**3. Diagnosis of HPV infections**

Despite the promising outcomes, vaccination does not exempt from performing periodic control visits, because the effects of the vaccine at 15-20 years and the role other genotypes with oncogenic capacity not included in the vaccine may play are still unknown. Furthermore, there is still a large population of women which has had no access to it. Then, secondary prevention by screening and treatment will continue to be crucially important in cervical cancer prevention programs. Moreover, the fact that infection by HPV provokes long-term symptomatology demands a close follow-up (screening) of those individuals susceptible to infection in order to avoid related problems.

Currently, cervical cancer screening is acknowledged as the most effective approach for cervical cancer control. The primary screening and diagnostic methods have been cytology

and histology, but two limitations of the Pap smear exist: low specificity leading to the need for repeat screening at relatively short intervals and cervical cancer screening, based on Pap smear, remains beyond the economic resources of nation in developing world. This econom‐ ic disparity has meant that cervical cancer incidence and mortality rates in the developing world have remained high, with large reductions in these rates being limited primarily to the industrialized world. Thus, the reduction of cervical cancer in developing nations remains an unmet need of high priority. Since the link between HPV and cervical cancer is known and numerous large scale studies have been done, molecular methods to detect HPV DNA in clinical specimens (vaginal, urethral, paraurethral, anal or pharyngeal exudates, biop‐ sies, and, especially, endocervical exudates) have been introduced into screening algorithms.

Increased sensitivity has important clinical outcomes because reduce mortality and an elongation of screening, and implies better compliance with screening and lower cost [47]. An Italian study showed that HPV-based screening is more effective than cytology in preventing invasive cervical cancer, by detecting persistent high-grade lesions earlier and providing longer low-risk period [48].

HPV serves as paradigm for the use of NAATs for its diagnosis and typification due to how difficult it is to obtain the virus via cell cultures or to develop indirect diagnosis techniques [49].

The first protocols for detect HPV were described about 20 years ago, using L1 consensus primers PCR systems, particularly MY09/11 and GP5+/6+ [50-52]. These primer systems have been widely used to study the natural history of HPV and their rule in the development of genital cancer [53-55]. Nowadays, several kits are commercially available which allow for the detection of the virus or the detection and typification of the most relevant HPVs: Amplicor HPV test and Linear array HPV Genotyping test (Roche Diagnostics, Switzerland), Innolipa HPV Genotyping Extra (Innogenetics, Belgium), Biopat kit (Biotools, Spain) or Clart Papillo‐ mavirus 2 (Genómica, Spain). The latter uses microarray technology to increase the number of hybridizations in a reduced space. Besides genome amplification, direct hybridization protocols on the sample (hybrid capture) approved by the FDA for diagnosing HPV in women (Hybrid Capture II, Digene, USA) is also used. These protocols identify high and low-risk genotypes without specifying the infecting genotype.

The sensitivity of such methods has left out cytological methods (Papanicolau), which are less sensitive and specific. This high degree of sensitivity allows to extending the period between control visits of women to 5 or 6 years [56, 57].

#### **3.1. Signal amplification systems**

as in lesions premalignant cervix. In cervical cancer, pRb is functionally inactivated from the initial stages of cervical carcinogenesis as a consequence of expression of HPV E7 gene. Genes E6 and E7 therefore act to remove two principle mechanisms of cell defence, and drive the cell replication machinery towards production of new virus particles. E6 and E7 are also known

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

On the other hand, integration of HPV-DNA into the host DNA is a well known topic in cervical cancer. Integration of HPV 16 DNA correlates with dysfunction of HPV E1 or E2 ORF, which are active during HPV replication. E2 loss of function allows up-regulation of E6 and E7

oncoproteins, because E2 is a repressor of E6 and E7. (Figure 2).

