Development and validation of a wart-associated human papilloma virus genotyping assay for detection of HPV in cutaneous warts.

Cutaneous warts are infectious disorders caused by human papillomavirus (HPV). A recent study revealed that the HPV genotype influences the natural course and response to treatment for plantar warts, suggesting that HPV genotyping could potentially be used to optimize wart treatment schemes. For this purpose, a wart-associated HPV genotyping assay was developed. The assay was subjected to an intensive validation process including, i.a., empiric determination of the annealing temperature, primer-probe optimization, evaluation of the analytical specificity and sensitivity, viral load quantification, and qualitative as well as quantitative analysis of intra-run repeatability and inter-run reproducibility. The newly developed assay was employed in a small-scale HPV genotyping study of wart biopsies (n = 50). The assay exhibited an analytical type-specific sensitivity and specificity of 100% (95% confidence interval [CI]: 83.9%-100%). The limit of quantification of the tested sequences corresponded to less than 17 viral copies/µl, while the limit of detection was less than 5 copies/µl. Very good to excellent agreements were gained between intra- and inter-run measurements (κ = 0.85-1.00) and coefficients of variation of the quantitative agreements were less then 3%. 22.5% (95% CI: 11%-39%) of the analyzed biopsies were negative for the tested HPV types, while 35% (95% CI: 21%-52%) contained multiple infections. The wart-associated HPV quantitative polymerase chain reaction assay was proven to be highly sensitive and specific. Multiple HPV infections were detected in 35% of lesions, contradicting the current literature claiming that in immunocompetent patients only 4%-16% of warts exhibit multiple HPV infections. This assay is qualified to be implemented in development of future genotype specific wart treatment strategies.

High-risk human papillomavirus detection in self-collected vaginal samples compared with healthcare worker collected cervical samples among women attending gynecology clinics at a tertiary hospital in Pretoria, South Africa.

BACKGROUND: In 2017, the South African National Department of Health (NDoH) Cervical Cancer Prevention and Control Policy was revised. Human papillomavirus (HPV) testing on self-collected samples may offer improved screening uptake. The objectives of the study were to compare the positivity of high-risk (hr)-HPV deoxyribonucleic acid (DNA) and hrHPV viral messenger ribonucleic acid (mRNA) between healthcare worker-collected cervical and self-collected vaginal samples and investigate the accuracy of the applicator-tampon-based self-collected samples in detecting hrHPV DNA and hrHPV mRNA. METHODS: A total of 527 women aged 18 years and older and seeking gynecology services at a tertiary hospital in Pretoria, South Africa, were enrolled. Vaginal samples were self-collected using SelfCerv applicator tampon, followed by cervical samples collected by a healthcare worker using a Cervex Brush® Combi. Both samples were tested with the Abbott m2000 analyzer for 14-hrHPV types and 285 paired samples were tested for hrHPV E6/E7 mRNA using the Aptima HR-HPV mRNA assay. The prevalence of hrHPV DNA and hrHPV E6/E7 mRNA was estimated and the positivity between the two collection methods was compared for the total group as well as per age group. RESULTS: HrHPV prevalence was 48.0% (95% CI 43.7-52.4) among healthcare worker collected samples and 47.6% (95% CI 43.3-52.0) among self-collected samples. There was no difference in positivity between healthcare worker collection (48.0%) and applicator-tampon-based self-collection, 47.6% (p-value = 0.90). The proportions of hrHPV were equal between the age groups as shown by the McNemar test (p = 0.9036) results for correlated proportions. The prevalence of hrHPV mRNA was 78.6% (95% CI 73.4-83.2) and 58.6% (95% CI 52.6-64.4) for healthcare worker- and self-collection, respectively. The McNemar test for correlated proportions was highly significant (p < 0.0001), indicating that the hrHPV mRNA proportions are not comparable, although this differed between age groups. CONCLUSIONS: Applicator-tampon-based self-collection has a comparable hrHPV DNA positivity rate as healthcare worker collection but different positivity rates for hrHPV mRNA. Self-sampling showed high concordance with healthcare worker-collected sampling for hrHPV DNA detection, especially regarding HPV 16/18 detection. HrHPV DNA was equally detected between the total group as well as per age group. Implementation of self-sampling using an applicator tampon as a primary screening tool may be considered.

