Human Papillomavirus DNA Testing Research Articles

Immunization and Screening Methods for Cervical Carcinoma in Developing Nations: A Detailed Review

Objective: Cervical cancer remains a significant public health challenge in developing countries, disproportionately affecting women in these regions due to limited access to effective screening and vaccination programs. This review synthesizes the current literature on the implementation strategies, effectiveness, and challenges of cervical cancer screening and Human Papillomavirus (HPV) vaccination programs in developing countries, aiming to highlight successful models and identify gaps in current practices. Screening, for cervical cancer in developing countries faces numerous obstacles, including logistical challenges, lack of awareness, and insufficient healthcare infrastructure. Despite these hurdles, strategies such as single-visit approaches, leveraging low-cost HPV DNA testing, and utilizing mobile health units have shown promise in increasing screening coverage and early detection rates. The prerequisites for effective screening highlighted include affordability, cultural acceptability, and the ability to implement large-scale screening with limited resources. HPV vaccination presents a primary prevention measure with the potential to significantly reduce the incidence of cervical cancer. However, vaccination programs in developing countries encounter challenges in vaccine procurement, distribution, and achieving high coverage rates among the target population. Innovations in vaccine delivery, education campaigns to raise awareness, and partnerships with international health organizations are crucial for overcoming these barriers.This comprehensive review underscores the need for a multifaceted approach to cervical cancer prevention in developing countries, encompassing improvements in screening and vaccination strategies, health system strengthening, community engagement, and international collaboration. By learning from successful models and addressing identified gaps, there is a significant opportunity to reduce the burden of cervical cancer in these regions and move closer to achieving global health equity.

Prevalence of anal HPV infection and anal HSIL among MSM 50 years and older living with or without HIV

Background: Anal cancer is caused by human papillomavirus (HPV), particularly HPV-16, and is preceded by anal high-grade squamous intraepithelial lesions (HSIL). The incidence of anal cancer is highest among men who have sex with men (MSM) living with HIV (MSMLWH) and increases with age. However, most previous studies of anal HPV infection and anal HSIL were performed on men under 50 years of age, and relatively little is known about HSIL among older MSMLWH or MSM not living with HIV (MSM-Not-LWH). Setting: We enrolled MSM who were aged 50+ during 2018-2022 in San Francisco, California. Methods: 129 MSMLWH and 109 MSM-not-LWH participated. All participants had anal HPV DNA testing (Atila Biosystems) and high-resolution anoscopy with biopsy of visible lesions. Results: Among MSMLWH, 47% had anal HSIL, 19% had HPV-16, and 51% had other oncogenic anal HPV types (excluding HPV-16). Among MSM-not-LWH, 37% had anal HSIL, 22% had HPV-16, and 34% had other oncogenic anal HPV types. Increasing age was not statistically associated with prevalent HSIL, HPV-16, or other oncogenic HPV infections in MSMLWH or MSM-not-LWH. HPV-16 (OR:45.1, 95% CI:15.8-129), other oncogenic HPV types (OR:5.95, 95% CI:2.74-12.9) were associated with increased odds of anal HSIL, adjusted for age, income, education, and HIV status. Conclusion: The prevalence of oncogenic anal HPV, anal HPV-16, and anal HSIL remain very high in older MSMLWH and MSM-not-LWH. With recent evidence showing that treating anal HSIL prevents anal cancer, MSM aged 50+ should be considered for anal cancer screening.

Cost-effectiveness of single-visit cervical cancer screening in KwaZulu-Natal, South Africa: a model-based analysis accounting for the HIV epidemic.

