Human papillomavirus negative high grade cervical lesions and cancers: Suggested guidance for HPV testing quality assurance.

BACKGROUND: Some high-grade cervical lesions and cervical cancers (HSIL+) test negative for human papillomavirus (HPV). The HPV-negative fraction varies between 0.03 % and 15 % between different laboratories. Monitoring and extended re-analysis of HPV-negative HSIL+ could thus be helpful to monitor performance of HPV testing services. We aimed to a) provide a real-life example of a quality assurance (QA) program based on re-analysis of HPV-negative HSIL+ and b) develop international guidance for QA of HPV testing services based on standardized identification of apparently HPV-negative HSIL+ and extended re-analysis, either by the primary laboratory or by a national HPV reference laboratory (NRL). METHODS: There were 116 initially HPV-negative cervical specimens (31 histopathology specimens and 85 liquid-based cytology samples) sent to the Swedish HPV Reference Laboratory for re-testing. Based on the results, an international QA guidance was developed through an iterative consensus process. RESULT: Standard PCR testing detected HPV in 55.2 % (64/116) of initially "HPV-negative" samples. Whole genome sequencing of PCR-negative samples identified HPV in an additional 7 samples (overall 61.2 % HPV positivity). Reasons for failure to detect HPV in an HSIL+ lesion are listed and guidance to identify cases for extended re-testing, including which information should be included when referring samples to an NRL are presented. CONCLUSION: Monitoring the proportion of and reasons for failure to detect HPV in HSIL+ will help support high performance and quality improvement of HPV testing services. We encourage implementation of QA strategies based on re-analysis of "HPV negative" HSIL+ samples.

Quality assurance in human papillomavirus testing for primary cervical screening.

The recommendation for cervical screening is that it should be based on human papillomavirus (HPV) molecular testing. For all screening programs, attention to quality assurance is required to fully realize the benefits. Internationally recognized quality assurance recommendations for HPV-based screening are needed that are ideally applicable for a variety of settings, including in low- and middle-income countries. We summarize the main points of quality assurance for HPV screening, with a focus on the selection, implementation, and use of an HPV screening test, quality assurance systems (including internal quality control and external quality assessment), and staff competence. While we recognize that it might not be possible to fulfill all points in all settings, an awareness of the issues is essential.

Implementing HPV testing in 9 Latin American countries: The laboratory perspective as observed in the ESTAMPA study.

Background: Replacement of cytology screening with HPV testing is recommended and essential for cervical cancer elimination. HPV testing for primary screening was implemented in 12 laboratories within 9 Latin American countries, as part of the ESTAMPA cervical cancer screening study. Our observations provide information on critical operational aspects for HPV testing implementation in diverse resource settings. Methods: We describe the implementation process of HPV testing in ESTAMPA, focusing on laboratory aspects. We assess the readiness of 12 laboratories to start HPV testing and their continuity capacity to maintain good quality HPV testing until end of recruitment or up to December 2021. Readiness was based on a checklist. Information from the study database; regular meetings and monitoring visits; and a questionnaire on laboratory operational aspects sent in May 2020 were used to assess continuity capacity. Compliance with seven basic requirements (readiness) and eight continuity requirements (continuity capacity) was scored (1 = compliant, 0 = not compliant) and totaled to classify readiness and continuity capacity as very limited, limited, moderate or high. Experiences, challenges, and enablers of the implementation process are also described. Results: Seven of 12 laboratories had high readiness, three moderate readiness, and of two laboratories new to HPV testing, one had limited readiness and the other very limited readiness. Two of seven laboratories with high readiness also showed high continuity capacity, one moderate continuity capacity, and the other four showed limited continuity capacity since they could not maintain good quality HPV testing over time. Among three laboratories with moderate readiness, one kept moderate continuity capacity and two reached high continuity capacity. The two laboratories new to HPV testing achieved high continuity capacity. Based on gained expertise, five laboratories have become part of national screening programs. Conclusion: High readiness of laboratories is an essential part of effective implementation of HPV testing. However, high readiness is insufficient to guarantee HPV testing high continuity capacity, for which a "culture of quality" should be established with regular training, robust monitoring and quality assurance systems tailored to local context. All efforts to strengthen HPV laboratories are valuable and crucial to guarantee effective implementation of HPV-based cervical screening.

