HPV prevalence and genotype distribution among women in eastern China during the Covid-19 pandemic.

Since March 2020, the pandemic caused by SARS-CoV-2 has affected nearly all aspects of daily life. In this study, we investigated the age-stratified prevalence and genotype distribution of human papillomavirus (HPV) among females in Shandong province (eastern China) and aimed to provide guidance on HPV-based cervical cancer screening and vaccination. The distribution of HPV genotypes was analyzed using PCR-Reverse Dot Hybridization. The overall infection rate of HPV was 16.4%, which was dominated by high-risk genotypes. The most prevalent genotype was HPV16 (2.9%), followed by HPV52 (2.3%), HPV53 (1.8%), HPV58 (1.5%), and HPV51 (1.3%). Among the positive cases with HPV infection, single-genotype infection was significantly higher than that of multi-genotype infection. In subgroup analyses by age (≤25, 26-35, 36-45, 46-55, >55), HPV16, 52, and 53 were consistently the three most common hrHPV genotypes in all age groups. The infection rate of multi-genotypes in the ≤25 and >55 age groups was significantly higher than that in other age groups. A bimodal distribution of HPV infection rate was observed in different age groups. Among lrHPV genotypes, HPV6, HPV11, and HPV81 were the three most common types in the ≤25 age group, while in other age groups, HPV81, HPV42, and HPV43 are the three most common lrHPV genotypes. This study provides basic information on the distribution and genotypes of HPV in the female population in eastern China, which could improve the application of HPV diagnostic probes and vaccines.

Circulating tumor HPV DNA as an alternative method to determine HPV status in oropharyngeal squamous cell carcinoma.

Human Papilloma Virus (HPV) testing is mandatory for all newly diagnosed oropharyngeal squamous cell carcinoma (OPSCC) due to its importance for prognostication and aiding in treatment decision making. Fine needle aspiration (FNA) is a widely used and accepted diagnostic tool for OPSCC. Although FNA can accurately determine histological diagnosis, results are often indeterminate or lack insufficient samples for HPV testing. For samples with an indeterminant FNA, we propose an alternate method for determining HPV status using circulating tumor tissue modified HPV DNA (ctHPVDNA). We report three cases that confirmed HPV status using ctHPVDNA following an indeterminate FNA. If validated, this non-invasive assay could prevent the need for repeat FNAs or operative biopsies for the sole purpose of determining HPV status.

Frequency of human papilomavirus and associated factors in gypsy and quilombola women : Human papillomavirus in gypsy and quilombola women.

BACKGROUND: The prevalence of Human Papillomavirus (HPV) infection in the general population is widely known, however, there are still few studies related to this infection in minority groups, Thus, the objective is to analyze the frequency of human papillomavirus and associated factors in quilombola and gypsy women. METHODS: Cross-sectional research with 145 quilombola and gypsy women from Caxias, Maranhão. Two Pap smear collections were performed and a questionnaire with 46 questions was applied between January, 2020 and March, 2021. Descriptive analysis and Odds Ratio with 95% confidence interval were performed. The research was approved by the ethics committee. RESULTS: There were 09 cases of atypia. The frequency of human papillomavirus was 41.37%, with a higher risk in quilombolas 55 (91.70%). Multiple infections were prevalent (53%) with high-risk genotypes 21 (35%). Types 16 and 18 together accounted for 42.85% of cases. CONCLUSIONS: The frequency of human papillomavirus infection was higher than those recorded in the Northeast and Brazil, and therefore type 16 predominated. Due to limitations, the virus lineages and sublineages were not evaluated. Quilombola women had a higher rate of infection than gypsies.

TaME-seq2: tagmentation-assisted multiplex PCR enrichment sequencing for viral genomic profiling.

