High-risk Human Papillomavirus Testing Research Articles

High-grade squamous intraepithelial lesion cervicovaginal paps with negative high-risk HPV testing, a prospective study with histological follow-up.

High-risk human papillomavirus (hrHPV) testing has become integral in the screening and treatment protocols for cervical neoplasia. Stand-alone HPV testing is advocated as a screening tool for cervical neoplasia. However, negative hrHPV tests with diagnosis of high-grade squamous intraepithelial lesion or worse (≥HSIL) have been reported. We report our experience with paps diagnosed as HSIL, negative hrHPV testing, and subsequent follow-up. Of 303 women with HSIL diagnosed on ThinPrep pap between 2019 and 2023, 84 (28%) were tested for hrHPV by Roche Cobas. Repeat testing was performed at a referral center. Immunohistochemistry (IHC) for p16 was performed on follow-up biopsies and hrHPV in-situ hybridization. Of 84 HSIL cases, 8 were hrHPV negative. Follow-up biopsies available in 7 cases were ≥HSIL (1 with invasive squamous cell carcinoma, 1 endocervical adenocarcinoma in situ). Follow-up HSIL was found on additional cases of HPV negative atypical glandular cells favor neoplastic and atypical squamous cells favor HSIL. IHC for p16 was positive on all biopsies. hrHPV FISH was negative. Our experience with hrHPV testing by Roche Cobas demonstrates that some morphologically diagnostic HSIL paps are hrHPV negative: 8.3% of HSIL paps with subsequent histological HSIL were HPV negative. The index case caused concern among our clinical colleagues. Positive staining for p16 is highly suggestive of HPV induced disease. Possible reasons for negative hrHPV testing could include non-hrHPV types, low HPV DNA levels, or HPV types not included in the Cobas testing panel.

Concurrent HPV DNA testing and a visual inspection method for cervical precancer screening: A practical approach from Battor, Ghana.

Cytology-based cervical cancer screening programs have been difficult to implement and scale up in developing countries. Thus, the World Health Organization recommends a 'see and treat' approach by way of hr-HPV testing and visual inspection. We aimed to evaluate concurrent HPV DNA testing and visual inspection in a real-world low-resource setting by comparing the detection rates of concurrent visual inspection with dilute acetic acid (VIA) or mobile colposcopy and hr-HPV DNA testing to standalone hr-HPV DNA testing (using the careHPV, GeneXpert, AmpFire, or MA-6000 platforms). We further compared their rates of loss to follow-up. This retrospective, descriptive cross-sectional study included all 4482 women subjected to cervical precancer screening at our facility between June 2016 and March 2022. The rates of EVA and VIA 'positivity' were 8.6% (95% CI, 6.7-10.6) and 2.1 (95% CI, 1.6-2.5), respectively, while the hr-HPV-positivity rate was 17.9% (95% CI, 16.7-19.0). Overall, 51 women in the entire cohort tested positive on both hr-HPV DNA testing and visual inspection (1.1%; 95% CI, 0.9-1.5), whereas a large majority of the women tested negative (3588/4482, 80.1%) for both and 2.1% (95% CI, 1.7-2.6) tested hr-HPV-negative but visual inspection 'positive'. In total, 191/275 (69.5%) participants who tested hr-HPV positive on any platform, as a standalone test for screening, returned for at least one follow-up visit. In light of factors such as poor socioeconomic circumstances, additional transportation costs associated with multiple screening visits, and lack of a reliable address system in many parts of Ghana, we posit that standalone HPV DNA testing with recall of hr-HPV positives will be tedious for a national cervical cancer prevention program. Our preliminary data show that concurrent testing (hr-HPV DNA testing alongside visual inspection by way of VIA or mobile colposcopy) may be more cost-effective than recalling hr-HPV-positive women for colposcopy.

CDKN2A/p16 evaluation in cytology specimens.

CDKN2A/p16 evaluation in cytology specimens.