Human papillomavirus genotyping using next generation sequencing (NGS) in cervical lesions: Genotypes by histologic grade and their relative proportion in multiple infections.

Sensitive and specific genotyping of human papillomaviruses (HPVs) is critical for the surveillance and monitoring of the vaccine effectiveness. Here, HPV genotypes were identified in 137 cervical samples with different histology (79 ≤CIN1 and 58 CIN3+) using Nested-PCR followed by Next-Generation sequencing (NGS) and relative proportions for each genotype in multiple infections were computed. All samples had been previously genotyped by PCR-Reverse Blotting Hybridization (PCR-RBH) thus allowing for a concordance analysis between both techniques. Multiple infections were present in 85% of ≤CIN1 cases compared to only 41% in CIN3+ cases (p<0.001). Among ≤CIN1 cases a towering genotypic diversity was observed, considering both low (LR-) and high risk (HR-) HPV genotypes; while among CIN3+, diversity was lower, HR-HPVs prevailing in most cases, especially HPV16. Furthermore, the predominance of HR-HPV genotypes in the proportions identified in each sample was higher in CIN3+ cases [(HPV16 (62.5%), followed by HPV31 and HPV58 (8.3% each)], than in ≤CIN1 cases [(HPV16 (17.7%), followed by HPV52 (14.7%) and HPV31 (10.3%)]. Agreement between PCR-RBH and NGS was higher than 90% for all genotypes (with an overall Kappa of 0.7), even though NGS identified eighty-nine positive results for HPV genotypes that had not been detected by PCR-RBH, evidencing its greater sensitivity. These results suggest that a reduction in genotypic diversity and/or an increase in the relative proportion of HR-HPVs in multiple infections can be considered as a biomarker for the potential risk of malignant progression.

Distribution of human papillomavirus genotypes by severity of cervical lesions in HPV screened positive women from the ESTAMPA study in Latin America.

The proportion of HPV16 and 18-associated cervical cancer (CC) appears rather constant worldwide (≥70%), but the relative importance of the other HR-HPV differs slightly by geographical region. Here, we studied the HPV genotype distribution of HPV positive Latin American (LA) women by histological grade, in a sub-cohort from the ESTAMPA study; we also explored the association of age-specific HPV genotypes in severe lesions. Cervical samples from 1,252 participants (854 ≤CIN1, 121 CIN2, 194 CIN3 and 83 CC) were genotyped by two PCRs-Reverse Blotting Hybridization strategies: i) Broad-Spectrum General Primers 5+/6+ and ii) PGMY9/11 PCRs. HPV16 was the most frequently found genotype in all histological grades, and increased with the severity of lesions from 14.5% in ≤ CIN1, 19.8% in CIN2, 51.5% in CIN3 to 65.1% in CC (p < 0.001). For the remaining HR-HPVs their frequency in CC did not increase when compared to less severe categories. The nonavalent vaccine HR-types ranked at the top in CC, the dominant ones being HPV16 and HPV45. HR-HPV single infection occurs, respectively, in 57.1% and 57.0% of ≤CIN1 and CIN2, increasing to 72.2% and 91.6% in CIN3 and CC (p<0.001). No association between age and HPV type was observed in CC, although the risk of HPV16 infection in CIN3 cases increased with age. Results confirm the relevance of HPV16 in the whole clinical spectrum, with a strong rise of its proportion in CIN3 and cancer. This information will be relevant in evaluating the impact of HPV vaccination, as a baseline against which to compare genotype changes in HPV type-specific distribution as vaccinated women participate in screening in LA region. Likewise, these data may help select the best HPV testing system for HPV-based efficient, affordable, and sustainable screening programmes.

