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Molecular Diagnostics of Human Papillomavirus

Ashley Arney MT(ASCP)CM, Katie M. Bennett PhD, MB(ASCP)CM
DOI: http://dx.doi.org/10.1309/LM75WGJSVMI7VVEF 523-530 First published online: 1 September 2010

Abstract

Purpose: Human papillomavirus (HPV) infection is the most common sexually transmitted infection in the United States. This review summarizes the molecular testing methods currently in use for the detection and genotyping of HPV DNA and discusses potential future approaches.

Summary: There are more than 100 different types of HPV, which are separated into low- and high-risk categories, depending on clinical presentation. Low-risk types can cause warts, while high-risk types are associated with cervical cancer. The available methods for HPV detection and genotyping include both target and signal amplification techniques.

Conclusion: Molecular diagnostic methods allow for the identification of the many unique types of high-risk HPV, which can lead to better diagnoses and treatment plans for patients. Use of these diagnostic techniques requires consideration of the consensus guidelines for management of women with normal and abnormal cervical screens, as well as consideration of assay clinical sensitivity and diagnostic utility.

Keywords:
  • HPV
  • cervical cancer
  • sexually transmitted disease
  • DNA
  • PCR
  • genotyping

Human papillomavirus (HPV) is the most common sexually transmitted infection in the United States, affecting men and women.1 It is estimated that more than 24 million men and women in the United States are currently infected with HPV.2,3 More than 4 million new HPV infections are reported in the United States annually, and about 1%–10% of the sexually active U.S. population is infected at any given time.4,5 There are 2 categories of HPV: those infections that are passed through skin-to-skin contact and those that are sexually transmitted. Typically, an infection with HPV is transient, and the immune system can clear the infection within 2 years.6 Currently, there are more than 100 different known HPV genotypes that have been grouped into low-risk and high-risk categories and designated as causing mucosal or cutaneous infections.7 The types of HPV passed through skin contact typically cause skin warts, especially in children. The most common sexually transmitted disease caused by HPV is genital warts (condyloma acuminatum), a wart-like growth on the cervical or vulval mucosa in females, or on the glans or prepuce in males.4 Warts are generally the result of infection by low-risk types of HPV, including 6, 11, 32, 40, 42, 44, 54, 55, 61, 62, 64, 71, 72, 74, 81, 83, 84, 87, 89, and 91.8,9 High-risk types of HPV include 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, 85, and IS39.8,9 The high-risk strains induce cervical dysplasia and can lead to the development of several types of cancers including cancer of the cervix, vulva, vagina, anus, and penis.10 The most common of these is HPV-associated cervical cancer.

Cervical Cancer and Pap Smear

The development of cervical cancer after an HPV infection involves a number of other circumstances, including gene mutations, and occurs many years after the initial HPV infection. Human papillomavirus is a necessary factor, but it is not sufficient for development of dysplasia or carcinoma. Disease generally develops when there is persistent HPV infection of the cervical epithelium.11,12 Infection with a high-risk strain of HPV is not a verdict for the development of cervical cancer, but the cervical dysplasia associated with HPV infection has the potential to become malignant.13 Often women are infected with HPV and never develop any serious or even apparent complications, although this should not diminish the need for regular cervical cancer screenings.

Papanicolaou (Pap) staining is the gold standard for detecting abnormal cervical epithelial cells, using microscopic analysis of conventional cervical smears or cell suspensions from liquid cytology medium.13 In the 1930s, cervical cancer was the No. 1 cause of cancer-related death for women in the United States. By 2000, the rate had been cut by almost 90%, largely because widespread adoption of regular cervical cytology exams, or Pap smears, led to a decline of cervical cancer.4 Currently cervical examinations and Pap tests remain the screening method of choice for most women. Morphological findings from a cytology analysis determine the level of risk for developing cervical malignancy. Cervical epithelial cells determined to be atypical or abnormal, but not yet defined as neoplastic, are given the term “atypical squamous cells of undetermined significance” (ASCUS) or “cannot exclude high-grade lesions” (ASC-H). Most cases of ASCUS signal the presence of low-grade squamous intraepithelial lesions (LSIL), which are generally associated with transient, self-resolving HPV infections. However, some ASCUS findings are associated with underlying high-grade disease, including cervical intraepithelial neoplasia (CIN).14 Women over age 30 who have a persistent infection with high-risk types of HPV have the greatest risk of developing cervical cancer.15 Molecular diagnostic tests for HPV can augment screening for cervical cancer when used in conjunction with the Pap smear.

