Codon usage bias of human papillomavirus type 33 and 58: A comprehensive analysis.

Cervical cancer is closely linked to specific strains of human papillomavirus (HPV), notably HPV-33 and HPV-58, which exhibit a significant prevalence among women in China. Nevertheless, the codon usage bias in HPV-33 and HPV-58 is not well comprehended. The objective of this research is to analyze the codon usage patterns HPV-33 and HPV-58, pinpoint the primary factors that influence codon preference. The overall preference for codon usage in two HPV genotypes is not significant. Both HPV genotypes exhibit a preference for codons that end with A/U. The GC3 content for HPV-33 is 25.43% ± 0.35%, and for HPV-58, it is 29.44% ± 0.57%. Out of the 26 favored codons in HPV-33 and HPV-58 (relative synonymous codon usage (RSCU) > 1), 25 conclude with A/U. Principal component analysis (PCA) shows a tight clustering of the entire genome sequences of HPV-33 and HPV-58, suggesting a similarity in their RSCU preferences. Moreover, an examination of dinucleotide abundance indicated that translation selection influenced the development of a distinctive dinucleotide usage pattern in HPV-33 and HPV-58. Additionally, a combined analysis involving an effective number of codons plot, parity rule 2, and neutrality analysis demonstrated that, for HPV-33 and HPV-58, the primary determinant influencing codon usage preference is natural selection. HPV-33 and HPV-58 exhibit a restricted set of favored codons in common with humans, potentially mitigating competition for translation resources. Our discoveries could provide valuable perspectives on the evolutionary patterns and codon usage preferences of HPV-33 and HPV-58 viruses, contributing to the development and application of relevant HPV subtype vaccines.

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 authors compared the clinical performance of DH3 human papillomavirus (HPV) assay, which detects 14 high-risk HPVs with 16/18 genotyping based on hybrid capture technique, and Hybrid Capture 2 (HC2) test for women undergoing cervical cancer screening. A total of 7, 263 residual cytology specimens from an adjudicated cohort with 3-year follow-up were tested by the DH3 assay and the HC2 test. Assay results were compared with each other and to histology review. The overall agreement between the DH3 assay and the HC2 test was 99.2% (κ = 0.938). At baseline, DH3 had the equal sensitivity to that of HC2 for cervical intraepithelial neoplasia (CIN) grade 2 or higher (CIN2+, n = 75) and CIN grade 3 or higher (CIN3+, n = 45), 98.67% and 97.78%, respectively. After 3 years of follow-up, the sensitivity for CIN2+ (n = 133) and CIN3+ (n = 74) were both similar between DH3 and HC2 (95.49% vs 94.74%, 95.95% vs 95.95%, respectively, all P > 0.05). The respective specificity for CIN2+ or CIN3+ did not differ between the two tests. A noninferiority test showed that both sensitivity and specificity of DH3 for CIN2+ and CIN3+ were noninferior to those of HC2 at baseline and after 3-year follow-up, respectively (all P < 0.001). When used in primary screening strategy, the DH3 assay would yield an immediate sensitivity of 92% for CIN2+. DH3 HPV performs equally to HC2 for the detection of high-grade lesions in cervical cancer screening and has a potential advantage in primary screening strategy due to HPV16/18 genotyping. IMPORTANCE 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. However, HC2 cannot discriminate HPV16 and 18 from the other hrHPV types, which greatly limited the application of HC2 in cervical cancer screening. The DH3 human papillomavirus (HPV) is a recently developed assay based on hybrid capture technique like to HC2, which can specifically identify HPV 16/18 on the basis of detecting the 13 hrHPV types targeted by HC2 as well as HPV66. This comparative study of the two assays for detection of hrHPV infection in residual cytology samples from cervical cancer screening setting reveals that DH3 HPV provides a perfect alternative to HC2 in detecting hrHPV infection and identifying cervical precancer, while allowing concurrent HPV 16/18 genotyping.

Human papillomaviruses: diversity, infection and host interactions.

