The diagnosis of human papillomavirus in the oral mucosa may be suspected when one inspects the lesion, and also through cytology and biopsy. The cytological aspect of the HPV infection is characterized by:

• A.

  Major criteria: classic coilocytes, perinuclear cytoplasmic halos and nuclear dysplasia.

• B.

  Minor criteria: dysceratocytes, atypical immature metaplasia, macrocyte and binucleation. This method bears limited sensitivity and does not type the HPV9,10.

The methods used to detect the HPV DNA in lesions vary broadly as far as their sensitivity and specificity are concerned11. These are further broken down in three categories:

Low Sensitivity: immunohistochemistry and in situ hybridization - because they only detect the virus when it is present in more than 10 copies of the viral DNA per cell.

Moderate Sensitivity: Southern blot, dot blot and reverse dot hybridizations, because they only detect the virus when present in 1 to 10 copies of the viral DNA per cell; and the High Sensitivity: PCR, because it detects the virus in less than 1 copy of the viral DNA per cell12.

Immunohistochemistry may detect the protein coat of HPV viral particles which are present in the lesions seen under light microscopy in paraffin bearing material or in cytological preparations, and in these cases, polyclonal antibodies are used against different types of HPV specific antigens9. This technique is hampered by the limited availability of antibodies against specific types of HPV, since the virus does not replicate in vitro. Commercially available antibodies are created against bovina papillomavirus capsule antigens, with an HPV crossed reaction13.

Hybridization tests are currently the methods of choice to detect HPV DNA or RNA in smears or tissue samples. They are made directly or after DNA and RNA amplification through PCR. The basic principle behind these hybridization techniques is the double formation between the DNA single strand, RNA molecules or cloned HPV types, denaturated DNA derivatives and viral nucleic acid molecules present in the cell, which represents the hybridization test target10.

The Southern blot hybridization is used in order to detect the HPV DNA from biopsies and is considered the “gold standard” to detect HPV genome. It is a sensitive and highly specific test, thus proving to be a valuable research tool, however it has no application in clinical routine tests because it takes longer and it requires harder work10.

Another recently developed hybridization method is the hybrid capture (HCA), which does not distinguish among the specific HPV types; and their applicability as a research method is limited, however it may represent a good test for routine clinical use10.

Polymerase Chain Reaction (PCR) is a technique that has revolutionized virology, because of its extremely high sensitivity5. It is characterized by the amplification of minute amounts of target DNA sequences in many million times. It is a cyclic thermal process that includes three stages: denaturation, in which the double DNA strand is separated in single strains; coupling, in which the primers couple specifically with their complementary single strand target-DNA sequences; and finally, the primer extension, in which a thermostable DNA polymerase generates DNA “offspring” strands which cross the region between two primers. From then on, recently generated double strands serve as models for a subsequent PCR cycle. The primers may be: type-specific primers, which detect a simple type of HPV, or the consensus primers (also called general or generic), which detect a whole array of different HPV types in a single reaction14.

PCR detection of HPV is usually carried out through one of the consensus primers, the MY09-MY11 or the GP5/GP610,15. Currently, PCR allows for a more in depth assessment of epidemiologic data, including the prevalence of latent of subclinical infections. However, it bears the following disadvantages: amplification of minute amounts of contaminating HPV DNA, which may lead to false-positive results. Each method is limited by its sensitivity, specificity, its practice, cost and its commercial availability. It is important and fundamental to assess the efficacy of different HPV detection techniques, in order to establish HPV etiology in oral lesions10,16.

HPV prevalence in the oral cavity and oropharynx is uncertain. Studies have shown dubious results, assessing a small number of patients and identifying some of the many HPV types found in mucosal lesions17.

So far, over 100 types of HPV have been identified18,19. Of these, 25 types have been associated to oral lesions (HPV-1, 2, 3, 4, 6, 7, 10, 11, 13, 16, 18, 31, 32, 33, 35, 40, 45, 52, 55, 57, 58, 59, 69, 72 and 73)20.

