Enhancing human papillomavirus (HPV) vaccine uptake rates to protect women's health is an important public health issue worldwide. China has taken a series of measures in recent years to promote HPV vaccination among school-aged girls, but the vaccine uptake rate remains low. Investigating the factors influencing vaccination-related decision-making of adolescent girls' parents is key to solving the problem. This study aimed to examine the influence of sociocultural-psychological predictors, including exposure to HPV-related stories (positive/negative), affective reactions (pride/regret), injunctive norms on the Internet and perceived moral obligation, on parents' HPV vaccination-related decision-making for girls aged 13–15 years in mainland China.A cross-sectional online survey using quota sampling was conducted in February 2022. Four hundred and five valid and qualified questionnaires were obtained. Partial least squares structural equation modeling was performed by SmartPLS 3 (i) to evaluate the reliability and validity of the measurement models of 11 constructs, and (ii) to test the effect relationships of the sociocultural–psychological predictors on parents' intention to vaccinate their daughters.t = 8.448, p < 0.001), which strongly predicted the attitude toward vaccination ; anticipated pride had more positive influence on vaccination-related decision-making  than anticipated regret. In terms of social influence, injunctive norms on the Internet had a significantly positive influence on vaccination intention , similar to descriptive norms . Perceived moral obligation at the cultural level was the strongest predictor of parental decision-making regarding HPV vaccination .The study findings showed that parental decision-making regarding HPV vaccination was influenced by sociocultural and psychological factors. At the level of individual psychological factors, exposure to positive stories was significantly associated with perceived vaccine effectiveness (β = 0.331, This study is the first in mainland China to systematically examine the sociocultural-psychological predictors of parents' decision-making to vaccinate their 13–15-year-old daughters against HPV. A new extended TPB model with a sociocultural-psychological approach was developed. This model can support the investigation of factors affecting HPV vaccine uptake rates in the mainland Chinese population and similar populations and help to understand the differences in vaccination-related decision-making between Eastern and Western cultures. Furthermore, the study provided some suggestions for HPV vaccination communication campaigns targeting adolescent girls' parents. As the country with the largest population, China had a crude cervical cancer incidence of 15.6 per 100,000 women in 2020 and 51,600 deaths due to cervical cancer in 2019 . MainlanAccording to the national routine vaccine report data, the number of HPV vaccine doses administered in mainland China increased from 3.417 million in 2018 to 12.279 million in 2020 .World Health Organization (WHO) suggested that 90% of girls should be fully vaccinated against HPV by 15 years of age by 2030 . In receThe theory of planned behavior (TPB) is a representative theory about the relationship between attitude and behavior in the field of social psychology. It posits that attitude, subjective norms, and perceived behavioral control (PBC) are the three mutually influencing factors that affect a person's behavioral intention, which is in turn the factor that most directly influences actual behavior. Attitude toward a behavior refers to an individual's overall evaluation of a behavior. Subjective norms are described as an individual's perceived social expectations for a behavior. PBC is defined as an individual's perceived confidence in performing it, and it is commonly measured as self-efficacy in performing a behavior .Since Ajzen suggested that the TPB model is open to expansion , many reA large number of studies in Western countries have confirmed the explanatory power of attitude, subjective norms, and PBC on parental HPV vaccination intention for their children. Other determinants, including media use, perceptions of HPV infection and vaccination risks, descriptive norms, and anticipated regret have also been included in extended TPB models to explore their impact on behavioral intention –33. TherChina has a different social culture from Western societies, as it emphasizes collectivism and child-centered family traditions, greater obedience to authority, and a higher tolerance for uncertainty . This leAttitude is the strongest predictor of vaccination intention in the TPB model. The established TPB model emphasizes the cognitive antecedents of vaccination attitude, e.g., risk perception, but ignores the factors affecting the cognitive antecedents. Recent studies have shown that exposure to messages from different channels can shape people's knowledge and attitudes toward HPV vaccines –39. HPV The variable of attitude in TPB model only emphasizes the cognitive component, and ignores the emotional component that influences health behavioral intention and decision-making –45. AntiThe variable of subjective norms in the TPB model emphasizes the social and cultural factors affecting individuals' health-related decision-making. It includes two aspects: descriptive norms (copying others' behavior) and injunctive norms (behaving as others expect). Descriptive norms has been proven to be a significant influence factor on vaccination attention , 51, whiAjzen suggested that moral norms, along with attitude, subjective norms, and PBC, directly affect intentions . SeveralBased on the local situation in mainland China, this study aimed to assess the impact of sociocultural-psychological predictors affecting mainland Chinese parents' decision-making regarding HPV vaccination for their adolescent daughters. We aimed to test the relationships between the influence of exposure to positive or negative stories and perceived vaccination risk, including vaccine effectiveness and vaccine side-effects, and attitude toward vaccination. Further, we aimed to explore the effects of three levels of psycho-emotional, social norm, and cultural influences, namely, anticipated emotional reactions (anticipated regret and pride), injunctive norms on the Internet, and perceived moral obligation, on parents' vaccination intention for their daughters. We proposed following hypothesis:H1: Exposure to positive stories has a positive influence on perceived vaccine effectiveness.H2: Exposure to negative stories has a positive influence on perceived vaccine side-effects.H3: Perceived vaccine effectiveness has a positive influence on attitude toward vaccination.H4: Perceived vaccine side-effects has a negative influence on attitude toward vaccination.H5: Perceived infection risk has a positive influence on attitude toward vaccination.H6: Attitude toward vaccination has a positive influence on vaccination intention.H7: Anticipated regret has a positive influence on vaccination intention.H8: Anticipated pride has a positive influence on vaccination intention.H9: Descriptive norms has a positive influence on vaccination intention.H10: Injunctive norms on the Internet has a positive influence on vaccination intention.H11: Perceived moral obligation has a positive influence on vaccination intention.H12: Self-efficacy for vaccination has a positive influence on vaccination intention.Based on Hypotheses 1–12 as proposed above, In February 2022, a cross-sectional anonymous online survey of mainland Chinese parents of adolescent girls aged 13–15 years was conducted on a professional online research platform (Survey Plus). Quota sampling based on the educational level and vaccination status (yes/no) was used to ensure that the samples adequately represented the specific demographic characteristics of general Chinese parents. At the beginning of the questionnaire, the participants were given an explanation of the study purpose and procedures. Participation in the study was voluntary, and the participants could withdraw at any time without any consequence.To obtain qualified data, we included several quality control procedures in the questionnaire system, including an Internet Protocol duplicate check avoiding multiple answers by one participant, an answering time of at least 15 min, an attention test preventing participants from answering indiscreetly, and a logical relationship check to identify unqualified questionnaires with contradictory answers. After rigorous data check and cleaning, 405 valid questionnaire responses were obtained. Of the total survey respondents, 30.4% were male and 69.6% were female. This sex ratio is reasonable as in Chinese families, mothers are often responsible for the daily lives and health of their children. The average age of the respondents was 39.1 years (standard deviation = 3.36). Adapted from previous research , the varThe parents indicated the content of negative stories describing people being harmed by HPV vaccine using a multiple-choice question with five response options: Have you heard the stories that HPV vaccine (1) had mild side-effects; (2) had serious temporary harms; (3) had serious long-lasting harms; (4) caused death; or (5) had other harms? We assigned a different score to each option according to its negativity: one point for the first and fourth options, two points for the second option, three points for the third option, and zero points for the fifth option. Finally, we used a summed total score for each parent as an index to measure the negativity of the HPV vaccination-related information to which each parent had been exposed.In addition to the above two variables, 11 other constructs were measured.Based on the findings in the literature, localized modifications were made to the scales, and the scales were translated into Chinese. Seven-point Likert scales were used. Items below the factor loading value threshold were eliminated. The details of the scales for the study constructs were provided in the R2) and the predictive correlation (Q2) were used to indicate the qualification of the four structure models. Variance inflation factors were adopted to examine the multicollinearity issue.Partial least squares structural equation modeling (PLS-SEM) has been demonstrated to be effective for verifying complex influence relationships in path models with latent variables . Our resThe first part shows the results of measurement model evaluation that indicate the rationality of the measurement models through reliability, convergent validity, and discriminant validity.All factor loadings ranged from 0.820 to 0.972, and the average variance extracted values were between 0.710 and 0.940, which are higher than 0.5 , 63. ThiThe results of the Fornell–Larcker criterion and heterotrait–monotrait ratio of correlation analyses revealed that the measurement model had good discriminant validity among these constructs . In TablR2) was used to confirm the effects of external variables on internal dependent variables (Q2) value was used to indicate whether the structural model could accurately predict the data , reflecting that the external variables in the model had a notable impact on the internal dependent variables , while exposure to negative stories was significantly associated with perceived vaccine side-effects . This confirmed our H1 and H2. The three variables of perceived vaccine effectiveness, perceived vaccine side-effects, and perceived infection risk were all significantly related to attitude toward vaccination. Among them, perceived vaccine effectiveness and perceived