The hepatitis C virus (HCV) causes both acute and chronic infection and continues to be a global problem despite advances in antiviral therapeutics. Current treatments fail to prevent reinfection and remain expensive, limiting their use to developed countries, and the asymptomatic nature of acute infection can result in individuals not receiving treatment and unknowingly spreading HCV. A prophylactic vaccine is therefore needed to control this virus. Thirty years since the discovery of HCV, there have been major gains in understanding the molecular biology and elucidating the immunological mechanisms that underpin spontaneous viral clearance, aiding rational vaccine design. This review discusses the challenges facing HCV vaccine design and the most recent and promising candidates being investigated. First discovered in 1989 . ChronicTherapeutic treatment of HCV has been vastly improved over the past decade due to the development of direct acting antivirals (DAAs). These compounds act as inhibitors of either the NS3/4a serine protease, NS5a or the NS5b RNA-dependent polymerase  and can The ability to effectively treat HCV infection has been a major achievement. However, there is growing evidence that HCV can leave lasting impacts upon its host post infection. For example, during infection extensive liver fibrosis can occur, which can persist for several years after viral clearance. Furthermore, persistent hyperfunctional CD8+ T cell phenotype has been reported following successful treatment with DAAs, suggesting continued immunological impairment . There aModelling based on viral kinetics in reinfected individuals with pre-existing immunity has shown that transmission risk can be greatly reduced when an immune response occurs, despite detectable virus RNA titres . This isVirus-host interactions determine the outcome of acute HCV infection. This interplay is complex and includes components of both the adaptive and innate host immune system. The most common scenario is progression to chronic infection. However, 25%–40% of individuals undergo spontaneous viral clearance (SVC) ,20 withiInnate immunity provides a first line of defence against viral infection. The liver presents a unique microenvironment that is enriched with cells that participate in this response, namely Kupffer cells, natural killer (NK) cells, hepatic stellate cells (HSCs) and macrophages. Initial innate responses are triggered by HCV-derived pathogen-associated molecular patterns (PAMPs) that are recognised by pattern recognition receptors (PRRs). Virion-associated PAMPs are the E1 and E2 glycoproteins ,26, whilIFNλ4 with SVC. In this case approximately 50% of individuals with a C/C genotype achieve SVC [Successful SVC is influenced by the outcome of innate immune responses. A major example is the association of single nucleotide polymorphisms (SNP) rs12979860 present in the type III IFN gene ieve SVC ,34. Addiieve SVC  due to aieve SVC ,37.FH) are required to establish a long-term antibody response [Cellular immunity has long been associated with spontaneous HCV clearance  and is mresponse ,43. HCV-response . During response ,46. The response . The reaThe importance of T cells to SVC was first demonstrated in experimentally infected chimpanzee in which HCV persistence was observed in the absence of either a CD4+ or CD8+ T cell response ,49. InteNeutralising antibodies (nAbs) in the context of HCV infection were first described by Farci et al. , althougVH1-69 gene which is found in potent cross-reactive nAbs that target antigenic region 3 (AR3) and is also present in nAbs targeting HIV and Influenza [Further insight into the humoral response has been obtained through characterising nAbs derived from patients. Bailey et al.  sequencenfluenza ,69,70,71nfluenza ,73. ThisThe HCV envelope glycoproteins, E1 and E2, are located at residues 192–746 of the polyprotein and are the targets of the humoral immune response, making them an attractive vaccine target ,76. The The E1E2 heterodimer mediates entry into the hepatocytes through interactions with four essential host receptors, CD81 , scavengAntibody-mediated neutralisation of HCV is achieved through targeting the E1E2 heterodimer on the surface of the virus. To date, most characterized nAbs target the E2 protein. The are several linear and discontinuous regions of E2 that are targeted by Abs and these have varying nomenclature, being referred to as ARs1-5 , Epitope−4 substitutions per site of the genome [As a species, HCV exhibits extensive genetic diversity is driven by a mutation rate in the order of 10e genome ,94. Mutae genome . Gt1 HCVe genome , and aree genome . In conte genome . Genotype genome . Gt1-7 ce genome . Rapid ee genome . The dive genome .The extent of genetic diversity gives rise to genotype-specific immune responses. The E1E2 sequence shows the greatest level of variation as a result of the selective pressure exerted by the host immune response , leadingThe ability of HCV to establish a chronic infection highlights the efficiency in which this virus can subvert the host immune response. This is achieved through multiple strategies. Firstly, immunodominant epitopes such as the HVR1 of E2 elicit a response of non-neutralising antibodies. Currently it is thought that HVR1 acts as a shield of the more conserved epitopes in the E2 core that contain CD81 binding residues, since removal of HVR1 increases virus susceptibility to neutralisation ,106. EviEpitope masking is also achieved through the glycosylation of envelope proteins to form a glycan shield . This meThe evasion of nAbs can also be attributed to the presence of host derived factors. One such factor is high-density lipoproteins which are present in human serum and has been shown to increase HCV infectivity via SR-BI interactions which in turn decreases the time available for nAbs to bind to their target ,117. AddSince the discovery of HCV, research was hampered by the lack of infection models both in vivo and in vitro. The use of models in vaccine research is essential for the assessment of sera and monoclonal antibodies arising from natural infection, experimental immunizations or vaccine trials. In the case of in vitro models, the key challenge was generating a cell culture-based system that could produce infectious HCV at titres suitable for further experimentation. This problem was solved with the discovery of two Gt2a