Abstract 1978: Community Genomic Health Promotion Program evaluates precision DNA methylation biomarkers for use in self-testing and collection kits in urine, to increase cervical cancer screening rates among Latina women

Abstract Cervical cancer incidence is 32% higher among Latina women in the United States (US) and 78% higher among women in Puerto Rico compared to non-Latina White (NLW) women, yet it is largely preventable through screening. Latina women with public insurance or uninsured are significantly less likely to have had a Pap test, undergo fewer Pap and high-Risk Human Papilloma Virus (HPV) testing than NLW and Black women, and are less likely than women of other races/ethnicities to return for recommended follow-up after an abnormal Pap test. Utilizing the PRECEDE-PROCEED model, Community Based Participatory Research strategies and the NIH I-Corps hypothesis-driven research method of customer discovery, we implemented the Community Genomic Health Promotion Program in Federally Qualified Health Centers to evaluate cervical cancer screening needs in six rural municipalities in Puerto Rico. We recently validated in clinical liquid cytology samples that the CervicalMethDx test, a precision DNA methylation test, can identify HPV positive women most likely to be diagnosed with Cervical Intraepithelial Neoplasia (CIN) grades 2 and 3. We hypothesized that a molecular pathology test which identifies advanced premalignant cervical lesions in urine, can be distributed by commercial channels targeting populations with low screening rates. The CervicalMethDx quantitative Methylation Specific PCR (qMSP) test was used to quantify ZNF516, FKBP6 and INTS1 promoter DNA methylation in well-characterized, discarded, liquid cytology clinical samples from Puerto Rico. We then tested the CervicalMethDx test concordance on paired cytobrush (n=82) and urine samples (n=82) from Chile. The CervicalMethDx test correctly classified 86% of discarded liquid cytology clinical samples from HPV-positive women, when comparing DNA methylation in CIN2/CIN3 samples (n=47) to samples with a cervical pathology diagnosis of No Intraepithelial Lesions or Malignancy (n=18), with 83% Sensitivity, 94% Specificity, Area Under the Curve (AUC) of 0.88, and 98% positive predictive value (PPV). The concordance of the CervicalMethDx test in paired brush and urine samples was moderate (rho=0.45). The Wilcoxon signed-rank test showed no statistically significant differences for the CervicalMethDx test in paired brush and urine samples (p=0.14) These encouraging preliminary results show the successful identification of advanced premalignant cervical lesions in liquid cytology media and urine samples from Latina women. The data suggests that urine should be explored as a biofluid for cervical cancer screening. Future research may lead to expanded screening options, enabling women with low access to health care (public insurance or uninsured) to collect a specimen at home, and either send it to a testing facility or perform the test at home. Citation Format: Carmen Ili, Priscilla Brebi, Jaime López, Fernando T. Zamunér, Ana Purcell-Wiltz, Amanda García-Negrón, Keilyn Vale-Lassalle, Keerthana Gosala, Mirla Otero-García, Camila Rivero-Maldonado, Teresa Torres-Rivera, Adriana Pérez-Vicente, Ashley Ramos-López, David Sidransky, Mariana Brait, Rafael E. Guerrero-Preston. Community Genomic Health Promotion Program evaluates precision DNA methylation biomarkers for use in self-testing and collection kits in urine, to increase cervical cancer screening rates among Latina women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1978.

Psychological impact of the announcement of an abnormal cervical smear: How to improve patient experience and information? COLANX: a multicenter study in Seine-Maritime

When an anomaly in the screening by cervical smear or by the HPV-HR test is detected, the women are called for a colposcopy. Waiting for colposcopy is often an anxiety-provoking situation for women, probably linked to a widespread ignorance of the value of screening and the pathophysiology of HPV infection. The COLANX was a multicentric study, in Seine-Maritime, conducted with 8 colposcopist practitioners. Self-questionnaires were distributed to volunteer women, summoned for a first abnormal smear result, at the exit of colposcopy and filled out immediately on site. This questionnaire evaluated: the psychological impact of the announcement of the abnormal cervico-uterine smear, its impact on the sexual quality of life, the epidemiological characteristics of the population studied, the modalities of the announcement made of the abnormal result, the satisfaction of the women and their desire for additional information. 131 questionnaires were included, from June 1, 2020 to January 18, 2021. 61.5% of responding women presented proven psychological distress, as evidenced by a GHQ-12 score ≥ 2. The state of psychological distress was significantly different depending on the time left by the practitioner between the announcement of the abnormal cytology result and the colposcopy appointment. The GHQ-12 score was significantly different according to the initial classification of the cervico-uterine smear, high in case of suspicion of high-grade lesions in particular. 36.3% of patients had a significant deterioration in their sexual quality of life, demonstrated by an ASEX score ≥ 18. The women were statistically more satisfied when the announcement of their abnormal screening result had been made by telephone or during a consultation, rather than by mail, but no significant difference was found on the presence or absence of distress psychological according to this mode of announcement. 52.3% of the women in this study expressed a request for additional information support, preferably written support (for 89.4% of these women), to be delivered when the abnormal screening was announced (for 76, 5% of these women). The main results of the COLANX study are consistent with those of the literature, in particular concerning the negative psychological impact in the announcement of an abnormal smear result. The new methods of organized screening will increase the use of colposcopy for many women. There is a significant impact on their psyche and their intimate life. The issuance of additional information during this announcement would probably reduce this anxiety.

