HPV multisite
Med Oral Patol Oral Cir Bucal. 2020 May 1;25 (3):e425-30.
Journal section: Oral Medicine and Pathology
Publication Types: Research
doi:10.4317/medoral.23462
http://dx.doi.org/doi:10.4317/medoral.23462
Oral Human Papillomavirus: a multisite infection
María Inés Criscuolo 1, Rosana A. Morelatto 1, Paola A. Belardinelli 1, Jessica Mosmann 2, Cecilia Cuffini 2,
Silvia A. López de Blanc 1
1
2
School of Dentistry, Universidad Nacional de Córdoba. Córdoba, Argentina
Virology Institute. School of Medicine.Universidad Nacional de Córdoba. Córdoba, Argentina
Correspondence:
School of Dentistry
Universidad Nacional de Córdoba
Haya de la Torre s/n, Córdoba, Argentina
maria.criscuolo@unc.edu.ar
Received: 24/09/2019
Accepted: 17/02/2020
Criscuolo MI, Morelatto RA, Belardinelli PA, Mosmann J, Cuffini C,
López de Blanc SA. Oral Human Papillomavirus: a multisite infection.
Med Oral Patol Oral Cir Bucal. 2020 May 1;25 (3):e425-30.
Article Number: 23462
http://www.medicinaoral.com/
© Medicina Oral S. L. C.I.F. B 96689336 - pISSN 1698-4447 - eISSN: 1698-6946
eMail: medicina@medicinaoral.com
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Abstract
Background: The Human Papillomavirus (HPV) has different strategies for persist in the cells. This characteristic
has led us to consider the presence of the virus in tissues of the oral cavity that had no clinical signs of infection.
The aim of this study was to detect the presence of DNA-HPV at multiple sites of the oral cavity.
Material and Methods: A case-control study was designed: Oral Squamous Carcinoma Group (OSCG), healthy
n=72 and Control Group (CG), n=72, healthy volunteers paired by sex and age with OSCG. Four samples were
taken from OSCG: saliva, biopsy, brush scraping of lesion and contralateral healthy side. In CG a saliva sample
and a scratch of the posterior border of tongue were collected. HPV was detected by PCR using Bioneer Accuprep
genomic DNA Extraction kit, and consensus primers MY09 and MY11. Chi square test was applied.
Results: 432 samples were obtained from 144 individuals. DNA-HPV was detected in 30 (42%) of OSCG subjects
and 3 (4%) of CG. Two or more positive samples were obtained in 67% of the OSCG, 67% in saliva and 60% in
biopsy; in CG 100% of the individuals were positive in the two samples.
Conclusions: HPV is frequently present in oral cavity as a multifocal infection, even without the presence of clinical lesions.
Key words: HPV, Oral cavity, Saliva, Oral cancer
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Introduction
cavity, which means, with any oral clinical lesions at the
time of this study, paired by sex and age (+/- 5) with the
OSCG. This group was relieved from a population study
titled “Survey of oral health parameters in the adult
population from Córdoba city 2014–15” done in the city
of Córdoba, Argentina. A multistage cluster sampling
was conducted, with random selection of census tracts,
probability proportional to size, blocks, households, and
persons; the same number of individuals per tract was
selected. The included subjects were asked to attend a
health center in the area. Pregnant women and subjects
under age 18 were excluded. The results were informed
by phone or via mail to patients and healthy controls and
they were also clinically controlled.
- Data collection
Clinical history of all patients was recorded, including
personal data, personal and hereditary records, risk factors and oral health state. Subjects were inspected by
personnel trained for clinical diagnosis. OSCG were
subjected to biopsy to obtain diagnostic certainty. The
data on tobacco consumption was made according the
number of pack year smoked; a subject that smoked fifteen or more pack years in twenty years was considered
a smoker (13). One alcohol unit a day (one drink) was
considered as regular alcohol consumption (14).
- Sample collection
Four samples per patient were obtained from the OSCG:
1-unstimulated whole saliva collected in sterile tube;
2-cytobrush of lesion; specimens were placed in sterile tube containing 2 ml phosphate buffer solution with
antibiotic and antifungal agents; 3-cytobrush of healthy
contralateral mucosa to the lesion site, as in 2; and 4biopsy of lesion; samples were placed in sterile tube
in fresh conditions. All samples were preserved at 4°C
until DNA extraction. The CG group was subjected to
saliva sampling and cytological analysis of the posterior
border of the tongue, using the same technique as in
OSCG. In this group, we chose this area for being one of
the most exposed to micro-wounds that would facilitate
the entry of the virus.
