Human Papillomavirus (HPV) encompasses a heterogeneous group of more than 200 viruses that predominantly infect the epithelial cells of the skin and mucosal membranes. It stands as one of the most prevalent sexually transmitted infections globally, affecting a substantial proportion of sexually active individuals during their lifetime. HPV types are classified into low-risk and high-risk variants based on their oncogenic potential. Particularly, high-risk variants such as HPV 16 and 18 exhibit a strong association with the pathogenesis of various malignancies, including cervical, anal, and oropharyngeal carcinomas.
HPV displays a non-enveloped morphology characterized by an icosahedral capsid measuring approximately 55 nm in diameter. The capsid consists of 72 capsomeres, with each capsomere composed of five units of the major capsid protein L1. The L1 protein is essential for viral infectivity and the induction of immune responses. In addition to L1, the minor capsid protein L2 assists in genome encapsulation and facilitates the entry of virions into host cells.
The genome of HPV is a circular double-stranded DNA molecule approximately 8,000 base pairs long. This genetic structure is organized into three main regions: the early (E) region, late (L) region, and long control region (LCR). Within the E region are critical genes responsible for viral replication and oncogenic processes, prominently including E6 and E7. The L region encodes structural proteins such as L1 and L2. Essential regulatory sequences governing viral replication and transcriptional regulation are located within the LCR.
Central to the oncogenicity of high-risk HPV types are the oncoproteins E6 and E7. The degradation of the tumor suppressor protein p53 is orchestrated by E6, thereby inhibiting apoptosis and promoting genetic instability. Concurrently, E7 disrupts cell cycle regulation through its interaction with the retinoblastoma protein (pRb), facilitating uncontrolled cellular proliferation. These molecular mechanisms underscore the potent oncogenic capabilities of high-risk HPV types and their significant role in carcinogenesis.
Understanding the structural and functional attributes of HPV elucidated here not only enhances comprehension of viral pathogenesis but also emphasizes the necessity for precise genotyping methodologies. These insights are essential for informed therapeutic strategies and the continual advancement of preventive measures aimed at reducing the global burden of HPV-associated diseases.
The structure of HPV. (José Veríssimo Fernandes et al,. 2012)
HPV is classified within the genus Alphapapillomavirus of the family Papillomaviridae. These viruses are non-enveloped and possess spherical DNA, known for their capacity to induce epithelial hyperplasia in human skin and mucous membranes. The taxonomy identifies over 200 distinct HPV types.
HPV types are categorized into high-risk and low-risk variants based on their association with cervical cancer and precancerous lesions.
High-risk HPV types encompass a group of 18 distinct strains, namely HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 82, and 73. These HPV types are associated with the etiology of low-grade cervical intraepithelial neoplasia, high-grade cervical intraepithelial neoplasia (which serve as precursors to malignancy), as well as various cancers including those affecting the cervix, anus, oral cavity, tonsils, vulva, penis, and bladder. The persistence of infection with high-risk HPV types is considered a prerequisite for the progression from cervical precancerous lesions to invasive cervical carcinoma in the absence of intervention.
Conversely, low-risk HPV types encompass 10 variants, specifically HPV 6, 11, 40, 42, 43, 44, 55, 61, 81, and 83. Infection with these low-risk types can result in manifestations such as genital warts and common warts. Notably, the overwhelming majority (90%) of genital warts are attributed to HPV types 6 or 11 among the low-risk category.
Cervical cancer is the sole human malignancy with an unequivocal etiology that can be both prevented and treated in its early stages. The 5-year survival rate for early intervention approaches 100%, whereas for late-stage treatment, it diminishes to 20-50%. The substantial correlation between persistent infection with high-risk HPV types and cervical cancer, in conjunction with the feasibility of contemporary genetic testing technology, has positioned HPV genotyping as a principal modality for cervical cancer screening. Timely and effective intervention for individuals with HPV infection is pivotal in the prevention of cervical cancer.
HPV genotyping assay identifies specific types of HPV infection, each associated with varying pathogenic potential. Distinct HPV types induce different diseases, with notable variations in pathogenicity among high-risk HPV types. Persistent infection with high-risk HPV is a prerequisite for the development of cervical neoplasia. However, the likelihood of progression to cervical lesions and invasive carcinoma, as well as the prognosis of such invasive cancers, differs by HPV genotype.
