When performing the molecular detection of HPV DNA from oral samples, attention should be focused on the low productivity of oral HPV infections. Indeed, studies based on polymerase chain reaction (PCR) assay have shown that, when compared to those from HPV-positive cervical specimens, HPV-DNA positive oral samples from both normal oral mucosa [
13,
48] and cancerous lesions [
49] produce weaker PCR products. Based on such data, it is then essential that, in clinical and research HPV testing, all procedures employed are highly sensitive, specific and reliable. In addition, the aim of enhancing the standardization of the approach, in terms of the type of oral specimen examined, sampling method applied and HPV molecular assay employed, should be pursued. As far as the oral sample to be examined is concerned, both tissue samples and superficial cells have been evaluated in literature [
48,
50,
51]. Biopsy tissue samples can be either frozen at -80°C and then minced without thawing for DNA analysis or they can be formalin-fixed and paraffin-embedded. Oral mucosa exfoliated cells can be obtained either by oral brushing or rinse, and advantages and disadvantages have been reported for each method. Tissue samples allow for the histo-pathological examination of the biopsies used for the HPV test, as well as permitting the
in situ hybridization and localization of HPV DNA in infected cells. However, the highest rate of HPV DNA is reported in DNA from frozen tissue, but the value is much lower for paraffin-embedded tissue samples [
48]. Oral scrapes or rinse samples, with their greater surface area of mucosa than with biopsies, are less invasive, and HR HPV detection in oral exfoliated cells is a reliable biomarker of an HPV-related HN cancer risk. A drawback is that not all patients who have HR-HPV types in oral exfoliated cells are detected with HPV DNA in the primary tumour [
50]. However, if HPV testing of oral exfoliated cells has been selected, the use of oral rinses would appear more efficient, in terms of cell yield and DNA-containing nucleated cells, compared with the superficial brushing/scraping of oral mucosa (by using a cotton swab, wooden spatula or a cytobrush) [
51,
52]. Of the several mouthwashes tested in the literature, commercial mouthwashes (
e.g., Cepacol
®, Listermint
®) would seem more efficient than sucrose, glucose and saline, in terms of DNA yield, quality and stability [
53]. As far as HPV detection methods are concerned, it has been reported that methods such as Southern blotting and
in situ hybridization should be avoided as they lead to lower HPV rates, compared with those obtained by using PCR assays [
54,
55].
As is the case with the most validated sampling procedures, it should be kept in mind that there are several other variables which may affect the efficiency and reliability of HPV detection in oral samples. For instance, the method of DNA purification also has a potentially large impact upon the ability to detect HPV DNA by PCR amplification [
56]. Additionally, considerable differences exist regarding the use of different PCR primers. In the majority of the PCR-based HPV detection systems, a broad spectrum of HPV types is amplified by consensus primers, followed by detection with type-specific probes. The consensus primers may be either degenerate (as in the MY09/11 systems), or they may contain mismatches (as in the GP5+/6+ system), or they may contain inosine residues at ambiguous base positions (as in the SPF primers), or sets of overlapping primers (as in the PGMY primers). These methods have different analytical and clinical characteristics [
57], and every method has its strengths and weakness. Although difficult to achieve, standardization and the use of validated procedures for HPV DNA is paramount in assisting physicians to provide more effective treatment and more efficient screening for patients.