Women living with human immunodeficiency virus (WLHIV) face elevated risks of human papillomavirus (HPV) acquisition and cervical cancer (CC). Coverage of CC screening and treatment remains low in low-and-middle-income settings, reflecting resource challenges and loss to follow-up with current strategies. We estimated the health and economic impact of alternative scalable CC screening strategies in KwaZulu-Natal, South Africa, a region with high burden of CC and HIV. We parameterized a dynamic compartmental model of HPV and HIV transmission and CC natural history to KwaZulu-Natal. Over 100 years, we simulated the status quo of a multi-visit screening and treatment strategy with cytology and colposcopy triage (South African standard of care) and six single-visit comparator scenarios with varying: 1) screening strategy (HPV DNA testing alone, with genotyping, or with automated visual evaluation triage, a new high-performance technology), 2) screening frequency (once-per-lifetime for all women, or repeated every 5 years for WLHIV and twice for women without HIV), and 3) loss to follow-up for treatment. Using the Ministry of Health perspective, we estimated costs associated with HPV vaccination, screening, and pre-cancer, CC, and HIV treatment. We quantified CC cases, deaths, and disability-adjusted life-years (DALYs) averted for each scenario. We discounted costs (2022 US dollars) and outcomes at 3% annually and calculated incremental cost-effectiveness ratios (ICERs). We projected 69,294 new CC cases and 43,950 CC-related deaths in the status quo scenario. HPV DNA testing achieved the greatest improvement in health outcomes, averting 9.4% of cases and 9.0% of deaths with one-time screening and 37.1% and 35.1%, respectively, with repeat screening. Compared to the cost of the status quo ($12.79 billion), repeat screening using HPV DNA genotyping had the greatest increase in costs. Repeat screening with HPV DNA testing was the most effective strategy below the willingness to pay threshold (ICER: $3,194/DALY averted). One-time screening with HPV DNA testing was also an efficient strategy (ICER: $1,398/DALY averted). Repeat single-visit screening with HPV DNA testing was the optimal strategy simulated. Single-visit strategies with increased frequency for WLHIV may be cost-effective in KwaZulu-Natal and similar settings with high HIV and HPV prevalence.

Correlation of VIA Positive Cases with Colposcopic and Histopathological Findings in Diagnosis of Precancerous Lesion of Cervix in A Tertiary Care Hospital

Background: Cervical cancer prevention strategies are evolving by integrating new screening modalities such as human papillomavirus (HPV) DNA testing and visual inspection with acetic acid (VIA). Colposcopy, as a diagnostic tool, plays a crucial role in the management of abnormal cytology smears, especially in developed countries. However, its utilization and efficacy in resource-limited settings like Bangladesh remain underexplored. Objective: This study aimed to assess the correlation between VIA positivity and colposcopic/histopathological findings in diagnosing precancerous cervical lesions among women in Bangladesh. Method: A cross-sectional study was conducted among 200 women aged 30-60 attending the Gynecology OPD of Dhaka Medical College and Hospital. VIA positivity was determined, and all positive cases underwent colposcopic evaluation. Histopathological examination was performed for tissue samples obtained during colposcopy. Result: Among the participants, 21% were VIA-positive. Colposcopic evaluation revealed inflammation (26.2%), CIN I (38.1%), CIN II (11.1%), CIN III (9.5%), and invasive carcinoma (9.5%). Histopathology findings included inflammation (28.6%), CIN I (40.5%), CIN II (7.1%), and invasive carcinoma (16.7%). True positive and false positive cases were identified, with percentages calculated accordingly. Conclusion: Detailed colposcopic evaluation with guided biopsy is crucial for detecting pre-invasive and early cervical cancer. Integrating colposcopy into screening programs in Bangladesh could significantly reduce morbidity and mortality among young women.

Cervical Cancer Treatment Update: A Society of Gynecologic Oncology Clinical Practice Statement

ABSTRACT Despite increased screening and human papillomavirus (HPV) vaccination in high-income countries, cervical cancer represents the fourth most diagnosed cancer in women worldwide and the second highest cause of cancer mortality among women in low- and middle-income countries. This clinical practice statement describes therapies for cervical cancer by treatment setting, as well as quality of life, financial toxicity, and disparities associated with the disease. Persistent HPV infection, particularly types 16 and 18, are the main factor in the development of cervical cancer. High-risk HPV variants integrate HPV DNA into the host genome to upregulate two viral oncoproteins, E6 and E7, which in turn inhibit the tumor suppressor proteins p53 and Rb. Certain driver mutations including PIK3CA, KRAS, and EGFR are frequently seen in cervical cancer, however further research is needed to identify an actionable biomarker based on targetable oncogenic alterations. Locally advanced cervical cancer is defined as clinically visible tumor exceeding 4 cm or invading beyond the cervix, extending to the pelvic sidewalls, vagina, bladder, rectum, or involving pelvic and/or paraaortic lymph nodes. Standard treatment for locally advanced cervical cancer is external beam radiation therapy and concurrent weekly cisplatin. Standard treatment for advanced, recurrent, or metastatic cervical cancer is cisplatin, paclitaxel, and bevacizumab (possibly adding pembrolizumab). Several studies have shown that checkpoint inhibitors, when combined or sequenced with chemoradiation, may provide survival benefit in both locally advanced and recurrent/metastatic cervical cancer. Given these findings, immunotherapy should be incorporated in treatment of advanced or recurrent disease. Negative effects on quality of life (QOL) in patients with cervical cancer often begin in people who receive negative Papanicolaou test results or positive HPV DNA tests. Negative body image, self-esteem, and relationships with partners contribute to an overall decrease in QoL. Surgical, radiation-induced, and chemotherapy-induced treatment effects further impact survivors’ physical health, body-image, and sexual function. Economic burdens of cancer diagnosis and treatment may disproportionally affect cervical cancer patients due to the intensity of curative treatment, decreases in QoL and functional status, and unique population including younger patients, those with poor access to health care at baseline, and those who identify as racial or ethnic minorities.

