**4. Human papillomavirus**

Human papillomavirus (HPV) is a pathogen associated to development of cervical cancer, which is the second more common cancer in women worldwide. These viruses typically infect the skin and mucosal surfaces of humans. HPV is a non-enveloped virus with a circular and supercoiled double-stranded DNA genome of approximately 8,000 bp long within an icosa‐ hedral coat or capsid comprised by 72 pentameric capsomers. The open reading frames (ORFs) in the viral genome are organized in three regions: the early expression region (E), the late region (L) and the long control region (LCR) that bears the origin of viral replication and transcription. The E region codes for proteins related to replication (E1) and to activation or repression of the viral DNA (E2), and the oncogenes E6, E7, E5 [82]. The L1 and L2 genes encode the mayor and minor capsid proteins. Late genes L6 and L7 code for structural capsid proteins which encapsidate the viral genome (Figure 4) [83]. The L1 gene is the region most conserved between individual types and used to analyze phylogenetic relationship.

Those HPVs with L1 sequence divergence of 2–10% are known as subtypes and less than 2%, variants. The term "genus" is used for the higher order clusters, named using the Greek alphabet, and within genus, small clusters are referred to as species and given a number [84-86]. There are more than 200 different types, of which approximately 100 are fully se‐ quenced and classified according to their biological niche, phylogenetic position and oncogenic potential [87]. About 40 can infect the anogenital mucosa of humans (mucosotrophic HPVs) [88-89]. According to their association with cervical cancer, there are low-risk HPV, which can cause benign or low-grade cervical cell changes, genital warts, and recurrent respiratory papillomatosis. High-risk HPV types act as carcinogens in the development of cervical cancer and other anogenital cancers [90]. Cervical cancer is a commonly-encountered malignant tumor in women. The surveys demonstrated that the increase of women with cervical cancer Molecular Diagnostics as an Indispensable Tool for the Diagnosis of Infectious Diseases of… http://dx.doi.org/10.5772/57581 101

in influenza diagnosis and others viruses such as parainfluenza, respiratory syncytial virus, human metapneumovirus, adenovirus, rhrinoviruses, coronaviruses causes pneumonia, which can be analyzed by multiplex PCR that assist in the discrimination of the etiologic agent [Pavia, 2011; Mahony, 2008]. The NGEN respiratory virus analyte-specific assay (Nanogen, San Diego, CA) detects influenza A, influenza B and others pathogens such as PIV type 1 (PIV-1), PIV-2, PIV-3, and RSV on a NanoChip 400 electronic microarray. The FilmArray Respiratory Panel (RP) is a PCR array test (Idaho Technology, Inc., Salt Lake City, UT) that can detect up to 21 viral and bacterial respiratory pathogens within about an hour as Adenovirus, Bocavirus, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, Coronavirus OC43, Human Metapneumovirus, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza virus 4, Rhinovirus/Enterovirus, Respiratory Syncytial Virus, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumonia and Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza A subtype H1 2009, Influenza B. This assay is a feasible alternative to viral culture in an acute clinical setting [80]. Recently, it was reported the method for the detection of genetic markers associated with high pathoge‐

Human papillomavirus (HPV) is a pathogen associated to development of cervical cancer, which is the second more common cancer in women worldwide. These viruses typically infect the skin and mucosal surfaces of humans. HPV is a non-enveloped virus with a circular and supercoiled double-stranded DNA genome of approximately 8,000 bp long within an icosa‐ hedral coat or capsid comprised by 72 pentameric capsomers. The open reading frames (ORFs) in the viral genome are organized in three regions: the early expression region (E), the late region (L) and the long control region (LCR) that bears the origin of viral replication and transcription. The E region codes for proteins related to replication (E1) and to activation or repression of the viral DNA (E2), and the oncogenes E6, E7, E5 [82]. The L1 and L2 genes encode the mayor and minor capsid proteins. Late genes L6 and L7 code for structural capsid proteins which encapsidate the viral genome (Figure 4) [83]. The L1 gene is the region most conserved

Those HPVs with L1 sequence divergence of 2–10% are known as subtypes and less than 2%, variants. The term "genus" is used for the higher order clusters, named using the Greek alphabet, and within genus, small clusters are referred to as species and given a number [84-86]. There are more than 200 different types, of which approximately 100 are fully se‐ quenced and classified according to their biological niche, phylogenetic position and oncogenic potential [87]. About 40 can infect the anogenital mucosa of humans (mucosotrophic HPVs) [88-89]. According to their association with cervical cancer, there are low-risk HPV, which can cause benign or low-grade cervical cell changes, genital warts, and recurrent respiratory papillomatosis. High-risk HPV types act as carcinogens in the development of cervical cancer and other anogenital cancers [90]. Cervical cancer is a commonly-encountered malignant tumor in women. The surveys demonstrated that the increase of women with cervical cancer

between individual types and used to analyze phylogenetic relationship.

nicity of influenza virus [81].

