Cervical cancer is a highly preventable disease. To not attend an organized cervical cancer screening program increases the risk for cervical dysplasia and cervical cancer. The aim was to investigate the participation rate in three different intervention groups for non- attendees in the Swedish national program for cervical screening. The participation in the recommended follow up, and the histology found were also examined.Population-based randomized control trial. It included10,614 women that had not participated in the cervical cancer screening programme during the last six years (ages 30–49) and the last eight years (ages 50–64) were randomised 1:1:1; an HPV self-sampling test only; or the routine procedure with a yearly invitation).In the intention to treat analysis the participation rates were 25.5% (N = 803/3146) vs 34.1% (N = 1047/3068) and 7.0% (N = 250/3538) (p<0.001) for telephone, HPV self-test and control groups respectively. In the by protocol analysis including women that answered the phone call the participation rates were 31.7% (N = 565/1784) vs 26.1% (N = 788/3002) and 7.0% (N = 250/3538) (p<0.001) for telephone, HPV self-test and control groups. The corresponding results in the by protocol analysis including women that did not answer the phone call was 19.7% (N = 565/2870) vs 26.1% (N = 788/3002) and 7.0% (N = 250/3538) (p< 0.001). The majority of the women 63,4% (1131/1784) who answered the telephone wanted to participate either by booking a visit for pap smear  or to be sent a HPV self- sampling test  (p<0.001). Women who chose an HPV self-test were older and gave anxiety/ fear as a reason to decline participation, and they were also less likely to participate in the follow-up if found to be HPV-positive compared to the women who chose a Pap smear. The attendance to the recommended follow-up after abnormality was in total 87%. The non-attendees had a three or eight times higher risk of having a cytology result of HSIL or suspected SCC respectively, in the index sample compared to women screened as recommended . A total of ten SCC and one adenocarcinoma were found in the histopathology results from the non-attendee group with a study intervention, while there was only one SCC in the non-attendee group without any study intervention .Our study suggests, according to intention to treat analysis, that the best intervention to get as many non-attendees as possible to participate is to send an HPV self-sampling test together with an invitation letter. Almost 90% of women in the study with an abnormal index sample attended follow-up. This is high enough to indicate that interventions to increase the participation among non-attendees are meaningful.International Standard Randomised Controlled Trial Number (ISRCTN)ISRCTN78719765.Registration number  In Sweden, 559 new cases of cervical cancer were identified in 2017, i.e. 11.2 cases per 100 000 women . There aprimary aim of this study was to evaluate the participation rate comparing different interventions among the non-attendee women and to describe the attendance to recommended follow-up . The secondary aim was to determine what intervention the non-attendees would choose if, in a telephone call with a midwife, they were given the possibility to choose between an HPV self -test outside the clinic and a visit for a Pap smear. We also hypothesised that we would find more abnormal index samples in non-attendees than in attendees. Our third aim was therefore to describe the cytological diagnoses found in the index Pap smear and compare them with those found in attendees during the same period. The histological diagnosis found in the follow-up will also be described.We hypothesised that the participation rate would increase if the non-attendees could choose between different test methods presented by a midwife in a telephone call (an HPV self-test or Pap smear in the clinic) or if they received an HPV self-test directly by post for sampling rather than yearly invitations (routine procedure). Therefore the Non-attendees were defined as women who had not participated in the cervical cancer screening programme during the last six years if aged 30–49, and during the last eight years if aged 50–64. This strategy was in accordance with the Swedish age-differentiated screening intervals at that time. The population was defined as non-attendees living in the region of Östergötland, Sweden on March 8th 2016. Data were extracted from the Swedish national cervical cancer screening registry (NKCx) which has almost 100% coverage of invitations. The results of Pap smears and biopsies can be extracted from the personal identification number, which is unique to each individual. The results from the HPV self-tests were extracted from a local registry in the clinic. Data concerning follow-up results were obtained until the 31th of December 2017 when the study period ended. The non-attendees’ addresses were obtained from the Swedish Population Register (SPR). For comparison, all women (N = 44 938) from the region of Östergötland that completed the last two previous cervical cancer screening rounds were included in the analysis concerning cytology results. They were defined as the attended group. The data were extracted from the Swedish national cervical cancer screening registry (NKCx).The non-attendees were randomly, 1:1:1, assigned to one of three different groups: telephone, HPV self-test or control with no intervention except for the yearly invitations (routine procedure). The study groups were computer randomised and adjusted for area code and age by a statistician uninvolved in the study. All three groups received the yearly invitations which were distributed equally over the study period. The first analysed sample, Pap smear or HPV test, in all groups is referred to as the index sample. We considered that the different interventions had an effect if an HPV self-test was ever returned during the study period or a Pap smear was analysed within six months from the date a study invitation letter was sent. Non-attendees randomised to the telephone group were offered a choice of different sampling options, as presented below. Invitation letters were posted between April 12th 2016 and 31st May 2017.Free of charge HPV self-tests (Cobas® PCR Media Uni Swab kit) were distributed between April 2016 and May 2017 to non-attendees randomised to the HPV self-test group, accompanied by an invitation letter with information about the aim of the study and instructions on how to correctly collect the sample, together with descriptive illustrations. The invitation letter gave information about recommended follow-up if the HPV self-test was positive. A pre-paid envelope was included in the invitation letter. The participants were instructed first to wash their hands, second to insert the swab at least 5 centimetres into the vagina, and rotate the swab for approximately 30 seconds. They were instructed then to place the swab into a test tube, avoiding contamination with the surrounding environment. The women were asked to carefully check that the personal identification number was correct before affixing the pre-printed labels onto the test tube. If the HPV self-test was not returned within a month a reminder letter was sent. If the HPV self-test returned was positive, the women were booked for a colposcopy at the outpatient clinic. Non-attendees with a negative HPV were informed of the result by letter and told that in the future they would be invited to attend screening according to the regular cervical cancer screening programme.Telephone numbers were collected by a medical secretary from different sources such as medical records, local registries and websites listing telephone numbers. Only when a telephone number was found, a study invitation letter with information was possible to send. The letters were sent between April 2016 and February 2017. In total, 24 midwives called non-attendees within a month from the date the invitation letter was sent, offering an appointment with a midwife for a Pap smear at the out-patient clinic, or the option to receive an HPV self-test at the address registered in the SPR, both options free of charge. The telephone call was presented with a number from the hospital if the women could see the sender on the display on their telephones. Three attempts were made to reach the non-attendees. Telephone calls were mainly carried out in the evenings between 5- and 8 pm. All midwives participating in the study underwent education about HPV and the cervical cancer screening programme. Questions about reasons for not participating in the cervical screening programme were also asked and all midwives followed a manual during the telephone call. Information about the association between HPV and cervical cancer was given. Non-attendees who chose an HPV self-test also received study information by a letter and instructions (see above). No reminder letter was sent to this group. The above described procedure concerning positive versus negative HPV results for the women choosing an HPV self-test was also applied with the telephone group. If an appointment for a Pap smear was chosen, the result of the index sample was handled according to the current medical guidelines. Non-attendees who did not choose any of the interventions were recommended to attend the cervical cancer screening programme or to visit the clinic for a Pap smear during drop-in times. If the non-attendee was found to have previously undergone a hysterectomy a special form was sent to an administrator to see if the women could be excluded from the screening program. Non-attendees who said they did not want to participate in the screening programme in the future received a special form by post to complete.The HPV self-test was analysed on the Cobas 4800 system . PreparaThe Pap smears were collected with the Surepath liquid-based Pap-test and diagnosed by cytologists in an accredited laboratory .Non-attendees with a positive HPV self-test or a Pap smear with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesions (LSIL) with positive HPV in the reflex test were booked for examination with colposcopy and possible biopsy at the outpatient