Early Region (E): It constitutes about 50 % of the viral genome and is one of the protein coding regions for early viral life cycle. E1 and E2 encode proteins for viral DNA replication and regulate the transcription of E6 and E7. E4 helps in the release of virions from infected cell. E5 interacts with various transmission proteins which promote cell growth. E6 and E7 are viral oncogenes, which induce cell immortalization and transformation of the host cell.
Late Region: It forms about 40 % of the viral genome and is expressed late in the viral life cycle. This region encodes two structural proteins of the viral icosahedral capsid. L1 is responsible for the formation of major capsid proteins and L2 for minor capsid proteins.
Long Control Region (LCR): It’s a noncoding regulatory region which constitutes approximately 10 % of the HPV genome. It controls DNA replication and transcription by protein coding regions, i.e., early and late regions.
The life cycle of HPV begins with the entry of virus into the basal epithelium of the host. The entry of virus requires mild abrasion or microtrauma. The virus replicates in the basal cell and gradually migrates upward to the surface epithelium. Late viral genes appear at this stage, and virions are released to restart the cycle. The viral genome remains extrachromosomal during replication in normal life cycle, in benign lesions, and in early dysplasia, but for development of precancer and invasive malignancies, viral DNA integrates into the host genome.
Approximately 190 different types of HPV viruses have been known. There are 30 types of HPV which target the genital mucosa; out of which 15 are high-risk or oncogenic types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) [3]. HPV types 16 and 18, together, account for more than 70 % of cervical precancers and cancer cases, followed by HPV 45, 31, and 33. Non-oncogenic low-risk types especially HPV 6 and HPV 11 account for 90 % of benign genital warts. Many HPV strains are structurally and functionally similar. HPV 16 is closely related to HPV 31, 33, 35, 52, and 58, and HPV 18 is related to HPV 45.
Most infections resolve spontaneously but may persist in some women leading to persistence and progression to precancerous lesions and invasive cancer in untreated women over a period of 5–15 years. The prevalence of HPV infection peaks at the age of 18–28 years, after which it declines. Approximately 90 % of lesions regress spontaneously within 12–36 months. The prevalence of hrHPV infections in women above 30 years of age is around 10 %. Older women with persistence are more likely to be at risk of invasive cancer.
Other factors influencing the progression towards cervical cancer are immunosuppression, long-term use of oral contraceptives, multiple sexual partners, early onset of sexual activity, and smoking.
6.2 Tests for HPV Detection
Detection of HPV in human cells has been strenuous because of two main reasons: the early proteins being expressed in low amounts and lack of specific antibodies against the viral proteins. Since HPV cannot be cultured, diagnostics rely mainly on the detection of viral nucleic acids in cervical smear samples. There has been constant evolution in detecting the presence of HPV in cervical smears. These techniques evolved from scoring of koilocytes to indicate the presence of HPV in the specimen to the most recently advanced signal and target-amplified nuclide acid hybridization tests.
For genome analysis, non-amplified nuclide acid hybridization tests, such as Southern blot for DNA molecules and Northern blot for RNA molecules, were used, but these tests are time-consuming and require well-preserved and full-sized molecules and hence cannot be done with specimens particularly those derived from fixed tissues containing degraded nucleic acid. Therefore, these tests cannot be used for large population studies.
6.2.1 In Situ Hybridization
It is based on the complementary pairing of a labeled probe to HPV antigens or nucleic acids in cells of cervical smear sample [4]. It demonstrates the localization of viral genome in individual cells by using chromogenic or fluorescence technique. The INFORM HPV [5] assay includes a low-risk (6, 11, 42, 43, and 44) and high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66) assay. The advantage of ISH is that it can be applied to tissues that have been fixed and processed. However, the clinical sensitivity of this technique is limited due to probe cross-hybridization.
