Microbiology Spectrum

Individuality and generality of intratumoral microbiome in the three most prevalent gynecological malignancies: an observational study

ABSTRACT Growing evidence have indicated the crucial role of intratumor microbiome in a variety of solid tumor. However, the intratumoral microbiome in gynecological malignancies is largely unknown. In the present study, a total of 90 Han patients, including 30 patients with cancer in cervix, ovary, and endometrium each were enrolled, the composition of intratumoral microbiome was assessed by 16S rDNA amplicon high throughput sequencing. We found that the diversity and metabolic potential of intratumoral microbiome in all three cancer types were very similar. Furthermore, all three cancer types shared a few taxa that collectively take up high relative abundance and positive rate, including Pseudomonas sp ., Comamonadaceae gen. sp ., Bradyrhizobium sp ., Saccharomonospora sp ., Cutibacterium acnes , Rubrobacter sp ., Dialister micraerophilus , and Escherichia coli . Additionally, Haemophilus parainfluenzae and Paracoccus sp . in cervical cancer, Pelomonas sp . in ovarian cancer, and Enterococcus faecalis in endometrial cancer were identified by LDA to be a representative bacterial strain. In addition, in cervical cancer patients, alpha-fetoprotein (AFP) (correlation coefficient = −0.3714) was negatively correlated ( r = 0.4, 95% CI: 0.03 to 0.7) with Rubrobacter sp . and CA199 (correlation coefficient = 0.3955) was positively associated ( r = 0.4, 95% CI: 0.03 to 0.7) with Saccharomonospora sp .. In ovarian cancer patients, CA125 (correlation coefficient = −0.4451) was negatively correlated ( r = −0.4, 95% CI: −0.7 to −0.09) with Porphyromonas sp. . In endometrial cancer patients, CEA (correlation coefficient = −0.3868) was negatively correlated ( r = −0.4, 95% CI: −0.7 to −0.02) with Cutibacterium acnes . This study promoted our understanding of the intratumoral microbiome in gynecological malignancies. IMPORTANCE In this study, we found the compositional spectrum of tumor microbes among gynecological malignancies were largely similar by sharing a few taxa and differentiated by substantial species owned uniquely. Certain species, mostly unreported, were identified to be associated with clinical characteristics. This study prompted our understanding of gynecological malignancies and offered evidence for tumor microbes affecting tumor biology among cancers in the female reproductive system.

Altered Gut Microbial Profile Accompanied by Abnormal Fatty Acid Metabolism Activity Exacerbates Endometrial Cancer Progression

A growing number of studies have shown the connection between gut microbiota, obesity, and cancer. However, to our knowledge, the association between gut microbiota and endometrial cancer progression in humans has not been studied.

Development and clinical validation of an ERA-CRISPR/Cas12a assay for the rapid detection of 14 high-risk HPV types

ABSTRACT Persistent infection with high-risk human papillomavirus (HR-HPV) is the leading cause of cervical cancer, highlighting the critical need for early detection to improve prevention. Although real-time quantitative polymerase chain reaction (RT-qPCR) remains the gold standard for HR-HPV detection, its dependence on sophisticated equipment, complex procedures, and trained personnel limits accessibility. Here, we developed a simplified assay for 14 HR-HPV types by integrating direct lysis, enzyme-mediated isothermal rapid amplification (ERA), and CRISPR-Cas12a-mediated cleavage into a streamlined workflow that requires only a basic isothermal heating device. The optimized system achieved a sensitivity of 50 copies per reaction with no cross-reactivity, while a refined lysis buffer containing 20% Chelex-100 minimized inhibition from vaginal swab samples, thereby enhancing detection performance. Validation with 152 clinical samples demonstrated 97.62% sensitivity and 100% specificity, confirming the reliability of the method. This user-friendly and cost-effective assay requires minimal equipment, enabling rapid and field-deployable HR-HPV detection, and offers a practical alternative to conventional laboratory-based approaches, particularly in resource-limited settings. IMPORTANCE High-risk human papillomavirus (HR-HPV) is the principal etiological agent of cervical cancer, and early detection remains central to effective disease prevention. Current PCR-based assays, however, rely on specialized laboratories and trained personnel, limiting their deployment in many settings. Here, we report a streamlined CRISPR-Cas12a assay that integrates direct sample lysis, ERA, and CRISPR-based detection into a single workflow operable with only a simple heating device to determine the presence of 14 HR-HPV types. The assay achieves high analytical sensitivity, strong specificity, and robust clinical performance while maintaining low cost and ease of use. This platform enables rapid HR-HPV detection and scalable screening, particularly in resource-constrained environments, with the potential to facilitate earlier intervention and reduce cervical cancer incidence.