**Figure 2.** The location in squamous epithelium of the main stages of the papillomavirus life cycle. [46]

Despite the promising outcomes, vaccination does not exempt from performing periodic control visits, because the effects of the vaccine at 15-20 years and the role other genotypes with oncogenic capacity not included in the vaccine may play are still unknown. Furthermore, there is still a large population of women which has had no access to it. Then, secondary prevention by screening and treatment will continue to be crucially important in cervical cancer prevention programs. Moreover, the fact that infection by HPV provokes long-term symptomatology demands a close follow-up (screening) of those individuals susceptible to

Currently, cervical cancer screening is acknowledged as the most effective approach for cervical cancer control. The primary screening and diagnostic methods have been cytology

to promote oncogenesis. [45]

**3. Diagnosis of HPV infections**

infection in order to avoid related problems.

The Hybrid Capture II system (HCII, Digene, USA) is a non radioactive signal amplifica‐ tion method based on the hybridization of the target HPV-DNA to labeled RNA probes in solution. The resulting RNA-DNA hybrids are captured onto microtiter wells and are detected by specific monoclonal antibody and chemiluminiscence substrate, providing a semiquantitative measurement of HPV-DNA. Two different probe cocktails are used, one containing probes for five low-risk gentypes: HPV 6, 11, 42,43 and 44 and the other contain‐ ing probes for 13 high-risk genotypes: HPV 16,18,31,33,35,39,45,51,52,56,58,59 and 68. However, HCII has some limitations. It distinguishes between the high-risk and low-risk groups but does not permit identification of specific HPV genotypes. Hybrid Capture II (HCII) has been shown to have similar analytic sensitivity to some PCR methods for HPV DNA detection [58], but present cross-reactivity of the two probe cocktails can reduce the clinical relevance of a positive result [59, 60].

patient follow up [69]. Over the last few years, virus genotyping has become an important way to approach cervical cancer. Then HPV genotype detection could increase specificity in a routine screening program or in post –treatment follow-up (i.e. test of cure) by differentiating

Molecular Diagnosis of Human Papillomavirus Infections

http://dx.doi.org/10.5772/55706

9

Population-based genotyping characterizations pre- and post-vaccination will be important to determinate vaccine effectiveness and potential unmasking of niche replacements by nonvaccines HPV types in cytologically normal women and women with low and high grade

Genotyping assays have been developed, like GP5+/6+ reverse line blot, or MY90/11 dot-blot. Based in these technologies, specific kits have been comercializated: PGMY09/11 linear array (Linear Array® HPV genotyping test; Roche Molecular Systems, Switzeland) and SPF10 LiPA 25 (Inno-LiPA® HPV test, Innogenetics, Belgium). The assays are based on consensus broad spectrum PCR which are subsequently differentiated by type-specific oligonucleotide probe hybrydizacion. These assays have the ability to identify multiple several viruses in cases of multiple infections. In the last years, others assays for HPV genotyping has been commercial‐ ised and introduced in clinical and research laboratories with full or partial automation (PapilloCheck HPV-Screening Test, Greiner Bio-One; Clart HPV2, Genomica, Infiniti HPV Genotyping assay, Autogenomics; Cobas 4800 HPV Test, Roche diagnostics; Real Time High

As already reported and in spite of its limitations, sequencing could be considered the gold standard for HPV genotyping, due to the possibility of identifying virtually all virus types without mistaken classifications through cross-reactions among similar types, which can occur using tests based on hybriditation [74, 75]. Nevertheless, it was disadvantaged at identifying genotypes in samples with multiple infections, in which viral sequences overlap and it is not

In any case, genotyping is a technology that has to be incorporated in the HPV surveillance. Waiting for massive sequencing, now the most promising field is automated methods, because simplifies the testing procedure, increases the sample processing capability, minimizes the human errors, facilitates the quality assurance, reduces the cost and can be developed in

Because molecular testing for HR-HPV DNA may detect infection too early in the process, with only a small subset of women developing disease that progresses to cancer, there is interest in defining secondary markers that have potential application in identification of women who need to be followed more closely because they are at higher risk of developing high-grade lesions [77]; especially, when the positive predictive value of current screening strategies will be diminished in a vaccinated population [78]. Then, the impetus for new screenig or progre‐ sion technologies in the developed world is thus predominately driven by the need to increase

**4. Screening and progression prognostic biomarkers technologies**

transient and sequential infection from persistent infection [70-72].

lesions.