infection risk had positive effects on the attitude toward vaccination, with path coefficients of 0.521  and 0.109 , respectively, while perceived vaccine side-effects had a negative effect on the attitude toward vaccination . Thus, our H3, H4, and H5 were supported. Regarding the cause–effect relationships between the seven independent variables and the dependent variable of HPV vaccination intention, except for the variable of anticipated regret, all of the other six variables showed significant effects on HPV vaccination intention. The path coefficients of attitude toward vaccination and anticipated pride to vaccination intention were 0.146  and 0.156  respectively, supporting H6 and H8; the path coefficients of descriptive norms and injunctive norms on the Internet to vaccination intention were 0.135  and 0.127 , supporting H9 and H10. H11 and H12 are also supported, as the path coefficients of perceived moral obligation and self-efficacy for vaccination to HPV vaccination intention were 0.193  and 0.170 , respectively. Moreover, multicollinearity was mainly detected by the variance inflation factor  was used to test the statistical significance of the variables. The results are shown in n factor . Appendin factor .Consistent with previous studies –31, 69, Both perceived infection risk and perceived risks associated with vaccination (vaccine effectiveness and vaccine side-effects) are important factors affecting attitude toward vaccination . This stStudies on parental decision-making regarding vaccination in Western countries have shown that anticipated negative affective reactions, such as anticipated regret, anticipated worry, and anticipated anxiety, were significantly associated with increased parental intention to have their children vaccinated , 71, 72.Some studies have shown that anticipated regret is a key factor affecting parents' decision to vaccinate their daughters in the case of mandatory vaccination or during highly contagious and devastating pandemics , 73. HPVIn addition to psychological factors, social rules and parenting culture are important factors affecting Chinese parents' decision-making regarding HPV vaccination for their daughters. The result of our study revealed the positive influence of descriptive norms on vaccination intention (Hypothesis 9), which is consistent with those of previous studies , 72, 77.Since the 1980's, China has entered the era of market economy, and material life has become extremely rich. The quality of life of children who grew up after the 1990's is much better than that of their parents. Because parents and grandparents place all of their expectations on these children and take care of them meticulously, many of the children are called “little emperors” by the media . As prenThe effectiveness of injunctive social norms depends on the Chinese culture of “Wangzichenglong.” Every Chinese parent has a strong sense of ethical responsibility, and the normative value of “everything is for the child” has been deeply embedded in their daily parenting practices. This study also has confirmed a positive relationship between the variable of perceived moral obligation and vaccination decision-making, with this variable showing the strongest impact coefficient among all of the influencing factors (Hypothesis 11). The salient effects of injunctive norms and perceived moral obligation also help us to understand the role of anticipated pride. When parents take the initiative to complete the non-mandatory HPV vaccination for their daughters, they meet the high requirements and expectations of society regarding parenting and fulfill the parental responsibility of “the best for the child.” This elicits a sense of pride— “I am a competent parent”—indicating that this positive affective reaction strengthens the parents' intention to vaccinate their daughters.First, the findings of our study should be verified in different contexts, such as different socioeconomic classes and regions. Although parents with low educational levels and from rural regions were included in this survey, the representation of these populations was insufficient as the majority of our sample belonged to the middle and higher socioeconomic classes. Parents of different socioeconomic classes tend to have different parenting views ; therefoSecond, the subject of this study was the parents' vaccination decision-making only for girls aged 13–15 years old. Decision-making regarding the vaccination of boys was not included. There is no HPV vaccine available for men in mainland China, and most members of the public believe that HPV vaccination is exclusively for women. However, we should be aware that it is a global trend to include men in HPV vaccination programs, which is beneficial to eliminate the adverse effects of HPV infection on human health worldwide. Therefore, future research needs to pay attention to the uniqueness of the vaccination decision-making behavior of parents of school-aged boys and the establishment of new explanatory models of factors affecting this process, and provide effective recommendations for future policy-making on HPV vaccination for the all age-qualified populations including men.Third, social, cultural, and psychological factors are known to be intertwined, and thus, their effects on parental decision-making regarding HPV vaccination are interdependent. Consistently, this study also found correlations between injunctive norms on the Internet, perceived moral obligation, and anticipated pride. Therefore, future research should further explore the specific relationships between these three variables to enrich the expanded TPB model.Finally, this study is the first to explore the factors influencing parental vaccination intention, but the findings are still insufficient to help improve the HPV vaccine uptake rate in the target population in mainland China, as there is always a gap between vaccination intention and vaccine uptake , 29. TheThe question of how to increase the willingness of parents to vaccinate their school-aged daughters against HPV is an important issue in mainland China. In this study, we found the following answers: (1) parents in mainland China are less exposed to positive stories about HPV vaccines, and their awareness of HPV vaccine efficacy is insufficient, resulting in an insufficiently strong desire to vaccinate their adolescent daughters; (2) parents' sense of ethical responsibility to be good parents and the anticipated pride brought about by their daughters' HPV vaccination has not been effectively encouraged; (3) parents have not felt the expectations and requirements of society, including information and views provided on the Internet, to adopt vaccination behaviors; and (4) the current supply of HPV vaccines in mainland China is not sufficient and the cost is relatively high, which together, discourage parents in mainland China from behavioral intentions of HPV vaccination for their daughters.This study is the first in mainland China to systematically examine the sociocultural–psychological predictors of parents' decision-making to vaccinate their 13–15-year-old daughters against HPV. An extended TPB model incorporating the variables of exposure to vaccination-related stories (positive/negative), anticipated affective reactions (regret/pride) injunctive norms on the Internet and perceived moral obligation was developed. The findings showed that the cultural predictor of perceived moral obligation had the strongest impact on parents' vaccination intention for their daughters. At the social level, this study newly discovered the positive relationship between the injunctive norms on the Internet and parental vaccination intention. Further, we found that the exposure to positive stories about HPV vaccination positively affected parents' perceived vaccine effectiveness, which in turn had a strong positive impact on their attitudes toward vaccination for their daughters. However, we found a weak impact of exposure to negative stories on parents' attitudes. Importantly, the study demonstrated that the impact of the positive affective response (anticipated pride) on parents' decision-making was stronger than the impact of their attitudes, whereas the negative affective response (anticipated regret) has no significant impact on vaccination intention. Self-efficacy for vaccination also was found to be a powerful factor influencing parental decision-making regarding vaccination.In a word, the research findings showed that the influence of the sociocultural-psychological predictors on parental decision-making regarding HPV vaccination in the context of China emphasizing collectivism and family culture of “children first.” The study contributed to the research of parents' HPV decision-making considering comprehensively local social culture and individual's affective emotions. Based on these findings, the following suggestions are proposed for future public health campaigns on HPV vaccination.Communication campaigns for HPV vaccination in mainland China should specifically target the parents of 13–15-year-old girls and actively disseminate positive yet scientific HPV vaccination-related stories mainly on social media or other channels. This could help to enhance parents' understanding of the effectiveness of HPV vaccines in preventing HPV infection and cervical cancer. Further, we found that although exposure to negative stories tended to strengthen parents' perception of vaccine side-effects, they did not have a strong negative impact on attitude toward vaccination; thus, scientific communicators need not worry too much about negative stories on social media.Furthermore, perceived moral obligation and anticipated pride were found to be the two main factors affecting parents' decision-making regarding vaccination for their daughters. Thus, we suggest that communication contents be designed around these two aspects. On the one hand, communications could highlight the vaccination of daughters against HPV as the responsibility of competent parents; on the other hand, communications could emphasize the sense of pride that daughters' vaccination could elicit in the parents, which would help to further stimulate parents' willingness to vaccinate their daughters.In terms of social influence, in addition to paying attention to the influence of traditional primary groups, such as relatives and friends, the influence of netizens, experts on the Internet, and social media should also be considered . In addiCurrently, the shortage of HPV vaccine supply and the high cost of these vaccines in mainland China are barriers to parental decision-making regarding vaccination. Therefore, public health policymakers should strive to eliminate these barriers by increasing the supply of vaccines and fully implementing free vaccination for school-aged girls to improve the self-efficacy of parents and enhance their intention to vaccinate their daughters.The original contributions presented in the study are included in the article/Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.LZ was responsible for the theoretical conceptualization and research design of the whole study, designed questionnaire, and sampling method. WK and LZ contacted the online survey platform, data collection, organized the database, and completed the data cleaning. JY performed the statistical analysis and contributed to the review and editing, in particular Sections Methods and Results. JY and LZ provided the financial support. LZ and YC wrote the original manuscript and completed the visualization. All authors have read and agreed to the published version of the manuscript.


