**3. Screening methods for detect HPV infection**

HPV cannot be cultured in conventional cell cultures. Other classical direct virological diagnostic techniques, such as immunohistochemistry, lack the sensitivity as well as specificity for the routine detection of HPV. Serological assays for the detection of anti-HPV antibodies have only limited analytical accuracy and their possible clinical utility is currently unresolved. Consequently, all HPV tests currently in diagnostic use rely, on the detection of HPV nucleic acids in clinical specimens.

discussed the problems of the low sensitivity of cytology and uncertain cytology triage (ASCUS) or low-risk lesions (LSIL) can lead to positive HPV tests with unknown clinical significance. However, the benefits of the HPV test result prior cytology have been demon‐ strated [49]. Currently there are proposals to make the detection of HPV followed by cytology, which has the best results of clinical sensitivity, reducing costs to distance the negative control women but also may increase if performed in populations with a high incidence of infection, as in young women [50]. Hence, several protocols established older than 30-35 years for a suitable cost-effectiveness. It remains to determine the usefulness of combining HPV screening combined with a new biomarkers triage as detection of HPV mRNA, p16-INK, and other

HPV Diagnosis in Vaccination Era http://dx.doi.org/10.5772/55818 63

This scenario has increased the range of methods and technologies for the diagnosis of HPV in clinical samples, whether exfoliated cells or biopsy, even paraffin. Extraction procedures of nucleic acids, methods of viral genome detection and automation necessary for a determi‐ nation which is becoming routine laboratories have acquired high levels of complexity for the most suitable choice [51]. The commercially available tests can be systematized in five groups [52]: 1) screening assays based on the detection of HPV DNA of high risk, which includes hybrid capture as the reference method because of the enormous experience gathered in previous studies, amplification using probes invader and PCR procedures, 2) similar screening tests for the detection of DNA of high-risk HPV concurrent determina‐ tions of genotypes or reflex HPV-16 and HPV-18 genotypes whose infection is a risk of developing cancer than other genotypes too, 3) genotyping assays based on HPV PCR using various technologies: reverse hybridization, microarray, array and RFLP suspension, which joins the pyrosequencing and other methods of mass sequencing, 4) based screening assays for the quantification of mRNA expression of E6/E7 high-risk HPVs, with the added value of its utility as a marker of progression, and 5 ) in situ hybridization, available but with

High-risk HPV-DNA-based screening assays represent a group of qualitative or semiquantitative multiplex assays in which the DNA of the targeted HPV types is detected using mixtures of probes (probe cocktails) for several HPV types with similar clinical characteristics. None of the assays from this group allow the exact determination of HPV type(s) present in a clinical specimen, but rather express the results of the tested group of HPV types as positive or negative. Until recently, such an approach has been widely accepted by the HPV community as the best way to present the results of hr-HPV testing to clinicians involved in primary screening for cervical carcinoma and management of patients with cervical intraepithelial neoplasia (CIN). The Hybrid Capture 2 (HC2) HPV DNA Test, originally developed by Digene Corporation (Gaithersburg, MD, USA) in 1997 and currently marketed by Qiagen, has been the most important HPV diagnostic assay for the last decade and is still the most frequently used diagnostic HPV test worldwide [53]. The main problems of the current version of HC2 are: analytical inaccuracy due to the cross-reactivity of its probe cocktails with untargeted HPV

histological progression markers (Ki-67, MCM2 and TOP2A).

limitations because of lower sensitivity.

