Detection Of Papillomavirus DNA Research Articles

Human papillomavirus DNA detection in cervical samples from women of reproductive age in Mogadishu, Somalia.

Somalia is among the countries with a high burden of human papillomavirus (HPV) infection in Sub-Saharan Africa. In 2022, 660,000 infections are reported among women globally, out of which 350,000 died from the disease. Most of the studies on HPV reported from Somalia are based on cytologic analysis which is a subjective and suboptimal assessment. The recent World Health Organization (WHO) guidelines for HPV recommend DNA-based testing as a better alternative to the traditional pap smear test. This study was undertaken to determine the prevalence of HPV based on the preferred HPV DNA assay on cervical samples of women of reproductive age in Mogadishu, Somalia. The HPV DNA detection was carried out using real-time quantitative polymerase chain reaction (RT-PCR) assays to identify high-risk oncogenic HPV16, HPV18, and other high-risk HPV types. Overall, 31.7% (60/189) of cervical samples were positive for HPV DNA. Out of this, 19.6% were high risk-HPV (hrHPV), 13.8% were HPV16, and 5.3% were HPV18. Moreover, age, income and education level were found to be significant risk factors for HPV infection. These results provide further proof that HPV continues to be a serious public health challenge in Somalia with the risk of progressing to fatal cervical cancer.

A novel human papillomavirus and host DNA methylation score and detection of cervical adenocarcinoma.

The widespread introduction of Pap testing in the 1960s was followed by substantial reductions in the incidence of cervical squamous cell cancer (SCC). However, the incidence of cervical adenocarcinoma (ADC) did not decrease, likely because of low Pap test sensitivity for ADC and adenocarcinoma in situ (AIS). This study assessed a novel human papillomavirus (HPV) and host DNA Methylation Score for AIS and ADC screening. We measured methylation levels at CpG sites in the L2/L1 open reading frames of HPV16, HPV18, and HPV45-as well as 2 human loci, DCC and HS3ST2. Specifically, we tested exfoliated cervicovaginal cells from women in the HPV Persistence and Progression (PaP) cohort who were positive for 1 of HPV16, 18, or 45, including: 1) 176 withAIS/ADC, 2) 353 with cervical intraepithelial neoplasia-3 (CIN3) or SCC, and 3) controls who either cleared (HPV-Clearers; n = 579) or had persistent HPV16, 18, or 45 infection (HPV-Persisters; n = 292). CpG site-specific methylation percentages were measured using our reported next-generation methods. The Methylation Score was the average methylation percentage across all 35 CpG sites tested. Each individual CpG site had higher methylation percentages in exfoliated cervicovaginal cells collected from patients with AIS/ADC, and as well as those with CIN3/SCC, relative to either control group (weakest P = .004). The Methylation Score for AIS/ADC had a sensitivity of 74% and specificity of 89%. The multivariate odds ratio (OR) between the Methylation Score (4th vs 1st quartile) for AIS/ADC was ORq4-q1 = 49.01 (PBenjamini-Hochberg = 4.64E-12), using HPV-Clearers as controls. CIN3/SCC had similar, albeit weaker, associations with the Methylation Score. HPV16/18/45-infected women with Methylation Scores in the highest quartile had very high odds of AIS/ADC, suggesting they may warrant careful histologic evaluation of the cervical transition zone (eg, conization or loop electrosurgical excision procedure [LEEP]).

Human Papilloma Virus DNA Detection in Clinical Samples Using Fluorogenic Probes Based on Oligonucleotide Gated Nanoporous Anodic Alumina Films.

In this work, fluorogenic probes based on oligonucleotide capped nanoporous anodic alumina films are developed for specific and sensitive detection of human papilloma virus (HPV) DNA. The probe consists of anodic alumina nanoporous films loaded with the fluorophore rhodamine B (RhB) and capped with oligonucleotides bearing specific base sequences complementary to genetic material of different high-risk (hr) HPV types. Synthesis protocol is optimized for scale up production of sensors with high reproducibility. The sensors' surfaces are characterized by scanning electron microscopy (HR-FESEM) and atomic force microscopy (AFM) and their atomic composition is determined by energy dispersive X-ray spectroscopy (EDXS). Oligonucleotide molecules onto nanoporous films block the pores and avoid diffusion of RhB to the liquid phase. Pore opening is produced when specific DNA of HPV is present in the medium, resulting in RhB delivery, that is detected by fluorescence measurements. The sensing assay is optimized for reliable fluorescence signal reading. Nine different sensors are synthesized for specific detection of 14 different hr-HPV types in clinical samples with very high sensitivity (100%) and high selectivity (93-100%), allowing rapid screening of virus infections with very high negative predictive values (100%).

