Infection by human papillomaviruses (HPVs) has been implicated in the aetiology of a variety of cancers. Studies evaluating the presence of HPVs in breast cancer (BC) have generated considerable controversy. To date, most studies have focused on the presence of viral DNA in BC; however there are important gaps in evidencing the role of HPV persistence in the invasiveness of BC. While these studies have been conducted in several countries, none, on the presence and biological activity of high risk (HR) HPV in BC has been done in the UK. Hence, we aimed to investigate these gaps by screening a total of 110 fresh breast tissue specimens from UK patients for the presence of twelve HR-HPV types DNA using PCR and Sanger sequencing. Samples positive for HPV-DNA were screened for viral oncoprotein expression using western blot and dot blot. Data obtained showed the presence of HR-HPVs in 42% of breast tissues of which the viral activity was only confirmed in a number of invasive carcinomas (5/26). This finding, the first to report in the UK, suggests that the selective expression of viral oncoprotein in invasive cases may propose a role for HR-HPVs in the development of some types of BC. Breast cancer is one of the main health problems worldwide, and remains a leading cause of mortality in women1Although it is well-known that multiple risk factors are associated with breast cancer development, in most cases the initiating cause has not been identified. This has led to studies to identify new factors related to this neoplasia1454The HPVs belong to a large family of common viruses that infect cutaneous and mucosal epithelial surfaces  and cause both benign and malignant hyperproliferative lesions891112131417Regardless, while the body is working to get the infection under control, the HPV can spread through sexual means and by skin to skin contact1515Investigating a relationship between HPV and breast cancer is a valid hypothesis for a number of reasons. The exposure of the mammary ducts to the external environment via the nipple areola complex could provide an entry point for HPV infection. Also, most breast cancers originate from mammary duct epitheliaet al. (1992) were the first to report the positive relationship between HPV and breast cancer, by demonstrating the presence of HPV in 29% of breast carcinoma. Since the first discovery, several studies have investigated and detected over 60% HPV in breast cancer tissue. On the contrary, opposing studies were reported the absence of HPV DNA in breast cancer and suggested it is improbable that HPV is associated with the development of breast carcinomas. Therefore, it is still not clear whether HPV is present in breast tissue and breast tumours2223242526To date, studies on the role of HPV in breast carcinogenesis have generated considerable controversy. Di Lonardo Despite breast cancer emerging as a major public health imperative in the UK, studies on the aetiology of breast cancer requires further attention. Given that HPV infection may be a possible risk factor in the development of the breast cancer which causes high mortality among the global female population, and in the UK, there are no studies, to our knowledge, that investigates the implication of HPV infection in breast cancer development. Therefore, this study aims to determine the presence and the expression of 12 High-risk (HR) HPV genotypes present in biopsies of normal, benign and malignant breast tissue from patients from a UK population using highly sensitive molecular biology approaches. We also correlated the expression of HPV oncoprotein in a number of invasive cases that had tested positive for HPV DNA.in situ carcinoma tissues, 31 invasive carcinoma and 29 invasive and in situ carcinoma samples). The remaining 36 samples were classified as normal or benign .The results of the breast tissue biopsy pathological diagnosis are collated in Tissue specimens from a total 110 fresh abnormal breast tissues (including cancerous and non- cancerous) from women aged 25–82 years old were examined for the presence of DNA of 12 HR-HPV types using type-specific PCR. The amplification of β-globin gene (fragment size of a 723 bp) was positive in all extracted DNA, indicating an adequate quality of DNA. As a standard procedure, positive and negative controls were included for every sample run of gel electrophoresis. As shown in In each case, a and b, are representative gel electrophoresis patterns of the analysis for each set of HR-HPV types of each breast tissue sample.Sequencing was performed on PCR products which confirmed the amplification of HPV DNA. The BLAST analysis revealed 99% and 98% concordance of the HPV DNA sequences in breast tissue samples tested with the reference sequences for high risk HPV types. Additionally, the data obtained from the sequencing are consistent with PCR results and this further validates our findings on the detection of HPV DNA.For each sample, the PCR experiment was repeated in triplicate to validate the accuracy of the obtained data. As shown in In cancerous cases, HR-HPV DNA was detected with a prevalence of 47% , in whicBenign and normal/cosmetic reduction specimens displayed the presence of HPV in 31%  and HPV in situ.As shown in First, we examined the expression of HPV E7 oncogenic protein that was involved in the oncogenesity of HPV using dot blot. Cell lysate from cervical cancer cell line (CaSki) that is known for expressing HPV E7 protein was used as a positive control and cell lysate from Human immortalised keratinocyte (HaCaT) cell line was used a negative control. As shown in To complement dot blot results, we next assessed and confirmed protein expression by western blotting. Antibodies against the HPV oncoprotein E7 were used to detect the presence of viral gene expression in HPV positive breast cancer samples. As shown in in situ and invasive malignancy demonstrated the presence of HPV-DNA. Furthermore, we were able to detect HPV E7 oncogenic protein in a number of invasive breast cancer tissues which may suggest a role for HR-HPV types in the development of some type of breast cancer.In summary, this study was able to detect the presence of 12 high-risk HPV gene sequences in fresh abnormal breast tissue. The obtained results demonstrated HPV-DNA was detected in 46 out of 110 breast tissue specimens using PCR with 47% of breast cancer testing positive for HPV DNA . Both inThe global breast cancer burden in women is markedly increasing. Breast cancer is the leading cause of cancer death among women worldwide. This emphasises the importance of identifying novel etiological risk factors that are associated with breast cancer development228A number of studies have investigated HR-HPV in breast cancer in which the detection of the HPV infection was based on testing for the presence of viral DNA using a range of molecular methods31in-situ and invasive carcinoma) and 36/110 (33%) from benign and normal cases. The results of high-risk HPV typing showed that HPV-DNA was found in 46/110 (42%) of abnormal breast tissue specimens.Histopathological results on the 110 fresh breast biopsies in our study revealed that 74/110 (67%) of abnormal breast biopsies were from cancer patients  calls for further investigations to fully understand the presence of HPV in these benign specimens. There exists the possibility that the benign cases may perhaps develop later to breast cancer and hence makes necessary the follow-up of the individual patients. Although we have shown the presence of HR-HPV in benign specimens, we were unable to confirm the expression of the HR-HPV protein in theses samples. While we did not find evidence of viral expression in benign cases which were positive for HPV DNA, others were able to show the expression of HPV oncoprotein in benign samples via immunohistochemistry (IHC) method36Our data also shows the presence of 12 HR-HPV DNA in 35/74 (47%) of breast carcinoma cases. These results validate the findings of other studies in different geographical regions worldwide including Asia and Europe, which identified the presence of high-risk HPVs in breast cancer samples. Across these studies, the presence of HR-HPVs was reported with HPV frequencies as high as 86% in some studies38In this study, the most prevalent HPV type detected in breast cancer samples positive for HPV was HPV 39 in 13 (20%), followed by HPV 18 in 8 (12%), HPV 45 in 8 (12%), and each of HPV type 16, 35 and 59 in 7 (11%) of HPV positive samples. Unlike our study, a number of reports have identified HPV types 16 and 18 as most prevalent types in breast cancer specimens, but these relied on screening a limited number of HR-HPV typesAdditionally, examination of 12 different high-risk HPV types in each breast samples allowed for the detection of HR-HPVs coinfection in 16/35 (46%) of cancerous samples . MultiplOur data also revealed that the prevalence of HPVs DNA in cancerous cases was higher among women with invasive ductal carcinoma and lowest in lobular carcinoma. This observation is consistent with other reports, which highlighted the highest HPV positivity rate in patients with ductal breast carcinoma, but lower rates of HPV prevalence in other histological classifications. The presence of HPV infection in the breast ducts is a logical association as the exposure of the mammary ducts to the external environment could possibly provide an entry point for HPV infection; this may serve as cogent explanation for most breast cancers originating from the mammary duct epitheliaThe detection of viral DNA has been used as a marker for the presence of the virus in related lesions, although this is not indicative of a productive infection. Hence, we investigated activity of the virus by examining the expression of HR-HPVs E7 oncoprotein using immunoblotting approaches. We were able to spot the biological activity of HR-HPVs in a number of breast cancer specimens (5/35). It is interesting to note the expression of HPV E7 oncogenic protein that is involved in the oncogenesity of HPV was only detected in invasive breast cancer specimens.et al.This is the first study that was able to document the expression of HR-HPV oncoprotein E7 in fresh breast cancer samples using immunoblotting techniques. We showed the expression of E7 in breast tissue from patients with breast cancer rather than in patients with early lesions or in healthy subjects. Regarding the association between the HR-HPV and tumour aggressiveness, Wazer HR-HPV oncogenes, E6 and E7, are expressed early in cervical carcinogenesis as they play an important role in the transformation process. Hence, these genes are expressed at accumulative levels throughout cancer progression as a result of viral DNA integration into the host genome and the subsequent effect of uncontrolled overexpression. And this ultimately leads to an abundant expression of E6 and E7 throughout the epithelia in high-grade cervical dysplasia and cervical cancer141However, due to the low viral load of HPV in breast cancer, we firmly believe that employing immunoblotting techniques to detect the HPV oncoprotein in breast cancer tissues can be