Characterization of cervical microbiota in cervical intraepithelial neoplasia and cervical cancer using low-coverage whole genome sequencing

ABSTRACT This study characterized compositional shifts in cervical microbiota across disease stages from benign conditions through cervical intraepithelial neoplasia (CIN) to cervical cancer (CC) and investigated interactions with high-risk HPV (hr-HPV) infection using species-resolution profiling to identify severity-associated biomarkers. Cervical exfoliated epithelial cells from 50 patients (eight normal/CIN1, 15 CIN2, 19 CIN3, 5 CC) were analyzed using Low-Coverage Whole Genome Sequencing combined with the Ultrasensitive Chromosomal Aneuploidy Detector (UCAD), a technology featuring a two-step normalization framework that systematically converts raw microbial reads into statistically validated abundance deviations. This enables quantitative identification of pathologically relevant microbiota through cohort-wide Z-score benchmarking. Microbial diversity, differential biomarkers, and HPV-microbiota interactions were assessed using Kruskal-Wallis tests, LEfSe, and Random Forest modeling. Results revealed progressive Lactobacillus depletion (e.g., Lactobacillus crispatus : 32.9% in ≤CIN2 vs. 8.8% in CC) and enrichment of pathobionts like Gardnerella and Bacteroides with lesion severity. CC exhibited the highest microbial diversity (Shannon index: CC vs. CIN2, P =0.045), dominated by HPV16 (11.8%), Bacteroides (55.4%), and Porphyromonas (25.2%). LEfSe identified HPV16, HPV35, Parvimonas micra , and Anaerococcus lactolyticus as CC-specific markers, while Random Forest highlighted Mobiluncus curtisii (importance score=2.0) and HPV16 as key discriminators. CC microbiota showed significant Bacteroidetes enrichment (82% at class level) and reduced Firmicutes abundance. These findings suggest carcinogenesis-associated microbial restructuring, marked by Lactobacillus loss, anaerobic proliferation, and HPV16/35 dominance, potentially modulating disease progression. The identified signatures may inform diagnostic development and microbiome-targeted therapies. IMPORTANCE Our study pioneers an LC-WGS/UCAD approach to characterize microbial across the spectrum from benign lesions through precancerous cervical intraepithelial neoplasia to invasive cervical carcinoma. By identifying lesion-specific microbial biomarkers and HPV-associated cofactors, this work advances mechanistic understanding of microbiota-driven oncogenesis and informs future strategies for microbiota-targeted cervical cancer prevention.

HPVPool-Seq: a genotype-guided pooling strategy for cost-effective next-generation sequencing detection of HPV integration in cervical samples