Diagnostic study of human papillomavirus DNA detection in cervical and vaginal samples using the filter paper card.

To determine the accuracy of high-risk human papillomavirus (HPV) DNA samples on filter paper in comparison to specimen transport medium (STM). This was a cross-sectional diagnostic study of 42 consecutive women who were prospectively recruited. Each had self-collected vaginal samples on filter paper, physician-collected cervical samples on filter paper, and physician-collected cervical samples in STM. HPV DNA testing was performed with a Hybrid Capture 2 system (Qiagen). Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and agreement of filter paper methods with the standard procedure were calculated. The overall prevalence of HPV in STM was 67.5%. Detection of HPV DNA in the physician-collected cervical samples on filter paper had a sensitivity of 77.8%, a specificity of 100%, a PPV of 100%, and an NPV of 68.4%. The patient's self-sampling on filter paper had a sensitivity of 66.7%, a specificity of 100%, a PPV of 100%, and an NPV of 59.1%. The agreement between STM method and physician-collected sample on filter paper was substantial, (κ = 0.695, P < 0.001), while the agreement between STM and self-collected samples on filter paper was moderate (κ = 0.565, P < 0.001). Most patients reported that self-collection was acceptable (100%), painless (95%), and not embarrassing (95%). Filter paper, with dried self-collected vaginal samples, can be used to detect high-risk HPV with acceptable accuracy.

Utilizing first void urine for high-risk HPV testing for cervical cancer screening in HIV-positive women in Katete, Zambia

BackgroundThe World Health Organization targets to screen 70% of women worldwide twice for cervical cancer by the year 2030, first by age of 35, and again by the age of 45. However, with the current low screening coverage in many developing countries, this may not be achieved because the invasive sampling method is unacceptable to some. In Zambia, for instance, despite the availability of free cervical cancer screening through the establishment of the Cervical Cancer Prevention Programme, some women are still reluctant to go for screening. First void urine sampling is non-invasive and thus has the potential to increase screening coverage. We aimed to determine the performance of first void urine for high-risk human papillomavirus DNA detection, the prevalence of high-risk HPV, and the acceptability of first void urine sampling.Materials and methodA comparative cross-sectional study was conducted among 100 HIV- infected women at St Francis’ Hospital in Zambia, attending the routine HIV/AIDS services and cervical cancer screening. 17 mL of first void urine sample collected by each participant was immediately mixed with 3 mL of 0.5 M EDTA preservative solution before cervical sample collection by the clinician. For testing, 2 mL of first void urine and 1 mL of the cervical sample were tested using the GeneXpert platform. An interview-based questionnaire was used to gather data on the acceptability of first void urine sampling. Data was analyzed using Stata version 17.ResultsThe mean age of the participants was 42.58 years (95% CI 40.98–44.19; SD 8.01). High-risk HPV prevalence was 34% (95% CI 24%-43.9%) in both cervical and first void urine samples. Sensitivity and specificity were 84.8% (95% CI 68.1%–94.9%) and 92.3% (83%–97.5%), respectively. There was 89.80% agreement between the samples (κ = 0.77; 95% CI 0.64–0.91). First void urine sampling was highly accepted.ConclusionHigh-risk HPV DNA can be detected in first void urine samples using the GeneXpert, with a substantial agreement with cervical samples. An affordable preservative such as Ethylenediamine tetraacetic acid can prevent DNA degradation. With optimization, first void urine sampling has the potential to increase screening coverage.

Study to Determine Concordance between High-Risk Human Papilloma Virus DNA Detection in Self Collected First Voided Urine Samples and Health-Care Worker Collected Cervical Samples in a Subset of Women with Proven Histopathological Precancerous and Cancerous Lesions of the Cervix.

