In Situ Colorimetric LAMP Based on One-Step Modified Filter Paper to Screen Human Papillomavirus (HPV)16/18 from Clinical Samples.

Cervical cancer is among the most common malignant tumors in women. The development of rapid screening techniques plays an important role in early screening for cancer treatment. We have developed an HPV screening method, which effectively combines the high-efficiency nucleic acid enrichment of chitosan-modified filter paper and the rapid visual detectability of colorimetric LAMP, along with the enhancement of the tolerance ability of the pH-sensitive LAMP reagent to acidic original samples, making the detection of HPV 16/18 easy to carry out and reliable, which is helpful for the epidemiological prevention and control strategies of HPV-induced cancer. This technique can simultaneously exhibit the "in situ amplification" capability of chitosan-modified filter paper and the nontemperature cycle dependence of visual LAMP detection. Therefore, DNA extraction and amplification can be performed efficiently and quickly within a single reaction where all DNA is concentrated in the QF paper disc. By embedding amino-modified filter paper into the plastic chip, a simple and reliable disposable chip was prepared for rapid HPV16 and HPV18 detection from clinical endometrial samples, and the results were 100% consistent with clinical diagnosis. More importantly, even after the sample was diluted 100-fold, HPV16/18-infected cells could be accurately identified, showing the advantages of the system in early cancer screening. Moreover, for endometrial samples containing plenty of cells, the filter paper could be used to enrich cells by filtration, preventing the acidic fluid from impacting pH-induced colorimetric LAMP detection and realizing direct amplification for HPV identification without nucleic acid extraction. This easy-to-operate system that can analyze a wide range of samples will be suitable for routine on-site HPV screening, dramatically extending the applications and utility for rapid, near-patient nucleic acid testing.

Immediate risk of cervical intraepithelial neoplasia and diagnostic value of colposcopy among cytology-negative women with oncogenic HPV: a retrospective study.

BACKGROUND: Cervical cancer screening results that are negative for cytology but positive for high-risk human papillomavirus (HR-HPV) are not uncommon. One-year follow-up is suggested for patients with no history of HPV positivity under the most recent American Society of Colposcopy and Cervical Pathology (ASCCP) guidelines (2019). The aim of this study was to evaluate the immediate risk of cervical intraepithelial neoplasia (CIN) among cytology-negative patients positive for HR-HPV. The diagnostic accuracy of colposcopy in these patients was investigated. METHODS: A retrospective study was conducted in patients who were cytology negative but HR-HPV positive and referred for colposcopy from January 2022 to August 2023. Patients were compared in terms of the immediate rate of CIN lesions among the HPV16-positive group, the HPV18-positive group and the non-16/18 HR-HPV-positive group. The distribution of CIN2 + lesions according to age was evaluated. The factors associated with the accuracy of colposcopy were evaluated using univariate and multivariate logistic regression. RESULTS: Among the 372 patients, 195 had chronic cervicitis, 131 had CIN1, 37 had CIN2/3, and nine had carcinoma. The immediate rates of CIN2 + lesions and CIN3 + lesions in patients who were not HR-HPV16/18-positive were comparable to those in patients who were HPV16/18-positive (P = 0.699). In addition, among patients diagnosed with CIN2 + lesions, 8 (17.39%) patients were women aged < 30 years. When pathological results were used as a reference, the consistency rate of colposcopy was 61.0% (227/372). Multivariate analyses revealed that age and the type of cervical transformation zone were independent factors affecting the accuracy of colposcopy (P < 0.001). CONCLUSIONS: In countries with limited resources, immediate colposcopy referral should be recommended for patients who are cytology negative but HR-HPV-positive (including non-16/18 HR-HPV-positive), and cervical cancer screening via cotesting should be suggested for women aged < 30 years. Colposcopy has moderate diagnostic value and can be affected by age and the type of cervical transformation zone.

HPV DNA status and clinical history of patients are supplements for accurate reporting of the cytological Pap smear.