ABSTRACT Integration of high-risk human papillomavirus (hrHPV) DNA is a critical event in carcinogenesis and a promising biomarker for risk stratification. However, the high cost of next-generation sequencing (NGS) limits its widespread clinical adoption. We developed HPVPool-Seq, an innovative pooling strategy that leverages the inherent diversity of HPV genotypes as natural barcodes, enabling cost-effective, scalable integration detection. Samples were pooled based on qPCR-derived HPV genotypes and viral loads prior to targeted NGS and bioinformatic decoding. A web-based automation tool was implemented to streamline pooling and decoding workflows. In a proof-of-concept study of 175 clinical specimens, HPVPool-Seq achieved 77.1% exact genotype concordance and 97.1% combined sensitivity compared with qPCR. Cost simulations demonstrated a 60% reduction in per-sample sequencing expenses. Self-correcting capability through targeted retesting further enhanced reliability. HPVPool-Seq offers a novel, traceable, and economically viable solution for high-throughput HPV integration profiling, balancing cost, scalability, and clinical precision. This strategy sets a new framework for molecular screening in HPV-associated cancers. IMPORTANCE Accurate detection of high-risk HPV integration is critical for identifying individuals at true risk of progression to malignancy. However, the high cost of next-generation sequencing (NGS) has limited its widespread clinical application. Here, we propose HPVPool-Seq, a novel pooling-based sequencing strategy that uses HPV genotypes as intrinsic barcodes to guide sample pooling without compromising detection sensitivity. This method dramatically reduces sequencing costs while maintaining genotype-level traceability and offers a built-in mechanism for selective retesting of discordant cases. By addressing both technical and economic barriers, our approach provides a scalable, clinically applicable solution for HPV integration profiling in large cohorts, with important implications for precision screening, triage, and epidemiological surveillance.

Multi-center evaluation of the Alinity m HR HPV assay with liquid-based cytology cervical specimens in the United States

ABSTRACT Incorporating molecular testing for human papillomavirus (HPV) into the screening of cervical specimens can improve risk stratification and, in turn, patient management. Infection with a high-risk (HR) HPV genotype is associated with greater risk for persistent infection, viral integration, and progression of cervical neoplasia. Current guidelines consider HPV 16 or HPV 18 clinically actionable with referral to colposcopy; however, 12 Other HR HPV genotypes have been associated with cervical cancer risk, suggesting a benefit of extended genotyping. In this multi-center study, we evaluated the performance of the Alinity m HR HPV assay, which reports HPV 16, 18, and 45 individually and aggregates of HPV 31/33/52/58 and HPV 35/39/51/56/59/66, compared with cobas HPV and Aptima HPV assays, across a variety of cytology result categories. A total of 746 de-identified residual cervical specimens, collected as part of routine cervical cancer screening programs, were tested using Alinity m HR HPV and at least one comparator assay. The overall percent agreement was ≥90.7% for results from the Alinity m HR HPV assay and cobas HPV assays and 90.5% for results from the Alinity m HR HPV and Aptima HPV assay. In patients with any abnormal cytology result, Alinity m identified 78 specimens with non-HPV 16/18 results, underscoring the benefit of detecting additional HR HPV genotypes to guide patient management more accurately. Among specimens with normal cytology, Alinity m detected 14 additional specimens with non-HPV 16/18 genotypes. Extended HR HPV testing can provide additional information to triage patients for appropriate testing and follow-up. IMPORTANCE Extended genotyping for high-risk human papillomavirus (HPV) types enhances diagnostic precision by identifying additional oncogenic HPV types beyond 16 and 18 therefore offering a more nuanced risk profile. This more comprehensive detection may aid in identifying persistent infections that are more likely to progress, thereby supporting future risk-based patient management strategies.

Clinical validation of the Roche cobas HPV test on the Roche cobas 5800 system for the purpose of cervical screening

ABSTRACT This study assessed the relative clinical sensitivity and specificity, as well as reproducibility, for high-risk HPV types of the Roche cobas HPV test when processed using the Roche cobas 5800 system. The results from this study demonstrate that the cobas HPV test using the cobas 5800 system fulfils the Meijer criteria for use in population-based cervical screening. This clinical validation study also examines the clinical sensitivity and specificity based on partial genotyping, with separate detection of HPV16 and HPV18, compared with the Roche cobas 4800 HPV test, a second-generation standard comparator assay. The cobas HPV test has a relative clinical sensitivity of 1.000, when compared with the cobas 4800 HPV test to detect histologically confirmed CIN2+ lesions in woman aged 30 years or older, with a relative clinical specificity of 0.995. The general intra- and inter-laboratory agreement for the cobas HPV test on the cobas 5800 system for finding a HPV positive result were 99.1% and 99.6%, respectively. IMPORTANCE This study demonstrates, for the first time, the clinical performance of the Roche cobas HPV test when processed using the new cobas 5800 system [cobas (5800)]. This study shows that the cobas (5800) demonstrates relative clinical sensitivity and specificity, when compared with a standard comparator HPV test, which meets the international HPV test validation requirements. Intra- and inter-laboratory reproducibility also fulfills these criteria. The current study demonstrates that the cobas (5800) can be used for primary HPV-based cervical screening on cervical specimens.