A randomized, controlled trial of two strategies of offering the home-based HPV self-sampling test to non- participants in the Flemish cervical cancer screening program.

We conducted a randomized, controlled trial to evaluate different strategies of offering an HPV-self sampling program, and compared this with two control groups. All total of 35,354 women who did not participate in the Flemish cancer screening program were included in the study: 9,118 received a HPV self-collection brush (RIATOL qPCR HPV genotyping test (qPCR [E6/E7]); 9,098 were offered the opportunity to order an HPV-selfsampling brush, 8,830 received the recall letter; 8,849 received no intervention. Within 12 months after the mailing, 18.7% of the women who had received the brush, participated by returning a self-sample sample, while 10.6% women allocated to the opt- in group did so. 10.5% women who received the standard recall letter, had a PAP smear taken within a period of 12 months; while 8% women did so without receiving an intervention at all. Participation in postmenopausal women was higher than in women younger than 50 in both self-sampling arms. Screening by means of the self-sample kit increased by age, contradictory when screening is performed by a PAP smear. Of those testing hrHPV positive (9.5%), 88.9% attended for follow up cytology. The mean DNA concentration, found in the self-sampler, decreased by age, causing a higher number of inconclusive results. Our results support the efficacy of a self-sampling strategy to increase participation in the Flemish screening program. Self-sampling seems particularly acceptable to postmenopausal non-responders. Future research should focus on the performance of different self-sampling devices in post-menopausal women as low DNA concentrations exponentially increased over age.

Validation of intra- and inter-laboratory reproducibility of the Xpert HPV assay according to the international guidelines for cervical cancer screening.

BACKGROUND: Cervical cancer screening with assays detecting DNA of high-risk human papillomavirus (hrHPV) types is more effective than cytology-based screening. This study completes the diagnostic accuracy assessment conducted previously within the framework of VALGENT-2 (Validation of HPV genotyping Tests) and aims to determine whether the reproducibility of Xpert HPV is in line with international validation criteria. METHODS: Validation of new hrHPV DNA assays requires demonstration of good reproducibility and non-inferior clinical accuracy for cervical precancer compared to a standard comparator assay. The international reproducibility criteria are: lower bound of 95% confidence interval of the intra- and inter-laboratory agreement regarding detection of high-risk HPV DNA exceeding 87% with kappa ≥0.5. RESULTS: The Xpert HPV assay showed high intra-laboratory reproducibility with an overall positivity/negativity agreement of 96.9% and a kappa of 0.925. Inter-laboratory testing showed an agreement of 97.8% with a kappa of 0.948. CONCLUSIONS: The Xpert HPV assay fulfills the HPV test reproducibility criterion requirement for use in cervical cancer screening.

Prevalence of high-risk human papillomavirus and abnormal pap smears in female sex workers compared to the general population in Antwerp, Belgium.

BACKGROUND: Although female sex workers (FSWs) are a well-known high-risk group for Human Papillomavirus (HPV) infections, few tailored intervention programmes for HPV have been established worldwide. The lack of reliable data on the prevalence of HPV and related cervical lesions hampers the establishment of evidence-based intervention programmes. The objectives of this study were to describe the prevalence of high-risk Human Papillomavirus (hrHPV) infections and abnormal pap smears in FSWs compared to a control group in Antwerp, Belgium. METHODS: HPV genotyping and cytology data were analysed from routine Pap smear tests that were collected from both FSWs and the general population (1334 samples for each group) between June 2006 and June 2010. Within the laboratory database, all FSWs were matched 1:1 for age and testing date to determine the ORs of hrHPV genotypes, DNA and cytology outcome. RESULTS: The prevalence of hrHPV DNA in FSWs was 41.7 % compared to 19.8 % in the age-matched controls with an overall OR of 2.8 (95 % CI: 2.3-3.4). Significant differences were observed in all age groups, and the most significant differences were observed in the cohort under 21 years of age (prevalence of 64.4 % in FSWs versus 14.8 % in controls; OR 10.3 (95 % CI: 5.0-21.2). Significantly more cervical lesions were observed in FSWs, particularly in the 17- to 21-year old age group (OR for LSIL or HSIL: 10.3 (95 % CI: 3.2-33.8). In both groups, HPV 16 was the most prevalent at 12.1 and 6.6 % in the FSW and control groups, respectively. HPV 18 was the 8(th) and 7(th) most frequent genotype at 5.0 and 2.5 % in the FSW and control groups, respectively. CONCLUSIONS: FSWs have a significantly higher prevalence of hrHPV and more abnormal Pap smears than does the general population in Antwerp, Belgium. The hrHPV prevalence in FSWs is similar to that reported in the literature. The need for tailored intervention programmes should be investigated further.