To improve communication of harm in publications of randomised controlled trials via the development of recommendations for visually presenting harm outcomes.Consensus study.TheBMJ.15 clinical trials units registered with the UK Clinical Research Collaboration, an academic population health department, Roche Products, and Experts in clinical trials: 20 academic statisticians, one industry statistician, one academic health economist, one data graphics designer, and two clinicians.measures A methodological review of statistical methods identified visualisations along with those recommended by consensus group members. Consensus on visual recommendations was achieved (at least 60% of the available votes) over a series of three meetings with participants. The participants reviewed and critically appraised candidate visualisations against an agreed framework and voted on whether to endorse each visualisation. Scores marginally below this threshold (50-60%) were revisited for further discussions and votes retaken until consensus was reached.28 visualisations were considered, of which 10 are recommended for researchers to consider in publications of main research findings. The choice of visualisations to present will depend on outcome type , and the scenario . A decision tree is presented to assist trialists in deciding which visualisations to use. Examples are provided of each endorsed visualisation, along with an example interpretation, potential limitations, and signposting to code for implementation across a range of standard statistical software. Clinician feedback was incorporated into the explanatory information provided in the recommendations to aid understanding and interpretation.Visualisations provide a powerful tool to communicate harms in clinical trials, offering an alternative perspective to the traditional frequency tables. Increasing the use of visualisations for harm outcomes in clinical trial manuscripts and reports will provide clearer presentation of information and enable more informative interpretations. The limitations of each visualisation are discussed and examples of where their use would be inappropriate are given. Although the decision tree aids the choice of visualisation, the statistician and clinical trial team must ultimately decide the most appropriate visualisations for their data and objectives. Trialists should continue to examine crude numbers alongside visualisations to fully understand harm profiles. Well designed graphics are an effective way of communicating messages to a range of audiences and help to identify patterns in data that might otherwise be missed.Advances in computer software have improved trialists’ capability to produce visualisations; however, little guidance exists on what and how to visually display complex harm data in journal articles. This has resulted in independent calls from the statistical community for direction on “how to decide which of many possible graphics to draw.”The BMJ. All these participants are experienced clinical trialists or have an interest in the visual representation of data, or fit into both categories. Against an agreed framework, the group reviewed and critically evaluated 28 plots proposed for visualising data for harm outcomes and refined these plots as necessary, predominantly focusing on clinical trials of an investigational medicinal product. Examples of each of the candidate plots was produced by use of data from one of four completed parallel arm randomised controlled trials and a synthetic dataset (see supplement 1 for further details). The group sought consensus on the plots to endorse and then developed recommendations. To support researchers analysing and interpreting harm outcomes, we present a decision tree to aid their choice of visualisations. We focused on static plots that allow a comparison between treatment groups, in line with the aims of randomised controlled trials that make such inferences. Supplement 1 provides details of the methods used for identification of the considered plots, the consensus process, and how the recommendations were developed. In this paper, we describe each of the endorsed plots, give an example interpretation, and provide our recommendation. We held a series of consensus meetings with 20 statisticians from 15 UK Clinical Research Collaboration registered clinical trial units, one health economist based at an academic population health department, one industry statistician, and one data graphics designer who is part of the multimedia team at This work forms part of a wider research project that was developed with input from a range of patient representatives. No patient representatives were directly involved in this work, but representatives with experience as clinical trial participants and patient and public involvement advisors reviewed the original proposal and patient and public involvement strategy. We did not speak to patients directly for this research because our focus was to identify the best plots to present in scientific journals with a predominantly scientific readership. The next step is to ask patients for feedback. We provide the endorsed visualisations that researchers should consider using in the publication of their main research findings according to outcome type and number of events , time-to-event outcomes , and continuous outcomes . We present endorsed visualisations, according to whether the entire harm profile is assessed or a direct message conveyed about a particular event or events of interest, alongside recommendations for use . To helpThe dot plot summarises both the absolute and the relative risk for multiple events .The relative risk, risk difference, odds ratio, or incident rate ratios (adjusted or unadjusted as desired) can be plotted as the measure of comparison in the central panel of this plot. Some researchers might also prefer to present the data table in the central panel so that it appears alongside the absolute summary. This plot can be presented in grayscale without any loss of meaning. A small number of additional arms can be added for multiarm studies through incorporation of multiple non-overlapping estimates on the same plot ; however, an increase in the number of active treatment groups can lead to incomprehensive distinction between arms.Confidence intervals around the relative differences are useful to identify potential signals  of harm for further investigation. However, confidence intervals should not be used as a proxy for hypothesis testing, which will increase the chance of finding spurious significant differences resulting from multiple hypothesis tests.https://www.ctspedia.org/do/view/CTSpedia/ClinAEGraph000). The SAS example does not include code to incorporate the data table.The dot plot can be produced in Stata by use of the aedot or aedots command, in R with the code available in supplement 4, and in SAS with the code available from the CTSpedia Wiki page .A bar chart presents information on the number or count of adverse events reported per participant .The Kaplan-Meier plot for single time-to-event outcomes shows the cumulative proportion of participants remaining event-free over time by treatment group  is recommended. Some generic limitations of using time-to-event plots in this setting are discussed laterhttps://github.com/sarwarislam/kmunicate_stata).Kaplan-Meier plots are easily implemented as standard plots across a variety of statistical packages. To incorporate the extended risk tables, trialists can use the R package KMunicate and a program for implementation in Stata  as a function of time  by treatment group  and risk table, or present separate plots for comparison of each intervention group with a common comparator, or separate plots for different events..For clarity in presentation, mean cumulative function plots are typically limited to one type of event at a time. More generic limitations and cautions of use of time-to-event plots in the harm setting are provided later in this paperThe mean cumulative function with confidence interval bands can be implemented using the SAS proc reliability procedure and mcfplot command.The measure of uncertainty  in the Kaplan-Meier plot and the plot of the mean cumulative function are within treatment groups and not between treatment groups, which is the inference of interest in comparative clinical trials. To incorporate an estimate of the between group difference with a measure of uncertainty, the survival ratio plot can be used (see later). Additionally, when time-to-event methods for harm data are used, trialists must remain aware of the limitations around competing risks and consider these when performing the underlying time-to-event analyses. More information on alternative strategies to account for competing risks can be found in Proctor and Schumacher-Meier and mean cumulative function plots, this plot allows a direct comparison between treatment groups  between treatment groups over time along with 95% confidence intervals. Unlike the KaplanThe survival ratio plot  depicts The survival ratio plot would be suitable for signal detection analysis for emerging events because it provides a between group comparison that can be used to spot departures from unity and helps to identify the time that such divergences occur, which can help detect potential signals for adverse drug reactions. For events of specific interest when the focus is on accurately estimating survival probabilities over time, this plot is less suitable. This plot can be presented alongside the Kaplan-Meier plot to show both a relative and an absolute measure.Our example displays the ratio of survival probabilities estimated using the Kaplan-Meier method; alternatively, the display could show the difference in survival probabilities. As with both the Kaplan-Meier and the mean cumulative function plots, multiple lines can be added to one graph to display estimates for different events or multiple treatment comparisons. As with Kaplan-Meier plots, the survival ratio plot allows for only time-to-first event; therefore, this graph is not suitable for recurrent events. The plot is also limited to one type of event; however, in some situations multiple estimates can be added to the same plot but with the same considerations as plotting multiple lines on the Kaplan-Meier plot. As with other time-to-event plots, competing risks are important to consider when performing the underlying time-to-event analysis, further details of which are discussed above. The confidence interval band of values around the relative differences are useful to detect signals of potential harm for further monitoring, but we are not encouraging hypothesis testing in this setting.The survival ratio plot can be implemented in R using the survRatio package with the drsurv function to take the time, censoring indicator, and treatment indicator as inputs. This package returns Kaplan-Meier survival estimates and corresponding confidence intervals to create an object of the survival ratio, survival difference, and pointwise (bootstrap) confidence bands. The ggsurv function is then used to create the plot of the survival ratio and confidence interval bands.In the line graph plot, markers display mean values and vertical lines indicate the standard deviation (not standard error) of raw values at each discrete time point, connected with a line to the point closest in time for each treatment group  rather than blood markers, where clinicians are more often interested in the tails of the distribution .The summary statistic displayed in this plot should be chosen to reflect each individual dataset and the purpose of the plot, for example, when interest is in presenting descriptions of the distributions, either means and standard deviations or medians and interquartile ranges can be plotted, and if interest is in drawing inferences of between group comparisons, then estimates from mixed effects models for repeated measures with 95% confidence intervals can be presented. This plot can easily incorporate multiple groups or outcomes and can be modified to exclude the use of colour.Changes in the tails of the distributions are usually of most interest when monitoring blood markers for harm, and such changes might be difficult to see using this plot. This graph is also unsuitable for skewed distributions; alternative plots for such data are presented below. Appropriate colours and line styles should be considered for clarity, particularly when adapting line graphs to multiarm trials.Line graphs are easily implemented as standard plots across the variety of statistical packages .The hollow circle marker on the violin plot indicates the median value, the narrow rectangular boxes indicate the interquartile range, and lines extend from the box to the minimum and maximum points for each group at each time point. These parts are overlaid with kernel density plots (see later), which summarise the distribution of the raw values (At time 0 (randomisation) the distributions were similar across treatment groups, but from week 2 onwards the distribution of