challenging, therefore extending this investigation with further approaches such as Immunohistochemistry would be beneficial for the detection of lower levels of HPV expression.The presence of high risk HPV viral sequences and the expression of viral protein in malignant breast tissue highlight the importance of further studies to investigate and assess the possible viral mechanism in the pathogenesis of breast cancer. We have established a clear rationale for this investigation and the prospect of investigating a role of vaccination against a broader range of HR-HPV subtypes, and not just HPV type 16 and 18, in the primary prevention of breast cancer. This can provide a strong impetus to advance research in delaying disease progression or in preventative initiatives in an important global health imperative involving women.in situ carcinoma, invasive carcinoma, and in situ and invasive carcinoma  REC reference: 12/LO/0837. All methods were carried out in accordance with the approved guidelines and regulations. Following informed consent, a total of 110 fresh breast tissue specimens were aseptically collected by one surgical team (over a period of 3 years) from patients immediately after excision in surgery in the Breast Surgery Department at Kingston Hospital, London, UK. All breast tissue specimens were formally reported by Kingston Hospital Pathology Department as part of the standard clinical diagnosis of the patients. The tumours were histopathologically classified into: benign, benign with previous cancer, arcinoma .All biopsies obtained following surgery were immediately preserved using Allprotect reagent from QIAGEN to stabilize DNA, RNA and protein, with aseptic handling of tissue in the theatre environment to avoid contamination of samples.Separate disposable items, such as gloves, surgical blades and tubes, were used during tissue handling to avoid cross-contamination between specimens. Cellular DNA, RNA and protein were extracted from the collected breast tissue samples using Allprep DNA/RNA/Protein Mini Kit (QIAGEN) in accordance with the manufacturer’s instructions. Briefly, to obtain genomic materials and protein, this entailed the following: The tissue was cut into small pieces and then homogenised in lysis buffer using Tissue Lyser (QIAGEN) and QIAshredder spin columns. The lysate of each sample was subjected to extraction steps to obtain the purified nucleic acid as per the manufacturer’s instructions. The concentration and the purity of nucleic acids were assessed using NanoVue plus spectrometer (GE Lifesciences).The research approach to the identification of HPVs was to use Polymerase Chain Reaction (PCR). HPV genotyping was performed using the HPV- HCR Genotype-Eph kit (AmpliSens) using the purified DNA extracts from the 110 benign and breast cancer tissue specimens (extracted at Kingston University laboratories-UK). The kit is based on simultaneous amplification of four targeted regions of the HPV gene of each of the four HPV DNA types in one tube (multiplex-PCR). The HPV DNA amplification was run in three tubes, in which 4 different HPV types were amplified in each tube as follows; HPV-16/31/33/35, HPV-18/39/45/59 and HPV-52/56/58/66. This enabled the detection of infections and co-infections of 12 high risk HPV genotypes in each collected tissue sample.To analyse and determine HPV genotypes, the amplified PCR products were electrophoresed along with type specific positive controls on a 3% (w/v) agarose gel stained using SYBR Safe (Invitrogen). This was then visualised under UV using a Gel Doc XR+ System (Bio-Rad). For each sample, the amplification of β-globin served as an internal positive control to determine the adequacy of the extracted DNA for amplification. PCR was performed in triplicate for each HPV positive sample to confirm the result.In order to confirm presence of HPV genes, PCR products of the HPV positive samples were subjected to direct sequencing. All PCR products were purified using a QIAquick PCR Purification Kit (Qiagen) and then sequenced using an Applied Biosystems 3730xL analyser. The results were evaluated using NCBI BLAST program to verify HPV sequences. The use of this method was used to add to the validity of results.A standard dot blotting method was employed to investigate whether HR-HPVs, expressed in samples positive for HPV DNA using anti-HPV E7 monoclonal antibody (Cervimax) – Valdospan GmbH, Austria, cross reacted with a number of other HR-HPV types. Equal amounts of purified protein were directly spotted onto a nitrocellulose membrane. The membranes were blocked with 2% (w/v) bovine serum albumin (BSA) in Phosphate-buffered saline (PBS)-Tween20 (0.05% (v/v) Tween20) at room temperature for 1 hour to reduce non-specific binding. These were then washed with PBS-Tween for 5 minutes three times. The blocked membranes were incubated with E7 mouse monoclonal antibody (CERVIMAX) (diluted at 1:500 in 1% (w/v) non-fat milk in PBS-T) at 4 °C for 3–4 hours. The membranes were subjected for incubation with secondary antibody Goat Anti-Mouse IgG - Alkaline Phosphatase conjugate antibody (Invitrogen) for 2 hours at room temperature and were then washed for 5 minutes 3 times. Finally, the membranes were developed using NBT and BCIP reagent Kit (Invitrogen) for 5 minutes and then washed twice in PBS-T.Li-Cor) at room temperature for 2 hours. The membrane was then visualised by OdysseyCLx Imaging System (Li-Cor).To validate the dot blot findings, the expression of HR-HPV proteins was also examined using a standard semi-dry western blotting method which was employed to investigate whether HR-HPVs, expressed in samples positive for HPV DNA using anti-HPV E7 monoclonal antibody (Cervimax) – Valdospan GmbH, Austria. Purified protein was subjected to western blot to examine the expression of HPV oncogenic protein E7. Equal amounts of protein were electrophoresed using Bolt™ 4–12% Bis-Tris gel (Invitrogen) and transferred on to 0.2 μm Nitrocelulose membrane using MES Buffer: 50 mM MES, 50 mM Tris-HCl, 0.1% (w/v) SDS, 1 mM EDTA, pH 7.3. Membranes were blocked with 5% (w/v) non-fat dry milk in TBS-T  Tween20) at room temperature for 2 hours to reduce non-specific binding. The membrane was then incubated with E7 mouse monoclonal antibody (CERVIMAX) (diluted at 1:500 in 1% (w/v) non-fat milk in TBS-T) at 4 °C overnight. The membrane was washed with TBS-Tween buffer for 20 minutes 3 times and then incubated with IRDye secondary antibody Donkey anti-Mouse IgG (diluted at 1:15000) .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Laticauda semifasciata  from the waters around southern South Korea—an area located outside its typical distribution range . We investigated the genetic origin of Korean specimens by analyzing mitochondrial cytochrome b gene (Cytb) sequences. Six individuals shared haplotypes with a group found in Taiwan-southern Ryukyu Islands, while the remaining six individuals shared haplotypes with a group encompassing the entire Ryukyu Archipelago. These results suggest L. semifasciata moved into Korean waters from the Taiwan-Ryukyu region via the Taiwan Warm Current and/or the Kuroshio Current, with extended survival facilitated by ocean warming. We highlight several contributing factors that increase the chances that L. semifasciata establishes new northern populations beyond the original distribution range.Marine reptiles are declining globally, and recent climate change may be a contributing factor. The study of sea snakes collected beyond their typical distribution range provides valuable insight on how climate change affects marine reptile populations. Recently, we collected 12  Reptiles, both terrestrial  and mariHydrophis platurus from California, USA [Laticauda semifasciata and L. laticaudata from mainland Japan [L. semifasciata and H. platurus from Russia [L. semifasciata from Korea [In this study, we focus on sea snakes. For marine reptiles, sea turtles are the common example for population declines , but seania, USA , Laticaund Japan , L. semim Russia  and L. som Korea –17. It hH. platurus, H. cyanocinctus, H. melanocephalus) were reported from Korea [L. semifasciata in Korean waters represents a new and recent appearance [Laticauda semifasciata is typically found in the waters around Japan, China including Taiwan, Philippines and Indonesia [L. semifasciata showed distinctive genetic structure between island groups in Taiwan and Japan. In this study, we use molecular methods to determine the geographic origin and cause of L. semifasciata recently collected from Korean waters.Historically, only three Hydrophiine species . L. semifasciata  were collected and donated by local fishermen—nine from Jeju Island and three from the Korean Peninsula. For convenience, we named the locality based on the nearest port to the capture site. All specimens were delivered to the Herpetological Lab of Kangwon National University (KNU). Upon arrival, we collected basic data on the specimens: sex (using a probe), snout-vent length  and body weight . For tissue collection, we took a tail clip  using scissors [L. semifasciata specimens  were included in our study to increase geographic sampling. Sequences from the Ryukyu Islands, Japan were obtained from GenBank [To obtain sea snake samples from Korean waters, we placed more than 200 posters and 50 banners around coastal towns of southern South Korea bordering the South Sea (including Jeju Island) between April 2015 and October 2016. A total of 12 scissors . Additio GenBank .Cytb because there are comparable data in GenBank. We amplified partial sequences of the Cytb gene using the primers L14910 and H16064 [® PCR Purification Kit . PCR products were sequenced in both directions using the same PCR primers at Macrogen  on a 3730xl DNA analyzer .We extracted whole genomic DNA from tissue samples using the QIAGEN DNeasy Blood & Tissue kit  following the manufacturer’s instructions. For this study, we targeted d H16064 . DNA wasd H16064 . We veriL. semifasciata, we constructed a phylogenetic tree using 16 new sequences (GenBank accession numbers KY445753 to KY445768), 11 Laticauda sequences from GenBank  and 3 Hydrophis sequences as outgroups. (http://tree.bio.ed.ac.uk/software/tracer). The first 500,000 generations were discarded as burn-in and the remaining trees were used to build a consensus tree.We edited and assembled sequences using Geneious v5.3.6. To verify our specimens were all tgroups. . Sequenchttp://popart.otago.ac.nz). The dataset analyzed is a combination of our data  with data from Tandavanitj et al. [For population genetic analyses, we built a haplotype network using a median joining method in PopArt 1.7.2  to be fasciata .Cytb haplotypes from our specimens, all which were previously reported from the Taiwan-Ryukyu Archipelago , which could be placed into two major groups based on geography: (1) Semi-1 and Semi-3 haplotypes originating from Taiwan-southern Ryukyu  and (2) Semi-5 haplotype originating from the entire Ryukyu Archipelago . Korean samples did not have any haplotypes restricted to either central or northern Ryukyus.L. semifasciata from Korean waters: one group  is