Research has demonstrated that approximately 70-80% of cervical carcinomas globally are attributable to HPV types 16 and 18. Specifically, HPV types 16, 18, 58, and 59 exhibit a strong association with squamous cell carcinoma, whereas HPV 18 is frequently implicated in adenocarcinoma, adenosquamous carcinoma, and small-cell carcinoma. Furthermore, HPV 16 is also a major contributor to the pathogenesis of adenocarcinomas. The utilization of HPV genotyping assays enables the stratification of individuals based on their risk of disease progression, thereby informing the timing and strategy for therapeutic interventions.
HPV genotyping serves to distinguish between single and multiple infections, a critical differentiation due to the increased duration of persistent HPV infection and augmented carcinogenic risk associated with multiple HPV genotypes. Consequently, the delineation of single versus multiple infections is imperative through HPV genotyping testing. Furthermore, HPV genotyping distinguishes between persistent and transient infections. Post-infection, the immune system clears the HPV virus in over 80-90% of cases within two years. However, numerous studies underscore that HPV type 16 exhibits a higher propensity for persistence and progression to Cervical Intraepithelial Neoplasia grade 3 (CIN3) and invasive carcinoma compared to other high-risk HPV types. Women positive for HPV type 18 demonstrate a heightened likelihood of developing CIN3, particularly adenocarcinomas and related precancerous lesions. The risk coefficient for persistent infection due to high-risk HPV significantly surpasses that for transient infections. Therefore, HPV genotyping is indispensable for the distinct management of persistent and transient infections, essential for preventing carcinogenesis and enhancing therapeutic outcomes.
Regular screening, aimed at early detection of precancerous lesions or early-stage cancers, is widely acknowledged as the most effective measure in oncological care to enhance patient survival rates and reduce mortality.
For eligible women, undergoing periodic HPV genotyping testing enables the timely establishment of scientifically effective preventive interventions and treatment strategies. This approach ensures early identification, prevention, and treatment, thereby lowering the incidence of cervical cancer in women.
HPV genotyping is a crucial component of HPV-related disease management and prevention. Advanced methods such as PCR, hybridization-based techniques, and next-generation sequencing offer precise and comprehensive identification of HPV genotypes.
PCR-based methodologies are extensively employed in HPV genotyping owing to their heightened sensitivity and specificity. These techniques facilitate the amplification of targeted segments within the HPV genome, thereby enabling the identification of diverse HPV genotypes.
Type-specific PCR entails the utilization of primers designed specifically for the DNA sequences characteristic of individual HPV types. This approach enables precise identification of the particular HPV types present in a given sample. The specificity inherent in these primers ensures detection of even minimal quantities of viral DNA, underscoring its efficacy in clinical diagnostic settings. For instance, Clifford et al. (2006) demonstrated the robust utility of type-specific PCR in accurately detecting HPV types 16 and 18 in their study published in the Journal of Clinical Microbiology.
Multiplex PCR enhances genotyping efficiency by enabling simultaneous amplification of multiple HPV types within a single reaction. This method employs a combination of type-specific primers, thereby economizing time and expenses associated with conducting individual PCR reactions for each HPV type. Notably, multiplex PCR proves particularly advantageous in screening large sample cohorts, facilitating prompt acquisition of comprehensive HPV genotype data. Research highlighted in Clinical Chemistry illustrated its capability to detect and differentiate up to 19 high-risk HPV types within a solitary assay (Schmitt et al., 2006).
HPV detection using real-time PCR (Dong Hyeok Kim et al,. 2022)
Hybridization-based techniques, such as DNA microarrays and line probe assays, rely on the complementary binding of labeled HPV DNA fragments to sequences immobilized on a solid support. These methods provide an alternative approach to HPV genotyping, leveraging the specificity of sequence hybridization for diagnostic purposes.
