Post-traumatic stress disorder (PTSD) is a pervasive disorder among both current and ex-serving Australian Defence Force (ADF) members. Studies have shown current psychological and pharmacological treatments for PTSD are suboptimal in veterans, with high dropout rates and poor adherence to treatment protocols. Therefore, evaluating complementary interventions, such as assistance dogs, is needed for veterans who may not receive the ultimate benefit from traditional therapies. The present longitudinal mixed-method study examined the effectiveness of Operation K9 assistance dogs among sixteen veterans with PTSD, specifically, their effects on suicidality, PTSD, depression, and anxiety from baseline to 12 months post-matching. Self-reported measures were completed prior to receiving their dog (baseline) and at three time points  following matching. The Clinician-Administered PTSD Scale for DSM-5 was used to assess the severity of every PTSD case. Veterans participated in a semi-structured interview 3 months post-matching. Whilst there was a reduction in the proportion of veterans reporting any suicidality, there was no significant change in the probability of veterans reporting suicidality between time points. There was a significant effect of time on PTSD, depression, and anxiety symptoms. Three major themes emerged from qualitative data analysis: life changer, constant companion, and social engagement. Qualitative data suggest assistance dogs can have a positive impact on important areas of daily life and support veterans in achieving some of the prerequisites for health, including access to services, transport, education, employment, and development of new and diverse social and community connections. Connections were key in improving health and wellbeing. This study exemplifies the power of human–animal relationships and adds emphasis to the need to take these seriously and create supportive healthy environments for veterans with PTSD. Our findings could be used to inform public health policy and service delivery, in line with the Ottawa Charter action areas and indicate that for veterans with PTSD, assistance dogs may be a feasible adjunct intervention. Post-traumatic stress disorder (PTSD) is a pervasive disorder among both current and ex-serving Australian Defence Force (ADF) members, resulting in many disabling negative outcomes . ApproxiPTSD has been associated with increased rates of morbidity, mortality, and suicidality  and is commonly comorbid with depression and anxiety in the general population ,4,5. In Between 2001 and 2018, there were 267 certified suicide deaths among ADF ex-serving personnel [Individuals with PTSD who do not engage in treatment are at risk of a chronic course of illness . TreatmeAs such, there is a clear need to evaluate the use of adjunct interventions as a supplement to traditional therapies. An emerging area is the use of assistance dogs as an adjunct intervention for veterans with PTSD. An assistance dog is specifically trained to perform a variety of tasks aimed at alleviating PTSD symptoms ,17. Taskn = 12) veterans 6 months after they received their assistance dog [A systematic review of the effect of assistance dogs on PTSD symptoms in veterans concluded that participants experienced improved wellness because the dog had a calming influence on their mental/emotional state in potentially stressful situations . Other sance dog .Qualitative studies frequently reported reductions in suicidality, with veterans accrediting their assistance dog with keeping them alive ,25,26,27Despite limited empirical evidence, the demand for and use of assistance dogs is increasing in Australia. Therefore, it is critical to investigate the effectiveness of assistance dogs as an adjunct intervention for veterans with PTSD within the Australian context. Whilst preliminary evidence supports the use of assistance dogs as an adjunct intervention, methodological differences and the small number of studies available make it difficult to compare results. Most studies to date also tend to rely on standardised self-reported measures that do not examine the lived experience of the veteran or are purely qualitative. Furthermore, few studies have used a longitudinal approach to measuring changes over time. A longitudinal mixed-method study design incorporating a more objective diagnostic measure of PTSD, such as the Clinician-Administered PTSD Scale (CAPS-5) ,28, woulThis study is the first in Australia to employ both quantitative and person-centred qualitative methods to investigate the effectiveness of an assistance dog program in helping Australian veterans with PTSD cope. The aim is to longitudinally examine the effectiveness of Operation K9 assistance dogs on mental health and wellbeing in veterans with PTSD over a 12-month period. This study will address the following research questions: 1. What is the effect of having an Operation K9 assistance dog on veterans’ mental health and wellbeing , as measured by both self-reporting and clinician-administered measures of mental health over a 12-month period? and 2. What are veterans’ perceptions and experiences of having an Operation K9 assistance dog in terms of mental health and wellbeing?This study analysed data from 16 veterans who received an assistance dog through the Royal Society for the Blind Operation K9 Program between October 2015 and March 2019.https://www.seedifferently.org.au/operation-k9-steps-up (accessed on 6 February 2020).The Operation K9 Program provides assistance dogs to Australian Defence Force (ADF) veterans (ex-serving) with PTSD. Individuals are signposted to the program either through a veteran’s support agency, medical and allied health professionals, or self-referral. A small breeding program supplies the Operation K9 Program. A considered matching process of client and dog ensures that working teams worked optimally and within their strengths. The rigorous application process takes many factors into consideration, including physical abilities, verbal capacity to interact with an assistance dog, and information from medical professionals, specialist skills of each dog, the needs of veterans, and the personalities of both dog and human. Whilst providing companionship, comfort, support, and increased social interaction, the Operation K9 assistance dogs are trained to meet the specific individual needs of each veteran. Given the process of breeding, training, and matching dogs is time- and resource-intensive, the total number of participants in this study was small due to constraints in the number of dogs available to be trained. Further information can be found on the Operation K9 website . The dependent variables were self-reported scores on suicidality, PCL-5 (PTSD Checklist), DASS-21 , depression and anxiety subscales, and clinician-assessed PTSD using the CAPS-5 . The research was approved by the Department of Defence and Veterans’ Affairs Human Research Ethics Committee  and the University of South Australia’s Human Research Ethics Committee .At baseline, 3, 6, and 12 months, all participants completed a self-report questionnaire containing questions on demographic information and validated measures of mental health and wellbeing. Suicidal ideation plans and attempts were examined using four questions from the National Survey of Mental Health and Wellbeing . ParticiThe PTSD Checklist (PCL-5) was used to assess PTSD symptom severity . The scaDepression and anxiety symptom severity were measured using the Depression Anxiety Stress Scale—Short Form (DASS-21) depression and anxiety subscales . The DASA clinical diagnosis of PTSD was obtained at baseline, 6, and 12 months via a structured diagnostic interview using the Clinician-Administered PTSD Scale for DSM-5 Past Month Version (CAPS-5) . PTSD diSemi-structured qualitative interviews were undertaken three months post-matching. Open-ended interview questions enabled unrestricted responses, therefore providing richness and depth of data . ParticiParticipants were provided with information regarding the study during their first meeting with Operation K9 staff and asked to provide consent to be contacted by the research team. A member of the research team contacted potential participants; consenting participants completed a participant consent form.Participants were asked to complete the self-report questionnaire at four time points: (1) treatment baseline (about to receive dog), (2) 3 months, (3) 6 months, and (4) 12 months post-matching. Participants were provided a list of resources and contacts within the questionnaire in case they felt distressed by any aspect of the study. All interviews were conducted in person or via telephone and audio-recorded to allow transcription. Interviews also included opportunities for feedback and for any concerns to be raised. The study’s interview protocol also included detailed procedures to support and manage potentially distressed and suicidal participants.p < 0.05. A sensitivity power analysis was performed using G*Power3 software [n = 16 to determine the minimum effect size the study was sensitive to. The study was sufficiently sensitive to detect a medium to large effect size. Continuous variables and residual errors were normally distributed with no outliers . Statistical significance was considered at software  with α =D values ).Total scores for PCL-5 and DASS-21 depression and anxiety subscales were calculated using mean imputation to replace missing data (<20%). Following imputation, one baseline PCL-5 score was missing, and so mixed-effect models, a model well suited for dealing with missing data, were conducted with the missing data points . For calGeneralised estimating equation models were conducted to examine changes in the probability of suicidality and severity of PTSD case (CAPS-5) across time. The models specified a dichotomous variable of suicidality and PTSD case where 1 = Yes and 0 = No, with a predictor variable of time (baseline measurement reference category) and panel variable of subject ID.d effect size between baseline and the other time points. Cut-offs for effect size were calculated using the Cohen’s d method with values of small (0.20), medium (0.50), and large (0.80).To account for repeated measurements of self-reported PTSD symptoms (PCL-5) and depression and anxiety symptoms , scores were compared over time using linear mixed-effects models. The main effect of time is reported. Planned comparisons were conducted to investigate statistically significant changes in scores and Cohen’s Reliable change and clinical significance were evaluated for PTSD symptom severity (PCL-5) from baseline (BL) to 3 months, 6 months, and 12 months to estimate the extent to which symptom changes could be considered reliable and clinically significant. Evidence suggests reliable change is represented by a 5–10-point change and a clinically significant change by a 10–20-point change in PCL-5 total score . The curQualitative analysis was used in a complementary way to elaborate on the results from the quantitative analysis and increase understanding and meaningfulness . To idenParticipant demographic data for sex, age, and service are presented in The proportion of participants reporting suicidality at each time point is presented in Means and standard deviations for the PCL-5 symptom severity score and DASS-21 depression and anxiety subscale scores are presented in F = 20.l29, p < 0.001. Planned comparisons revealed a significant mean difference between baseline and 3, 6, and 12 months with effect sizes ranging from 0.43 to 0.94  = 4.461, p = 0.008. Planned comparisons revealed a significant mean difference between baseline and 3, 6, and 12 months  = 11.642, p < 0.001). Planned comparisons revealed a significant mean difference between baseline and 3, 6, and 12 months with large effect sizes  participants met the diagnostic criteria for PTSD, while at 6 months there were 7 (47%) and at 12 months a total of 5 (31%). Overall, there was a 50% reduction in the number PTSD cases between baseline and 12 months with a significant change observed .Data from qualitative interviews were analysed to examine veterans’ perceptions and experiences of having an Operation K9 assistance dog. Three major themes emerged from the data analysis: life changer, constant companion, and social engagement. Themes, findings, and illustrative data examples are presented.This theme captured the life-changing impact of assistance dogs in improving quality of life for veterans and providing support for symptom management:‘See I didn’t even think it was possible for me to ever, ever improve. I thought this was going to be my lifestyle for the rest of my life, but you know this has just absolutely proven me wrong, it’s turned me right around 180 degrees’.