values in the mepolizumab group was narrower than in the placebo group . The disThis plot is an alternative to the line graph to describe continuous data that can be used even if the outcome of interest is not normally distributed. Outlying values are displayed and these can be labelled to highlight participants who persistently record values of concern.In the current format, information is duplicated because the kernel density plot is mirrored. Presenting only one kernel density would improve clarity and produce a more space efficient plot.The violin plot only allows for informal between group comparisons of distributions and does not allow for presentation of formal between group inferences such as the estimates from mixed effects models, which can be presented in a line graph. Adaptations to multiarm trials are not as space efficient as for the line graph. Kernel density estimates for some data might extend to values outside the plausible range—for example, some kernel densities are estimated to be below 0 for eosinophil counts, which is not feasible clinically.The violin plot can be implemented in Stata by use of vioplot or by use of the ggplot2 package in R with geom_violin or SAS proc sgpanel.The kernel density plot displays the distribution of a continuous outcome. Data can be for a single time point or a derived change score—for example, the difference between the baseline value and maximum value while receiving treatment  and the participants who have improved would be in the bottom right . If more participants from the intervention group than control group were in the top left quadrant this would be cause for concern. In The scatterplot matrix is recommended in an exploratory setting to identify any outliers or patterns of interest. We suggest labelling outlying values with a participant identifier, as shown in This plot could be used to explore two continuous measures at any time point over study follow-up. Variations in symbol style and colours should be used to help separate overlapping measurements between groups. Reference lines could be included to indicate both upper and lower limits of normal for each outcome.This plot presents several visual problems. Use of solid colours results in occlusion, making it difficult to distinguish individual points; transparency options could help with this issue.Scatterplots are easily implemented as standard plots across the variety of statistical packages. For example, use of twoway scatter in Stata to produce the individual plots and the graph combine command or use of the grc1leg command to produce the scatterplot matrix.Among the visualisations considered for displaying multiple time-to-event outcomes, the options available were judged to be poor. Although multiple Kaplan-Meier plots could be used to display information on a limited number of prespecified events of interest, a gap remains in how to visualise multiple time-to-event outcomes simultaneously on the same plot. We discussed the development of novel plots in this setting and we will pursue this in future work.Randomised controlled trials provide a valuable source of data to compare harm outcomes between treatment groups and can help to identify potential signals for adverse drug reactions. However, evidence suggests that practices of reporting data for harm outcomes in clinical trial manuscripts are suboptimal. The CONSORT harms extensionOur aim was to provide consensus recommendations developed over a series of virtual meetings with researchers responsible for producing clinical trial manuscripts, including clinical trial statisticians and researchers from both academia and industry, as well as clinicians. We have provided examples of the endorsed visualisations to communicate risks of harms in the randomised controlled trial setting that can be used as an alternative to the widely used contingency tables. Our purpose was to increase the use of visualisations for harm outcomes in clinical trial manuscripts and reports and promote presentation of clearer and more informative information on harm outcomes to aid interpretation. Each of the endorsed visualisations can be constructed in standard statistical software and we have signposted to accessible code, when available, for implementation, with the aim of supporting adoption and to ensure efficient application of the recommendations. Trialists can implement our recommendations alongside the CONSORT harms extensionThe choice of visualisation will depend on the outcome type , the scenario , the trial design , and the purpose of the plot . It is for the statistician and clinical trial team to decide the most appropriate visualisation or visualisations for their data and objectives. A combination of plots is likely to be necessary—for example, presenting the traditional Kaplan-Meier plot alongside the survival ratio plot for prespecified harm outcomes to explore the temporal relationship, in addition to the dot plot to summarise the overall harm profile. Researchers can use the decision tree  to suppoAlthough these recommendations give a clear steer on the type of visualisations to consider, with some guiding principles on format, users can vary many aspects of plot design. For example, colours and symbols used, axis scales and limits, text formatting, appropriate use of labels, and number of groups being compared at once can all affect interpretation and understanding. Much has been written on these aspects, and we refer readers to the articles by Unwin and Muth,The predominance of statisticians over other researchers in the consensus group could be deemed a limitation of this work. Statisticians are, however, typically responsible for producing information on harms, such as in tables or visualisations, and thus the implementation of these recommendations. We therefore deemed their inputs and opinions highly relevant to the process. In addition to statisticians, a graphic designer was present across all meetings, and feedback was sought from each continually throughout the project. To ensure breadth of input, we worked with clinicians with experience in clinical trials to seek their feedback on the endorsed plots and to ensure understanding of each plot because they are likely to be the main consumers of such information. This collaboration with clinicians allowed us to incorporate clarifications into the recommendations where necessary. We hope that choosing clinicians who are active trialists will help to assist with dissemination of our findings and help us to increase the likelihood of these plots being used in practice. Patients were not involved in this work because our focus was to identify the best plots to present in scientific journals with a predominantly scientific readership. Our aim was to first provide guidance and tools to the authors of reports of randomised controlled trials. The next step that needs to be addressed is patient feedback. We did not consider use of interactive visualisations in these recommendations because we believe that these are in their own separate domain and require different considerations for appraisal , but the appraisals showed their inadequacies and a preference for more traditional plots. Endorsement was given for two less commonly used plots—the survival ratio plot and the plot of the mean cumulative frequency, and we encourage use of such plots with clear explanations to ease interpretation. We particularly encourage use of the mean cumulative function plot as a summary of the overall burden of harm in place of, or in addition to, summaries of time to discontinuation that are often reported as a proxy for harm. Given the scarcity of visualisations for presenting data for harm outcomes for randomised controlled trials, use of any visualisation of these harms is arguably novel, especially for emerging events. Once the use of visualisations for harm outcomes is more common in scientific publications, the desire for more innovative plots might increase.Although we suggest amendments to existing plots, the purpose of this work was not to develop new plots. However, it was clear that new approaches are needed for some scenarios, particularly when the visualisation of multiple time-to-event outcomes or multiple continuous outcomes is of interest, or when consideration of duration of events is important. Development of new plots will be undertaken in future work and we will seek to update guidelines to reflect any future progress. With a high likelihood of future updates being required, development of a website that can be more readily updated over time without need for new publications is one further thing to explore and has previously been advocated by Chuang-Stein and Xia.Visualisations provide a powerful tool to communicate harms in clinical trials, offering an alternative perspective to the traditional frequency tables. Implementation of the recommendations in this article should improve reporting of harm outcomes in clinical trial manuscripts and enable clearer presentation of harm profiles, and should help to identify potential signals for adverse drug reactions for further monitoring. We endorse each of the plots presented; however, we also highlight the limitations of each plot and provide examples of when their use would be inappropriate. We also caution users to practise care when creating and interpreting each plot. Although the decision tree aids the choice of visualisation, statisticians and clinical trial teams must ultimately decide the most appropriate visualisations for their data and objectives. We recommend trialists continue to examine crude numbers alongside visualisations to fully understand harm profiles. This information should also be reported in supplementary appendices so that readers of trial manuscripts can also appraise this information and so that the data are available to researchers who want to undertake systematic reviews and meta-analyses of harms.Harm outcomes data are complex, but visualisations can provide a clear summary of the harm profile and help identify potential adverse drug reactionsReporting data for harm outcomes in clinical trial manuscripts can be suboptimalResearchers have requested guidance on appropriate visualisations for harm outcomes and case studies detailing examples of useTo aid researchers in their choice of visualisations, this study undertook a consensus and endorsed visualisations, presented alongside a decision tree, to communicate harms in the randomised controlled trial setting that can be used as alternatives to the widely used contingency tablesThe choice of visualisation will depend on outcome type , scenario , trial design , and purpose of the plot Increasing the use of visualisations will provide clearer presentation of information on harm outcomes and thus enable informative interpretation, especially for assessing the harm profile


Secondary prevention measures have strongly contributed to the reduction of incidence and mortality of cervical cancer (CC) identifying women at high risk of developing it. This cross-sectional study aimed to investigate the acceptability of a home-based self-sampling methodology for Human Papilloma Virus (HPV) testing and the factors that may influence women’s preference.A random sample of women over the age of 50 years has been selected in Southern part of Italy. Data was collected through an anonymous self-administered questionnaire and included socio-demographic characteristics, knowledge of HPV infection and prevention measures, and attitudes towards the acceptability of self-collected cervico-vaginal sampling (CVS) and urine sampling (US).Among the 321 women who completed the survey, more than two-thirds (73.7%) knew that CC is caused by HPV, only 68.9% knew that the HPV screening is useful for an early detection and diagnosis of CC, and 17% never had Pap-test or HPV-DNA test. Of the respondents, 67.9% declared that they preferred self-collected US for future HPV testing compared with clinician-taken cervical samples (CCS). The most common reasons reported for preferring US included that it was easier (54.8%), more convenient (28.7%), and less embarrassing (21.7%). Among those women who showed negative attitude towards self-collected US, 8 out of 10 (77.3%) expressed scepticism about its diagnostic performance. Only 37% of the sample preferred CVS, and this attitude is mainly attributable to the fear of not carrying out a correct self-sampling (71.2%) and to its underrated diagnostic performance (33.7%).The preliminary results suggest that US is more acceptable than CCS and CVS in Italy. Urinary HPV test presents similar accuracy of the latter tests to detect CC and its supply in the context of population-based screening programmes could improve adherence, reducing the cost and burden on physicians.• Urine self-sampling could represent an innovative early detection approach to increase adherence to cervical cancer screening programmes.• Further research is needed to assess whether the screening status and the strategy of self-samplers distribution could act as predictors of screening uptake.