- Sample processing
DNA-HPV was detected via PCR using Bioneer AccuPrep genomic DNA Extraction kit, and consensus primers MY09 and MY11. These primers amplified a fragment of approximately 450 base pairs corresponding to
the highly conserved L1 region of the virus genome.
The method involved a 50 μl reaction mix containing 10
μl DNA, 1.5 mM MgCl2, 200 μM of each dNTP (dATP,
dCTP, dGTP and dTTP), 10 pmoles of each oligonucleotide primer and 1 U Taq DNA polymerase (Promega
Corporation, Madison, USA). The temperature profile
used for amplification constituted an initial denaturation at 94°C for 3 min followed by 35 cycles with denaturation at 94°C for 1 min, annealing at 55°C for 1 min
and extension at 72°C for 1 min which was extended
The first cells to become in contact with the human papillomavirus (HPV) in the oral cavity, as in the genital
mucosa, are undifferentiated cells of the basal layer of
the epithelium. This contact implies the previous existence of ulceration or micro-wounds on the epithelial
surface that allows its entry (1-3). Viral DNA and RNA
transcriptions occurs in the basal layer, whereas viral
replication occurs in the most differentiated layers of
the epithelium (4,5). This association between viral replication and progressive differentiation would involve a
strategy to persist in a host with high cell proliferation
(6). In addition, the virus can evade the innate immune
system, delaying the adaptive immune response. Infected cells are pushed out towards the epithelial surface,
away from the circulating immune system, and therefore no viremia occurs. In conclusion, the lack of cell
death and inflammatory response makes HPV a “successful microorganism” for infection (7). This characteristic of the virus has led us to consider the possible
presence of HPV in saliva or samples of other tissues
of the oral cavity that had no clinical signs of infection
and to think whether the presence of the virus could
occur silently or latently in multiple sites of the oral cavity simultaneously. We also consider, as other authors,
that areas of constant rubbing in the mouth, such as the
lateral border of the tongue, could be the first site of
exposure to the virus (8).
We do not find in the current literature at our disposal,
studies on the presence of simultaneous HPV infection
in different tissues of the oral mucosa. Nevertheless,
some authors reported the presence of HPV in different
sites of the anogenital tract without clinical symptoms
of infection. These studies report cases of simultaneous
infection of the oral and genital mucosa (9-11). Studies on the prevalence of oral HPV infection in healthy
individuals have revealed a high variability in different populations, with values ranging between 0.2% and
20.7% (12). Tables shows prevalence of oral HPV infection in healthy individuals of different populations.
The objective of this work was to detect the presence of
DNA-HPV in multiple sites of the oral cavity.
Material and Methods
A case-control study was designed and conducted between 2010 to 2017, which was approved by the Institutional Committee of Ethics in Health Research
from the School of Dentistry, Universidad Nacional de
Córdoba (CIEISN°6/2010). All the subjects signed an
informed consent.
- Study groups
Two groups were considered:
a- Oral Squamous Carcinoma Group (OSCG): 72 patients with diagnosis of oral squamous cells carcinoma;
b- Control Group (CG):72 clinically healthy at the oral
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for 5 min in the final cycle. Typing was performed by
means of the analysis of the amplified product using
the enzyme restriction fragment length polymorphism
(RFLP) with the following restriction enzymes: Bam
H1, Dde I, Hae III, Hinf I, Pst I, Rsa I, and Sau3 AI. A
mixed of PCR product and buffer was prepared to each
enzime separately for subsequent addition of individual restriction enzymes. The mix included 6.5 µl PCR
product, 2 µl buffer, 1.5 µl restriction enzyme, followed
by incubation 40ºC for 60 min. The result was a characteristic banding pattern visualized with UV light (15).
Subjects with at least one positive sample were considered HPV+, this means an HPV infection of the sample.
- Statistical analysis
Data were expressed as relative frequencies; data association was analyzed using the chi square test, and OR
was used to reveal the intensity of the association.
Results
Two study groups were formed with 144 subjects, corresponding 72 to each group. The total of samples obtained
from both was 432. Table 1 describes the general characteristics of both groups or of each of the two groups.
- Sample analysis: DNA-HPV was detected in 30 (42%)
patients of OSCG and 3 (4%) in subjects of CG. The
analysis and comparison of the results of OSCG with
controls showed a highly significant difference, with an
OR of 16.43 (CI: 5.10-52.94), p<0.0001 (Table 2).
- DNA-HPV at multiple sites: 67% of the HPV+ patients
of OSCG had two or more positive samples. In the case
of CG, 100% of the cases were positive in both samples
Table 3 describes the distribution of positive samples in
both groups. Four positive samples prevail in OSCG,
where the most frequent positive sample was in saliva
(67%), followed by biopsy (60%).