Human papillomavirus testing on self-collected samples to detect high-grade cervical lesions in rural Bhutan: The REACH-Bhutan study.

"REACH-Bhutan" aimed to evaluate the feasibility and clinical performance of a community-based screening program for cervical cancer in rural Bhutan using self-collected samples for high-risk human papillomavirus (HR-HPV) testing. In April/May 2016, 2590 women aged 30-60 years were screened across rural Bhutan by providing a self-collected sample for careHPV testing. All careHPV-positive women, plus a random sample of careHPV-negative women, were recalled for colposcopy and biopsy. Self-samples also underwent GP5+/6+ polymerase chain reaction (PCR)-based HR-HPV DNA detection and genotyping. Cross-sectional screening indices were estimated against histological high-grade squamous intraepithelial lesions or worse (hHSIL+), including imputation of hHSIL+ in women without colposcopy. HR-HPV positivity was 10.2% by careHPV and 14.8% by GP5+/6+ PCR. Twenty-two cases of hHSIL+ were histologically diagnosed, including one invasive cancer; an additional 7 hHSIL+ were imputed in women without colposcopy. HR-HPV testing by GP5+/6+ showed higher sensitivity for hHSIL+ (89.7%, 95% CI 72.6-97.8) than careHPV (75.9%, 95% CI 56.5-89.7). Negative predictive value was also slightly higher for GP5+/6+ (99.9%, 95% CI 99.6-100) than careHPV (99.7%, 95% CI 99.4-99.9). Specificity, however, was lower for GP5+/6+ (86.1%, 95% CI 84.6-87.4) than careHPV (90.6%, 95% CI 89.4-91.7), as was positive predictive value (6.9%, 95% CI 4.5-9.9 vs. 8.5%, 95% CI 5.4-12.6). Of 377 HR-HPV-positive women by GP5+/6+, 173 (45.9%) were careHPV-positive, including 54.7% HPV16-positive and 30.2% HPV18-positive women. The final REACH-Bhutan results show that screening for cervical cancer with self-collection of samples and HR-HPV testing, in addition to our previous report of achieving high participation, can also perform well to detect women with hHSIL+.

Overview of organisational methods of primary cervical lesion screening programmes that use human papillomavirus testing.

Many factors need to be considered when planning and managing a screening programme for the early detection of cervical cancer (CC). A non-systematic international review of the organisation of CC screening using high-risk human papillomavirus (HPV-HR) testing, aimed at identifying the organisational methods of these programmes, was conducted with a view to supporting the future of the French system in the context of the transition to HPV-HR testing. In countries where HPV testing has been implemented or planned, the initial reflection process has provided an opportunity to rethink the previous (cytological) screening organisation. Despite considerable differences between countries, a nationally or regionally centralised organisational model appears to be the preferred option in most countries. This model is based on a national/regional structure tasked with all invitations, reminders, follow-up and coordination, centralised laboratories integrating both biology and pathology laboratories, and a unified information system integrated with routine health management tools used by health practitioners and nurses. Besides quality considerations, grouped purchasing makes it possible to implement a public procurement policy that includes price negotiations with suppliers. Discussions around the introduction of HPV testing have resulted in most countries reviewing or creating information systems and quality assurance processes. While the WHO seems to recommend the systematic use of vaginal self-sampling, very few countries have considered this option. More and more countries are planning to implement vaginal self-sampling, but no clear organisational model has emerged from the countries where it has been implemented to date.