In order to explain the HPV infection relationship with many oral lesions, it is important to investigate HPV prevalence in the normal oral mucosa.

Papillomavirus in normal oral mucosa must be investigated throughout studies on oral cavity HPV infection natural history. Its prevalence in the normal oral mucosa is controversial21.

In the normal oral mucosa we have seen a great variation in HPV rates detected, from 22% to 60%17; from 0% all the way to 81.1% in studies using different methods, and with a limited number of individuals, and this seems to depend on the population studied and the choice of method16,21. It has been suggested that HPV prevalence in the normal mucosa includes subclinical and/or latent infections, and that the infection with a low number of virus copies is common in the oral cavity8.

Table 1 depicts the techniques and the results obtained by different authors as to the identification of the HPV type in the normal oral mucosa.

Human papillomavirus prevalence in benign lesions (squamous cells papilloma, condiloma acuminata, common wart and focal epithelial hyperplasia of the oral cavity and oropharynx related to the virus).

Oral and oropharynx squamous cells papilloma are benign tumors that occur mainly between 30 and 50 years of age, although they may also occur below 10 years of age22. They represent about 8% of oral tumors in children23.

They usually involve the soft palate, tongue, frenulun linguae and the lower lip. In most of the cases, the papillomas are single and small (<1cm)24. They have an exophytic growth and show up both as a broad based ovoid bulging, or a pedicled lesion. The surface may present small finger-like projections, giving it a rough verruca contour. The color varies from white to pinkish, depending on the levels of keratinization and vascularization25.

Oral condiloma acuminata is usually considered a sexually transmitted disease, acquired by oral sexual contact. Currently, the trend is to consider that the oral condiloma may be acquired not only by oral Sex, but also by self inoculation or as a result of maternal transmission20,25,26.

Clinical and histological similarities between squamous cells papilloma, condiloma acuminata and verruca in the oral cavity are clear27. This differentiation with PCE is difficult and purely academic. In the mouth they are usually present as small pinkish or whitish nodules, which spread in papillary projections and may be pedicled or sessile. The surface contour, in most of the cases, is cauliflower-like rather than papillomatous24. They occur alone or in multiple form, frequently on the tongue, lip, palate and floor of the mouth28.

In the genital tract, the terms papilloma and condiloma were used separately, until the year 1970; since then, both lesions are called condiloma. This modification may also be applied to oral lesions, because both the papilloma and the condiloma have the same HPV types found in genital condilomas20.

The human papillomavirus was detected in condilomatous oral lesions, initially by immunohistochemistry, and later by hybridization techniques, with a positiveness varying between 75% to 85% for HPV 6 and 1120,24.

In 1991, Zeuss et al.29, considered HPV 6 and 11 as being the types most frequently associated to benign papillomatous oral lesions.

The common wart is one of the most common skin lesions, specially in children24. They are clinically undistinguished from PCE and condiloma, appearing as a sessile papillomatous whitish lesion of rough surface25. They are frequently located on the lips, hard palate, gums and tongue dorsum30. The diagnosis of an oral wart must be restricted to a lesion with clinical and histological characteristics of a common skin wart, and confirmed by seeing the HPV types in the skin wart20.

Many authors showed the presence of the virus in oral warts by immunohistochemistry and hybridization tests. They observed a variation in the HPV detection rates from 43% to 100% in oral warts29, 31,32.

HPV prevalence in oral warts seems to be HPV 2, followed by the HPV 57, although more studies are necessary to identify the broad spectrum oral infectiosn caused by skin HPV types20,33.

Table 2 shows the techniques and the results of the studies about papillomas, condilomas and warts in the oral cavity and oropharynx.

The term focal epithelial hyperplasia (FEH), or Heck’s disease, introduced by Archard et al.34 in 1965, to describe multiple nodular elevations in the oral mucosa observed in Alaska Eskimos and North and South American Indians. It is rarely observed in caucasians35. It has also been described in Israel, South Africa and Sweeden36.