Feasibility and Acceptability of Self-sampling for Human Papillomavirus DNA Testing Among Asymptomatic Women in Rural Delta State, Nigeria

Background: Self-sampling and human papillomavirus (HPV) DNA testing can be vital tools in cervical cancer screening for hard-to-reach women with limited access to health care in sub-Saharan Africa. This study explored the feasibility and acceptability of self-sampling for HPV testing among asymptomatic women in Orhuwhorun community in Delta State. Methods: This was a cross-sectional study of 230 women aged between 30 - 65years, enrolled by a multi-stage sampling method. Recruited women were asked to provide self-collected vaginal samples between May and June, 2021. HPV detection and genotyping was done using 21 HPV Geno-Array Diagnostic kit. Participants were asked to complete questionnaires after self-sampling to point out their experiences and acceptability of HPV self-sampling. Results: An excellent feasibility of self-sampling (95.2%) was observed. The acceptability rate of self-sampling was 93.0%, and 99.6% (229/230) of the participants were confident that they used the device correctly. The quality of self-sampling was satisfactory in 100% of the samples; 21.1% (48/228) of the samples were positive for HPV, including 12.3% (28/228) with high-risk HPV types,2.6% (6/228) with probable high risk HPV types and 1.8% (4/228)with low-risk HPV types. Multiple HPV infection occurred in 10 cases (4.4%). Conclusion: This study indicates that self-sampling is a feasible and acceptable approach for cervical cancer screening among women in rural Delta State. Thus, the government should consider self-sampling as a valuable strategy in implementing national cancer screening programme.

The Epidemiology of Anal Human Papillomavirus (HPV) in HIV-Positive and HIV-Negative Women and Men: A Ten-Year Retrospective Observational Study in Rome (Italy).

Human papillomaviruses (HPVs) commonly infect the anogenital mucosa; most infections are transient, but a fraction of those caused by high-risk (HR) types persist and may lead to anogenital cancer. The epidemiology of HPV genotypes in anal infections in groups at different risk for anal cancer has not been well described in Italy. This retrospective study reports the results of HPV DNA testing and complete genotyping performed on anal swabs from 691 female and male patients attending proctology clinics in Rome during 2012-2021; one-third had repeated testing. Cumulative HPV positivity in 1212 anal swabs was approximately 60%, was not age related, and showed an increasing trend over the study period. HPV rates differed significantly by sex and HIV status: HIV-negative women had the lowest (43.6%) and HIV-positive men the highest (83.5%) HPV prevalence. HIV-positive men had more oncogenic HPV genotypes detected, more multiple infections, and the highest frequency of persistent infections. Two-thirds of all infections were vaccine-preventable. This study found that anal HPV infection rates are still elevated and even increasing in groups at low and high risk of developing anal cancer. Prevention programs need to be improved to reduce rates of anal infection in young women and men.

Screening and prevention of HPV-related anogenital cancers in women living with HIV in Europe: Results from a systematic review.