The present study was aimed at showing the importance of HPV DNA status and the clinical history of the patients required by the cytologist for accurate reporting. A total of 1250 symptomatic women who attended the gynaecology outpatient department of the Mahavir Cancer Sansthan and Nalanda Medical College, Patna, for pap smear examinations were screened and recruited for the study. Due to highly clinical symptoms out of the negative with inflammatory smears reported, one hundred and ten patients were randomly advised for biopsy and HPV 16/18 DNA analysis by a gynaecologist to correlate negative smears included in the study. Pap smear reports revealed that 1178 (94.24%) were negative for intraepithelial lesions (NILM) with inflammatory smears, 23 (1.84%) smears showed low-grade squamous intraepithelial lesions (LSIL), 12 (0.96%) smears showed high-grade squamous intraepithelial lesions, and 37 (2.96%) smears showed an atypical squamous cell of undetermined significance (ASC-US). A biopsy of 110 out of 1178 (NILM) patients revealed that 15 (13.63%) women had cervical cancer, 29 women had CIN I, 17 women had CIN II + CIN III, 35 women had benign cervical changes, and 14 women had haemorrhages. On the other hand, HPV 16/18 DNA was detected as positive in 87 out of 110. The high positivity of HPV in biopsied cases where frank cervical cancer and at-risk cancer were also observed in the negative smear-screened patients reveals that the HPV status and clinical history of the patients will be quite helpful to the cytologist for accurate reporting, and suggests that a negative HPV DNA result may be a stronger predictor of cervical cancer risk than a negative Pap test.

PAX1 methylation as a robust predictor: developing and validating a nomogram for assessing endocervical curettage (ECC) necessity in human papillomavirus16/18-positive women undergoing colposcopy.

OBJECTIVE: The major challenge in routine endocervical curettage (ECC) among Human Papillomavirus (HPV) 16/18-positive patients is that only a small fraction benefit. Nevertheless, current reported models often overestimate the validity and necessity of ECC, making it difficult to improve benefits for patients. This research hypothesized that assessing paired boxed gene 1 methylation levels (PAX1m) and clinical characteristics could enhance the predictive accuracy of detecting additional high-grade squamous intraepithelial lesions or worse (HSIL +) through ECC that were not identified by colposcopy-directed biopsy (CDB). METHODS: Data from 134 women with HPV16/18 positivity undergoing CDB and ECC between April 2018 and April 2022 were collected and analyzed. Quantitative methylation-specific polymerase chain reaction (qMSP) was utilized to measure PAX1m, expressed as ΔCp. Univariate and multivariate regression analyses were conducted to screen variables and select predictive factors. A nomogram was constructed using multivariate logistic regression to predict additional HSIL + detected by ECC. The discrimination, calibration, and clinical utility of the nomogram were evaluated using receiver operating characteristic curves (ROC) and the calibration plot. RESULTS: Age (odds ratio [OR], 5.654; 95% confidence interval [CI], 1.131-37.700), cytology (OR, 24.978; 95% CI, 3.085-540.236), and PAX1 methylation levels by grade (PAX1m grade) (OR, 7.801; 95% CI, 1.548-44.828) were independent predictive factors for additional detection of HSIL + by ECC. In HPV16/18-positive women, the likelihood of additional detection of HSIL + through ECC increased with the severity of cytological abnormalities, peaking at 43.8% for high-grade cytological lesions. Moreover, when cytological findings indicated low-grade lesions, PAX1 methylation levels were positively correlated with the additional detection of HSIL + by ECC (P value < 0.001). A nomogram prediction model was developed (area under curve (AUC) = 0.946; 95% CI, 0.901-0.991), demonstrating high sensitivity (90.9%) and specificity (90.5%) at the optimal cutoff point of 107. Calibration analysis confirmed the model's strong agreement between predicted and observed probabilities. CONCLUSION: The clinical nomogram presented promising predictive performance for the additional detection of HSIL + through ECC among women with HPV16/18 infection. PAX1 methylation level could serve as a valuable tool in guiding individualized clinical decisions regarding ECC for patients with HPV 16/18 infection, particularly in cases of low-grade cytological findings.

Advances in human papillomavirus detection and molecular understanding in head and neck cancers: Implications for clinical management.

Head and neck cancers (HNCs), primarily head and neck squamous cell carcinoma (HNSCC), are associated with high-risk human papillomavirus (HR HPV), notably HPV16 and HPV18. HPV status guides treatment and predicts outcomes, with distinct molecular pathways in HPV-driven HNSCC influencing survival rates. HNC incidence is rising globally, with regional variations reflecting diverse risk factors, including tobacco, alcohol, and HPV infection. Oropharyngeal cancers attributed to HPV have significantly increased, particularly in regions like the United States. The HPV16 genome, characterized by oncoproteins E6 and E7, disrupts crucial cell cycle regulators, including tumor protein p53 (TP53) and retinoblastoma (Rb), contributing to HNSCC pathogenesis. P16 immunohistochemistry (IHC) is a reliable surrogate marker for HPV16 positivity, while in situ hybridization and polymerase chain reaction (PCR) techniques, notably reverse transcription-quantitative PCR (RT-qPCR), offer sensitive HPV detection. Liquid-based RT-qPCR, especially in saliva, shows promise for noninvasive HPV detection, offering simplicity, cost-effectiveness, and patient compliance. These molecular advancements enhance diagnostic accuracy, guide treatment decisions, and improve patient outcomes in HNC management. In conclusion, advances in HPV detection and molecular understanding have significant clinical management implications. Integrating these advancements into routine practice could ultimately improve patient outcomes.

