Diagnostic Microbiology and Infectious Disease

High throughput HPV genotyping by next generation sequencing for detection of 28 HPV types and 13 sexually transmitted infections: A first community-based cervical cancer screening study from India

High-risk human papillomavirus (HR-HPV) infection is the primary cause of cervical cancer. However, conventional PCR-based assays provide limited HPV genotyping, and cytology alone has low sensitivity for detecting subclinical infections. Comprehensive molecular approaches are needed to better characterize HPV genotypes and co-infections in community screening settings. This study evaluated the analytical performance of a next-generation sequencing (NGS)-based HPV-STI assay in comparison with the clinically validated Roche Cobas The NGS assay showed high agreement with the Cobas A substantial proportion of women with abnormal cytology in this community cohort harboured non-HPV16 and HPV18 high-risk infections, highlighting the importance of extended HPV genotyping. NGS-based HPV genotyping was concordant with the comparator assay used and had simultaneous STI identification, supporting its potential utility as a comprehensive tool for community-based cervical cancer screening and risk stratification.

Comparative efficacy of HPV 16/18 DNA and E6/E7 mRNA testing in detecting high-grade cervical lesions (CIN2+) in women with cervical biopsies

The study evaluated the efficacy of HPV 16/18 E6/E7 mRNA detection in women with abnormal cervical histology. A total of 99 cervical biopsy samples were analyzed, including 49 benign, 16 with cervical intraepithelial neoplasia grade 1 (CIN1), 9 with CIN2/3, and 25 with cervical cancers. Samples were tested for HPV 16/18 using both DNA and mRNA RT-PCR methods. The findings revealed a sensitivity of 85.3 % (29/34) for the HPV DNA test and 76.5 % (26/34) for the mRNA test in detecting CIN2+ lesions. Notably, the E6/E7 mRNA test demonstrated greater specificity for CIN2+ at 75.4 % (49/65), compared to 52.3 % (34/65) for the DNA test. The prevalence of positive results for both tests increased with the severity of squamous cell abnormalities. However, the HPV 16/18 E6/E7 mRNA test provided superior specificity, making it a more effective method for cervical cancer screening in this region, offering more precise results than DNA testing alone.

Clinical vaginal-microecology testing using double-fluorescence staining in patients with high-risk human papillomavirus infection

High-risk human papillomavirus (hrHPV) infection is associated with cervical cancer; imbalanced vaginal microecology may contribute to HPV progression. Currently used methods for clinical vaginal-microecology (CVM) testing are associated with several disadvantages, such as low accuracy and complicated operation. This retrospective study presents a novel testing method to examine vaginal microecology via double-fluorescence staining and explores the relationship between hrHPV and CVM. We analyzed 1242 patients who underwent hrHPV testing at our hospital over a two-month period; of these, 695 also underwent clinical vaginal-microecology testing (CVMT). Patients underwent routine leukorrhea detection (n=322), functional testing (n=277), and our novel double-fluorescence staining-based CVMT approach (n=376). Patients with hrHPV exhibited more epithelial cells, miscellaneous bacteria, and hyphae than those without hrHPV on double-fluorescence staining-based CVMT approach. Double-fluorescence staining was effective in identifying indicators of hrHPV infection and may serve as an auxiliary tool for clinical hrHPV screening.

Factors associated with the persistence of human papillomavirus after surgery in patients with cervical cancer

To determine the rate of human papillomavirus (HPV) persistence after surgery in patients with cervical cancer, and to analyze the factors associated with HPV persistence and viral load after surgery. Medical records of women who underwent surgery for treatment of cervical cancer between 1 January 2018 and 30 June 2019 at Obstetrics and Gynecology Hospital of Fudan University in Shanghai, China, were retrospectively analyzed. Patients with persistent HPV infection after 2 years of follow-up were identified. Univariate and multivariate analyses were employed to determine the impact of various factors including patient age, menopausal status, parity, and surgical margin status on HPV persistence. The Wilcoxon test was used to analyze the factors that influenced postoperative HPV viral load. Altogether, 607 women were eligible for the final analysis. The persistence rates of HPV at 6 months, 1 year, and 2 years after surgery were 17.3, 13.7, and 10.2 %, respectively. In univariate analysis, the factors that were predictive of the persistence of HPV infection were old age, postmenopausal status, and positive vaginal incision margin with cancer. In multivariate analysis, the significant independent predictive factors were postmenopausal status and positive vaginal incision margin with cancer (P < 0.05, odds ratio (OR) = 2.289, 95 % confidence interval (CI): 1.262-4.150 and OR = 3.271, 95 % CI: 1.253-8.537, respectively). A vaginal lesion with cancer or squamous intraepithelial lesion (SIL) and positive vaginal incision margin influenced HPV viral load at 6 months after surgery (P < 0.05). Postmenopausal patients and those with positive vaginal incision margin with cancer are at an increased risk of HPV persistence after surgical treatment for cervical cancer. Vaginal lesions with cancer or SILs and positive vaginal incision margin are risk factors for high HPV viral load after surgery.