isolates, JFH1 and J6 which produce high titres of infectious HCV from cultured Huh 7.5 cells . FollowiViral pseudotyping has also been used to study the entry mechanism of HCV. This approach utilizes the ability of retroviral gag/pol proteins to self-assemble into enveloped virus particles. Simultaneous expression of foreign viral envelope proteins in the same cells leads to the incorporation on to the surface of the retroviral particle whose entry properties are dictated by the envelope glycoprotein. Infectivity can be measured by the incorporation of a reporter gene, usually luciferase, which is introduced into target cells and expressed. Methods to generate HCV pseudoparticles (HCVpps) commonly use murine leukemia virus or HIV derived vectors ,128. OurThe ability to generate both HCVcc and HCVpp provide essential tools for the in vitro study of HCV and assessing the neutralising breadth of sera and monoclonal nAbs. The importance of isolate panels for the validation of vaccine candidates is exemplified in the field of HIV vaccine research, in which candidates have been tested against an extensive panel of 109 isolates developed by Seaman et al. , which aHumans are the natural host of HCV, and therefore it is imperative that suitable in vivo models are used in order to test the efficacy and safety of preclinical vaccine candidates. Arguably, the most successful model has been the chimpanzee, which is permissive to HCV infection under experimental conditions. However, ethical concerns over the use of this species has led to a ban on its use in experimental research. An alternative has been the use of chimeric humanized or transgenic mouse models which have humanized livers such as Alb-uPA/SCID mice ,137 or tHepacivirus genus for use as analogues. At the time of writing there are two species of particular interest, the Rodent Hepacivirus (RHV) and the Equine Hepacivirus (EqHV). RHV was initially discovered in the species Rattus norvegicus in 2014 [In light of the limited options available for the in vivo modelling of HCV, there has been increasing interest in other members of the  in 2014 . Experim in 2014 . There a in 2014 .EqHV was initially discovered in canines and subsequent serology testing identified equines as the natural host ,145. ThiIgVH 1-69 gene with an extended CDRH3 region has previously been described as a feature of anti-HCV specific nAbs that are elicited in SVC and therefore it is logical that a vaccine candidate could be assessed based on its ability to elicit these types of nAbs. With this in mind it is worth considering how comparable the antibodies of animal model species are to those of humans. One such difference is the length of the CDRH3 regions which are typically 16 residues long [The use of analogous Hepacivirus species will allow for the testing of vaccine strategies and the experimental challenge in the natural host of that species, a feat that cannot currently be achieved for HCV. Whilst these approaches are likely to prove highly valuable to the field of HCV vaccine research it is important to acknowledge the differences in mammalian immune systems and how this will impact on our interpretation of experimental data. For example, the ues long , howeverues long  and equiues long , respectThe informed, rational design of immunogens will be vital in order to produce a successful HCV vaccine. As advances in the analysis of antibody repertoires are allowing deeper insight into the types of nAbs that are associated with SVC, there is potential to use this information to develop immunogens that favour the production of these types of antibodies. An example of this can be seen with the E2 epitope I region or antigenic site 412 (AS412). This linear epitope is structurally flexible and when in complex with nAbs and adopts three distinct conformations. Firstly epitope I forms an extended conformation in complex with the rat nAb 3/11, a β-hairpin conformation is observed when bound to AP33 or the human nAb HCV-1, and an intermediate conformation occurs when in complex with the human nAb HC33.1 . As prevDespite the challenges faced in HCV vaccine design, there have been a variety of different vaccine approaches investigated with a small number of these candidates reaching human trials (summarized in Recombinant subunit vaccines are an attractive technology as they have previously been utilized for a variety of pathogens  and are Δ123) which can be expressed in a correct conformation which interacts with CD81 and conformation sensitive antibodies [Δ123 in mammalian cells results in biochemically and immunogenically distinct isoforms of this protein that exist as monomeric, dimeric, pentameric and a high molecular weight aggregate . Immunisation of guinea pigs with different isoforms showed that HMW1 elicited greater titres of cross-reactive nAbs that could neutralise isolates from Gt2a, 4a, 5a, 6a and 7a expressed in a HVCcc system [Δ123 reduced homologous neutralisation activity, a likely result of the removal of HVR1 which was also observed by Law and colleagues [Another subunit vaccine approach has been the use of truncated soluble E2 (sE2) which results in a secreted form of the E2 ectodomain. A candidate of particular note is the H77 derived sE2384-661 construct with deleted HVR1, HVR2 and IgVR  are a promising area of vaccine research and have been investigated extensively to design a HCV vaccine . To dateHCV-LPs were first described by Baumert et al.  in whichViral vectors have the capacity to induce high level cellular immune responses , making In the 30 years since the discovery of HCV, it has been apparent that this virus is highly complex and presents a major public health challenge. Encouragingly, efforts toward a vaccine continue and investigation into a range of approaches have yielded interesting results. The vast improvements in in vitro analysis of HCV have greatly aided our understanding of how nAbs work, informing rational vaccine design. In this review, we have highlighted the potential advantages of adopting reference panels of patient-derived E1E2 sequences in order to assess vaccine candidates for their potential to induce nAbs capable of targeting circulating HCV strains. Adopting this approach will provide a more rigorous tool for evaluating the nAb response produced by novel HCV vaccine candidates a characteristic that will likely be essential in a successful HCV vaccine.