DNA microarrays employ immobilized probes corresponding to various HPV genotypes on a chip. Upon application of a sample containing HPV DNA to the chip, hybridization occurs between the DNA and complementary probes, generating a detectable signal. This methodology enables concurrent identification of multiple HPV types within a single assay, rendering it a potent tool for expansive epidemiological investigations. Gravitt et al. (2003) demonstrated in the Journal of Clinical Microbiology the high-throughput capacity of DNA microarrays, successfully identifying 37 distinct HPV genotypes in clinical samples.
LPAs entail the hybridization of amplified HPV DNA to specific probes affixed onto a strip. Detection of hybridized DNA is achieved through a colorimetric or chemiluminescent substrate. LPAs exhibit high sensitivity and discriminatory capability among closely related HPV genotypes, making them prevalent in clinical laboratories due to their simplicity and rapid analytical response. Eklund et al. (2010) documented in the Journal of Clinical Microbiology the efficacy of LPAs in accurately detecting and distinguishing 25 high-risk HPV types.
Principles of molecular HPV Hybrid Capture tests (Katarzyna Sitarz et al,. 2019)
Sequencing (NGS) has revolutionized HPV genotyping through its high-throughput and detailed analysis of the HPV genome. This technology enables the identification and characterization of multiple HPV genotypes in a single sequencing run, providing unmatched resolution and accuracy.
Whole genome sequencing (WGS) of HPV encompasses the sequencing of the complete viral genome, offering comprehensive insights into the genetic composition of the virus. This method enables detection of new mutations and variations that could affect the virus's pathogenicity and transmission dynamics. Research featured in PLOS ONE illustrated the capability of WGS to provide in-depth understanding of HPV's genomic diversity, thereby enhancing insights into its epidemiology. The study examined the entire genomes of numerous HPV16-positive samples, revealing significant genetic diversity within the type and novel variants potentially influencing viral behavior and its correlation with cervical cancer (Chen et al., 2014).
Targeted sequencing directs attention to particular segments of the HPV genome, notably the L1 or E6/E7 genes, extensively employed in genotyping practices. This approach proves cost-effective and yields adequate resolution for precise identification of HPV types. Targeted sequencing holds particular relevance in clinical settings demanding detailed information on high-risk HPV types. Research featured in the Journal of Virological Methods underscored the effectiveness of targeted sequencing in accurately identifying high-risk HPV types with exceptional precision (Wang et al., 2010).
Capture-NGS method to identify HPV genotypes and integration sites. (Holmes Allyson et al,. 2016)
CD Genomics leverages these cutting-edge technologies to provide reliable HPV genotyping services, supporting research efforts in the fight against HPV-associated diseases. With ongoing advancements in genotyping techniques, the ability to accurately detect and differentiate HPV types will continue to improve, enhancing our understanding and control of HPV infections.
Service
HBV/HPV/EBV Capture Sequencing Service
Product
HPV genotyping comprises a sequential procedure aimed at identifying distinct strains of HPV within clinical samples. Each phase of this process plays a pivotal role in guaranteeing precise and dependable outcomes, influencing clinical judgments and contributing significantly to epidemiological investigations.
Precise HPV genotyping commences with meticulous sample collection, typically from the cervix using a cytobrush or swab during pelvic examination. Ensuring adequate cellular material and viral DNA content is critical for successful genotyping. Subsequently, collected samples are preserved in a stabilizing solution like ThinPrep or SurePath, which safeguards cellular integrity and viral DNA until analysis.
Following sample collection, DNA extraction is conducted to isolate HPV DNA from the collected cells. The use of high-quality DNA extraction methods is crucial to achieve pure and intact viral DNA. Commonly employed techniques include silica-based column extraction and magnetic bead-based purification, both effective in eliminating contaminants and inhibitors that may interfere with subsequent amplification and genotyping procedures. Ensuring the integrity of DNA extraction is pivotal for the success of subsequent processes.
The extracted HPV DNA undergoes amplification using PCR or alternative amplification methodologies. This process augments the quantity of viral DNA, facilitating its detection and genotyping. PCR represents the predominant approach, focusing on specific segments of the HPV genome like the L1 gene. The selection of amplification techniques may vary based on the subsequent genotyping method employed. Real-time PCR, renowned for its heightened sensitivity and specificity, is frequently utilized to detect minimal quantities of viral DNA.