6.2.2 Polymerase Chain Reaction (PCR)
It can detect HPV in samples with few cells containing few viral copies with poor DNA quality. PCR can produce one billion copies from a single dsDNA molecule after 30 cycles of amplification. Also the reaction mix includes internal controls to decrease false-positive and false-negative results. Since integration of the HPV genome into the human chromosomes may result in loss of the L1 region, PCR tests can have false-negative results. Furthermore, as PCR can produce millions of copies of a DNA target from a single molecule, hence the environment is extremely vulnerable to contamination with HPV sequences from aerosolized reaction mixtures [6]. The size of the amplified product remains the same regardless of the HPV type; therefore, electrophoresis cannot detect the actual type of HPV. Studies have shown the sensitivity and specificity for detecting CIN 3 or higher with PCR testing to be 88.2 % (78.9–93.8 %) and 78.8 % (77.9–79.7 %), respectively [7]. Recently Roche Diagnostics developed the AMPLICOR HPV kit test that amplifies a smaller fragment of the L1 gene; this short PCR fragment (SPF)-PCR can discriminate a broad spectrum of HPV types and is considered to be more sensitive and usable for less-preserved specimens.
6.2.3 Hybrid Capture
Digene Corporation (now known as Qiagen Corporation) developed signal amplification technique that detects nucleic acid targets directly [8]. It has developed two tests:
Hybrid capture tube (HCT) test: It is a US Food and Drug Administration (FDA)-approved semiquantitative measure of viral load relative to 10 pg/ml and uses RNA probes that react with nine high-risk HPV types (16, 18, 31, 33, 35, 45, 51, 52, and 56).
HPV hybrid capture test (HCII): In 1999, FDA approved the second generation of HCT. It detects viral load up to 1 pg/ml and four additional hrHPV (39, 58, 59, 68). The test is based on hybridization, in a solution of long synthetic RNA probes complementary to the genomic sequence of 13 high-risk (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and 5 low-risk (6, 11, 42, 43, 44) HPV types. It takes around 6–7 hours for detection of HPV; about 90 patients’ samples can be processed simultaneously on one microtiter plate. The test result is expressed as relative light units (RLU). The FDA recommended the cutoff value for test-positive results to be 1.0 RLU (equivalent to 1 pg of HPV DNA per 1 ml of sampling buffer). As HCII is based upon signal amplification, it is less prone to cross specimen contamination as compared to PCR. However, there are false-negative and false-positive results because of the absence of internal control for the amount of input of DNA and inability to identify specific HPV types.
6.2.4 RNA-Based Amplification Techniques
Of late, HPV RNA is considered as an important target for molecular diagnosis of HPV infections. Unlike HPV DNA assays that detect only the presence of viral genomes, testing for viral RNA evaluates the HPV genome expression and viral activity in the infected cells. Detection of HPV E6/E7 mRNA in cervical smear samples can be performed by reverse transcription (RT)-PCR or by nucleic acid sequence-based amplification (NASBA) [9] (PreTect HPV-Proofer; Norchip). It detects E6/E7 transcripts from the five common hrHPV types in cervical carcinoma (16, 18, 31, 33, and 45). In this, single-stranded nucleic acids (viral genomic RNA, mRNA, or rRNA) are amplified in a background of dsDNA.
Gen-Probe has developed the APTIMA HPV [10] Assay, targeting E6/E7 mRNA from 14 carcinogenic HPV genotypes. A prototype of this assay was evaluated in 536 women with histological outcomes. Detection of E6/E7 mRNA was strongly correlated with severity of the lesion; all five carcinomas and 90 % of CIN 3 cases revealed E6/E7 mRNA [10].
A Norwegian hospital-based, cross-sectional study has shown that PreTect HPV-Proofer [11] is positive in 89 % of cervical cancer and in 77 % of high-grade precursor lesions. High-grade histology (CIN 2+) was found in 83 % of women with normal cytology and positive PreTect HPV-Proofer. Though the predictive value of HPV testing was not calculated in this study, the specificity of mRNA testing seems to be better compared to HPV DNA testing.
