Background: Human papillomaviruses (HPV) are implicated in cervical cancer and, recently in oral cancer. In Mexico, there are few studies on oral cancer, therefore the interest in identifying the HPV frequency of low and high risk in samples of the oral and cervical cavities, and determining some risk factors. Objective: To determine the frequency of high and low risk HPV infection in the oral cavity of women with cervical HPV , and to correlate the infection site with risk factors. Materials and Methods: Eighteen female patients between 24 and 53 years, with antecedents of genital HPV infection were included. Both samples of oral cavity and cervix were obtained. DNA extraction from the epithelial cells was performed using the Qiagen kit. PCR was done and the amplicon was observed in 2% agarose gels stained with ethidium bromide. A correlation of HPV infection and risk factors was done. Results: HPV-DNA was detected in the 67% of both samples. The frequency of oral and cervix low risk HPV-DNA was 50%, while high risk HPV-DNA in oral cavity was detected in 17%, and 39% in the cervix. The study of the risk factors involved in HPV infection showed that the participants had the habits of smoking 39%; alcohol drinking 28%; and 78% oral sex. Conclusion: The results showed a high frequency of HPV (67%) infection in the oral and genital mucosas, suggesting that patient’s habits could contribute to the infection; t he presence of HPV in the oral mucosa may act as reservoir for new HPV infections.
The human papillomavirus (HPV) infection can be grouped into cutaneous and mucosotropic types. The mucosotropic HPVs are typically found in the anogenital mucosa and oral mucosa. Genital infection can be transmitted to oral mucosa through autoinoculation, oral sex, or oral contact [1,2]. The HPV related genital lesions are more frequently in adults. In Mexico, cervical-uterine cancer (CUC) is the most frequent sexually transmitted viral disease and cause of death in women older than 25 years [3,4]. A link between HPV and squamous cell carcinoma of the head and neck was suggested more than 20 years ago [
More than 130 types of HPV have been identified according to the nucleotide sequence alignment of its open reading frames [
We studied 18 women, aged between 24 and 52 years, who came to the Gynecology and Obstetrics Services of the Hospital Español with background of genital infection by HVP, and with clinically normal oral mucosa.
Smears of eighteen patients positive to HPV-DNA at the cervix with clinical and histopathology confirmation of high-grade squamous intraepithelial lesion were taken. Ten out of eighteen patients had previous treatment with cryotherapy between November 1991 to February 2006, and eight women were newly diagnosed with HPV. Cryotherapy was performed in ten patients using a single freeze technique with nitrous oxide as refrigerant, and it was applied to the cervix. The details of the study were explained to all participants and also an informed consent was provided before entering into the study. Information on age, education, smoking/alcohol drinking habits and sexual behavior was collected. A questionnaire was applied to gather information on personal and clinical data to determine some risk factors.
Two samples were taken per patient. Cervical cells were obtained by using Ayre’s spatula and an endocervical cytobrush, oral samples were collected by brushing (Oral CDX; CDx Labs). Ten complete backward and forward brushes at each oral site (upper and lower gum, cheeks, and dorsum of tongue) were performed. The brush was squeezed immediately into the side of a tube containing 1 ml of cold phosphate-buffered saline solution stored at −20˚C until use. After collection, both cervical and oral specimens were centrifuged at 2000 g × 10 min and washed with saline solution, centrifuged at 12,000 g × 10 min, and the pellet stored at −20˚C.
For DNA extraction, samples were centrifuged at 2000 × g for 10 min and DNA was extracted from the pellets using the QIAamp DNA Mini kit (Qiagen Maryland, USA), according to the manufacturer’s instructions. To adsorb the nucleic acids of DNA a silica-gel column containing buffer of (10 mM Tri-HCl; 0.5 mM EDTA), and stored at −70˚C until used.
In order to identify HPV-DNA of high or low oncogenic potential, we utilized two consensus sequence primer pairs within the E6 and E7 open reading frames (ORF) to amplify HPV DNA [
DNA from HeLa and CaSki cells was used as positive controls of high risk, and DNA genotype 6 as low risk control. And as negative control DNA from HEp-2 cells or distilled water instead of DNA was used. All controls were obtained from the American Type Culture Collection (ATCC) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as internal control. PCR samples was separated by electrophoresis in a 2% agarose gel (Sigma-Aldrich) containing ethidium bromide (Sigma), and the gels were visualized under UV light with a transiluminator (UltraLum, California, USA).
The following primers was used to identify HPV-DNA; 1ul (100 mM) HPV high risk sense: (5’-TGT-CAA-AAACCG-TTG-TGT-CC-3’); HPV-DNA high risk antisense: of 1 ul (100 mM); (5’-TGC-TAA-TTC-GGT-GCT-ACCTG-3’);
HPV low risk sense: 1 ul (100 uM) (5’-GAG-CTGTCG-CTT-AAT-TGC-TC-3’), HPV low risk antisense: 1ul (100 mM) (5’-TGC-TAA-TTC-GGT-GCT-ACC-TG -3’) [
The association between HPV infection and potential risk factors (smoking/drinking habits and oral sex) was evaluated with Chi square test/Fisher’s exact test with software GraphPad Prism 5. The predetermined level of significance was p < 0.05.
