Pathology Research and Practice 209 (2013) 399403

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Pathology Research and Practice

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Original article

Oral epithelial changes in HIV-positive individuals

Elia C.S. Almeida a, , Renata M. Etchebehere b , Benito A.S. Miranzi c ,
Vitorino M. Santos d , Maria G. Reis e
a

General Pathology Division, Federal University of Uberaba (UFTM), Uberaba, MG, Brazil
Division of Surgical Pathology UFTM, Uberaba, MG, Brazil
Discipline Biostatistics, University of Uberaba (UNIUBE), Uberaba, MG, Brazil
d
Department of Internal Medicine Armed Forces Hospital (HFA), and Catholic University of Braslia, Braslia-DF, Brazil
e
Discipline of Histology UFTM, Uberaba-MG, Brazil
b

a r t i c l e

i n f o

Article history:
Received 19 March 2012
Received in revised form 4 September 2012
Accepted 4 March 2013
Keywords:
Acquired immunodeciency syndrome
Autopsy
Mouth
Epithelium
Buccal mucosa

a b s t r a c t
HIV infections frequently affect the oral cavity, and local changes may be utilized as indicators of
immunosuppression in HIV-positive patients. Morphometric and morphological features of the lining,
masticatory, and specialized epithelium of the oral mucosa were studied in 12 HIV-positive and 12 HIVnegative patients autopsied from 2007 to 2010. Mucosal samples from the cheek, gingival, and tongue of
24 individuals were xed in Carnoy solution and stained with hematoxylineosin. Various morphometric characteristics (epithelial thickness, number of cell layers, mean cell diameter) and morphological
parameters (basal layer hyperplasia, exocytosis of inammatory cells, glycogenic acanthosis, cell ballooning degeneration) were then measured. The HIV-positive group had a greater epithelial thickness
(mean: 304.4 m) and a higher mean cell diameter (11.84 m), whereas the HIV-negative group had
more epithelial layers (26.7). Basal layer hyperplasia did not differ signicantly between the two groups,
but exocytosis of inammatory cells, glycogenic acanthosis, cell ballooning, and spongiosis were more
prevalent in the HIV-positive group. Our ndings demonstrate that HIV infection causes diverse epithelial changes in the oral cavity, including thickening, increased cell diameter, increased migration of
inammatory cells, and inter- and intra-cellular edema.
2013 Elsevier GmbH. All rights reserved.

Introduction
The Brazilian Ministry of Health estimated the incidence of HIV
infection in Brazil in 2010 at 17.8/100,000 inhabitants, with a prevalence of 0.6% in the whole population [14]. Diverse manifestations
of HIV infection occur in the oral cavity, and can disclose immunosuppression associated with HIV/AIDS [5,8,12]. Oral mucosa is
covered by a layer of stratied (keratinized or non-keratinized)
epithelium that, through variable differentiation, is able to diversify its morphological and biochemical characteristics depending
on local and regional needs [2,11]. On the inner surface of the lips,
oor of the mouth, cheeks, alveolar mucosa, the ventral surface of
the tongue, and the soft palate, the epithelium is non-keratinized.
The specialized mucosa of the dorsum of the tongue is covered

Corresponding author at: Department of Biological and Natural Sciences, Tringulo Mineiro Federal University, Praca Manoel Terra s/n, Uberaba, ZIP 38050-015
MG, Brazil. Tel.: +55 34 33185449; fax: +55 34 3318 5462.
E-mail addresses: eliacsa@dcb.uftm.edu.br (A. Elia C.S.),
renataetch@hotmail.com (E. Renata M.), bmiranzi@mednet.com.br (M. Benito A.S.),
vitorinomodesto@gmail.com (S. Vitorino M.), mgreis.hist@dcb.uftm.edu.br
(M.G. Reis).
0344-0338/$ see front matter 2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.prp.2013.03.001

either by an ortho-keratinized epithelium (on the liform and foliate papillae) or a para-keratinized epithelium (on the fungiform and
circumvallate papillae). The masticatory epithelium, found over the
gingivae and hard palate, is keratinized [2,11]. We performed morphological and morphometric studies in samples of lining (cheek),
masticatory (gingiva), and specialized (tongue) mucosal epithelia
from the oral cavity of HIV-positive individuals and controls.