6.2.5 Newer Tests
Khan et al. [12] reported that 21 % of cytology-negative, HPV 16-positive women developed CIN 3+ over a period of 10 years, while 18 % who were cytology negative and HPV 18 positive developed CIN 3+ during this period; for all other high-risk HPV types combined, only 1.5 % developed CIN 3+, reinstating the importance of HPV genotyping. The FDA has approved two tests for HPV genotyping: Cervista HPV 16/HPV 18 (Hologic, Bedford, MA) and cobas HPV Test (Roche Diagnostics).
Cervista HPV 16/HPV 18 [13] is a qualitative, in vitro diagnostic test for the detection of DNA from high-risk HPV types: 16 and 18. The CervistaTM HPV 16/HPV 18 test uses signal amplification method for detection of specific nucleic acid sequences. It uses two types of isothermal reactions: a primary reaction that occurs on the targeted DNA sequence and a secondary reaction that produces a fluorescent signal. A final positive, negative, or indeterminate result for any particular sample is generated based on the analysis of two separate reaction wells.
The cobas® HPV Test [14] is based on automated specimen preparation to simultaneously extract HPV and cellular DNA followed by PCR amplification of target DNA sequences using both HPV and beta-globin-specific complementary primer pairs. The amplified signal from 12 high-risk HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) is detected using a common fluorescent dye, while HPV 16, HPV 18, and beta-globin signals are each detected with their own dedicated fluorescent dye. The results are determined as “positive,” “negative,” or “invalid” in each channel based on predefined parameters and Ct ranges. The ultimate result is determined as a combination of results from all four detection channels according to a predefined table.
careHPV test [15]: This is a new test which has been developed by Qiagen to detect high-risk HPV DNA. It is a screening test that is accurate and affordable. It can detect DNA from 14 high-risk types of HPV with the test results being available in about 2.5 hours. The test is based on the same principle of signal amplification as hybrid capture 2 and is only slightly less sensitive than it. As the test requires no electricity, no running water, and only 2.5 h to conduct, it is cheap and is a promising option in the low-resource settings.
6.3 Sample Collection
Studies have shown that HPV testing of self-collected vaginal swabs is less sensitive but as specific as HPV DNA for detecting high-grade cervical disease [16], with provider-collected cervical sample resulting in highest HPV DNA sensitivity of 84–100 % and sensitivity of 66–88 %. A cross-sectional mixed method study was conducted within the context of a cervical cancer screening demonstration project, the Screening Technologies to Advance Rapid Testing-Utility and Program Planning (START-UP) [17] project in India, Nicaragua, and Uganda, with the objective to generate evidence comparing screening options implemented by public health systems in regionally representative developing country settings. The studies show that self-sampling was highly acceptable and that a majority of women preferred self-sampling because it was more comfortable and less painful.
6.4 Clinical Application of HPV Testing
Initially in 1999, FDA approved HCII to be used as an adjunctive test for the triage of patients with equivocal cytology results (ASCUS) so as to determine the need for referral to colposcopy and later in 2003 approved it as primary screening together with cytology in women aged 30 years and older. Presently there are three clear indications for HPV testing:
- 1.
Primary screening modality
- (a)
Co-testing with cytology
- (b)
hrHPV DNA testing alone
- (a)
- 2.
Triage of minor cytological abnormalities
- 3.
Follow-up after treatment of CIN
6.4.1 Primary Screening Modality
Strategies of HPV testing for primary screening include:
6.4.1.1 Co-testing with Both HPV and Pap Smear
This is the most acceptable screening method at present. If both tests are negative, there is a very strong negative predictive value, and the tests need to be repeated after 5 years. As more data becomes available, this testing interval might be increased to 10 years.
The International Agency on Research on Cancer (IARC) recommends that the age at which screening begins should aim to maximize the detection of cervical precancer cases while avoiding the large number of transient HPV infections. ACOG and ASCCP both reinforce the same and recommend the following (level A evidence):
Cervical cancer screening should begin at age 21 years.
Pap cytology screening is recommended every 3 years for women between the ages of 21 years and 29 years.
For women aged 30–65 years, co-testing with cervical cytology screening and HPV testing is preferred and should be performed every 5 years.
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