To determine the feasibility of conducting a randomised trial of the effectiveness of cranberry extract in reducing antibiotic use by women with symptoms of acute, uncomplicated urinary tract infection (UTI).Open-label feasibility randomised parallel group trial.Four general practices in Oxfordshire.Women aged 18 years and above presenting to general practice with symptoms of acute, uncomplicated UTI.Women were randomly assigned using Research Electronic Data Capture in a 1:1:1 ratio to: (1) immediate antibiotics alone (n=15); (2) immediate antibiotics and immediate cranberry capsules for up to 7 days (n=15); or (3) immediate cranberry capsules and delayed antibiotics for self-initiation in case of non-improvement or worsening of symptoms (n=16).The primary outcome measures were: rate of recruitment of participants; numbers lost to follow-up; proportion of electronic diaries completed by participants; and acceptability of the intervention and study procedures to participants and recruiters. Secondary outcomes included an exploration of differences in symptom burden and antibiotic use between groups.Four general practitioner practices (100%) were opened and recruited participants between 1 July and 2 December 2019, with nine study participants recruited per month on average. 68.7% (46/67) of eligible participants were randomised (target 45) with a mean age of 48.4 years . 89.1% (41/46) of diaries contained some participant entered data and 69.6% (32/46) were fully complete. Three participants (6.5%) were lost to follow-up and two (4.4%) withdrew. Of women randomly assigned to take antibiotics alone (controls), one-third of respondents reported consuming cranberry products . There were no serious adverse events.It appears feasible to conduct a randomised trial of the use of cranberry extract in the treatment of acute, uncomplicated UTI in general practice.ISRCTN Registry (ID: 10399299). UTIs are one of the most common bacterial infections seen in primary care,3 6 7acute UTIs, alone, or in combination with antibiotics. There are some mechanistic data to support this approach; urine collected within 6 hours of consumption of cranberry powder containing standardised amounts of proanthocyanidins (PAC) has been shown to exert an anti-adhesion effect against Escherichia coli in vitro, in a dose-dependent fashion.10Cranberry has been extensively evaluated as a preventative agent for UTIs, with mixed results. Up to 27% of women report using cranberry to help treat the symptoms of an acute UTI.The primary objective of this feasibility trial was therefore to determine the feasibility of conducting a randomised clinical trial to determine the effectiveness of cranberry extract in reducing antibiotic use by women with symptoms of acute, uncomplicated UTI in general practice.This was an open-label, pragmatic, feasibility trial with randomisation in a 1:1:1 ratio between treatment groups in UK primary care. The eligibility criteria have been described in detail elsewhere.et alParticipants were emailed a link to an electronic symptom diary and were asked to complete it daily for 2 weeks. They were asked to rate a number of symptoms on a scale of 0–50, and to record any UTI treatments (study interventions or otherwise) that they were taking. The list of symptoms in the daily diary has been published with the feasibility trial protocol.0=normal/not affected.1–9=very little problem.10–19=slight problem.20–29=moderately bad.30–39=bad.40–49=very bad.50=as bad as it could be.We used this wider scale as part of the feasibility testing to see whether participants liked having more rating options. We also wondered whether the wider scale might enable better detection of within-participant and between-participant rating variation. A follow-up phone call took place at 2 weeks to encourage diary completion and obtain a minimum data set from participants whose diaries were incomplete. Women were invited for interview at a later date if they had provided consent at the point of trial recruitment to receive information about the interview study. A notes review took place at 4 weeks to record any clinical contacts that were believed to be related to the original UTI episode, as well as the urine culture result.Adverse event data were collected through symptom diary entries, through participant reports at the 2-week follow-up call and through notes reviews.E. coli.Women in group 1 (control) received a prescription for first-line immediate antibiotics alone. In keeping with pragmatic trial design, the antibiotic prescribed and the duration was at the discretion of the recruiting clinician. In the UK, the usual recommended first-line treatment for acute uncomplicated UTI is a 3-day course of nitrofurantoin.The primary outcomes were feasibility measures,11Although not made explicit in the protocol, a comparison of the time to feeling fully recovered between groups was made, as prespecified in the statistical analysis plan. These data were already being collected through the electronic diary, which had received ethical approval. A decision to conduct an exploratory analysis of the proportion of participants experiencing ‘double sickening’  was made after the statistical analysis plan had been completed, but before the trial data had been reviewed or analyses commenced. The concept of double sickening has been noted in acute respiratory illnessA sample size of 45 participants was chosen to enable estimation of a loss-to-follow-up rate of 20% with a CI of 9.6% to 34.6%.The randomisation sequence was generated using Stata V.15.1  by a statistician at the Nuffield Department of Primary Care Health Sciences, University of Oxford, using computer-generated block randomisation with variable block size. The sequence was password protected and accessible only by the statistician. Participants were randomised by recruiting clinicians through the click of a button on the electronic trial registration page, which was created using Research Electronic Data Capture.Unless otherwise stated, primary outcomes are presented as proportions and percentages for each trial group and for the entire trial population. Participants in group 2 and group 3 were compared separately with participants in group 1 (the control group).The number of antibiotic courses (or part thereof) consumed by participants in each group at 2 weeks and 4 weeks was compared using Poisson regression and reported using incidence rate ratios. The difference in the duration of symptoms rated moderately bad or worse between