(Veteran #12)‘cause otherwise I wouldn’t be here’ (Veteran #6). Another veteran, talking about suicidality, said, ‘this has now alleviated all of that, I don’t even think about it anymore’ (Veteran #12). Furthermore, veterans expressed a sense of responsibility to care for their dog as providing a reason to live, for example:Three veterans reported the role of their dog in suicide prevention, with one stating ‘You just sort of look at (dog) and you think, well, you know, at least she’s worth living for and she’s there … it sort of reminds you constantly, you know, when you’re all by yourself, there’s always someone there.’(Veteran #6)‘I need to stay alive now otherwise, (dog) would miss me … who would look after (dog), he gives me that right to live again’ (Veteran #12). For many veterans their assistance dog had given them a sense of purpose. One said their dog gave them ‘a reason to be’ (Veteran #2), while another ‘it gives me a reason and purpose now, so I’ve got to stay well and fit enough now to look after him, so he’s now my family’and (Veteran #12).‘I notice I don’t do it as much (hypervigilance) and even my seating its, you know, I just go oh there’s some empty seats, we’ll go sit there, you know have my back exposed …’ (Veteran #14). While another said, ‘if I’m feeling a bit depressed or whatever he’s there, and I know he’s there and it just takes my mind off things makes life a lot easier’ (Veteran #9). Veterans reported their dog actively seeking them out to interrupt symptoms provided comfort, resulting in them feeling calm and relaxed. One described their dog as their ‘comfort blanket’ (Veteran #4), while another noted:Veterans reported a sense of having reclaimed their life, giving them independence and a way of managing their emotional and mental states and coping with a range of symptoms including anxiety, low mood, and hypervigilance. One veteran said, ‘He calms me and he knows, he really does know when you’re having a bad day or a problem... he just lays and puts his head on my foot and it is the most calming, relaxing feeling and it’s a confidence thing you know, it just makes me feel good all the time’.(Veteran #12)This theme reflected upon the unique bond that is characterised by the human–dog relationship. Veterans highly valued the constant physical and emotional presence of their dog, appreciating how their dog was ‘always there’ for them. Assistance dogs were described as providing a stable base of support for veterans and providing a buffer against stressful and anxiety-producing situations.‘in safe hands’ (Veteran #12), while another referred to their dog as their ‘security blanket’ (Veteran #5). Veterans reported the sense of being looked after by their dog, for example: ‘… constant sort of watching on her part and constant support’ (Veteran #1).A number of veterans also reported the presence of their dog provided a sense of safety and security, one stating they are ‘giving me more reassurance and confidence … the ability to rest easier at night, the comforting of just walking around with me during the day … it is all been absolutely amazingly positive’ (Veteran #13).The presence of their dog seemingly provided a calming and therapeutic effect. Many veterans discussed feeling increased self-confidence due to the presence of their dog, and how this has enhanced their communication skills, while others said their dog was a source of reassurance, for example, ‘I was a recluse and didn’t leave my home for many, many years and now every day is an adventure … and gives me something to look forward to’ (Veteran #12). Having their assistance dog resulted in positive interactions with family, friends, and members of the community, leading them to feel more comfortable in public and allowing them to spend increased time in the community and engage more socially. For example, one veteran noted ‘I’ve just felt a lot more comfortable going out in public … kind of broken that barrier where I used to … try and avoid the public at all costs.’ (Veteran #9). Another stated that ‘I can like sit in a cafe and not feel self-conscious anymore … I won’t race off I’ll just linger and try and relax …’ (Veteran #1).Prior to receiving their assistance dog, many veterans reported difficulties with isolation and disengagement from family, friends, and community: The aim of this study was to longitudinally examine the effectiveness of Operation K9 assistance dogs on mental health and wellbeing in veterans with PTSD over a 12-month period. Although there was no significant change in suicidality, there was a significant reduction in the proportion of veterans meeting clinician-diagnosed PTSD and reduced severity of self-reported PTSD, depression, and anxiety. Veterans’ perceptions of their assistance dog’s role in supporting their mental health and wellbeing were classified into three main themes: life changer, constant companion, and social engagement.This is the first longitudinal mixed-method study investigating an Australian assistance dog program for veterans with PTSD. Only one other study has investigated changes in suicidality of participants after receiving an assistance dog , with reA contribution and strength of the current study is the rich personal experiences provided by veterans about the impact of their assistance dog on their mental health and wellbeing. In relation to suicidality, veterans reported that their assistance dogs had given them a sense of purpose and responsibility, a ‘reason to be’, and a reason to live. Three veterans explicitly discussed the role of their dog in reducing suicidality. This is consistent with previous qualitative studies that have reported the role of an assistance dog as a protective factor for suicidality ,26. InteThe current study is the first study to suggest that assistance dogs may provide a sustained reduction in symptoms of PTSD, depression, and anxiety over a 12-month period. Findings are consistent with previous quantitative studies that found a significant reduction in self-reported PTSD, depression, and anxiety symptoms in participants 3 to 6 months after receiving an assistance dog ,21,22,23To account for potential self-report response bias for PTSD, the CAPS-5 was used in the current study to provide an objective, clinical measure of PTSD based on DSM-5 criteria. This is the first study to use a validated clinician assessment of PTSD in an evaluation of assistance dogs. Our results suggest that whilst veterans may still have some symptoms of PTSD, depression, and anxiety, they may experience reduced symptom severity and distress due to the presence of their assistance dog. The significant decreases seen in PTSD, depression, and anxiety symptoms may be contributing to increased functioning, independence, and social engagement. For example, veterans acknowledged that even though they may be feeling depressed or anxious, having their dog there ‘just makes it easier’. Veterans emphasised their relationship with their dog above all else, describing their dog as a constant, stable base of support, providing an overall calming effect. These findings are consistent with those of Van Houtert et al. (2018), which suggest that improvements in PTSD symptoms are due to the dogs’ calming influence on mental and emotional states in stressful situations . Whilst A limitation of the current study was the lack of a control group of veterans with PTSD not receiving an assistance dog. Recruiting such control groups is, however, extremely difficult in longitudinal research, particularly when participants are required to complete assessments over multiple time points, with no benefits to them. Future studies could consider including a waitlist control group to provide a comparison group and enable an investigation of the efficacy of assistance dogs.The veterans recruited for this study were considerably motivated to have an assistance dog to help manage their PTSD-related symptoms, and as a result may not be representative of all veterans with PTSD. As noted by O’Haire and Rodriguez (2018) in their paper, this limits generalisability to those who are amenable to receiving an assistance dog. Furthermore, the small sample size due to low availability of assistance dogs limits the generalisability of the findings from this study to other groups of veterans. While the current study was adequately powered for large effects, future research should aim to use a larger sample. The cost and time it takes to train an assistance dog may impact the extent to which studies could be implemented in the future.The current study did not specifically ask veterans what challenges they experienced as a result of having an assistance dog. Future research should explore this in more detail and be used to inform policy and service delivery .Given the high attrition rates in other interventions for veterans’ mental health ,15, furtOne concern is that the decreases seen in the current study are partly due to a ‘repeated measurement effect’. This has been observed in psychological research when measures of the self-reported negative effect decrease over time, even without treatment . While sThe use of a dichotomised variable for suicidality was somewhat limiting in terms of statistical power. Questions about suicidality were not explicitly asked in the qualitative interviews but rather, related content emerged organically. This may mean that some veterans did not speak about the role of their assistance dog in terms of reducing suicidal thoughts and behaviour potentially due to reluctance to disclose such thoughts and behaviours. Future studies could include a more comprehensive continuous measure of suicidality to increase the statistical power to observe changes. It may also be of interest to include specific questions about suicidality in qualitative interviews to understand veterans’ perceptions and experiences more clearly in relation to this construct.In Australia, 24.9% of recently transitioned veterans will have a diagnosis of PTSD in their lifetime . This st