In 2006, the American Society for Colposcopy and Cervical Pathology (ASCCP) published guidelines for the management of women with cervical neoplasia or abnormal cervical cancer screening tests.16 These recommendations were largely based on the results of the National Cancer Institute ASCUS/LSIL Triage Study (ALTS), which was a large, multisite clinical trial designed to evaluate 3 methods of management: immediate colposcopy (cervical exam), cytologic follow-up, and triage by HPV DNA testing.17 The ASCCP consensus guidelines state that a woman over age 30 with a negative cytology report should be screened for HR-HPV infection. If positive for a high-risk strain, rescreening and Pap cytology should be repeated in 12 months, followed by colposcopy if positive HPV status persists. In 2009, after the approval of the first HPV genotyping test, the ASCCP suggested that a woman 30 years or older who is HR-HPV positive but cytology negative would benefit clinically from an assay that specifically detects HPV genotypes 16 and 18. If positive for HPV 16/18, colposcopy should be done immediately, and if negative, follow-up cytology and a high-risk HPV assay should be done in 1 year. Women over age 30 with no sign of high-risk HPV DNA should be rescreened for HPV in 3 years. High-risk HPV DNA testing and genotyping is not recommended in adolescents under 20 years old or for women 21 and older with ASC-H, LSIL, or high-grade squamous intraepithelial lesion (HSIL) cytology.16 This recommendation is based on the results from the ALTS study that concluded that HPV testing was not of value in the clinical management of women with LSIL, and these women should undergo immediate colposcopy instead.18 Human papillomavirus testing is also not indicated in women considering vaccination against HPV, for routine STD screening, or as part of a sexual assault case.16

Human papillomavirus screening is less clinically effective for younger women, because women under age 30 have a high incidence of HPV infection, of which the majority will clear spontaneously and never cause cancer. According to population-based data from the National Health and Nutrition Examination Survey (NHANES) from 2003–2004, the overall HPV prevalence, including high- and low-risk types, was 26.8% among U.S. females aged 14 to 59 years, with a peak at 44.8% at 20 to 24 years.19 Therefore, screening a young female for HPV will often reveal HPV infection but has little correlation with the incidence of cervical abnormalities or cancer. A more effective approach for managing reproductive health in this age group is prevention of HPV infection by vaccination.

HPV Vaccine

In 2006 the Food and Drug Administration (FDA) approved a prophylactic HPV vaccine. The Gardasil (Merck, Whitehouse Station, NJ) vaccine is a series of 3 shots given over a 6-month period that helps to guard against 4 common types of HPV: 6, 11, 16, and 18. Types 6 and 11 are known to cause around 90% of genital warts cases,20 and types 16 and 18 are associated with up to 70% of cervical cancer cases.21,22 The vaccine is indicated for females aged 9 to 26. The vaccine can be given before or after becoming sexually active. As of October 2009, the vaccine is also indicated for the prevention of genital warts in boys and men 9 through 26 years of age.23 Also in October 2009, a new HPV vaccine was approved by the FDA, called Cervarix (GlaxoSmithKline, London, UK). Cervarix prevents only against the 2 high-risk strains, HPV 16 and 18, and is indicated for prevention of cervical cancer and abnormalities in females 10 through 25 years of age.24 Cervarix has been available in Europe since 2007 but does not protect against HPV types 6 and 11, the most common causes of genital warts. Medical organizations such as the American Academy of Pediatrics, the Centers for Disease Control and Prevention (CDC), and the American College of Obstetricians and Gynecologists recommend HPV vaccination for the prevention of cervical cancer, although regular cervical cancer screenings are still necessary after vaccination.