BACKGROUND: Previously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the human papillomavirus (HPV) DNA consensus sequence, low-frequency variable sites, and chromosomal integration events. The method has been successfully validated and applied to the study of five carcinogenic high-risk (HR) HPV types (HPV16, 18, 31, 33, and 45). Here, we present TaME-seq2 with an updated laboratory workflow and bioinformatics pipeline. The HR-HPV type repertoire was expanded with HPV51, 52, and 59. As a proof-of-concept, TaME-seq2 was applied on SARS-CoV-2 positive samples showing the method's flexibility to a broader range of viruses, both DNA and RNA. RESULTS: Compared to TaME-seq version 1, the bioinformatics pipeline of TaME-seq2 is approximately 40× faster. In total, 23 HPV-positive samples and seven SARS-CoV-2 clinical samples passed the threshold of 300× mean depth and were submitted to further analysis. The mean number of variable sites per 1 kb was ~ 1.5× higher in SARS-CoV-2 than in HPV-positive samples. Reproducibility and repeatability of the method were tested on a subset of samples. A viral integration breakpoint followed by a partial genomic deletion was found in within-run replicates of HPV59-positive sample. Identified viral consensus sequence in two separate runs was > 99.9% identical between replicates, differing by a couple of nucleotides identified in only one of the replicates. Conversely, the number of identical minor nucleotide variants (MNVs) differed greatly between replicates, probably caused by PCR-introduced bias. The total number of detected MNVs, calculated gene variability and mutational signature analysis, were unaffected by the sequencing run. CONCLUSION: TaME-seq2 proved well suited for consensus sequence identification, and the detection of low-frequency viral genome variation and viral-chromosomal integrations. The repertoire of TaME-seq2 now encompasses seven HR-HPV types. Our goal is to further include all HR-HPV types in the TaME-seq2 repertoire. Moreover, with a minor modification of previously developed primers, the same method was successfully applied for the analysis of SARS-CoV-2 positive samples, implying the ease of adapting TaME-seq2 to other viruses.

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.

IPVS policy statement on HPV nucleic acid testing guidance for those utilising/considering HPV as primary precancer screening: Quality assurance and quality control issues.

We advise that only clinically validated HPV assays which have fulfilled internationally accepted performance criteria be used for primary cervical screening. Further, assays should be demonstrated to be fit for purpose in the laboratory in which they will ultimately be performed, and quality materials manuals and frameworks will be helpful in this endeavor. Importantly, there is a fundamental shortage of well validated, low-cost, low complexity HPV tests that have demonstrated utility in a near-patient setting; representing a significant challenge and focus for future development in order to reach the WHO's goal of eliminating cervical cancer.

Clinical Validation of the Fully Automated NeuMoDx HPV Assay for Cervical Cancer Screening.

The NeuMoDx HPV assay is a novel fully automated, real-time PCR-based assay for the qualitative detection of high-risk human papillomavirus (HPV) DNA in cervical specimens. The assay specifically identifies HPV16 and HPV18 and concurrently detects 13 other high-risk HPV types at clinically relevant infection levels. Following the international guidelines, the clinical performance of the NeuMoDx HPV assay for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) against the reference standard Hybrid Capture 2, as well as intra- and inter-laboratory reproducibility were assessed on PreservCyt samples. The clinical accuracy of the assay was additionally evaluated against the clinically validated Alinity m HR HPV and COBAS 4800 HPV Test on PreservCyt samples, and against the clinically validated HPV-Risk assay on SurePath samples. The NeuMoDx HPV assay performance for CIN2+ was non-inferior to the reference methods on both sample types (all p < 0.05), and showed excellent intra- and inter-laboratory reproducibility (95.7%; 95% CI: 93.9-97.3; kappa value 0.90 (95% CI: 0.86-0.94); and 94.5%; 95% CI: 92.6-96.2; kappa value 0.87 (95% CI: 0.82-0.92), respectively). In conclusion, the NeuMoDx HPV assay meets international guideline criteria for cross-sectional accuracy and reproducibility, and performs equally well on cervical screening specimens collected in two widely used collection media. The NeuMoDx HPV assay fulfils the requirements to be used for primary cervical screening.

Role of Self-Sampling for Cervical Cancer Screening: Diagnostic Test Properties of Three Tests for the Diagnosis of HPV in Rural Communities of Cuenca, Ecuador.