Human papillomaviruses (HPVs) are an ancient and highly successful group of viruses that have co-evolved with their host to replicate in specific anatomical niches of the stratified epithelia. They replicate persistently in dividing cells, hijack key host cellular processes to manipulate the cellular environment and escape immune detection, and produce virions in terminally differentiated cells that are shed from the host. Some HPVs cause benign, proliferative lesions on the skin and mucosa, and others are associated with the development of cancer. However, most HPVs cause infections that are asymptomatic and inapparent unless the immune system becomes compromised. To date, the genomes of almost 450 distinct HPV types have been isolated and sequenced. In this Review, I explore the diversity, evolution, infectious cycle, host interactions and disease association of HPVs.

Human papillomavirus 68 prevalence and genetic variability based on E6/E7 genes in Sichuan.

HPV68 is a common HR-HPV, its persistent infection is closely related with the occurrence of cervical cancer. In this study, 2939 (27.60%, 2939/10650) positive samples were detected, and 174 (5.92%, 174/2939) were HPV68. 150 HPV68 E6-E7 were successful sequenced, 4 non-synonymous mutations were detected in E6, and E7 were 12. N133S non-synonymous mutations of HPV 68 E6 and C67G, T68 A/M of HPV68 E7 are E6, E7 positive selection sites, they all located in the key domains and major motifs of E6/E7 protein, the above amino-acid substitutions changed the protein structure, disturbed the interaction with other protein or cellular factors and make a difference in epitopes affinity, may affect the pathogenicity and adaptability of HPV68 to the environment. The enrichment of HPV68 data is of great significance for understanding the inherent geographical and biological differences of HPV68 in China.

Association between Human Papillomavirus and Chlamydia trachomatis genital infections in male partners of infertile couples.

The prevalence of HPV infection and its relationship with other sexually transmitted infections was analyzed in a cohort of 117 male partners of infertile couples from Cordoba, Argentina. Semen samples and urethral swabs were obtained and the infection with HPV, Chlamydia trachomatis, HSV1, HSV2, Mycoplasma hominis and Ureaplasma urealyticum was analyzed. A prevalence of HPV infection of 27.4% was found. Interestingly, infections by exclusively low risk HPV genotypes or high/intermediate risk HPV genotypes were present in 64.5% and 22.6% of cases, respectively. Low risk-HPV6 was the most frequently detected genotype. Remarkably, HPV and C. trachomatis infections were significantly associated to each other (OR: 11.55, 95% CI 1.14-117.06). No significant differences in sperm quality were found between HPV-positive and HPV-negative patients indicating that HPV male urogenital infection does not impair sperm quality. Our results show a high prevalence of HPV urogenital infection among male partners of infertile couples, and that HPV and C. trachomatis infections are reciprocal risk factors of their co-infection. Moreover, our results suggest that men constitute a reservoir for continued transmission of C. trachomatis and HPV to women highlighting the need for routine screening for these two pathogens in male partners of infertile couples.

Focal Epithelial Hyperplasia.

Focal epithelial hyperplasia (FEH) or Heck's disease is a rare, benign, oral condition that is associated with infection by human papillomavirus type 13, 32 or both. The whiteish to mucosal-colored, soft, papular or nodular elevated lesions in the oral cavity are normally asymptomatic but can grow to a size or at a location where treatment is needed. The diagnosis is often based on clinical presentation and histopathology, and the HPV genotype can be determined using PCR utilizing specific primers or DNA sequencing. While FEH was reported to often affect several members of the same family and exist primarily among indigenous populations around the world, the number of reported cases within the European region is increasing. This contemporary review summarizes the main findings in relation to HPV genotypes, impact of superinfection exclusion and vaccination, transmission, diagnosis, geographical and ethnical distribution, comorbidities and treatment of FEH with an emphasis on including the most recent case reports within the field. Furthermore, we describe for the first time a FEH lesion infected with the low-risk HPV90.

Prevalence characteristics of human papillomavirus (HPV) infection among women receiving physical examinations in the Shangcheng District, Hangzhou city, China.