Table 1: Prevalence of oral HPV infection in healthy individuals.
Year
Country
Sample
1994-96
Grace
cytobrush
2000-02 Miyako Island, Japan cytobrush
2006
SJR Preto, Brazil
cytobrush
2000-06
Ohio, USA
oral rinse
2007
Sardinia, Italy
saliva
2006-7
Brazil
cytobrush
2009-10
Ohio, USA
oral rinse
2009-11
China
oral swabs
2012
Modena, Italy
cytobrush.
2013
Australia
oral rinse
Sample
size n
169
668
50
542
164
100
5579
5351
81
307
14-85
3-85
16-52
25-87
4-77
20-31
14-69
25-65
49-77
18-35
Prevalence
n (%)
19 (9.5)
4(0.6)
7(14)
22(4.06)
30(18.3)
0
385(6.9)
36 (0.67)
1(1.2)
7(2.3)
Age
2014-15
Treviso, Italy
oral rinse
500
19-35
20(4.0)
2014-15
Córdoba, Argentina
cytobrush
401
18-87
13(3.3)
Genotypes
Author
6/16/11
Lambropoulos et al.
16/53/71/12
Kurose et al.
18/18/52/61
Do Sacramento et al.
16/51/56/59/6/1
D Souza et al.
1/42/16/31
Montaldo et al.
NS
Esquenazi et al.
16/62/55/84/72
Guillison et al.
16/45/58
Hang et al.
16
Migaldi et al.
18/16/67/69/90
Antonsson et al.
16/56/6/11/81/5
Lupato et al.
2/30/53/90
11/84/6/81
Criscuolo et al.
MI Criscuolo, et al. Translational Research in Oral Oncology 3: (1–8) 2018.
Table 2: General characteristics of the study groups.
Characteristics
Gender
F
M
Mean age
Tabaco
Yes
No
Alcohol
Yes
No
HPV
Yes
No
Total
TOTAL
n (%)
OSCG
n (%)
CG
n (%)
66(46)
78(54)
62
33(46)
39(54)
64,5
33(46)
39(54)
63
61(42)
83(58)
37(51)
35(49)
24 33)
48(67)
71(49)
73(51)
35(49)
37(51)
36(50)
36(50)
33(23)
111(7)
144
30(42)
42(58)
72
3 (4)
69 (96)
72
OSCG: Oral Squamous Cancer Group, CG: Control Group, OR: Odd Ratio
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OR
P
------
------
------
------
2(1,08-4,12)
p<0,03
0,92(0,48-1,76)
p<0,87
16.43(5.10-52.94)
p<0.0001
HPV multisite
Med Oral Patol Oral Cir Bucal. 2020 May 1;25 (3):e425-30.
Table 3: Distribution of HPV in different sites.
GROUP
OSCG
One site
2 sites
3 sites
4 sites
CG
One site
2 sites a
a
Saliva
n (%)
Biopsy
n (%)
C.lesion
n (%)
C.healthy
n (%)
TOTAL
n (%)
3(30)
6(33)
5(33)
6(25)
2(20)
7(39)
3(20)
6(25)
3(20)
3(17)
4(27)
6(25)
2(20)
2(11)
3(20)
6(25)
10 (15)
18 (27)
15 (22)
24 (36)
3(50)
-
-
3(50)
6 (100)
In CG samples were taken only from saliva and posterior border of the tongue
When positive patients in two or more sites in the OSCG
were analyzed and was related with the tumors site, we
observed that the 65% were located at the border of the
tongue and oropharynx.
- Genotypes: The genotypes were 16/ 6/ 11/ 31/ 33/ 45/
52/ 53/ 69, with 16/ 6/ 11 being the most frequent. In
nine samples of the OSCG it was not possible to identify
the genotype. In the OSCG, 12 patients (40%) had highrisk genotypes: 16/ 52/ 45/ 31/ 69 and 33, with 50% of
these being HPV 16. Of the remaining positive types,
9 patients (30%) were of low risk (6/11); one of these
patients had combination of genotypes 6 and 11, and of
the remaining nine patients (30%) were not genotyped.
In CG all genotypes were of low risk (11/84).
the samples taken in the present work in the carcinoma
group. It should be noted that although they obtained
similar percentages in both sexes, 51% of HPV positive
males in the oral cavity had simultaneous genital infection, resulting in a significant association, with an OR of
3.63 (CI 1.37-9.58) (9). On the other hand, Ucciferri et al.
studied HPV in 90 males, 45 HIV+ and 45 HIV-, in oral,
anal, urethral mucosa and coronal sulcus samples. The
results showed that 59% of the subjects had infection at
multiple sites, 34% at two sites and 22% at three sites.