Replacement of cytology screening with HPV testing is recommended and essential for cervical cancer elimination. HPV testing for primary screening was implemented in 12 laboratories within 9 Latin American countries, as part of the ESTAMPA cervical cancer screening study. Our observations provide information on critical operational aspects for HPV testing implementation in diverse resource settings.readiness of 12 laboratories to start HPV testing and their continuity capacity to maintain good quality HPV testing until end of recruitment or up to December 2021. Readiness was based on a checklist. Information from the study database; regular meetings and monitoring visits; and a questionnaire on laboratory operational aspects sent in May 2020 were used to assess continuity capacity. Compliance with seven basic requirements (readiness) and eight continuity requirements (continuity capacity) was scored  and totaled to classify readiness and continuity capacity as very limited, limited, moderate or high. Experiences, challenges, and enablers of the implementation process are also described.We describe the implementation process of HPV testing in ESTAMPA, focusing on laboratory aspects. We assess the high readiness, three moderate readiness, and of two laboratories new to HPV testing, one had limited readiness and the other very limited readiness. Two of seven laboratories with high readiness also showed high continuity capacity, one moderate continuity capacity, and the other four showed limited continuity capacity since they could not maintain good quality HPV testing over time. Among three laboratories with moderate readiness, one kept moderate continuity capacity and two reached high continuity capacity. The two laboratories new to HPV testing achieved high continuity capacity. Based on gained expertise, five laboratories have become part of national screening programs.Seven of 12 laboratories had High readiness of laboratories is an essential part of effective implementation of HPV testing. However, high readiness is insufficient to guarantee HPV testing high continuity capacity, for which a “culture of quality” should be established with regular training, robust monitoring and quality assurance systems tailored to local context. All efforts to strengthen HPV laboratories are valuable and crucial to guarantee effective implementation of HPV-based cervical screening. More than 600,000 new cases and 300,000 cervical cancer deaths occur every year; over 90% of these are in low-income and middle-income countries (LMIC) .Cytology-based screening has successfully reduced cervical cancer rates in places where it has been systematically implemented . HoweverPersistent infection with high-risk HPV is the leading cause of cervical cancer . SeveralIn the World Health Assembly  adopted Several tools are available to support countries with the implementation of HPV testing within cervical cancer screening –33. In pESTAMPA is a multicentric study of cervical cancer screening with HPV testing conducted in 12 study centers’ laboratories (SC1-12) in nine countries in Latin America. The study aims to evaluate the performance of different techniques and approaches to triage HPV positive women and to inform on how best to implement affordable and sustainable HPV-based screening programs in LMIC.The study protocol has been previously published . BrieflyDigene HC2 HPV DNA test  or COBAS HPV test . After HPV testing, aliquots of PC were prepared for future molecular triage. Digene HC2 sample conversion kit was used for conversion of samples collected on PC before HC2 HPV testing. HPV (HC2 or COBAS) was done following manufacturer instructions and ESTAMPA standard operating procedures (SOPs). For quality control (QC) of HPV testing, in some laboratories, about 10% of samples tested with HC2 or COBAS were retested either at the same laboratory using a different HPV technique or at and international hub with COBAS. In one center, samples from the first 900 recruited women were stored in the Digene Standard Transport Medium (STM) and subsequently tested with HC2. In this center, a sub-study evaluating the impact of operational factors on HPV positivity of HPV assays including HC2, COBAS and APTIMA  was conducted and COBAS results were assumed as QC of HC2 and APTIMA  were referred to colposcopy, 2-3 biopsies were collected from any observed lesions, and women diagnosed with cervical intraepithelial neoplasia (CIN) grade 2 or worse (CIN2 +) were treated with LLETZ. At this colposcopy visit, a cervical sample was collected for HPV testing and other molecular triage tests. Women with negative colposcopy or histology showing lesions less severe than CIN2 (< CIN2) were invited for a follow-up visit at 18 months for a second HPV screen to complete ascertainment of disease. Women positive for HPV at this visit were referred to colposcopy and clinically managed as needed.Overall, women could have one, two, three or four HPV tests during the study, depending on their screening results and associated study visits  and on the sample being selected for QC.The main steps carried out to implement HPV testing in ESTAMPA laboratories  are descreadiness for HPV testing implementation in the laboratory. Before launching the study, local principal investigators willing to take part, were asked to identify a laboratory with adequate facilities as to carry out HPV testing for primary screening. Laboratory-based investigators could propose their own facilities or others thought more suitable for future participation in national HPV-based cervical screening program.(1) Assessment of Next, a checklist  was usedLaboratories were also asked whether they had personnel to perform the test; and whether they had established logistics for transport and storage of samples, management of reagents validity and delivery of results. We did not request specific arrangements for external quality assurance (EQA) in this checklist, but we proposed different QA measures as the study progressed.Based on this initial assessment, each study center developed a road map to address limitations and joined the project when ready to launch recruitment.(2) Initial site visit to start recruitment and HPV testing. The study covered the cost of HPV reagents and additional consumables where needed. Procurement was done internationally or in-country, depending on availability of local distributors and registration of products. Once consumables were in place  and laboratories were ready, a site visit by IARC researchers to launch the ESTAMPA study was organized.During this initial visit, training on the study protocol, study’s SOPs and good clinical practices was given to all study personnel over 1-2 days, followed by real-life recruitment. In parallel, the selected HPV testing laboratory was visited to verify that conditions were ready to start testing. When enough samples for a full- or a half-testing batch  had been collected, additional training on the study protocol, laboratory SOPs and overall sample handling from reception at the laboratory to delivery of results, was provided to all laboratory personnel.Next, using recently collected samples, hands-on training was given to 1-2 technicians on: (i) Running HPV testing if no previous experience, (ii) Performing conversion of samples collected in PC if HC2 testing was used, (iii) Preparing aliquots from PC vials, and (iv) Recording results on the study database. Lead investigators at each center made arrangements to repeat this training as needed when new laboratory personnel joined the study.As the study progressed initial and monitoring site visits were done by a team mostly composed by local researchers selected from study centers already recruiting with support from ESTAMPA researchers.continuity capacity of laboratories to perform good quality HPV testing. Laboratories’ continuity capacity was assessed throughout the study. Sources of information used for the assessment included: minutes from meetings with local investigators and laboratory managers, reports from monitoring visits, data provided by the study centralized web-system, and from a questionnaire on laboratory operational aspects ((3) Assessment of  aspects  sent to As laboratories joined the study, implementation of quality assurance processes was recommended, such as providing regular training to technicians on HPV testing and study’s SOPs, retesting of a subset of samples and annual subscription to EQA. The study covered the first year EQA fees for new laboratories which were encouraged to ensure funding for next subscriptions.The centralized web-system specifically developed for the study allowed regular checking on the progress of recruitment, the number of HPV tests carried out , the speed of uploading results, and statistics on clinical activities. Thus, the web-system provided effective indicators of the HPV testing capacity of laboratories, such as the volume of HPV testing and the HPV positivity over time. As over 75% of samples tested in each laboratory were collected at the initial screening visit, the number of recruited women per study center was used as a proxy of HPV testing volume, and the positivity of HPV testing at the time of enrolment was used as a proxy of overall HPV positivity, independently of the HPV detection technique used . Based on previously reported HPV prevalence among Latin American women –38, we aThe questionnaire sent to laboratory managers requested current details on HPV equipment status, annual subscriptions to EQA, training on HPV testing for current and new technicians, preparedness for procurement process of HPV and related laboratory supplies, availability of tracking systems for HPV reagents and laboratory supplies and for samples transport and storage and turnaround time of HPV results.As recruitment progressed, many logistic activities were smoothly handled over to local investigators, including procurement of reagents and consumables if they became available in the country, and responsibility for maintaining adequate stocks. This transition facilitated implementation or expansion of HPV-based cervical screening programs.All collected data were used to assess whether laboratories were able to continue performing good quality HPV testing over time, from the first batch of testing up until end of recruitment or December 2021.readiness for HPV implementation. Monitoring, and operational questionnaires classifying eight continuity requirements (within the six domains), were used to assess the continuity capacity of laboratories to perform good quality HPV testing over time.Collected data from the readiness and continuity capacity assessments were summarized in six main domains: characteristics of the laboratory facility, personnel, procurement of reagents and consumables, tracking systems for reagents expiry and supplies, logistics for transport and storage and delivery of testing results. Responses to the initial checklist were classified into seven basic requirements (within the above six domains) and were used to assess the laboratory very limited (score ≤ 3), limited (score = 4-5), moderate (score = 6) and high (score = 7). Similarly, the continuity capacity of laboratories to perform HPV testing