This paper aimed to investigate the characteristics of female HPV infection in the Shangcheng District, Hangzhou city, China. The retrospective study was designed to analyze the HPV prevalence rate of 22,382 women receiving physical examinations from 2016 to 2020 in the Shangcheng District of Hangzhou city in China. A commercial kit was designed to detect the HPV genotypes. Trends were examined for age-specific groups (≤ 30 years, 31-44 years, 45-54 years, 55-64 years, ≥ 65 years). A receiver operating characteristic (ROC) analysis was used to assess the correlation of age classification in high risk HPV (HR-HPV) infection. 22.41% (5015/22,382) of samples were HPV positive, 91.28% (4578/5015) of HPV positive women were infected by HR-HPV. The most prevalent HR-HPV genotypes were 16, 52, 18, 58, 56, and 51. The trend of HPV prevalence showed the significant differences in age-specific groups (χ2 = 164.70, P < 0.001). Moreover, the areas under ROC curve (AUC) was 0.712 in 55-64 years group which showed a strong contribution of age classification for HR-HPV infection. This study provided baseline data on the prevalence characteristics of HPV infection and the critical age group of HR-HPV prevalence rate was 55-64 y among the samples receiving physical examinations.

Relevance of Human Papillomaviruses in Head and Neck Cancer-What Remains in 2021 from a Clinician's Point of View?

Human papillomaviruses (HPV) cause a subset of head and neck cancers (HNSCC). HPV16 predominantly signs responsible for approximately 10% of all HNSCC and over 50% of tonsillar (T)SCCs. Prevalence rates depend on several factors, such as the geographical region where patients live, possibly due to different social and sexual habits. Smoking plays an important role, with non-smoking patients being mostly HPV-positive and smokers being mostly HPV-negative. This is of unparalleled clinical relevance, as the outcome of (non-smoking) HPV-positive patients is significantly better, albeit with standard and not with de-escalated therapies. The results of the first prospective de-escalation studies have dampened hopes that similar superior survival can be achieved with de-escalated therapy. In this context, it is important to note that the inclusion of p16INK4A (a surrogate marker for HPV-positivity) in the 8th TMN-classification has only prognostic, not therapeutic, intent. To avoid misclassification, highest precision in determining HPV-status is of utmost importance. Whenever possible, PCR-based methods, still referred to as the "gold standard", should be used. New diagnostic antibodies represent some hope, e.g., to detect primaries and recurrences early. Prophylactic HPV vaccination should lead to a decline in HPV-driven HNSCC as well. This review discusses the above aspects in detail.

Genetic and Epigenetic Variations of HPV52 in Cervical Precancer.

The goal of this study was to identify human papillomavirus (HPV) type 52 genetic and epigenetic changes associated with high-grade cervical precancer and cancer. Patients were selected from the HPV Persistence and Progression (PaP) cohort, a cervical cancer screening program at Kaiser Permanente Northern California (KPNC). We performed a nested case-control study of 89 HPV52-positive women, including 50 cases with predominantly cervical intraepithelial neoplasia grade 3 (CIN3) and 39 controls without evidence of abnormalities. We conducted methylation analyses using Illumina sequencing and viral whole genome Sanger sequencing. Of the 24 CpG sites examined, increased methylation at CpG site 5615 in HPV52 L1 region was the most significantly associated with CIN3, with a difference in median methylation of 17.9% (odds ratio (OR) = 4.8, 95% confidence interval (CI) = 1.9-11.8) and an area under the curve of 0.73 (AUC; 95% CI = 0.62-0.83). Complete genomic sequencing of HPV52 isolates revealed associations between SNPs present in sublineage C2 and a higher risk of CIN3, with ORs ranging from 2.8 to 3.3. This study identified genetic and epigenetic HPV52 variants associated with high risk for cervical precancer, improving the potential for early diagnosis of cervical neoplasia caused by HPV52.

Lineage analysis of human papillomavirus type 39 in cervical samples of Iranian women.