Women living with HIV (WLWH) are at increased risk of human papillomavirus (HPV)-related cancers. Throughout Europe, there is great heterogeneity among guidelines for screening programmes, access to HPV testing and HPV vaccination. The aim of this systematic review is to summarize available data on screening and prevention measures for HPV-related anogenital cancers in WLWH across the WHO European Region (WER). The systematic review followed the PRISMA guidelines and was registered on Prospero. PubMed, Embase and Web of Science databases were searched to identify available studies, written in English and published between 2011 and 2022. A metanalysis was conducted using random-effects models to calculate pooled prevalence of HPV. Subgroup analyses were conducted according to country and HPV testing. Thirty-four articles involving 10 336 WLWH met the inclusion criteria. Studies were heterogenous in their methodology and presentation of results: 73.5% of studies focused on cervical cancer prevention, and only 4.4% on anal cancer; 76.5% of studies conducted HPV testing as a routine part of screening. The prevalence of high-risk HPV was 30.5-33.9% depending on the detection method used. A total of 77% of WLWH had cervical cytology results reported. Six studies reported the positive association of CD4 cell count <200 cells/μL with HPV prevalence and cervical abnormalities. Anal HPV testing was conducted in <8% of participants. HPV vaccination was completed in 5.6% of women (106/1902) with known vaccination status. There was no information about the vaccination status of the majority of women in the analysed studies (8434/10336). Data about screening of HPV-related anogenital cancer in WLWH in Europe are heterogenous and lacking, especially in relation to anal cancer. HPV DNA testing is not routinely done as part of screening for HPV-related cancer; guidelines should include indications for when to use this test. Low CD4 count is a risk factor for HPV infection and cytological abnormalities. HPV vaccination data are poor and, when available, vaccination rates are very low among WLWH in Europe. This review concludes that significant improvements are required for data and also consistency on guidelines for HPV screening, prevention and vaccination in WLWH.

Detection and typization of HPV genotypes in subjects with oral and upper respiratory tract lesions, Milan, Italy

Background Oral human papillomavirus (HPV) is common among healthy individuals but causes and implications of persistent infections are under evaluation in the pathogenesis of head and neck neoplasms. Methods This was a retrospective study evaluating the prevalence of high-risk (HR), probable HR and low-risk (LR) HPV types in patients reporting signs/symptoms of oral and upper respiratory tract lesions. Individuals attending between 2019 and 2022 a University Hospital in Milan, Italy, with risk factors for HPV (unprotected oral sex and/or previous documentation of HPV infection in oral and upper respiratory tract and/or another anatomical site) were included. Results Fourteen out of 110 (12.7%) individuals tested positive for HPV DNA. The prevalence of HR-HPV and LR-HPV was 3.6% (4/110) and 9.1% (10/110), respectively. No probable/possible HR-HPV was detected. Specifically, 10/110 (9.1%) were diagnosed with 1 LR-HPV genotype, 3/110 (2.7%) were infected with 1 HR-HPV and 1/110 had 3 concomitant HR-HPV types. HPV 16 (2.7%, 3/110) and 6 (4.5%, 5/110) were the most common HR and LR types, respectively. One patient positive for HPV 16, 33 and 35 was diagnosed with cancer at the base of the tongue. Two individuals among those who tested positive for HPV DNA reported previous HPV vaccination. Conclusions Our data, in line with observations from previous prevalence studies, support the potential role of HPV in head and neck neoplasms. HPV DNA testing should be performed in patients presenting lesions in oral/respiratory tracts and risk factors for HPV. Improvement in HPV vaccination coverage is warranted.

Determinants of Type-Specific Human Papillomavirus Concordance Across Anatomic Sites in Young Men Who Have Sex With Men and Transgender Women, 3 U.S. Cities, 2016-2018.

Among men who have sex with men (MSM) and transgender women (TGW), the dynamics of human papillomavirus (HPV) infections at different anatomical sites are not well understood. Information on HPV concordance between anatomic sites can inform the extent of autoinoculation, and susceptibility of different anatomic areas to HPV infection. We described and assessed correlates of HPV concordance across anal, oral, and genital samples. We enrolled 1876 MSM and TGW aged 18 to 26 years in 3 US cities. Oral, genital, and anal samples were self-collected for type-specific HPV DNA testing (37 types). Demographics, sexual behaviors, and health history were self-reported. Kappa statistics based on percent positive agreement (kappa+) and generalized estimating equations were used to describe and identify correlates of HPV type-specific concordance between anatomic sample pairs. Any HPV was detected in 69.9%, 48.6%, and 7.4% of anal, genital, and oral samples, respectively. Detection of any HPV (concurrence) was most common in anal-genital pairs (40.9%) and uncommon in oral-genital and oral-anal pairs (3.4% and 6.5% respectively). Type-specific concordance was poor across all sample pairs (kappa+ <0.20). Younger age and older age at first sex were positively associated with type-concordant anal-genital infections. Sexual behaviors were unassociated with concordance. Poor oral/anogenital concordance suggests the oral mucosa has different susceptibility to HPV infection, differential clearance and/or autoinoculation between oral and anogenital sites is unlikely. There was some observed concurrence and concordance between anal and genital sites, unassociated with sexual behavior, suggesting autoinoculation. Longitudinal studies are necessary to further elucidate mechanisms of multisite infections.