clinic. All non-attendees with cytology that could not exclude a high-grade squamous intraepithelial lesion were also investigated with colposcopy and biopsy, but without an HPV test according to the medical guidelines. The results from the colposcopy and biopsy determined future investigations and treatments.Data were analysed with descriptive statistics. To reach a power of 80% regarding the participation rates in each study arm, 723 non-attendees were needed in each study arm. The rejection of the null-hypothesis was set to 0.05 (two-sided) in all statistical analyses. A student’s t-test was used to test differences between quantitative variables. A Pearson’s Chi-square test was used for testing differences in proportions between categories. Odds ratios were used as a measure of association, and are presented with exact confidence limits.The intention to treat analysis included the study population randomized to each group excluding the individuals that had given a Pap smear before the invitation letter was sent .The by protocol analysis included the study population randomized to each group, excluding the individuals that had given a left Pap smear before the invitation letter was sent or who did not have an address (the HPV self-test group and Telephone group) and did not have phone number (the Telephone group). A comparison was made in regard to whether the women answered the phone call from the midwife or not (the Telephone group) .All statistical analyses were carried out done with Stata v 15.1, Stata Corp LLC, College Station, TX, USA. Regional Ethical review board concerning the information to the study population, came after the randomization had taken place but before the invitation letters were sent. The consent was giving verbally and/or confirmed by taking a sample (Pap smear or HPV self-test) after the study information was given. The attending group of women were extracted anonymously from the Swedish national cervical cancer screening registry for comparison reasons. The trial was not registered in ISRCTN or ClinicalTrials.gov before the enrolment of participants started because this was not recommended by the ethical board.This study was approved by the Regional Ethical Review Board in Linköping, Sweden (Dnr 2015/480-31 dated 27/01/2016 and 12/04/2016) which determined that written informed consent by the study participants was not required. The final approval from the The study population is shown in The HPV self-test group consisted of 3538 women, but since 470 women had provided a Pap smear between the date of randomisation and the date the study invitation letter with the HPV self-test was sent they were excluded. The intention to treat group consisted of 3068 (3002 + 66 without address) women and 788 HPV self-tests were returned . In this group, 259 women provided a Pap smear within six months after the date the invitation letter with the HPV self-test was sent instead of returning the HPV self-test, making 1047 (34.1%) index samples in total. Of the women that received an HPV self-test, 26.2% returned a sample (788/3002 by protocol) (rotocol) .Of the HPV self-tests 36.8% (N = 290/788) were received after a reminder letter was sent . In totaThe telephone group consisted of 3538 women. The intention to treat group consisted of 3146 women since 392 women had provided Pap smears before the study invitation letter was sent. Another 668 were excluded for different reasons. In total, 2870 women received a phone call from a midwife and 62.2% (1784/2870) answered the phone  index samples, Pap smears (N = 642) or HPV self-tests (N = 161) were analysed . Of the The mean age of the women who answered the telephone call was 49.9 years and for those who did not it was 48.7 years (p = 0.005). After one telephone call, 58% (N = 1035) of the women answered and 27% (N = 482) respectively 15% (N = 267) after the second and third calls. The outcome of the phone call is shown in About two thirds  of the women who answered the phone wanted to participate, either by booking a visit for a Pap smear (38.5% 686/1784) or by being sent an HPV self-test (24.9% 445/1784) (p<0.001). The preThe women in the 50–65 years age group preferred an HPV self-test while the women in the 30–39 age group preferred the Pap smear (p<0.001). Of the 686 women that were booked for a Pap smear, 58.9% (N = 404) came to the visit, and of the 445 women who had chosen an HPV self-test, 36.2% (N = 161) returned it (p<0.001) . Reasonscontrol group, 250 Pap smears were collected between the 8th of March 2016 and six months after that  (In the 0/ 3538) , Table 1A comparison between the three study groups regarding index samples is shown in The women that had a telephone call before the HPV self-test was sent returned it (36.1% N = 161/445) more often compared with the women that only had the HPV self-test sent by post with or without a reminder letter (16.6% N = 498/3002) (p<0.001) (9.7% N = 290/3002) (p<0.001).In the HPV self-test group, 13.1% of the tests (N = 103/786) were positive for HPV, and 84.5% of the women (N = 87/103) came to a follow-up visit. In the telephone group, 15.0% of the women who returned an HPV self-test were positive for HPV (N = 24/160), and 70.1% (N = 17/24) came for a follow-up. In total, 81.5% (N = 104/128) of women with positive HPV (13.4% 127/946) came for a follow-up. The distribution of the positive HPV results is shown in The cytology diagnoses from the index Pap smears in the telephone group and the control group are shown in Of the 642 index Pap smears collected in the telephone group, 7.8% (N = 50/641) had dysplasia . All of For comparison, a group of women (N = 44938) that had completed the two previous cervical cancer screening procedures were included in the analysis. Non-attendees had a three or eight times higher risk of having a HSIL or suspected SCC in cytology respectively compared to those women  . ResultsBiopsies from the cervix were taken from 66.3% (N = 69/104) of non-attendees with positive HPV and from 72% (N = 36/50) with cervical dysplasia in their Pap smears. In histopathology a total of ten SCCs and one adenocarcinoma were found in the non-attendee group with a study intervention compared to one SCC in the non-attendee control group . The distribution among the non-attendees concerning histopathology diagnosis was similar ... Another recently published study has shown that the response rate after receiving an HPV self-test was slightly lower compared to our results (13.2%) but increased with age Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: NoReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Long-term non-attenders to cervical screening have a higher risk of cervical cancer; therefore, determining effective ways to reach women who do not respond to routine invitations is important for the effectiveness of the screening program. This paper examines the effect on participation, follow-up, and disease detection of two active interventions involving self-sampling with the routine practice of sending a reminder invitation. The authors have also compared to a population that routinely attends cervical screening. While the premise is important, there are several aspects of the paper that need clarification and structuring to better highlight the study protocol and the results.1) The materials and methods section needs to be reorganized for clarity – statements regarding registration of the trial can be moved to the end; information on the extraction of the comparison group can be included under the “attended group” heading along with further details on this population and reasoning on the selection criteria; a description of the ITT and per protocol population definitions should be included in the analysis section.2) In the second paragraph of the methods, the authors state that all three groups received the yearly invitations. Is it possible to separate the effects of the intervention and the routine yearly invitations? Did they occur simultaneously or sequentially?3) More details on the interventions would be helpful for reproducibility and understanding effects. What information did women receive about the intervention procedure? More details on the HPV self-sampling are needed – what test was used, were instructions included?4) The authors have explored a series of outcomes related to the screening process but what is most interesting is the comparison between the interventions at each stage. That said, it would be helpful for the reader if the results were organized by outcome rather than a mix of intervention arm and outcome. As it stands, the results are somewhat hard to follow. The first stage in the results are framed as “obtaining an index sample”, would it be fair to say that this reflects “participation” and that the interventions are differently effective in increasing participation as compared to the control group? This interpretation might be more understandable to the reader and corresponds better to the screening process reflected in the choice of the other outcomes.5) Perhaps it should be noted again that Table 5 reflects cytological results, and therefore *suspected* cancer, correct? If I’ve understood it correctly, these are not histopathologically confirmed cancer whereas Table 6 reflects results that are histopathologically confirmed. Furthermore, the description of Table 6 needs to be edited. The OR mentioned in line 295 is from Table 5 not Table 6 (this matters for the results summarized in the abstract as well). This needs to be clarified in the final paragraph of the Discussion (starting at Line 382).6) While the authors have detected more underlying disease (and suspected cases of cancer) in the intervention arms as compared to the general population that attends screening regularly, the authors have made this conclusion based on cytology results which precludes information on staging. Therefore, statements on the downstaging effect of the interventions should be made with more caution at the end of the Discussion.Smaller edits1) Ref 4 - HPV16/18 cause 70% of all cervical cancer, not just SCC.2) Please read through the text again – there are several typos that make the text less readable.3) I may