Clinical evaluation of primary human papillomavirus (HPV) testing with extended HPV genotyping triage for cervical cancer screening: A pooled analysis of individual patient data from nine population-based cervical cancer screening studies from China.

OBJECTIVE: To assess the clinical values of extended human papillomavirus (HPV) genotyping in triage of high-risk HPV-positive women, focusing on the trade-off between cervical precancer detections and colposcopy referrals. METHODS: A bivariate random-effects model was used to estimate the diagnostic accuracy of primary HPV screening with following triage strategies to detect cervical precancers: (i) partial genotyping for HPV16/18 combined with cytological testing at atypical squamous cells of undetermined significance threshold (used as the comparator), (ii) genotyping for HPV16/18/58/52, (iii) genotyping for HPV16/18/58/52/33, (iv) genotyping for HPV16/18/58/33/31, (v) genotyping for HPV16/18/58/52/33/31, and (vi) genotyping for HPV16/18/58/52/33/31/39/51. Internal risk benchmarks for clinical management were used to evaluate the risk stratification of each triage strategy. RESULTS: A total of 16,982 women (mean age 46.1 years, range 17-69) were included in this analysis. For CIN3+ detection, triage with HPV16/18/58/33/31 genotyping achieved lower positivity (6.85% vs. 7.35%, p = 0.001), while maintaining similar sensitivity (91.35% vs. 96.42%, p = 0.32) and specificity (94.09% vs. 93.67%, p = 0.56) compared with the comparator strategy. Similar patterns were observed for CIN2+ detection. Women with a positive HPV16/18/58/33/31 genotyping test had high enough risk for CIN3+ for colposcopy referral, while the risk for women with a negative test was below the 1-year return decision threshold according to internal benchmarks. CONCLUSIONS: Our findings suggested extended HPV genotyping is of potential to be used as a triage technique integrated into HPV-based cervical cancer screening, leading to reduced need for colposcopy referral while maintaining similar disease detection and efficient risk stratification.

Screening outcome of HPV-vaccinated women: Data from the Danish Trial23 cohort study.

BACKGROUND: Danish women-who were HPV-vaccinated as girls-are now reaching an age where they are invited to cervical cancer screening. Because of their expected lower cervical cancer risk, we must reassess our screening strategies. We analyzed Danish HPV-vaccinated women's outcomes after the first screening test at age 23. METHODS AND FINDINGS: Our study was embedded in Danish routine cytology-based screening. We conducted an observational study and included women born in 1994, offered the 4-valent HPV vaccine at age 14, and subsequently invited to screening at age 23. Cervical cytology was used for diagnostics and clinical management. Residual material was HPV tested with Cobas® 4800/6800. The most severe histology diagnosis within 795 days of screening was found through linkage with the Danish National Pathology Register. We calculated the number of women undergoing follow-up (repeated testing and/or colposcopy) per detected cervical intraepithelial neoplasia (CIN2+). A total of 6021 women were screened; 92% were HPV-vaccinated; 12% had abnormal cytology; 35% were high-risk HPV-positive, including 0.9% HPV16/18 positive, and 20% had follow-up. In women that were cytology-abnormal and HPV-positive (Cyt+/HPV+), 610 (98.5%) had been followed up, and 138 CIN2+ cases were diagnosed, resulting in 4.4 (95% CI 3.9-5.2) women undergoing follow-up per detected CIN2+. In contrast to recommendations, 182 (12.2%) cytology-normal and HPV-positive (Cyt-/HPV+) women were followed up within 795 days, and 8 CIN2+ cases were found, resulting in 22.8 (95% CI 13.3-59.3) women undergoing follow-up per detected CIN2+. CONCLUSION: Overall, HPV prevalence was high in HPV-vaccinated women, but HPV16/18 had largely disappeared. In the large group of cytology-normal and HPV-positive women, 23 had been followed up per detected CIN2+ case. Our data indicated that primary HPV screening of young HPV-vaccinated women would require very effective triage methods to avoid an excessive follow-up burden. TRIAL REGISTRATION: Trial registration number: NCT0304955.

Dual lateral flow assay based on PdRu nanocages for human Papillomavirus detection.