Comparison of the Clinical Performance of Aptima HPV Assay and the Cobas 4800 Platform in Women with Normal Cytology and Positive High-Risk HPV

Introduction: According to the current American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines, patients with normal cytology results may be referred for colposcopy according to their high-risk human papillomavirus (hrHPV) test results. A higher positive predictive value (PPV) of hrHPV has significance for preventing unnecessary colposcopic examinations. Several studies have compared the performance of the Aptima assay and the Cobas 4800 platform among patients who had minor cytologic abnormalities. However, in our English literature search, we found no other study that had been conducted to compare these two methods in patients with normal cytology. We thus aimed to compare the PPV of the Aptima assay and the Cobas 4800 platform among women with normal cytology. Methods: Between September 2017 and October 2022, we retrospectively identified 2,919 patients who had normal cytology and hrHPV positivity and had been referred for a colposcopy. Among them, 882 agreed to undergo a colposcopy; on examination, 134 had target lesions revealed and underwent a colposcopic punch biopsy. Results: Among the patients who underwent a colposcopic punch biopsy, 49 (38.9%) were tested with Aptima, and 77 (61.1%) were tested with Cobas. In the Aptima group, 29 (59.2%) patients showed benign histology, 2 (4.1%) patients had low-grade squamous intraepithelial lesions (LSILs), and 18 (36.7%) patients had ≥high-grade squamous intraepithelial lesions (HSILs) biopsy results. The false-positivity rate and PPV of Aptima were 63.3% (31/49) and 36.7% (95% confidence interval [CI]: 0.232–0.502), respectively, for a histopathologic diagnosis of ≥HSIL. In the Cobas group, 48 (62.3%) biopsies were benign, 11 (14.3%) reported an LSIL, and 18 (23.4%) biopsies were ≥HSIL. The false-positivity rate and PPV of Cobas were 76.6% (59/77) and 23.4% (95% CI: 0.139–0.328), respectively, concerning a ≥HSIL tissue diagnosis. The false-positivity rate of Aptima HPV 16 positivity was 40% (4/10). The false-positivity rate of Cobas HPV 16 positivity was 61.1% (11/18). The PPVs of HPV 16 positivity for Aptima and Cobas were 60% (95% CI: 0.296–0.903) and 38.9% (95% CI: 0.163–0.614), respectively, concerning ≥HSIL tissue diagnosis. Conclusion: We recommend analyzing the performances of hrHPV platforms in future, larger studies in patients with normal cytology rather than only in cases with abnormal cytology.

Comparison of the AmpFire and MA-6000 polymerase chain reaction platforms for high-risk human papillomavirus testing in cervical precancer screening

High-risk human papillomavirus (hr-HPV) testing for primary cervical precancer screening offers an opportunity to improve screening in low-middle income countries (LMICs). This study aimed to compare the analytic performances of the AmpFire and MA-6000 platforms for hr-HPV DNA testing in three groups of women screened for hr-HPV types in Ghana: group 1 with 33 GeneXpert-archived ThinPrep/liquid-based samples subjected to both tests, group 2 with 50 AmpFire-archived dry brush samples subjected to MA-6000 testing, and group 3 involving 143 cotton swab samples simultaneously subjected to both tests without archiving. The overall agreement rates were 73 %, 92 %, and 84 %, for groups 1–3, respectively, and 84 % (95 % CI, 78.6–88.6) for the entire group. Neither AmpFire nor MA-6000 was more likely to test hr-HPV positive in all three groups and the combined group. Group 1 showed fair agreement without statistical significance (κ = 0.224, 95 % CI, −0.118 to 0.565), while group 3 showed significant moderate agreement (κ = 0.591, 95% CI, 0.442–0.741). Group 2 showed an almost perfect significant level of agreement (κ = 0.802; 95 % CI, 0.616–0.987). Thus, both platforms showed statistically significant moderate to near-perfect agreement for detecting hr-HPV in cervicovaginal samples, with variation according to archiving conditions and duration between sample collection and retesting. For LMICs using these platforms for COVID-19 testing, as the COVID-19 pandemic subsides, the platforms can become available for running other tests such as hr-HPV DNA testing for cervical precancer screening.