have missed something, but the dates for the interventions described in the second paragraph of the materials and methods section don’t line up with the dates described for each intervention separately.4) I suggest using “abnormality” or “positive screening result” to describe the index tests that needed further follow-up rather than “pathology”. Also, I suggest replacing “PAD” in Table 6 with “histopathological results”. Furthermore, it can be noted that HSIL/LSIL terminology is used for both cytology and histology in this context to clarify for the reader.Reviewer #2: Lilliecreutz et al., studied outcome of two intervention arms and one control group among non-attendees in cervical cancer screening.They randomized 10.500 women to three groups among those that had not participated in > 6 years. Among the group of women who were randomized to get a HPV self-sample kit, 34.1% sent in an index sample (either HPV-sample or a pap-smear). Corresponding figures were 25.5% and 7.0% (cytology) for women that was invited by telephone and by routine reminder by post, respectively. The majority of the women 63.4% who answered the telephone wanted to have a pap smear (38.5%) or to be sent a HPV self- sampling kit (24.9%) (p<0.001). Women who chose an HPV self-test were older and gave anxiety/ fear as a reason to decline participation, and they were also less likely to participate in the follow-up if HPV-positive compared to the women who chose a Pap smear. In the by protocol analysis the telephone group (among those who answered) was the most effective regarding collection of an index sample compared to the HPV self-sampling test and control group with 31,7% vs 26,1% and 7,0% ( p<0.001) respectively in each group. The attendance to the recommended follow-up in total was 87%. Non-attendees had a three respectively eight times higher risk of having a HSIL or SCC compared to women screen as recommended . A total of ten SCC and one adenocarcinoma were found in the non-attendee group with a study intervention compare to one SCC in the non attendee group without any study intervention .They concluded that the best strategy to get more non-attendees to participate is to send a HPV-self sample kit.The study is interesting due to its size of invited population based women and the engagement of 24 midwifes in order to perform the telephone arm.CommentsThe manuscript will benefit from improvements as mentioned below.1. Abstract. Please clarify which result that was used to conclude that the best intervention method to get as many non-attendees as possible to leave a sample is to send a HPV self sampling test together with an invitation letter? In contrast (line 344) in the discussion you favour personal contact with a midwife with an option for the woman to choose sampling method.2. Please describe that “intention to treat statistical analysis” was performed in both abstract and materials and methods.3. Please clarify “by protocol” in the Material and Method section.4. Line 179 ASC-H? Please change to ” that cannot exclude a high-grade squamous intraepithelial lesion”5. Line 368. Please clarify the term “majority”?6. Line 337. Please consider a recent study which also observed increased return rates of self-samples by increasing age (PMID: 32082413).7. Table 1. Please give explanations (footnotes) of “Intention to treat”, “Index sample”, “By protocol” and add headings in the two left bottom squares.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  22 Apr 2020Dear Academic Editor, Professor DillnerThank you for valuable comments on our manuscript “Participation in interventions and recommended follow-up for non-attendees in cervical cancer screening -taking the women’s own preferred test method into account - a Swedish randomised controlled trial”.We have tried to change the manuscript in accordance to the suggestions made by the reviewers and yourself, please see below. If you still think there are modifications that need to be done in order to make the manuscript publishable, please do not hesitate to contact us. But our sincere hope is that you will find the manuscript satisfactory for publication.The paper describes that the participants were enrolled before an IRB approval was obtained. This could have been a cause for a rejection without reviews, but I decided to give you the opportunity to explain. Please include this both in the accompanying letter when you resubmit the paper and in the revised manuscript itself.The first decision from IRB came 27 th of January 2016 with a request that we needed to change some of the content in the information to the study population. The final approval from the IRB came 12th of April 2016 after the time of the randomization took place (March 2016) but before the invitations were send to the patients. Revisions and clarifications has been made in the manuscript.From the comments, I am concerned about that it is not clear if the outcome is measured by cytology or histopathology, Thank you for your comment. We have clarified the outcomes concerning cytology and histopathology throughout the manuscript.that it is not clear what the difference is between per protocol and intention to treat andWe have added a definition in the statistic section and hope that it will be to your satisfactionhow these 2 analyses could even have different conclusions. Since we only included women who answered the phonecall in the by protocol analyses, the studygroup is much smaller than in the intention to treat group, which contribute to the differences in the conclusions. We have added measures in the results-section and clarified this in the discussion section. We also made changes in table 1.Please give exact counts (not only percentages) already in the abstract. Thank you for the comment. This has been added.Finally, please have a native English speaker revise the paper before you resubmit.https://www.anchorenglish.com who has revised the manuscript once againWe have sent it back to Anchor English, - Proofreading Services  Journal Requirements:We note that you have reported significance probabilities of 0 in places. Since p=0 is not strictly possible, please correct this to a more appropriate limit, eg 'p<0.0001'. Changes has been done in the manuscriptWe suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. https://www.anchorenglish.comThe manuscript has been copyedit for language, spelling and grammar by Anchor English - Proofreading Services Caroline Lilliecreutz and Anna-Clara Spetz-Holm has been editing the manuscript. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified whether consent was written or verbal/oral. If consent was verbal/oral, please specify how verbal/oral consent was recorded.The consent was giving verbal and confirmed by taking a sample (pap smear or HPV self-test) after study information was given. Revisions had been made.Please include in the Methods the rationale behind patients being enrolled prior to obtaining IRB approval.This has been clarified. Thank you.http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.6. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see In your revised cover letter, please address the following prompts:a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail  and who has imposed them . Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. mats.fredrikson@liu.se. Registries used for this study include the Swedish National Cervical Cancer Registry and A local database (HPV data). Address to the ethics committee: www. Etikprovningsmyndigheten .se. E- mail; registrator@etikprovning.se.Data is available on request for any researchers to allow replication of results provided all ethical and legal requirements are met. Data can be requested through Forum Östergötland, Linköping University, with contact person Mats Fredrikson Mats http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see See comment aboveWe will update your Data Availability statement on your behalf to reflect the information you provide.http://journals.plos.org/plosone/s/supporting-information.Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: We do not have any supporting information files to publish. If we have misunderstood the instruction, please let us know.Review Comments to the AuthorReviewer #1: Long-term non-attenders to cervical screening have a higher risk of cervical cancer; therefore, determining effective ways to reach women who do not respond to routine invitations is important for the effectiveness of the screening program. This paper examines the effect on participation, follow-up, and disease detection of two active interventions involving self-sampling with the routine practice of sending a reminder invitation. The authors have also compared to a population that routinely attends cervical screening. While the premise is important, there are several aspects of the paper that need clarification and structuring to better highlight the study protocol and the results.1) The materials and methods section needs to be reorganized for clarity• Statements regarding registration of the trial can be moved to the end. Revisions has been made.