Cervical cancer is one of the most common gynecological malignancies, with the vast majority of which being caused by persistent infection with Human Papillomavirus (HPV) 16 and 18. The current available HPV detection methods are sensitive and genotyped but are restricted by expensive instruments and skilled personnel. The development of an easy-to-use, rapid, and cost-friendly analysis method for HPV is of great need. Herein, hollow palladium-ruthenium nanocages modified with two oligonucleotides (PdRu capture probes) were constructed for genotyping and simultaneous detection of target nucleic acids HPV16 and HPV18 by dual lateral flow assay (DLFA). PdRu capture probes were endowed with bi-functions for the first time, which could be used to output signals and hybridize target nucleic acids. Under optimized conditions, the PdRu based-DLFA with detection limits of 0.93 nM and 0.19 nM, respectively, exhibited convenient operation, and high sensitivity. Meanwhile, the DLFA achieved excellent rapid detection within 20 min, which was attributed to capture probes that can be directly bound to amplification-free target nucleic acids. Therefore, the development of PdRu-based DLFA can be utilized for rapid, sensitive, and simultaneous genotyping detection of HPV16 and HPV18, showing great application for nucleic acid detection.

Decoding Fujian's cervical HPV landscape: unmasking dominance of non-16/18 HR-HPV and tailoring prevention strategies at a large scale.

Background: Persistent HR-HPV causes cervical cancer, exhibiting geographic variance. Europe/Americas have higher HPV16/18 rates, while Asia/Africa predominantly have non-16/18 HR-HPV. This study in Fujian, Asia, explores non-16/18 HR-HPV infections, assessing their epidemiology and cervical lesion association for targeted prevention. Methods: A total of 101,621 women undergoing HPV screening at a hospital in Fujian Province from 2013 to 2019 were included. HPV genotyping was performed. A subset of 11,666 HPV-positive women with available histopathology results were analyzed to characterize HPV genotype distribution across cervical diagnoses. Results: In 101,621 samples, 24.5% tested positive for HPV. Among these samples, 17.3% exhibited single infections, while 7.2% showed evidence of multiple infections. The predominant non-16/18 high-risk HPV types identified were HPV 52, 58, 53, 51, and 81. Single HPV infections accounted for 64.1% of all HPV-positive cases, with 71.4% of these being non-16/18 high-risk HPV infections. Age-related variations were observed in 11,666 HPV-positive patients with pathological results. Cancer patients were older. In the cancer group, HPV52 (21.8%) and HPV58 (18.6%) were the predominant types, followed by HPV33, HPV31, and HPV53. Compared to single HPV16/18 infection, non-16/18 HPV predominated in LSIL. Adjusted odds ratios (OR) for LSIL were elevated: multiple HPV16/18 (OR 2.18), multiple non-16/18 HR-HPV (OR 2.53), and multiple LR-HPV (OR 2.38). Notably, solitary HPV16/18 conferred higher odds for HSIL and cancer. Conclusion: Our large-scale analysis in Fujian Province highlights HPV 52, 58, 53, 51, and 81 as predominant non-16/18 HR-HPV types. Multiple HPV poses increased LSIL risks, while solitary HPV16/18 elevates HSIL and cancer odds. These findings stress tailored cervical cancer prevention, highlighting specific HPV impacts on lesion severity and guiding region-specific strategies for optimal screening in Asia, emphasizing ongoing surveillance in the vaccination era.

Prevalence of high-risk human papillomavirus in oral squamous cell carcinoma with or without chewing habits.

Oral cancer (OC) is the most common cancer in Pakistani males and the second most common in females. Major risk factors include peculiar chewing habits, human papillomavirus (HPV) infection and molecular pathways. However, less data is available for this avertible cancer regarding its association with high-risk HPV (HR-HPV) and chewing habits in this region. Therefore, this study was done to determine the prevalence of HR-HPV in oral squamous cell carcinoma (OSCC) and its correlation with p16 and chewing habits. Formalin-fixed paraffin-embedded (FFPE) biopsy specimens of 186 samples were tested for HR-HPV type 16/18 by PCR, followed by p16 immunostaining (IHC) in a subset of cases (n = 50). Appropriate statistical tests were applied to find the association between HR-HPV/p16 and peculiar chewing habits with significance criteria of p<0.05 with 95% CI. HR-HPV (type 16 &18) was present in seven out of 186 cases (3.8%). Of these seven cases, five were positive for HPV16, whereas two were positive for HPV16/18. The overall expression of p16 protein in 50 samples was 38% (n = 19), and among these 19-IHC positive samples, 26% were positive for HR-HPV DNA. No significant association was found between HR-HPV positivity and p16 and chewing habits (p>0.05). It was concluded that HR-HPV prevalence in OSCC was very low in our population, with no statistically significant correlation with p16 and chewing habits. These results suggest the role of HR-HPV as an independent risk factor in OSCC in the local setting.

Preliminary outcomes of the Cervical Cancer Screening Program of Northern Portugal: A snapshot.