groups was compared using negative binomial regression; incidence rate ratios are reported and a Kaplan-Meier graph plotted . The mea10.1136/bmjopen-2020-046791.supp1Supplementary dataA risk ratio and 95% CI was used to estimate the difference in the proportion of participants consuming a course of antibiotics (or part thereof) between groups at 2 weeks and 4 weeks, the difference in the proportion of adverse events between groups at both 2 weeks and 4 weeks, and to compare the proportion of participants in each group experiencing double sickening. It was not possible to use log binomial regression, as originally planned, to compare the proportion of participants consuming antibiotics between groups as the model would not converge. A risk ratio and 95% CI was used in lieu of Fisher’s exact test  to estimate the difference in proportion of adverse events between groups in order to generate 95% CIs rather than p values. As this study was not powered to detect differences between groups, we had prespecified that p values would not be reported.Intention-to-treat analysis was conducted for all analyses and estimates are presented with their 95% CIs. Due to the small sample size and the fact that this is a feasibility study, no subgroup or sensitivity analyses were conducted.Four patient and public involvement (PPI) contributors have been closely involved with the study from the outset, two of whom sit on the Trial Steering Committee. During trial conception and development, they confirmed that the topic chosen was important. They critiqued all public-facing trial documentation to ensure that it was free of jargon, and their feedback was incorporated. They were also involved in the design of the electronic diary, helping to ensure that it was user-friendly. PPI contributors unanimously felt that the diary would not be onerous for participants; and suggested it may make the experience of participating in the trial more meaningful, stating “…completing the symptom diary hooks people in. It makes them feel that they are contributing.” The results of the study have been shared with PPI contributors, allowing them to give their views on ways to explain the findings, which have been incorporated into the discussion. PPI contributors are also helping to inform the dissemination of the study findings.Baseline information was completed electronically by participants upon accessing the electronic diary on day 1 of enrolment, prior to inputting their day 1 symptom ratings. The mean age of participants was 48.3 years (range: 18–81 years). The mean age of women in the immediate cranberry and delayed antibiotics group was lower than the other two groups. Most women had experienced a previous UTI . A total of 75.6% (31/41) of respondents were expecting antibiotics. Sixty-one per cent (25/41) believed that cranberry could help their symptoms, while 24.4% (10/41) believed that their symptoms could improve without antibiotics .Forty-six women were recruited to the trial, surpassing the target of 45 see . Of the A total of 89.1% (41/46) of electronic diaries had some data entered by participants, while 69.6% (32/46) of electronic diaries were fully complete . There wOnly one participant reported a side effect or problem related to consuming cranberry capsules in the electronic diary . Few parThree participants were lost to follow-up . Loss toTwo participants withdrew from the study. One participant was consented and randomly assigned to immediate cranberry capsules with delayed antibiotics. As soon as she received this assignment, she decided to withdraw from the study and did not receive the cranberry capsules. Another participant was consented and also randomised to immediate cranberry and delayed antibiotics. The day after being enrolled, she emailed the trial team asking to be removed from the mailing list (links to the electronic diary were sent by email). In light of this, the chief investigator withdrew the participant from the study.Participants were able to provide free-text comments on the final day (day 14) of diary completion. Free-text boxes allowed women to provide general comments about the trial, as well as comments specifically about difficulties using the electronic diary, difficulties using the rating scale and any problems related to consuming cranberry capsules. Fourteen women provided comments.. Another participant mentioned that she did not receive a link to complete the final set of diary questions, and one woman was unclear about the guidance regarding the need to continue completing the diary once symptoms had resolved for 2 consecutive days. Two women commented that the scale of 0–50 was ‘too wide a range’.Some women provided general feedback, including being ‘happy to help’ with the study and finding the diary ‘very easy to complete’. Two participants described some difficulty keeping up with filling in the diary ‘Just time and keeping up with it that is all. Happy to have taken part.’ —‘Nice to only take three days of antibiotics not a week’ . One woman stated that despite starting with cranberry, she had to take antibiotics on day 2 as she was in pain .One woman stated that she was sceptical about cranberry at first, but found that it ‘helped a lot’ . One participant found that the combination of antibiotic with cranberry cleared her infection ‘quite quickly’ , while another found that the antibiotic/cranberry combination resulted in her taking a shorter course of antibiotics than usualThree adverse events were picked up from free-text comments (see the Harms section below).Approximately one-third of respondents reported contacting a healthcare practitioner within 2 weeks of being enrolled . Most of these women contacted their GP practice (GP or nurse at their surgery)—one woman reported liaising with a pharmacist, and another contacted an out-of-hours GP. No participants reported requiring Accident and Emergency or secondary care input. Just over half  of the urine samples at the time of enrolment were positive (organism growth greater than 10⁵/CFU (colony-forming unit)).Exploratory analyses of effectiveness were conducted, while recognising that the study was not powered for these analyses . For thiThere was a 20% reduction in the risk of consuming a course of antibiotics (or part thereof) in the immediate cranberry group (group 3), compared with the immediate antibiotics alone group (group 1—controls). This was the case at both 2 weeks and 4 weeks . The number of courses of antibiotics consumed at 2 weeks and at 4 weeks was