Unravelling the Impact of Human Papillomavirus (HPV): A Comprehensive Exploration of its Role in Cancer Progression and Global Health Challenges

HPV represents a large group of double stranded DNA viruses that are highly involved with different types of human cancers. This synopsis describes the complexities surrounding HPV virology, classifications, and genomic variation, especially focusing on high-risk strains such as HPV16 and HPV18, the main causative factors for cervical cancers. The International Committee on Taxonomy of Viruses specifies more than 200 HPV types each associated with a respective disease and indicated in Table 1. HPV spread is mostly due to direct skin-to-skin contact between individuals’ sex organs causing infections on the mucous membrane and keratinized epithelial cells. Although majority of the infections are asymptomatic, some chronic HRHPV infections may progress into cancers because of oncoproteins E6 and E7. The high-risk variants of HPV lead to cervical, anal, and oropharyngeal cancers whereas the low-risk types cause harmless genital warts. While integrating HPV DNA into the host genome, orchestrated by oncoproteins E6 and E7 disrupts the cell regulatory mechanism; this leads to abnormal control over cell multiplication–one of the major elements of the process that develops to HPV induced cancer. A classic case is cervical cancer that has been extensively investigated as one of the highly documented HPV associated malignancies. Pap smears and HPV DNA tests are among the screening means that lower the incidents and deaths associated with cervical cancers. HPV-associated cancers of the head and neck, anus, penis, vulva, and vagina all show a characteristic profile in terms of the pathogen aetiology and risks involved. HPV-related head and neck cancers affect non-smokers and show good response to standard therapies. HPV poses increased anal cancer risk for immunocompromised individuals highlighting the complexity of interdependence of immunity and cancer development. Penile cancer results from poor hygiene and non-circumcision. Vulvar and virginal cancer mostly affects women, and the risk factors involve HPV infection and smoking. Therefore, various types of multidisciplinary approach that may include surgeries, radiotherapy, and chemotherapy are necessary to ensure proper treatment. Certain cancers are strongly associated with some high-risk HPV genotypes, such as HPV-16 and HPV-18; thus, vaccination is important. HPV infection outcomes are determined by the immune response as well as clearance of HPV infection. Screening and immunological understanding for early detection of HPV related health risks is fundamental. However, this encompassing review highlights the multi-dimensional impact of HPV encompassing virology of HPV, cancer specific presentation of HPV and control including prevention of HPV infection, screening, and research on cancer attributable by HPV and strategies towards mitigation of this global health problem.

Human papillomavirus DNA testing with the urine sample is not yet available: the accuracy of two distinct kits.

The aim of this study was to assess the results and efficiency of two real-time polymerase chain reaction procedures for detecting human papillomavirus utilizing urine samples. This study comprised 151 patients who had previously tested positive for human papillomavirus in their cervical samples. Two different commercial real-time polymerase chain reaction techniques were used for identification and genotyping human papillomavirus in urine specimens. The urine samples of 151 patients were evaluated via the Roche Cobas test, and the urine samples of 91 patients were also evaluated via the Qiagen test. The overall consistency of urine and cervical swab specimens for the identification of human papillomavirus in Roche Cobas and Qiagen tests were 44.8 and 44%, respectively. The rates of positive human papillomavirus results from urine samples were 57 and 70.3%, respectively. The overall concordance among Roche Cobas and Qiagen tests utilizing urine samples for human papillomavirus type 16/18 was 84.3% with a kappa value of 0.675, and for other high-risk-human papillomavirus, it was 75.60% with a kappa value of 0.535. Roche Cobas showed high concordance with Qiagen test. human papillomavirus positivity was not detected in all urine samples. It is still inappropriate to recommend the use of urine liquid biopsy for the accurate and reliable detection of human papillomavirus. Due to the lack of a standardized tool, the utilization of urine samples as a screening human papillomavirus test remains a challenge.