• Information on the extraction of the comparison group can be included under the “attended group” heading along with further details on this population reasoning on the selection criteria;Thank you for the comment. The description of the control group is described earlier in the methods section. We added more information of the attendee-group earlier in the methods section. Due to these changes we omitted sections “control group” and “attended group”. • a description of the ITT and per protocol population definitions should be included in the analysis section.Thank you, we have added information about this in the statistics section under Material and Methods2) In the second paragraph of the methods, the authors state that all three groups received the yearly invitations. Is it possible to separate the effects of the intervention and the routine yearly invitations? Did they occur simultaneously or sequentially? Since all three groups received the routine yearly invitations the effect of this invitation exists in all groups. The study interventions are in addition. A clarification has been made in the discussion section and we also added information in the Methods section.3) More details on the interventions would be helpful for reproducibility and understanding effects. What information did women receive about the intervention procedure? More details on the HPV self-sampling are needed – what test was used, were instructions included?More details about the HPV self-sampling has been added- se Method section.4) The authors have explored a series of outcomes related to the screening process but what is most interesting is the comparison between the interventions at each stage. That said, it would be helpful for the reader if the results were organized by outcome rather than a mix of intervention arm and outcome. As it stands, the results are somewhat hard to follow. The first stage in the results are framed as “obtaining an index sample”, would it be fair to say that this reflects “participation” and that the interventions are differently effective in increasing participation as compared to the control group? This interpretation might be more understandable to the reader and corresponds better to the screening process reflected in the choice of the other outcomes.Thank you, we understand the complexity of our results. We have made some changes in the results section and changed the headlines to better clarify this. We hope this makes it clearer. We choose to keep “index sample” in some instances we found appropriate, and changed to “participation” when suitable. 5) Perhaps it should be noted again that Table 5 reflects cytological results, and therefore *suspected* cancer, correct? This has been added. If I’ve understood it correctly, these are not histopathologically confirmed cancer whereas Yes that’s correctTable 6 reflects results that are histopathologically confirmed. Yes that is correctFurthermore, the description of Table 6 needs to be edited. We have changed the description to this TableThe OR mentioned in line 295 is from Table 5 not Table 6 (this matters for the results summarized in the abstract as well). This needs to be clarified in the final paragraph of the Discussion (starting at Line 382).The OR is from Table 5 and we have clarified that in the results section concerning cytology (suspected SCC) and not histopathology. We have also added clarifications concerning this in the abstract and discussion.6) While the authors have detected more underlying disease (and suspected cases of cancer) in the intervention arms as compared to the general population that attends screening regularly, the authors have made this conclusion based on cytology results which precludes information on staging. Therefore, statements on the downstaging effect of the interventions should be made with more caution at the end of the Discussion.Thank you, sorry for confusion in the manuscript, we have made changes in the end of the discussion section to clarify this. Since the results from our study groups reflects histopathology we think that the conclusion that this indicates that the non-attendees has a higher risk of SCC than attendees is fair enough. Smaller edits1) Ref 4 - HPV16/18 cause 70% of all cervical cancer, not just SCC. Thank you. Revision has been made.2) Please read through the text again – there are several typos that make the text less readable.https://www.anchorenglish.com who has revised the manuscriptWe have once again sent it back to Anchor English, - Proofreading Services 3) I may have missed something, but the dates for the interventions described in the second paragraph of the materials and methods section don’t line up with the dates described for each intervention separately.Thank you, we have changed, however, the distribution of the invitation letters differed a little between the telephone group and the HPV self-test group.4) I suggest using “abnormality” or “positive screening result” to describe the index tests that needed further follow-up rather than “pathology”. Changes has been made to meet thisAlso, I suggest replacing “PAD” in Table 6 with “histopathological results”. Changes has been madeFurthermore, it can be noted that HSIL/LSIL terminology is used for both cytology and histology in this context to clarify for the reader.Thank you for your comment, however vi have tried to clarify this throughout the manuscript.Reviewer #2: Lilliecreutz et al., studied outcome of two intervention arms and one control group among non-attendees in cervical cancer screening.They randomized 10.500 women to three groups among those that had not participated in > 6 years. Among the group of women who were randomized to get a HPV self-sample kit, 34.1% sent in an index sample (either HPV-sample or a pap-smear). Corresponding figures were 25.5% and 7.0% (cytology) for women that was invited by telephone and by routine reminder by post, respectively. The majority of the women 63.4% who answered the telephone wanted to have a pap smear (38.5%) or to be sent a HPV self- sampling kit (24.9%) (p<0.001). Women who chose an HPV self-test were older and gave anxiety/ fear as a reason to decline participation, and they were also less likely to participate in the follow-up if HPV-positive compared to the women who chose a Pap smear. In the by protocol analysis the telephone group (among those who answered) was the most effective regarding collection of an index sample compared to the HPV self-sampling test and control group with 31,7% vs 26,1% and 7,0% ( p<0.001) respectively in each group. The attendance to the recommended follow-up in total was 87%. Non-attendees had a three respectively eight times higher risk of having a HSIL or SCC compared to women screen as recommended . A total of ten SCC and one adenocarcinoma were found in the non-attendee group with a study intervention compare to one SCC in the non attendee group without any study intervention .They concluded that the best strategy to get more non-attendees to participate is to send a HPV-self sample kit.The study is interesting due to its size of invited population based women and the engagement of 24 midwifes in order to perform the telephone arm.CommentsThe manuscript will benefit from improvements as mentioned below.1. Abstract. Please clarify which result that was used to conclude that the best intervention method to get as many non-attendees as possible to leave a sample is to send a HPV self sampling test together with an invitation letter? Thank you for your comment. We have clarified this in the abstract and this is according to the intention to treat-analyse.In contrast (line 344) in the discussion you favour personal contact with a midwife with an option for the woman to choose sampling method.Thank you, this is according to by protocol analyse, we have clarified this in the discussion. 2. Please describe that “intention to treat statistical analysis” was performed in both abstract and materials and methods.Thank you, we have added information in the abstract as well as in the material and methods section.3. Please clarify “by protocol” in the Material and Method section.We have clarified this in the Methods section under the section Statistics4. Line 179 ASC-H? Please change to ” that cannot exclude a high-grade squamous intraepithelial lesion” Thank you. Changes has been made.5. Line 368. Please clarify the term “majority”? Clarifications has been made.6. Line 337. Please consider a recent study which also observed increased return rates of self-samples by increasing age (PMID: 32082413). Thank you. Very interesting data. This reference has been added.7. Table 1. Please give explanations (footnotes) of “Intention to treat”, “Index sample”, “By protocol” and add headings in the two left bottom squares. Thank you this has been added. Definitions is also included in the statistics section.AttachmentResponse to Reviewers April 2020.docxSubmitted filename: Click here for additional data file. 11 Jun 2020Participation in interventions and recommended follow-up for non-attendees in cervical cancer screening -taking the women´s own preferred test method into account- a Swedish randomised controlled trialPONE-D-20-00773R1Dear Dr. Lilliecreutz,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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Transcription of human papillomavirus (HPV) genes proceeds unidirectionally from multiple promoters. Direct profiling of transcription start sites (TSSs) by Cap Analysis Gene Expression (CAGE) is a powerful strategy for examining individual HPV promoter activity. The objective of this study was to evaluate alterations of viral promoter activity during infection using CAGE technology. We used CAGE-based sequencing of 46 primary cervical samples, and quantitatively evaluated TSS patterns in the HPV transcriptome at a single-nucleotide resolution. TSS patterns were classified into two types: early promoter-dominant type (Type A) and late promoter-dominant type (Type B). The Type B pattern was more frequently found in CIN1 and CIN2 lesions than in CIN3 and cancer samples. We detected transcriptomes from multiple HPV types in five samples. Interestingly, in each sample, the TSS patterns of both HPV types were the same. The viral gene expression pattern was determined by the differentiation status of the epithelial cells, regardless of HPV type. We performed unbiased analyses of TSSs across the HPV genome in clinical samples. Visualising TSS pattern dynamics, including TSS shifts, provides new insights into how HPV infection status relates to disease state. Persistent infection with high-risk human papillomaviruses (HR-HPVs) is the main cause of cancer development4. During the last two decades, HPV-induced carcinogenesis has been extensively studied. The HPV-derived E6 and E7 oncoproteins inactivate the p53 and pRb tumour suppressor proteins, respectively, which results in resistance to apoptosis and promotion of cell proliferation. Continuous high expression of E6 and E7 is the most important factor in cervical cancer progression6.Uterine cervical cancer is the second most commonly diagnosed cancer, and the third leading cause of mortality among women in developed countries7. These transcripts overlap, complicating the