Cervical Squamous Intraepithelial Lesions Are Associated with Differences in the Vaginal Microbiota of Mexican Women

Human papillomavirus (HPV) plays a critical role in cervical carcinogenesis but is not sufficient for cervical cancer development, indicating the involvement of other factors. The vaginal microbiota is an important factor in controlling infections caused by HPV, and, depending on its composition, it can modulate the microenvironment in vaginal mucosa against viral infections.

Association of Cervical Dysbacteriosis, HPV Oncogene Expression, and Cervical Lesion Progression

The main findings of this study were that we clarified the associations of the different bacterial species with the expression of human papillomavirus (HPV) oncogenes at different stages of cervical cancer. Along with the severity of cervical lesions, the abundance of the genus and species of Lactobacillus obviously declined, while the aerobic and anaerobic bacteria, as well as the prevalence and expression of HPV E6/E7 oncogene, were increased dramatically.

Performance of BD Onclarity HPV assay on FLOQSwabs vaginal self-samples

ABSTRACT This study assessed the accuracy of high-risk human papillomavirus testing of BD Onclarity HPV (Onclarity) assay on vaginal self-collected FLOQSwab versus cervical samples to ensure similar accuracy to detect cervical intraepithelial neoplasia. Testing was performed on two automated platforms, BD Viper LT and BD COR, to evaluate the effect of machine and using two vaginal self-samples to analyze the influence of collection, transport, and freezing-unfreezing on the results. A cervical sample and two self-samples were collected from 300 women. The first collected vaginal and the cervical sample were tested on BD Viper LT, and the second swab was frozen and subsequently tested on both automated systems. Test results on vaginal and cervical specimens were considered the index and comparator, respectively; colposcopy and histology were reference standards. Relative sensitivity for ≥CIN2 on vaginal samples analyzed versus the cervical sample was 1.01 (0.97–1.06), 1.01 (0.97–1.06), and 1.00 (0.95–1.05), for the first, second self-collected sample tested on BD VIPER LT, and second self-collected sample tested on BD COR, respectively. Relative specificity was 0.83 (0.73–0.94), 0.76 (0.67–0.87), and 0.82 (0.73–0.92) using the three different workflows. Cut-off optimization for human papillomavirus (HPV) positivity defined at Ct ≤38.3 for HPV16, ≤ 34.2 for HPV18, and ≤31.5 for all other types showed an increased relative specificity with similar sensitivity. No significant difference was observed between self-samples tested with the two platforms and between first- and second-collected swabs. Onclarity assay on FLOQSwab using both platforms showed similar sensitivity but lower specificity to detect ≥CIN2 compared to cervical samples. By cut-off optimization, non-inferior specificity could be reached. IMPORTANCE Human papillomavirus (HPV) testing on self-collected vaginal samples has been shown to improve women’s participation to cervical cancer screening programs, particularly in regions with limited access to health care. Nevertheless, the introduction of self-sampling in cervical cancer screening programs requires prior clinical validation of the HPV assay in combination with a self-sample collection device, including also the laboratory workflow and automation required for high-throughput testing in screening. In this study, the performance of BD Onclarity HPV on FLOQSwab-collected vaginal self-samples has been compared to clinician-taken liquid-based cytology samples, to detect high-grade cervical intraepithelial neoplasia using two high-throughput platforms, BD Viper LT and BD COR. The study findings have shown a similar performance of BD Onclarity on testing self-collected samples, confirming the validation of the proposed pre-analytical and analytical protocols for their use in cervical cancer screening programs based on self-collected vaginal samples.