Self-sampling for human papilloma virus DNA as a strategy to increase access to cervical cancer screening: patients' and providers' perspectives on facilitators and barriers to scale.

Self-sampling represents a high accuracy approach to human papilloma virus DNA (HPV-DNA) testing that allows for privacy and autonomy. As part of a larger study to evaluate cervical cancer screening in Mozambique, we explored user-driven facilitators and barriers to, and provider perspectives on, self-sampling. Our study was conducted in 4 public health facilities in 2 districts in Mozambique. Women aged 30-49 were prospectively enrolled for HPV screening and were offered provider or self-collected sampling. We used enrolment data from 9014 participants to examine characteristics of women choosing self-sampling and conducted in depth interviews with 104 women and 15 providers to understand facilitators and barriers to self-sampling. 97.5% of participants chose self-sampling over provider sampling. Participant-reported barriers included fear about technique, discomfort and inadequate training. Facilitators to self-sampling included increased privacy and having been exposed to a peer who previously self-sampled. Providers expressed concern about their limited role in the screening process with a self-sampling technique. Self-sampling for HPV is an acceptable approach to cervical cancer screening but barriers such as fear of sampling incorrectly and discomfort with their bodies remain.

Liquid-Based Cervical Cytology: Monitoring the Laboratory Quality Indicators.

Many developments in cervical cancer screening have happened in the past century, helping women in earlier detection of cervical cancer and its precursors. Cytology still holds the fort as being a specific test, though it suffers in sensitivity. As a part of the quality control program, the aim of the study is to determine the total number of abnormal liquid-based cervical cytology (LBC) at our center and correlate the abnormal LBC with histology and human papillomavirus (HPV) DNA test results. Retrospective analysis of 4286 LBC screening cases was carried out over a period of 5 years. For cytology-histology correlation, cervical biopsy and cytology test results were analyzed. The two-tier grading system for biopsy interpretation was used. HPV DNA test results wherever available were correlated. Of the 4286 LBC cases, 157 samples (3.7%) were unsatisfactory for evaluation, 3915 samples (91.3%) were negative for intra-epithelial lesion or malignancy, and 214 samples (5%) showed epithelial cell abnormality. ASC-US was reported in 60 cases (1.4%), ASC-H in 35 cases (0.8%), LSIL in 47 cases (1.1%), HSIL in 41 cases (1.0%), squamous cell carcinoma in a single case (0.02%), and atypical glandular cells in 30 cases (0.7%). The ASC/SIL ratio was 1.07:1. The CHC major discrepancy was calculated as 16.2%. The concordance of HSIL on cytology and biopsy as a measure of PPV is 94.4%. Of the epithelial cell abnormalities, 24 cases were positive for high-risk HPV (hrHPV). Molecular test results of 2737 samples showed HPV detected in 50 cases, of which 24 cases were positive for hrHPV. The study helped us to analyze the quality parameters of our cytopathology laboratory which are within the acceptable limits.

External quality assessment program for human papillomaviruses DNA testing in Thailand

BackgroundSince 2020, the National Health Security Office includes the human papillomavirus DNA testing for cervical cancer screening in the government's healthcare schemes. HPV DNA testing has become primary screening in many laboratories in Thailand. External quality assurance scheme is crucial for assessment of laboratory performance. ObjectivesThe aim of this study was to develop a pilot program using LBC samples for the EQA of molecular methods and to review the methods used by participants to detect the presence of high risk HPV genotypes. Study designFour pilot distributions were shipped between December 2021 and May 2023, six months apart of two panels, each consisting of five different specimens. ResultsAll participants achieved 100 % accuracy in correctly identifying the presence or absence of high-risk genotypes in all 5 EQA samples. The most used HPV DNA test for detecting the presence of high-risk HPV DNA was the two specific high-risk genotypes and 12 other high-risk HPV genotypes. There was an observed increase in the use of assays that could detect 14 HPV HR genotypes. It suggests expanding testing methods to include a broader range of high-risk HPV genotypes, which could improve the comprehensiveness of the testing. ConclusionsThe HPV DNA testing scheme provides a standardised, homogeneous and characterised clinical specimen. These results indicate that the LBC samples are suitable for utilisation in an EQA scheme. EQA of HPV molecular screening programme is essential for monitoring the performance of laboratory networks.