A comprehensive HPV-STI NGS assay for detection of 29 HPV types and 14 non-HPV sexually transmitted infections.

BACKGROUND: Sexually transmitted infections (STIs) are prevalent throughout the world and impose a significant burden on individual health and public health systems. Missed diagnosis and late treatment of STIs can lead to serious complications such as infertility and cervical cancer. Although sexually transmitted co-infections are common, most commercial assays target one or a few STIs. The HPV-STI ChapterDx Next Generation Sequencing (NGS) assay detects and quantifies 29 HPVs and 14 other STIs in a single-tube and single-step PCR reaction and can be applied to tens to thousands of samples in a single sequencing run. METHODS: A cohort of 274 samples, previously analyzed by conventional cytology/histology and Roche cobas HPV Test, were analyzed by ChapterDx HPV-STI NGS assay for detection of 43 HPV and STI. A set of 43 synthetic control DNA fragments for 43 HPV and STI were developed to evaluate the limit of detection, specificity, and sensitivity of ChapterDx HPV-STI NGS assay. RESULTS: The assay was evaluated in this study, and the limit of detection was 100% at 50 copies for all targets, and 100%, 96%, 88% at 20 copies for 34, 8, and 1 target, respectively. The performance of this assay has been compared to Roche cobas HPV test, showing an overall agreement of 97.5% for hr-HPV, and 98.5% for both, HPV16 and HPV18. The assay also detected all HPV-infected CIN2/3 with 100% agreement with Roche cobas HPV results. Moreover, several co-infections with non-HPV STIs, such as C. trachomatis, T. vaginalis, M. genitalium, and HSV2 were identified. CONCLUSIONS: The ChapterDx HPV-STI NGS assay is a user-friendly, easy to automate and cost-efficient assay, which provides accurate and comprehensive results for a wide spectrum of HPVs and STIs.

Biomarkers of human papillomavirus (HPV)-driven head and neck cancer in Latin America and Europe study: Study design and HPV DNA/p16INK4a status.

BACKGROUND: Human papillomavirus (HPV)-driven head/neck squamous cell carcinomas (HNSCC) prevalence varies globally. We evaluated HPV DNA and p16INK4a in formalin fixed paraffin embedded (FFPE) HNSCC from Argentina, Brazil, Colombia, and Peru. METHODS: HPV was genotyped by PCR-hybridization. All HPV DNA positive and some HPV DNA negative cases underwent p16INK4a immunohistochemistry. RESULTS: HPV DNA was detected in 32.8%, 11.1%, and 17.8% of oropharyngeal (OPC), oral cavity (OCC) and laryngeal (LC) cancers, respectively. OPC HPV prevalence was higher in Colombia (94.7%), and Argentina (42.6%) compared to Brazil (10.6%) and Peru (0.0%). HPV-16 was the most detected. Other HPVs were found in LC. Higher rates of p16INK4a positivity were observed among HPV positive OPC/OCC cases compared to LC cases. CONCLUSIONS: Our results support a role for HPV-16 in a subset of HNSCC, corroborate the heterogeneity observed in samples from different countries, and contribute additional etiological and biomarkers information in tumors of significant impact worldwide.

Baseline prevalence and type distribution of Human papillomavirus in sexually active non-vaccinated adolescent girls from Argentina.