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

ABSTRACT 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.

The Significance of Human Papillomavirus Receptors Related Genetic Variants in Cervical Cancer Screening

Virus receptors are known to mediate virus attachment and further lead to virus infection of the host cells. In the current study, we investigated the relationship between single nucleotide polymorphisms (SNPs) in human papillomavirus (HPV) receptor associated genes and HPV susceptibility and clinical outcomes in Chinese women, and to explore the new triaging strategy for non-16/18 high-risk HPV infection.

Microbial and metabolic profiles associated with HPV infection and cervical intraepithelial neoplasia: a multi-omics study

ABSTRACT Cervical cancer is the most common malignancy of the female reproductive system, with the incidence of human papillomavirus (HPV) being a crucial factor in its pathogenesis. Emerging evidence indicates that cervicovaginal microbiota may influence HPV persistence and cervical intraepithelial neoplasia (CIN). However, the interplay between cervicovaginal and cervical tissue microbiomes and their association with HPV infection and CIN remains poorly understood. In this cross-sectional study, we analyzed the microbiota profiles of cervicovaginal and cervical tissue via five-region 16S rRNA gene metabarcoding, along with cervicovaginal metabolites, including short-chain fatty acids (SCFAs) and non-targeted metabolomic data, from 94 women. Key species, particularly Lacticaseibacillus and various anaerobes, are vital components of the microbiota found in both cervicovaginal secretions and cervical tissue, despite notable differences in microbial composition. The CIN group exhibited significant differences in microbial diversity and composition compared to the control groups, with key species such as Lacticaseibacillus iners and Prevotella bivia associated with HPV status and CIN progression. Metabolomic analysis revealed alterations in glycerophospholipid metabolism, but not in SCFAs, with correlations observed between metabolites and HPV status. Notable associations, including P. bivia –PE(18:1/0:0)–HPV and Fusobacterium periodonticum –PI(40:6)–HPV, were identified. Our findings emphasize the critical roles of cervicovaginal and cervical tissue microbiomes in HPV infection and CIN development, highlighting specific microbial species and metabolic pathways for early detection and therapeutic targets. IMPORTANCE Cervical cancer is the most prevalent malignancy in the female reproductive system, with human papillomavirus (HPV) persistency being a critical factor in its pathogenesis. This study highlights the significant yet often overlooked role of cervicovaginal secretion and cervical tissue microbiota in influencing HPV infection and the progression of cervical intraepithelial neoplasia (CIN). By employing a multi-omics approach, we elucidated distinct microbiota profiles in cervical tissues compared to cervicovaginal secretions, revealing a complex interplay between specific bacterial species (notably Lacticaseibacillus and anaerobes) and metabolomic changes associated with glycerophospholipid metabolism. Our findings address a significant gap in understanding the interplay between cervicovaginal secretion and cervical intratissue microbiomes, HPV infection, and CIN.

Comparative clinical performance of Alinity m HR HPV, cobas 4800 HPV, and cobas 6800 HPV for cervical cancer screening

ABSTRACT Primary high-risk human papillomavirus (HPV) testing is recommended for cervical cancer screening due to its sensitivity and high negative predictive value. Most of the cervical cancers are caused by HPV16 and HPV18, and their presence has been used to guide patient management. Here, we compared the clinical performance of the Alinity m HR HPV, cobas 4800 HPV, and cobas 6800 HPV assays in the context of cervical cancer screening. Clinical sensitivity and specificity were evaluated with 125 ≥CIN3 (cervical intraepithelial neoplasia grade 3) cases and 244 controls (≤CIN1). The genotype agreements between the assays were also evaluated for the case and control groups. The clinical sensitivities were 96.0% for Alinity m and cobas 6800 assays, and 95.2% for cobas 4800 assay. The clinical specificities observed were 67.6%, 68.0%, and 68.4% for Alinity m, cobas 6800, and cobas 4800 assays, respectively. Overall, the three HPV assays demonstrated similar clinical performance. In the ≥CIN3 group, genotype-specific positive agreement was ≥98.4% between Alinity m and cobas 4800 assays, and ≥85.7% between cobas 6800 and cobas 4800 assays. In the ≤CIN1 group, overall positive agreement among the three assays was ≥94.8%. This study showed similar clinical sensitivity and specificity for Alinity m HR HPV, cobas 4800 HPV, and cobas 6800 HPV assays. Alinity m was more specific in detecting HPV16 and HPV18, which could reduce unnecessary immediate referrals of women to colposcopy. IMPORTANCE This study provides evidence that the Alinity m HR human papillomavirus (HPV) assay has similar clinical performance in comparison with the cobas 4800 HPV and cobas 6800 HPV, two widely used tests. Validation of HPV assays in a clinical setting is crucial to ensure that they can provide a balanced sensitivity and specificity for detecting high-grade cervical intraepithelial neoplasia, potentially improving patient management by enabling proper follow-up or treatment and avoiding unnecessary procedures.