100 Trends in Infectious Diseases

**4. Human papillomavirus**

**Figure 4.** Linear representation of Human Papillomavirus (HPV) genome. The circular HPV genome has approximately 7900 base pairs, in the schematic with eight overlapping reading frames. L1 is signaled as the most used genomic tar‐ get for detection of HPV.

less than 35 years old is particularly significant. The possible causes that result in cervical cancer to tend to occur in young include remarkable increase of HPV infection rate, especially the high-risk HPV 16 and 18 infection closely related to cervical cancer [91]. The primary screening and diagnostic methods have been cytology and histology. Papanicolaou (Pap) staining is the gold standard for detecting abnormal cervical epithelial cells, using microscopic analyses of conventional cervical smears or cell suspensions from liquid cytology medium. The limitation of Pap smear are low specificity, is need repeat the screening at short intervals [92]. Morphological findings from a cytology analysis determine the level of risk for develop‐ ing cervical malignancies. Cervical epithelial cells atypical or abnormal are known as "atypical squamous cells of undetermined significance" (ASCUS). Some ASCUS signals the presence of low-grade squamous intraepithelial lesions (LSIL). However, some ASCUS are associated with underlying high-grade disease, including cervical intraepithelial neoplasia (CIN) [93-95]. HPV serology is not used diagnostically. Detection of the humoral antibody response is type-specific and first detected 6–18 months after infection. The response is weak and only between 50–60% of patients positive to HPV DNA-positive mount a measurable antibody response [96]. Although, the role played by the humoral immune response during the HPV infection is not very well understood; it was observed that this response is generated all throughout the malignant process; 54%-69% of women with incident HPV 16, 6, or 18 infections had antibod‐ ies. Serum antibodies against many different viral products of HPV have been detected by ELISA. The best characterized and most type-specific antibodies are those directed against conformational epitopes of the L1 capsid protein assembled as VLPs (virus-like particles) such as HPV-16 L1 VLP (virus-like particle)-based ELISA [97]. There is some high-throughput single-serum-dilution enzyme-linked immunoassay (ELISA) system for determining anti-HPV antibody titers following vaccination against HPV [98].

At present there is no "gold standard" for HPV detection. Guidelines for the management of women with cervical neoplasia or abnormal cervical cancer screening tests indicates the immediate colposcopy (cervical exam), cytologic follow-up, and triage by HPV DNA testing [99]. HPV serves as paradigm for the use of NAATs due to how difficult it is to obtain the virus via cell cultures or to develop indirect diagnosis techniques [100]. There are several molecular techniques for HPV DNA detection (Table 3). They include DNA hybridization, PCR-RFLP, reverse-line hybridization and hybrid capture assay. The method most commonly used is the polymerase chain reaction (PCR). All target amplification techniques such as PCR for HPV virus detection currently use consensus or type-specific primers group-or type-specific conventional PCR, to amplify a broad-spectrum of HPV genotypes by targeting a conserved region within the HPV genome [101, 102]. L1 and E1 regions are the most conserved parts of the genome [103]. Many assays use primers targeted to the viral capsid L1 gene, which can detect numerous HPV types [104]. There are several PCR primer sets as GP5+/6+that amplify a 140 bp region in the L1 gene allowing the identification of 30 HPV genotypes. This method is useful in predicting high-grade cervical intraepithelial neoplasia. The MY09/11 system identifies high-risk HPV genotypes by amplifying a 450 bp sequence in the conserved L1 region. The MY09/11 primer set uses degenerate bases to reduce variability due to different genotypes. The SPF10 system primers amplify a 65 bp sequence from a highly conserved region of the viral L1 gene for the identification of 16 different genotypes of the human papillomavirus (HPV) [105]. Commonly used L1 consensus primer sets include PGMY09/11, GP5+/6+, and SPF10, having the ability to identify a large range of HPV types with 1 amplification [106, 107]. Hybridization on PCR products (Cervista HPV HR Test, INNO-LiPa HPV Genotyping, Linear Array HPV Genotyping Test, Digene HPV Genotyping RH Test) [Estrade et al., 2011; Jeney et a., 2007; Chan et a., 2012]. Linear Array HPV Genotyping Test (Roche Diagnostics, Indianapolis, IN) is able to identify 37 types of HPV, 14 are high-risk genotypes. Linear Array also includes PGMY primers and is a commonly used method for genotyping HPV using Probes for multiple HPV types are fixed on a membrane strip, and the PCR product is hybridized to the strip, followed by visual detection [108]. Other assay so used is INNO-LiPA HPV Genotyping Extra (Innogenetics, Ghent, Belgium). This kit amplifies HPV DNA with SPF10 primers at the L1 region. The probes are fixed to membrane strips in sequence-specific lines and visualized as purple/brown bands. The test can detect and distinguish 24 low-and high-risk HPV types. [INNO-LiPA HPV Genotyping Extra. Ghent, Belgium: Innogenetics; 2007. A HPV viral target to detect is the L1 gene to amplify a broad spectrum of HPV types with a single set of degenerated primers or a cocktail of primers (Amplicor HPV Test) [Sepehr et al., 2012] and HPV mRNA amplification against HPV E6/E7 mRNA (APTIMA HPV Assay, NucliSENS EasyQ HPV, PreTect HPV-Proofer). Another test is the Multiplex Genotyping Kit (Multimetrix, Heidelberg, Germany). The test is a PCR-based fluorescent bead array that can detect 24 low-and high-risk HPV types [109]. The quadruplex quantitative PCR method (AllGlo fluorescent probes) was established to simultaneously detect and differentiate HPV 6, 11, 16 and 18 [110]. Between the signal amplification methods are liquid-phase or in situ hybridization as Hybrid Capture 2 HPV DNA Test for to detect 13 high-risk types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) and five low-risk types [111, 112]. Microarray on PCR products is other molecular technique to detect influenza virus such as Infiniti HPV-HR QUAD Assay, PapilloCheck or HybriMax for HPV genotyping [113]. PapilloCheck (Greiner Bio-One, Monroe, NC) for HPV genotyping PapilloCheck identifies 24 types of low-and highrisk HPV with a high specificity and sensitivity [114]. Genotyping with this method is based