evaluation of expression levels of each transcript. There are two major promoters in the HPV genome: the early promoter, located in the long control region (LCR); and the late promoter, located in the E7 gene downstream of the early promoter. The early promoter controls the expression of E6 and E7, while the late promoter regulates the expression of E1, E2, E4, and E5 , as well as the expression of L1 and L2 capsid protein genes8. The activity of these promoters is regulated by cellular transcription factors, or by the epigenetic alteration of the viral genome.HPV transcription is unidirectional and generates numerous viral transcripts via differential RNA splicing. At least 13 transcripts from eight HPV genes were identified in HPV-16–infected W12E cells9 can be used to determine the 5′-terminal sequence of RNA, allowing for promoter detection and quantitative measurement of promoter activity. The two main CAGE protocols currently used are no-amplification non-tagging (nAnTi)-CAGE10, which does not involve PCR amplification, and nanoCAGE11, which is designed to process samples that yield nanograms of RNA. nanoCAGE is based on PCR amplification, with the PCR bias removed through the use of unique molecular identifiers12. We previously reported that nAnTi-CAGE may be used to identify precise transcription start sites (TSSs) in the HPV genome, and have used this technology to quantify the activity of multiple promoters in three cell lines and one patient sample13. Direct evaluation of TSSs may represent a novel diagnostic strategy to assess HPV infection status and disease progression.A transcriptome profiling method known as Cap Analysis Gene Expression (CAGE)15. In the upper epithelial layers, the viral late genes L1 and L2 are expressed to allow viral capsid assembly, packaging, and shedding from the superficial layer of the epithelium. As the viral late gene expression is promoted, E2 suppresses the activity of the early promoter by binding to the E2 binding sites (E2BS) of the LCR18. Thus, in the late stages of epithelial differentiation, HPV early promoter activity is relatively suppressed. As the severity of cervical intraepithelial neoplasia (CIN) increases, sustained high expression of E6 and E7 is driven by the early promoter, and, conversely, L1 gene expression is suppressed19. Several methods have been devised to evaluate the expression of late genes, such as L1 or E4, as biomarkers for CIN progression25. Precise evaluation of the late gene expression patterns could support their use as novel biomarkers for cervical cancer progression.HPV genes are differentially expressed in parallel with the differentiation programme of the cervical epithelium. At the initial stage of HPV infection, the copy number of the viral genome in cells in the basal layer of the cervical epithelium is very low. Viral DNA replication proceeds along with epithelial differentiationIn this context, we propose that a quantitative assessment of promoter activity, by evaluating TSS activity, would allow for classification of HPV status, as well as CIN severity. In the present study, we developed a novel approach for the evaluation of differences of viral promoter activity at the single-nucleotide level using CAGE technology in clinical HPV samples.26. We identified two TSS patterns when focusing on the most activated TSS clusters: the early promoter-activated pattern and the late promoter-activated pattern, which were designated Type A and Type B, respectively. To analyse multiple HPV subtypes in parallel, we defined broad windows containing the early and late promoters in any HPV genome: from nucleotide 80 to 110, and from nucleotide 600 to 950, respectively. We discovered TSS patterns indicative of the early and late promoters, and we subsequently refined the TSS pattern definitions so that Type A included samples where one-third of early promoter activity ≥ late promoter activity; Type B, one-third of early promoter activity < late promoter activity . As the principle of both nAnTi-CAGE and nanoCAGE is highly similar, we first performed nAnTi-CAGE analysis for 9 samples, and we used 37 samples for nanoCAGE analysis, which is a novel technology developed after nAnTi-CAGE to meaningfully observe TSS pattern dynamics with CAGE analysis. The HPV TSS patterns were classified into broad TSS types. First, we visualised TSS activity at a single-nucleotide level using ZENBU software27 to investigate the accuracy of the classified HPV-derived TSS types. Among 37 samples analyzed by nano-CAGE, 33 samples of which HPV-derived TSS was detected were included in this study. Of them, 2 samples were co-infected with two HPV genotypes. Thirty-five HPV-derived TSS types were classified by Gaussian mixture models, and compared to the types of HPV-derived TSSs classified according to the averaged difference in expression between the early and late promoters, defined as (early – late)/(early + late). The model with the highest likelihood was univariate, with two components and unequal variance: this corresponded closely to Type A and the union of Type B, since only one Type A sample (sample #27) was classified as Type B  for the early and late promoters. The HPV transcriptome represents only a fraction of the available sequence libraries, varying roughly between 1 per million and 1 per cent. Therefore, either new samples should be sequenced at a depth of 10 to 20 million reads, or an enrichment method should be developed to address this issue. A limitation of the present study was that the number of clinical samples was not sufficient to allow statistical validation of the association between different TSS patterns and the severity of CIN lesions, or the differentiation status of the epithelium.In conclusion, in this study we demonstrated the feasibility to analyse TSS activity at the single-nucleotide level using CAGE technology in clinical HPV samples. Further work on a larger cohort following the same patients over time will be needed for determining the sensitivity and specificity of the quantification of dynamic changes of TSS patterns as a biomarker of disease progression.A260/A290 and A260/A230 ratios were > 1.7.HPV-infected cervical tissues were obtained from biopsy or surgery samples. Diagnosis was confirmed by experienced pathologists and gynaecological oncologists through pathological and colposcopic examination at the University of Tokyo Hospital. HPV-infected cervical tissues were also examined by H&E staining, and the extent of dysplasia was evaluated. Cervical intraepithelial neoplasia was categorised as grade 1, 2, and 3  depending upon the proportion of abnormal cell thickness. Then, experienced gynaecological oncologists confirmed the biopsied samples as CIN or a cervical cancer lesion. The samples for CAGE analysis were taken from the same area that met the diagnostic criteria of a cervical lesion. All experimental procedures were approved by the institutional review board at The University of Tokyo , and signed informed consent for the use of the tissues and genomic data was obtained from each participant. Preparation of nanoCAGE libraries at RIKEN was approved by the institutional review board at the Yokohama Campus . For the analysis, RNA (5 µg for nAnTi-CAGE and 500 ng for nanoCAGE) was extracted from each sample using an miRNeasy kit . RNA quality was assessed using a Bioanalyzer (Agilent) and standardised to an RNA integrity number (RIN) of > 7.0 for nAnTi-CAGE or > 5.0 for nanoCAGE. The purity of RNA samples was assessed using NanoDrop analysis, which confirmed that the This study was approved by the institutional review board at The University of Tokyo  in accordance with the Declaration of Helsinki. All patients provided written informed consent for study participation.35. The sequenced CAGE tags were mapped to the HPV-16 and HPV-52 genome based on the infected HPV genotypes using BWA software (v0.5.9), discarding ribosomal or non-A/C/G/T base-containing RNAs. For the HPV-16 genes, CAGE tag 5′-coordinates were used for Paraclu clustering36 with the following parameters: (i) minimum five tags per cluster; (ii) (maximum density/baseline density) ≥ 2; and (iii) 100-bp maximum cluster length. Tag numbers < 5 were designated as negative for each TSS cluster.First-strand cDNA was transcribed to include the 5′-end of capped RNA, and CAGE ‘barcode’ tags were attached as previously described12, with some modifications. The reverse-transcription products were eluted in 40 µL, and qPCR was conducted using the SYBR Premix Ex Taq kit (TaKaRa). Cycle numbers were estimated as Ct + 4 cycles, and PCR was conducted to generate cDNA using the Ex Taq enzyme (TaKaRa). PCR products were eluted in 30 μL of sterile distilled water after purification, and 0.3 ng of each sample was tagmented individually at 55 °C for 5 min. The extension time of the final PCR was 30 s, and the final purification was achieved using one volume of AMPure reagent , with the products eluted in 25 μL of reaction mixture. The multiplexed libraries were then paired-end sequenced in five lanes of a HiSeq 2000 sequencer (Illumina) and aligned to human genome version hg38 supplemented with all the HPV genomes available on the Papillomavirus Episteme database37 on 5 Sep, 2016, using the CAGEscan pipeline v3.0 , which assembles overlapping pairs originating from the same molecule and maps them to the genome using the LAST aligner38.nanoCAGE libraries were constructed from isolated RNA as previously described27 was used to compare the likelihood of different Gaussian mixtures. After defining a score as the average difference between the expression levels of the early and late promoters, we fitted Gaussian mixture models to these scores. The TSS patterns were classified according to the averaged difference in expression between the early and late promoters, calculated as (early—late)/(early + late). The minimum number of samples required for achieving confidence < 0.25 (i.e. to distinguish between Types A and B) was determined using the ‘ciss.wald’ function in the R package binomSamSize v0.1–5 . A p value < 0.05 was considered statistically significant. The ‘densityMclust’ function in the R package mclust v5.41–5 Fig. .