Urinary detection of high-risk HPV DNA to enhance cervical cancer screening in developing countries

ABSTRACT To increase cervical cancer screening capacity and participation, we evaluated the performance of the newly developed high-risk human papillomavirus (hrHPV) ReadyMix qPCR Kit for detecting hrHPV in urine samples while genotyping for HPV-16, HPV-18, and HPV-52. A total of 876 samples were used to assess the performance of the hrHPV ReadyMix qPCR Kit in detecting hrHPV in standard cervical swab samples compared with that of the Roche Cobas 6800 HPV system. hrHPV detection in urine was compared with that in corresponding paired cervical swab samples. The sensitivity of the hrHPV ReadyMix qPCR Kit for HPV detection in cervical swab samples was 96.55%, and the specificity was 99.87%. Despite higher cycle threshold (Ct) values, urine samples demonstrated 80.88% sensitivity and 100.00% specificity compared with cervical swab samples. Our method enables population-based hrHPV analysis, with a 6.62% HPV prevalence determined via cervical swab samples and a 6.28% HPV prevalence determined via urine samples. Furthermore, the hrHPV ReadyMix qPCR Kit presented comparable HPV type distributions in urine samples and the ability to genotype HPV-16 and HPV-18 to those obtained via the Roche Cobas 6800 HPV system. Self-collected urine samples tested using the hrHPV ReadyMix qPCR Kit demonstrated a diagnostic accuracy of 98.48% for detecting hrHPV. This study highlights the potential of the hrHPV ReadyMix qPCR Kit to enhance cervical cancer screening, offering valuable insights for future interventions. IMPORTANCE This study demonstrated that urinary detection of high-risk HPV DNA using the hrHPV ReadyMix qPCR Kit is an effective alternative for hrHPV screening. This non-invasive approach holds significant potential for large-scale screening, particularly in underserved populations. Integrating urine-based hrHPV testing into screening programs could improve early detection efforts, thereby enhancing the prevention and control of cervical cancer in low-resource settings.

Evaluation of the Allplex HPV assay’s adherence to international guidelines for cervical cancer screening in clinician-collected samples