BACKGROUND: Cervical cancer screening remains an essential preventive tool worldwide. First line high-risk Human Papillomavirus (HrHPV) genotyping became gold standard for cervical cancer screening, and has been adopted by several countries, including Portugal. Herein, we aimed to assess the early outcomes of the regional Cervical Cancer Screening Program of Northern Portugal. METHODS: The analysis of a representative set of cases evaluated during a one-month period (January 2020), with adequate follow-up was performed. Descriptive analysis was performed. RESULTS: Overall, 7278 samples were received, of which 15.2% were HrHPV positive, most of these disclosing a negative result in subsequent liquid-based cytology. Nearly half of the HrHPV-positive women were referred to colposcopy. Within this group, HPV16/18+ cases depicted the higher frequency of high-grade squamous intraepithelial lesion (HSIL) or worse, compared with abnormal cytology or persistent HrHPV infection. Among women with non-HPV16/18 HrHPV infection and negative cytology, which are eligible for repeat sampling in one year, 65% were re-tested. Importantly, nearly half of these cleared HrHPV infection. Furthermore, referral to colposcopy due to HPV16/18 infection and/or abnormal cytology results were associated with > 40% risk for HSIL or worse lesion. CONCLUSIONS: Our study confirmed the reliability and effectiveness of first line HrHPV genotyping in the Cervical Cancer Screening Program of Northern Portugal. Nonetheless, it also raised concerns about excessive referral to colposcopy, with the inherent human and financial costs. Thus, further improvement and optimization are key to ensure the sustainability of the program.

Cervical microbiota dysbiosis associated with high-risk Human Papillomavirus infection.

High-risk Human Papillomavirus (HR-HPV) genotypes, specifically HPV16 and HPV18, pose a significant risk for the development of cervical intraepithelial neoplasia and cervical cancer. In the multifaceted cervical microenvironment, consisting of immune cells and diverse microbiota, Lactobacillus emerges as a pivotal factor, wielding significant influence in both stabilizing and disrupting the microbiome of the reproductive tract. To analyze the distinction between the cervical microbiota and Lactobacillus-dominant/non-dominant status of HR-HPV and non-infected healthy women, sixty-nine cervical swab samples were analyzed, included 44 with HR-HPV infection and healthy controls. All samples were recruited from Human Papillomavirus-based cervical cancer screening program and subjected to 16s rRNA sequencing analysis. Alpha and beta diversity analyses reveal no significant differences in the cervical microbiota of HR-HPV-infected women, including 16 and 18 HPV genotypes, and those with squamous intraepithelial lesion (SIL), compared to a control group. In this study we identified significantly lower abundance of Lactobacillus mucosae in women with HR-HPV infection compared to the control group. Furthermore, changes in bacterial diversity were noted in Lactobacillus non-dominant (LND) samples compared to Lactobacillus-dominant (LD) in both HR-HPV-infected and control groups. LND samples in HR-HPV-infected women exhibited a cervical dysbiotic state, characterized by Lactobacillus deficiency. In turn, the LD HR-HPV group showed an overrepresentation of Lactobacillus helveticus. In summary, our study highlighted the distinctive roles of L. mucosae and L. helveticus in HR-HPV infections, signaling a need for further research to demonstrate potential clinical implications of cervical microbiota dysbiosis.

The Prevalence of Epstein-Barr Virus in Normal, Premalignant, and Malignant Uterine Cervical Samples in Iran.

INTRODUCTION: It is suggested that Epstein-Barr virus (EBV) may play an important role in cervical cancer development. Most studies found a higher rate of EBV in cervical cancer samples in comparison to premalignant and normal groups. In this regard, this study aimed to investigate the prevalence of EBV in cervical samples. METHODS: In total, 364 samples from 179 healthy subjects, 124 women with premalignant lesions, and 61 patients with cervical cancer were investigated using nested-PCR. RESULTS: The mean age ± SE was 54.1 ± 13.4 in women with cervical cancer, 36.1 ± 9.4 among women with premalignant lesions, and 36.6 ± 11.5 in healthy individuals. In total, 290 out of 364 samples were human papillomavirus (HPV) positive and the following HPV genotypes were detected among them: HPV 16/18 was found in 43.1%, 23.9%, and 65.5% of normal, premalignant, and malignant samples, respectively, and other high-risk types were detected in 56.9% of normal, 76.1% of premalignant, and 34.5% of malignant samples. The prevalence of EBV was found to be 9.8%, 2.4%, and 2.8% in cervical cancer, premalignant lesions, and normal specimens, respectively, and the difference was statistically significant (p = 0.028). The overall frequency of coinfection between EBV and HPV was shown to be 3.6%. The coinfection was more prevalent among HPV 16/18-infected samples than other high-risk HPVs (6.6 vs. 2.9%) although the difference was not reached a statistically significant difference (p = 0.23). CONCLUSION: Our findings indicated that EBV could play an important role as a cofactor in the progression of cervical cancer. However, future studies with larger sample sizes and the expression analysis of EBV transcripts or proteins are mandatory.