closely related to the entire Ryukyu Archipelago (Semi-5), while the second group  has genetic affinity to Taiwan and the southern Ryukyu Islands . For the first group, there is not enough genetic structure to infer the specific geographical origin beyond the Ryukyu Islands. However, for the second group, the available data suggest that the genetic origin of these individuals is either Taiwan or the southern Ryukyus. It is possible, based on the pathways of the Kuroshio Current [L. semifasciata. The presence of L. semifasciata in Korea was previously undetected, but becoming more common, and we believe that these currents along with recent climate change have contributed to increased records.We identify two genetic groups of  Current , that th Current –32, and L. semifasciata sightings has increased, while H. platurus observation and capture has been relatively steady [L. semifasciata in Korean waters, we consider three contributing factors: 1) increased effort, 2) northward expansion of prey fish and 3) higher ocean temperatures in Korean waters.In the past, only Hydrophiine species were known to occur in Korea, with fewer than seven observations ,21, whily steady ,33. To eH. platurus, while we collected 12 L. semifasciata and had greater than six observations. The increased numbers of L. semifasciata in Korean waters is likely a true pattern.First, increased observation of Laticaudine snakes may be attributed to increased effort in sea snake research relative to the past. We do not believe this to be a major factor because increased effort should also increase observations of Hydrophiine sea snakes known to exist in Korea; during our study period, we had only two reports of Chromis notatus and Haliechoeres tenuispinis (family Labridae). Previous studies found L. semifasciata consume these two fish species [L. semifasciata are in waters around Korea, they can feed on familiar prey fish, resulting in longer survivorship and more sea krait records.Second, the northward expansion of prey fish species due to climate change in the East China Sea might increase survival of the sea kraits in northern areas by providing suitable prey. Previous studies showed that global climate change actively or passively expanded various marine fish species beyond their typical distribution ranges –36. In t species ,40. So, L. semifasiata in northern waters. In such a situation, L. semifasciata are occasionally dispersed northward, but it is only recently with warmer temperatures that individuals could survive, at least temporarily. The annual mean ocean surface temperature around Jeju Island has increased at a rate of 0.024°C/ year (from 17.9°C to 18.6°C) between 1971 and 2000. Winter temperatures are colder and more critical for survival, and these temperatures have also increased  [L. semifascaita that moved into Korean waters should survive for a longer period than previous, resulting in increased observation of sea kraits.Third, higher ocean temperatures could contribute to survival of L. semifasciata the only collected sea krait species? and (2) why were more female L. semifasciata captured? We provide potential explanations for these questions. There are three Laticauda species found in Northeast Asia: L. semifasciata, L. colubrina and L. laticaudata [L. semifasciata in Korean waters might be due to the geographic distribution, habitat use and physical characteristics of the three species. At a global scale, L. colubrina and L. laticaudata have a wide regional distribution throughout Southeast Asia, whereas L. semifasciata is has a more restricted, northeastern distribution  [L. semifasciata are located in Taiwan and the Ryukyu Islands [L. semifasciata should have the highest chance to move into Korean waters. In addition, of the three Laticauda species, L. semifasciata is the most adapted to marine environments: spends the more time in the water [L. semifasciata drifts or moves into ocean currents.Our study raises two additional questions: (1) why was icaudata –44. The donesia) ,45. The  Islands . If the he water ,45,46, hhe water  and has L. semifasciata captured? Of the 12 total sea kraits collected in this study, 11 sea kraits were female (92%). We hypothesize that different feeding behavior and abilities contribute to the skew of females found in Korean waters. Laticauda species are benthic feeders and often make trips to the deep bottom (> 80 m) of the ocean for an extended time (> 130 min) to obtain their food [L. semifasciata more frequently drifting into the rapid ocean currents or seasonal typhoons [Why were more female eir food ,49. Becaeir food , femalestyphoons .L. semifasciata is a real phenomenon, and has the potential to establish a new population beyond the typical distribution range. Second, relatively long-distance dispersal of L. semifasciata confirms the importance of ocean currents in the dispersal of marine animals, as recently shown in H. platurus [L. semifasciata are venomous sea kraits  [L. semifasciata in Korea originate from specific population or various populations, how long L. semifasciata survive at the new northern sites, whether new breeding populations are being established, and how this species as a new predator impact local marine ecosystems [L. semifasciata in Korea and other worldwide sea snake populations beyond typical the distribution range will help clarify these issues.Findings from this study have three important ecological implications. First, northward dispersal of platurus . Third, to bite) , approprosystems . DetaileS1 FigLaticuada semifasciata. Numbers at the end of the taxon name refer to GenBank accession numbers. Numbers on the branches represent support values for the major groups—ML bootstrap support, followed by Bayesian posterior probabilities.The new specimens collected in our study  are in larger, bold font. All were identified as (TIFF)Click here for additional data file.S1 TableCytb are based on [The haplotype names of based on .(DOCX)Click here for additional data file.


Worldwide use of formalin-fixed paraffin-embedded blocks (FFPE) is extensive in diagnosis and research. Yet, there is a lack of optimized/standardized protocols to process the blocks and verify the quality and presence of the targeted tissue. In the context of an international study on head and neck cancer (HNC)—HPV-AHEAD, a standardized protocol for optimizing the use of FFPEs in molecular epidemiology was developed and validated. First, a protocol for sectioning the FFPE was developed to prevent cross-contamination and distributed between participating centers. Before processing blocks, all sectioning centers underwent a quality control to guarantee a satisfactory training process. The first and last sections of the FFPEs were used for histopathological assessment. A consensus histopathology evaluation form was developed by an international panel of pathologists and evaluated for four indicators in a pilot analysis in order to validate it: 1) presence/type of tumor tissue, 2) identification of other tissue components that could affect the molecular diagnosis and 3) quality of the tissue. No HPV DNA was found in sections from empty FFPE generated in any histology laboratories of HPV-AHEAD consortium and all centers passed quality assurance for processing after quality control. The pilot analysis to validate the histopathology form included 355 HNC cases. The form was filled by six pathologists and each case was randomly assigned to two of them. Most samples (86%) were considered satisfactory. Presence of >50% of invasive carcinoma was observed in all sections of 66% of cases. Substantial necrosis (>50%) was present in <2% of samples. The concordance for the indicators targeted to validate the histopathology form was very high (kappa > 0.85) between first and last sections and fair to high between pathologists (kappa/pabak 0.21–0.72). The protocol allowed to correctly process without signs of contamination all FFPE of the study. The histopathology evaluation of the cases assured the presence of the targeted tissue, identified the presence of other tissues that could disturb the molecular diagnosis and allowed the assessment of tissue quality. For molecular studies, it is critical to avoid sample cross-contamination during the processing (e.g. sectioning) of the FFPE tissue blocks and to obtain the highest quality of the specimens in order to perform all laboratory assays. The validation of the original histopathological diagnosis is essential to ensure that the study specimens correspond to the pathology targeted by the study and that there is sufficient representation.In the last decade an enormous number of molecular biology techniques have been developed, allowing for analysis of a wide spectrum of biomarkers in human specimens. Importantly, this high throughput technology permits the design of retrospective studies that are based on the use of archived material, such as formalin-fixed paraffin-embedded (FFPE) tissue samples. The conduction of worldwide multicenter studies using FFPE tissue samples has become a common practice in molecular epidemiology –6. Yet, Studies aiming to estimate the attributable fractions (AFs) of infections-related cancers in different geographical areas are nowadays frequent. Relevant examples are the studies on mucosal high-risk (HR) human papillomaviruses (HPV) that are associated with cervical cancer and a proportion of other ano-genital cancers and oropharyngeal cancers –6. In suWe have recently conducted a HNC case study that involved many centers in Europe and India, i.e. “Role of human papillomavirus infection and other co-factors in the aetiology of head and neck cancer in Europe and India” (HPV-AHEAD). The HPV-AHEAD consortium comprised nine partners from six European countries  and one partner from India ,8. The mDue to the multi-centric nature of the study, it was essential to develop several standardized protocols to efficiently achieve the planned goal. These protocols were to be performed simultaneously in different centres in Europe and India and included processing of human specimens for the several laboratory assays and the histopathologic review of the cases. In order to validate the standardization of the protocols, two main objectives were established. The first one was to assure that there had not been contamination when sectioning the blocks at the different centers. The second one was to confirm that the histopathology evaluation form developed by an international panel of pathologists could: 1) assure the presence of the tumor tissue, 2) identify other tissues that could invalidate the diagnosis of interest  and 3) validly assess the quality of the tissue. Here, we describe all protocols and procedures for the processing of the archived FFPE tissue specimens that have been developed in the context of the HPV-AHEAD consortium and that can be adopted by other multicentric studies. We also report the results of a pilot study on the achievement of the objectives to validate it.One of the major goals of the HPV-AHEAD study was to collect approximately 8000 FFPE HNCs in Europe and India and to determine the