BACKGROUND: The data with regards to the regional variants of distinct HPV types is of great value. Accordance with this, this study aimed to investigate the sequence variations of E6 gene and long control region of HPV 39 among normal, premalignant and malignant cervical samples in order to characterize the frequent HPV 39 variants circulating in Tehran, Iran. METHODS: In total, 70 cervical samples (45 normal, 16 premalignant, and 9 malignant samples) infected with HPV 39 were analyzed by nested-PCR and sequencing. RESULTS: Our results revealed that all samples belonged to A lineage. Almost all sequences (98.6%) were classified in A1 sublineage and only one sample (1.4%) was A2 sub lineage. CONCLUSIONS: Our findings showed that lineages A, sublineage A1, is dominant in Tehran, Iran. However, the small sample size was the most important limitations of this study. Further studies with larger sample size from different geographical regions of Iran are necessary to estimate the pathogenicity risk of HPV 39 variants in this population.

High human papillomavirus prevalence among females attending high school in the Eastern Cape Province of South Africa.

As part of the human papillomavirus (HPV) vaccination strategy in South Africa, it is essential to have information on HPV prevalence, and HPV types distribution among the unvaccinated population. Information on the prevalence of HPV and the distribution of HPV types in adolescents and young women in South Africa's Eastern Cape Province is minimal. Therefore, this study investigates the prevalence, distribution of HPV types, and factors associated with HPV infection amongst unvaccinated female learners. A sample composed of 213 sexually active female learners attending high schools in the Eastern Cape Province of South Africa; median age 18 years, who provided self-collected vaginal specimens. Roche Linear Array HPV genotyping assay that detects 37 HPV genotypes was used to detect HPV infection. HPV infection was detected in 76.06% (162/213) of participants. Of these 14.55% (31/213) were positive for HPV types targeted by the Cervarix® HPV vaccine (HPV-16 and/or 18), 20.66% (44/213) by Gardasil®4 (HPV-6, -11, -16 and/or -18) and 37.09% (79/213) by Gardasil®9 (HPV-6, -11, -16, -18, -31, -33, -45, -52 and/or -58). HPV-35, commonly detected in cervical cancer cases among women of African ancestry, was frequently detected (9.40%). Participants who reported to have ever consumed alcohol had a significantly higher risk of HPV infection (OR: 2.91, 95% CI: 1.38-6.11, p = 0.005). High HPV prevalence was observed among participants. The high prevalence of HPV types targeted by the Gardasil®9 vaccine encourages the introduction of the Gardasil®9 vaccine. Data from this study will inform both vaccination campaigns and monitor the impact on HPV types after vaccination.

The p16/ki-67 assay is a safe, effective and rapid approach to triage women with mild cervical lesions.

OBJECTIVE: The aim of this study was to evaluate the diagnostic accuracy and efficiency of p16/ki-67 dual stain in the identification of CIN2+ lesions, in Greek women with ASCUS or LSIL cytology. METHODS: A total of 200 women, 20 to 60 years old, were enrolled in the study. All samples were cytologically evaluated and performed for p16/ki-67 and high-risk HPV (HR-HPV) test. All patients were referred to colposcopy for biopsy and histological evaluation. Three cervical cancer (CC) screening strategies were designed and the total direct medical costs of the procedures during our clinical trial were evaluated, from a healthcare perspective. RESULTS: HPV 16 as expected was the most common HR-HPV type followed by HPV 31 and HPV 51. The risk for CIN2+ was significantly higher in HPV 16/18 positive cases. p16/ki-67 demonstrated a high sensitivity for CIN2+ identification in both ASCUS and LSIL groups (90.4% and 95%, respectively). HR-HPV test with sensitivity 52.3% and 65.5%, as well as colposcopy with sensitivity 14.3% and 36% respectively in ASCUS and LSIL group, showed inferior results compared to p16/ki-67. The specificity of p16/ki-67 for ASCUS and LSIL was 97.2% and 95.2% respectively, inferior only to colposcopy: 100% and 100%, lacking however statistical significance. HR-HPV test instead, presented the lowest specificity: 76.4% and 71.4% respectively in comparison to the other two methods. From a healthcare perspective, the costs and benefits of the tests implementation for the annual screening and triaging, in three CC screening strategies, were also calculated and discussed. CONCLUSIONS: The results of the study indicate that p16/ki-67 is a safe and rapid assay that could be used to detect CIN2+ among women with mild cervical lesions, presenting both high sensitivity and specificity and could minimize the psychological and economic burden of HPV screening.