It is important to note that in oral mucosa HPV positive
patients did not exhibit different values between HIV
positive and negative subjects (10). In a similar study
(20) where studied HPV at oral, genital and anal mucosa
in HIV positive and negative males, observed that the
virus was detected in the three samples simultaneously
in 14% and 10% respectively. In Peru, Blas et al. studied the detection of HPV at multiple sites in men who
had sex with men, HIV positives and negatives; they
obtained samples from oral, anal, and genital mucosa
and blood. Although the report does not provide the percentage of HPV positive cases at several sites simultaneously, at the oral cavity level the values were similar
in both groups, with 8.5% in HIV positive subjects and
9.9% in HIV negative subjects. The highest HPV percentage recorded was in anal mucosa of HIV positive
patients; high risk genotypes were the most prevalent
(86%) in this group and at this site, and HPV 16 was the
most frequent. In oral mucosa, HIV positive cases had
a higher percentage of high-risk genotypes than HIV
negative cases (21,22). In our study, the most frequent
genotypes were 16/6/11 and the oral cancer group was
the one with highest percentage of high-risk HPV and,
as in the previously mentioned study, HPV 16 was the
most frequent genotype. In the current clinical cases,
we do not find HPV 18, a high-risk genotype that is frequently found in the literature (23). Table 4 compares
the prevalence of HPV in OSCC patients of different
studies and the highest frequency of HR-HPV in these
groups (24).
Discussion
The results of the present work show the prevalence of
positive samples in more than one site of the oral cavity
in DNA-HPV positive subjects of the groups studied.
We also observed that in healthy mucosa the virus was
present despite the lack of clinical signs of infection.
In fact, 100% of controls and 43% of patients with oral
cancer were positive in healthy mucosa.
Some authors assume that HPV could present as a multifocal infection in different mucous membranes (1619). Our results effectively confirm that oral DNA-HPV
appears as a simultaneous infection in different sites of
the mouth.
In spite of not finding in the current literature, studies like ours, we found some authors who had studied
the virus in different conditions such as HIV positive
patients or groups at risk for diseases such as sexually transmitted diseases. Authors like Ciccarese et al.
studied the HPV in a risk group of sexually transmitted diseases, and evaluated the presence of the virus at
different sites with no clinical signs of infection. They
took samples from the oral, genital and anal mucosa.
The highest values were recorded in genital samples,
and 37% of HPV positive values were obtained in the
oral cavity. This result is similar to the values found in
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Med Oral Patol Oral Cir Bucal. 2020 May 1;25 (3):e425-30.
Table 4: Prevalence of HPV in OSCC patients.
Year
2003
2004
2008
2009
2016
2010
2010
2011
2016
2018
2019
Sample Prevalence
HR-Gen
LR-Gen
size n
n (%)
n (%)
n (%)
Germany
118
51 (43.2) 41 (34.7) 54 (100) 10 (4.2)
Shanghai, China
73
54 (74)
14 (58)
--Madrid, Spain
33
24 (73)
29 (100)
10 (42)
India
45
29 (64.5)
--Khon Kaen, Thailand
91
27 (29.7)
24 (92)
3 (7)
Thailand
62
15 (24.19)
10 (67)
5 (33)
India
222
154 (74)
154 (100)
--Allahabad, India
45
39 (87)
39 (100)
--China
178
26 (14)
26 (100)
--Guntur, India
20
11(55)
11(100)
--Jakarta, Indonesia
78
14(17.9)
14(100)
--Country
We believe important that these results of virus detection and the expression of predominant genotypes in
both sexes are useful to support educational campaigns
and the need to apply HPV preventive vaccines. Although the World Health Organization recommends the
inclusion of males in HPV vaccination strategies, only
in some countries like Argentina these are applied preventively since 2017, in both sexes (25).
Regarding our findings of a high positive DNA-HPV
percentage in saliva, similar to that in tissue subjected
to biopsy, several authors have discussed the utility of
detecting the virus in this fluid. Zhao et al. analyzed
HPV 16 in saliva of patients with head and neck carcinoma and compared it with a healthy control group;
they found significant differences between groups (26).
In our results, saliva samples of all the HPV positive
cases of the healthy volunteer group were positive, despite the lack of lesions suggestive of DNA-HPV infection.
Genotypes
Author
16/18/6/11
16/18
16/31/6/11
16/18
16/45/13/18
16/18/6/11
16
16/18
16/18
16/18
16/18
Ostwald et al.
Zhang et al.
Llamas-Martínez et al.
Mathew et al.