It is a benign lesion that may be located in the oral mucosa, lips and tongue, and most notably in the lower lip24, 37, 38. It is clinically characterized by multiple painless and soft papules, of color that varies between pale pink and the normal mucosal color. A strong family history has been suggested by a number of authors24,25,50.

Viral etiology has been initially shown by immunohistochemistry and later on by hybridization techniques identifying HPV 13 and 32, which were detected in 75-100% of the cases32.

HPV 13 and 32 were considered specific in focal epithelial hyperplasia, however, HPV 32 was also found in other oral lesions, but never outside the oral cavity36,39,40.

The studies and their results are depicted on table 3.

Unfortunately, oral cancer still bears high mortality rates. Its incidence rate varies from one region to another, being high in India, Sri Lanka, Vietnam, Filipinas, Hong Kong and Taiwan, where about 30% of all the cancers occur in the oropharynx. India has approximately 56,000 new cases per year, and is probably among the highest incidence rates in the world41.

It is a disease that affects patients in their 50th decade of life, being more evident between 60 and 70 years of age. Oral cancer includes malignant neoplasms in the lips, oral cavity and oropharynx42. Oral cancer is a severe and growing public health concern in Brazil, corresponding to 4% of all cancer types, occupying the eighth place among the tumors that affect men, and the 11th among women43.

The most common location for oropharynx cancers, is the palatine tonsils. Notwithstanding, lingual cancer corresponds to 30% of these44. About 90% of these lesions are located in soft tissues and are originated from squamous epithelium20.

Cigarette smoking and alcohol are the main causes of oral cancer. They act in synergy and are a dose-dependent12,17. However, part of the population develops oral cancer without having been exposed to these risk factors, suggesting other causes, such as: genetic predisposition, diet and viral agents, most specifically HPV 345.

The simultaneous presence of chemical agents and HPV infection in the oral mucosa may favor the malignant transformation46. Notwithstanding, the HPV role as an etiological agent in the oral cancer is less than that from alcohol and tobacco use because HPV infection prevalence is less than alcohol and tobacco use in the genesis of oral cancer20,47.

Syrjänen et al.48, in 1983, suggested that HPV may be involved in oral cavity squamous cell carcinoma, when they described HPV cytopathological alteration in oral cancer, identical to those previously found in uterine cervix cancer, although we still lack proof by other DNA hybridization methods.

Many studies have shown HPV 16 to be the most prevalent type in oral cancer, as it happens in anogenital cancer24,25,49.

In a 2001 metanalysis, Miller & Johnstone47, showed an HPV increase in oral epithelial carcinoma and dysplasias when compared to the normal oral mucosa, mainly in high risk genotypes. Results show HPV to be an independent risk factor for squamous cell oral carcinoma. In 2003, Herrero et al.50, studied HPV in oral cancer and observed a higher prevalence of HPV 16 in the oral cavity and the oropharynx among patients that had more than one sex partner and/or who practiced oral sex, while the lowest prevalence was seen among smokers.

According to many studies, the HPV prevalence rate in oral cancer varied from 0-100%17,25,29,51–54. This broad variation in HPV detection rate is explained by the different detection methods used in the HPV investigation25.

One of the greatest difficulties in identifying HPV in oral cancer patients is the presence of this virus in only one subpopulation of cells and the reduced number of copies of these cells which are infected. That is why we need high sensitivity detection methods55.

The verrucous carcinoma was first described as a variant of the squamous cells carcinoma, which originates in the oral cavity.

Also known as Ackerman’s tumor, its growth is exophytic, slow and invasive -only superficially, with a low metastasis rate, and it may be treated with simple excision. Many authors have reported the HPV presence in the verrucous carcinoma20,32,46,56–58.

Encompasses about 95% of all the oral cancers. Its clinical aspects vary from a nodule up to a chronic ulcer. Many researchers have shown HPV present in SCC18,59–62. Initially we did microscopy, immunohistochemistry and, more recently, molecular biology tests20.

Studies have shown the important role of HPV in head and neck cancers, and also suggest that HPV 16 may be involved in the development of some oral carcinomas56,60,63.

Table 4 shows oral carcinoma studies with their results and techniques.