Laser micro-dissection and qPCR for identifying specific HPV types responsible for malignancy in penile lesions.

The aim of the study was to identify specific human papillomavirus (HPV) type responsible for malignancy in penile tissue samples using laser micro-dissection and TaqMan quantitative real-time PCR (qPCR). The study was based on two pre-malignant and seven malignant penile tissue samples and laser micro-dissection was performed on all. Genotyping was performed on whole tissue sections and laser micro-dissection samples using qPCR. Two whole tissue section samples were HPV negative while seven were HPV positive. In four samples that were single HPV infections with whole tissue section PCR, identical HPV types were confirmed with laser micro-dissection PCR. Clearly confirming that the single HPV type detected is responsible for malignancy. In two samples that had multiple HPV infections with whole tissue section PCR, only one HPV type with the highest viral load was detected with laser micro-dissection PCR, suggesting that the HPV type with the highest viral load is most likely the cause of that particular lesion. HPV 11 and/or HPV 16 were the only types detected with laser micro-dissection PCR in these cases, compared to multiple HPV types (HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, and HPV 39) initially detected with whole tissue section PCR. HPV 11 was associated with verrucous lesions while HPV 16 was associated with squamous cell carcinoma and PIN 3 lesions. This study confirms that laser micro-dissection and qPCR are essential tools in identifying the HPV types responsible for malignancy in penile lesions, particularly in samples with multiple infections.

Detection, genotyping and quantitation of multiple hpv infections in south African women with cervical squamous cell carcinoma.

In Africa, data is limited on quantitation of human papillomavirus (HPV) types in women with multiple infections. This study applied a real time PCR (qPCR) assay for detection, genotyping and quantitation of multiple HPV infections in 90 tissue blocks of South African women with cervical squamous cell carcinoma. One sample with multiple HPV types was subjected to laser micro-dissection and qPCR. Four samples were negative for ß-globin and these were excluded from the analysis. The HPV DNA positivity rate was 93.0% (80/86). All 80 positives showed the presence of HR HPV types; HPV 68 was the only type negative in all the samples. Overall, HPV 16 was positive in most of the samples (88.8%), followed by HPV 56 (28.7%), HPV 18 (20.0%) and HPV 39 (18.7%). More than half of the samples (65.0%) had multiple infections. HPV 16 was present in majority of single (85.7%) and multiple infections (90.4%). HPV 16 showed higher viral loads in 70.3% of the HPV 16 co-infected samples. In one multiple infected sample laser micro-dissection and qPCR identified HPV 18 with higher viral load as the most likely cause of the invasive lesion. There is large number of multiple HPV infections in South African women with cervical squamous cell carcinoma. HPV 16 is the most frequently detected type and often presents with higher viral load, suggesting it could be responsible for pathogenesis of the lesions in the majority of cases.

Prevalence and viral load of 51 genital human papillomavirus types and three subtypes.