Figure 1

HPV Genome. The HPV genome is a circular molecule of approximately 7900 base pairs. The schematic shows the approximate location of the 8 overlapping reading frames in a linear orientation. The L1 gene, which codes for viral capsid protein, is the primary target for amplification and detection of HPV DNA. The E6 and E7 genes are responsible for the role of HPV in cervical cancer.

View this table:
Table 1

Summary of HPV Molecular Diagnostic Techniques

TestPrincipleCommentsLow-Risk StrainsHigh-Risk Strains
Reverse Line Blot (Roche)Target amplification; genotyping; consensus PCR and line blotResearch use only6, 11, 61, 62, 64, 67, 69, 72, 81, 8916,18, 26, 31, 33, 35, 39, 40, 42, 45, 51 to 59, 66, 68, 73, 82, 83, 84
LINEAR ARRAY HPV Genotyping Test (Roche)Target amplification; genotyping; PCR followed by line hybridizationCE-Marked for use in Europe6, 11, 40, 42, 53, 54, 55, 61, 62, 64, 67, 69, 70, 71, 72, 81, 84, IS39, CP610816, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, 82, 83
INNO-LiPA HPV Genotyping Extra (Innogenetics)Target amplification; genotyping; SPF10 primers at L1 region, reverse hybridizationCE-Marked for use in Europe6, 11, 40, 43, 44, 54, 7016, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 71, 73, 74, 82
AMPLICOR HPV (Roche)Target amplification; detection; PCR and nucleic acid hybridizationCE-Marked for use in EuropeN/A16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68
PapilloCheck (Greiner Bio-One)Target amplification of E1 for genotyping; PCR/DNA-arrayCE-Marked for use in Europe6, 11, 40, 42, 43, 4416, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82
Multiplex HPV Genotyping Kit (Multimetrix)Target amplification; genotyping; PCR and fluorescent bead arrayResearch use only6, 11, 42, 43, 44, 7016, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82
GenoID Real-Time HPV Assay (GenoID)Target amplification for detection or semi-genotyping; real-time PCRCE-Marked for use in Europe6, 11, 42, 43, 44 (Lightcycler only)16, 18, 31, 33, 35, 39, 45, 51,52, 56, 58, 59, 66, 68
Digene Hybrid Capture II (HC2) HR HPV DNA Test (Digene/Qiagen)Signal amplification for detection; hybrid capture, semi-quantitativeFDA-approvedN/A16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68
Digene Hybrid Capture II (HC2) HPV DNA Test (Digene/Qiagen)Signal amplification for detection; hybrid capture, semi-quantitativeFDA-approved6, 11, 42, 43, 4416, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68
CareHPV (Qiagen)Signal amplification for detection; rapid test related to HC2For use in developing countriesN/A16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68
Cervista HPV HR (Hologic)Signal amplification for detection; Invader technologyFDA-approvedN/A16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68
Cervista HPV 16/18 (Hologic)Signal amplification for genotyping; Invader technologyFDA-approvedN/A16, 18

Molecular Diagnostics

For those women who are deemed to benefit from HPV screening, there are a number of molecular diagnostic tests available to detect HPV. Due to the inability to culture HPV in a laboratory, molecular techniques are the only method available to detect HPV DNA. The HPV genome is double stranded and circular with approximately 7900 bp.13 Figure 1 is an illustration of a generalized HPV genome, shown in linear form. The HPV virus has 8 overlapping reading frames, or genes, categorized as either early or late, depending on when they are expressed. Early genes, E1 and E2, participate in genome replication and transcription while the E4 gene promotes the productive phase of the viral life cycle. The oncoproteins, E6 and E7, form complexes with tumor suppressors,13,25 and thus can lead to host cell transformation and progression to cervical cancer.26 The L1 and L2 late genes encode viral capsid proteins.25 Most HPV assays target the L1 region for classification of HPV genotypes, although the E1 gene is also used.