Interest of high-risk HPV typing by hybrid capture 2 in severe cervical dysplasias (CIN2/3) in Algeria

Persistent infection with high-risk human papillomavirus (HR-HPV) is the leading cause of cervical precancerous lesions and cervical cancer. The aim of the study was to evaluate the prevalence of high-risk HPV (HR-HPV) types among women with cervical intraepithelial neoplasia (CIN 2-3) and to evaluate the interest of high-risk HPV typing by hybrid capture 2 (HC2) in severe cervical dysplasias. 110 women were subjected to cyto-histological diagnostics and to the HPV test by HC2. The HC2 performance tests included Chi 2. Our results showed that the average age at the gynecological consultation in relation to cervical lesions was 38.75 ± 9.59 (22-72 years). The overall prevalence of HPV infection was 41.8%. High-risk HPV (HR-HPV) was detected in 31.8% of the total patients. In comparison with the other cytopathological stages, the HC2 test made it possible to evaluate the rate of HR-HPV at the CIN2/3 stage at 86% (p<0.0061). In conclusion the prevalence of HR-HPV associated with cervical lesions is comparable to those in the literature. HC2 Allows good detection of HR-HPV in severe cervical dysplasias and can be used to compensate for the absence of vaccines in our country.

Efficacy of optical coherence tomography in the triage of women with minor abnormal cervical cytology before colposcopy.

To evaluate the efficacy of Optical Coherence Tomography (OCT) for detecting cervical lesions in women with minor abnormal cytology results (atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LSIL)). A prospective study was conducted at gynecologic clinic from Mar 2021 to Sep 2021. The recruited women with cervical cytological findings of ASC-US or LSIL were inspected with OCT before colposcopy-directed cervical biopsy. The diagnostic performance of OCT, alone and in combination with high-risk human papillomavirus (hrHPV) testing were evaluated to detect cervical intraepithelial neoplasia of grade 2 or worse (CIN2+)/CIN3 or worse (CIN3+). The rate of colposcopy referral and the immediate risk of CIN3+ of OCT were calculated. A total of 349 women with minor abnormal cervical cytology results were enrolled. For detection of CIN2+/CIN3+, the sensitivity and NPV of OCT were lower than those of hrHPV testing (CIN2+: 71.3% vs. 95.4%, 89.0% vs. 91.1%, P < 0.001; CIN3+: 75% vs. 93.8%, 96.5% vs. 95.6%, P < 0.001), but the specificity, accuracy and PPV were higher than those of hrHPV testing (CIN2+: 77.5% vs. 15.6%, 75.9% vs. 35.5%, 51.2% vs. 27.3%, P < 0.001; CIN3+: 69.4% vs. 13.6%, 69.9% vs. 20.9%, 19.8% vs. 9.9%, P < 0.001). OCT combined with hrHPV testing (CIN2+: 80.9%; CIN3+: 72.6%) showed higher specificity than that of OCT alone (P < 0.001). The colposcopy referral rate base on OCT classification was lower than that based on hrHPV testing (34.7% vs. 87.1%, P < 0.001). Patients with hrHPV-positive ASC-US and hrHPV-negative LSIL cytology, the immediate CIN3+ risk in OCT negative cases was less than 4%. OCT alone or combination with hrHPV testing shows good performance for detecting CIN2+/CIN3+ in patients with ASC-US/LSIL cytology. OCT is an effective method for colposcopy triage in women with hrHPV-positive ASC-US and hrHPV-negative LSIL cytology.

Association of TLR4 and TLR9 gene polymorphisms with cervical HR-HPV infection status in Chinese Han population