reduced in groups 2 (immediate antibiotics and immediate cranberry) and 3 (immediate cranberry and delayed antibiotics), compared with the control group . Participants in groups 2 and 3 had a longer duration of symptoms rated moderately bad or worse, compared with those in the control group . The mean symptom burden (from day 2 to day 4 inclusive) was reduced in group 2 participants  and increased in group 3 participants , compared with the control group. Similarly, the time to feeling recovered was reduced in group 2  but increased in group 3 , compared with controls. Of women randomly assigned to take antibiotics alone (controls), one-third of respondents reported consuming cranberry products  .An exploratory analysis was conducted to compare the proportion of participants experiencing ‘double sickening’ between groups at 2 weeks. Double sickening was defined as a participant selecting the relevant checkbox in the electronic diary to confirm that they felt fully recovered, then subsequently indicating through checkbox responses that they no longer felt fully recovered. Given the short period of time over which this was assessed (2 weeks from enrolment), it is likely that these double sickening episodes represent incompletely treated infection, rather than a new infection. Compared with the control group, group 2 participants had a reduced risk of experiencing double sickening , while none of the group 3 participants experienced double sickening.possible pyelonephritis (numbers 3 and 7). However, one of these adverse events occurred in a participant whose midstream urine was negative, who reported feeling fully recovered on day 5 of the study and who did not contact a healthcare practitioner up to 28 days after enrolment. The second occurred in a participant who subsequently contacted her GP practice, however, her clinical syndrome was not reported as pyelonephritis by the consulting healthcare practitioner and the participant’s symptoms settled with increased fluid intake alone. There were no serious adverse events.Seven adverse events occurred . AdverseThe difference in the proportion of adverse events between groups was compared, although the feasibility trial was not powered to detect differences between groups. At 2 weeks, the point estimates indicated an increased risk of experiencing an adverse event for participants in group 2  and group 3  compared with the control group, although the 95% CIs were wide. At 4 weeks, the risk of experiencing an adverse event in group 2 was lower than for the control group . The risk remained higher for participants in group 3 compared with controls at 4 weeks  .10.1136/bmjopen-2020-046791.supp2Supplementary dataIn this open-label, feasibility randomised trial, we recruited beyond target and at a rate faster than predicted. Overall, participants found the trial procedures and interventions acceptable; few reported problems consuming cranberry capsules or using the electronic symptom diary.acute treatment for UTI. The trial processes were designed, in discussion with PPI contributors, to be minimally burdensome for participants and recruiters, which may have contributed to the faster than anticipated recruitment, and to recruiting beyond target.To our knowledge, this is the first trial to use cranberry capsules alone and combined with antibiotics as an Using a participant-completed diary led to missing data, and in particular, incomplete baseline data. We also collected very limited demographic data. Missing data means that we do not have a full picture of all of the participants’ experiences and outcomes. However, insights gained from the trial combined with the qualitative interviews (to be reported elsewhere) should help to optimise future trial design and minimise missing data in a future study.Some may describe the open-label design as a weakness. However, this was designed to be a pragmatic trial with procedures closely aligned with real-world practice. We were therefore not prescriptive about the antibiotic issued, nor the duration, recognising that clinicians’ prescribing is influenced by a host of factors, including a patient’s medical history and UTI history. Despite this, we appreciate that there is a lack of standardisation of antibiotic prescribing between groups. Furthermore, participants who missed diary entries were able to complete the diary retrospectively; retrospective entries are likely to be less accurate than real-time diary completion.We did not control participants’ consumption of other sources of PAC with type A linkages, namely blueberry. However, unless participants regularly consumed large amounts of blueberry, it is unlikely that this would have had a significant impact on the results. Interestingly, despite advice not to, one-third of women in the immediate antibiotics alone group reported consuming cranberry products during the trial. This highlights the challenge of trying to control external influences in a trial, particularly when this is in conflict with a participant’s usual behaviour.No study participants developed pyelonephritis. A number of trials of non-antibiotic treatments have reported higher levels of pyelonephritis in participants not assigned to an antibiotic arm.4The proportion of participants lost to follow-up or withdrawing from this feasibility trial (11%) is roughly comparable with analogous trials, which report between 1.3%et alWhile not powered to detect differences between groups, in line with the findings of similar trials, participants in this feasibility trial not receiving immediate antibiotics (group 3) consumed fewer antibiotics but had a higher symptom burden. However, participants in group 3 were less likely to experience double sickening. Interestingly, Gagyor recurrent UTIs .A Cochrane review in 2012 found that overall, treatment with cranberry did not significantly reduce This feasibility trial was not powered to detect differences between groups, thus, no definite inferences about the effectiveness or safety of cranberry in the context of acute uncomplicated UTI can be made. If cranberry extract is found to be safe and effective in the management of acute UTIs through robust and adequately powered trials, it could represent an important health intervention for this common condition and has the potential to significantly reduce antibiotic consumption.There is a need for adequately powered, randomised clinical trials to provide definitive evidence of whether cranberry extract is effective in reducing antibiotic use and managing symptoms of acute UTI. While this feasibility trial does not provide evidence of the effectiveness or safety of cranberry extract in the context of acute UTI, the exploratory analyses suggest that it would be worthwhile to explore these as yet unanswered questions through an adequately powered trial. Such trials should use cranberry products with standardised amounts of PAC. Participants should be willing to stop all non-trial cranberry products for the duration of the study; as a minimum, the use of non-trial cranberry products by participants should be captured.may have a beneficial impact on certain measures of antibiotic usage and symptoms. These findings support the conduct of further, definitive research in this area.It is feasible to conduct a randomised trial of similar design to this study to assess whether cranberry extract can help reduce antibiotic use for symptoms of acute, uncomplicated UTI. The exploratory, underpowered analyses suggest that cranberry