Electronic healthcare records (EHRs) are a rich source of information with a range of uses in secondary research. In the United Kingdom, there is no pan-national or nationally accepted marker indicating veteran status across all healthcare services. This presents significant obstacles to determining the healthcare needs of veterans using EHRs. To address this issue, we developed the Military Service Identification Tool (MSIT), using an iterative two-staged approach. In the first stage, a Structured Query Language approach was developed to identify veterans using a keyword rule-based approach. This informed the second stage, which was the development of the MSIT using machine learning, which, when tested, obtained an accuracy of 0.97, a positive predictive value of 0.90, a sensitivity of 0.91, and a negative predictive value of 0.98. To further validate the performance of the MSIT, the present study sought to verify the accuracy of the EHRs that trained the MSIT models. To achieve this, we surveyed 902 patients of a local specialist mental healthcare service, with 146 (16.2%) being asked if they had or had not served in the Armed Forces. In total 112 (76.7%) reported that they had not served, and 34 (23.3%) reported that they had served in the Armed Forces . The MSIT has the potential to be used for identifying veterans in the UK from free-text clinical documents and future use should be explored. The United Kingdom’s (UK) veteran population, defined by the British Government as those who have served in the military for at least one day , is estiWhilst most veterans transition out of the military without experiencing any difficulties, a sizeable minority report a range of mental health problems ,11,12. SThe ability to identify those who serve in the military would allow a comparison between those with and without military experience in relation to their demographic characteristics, physical and mental health outcomes, and healthcare utilization. Prior research has similarly conducted comparisons between the general population and specific cohorts, such as those with chronic mental illness ,18, and Veterans were manually identified using the South London and Maudsley (SLaM) Biomedical Research Centre (BRC) Clinical Record Interactive Search (CRIS) database holding secondary mental healthcare electronic records for the SLaM NHS Foundation Trust. An iterative approach was then followed. First, a structured query language (SQL) method was developed, which was refined using NLP and machine learning to create the MSIT, a tool designed to identify if a patient was a non-veteran or veteran ,22. We oGiven the large-scale use of free text in patient record systems and the vital role they play in clinical decision-making and describing patient characteristics, it is important this recording is accurate. Variations in the methods of notetaking among different healthcare professionals—combined with local, regional and national differences—have created data quality issues that could impact the use of automated text analysis tools ,24. The Routinely collected EHRs can be used to evaluate disease prevalence, monitor disease spread, facilitate epidemiological analyses , and impIn recent years, there has been a growth in the use of EHRs in the field of health data analytics either via NLP, or machine learning . The useIn the context of NLP algorithms, the two main themes specified are used to perform syntactic representation , extract specific information of themes , capturFor example, Downs et al.  used NLApproaches in recent years have also used machine learning combined with NLP to analyze large datasets. Kapadi et al.  developEHRs are a powerful resource when combined with NLP and machine learning; this approach may enable health practitioners, decision-makers and researchers to monitor and improve admissions, clinical decision-making patient outcomes, and quality of care. These algorithms also have the potential to identify current and former occupations, including military service, thus enabling a suite of research that could examine the health characteristics of different occupational groups.The Clinical Record Interactive Search (CRIS) system provides de-identified EHRs from the SLaM NHS Foundation Trust, a secondary and tertiary mental healthcare provider serving a geographical catchment of approximately 1.3 million residents across 4 south London boroughs  .https://github.com/DrDanL/kcmhr-msit, accessed on 8 February 2023). For this article, we provide a brief overview of the MSIT.A machine learning classification framework underpins the MSIT and is responsible for making predictions. It was developed in Python using the Natural Language Processing Toolkit (version 3.2.5)  and Scik(1)punctuations (using regular expressions);(2)words/phrases related to another individual’s military service;(3)stop words and frequently occurring terms (except military terms); and(4)word/phrases that may cause confusion with correctly identifying a veteran.A gold standard manually labeled dataset was created to train the MSIT. This labeled dataset included terms such as “veteran”, “army”, and “served in the forces”. Once documents had been identified, free-text documents were preprocessed to remove:The remaining features were then converted into term frequency-inverse document frequency features. The classification framework was trained to identify veterans based on the use of military terms and phrases with the outcome being binary .To improve the true positive rate of the MSIT and reduce the potential for false positives, a postprocessing of the outcome was applied based on a set of rules. For each document that was classified as containing indicators of military service, an SQL operation was performed to ensure the document used a military term or phrase.Sample: The MSIT was run over a batch of 779,944 records from patients who had used SLaM NHS Foundation Trust services since January 2017. This month marked the inception of the Consent for Consent (C4C) mechanism that records patients who consent to be contacted for research purposes. From these records, the BRC identified patients who were listed (1) as “Alive”; (2) aged 18 years or older; (3) had given C4C; (4) did not have indicators of dementia or psychosis according to their diagnostic codes; (5) had an email address or mobile telephone number; (6) were able to communicate in English without an interpreter; and (7) if they were active patients, were approved by their care coordinator to take part in the study.MSIT Execution: Once the sample had been identified, all free-text clinical documents relating to their care in SLaM were analyzed using the MSIT. Each patient was evaluated as being a military veteran or non-veteran based on their medical notes.Recruitment: Recruitment spanned February to June 2022. The research team performed another manual screening to detect any changes or further details regarding patients’ eligibility criteria . Participants were contacted in the first instance by email and, if this was not available, by text. Reminder emails and texts were sent and if participants requested not to be contacted, the researchers updated their C4C status.Data collection: Patients were invited to take part in the study via email or text, which included the survey link. After consenting, participants were asked a single question: “Have you ever served in the Armed Forces (military)?” to which they could reply “Yes” or “No”. If participants endorsed having served in the Armed Forces, they were asked follow-up questions about their military characteristics to further develop the MSIT, and to provide context for understanding any inaccurate misclassifications. These questions collected branch of service, rank, length of service, and regular/reserve status. Data relating to patients’ EHRs accessible via SLaM Electronic Patient Journey System (ePJS) were kept separately from their survey responses.Prize draw: Participants who completed the survey were entered into a prize draw for 26 e-vouchers comprised of 20 × £10, 5 × £20, and 1 × £50.Whilst the MSIT has high precision, it is important to ensure that the MSIT can identify “true” military veterans and that the underlying data used to train the MSIT are accurate. This was achieved by sending an online survey to a sample of patients in the SLaM NHS Foundation Trust to determine their self-reported veteran/non-veteran status and to compare this to their MSIT classifications. Responses allowed us to validate the MSIT algorithm’s predictive performance. Incorrect classifications would provide further information with which to refine the algorithm and improve its accuracy. To undertake this evaluation, the following steps were followed:All analyses were performed using STATA 16.1 MP . The positive predictive value was defined as the proportion of correctly identified true veterans over the total number of true veterans identified by the MSIT. Sensitivity was defined as the proportion of non-veterans identified by the MSIT over the total number of actual non-veterans (identified by patient report); specificity was determined as the proportion of veterans identified by the MSIT over the total number of actual veterans. Accuracy was measured using the Youden Index , which cEthical approval was given by the East of Scotland Research Ethics Service within the NHS Research Ethics Service (Ref: 20/ES/0060). Approvals were obtained by the SLaM Research and Development Office at King’s College London (Ref: R&D2020/029).Each stage of sampling, recruitment and inclusion/exclusion is depicted in After applying the automated exclusion criteria listed in n = 122/146). A sensitivity and specificity analysis were performed to determine how many veterans and non-veterans were misclassified. Overall, 23 true non-veterans were inaccurately categorized by the MSIT as veterans, and 1 veteran was inaccurately categorized by the MSIT as non-veteran. We found:The sensitivity of the MSIT was 0.83.The specificity of the MSIT was 0.92.In total, 146 participants provided eligible responses to the questionnaire see . Of thesA manual investigation of inaccurate classifications found that the MSIT had a high degree of accuracy with some exceptions. In minor examples of misclassification, the MSIT may be prone to assigning non-veterans as veterans. The most common reasons for misclassification included the mentioning of military family members and support received by the Salvation Army.This validation study has demonstrated that it is possible to identify veterans from free-text clinical notes using the MSIT. The MSIT tool performed well, as indicated by its high sensitivity and specificity. To the authors’ knowledge, this is the only study to have developed, applied, tested, and validated an NLP and machine learning framework for the identification of veterans in the UK using a large psychiatric database. Notably, by assessing the validity of the underlying free-text record, we have also demonstrated accuracy in the identification of records belonging to veterans.In the absence of a universal marker denoting prior military service, there has been no systematic way to identify and to thus examine veteran populations in mental healthcare services. The current Veterans’ Strategy Action Plan reinforces the importance of better understanding the health and well-being needs of the veteran community . The abiEHR-based Case Registers, such as CRIS, function as single, complete, and integrated electronic versions of traditional paper health records . These rEHRs in mental healthcare provide extremely rich material and analyses of their data can reveal patterns in healthcare provisions, patient profiles and mental and physical health problems ,42. ThisThis study has demonstrated that it is possible to identify veterans who accessed secondary mental healthcare services in the UK by using a Case Register. The MSIT was able to identify potential veterans with high precision, with 84.2% of cases correctly identified when corroborated against patients’ self-reported status. A key strength of the MSIT was the exploitation of NLP and the annotation of a large corpus of medical records. This is advantageous for automating the process of identifying veterans, as well as reducing the possibility of human error, and conscious/unconscious biases, and overcoming challenges when using military cohorts linked to Case Registers .At present, the MSIT is the only tool implemented by CRIS using C4C to validate the integrity of the patient record and validate reported status . The metThe MSIT does not rely on any coding structure or predefined fields and solely uses free-text documents, which broadens its potential applications to areas like diagnoses, occupations, and identifying ethnicity. This may allow studies to assess the specific characteristics of veterans in the SLaM sample compared to their non-veteran counterparts, as undertaken by Mark et al. . This was not deemed a substantial problem since the research team deliberately sought to oversample non-veterans. This was decided because erroneous classifications of non-veterans would contaminate a “true” veteran sample for future analyses. In addition, the smaller sample size of veterans reflects the reality that non-veterans outnumber veterans in the general population.Despite maximizing the sampling pool to improve our chances of finding eligible veterans, our attempts did not yield a large veteran sample . This may prohibit specific individuals, e.g., those with more complex mental health issues who may not consent to participate in research, from being represented in future analyses. This is an important consideration for future research.To address the inconsistencies in whether veteran status is recorded, a military service marker in the case registers and similar NHS databases could be implemented. Although time-consuming, it is possible that this could be verified by referencing UK Ministry of Defence records. This could be accompanied by broader educational efforts to help clinicians better support veterans in their care, to have a more in-depth understanding of the unique mental health needs of this population, and to be aware of the benefits of recording veteran status for referring into bespoke and specialist services. Any endeavor to ascertain veteran status must acknowledge that this can be a highly sensitive and personal attribute, especially in areas such as Northern Ireland .Additionally, the MSIT could be customized and further tested to identify other occupational groups, including those that are similarly exposed to potentially traumatic events, such as first responders; who have similar work-related patterns, such as oil rig workers, or who experience specific mental health issues, such as suicide among construction workers .Whilst the MSIT was successful in the context of the SLaM NHS Foundation Trust, further work is required to refine the tool to function on other datasets from other NHS Trusts. To that end, we have released the source-code of the tool. As with any other effort to access and test tools on EHRs, the researchers encountered many bureaucratic and governance hurdles, making the research an extensive and lengthy endeavor. This is noteworthy for future research seeking to test, develop and utilize the MSIT and other applications. The potential benefits of the MSIT, however, remain. Principally, the MSIT allows for the identification of veterans until a mandatory field is introduced, and for its continued use in retrospective analyses.We have shown that it is possible to identify veterans using NLP in electronic healthcare records of a secondary mental health service. The MSIT was able to identify military veterans with high accuracy and precision. This work has demonstrated that the MSIT can be used in a healthcare setting and was successful in exploiting 800,000+ free-text medical records. With the ability to identify military veterans, we can now conduct analyses comparing the health and well-being needs of this important cohort with the public, and other high-intensity occupations. With further refinement, the MSIT can be implemented in other electronic healthcare systems and to possibly identify other occupational groups.