BackgroundToll-like receptors (TLRs) may be involved in the natural history of human papillomavirus (HPV) infection. In our study, we aimed to investigate the association of TLR4 (rs10116253, rs1927911, rs10759931) and TLR9 (rs187084, rs352140) gene polymorphisms with cervical persistent high-risk HPV (HR-HPV) infection, as well as multiple HR-HPV infections.MethodsA total of 269 study subjects were enrolled and grouped by retrospectively analyzing the HR-HPV testing results and other clinical data of 2647 gynecological outpatients from Jingzhou Hospital Affiliated to Yangtze University. We conducted a case–control study to compare the role of TLR4/TLR9 gene polymorphisms between HR-HPV transient and persistent infections, as well as between HR-HPV single and multiple infections. HR-HPV genotypes were detected using Real-time polymerase chain reaction (RT-PCR). PCR-restriction fragment length polymorphism (PCR–RFLP) was used to determine TLR4 and TLR9 gene polymorphisms. Analyses of the different outcome variables (HR-HPV infection status and time for HR-HPV clearance) with respect to TLR4/TLR9 polymorphisms were carried out. Logistic regression analysis was used to determine the association of TLR4/TLR9 genotypes and alleles with HR-HPV infection status. The Kaplan–Meier method with the log-rank test was used to analyze the relationship between TLR4/TLR9 genotypes and the time for HR-HPV clearance.ResultsThe mutant genotypes of TLR9 rs187084 and rs352140 were associated with persistent (rs187084: CT and CT+CC; rs352140: CT and CT+TT) and multiple (rs187084: CT and CT+CC; rs352140: CT+TT) (all P < 0.05) HR-HPV infection. However, no association was found between TLR4 polymorphisms and HR-HPV infection status. Kaplan–Meier time to HR-HPV clearance analysis demonstrated that women carrying rs187084 and rs352140 mutant genotypes take longer duration to clear HR-HPV infection compared with wild-type genotype carriers (P1 = 0.012; P2 = 0.031).ConclusionOur results suggested that TLR9 polymorphisms, but not TLR4, were associated with cervical persistent and multiple HR-HPV infections, which could be useful as a potential predictor of HR-HPV infection status.

Screening Tests and Support in Positive Human Papilloma Virus Tests

Screening for the detection of Cervical Cancer (CC) is moving from cytology or Papanicolaou (Pap) test to the detection of the primary highrisk Human Papillomavirus (HPV) test, based on evidence for this change, with support or triage options for positive HPV test results with better access innovations. The test of higher specificity such as cytology makes it an option for triage in positive HPV tests; but cytology and genotyping have been recommended for all positive HPV tests, especially for the other pool-positive hr-HPVs; but HPV-16/18 negative. Screening with primary HPV testing has been shown to be effective, affordable, and acceptable to women, especially in emerging countries; the use of Dual Staining (DS) to classify positive HPV results has higher sensitivity and specificity than HPV-16/18 genotyping or cytology at triage.

Predictors of CIN2+ in Patients with PAP III-P (ASC-H): A Cross-Sectional Study

This study aims to investigate whether specific characteristics of the patient group with ASC-H (PAP III-p) findings increase the likelihood of clinically significant disease (CIN2+), offering implications for risk-adapted clinical management. 225 patients with an ASC-H smear presenting to our colposcopy unit between 2014 and 2021 were identified and examined retrospectively. A total of 203 patients were included in the cross-sectional study using multivariate logistic regression. Of the 12 variables that entered the regression model, transformation zone (p = 0.045) and HPV infection (p = 0.005) contributed significantly to predicting CIN2+. A T3 transformation zone had a protective effect, reducing the likelihood of significant pathology. Infection with HPV high risk (non 16/18) or HPV high risk (16/18), on the contrary, increased the likelihood of CIN2+ four times and seven times, respectively, compared to the lower-risk category. A differential approach in the workup of ASC-H cytology is advisable. Younger, premenopausal patients with positive HPV high-risk findings are at a significantly higher risk for CIN2+ lesions than older postmenopausal women with negative high-risk HPV test results. As the risk increases from HPV high-risk non 16/18 to HPV 16/18 infections, using HPV PCR testing is advisable.

Multiplex single-cell droplet PCR with machine learning for detection of high-risk human papillomaviruses

High-risk human papillomavirus (HPV) testing can significantly decline the incidence and mortality of cervical cancer. Microfluidic technology provides an effective method for accurate detection of high-risk HPV by utilizing multiplex single-cell droplet polymerase chain reaction (PCR). However, current strategies are limited by low-integration microfluidic chip, complex reagent system, expensive detection equipment and time-consuming droplet identification. Here, we developed a novel multiplex droplet PCR method that directly detected high-risk HPV sequences in single cells. A multiplex microfluidic chip integrating four flow-focusing structures was designed for one-step and parallel droplet preparation. Using single-cell droplet PCR, multi-target sequences were detected simultaneously based on a monochromatic fluorescence signal. We applied machine learning to automatically identify the large populations of single-cell droplets with 97% accuracy. HPV16, 18 and 45 sequences were sensitively detected without cross-contamination in mixed CaSki and Hela cells. The approach enables rapid and reliable detection of multi-target sequences in single cells, making it powerful for investigating cellular heterogeneity related to cancer diagnosis and treatment.