Background: Our understanding about knowledge, attitudes and perceptions (KAP) of immigrants regarding human papillomavirus (HPV) vaccine is poor. We present the first systematic review on KAP of immigrant parents towards HPV vaccine offered to their children. Methods: Major bio-medical databases  were searched using a combination of keyword and database-specific terms. Following identification of studies, data were extracted, checked for accuracy, and synthesised. Quality of the studies was assessed using the Newcastle Ottawa Scale and the Joanna Briggs Institute Qualitative Assessment tool. Results: A total of 311 titles were screened against eligibility criteria; after excluding 292 titles/full texts, 19 studies were included. The included studies contained data on 2206 adults. Participants’ knowledge was explored in 16 studies and ranged from none to limited knowledge. Attitudes about HPV vaccination were assessed in 13 studies and were mixed: four reported negative attitudes fearing it would encourage sexual activity; however, this attitude often changed once parents were given vaccine information. Perceptions were reported in 10 studies; most had misconceptions and concerns regarding HPV vaccination mostly influenced by cultural values. Conclusion: The knowledge of HPV-related diseases and its vaccine among immigrant parents in this study was generally low and often had negative attitude or perception. A well-designed HPV vaccine health educational program on safety and efficacy of HPV vaccination targeting immigrant parents is recommended. Human papillomavirus (HPV) infection is a sexually transmitted disease and both women and men are rapidly exposed to it after the onset of sexual intercourse ,2. OncogCervical cancer is the fourth most common cancer found in women and the third most frequent cause of death with approximately 570,000 cases and 311,000 deaths in 2018 worldwide . In deveHPV vaccination is the most effective method of preventing HPV infection . The immKnowledge and understanding of HPV infection and HPV vaccine are important factors in decision-making about disseminating the vaccine . Since tLiterature searches were performed using OVID Medline (1946–April 2019), OVID Embase Classic (1947–April 2019), PsycINFO (1806–May 2019) and SCOPUS (1945–May 2019). The searches used a combination of data base-controlled vocabulary terms and text word terms. These included “Papillomavirus vaccines”, “Human Papillomavirus vaccine”, “knowledge, attitudes, perceptions”, “emigrants”, “immigrants”, “population groups”, “ethnic groups”, “refugees”, “mothers”, “fathers” and “parents”. Searches were conducted from 2007 to 2019. The final search was conducted on 1 May 2019. No language or date restrictions were applied. The OVID Medline search strategy used is available upon application to authors. We additionally searched the reference lists of review articles to identify original research articles describing knowledge, attitudes and perceptions of HPV vaccine among immigrant parents.http://www.ohri.ca/programs/clinical_epidemiology/oxford.asp and by Joanna Briggs Institute (JBI) Critical Appraisal tools for use in JBI Systematic Reviews Checklist for Qualitative Research https://joannabriggs.org/sites/default/files/2019-05/JBI_Critical_Appraisal-Checklist_for_Qualitative_Research2017_0.pdf.For inclusion in this review, papers needed to discuss knowledge or attitudes or perceptions of immigrant parents  and/or primary immigrant caregivers towards HPV vaccine. Papers were excluded if they did not include the views of parents or only discussed other childhood vaccines. Perception was defined as how parents interpreted/perceived HPV vaccine in light of their life experiences, and attitude was defined as their reactions to those perceptions. After screening the titles, full texts were retrieved and reviewed, and data were extracted in an Excel sheet by the first author. The data collection form included the author, year, country of study, method, population, result of the study. Another author (HR) checked data abstraction and any discrepancy was resolved through discussion then data were synthesised. The quality of included studies was assessed by Newcastle Ottawa Scale (NOS) for assessing the quality of nonrandomised studies in meta-analyses In this systematic review, 311 titles from four databases were retrieved in total. There were 134 duplicates leaving 177 records to be screened. Of 177 titles, 121 were excluded for not meeting inclusion criteria. The full texts of the remaining 56 titles were assessed. Of these 36 studies were determined to be out of scope of this systematic review and excluded with reasons, the remaining 19 articles met the eligibility criteria of the systematic review as shown in the PRISMA flowchart . There wTotal number of participants in all included studies was 2206  with a male to female ratio of 1:27, where data were provided. Where age of interviewees was mentioned, the range varied from 18 to 66 years. Twelve studies were conducted in the USA, three in the UK, one in the Netherlands, one in Denmark, one in Sweden, and one in Puerto Rico. Six studies were conducted in community organizations including faith-based centres like churches and mosques ,24,25,26Of the 19 studies, 16 reported on knowledge of the immigrant parents about HPV vaccine , 13 repoAll included studies discussed the KAP of immigrant populations. If the study author(s) used the term “ethnic minority” to represent, we have similarly reported this term in the result tables.For knowledge, the level of parents’ knowledge about HPV disease and HPV vaccine ranged from no knowledge in 11 studies ,35,37,39In regards to attitudes towards HPV disease and HPV vaccine , a numbeParticipants had misperceptions about HPV vaccine. The main reasons for declining HPV vaccine were their religious belief and culture; in particular, their belief that abstinence from sex before marriage would provide protection from disease ,31,36. AMost studies were of generally good quality. When scored against the checklist used, ten qualitative studies received eight out of a possible 10 points, and one 10 of 10 . Four ofThis systematic review identifies gaps in knowledge, attitudes, and perceptions about HPV infection and its vaccine among immigrant parents in western countries. Our analyses indicate that although HPV vaccine has been in use for over a decade, information about this vaccine, and HPV infection in general, and its relation to cancer in particular, does not appear to have been well disseminated to immigrant parents. Most participants in 12 included studies had no knowledge about HPV vaccine , one thiIn concordance with our systematic review findings, semi-structured interviews conducted with non-parent immigrant participants also showed limited knowledge about HPV infection its vaccine. For example, a study conducted in a Western Canadian province, found participants had limited knowledge about HPV. Most women perceived their risk of HPV to be low but reported willingness to receive the vaccine when recommended by their doctors . SimilarFurthermore, another systematic review that explored knowledge and attitudes of Iranian people towards HPV vaccination found that the overall knowledge and awareness about HPV vaccination was low; however, their attitude toward HPV vaccination was positive and strong . This coUnlike the immigrants, mainstream populations of USA had better knowledge and more positive attitudes toward HPV vaccine. A quantitative study conducted in Southern California compared knowledge and acceptability between US-born African Americans and African immigrants, and between US-born Latinas and Latina immigrants. African and South American immigrants were less likely to know where they can get/refer for HPV vaccine and less likely to have heard about HPV vaccine than South Americans and US-born Africans . SimilarA study in 2018 indicated that the increase in refusal and hesitancy of Muslim parents to accept childhood vaccination was identified as one of the contributing factors in the increase of vaccine-preventable diseases cases in several countries such as Afghanistan, Malaysia and Pakistan. News disseminated via some social media outlets claiming that the vaccine has been designed to weaken Muslims, reinforced the suspicion and mistrust of vaccines by parents . A qualiAttitudes toward HPV vaccine are important in HPV vaccine uptake. Our systematic review revealed certain attitude-related barriers to vaccine acceptability for adolescents, particularly vaccine hesitancy among some mothers. A qualitative study reported that Latin American immigrant mothers of adolescent daughters expressed more hesitancy regarding adolescent vaccines compared to childhood vaccines expressed an increased sense of belief in their ability to determine what is best for their children . In contTo our knowledge this is the first systematically conducted review of HPV vaccination knowledge, attitudes and perceptions among immigrants. Most included studies were of acceptable quality. We failed to identify research regarding knowledge, attitudes and perceptions of immigrant parents towards HPV vaccine in developing countries. Some papers did not clearly distinguish between attitudes and perceptions as outcomes. However, these studies suggest that tailored educational programs to improve KAP on HPV vaccine among immigrant parents may be a valuable intervention for HPV vaccination uptake.Parental knowledge and attitudes towards HPV vaccine have been examined in many recent studies and lower uptake of HPV vaccine among immigrants, refugees and ethnic minorities has been documented. Our results support the pressing need to develop an intervention aimed to improve HPV vaccination uptake in these populations. More research is needed in the design and evaluation of tailored educational resources for ethnic minority groups, particularly in the framework of the vaccination programme.


Women who have received cervical cancer screening had significantly greater knowledge about cervical cancer and HPV than those with no screening (p < 0.05). Women in northeastern China have little knowledge of cervical cancer, HPV, and HPV vaccine, lack disease knowledge, and hold a skeptical attitude about HPV vaccination. Medical institutions are the main channel providing information to these women.This study aimed to research the understanding and knowledge of cervical cancer, human papilloma virus (HPV), and HPV vaccination, and the acceptance of HPV vaccination, among a population of women in northeastern China. A cross-sectional survey was carried out by questionnaire to investigate knowledge of cervical cancer, HPV, and HPV vaccination. The 230 female participants were native residents of northeastern China, and their ages ranged between 18 and 65 years. Questionnaires were randomly acquired by the respondents from online and paper questionnaire distribution. The questionnaire included questions on three major aspects to record people’s perceptions of cervical cancer, HPV, and vaccines. Of the sample of 230 women surveyed, 80.9% had heard of cervical cancer, but understanding was only 15.7%; 38.3% knew about HPV; 20% knew about HPV vaccine; 39.6% agreed to receive HPV vaccination, and the remainder were mainly concerned about its safety and effectiveness. Data analysis showed that age, family income, and whether there was experience of screening all influenced knowledge of cervical cancer, but this was not statistically significant. The level of education had no obvious effect on the degree of knowledge about cervical cancer; however, with an improvement in education, women’s awareness of HPV vaccine improved significantly ( Cervical cancer (CC) is the third highest cause of cancer-related death for females in low- and middle-income countries , 2. OverA total of 400 persons, including hospital visitors and outpatients without cervical disease or relevant history, were treated in the Nanhu Branch and Huaxiang Branch of the Shengjing Hospital between October 2015 and April 2016. In addition, on-line participants were included in the study (300 completed paper questionnaires and 100 online questionnaires were received). Inclusion criteria are as follows: women aged 18–65 years; native to the three northeastern provinces ; questionnaire completion rate > 95%. Exclusion criteria are as follows: age < 18 years old or > 65 years old; not native to the three northeastern provinces; questionnaire completion rate < 95%; cannot complete the questionnaire independently due to low educational level or other conditions.