The cervical epithelial tissue lipidome at different stages of cervix neoplastic transformation was studied by a shotgun label-free approach. It is based on electrospray ionization mass spectrometry (ESI-MS) data of a tissue extract. Lipidomic data were processed by the orthogonal projections to latent structures discriminant analysis (OPLS-DA) to build statistical models, differentiating stages of cervix transformation. Significant differences in the lipid profile between the lesion and surrounding tissues were revealed in chronic cervicitis, LSIL, HSIL, and cervical cancer. The lipids specific for HPV-induced cervical transformation mainly belong to glycerophospholipids: phosphatidylcholines, and phosphatidylethanolamines. The developed diagnostic OPLS-DA models were based on 23 marker lipids. More than 90% of these marker lipids positively correlated with the degree of cervix transformation. The algorithm was developed for the management of patients with HPV-associated diseases of the cervix, based on the panel of 23 lipids as a result. ESI-MS analysis of a lipid extract by direct injection through a loop, takes about 25 min (including preparation of the lipid extract), which is significantly less than the time required for the HPV test . This makes lipid mass spectrometric analysis a promising method for express diagnostics of HPV-associated neoplastic diseases of the cervix.A dramatic increase in cervical diseases associated with human papillomaviruses (HPV) in women of reproductive age has been observed over the past decades. An accurate differential diagnosis of the severity of cervical intraepithelial neoplasia and the choice of the optimal treatment requires the search for effective biomarkers with high diagnostic and prognostic value. The objective of this study was to introduce a method for rapid shotgun lipidomics to differentiate stages of HPV-associated cervix epithelium transformation. Tissue samples from 110 HPV-positive women with cervicitis ( A significant increase in cervical diseases caused by human papillomavirus (HPV) in women of reproductive age has been observed recently. HPV persistence often leads to malignancy and cervical cancer, which is one of the leading cancers and causes of death among women .The cause of cervical cancer in 96% of cases is HPV of high carcinogenic risk . AccordiPrimary screening for precancerous and malignant diseases of the cervix in developed countries includes cytological examination of cervical smears and HPV testing . ExtendeThe cytological method is highly specific for the detection of severe lesions of the cervix uteri  . ReplaciThe results of long-term follow-up of 176,464 women aged 20–64 years from Sweden (Swedescreen), the Netherlands (POBASCAM), England (ARTISTIC), and Italy (NTCC) clearly showed that the most effective and progressive direction is the use of the HPV test in the primary manifestations of precancer and cervical cancer . CompareThe immunocytochemical (ICC) and histochemical (IHC) study of the expression of oncoproteins p16 and Ki-67 in scrapings and biopsy samples make it possible to determine the histogenesis of individual tumors and clarify the source of metastasis. This study takes a long time  and is quite costly. Thus, the search for effective and accurate minimally invasive biomarkers with high diagnostic and prognostic value, requiring minimal time and material costs, remains relevant.More than 90% of malignant neoplasms result from genome damage—mutations. These are multiple mutations in the somatic cells of individual organs and tissues . ResearcMetabolomics (lipidomics) approaches have several advantages over proteomics and genomics presenting the distinct molecular phenotype of a particular pathological condition in real time . The hign = 30), LSIL (n = 30), HSIL (n = 30), and squamous cervical cancer . Comparative analysis of anthropometric data, menstrual function, hereditary, and obstetric anamnesis also did not reveal significant differences between the groups  . Women with severe precancerous and malignant diseases of the cervix (HSIL and SCC) had a significantly greater number of sexual partners compared with chronic cervicitis and LSIL groups. HPV infection in history was found in more than half (53%) of the patients included in the study. Patients with HSIL had significantly more frequent HPV-infections in history, compared with the chronic cervicitis group .The average age (33.4 ± 7.0) did not differ statistically significantly in the groups studied: chronic cervicitis with HPV infection  . Vulvovan = 110) underwent a cervical biopsy  and SCC  groups, the presence of two or more HPV types was significantly more common than in the ChC group.The results of HPV typing are presented in p-value < 0.05).The average HPV viral load was 5.3 Lg (1.6–8.8 Lg). More than half (53%) of the patients had a high viral load (more than 5 Lg): 34% of LSIL, 63% of HSIL, and 71% of SCC cases were due to long-term persistence of high-risk HPV with high viral load. HPV with a high viral load was significantly more common in the groups with severe dysplasia (HSIL) and SCC compared to the group with chronic cervicitis and LSIL (p = 0.014) and by 6.6 times (p = 0.001) higher in cervical cancer groups compared with chronic cervicitis. Pronounced changes were found in genes expression for cervical cancer compared to chronic cervicitis: a 7.3 times increase for MKI67 , a 12 times decrease for PGR , a 6.5 times decrease for OSM , a 4 times decrease for PTGS2 , and a 1.6 times decrease for PTEN .Seven of the 13 genes studied had comparable expression levels in all groups. Statistically significant differences were obtained for the genes MKI67, CDKN2A, PGR, PTGS2, OSM, and PTEN . The levp-value < 0.05) correlation with the degree of neoplastic transformation was found. The expression level of PGR, PTEN, and TLR7 changed most markedly with a histological diagnosis worsening.For 10 genes , a significant (p = 0.024), HSIL (p = 0.024), and cervical cancer (p = 8.8 × 10−6) groups compared with the cervicitis group  at risk for developing cervical cancer: 14 (47%) from the LSIL group, 9 (30%) from the HSIL group, 22 (73%) patients from the cervical cancer group, and none of the patients from the group with cervicitis. At the same time, there was a statistically significant increase in the median risk index in the LSIL  for pairwise comparisons of pathological and surrounding tissues from patients of all considered groups. OPLS-DA analysis of the lipid profile of the lesion vs. the surrounding tissue revealed significant differences for each separate group  (Characteristic positive ion mass spectra obtained by electrospray ionization mass spectrometry (ESI-MS) that averaged over 100 scans are shown in and SCC) .The lipids with the maximum contribution to the OPLS-DA model division were identified for each group . Lipid sOPLS-DA models were built to differentiate between benign (ChC), precancerous (LSIL and HSIL), and cancerous tissues (SCC) for the affected and surrounding tissues. The value of the dependent variable (“y”) in mathematical models was set in accordance with the severity of neoplastic transformation of the cervical epithelium . In the case of surrounding tissues, the SCC samples were grouped separately from the ChC, LSIL, and HSIL sample cluster . Thus, tA significant similarity between the HSIL and SCC lipidome was found for dysplasia cervical tissues . The levs = 0.4; p-value < 0.001) was found between the MS prognosis calculated using the OPLS-DA model and the severity of intraepithelial lesions. Moreover, almost all marker lipids (85%) significantly  negatively correlated with the degree of cervix transformation  negatively regulate intracellular levels of phosphatidylinositol-3,4,5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating the AKT/PKB signaling pathway. It is also one of the most frequently mutated genes in human cancer. PTEN regulates many cell processes: growth, apoptosis, migration, adhesion, and invasion ,48,49. LOncostatin-M (OSM) utilizes JAK–STAT3 and PI3K–AKT–mTOR pathways to promote EMT-associated cancer cell invasion and metastasis . One of Cyclooxygenase COX-2  is an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins and modulates several cellular processes: cell cycle regulation, apoptosis, extracellular matrix deposition, and angiogenesis. Prostaglandins have an important role in cancer-related progression and inflammation. PTGS2 over-expression has been considered as an indicator of invasiveness, aggressiveness, and metastatic potential in different malignancies including cervical carcinoma ,55. In oThe lipidomic analysis of cervical tissues neoplastic changes is considered as a highly informative method for biomarker search ,25. LipiA significant increase in the level of phosphatidylcholines was found in cervical cancerous tissues. T. Altadill and colleagues obtained similar results for endometrial tumor tissues. The possibility of differentiating the stages of endometrial cancer (according to FIGO) by the lipid profile of endometrial tissue samples has been demonstrated also .The increase in the PC 34:1 level during cervical epithelium neoplastic transformation was consistent with data from other studies. In particular, an increase in the level of this lipid in cancerous tissues of the breast, endometrium, and