over time was classified as very limited (score ≤ 3), limited (score = 4-5), moderate (score = 6-7) and high (score = 8)  or non-compliant , and values were summed up to generate a total score (range = 0-7 for readiness and 0-8 for continuity capacity). Using the scores, the readiness of laboratories to start HPV testing at the time of initial assessment was classified as ore = 8) , 2.Twelve study centers with corresponding HPV laboratories, located in nine Latin American countries  participated in the ESTAMPA study. Eleven laboratories were part of public systems , and one offered service under a private system. Six laboratories had staff with experience in detecting HPV for clinical diagnosis and research at the initial assessment. Two of the remaining laboratories had experience in detecting HPV for research only, and two had experience in HPV detection for diagnosis but not for research. One laboratory was set up in a regional hospital, but the study team was based on a high-level expertise (diagnostics and research) HPV reference laboratory. Finally, one laboratory did not have previous experience in HPV detection or any other molecular diagnostics .Despite differences in recruitment targets  and year when recruitment and HPV testing started (2012 to 2017), among 42,502 women with valid HPV results recruited until December 2021, the overall yearly HPV positivity consistently ranged between 12 and 16% over time  .As the study provided all relevant consumables at the start, centers did not need to engage in direct procurement activities. However, all teams and particularly laboratory leads demonstrated awareness of current local regulations for purchasing in-country, and the ability to deal with importation of non-registered in-country consumables or to negotiate prices of testing kits and machine loaning as SC7.All laboratories had established logistics for transporting and storage samples (as per protocol), only (SC12) did not have a system to control the validity of laboratory reagents, and in addition to SC2 and SC3 did not have a system in place to return results to women, did not have a standard report template nor instructions on how to share results with participants.high readiness at initial assessment and three  had moderate readiness with only one essential requirement not fulfilled. Of two laboratories totally new to HPV testing, the one (SC7) supported by a high-level laboratory had limited readiness and the other one (SC12) very limited readiness.In summary, seven laboratories  had All laboratories offered appropriate training in HPV testing and laboratory study SOPs to new laboratory personnel, even if they were not assigned to HPV testing and gained or improved the ability to ensure timely procurement of HPV reagents and other consumables.Laboratories developed procurement plans, independently of whether the planning only involved preparing paperwork for customs clearance in advance or dealing with local distributors or purchasing the consumables . However, two laboratories  had difficulties coordinating supplies’ importation all over recruitment.Laboratory management systems  for tracking of reagents expiry and handling of samples (transport and storage) were incorporated or improved by all laboratories. However, three laboratories  needed the central team to send regular reminders to verify expiration dates throughout the study.Six laboratories struggled to consistently deliver results within one month. Four of them  had additional challenges linked to procurement of consumables, while the delays for the other two laboratories (SC2 and SC6) were largely related to the study setting. SC2 recruited in a remote location, and samples were shipped by air only 1-2 times per month. SC6 recruited in several centers concurrently in order to reach its recruitment target of 10,000 women. This meant that samples arrived at both laboratories in large batches and accumulated, causing delays with processing and turnaround of HPV results. In such cases, different measures were applied when to guarantee adequate follow-up of screen-positives .high continuity capacity as they fulfilled all criteria; two (SC2 and SC6) had moderate continuity capacity having only not fulfilled one requirement and four  showed limited continuity capacity.Overall, six laboratories  showed high readiness also showed high continuity capacity, one (SC6) showed moderate continuity capacity, and the other four  were not able to maintain good-quality HPV testing over time after an excellent start and showed limited continuity capacity. Among three laboratories with moderate readiness, one (SC2) kept moderate continuity capacity and two (SC3 and SC4) reached high continuity capacity. The two laboratories new to molecular testing  with subsequent limited and very limited readiness, respectively, achieved high continuity capacity.n = 497) having invalid HPV results. Immediate correcting measures to guarantee the safety of participants  were implemented in these three SCs.Five of the 12 laboratories are now or soon will be part of their national HPV-based screening program, including SC1 and SC7 with high continuity capacity, SC6 (moderate) and despite challenges during the study, SC9 and SC10 with limited continuity capacity based on their nature  and on the experience, increased installed capacity and lessons learnt throughout the study.The WHO recommends screening women with a high-performance test, such as HPV DNA testing, twice by age 35 and 45, and encourages countries to move from cytology- or VIA- to HPV-based cervical screening programs. However, most cervical screening worldwide is done with cytology and transition to HPV-based cervical screening should be carefully planned. There are several important factors to consider, such as the size of target population, the screening approach, women’s acceptance and participation in HPV testing, local availability of HPV testing platforms , laboratory installed capacity, availability of trained personnel, quality assurance, and other operational aspects.In our study, two main factors enabled successful performance of HPV laboratories as part of HPV introduction: the initial assessment of existing laboratory capacity and the regular support offered to participating laboratories throughout the study. The initial assessment identified limitations in the laboratory’s ability to perform HPV testing and helped laboratory teams to develop a road map  to address shortcomings before launching the study. In all laboratories, rapid training of new technicians, regardless of their role, was provided in order to minimize disruption of activities. The simple hands-on processing of both HPV tests used in the study (HC2 and COBAS) also helped train new personnel. The training was usually completed within a week, even where staff had no previous HPV testing experience.The ESTAMPA study aims to evaluate triage techniques for HPV positive women and contribute to the implementation of HPV-based cervical cancer screening in Latin America. Two baseline cervical samples were collected using a cytobrush washed in vials containing PreservCyt medium (PC) to assess the performance of different molecular triage techniques. HC2 testing is incompatible with PC and uses its own collection medium . This meant that study centers using HC2 had to prepare samples for testing using a conversion kit, adding on average 1.5 full working days per testing batch of 88 samples. Additionally, all laboratories (independently of the HPV test used) were requested to produce aliquots from both PC vials for future evaluation of molecular triage techniques. This process could take up to one full working day. Additionally, screened positives attended a colposcopy visit at which another sample for HPV testing was collected, and those not receiving treatment based on colposcopy and histology results were recalled at 18 months and had another HPV test, and aliquots were produced from samples once HPV tested. In real-life settings, laboratories may have more capacity for HPV testing as these extra research activities will not be required.The average HPV positivity over time was 14.1%, with some fluctuations related to debut and end of recruitment and testing at each center along the study. We detected HPV positivity values below 10% or above 16% (the inspection threshold) in some centers over time. This triggered an immediate evaluation of the entire screening process by using the study centralized web-system, followed by a site visit to reinforce training and ensure safety of participants as needed. In most cases, low or high HPV positivity was usually associated with characteristics of the included study population. For instance, one center reported a consistently high positivity (>16%), most likely due to the recruitment of a referral population  (data not shown). Assessments suggested HPV testing problems only in one laboratory, where immediate evaluation of the HPV equipment and a plan to maintain good cleaning and calibration of laboratory equipment were requested. This laboratory could not comply with these measures and the study had to stop before reaching the recruitment target.high readiness requirements. Once testing started, laboratories faced challenges in achieving high continuity capacity for HPV testing. In some study centers, the recruitment rate was unexpectedly low at times, leading to several weeks of delay before enough samples for a whole testing batch were collected, sometimes leading to expiry of reagents before HPV testing and impacting the turnaround of HPV results. Corrective measures such as testing half batches were introduced; however, sometimes, even half-batch testing was not achievable, particularly during the COVID pandemic, and inevitably led to wastage of reagents. In addition, as HPV testing had not been rolled out in almost all participating countries during the study, collection medium and HPV testing reagents needed to be imported as local procurement was not possible. Local principal investigators had to quickly learn regulations to import unregistered products, logistics associated with customs clearance, and different aspects of the procurement process, including price negotiation and management of expiry and stock levels of consumables. Laboratories managers were encouraged to subscribe to international EQA schemes. Six laboratories had yearly subscription to EQA schemes before the start of the study as required by local policies. The study covered the first-year fees for other four laboratories, but only one of them managed to continue with subscriptions afterward. Fees for the remaining two laboratories were not provided by the study nor covered by the laboratory. The main reason for not ensuring funds for EQA, in addition to limited resources, was the lack of a “culture of quality”, as managers did not understand the importance of maintaining regular EQA not only for HPV testing but for other laboratory activities.Readiness for HPV testing was measured ahead of starting recruitment, and study teams whose laboratories were not fully ready to start, took several months (up to 3 years) to deal with shortcomings and they only started recruitment and testing once they fulfilled We did not observe any relationship between laboratory high continuity capacity and attainment and