ABSTRACT Clinically validated human papillomavirus (HPV) assays are crucial in cervical cancer screening. In this study, we evaluated the Allplex HPV HR Detection assay (Seegene, SouthKorea) for its clinical accuracy and reproducibility according to the international criteria, using the RealTime High Risk HPV m2000 assay (Abbott, USA) as standard comparator. The Allplex HPV HR assay exhibits significant non-inferior sensitivity to detect cervical intraepithelial neoplasia grade (CIN) 2 or worse (CIN2+) with a ratio of 1.00 (95% CI: 0.97–1.03, P = 0.006), insignificant non-inferior sensitivity to detect CIN3+ with a ratio of 1.00 (95% CI: 0.88–1.13, P = 0.098), and non-inferior specificity to exclude CIN2+ with a ratio of 0.99 (95% CI: 0.99–1.00, P < 0.001) compared to the standard comparator. In addition, the assay shows an excellent reproducibility within the same laboratory [96.5% (95% CI: 94.6–97.9) with a kappa value of 0.91 (95% CI: 0.87–0.95)] and between laboratories [96.7% (95% CI: 94.8–98.0) with a kappa value of 0.91 (95% CI: 0.87–0.95)] for overall high-risk HPV positivity as well as for each individual HPV type. Pooling our study data with those of another independent study supports the consistency of our findings. We conclude that both the clinical accuracy to detect cervical precancer and the reproducibility of Allplex HPV HR Detection assay fulfill the international validation criteria of use in cervical cancer screening. IMPORTANCE The clinical validation of human papillomavirus (HPV) assays in accordance with well-established international guidelines is crucial to ensure that only validated assays are used in the context of screening (Meijer et al., Int J Cancer, 2009). The guidelines, developed by an international consortium, require that a novel HPV assay has non-inferior accuracy against a standard comparator test for the detection of cervical intraepithelial neoplasia grade (CIN) 2 or worse (CIN2+). Additionally, a new HPV assay should meet specific criteria for both intra- and inter-laboratory reproducibility to ensure the assay consistently exhibits technical precision and robust performance. Pooling our study data with those of another independent study supports the consistency of our findings. In conclusion, both the clinical accuracy to detect cervical precancer and the reproducibility of Allplex HPV HR Detection assay fulfill the international validation criteria of use in cervical cancer screening.

Extended HPV genotyping by the BD Onclarity assay: concordance with screening HPV-DNA assays, triage biomarkers, and histopathology in women from the NTCC2 study

ABSTRACT The use of clinically validated human papillomavirus (HPV) assays is recommended in cervical cancer screening, and extended genotyping is getting attention as a triage biomarker because of the different oncogenic risk of the high-risk HPV genotypes. We compared the results of the Becton & Dickinson (BD) Onclarity HPV assay, on the residual baseline cervico-vaginal specimens of the NTCC2 trial, to those of the screening HPV-DNA assay (Cobas 4800 or HC2) and to cytology, p16/ki67 and E6/E7 mRNA triage results. We genotyped virtually all HPV-positive women and a consecutive sample of HPV-negatives. Among the 3,129 baseline-positives, 75.5% ( k = 0.368) were BD-positive, as were 5 of the 333 baseline-negatives (1.5%). The concordance between BD and HPV-DNA screening test was 87% for Cobas (1,250/1,436) and 65.9% for HC2 (1,115/1,693). A higher than the recommended positivity threshold for Onclarity would increase the agreement but would not improve concordance in the overall screening population. Among the baseline-positive cases, we observed an increasing trend of BD positivity with cytology severity (from 71.6% in negative for intraepithelial lesion of malignancy to 95.1% in ASC-H+ samples), with histologically confirmed CIN3 (96.9%), with p16/ki67 dual staining positivity (90.9% among the positive and 69.6% among the negative specimens), and with E6/E7 mRNA positivity (93.4% in the mRNA-positive cases vs 39.7% among the mRNA-negatives). Our findings confirm some disagreement among different HPV assays used for screening. Nevertheless, the agreement is substantial for women with high-grade cytology, histologically confirmed CIN3, and p16/ki67 or mRNA positivity at triage, thus confirming a good clinical performance of all the tests used. The NTCC2 trial is registered as Clinicaltrials.gov identifier NCT01837693 . IMPORTANCE Large randomized clinical trials have demonstrated that human papillomavirus (HPV) testing for high-risk types is more effective than cytology in detecting pre-cancerous lesions and preventing cervical cancer. Its use is being implemented in cervical cancer screening in several countries. The most recent guidelines recommend a risk-based management. It is therefore important to assess the individual risk of having/developing high-grade lesions of women testing high-risk HPV-positive. A crucial viral factor influencing the risk is the HPV genotype since different types are associated to different carcinogenetic risks. Understanding the degree of concordance among different assays targeting either HPV presence/type(s) or cellular morphology and proteins’ expression provides knowledge useful to better define how these tests can be used in screening protocols for an effective triage and to anticipate the possible implementation issues. Our study shows that the concordance between tests is higher when the infections have a higher probability of producing a clinically relevant lesion.