A novel tailed primer nucleic acid test for detection of HPV 16, 18 and 45 DNA at the point of care

Cervical cancer is a leading cause of death for women in low-resource settings despite being preventable through human papillomavirus (HPV) vaccination, early detection, and treatment of precancerous lesions. The World Health Organization recommends high-risk HPV (hrHPV) as the preferred cervical cancer screening strategy, which is difficult to implement in low-resource settings due to high costs, reliance on centralized laboratory infrastructure, and long sample-to-answer times. To help meet the need for rapid, low-cost, and decentralized cervical cancer screening, we developed tailed primer isothermal amplification and lateral flow detection assays for HPV16, HPV18, and HPV45 DNA. We translated these assays into a self-contained cartridge to achieve multiplexed detection of three hrHPV genotypes in a disposable cartridge. The developed test achieves clinically relevant limits of detection of 50–500 copies per reaction with extracted genomic DNA from HPV-positive cells. Finally, we performed sample-to-answer testing with direct lysates of HPV-negative and HPV-positive cell lines and demonstrated consistent detection of HPV16, HPV18, and HPV45 with 5000–50,000 cells/mL in < 35 min. With additional optimization to improve cartridge reliability, incorporation of additional hrHPV types, and validation with clinical samples, the assay could serve as a point-of-care HPV DNA test that improves access to cervical cancer screening in low-resource settings.

HPV testing versus p16 immunohistochemistry in oropharyngeal squamous cell carcinoma: results from the DAHANCA 19 study

Introduction The prognosis after primary (chemo-)radiotherapy for oropharyngeal squamous cell carcinoma (OPSCC) is affected by Human Papillomavirus (HPV) status, with a better prognosis in HPV-positive OPSCC. HPV-status is routinely assessed by p16 immunohistochemistry (IHC), but additional HPV DNA testing is debated. Also, there are numerous HPV genotypes, which prognostic role may need clarification. The purpose of this study was: (1) to test a custom-made targeted HPV next generation sequencing (NGS) panel in OPSCC, (2) to determine correlation with p16 IHC, and (3) to assess the impact of HPV DNA testing on outcome in the prospectively randomized clinical trial DAHANCA 19. Materials and methods We included 271 patients with OPSCC treated with primary (chemo-)radiotherapy in the DAHANCA 19 trial. Of these, 199 (73%) were p16-positive. HPV-status was determined by targeted HPV next generation sequencing (NGS), using a custom-made HPV genotyping panel. Results HPV was detected in 194 tumor samples. p16 IHC and NGS HPV status were concordant in 265 (98%) of 271 patients, whereas we did not detect HPV DNA in 5 p16-positive tumors. HPV16 accounted for 169 of 194 HPV-positive cases (87%). HPV genotypes 18, 31, 33, 35, and 59 were also detected. Loco-regional failure and overall survival were similar whether patients were separated by p16 IHC, or HPV DNA status (p < 0.0001 for all) and did not depend on HPV genotype (p = 0.9 and p = 0.7). Conclusion In the present study, HPV DNA testing or typing in a Danish OPSCC cohort did not add additional information to p16 IHC, the most widely used and accepted prognostic indicator.

Circulating tumor-tissue modified HPV DNA testing in the clinical evaluation of patients at risk for HPV-positive oropharynx cancer: The IDEA-HPV study

ObjectivesWhile survival outcomes are favorable for Human Papillomavirus (HPV)-positive oropharyngeal squamous cell carcinomas (OPSCCs), early diagnosis may minimize treatment-related morbidity and mortality. This study evaluated circulating tumor tissue-modified viral (TTMV)-HPV DNA plasma testing to facilitate early diagnosis of HPV-positive OPSCCs. MethodsIn this prospective exploratory cohort study, patients presenting to an Otolaryngology-Head and Neck Surgery clinic with unexplained signs or symptoms considered high-risk for HPV-positive OPSCC were recruited between March 2021-October 2022. Circulating TTMV-HPV DNA testing was performed, and results were shared with subjects and treating clinicians. Clinicians were surveyed regarding the perceived clinical utility of the test. ResultsThirty-nine subjects were included. Most subjects were women (N = 23, 59 %), white (N = 32, 82 %) and never-smokers (N = 20, 51 %) with median age 60 years. Circulating TTMV-HPV DNA was detected in 2/39 subjects, both subsequently diagnosed with HPV-positive OPSCC. Both were white men aged 70–80 years with a neck mass. One subject with undetectable TTMV-HPV DNA was also diagnosed with HPV-positive OPSCC through excisional neck mass biopsy. Other eventual diagnoses included 3 HPV-negative head and neck squamous cell carcinomas and 4 other malignancies. Testing was perceived as helpful in clinical decision-making for 26/38 (68 %) subjects, and useful for similar future patients for 32/37 (86 %) subjects. ConclusionCirculating TTMV-HPV DNA testing is feasible and holds potential as a diagnostic aid for HPV-positive OPSCC alongside standard clinical workup. Clinicians should be cognizant of its limitations, as a negative test does not necessarily indicate the absence of disease. Further studies to evaluate its utility are warranted.