In 2011, Argentina launched a government-funded national Human papillomavirus (HPV) immunization program incorporating a bivalent HPV vaccine, with a 0-1-6-month schedule, for girls 11 years of age, born after January 2000. Monitoring the changes of HPV infection prevalence among young women has been proposed as an endpoint for early assessment of HPV vaccination programs. However, the data on HPV prevalence at young ages are very limited. The aim of this work was to determine the prevalence of HPV infection and type-specific distribution in sexually active 15-17-year-old non-vaccinated girls. Cervical samples from 1073 adolescents were collected for HPV detection and genotyping using the BSGP5+/GP6+PCR-reverse line blot (RLB) assay. Out of 957 specimens analyzed, 56.3% were positive for any HPV type; 42.2% harbored at least one high-risk HPV (HR-HPV) type and 30.8% low-risk HPV (LR-HPV) types. Multiple and single infections were identified in 36.3% and 20.0% of the samples respectively. The 6 most common HR-HPV types were HPV16 (11.1%), HPV52 (10.8%), HPV56 (8.3%), HPV51 (7.4%), HPV58 (7.3%) and HPV31 (7.1%). The prevalence of HR-HPV-16/18 was 15.2%. In conclusion, results confirm that HPV (particularly HR-types) are very common among sexually active adolescents, and prevalence rises quickly after their sexual debut. Our HPV type-specific prevalence baseline may be used to monitor post-vaccinal longitudinal changes in Argentina.

Cutaneous human papillomavirus genotypes in different kinds of skin lesions in Argentina.

Cutaneous human papillomaviruses (HPVs) comprise a large and highly heterogeneous virus group. Some of the cutaneous HPVs of the genus Beta have been suggested as a co-factor in the development of non-melanoma skin cancer (NMSC). The aim of this study was to determine cutaneous HPV prevalence and type-specific distribution in different kinds of skin lesions from Argentine patients visiting Dermatology Departments of three hospitals from Buenos Aires. A cross-sectional analysis was performed. HPV DNA was analyzed in (i) 3 patients with Epidermodysplasia verruciformis (EV) harboring benign lesions (BL) (n = 1) and squamous cell carcinoma (SCC) (n = 4); (ii) 240 non-EV patients harboring: (a) BL (n = 38), (b) Actinic Keratosis (AK) (n = 83), (c) SCC (n = 74), and (d) basal cell carcinoma (BCC) (n = 96). Detection and genotyping of 35 cutaneous HPV DNA was carried out by BGC-PCR and GP5+/6 + PCR followed by reverse line blot assay. In EV patients, Beta types were found in all lesions (5/5), including the potentially high-risk HPV types 5 and 8, mostly in multiple infections. In non-EV patients, cutaneous types were found in 50.0% of BL, 43.4% of AK, 31.1% of SCC, and 16.7% of BCC. Beta HPVs were the most frequently found in all lesions, being present in all AK and SCC cases that were positive for HPV. No type-specific correlation with lesion severity was found. In our series, a wide spectrum of cutaneous HPV types was detected in different skin lesions. A possible role for these HPVs in skin carcinogenesis deserves further study. J. Med. Virol. 89:352-357, 2017. © 2016 Wiley Periodicals, Inc.

Epstein Barr virus genotypes and LMP-1 variants in HIV-infected patients.

UNLABELLED: Two Epstein Barr virus (EBV) genotypes: EBV-1 and EBV-2 have been described. A 30-bp deletion in latent membrane protein-1 gene (del-LMP-1) has been identified in various pathologies. The aim of this study was to determine EBV genotypes and 30-bp deletion frequency in HIV-infected patients from Argentina. The study was performed on 258 individuals: CASES: 144 HIV-infected patients that included: (a) 7 AIDS patients with primary central nervous system lymphoma (PCNSL), (b) 62 AIDS patients, and (c) 75 asymptomatic HIV-infected patients. CONTROLS: 114 HIV-negative individuals. EBV genotypes and variants in LMP-1 gene were detected by polymerase chain reaction (PCR)-Southern blot on DNA extracted from peripheral blood mononuclear cells and brain biopsies. In PCNSL, the presence of EBV was confirmed by EBER RNA in situ hybridization, and DNA sequencing of 3' end LMP-l gene of PCR products was performed. In HIV-infected patients, EBV-1 was detected in 48.6%, EBV-2 in 18.8%, and co-infection with both genotypes in 32.6%. In control group, EBV-1 was present in 74.3%, EBV-2 in 12.4%, and co-infection in 13.3%. Del-LMP-1 was found in 44.4% of HIV-infected patients samples (20.7% alone and 23.7% co-infection with non-deleted form) while it was found in 25.3% (6.3% alone and 19% with co-infection) in HIV-negative individuals. In HIV-infected patients EBV-2, co-infection and 30-bp deletion are more prevalent than in control group. In all, PCNSL brain biopsies samples, del-LMP-1 always was detected with EBV-2, but more cases would have to be included to draw definitive conclusions.