Development and evaluation of a new non-competitive Luminex immunoassay detecting antibodies against human papillomavirus types 6, 11, 16 and 18.

Serum antibody levels can be used to measure the humoral immune response against human papillomaviruses (HPV). We developed and validated a rapid, technically simple and relatively inexpensive multiplex non-competitive Luminex-based immunoassay (ncLIA) to measure total IgG antibody levels against four HPV types. For the assay's solid phase, virus-like particles (VLPs) of HPV6, 11, 16 and 18 were bound to heparin-coated beads. HPV serum antibody levels binding to the VLPs were quantified using a phycoerithrin-conjugated secondary polyclonal donkey anti-human IgG antibody. Standardization and validation of the ncLIA were performed using 96 paired serum and genital samples from participants in the HITCH cohort study, including young women (aged 18-24 years) and their male sexual partners (aged 18+) in Montreal, Canada. Results from the ncLIA were compared to a validated Luminex immunoassay from PPD laboratories using Pearson's correlation coefficients, receiver operating characteristic curves and logistic regression. Our assay had good inter- and intra-assay variability. The correlation of serum antibody levels between the ncLIA and validation assay was highest for HPV16 and HPV11 (r=0.90), followed by HPV6 (r=0.86) and HPV18 (r=0.67). The ncLIA was better able to predict HPV DNA positivity in genital samples than the validation assay for HPV16 [area under the curve (AUC) 0.65 versus 0.52, P=0.001] and HPV18 [AUC 0.71 versus 0.57, P=0.024]. AUCs for HPV6 and HPV11 were similar between the two assays (0.70 versus 0.71, P=0.59, and 0.88 versus 0.96, P=0.08, respectively). The developed ncLIA is useful for measuring total IgG antibody response following natural infection or vaccination against four HPV VLPs included in the quadrivalent vaccine.

Prevalence of human papillomavirus in young men who have sex with men after the implementation of gender-neutral HPV vaccination: a repeated cross-sectional study.

BACKGROUND: Anal infection with high-risk human papillomavirus (HPV) genotypes 16 and 18 and anal cancer are overrepresented in men who have sex with men (MSM). This study investigated HPV prevalence in young MSM before and after the implementation of a school-based quadrivalent HPV (genotypes 6, 11, 16, and 18) vaccination programme for boys in Australia in 2013. METHODS: In this repeated cross-sectional study, MSM aged 16-20 years were recruited from two successive birth cohorts via sexual health clinics and the community in Melbourne, Australia. The first cohort was before the implementation of gender-neutral vaccination (HYPER1 study, done in 2010-12, NCT01422356), and the second was the post-vaccination cohort (HYPER2 study, done in 2017-18, NCT03000933). Men who self-identified as being same-sex attracted were enrolled, and those recruited via the HYPER2 study had to be resident in Australia since 2013 to ensure eligibility. Study procedures were done in the Melbourne Sexual Health Centre. A clinician-collected anal swab and self-collected penile swab and oral rinse were tested for 28 HPV genotypes, and data on demographics and sexual health practices were collected via questionnaires. Only assessable samples were included in the analyses. We compared anatomical site-specific prevalence of HPV genotypes between cohorts by calculating the prevalence ratio, adjusting for age, circumcision, and sex with women. Herd protection was also assessed, by calculating the adjusted prevalence ratios by vaccination status. FINDINGS: 400 MSM, 200 per cohort, were included in the study. In both cohorts, the median number of lifetime male partners was ten (IQR 5-25). The prevalence of any anal quadrivalent vaccine-preventable HPV genotype was higher in the pre-vaccination cohort (54 [28%] of 193) than in the post-vaccination cohort (14 [7%] of 193; adjusted prevalence ratio [PR] 0·24, 95% CI 0·14-0·42), largely driven by decreases in HPV6, followed by HPV11, 16, and 18. Nevertheless, there was also a significant reduction in anal HPV16 and 18 in the post-vaccination cohort from the pre-vaccination cohort (0·31, 0·14-0·68). The prevalence of any penile quadrivalent vaccine-preventable HPV genotype was also higher in the pre-vaccination cohort (21 [12%] of 177) than in the post-vaccination cohort (11 [6%] of 179; 0·48, 0·24-0·97), driven by decreases in HPV 6 and 11, but not by 16 and 18. The prevalence of any oral quadrivalent vaccine-preventable HPV genotype was higher in the pre-vaccination cohort (seven [4%] of 200) than in the post-vaccination cohort (one [1%] of 199; 0·10, 0·01-0·97); there were no cases of oral HPV6 or 11 detected in HYPER2. Comparing the pre-vaccinated cohort with the 149 confirmed vaccinated men from HYPER2 showed a reduction in any quadrivalent vaccine-preventable HPV genotype for anal (0·09, 0·03-0·25) and penile (0·18, 0·05-0·59) infection but not for oral infection (0·17, 0·03-1·08). INTERPRETATION: A reduction in anal, penile, and oral quadrivalent vaccine-targeted genotypes occurred in young MSM following the implementation of a school-based gender-neutral HPV vaccination programme. The fall in anal HPV16 and 18 may lead to a reduction in the incidence of anal cancer. FUNDING: Merck and the Australian Government Department of Health.