size of recruitment goals. Of six laboratories with high continuity capacity, two exceeded their original recruitment targets of 10,000 and 5,000 by 10% and 27%, one reached its target , one is still recruiting  and two aiming to recruit 5,000 had to stop at 67% and 88% attainment. The two with moderate continuity capacity reached their targets ; and none of the four showing limited continuity capacity completed recruitment, two originally targeted 500 and the other two 5,000 women. Of importance, independently of the level of continuity capacity, five laboratories will become or are already part of their national screening program. For instance, one laboratory with high continuity capacity that stopped recruitment because the HPV testing platform loan ended, and the equipment was retrieved by the manufacturer company is now part of the national program though using a different HPV testing platform but maintaining the laboratory staff trained on HPV testing for the study. The incorporation of these five laboratories into national programs demonstrates that all efforts in training, continued support, and engagement with and between local and regional stakeholders during the study, are certainly contributing to the implementation of HPV-based screening programs in Latin America.Most difficulties faced by laboratories reflect the early days of the HPV testing market. Currently, more than 250 HPV tests are available on the market, although most of them are not yet adequately validated , 39. It The main strengths of our results are: (1) The diversity of laboratories where HPV testing was performed, contributing with valuable information on the multiple challenges that countries may face during transition to, implementation and scale-up of HPV testing in primary screening; (2) The use of regular feedback given by the central coordinating team and most importantly through exchange of experiences and lessons through the strong ESTAMPA study network, which facilitated installing a “culture of quality” in laboratories, and (3) The demonstration that readiness and continuity capacity are good indicators of how HPV implementation is ongoing in terms of laboratory management and quality of HPV testing, and can be assessed using simple checklists and questionnaires. On the other hand, two limitations of our study are related to these simple tools. First, information provided by laboratory researchers or managers may be prone to reporting bias, and second, continuity capacity was assessed based on responses to a single-time questionnaire. Nevertheless, in our study, these limitations were mitigated by completing and verifying information through monitoring visits to all teams and their participating laboratories over time. However, it should be noted that in the context of a screening program, regular monitoring visits may not be feasible and other strategies may be needed to complete implementation assessment.The 12 laboratories participating in ESTAMPA were essential for study centers screening and overall study achievements, despite several of them being new to HPV testing or to its use in cervical screening and having faced challenges to adopt the technique into daily activities. The strong support provided by the study network facilitated overcoming most difficulties leading to 42,502 HPV screened women across 12 study centers. We have described here the process of introducing HPV testing in 12 laboratories across Latin America, we further plan to use data per HPV testing batch to evaluate and compare HPV performance across laboratories, over study visits  and over time.high readiness is essential for successfully implementing HPV testing in laboratories; however, high readiness is not sufficient to guarantee HPV testing high continuity capacity. Several aspects to achieve this high continuity capacity should be considered:In summary, our assessments confirm that as (1)A “culture of quality” in laboratories and across the cervical cancer screening spectrum should be established, including regular training on SOPs, robust monitoring and quality assurance systems  tailored to the installed capacity and local resources(2)Using a friendly and tailored to local context database, if possible centralized and web-based, to allow regular monitoring of the overall screening process, including laboratory and clinical activities, and feedback for improvement(3)Training on preparation of appropriate plans for HPV supplies procurement and stocking and on local regulations on products registration and importation of laboratory supplies should be provided to laboratory managers and leaders of cervical screening programs(4)Strategies to guarantee reasonable turnaround of HPV results to women should be in place to ensure that any screened woman receives her HPV results and that screened positives receive adequate and timely treatment and follow-up(5)Exchange of experiences and lessons learnt between multidisciplinary implementers from different settings and countries should be encouraged to apply most suitable implementation strategies according to context and define additional implementation research needs.The original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the International Agency for Research on Cancer (IEC Project 12-27-A7), the Pan American Health Organization (PAHO) Ethics Committee and Ethical Committees in each of the study participating centers. The informed consent include details on the background, procedures of the study, risks and benefits, statement of confidentiality, specimen use and study staff to contact. The study is considered minimal risk as the procedures are standard practice in cervical cancer screening programs. The patients/participants provided their written informed consent to participate in this study.MA and RH conceived the ESTAMPA study and are the principal investigators responsible for its overall conduction. MAP, AF, GS, LM, CT, LF, VV, GV, AC-V, GR, AC, and CW are the local principal investigators responsible for recruitment, clinical management, and data collection. MAP, AF, and GS conceptualized the laboratory aspects of the study and together with MLR, MH, LM, MRP, CT, PH-N, FD, JA-S, MZ, and VV coordinated all laboratory activities including training of new technicians as needed. JL, AP, MB, LM, PM, YC, RC, DC, LG, AR, MR, MLB, and LC performed HPV testing. JV and AB provided statistical support and together with MA, MH, MLR, EL, and AR centrally coordinated the study. MA, RH, NB, and SL contributed to the funding acquisition and local capacity building. MLR and MA wrote the manuscript. MH, AB, JV, SL, NB, and RH contributed to the finalization of the manuscript. All authors reviewed and approved the final version of the manuscript.


Human papillomavirus (HPV)-based screening programs still use one-size-fits-all protocols but efficiency and efficacy of programs may be improved by stratifying women based on previous screening results.We studied the association between cervical intraepithelial neoplasia grade 3 or cancer (CIN3+) and previous screening results in the Population-Based Screening Study Amsterdam (POBASCAM) trial, performed in the Netherlands in the setting of regular screening, where women aged from 29 to 61 years old were invited to cytology and HPV co-testing at enrolment in year 1999/2002 and at the next round in 2003/2007. We selected 18,448 women  who tested HPV–negative in 2003/2007 and did not have cervical intraepithelial neoplasia grade 2 or worse (CIN2+) or hysterectomy after enrolment. Follow-up was collected until 14 years after the 2003/2007 screen, covering 4 rounds of screening. Risk of CIN3+ and CIN2+ among women with an HPV–negative test, irrespective of previous round results and stratified according to previous round HPV and cytology results, were calculated by the Kaplan–Meier method.p < 0.001) or co-test  and, equivalently, decreased in women with a previous negative HPV test  or a negative co-test . The CIN3+ risk was not influenced by the previous cytology result. The CIN3+ risk among HPV–negative women was increased after both a previous HPV16–positive test  and a previous HPV16–negative/HPVother–positive test . For endpoint CIN2+ (147 cases), findings were similar except that the CIN2+ risk was increased after previous abnormal cytology . The presented risk estimates were calculated by tracking histological results through the Dutch nationwide pathology archive (PALGA) and were not adjusted for non-compliance with the colposcopy referral advice.During 14 years of follow-up, 62 CIN3+ cases (24 in the intervention group and 38 in the control group) were detected. HPV–negative women had a 14-year CIN3+ risk of 0.48%  and CIN2+ risk of 1.17% (0.99 to 1.38). The CIN3+ risk among HPV–negative women was increased in women with a previous positive HPV test  trial with enrollment in 1999/2002 and 19 years of follow-up, selected 18,448 women with a negative HPV result at the second study-related screening round (5 years after enrolment) and stratified them according to screening results at the time of study enrolment.We estimated the 14-year stratum-specific risk of CIN3+ and CIN2+, covering 3 rounds of screening.The 14-year CIN3+ risk among HPV–negative women was 0.48%, was slightly lower when the HPV test in the previous round was negative (0.43%), but was markedly higher when the HPV test in the previous round was positive (2.36%).The 14-year CIN3+ risk among HPV–negative women was not increased when the cytology test in the previous round was positive.Our results confirmed the safety of extending the screening interval from 5 to 10 years when switching from cytology-based screening to HPV-based screening.A screening extension from 5 to 10 years can be maintained at the second HPV-based screening round when the HPV result in the previous round was negative.A history of HPV positive screening results is associated with an increased future risk of precancer also when the current HPV result is negative.HPV results from multiple screening rounds should be considered when determining the time to the next screening invitation. Human papillomavirus (HPV) causes virtually all cases of cervical cancer and its precursors. Testing for HPV DNA has demonstrated earlier detection of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) and greater protection against invasive cervical cancer than cytology –3. This HPV testing is less specific than cytological testing, but HPV-based screening programs may achieve a higher level of efficiency by extending the screening interval after a negative primary HPV test. The new Dutch HPV-based cervical screening program, which screens women aged from 30 to 60 years old every 5 years, was implemented in 2017 with an extension of the screening interval from 5 to 10 years for HPV–negative women aged 40 or 50 years old ,9. The qIn order to gain insight into the safety of HPV-based screening intervals longer than 5 years by previous screening results, we collected follow-up data from the POBASCAM (Population-Based Screening Study Amsterdam) trial. This HPV-based screening trial offers a unique opportunity to anticipate risk estimation based on longitudinal HPV-based screening data, because participating women received HPV testing in 2 consecutive screening rounds, the first at enrolment in 1999/2002 and the second 