral cancer.
For this purpose, two groups were evaluated: study group
(HIV patients under HAART) and control group (patients
with no HIV infection). The conception of the study design
included a third group: HIV patients before HAART
treatment. Nevertheless, in Brazil, patients fortunately start
treatment at the moment of diagnosis. This is a huge step to
a developing country. The absence of a non-HAART HIV
patient group is a weakness in this study which leads to
difficult in the dissociation of the effects of HIV and
HAART in oral mucosa separately.
Another point regarding the difficulties of the inclusion of
non HAART HIV patients is the presence of infectious
lesions, usual in this group untreated, that can result in false
positive for cytogenetic damage (24). Infected patients fall
within the exclusion criteria. The immunocompromised
status of patients without HAART predisposes to opportu-
nistic oral lesions. Herpesvirus family might be listed as one
example.

J Oral Pathol Med


Table 3 Frequency of MN according to the use of mouthwashes in both groups
Single MN ˃01 MN
MN
Groups HIV Control HIV Control
Median 0 0 0 2
IQI 0 1 0.75 1.5
CV (%) * 223.61 200.0 63.89
P-value 0.217 * 0.088
IQI, interquartile range; CV, coefficient of variation.
*Could not be calculated.
The human herpes viruses (HHV) include different
viruses caused by herpes simplex virus (HSV), human
cytomegalovirus (HCMV), Epstein–Barr virus (EBV) and
KS-associated herpesvirus (KSHV) (4, 24–26). EBV infec-
tions are related to the development of malignancies in
immunosuppressed patients, and they are an interfering
factor in the cytogenetic analysis. EBV DNA tumour virus
can result in a latent infected cell and undergo clonal
expansion in some cases (4, 24).
The role of HIV infection within the oral carcinogenesis
process has not been established, but the immunosuppres-
sion leads to increased susceptibility to carcinogens and MN
formation (4–6). The subjects to study were selected from a
HAART therapy in a follow-up programme and were
monitored for HIV infection and the development of
correlated diseases. The effectiveness of HAART therapy
can be observed when the results of viral loads are below 50
copies/ml (27, 28), which is considered undetectable. This
situation was observed in 83.33% of the evaluated HIV+
group. Furthermore, the nutritional control, part of the
treatment programme, is an important factor to control the
changes in DNA synthesis (23), which could lead to
alteration in the MN frequency.
There was no difference among HIV+ patients and
control for total MN amount. However, for MN analysis, the
presence of single or multiple MNs in the cells has to be
considered. This reflects the difference of MN formation
due to the intensity of genotoxic agents produced by direct
or indirect clastogenic effect (23). The MN formation can
occur as a result of genetic material lost during mitosis due
to chromosome breakage and spindle disturbances (15, 23).
In the present study, single MN was increased in the
control group when compared to the study group. The
exposure to environmental carcinogens in the control group
could lead to chromosome breakage on cells and result in
single MN formation (17).
Although not statistically significant, MN count in the
study group showed superiority in the mean of multiple MNs
when compared to control group. The multiple MN formation
could be related to genotoxic agents with direct action in
cellular cycle. The Vpr gene action, an accessory gene of HIV,
induces alterations that accumulate in the phase G2/M of the
cell cycle (14, 15), leading to spindle disturbances due to
indirect and smooth action of genotoxic agents. Nevertheless,
for the best of our knowledge, there are no reports analysing
MN frequency in HIV+ patient’s oral mucosa cells in the
current literature to corroborate these theories.
Changes in oral mucosa of HIV patient
Lima et al.
647
Micronucleated cells Total of MN
HIV Control HIV Control
0 2 0 2
0.75 1 0.75 2
200.0 55.90 200.0 91.29
0.060
Recently, more theories have pointed out the presence of
HIV virus accessory genes and infection by oncovirus from
HHV and HPV. The oncogenic process can be explained by
the action of the HIV virus accessory gene Vpr which leads
to abnormalities in the cell cycle and accumulates in the G2/
M cell division phase culminating in ploidy alterations (14,
15). Although there are strong evidences that the action of
HPV can be associated with HIV infection, leading to
changes in DNA, this process is restricted to oropharyngeal
cancer (29), and it is not applied to our study design.
High MN frequency was observed when the CD4+
lymphocyte count was equal or less than 200 CD4+ cells/
mm3. No statistical correlation was observed between CD4+
lymphocyte count and frequency of MN. For a better
correlation analysis, it would be necessary to include a
group with low immunity. This study was conducted in a
referral centre for the treatment of HIV patients, and for this
reason, all participants had a low viral load and appropriate
levels of CD4+ lymphocytes. Mendes et al. (15) evaluated
MN frequency of uterine cervix from HIV+ women, in the
AIDS phase of the disease, and concluded that the immune
condition is inversely proportional to the MN quantity.
The use of mouthwashes evaluated in both of groups
produced no impact on the frequency of MN (P = 0.06).
These findings corroborate those reported by Ros-Llor and
Lopez-Jornet (30), although their evaluation had been
performed on healthy individuals, and it is contradictory to
the results of Diniz-Freitas et al. (31) who observed higher
frequency of MN in mouthwash users in the evaluation of
smokers, alcoholic smokers and healthy subjects.
The MN test was performed in accordance with
staining and analysis described in the literature (25, 27–
31). The staining and analysis of smears has a great
importance to determine MN frequency. The use of a
DNA-specific staining, such as Feulgen, supports the
results (27), as the use of other staining may cause
difficulties in the smears assessment, leading to false
results due to the confusion with keratin bodies. The
analysis must follow the criteria described by Tolbert
et al. (22) and improved by Holland et al. (28) in the
HUMN project, where the MN evaluation should be
performed on a minimum of 1000–3000 cells.
This study demonstrated that HIV patients under HAART
presenting low viral load values showed higher mean frequency
of multiple MNs, although not statistically significant, which
may be due to the action of the Vpr gene. These results
corroborate the theory of HIV infection cytogenetic damage.
J Oral Pathol Med
 Changes in oral mucosa of HIV patient
Lima et al.
648
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Acknowledgements
This work was supported by S~ao Paulo Research Foundation, FAPESP 2012/
05062-1.
Conflict of interest
The authors state that they have no potential conflict of interest.
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