Circulating Epstein-Barr virus (EBV) in HIV-infected patients and its relation with primary brain lymphoma.

OBJECTIVE: To analyze Epstein-Barr virus (EBV) load at different HIV infection stages and its relation with brain lymphoma. DESIGN: A cross-sectional study was conducted on 172 HIV-infected individuals: 62 asymptomatic HIV carriers (group A), 30 HIV progressors (group B), 73 AIDS patients (group C), seven AIDS patients with brain lymphoma (group C-BL); and 26 blood donors (group BD) as healthy carriers. EBV load was measured in peripheral blood mononuclear cells (PBMC) and plasma samples using a semi-quantitative PCR method. RESULTS: PBMC-EBV levels in HIV-infected patients were higher than in the blood donors (p<0.05). No differences in PBMC-EBV loads were found in groups A, B, or C (p>0.05), while the C-BL group had significantly lower levels (p<0.05). Similar PBMC-EBV loads were seen in HIV-infected patients with CD4+ T cell counts higher than 50/mm(3) (p>0.05), while significantly lower levels were found in cases with less than 50 cells/mm(3) (p<0.05). In all HIV-infected patients, plasma-EBV load was lower than, or similar to, PBMC-EBV load, unlike 2/7 HIV-positive brain lymphoma patients. CONCLUSIONS: During HIV infection PBMC-EBV load rises in comparison to healthy carriers, but decreases when immunosuppression progresses and CD4+ T cell count becomes <50/mm(3). Circulating EBV is mainly cell-associated in the HIV-infected population. Neither PBMC-EBV nor plasma-EBV loads would be useful to diagnose brain lymphoma in AIDS patients.

Epstein-barr virus (EBV) in healthy carriers: Distribution of genotypes and 30 bp deletion in latent membrane protein-1 (LMP-1) oncogene.

There are two types of Epstein Barr virus (EBV): EBV-1 and EBV-2, distinguished by genomic polymorphism in the genes encoding the nuclear antigens (EBNA-2, -3A, -3B, -3C). Latent membrane protein 1 (LMP-1) is an EBV protein with known oncogenic properties. Different variants had been described; among them, a 30 base pair (bp) deletion (del-LMP-1) had been reported in benign and malignant pathologies, but there is little information about its frequency in healthy populations. The aim of this study was to determine the distribution of the EBV genotypes and the 30 bp deletion frequency, in EBV healthy carriers from Argentina. Analysis of EBNA-3C and LMP-1 genes were done by polymerase chain reaction (PCR) followed by Southern blot hybridization on DNA of peripheral blood mononuclear cells (PBMCs) from blood bank donors. EBV-1 was present in 75.9% of samples, EBV-2 in 14.6%, and co-infections with both types in 6.5%. The deleted LMP-1 variant was found in 7.4% of analyzed samples, corresponding 3.2% to deleted variant alone and 4.2% to co-infections with non-deleted form. The non-deleted variant was found in 64.6% whereas in the remaining 28%, no PCR product was detected. These results showed that EBV-1 was the more prevalent type in healthy carriers of Argentina, similar to reports from others countries. A predominance of the non-deleted LMP-1 variant was observed. The presence of co-infections with both types and variants demonstrated that healthy individuals may also harbor multiple EBV infections.