5 years later [N = 44,102) aged from 29 to 61 years old were randomly assigned (1:1) to receive HPV and cytology co-testing (intervention) or cytology-only with blinded HPV (control). In the intervention group, women with normal cytology and HPV–negative result were invited to the next screening round after 5 years. Women with high-grade abnormal cytology (HSIL) were referred for colposcopy irrespective of the HPV result. Women with borderline or low-grade cytology (ASCUS/LSIL) and those with normal cytology and HPV–positive result were advised to undergo repeat co-testing at 6 or 18 months, and referred for colposcopy in case of HSIL and/or HPV–positive result. Women without HSIL and HPV–negative result were invited at the next round after 5 years. In the control group, women with normal cytology were invited to the next round after 5 years. Women with HSIL cytology were referred for colposcopy. Women with ASCUS/LSIL were advised to repeat cytology after 6 or 18 months and referred for colposcopy in case the repeat cytology result was abnormal (ASCUS+). Women with normal repeat cytology were invited at the next round after 5 years. Post-colposcopy management was done by cytology at 6, 12, and possibly 24 months after colposcopy and, if no lesion was detected, women were referred back to routine screening. At the second screening round after 5 years, women in both study groups were managed according to the intervention protocol (HPV and cytology co-testing). At the third screening round after 10 years, women in both study groups were managed according to the control protocol (cytology-only), which was in line with the screening program at that time. At the fourth screening round after 15 years, women invited before January 2017 were managed according to the control protocol and women invited at a later time were managed according to the new primary HPV screening program. In the new program, HPV–negative women are invited to the next screening round after 5 to 10 years and HPV–positive women are tested by cytology. HPV–positive women with ASCUS+ at baseline are directly referred for colposcopy, while HPV–positive women with normal cytology are invited for repeat cytology testing after 6 months, and referred for colposcopy when ASCUS+. Women with normal repeat cytology are invited at the next round after 5 years.This is a longitudinal study in which we collected passive follow-up of women who participated in the POBASCAM trial. The POBASCAM trial  was conducted in the setting of the regular (cytology) cervical cancer screening program in the Netherlands that invites women aged from 30 to 60 years old every 5 years. The design has been published previously ,13. In bIn the POBASCAM trial, a conventional cervical smear test was prepared on a glass slide for cytology reading after which the brush was placed in a vial for HPV DNA testing done using high-risk HPV GP5+/6+ PCR-enzyme immunoassay (EIA)  that detThe POBASCAM trial was approved by the VU University Medical Centre (no 96/103) and the Ministry of Public Health (no 328650). This study was approved by the scientific committee of PALGA.We included women from the intervention and control group of the POBASCAM trial who were invited for co-testing and had a negative HPV test result at the second screening round. Hereby, we will refer to these women as HPV–negative women or as the total study sample. Those with a CIN2+ diagnosis or hysterectomy before the start of the second screening round were excluded. Follow-up started counting at the second screening round visit and ended on the date of a CIN3+ diagnosis or hysterectomy. As detection of CIN3+ after a positive screen can take up to 4 years , the 4-year cutoff corresponds with the risk of prevalent disease, the 9-year cutoff corresponds with the risk of CIN3+ because of a positive screen after 5 years, and the 14-year cutoff represents the risk of CIN3+ because of a positive screen after 10 years. If the second screening round was the last attended screening round and no CIN3+ or hysterectomy was reported, follow-up was censored 4 years after the starting date of the second round. If the third screening round was the last attended and no CIN3+ or hysterectomy was reported, follow-up was censored 9 years after the starting date of the second round. In all other women, follow-up ended 14 years after the start of the second round or on 31 January 2018, whichever came first. The approach for censoring the data was also used in previous follow-up analyses of the POBASCAM trial  as previous test result. HPV was labeled as positive if the result was positive at the generic HPV test and negative otherwise. Cytology was labeled as positive if the result was borderline or worse (ASCUS+) and negative otherwise. Co-test was labeled as positive if HPV and/or cytology were positive and negative if they were both negative.p-values for the comparison of CIN3+ risks after a positive and negative screening result in the previous round. Separate risk comparisons were made for previous round HPV, cytology, and co-test results. The 95% CI were calculated by parametric bootstrapping and p-values were calculated by Wald testing, assuming a normal distribution for the logarithmic risk. Separate estimates were reported for intervention and control group, and for the pooled estimate over the 2 study groups. Finally, we calculated separate CIN3+ risks for age strata <39 years, 39 to 48 years, and ≥49 years at the second round, defined as matching with the screening invitations, and genotype results at the previous round, using hierarchical ranking based on HPV genotype groups HPV16, HPV18/45, HPV31/33/35/52/58, and other high-risk HPV genotypes. Statistical significance was considered at a significance level of 5%. All analyses were repeated for endpoint CIN2+ and the main analysis was repeated for endpoint cancer. All analyses were performed using Microsoft Excel 2016 and STATA/SE 14.1, except for bootstrapping and visualization that were performed using R version 4.0.3. We followed the STROBE guidelines for reporting of observational studies . We also reported risk differences (RDs) with 95% CI and  studies .The screening profile and characteristics of women who participated in the POBASCAM trial and had an HPV–negative study-related second screening test is presented in p < 0.001) or a positive co-test . Equivalently, a decreased CIN3+ risk was observed among HPV–negative women who in the previous round also had a negative HPV test  or a negative co-test . We did not find an increased CIN3+ risk in HPV–negative women who in the previous round had a positive cytology test  or, equivalently, not a significantly decreased CIN3+ risk in those who in previous round had a negative cytology test . Intervention and control group-specific estimates of the absolute CIN3+ risks were similar despite the different screening management in the first round after enrolment  or a previous positive co-test .Women with an HPV–negative result had a 14-year cumulative CIN3+ risk of 0.48% (95% CI 0.37 to 0.62) . A markenrolment . The 14-In second-round age strata <39 years, 39 to 48 years, and ≥49 years, the 14-year CIN3+ risks were 0.95% (95% CI 0.60 to 1.51), 0.54% (95% CI 0.39 to 0.74), and 0.17% (95% CI 0.07 to 0.40) (Table A in p < 0.001) and HPV16–negative/HPVother–positive . The CIN3+ risk was also increased among women who, in the previous round, were HPV31/33/35/52/58–positive but HPV16/18/45–negative , or positive for high-risk HPV genotypes other than HPV16/18/45/31/33/35/52/58 . The CIN3+ risk was not significantly increased among women who, in the previous round, tested HPV18/45–positive but HPV16–negative .The 14-year CIN3+ risk among HPV–negative women was increased in all genotype-specific subgroups with a previous round positive HPV test: HPV16–positive  and, equivalently, decreased after a negative cytology test in the previous round . The 14-year CIN2+ risk was increased in all genotype-specific subgroups with a previous round positive HPV test, including the HPV18/45–positive but HPV16–negative subgroup.The analyses were repeated for endpoint CIN2+ and similar estimates were found when stratifying according to HPV and co-test results in the previous round . Howeverp < 0.001) or a previous positive co-test , and decreased in those with a previous negative HPV test  or a previous negative co-test . In our sample, there were no cancer cases among HPV–negative women with a previous positive cytology test.Similar estimates were found for endpoint cancer. The overall 14-year absolute risk of cancer was 0.03% (95% CI 0.01 to 0.08). The 14-year cancer risk was increased in HPV–negative women with a previous positive HPV test cancer ,16,23–27Organized screening programs have nowadays linked digitalized screening registries and invitation systems , meaningOur study confirmed the safety of extending the screening interval from 5 to 10 years among HPV–negative women who were also screen-negative at the previous round. A history of HPV positivity, even when the current result is negative, is associated with a long-term increased precancer risk that warrants a re-evaluation of the extended 10-year screening interval. Altogether, our results indicate that HPV results from multiple screening rounds should be considered when determining the time to the next screening invitation.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


For many decades, the thiazole moiety has been an important heterocycle in the world of chemistry. The thiazole ring consists of sulfur and nitrogen in such a fashion that the pi (π) electrons are free to move from one bond to other bonds rendering aromatic ring properties. On account of its aromaticity, the ring has many reactive positions where donor–acceptor, nucleophilic, oxidation reactions, etc., may take place. Molecules containing a thiazole ring, when entering physiological systems, behave unpredictably and reset the system differently. These molecules may activate/stop the biochemical pathways and enzymes or stimulate/block the receptors in the biological systems. Therefore, medicinal chemists have been focusing their efforts on thiazole-bearing compounds in order to develop novel therapeutic agents for a variety of pathological conditions. This review attempts to inform the readers on three major classes of thiazole-bearing molecules: Thiazoles as treatment drugs, thiazoles in clinical trials, and thiazoles in preclinical and developmental stages. A compilation of preclinical and developmental thiazole-bearing molecules is presented, focusing on their brief synthetic description and preclinical studies relating to structure-based activity analysis. The authors expect that the current review may succeed in drawing the attention of medicinal chemists to finding new leads, which may later be translated into new drugs. In the list of five-membered heteroaryl ring systems, thiazole is a unique ring that carries nitrogen and sulfur atoms, which makes it a versatile entity in actions and reactions. Though free thiazole cannot be spotted in nature, the ring of thiazole is detected in several natural compounds, such as peptide alkaloids, metabolites, and cyclopeptides . The lonThe thiazole moiety has received much attention in recent decades, with multiple review articles stressing the significance of