Development and evaluation of a CRISPR/Cas12a-based diagnostic test for rapid detection and genotyping of HR-HPV in clinical specimens

ABSTRACT Persistent infection with high-risk human papillomavirus (HR-HPV) is the principal etiological factor of cervical cancer. Considering the gradual progression of cervical cancer, the early, rapid, sensitive, and specific identification of HPV, particularly HR-HPV types, is crucial in halting the advancement of the illness. Here, we established a rapid, highly sensitive, and specific HR-HPV detection platform, leveraging the CRISPR/Cas12a assay in conjunction with multienzyme isothermal rapid amplification. Our platform enables the detection and genotyping of 14 types of HR-HPV by using type-specific crRNAs. The outcomes of the detection can be interpreted either through a fluorescence reader or visually. Furthermore, we achieved one-tube multiplex detection of 14 HR-HPV types through the use of multiple amplifications and a crRNA pool. The detection sensitivity of this method is 2 copies/μL with no cross-reactivity, and the results can be obtained within 30 minutes. This method exhibited 100% clinical sensitivity and 100% clinical specificity when applied to 258 clinical specimens. Based on these findings, our CRISPR/Cas-based HR-HPV detection platform holds promise as a novel clinical detection tool, offering a visually intuitive and expedited alternative to existing HPV infection diagnostics and providing fresh perspectives for clinical cervical cancer screening. IMPORTANCE This study developed a novel high-risk human papillomavirus (HR-HPV) detection platform based on CRISPR/Cas12a technology. This platform not only enables the rapid, highly sensitive, and specific detection and genotyping of 14 types of HR-HPV but also achieves single-tube multiplex detection of 14 HR-HPV types through ingenious design. The outcomes of the detection can be interpreted either through a fluorescence reader or visually. To the best of our knowledge, this is the first paper to utilize CRISPR/Cas diagnostic technology for the simultaneous detection of 14 types of HPV and to evaluate its feasibility in clinical sample detection using a large number of clinical samples. We hope that this work will facilitate the rapid and accurate detection of HPV and promote the broader application of CRISPR/Cas diagnostic technology.

Clinical Performance of the RealTi m e High Risk HPV Assay on Self-Collected Vaginal Samples within the VALHUDES Framework

Self-samples are becoming a crucial part of HPV-based cervical cancer screening programs to reach nonattendee women and increase screening coverage. Therefore, the VALHUDES framework was established to validate and evaluate HPV tests and devices on self-samples.

Deep Sequencing of HPV16 E6 Region Reveals Unique Mutation Pattern of HPV16 and Predicts Cervical Cancer

The study provides evidence for the first time that HPV16 E6 sequences contain vital mutation features in predicting the high‐grade squamous intraepithelial lesion and can reduce even more unneeded colposcopies without a loss of sensitivity to detect cervical cancer. Moreover, the D32E and H85Y variants of E6 exhibited a significantly higher ability to degrade p53, which may play a vital role in the development of cervical cancer.

Head-to-Head Comparison of DH3 HPV Test and HC2 Assay for Detection of High-Risk HPV Infection in Residual Cytology Samples from Cervical Cancer Screening Setting: Baseline and 3-Year Longitudinal Data

The benefits of testing for high-risk human papillomavirus (hrHPV) in cervical cancer screening have already been demonstrated. Hybrid Capture 2 (HC2) is the best validated HPV assay and has been considered the gold standard for hrHPV testing.

Detection of Circulating HPV16 DNA as a Biomarker for Cervical Cancer by a Bead-Based HPV Genotyping Assay

The validity of HPV ctDNA as a marker of HPV-driven cancers has been previously reported. Herein we validated an alternative to ddPCR for HPV16 ctDNA detection, using a bead-based HPV genotyping assay that offers the potential advantage of reducing the cost of clinical management due to the multiplex capability of the test, thus facilitating its use in clinical settings.