The phased withdrawal of oral polio vaccine (OPV) and the introduction of inactivated poliovirus vaccine (IPV) is central to the polio ‘end-game’ strategy.We analyzed the cost implications in Chile of a switch from the vaccination scheme consisting of a pentavalent vaccine with whole-cell pertussis component (wP) plus IPV/OPV vaccines to a scheme with a hexavalent vaccine with acellular pertussis component (aP) and IPV (Hexaxim®) from a societal perspective. Cost data were collected from a variety of sources including national estimates and previous vaccine studies. All costs were expressed in 2017 prices (US$ 1.00 = $Ch 666.26).The overall costs associated with the vaccination scheme  from a societal perspective was estimated to be US$ 12.70 million, of which US$ 8.84 million were associated with the management of adverse events related to wP. In comparison, the cost associated with the 4-dose scheme with a hexavalent vaccine (based upon the PAHO reference price) was US$ 19.76 million. The cost of switching to the hexavalent vaccine would be an additional US$ 6.45 million. Overall, depending on the scenario, the costs of switching to the hexavalent scheme would range from an additional US$ 2.62 million to US$ 6.45 million compared with the current vaccination scheme.The switch to the hexavalent vaccine schedule in Chile would lead to additional acquisition costs, which would be partially offset by improved logistics, and a reduction in adverse events associated with the current vaccines. Haemophilus influenzae type b (Hib) and malaria  vaccines to 90% in seventy-two of the world’s poorest countries between 2011 and 2020 would save the lives of an estimated 6.4 million children aged < 5 years, representing $231 billion in the value of statistical lives saved [Vaccines and associated pediatric immunization programs have been a vital public health intervention in reducing morbidity and mortality associated with many communicable diseases worldwide. These interventions not only protect those immunized, but also provide community-wide protection by reducing the spread of disease. In addition, the reduction in associated disease burden promotes healthy well-being which supports economic growth and poverty reduction , 2. ImprPoliomyelitis is an acute paralytic disease caused by three poliovirus serotypes , 5. The OPV has been the vaccine of choice for the Global Polio Eradication Initiative (GPEI) of the World Health Organization (WHO) because of its low cost, ease of administration and superiority in inducing mucosal immunity , 9. HoweVaccination against polio in Chile began in 1961 , and by Combination vaccines have been developed and introduced in routine childhood immunization programs, allowing individuals to be simultaneously vaccinated against multiple infectious diseases including diphtheria, tetanus, pertussis, poliomyelitis, Hib and hepatitis B (HepB) , 18. ComChile introduced a combined bivalent vaccine for diphtheria and pertussis (DP) into the routine childhood immunization program in 1950, which was later replaced by the trivalent vaccine that included pertussis (DTwP) in 1975. The tetravalent vaccine including Hib (DTwP-Hib) was introduced in 1996 and the switch to the pentavalent vaccine including HepB (DTwP-Hib-HepB) occurred in 2006. In 2015, Chile started to replace the 2-month OPV dose  with IPV in line with WHO-SAGE recommendations to support the polio eradication endgame strategy, with the vaccination series completed with OPV  administered at 4, 6 and 18 months. Then in February 2018, the hexavalent vaccine including IPV (DTaP-IPV-HepB-Hib) was introduced at 2 and 4 month and the vaccination series completed with the pentavalent (DTwP-Hib-HepB) vaccine and OPV both administered at 6 and 18 months . More reIn Latin America, there is an increasing role for economic evaluations in supporting evidence-based decision making. This has also applied to the assessment of new vaccines or immunization programs  vaccine scheme at 2, 4, 6 and 18 month of age. Because the new scheme represents a replacement of antigens, for both OPV to IPV and wP to aP, without any significant difference in the efficacy of the switched antigens, we considered a cost-minimization analysis as the most appropriate economic evaluation methodology to utilize.The objective of the current study was to describe and analyze the cost differences between switching from the current vaccination scheme to a new scheme, based on a fully-liquid ready-to-use hexavalent vaccine. We considered a full switch between schemes to the new hexavalent program comprising 4 doses (three primary and one ‘booster’ doses). The programs were analyzed from the societal perspective.The study cohort (infants eligible for vaccination) was taken from national statistics produced by the Chilean government . The vacAdverse event rates, associated with vaccination, were taken from previously published studies , 31 and It was assumed that some of the more serious adverse events result in costs being incurred, for example through medical consultations, absenteeism and medication. Those events which were deemed to result in medical treatment were severe injection site edema > 20 mm; severe pain; severe irritation due to agitation or persistent acute crying; and drowsiness. Based upon expert opinion, we assumed that 20% would be the appropriate consultation rate. For those suffering from some more serious neurological manifestations  it was assumed that all cases consulted were hospitalized for 1.5 days, and had a single