Impact of high-performance human papillomavirus testing to improve cervical cancer screening in China: a prospective population-based multicentre cohort study.

OBJECTIVES: The aim of the study was to evaluate the clinical performance of HBRT-H14, a real-time PCR-based assay that separates human papillomavirus (HPV) 16 and HPV18 from 12 other high-risk (HR) HPV types, in population according to Chinese guideline. METHODS: A total of 9829 eligible women aged 21-64 years from Henan, Shanxi, and Guangdong provinces were performed by HBRT-H14 testing and liquid-based cytology (LBC) screening at baseline and followed up for 3-year. The sensitivity, specificity, positive predictive value (absolute risk), and negative predictive value of LBC diagnosis and HPV testing were calculated for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) Lesions. RESULTS: At baseline, 80 (0.81%) participants were diagnosed with CIN2+. HR-HPV with reflex LBC had a significantly higher sensitivity (78/80, 97.50% [95% CI, 91.34-99.31%] vs. 62/80, 77.50% [67.21-85.27%], McNemar's test p < 0.001), and a slightly lower specificity (8528/9749, 87.48% [86.80-88.12%] vs. 8900/9749, 91.29% [90.72-91.83%], McNemar's test p < 0.001) than LBC with reflex HR-HPV for CIN2+. 7832 (79.6%) participants completed 3-year follow-up and 172 (2.20%) participants were cumulatively diagnosed with CIN2+. Compared with LBC with reflex HR-HPV, HR-HPV with reflex LBC significantly increased the sensitivity (161/172, 93.60% [88.91-96.39%] vs. 87/172, 50.58% [43.18-57.96%], McNemar's test p < 0.001), but marginally decreased the specificity (6776/7660, 88.46% [87.72-89.16%] vs. 6933/7660, 90.51% [89.83-91.15], McNemar's test p < 0.001). In addition, the absolute 3-year risk of CIN2+ in HPV16/18-positive individuals was as high as 33% (80/238), whereas the risk in the HPV-negative population was only 0.16% (11/6787), much lower than those in the negative for intraepithelial lesion or malignancy population (1.21%, 85/7018). Moreover, similar results were found in women ≥30 years old. DISCUSSION: The study has indicated that HBRT-14 has a reliable clinical performance for use in cervical screening. The validated HPV test would improve the quality of population screening.

Enhanced CRISPR/Cas12a-based quantitative detection of nucleic acids using double emulsion droplets.

Pairing droplet microfluidics and CRISPR/Cas12a techniques creates a powerful solution for the detection and quantification of nucleic acids at the single-molecule level, due to its specificity, sensitivity, and simplicity. However, traditional water-in-oil (W/O) single emulsion (SE) droplets often present stability issues, affecting the accuracy and reproducibility of assay results. As an alternative, water-in-oil-in-water (W/O/W) double emulsion (DE) droplets offer superior stability and uniformity for droplet digital assays. Moreover, unlike SE droplets, DE droplets are compatible with commercially available flow cytometry instruments for high-throughput analysis. Despite these advantages, no study has demonstrated the use of DE droplets for CRISPR-based nucleic acid detection. In our study, we conducted a comparative analysis to assess the performance of SE and DE droplets in quantitative detection of human papillomavirus type 18 (HPV18) DNA based on CRISPR/Cas12a. We evaluated the stability of SEs and DEs by examining size variation, merging extent, and content interaction before and after incubation at different temperatures and time points. By integrating DE droplets with flow cytometry, we achieved high-throughput and high-accuracy CRISPR/Cas12a-based quantification of target HPV18 DNA. The DE platform, when paired with CRISPR/Cas12a and flow cytometry techniques, emerges as a reliable tool for absolute quantification of nucleic acid biomarkers.