Materials and methods

This cross-sectional study was performed at the Clinics Hospital
of Tringulo Mineiro Federal University (UFTM) and at the Clinics
Hospital of Ribeiro Preto, Faculty of Medicine So Paulo University (URP/USP) between 2007 and 2010. The study methods were
approved by the appropriate Research Ethics Committees at these
institutions.
Samples of oral mucosa from 24 autopsied patients (12 HIVpositive individuals and 12 HIV-negative controls; male:female
ratio in both groups, 1:1) were evaluated. All tissue samples were
collected from areas of healthy oral mucosa with no evidence of
macroscopic lesions. Oral mucosa samples were obtained from
cheek, gingival, and tongue, with a maximal extension of 510 mm.

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A. Elia C.S. et al. / Pathology Research and Practice 209 (2013) 399403
Table 1
Comparative data for HIV-positive and -negative patients necropsied at the study
institutions between 2007 and 2010.
Parameters

HIV-positive (n = 12)

HIV-negative (n = 12)

Mean age*
Mean BMI
Race (nonwhite:white)
Alcoholism
Smokers
Basic infectious disease

41.7 6.5 years

22.4 kg/m2
2:1
8
6
11

34.2 8.8 years

23.9 kg/m2
1:1
6
5
2

Fig. 1. Correlation between the thickness of the epithelium in the gingivae group
with AIDS and viral load.

Fig. 2. Correlation between thickness of the epithelium in the tongue group with
AIDS and viral load.

Samples from the cheek were obtained above the line of mastication and close to the maxillary premolars. Tongue samples were
taken from the anterior third, while gingival samples were taken
from the anterior region between the maxillary right canine and
rst premolar. The fragments were processed by parafn inclusion
techniques and stained with hematoxylin-eosin (HE) for histological evaluation. A standard laboratory microscope (Olympus BX41)
and ImageJ software were utilized for morphologic analysis and
processing. Analysis included the evaluation of basal layer hypertrophy, interpapillary crests, glycogenic acanthosis, cell ballooning,
spongiosis, and exocytosis of inammatory cells (Figs. 1 and 2).
The number of papillae and degree of keratinizatization were also
evaluated in the tongue samples. Morphometric analysis included
the measurement of epithelial thickness, the number of cell layers, and measurement of the mean cell diameter. The statistical
analysis employed the MannWhitney test, paired t-test, analysis
of variance (ANOVA), chi-square test, and Pearsons and Spearman
correlation, with the signicance level set at 5% (P < 0.05).
Results
Table 1 shows comparative data for the HIV-positive and negative groups, including age, body mass index (BMI), race, alcohol

p < 0.05.

intake, smoking status, and basic infectious disease. Except for the
age of individuals, there was no signicant difference between the
study groups. Based on their BMI, most patients were classied as
eutrophic, although one was classied as marasmic.
Categorization by race in the infected group showed a nonwhite:white ratio of 2:1, whereas in the HIV-negative group, this
ratio was 1:1. Most HIV-positive patients were alcoholics, and
approximately 50% in both groups were smokers. The necropsy
request-related data were highly variable, to the extent that they
could not be used for comparisons between the groups. Patients
were thus classied as having (or not) any basic infectious disease. The diagnosis of infectious disease at autopsy was performed
in 91.6% of patients in the HIV-positive group, but only in 16.6%
in the HIV-negative group. For all patients, an evaluation of the
oral cavity was performed encompassing evidence of caries, dental plaque, teeth requiring extraction, gingivitis, periodontitis, and
oral lesions. These characteristics were ranked collectively as good,
intermediate, or poor on a scale of oral health and hygiene. Most
patients had mild-to-moderate gingivitis, but moderate-to-severe
gingivitis was more common in the HIV-positive group. However,
only three patients had conspicuous periodontitis. To characterize
the health of the hard tissues, patients were classied as having lost up to one quarter, up to half, or up to three quarters
of their of teeth. In this study, only one patient had a complete
and healthy dentition; all others exhibited some degree of tooth
loss.
Individuals in the HIV-positive group had variable viral load and
CD4/CD8 levels (Table 1). Furthermore, only two infected patients
used antiretroviral therapy (patient numbers 9 and 11 in Table 2).
These data were not available for some patients given their initial general condition, while some had not had these tests or had
died before knowing their results. Most patients had no regular
monitoring for the disease and had low levels of CD4/CD8.