on PCR amplification of the E1 gene by a group of new E1-specific primers, followed by hybridization to a DNA chip with immobilized HPV oligoprobes. A novel assay for molecular diagnostics and typing application known as Sequencing Bead Array (SBA) is an alternative method to HPV diagnosis. SBA is a digital suspension array using Next-Generation Sequenc‐ ing (NGS) that in the case of HPV could distinguish ten Human Papillomavirus (HPV) genotypes associated with cervical cancer progression. This is a robust system capable to identify genetic signatures or single nucleotide polymorphisms (SNPs). SBA has the potential to change the probe-based diagnostics, and allow for a transition towards the technology by genomic sequencing [115]. Some groups are studying other molecular factors as possible markers of infection by HPV as a complementary diagnostics. The overexpression of the HPV E6 and E7 genes is indicated in HPV-induced carcinogenesis, making these genes a potential measure of virulence. Monitoring the expression levels of these genes may allow for screening

Molecular Diagnostics as an Indispensable Tool for the Diagnosis of Infectious Diseases of…

**NAME TEST GENOTYPES AND TARGET DETECTED**

gene.

gene.

14 high risk HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and to partially genotype 16, 18 from other 12 high risk genotypes using L1

http://dx.doi.org/10.5772/57581

103

HPV 16 and HPV 18 and concurrently detecting the rest of the high risk types (31, 33, 35, 39, 45, 51, 52,

High-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52,

HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58,

Qualitative detection of E6/E7 viral messenger RNA (mRNA) from 14 high-risk types of HPV: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.

Detection of HPV subtypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82 using L1

Detection of at least 54 HPV types using L1 gene.

Linear Array detects thirty seven anogenital HPV DNA genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66,

56, 58, 59, 66 and 68), using L1 gene

56, 58, 59 and 68 using all genome.

59 and 68 using L1 gene.

and monitoring of cancer progression [116].

Real time Real Time High risk HPV test

Hybrid capture Hybrid Capture 2 High-Risk HPV DNA Test

(Hologic)

(Innogenetics)

Reverse hybridization Digene HPV Genotyping RH Test (Qiagen)

PCR and hybridization AMPLICOR Human

TMA APTIMA HPV assay

(Abbott Molecular)

Cobas 4800 HPV Test Amplification/Detection kit (Roche Molecular Diagnostics)

Papillomavirus Test (Roche Molecular Diagnostics)

INNO-LiPA HPV Genotyping Extra

Linear Array HPV Genotyping test (Roche Molecular Diagnostics)