the thiazole nucleus in the design and optimization of newer bioactive drug candidates ,23,24,252R) receptor suppressor that reduces the formation of stomach acid and is often used to control peptic ulcers and gastroesophageal reflux disease -N-(2-trifluoromethylphenyl)acetamide, and 5 -thiazolidin-3-yl)acetic acid ethyl ester demonstrated strong anticonvulsant action in both models. The effect of compound 4b was similar to or higher than the reference drug sodium valproate -1H-1,2,4-triazole and 7b 1- on the benzylidene portion as observed in 43a and 43c, the presence of an electron-withdrawing group [–N(CH3)2] on the phenyl nucleus in 43b, and 2-OH naphthaldehyde, as found in 44, was essential for activity substiuted-2H-chromen-2-one derivatives. Among the compounds examined, 53 showed excellent anti-tubercular action with an MIC of 0.09 µg/mL. The structure-based activity study indicated that the presence of a chloride ion (Cl)-linked phenyl group was essential for anti-tubercular activity  thiazole-5-carboxylic acid and (3-fluoro-4-(4-(5-(trifluoromethyl) pyridin-2-yl)piperazin-1-yl)phenyl)methanamine were dissolved in dry CHactivity . The newe moiety . Some nerculosis . Multiplctively) 71 is significantly active in reducing inflamed animal paws compared to diclofenac. A quick look at the structure-based activity study revealed that the presence of decylamine was an indispensable requirement for the anti-inflammatory activity thiazole, and 2-naphthol or 6-hydroxy quinoline reacted in the presence of NaCl in water via the microwave method. The final compounds obtained out of these reactions were checked for anti-repellent activity against Anopheles arabiensis mosquitoes. Among these analogous, ompounds . In vitrotential .In this review article, the chronology of thiazole-derived compounds has been organized and orderly presented. This review is divided into three major sections: (1) Thiazoles as treatment drugs, (2) thiazoles in clinical trials, and (3) thiazoles in preclinical and developmental stages. The preclinical investigation section included research since 2010, with some exceptions. The preclinical investigation section listed 11 subsections: Thiazole as anticonvulsant, antimicrobial, antitubercular, anti-inflammatory, antimalarial, antiviral, anti-Alzheimer, antidiabetics, anti-A1 receptor, and bioactive agents. Numerous thiazole compounds included in this review appeared to be more potent than reference medications, suggesting that they are excellent candidates for further modification and development, which may lead to future drugs.The list of prescription drugs bearing thiazole ring is long, and only a few have been presented in The second important section in this review is thiazoles in the stages of clinical trials. Some thiazole-bearing drugs have recently completed clinical trials and been approved by the USFDA. For example, mirabegron has recently been approved to treat overactive bladder. Ziritaxestat (GLPG1690), an autotaxin inhibitor, was considered a potential therapeutic candidate for the treatment of idiopathic pulmonary fibrosis. A recent report from “clinical trials.gov” showed that the Ziritaxestat phase 3 trial had been discontinued due to the benefit–risk profile. SRT2104 (GSK2245840) is a selective sirtuin 1(SIRT1) activator engaged in energy homeostasis management. This drug candidate is being investigated for the clinical use of atrophy, sepsis, psoriasis, type 2 diabetes mellitus, and muscular dystrophy. The clinical trial results (NCT01154101 and NCT01031108) and a good number of high-quality research indicated the potential of SIRT1 in the treatment of many diseases. Similarly, IRAK inhibitor 6 is a selective renal carcinoma antigen NY REN 64 blocker that is possibly used to manage bone degradation and rheumatoid arthritis-induced joint inflammation. This drug is associated with several clinical trials (NCT04440410 and NCT04815811) for other investigations. The other drugs, for example Fatostatin, WEHI-539, O4I2, TP0427736, SC75741, SRT3025, and UM-164, are being optimized for clinical trials.3a, 3b, 3c, 3d, 3e, 3f, and 3g showed significant anticonvulsant action with a median effective dose of less than 20 mg/kg, which was approximately seven-times lower than the standard medication, ethosuximide. In other study analogs, 4a, 4b, and 5 demonstrated strong anticonvulsant action in both models. The effect of compound 4b was similar to or higher than the reference drug sodium valproate. The third preclinical and development stage has described many promising agents, which can further be investigated for their potential in clinical translation. For example, in the subsection “thiazole as anticonvulsants,” many agents showed potential better than the standard drugs. Compounds 13, 14, 15, 16, and 17 have significant anti-Bcl-2 Jurkat and anti-A-431 activity. Molecule 13 was equipotent against both cell lines, with IC50 against both cell lines being lesser than the reference drug doxorubicin. In another study, one of the thiazole-pyridine hybrids 23 showed better anti-breast cancer efficacy (IC50 5.71 μM) than the standard drug 5-fluorouracil (IC50 6.14 μM). Some 5-(1-(2-(thiazol-2-yl)hydrazono)ethyl)thiazole analogs, such as 25a, 25b, and 26, were also found to have a better antitumor profile than the standard drugs harmine and cisplatin. In “thiazoles as antitumor agents,” some thiazole-bearing compounds showed excellent antitumor activities. For example, studies found some unique structurally diverse thiazole analogs as potential anti-cancer drug candidates. A-431, ARPE-19, and Bcl-2-Jurkat cultures were employed to screen the newly prepared thiazole derivatives. Analogs 39a and 39b demonstrated a better activity profile when compared to the reference drugs ampicillin and streptomycin. In another study, one novel thiazole compound 40 was found equipotent to chloramphenicol against S. aureus (MIC 3.125 g/mL) and considerable activity against B. thuringiensis (MIC 6.25 micro-g/mL). In a separate study, the derivative 5- against Staphylococcus epidermidis. In one important study, novel thiazole-based chalcones 50 appeared to be 10- and 56-fold more potent compared to streptomycin and ampicillin, respectively. Another study reported the compound 3-(4-(adamantan-1-yl)thiazol-2-yl)-2-thiazolidin-4-one 52 to havee MICs of 0.9–6.25 µmol mL−1 × 10−2 and MBCs of 1.53–12.5 µmol mL−1 × 10−2. Compound 52 also displayed high fungistatic activity against all of the investigated fungi, with MIC values as low as 0.021–0.042 µmol mL−1 × 10−2 and MFC values as low as 0.06 µmol mL−1 × 10−2.Similarly, in “thiazole as antimicrobial agents,” many thiazole ring-containing compounds showed higher antimicrobial activity than the standard drugs used. For example, in one study, synthesized molecules us MIC 3.25 g/mL a70a (MIC = 15.28 µM) and 70b (MIC = 17.03 µM) showed almost three times stronger Mtb growth inhibitory action than NTZ and were free of cytotoxicity .Thiazoles as antitubercular agents have received wider attention. Many new derivatives were synthesized to combat multidrug-resistant Mycobacterium tuberculosis. A majority of the compounds reported in this review displayed moderate to good activity in combating the resistant species. In one study, substituted 4-arylthiazol-2-amino derivatives as modified analogs of nitazoxanide (NTZ) 73a, 73b, 73c, 73d, 73e, and 73f exhibited better anti-inflammatory efficacy than the reference drug celecoxib. In another study, a different class of tetrahydronaphthalene–thiazole-coumarin multi-nucleus derivative (E)-3-thiazol-2-yl)hydrazono)methyl)-4H-chromen-4-one 76 displayed higher anti-inflammatory and analgesic activity than indomethacin.In pursuit of finding new anti-inflammatory agents, several thiazole derivatives were synthesized during the last decade. Many of them were moderate to good in controlling inflammation; however, some displayed better results than the standard drugs. In one report, some new substituted pyrazoles containing thiazolyl and thiazolidinonyl derivatives 89 along with a few other compounds.In the subsection “thiazoles as antimalarial agents,” pyrazole-linked thiazole analogs were found to be promising against Plasmodium berghei and chloroquine-inactive (RKL9) Plasmodium falciparum strains. The results highlighted the greatest potency was achieved by molecule 101c, and 101d showed superior antiviral activity against EV71, and compounds 101a, 101b, 101c, 101d, 102a, and 102b had superior antiviral activity against CVB3, when compared to the reference drugs ribavirin or pirodavir.Though many thiazole-bearing antiviral derivatives were synthesized in the recent past, the activities of the majority of the derivatives were not found to be superior to standard antiviral drugs. In a report, compounds 101a, 111, with an IC50 of 0.011 µM, whereas the IC50 value of the conventional medication donepezil was 0.054 µM. In the other study, compound 123 was found to be the most active agent in the series with an IC50 value of 0.028 ± 0.001 µM, which indicated an inhibition profile similar to the reference drug, donepezil.In the subsection thiazoles as anti-Alzheimer agents, some newly synthesized derivatives were moderately good at eliciting anti-Alzheimer’s activity. The most active molecules against AChE were reported to be 134 were observed to be a more potent inhibitor of DPP-4 (2.32 nM) than the standard alogliptin (3.56 nM).In the subsection of thiazoles as anti-diabetic agents, the majority of the newly synthesized compounds exhibited good activity. After a critical analysis, 141 showed very strong A2AR selectivity in comparison to the conventional A2AR antagonist SCH58261. In another study, compound 142 was recommended as a lead structure for the development of second-generation non-xanthine-derived A1antagonists with innovative therapeutic potential.Many thiazole-bearing compounds displayed moderately good adenosine receptor inhibitory activity. However, compound 145a, 145b, 145c, and 145d were proven to have better antioxidant activity (AO) and lower IC50 values than the reference drugs. In a separate study, disubstituted thiazole derivative 147 displayed higher antioxidant properties than the standard BHT. In another study on insect repellent activity, analogous 152a, 152b, and 152c exhibited the highest repellent activity comparable to the positive control DEET. In a separate study, a thiazole derivative called 4,5-bis(p-methoxyphenyl)-2-(trifluoromethyl)-thiazole 153 was found to be a potent suppressor of collagen-induced platelet aggregation. At a concentration of 1 ng/mL, it was effective in human platelet-rich plasma, which was superior to the standard drug flurbiprofen.In the last section, we reported some bioactive compounds with varied biological activities. Some compounds were found to be better than the respective reference drugs. For example, the selenourea derivatives of thiazole The review at a glance indicated that the thiazole moiety alone displayed a host of diverse pharmacological activities. All these thiazole-bearing derivatives needed further investigation and optimization. The toxicity profile should also be investigated as, in many reports, a toxicity study was missing. However, further investigations of some of the highly potent derivatives may lead to the generation of drug candidates.