Development and validation of real-time recombinase polymerase amplification-based assays for detecting HPV16 and HPV18 DNA.

Cervical cancer is the fourth leading cause of cancer-related death among women worldwide. Persistent human papillomavirus (HPV) infection is the principal cause of cervical cancer, with HPV16 and HPV18 accounting for about 70% of cases worldwide. Cervical screening is an effective secondary measure for preventing cervical cancer. Testing for HPV DNA is becoming increasingly important in cervical cancer screening. The existing PCR-based HPV detection technology has several disadvantages: the assays are time-consuming, and sophisticated equipment is required to control the temperature cycles. These drawbacks have led to the development of detection technologies based on isothermal amplification. Here, we present real-time recombinase polymerase amplification (RPA-exo)-based assays for single genotyping of either the E7 or the L1 segment of HPV16 or HPV18. These assays were highly sensitive, able to detect HPV in all clinical samples with Ct values below 34, and yielded results within 25 min. A dual-detection system capable of detecting both HPV16 and HPV18 in a single reaction was also developed based on the L1 gene. It has a limit of detection of approximately 10,000 copies of each genotype per reaction. The assays were validated with DNA extracted from 36 biopsy specimens and 42 exfoliated cell samples from 43 patients with cervical lesions at different stages. The RPA-exo system is a promising clinical detection platform with the advantages of yielding results rapidly and operating at a constant temperature, while being cost-effective and easy to use. IMPORTANCE HPV DNA screening is an effective approach for the prevention of cervical cancer. The novel real-time recombinase polymerase amplification-based HPV detection systems we developed constitute an improvement over the HPV detection methods currently used in clinical practice and should help to extend cervical cancer screening in the future, particularly in point-of-care test settings.

Circulating Tumor HPV DNA in Patients With Head and Neck Carcinoma: Correlation With HPV Genotyping.

Circulating tumor human papillomavirus DNA (ctHPVDNA) testing using digital-droplet polymerase chain reaction (PCR) detects fragments of tumor-modified human papillomavirus (HPV) in the plasma of patients with HPV-associated head and neck squamous cell carcinomas (HNSCCs). Its impact on tumor surveillance and primary diagnosis is limited by unresolved issues relating to sensitivity and specificity. The study population consisted of patients with HNSCC who had undergone ctHPVDNA testing. HPV status was determined by p16 immunohistochemistry and PCR-HPV genotyping on the tumor samples. For discrepant cases (HPV-positive/ctHPVDNA-negative), HPV status was confirmed by RNA in situ hybridization and, when possible, targeted single-nucleotide polymorphisms genotyping. A total of 167 patients had ctHPVDNA testing, and 141 tumors were HPV positive by p16 immunohistochemistry and PCR genotyping. Genotypes included types 16 (91.5%), 33 (4.3%), 35 (2.1%), and 18 (2.1%). ctHPVDNA was detected in 133 (94.3%) of HPV-positive HNSCCs but in none of the HPV-negative HNSCCs. Four of the 5 p16-positive cases that were negative by PCR and ctHPVDNA were positive by RNA in situ hybridization, and in 2 of these cases, rare high-risk genotypes were identified. ctHPVDNA had a sensitivity of 91.7%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 63.6%. The likelihood that patients with HPV-positive HNSCC have detectable ctHPVDNA is high. Non-HPV16 genotypes contribute to discrepancies but only in a small subset of cases. This finding validates ongoing efforts to use ctHPVDNA as a surveillance tool, and even as a primary diagnostic assay in patients presenting with masses in the neck and/or oropharynx.