(1) Some participants completed the hard-copy of the questionnaire on-site; (2) online questionnaires were distributed through the mobile app called WeChat, and the participants completed the questionnaire using their smartphone and then submitted it; (3) each questionnaire was coded and the date were recorded to ensure authenticity, validity, and completeness.The study used a questionnaire survey. The questionnaire was designed on the basis of the cultural background, customs, cognitive channels, and other factors in northeastern China. A large number of domestic and international survey studies were also referenced during the design of the questionnaire. The instrument consists of three major parts. The first part of the survey collected demographic information. The first part enquires about the basic situation of the women and obtains basic information about the participants, such as their native place, age, fertility history, and gynecological examination history.The second part investigates participants’ awareness and sources of information of CC. This part includes, have you heard of cervical cancer? Have you heard of precancerous lesions of the cervix? The high age of cervical cancer? What is the frequency of screening for cervical cancer? Etc Table . After dThe third part investigated knowledge of HPV and HPV vaccines. These items included whether the participant had heard of HPV, how they had heard about HPV, HPV virus transmission route, whether they had heard of HPV vaccine, how they had heard about HPV vaccine, and whether the HPV vaccine can prevent CC. The following questions were raised regarding the acceptance of HPV vaccines: acceptance of HPV vaccine, acceptable price, acceptable vaccination age, vaccination interval, etc. All items were scored quantitatively. After descriptive analysis, these responses were dichotomized as “correct” or “yes” (1 point) and “incorrect” or “do not know” or “no”(0 point). The total number of points available was 10. Achieving less than 1/3 of the total points (0–3) indicated that the participants had no knowledge of HPV; 1/3–2/3 of the total points (4–7 points) represented moderate knowledge and more than 2/3 (8–10 points) represented full knowledge.http://www.epidata.dk/) was used to input the data. Dual data entry and consistency testing were applied to ensure the accuracy and reliability of the data. All data were analyzed using SPSS18.0 software . Measurement data were expressed as mean ± SD, enumeration data were expressed as rates, and three groups of measurement data were analyzed by one-way analysis of variance (one-way ANOVA). The enumeration data were subjected to the χ2 test or Fisher’s exact test. p < 0.05 indicated a statistical significance.Epidata 3.1; Jilin province accounted for 29% ; and Heilongjiang province accounted for 26% . There was no significant difference in the age of the respondents among the three provinces ; 100 on-line questionnaires were returned, including 73 valid questionnaires . There were a total of 230 valid questionnaires and the overall validity rate was 75.4%. The age range of the respondents was from 19 to 68 years old (39.8 ± 11.8 years old) (mean ± SD). Liaoning province accounted for 45% (p = 0.8). Urban residents showed slightly greater knowledge of CC than rural residents but the difference was also not statistically significant (p = 0.48). Additionally, educational level did not significantly contribute to the differences in the degree of knowledge (p = 0.97). With an increase in family income, the knowledge of CC increased accordingly: it was highest in women with family annual income of 110–200 thousand yuan; however, the difference in the knowledge of CC among the women with different levels of family income did not achieve statistical significance (p = 0.06). The results showed that marital status, the number of sexual partners, and a history of gynecological examination did not significantly affect the knowledge of CC (p = 0.827/0.981/0.42). Only 30% of the participants had once received CC screening. The women who had received CC screening had significantly greater knowledge of the disease than those who had never undergone screening (p = 0.002)  compared with Liaoning province (14.6%) and Jilin province (14.9%), the difference was not statistically significant (p = 0.77), suggesting the knowledge of HPV among the women in northeastern China is independent of region. Educational level was associated with knowledge of HPV. The respondents with a high level of education had greater knowledge of HPV and HPV vaccine (p < 0.05). Moreover, age was also associated with the knowledge of HPV. Women in the age range of 31–40 years had greater knowledge of HPV than women in other age ranges; women who were older than 50 years old had the lowest level of knowledge of HPV (p = 0.003). Other factors, including living environment , the number of sexual partners, marital status, and gynecological examination history, did not show a significant association with knowledge of HPV (p = 0.34/0.81/0.52/0.09). Among the women investigated, 38% (n = 88) had heard of HPV: 74% of these women had learned about it from medical institutions, and 11% and 5% had learned of it from the media or from friends or relatives, respectively  of women had heard of HPV vaccine; 43 respondents provided information on the channels through which they knew about HPV vaccine: 67% (n = 29) learned it from medical institutions, 23% (n = 10) from mass media, 5% (n = 2) from friends, and 5% (n = 2) from other channels  of women chose “acceptable”, 27.8% (n = 64) held a negative attitude, and 32.6% (n = 75) held a “wait-and-see” attitude and did not make a choice. Notably, among the women surveyed, none had received HPV vaccination. Although the HPV vaccine had been introduced 10 years previously, the vaccination rate in this region was extremely low. According to our survey, 54.4% respondents agreed with the recommendation of vaccination in adolescents, 45.6% respondents disagreed with the recommendation of vaccination in adolescents, and 21.3% of the respondents were concerned about the long-term effects and side effects of the vaccine  hoped that HPV vaccine would be listed in the national immunization plan. The women who had a high knowledge of CC were more likely to hope that HPV vaccine would be included in the national immunization plan, although the association was not statistically significant.In the population surveyed, 20%  reported that 4030 patients died of CC among the 12,340 with newly diagnosed CC. The incidence of squamous cell carcinoma, which is sensitive to radiotherapy and has a good prognosis, is gradually decreasing, but the incidence of adenocarcinoma is increasing. In particular, the incidence of CC in younger women shows an increasing trend which is more obvious in developing countries. In China, the incidence of CC is second only to that of breast cancer as the disease which most seriously threatens women’s health. Since 2004, the Chinese government has been actively promoting and carrying out a CC screening program, including establishing pilot and demonstration sites for early intervention in patients with CC. Up to 2009, CC pilot sites had been established in 221 counties in 31 provinces or autonomous regions. Large-scale education and screening have significantly decreased the incidence and mortality of CC in China . HoweverFortunately, CC has a good prognosis if it can be screened, detected, and treated early. In this study, 43% of women said they could accept the current price of CC screening, and those who had undergone CC screening had higher recognition of CC and HPV than women who had not had CC screening. Therefore, the price of CC screening plays an important role in women’s awareness of CC and HPV. The price of CC screening varies among hospitals. This study hopes to provide some data to guide the pricing policy for CC screening. Appropriately reducing the price of CC screening or including it in outpatient health insurance coverage would allow more people to have the opportunity to undergo CC screening. At the same time, promoting relevant knowledge from all aspects will ensure that most people understand the screening and regularly attend a hospital for examination. There is a long way to go to achieve universal coverage of CC screening in China.In 1976, Harald Zur Hausen put forward the association between HPV and CC for the first time ; this enn = 46) of women had heard of HPV vaccine. This finding is in line with a study reported in 2017 in which the rate of awareness of HPV vaccine was 17.13% [n = 105) of women disagreed to recommend HPV vaccine to adolescents. The reason why vaccination is not recommended for adolescents is mainly concerned with the long-term effects and side-effects of the vaccine. It has been reported in the literature that about 6–12% of parents in Europe and the USA worry that vaccination will promote premature sexual behavior among adolescents [n = 125) of women agreed to recommend HPV vaccine to adolescents, but nearly half of the women considered that the most suitable age for vaccination was over 18 years old. The American Academy of Pediatrics (AAP) recommends that HPV vaccination should be performed at 11–12 years old for both boys and girls since HPV vaccines are most effective before adolescents begin sexual activity [Women are not only susceptible to HPV infection, the cause of CC, but are also the guardians of children. They play a decisive role in whether or not to vaccinate. Parents’ knowledge of HPV and health awareness directly affects their willingness to vaccinate their children. Therefore, it is of great significance to investigate women’s awareness of CC and HPV, and of the corresponding vaccine , 10. In s 17.13% . The surlescents , 17. In The price of the HPV vaccines launched is about RMB1000/dose and it is recommended to use three doses. In our survey, few of the women investigated could accept such a price. The price which could be accepted by most respondents was below RMB500. Manufacturers should consider speeding up vaccine development, reducing vaccine prices to make them affordable to more people, increasing the number of vaccines, and enabling people to be vaccinated in order to greatly reduce CC incidence. In addition, the HPV vaccine should be included in the scope of medical insurance reimbursement, and the reimbursement rate should be increased as much as possible. In recent years, some provinces and cities in southern China have included it in the scope of medical insurance reimbursement, but, owing to the limited number of vaccines and large population, it is difficult to popularize each one that is suitable for women. In the procurement of vaccines, the health department has signed a procurement agreement to ensure the supply of future products and to reduce the sales price as much as possible . Given tCurrent HPV vaccines include prophylactic and therapeutic vaccines. Therapeutic HPV vaccines are still being investigated. Most HPV vaccines, including those launched in China, are prophylactic vaccines. There are no reports of serious adverse reactions so far . HoweverMany studies in China and abroad have shown that people’s awareness of CC, HPV, and its vaccine can effectively improve the vaccination rate of HPV , 6, 9, rAt present, studies involved in disseminating knowledge of CC and HPV in northeastern China, particularly large-sample studies to identify the reasons for the low knowledge of the disease in this region, as well as providing potential solutions, are still lacking. To improve knowledge of the disease among the women of this region and increase the HPV vaccination rate, on the one hand, requires appropriate education and follow-up from medical staff, and publicity via the media or internet. On the other hand, the support of vaccine suppliers is important, for example, in developing low-price vaccines and launching corresponding policy incentives , 21. In The survey showed that women in northeastern China had a low level of knowledge of CC, HPV infection, and HPV vaccine; 83.4% of the women lacked knowledge of the disease and had a skeptical attitude toward HPV vaccine. It is important to strengthen the knowledge of the disease and HPV vaccine, and to increase the HPV vaccination rate among the women in this region. Medical institutions are the primary channel used by the women in this region to acquire relevant information; therefore, enhancing the education and propaganda offered by medical institutions is an important avenue by which to prevent CC. Meanwhile, reducing the HPV vaccine price and increasing its coverage are crucial for improving HPV vaccination rates.