To investigate the human papilloma virus (HPV) infection status, main subtypes and age distribution characteristics of women in the Putuo area of Shanghai.A total of 13,936 subjects were enrolled in this study. These subjects were 15–89 years old, with a mean age of 41.68. Real-time fluorescence quantitative polymerase chain reaction technology was used to detect 21 types of HPV.A total of 2,500 subjects with HPV infections were detected in 13,936 cervical exfoliated cell specimens . There were 15 people aged below 20,486 people aged 21-30,876 people aged 31-40,484 people aged 41–50, 338 people aged 51–60, and 301 people aged > 60. In total, 1,893 (75.7%) subjects had a single type of HPV infection, 424 (16.9%) had a double infection, and 183 had triple or more infections (7.4%). The top 6 subtypes of HPV infection in the Shanghai Putuo District were HPV 52 (3.81%), HPV 58 (2.46%), HPV 16 (2.43%), HPV 53 (2.30%), HPV 81 (1.74%) and HPV 39 (1.5%). The number of high-risk HPV infections was 1,978, and the total infection rate was 14.19%. The number of intermediate-risk HPV infections was 578, and the total infection rate was 4.15%. The number of low-risk HPV infections was 338, and the total infection rate was 2.43%.The top 3 populations with HPV infection rates in the Putuo District, Shanghai, were ≤ 20 years old, older than 60, and 21–30 years old. The infection rate of HPV in cervical outpatient clinics was significantly higher than that of other departments. The 9-valent vaccine is recommended for HPV vaccination in this area. Human papilloma virus (HPV), belonging to the papilloma vacuolar virus, is a small covalent double-stranded circular DNA virus mainly present in human skin, mucosa and female cervical epithelial cells. More than 200 kinds of HPV have been found –3. StudiAt present, laboratory testing methods mainly include nucleic acid testing, cytology testing, pathological histology testing, and so on. The current HPV vaccination campaign is also in full swing. Some experts also believe that the HPV subtype infection situation and the region’s age factors should be considered for HPV vaccination. Among them, the HPV high-risk type tends to cause intraepithelial neoplasia of cervical cells. In contrast, the HPV low-risk type of infection causes genital tract warts. The HPV high-risk types include HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 59, and HPV 68, five intermediate-risk types including HPV 26, HPV 66, HPV 53, HPV 73, and HPV 82, and three low-risk types including HPV 6, HPV 11, and HPV 81 [In this study, with patients in the department of gynaecology, the cervical department, the obstetrical department, and the physical examination department in the Putuo District, Shanghai, as study subjects, 21 subtypes of HPV infection were tested, using real-time quantitative polymerase chain reaction  technology, aimed to understand the epidemiological characteristics of HPV in women in Shanghai in detail, analyse and evaluate the pathogenic risk of cervical lesions of different high-risk or suspected high-risk HPV, and provide a reference for developing cervical cancer screening strategies and the research, development and distribution of HPV vaccines suitable for China's national conditions.This study is a retrospective, single-center study. A total of 13,936 females were selected to voluntarily undergo HPV examination in the departments of gynaecology, cervical, obstetrical and physical examination of the Shanghai Putuo District Maternal and Child Health Hospital from June 2021 to June 2022.The study subjects met the requirements: (1) the non-menstrual period; (2) Intravaginal irrigation and medication should not be performed 24 h before sampling. Cervical exfoliated cells were collected by routine gynaecological examination with a vaginal speculum to enlarge the vagina and to rotate the cervical sampler brush clockwise or counter clockwise for three to four laps. After sampling, it was placed in the storage solution of cervical exfoliated cell samples and sent to the specimen receiving laboratory.For HPV testing, an HPV typing testing kit  was used. The SLAN-96P fluorescence quantitative PCR instrument  was used for PCR analysis. HPV detection and typing method: Real-time quantitative polymerase chain reaction  technology was used to target the human papillomavirus genome L1 region, 21 subform-specific primers and probes were designed, and the corresponding subtypes were marked with FAM, HEX and ROX, respectively. The probes were oligonucleotides, including the 5′-end reporter dye and the 3′-end quencher dye. During PCR amplification, when the probe is complete, the fluorescence emitted from the reporter dye is absorbed by the quencher dye due to its proximity to the reporter dye and emits no fluorescence signal. When the primer was extended, the probe bound to the template was cut off by the Taq enzyme (5′ → 3′ exonuclease activity). The reporter dye was separated from the quencher dye to produce the fluorescence signal. The quantitative PCR instrument automatically drew the real-time amplification curve according to the detected fluorescence signal to realise the qualitative detection of human papillomavirus on the nucleic acid level. At the same time, reference genes can be used to monitor and exclude false negatives due to instrument failure, reagent factors, improper manipulation, or inhibitors in the sample. The instrument automatically saves the results after the reaction. The baseline start point, endpoint, and threshold can be adjusted according to the analysed image. The result can also be automatically read by the instrument . Click Analysis to automatically obtain the analysis results and find the results in the same interface.Positive plasmids and blank controls were used to monitor the amplification reactions during the amplification process. The experiment participated in the Shanghai temporary inspection centre room quality control as a laboratory quality assurance. Blank control: No typical S-type amplification curve is shown. Positive control: the HPV 21 subtypes and the reference gene test showed a typical S-type amplification curve and a CT value ≤ 30. Reference gene: The FAM channel amplification curve in group H has a typical S-type curve and CT ≤ 36.7, otherwise, it is related to errors caused by sampling, transportation, preservation conditions and experimental operation. The above requirements should be met simultaneously in the same experiment. Otherwise, the qualitative results of this experiment were invalid.P < 0.05 was considered a statistically significant difference. The sample size was calculated using the PASS software  with the module of confidence interval for one proportion. A sample size of 715 produces a two-sided 95% confidence interval with a width equal to 0.060 when the sample proportion is 0.200.Statistical analysis was performed using SPSS17.0 . Measurement data were tested for normality using the Shapiro–Wilk test. Data following normal distribution were described by the mean ± standard deviation. The count data were statistically described by number (%). The rate of HPV infection was compared using the chi-square test. A total of 13,936 study subjects were included, aged 15–89, with a mean age of 41.7 ± 12.3 years. Subjects aged 31–40 accounted for the highest proportion (36.17%). All patients were from urban areas and had experienced sexual activity but were not HPV vaccinated. Most patients were married (89.16%) and were educated to the level of junior high school or above (82.71%). In addition to the presence or absence of HPV infection, some patients were infected with other sexually transmitted diseases, such as Ureaplasma urealyticum (47.93%) , HPV 58 (2.46%), HPV 16 (2.43%), HPV 53 (2.30%), HPV 81 (1.74%) and HPV 39 (1.52) , 486 patients between the ages of 21 and 30, 876 patients between the ages of 31 and 40, 484 patients between the ages of 41 and 50, 338 patients between the ages of 51 and 60, and 301 patients over the age of 60 (infection rate: 20.70%). The infection rates among the age categories varied significantly . Of all the 13,936 specimens, the numbers of high-risk, moderate risk, and low-risk HPV infections were 1,978 (14.19%), 578 (4.15%), and 338 (2.43%), respectively.Of the 2,500 HPV-positive specimens, 1,893 (75.7%) were single infections, 424 (16.9%) were double infections, and 183 (7.4%) were triple or more infections (Fig. This study shows that the top three ages with a high incidence of HPV are ≤ 20 years old, > 60 years old and 21–30 years old. Therefore, we should not only vaccinate these people with the HPV vaccine in advance but also pay attention to the importance of reproductive health and HPV vaccination in our daily science popularisation work. Investigating the prevalence of HPV infection according to different age populations is of great importance to achieve the precision prevention and establish appropriate therapeutic strategies , 13. ForOur findings provided further insights into the prevalence of HPV infections according to different subtypes, which was useful to establish the epidemiological distribution pattern of HPV in the local area and provide guidance for secondary and tertiary prevention. In this study, the total infection rate of 13,936 women was 17.9%, lower than the 22.6% reported in the Beijing area  and loweHPV 52 had a higher incidence of cervical lesions compared to HPV 16, HPV 53, and HPV 58. The findings suggested that HPV 56, HPV 58, and HPV 52 were associated with an increased incidence of cervical lesions compared to HPV 16, and that HPV 53/58/52 infection individuals may also be sent for colposcopy in addition to HPV 16 and 18. There are few related studies on other HR-HPV cancer-causing forces, and no unified cognition has been formed. Wang et al.  reportedThe present study has some research limitations. First, the source of our sample is relatively limited, only for population screening in key departments, and cannot represent the screening results of the whole population, which may have some bias in the extrapolation of the results. Second, we have only analysed the infection status of HPV and have not yet focused on the relationship between HPV infection and its outcome shunt, which should be further deepened in future studies. Third, the generalizability of our results is limited due to our single-center design. The validity of this study would increase if the rural population was also included. Finally, we have not analysed the association between individual basic characteristics and HPV infection, and therefore, we will analyse the association in more depth in subsequent studies.In conclusion, HPV infections in the Putuo area, Shanghai, is still at a high level, and the top-ranked HPV infection subtypes are HPV 52, HPV 58, HPV 16, and HPV 53. We should take more proactive measures against HPV 52/58/53 infection with the cervical cancer screening strategy, vaginal referral, and the future development of the HPV vaccine in China, as it serves as a reminder that the HPV vaccine vaccination is still the primary method of preventing and controlling cervical cancer at our primary level. Further studies are warranted to elucidate the mechanism behind the association with HPV infection and risk of cervical lesions in this area with a long-term follow-up.