The objective of our study was to assess whether urinary samples for human papilloma virus (HPV) detection are a good predictive marker of cervical cancerous and precancerous lesions, by comparing against results from cervical scrapings as the gold standard test. The study is a hospital-based cross-sectional study wherein symptomatic women were screened at the colposcopy clinic. Paired samples-cervical scrapings/washings and urine samples were tested for hr-HPV for women who were found to harbor premalignant and malignant lesions of the cervix in histopathological lesions, by multiplex real-time polymerase chain reaction and HPV genotyping. Diagnostic accuracy was tested by calculating concordance with Cohen's kappa with hr-HPV detection in cervical samples as the gold standard. A total of 295 patients undergoing colposcopy were recruited in the study, out of which 54 had histopathological-proven premalignant and malignant lesions of the cervix. Overall, positivity rate in urinary samples for both HPV 16 and 18 combined is 64.81%, whereas for cervical samples is 68.51%. HPV 16 was seen in 30 (55.5%) and 32 (59.3%) cervical and urinary samples, respectively, whereas HPV 18 was seen in 7 (12.9%) and 6 (11.1%) samples, respectively. There was substantial concordance between the cervical samples and first-void urinary samples results with Cohen's k: 0.6988 (95% confidence interval: From 0.507 to 0.891). There was 85.96% agreement among all the tests that were performed with only 14.04% disagreement. The study showed that HPV DNA detection from the urine and cervical samples showed significant agreeability for the detection of precancerous and cancerous lesions of the cervix among women with abnormal histology results. Thus, urinary sampling can be done as a potential replacement for cervical sampling methods with the added benefit as it can be used in females reluctant to provide cervical samples, if there is no availability of skilled workforce for collecting samples, for mass screening, and for the follow-up of vaccination programs.

Multiplexed Detection of Human Papillomavirus Based on AzaBODIPY-Doped Silica-Coated Polystyrene Microparticles

Human papillomavirus (HPV) DNA detection can enable the early diagnosis of high-risk HPV types responsible for cervical cancer. HPV detection is also essential for investigating the clinical behavior and epidemiology of particular HPV types, characterization of study populations in HPV vaccination trials and monitoring the efficacy of HPV vaccines. In this study, two azaBODIPY dyes (1 and 2) were used as references and were doped into polystyrene particles (PS40), while a short HPV DNA single strand was used as a target molecule and was covalently bound to the silica shell. These particles were employed as optical probes in 1:1 hybridization assays, and their potential applicability as a tool for multiplex assays for the detection of different strands of HPV was evaluated using flow cytometry. A good separation in the fluorescence of the four different concentrations prepared for each dye was observed. To perform the hybridization assays, HPV18, HPV16, HPV11 and HPV6 single strands were attached to the particles through EDC-mediated coupling. The c-DNA-1-PS40 and c-DNA-2-PS40 particles exhibited low limit of detection (LOD) and quantification (LOQ) values for HPV11, and a narrow detection range was obtained. Multiplexed assay experiments were successfully performed for both particles, and the results proved that c-DNA-1-PS40 could potentially be used as a tool for multiplexing assays and merits further in-depth study in this context.

High-Risk Human Papillomavirus (HR-HPV) DNA Detection in Mouthwashes for Diagnosis of HPV-Driven Oropharynx Cancer and Its Curative Therapy-A Feasibility Study.

Detection of p16 through immunohistochemistry (IHC) is the standard for determining the HPV status of the tumor according the TNM eighth edition released in 2017 and has become crucial for determining the HPV status of oropharyngeal squamous cell carcinomas (OPSCC) with direct impact on staging and prognostication. In recent years, detection of HPV DNA in mouthwashes has been proposed as a noninvasive alternative, both for OPSCCs and for other head and neck squamous cell carcinomas (HNSCCs). However, the prospect of using the mouthwashes to monitor the response to therapy is unclear. To evaluate the effect of curative therapy on the detection of HPV DNA, we performed a prospective study comparing the detection frequency of high-risk HPV DNA (HR-HPV-DNA) in pre- and post-therapy mouthwashes. We collected 137 mouthwashes from 88 pathologically confirmed HNSCC patients for DNA isolation and HPV genotyping with the Inno-LiPA assay. We show that HPV DNA in pretherapeutic mouthwashes can detect HPV-driven HNSCCs with a sensitivity of 50.0% and specificity of 85.4%, alongside a high negative predictive value of 79.5% and an accuracy of 74.5%. Furthermore, we observed a notable decrease in the detection frequency of HR-HPV-DNA after successful treatment (pre-therapy 50.0% (9/18) versus post-therapy 9.7% (3/28)). However, the comparatively low sensitivity regarding detection of HPV-driven OPSCC argues against its use in clinical routine.