Detección e integración del virus del papiloma humano de alto riesgo en lesiones pre-neoplásicas en mujeres del sur de Chile: un estudio transversal / High-risk human papillomavirus detection and integration in preneoplastic lesions among women in Southern Chile: a cross-sectional study

INTRODUCCIÓN: El virus del papiloma humano de alto riesgo (VPH-AR) es responsable del cáncer de cuello uterino y sus lesiones preneoplásicas. Los genotipos VPH16 y VPH18 son los más frecuentes en este cáncer. La integración del VPH-AR en el genoma de la célula hospedera es crucial en la carcinogénesis cervical, pero la etapa en que ocurre en la población chilena es incierta. OBJETIVO: Evaluar la integración de VPH16 y VPH18 en lesiones pre-neoplásicas de cuello uterino. MÉTODOS: Se analizaron 108 muestras de raspados cervicales. El VPH se genotipificó mediante reacción de polimerasa en cadena (RPC) e hibridación no radiactiva. La integración de VPH16 y VPH18 se determinó por presencia del gen E2 mediante RPC. RESULTADOS: VPH16 y VPH18 se detectaron en 36,1% y 12,0% de las muestras, respectivamente. El VPH16 se integró en 23,1% de los casos de VPH16, mientras que VPH18 se integró en 100% de las muestras positivas para este genotipo. CONCLUSIONES: La integración VPH-AR es un evento temprano en la carcinogénesis cervical que ocurre en casi la mitad de las lesiones pre-neoplásicas y es más frecuente en VPH18 que en VPH16. La evaluación de la integración VPH-AR puede ser una herramienta útil para detectar el virus en la población chilena.

BACKGROUND: High-risk Human Papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer and its preneoplastic lesions. HPV16 and 18 are the most frequent HR-HPV genotypes detected in cervical cancer. HR-HPV genome integration into the host cell is an important event in the carcinogenic process. However, it remains uncertain which stage of cervical carcinogenesis HPV16 and 18 integration occurs in the Chilean population. AIM: The goal of this study was to evaluate HPV16 and HPV18 integration in preneoplastic lesions of the cervix. METHODS: DNA was extracted from 108 cervical scrape samples with preneoplastic lesions. HPV was genotyped using PCR and non-radioactive hybridization. The integration status of HPV16 and HPV 18 was determined by evaluating the E2 gene presence through PCR. RESULTS: HPV16 and HPV18 tested positive in 36.1% and 12.0% of samples, respectively. HPV16 was found integrated in 23.1% of HPV 16 cases, while HPV 18 in 100% of samples positive for this viral genotype. CONCLUSIONS: HR-HPV integration is an early event in cervical carcinogenesis, occurring in nearly half of preneoplastic lesions and being more frequent in HPV18 than in HPV16. The evaluation of HR-HPV integration can be utilized as a complementary tool for detecting HPV in the Chilean population.

Clinical efficacy of primary human papillomavirus (HPV) screening with partial genotyping for HPV-16 and HPV-18 subtypes in women from 25 years old.

Introduction: Cervical screening programmes differ in the age of women recommended for primary human papillomavirus (HPV) testing. This study aims to determine the clinical efficacy and impact of 14-high-risk HPV DNA testing for women from 25 years old. Method: This was a retrospective analysis of data collected prospectively from women 25 years or older who attended hospital-based gynaecology clinics for cervical screening. Women with history of cervical neoplasia or abnormal cytology were excluded. High-risk HPV DNA testing with partial genotyping for HPV-16 and HPV-18 were performed on cobas 4800 System (Roche Diagnostics International AG, Rotkreuz, Switzerland). Women tested positive for the 12 other high-risk HPV subtypes (HPV-12 other) had a reflex cytology test. Positive screening included positive for HPV-16 and/or HPV-18, HPV-12 other with cytology abnormalities equal to or greater than atypical squamous cells of undetermined significance, and repeated positive HPV at 12 months. HPV detection and colposcopy referral rates, and detection of high-grade neoplasia were determined. Results: Of 10,967 women studied, 822 (7.50%) were HPV DNA positive. The overall discharge rate to routine screening according to screening protocol was 93.1%. Colposcopy referral rate was 4.4%. The screening detected 41 cervical intraepithelial neoplasia grade 2+ (CIN2+) (0.37%) and 31 (0.28%) CIN3+. The number of colposcopies needed per case of CIN2+ was 9.5, similar for women below and above 30 years old. The number of colposcopies needed per case of CIN3+ for HPV-16 positivity was 8.5, compared to 17.0 for other categories (P=0.040). Colposcopy efficacy was similar for HPV-18 and HPV-12 other positivity with abnormal cytology. Conclusion: Taking CIN2+ detection and colposcopy referral rate as endpoints, HPV testing in Singapore can be extended to include women from 25 years old.

Results from a cervical cancer screening program in Samsun, Turkey.