Risk HPV test, Abbott Molecular) [73]

multiples laboratories.

possible to distinguish the various types [74, 76].

The Hybrid Capture III (HCIII, Digene, USA) is being evaluated as the next generation of hybrid capture clinical assays. A primary technical distinction between HCIII and HCII is that HCIII employs a biotinylated DNA oligonucleotide specific for selected HPV DNA sequences (HPV16 and HPV18) for the capture of the DNA-RNA complexes on streptavidin-coated wells, to reduce false positivity [59].

### **3.2. Target amplification systems (PCR)**

Type specific primers designed to amplify exclusively a single HPV genotype can be use but multiple type-specific PCR reactions must be performed separately to detect the presence of HPV in a sample. This method is labor-intensive, a little bit expensive and the type –specificity of each PCR primer set should be validated. Alternatively, consensus or general PCR primers can be used to amplify a broad-spectrum of HPV types: genome amplification protocols (PCR) with degenerate primers targeted towards the L1 gene fragment (MY09/MY11) allow for the detection of a wide range of viral subtypes, which are then identified with specific probes [50, 61]. Other consensual primers (PGMY, GP5+/GP6+ or SF10) used on the same target enhance diagnostic sensitivity [52, 62, 63]. Thanks to these protocols, the low and high cancer progres‐ sion risk genotypes were identified [25].

Amplification protocols have also experimented great advancements with the application of real-time PCR, which reduces reaction times (e.g. HPV RealTime test, Abbot, USA; GenoID, Hungary). In fact, it is now possible to automate the whole process (Cobas® 4800 HPV Test with 16/18 Genotyping, Roche Diagnostics, Switzerland).

Type-specific PCR primers can be combined with fluorescent probes to real-time detection [64-66]althoughmultiplexingseveraltype specificprimerswithinone reactioncanbe technical‐ ly difficult. Broad-spectrum PCR primers have also been used in real-time PCR [67, 68].

The HCII method and consensus PCR assays are currently the most frecuently applied. In last years, RT-PCR is being introduced in clinical microbiology laboratories.

#### **3.3. Full spectrum genotyping**

About 40 different HPV types (involved in human genital infections) have been identified based on DNA sequence analysis so far, with a subset of these being classified as high risk. DNA of these types is found in almost all cervical cancers, however, regional variation in the distribution of certain HPV types should be taken into account in the composition of screening "cocktails" for high-risk HPV types from different populations [29]. The diversity of virus types and the incidence of multiple infections have made it necessary to develop reliable methods to identify the different genotypes, for epidemiological studies as well as for the patient follow up [69]. Over the last few years, virus genotyping has become an important way to approach cervical cancer. Then HPV genotype detection could increase specificity in a routine screening program or in post –treatment follow-up (i.e. test of cure) by differentiating transient and sequential infection from persistent infection [70-72].

However, HCII has some limitations. It distinguishes between the high-risk and low-risk groups but does not permit identification of specific HPV genotypes. Hybrid Capture II (HCII) has been shown to have similar analytic sensitivity to some PCR methods for HPV DNA detection [58], but present cross-reactivity of the two probe cocktails can reduce the

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

The Hybrid Capture III (HCIII, Digene, USA) is being evaluated as the next generation of hybrid capture clinical assays. A primary technical distinction between HCIII and HCII is that HCIII employs a biotinylated DNA oligonucleotide specific for selected HPV DNA sequences (HPV16 and HPV18) for the capture of the DNA-RNA complexes on streptavidin-coated wells,