possible presence of HPV and cellular gene transcripts expression by different laboratory assays. Due to the fact that several collecting centers could not deliver the FFPE blocks to central lab of the project at the International Agency for Cancer Research (IARC), we developed a protocol for FFPE sectioning that was distributed to the collecting centers. The protocol describes the generation of several sections for laboratory assays and histological analyses, as schematically shown in INK4a immunohistochemistry (IHC) analysis; The slides to be used for viral DNA and RNA were optimally chosen as those in between the slides for histology and p16INK4a staining: the S3 to S5 sections for HPV mRNA analysis; the S6 to S8 sections for detection of and genotyping HPV DNA. If there was sufficient material, additional slides (S11-S30) were generated for additional IHC and real-time RT-PCR analyses with other cellular proteins. The procedure was well validated in the context of the HPV-AHEAD study, and resulted in the ideal use of FFPE tissue blocks without extensive manipulation.Only specimens fixed in neutral buffered formalin were included in the study. Specimens fixed in Bouin were not considered suitable for the study and thus excluded since the acidic environment of this fixative is a major cause for DNA degradation and makes extraction very difficult . The totDue to the different nature of the routine work performed in histology laboratories, it was essential to train each collecting center in preventing cross-contamination among the different FFPE blocks during their processing. Before starting to process the FFPE blocks, each histology laboratory received a test panel comprised of 25 alternate HPV-positive and HPV-negative FFPE blocks. To avoid the use of human material and to have unlimited source, the test panel included FFPE skin tissue of wild-type or HPV 16 E6/E7 transgenic mice. Sections were generated in the histology laboratories blinded to the HPV result and shipped to IARC for HPV genotyping. Only after performing the quality test for the generation of sections with acceptable results, each collecting center was allowed to start processing FFPE HNC tissue blocks.The protocol also described several procedures to minimize and monitor possible cross-contamination among the different FFPE see . For insThe HPV-AHEAD study implied to retrieve cases from archives of European and Indian hospitals. Although all the HNC tissues were already subjected to histological diagnosis, they were re-analysed in the context of the HPV-AHEAD study. A panel of six pathologists with a regional balance was created. The panel established the general criteria for a standardized histological review  and geneFrom the 14 items included in the histopathology evaluation form, four were selected to be explored between the first and last sections and between pathologists’ reviews in a pilot study of 355 cases in order to validate the form by assuring the presence of the targeted tissue, identifying other tissues that could contaminate the results and validly assessing the quality of the tissue. Those were: quality of tissue , percentage of invasive carcinoma , percentage of tumor necrosis  and presence of normal epithelium . Indeed, optically normal mucosa may carry HPV and could contaminate or give false information regarding HPV types. For example, when multiple HPV types are detected in a PCR from a piece of tissue with tumor and normal or displastic epithelium, by means of laser-microdissection it is possible to demonstrate that tumor harbours one HPV type, and benign mucosa other [Data were analyzed with STATA version 13.1. The main diagnoses of the pathologists were presented as frequencies to the selected items stratified by section (first or last) and pathologist. The differences of diagnoses between type of tissue  were explored by Pearson’s chi-square test or Fisher’s exact test when appropriate. Statistical significance for all the analyses was set at the 0.05 level. Concordance percent and kappa statistics were calculated to evaluate the agreement intra-pathologist (i.e. between reading of first and last sections done by the same pathologist) and inter-pathologist (i.e. between reading of first section of pathologist 1 and 2). The Kappa statistic characterization was established as following: <0: No agreement; 0–0.20: slight agreement; 0.21–0.40: fair agreement; 0.41–0.60: moderate agreement; 0.61–0.80: substantial agreement; 0.81–1.0: almost perfect agreement. Prevalence adjusted bias adjusted kappa (Pabak) statistic was explored when Kappa was not valid  but low kappas are observed). McNemar test p-value was also calculated to evaluate the distribution among the discordant cases.Ethical clearance for the investigations reported in this study was obtained from the Institutional Ethical Committees of MGIMS, Sevagram, India and IARC, Lyon, France. Study implied the use of archival material only, and it did not envisage any contact with the patients. Adequate measures to ensure data protection, confidentiality, patients’ privacy and anonymization were taken into account. No informed consent was available due to the retrospective design of the study and the large proportion of deceased and untraceable patients.In Europe, the HPV-AHEAD consortium retrieved approximatively 5000 FFPE HNC from 44 hospital archives, while in India 3000 FFPE HNC were retrieved in three rural and three urban hospitals. They were distributed for processing to six different histology laboratories from France, Belgium, Italy, Spain and India. As first step, each histology laboratory received the test panel of HPV-positive and HPV-negative FFPE tumor blocks containing skin tissue of wild-type or HPV 16 E6/E7 transgenic mice together with the detailed protocol for sectioning and for preventing cross-contamination of the specimens see . SectionAfter completion of the training phase, each center processed the FFPE blocks according to the HPV-AHEAD protocols and sections were sent to the different laboratories for additional analysis. The sectioning of all FFPE tissue blocks was completed in the following histology laboratories: (i) European Institute of Oncology (n = 1075); (ii) University of Antwerp (n = 1084); (iii) Catalan Institute of Oncology n = 1364); (iv) IARC n = 1524) and (v) RGCB (n = 389). HPV genotyping was performed blinded to the anatomical site by a very sensitive Luminex-based assay at IARC , 12. Alt64; (iv) 24 and (vINK4a). Inter-pathologist (i.e. between diagnoses of pathologists 1 and 2) differences in the diagnoses targeted to validate the form between biopsies and surgical pieces were explored. However, the results were inconsistent (data not shown), probably due to the small number of surgical pieces.The analysis of the 355 HNC histopathology evaluation forms showed that most of samples (88.7%) were biopsies and 11.0% surgical pieces (for one sample the type of tissue was not recorded). For 75.5% of the samples, S1&S31 could be evaluated (i. e. there was enough tissue for complete sectioning). This percentage was higher in surgical pieces than biopsies . Most biopsies (86%) were small with a median size of 7 mm. However, on average 86% of samples were considered satisfactory in both first and last sections irrespective of being a biopsy or a surgical piece. More than 60% of the samples showed >50% of invasive carcinoma in the section. Substantial necrosis (>50%) was present in less than 2% of the samples, and thus should not affect the quality of molecular analyses. Normal epithelial components were present in approximately 61% of the samples. This could be checked to justify discordance between biomarkers (e.g. cases positive for HPV DNA but negative for p16The results of the intra-pathologist (i.e. between diagnoses of first and last slides) and inter-pathologist concordance analyses of this pilot are shown in The concordance inter-pathologist was substantial for the diagnosis of tissue quality : Percentage of concordance of 86%, kappa 0.34 (pabak 0.72). In contrast, the concordance in the diagnosis of percentage of invasive carcinoma was low: 37%, kappa 0.11; when grouping the diagnoses of percentage of invasive carcinoma  the concordance slightly improved: 67%, kappa 0.25. The concordance in diagnosis of percentage of tumoral necrosis was fair: 61%, kappa 0.25. In this case, when grouping the diagnoses  the concordance improved substantially: 98.3%, kappa 0.44 (pabak 0.97). Finally, the concordance in diagnosis of presence of normal epithelium was moderate: 74%, kappa 0.44.To the best of our knowledge, this is the first attempt to develop and report a standardized sectioning and histopathology review protocol for optimizing the use of HNC paraffin blocks in multi-centric molecular epidemiological studies. Moreover, this protocol could also be extended for other cancer sites in which cross contamination and precise diagnosis are crucial. An equivalent histopathology protocol for breast cancer in situ has been described . AnotherThe assessment of the histopathology evaluation form developed by an international panel of pathologists confirmed its validity to assure the presence of the targeted tissue, identify other tissues that could contaminate the diagnosis and assess the quality of the tissue. The concordance between first and last slides was very high, confirming no loss of targeted tissue throughout the different sections. Most cases were considered satisfactory regardless of the type of sample . The concordance between pathologists’ diagnoses was fair to high and improved when grouping the categories of the items to more general diagnoses. Macrodisssection was not among the required procedures in our protocol and thus, the objective of assessing percentage of invasive carcinoma did not need to be as accurate as in other studies . Thus, tIt is expected that such standardized and validated sectioning and histopathology protocol can be adapted to any multi-centric study with similar aims as the ones observed in the HPV-AHEAD study. Less stringent protocols using this scheme as backbone could also be applied to other molecular studies, including high-throughput and next generation genomic studies. The versatility of the histopathology evaluation form, which was developed specifically for the study and not for clinical practice, allows for a wide range approach studies. The protocol as presented can be easily performed in any location in the world. The herein developed procedure for simultaneous on-line revision of the samples facilitates and enhances the establishment of international panel of experts of any pathology.In an era of increasing technological developments that allow running a wide range of biomarkers in a single sample, as well as of increase of international collaborative projects to tackle and provide important insights of a wide range of diseases, we believe that the sectioning and histopathology protocol that we have developed and validated in the context of the HPV AHEAD study may be of great interest and applicability.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.