BACKGROUND: HPV primary screening has shown effectiveness for cancer prevention; however, gynaecological examination is considered uncomfortable. Self-sampling methods increase the acceptance of screening. The aim of this study is to compare the sensitivity and specificity of clinician sampling versus vaginal and urine self-sampling for HPV diagnosis. METHODS: A diagnostic test study was conducted in a rural parish of Cuenca, Ecuador. A total of 120 women participated. Each participant self-collected urine and vaginal samples and underwent clinician sampling for HPV testing. The latter was considered as the golden standard. All three samples were processed with the same amplification and hybridization protocol for HPV detection (Hybribio) following the manufacturer's instructions. RESULTS: Characteristics of the participants were: median age 35 years; 40.8% married; 46.7% had a primary level of education; and median age of sexual onset, 17.6 years. The prevalence of any type of HPV with clinician sampling was 15.0%, 17.5% with urine sampling and 18.3% with vaginal self-sampling. Self-sampling sensitivity reached 94.4% (IC 74.2-99.9), and specificity 92.1% (IC 85.2-95.9). Urine sampling had a sensitivity of 88.8% (IC 67.2, 96.9), and specificity 94.1% (IC 67.2-96.9). The negative predictive value was 98.9% (IC 94.2-99.8) for vaginal self-sampling and 97.6% (IC 92.6-99.4) for urine sampling. CONCLUSIONS: This study shows that vaginal and urine self-sampling methods have similar sensitivity and specificity compared with clinician sampling for the diagnosis of HPV. The correlation between HPV genotypes among the three tests is satisfactory.

Impact of Collection Volume and DNA Extraction Method on the Detection of Biomarkers and HPV DNA in First-Void Urine.

The potential of first-void (FV) urine as a non-invasive liquid biopsy for detection of human papillomavirus (HPV) DNA and other biomarkers has been increasingly recognized over the past decade. In this study, we investigated whether the volume of this initial urine stream has an impact on the analytical performance of biomarkers. In parallel, we evaluated different DNA extraction protocols and introduced an internal control in the urine preservative. Twenty-five women, diagnosed with high-risk HPV, provided three home-collected FV urine samples using three FV urine collection devices (Colli-Pee) with collector tubes that differ in volume (4, 10, 20 mL). Each collector tube was prefilled with Urine Conservation Medium spiked with phocine herpesvirus 1 (PhHV-1) DNA as internal control. Five different DNA extraction protocols were compared, followed by PCR for GAPDH and PhHV-1 (qPCR), HPV DNA, and HBB (HPV-Risk Assay), and ACTB (methylation-specific qPCR). Results showed limited effects of collection volume on human and HPV DNA endpoints. In contrast, significant variations in yield for human endpoints were observed for different DNA extraction methods (p < 0.05). Additionally, the potential of PhHV-1 as internal control to monitor FV urine collection, storage, and processing was demonstrated.

Footprint of the host restriction factors APOBEC3 on the genome of human viruses.

APOBEC3 enzymes are innate immune effectors that introduce mutations into viral genomes. These enzymes are cytidine deaminases which transform cytosine into uracil. They preferentially mutate cytidine preceded by thymidine making the 5'TC motif their favored target. Viruses have evolved different strategies to evade APOBEC3 restriction. Certain viruses actively encode viral proteins antagonizing the APOBEC3s, others passively face the APOBEC3 selection pressure thanks to a depleted genome for APOBEC3-targeted motifs. Hence, the APOBEC3s left on the genome of certain viruses an evolutionary footprint. The aim of our study is the identification of these viruses having a genome shaped by the APOBEC3s. We analyzed the genome of 33,400 human viruses for the depletion of APOBEC3-favored motifs. We demonstrate that the APOBEC3 selection pressure impacts at least 22% of all currently annotated human viral species. The papillomaviridae and polyomaviridae are the most intensively footprinted families; evidencing a selection pressure acting genome-wide and on both strands. Members of the parvoviridae family are differentially targeted in term of both magnitude and localization of the footprint. Interestingly, a massive APOBEC3 footprint is present on both strands of the B19 erythroparvovirus; making this viral genome one of the most cleaned sequences for APOBEC3-favored motifs. We also identified the endemic coronaviridae as significantly footprinted. Interestingly, no such footprint has been detected on the zoonotic MERS-CoV, SARS-CoV-1 and SARS-CoV-2 coronaviruses. In addition to viruses that are footprinted genome-wide, certain viruses are footprinted only on very short sections of their genome. That is the case for the gamma-herpesviridae and adenoviridae where the footprint is localized on the lytic origins of replication. A mild footprint can also be detected on the negative strand of the reverse transcribing HIV-1, HIV-2, HTLV-1 and HBV viruses. Together, our data illustrate the extent of the APOBEC3 selection pressure on the human viruses and identify new putatively APOBEC3-targeted viruses.