thyroid gland was noted ,22,23,65In our study, the main changes in malignant transformation of the cervical epithelium affected glycerophospholipids. This class of lipids has previously been proposed as potential tumor markers for other types of cancer  ,32,33,34L. Krasny et al. studied tissue lipidomics in cancer and benign diseases of the parotid gland . ParotidAcetonitrile, 2-propanol, chloroform, methanol, water, and NaCl were of high purity grade and purchased from Sigma–Aldrich . PREP-NA, Proba-GS, PCR HPV “Kvant-21” kits were purchased from DNA-TECHNOLOGY LLC, Russia.n = 110) read and signed voluntary informed consent. The study was approved by the Ethical Committee of the National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov (protocol No. 4 of October 2017). All patients were HPV-positive. Four groups were formed depending on the results of histological examination of cervical biopsies: chronic cervicitis with HPV infection , LSIL (n = 30), HSIL (n = 30), and squamous cervical cancer .All patients (The inclusion criteria were the following: reproductive age (21–45 years); HPV infection; intraepithelial lesions of the cervix, confirmed by a histological study; regular menstrual cycle; ability to comply with the protocol requirements; and written informed consent. Pregnancy, lactation, postpartum period, hormonal therapy, acute inflammatory diseases of the pelvic organs, impaired renal, liver, lung function in the stage of decompensation, and neuropsychiatric diseases were exclusion criteria.Evaluation of cytological smears (Cervix-brush type) from the cervix was carried out according to the Terminology Bethesda System (TBS) proposed by the US National Cancer Research Institute (last revised in 2014). Cytological smears were divided into three categories: normal (NILM), smears of undetermined value , intraepithelial lesions (precancerous), low , and high  degree.Cervical biopsies were scored as follows: mild dysplasia (CIN1), moderate dysplasia (CIN2), severe dysplasia (CIN3), and cervical cancer. Biopsy material was assessed using Lower Anogenital Squamous Terminology (LAST) (2012), where CIN1 is equivalent to LSIL and CIN2 and CIN3 are equivalent to HSIL .The Proba GS kit  was used to isolate DNA. Cells were lysed with a strong chaotropic agent, nucleic acids from the cells were adsorbed on a sorbent, washed, and eluted. The volume of samples after isolation was 100 μL.Amplification of type-specific DNA fragments of human papillomavirus and human DNA used to control sampling was carried out using the Kvant-21 reagent kit  for the detection, typing, and quantitation of HPV by PCR. Amplification was performed in real time using a DT-964 instrument . The fluorescence level was measured at each cycle of amplification in the FAM, HEX, ROX, and Cy5 channels. The processing of the results was carried out automatically using the software for the DT-964.The level of mRNA expression of 13 genes  in scrapings of the cervical epithelium was determined by the reverse transcription and polymerase chain reaction (RT-PCR). The material was taken with a cytobrush from the cervix, with the obligatory capture of the transformation zone . To prevent mRNA degradation, the brush was immediately immersed in a test tube with guanidine thiocyanate solution , rotated in a test tube for 10–15 s, and removed by squeezing thoroughly. The samples were stored at −20 °C for one month or at −70 °C for one year.RNA isolation was performed using the PREP-NA kit . The method is based on lysis in a guanidine thiocyanate solution and precipitation of nucleic acids. Reverse transcription was performed at 40 °C for 30 min and then at 95 °C for 5 min. PCR was carried out by thermocycler RealTime system DTprime 4X1 in the “real time” mode in a volume of 12 μL according to the program: 15 cycles-80 °C 5 s, 94 °C 5 s; 1 cycle-94 °C 5 min; 50 cycles-94 °C 20 s, 64 °C 20 s; 10 °C-storage. The following reagents from DNA-Technology, Russia were used: RT-buffer, PCR-buffer, dNTPs, reverse transcriptase, Taq-polymerase, oligonucleotide RT primers and PCR primers, and fluorescent-labeled TaqMan-probes corresponding to each gene . The fluorescence level was measured on each cycle at a temperature of 64 °C along the FAM fluorescence channel. To increase the sensitivity and specificity of PCR, a “hot start” was used, which was provided by polymerase with antibodies (TechnoTaq).v/v), incubation for 10 min, and filtration using filter paper. A volume of 800 μL of an aqueous solution of NaCl (1 mol/L) was added to the filtrate to induce phase separation. The mixture was centrifuged at 400× g for 5 min at room temperature. The lower organic layer containing lipids was collected and dried in a stream of nitrogen, then redissolved in 40 μL of acetonitrile-2-propanol  for mass spectrometric analysis. The samples of tissue were very small (punch biopsy) in order to minimize trauma to the cervix, especially with small lesions of the epithelium. Thus, the final sample volume was 40 µL and was only sufficient for analysis in one ion mode.Part of the punch biopsy specimen for histological examination (10 mg) was frozen in liquid nitrogen and stored at −80 °C for further lipidomic research. Lipid extracts were obtained in accordance with the modified Folch method . A biopsm/z 400–1000 with resolution of 50,000 and the following ion source settings: capillary voltage 4.1 kV, spray gas pressure 0.7 bar, drying gas flow rate 6 L/min, and the temperature of the drying gas was 200 °C. Due to the low sample volume the positive ion mode was chosen for this study only.Molecular composition of the samples was determined by flow injection analysis (FIA) electrospray ionization mass spectrometry using Maxis Impact qTOF mass spectrometer  ,23,24,25Tandem mass spectrometry was done using data-dependent analysis with the following characteristics. Five of the most abundant peaks were chosen after full mass scan and subjected to MS/MS analysis with collision induced dissociation applying 35 eV collision energy, 1 Da isolation window, and 1 min of mass exclusion time.m/z table for further processing , and transformed into the abundance-ocessing . The sprm/z values with theoretical computer-generated values. The code searched a record within 10 ppm from the experimental m/z. Proton and sodium ion adducts were considered. More precise identification was done based on the MS/MS data for the peak under consideration, if it had undergone MS/MS analysis. Lipids nomenclature through the paper is in accordance with LIPID MAPS /[PGR]—a ratio of mRNA expression levels MKI67 and PGR,[CDKN2A]/[BCL2]—a ratio of mRNA expression levels of CDKN2A and BCL2.The IR threshold was previously calculated using ROC analysis. It is equal to 57 units on a 100-point scale.2) and predict (Q2) variance of the data. The Q2 parameter was calculated by 7-fold cross-validation. Permutation test with 100 permutations was used to validate models and estimate the significance of Q2 and R2.ESI-MS data were analyzed using multivariate latent structures discriminant analysis (OPLS-DA) for pairwise comparisons of pathological and surrounding tissue categories and to compare surrounding tissues and pathological tissues with each of the diagnoses  ,75. Thesp-value < 0.05) were used to construct OPLS-DA models for categorical prediction of the diagnosis [The degree of correlation of potential marker lipids for a neoplastic process with histological diagnosis and expression of 13 studied genes was assessed using Spearman’s rank correlation . Lipids iagnosis . To deteShotgun lipidomics were proven to have a high potential for accurate, fast, and minimally invasive early and differential diagnosis of HPV-associated cervical diseases. Significant differences in the lipid profile between the affected and surrounding tissues were revealed in chronic cervicitis, LSIL, HSIL, and cervical cancer. ESI-MS analysis of lipid extract by direct injection takes about 25 min (including preparation of the lipid extract), which is significantly less than the time required for the HPV test . This makes lipid mass spectrometric analysis a promising method for the express diagnostics of HPV-associated neoplastic diseases of the cervix.In this study, the lipids specific for HPV-induced cervical transformation mainly belong to glycerophospholipids: PC and PE. These classes of lipids are associated with apoptosis suppression, impaired cell metabolism, and the stimulation of proliferative processes. An increase in the level of PC and a decrease in the level of LPC in tissues with neoplastic transformation, were shown for tumor tissues of other organs.p-value < 0.05) with the degree of cervix transformation. Thus, the ESI-MS of changed tissue organic extract demonstrates high accuracy in the classification of neoplastic lesions of the cervix, comparable to the histological research method, the gold standard of diagnosis.The lipid composition of tissues adjacent to the transformation zone undergoes significant changes. At the same time, the lipid spectra of the affected tissues in severe lesions (HSIL) and cervical cancer overlapped in many ways. Diagnostic OPLS-DA models were developed based on 23 marker lipids. More than 90% of these marker lipids positively correlated , as well as a decrease in the unnecessary use of destructive treatment techniques.