The study group comprised 18 women (mean age: 36 years; range: 20 - 53 years) with cervical HPV infection.
The analysis of HPV-DNA specimens are shown in
In the oral and cervical cavities of the eighteen patients low HPV-DNA was detected in 8/18 (44%); high risk HPV in both cavities 2/18 (11%); in the cervix of one patient 1/18 (6%) was detected either low and high risk HPV-DNA, whereas, one patient exhibited high risk HPV-DNA in the oral cavity (6%) but not in cervix; and in 3/18 (17%) was detected only high risk HPV-DNA in the cervix not in the oral cavity (
not detected in 3/18 (17%) neither oral cavity nor cervix; and 8/18 (44%) were newly HPV-DNA diagnosed.
The analysis of smoking/alcohol drinking/sex habits in the eighteen patients showed that 7/18 (39%) presented smoking habit; 5/18 (28%) alcohol drinking habit; 14/18 (78%) practiced oral sex (p < 0.02). Regarding the participants age we observed that the most affected group with HPV-infection was the age group between 42 - 53.
Molecular, clinical and epidemiological studies have verified that the HPV is the main etiological agent of CUC [11, 27-30]. HPV infections represent one of the most common sexually transmitted viral diseases with frequencies going from 30% to 50% in sexually active women [
HPV-DNA virus.
The results of our study showed that high risk HPVDNA was more prevalent in the cervix of women 39%, whereas oral high risk HPV-DNA was detected only in 17%. A predominance of low risk HPV-DNA in oral cavity and cervix (50%) was observed (
A study was conducted to determine the HPV prevalence and concurrent infection in the cervix and oral cavity of 577 pregnant women, found 29% positivity in the cervix and 2.4% positivity in the oral cavity. No association was found between HPV positivity and its types detected in the cervix and oral cavity of these women, suggesting the author that selfinoculation was uncommon [32-34].
The finding that the high risk HPV-DNA in oral cavity was found in less proportion (17%) than in cervix (39%) could have the follow explanation (
Our results showed that the higher of HPV positivity was in the oldest group age participants (43 to 53 years) was infected with low risk and high risk HPV, this could be explained partially because the infection with high risk genotypes (16 and 18) involves factors that influence the acquisition and progression of the injury degrees caused by HPV infection, the most common ones are: early start of sexual activity, the number of sexual encounters (in women with only one partner, HPV is observed in 17% to 21% and increases to 69% - 83% in those with more than five sexual partners), an inadequate immune response, hormonal action (including the prolonged use of oral anticonceptives associated with a higher susceptibility to malignant progression), nutritional deficiencies, other sexually transmitted infections, and genetic predisposition (haplotypes of the major histocompatibility system associated to a greater susceptibility to malignant progression) [35-39]. Other possibility is that this finding could be the result of an accumulated lifetime exposure to the viruses, with the added risk of sexual activity. In this work the statistical analysis of risk factors showed that oral sex habit (p < 0.02) is a dependent variable that could promote the HPV establishment in the oral cavity, so these data suggest that oral cavity may be a reservoir of HPV in these patients. In our study we found that the oral sex habit was present in the oral cavity of women with cervical HPV (p < 0.02), if subjects with cervical HPV practice oral sex, then HPV could be established in the oral cavity with any problem.
Our results showed a high prevalence from 78% (14/18) of oral sex habits, so probably this risk factor could be relevant in the Mexican population, but it has to be analysed a higher number in order to conclude the importance of the oral sex habit in the HPV transmission. Interestingly, 7 out of 10 patients treated with cryotherapy between November 1991 to February 2006 and HPVDNA positive at the moment of the study, present the oral sex habit 6/7 (60%) so it could be important to determine if the oral cavity is an important reservoir for reinfections or cryotherapy is not an adequate treatment to eliminate HPV.
On the other hand, studies have shown that smoking has potential to alter oral epithelium, thus it has and influence on HPV expression in oral cavity [
D’Souza et al. [
It has been theorized that oral infections are likely the result of oral-genital contact. The present study suggests and could expands on that theory by demonstrating that not only are oral-sexual behaviours important, but more common habits, such as smoking or drinking alcohol, may also contribute to the HPV infection to the oral region. It could be interesting to evaluate whether the oral cavity could be not only the reservoir for HPV, but for other pathogens [
Funding: This work was supported by the Departamento de Microbiología y Parasitología, Facultad de Medicina UNAM and the Instituto Nacional de Enfermedades Respiratorias (INER).
The Ethical Approval was given by the Ethics committee of the INER with the reference number C54-11.