Of the 120 known human papillomaviruses (HPV), 51 HPV types infect the genital mucosa. Very little is known about the prevalence and viral load of the majority of these low-risk (Lr-) HPV types in screening populations. We determined the prevalence of 51 HPV types and three subtypes in 999 consecutive BD-SurePath™ liquid-based cervical cytology samples collected during routine gynecological health checks from Belgian women. This series of screening samples was enriched with ASC-US (n = 100), low-grade squamous intraepithelial lesion LSIL (n = 100) and high-grade squamous intraepithelial lesion (HSIL) (n = 97) and analyzed by BSGP5+/6+-PCR/MPG assay for 51 HPV types and three subtypes. In consecutive screening samples, any of the 54 genital HPV (sub)types was found in 37.1%; Hr-HPV types were detected more frequently (26.8%) than the 31 Lr-HPV types (16.4%) and the six possibly high-risk types (6.6%). High viral load infections were present in 17.0% of the screening population. Among the women with cytological abnormalities, the prevalence of high viral loads of Hr-HPV types increased from negative for intraepithelial lesion or malignancy (NIL/M) over ASC-US, LSIL to HSIL (5.3, 47.1, 84.2 and 91.8%, respectively). The prevalence of possibly Hr and Lr-HPV types increased from NIL/M to LSIL but declined to HSIL. From NIL/M to HSIL, Hr-HPV infections showed an increasing frequency of high viral loads compared to total DNA positivity, but the increase between LSIL and HSIL was small. Type-specific analyses revealed substantial differences between individual HPV types in these groups. Our study provides quantitative data for the whole spectrum of genital HPV in a Belgian screening population and in a representative set of women with cervical abnormalities.

Multiple human papillomavirus infections with high viral loads are associated with cervical lesions but do not differentiate grades of cervical abnormalities.

Multiple human papillomavirus (HPV) genotypes often coexist within cervical epithelia and are frequently detected together in smears of different grades of cervical neoplasia. Describing the association between multiple infections and cervical disease is important in generating hypotheses regarding its pathogenesis. We analyzed the prevalence of multiple HPV infections and their attribution to cervical disease in a screening population of 999 consecutive BD SurePath liquid-based cervical cytology samples enriched with atypical squamous cells of undetermined significance (ASCUS) (n = 100), low-grade squamous intraepithelial lesions (LSIL) (n = 100), and high-grade squamous intraepithelial lesions (HSIL) (n = 97). HPV genotyping was performed only on cytology specimens using a broad-spectrum GP5(+)/6(+)-PCR/multiplex HPV genotyping (BSGP5(+)/6(+)-PCR/MPG) assay that detects and quantifies 51 HPV genotypes and 3 subtypes. Using a recently defined high viral load cutoff, the quantitative data were scored as high or low viral load. In the 36-month follow-up, 79 histologically confirmed cervical intraepithelial neoplasia grade 2 or greater (CIN2+) cases were identified. In the screening population, there was a trend of having more multiple infections at a younger age. Multiple HPV infections were common. Multiple HPV types were most prevalent in LSIL (75.9% of HPV positives), followed by HSIL (65.5%), ASCUS (64.6%), and negative for intraepithelial lesion or malignancy (NILM) (36.8%). On average, 3.2 and 2.5 HPV types were detected per LSIL and HSIL sample, respectively. Multiple HPV types with high viral loads were most prevalent in LSIL (62.6% of high viral load positives), followed by HSIL (51.9%), ASCUS (40.7%), and NILM (19.3%). Patients with multiple high viral loads showed a 4- to 6-fold-higher risk of having cervical precancerous cytological lesions than did patients with single high viral loads. Compared to NILM, multiple infections, especially with multiple high viral loads, were significantly associated with cytological precancerous lesions. However, the presence of multiple infections did not distinguish low-grade from high-grade cytological lesions.

Evolution of human papilloma virus prevalence in a highly vaccinated region in Belgium: a retrospective cohort study in Flemish women (2010-2019).