The major diagnostic techniques for HPV detection and genotyping are target amplification, signal amplification, and probe amplification. Target amplification duplicates fragments of DNA from a targeted gene sequence. The most well-known example of this is polymerase chain reaction (PCR). Signal amplification uses branched DNA technology or hybrid capture to increase the DNA-proportional signal to detectable levels. Probe amplification includes technologies such as ligase chain reaction, which amplify the probe.27 Traditional molecular non-amplified techniques include Southern blot hybridization, in situ hybridization, and dot blot. These non-amplified techniques typically demand more when it comes to time, skill level, and necessary equipment and are being surpassed by more sensitive and reliable methods. This review focuses on the most commonly used techniques: PCR-based and signal amplification methods. Table 1 is a summary of molecular diagnostics used in HPV detection and genotyping.

Target Amplification Techniques

Polymerase Chain Reaction

Polymerase chain reaction is the most commonly used tool in the detection of HPV DNA. In theory, PCR can take a single double-stranded piece of DNA and amplify it to 1 billion copies after 30 cycles.18 Polymerase chain reaction consists of the following 3 basic steps: denaturation, annealing, and extension. Denaturation occurs at 95°C and melts the double-stranded DNA into 2 separate strands. The annealing step takes place at approximately 55°C and involves the binding of primers to target DNA. For the extension step, the temperature is raised to 72°C and complementary strands of DNA are produced by DNA polymerase. Typically, PCR procedures for HPV detection use primers targeted to the viral capsid L1 gene, which can detect numerous HPV types.18 Commonly used L1 consensus primer sets include PGMY09/11, GP5+/6+, and SPF10, along with a number of proprietary primers having the ability to identify a large range of HPV types with 1 amplification.13,28 Type-specific PCR, as the name implies, amplifies a single genotype of HPV by targeting a type-specific DNA sequence. For type-specific PCR, several repeats of PCR may be necessary in order to determine the specific sequence existing in the sample.28 Various applications of target amplification for HPV detection and genotyping are discussed below.

Reverse Line Blot and Linear Array

The reverse line blot assay from Roche Molecular Systems (Alameda, CA) was 1 of the first widely used prototype methods. The line blot assay uses L1 consensus primer-based PCR with PGMY09/11 primers. Probes for multiple HPV types are fixed on a membrane strip, and the PCR product is hybridized to the strip, followed by visual detection. The assay detects 27 different HPV types and the extended edition adds 11 low-risk types, which include 61, 62, 64, 67, 69 to 72, 81, 82, and 89.28 The line blot assay is a research use only test, and the original assay is now commercialized as the Linear Array HPV Genotyping Test.29 The Linear Array HPV Genotyping Test (Roche Diagnostics, Indianapolis, IN) is able to identify 37 types of HPV, 14 of which are high-risk genotypes. Linear Array also includes PGMY primers and is a commonly used method for genotyping HPV using strips for hybridizing. The results are read with the unaided eye based on a visible band in specific areas of the hybridization strip. This subjectivity has the potential to affect the interpretation of results, and there are currently automation techniques being evaluated to eliminate the subjectivity involved in reading these results.30 The Linear Array Test is CE-Marked for in vitro diagnostic use in Europe. This test, along with another HPV detection method from Roche (Amplicor, below), has been accepted for review by the FDA but has not yet been approved.

Another commercially available test, INNO-LiPA HPV Genotyping Extra (Innogenetics, Ghent, Belgium), uses the principles of reverse line blot hybridization. The INNO-LiPA test 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 INNO-LiPA test can detect and distinguish 24 low- and high-risk HPV types.31 INNO-LiPA HPV is CE-Marked for use in Europe.

Amplicor HPV

The Amplicor HPV test from Roche Molecular Systems is a PCR-based kit that can identify 13 high-risk HPV types. This system amplifies target DNA using PCR followed by nucleic acid hybridization.32 A 96-microwell plate is used and requires only a small sample of 250 μL for testing. The Amplicor assay will detect HPV but will not identify the specific genotype. In a study comparing Amplicor to the Hybrid Capture II Assay (HC2, discussed below) for the detection of these specific 13 high-risk HPV types, the concordance was found to be 83.3%, with Amplicor finding more positives than HC2 (39.3% vs 32.1%), but the overall agreement of the 2 was shown to be 97.5%.32 As mentioned, Amplicor is awaiting FDA approval, but is currently CE-Marked in Europe.