Type specific primers designed to amplify exclusively a single HPV genotype can be use but multiple type-specific PCR reactions must be performed separately to detect the presence of HPV in a sample. This method is labor-intensive, a little bit expensive and the type –specificity of each PCR primer set should be validated. Alternatively, consensus or general PCR primers can be used to amplify a broad-spectrum of HPV types: genome amplification protocols (PCR) with degenerate primers targeted towards the L1 gene fragment (MY09/MY11) allow for the detection of a wide range of viral subtypes, which are then identified with specific probes [50, 61]. Other consensual primers (PGMY, GP5+/GP6+ or SF10) used on the same target enhance diagnostic sensitivity [52, 62, 63]. Thanks to these protocols, the low and high cancer progres‐

Amplification protocols have also experimented great advancements with the application of real-time PCR, which reduces reaction times (e.g. HPV RealTime test, Abbot, USA; GenoID, Hungary). In fact, it is now possible to automate the whole process (Cobas® 4800 HPV Test

Type-specific PCR primers can be combined with fluorescent probes to real-time detection [64-66]althoughmultiplexingseveraltype specificprimerswithinone reactioncanbe technical‐ ly difficult. Broad-spectrum PCR primers have also been used in real-time PCR [67, 68].

The HCII method and consensus PCR assays are currently the most frecuently applied. In last

About 40 different HPV types (involved in human genital infections) have been identified based on DNA sequence analysis so far, with a subset of these being classified as high risk. DNA of these types is found in almost all cervical cancers, however, regional variation in the distribution of certain HPV types should be taken into account in the composition of screening "cocktails" for high-risk HPV types from different populations [29]. The diversity of virus types and the incidence of multiple infections have made it necessary to develop reliable methods to identify the different genotypes, for epidemiological studies as well as for the

clinical relevance of a positive result [59, 60].

to reduce false positivity [59].

**3.2. Target amplification systems (PCR)**

sion risk genotypes were identified [25].

**3.3. Full spectrum genotyping**

with 16/18 Genotyping, Roche Diagnostics, Switzerland).

years, RT-PCR is being introduced in clinical microbiology laboratories.

Population-based genotyping characterizations pre- and post-vaccination will be important to determinate vaccine effectiveness and potential unmasking of niche replacements by nonvaccines HPV types in cytologically normal women and women with low and high grade lesions.

Genotyping assays have been developed, like GP5+/6+ reverse line blot, or MY90/11 dot-blot. Based in these technologies, specific kits have been comercializated: PGMY09/11 linear array (Linear Array® HPV genotyping test; Roche Molecular Systems, Switzeland) and SPF10 LiPA 25 (Inno-LiPA® HPV test, Innogenetics, Belgium). The assays are based on consensus broad spectrum PCR which are subsequently differentiated by type-specific oligonucleotide probe hybrydizacion. These assays have the ability to identify multiple several viruses in cases of multiple infections. In the last years, others assays for HPV genotyping has been commercial‐ ised and introduced in clinical and research laboratories with full or partial automation (PapilloCheck HPV-Screening Test, Greiner Bio-One; Clart HPV2, Genomica, Infiniti HPV Genotyping assay, Autogenomics; Cobas 4800 HPV Test, Roche diagnostics; Real Time High Risk HPV test, Abbott Molecular) [73]

As already reported and in spite of its limitations, sequencing could be considered the gold standard for HPV genotyping, due to the possibility of identifying virtually all virus types without mistaken classifications through cross-reactions among similar types, which can occur using tests based on hybriditation [74, 75]. Nevertheless, it was disadvantaged at identifying genotypes in samples with multiple infections, in which viral sequences overlap and it is not possible to distinguish the various types [74, 76].

In any case, genotyping is a technology that has to be incorporated in the HPV surveillance. Waiting for massive sequencing, now the most promising field is automated methods, because simplifies the testing procedure, increases the sample processing capability, minimizes the human errors, facilitates the quality assurance, reduces the cost and can be developed in multiples laboratories.