Table 2
Comparative data between viral load, CD4 and CD8 levels, and the relationship
between CD4 and CD8 in HIV-positive patients necropsied at the study institutions
between 2007 and 2010.
Number

Viral load
(copies)

CD4 (cells or %)

CD8 (cells)

Relationship
CD4/CD8

1
2
3b
4
5
6b
7
8b
9
10
11
12

58.440
17.501

96.733
106.269

109.813

1000
0
15.589
500

2%a
37%a

241
91
61
9

151
55
190
11.55%a

114
937

480
983
1718

445
523
403
223

0.02
0.04

0.5
0.09
0.04

0.34
0.11
0.48
0.18

a
Values for absolute cell numbers are absent from patient records; under these
circumstances, percentage values were used instead.
b
Data was absent entirely.

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401

Table 3
Comparison of epithelial thickness, number of cell layers, and mean cell diameter in samples of epithelia from the cheek, gingivae, and tongue of HIV-positive and -negative
patients, autopsied at the study institutions between 2007 and 2010.
Groups

Epithelial thickness (m)

Number of cell layers

Mean cell diameter (m)

Pearsons correlation
R
P

C+
C
G+
G
T+
T

12
12
8
8
12
12

278.6 m
306.8* m
304.0* m
233.2 m
330.6* m
279.1 m

24.5
26.7*
26.2*
18.8
24.0*
22.5

11.1 m
11.3 m
10.9 m
11.5 m
13.5* m
11.6 m

0.1095
0.1182
0.1025
0.3959
0.1279
0.3535

0.01
0.0074
0.06
0.0001
0.0049
0.0001

Group denitions: C, cheek epithelium; G, gingival epithelium; T, tongue epithelium; +, HIV-positive group; , HIV-negative group.
*
p < 0.05.

Fig. 3. Morphological analysis of HE-stained epithelial samples from cheeck (A and B), gingiva (C and D) and tongue (E and F) of HIV-negative and HIV-positive patients
(Magnication: A, B, E and F 100x, 400x C and D).

Following morphometric analyses, the thickest epithelia were

found in the tongue and cheek. Between the groups, the HIVpositive patients displayed thicker epithelia in the tongue and
gingivae than HIV-negative subjects. Neither the number of cell
layers nor the cell diameter was correlated with epithelial thickness. However, tongue samples from the HIV-positive group (T+)
had, on average, a higher number of cell layers and wider cell diameter. Pearsons correlation coefcient between the number of cell
layers and cell diameter was positive and statistically signicant in
all comparisons, except for gingival samples from the HIV group
(G+), as shown in Table 3.
Correlating the thickness of the epithelium in the gingivae group
with AIDS viral load, we obtained results as the Spearman coefcient (rs) = 0.3929; t = 0.9553; p = 0.3833 (Fig. 1).
The above data are not parametric. By applying Spearman correlation between the thickness of the epithelium and viral load in the
gingivae group with AIDS, this proved to be positive and not signicant. Thus, the higher the viral load, the greater the thickness of
the epithelium.
Correlating the thickness of the epithelium in the tongue group
with AIDS viral load, we obtained results as the Spearman coefcient (rs) = 0.3333; t = 0.8660; p = 0.4197, pairs numbers = 8 (Fig. 2).
A Spearman correlation between the thickness of the epithelium
group with AIDS with viral load was not signicant and negative.
The data suggest a trend inversely proportional to the gingivae seen
in the group with AIDS.
Following morphological and morphometric analyses
(Figs. 3 and 4), the HIV-positive group exhibited attened

interpapillary crests at the interface between the epithelium and connective tissue and a reduced number of tongue
papillae, these being almost absent in some cases. Desquamation of the gingival epithelium was also a common
nding in this group, as was increased cell diameter in all
regions.
Glycogenic acanthosis was observed in both groups, with
variable intensity and extension, but was more conspicuous
in the HIV-positive group. Cell ballooning had similar intensity and extension in both groups, while spongiosis was more
severe in the HIV-positive group, although more widespread
severe in the control group. The gingival epithelium showed
no remarkable variation in glycogenic acanthosis; cell ballooning was intense and extensive in both groups; and spongiosis
was more frequent, severe, and widespread in the HIV-positive
group.
Tongue epithelia generally displayed less severe and less
widespread glycogenic acanthosis than other epithelial types,
whereas spongiosis was most prevalent in gingival epithelia
(although a common feature in all types). Papillae were observed
in all tongue samples.
Exocytosis of inammatory cells was more frequent in gingival epithelia and less frequent in cheek and tongue epithelia.
Furthermore, exocytosis was less frequent in the HIV-negative
group, to the extent that there was no exocytosis in some tongue
epithelium samples from this control group. Basal layer hyperplasia was observed in only two tongue samples, one from each
group.