Phusingha P et al.
Laouli et al.
Chaudhary et al.
Khanna et al.
Chen et al.
Chowdary SD et al
Purwanto DJ et al.
6. Molijn A, Kleter B, Quint W, van Doorn LJ. Molecular diagnosis of
human papillomavirus (HPV) infections.J Clin Virol. 2005;32:43-51.
7. Okami K. Clinical features and treatment strategy for HPV-related
oropharyngeal cancer. J ClinOncol. 2016;21:827-35.
8. Lee SY, Cho EC, Baek SJ, Kim WS, Shin DH, Kim SH. Relevance
of human papilloma virus (HPV) infection to carcinogenesis of oral
tongue cancer. Int J Oral MaxillofacSurg. 2010;39:678-83.
9. Ciccarese G, Herzum A, Rebora A, Drago F. Prevalence of genital, oral, and anal HPV infection among STI patients in Italy. J Med
Virol. 2017;89:1121-4.
10. Ucciferri C, Tamburro M, Falasca K, Sammarco ML, Ripabelli
G, Vecchiet J. Prevalence of anal, oral, penile and urethral Human
Papillomavirus in HIV infected and HIV non infected men who have
sex with men. J Med Virol. 2018;90:358-66.
11. Mendez-Martinez R, Rivera-Martínes NE, Crabtree-Ramirez B,
Sierra-Madero JG, Caro-Vega Y, Galvan SC, et al. Multiple human
papiloma infections are highly prevalent in the anal canal of human
immunodeficiency virus-positive men who have sex with men. BMC
Infectious Diseases. 2014;14:671-77.
12. Montaldo C, Mastinu A, Quartuccio M, et al. Detection and genotyping of human papillomavirus DNA in samples from healthy Sardinian patients: a preliminary study. J Oral Pathol Med. 2007;36:482-7.
13. Palmer EL, Hassanpour S, Higgins J, Doherty JA, Onega T.
Building a tobacco user registry by extracting multiple smoking behaviors from clinical notes. BMC Medical Informatics and Decision
Making. 2019;19:141.
14. Pentenero M, Broccoletti R, Carbone M, Conrotto D, Gandolfo
S. The prevalence of oral mucosal lesions in adults from the Turin
area. Oral Dis. 2008;14:356-66.
15. López-Revilla R, Martínez-Contreras LA, Sánchez-Garza M.
Prevalence of high-risk human papillomavirus types in Mexican
women with cervical intraepithelial neoplasia and invasive carcinoma. Infectious Agents and Cancer. 2008;3:3.
16. Syrjänen S. Human papillomavirus (HPV) in head and neck cancer. J Clin Virol. 2005;32:59-66.
17. Barzon L, Militello V, Pagni S, et al. Distribution of human papillomavirus types in the anogenital tract of females and males. J Med
Virol. 2010;82:1424-30.
18. Esquenazi D, Filbo IB, Da Costa Carvalbo MG, Souza de Barros F. The frequency of human papillomavirus findings in normal
oral mucosa of healthy people by PCR. Braz J Otorhingolaryngol.
2010;6:78-84.
19. Brouwera AF, Mezaa R, Eisenberg MC. Transmission Heterogeneity and Autoinoculation in a Multisite Infection Model of HPV.
Math Biosci. 2015;270:115-25.
20. van Rijn VM, Mooij SH, Mollers M, Snijders PJ, King AJ, de
Vries HJ, et al. Anal, Penile, and Oral High-Risk HPV Infections and
HPV Seropositivity in HIV-Positive and HIV-Negative Men Who
Have Sex with Men. PLoS ONE. 2014;9:1-9.
Conclusions
Oral cavity HPV often occurs as a multifocal infection,
even without the presence of clinical lesions. The current vaccination scheme in Argentina prevents against
the genotypes circulating in our medium. Future studies
are necessary to assess the clinical and epidemiological
implications of the virus presence in saliva.
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Acknowledgements
The authors are grateful with the authorities of the Municipal Dentistry Institute who facilitated the health centers for the oral examination of the group of healthy volunteers. We also say thanks to the
personnel of the Department of Statistics and Demography of the
Faculty of Economic Sciences and the University Statistics Program,
especially Professor Martin Saino; and to the Secretariat of Academic Affairs of Universidad Nacional de Córdoba for their advice
regarding the population sampling.
Funding
This study was financially supported by SECyT, Universidad Nacional de Córdoba, Córdoba Argentina.
Conflicts of interest
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Ethics
Approved by the Institutional Committee of Ethics in Health Research from the School of Dentistry, Universidad Nacional de Córdoba (CIEISN°6/2010).
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