OBJECTIVE: In order to lower the incidence of cervical cancer, vaccines against high-risk types of the human papilloma virus (hrHPV) were approved and brought on the market in 2007, with a partial reimbursement for Belgian citizens younger than 18 years old. Since 2010, a school-based vaccination program ensures a high vaccination coverage in young women. In this study, the impact of the Belgian vaccination program on the prevalence of HPV 16/18 is studied, together with the evolution of the prevalence of other hrHPV types and precancerous lesions. METHODS: Results of HPV typing and cytology in papanicolaou-smears from women aged 20-23 years taken between 2010 and 2019 were used. An older, nonvaccinated group of women of 40-45 years old served as a control group. RESULTS: A significant decrease in prevalence of HPV types 16 and 18 was found in the 20-23-years-old women, whereas no decrease was found in the age group 40-45. Alongside this decrease, a significant decrease in prevalence of subtypes 6, 11 and 31 was observed, whereas type 31 is not included in the administered vaccines. Remarkably, there was no decrease in prevalence of cytological abnormalities in the study group during this study. There was even an increase in prevalence of high-risk types 53, 58 and 67. CONCLUSION: These findings emphasise the need to maintain the screening programs, even in areas with high vaccination coverage.

Changes in type-specific human papillomavirus load predict progression to cervical cancer.

Persistent high-risk human papillomavirus (HPV) infection is strongly associated with the development of high-grade cervical intraepithelial neoplasia or cancer (CIN3+). However, HPV infection is common and usually transient. Viral load measured at a single time-point is a poor predictor of the natural history of HPV infection. The profile of viral load evolution over time could distinguish HPV infections with carcinogenic potential from infections that regress. A case-cohort natural history study was set-up using a Belgian laboratory database processing more than 100,000 liquid cytology specimens annually. All cytology leftovers were submitted to real-time PCR testing identifying E6/E7 genes of 17 HPV types, with viral load expressed as HPV copies/cell. Samples from untreated women who developed CIN3+ (n = 138) and women with transient HPV infection (n = 601) who contributed at least three viral load measurements were studied. Only single-type HPV infections were selected. The changes in viral load over time were assessed by the linear regression slope for the productive and/or clearing phase of infection in women developing CIN3+ and women with transient infection respectively. Transient HPV infections generated similar increasing (0.21 copies/cell/day) and decreasing (-0.28 copies/cell/day) viral load slopes. In HPV infections leading to CIN3+, the viral load increased almost linearly with a slope of 0.0028 copies/cell/day. Difference in slopes between transient infections and infections leading to CIN3+ was highly significant (P < .0001). Serial type-specific viral load measurements predict the natural history of HPV infections and could be used to triage women in HPV-based cervical cancer 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.

Evaluation of Urine and Vaginal Self-Sampling versus Clinician-Based Sampling for Cervical Cancer Screening: A Field Comparison of the Acceptability of Three Sampling Tests in a Rural Community of Cuenca, Ecuador.

Self-sampling methods for HPV testing have been demonstrated to be highly sensitive and specific. The implementation of these methods in settings with a lack of infrastructure or medical attention has been shown to increase the coverage of cervical cancer screening and detect cervical abnormalities in the early stages. The aim of this study is to compare the acceptability of urine and vaginal self-sampling methods versus clinician sampling among rural women. A total of 120 women participated. Each participant self-collected urine and vaginal samples and underwent clinician sampling for Pap smear and HPV testing. After the sample collection, a questionnaire to qualify the device, technique, and individual acceptability was applied, and the additional overall preference of three sample tests was evaluated. Results: The characteristics of the participants were as follows: median age of 35 years; 40.8% were married; 46.7% had a primary level of education; median age of sexual onset of 17.6 years. Compared with clinician sampling, both vaginal self-sampling, OR 20.12 (7.67-52.8), and urine sampling, OR 16.63 (6.79-40.72), were more comfortable; granted more privacy: vaginal self-sampling, OR 8.07 (3.44-18.93), and urine sampling, OR 19.5 (5.83-65.21); were less painful: vaginal self-sampling, OR 0.07 (0.03-0.16), and urine sampling, OR 0.01 (0-0.06); were less difficult to apply: vaginal self-sampling, OR 0.16 (0.07-0.34), and urine sampling, OR 0.05 (0.01-0.17). The overall preference has shown an advantage for vaginal self-sampling, OR 4.97 (2.71-9.12). No statistically significant preference was demonstrated with urine self-sampling versus clinician sampling. Conclusions: Self-sampling methods have a high acceptance in rural communities. Doubts on the reliability of self-sampling often appear to be a limitation on its acceptability. However, the training and education of the community could increase the uptake of these methods.

