**4. HPV-DNA screening methods**

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

62 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

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, 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

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

acids in clinical specimens.

screening, triage, diagnosis and treatment [47].

was a major component of this benefit [48].

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 types; and lack of internal control to evaluate specimen adequacy or the presence of potentially interfering substances. In order to resolve the current problems of analytical inaccuracy and to improve HC2 throughput, a next-generation diagnostic system has been developed [54]. Significant improvements have been engineered into the next-generation hybrid-capture system, which builds on the current advantages of the FDA-approved HPV screening tech‐ nology, HC2. Central to the improved chemistry in the NextGen assay on the QIAensemble automated system is the new collection medium, DCM [54].

that of HC2 (97.6% vs 95.1%), whereas RealTime demonstrated significantly higher analytical

HPV Diagnosis in Vaccination Era http://dx.doi.org/10.5772/55818 65

The cobas 4800 HPV Test (Roche Molecular Diagnostics, Pleasanton, CA, USA) is a real-time PCR assay based on concurrent individual genotyping for HPV-16 and HPV-18 and pooled detection of 12 other HPVs: HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66 and HPV-68 [63-65]. An excellent agreement (>93%) was obtained between cobas 4800 HPV and HC2 or Linear Array (LA) for the detection of HR HPV DNA in CIN [66]. The cobas 4800 HPV test will refer fewer women to colposcopy than automatic referral of all women with ASC-US. It will also distinguish women infected with HPV16 or HPV18 from those infected with other HR HPV genotypes, although this feature is not yet included in clinical guidelines for the management of women with ASC-US. When the cobas 4800 HPV was utilized by a diagnostic laboratory, its performance was equivalent to that of HC2 and show comparable levels of performance including results for women >30 years old with ASCUS cytology [67]. Gage et al found agreement between Cobas and LA to be very

The Cervista HPV 16/18 Test (Hologic) is a signal amplification qualitative test based on the

HPV DNA-based genotyping as says, which allow exact determination of several alpha-HPV types present in a clinical sample, are the largest group of currently available HPV commercial assays.IncontrasttothepreviouslydescribedtwogroupsofcommercialHPVassays,theclinical value of HPV DNA-based genotyping assays has still not been finally determined [70-72]. Currently, HPV genotyping methods are indispensable as research tools for the study of the natural history, transmission, pathogenesis and prevention of HPV infection. However, it is highly likely that genotyping methods will also have a role in clinical management in the near future [70, 73, 74]. As the use of prophylactic HPV vaccines becomes more widespread, surveillance for population-level effectiveness will become an increasingly important activity, which will require the use of a HPV genotyping method. If HPV genotyping is to be used for diagnostic applications and not just as a research tool, it will require standardized and validat‐ ed methods for the specific detection and identification of a defined spectrum of HPV types. Although DNA sequencing is still considered to be the 'gold standard' for HPV genotyping, it is costly, time-consuming and difficult to apply in routine diagnostic settings. Thus, in daily practice, the majority of laboratories use nonsequencing based methods for HPV genotyping, However the next generation sequencing can be implemented in the HPV detection [75]. This methodology also provides a tumor genomic copy number karyogram, and in the samples analyzed here, a lower level of chromosome instability was detected in HPV-positive tumors compared to HPV-negative tumors, as observed in previous studies. Thus, the use of nextgeneration sequencing for the detection of HPV provides a multiplicity of data with clinical

Invader chemistry specifically designed to detect HPV-16 and HPV-18 [69].

specificity compared with HC2 [61, 62].

good, better than that between Cobas and HC2 [68].

**6. HPV-DNA genotyping methods**

significance in a single test.

TheAmplicorHPVTest(Amplicor;RocheMolecular Systems,Branchburg,NJ,USA),launched on the European market in 2004, is a qualitative PCR-based test designed to detect the same 13 HPV types as HC2: HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52,HPV-56,HPV-58,HPV-59andHPV-68.SimilarlytoHC2,Amplicorexpressestheresults of the tested group of hr-HPV types as positive or negative. Amplicor is based on standard PCR amplification and detection of PCR products on microwell plates [55]. The Cervista HPV HR Test (Cervista; Hologic) is another FDA-approved signal amplification-based qualitative test for the routine detection of 14 HPVs. In March 2009, the FDA approved Cervista for two indications: to screen patients with ASC-US cervical cytology results to determine the need for referral to colposcopy; and to be used adjunctively with cervical cytology to screen women 30 years of age andolderto assess thepresence or absence of hr-HPV types [56].In contrastto HC2, Cervista is based on the Invader chemistry, a signal amplification method for detecting specific nucleic acid sequences. With support from the Bill and Melinda Gates Foundation, a careHPV Test (Qiagen), based on simplified HC2 technology, has been recently developed to detect the 13HPVtypesincludedintheoriginalHC2plusHPV-66,inapproximately3h.Suchrapid,simple and affordable HPV tests, whereby results can be given to a patient within the same visit, are anticipated to have the greatest impact in countries in which cervical cancer screening pro‐ grams do not exist or in countries in which substantial loss to follow-up impairs the effective‐ ness of cervical cancer screening programs [57].