Human papillomavirus (HPV) testing has been employed by several European countries to augment cytology-based cervical screening programs. A number of research groups have demonstrated potential utility of next-generation sequencing (NGS) for HPV genotyping, with comparable performance and broader detection spectrum than current gold standards. Nevertheless, most of these NGS platforms may not be the best choice for medium sample throughput and laboratories with less resources and space. In light of this, we developed a Nanopore sequencing assay for HPV genotyping and compared its performance with cobas HPV Test and Roche Linear Array HPV Genotyping Test (LA).Two hundred and one cervicovaginal swabs were routinely tested for Papanicolaou smear, cobas HPV Test and LA. Residual DNA was used for Nanopore protocol after routine testing. Briefly, HPV L1 region was amplified using PGMY and MGP primers, and PCR-positive specimens were sequenced on MinION flow cells (R9.4.1). Data generated in first 2 h were aligned with reference sequences from Papillomavirus Episteme database for genotyping.n = 7). For LA, Nanopore revealed higher concordance for high-risk (κ: 0.93) than non-high risk types (κ: 0.83), and with similar high-risk positivity in each cytology grading. Nanopore also provided better resolution for HPV 52 in 3 specimens co-infected with HPV 33 or 58, and for HPV 87 which was identified as HPV 84 by LA. Interestingly, Nanopore identified 5 additional HPV types, with an unexpected high incidence of HPV 90 (n = 12) which was reported in North America and Belgium but not in Hong Kong.Nanopore detected 96 HPV-positive (47.76%) and 95 HPV-negative (47.26%) specimens, with 10 lacking β-globin band and not further analyzed (4.98%). Substantial agreement was achieved with cobas HPV Test and LA (κ: 0.83–0.93). In particular, Nanopore appeared to be more sensitive than cobas HPV Test for HPV 52 , and with comparable or better performance than 2 reference methods in the market. Future prospective study with larger sample size is warranted to further evaluate test performance and streamline the protocol. Human papillomavirus (HPV) is generally accepted as the causative agent of cervical cancer (CC) , which wTwo hundred and one cervicovaginal swabs were collected from March to July, 2019 in Hong Kong Sanatorium & Hospital. The swabs were preserved in SurePath preservative fluid  and routinely tested for Papanicolaou smear , cobas HPV Test and LA . Routine test results are shown in Table DNA extraction and cobas HPV Test were performed using cobas 4800 system . Briefly, 500 μL of cervicovaginal specimen was added to 500 μL of sample preparation buffer and heated at 120 °C for 20 min. The mixture was brought to ambient temperature for 10 min and processed on cobas × 480 using ‘high-risk HPV DNA PCR’ protocol. Real-time polymerase chain reaction (PCR) was performed on cobas z 480. Fifty microliter of DNA extract was used for LA according to manufacturer’s recommendations. Residual DNA was used for Nanopore protocol after routine testing.For each specimen, L1 region of HPV genome was amplified in 2 separate PCRs using PGMY and MGP primer sets , 14. PriPGMY and MGP PCR amplicons of each positive specimen were pooled and purified using AMPure XP beads . Nanopore sequencing libraries were prepared from purified amplicons using Ligation Sequencing Kit 1D (SQK-LSK109) and PCR-free Native Barcoding Expansion Kit (EXP-NBD104/114) . The barcoded libraries were loaded and sequenced on MinION flow cells  after quality control runs.Data from first 2 h of sequencing runs was analyzed. FASTQ files generated by live basecalling (MinKNOW version 2.0) were demultiplexed using ‘FASTQ Barcoding’ workflow on EPI2ME  with default minimum qscore of 7, ‘auto’ and ‘split by barcode’ options. FASTQ files of each specimen were concatenated into a single file and analyzed using a 2-step custom workflow on Galaxy bioinformatics platform. Briefly, FASTQ files were converted into FASTA format, followed by aligning sequences against HPV reference genomes from PaVE database using NCBI BLAST+ blastn . PGMY and MGP reads were sorted based on sequence length and analyzed individually. Threshold of each run was derived from average number of background reads plus 10 standard deviations, which were calculated using interquartile rule, excluding first and last quartiles. A positive HPV call was based on either (1) the number of reads for a particular HPV type was above threshold, or (2) the specimen had the highest number of reads for a particular HPV type. All positive calls were further assessed by aligning FASTQ sequences against HPV reference genomes using minimap2 , and consensus sequences were built from BAM files using Unipro UGENE (version 1.29.0) for determining their percentage of identity to reference genomes.As HPV 66 is categorized as ‘other high-risk’ by cobas HPV Test, all calculations were based on this grouping, albeit HPV 66 was found as a single infection in cancers with extreme rarity and re-classified as possible carcinogen (Group 2B) by IARC Monographs Working Group [The results are summarized in Table Table n = 8) and 18 (n = 1) were 87.5 and 100%, respectively. Positive agreement was 75–100% for high-risk HPV 31, 33, 35, 39, 51, 52, 56, 58, 59 and 66, and 20% for HPV 68 (n = 5). For non-high risk HPVs, positive agreement was 37.5–100% for HPV 6, 11, 40, 42, 53, 54, 55, 61, 62, 70, 72, 73, 81, 82, 83, 84 and 89. There were 2 non-high risk types with 0% positive agreement (HPV 26 and 71). HPV 26 (n = 1) was only detected by Nanopore workflow, whereas HPV 71 (n = 2) was only detected by LA.Table Table Table Hong Kong has been one of the Asian regions with the lowest incidence and mortality rate of CC . This miCytology and HPV testing have their own value for CC screening. High quality cytology has high specificity for CC, but with lower sensitivity ranging from 50% suggested by cross-sectional studies to 75% estimated longitudinally . For HPVn = 34) not detectable by LA  which was reported in North America and Belgium but not in Hong Kong [Compared with HPV assays in the market, HPV genotyping by NGS offers a broader detection spectrum which, despite minimal benefit of non-high risk HPV information for CC screening, may provide important etiologic clues for other HPV-associated infections and a more complete picture of HPV epidemiology. For the latter, Nanopore identified more HPV types per sample Fig.  and 5 ex LA Fig. , with anong Kong , 21. Anoong Kong –25. Thesn = 7) and 59 (n = 4), with 81.82% (9/11) of these discrepant results matched with LA. Compared with LA, concordance for high-risk HPV was higher than non-high risk types. Among the 37 discrepant results, 22 were false negatives by Nanopore and 15 were not detected by LA.In general, Nanopore had substantial agreement with cobas HPV Test and LA. Compared with cobas HPV Test, Nanopore appeared to be more sensitive for HPV 52  were mixed infections, and similar finding was reported by other research groups using HPV consensus primers for NGS-based genotyping , 11. OthFor the 15 HPV types missed by LA, the average identity of Nanopore consensus sequences was 98.27% with an average difference of 16% from second BLAST hits , was less satisfactory for evaluating type-specific performance. Second, as residual DNA was used after routine testing, DNA input for PGMY and MGP PCRs was constrained which might lower the sensitivity. In addition, as flow cells with suboptimal number of active pores were used, sequencing time and depth might be further improved if new flow cells were used.Our study had several limitations. First, the sample size of certain HPV types, for example, HPV 18  for HPV genotyping referral service in our region Table . The pro