**4. HPV-DNA screening methods**

Having relied on cervical cytology effectively for several decades, primary human papillo‐ mavirus (HPV) testing is increasingly and widely recognised as the means to deliver effective cervical screening and the prevention of cervical cancer. Despite differences in the interven‐ tions amongst the main primary screening studies [43-46], the key message seems to be clear and consistent: a primary HPV test will increase the sensitivity for detecting *cervical intraepi‐ thelial neoplasia, grade 2+* (CIN2+), compared with cytology, and will allow the screening interval to be extended with fewer life-time tests for women. Conventional cytology-based screening is very effective in reducing cervical cancer when delivered in well-managed programmes with high population coverage and robust quality assurance, but is costly in terms of work‐ force, finance and infrastructure. These programmes will be more difficult to sustain with lower detection rates of abnormalities in HPV-immunised populations. Yet cervical screening needs to sit alongside HPV immunisation to optimise cancer prevention. Screening pro‐ grammes will differ between different resource settings: e.g. introducing basic screening where no previous programme has existed in low- or medium-resource settings, or providing more clinically effective prevention in highly resourced countries with organised or opportunistic screening. In protocols of prevention and treatment of cervical cancer involved, in addition to cytology and HPV detection, other diagnostic tools such as colposcopy, histological categori‐ zation and use of new biomarkers, with the correct risk stratification parameter that must be used to choose the appropriate procedures in the different phases of the clinical process: screening, triage, diagnosis and treatment [47].

Screening, defined as the preventive activity that can diagnose the disease in healthy popula‐ tion a priori, should have as target detecting cervical intraepithelial neoplasia grade 3 (CIN3), or being even stricter CIN2, histological stages of the disease are even preventable. Screening by cervical cytology detects only 50-60% of cases of CIN3, including cytological revisions very close in time. The analytical sensitivity of the detection of HPV DNA in exfoliated cells is much higher, above 90%, but the clinical specificity is low because of virological diagnostic predicts non persistence, necessary to cause the development of cancer. This limitation is accentuated in young women with a good chance of getting an HPV infection self-limited. However, the negative predictive value of HPV testing is very high and would, in patients free of viruses could increase the periods between two revisions in years. Using both techniques simultane‐ ously (cytology + HPV), as has been done in some trial cohort (Northern California Kaiser Permanent), increases the control intervals to three years with good results. Implementation of HPV DNA testing in cervical screening leads to earlier detection of clinically relevant CIN grade 2 or worse, which when adequately treated, improves protection against CIN grade 3 or worse and cervical cancer. Early detection of high-grade cervical legions caused by HPV16 was a major component of this benefit [48].

The costs are reduced in the triage by using a technique followed by another in accordance with the results of the first. The cytology followed by HPV testing has been used but as already discussed the problems of the low sensitivity of cytology and uncertain cytology triage (ASCUS) or low-risk lesions (LSIL) can lead to positive HPV tests with unknown clinical significance. However, the benefits of the HPV test result prior cytology have been demon‐ strated [49]. Currently there are proposals to make the detection of HPV followed by cytology, which has the best results of clinical sensitivity, reducing costs to distance the negative control women but also may increase if performed in populations with a high incidence of infection, as in young women [50]. Hence, several protocols established older than 30-35 years for a suitable cost-effectiveness. It remains to determine the usefulness of combining HPV screening combined with a new biomarkers triage as detection of HPV mRNA, p16-INK, and other histological progression markers (Ki-67, MCM2 and TOP2A).

This scenario has increased the range of methods and technologies for the diagnosis of HPV in clinical samples, whether exfoliated cells or biopsy, even paraffin. Extraction procedures of nucleic acids, methods of viral genome detection and automation necessary for a determi‐ nation which is becoming routine laboratories have acquired high levels of complexity for the most suitable choice [51]. The commercially available tests can be systematized in five groups [52]: 1) screening assays based on the detection of HPV DNA of high risk, which includes hybrid capture as the reference method because of the enormous experience gathered in previous studies, amplification using probes invader and PCR procedures, 2) similar screening tests for the detection of DNA of high-risk HPV concurrent determina‐ tions of genotypes or reflex HPV-16 and HPV-18 genotypes whose infection is a risk of developing cancer than other genotypes too, 3) genotyping assays based on HPV PCR using various technologies: reverse hybridization, microarray, array and RFLP suspension, which joins the pyrosequencing and other methods of mass sequencing, 4) based screening assays for the quantification of mRNA expression of E6/E7 high-risk HPVs, with the added value of its utility as a marker of progression, and 5 ) in situ hybridization, available but with limitations because of lower sensitivity.