**MOLECULAR DIAGNOSIS METHOD**

on PCR amplification of the E1 gene by a group of new E1-specific primers, followed by hybridization to a DNA chip with immobilized HPV oligoprobes. A novel assay for molecular diagnostics and typing application known as Sequencing Bead Array (SBA) is an alternative method to HPV diagnosis. SBA is a digital suspension array using Next-Generation Sequenc‐ ing (NGS) that in the case of HPV could distinguish ten Human Papillomavirus (HPV) genotypes associated with cervical cancer progression. This is a robust system capable to identify genetic signatures or single nucleotide polymorphisms (SNPs). SBA has the potential to change the probe-based diagnostics, and allow for a transition towards the technology by genomic sequencing [115]. Some groups are studying other molecular factors as possible markers of infection by HPV as a complementary diagnostics. The overexpression of the HPV E6 and E7 genes is indicated in HPV-induced carcinogenesis, making these genes a potential measure of virulence. Monitoring the expression levels of these genes may allow for screening and monitoring of cancer progression [116].

[99]. HPV serves as paradigm for the use of NAATs due to how difficult it is to obtain the virus via cell cultures or to develop indirect diagnosis techniques [100]. There are several molecular techniques for HPV DNA detection (Table 3). They include DNA hybridization, PCR-RFLP, reverse-line hybridization and hybrid capture assay. The method most commonly used is the polymerase chain reaction (PCR). All target amplification techniques such as PCR for HPV virus detection currently use consensus or type-specific primers group-or type-specific conventional PCR, to amplify a broad-spectrum of HPV genotypes by targeting a conserved region within the HPV genome [101, 102]. L1 and E1 regions are the most conserved parts of the genome [103]. Many assays use primers targeted to the viral capsid L1 gene, which can detect numerous HPV types [104]. There are several PCR primer sets as GP5+/6+that amplify a 140 bp region in the L1 gene allowing the identification of 30 HPV genotypes. This method is useful in predicting high-grade cervical intraepithelial neoplasia. The MY09/11 system identifies high-risk HPV genotypes by amplifying a 450 bp sequence in the conserved L1 region. The MY09/11 primer set uses degenerate bases to reduce variability due to different genotypes. The SPF10 system primers amplify a 65 bp sequence from a highly conserved region of the viral L1 gene for the identification of 16 different genotypes of the human papillomavirus (HPV) [105]. Commonly used L1 consensus primer sets include PGMY09/11, GP5+/6+, and SPF10, having the ability to identify a large range of HPV types with 1 amplification [106, 107]. Hybridization on PCR products (Cervista HPV HR Test, INNO-LiPa HPV Genotyping, Linear Array HPV Genotyping Test, Digene HPV Genotyping RH Test) [Estrade et al., 2011; Jeney et a., 2007; Chan et a., 2012]. Linear Array HPV Genotyping Test (Roche Diagnostics, Indianapolis, IN) is able to identify 37 types of HPV, 14 are high-risk genotypes. Linear Array also includes PGMY primers and is a commonly used method for genotyping HPV using Probes for multiple HPV types are fixed on a membrane strip, and the PCR product is hybridized to the strip, followed by visual detection [108]. Other assay so used is INNO-LiPA HPV Genotyping Extra (Innogenetics, Ghent, Belgium). This kit amplifies HPV DNA with SPF10 primers at the L1 region. The probes are fixed to membrane strips in sequence-specific lines and visualized as purple/brown bands. The test can detect and distinguish 24 low-and high-risk HPV types. [INNO-LiPA HPV Genotyping Extra. Ghent, Belgium: Innogenetics; 2007. A HPV viral target to detect is the L1 gene to amplify a broad spectrum of HPV types with a single set of degenerated primers or a cocktail of primers (Amplicor HPV Test) [Sepehr et al., 2012] and HPV mRNA amplification against HPV E6/E7 mRNA (APTIMA HPV Assay, NucliSENS EasyQ HPV, PreTect HPV-Proofer). Another test is the Multiplex Genotyping Kit (Multimetrix, Heidelberg, Germany). The test is a PCR-based fluorescent bead array that can detect 24 low-and high-risk HPV types [109]. The quadruplex quantitative PCR method (AllGlo fluorescent probes) was established to simultaneously detect and differentiate HPV 6, 11, 16 and 18 [110]. Between the signal amplification methods are liquid-phase or in situ hybridization as Hybrid Capture 2 HPV DNA Test for to detect 13 high-risk types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) and five low-risk types [111, 112]. Microarray on PCR products is other molecular technique to detect influenza virus such as Infiniti HPV-HR QUAD Assay, PapilloCheck or HybriMax for HPV genotyping [113]. PapilloCheck (Greiner Bio-One, Monroe, NC) for HPV genotyping PapilloCheck identifies 24 types of low-and highrisk HPV with a high specificity and sensitivity [114]. Genotyping with this method is based