post-discharge outpatient consultation.Cost data was taken from a variety of sources including PAHO, previous vaccine studies and national estimates and included: antipyretics use, patient transfers/travel expenses, medical consultation, hospitalization, lost work time/days for parental care, time to administer the vaccine dose , cost ofAll costs are in 2017 prices. Prices were converted from Chilean pesos into US$ using an exchange rate of $Ch 666.26 = US$1 (April 2017) .Four scenarios were modelled Table ; all werScenario 2 compared the current pentavalent vaccination plan and an additional 4-dose full IPV scheme versus vaccinating with hexavalent vaccine. The cost of the program with hexavalent vaccine was estimated based on its reference price in the Revolving Fund of PAHO of 2017 and compared with the cost of the current plan modified with the inclusion of a 4-dose full IPV scheme, which includes expenditure on the pentavalent vaccine (reference price of the tender in place), plus the average price of IPV in the PAHO Revolving Fund for 2017 for a 4-dose full IPV scheme and the additional costs which assumes the simultaneous scheme with the pentavalent vaccine. In this scenario, the price of IPV was the average of the prices of two different vaccines presentations in the Revolving Fund since there is a real possibility that the distribution of this vaccine through the Revolving Fund may vary due to supply issues.Scenario 3 was similar to Scenario 2 above, but used the lowest reported price for IPV (US$ 2.10) reported in the PAHO revolving fund. Scenario 4, was also based on Scenario 2 above, but used the highest reported price for IPV (US$ 5.10) reported in the PAHO revolving fund.The population of Chile was found to be relatively stable in the period 2010–2017 Table . We usedFrom a societal perspective, the costs associated with the management of adverse events with the 2016 vaccination scheme  are US$ 8.84 million Table  and thosThe difference of 30,944 doses in the amount of hexavalent and the other vaccines is based on 7736 children in the private market receiving four doses of Hexavalent (3 + 1 scheme), and was assumed that the ministry of health would reimburse the vaccine for the whole cohort.In Scenario 4, the switch to the hexavalent scheme would cost US$ 2.62 million.In this study, we estimated and compared the difference in costs generated by two schemes with similar health outcomes but differences in the costs and safety, which were largely associated with logistics and adverse events management. The four scenarios analyzed considered two different approaches towards polio immunization, moving from a representation of Chile’s 2016 approach  towards the GPEI objective in which OPV should be suspended as soon as possible, which means that the complete IPV scheme  would become the preferred option. Several countries in Latin America have already expressed interest in a complete IPV scheme, similar to that introduced in Uruguay in 2012, but the supply of IPV has not been adequate or sufficiently reliable to fully comply with that policy. Based upon the PAHO reference prices, use of the hexavalent vaccine implies a greater expense versus simultaneous vaccination using the current pentavalent plus polio vaccines. However, the existence of more adverse events in the current scheme associated with wP compared to the aP formulation of the hexavalent scheme minimizes the cost difference between the two schemes.There have been relatively few published assessments of the economic impact of hexavalent vaccines. A study in France (where the hepatitis B vaccine coverage was low) of a hexavalent combination vaccine, found that the public price associated with a break-even point would be €53.77. The annual additional reimbursed cost of protecting an infant against the risk of hepatitis B was €28.20 per child, or about €21 million for an annual cohort of 760,000 births  . Whilst Our findings are supported by a previous study conducted in Latin America. An earlier study in Mexico examined the impact of moving from OPV to IPV vaccination schedule. The authors found that changing from the existing OPV-based routine schedule and intensive supplementary activities to a sequential IPV-OPV routine schedule using a pentavalent and IPV/OPV vaccines would save US$ 14.52 per vaccinated child, and changing to a full IPV routine schedule would save US$ 9.41 per vaccinated child . This laA similar cost-minimization analysis has been performed for hexavalent vaccine introduction in South Africa, which analyzed replacing aP pentavalent vaccine and Hep B by the hexavalent combination. The authors concluded that implementing a hexavalent vaccine in South Africa was highly recommended, because it reduces healthcare provider costs by simplifying logistics and delivery infrastructure: reduced clinic visits, vaccination errors, number of injections and side effects, which can be expected to translate to better acceptability, convenience and increased compliance .The strengths of our analysis are that we utilized nationally available data, or taken from peer-reviewed scientific publications. Our analysis also produced results which are consistent with other previous analyses reported. One of the possible weaknesses of our analysis is that it relies on the assumption that the hexavalent vaccine has similar efficacy to the currently used vaccines. If this assumption were subsequently shown to be inaccurate, then the approach we took here (cost-minimization) would no longer be appropriate.Contradictory evidence exists on the aP vs. wP benefits. On one side, concerns about the immunity conferred by aP vaccines not being as complete or as long lasting as that with wP vaccines , and it Our analysis may have underestimated the benefits of switching to a new hexavalent scheme since the reduction in immunizations required, might well be valued by the parents involved, leading to increased coverage and/or improved timeliness of infants immunization, which isn’t captured in our analysis –22.The introduction of a hexavalent (DTaP-IPV-HepB-Hib) vaccine into the immunization schedule in Chile would lead to additional acquisition costs, which would be partially offset by improved logistics, and a reduction in adverse events associated with the 2016 vaccines. This analysis provides decision makers with additional information on the potential impact of changes to the vaccination schedule caused by technical innovations, whilst reflecting the likely supply of alternative vaccines.