Human papillomavirus (HPV)-induced neoplasms have long been considered to originate from viral infection of the basal cell layer of the squamous mucosa. However, this paradigm has been recently undermined by accumulating data supporting the critical role of a discrete population of squamo-columnar (SC) junction cells in the pathogenesis of cervical (pre)cancers. The present review summarizes the current knowledge on junctional cells, discusses their high vulnerability to HPV infection, and stresses the potential clinical/translational value of the novel dualistic model of HPV-related carcinogenesis. Cancer of the uterine cervix is generally observed near the squamo-columnar (SC) junction and is almost always caused by carcinogenic human papillomaviruses (HPV). However, the nature of cervical epithelial cells initially infected by HPV has been a matter of debate/speculation for a long time. Classically, the HPV-target cells are presumed to be the basal keratinocytes of the pluristratified mucosa lining the outer part of the cervix (ectocervix) and the region where the endocervical columnar epithelium, sensitive to the acidic vaginal pH, has undergone a squamous metaplastic transformation during puberty (transformation zone (TZ)). According to this theory, microtrauma, ulcerative lesions, or abrasions into the squamous epithelium would render the proliferating basal cell layer uniquely vulnerable to viral infection . This hyIn the early 2000s, endocervical reserve cells were also proposed as the cell of origin for cervical cancer . HoweverConsequently, for years, it has been thought that the ecto-endocervical junction microenvironment contains unique epithelial cells highly susceptible to high-risk HPV infection and related (pre)cancer development. However, the topographical preference of cervical neoplasia for the SC junction remained unexplained. In 2012, a discrete population of cells residing within, or in close proximity to, the cervical SC junction was identified  Figure . DisplaySimilar to the skin or other mucosal surfaces, the gynecological tract expresses a distinct set of host–defense peptides adapted specifically for limiting pathogen invasion and replication within this unique environment . InvolveAlthough some unclear points remain  or the pathway(s) involved in HPV entry), the mechanisms governing HPV entry into host cells is now relatively well-characterized . After iAll mucosal HPVs are characterized by a circular DNA genome of approximately 8000 base pairs coding for eight major proteins . Viral gene expression has been shown to be regulated at the level of transcription  between the former and the new SC junction has been shown to be driven by the junctional cell-dependent production of squamous metaplastic (reserve) cells . In gene+ antigen-presenting cells (Langerhans cells (LC) and dendritic cells (DC)), several studies showed that LC/DC density is largely decreased in both the SC junction microenvironment and the cervical (pre)neoplastic lesions compared to the surrounding HPV-uninfected ectocervical/vaginal squamous mucosa [Prior to and following the discovery of SC junction cells, we, and others, reported altered local immune responses within the SC junction or in metaplastic areas (TZ) located in close proximity. Focusing on CD1as mucosa ,44,45. Bs mucosa . The ress mucosa ,47. Mores mucosa . The sigs mucosa . Similars mucosa . The alts mucosa . In addis mucosa . AltogetIn pathology practical examination, it is well established that HPV genotypes impact the morphology of (pre)neoplastic lesions. Low-risk HPVs (mainly HPV6 and 11) are typically related to condylomata acuminata  development and viral cytopathic effect ,52. In cIn addition to lesions demonstrating a squamous phenotype , adenocarcinoma, as well as intriguing lesions that display both squamous and columnar differentiation are observed within the SC junction microenvironment ,58,59. Tink4, the HPV viral load and genotyping were the most investigated. Although, all gave some indications on high-risk patients, a lack of concordance has been extensively reported and all failed to precisely predict CIN, which will finally progress to cancer [Although bi- or quadrivalent HPV vaccines have been on the market for a decade and have been shown to be highly efficient to prevent HPV16/18-related high-grade anal/cervical lesions ,62,63,64o cancer ,73,74,75ink4 expression, full-thickness Krt7 immunoreactivity demonstrated the highest correlation with lesion progression [Supported by the very large discrepancy, in terms of cancer risk, between the uterine cervix and the vagina/vulva, it is now very likely that the nature of the HPV-infected cells strongly influences disease outcome. Recently, four studies analyzed the predictive value of junctional biomarkers  and, interestingly, all showed that low-grade CIN arising from the SC junction have a significantly higher risk to progress to CIN2/3 compared to their counterparts observed in the transformation zone/ectocervix ,13,14,15gression ,77. In aHPV vaccines, recommended for young sexually-naive women, hold promise to make a major impact on cervical cancer mortality in the future. However, a large number of older and/or vulnerable women are in need of a cancer preventive. The discovery of SC junction cells might point the way to a different, low-cost, and simple way to prevent cervical cancer in underserved populations: by removing the vulnerable cells that are its source. This recently-proposed clinical perspective is supported by historical data ,79,80. OWith the development and commercialization of two vaccines, as well as the Nobel Prize attributed to Harald Zur Hausen, experimental research on HPV-related carcinogenesis was supposed to be  over in the late 2000s. Three major findings/confirmations have recently raised the interest of the whole HPV community: (1) the involvement of beta HPV genotypes in skin cancer development ,92; (2)


Despite the introduction of Human papillomavirus (HPV) vaccination in national immunization programs (NIPs), vaccination rates in most countries remain relatively low. An understanding of the reasons underlying decisions about whether to vaccinate is essential in order to promote wider spread of HPV vaccination. This is particularly important in relation to policies seeking to address shortfalls in current HPV campaigns. The aim of this study was to explore prevailing perspectives concerning HPV vaccination among girls, boys, and parents, and so to identify potential determinants of HPV vaccination decisions in these groups.Perspectives were explored using Q-methodology. Forty-seven girls, 39 boys, and 107 parents in the Netherlands were asked to rank a comprehensive set of 35 statements, assembled based on the health belief model (HBM), according to their agreement with them. By-person factor analysis was used to identify common patterns in these rankings, which were interpreted as perspectives on HPV vaccination. These perspectives were further interpreted and described using data collected with interviews and open-ended questions.The analysis revealed four perspectives: “prevention is better than cure,” “fear of unknown side effects,” “lack of information and awareness,” and “my body, my choice.” The first two perspectives and corresponding determinants of HPV vaccination decisions were coherent and distinct; the third and fourth perspectives were more ambiguous and, to some extent, incoherent, involving doubt and lack of awareness and information (perspective 3), and overconfidence (perspective 4).Given the aim of publically funded vaccination programs to minimize the spread of HPV infection and HPV-related disease and the concerns about current uptake levels, our results indicate that focus should be placed on increasing awareness and knowledge, in particular among those in a modifiable phase. Human papillomavirus (HPV) is a common sexually transmitted disease in men and women. Most sexually active women and men become infected at some point in their lives, and infections may also reoccur . DespiteIn 2006, the European Medicines Agency and the US Federal Drug Administration approved the first vaccines against HPV. Because these were the first vaccines aimed at preventing cancer, wide policy debate about whether they should be included in national immunization programs (NIPs) ensued. The Netherlands, where the current study was conducted, was among the first countries to include the vaccine in its NIP, in 2008. However, this was only for girls and not for boys, which is currently still the case. Some countries that introduced the vaccine to their NIPs later also included HPV immunization for boys . DespiteAs the rationale behind publically funded vaccination is to achieve high coverage and, thereby, herd immunity, understanding the reasons underlying the decisions of children and their parents about whether to vaccinate is important to achieve greater permeation of the practice. Such understanding is particularly important for policies aiming to address shortfalls in current HPV campaigns. Furthermore, the novelty of HPV vaccination, as the first immunization measure aimed at preventing cancer, serves as an interesting case providing insight into how this “new generation” of vaccines is valued among designated users. Therefore, the aims of this study were to explore prevailing perspectives on HPV vaccination among girls, boys, and parents, and to identify underlying determinants shaping decisions about whether to vaccinate against HPV.perceived benefits of the vaccine have considerable effects on decisions about whether to vaccinate; the intention to vaccinate increases with the perceived benefits. Commonly reported benefits involve the perceived effectiveness of the vaccine . The average age was 45 (range 29–59) years among parents, and 13 (range 10–16) years among children. Forty-seven parents and children were from the same families . Four children were excluded from the study because a) they did not complete the exercise, b) understand the instructions after repeated explanation, or c) understand the majority of statements.Inspection of the solutions supported by the data yielded a four-factor solution. These factors were sufficiently distinct and clearly interpretable, and explained 50% of the variance. Table This pro-vaccination perspective, with which 45 participants  were associated significantly, emphasized the perceived threat of HPV and HPV-related diseases. An urgency to take preventive measures against HPV prevailed, as reflected in the rankings of statements describing the perceived severity of and susceptibility to HPV and HPV-related diseases. Participants with this perspective believed that they were likely to be exposed to HPV infection (statement 14 was ranked −1), were concerned about its potential consequences to their health , and placed importance on prevention through vaccination .From this perspective, the benefits of HPV vaccination clearly outweighed potential barriers. Vaccination was considered to be a positive means of preventive care to protect oneself and others , with more advantages than disadvantages , giving people the direct ability to influence their health . Taking preventive measures was seen as a small sacrifice relative to the potential consequences of HPV-related diseases, as highlighted in several answers to open-ended questions with the recurring idiom of “prevention is better than cure.”who should make the vaccination decision . Parents stated that they would rather announce this decision to their children than negotiate it with them. They felt strongly that children receiving formal invitation letters for local vaccination sessions could not understand such complex information and were incapable of making informed decisions.Another theme that emerged within this viewpoint was the perceived complexity of HPV and HPV-related diseases, reflected in beliefs about Given the perceived complexity of the topic, participants with this perspective valued professional advice from trusted sources, such as governmental recommendations . This perception may explain the minimal value placed on social influences : one mother said, “I do not rely on the opinion of my neighbor or sister-in-law; I collect good information from the Internet about pros and cons and then make my choice.” These participants’ trusting relationships with the government may additionally explain their generally favorable opinion of HPV vaccination. In ex-post interviews, respondents opined that the government would not add a vaccine to the NIP without proper and relevant justification. In the context of healthcare and the “medical world” in the Netherlands, participants with this perspective trusted the government to make good, wise choices with people’s best interests in mind.This perspective, with which 46 participants  were associated significantly, did not involve the strength and force to take action against HPV through vaccination, as the perceived severity and susceptibility of HPV-related disease was minimal. The perceived likelihood of exposure to HPV-related disease was minimal  and concern about contracting HPV-related disease was negligible . Hence, respondents with this perspective did not perceive HPV to be sufficiently important to warrant vaccination .Perceived barriers, particularly fear about the potential unknown long-term side effects of HPV vaccination , dominated this perspective. In this way, HPV vaccination was distinct from other childhood vaccinations . One mother stated, “[I have] concerns about the side effects in the long term. The vaccine was developed only recently. The possible long-term side effects cannot be known yet.” Respondents also referred to the effectiveness of the vaccine, emphasizing that the current vaccine does not protect against all cancer-causing HPV strains. One interviewee also questioned the duration of the vaccine’s protection against HPV infection.Respondents’ fears were also linked to mistrust of pharmaceutical companies, perceived as profit-making entities that wanted to sell their products, even at the cost of others’ well-being . Participating mothers stated, “Ah yes, surely they make plenty of money with it. Without really telling the truth about the severe side effects” and “This is nothing new. The pharmaceutical companies often are big crooks with dollar signs in their eyes. The humane element in the pharmaceutical industry has gone, regrettably.” This mistrust of the pharmaceutical industry and the prevailing fear about potential negative consequences made respondents perceive children vaccinated against HPV as test objects; one mother said, “I don’t really want my daughter to act as a guinea pig or for people to make money out of her at the cost of her health.” Thus, these participants considered it better to be safe and not get vaccinated, than to regret vaccination due to potential future side effects . However, they also expressed that only the future could tell whether they had made the right decision.This perspective also involved a lack of trust in the government to give advice about HPV vaccination . One interviewee referred to the government’s hasty choice to include the HPV vaccine in the NIP. In addition, these respondents placed little value on the role of GPs relative to those with other perspectives .Participants with this perspective perceived that adequate and objective information on HPV vaccination was difficult to find , which may partly explain the perception that they were inadequately informed about HPV and related diseases . In addition, they perceived HPV as a complex topic and thus felt that parents should make vaccination decisions . One respondent emphasized that her daughter was very compliant, with a constantly altering opinion depending on with whom she had spoken.The defining feature of this perspective, with which 38 participants  were associated significantly, was lack of awareness and information about HPV and HPV-related disease, which posed a major barrier to informed decision making . Many participants stated that the topic of HPV was completely unclear for them, and some had not heard about HPV before this study. Interviewees linked HPV with cervical cancer, albeit diffusely. Given their limited awareness and information, they pondered the advantages and disadvantages of vaccination, being unclear about its potential side effects  while perceiving the benefits of vaccination in general, and HPV vaccination specifically, as important .This perspective involved the least perceived severity among viewpoints of the potential consequences of HPV-related disease  and a neutral perception of susceptibility . Yet, interviewees stated that cervical cancer could be severe. This contradictory view could be explained by the expressed lack of information, awareness, and clarity regarding the link between HPV and  cancer.Respondents valued friends’ and family members’ opinions about the HPV vaccine , and thus appeared to be influenced more easily by the social environment. In contrast to participants with other perspectives, those with this viewpoint valued GPs’ advice highly . A 14-year-old girl said, “I think advice from the GP is important because you don’t know the drawbacks of an HPV shot. There is not enough information about the HPV shot” and a father stated, “Explanation by a GP gives you some more certainty! He is a specialist after all.”Interviewees wished that they knew more about HPV and HPV-related disease. When asked how information should be distributed, one respondent suggested more active communication about the vaccine through spot advertisements and increasing awareness through GPs and informational pamphlets distributed with formal invitations for other vaccinations.In addition, many children holding this view appeared to trust and rely on their parents’ decision, but felt that they should have a say in this decision despite their limited knowledge about the HPV vaccine. A 13-year-old girl, who had declined vaccination, stated that she would have liked to know more about HPV before making a decision and expressed anticipated regret if she were to acquire an HPV-related disease later in life. However, she declined having plans to rethink her decision.The 22 participants , in large majority girls, associated significantly with this perspective did not perceive HPV-related diseases as a particular threat to health. Perceived susceptibility was especially low, as respondents did not seem to worry about exposure to HPV and related diseases compared with those with other perspectives . One 14-year-old girl stated that she had decided to decline the HPV vaccine, but not other childhood vaccinations: “…these I felt were important and therefore I accepted them.” This viewpoint was further justified by the fact that common childhood vaccinations prevent diseases perceived as more common than HPV-related disease.Yet, respondents felt clearly that making the decision to vaccinate before becoming sexually active was important . Although they believed that vaccination in general benefitted oneself and others , respondents representing this perspective seemed to be undecided about whether HPV vaccination has more disadvantages than advantages . One interviewee referred to unknown side effects, and another referred to the effectiveness of the vaccine, stating she had heard that the vaccine’s ability to protect against cervical cancer was uncertain.The most important aspect of this viewpoint was control over the decision of whether to vaccinate. Children believed that they should make this decision , positioning themselves (as vaccine recipients) centrally in responses to open-ended questions and in ex-post interviews; a 15-year-old girl stated, “It’s your body so I think you may take the decision yourself.” Children also expressed that their opinions were equally important as their parents’, if not more important: 14- and 13-year-old girls stated, “Well, they thought my opinion was really important; so I didn’t have to get it if I didn’t want it” and “Yes, [my parents] thought my opinion was more important.”However, children also valued their parents’ knowledge and saw the decision as joint : 12- and 14-year-old girls said, “But it may be that as a child you are not too sure about what it involves, so therefore I find it important that parents also have a say in this” and “As a child you don’t know much about this but your parents just a bit more, so it’s useful to have them help decide.”.A probable cause for this rather contradictory view on who should make the decision is that children want to be able to decide who can influence the decision: preferably only people from their “circle of trust” . Yet, those with this perspective greatly appreciated GPs’ recommendations ; a 13-year-old girl said of her GP, “He knows me,” and referred to GPs’ medical knowledge about their clients. Yet, neither girl with this perspective who was interviewed had actively reached out to her GP for advice, despite having decided to decline the vaccination. Expert advice from other sources, such as the government, was not considered to be relevant . However, most children did not appear to fully understand the term “government” or what it entailedLack of information did not appear to be a barrier for respondents with this perspective , even those who expressed that they knew little about HPV and HPV-related disease. Compared with participants with other perspectives, they felt that knowledge, when needed, was easily obtainable from parents or GPs .Although many studies have explored beliefs regarding the HPV vaccine, this study is the first to investigate jointly the prevailing perspectives of girls, boys, and parents, and to identify determinants underlying HPV vaccination decisions. The results revealed four perspectives: “prevention is better than cure,” “fear of unknown side effects,” “lack of information and awareness,” and “my body, my choice.”what information would be adequate and by whom – other than government or the pharmaceutical industry – it should be provided to satisfy them. In both of these perspectives, the vaccination decision seems to be well considered and clear.Our results suggest that the first two perspectives were coherent and very distinct. Those with perspective 1 favoured vaccination, as it is not only promoted by a trusted source (the government), but is also an effective means of preventing HPV-related disease, thereby minimizing perceived susceptibility and severity. These determinants coincide strongly with previous findings about HPV vaccine uptake , 27, 33.In contrast, the determinants underlying perspectives 3 and 4 appear to be ambiguous and, to some extent, incoherent. Those with perspective 3 clearly showed a lack of awareness and information needed to make informed decisions about HPV vaccination. This perspective appears to involve doubt about vaccination, as contracting HPV infection or HPV-related disease was not perceived as a particular threat, and neither benefits nor barriers clearly carried more weight. Previous studies have shown that knowledge has different effects on vaccination decisions. Lack of knowledge or information can be a reason to decline vaccination , but it In relation to the HBM, the vaccination decision in perspective 3 can be interpreted as being in a modifiable phase, given the clear lack of vaccination preference and prevailing emotions regarding the topic. Those with this perspective are not likely to vaccinate against HPV, as they feel no sense of urgency in terms of perceived threat or benefit. Contrary to those with the first two perspectives, the deliberations of those with perspective 3 appear, at best, to be characterized by doubt. Those with perspective 4 (largely children) are arguably also in a modifiable phase, with minimal awareness and perceived severity and susceptibility of HPV and HPV-related disease, and no clear idea of HPV immunization benefits and barriers. The combined lack of awareness and focus on children’s roles in vaccination decision suggest that these individuals are overconfident in children’s ability and willingness to acquire and weigh the required information to make well-considered decisions about HPV vaccination.Interestingly, and significant for the future uptake of this new generation of vaccines of which HPV is considered to be the first, the importance of individual deliberation and choice in immunization programs and vaccination policies is increasing . In manyAlthough the participants in this study were sampled purposively, exploring associations between perspectives and background characteristics may generate interesting ideas for further research. Firstly, most parents held perspectives 1 and 2. Most children held perspectives 3 and 4, or were not associated with any perspective, possibly indicating lack of clear perspective about this topic. Secondly, perspectives were not associated with parents’ gender, but more girls were associated with perspective 4 and more boys with perspective 1. Thirdly, perspectives were not associated with parents’ education level, but children with lower education more often associated with perspectives 1, 4, or had no clear perspective. Fourthly, participants with perspectives 1 and 2 scored higher on personality trait conscientiousness, while those with perspective 4 scored lower; other Big Five Inventory  personalThe questionnaire also included questions about HPV vaccination. Firstly, parents who had received the formal invitation letter for their child’s vaccination more often held perspectives 2 and 4, whereas more of those who had not yet received this letter associated with perspectives 1 and 3, or no perspective. Secondly, those with perspective 2 more often had actively searched for information about HPV vaccination, and those with perspective 3 or no clear perspective less often. Thirdly, those with perspective 2 scored higher on a knowledge of HPV scale, adapted from Gefenaite et al. . This coThe findings of this study are important for several reasons. First, the modifiable-phase status of those with perspectives 3 and 4, who have not (yet) made well-considered decisions about HPV immunization, emphasizes the need for investment in tailor-made independent communication policies  to enhance HPV immunization. A focus on increasing awareness and knowledge is particularly important among those with perspectives 3 and 4, who appeared to be rather unfamiliar with the topic of HPV. The involvement of adolescents in decision-making is a particularly important opportunity to provide HPV education. HPV awareness could be increased by incorporating informational sessions in schools’ sexual health education classes. When providing information to those with perspectives 3 and 4, a focus on solidifying the link between HPV and cervical cancer, and emphasis that cervical cancer is only one of many severe consequences of persistent HPV, would be beneficial. Second, as suggested by Griffioen et al. , there aThis study has some limitations. First, as the aim of a Q-methodological study is to describe prevailing perspectives, the current data do not indicate how common these perspectives are in society; such information, acquired with a survey approach , could bOur study identified four prevailing perspectives on HPV vaccination among girls, boys and parents. Given the aim of publically funded vaccination programs to minimize the spread of HPV infection and HPV-related disease and concerns about current uptake, knowledge of existing perspectives on HPV vaccination in the public is crucial for policies wishing to promote uptake. HPV vaccine clearly deviates from other childhood vaccinations in NIP’s, as vaccinating against HPV is not necessarily considered “the natural thing to do”. Increasing awareness and knowledge by investing in tailored-made communication policies could help promote the uptake among those not familiar with the vaccine. This is especially important among those holding perspective 3 or 4, who appeared to be in a modifiable phase. Despite the non-generalizability of this study, the methods and results of this study could contribute to the development of campaigns wishing to address uptake of HPV vaccination.