HPV E6/E7 mRNA combined with thin-prep cytology test for the diagnosis of residual/recurrence after loop electrosurgical excision procedure in patients with cervical intraepithelial neoplasia

To evaluate the diagnostic value of combining HPV E6/E7 mRNA testing with Thin-Prep cytology (TCT) for residual/recurrence detection, a total of 289 patients who underwent loop electrosurgical excision procedure (LEEP) for high-grade cervical lesions were included. Patients were followed up at different time points, and residual/recurrent lesions were confirmed through vaginoscopy. TCT, HPV-DNA, and HPV E6/E7 mRNA tests were conducted. Diagnostic performance, including sensitivity, specificity, positive predictive value, negative predictive value, and accuracy, was assessed. Among the patients, 76 cases showed residual lesions/recurrence, while 213 cases showed no residual/recurrence. Positive margins in the cervical-vaginal and cervical canal areas were associated with a higher risk of residual/recurrence. The combined HPV E6/E7 mRNA and TCT test showed higher diagnostic efficacy than individual tests at 6-, 12-, and 24-months follow-up. The combined test consistently demonstrated higher specificity and sensitivity, with significantly larger area under the curve (AUC) values compared to the individual tests.

Methylation testing versus cervical cytology for triage of HPV-positive women: A comparative study

In many countries, HPV testing has replaced cervical cytology as the primary screening method for cervical cancer. While HPV testing has high sensitivity, its limited specificity necessitates triage to reduce unnecessary colposcopy referrals. This study evaluates the performance of cervical cytology (TCT) and the GynTect® six-gene methylation test as triage methods for HPV-positive women. A total of 276 women from China were categorized into four pathological groups: cervical squamous cell carcinoma (CSCC), CIN3, CIN2, and CIN1. The diagnostic performance of TCT and the GynTect test was assessed for detecting CIN3+, with sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) calculated for both methods. Abnormal cytology (ASC-US+) had a sensitivity of 71.9% and specificity of 75.0% for detecting CIN3+, with a PPV of 57.5% and NPV of 85.0%. In comparison, the GynTect test demonstrated superior diagnostic accuracy, with a sensitivity of 88.5%, specificity of 87.2%, PPV of 78.7%, and NPV of 93.5%. The GynTect test outperformed cytology in identifying CIN3+ while reducing unnecessary colposcopies in women with CIN2- lesions. The GynTect six-gene methylation test provides higher diagnostic accuracy than cervical cytology in the triage of HPV-positive women. Its superior sensitivity and specificity make it a valuable tool for improving risk stratification in HPV-based cervical cancer screening. These findings support the integration of methylation-based testing into screening programs to enhance diagnostic precision and optimize patient management.

The TP53 “rs 1042522″ Polymorphism and its association with precancerous cervical lesions progression among Tunisian women

The etiological agent of cervical cancer is the infection with High- risk Human Papillomavirus. The oncoproteins E6 and E7 are responsible for the pathogenicity of this virus. The HR-HPV E6 protein binds to the tumor suppressor protein p53 via the E6AP ubiquitin ligase, inducing p53's ubiquitination and subsequent degradation by the proteasome. In this study, we investigate the association between TP53 rs1042522 polymorphism and the risk of cervical cancer among women in Tunisia and its potential role in the progression of cervical intraepithelial neoplasia. A total of hundred and eight cases including ninety-eight swabs and ten tissue samples were collected, between April 2023 and April 2024, from female participants addressed to the Pathology Department of the Pasteur Institute for HPV screening. Sequencing analysis identified three rs1042522 genotypes corresponding to wild (CC), and two mutant types, GG and CG. We found that women exhibiting mutant genotypes (CG and GG) had increased risk to cervical intraepithelial lesion progression. Interestingly, almost all patients diagnosed with cervical cancer had the mutated genotype. The association between TP53 rs1042522 and the development of cervical intraepithelial neoplasia was significant, P = 0.037; P < 0.05. Our results suggest this polymorphism as a potential biomarker linked to the progression of cervical precancerous lesions in the Tunisian women.

Distribution of high-risk human papillomavirus in women with ASC-US or LSIL in Tunisian women: Place of HPV testing for women triage in Tunisia

The present study aimed to evaluate the distribution of oncogenic HPVs in Tunisian women diagnosed with ASC-US or LSIL in order to highlight the importance of HPV testing in the management of women with minor cytological lesions. The study involved 213 cervical samples from women aged from 18 to 82 years and diagnosed with ASC-US or LSIL. HPV detection and genotyping was performed by nested PCR followed by reverse Line Blotting. HPV DNA was identified in 161 cases (76.3%). Oncogenic HPV genotypes were detected in 53.1% of cases. The most frequent high-risk genotypes in this study were HPV16 (28.8%) followed by: HPV51 (9.6%), HPV18, HPV31 HPV56 (7.1%) and HPV45 (5.1%). Thus, 24 % of studied women were not infected by HPV and about 47% of infections are without oncogenic HPV. These results highlight the value of HPV testing in the decision algorithm of management of minor abnormalities lesions.