Type-specific HPV geno-typing improves detection of recurrent high-grade cervical neoplasia after conisation.

The aim of this case-control study was to examine if type-specific human papillomavirus (HPV) DNA geno-typing before and after treatment of high-grade cervical intra-epithelial neoplasia (CIN) improves prediction of recurring or persisting CIN 2 or 3 compared with follow-up cytology or high-risk (hr)HPV testing. Women with biopsy-proven recurrence of CIN 2 or 3 (cases) in a follow-up period of at least 24 months after treatment of high-grade CIN were compared with women without recurrence (controls). These cohorts were identified by a database search of the Riatol Laboratoria (Antwerp, Belgium). In a cohort of 823 women treated with conisation for high-grade CIN between January 2001 and December 2007, 21 patients with a histologically proven recurrence of CIN2+ were identified. A group of women (n=42) from the same cohort without recurrence was randomly chosen. We found that hrHPV testing at 6 months post-treatment is significantly more sensitive compared with follow-up cytology (ratio: 1.31, 95% confidence interval (CI): 1.10-1.54), but less specific (ratio: 0.85, 95% CI: 0.81-0.90) to predict failure of treatment. When compared with hrHPV testing, HPV geno-typing is more efficient (equal sensitivity, but higher specificity, ratio: 1.43, 95% CI: 1.280-1.62). When compared with follow-up cytology, HPV geno-typing is more sensitive (ratio: 1.31, 95% CI: 1.10-1.54) and more specific (ratio: 1.22, 95% CI: 1.14-1.36). All women who developed a recurrence tested positive for hrHPV. The negative predictive value in the absence of hrHPV DNA was 100%. Six months after treatment HPV geno-typing is the most sensitive and specific method to predict recurrent or persistent CIN 2-3 in the next 24 months.

Prior knowledge of HPV status improves detection of CIN2+ by cytology screening.

OBJECTIVE: The objective of the study was to investigate whether knowledge of human papillomavirus (HPV) deoxyribonucleic acid test results increases sensitivity of guided cytology screening for the detection of cervical intraepithelial neoplasia (CIN)-2 or higher-grade cervical lesions. STUDY DESIGN: This was a prospective colposcopy-controlled study of 2905 BD SurePath samples to identify cases with CIN2+ within a 24 month follow-up period. Sensitivity and specificity to detect CIN2+ was evaluated, comparing guided cytology screening with and without prior knowledge of HPV status. RESULTS: Prior knowledge of HPV status resulted in significantly higher detection rate of CIN2+ compared with screening blinded to HPV status (P = .005) with limited loss of specificity (P = .026). Gain in sensitivity is higher in older women (43.8%, P = .008) vs in younger women (10.2%, P = .317), whereas loss of specificity is more pronounced in younger women (P < .001) vs older women (P = .729). CONCLUSION: Guided cytological screening performed with prior knowledge of HPV status results in an improved detection of CIN2 or higher-grade lesions.

High-throughput detection, genotyping and quantification of the human papillomavirus using real-time PCR.