Survival With Cemiplimab in Recurrent Cervical Cancer

(Abstracted from N Engl J Med 2022;386(6):544–555) Despite widespread implementation of cervical cancer screening and prevention programs including cytologic assessment and high-risk human papillomavirus DNA detection as well as effective vaccines, the incidence of cervical cancer remains high. Treatment varies based on stage and grade and involves a combination of surgery, platinum-based chemotherapy, bevacizumab, chemoradiation, and intracavitary brachytherapy.

Clinical Significance of Detection of Human Papilloma Virus DNA and E6/E7 mRNA for Cervical Cancer Patients.

This study aimed to explore the clinical significance of detection of human papillomavirus (HPV) DNA and E6/E7 mRNA for cervical cancer patients. For this purpose, a total of 300 patients with cervical lesions who performed the colposcopy examination for the cervix between January 2018 and January 2020 were divided into three groups according to the level of cervical intraepithelial neoplasia (CIN): Low-level group (Group A, n = 101), high-level group (Group B, n = 149), cervical cancer group (Group C, n = 50) and gynecological inflammation group (Group D, n = 60). Tissue samples were collected from subjects above to perform the immunohistochemistry for E6/E7 protein and detection of HPV DNA and HPV E6/E7 mRNA. The results showed that HPV DNA copies and the positive rates of protein, DNA and mRNA of HPV E6/E7 in Groups A, B and C were significantly higher than those in Group D (all P<0.05), while no significant difference was identified in the comparison of the positive rate and copies of DNA among Group A, B and C (P> 0.05); in the Group A, B and C, patients had a higher positive rate of E6/E7 mRNA than those in the Group D (P<0.05), while in the Group B and C, the positive rate and copies of mRNA of HPV E6/E7 and HPV E6/E7 proteins were all higher than those in the Group A (P<0.05). In addition, the specificity of HPV E6/E7 mRNA was inferior to HPV DNA in the detection of the low-level intraepithelial neoplasia, but it performed better in the detection of the high-level intraepithelial neoplasia and cervical cancer. In general, HPV E6/E7 mRNA shows promising value in the detection of the development and progression of cervical cancer.

ThinPrep cytology combined with HPV detection in the diagnosis of cervical lesions in 1622 patients.

The timely detection of precancerous lesions and early intervention can greatly reduce cervical cancer occurrence. The current study aimed to assess the diagnostic value and accuracy of different methods of cervical lesion screening. A total of 1622 females who visited the Outpatient Department of Xinjiang Uyghur Autonomous Region People’s Hospital between January and December 2018 were consecutively enrolled. All participants underwent separate high-risk human papilloma virus (HR-HPV) DNA detection, ThinPrep cytology testing (TCT) and colposcopic biopsy. Their medical records were retrospectively analyzed. While considering biopsy outcomes as the gold standard, the diagnostic values of TCT, HR-HPV testing, and TCT+HR-HPV testing for cervical cancer screening were compared. The sensitivity, specificity and Youden index of each method were calculated. Among the different methods, TCT+HR-HPV testing had the highest sensitivity (89.8%), followed by TCT (79.9%) and HR-HPV testing (49.2%). The combined method also had the highest Youden value, and its screening outcomes exhibited the highest consistency with those of biopsy. In addition, the combined method had the largest area under the receiver operating characteristic (ROC) curve, which was 0.673 (0.647, 0.699), compared with any other screening method. Compared with TCT or HR-HPV testing alone, TCT+HR-HPV testing serves as a better screening method for cervical cancer and precancerous lesions.

A comparison of high-risk human papillomavirus DNA detection between urine and cervical sample testing in women with abnormal Pap smears.