PapilloCheck

Recently, a commercial DNA-array based test, called PapilloCheck (Greiner Bio-One, Monroe, NC), has been made available for HPV genotyping. PapilloCheck identifies 24 types of low- and high-risk HPV. PapilloCheck has the ability to detect and identify all 24 types simultaneously, and the company advertises a high specificity and sensitivity.33 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. The PapilloCheck DNA-chip setup allows for testing of 12 samples at a time and helps to eliminate false-positive and false-negative results. A laser scanner, CheckScanner, is used to detect excitation from fluorescently labeled probes that bind to the HPV primers. The reporting software, CheckReport, automatically reads and reports the results.33 The PapilloCheck assay was found to be comparable to the Roche Linear Array test, although there were variations between the tests in the detection rate of certain genotypes.34

Multiplex HPV Genotyping Kit

Another novel genotyping test is the Multiplex Genotyping Kit (Multimetrix, Heidelberg, Germany). The Multiplex test is a PCR-based fluorescent bead array that can detect 24 low- and high-risk HPV types. The PCR products are mixed with the Multiplex HPV Genotyping Kit bead mix. This bead mix has 26 populations of beads attached to 24 HPV probes, 1 β-globin probe, and 1 control probe. After the PCR products are hybridized, they are labeled using R-phycoerythrin marked streptavidin and read on the Luminex analyzer.35 The individual signatures of the beads can discern the types of HPV. Currently, the Multiplex HPV Genotyping Kit is only available for research purposes, but it has shown a high sensitivity with applications in large-scale epidemiological studies and potential future use in routine diagnostics of HPV.36

Real-Time PCR

Real-time PCR is a highly sensitive target amplification technique available for HPV-DNA detection. Real-time PCR combines fluorescent probes with PCR primers, allowing for accurate quantification of virus present in a sample. Viral load estimation of HPV is a particular advantage of real-time PCR, using the nuclear genome to control for cellular content of the sample.18 However, viral load significance is still being determined for HPV. A commercially available kit using real-time PCR is the GenoID assay. This kit identifies 14 high-risk and 5 low-risk types and takes under 3 hours to complete. The GenoID assay has been developed for 3 different real-time PCR platforms: Roche LightCycler 2, Applied Biosystems 7900 HT, and Corbett Rotor-Gene 6600. Like all of the previous commercially available target amplification assays, the GenoID kit is not FDA approved. However, it has been CE-Marked for in vitro diagnostic use in Europe.

Signal Amplification Techniques

Hybrid Capture Assay

Hybrid capture is a signal amplification technique, meaning that the chemiluminescent or fluorescent signal is amplified to aid detection, rather than the target DNA being amplified by PCR. The original Hybrid Capture Assay (HC1) was approved by the FDA in 1995. Digene developed its second version, HC2,37 which was also FDA approved in 1999. Hybrid Capture II remains the current gold standard in HPV diagnostics and is the most widely used HPV assay in clinical laboratories in the United States. The HC2 assay uses nucleic acid hybridization and microplate chemiluminescent detection. Specimens containing HPV DNA are hybridized with a HPV-specific RNA probe, creating a DNA:RNA hybrid molecule. The microplate well is coated with antibodies that bind DNA:RNA hybrids, thus capturing the hybrid molecules to the microplate. Alkaline phosphatase-conjugated antibodies bind the hybrid molecules, and a signal is detected on the addition of a chemiluminescent substrate. Signal amplification occurs because several alkaline phosphatase molecules are conjugated to each antibody, and multiple conjugated antibodies can bind each captured hybrid. Figure 2 shows a schematic of the principle of hybrid capture technology. There are 2 Digene assays: 1 that detects 5 low- and 13 high-risk types,38 and another that detects only 13 high-risk types.39 These assays are only useful for detection of HPV and not specific genotyping. Digene is now merged with Qiagen, but the tests retain the Digene prefix. The most commonly used assay is the high-risk HC2 assay, because of its clinical utility in detecting cervical cancer. Hybrid Capture II is validated for use with ThinPrep liquid cytology medium or Digene’s cervical collection kit. SurePath liquid cytology medium has been used with HC2, although its use is considered off-label and requires thorough validation by the laboratory until pending FDA approval is complete.