AACC Guidance Document on Cervical Cancer Detection: Screening, Surveillance, and Diagnosis.

Persistent genital infection with high-risk human papilloma virus (hrHPV) causes the vast majority of cases of cervical cancer. Early screening, ongoing surveillance, and accurate diagnosis are crucial for the elimination of cervical cancer. New screening guidelines for testing in asymptomatic healthy populations and management guidelines for managing abnormal results have been published by professional organizations. This guidance document addresses key questions related to cervical cancer screening and management including currently available cervical cancer screening tests and the testing strategies for cervical cancer screening. This guidance document introduces the most recently updated screening guidelines regarding age to start screening, age to stop screening, and frequencies of routine screening as well as risk-based management guidelines for screening and surveillance. This guidance document also summarizes the methodologies for the diagnosis of cervical cancer. Additionally, we propose a report template for human papilloma virus (HPV) and cervical cancer detection to facilitate interpretation of results and clinical decision-making. Currently available cervical cancer screening tests include hrHPV testing and cervical cytology screening. The screening strategies can be primary HPV screening, co-testing with HPV testing and cervical cytology, and cervical cytology alone. The new American Society for Colposcopy and Cervical Pathology guidelines recommend variable frequencies of screening and surveillance based on risk. To implement these guidelines, an ideal laboratory report should include the indication for the test (screening, surveillance, or diagnostic workup of symptomatic patients); type of test (primary HPV screening, co-testing, or cytology alone); clinical history of the patient; and prior as well as current testing results.

The Value of Immunocytochemical Staining for the HPV E7 Protein in the Diagnosis of Cervical Lesions.

To investigate the value of immunocytochemical (ICC) staining for human papillomavirus (HPV) E7 protein (E7-ICC staining) as a new-generation immunological method in the cytological diagnosis of cervical lesions. The exfoliated cervical cell samples of 690 women were subjected to a liquid-based cytology test (LCT), high-risk HPV (HR-HPV) test, E7-ICC staining, and cervical biopsy for pathological diagnosis. E7-ICC staining as a preliminary screening scheme for cervical precancerous lesions was comparable to the HR-HPV test in sensitivity and to the LCT in specificity. E7-ICC staining was advantageous in facilitating the secondary triage of HR-HPV-positive patients; therefore, this method can be used as an auxiliary scheme to routine LCT for diagnostic grading to improve the accuracy of cervical cytology. E7-ICC staining as a primary or auxiliary cytological screening scheme can effectively reduce the colposcopy referral rate.

Feasibility study of 5-aminolevulinic acid mediated photodynamic therapy of persistent vaginal high-risk HPV infection of post-hysterectomy patients.

Patients after hysterectomy are at higher risk for developing VaIN. However, there are no standard treatments for HPV infection and VaIN after hysterectomy and relative studies are limited. Thus we aim to evaluate the feasibility of 5-aminolevulinic acid photodynamic therapy (5-ALA-PDT) for the treatment of persistent vaginal infection with high-risk HPV (HR-HPV) in post-hysterectomy patients. Thirty-eight patients aged 43-70 years old with persistent vaginal stump HR-HPV infection with or without histological vaginal intraepithelial neoplasia (VaIN1) during follow-up after hysterectomy were recruited. Twenty patients received three times of ALA-PDT (PDT Group). Eighteen patients did not receive any treatments (Control Group). HPV testing, cytology, and colposcopy were performed in all patients 4-6 months and 12 months after treatment. HR-HPV remission rates of the PDT Group were 40% (8/20) and 66.67% (12/18) at the 4-6 month and 12-month follow-up, respectively, both were significantly higher than that of the Control Group (11.11% (2/18) and 6.23% (1/16), respectively; P<0.05). For the PDT Group, the regression rate of VaIN1 was 100% (7/7) at 4-6 months, while VaIN1 recurred in two cases (28.6%) at the 12-month due to persistent HR-HPV infection. No persistence or progression of VaIN1 was noted. For the Control group, the spontaneous regression rate of VaIN1 was 50% (3/6) at 4-6 months and one patient progressed into VaIN2. At the 12-month follow-up point, one patient reccurred and the disease regression, persistence and recurrence rates were 40% (2/5), 40% (2/5) and 20% (1/5), respectively. Adverse reactions were mild after PDT treatment. 5-ALA-PDT is a safe, non-invasive, and effective option for post-hysterectomy patients who have persistent HR-HPV infection.