To compare the clinical performance of high-risk human papillomavirus (hrHPV) DNA detection between urine and cervical samples collected from the same patient for the detection of CIN2+ lesions (high-grade squamous intraepithelial lesions or cervical cancer lesions). The secondary objectives were to evaluate agreement among hrHPV genotypes and to compare patient satisfaction between urine and cervical sample collection. This prospective cross-sectional study enrolled 96 women with abnormal cervical cytology who attended the colposcopy clinic at Siriraj Hospital (Bangkok, Thailand) between July 2016 and January 2017. Self-collected random-voiding and first stream urine samples were collected into a universal sterile urine container and immediately mixing with preservative before the pelvic examination. Cervical tissue sampling was performed according to standard treatment guidelines. Both specimens were sent for extraction and detection of hrHPV by Anyplex II HPV high-risk testing. Study patients were surveyed to compare patient satisfaction between urine and cervical sample collection. Carcinogenic hrHPV positive rate was 73% in urine samples and 81% in cervical samples. The sensitivity for HPV in the detection CIN2+ was high in both the urine and cervical groups at 86.2% and 94.8%, respectively. Agreement between the urine and cervical groups for HPV 16 or 18 detection was high, with kappa values of 0.86 for subtypes 16/18. Urine specimen collection had significantly higher satisfaction and acceptability than cervical specimen collection. Urine hrHPV testing by real-time polymerase chain reaction demonstrated high sensitivity and accuracy for the detection of CIN2+ lesions, with very good agreement when compared with cervical sample testing.

Performance of human papillomavirus DNA detection in residual specimens taken for Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acid amplification testing in men who have sex with men

ObjectivesRectal swab specimens, either alone or pooled with first-void urine (FVU) and pharyngeal swab specimens, are used to test for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infection in men...

Acquired epidermodysplasia verruciformis or generalized verrucosis? A clinical and virological comparative study.

Few articles have described the difference between epidermodysplasia verruciformis (EV) and generalized verrucosis (GV). This study aimed to analyze the clinical findings and virology of the two diseases. The study enrolled patients diagnosed with EV and GV by clinical and histopathological findings. The demographic information, clinical manifestation, treatment, and therapeutic outcome were analyzed. All of the biopsy specimens of enrolled patients were sent for β-human papillomavirus (HPV) DNA detection. A total of 13 cases were included in the present study, including five EV cases and eight GV cases. Legs and feet were significantly affected in GV cases (p=0.035). All acquired EV cases had immunocompromised conditions, while only three GV cases could be identified as having an immunocompromised history. All EV cases were detected with β-HPV infection, especially HPV5, but none of the GV cases were found to be infected with β-HPV. The therapeutic response was refractory and often relapsed after discontinuation of treatment in both groups.

Onclarity Performance in Human Papillomavirus DNA Detection in Formalin-Fixed Paraffin-Embedded Cervical Samples.

Diagnosis of HPV infection is usually performed from cervical liquid-based cytology specimens (LBC), but these often contain a large amount of human papillomavirus (HPV) genotypes, most of which might cause transient infections. The aim of the study was to evaluate the performance of BD Onclarity HPV test genotyping method on formalin-fixed, paraffin-embedded (FFPE) cervical specimens compared with genotyping results from LBC. Formalin-fixed, paraffin-embedded specimens from women surgically treated for cervical intraepithelial lesions (CINs) at the European Institute of Oncology, Milan, from September 2012 to June 2013 were retrieved from the archives of the Department of Pathology of the European Institute of Oncology. The FFPE and LBC specimens were genotyped using the same extended genotyping Onclarity assay. We collected 99 samples (26 CIN 1, 30 CIN 2, and 43 CIN 3+), but 15 were excluded from the analysis: these 84 samples show an overall agreement of 89% for HPV status between FFPE Onclarity samples versus LBC samples. The FFPE and LBC samples showed identical genotype in 75% samples, compatible genotype (at least 1 of the genotypes detected in LBC sample was found in the tissue sample) in 14% specimens, and discrepant genotype in 11% samples. Our data demonstrate a very good concordance between HPV genotypes found in cytological and tissue samples, suggesting that the Onclarity method could also be used to detect HPV in tissue samples and that the HPV genotype detected in FFPE samples is one of the HPV detected in cytological samples, supporting the thesis that one lesion is caused by one HPV genotype.