Polymerase chain reaction-based Xpert human papillomavirus (HPV) assay is a rapid test that detects high-risk HPV (hrHPV) infection. This point-of-care test is usually performed by collecting a cervical specimen in a vial of PreservCyt® transport medium. We compared HPV test positivity and accuracy between self-collected sample with a dry swab (s-DRY) versus physician-collected cervical sampling using a broom like brush and immediate immersion in PreservCyt (dr-WET).In this cross-sectional study, we recruited 150 women ≥ 18 years old attending the colposcopy clinic in the University Hospital of Geneva. Each participant first self-collected a vaginal sample using a dry swab and then the physician collected a cervical specimen in PreservCyt. HPV analysis was performed with Xpert. Part of the PreservCyt-collected sample was used for hrHPV detection with the cobas® HPV test. HPV test positivity and performance of the two collection methods was compared.HPV positivity was 49.1% for s-DRY, 41.8% for dr-WET and 46.2% for cobas. Good agreement was found between s-DRY and dr-WET samples (kappa±Standard error (SE) = 0.64±0.09,), particularly for low-grade squamous intraepithelial lesions (LSIL+) (kappa±SE = 0.80±0.17). Excellent agreement was found between the two samples for HPV16 detection in general (kappa±SE = 0.91±0.09) and among LSIL+ lesions (kappa±SE = 1.00±0.17). Sensitivities and specificities were, respectively, 84.2% and 47.1%(s-DRY), 73.1% and 58.7%. (dr-WET) and 77.8% and 45.7% (cobas) for CIN2+ detection. The median delay between sampling and HPV analysis was 7 days for the Xpert HPV assay and 19 days for cobas. There were 36 (24.0%) invalid results among s-DRY samples and 4 (2.7%) among dr-WET (p = 0.001). Invalid results happened due to the long interval between collection and analysis.Self-collected vaginal dry swabs are a valid alternative to collecting cervical samples in PreservCyt solution for HPV testing with the Xpert HPV assay.HPV self-collection with dry cotton swabs might assist in the implementation of an effective screening strategy in developing countries.ISRCTN83050913International Standard Randomized Controlled Trial Number Registry  In recent years, the development of high-risk (hr) human papillomavirus (HPV) tests has created an important change in our approach to cervical cancer (CC) screening. Overwhelming evidence from several randomized trials has shown that HPV screening is more effective than cytology in preventing CC, with better sensitivity and less frequent screening , 2.Developed countries are progressively incorporating HPV testing into their national screening programs and updating their current guidelines . DevelopThe introduction of a rapid point-of-care non-batch assay, which facilitates same-day screen and management strategies, is essential in developing countries, especially for screen-and-treat strategies. This approach minimizes the need for repeated visits, encouraging greater numbers of eligible women to participate in the program. The ideal system should be modular and easily integrated into low and medium-income countries (LMIC). Furthermore, real-time polymerase chain reaction (PCR)-based hrHPV tests with a high analytic sensitivity are preferable to ensure similar accuracy between clinician- and self-collected samples .The Xpert HPV assay from Cepheid  is a non-batch qualitative real-time PCR assay that has the capability to perform point of care hrHPV testing. The assay is formatted in a single-use test cartridge and can be completed in 1 hour, meaning it can be used in a screen-and-treat strategy adapted to the LMIC context.Xpert was previously evaluated for CC screening with specimens collected into a vial of PreservCyt® transport medium  –10. HoweIn a previous self-HPV study we found that swabs transported in a dry state provided test results that were comparable to those obtained with swabs shipped in a wet transport medium, in terms of quality of results . The posThe aim of this study was 1) to compare HPV test positivity between self-HPV with a dry swab (s-DRY) versus physician-collected cervical sampling using a broom like brush and immediate immersion in PreservCyt (dr-WET) and 2) to assess the accuracy and agreement between the two collection methods.From March 4, 2015 to October 31, 2015, 150 women were consecutively recruited from the colposcopy clinic of Geneva University Hospitals, Switzerland. Patients who attend this clinic are usually women with abnormal cytological results, persistent HPV infection, or follow-up of an untreated CIN1 lesion. Women were eligible if 1) they were at least 18 years old, 2) they understood the study procedures, and 3) voluntarily agreed to participate by signing a written informed consent form. Pregnant women and those with a history of total hysterectomy were excluded. The Cantonal Human Research Ethics Commission of Geneva approved the study .The protocol for this trial and supporting STARD and TREND Statement checklists are available as supporting information; see In this cross-sectional study, all women completed a self-administered questionnaire on demographics data. Eligible women were asked to perform self-HPV (s-DRY). Each participant received a package containing a specimen collection cotton swab in a plastic tube and instructions for use. They were instructed to wash their hands before the procedure. The instructions stated to hold the plastic head of the swab and insert the cotton tip of the swab into the vagina, avoiding contact with the external genitalia, until it met resistance. Holding the swab inside the vagina it should be gently turned 3 to 5 full rotations. After taking out the swab from the vagina and always holding the swab from the plastic head it should be placed inside the plastic tube. Following the self-collection and before colposcopic examination, the physician collected a cervical specimen using a Cervex brush , which was immediately placed in PreservCyt (dr-WET).Roche Diagnostics, Basel, Switzerland). Then, with the remaining PreservCyt sample, a cytological preparation was performed if a Pap smear result was not available from the last 3 months.The two specimens (s-DRY and dr-WET) were tested for the same pathogens (hrHPV) using the same diagnostic test (Xpert). In addition, a part of the PreservCyt-collected sample (dr-WET) was used for hrHPV detection with the Food and Drug Administration (FDA) approved cobas® HPV Test . The study protocol is available in After specimen collection was performed in the colposcopy clinic, both samples were stored at room temperature before being analyzed. In some cases, sampling and analysis was not performed on the same day, since the Xpert instrument only had 3 operative modules.In the laboratory, dry swabs (s-DRY) were rinsed into tubes with 3 ml of 0.9% NaCl and vortexed 3 times for 15 seconds each. Then, 1 ml of each sample was transferred to the cartridge and a four-module Xpert assay was performed. PreservCyt vials containing dr-WET samples were also vortexed 3 times for 15 seconds each. Next, 1 ml of each sample was transferred to the cartridge and an Xpert assay was performed. The s-Dry and dr-WET aliquots from each patient were simultaneously analyzed by the Xpert HPV Assay.HMBS [hydroxymethylbilane synthase]) and an internal Probe Check Control (PCC). The PCC verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity and dye stability. Threshold (Ct) values reflect the relative number of cells in the sample. A lower Ct-value represents a relatively higher number of cells.Xpert is a multiplexed real-time PCR test that targets the E6 and E7 oncogenes of 14 cancer-related HPV types. It uses as an internal assay control for specimen adequacy the detection of a human reference gene . The assay utilizes six fluorescent channels for the detection of individual types of HPV, groups of HPV, and the human reference gene. Each fluorescent channel has its own cut-off parameters for target detection/validity. These fluorescent channels detect: 1) HPV 16; 2) HPV 18 and HPV 45; 3) HPV 31, 33, 35, 52, and 58; 4) HPV 51 and HPV 59; 5) HPV 39, 56, 66, and 68; and 6) is also detected. Samples were analyzed according to the algorithm of Roche Diagnostics. This test allows the specific identification of HPV 16 and HPV 18 and detects 12 pooled hrHPV genotypes  at clinically relevant infection levels.part of the sample immersed in PreservCyt (dr-WET) was tested for HPV DNA using cobas at Biopath Lab, Lausanne. DNA was extracted from 400 μL of each sample. The cobas is a qualitative real time PCR assay using the cobas R4800 System. The detection is based on amplification of the L1 gene using TaqMan probes. The human reference gene ß-globin Cytologic preparations were made using the ThinPrep (Hologic) technology for all women. Cytotechnologists read the slides and a pathologist verified abnormal results for a final diagnosis. The results were classified according to the Bethesda System for cervical cytology.Histopathological preparations of biopsies and endocervical brushings were interpreted at the Department of Pathology and Immunology, University of Geneva. Patient management was provided according to the Swiss standards in the colposcopy clinic of Geneva.Data were analyzed