Multi-centre clinical validation of the NeuMoDx HPV assay for ASC-US / LSIL triage

The NeuMoDx™ HPV assay (QIAGEN, MI, USA) is a PCR assay that detects 15 high-risk human papillomavirus (HPV) types in cervical samples, with separate identification for HPV16 and HPV18. Although it has been validated for primary HPV-based cervical cancer screening, its performance in the triage of atypical squamous cells of undetermined significance (ASC-US) or low-grade squamous intraepithelial lesion (LSIL) in cytology-based screening algorithms has not yet been evaluated. This multicentre study assessed the clinical performance of the NeuMoDx assay for ASC-US/LSIL triage. A total of 447 residual clinician-collected cervical samples were analysed. At site 1 (Ljubljana, Slovenia), NeuMoDx was compared with the Alinity m HR HPV assay (Abbott Molecular, IL, USA), and at site 2 (Ghent, Belgium), it was compared with the APTIMA HPV assay (HOLOGIC, MA, USA). NeuMoDx demonstrated 91.3% (95% CI: 85.8-94.7%) concordance with Alinity and 96.2% (95% CI: 93.3-97.8%) concordance with APTIMA, with excellent kappa values of 0.824 (95% CI:0.737-0.912) for site 1 and 0.779 (95% CI: 0.653-0.905) for site 2. The relative sensitivity of NeuMoDx for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was 0.91 and 1.08 compared to Alinity and Aptima, respectively. Relative clinical specificity of NeuMoDx was 1.05 compared to Alinity and 1.00 compared to Aptima. In conclusion, the NeuMoDx HPV assay showed reliable clinical performance in triaging ASC-US/LSIL samples.

High-risk HPV genotype distribution and prevalence in cervical swabs from Western Turkey

This retrospective study analyzed HPV-DNA genotyping, cytological, and histopathological findings from cervical swab samples of women aged 18 and older who attended the Gynecology and Obstetrics outpatient clinic at Balıkesir University Health Practice and Research Hospital between October 2022 and March 2024. A total of 1,447 samples from women aged 19-82 were evaluated. High-risk human papillomavirus (HPV) DNA was detected in 144 cases (9.9 %), including 67 single-type and 77 multiple-type infections. The most frequent types were multiple infections without HPV16/18 (22.92 %) and HPV 16 only(18.75 %). The highest positivity rate (18.7 %) was observed in the 35-39 age group. Cytological abnormalities were found in 52 cases (38.30 %), including infection (24.11 %), atypical squamous cells of undetermined significance (ASC-US, 23.07 %), low-grade squamous intraepithelial lesion (LSIL, 5.77 %), atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion (ASCH), high-grade squamous intraepithelial lesion (HSIL), and atypical glandular cells (AGC), each at 1.92. Among HPV-positive patients who underwent biopsy, 6.25 % low-grade squamous intraepithelial neoplasia (LSIL/CIN-I), 4.17 % high-grade squamous intraepithelial neoplasia (HSIL/CIN-II) and 2.78 % HSIL/CIN-III. The results highlight the value of regional HPV genotype surveillance in guiding cervical cancer screening and vaccination strategies.

Performance evaluation of the Allplex HPV HR Detection assay in comparison with the Cobas HPV test for high-risk HPV genotyping

Molecular testing for high-risk human papillomavirus (hrHPV) genotypes is important in screening for cervical cancer. In this study, we evaluated the performance of a newly developed Allplex HPV HR Detection assay in comparison with the Cobas HPV Test. A total of 1,275 cervical specimens obtained from a healthcare center between August 2021 and May 2022 were analyzed. The overall agreement for hrHPV detection was 98.4%, with higher agreement observed for HPV-16 (99.7%) and HPV-18 (99.8%) compared to other hrHPV genotypes (97.2%). Sequencing revealed that the majority of discrepancies was genotyped accurately by the Allplex HPV HR Detection assay with the exception of one false positive for HPV-16 and two false positives for other hrHPV genotypes. The Allplex HPV HR Detection assay showed almost perfect agreement with the Cobas HPV test, emphasizing its utility in hrHPV screening and monitoring.

Comparable detection of HPV using real-time PCR in paired cervical samples and concentrated first-stream urine collected with Colli-Pee device

We compared high-risk human papillomavirus (HPV) detection on first-stream urine from self-sampled collection device (Colli-Pee) and same-day clinician-collected cervical swab in 240 women. Testing with automated cobas 4800 system showed 96.7 % concordance (198 concordant-negative, 34 concordant-positive, Cohen's kappa=0.87). HPV testing on Colli-Pee urine offers advantages for acceptable non-invasive HPV screening.