Detection and quantification of bovine papillomavirus DNA by digital droplet PCR in sheep blood

Highly pathogenic bovine papillomaviruses (BPVs) were detected and quantified for the first time using digital droplet polymerase chain reaction (ddPCR) by liquid biopsy in 103 clinically healthy sheep. Overall, ddPCR detected BPVs in 68 blood samples (66%). BPV infection by a single genotype was revealed in 61.8% of the blood samples, and BPV coinfection by double, triple or quadruple genotypes was observed in 38.2% of liquid biopsies. The BPV-2 genotype was most frequently seen in sheep, whereas BPV-1 was the least common. Furthermore, ddPCR was very useful for detection and quantification; the BPV-14 genotype was observed for the first time in ovine species, displaying the highest prevalence in some geographical areas (Apulia). In 42 of the positive samples (61.8%), a single BPV infection was observed, 26 of which were caused by BPV-2 (61.9%) and 7 by BPV-13 (16.7%). BPV-14 was responsible for 7 single infections (16.7%) and BPV-1 for 2 single infections (4.7%). Multiple BPV coinfections were observed in the remaining 26 positive samples (38.2%), with dual BPV-2/BPV-13 infection being the most prevalent (84.6%). BPV infection by triple and quadruple genotypes was also observed in 11.5% and 3.8% of cases, respectively. The present study showed that ddPCR, a biotechnological refinement of conventional PCR, is by far the most sensitive and accurate assay for BPV detection compared to conventional qPCR. Therefore, ddPCR displayed an essential diagnostic and epidemiological value very useful for the identification of otherwise undetectable BPV genotypes as well as their geographical distributions and suggesting that animal husbandry practices contribute to cross-species transmission of BPVs.

DNA Recovery Using Ethanol-Based Liquid Medium from FTA Card-Stored Samples for HPV Detection

Introduction: Alternative methods of dry storage and transportation may be a viable alternative to the use of liquid storage medium for cervical samples, especially for screening programs in places with few resources. Objective: The objective of this study is to verify the viability and efficacy of human papillomavirus DNA (HPV-DNA) detection in cervical cell samples collected and stored on a Flinders Technology Associates (FTA) card (Whatman Indicating FTA^® Elute Micro Card) and subsequently recovered in ethanol-based liquid medium and to compare the results to those obtained using samples stored directly in ethanol-based liquid medium. Study Design: Thirty-four women submitted to ETZ (excision of the transformation zone of the cervix) were included in this study. Before ETZ, 2 samples of exfoliated cervical cells were collected from each woman by a doctor and stored in ethanol-based liquid medium and on an FTA card. DNA recovery from FTA samples was performed using ethanol-based liquid medium. Detection of HPV-DNA in the samples was performed using the Cobas® 4800 HPV Test Platform. Results and Conclusions: The HPV-DNA detection positivity rates were 70.6% for the samples collected directly in liquid medium and 64.7% for the samples stored on the FTA card, with high detection accuracy in the DNA samples recovered from the FTA card (area under the curve = 0.958; 95% confidence interval = 0.890–1.000). The concordance between the results obtained using the 2 storage media was 94.1% (Kappa = 0.866). These preliminary results suggest that collection of cervical material on an FTA card may be an alternative to storage in liquid medium since the liquid medium has some limitations. In addition, DNA recovery from the card using ethanol-based liquid medium streamlines the workflow in the laboratory and reduces the cost associated with reagents, thereby facilitating access to the HPV test in places with few resources and potentially improving cervical cancer screening.

The diagnostic value of the droplet digital PCR for the detection of bovine deltapapillomavirus in goats by liquid biopsy.