102 Trends in Infectious Diseases



and hepatocellular carcinoma being a global public health as chronic diseases, cause of infectious disease mortality globally, each year causing approximately 1.4 million deaths. In addition, viral hepatitis are cause of liver cancer and the most common reason for liver transplantation [119]. The viruses more common are Hepatitis A, Hepatitis B, and Hepatitis C. Approximately 400 million persons living with chronic hepatitis B virus (HBV) or hepatitis C virus infection who die from cirrhosis or liver cancer, years and decades after of their infection [120]. Hepatitis B is caused by infection with HBV, which may lead to acute or chronic hepatitis. HBV is the 9th leading cause of death worldwide. It causes cirrhosis, liver failure and hepatocellular carcinoma. HBV is a small nonenveloped DNA virus that is a member of the Hepadnaviridae family, HBV contains a 3.2-kb partially double-stranded DNA genome with 4 open reading frames encoding 7 proteins (P/viral polymerase, S/Surface antigen proteins/HBsAg, C/core protein, HBeAg y X/HBx protein) [121]. Eight genotypes of HBV (designated-H) have been identified by sequence divergence of >8% over the entire genome of HBV DNA [122]. HBV is efficiently transmitted by percutaneous or mucous membrane exposure to infectious blood or body fluids that contain blood. Acute HBV infection can be either asymptomatic or symptomatic. Symptoms in acute HBV infection are clinically indis‐ tinguishable from those in other acute viral hepatitis infections [123]. Diagnosis of hepatic viral infection is carried out by studying biochemical as liver function tests, serologic assays and histological parameters. Serological HBV diagnosis identifies virally-encoded antigens and their corresponding antibodies in serum. Three clinical useful antigen-antibody systems have been identified for hepatitis B: hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti-HBs), antibody (anti-HBc IgM and anti-HBc IgG) to hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg) and antibody to HBeAg (anti-HBe) [124]. B surface antigen (HBsAg) is the first serological marker to appear during the course of HBV infection, is present in both acute and chronic infection. The immunoassays to detect HBsAg are highly spectific and show a sensitivity, allowing the detection of <0.15 ng/ml of HbsAg [125]. Detection of HBsAg allowed for the first time screening of inapparently infected blood donors for a dangerous pathogen. The simultaneous detection of antibodies against HBsAg (anti-HBs) and HBcAg (anti-HBc) can be useful to know the evolution of disease. The presence of IgM antibody to hepatitis B core antigen (IgM anti-HBc) is diagnostic of acute or recently acquired HBV infection. Antibody to HBsAg (anti-HBs) is produced after a resolved infection and is the only HBV antibody marker present after immunization. The presence of HBsAg and total anti-HBc, with a negative test for IgM anti-HBc, indicates chronic HBV infection. The presence of anti-HBc alone might indicate a false-positive result or acute, resolved, or chronic infection [126, 127]. Chronic HBV infection is defined by the persistence of serum HBsAg for more than 6 months [128]. Serologic assays for HBV are the mainstay diagnostic tools for HBV infection. The clinically silent HBV infections are a strong driving force in the development of modern virus diagnostics to analyze the HBV replication profile, HBV DNA levels and the viral protein expression [129]. When serological testing could be inconclusive for the diagnosis of a HBV infection (due to the presence of genetic variants of HBV), the molecular detection of HBV DNA may help to resolve the uncertainties. The NAT assays in plasma or serum can detect to 10 copies/mL [130]. The viral genomes can be detected and quantified by polymerase chain reaction (PCR), transcription-mediated amplification (TMA), and with signal amplification

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105

Information based on Kroupis C, et al., 2011 [90]; Arney A, et al., 2010 [100].

**Table 3.** Principal methods of molecular diagnosis of HPV.

Analysis of the host factor p16INK4a (p16), a cyclin-dependent kinase inhibitor could be a molecular marker to HPV infection. The increase of p16 indicates removal of the negative feedback control supplied by the retinoblastoma gene, pRB. When oncogenic HPV E7 proteins bind to pRB, p16 is overexpressed and elevated, representing active expression of HPV oncogenes [117]. The sialylation modification observed during oncogenic transformation, tumor metastases and invasion, has been associated with enhanced sialyltransferases (STs) transcription such as ST3Gal III, ST3Gal IV and ST6Gal I in CIN [118].