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A. Elia C.S. et al. / Pathology Research and Practice 209 (2013) 399403

Fig. 4. Glycogenic acanthosis, ballon cells, spongiosis and in samples from cheek (A, B and C) gingiva (D, E and F) and tongue (G, H and I). Assessment of tongue papillae (J)
and exocytosis of inamatory cells (K) (HE-stained cells. Magnication: 400x).

Discussion
In the present study, the mean subject age and BMI were similar to those described in previous studies [13,15]. Fry et al. [16]
reported an association between infection and race that was purported to be a reection of social inequality in Brazil. Although
the inuence of alcohol and tobacco use has already been widely
debated in the scientic community, it was not possible to analyze their inuence in this study because the use of tobacco and
alcohol was common to both groups [1719]. Epithelial thickness
was greater in T+, which is in agreement with previous studies
showing that the oral epithelium is thicker in the tongue and cheek
[6]. Montebugnoli et al. described increased epithelial thickness in
association with glycogenic deposits [10]. Moreover, ionic disorders may contribute to an increased cellular water content [20].
With regard to the number of cell layers, these were raised in G+
and T+, in contrast to the ndings of Rocha et al. in their study
of epithelia in various tissue regions in similar population groups
[13], indicating the diversication that oral epithelia can undergo
when responding to local stimuli. The mean cell diameter was

elevated in T+, a phenomenon probably due to the HIV disease

itself, which can result in increased potassium and sodium ion levels in infected cells. This enhanced ionic inux drags water with
it to maintain cellular osmolarity, and the consequent increase
in cell volume could indirectly contribute to increased epithelial
thickness, as already described for HIV-infected individuals [4,15].
The correlations between viral load and thickness of the epithelium in the tongue and gingivae group with AIDS, although not
statistically signicant, could be justied by the different types
of epithelium in two regions, one masticatory and the other specialized with cornication only in lingual papillae. We believe
that a stronger correlation could be found with a large sample.
Our morphometric analysis showed that glycogenic acanthosis was a common nding in the superior epithelial layers,
which may constitute a tissue response to non-specic injuries
[10]. Cell ballooning occurs because of deposits of plasma
proteins in the cytoplasm of epithelial cells, and may be a
consequence of tissue injury [7]. Spongiosis is the accumulation of uid in the intercellular space separating neighboring

A. Elia C.S. et al. / Pathology Research and Practice 209 (2013) 399403

cells, which subsequently appears like a sponge, and may

be caused by the action of noxious agents on the stratied epithelia [3,9]. In the present study, only two patients
showed hyperplasia of the epithelial basal layer. Excessive
proliferation of this layer constitutes a compensatory mechanism
for large losses of supercial cells, and is a common response in
esophageal stratied epithelia [1].
Conclusion
HIV infection can cause morphometric changes, including
epithelial thickening (due to increased cell numbers) and a higher
number of cell layers in the tongue and gingivae. The majority
of HIV-positive individuals had more epithelial layers, although
this difference was not signicant when compared with the
HIV-negative group. Notably, cells from the T+ samples exhibited increased cell diameter, and the tongue showed the most
intense changes among the various oral regions. HIV/AIDS infection is associated with morphological epithelial changes in the
cheek, gingivae and tongue, including intracellular deposits (glycogenic acanthosis and ballooning) and intercellular spongiosis.
The data suggest an association with viral load and thickness
of epithelium. HIV infection does not seem to play a signicant role in basal layer hyperplasia or exocytosis of inammatory
cells. These are interesting trends but were often not statistically signicant, likely because of the relatively small size of the
samples studied. This highlights the need for this work to be
extended into larger groups of patients with similar characteristics.
Conicts of interest
The authors had full freedom of manuscript preparation, and
there were no potential conicts of interest.
Acknowledgments
This work was supported by National Council for Scientic and
Technological Development (CNPq), Coordination for the Improvement of Higher Education Personnel (CAPES), the Minas Gerais State
Research Foundation (FAPEMIG), and the Teaching and Research
Foundation of Uberaba (FUNEPU).

403

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