In the present study, the highly pathogenic bovine deltapapillomavirus (δPV) was investigated by liquid biopsy in blood samples of 168 clinically normal goats using both droplet digital PCR (ddPCR) and quantitative real-time PCR (qPCR). Overall, ddPCR discovered BPV E5 DNA in~61.3% of the blood samples examined, while real-time qPCR revealed the virus in~10.7% of the same samples. Moreover, ddPCR showed BPV E5 DNA in~78.8% of blood samples from goats that were in close contact with cattle and in 20% of blood samples from goats living in closed pens without any contact with cattle. In addition, ddPCR unmasked a single BPV genotype in~59.2% and multiple genotypes in~40.8% of goats harbouring BPV DNA, while real-time qPCR detected single genotypes in~17% and multiple genotypes in~1%. Of the BPV co-infections detected by ddPCR, 28 (~67%) involved two genotypes, eight (~19%) three genotypes and six (~14%) four genotypes. In contrast, real-time qPCR revealed BPV co-infection by two genotypes in only one sample and failed to detect co-infection by three or four genotypes. BPV2 and BPV13 were the most prevalent viruses responsible for single and multiple co-infections, respectively. The present study showed that ddPCR is promising method for circulating bovine papillomavirus DNA detection and quantification and suggested that animal husbandry practices contribute to cross-species transmission of BPVs.

Human Papillomavirus DNA Detection by Droplet Digital PCR in Formalin-Fixed Paraffin-Embedded Tumor Tissue from Oropharyngeal Squamous Cell Carcinoma Patients.

High-risk human papillomavirus infection impacts staging and prognosis of oropharyngeal squamous cell carcinomas (OPSCCs). Determination of HPV status in tumor tissue by p16-immunohistochemistry (p16-IHC) can be challenging; therefore, complementary methodologies could be useful in a clinical setting. To test for accuracy and clinical relevance of HPV-DNA detection in formalin-fixed and paraffin-embedded (FFPE) tumor samples by droplet digital PCR (ddPCR). Fifty OPSCCs were tested for p16-IHC status followed by HPV-16/18 DNA detection/quantification in FFPE-recovered DNA using ddPCR. Accuracy for HPV status determination and association with patient information were also evaluated. 32.0% (16/50) of the cases were p16-IHC positive (p16 +), 42.0% (21/50) had detectable levels of HPV-16 DNA, and none were positive for HPV-18 DNA. A higher median viral load of HPV-16 DNA was observed in p16 + cases (p <0.0001). Concordance between p16-IHC and HPV-16 DNA ranged from 78.0 to 86.0% and accuracy rates were between 78.0 and 86.0%. P16-IHC and HPV-16 DNA detection was associated with gender, smoking status, and tumor subsite, while only HPV-16 DNA was associated with cT stage. The combination of HPV positivity by p16-IHC and ddPCR showed higher overall survival rates in comparison with p16 + /HPV-DNA- and p16 - /HPV-DNA- results. Type-specific HPV-DNA detection by ddPCR is highly specific but moderately sensitive for the determination of HPV status and showed clinical relevance, mainly when associated with p16-IHC status. Results highlight the importance of performing HPV-DNA testing in combination with p16-IHC for proper identification of HPV-associated OPSCC and to improve clinical management of OPSCC patients.

The Prevalence of Thrice, Twice, and Once Human Papillomavirus DNA Positivity in Older Chinese Women.

Background: Human papillomavirus (HPV) DNA detection in cervical samples is widely used to identify HPV infection; however, there is little detailed evaluation of the characteristics of HPV prevalence by repeated DNA detection in community populations.Methods: Beginning in 2014, a Papanicolaou (Pap) smear and HPV cotesting program was implemented among older women living in the Minhang district of Shanghai. This report uses information from 225,000 participants, who provided person-time data. Of these, 632 subjects had 3 repeated visits and at least one HPV DNA-positive result in the last 5 years.Results: All 16 genotypes of HPV displayed thrice, twice, and once positivity results in 3 repeated tests and differed by proportions among and within genotypes. HPV52 and 58 are the two most dominant genotypes in total and in thrice positive person-time. The thrice positive person-time exceeded 50% in each of HPV58-, 35-, 52-, 56-, 18-, 68-, 31-, and 16-infected women. The single positive person-time ratio ranged from 7.9% (HPV35) to 38.9% (HPV11). Age differed among and within genotypes in thrice, twice, and once positive women. The average age of HPV-free controls was 59.0 ± 7.2 yo, which is close to the median of average ages for thrice and twice positive women and is older than most average ages for once positive women. The percentages of negative results for intraepithelial lesion or malignancy (NILM) for thrice HPV52-, 58-, 16-, 56-, and 59-positive women were significantly lower than the percentage of NILM for HPV-free women.Conclusion: Thrice and/or twice HPV DNA positivity are common in HPV-infected women and tend to occur in older women.