Figure 2

Hybrid Capture Schematic. The schematic shows the basic steps of a hybrid capture assay. (A) The double-stranded DNA of the target is denatured, (B) followed by hybridization of a target-specific RNA probe, forming a DNA:RNA hybrid. (C) The hybrid is applied to a microwell plate coated with antibody binding DNA:RNA complexes, thus capturing the hybrid. (D) Alkaline phosphatase conjugated antibodies bind the captured hybrids. (E) A chemiluminescent substrate is added, which results in a detectable signal proportionate to the amount of target DNA.

CareHPV

In recognition of the lack of adequate cervical cancer screening in developing countries,27 Qiagen has developed a test called CareHPV, which is a spin-off of HC2. Currently the most common type of cervical cancer screening in underdeveloped nations is visual inspection of the cervix with vinegar. CareHPV provides a fast, accurate alternative to this less-standardized method. CareHPV takes roughly 2 hours and 30 minutes and is designed to be used by minimally trained individuals in less than ideal conditions in developing countries. It includes its own water source, allows flexible temperature ranges, and requires no expensive equipment. The short run time allows clinicians to follow-up with patients within the same visit, increasing the level of subsequent care. CareHPV has been shown to be 90% accurate and has been made much more affordable.40 There are currently ongoing pilot studies evaluating CareHPV in different parts of the world, many funded by the Bill and Melinda Gates Foundation.

Cervista HPV HR and Cervista HPV 16/18

After a 10-year lull in FDA approval of HPV tests, in March 2009 the Cervista HPV HR test (Third Wave Technologies, now Hologic, Bedford, MA) was approved by the FDA to detect 14 high-risk HPV types. Also approved was the Cervista HPV 16/18 genotyping test. The FDA approval of the Cervista HPV 16/18 test marks the first FDA-approved HPV genotyping test.16 The Cervista HPV HR and Cervista HPV 16/18 are approved for screening women with ASCUS and in conjunction with cytology for patients over age 30 to determine if high-risk types are present.

The Invader technology of the Cervista test is a unique signal amplification technique using 2 simultaneous isothermal reactions. Figure 3 summarizes the sequence of events in the detection of a target DNA sequence using Invader technology. A DNA probe including a sequence-specific region binds to the HPV DNA molecule. In addition, the proprietary Invader probe also associates with the target sequence, resulting in a 1-base overlapping structure at the key nucleotide. Proprietary enzymes cleave the probe, releasing a 5’ oligo flap.41 Multiple probes are cleaved for each molecule, causing signal amplification. Meanwhile each flap behaves as an Invader oligo in association with a florescence resonance energy transfer (FRET) probe. The FRET probe includes a fluorophore molecule in close proximity to a quencher molecule. Upon association of the flap with the FRET probe, another cleavage reaction occurs, releasing the fluorophore from its quencher. This results in a detectable fluorescent signal.42

A number of studies have shown the Invader technology used in the Cervista test is at least comparable to HC2 in the ability to accurately detect high-risk HPV, and the Cervista assay may additionally show increased specificity.4345 Some advantages of the Cervista assay include low to no cross-reactivity with low-risk HPV genotypes, addition of an internal control to assess DNA content and integrity, and lower required sample volumes.

Discussion and Future Directions

Researchers benefit from having a variety of molecular diagnostic tests at their fingertips; however, the clinical laboratories in the United States have a more limited selection of FDA-approved tests for HPV. Many of the HPV diagnostic kits available in regions such as Europe and Canada have not been approved for clinical use in the United States. The Amplicor HPV test, the Linear Array HPV Genotyping test, the PapilloCheck test, and the INNO-LiPA HPV test are examples of kits approved for use elsewhere in the world that have yet to be approved in the United States. However, in 2009 the FDA approved the first high-risk HPV DNA test in more than a decade. The approval of Cervista HPV HR and Cervista HPV 16/18 provides a stepping stone for incorporation of more FDA-approved kit-based assays for the detection of high-risk HPV types. Meanwhile, laboratories may choose to use non-approved tests as analyte specific reagents (ASRs) or home brews, although more extensive validation is required in these cases.