After amplification, the genotyping process begins. Various methods can be employed to identify the specific HPV types present in the sample, each with its own advantages and protocols.
Hybridization-based methodologies, including DNA microarrays and LPAs, entail the hybridization of amplified HPV DNA with complementary probes. DNA microarrays feature probes affixed to a chip, facilitating the concurrent identification of numerous HPV types. This capacity for high-throughput analysis proves advantageous in expansive research endeavors. In contrast, LPAs employ probes immobilized on a strip, detecting hybridized DNA through colorimetric or chemiluminescent substrates. LPAs are esteemed for their heightened sensitivity and proficiency in distinguishing closely related HPV types.
Sequencing-based methodologies, exemplified by NGS and WGS, afford extensive insights into the genetic composition of the virus. NGS facilitates the simultaneous detection of multiple HPV types within a single operation, rendering it invaluable for research and clinical diagnostic applications. Conversely, WGS provides in-depth analyses of HPV genomic diversity, encompassing newfound mutations and variations potentially influencing pathogenicity and transmission dynamics. These approaches boast high accuracy but necessitate more substantial time and resource allocations relative to hybridization-based techniques.
The ultimate stage of the HPV genotyping test procedure entails result interpretation, which involves analyzing data to ascertain the HPV genotypes within the sample. In clinical contexts, these outcomes inform patient management and treatment decisions. For example, detecting high-risk HPV types like HPV 16 and 18 may prompt more vigilant monitoring and intervention due to their heightened association with cervical cancer risk. Trained professionals must conduct the interpretation to ensure precision and relevance of the findings.
| Cat. No. | Product Name | Brief Description |
|---|---|---|
| PM097 | CD Human Parvovirus B19 Real-time PCR Kit | CD Human Parvovirus B19 Real-time PCR Kit can be used to detect HPV B19 in the sample based on probe-based real-time PCR technology. |
| PM174 | CD 15 High-Risk Human Papillomavirus (HPV) Real-time PCR Kit | CD 15 High-Risk Human Papillomavirus (HPV) Real-time PCR Kit can be used to detect 15 types of High-risk HPV Genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 82) based on probe-based real-time PCR technology. |
| RTPK040 | CD Human Papillomavirus (HPV) Detection Kit | The CD Human Papillomavirus (HPV) Detection Kit is used for the quantification and detection of Human Papillomavirus (HPV). |
| PM003 | CD 14 High Risk HPV Multiplex Real-time PCR Kit | CD 14 High Risk HPV Multiplex Real-time PCR Kit contains multiple pairs of primers for the detection of high-risk HPV genotypes. HPV16,18,31,33,35,39,45,51,52,56,58,59,66,68 |
| HNATQC013 | CD Human Papillomavirus (HPV66) gDNA Quality Control Product | CD Human Papillomavirus (HPV66) gDNA Quality Control Product is used for performance verification of HPV typing and nucleic acid detection kits, and indoor quality control. |
| HNATQC025 | CD Human Papillomavirus (HPV83) gDNA Quality Control Product | CD Human Papillomavirus (HPV83) gDNA Quality Control Product is used for performance verification of HPV typing and nucleic acid detection kits, and indoor quality control. |
| HNATQC014 | CD Human Papillomavirus (HPV68) gDNA Quality Control Product | CD Human Papillomavirus (HPV68) gDNA Quality Control Product is used for performance verification of HPV typing and nucleic acid detection kits, and indoor quality control. |
| HNATQC015 | CD Human Papillomavirus (HPV73) gDNA Quality Control Product | CD Human Papillomavirus (HPV73) gDNA Quality Control Product is used for performance verification of HPV typing and nucleic acid detection kits, and indoor quality control. |
| HNATQC016 | CD Human Papillomavirus (HPV82) gDNA Quality Control Product | CD Human Papillomavirus (HPV82) gDNA Quality Control Product is used for performance verification of HPV typing and nucleic acid detection kits, and indoor quality control. |
| HNATQC017 | CD Human Papillomavirus (HPV11) gDNA Quality Control Product | CD Human Papillomavirus (HPV11) gDNA Quality Control Product is used for performance verification of HPV typing and nucleic acid detection kits, and indoor quality control. |
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