Prevalence of Human Papillomavirus (HPV) DNA among Men with Oropharyngeal and Anogenital Cancers: A Systematic Review and Meta-Analysis.

OBJECTIVE: The term ''Human Papillomavirus'' or ''HPV'' has become synonymous with uterine cervical cancer leading to feminisation of all the preventive measures, especially immunisation. Taking into consideration the rising number of HPV associated cancers among men in many developed countries and the risk of transmission to women, male HPV infection is a serious concern. A systematic review and meta-analysis of literature was performed to determine the global prevalence of HPV among men with oropharyngeal and anogenital cancers. METHODS: A systematic review and meta-analysis of literature was performed searching electronic databases for published articles in English between January 1984- April 2020 based on standard systematic review guidelines. The meta-analysis component was modified appropriately for the synthesis of prevalence study results. National Institutes of Health checklist for observational, cohort and cross-sectional studies was used to assess the quality of the studies selected after the abstract and content review. The meta-analysis was performed in STATA version 13.0 (College Station, Texas 77,845 USA) and the forest plots were constructed using metan package in STATA. RESULTS: Through the electronic search of databases, 3486 original articles were screened for eligibility. Fifty-eight articles were systematically reviewed and 42 articles were qualified for meta-analysis including 4,250 men with oropharyngeal, penile and prostate cancers. The pooled prevalence of HPV DNA in oropharyngeal cancers was 45% (95%CI 24.0%-66.0%). Meanwhile the pooled prevalence rates of 48% (CI 40.0%- 57.0%) and 19% (CI 10.0%-29.0%) were observed in penile and prostate cancers respectively. Even though, articles regarding HPV prevalence in anal cancers were systematically reviewed, none of the studies were qualified for meta-analysis. CONCLUSION: Higher pooled prevalence of HPV DNA was observed among men with oropharyngeal and penile cancers. Multicentric molecular studies investigating the prevalence of HPV in prostate cancers have to be planned in future.

Transcription of human papillomaviruses in nonmelanoma skin cancers of the immunosuppressed.