In order to meet the WHO 2030 cervical cancer elimination program, evaluation and utilization of sensitive testing method, and feasible sampling technique is a paradigm for enhancing cervical cancer screening coverage. Self-sampling for screening of HPV DNA testing is one of the easiest and sensitive techniques, though the evidence was limited in the Ethiopian context. This study aimed to compare the performance of self-collected vaginal specimen versus clinician collected cervical specimen for detection of HPV among HIV positive women in Ethiopia.p < 0.05 considered as statistically significant.We conducted a comparative cross-sectional study design to collect cervicovaginal specimens among HIV positive women of age older than 24 years. Data were collected from six government hospitals from January to October 2021. A total of 994 cervicovaginal specimens was collected by clinicians and HIV positive women themselves in the cervical cancer screening unit using Abbott Cervi-Collect Specimen Collection Kit, and molecular HPV testing was conducted. Data were entered into an Excel spreadsheet and analyzed using SPSS version 25. Sensitivity, specificity and kappa were reported with p < 0.001). The discriminatory power of the test as true positive and negative was excellent with an area under the curve of 0.86.The prevalence of high-risk HPV was 29.4% among self-sampled specimen and 23.9% among clinician collected specimens. The overall concordance of the test result was 87.3%. Oncogenic HPV types, other than HPV16&18 were predominant in both sampling techniques, 19.9% from vaginal self-collected specimen and 16.7% of clinician collected cervical specimens. The sensitivity and specificity of self-sampled HPV test was 84.0% and 88.4%, respectively. The level of agreement was good (k = 0.68) and statistically significant (The magnitude of oncogenic HPV was higher in self-collected samples than the clinician collected specimen with good agreement between the two sampling methods. Thus, we recommend the Ministry of Health in Ethiopia to expand utilization of the self-sampled technique and enhance the coverage of screening in the country. Since the 1970s, several countries instituted screening and treating cervical cancer programs, however, still significant gaps and challenges persist in reducing morbidities and mortalities from the disease , and repSince the burden of cervical cancer is increasing, how does the global strategy to eliminate cervical cancer by 2030 is progressing so far? The pillar of the elimination program is set-forth 90–70–90 programmatic directions, meaning 90% of girls fully vaccinated with the human papillomavirus (HPV) vaccine by age 15 years, 70% of women are screened with a high-performance test, and 90% of women identified with the cervical disease receive treatment .Per the strategy of screening using high-performance test and easy technique, what did previous reports about self-sampling for HPV detection tell us? According to a study, carcinogenic HPV infections were identified in 23.2% of patient collected specimens as compared to 34.9% of the samples collected by the clinician, with more important carcinogenic HPV types being identified by the clinician collected samples , and mosThe aforementioned results, however, had reported some controversies and necessitate further study. In addition, such kind of studies was very limited in Ethiopia. And, therefore, the current study was aimed to examine the performance of self-collected vaginal specimens versus clinician collected cervical specimens, and to set forth a guideline, and recommend a very sensitive method with an easy sampling technique in the country which is supposed to enhance coverage screening.We conducted a comparative cross-sectional study to collect cervicovaginal specimens among HIV positive women of age older than 24 years in Addis Ababa, Ethiopia.This study was conducted among selected government hospitals that employed the new HPV DNA molecular method for screening cervical cancer from January to October 2021. The facilities enrolled included the Zewditu Memorial Hospital, St. Paul Hospital, ALERT hospital, Minilik Hospital, Yekatit-12 and St. Peter Hospital. The Zewditu Memorial Hospital is located in the Kirkos sub-city, St. Paul’s Hospital and St. Peter Hospital are located in the Gulele sub-city, Yekatit-12 and Menelik-II Hospitals in Arada sub-city, Alert Hospital located in Kolfe Keranio. Ethiopia is a country in the horn of Africa, and has eleven Regional States , BenishangulGumuz, Gambela, Sidama, Somalia, Harari, and South West) and two City administrations (Addis Ababa and Dire Dawa). Addis Ababa is the capital city of the country and it is one of the two city administrations. The city is divided into 11 sub-cities, and geographically located at 9°1′48″N38°44′24″E, covering an approximate land area of 526.46 square km and lying at an elevation of 2355 m (7726 feet) .All human immunodeficiency virus (HIV) positive women that had antiretroviral therapy (ART) follow-up and age older than 24 years, who volunteered to participate in the study, and signed consent were counseled and included in the study. However, pregnant women, women in menstrual period during the time of specimen collection, no history of sexual activity, received pelvic radiation therapy, period of genital tract infection  or acute inflammation, poor labeling or mislabeling of the specimen were the exclusion criteria used.Considering all the eligibility requirements, a total of 497 self-collected specimens and about the same number of clinicians collected cervical specimens with an overall total of 994 specimens collected from both groups for molecular HPV testing.Data were collected by trained clinicians and participants themselves for self-sampling. Intensive counseling was given to HIV positive women attending cervical cancer screening unit on self-sampling. Two transport tubes were used to collect cervicovaginal specimens for each woman (one for self-sampling and another for clinician’s collection) and were labeled. The tube labeled self (s) and one cervical brush were given to the woman to collect the specimen. Then clinicians collected cervical specimen using speculum examination immediately. After cervicovaginal sample collection, screening using visual inspection with acetic acid (VIA) was done by trained staff. The specimens were collected using Abbott Cervi-Collect specimen collection kit (List No. 4N73). Each kit contains; One Cervical Brush and one Transport Tube containing 2.4 ml Specimen Transport Buffer (guanidine thiocyanate in a Tris buffer) which stabilize DNA till sample preparation. The collected specimens were transported to the testing laboratory, and trained laboratory professionals and the principal investigator conducted laboratory testing using Abbott real time polymerase chain reaction (PCR) (m2000rt/SP) instrument and its molecular HPV testing assays . The principles of the procedure were described in a previous publication .Training was given for data collectors and the quality of data was maintained with continuous follow-up on the process. The recruited data collectors were experienced in conducting cervical cancer screening and treatment. The laboratory test was done by trained lab professionals and the principal investigator who are working at the accredited laboratory by the Ethiopian National Accreditation Office . The positive and negative external quality control included in each run to monitor the quality of test parameter. The entire process from extraction to amplification and detection was maintained using the endogenous human beta globin, internal control (IC).p < 0.05 was considered statistically significant.The collected data were entered into an Excel spreadsheet, coded and exported to SPSS version 25 for analysis. Descriptive statistics were applied to determine the performance measures, including sensitivity, specificity and kappa values. According to Altman (1991), the interpretation of the kappa value or the strength of the agreement was reported as poor (< 0.2), fair (0.2–0.4), moderate (0.4–0.6), good (0.6–0.8), and very good (0.8–1.0) . The senOf the total 497 women recruited in this study, 994 specimens were collected, both vaginal and cervical. From the given total, 16 (3.2%) samples were missing. Of which two specimens were failed lab result and the remaining’s were with no HPV DNA result recorded on the worksheet due to mislabeling from sample transaction sheet and sample tube. Demographic characteristics of the participants were described in a previous publication elsewhere .The findings of clinician collected specimen indicated that 363(73.0%) HIV positive women were negative for HPV and 119 (23.9%) were high-risk (HR) HPV positive, whereas, the finding of self-collected vaginal specimens indicated 349 (70.2%) HIV positive women were negative for HPV while 146 (29.4%) were HR HPV positive women. More frequent oncogenic HPV types identified in both specimen types were non-HPV16 and 18 (other HR HPV), i.e., 16.7% (83/497) from clinician collected cervical specimens vs. 19.9% (99/497) from vaginal self-collected specimen. In addition, 3.8% (19/497) oncogenic HPV16 from clinician collected vs. 4.8% (24/497) of self-sampled, and mixed HPV16 and other HR HPV of 2.4% (12/497) vs. 3.2% (16/497) of clinician and self-collected specimens, respectively of HPV burden were observed. One self-collected specimen (0.2%) indicated oncogenic HPV16, 18 and other HR HPV types  of the cervicovaginal specimens tested. In addition, 12.7% (61/481) discordant results were identified which need further investigation.p < 0.001) and a good agreement was observed in both sampling methods with k = 0.68 , indicating that the discrimination power of the test method as true positive and negative results was excellent.In this study, we tried to see the performance measures of a comprehensive evaluation of classification models, considering ROC and AUC. The true positive rate called sensitivity on the y-axis and the false positive rate (1-specificity) on the x-axis were also measured  and early proteins, E6/E7 and the PCR technique was different , and theA review paper in low- and middle-income countries revealed that the concordance rate of self-sampling with clinician collected sampling was 87.0–97.5% , which wThe Indian study depicted that the sensitivity and specificity of both self-sampling and clinician collected specimen were more than 98.0% . SimilarOncogenic HPV types were detected with higher prevalence of self-samples than clinician collected cervical specimen in the current study, of which, HR HPV types other than HPV16 and 18 were predominant, 19.9% of self-collected specimens versus 16.7% of clinician collected specimens. The reported finding on the genotypic distribution of HR HPV types other than HPV16 and 18 were similar with the previous finding in Ethiopia . AnotherThe women preferred self-sampling compared with clinician collected samples primarily due to privacy, although nurse assistance was needed . When coImplementing sensitive screening modalities combined with easy sampling methods with minimum pain or discomfort, such as self-sampling of vaginal and urine samples are highly encouraged and widely supported by the scientific community . Our finThe HR HPV detection using a real time PCR among vulnerable population, which was women with HIV with sufficient sample size in a resource limited setting, and the highlighting of the importance of self-sampling in the population of Ethiopia might be taken as a strength of the study. However, the study had limitations, that include, lack of further clinical investigation and follow-up of HPV positive women to clearly identify the probability of oncogenic HPV that causes cervical cancer.The magnitude of oncogenic HPV was higher from self-collected samples than the clinician collected specimen with good agreement between the two sampling methods. Therefore, the findings of the study may be considered by the Ministry of Health in Ethiopia and its stakeholders to expand the utilization of the self-sampling method in the country. Self-sampling might increase the screening coverage at national level to meet the World Health Organization (WHO) 2030 global cervical cancer elimination program.