using Stata Statistical Software Release 13 . Total agreement, percent positive agreement between s-DRY and dr-WET samples for all hrHPV types and each HPV channel, overall and stratified on pathological cytology, were measured. The Cohen's kappa coefficients were also determined. The proportion of positive agreement between paired s-DRY and dr-WET samples was calculated using 2a/(f1 + g1), where a is the number of samples that were positive for HPV in both tests, f1 is the number of samples that were positive for s-DRY and g1 is the number of samples that were positive for dr-WET.HMBS adequacy test were calculated for the two collection modalities. We also analyzed invalid test results from Xpert and the effect of a delay between self-collection and HPV analysis.We used low-grade squamous intraepithelial lesions or worse (LSIL+) as a cut-off, which includes LSIL, high-grade squamous intraepithelial lesions (HSIL), adenocarcinoma in situ, and invasive cervical cancer (CC). Sensitivity and specificity and positive and negative predictive values were also calculated for each method, using cobas results as the reference standard for hrHPV, since it is a FDA approved HPV test. Histological results  were used as the reference standard for cervical lesions. McNemar’s two-tailed test was used for mutual comparison of sensitivity and specificity. P values less than 0.05 were considered statistically significant. The mean Ct-values for the Kaplan-Meier curves were done to examine the association between the number of invalid HPV test results and the elapsed time for both s-DRY and dr-WET samples.Invalid HPV tests were excluded in the agreement and accuracy analysis.A sample size of 150 women was calculated to provide a 10% precision to estimate the kappa coefficient, if the kappa is 50% . Assuming a 40% prevalence of the HPV infection in our selected population, the precision of other measures will be more or less 15%.Fig 1. The median age of the participants was 32 years (interquartile range [IQR] 27–41), and the majority had a partner (80.0%). Sociodemographic data are represented in Table 1. The median delay between sampling and Xpert HPV assay analysis was 7 (IQR 2–23) days. The median delay between sampling and cobas HPV analysis was 19 (IQR 11–25) days. The greater delay between collection and analysis for the cobas test was due to the fact that the sample needed to be transported to another laboratory outside the Geneva University Hospitals in order to be processed. Moreover, the cobas is a batch test, which means that we needed to have enough samples to run the analysis. The Xpert HPV assay is a non-batch test and it was located next to the Colposcopy clinic, which facilitated the whole process.Overall, 150 patients were included in the study. The study flowchart is shown in HPV positivity was 56 (49.1%) for s-DRY samples, 61 (41.8%) for dr-WET samples and 67 (46.2%) for cobas. Among dr-WET samples, HPV positivity was 23 (15.8%) for HPV 16, 10 (6.9%) for HPV 18/45 and 28 (26.0%) for other hrHPV types. Among s-DRY samples, HPV positivity was 21 (18.4%) for HPV 16, 7 (6.1%) for HPV 18/45 and 34 (29.8) for other hrHPV types. Using the cobas test, HPV positivity was 25 (17.2%) for HPV 16, 5 (3.5%) for HPV 18 and 47 (32.4) for other hrHPV types.The mean Ct-values for the β-globin gene were 25.1 ± 14.3 for s-DRY samples and 30.5 ± 5.0 for dr-WET samples (p < 0.001). A lower Ct-value represents a relatively higher number of cells.Fig 2, a Kaplan–Meier failure estimate shows a clear increase in the proportion of invalid tests with increasing time elapsed between s-DRY samples.There were 36 (24.0%) invalid results among s-DRY samples and 4 (2.7%) among dr-WET (p = 0.001). The invalid results among s-DRY samples were due to the long interval between collection and analysis; all invalid results occurred in samples that waited 6 or more days before processing. A delay increased invalid results from 1.6% in the first week to 19.5% during the second week and 56.3% after more than 2 weeks (p < 0.001) among s-DRY samples. The median interval between sampling and analysis was 5.5 (2–10) days among valid results and 25 (15.5–35) days among invalid results (p < 0.001). In Fig 1). Forty-eight (32.2%) patients had LSIL+. A total of 74 (49.3%) patients were biopsied. There were 20 (27.0%) negative results, 27 (36.5%) CIN1, 16 (21.6%) CIN2, 9 (12.2%) CIN3 and 2 (2.7%) CC cases. Twenty-seven (36.5%) patients had CIN2+ (Table 2). The test positivity increased with increasing severity of diagnosis for both s-DRY and dr-WET methods.Among the 149 (99.3%) patients with cytological results, there were 73 (49.0%) with negative cytology, 22 (14.8%) with atypical squamous cells of undetermined significance (ASC-US), 6 (4.0%) with atypical squamous cells-cannot rule out high grade (ASC-H), 35 (23.5%) with LSIL, 12 (8.1%) with HSIL and 1 (0.7%) with CC , for hrHPV detection and for each HPV channel. The kappa (95% CI) between s-DRY and dr-WET for hrHPV was 0.64 (0.50–0.78) in general, 0.53 (0.34–0.73) among those with less than LSIL and 0.80 (0.59–1.00) among LSIL+ cases. The kappa was higher for the detection of HPV 16: 0.91 (0.80–1.00) in general, 0.84 (0.65–1.00) among those with less than LSIL and 1.00 among LSIL+ cases. The kappa was also excellent among LSIL+ cases for the detection of HPV 18/45 (1.00).Overall, the agreement ranged from 82.0% to 97.3% and the positive agreement ranged from 50.0% to 92.3%. Among those with less than LSIL, the agreement ranged from 77.9% to 96.1% and the positive agreement ranged from 33.3% to 85.7%. Among those with LSIL+, the agreement ranged from 90.9% to 100% and the positive agreement ranged from 57.1% to 100%.Overall, 111 women had a valid s-DRY and dr-WET test result. Three women had s-DRY valid test but dr-WET invalid, 35 women had dr-WET valid test but s-DRY invalid and 1 woman had both tests invalid.Table 4).Sensitivity of the s-DRY method for hrHPV using the cobas results as the reference standard was 79.3% (95% CI: 65.8–88.3), compared with 78.8% (95% CI: 50.7–67.0) for the dr-WET method (p = 0.763). We found specificities of 80.4% (95% CI: 67.4–89.0) and 93.3% (95% CI: 84.7–97.3) (p = 0.034) for the s-lDRY and dr-WET methods, respectively  and specificity of 47.1% (95% CI: 30.4–64.4); dr-WET sensitivity was 73.1% (95% CI: 51.7–87.3) with specificity of 58.7% (95% CI: 43.6–72.3).The Xpert HPV assay has been previously evaluated –10. To oHPV positivity was higher for vaginal dry samples than cervical wet samples (49.1% vs 41.8%). The mean Ct-values for the β-globin gene were 25.1 ± 14.3 for s-DRY samples and 30.5 ± 5.0 for dr-WET samples, which means that dry samples had a relatively higher number of cells comparing to wet samples (p < 0.001). Moreover, good agreement was found between s-DRY and dr-WET samples. The agreement improved in pathological cytology cases (LSIL+). Better agreement between the two samples was also found for HPV 16 and HPV 18/45. Agreement was only fair between the two samples for genotypes 39, 56, 66 and 68. These results are consistent with previous studies comparing dry and wet samples using different HPV DNA tests such as the Roche Linear Array , SeegeneDry samples had a clinical performance similar in terms of sensitivity to wet samples for hrHPV using cobas as the reference standard. However, for detecting CIN2+, there was a trend towards a better performance with s-DRY as compared to dr-WET or cobas, although these differences did not reach statistical significance. Equivalent and even improved sensitivity of self-HPV was found in previous studies when PCR-based HPV tests were used , 15, 16.We acknowledge some limitations of our study. We enrolled a population of women referred to the colposcopy clinic due to abnormal cytological results, persistent HPV infection, or follow-up of an untreated CIN1 lesion. This led to a high HPV prevalence , resultiIn the present study, invalid results increased as the storage interval increased, from 1.6% in the first week to 19.5% during the second week and 56.3% after more than 2 weeks (p < 0.001). Consequently, dry samples should be analysed within 1 week of collection in order to guarantee valid results. Wet samples were much less affected by a delay, with only four invalid samples, which is not surprising since PreservCyt medium is known for its long-term stability . The XpeThe strengths of our study include the ability to compare the s-DRY and dr-WET samples within categories of cytological and histological diagnoses. Moreover, we have also assessed agreement according to HPV channel and evaluated the performance of s-DRY, dr-WET and cobas among women with CIN2+ diagnoses.In conclusion, our data indicate that HPV self-collection with dry cotton swabs is a valid alternative to wet-collected cervical samples for the Xpert HPV assay if the samples are tested within a short period (less than 1 week) and might assist in the implementation of an effective screening strategy in developing countries. Further validation with real-world testing and large population-based prospective cohort studies is warranted to confirm our results.S1 Checklist(PDF)Click here for additional data file.S2 Checklist(DOCX)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.