BACKGROUND: Cervical cancer is a preventable disease. This study aimed to share the results of the national cervical cancer screening program performed in primary health care institutions in Samsun between 2015 and 2019. METHODS: Women aged 30-65 years who were screened for cervical cancer in screening centers of Samsun between January 01, 2015, and December 31, 2019, were included in this descriptive study. The data were obtained from the automation program of the "National Human Papilloma Virus (HPV) Laboratory Application" used by the Provincial Directorate of Health Cancer Unit through filtering the completion time of the tests, and all results were evaluated without sampling. Thus, data were presented using descriptive statistics. RESULTS: The mean age of 89,302 women included in the cervical cancer screening program was 45.9 ± 9.0 years. Of the samples obtained from the participants, 1.0% were determined as insufficient material, 94.1% as HPV-negative, and 4.9% as HPV-positive. The most common HPV genotypes were 16, 51, 31, and 52. Of the 4337 HPV-positive women, 74.7% of the pap smear results were negative (including infection, 36.5%), and the most common premalignant lesions were atypical squamous cells of undetermined significance in 7.1% and low-grade squamous intraepithelial lesions in 6.9%. HPV 16/18 was also observed in 31.7% of HPV-positive women. Seven hundred ninety-five women were referred to a specialist physician for further examination and treatment within the scope of the screening algorithm. CONCLUSION: Detecting HPV-positivity by reaching more women within the national cervical cancer screening program's scope is vital in fighting against this disease. The effectiveness of cancer screening programs should be increased by ensuring community participation through awareness activities.

Accuracy of different triage strategies for human papillomavirus positivity in an Italian screening population.

How to manage human papillomavirus (HPV)-positive women in cervical cancer screening remains debated. Our study compared different strategies to triage HPV positivity in a large cohort of women participating in a population HPV-based screening program. Women were tested for HPV (Cobas 4800; Roche), and those positive were triaged with cytology; cytology-positives were referred to colposcopy, while negatives were referred to 1-year HPV retesting. All HPV-positive women were also evaluated with p16/ki67 dual staining (Roche). All lesions found within 24 months of follow-up were included in the analyses. Of the 70 146 women tested, 4757 (6.8%) were HPV-positive. Of these, 1090 were cytology-positive and were referred to colposcopy. Of the 2958 HPV-positive/cytology-negative women who presented at 1-year retesting, 1752 (59.9%) still tested positive. Cumulatively, 532 CIN2+ (including 294 CIN3+) were found. The sensitivity of cytology, HPV16/18 and p16/ki67 as triage test for CIN3+ was 67.9%, 56.0% and 85.0%, respectively. The positive predictive value (PPV) for immediate colposcopy referral was 21.0%, 15.8% and 22.9%, respectively. Combining cytology with typing increased sensitivity to 83.9% and lowered PPV to 14.8%, while combining p16/ki67 and typing increased sensitivity to 91.1%, lowering the PPV to 15.9%. Women negative to p16/ki67 triage presented a cumulative 1-year CIN3+ risk of about 1%. In conclusion, when triaging HPV positivity, p16/ki67 performed better than cytology with or without HPV16/18 genotyping. The strategies that included dual staining achieved sensitivity and low 1-year risk for CIN3+ sufficiently high enough to permit considering extending the surveillance interval to 2 to 3 years for HPV-positive/triage-negative women.

Clinical performance of methylation as a biomarker for cervical carcinoma in situ and cancer diagnosis: A worldwide study.

The shift towards primary human papillomavirus (HPV)-based screening has necessitated the search for a secondary triage test that provides sufficient sensitivity to detect high-grade cervical intraepithelial neoplasia (CIN) and cancer, but also brings an improved specificity to avoid unnecessary clinical work and colposcopy referrals. We evaluated the performance of the previously described DNA-methylation test (S5) in detecting CIN3 and cancers from diverse geographic settings in high-, medium- and low-income countries, using the cut-off of 0.80 and exploratory cut-offs of 2.62 and 3.70. Assays were performed using exfoliated cervical specimens (n = 808) and formalin-fixed biopsies (n = 166) from women diagnosed with cytology-negative results (n = 220), CIN3 (n = 204) and cancer stages I (n = 245), II (n = 249), III (n = 28) and IV (n = 22). Methylation increased proportionally with disease severity (Cuzick test for trend, P < .0001). S5 accurately separated women with negative-histology from CIN3 or cancer (P < .0001). At the 0.80 cut-off, 543/544 cancers were correctly identified as S5 positive (99.81%). At cut-off 3.70, S5 showed a sensitivity of 95.77% with improved specificity. The S5 odds ratios of women negative for cervical disease vs CIN3+ were significantly higher than for HPV16/18 genotyping at all cut-offs (all P < .0001). At S5 cut-off 0.80, 96.15% of consistently high-risk human papillomavirus (hrHPV)-negative cancers (tested with multiple hrHPV-genotyping assay) were positive by S5. These cancers may have been missed in current primary hrHPV-screening programmes. The S5 test can accurately detect CIN3 and malignancy irrespective of geographic context and setting. The test can be used as a screening and triage tool. Adjustment of the S5 cut-off can be performed considering the relative importance given to sensitivity vs specificity.