BACKGROUND: The establishment of the causal relationship between high-risk human papillomavirus (HR-HPV) infection and cervical cancer and its precursors has resulted in the development of HPV DNA detection systems. Currently, real-time PCR assays for the detection of HPV, such as the RealTime High Risk (HR) HPV assay (Abbott) and the cobas® 4800 HPV Test (Roche Molecular Diagnostics) are commercially available. However, none of them enables the detection and typing of all HR-HPV types in a clinical high-throughput setting. This paper describes the laboratory workflow and the validation of a type-specific real-time quantitative PCR (qPCR) assay for high-throughput HPV detection, genotyping and quantification. This assay is routinely applied in a liquid-based cytology screening setting (700 samples in 24 h) and was used in many epidemiological and clinical studies. METHODS: The TaqMan-based qPCR assay enables the detection of 17 HPV genotypes and ß-globin in seven multiplex reactions. These HPV types include all 12 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), three probably high-risk types (HPV53, 66 and 68), one low-risk type (HPV6) and one undetermined risk type (HPV67). RESULTS: An analytical sensitivity of ≤100 copies was obtained for all the HPV types. The analytical specificity of each primer pair was 100% and an intra- and inter-run variability of <6.4% was observed. CONCLUSIONS: The type-specific real-time PCR approach enables detection of 17 HPV types, identification of the HPV type and determination of the viral load in a single sensitive assay suitable for high-throughput screening.

Evaluation of ILEX SelfCerv for Detection of High-Risk Human Papillomavirus Infection in Gynecology Clinic Attendees at a Tertiary Hospital in South Africa.

BACKGROUND: The SelfCerv Self-Collection Cervical Health Screening Kit (Ilex Medical Ltd., Johannesburg, South Africa) is an applicator tampon designed for self-collection of vaginal samples for the detection of human papillomavirus (HPV) deoxyribonucleic acid (DNA) and E6/E7 messenger ribonucleic acid (mRNA). The study aimed to evaluate the performance of the SelfCerv applicator tampon for the detection of hr-HPV for cervical cancer screening, and further to investigate women's experiences and preferences regarding self-sampling. METHODS: Vaginal samples were collected from 527 gynecology clinic attendees aged ≥18 years at a tertiary hospital in Gauteng Province, South Africa. Self-samples were collected using the SelfCerv kit, followed by endocervical samples collected by a healthcare professional using Cervex-Brush® Combi. Participants completed a self-administered questionnaire on self-sampling experiences and preferences. Both samples were tested for 14 high-risk (hr) HPV types and E6/E7 mRNA using the Abbott RealTime HR-HPV and Aptima HR-HPV mRNA assays, respectively. RESULTS: The overall agreement for hr-HPV typing between 527 paired samples was good (87.1%; κ =0.74) with high sensitivity (86.2%) and specificity (88.0%). HPV-16 (96.4%; κ = 0.83) had higher agreement rate than HPV-18 (96.8%; κ = 0.72) and the other 12 hr-HPVs (86.5%; κ = 0.72). Two hundred and eighty-five (285) sample pairs tested for E6/E7 mRNA showed fair agreement (70.2%; κ= 0.34). Furthermore, self-sampling was reported as comfortable (90.5%) and painless (86.7%), with 88.4% of women preferring self-collection. CONCLUSIONS: Self-collected samples had good agreement with the healthcare professional-collected samples for the detection of hr-HPV DNA and the procedure was highly preferred by women. Self-sampling using SelfCerv can be used as an alternative to healthcare professional sampling in clinic-based routine cervical cancer screening.

Nucleic acid sequence-based amplification assay for human papillomavirus mRNA detection and typing: evidence for DNA amplification.

Human papillomavirus (HPV) E6/E7 mRNA has been proposed as a more specific marker for cervical dysplasia and cancer than HPV DNA. This study evaluated the RNA specificity of nucleic acid sequence-based amplification (NASBA)-based HPV detection using HPV DNA plasmids (HPV type 16 [HPV16], HPV18, HPV31, HPV33, and HPV45) and nucleic acid extracts of several cell lines, which were systematically subjected to enzymatic treatments with DNase and RNase. HPV plasmid dilutions (10(6) to 10(0) copies/microl) and nucleic acid extracts (total DNA, RNA-free DNA, total RNA, and DNA-free RNA) of unfixed and fixed (PreServCyt and SurePath) HaCaT, HeLa, and CaSki cells were tested with the NucliSENS EasyQ HPV test. The RNA-free DNA extracts of HeLa and CaSki cells could be amplified by HPV18 and -16 NASBA, respectively. Fixation of the cells did not influence NASBA. All HPV plasmids could be detected with NASBA. Based on the plasmid dilution series, a lower detection limit of 5 x 10(3) HPV DNA copies could be determined. Our study identified viral double-stranded DNA as a possible target for NASBA-based HPV detection. The differences in diagnostic accuracy between the NASBA-based tests and conventional HPV DNA detection assays seem to be attributable not to the more specific amplification of viral mRNA but to the limited type range and the lower analytical sensitivity for HPV DNA.