Another potential approach for the screening of cervical cancer is the use of reverse-transcriptase (RT)-PCR to detect HPV mRNA. Reverse-transcriptase-PCR incorporates an RT step prior to performing basic endpoint or real-time quantitative PCR. Using this technique, the level of ultimate protein production of the gene of interest can be elucidated. 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.46 Reverse-transcriptase-PCR can detect mRNA at much smaller quantities than other mRNA techniques, such as northern blot. However, at the present time, RT-PCR techniques are not optimized for use in large-scale HPV testing.47 The majority of widely used detection technologies for HPV are DNA based, yet detecting expression of HPV oncogenes has the potential for considerable clinical importance.13

Regardless of the technical method used, careful consideration is necessary in the evaluation of diagnostic techniques for HPV screening. Most infectious disease tests strive for the highest possible analytical sensitivity, and PCR is typically the optimal method to achieve that standard. However, the more important standard for HPV screening is not analytical sensitivity but clinical sensitivity and specificity. Clinical utility of HPV screening is based on the prediction of cervical cancer, not simply the presence of the virus. Especially in young adult populations, detecting the HPV virus has little clinical use because the vast majority of these cases will self-resolve and never develop into cancer. The high sensitivity of PCR is thus a detriment in HPV screening, because PCR can detect even miniscule amounts of virus that may have no clinical significance. Polymerase chain reaction methods require that laboratories determine a threshold of detection representing a clinically significant result. Likewise, the detection of non cancer-causing, low-risk strains of HPV has virtually no clinical utility. Knowing that a low-risk HPV strain is present does not have an impact on the clinical management of a patient with cutaneous or mucosal warts. In order to prevent superfluous laboratory testing, clinicians should also heed the ASCCP guidelines for the management of women with or without cytological abnormalities. New assays for high-risk HPV must also be well validated, as suggested by Stoler and colleagues, to have sufficiently high clinical sensitivity to detect cervical abnormalities.48

Figure 3

Invader Technology Schematic. The schematic shows the major steps of a signal amplification assay using Invader technology. The target DNA is denatured, and a sequence-specific probe binds in conjunction with an Invader oligo. (A) The formation of this complex results in cleavage at the indicated site. (B) Cleavage releases a free flap of the probe molecule. Multiple flaps are generated from each target DNA molecule. (C) The flap then associates with a FRET probe, resulting in cleavage at the indicated site. (D) The fluorophore, F, is released from the quencher, Q, causing a fluorescent signal proportionate to the amount of target DNA.

The clinical laboratory must evaluate many factors in the adoption of an appropriate HPV test, including consideration of the population being served. In underprivileged areas, for example, HPV screening tests with less than optimal clinical sensitivities and specificities may still far surpass current cervical cancer screening methods. As new data emerge from recently established HPV screening methods, researchers and clinicians will continue to strive toward the goal of early and accurate detection of cervical cancer.

Abbreviations

HPV
human papillomavirus
Pap
Papanicolaou
ASCUS
atypical squamous cells of undetermined significance
ASC-H
cannot exclude high-grade lesions
LSIL
low-grade squamous intra-epithelial lesion
CIN
cervical intraepithelial neoplasia
ASCCP
American Society for Colposcopy and Cervical Pathology
ALTS
ASCUS/LSIL Triage Study
HSIL
high-grade squamous intraepithelial lesion
NHANES
National Health and Nutrition Examination Survey
CDC
Centers for Disease Control and Prevention
PCR
polymerase chain reaction
HC1
Hybrid Capture Assay
HC2
Hybrid Capture II
FRET
florescence resonance energy transfer
FDA
Food and Drug Administration
ASRs
analyte specific reagents
RT
reverse-transcriptase

References

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