Nonmelanoma skin cancer (NMSC) has a greatly increased incidence among the immunosuppressed and the DNA of human papillomavirus (HPV) is commonly found in these tumors. To investigate if there are any actively transcribed HPV infections in these tumors, we identified all skin cancers diagnosed after solid organ transplantation in Sweden during 1964-2011 (n = 7614 NMSCs) and requested the diagnostic tumor blocks from the corresponding pathology archives. For the present study, we selected diagnostic specimens from 345 NMSC and performed whole genome transcriptome analysis using NovaSeq (Illumina), in comparison with three cervical cancers. Although we obtained an abundance of high-quality paired reads per sample (median of 35 million reads), only 15 NMSC specimens contained HPV transcription. Three specimens had transcription of oncogenic anogenital HPVs (HPV16 and 56), six tumors had transcription of HPVs from the beta-2 species (three HPV38, two with HPV23 and one with HPV107) and then there was one observation each of transcription of HPVs 3, 26, 57, 147, 158, 168 and of two nonestablished HPV types belonging to the gamma genus. In conclusion, transcription of specific HPV types can be found in NMSC among the immunosuppressed, but this is not common.

NanoString Technology for Human Papillomavirus Typing.

High-throughput HPV typing assays with increased automation, faster turnaround and type-specific digital readout would facilitate studies monitoring the impact of HPV vaccination. We evaluated the NanoString nCounter® platform for detection and digital readout of 48 HPV types in a single reaction. NanoString (NS) used proprietary software to design CodeSets: type-specific probe pairs targeting 48 HPV types and the globin gene. We tested residual DNA extracts from epidemiologic specimens and defined samples (HPV plasmids at 10 to 104 copies/reaction) directly (No-PCR) as well as after L1 consensus PCR of 45 (PCR-45) or 15 cycles (PCR-15). Assay and interpretation followed NS recommendations. We evaluated analytic performance by comparing NanoString results for types included in prior assays: Roche Linear Array (LA) or HPV TypeSeq assay. No-PCR results on 40 samples showed good type-specific agreement with LA (k = 0.621) but sensitivity was 65% with lower limit of detection (LOD) at 104 plasmid copies. PCR-45 results showed almost perfect type-specific agreement with LA (k = 0.862), 82% sensitivity and LOD at 10 copies. PCR-15 results on 75 samples showed substantial type-specific agreement with LA (k = 0.796, 92% sensitivity) and TypeSeq (k = 0.777, 87% sensitivity), and LOD at 10 copies of plasmids. This proof-of-principle study demonstrates the efficacy of the NS platform with HPV CodeSet for type-specific detection using a low number of PCR cycles (PCR-15). Studies are in progress to evaluate assay reproducibility and analytic validation with a larger number of samples.

Infectious titres of human papillomaviruses (HPVs) in patient lesions, methodological considerations in evaluating HPV infectivity and implications for the efficacy of high-level disinfectants.

BACKGROUND: Recent publications from a single research group have suggested that aldehyde-based high-level disinfectants (HLDs), such as ortho-phthalaldehyde (OPA), are not effective at inactivating HPVs and that therefore, patients may be at risk of HPV infection from medical devices. These results could have significant public health consequences and therefore necessitated evaluation of their reproducibility and clinical relevance. METHODS: We developed methods and used standardised controls to: (1) quantify the infectious levels of clinically-sourced HPVs from patient lesions and compare them to laboratory-derived HPVs, (2) evaluate experimental factors that should be controlled to ensure consistent and reproducible infectivity measurements of different HPV genotypes, and (3) determine the efficacy of select HLDs. FINDINGS: A novel focus forming unit (FFU) infectivity assay demonstrated that exfoliates from patient anogenital lesions and respiratory papillomas yielded infectious HPV burdens up to 2.7 × 103 FFU; therefore, using 2.2 × 102 to 1.0 × 104 FFU of laboratory-derived HPVs in disinfection assays provides a relevant range for clinical exposures. RNase and neutralising antibody sensitivities were used to ensure valid infectivity measures of tissue-derived and recombinant HPV preparations. HPV infectivity was demonstrated over a dynamic range of 4-5 log10; and disinfection with OPA and hypochlorite was achieved over 3 to >4 log10 with multiple genotypes of tissue-derived and recombinant HPV isolates. INTERPRETATION: This work, along with a companion publication from an independent lab in this issue, address a major public health question by showing that HPVs are susceptible to HLDs. FUNDING: Advanced Sterilization Products; US NIH (R01CA207368, U19AI084081, P30CA118100).