The vaginal microbiota among the different status of human papillomavirus infection and bacterial vaginosis.

Although human papillomavirus (HPV) infection plays a decisive role in causing tumors, its infection is insufficient for independently promoting cancer development and other co-factors facilitate the carcinogenic process. The objective of this study was to demonstrate the association between vaginal microbiota and high-risk human papillomavirus (HR-HPV) infection in women with and without bacterial vaginosis (BV). The study included 1015 women aged 21-64 who participated in cervical cancer screening in two areas of China from 2018 to 2019. Women were collected cervical exfoliated cell specimens and reproductive tract secretions samples for HR-HPV, BV and microbial composition testing. From the non-BV& HPV- group (414 HPV-negative women without BV) to the non-BV& HPV+group (108 HPV-positive women without BV), to the BV& HPV-group (330 HPV-negative women with BV) and then to the BV& HPV+group (163 HPV positive-women with BV), microbial diversity increased. The relative abundance of 12 genera, including Gardnerella, Prevotella, and Sneathia increased, while Lactobacillus declined. Correlation networks of these genera and host characteristics were disrupted in the non-BV& HPV+group, and the network trended more disordered in the BV& HPV+group. Besides, multiple HPV infection, certain HPV genotype infection and cervical intraepithelial neoplasia (CIN) status were associated with some microbes and higher microbial diversity. HPV shifted the composition and diversity of vaginal microbiota, and BV further reinforced the trend. The relative abundance of 12 genera increased and 1 genus decreased on account of BV and HPV infection, and some genera including Lactobacillus, Prevotella, and Sneathia were associated with some specific HPV genotypes infection and CIN.

Promoter hypermethylation analysis of host genes in cervical intraepithelial neoplasia and cervical cancers on histological cervical specimens

BackgroundDNA methylation is an essential factor in the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer. The aim was to investigate the diagnostic value provided by methylation biomarkers of six tumor suppressor genes (ASTN1, DLX1, ITGA4, RXFP3, SOX17 and ZNF671) for cervical precancerous lesions and cervical cancer.MethodsThe histological cervical specimens of 396 cases including 93 CIN1, 99 CIN2, 93 CIN3 and 111 cervical cancers were tested for methylation-specific PCR assay (GynTect®) of score and positive rate. Among them, 66 CIN1, 93 CIN2, 87 CIN3 and 72 cervical cancers were further used for paired analysis. A chi-square test was used to analyze the difference of methylation score and positive rate in cervical specimens. The paired t-test and paired chi-square test were for analyzing the methylation score and positive rate in paired CIN and cervical cancer cases. The specificity, sensitivity, odds ratio (OR) and 95% confidence interval (95% CI) of the GynTect® assay for CIN2 or worse (CIN2 +) and CIN3 or worse (CIN3 +) were evaluated.ResultsAccording to the chi-square test trend, hypermethylation increased with severity of the lesions as defined by histological grading (P = 0.000). The methylation score above 1.1 was more common in CIN2 + than in CIN1. The DNA methylation scores in the paired groups of CIN1, CIN3 and cervical cancer were significant differences (P = 0.033, 0.000 and 0.000, respectively), except for CIN2 (P = 0.171). While the positive rate of GynTect® in each paired group had no difference (all P > 0.05). The positive rate of every methylation marker in the GynTect® assay showed differences in four cervical lesion groups (all P < 0.05). The specificity of GynTect® assay for detection of CIN2 + /CIN3 + were higher than high-risk human papillomavirus test. With CIN1 as a reference, the positive status of GynTect®/ZNF671 were significantly higher in CIN2 + : odds ratio (OR) 5.271/OR 13.909, and in CIN3 + : OR 11.022/OR 39.150, (all P < 0.001).ConclusionThe promoter methylation of six tumor suppressor genes is related to the severity of cervical lesions. The GynTect® assay based on cervical specimens provides diagnostic values for detecting CIN2 + and CIN3 + .