Efficacy of AV2-Salicylic acid combination therapy for cutaneous warts: Study protocol for a single-center randomized controlled trial.

Cutaneous warts comprise an extremely common condition caused by infection with the human papillomavirus (HPV). Although most verrucae will disappear spontaneously, many patients do seek treatment. Current wart treatments do not target the cause of the lesion directly, resulting in variable treatment efficacies and high wart recurrence rates. AV2 is a broad-spectrum antiviral drug, that is capable of deactivating HPV. It is however not able to destruct the already infected cells, which raises the need for an additional ablative treatment i.e. salicylic acid (SA). Implementation of AV2-Salicylic acid (AV2-SA) combination therapy would ensure permanent lesion clearance by on the one hand inactivation of HPV by AV2, and on the other hand elimination of the lesion by SA treatment. The primary aim of this study is to assess the efficacy of AV2-SA treatment versus standard SA treatment, by comparing cure and recurrence rates of cutaneous warts between the two treatment groups (at 12 weeks and six months after randomization). The second aim is to assess the safety and tolerability of AV2-SA therapy. The third aim is to identify subgroups of cutaneous warts that have favorable response to treatment, by comparing cure rates in an HPV genotype-specific manner. This randomized controlled trial will enroll 260 participants with cutaneous warts who will either receive the AV2-SA combination therapy or SA control treatment. Real time monitoring will be possible by daily photographs sent via WhatsApp™ (a messaging application) as well as online follow-up questionnaires administered on several occasions. HPV genotyping will be performed on swab self-samples.

Human Papillomavirus Prevalence in Oral and Oropharyngeal Rinse and Gargle Specimens of Dental Patients and of an HIV-Positive Cohort from Pretoria, South Africa.

INTRODUCTION: Studies on HPV prevalence in the head and neck region of South Africans are sparse. Of the available reports in the literature, there were no studies on the association between HPV-DNA presence in the mouth and oropharynx in relation to high-risk behaviours such as oral sex practice or tobacco and alcohol use. MATERIALS AND METHODS: Following ethical clearance and informed consent, patients attending a regional HIV-management clinic and patients attending a dental hospital were recruited to this study. The participants completed an interview-based questionnaire obtaining demographic information, data on HIV serostatus, and behavioural data including sexual practices and tobacco and alcohol use, and a rinse-and-gargle specimen was taken. Specimens were analysed for HPV DNA on 3 separate PCR/qPCR platforms. Statistical analyses were performed for associations between the study group and categorical variables, HPV status, and data from the questionnaires. RESULTS: Of 221 participants, 149 were from a general population and 72 from the HIV-management clinic. Smokers comprised 29.4% of the sample, and 45.2% of participants reported to have ever used alcohol. Open mouth kissing during teenage years was confirmed by 64.7% of participants, 40.3% have given oral sex with their mouth, and 44.8% confirmed to have received oral sex from their partner's mouth. Seven participants (3.2%) had detectable α-HPV DNA, and 1 (0.4%) had detectable ß-HPV DNA in their rinse-and-gargle specimens. Two participants were from the HIV-management clinic and 6 from the general dental population (overall 3.6%). CONCLUSION: Five high-risk HPV, 2 low-risk HPV, and one ß-HPV types were detected. The low prevalence of 3.6% compares well to similar studies in different cohorts studied in South Africa and falls within the global oral/oropharyngeal prevalence spectrum. Only 4 participants, all from the HIV-management clinic, had palatine tonsils. No significant relationships were found between HPV presence and demographic data or sexual, oral sexual, tobacco use, or alcohol use, and no associations were seen with numbers of sexual and oral-sex partners.