Railway inspection has always been a critical task to guarantee the safety of the railway transportation. The development of deep learning technologies brings new breakthroughs in the accuracy and speed of image-based railway inspection application. In this work, a series of one-stage deep learning approaches, which are fast and accurate at the same time, are proposed to inspect the key components of railway track including rail, bolt, and clip. The inspection results show that the enhanced model, the second version of you only look once (YOLOv2), presents the best component detection performance with 93% mean average precision (mAP) at 35 image per second (IPS), whereas the feature pyramid network (FPN) based model provides a smaller mAP and much longer inference time. Besides, the detection performances of more deep learning approaches are evaluated under varying input sizes, where larger input size usually improves the detection accuracy but results in a longer inference time. Overall, the YOLO series models could achieve faster speed under the same detection accuracy. Automatic inspection of railway track has always been attached great significance to the massive construction of railway network. In the research on railway inspection system, many scholars focused on infrastructure systems , surfaceIn recent years, the deep learning methods have attracted much attention from the research world due to the rapid development of graphics processing units (GPUs), which increases the computing power and greatly shortens the training time. The emergence of deep learning methods makes it more achievable to obtain state-of-art results on railway track component detection. Convolutional neural network (CNN), as a category learning method, can map data into nonlinear space and automatically extract potential features, which are more complex and more robust to images containing noises . Some clUp till now, the two-stage object detection algorithms have realized high detection accuracy, but the inference time is still intolerable for certain applications, such as real-time pedestrian detection  and videSo far, the one-stage detection models have achieved great success on classic image datasets, but few of them were put into applications on specific railway detection tasks. This paper focuses on railway track component detection based on YOLO family models. Different from the above works applied to large and general datasets, our model focuses on small railway track image dataset. By optimizing the parameters and comparing between other deep learning models, our work can obtain the best detection performance on railway track component detection in a limited dataset. The railway track images utilized in this study are provided by the Hong Kong Metro Corporation (MTR). To improve object detection results, we adjusted the detection layers of one-stage network to enhance the performance and optimize the parameters. The different size of anchors and inputs are also discussed to make the conclusion more reasonable.According to experiments, the advantages and limitations of the state-of-art YOLO models are revealed. The YOLOv3 shows no advantage in the component detection even it has finer feature extraction and more complicated structure of network.This paper provides a novel view of the research field, as it differs from existing papers in terms of the specific application on railway component detection. The success of representative one-stage models on railway track components detection can offer practical guidance on other industrial detection tasks such as track defects diagnosis.The major contributions of this work are summarized as follows:The models used in railway track component detection are all one-shot detection models which require only a single pass through the neural network and predicts all the bounding boxes in one go. In this section, some representative one-stage detectors such as YOLO, YOLOv2, and YOLOv3 are introduced. Based on the YOLO series models, we proposed YOLOv2 and YOLOv3 with anchors adjusted to batter adapt to our dataset.The YOLO model is built on the basis of Darknet which uses a modified GoogLeNet as the backbone. Darknet follows the general design of 3 × 3 filters to double the number of channels at each pooling layer and 1 × 1 filter to periodically compress the feature representation throughout the network. Most of the convolution layers have been pre-trained with the ImageNet dataset before adapted to the detection task. After 24 convolution layers, 2 fully connected layers are followed to integrate the predictions. YOLO algorithm simply splits image into fixed S × S cells and each cell predicts fixed B bounding boxes. So the output of final layer is a YOLOv2 is an enhanced version of the basic YOLO model. It adopts the concept of anchor priors in SSD. Rather than expecting the model to directly predicted bounding box descriptors, a collection of anchor boxes with varying aspect ratios that indicate some prior information about the size of objects are defined. Except for the improvement on anchors, YOLOv2 also randomly resized every 10 batches to make multiscale training possible in YOLOv2, which helped to train the model to be robust to images of different sizes. Another essential change is that YOLOv2 had less layers than the original YOLO, with only 19 convolution layers composed of 3 × 3 and 1 × 1 filter. Max pooling layers following each convolution layer are for reducing the feature map dimension. Instead of fully connected layers, YOLOv2 applies route layer and reorg layer on the feature maps to get fixed dimension of output. The route layer is to bring finer grained features in the network from earlier; the reorg layer is to make these features match the feature map size at the later layer.YOLOv3 adds a bunch of design tricks inspired by latest advances in the object detection field. First of all, the new Darknet-53 architecture still relies on successive 3 × 3 and 1 × 1 filters but has residual blocks inspired from ResNet  added. Sk-means clustering method was adopted on discovering the best aspect ratios of prior boxes with the distance metric changed to 1-IoU. Since the number of clusters k needs to be chosen according to manual experience, average IoU between ground truth boxes and the closest centroid under different k is calculated in k increasing, the average IoU increases because of more prior choices. Considering the trade-off between model complexity and difficulty of predicting, 6 anchors are chosen for YOLOv2 and 9 anchors are set for YOLOv3. With these anchors, coordinates are predicted as the offsets of the center of anchor boxes, widths and heights are predicted as the scale of anchor boxes, which is much easier than directly predicting from scratch in YOLO model. Plus, predicted bounding boxes will not share the same set of classification probabilities anymore, meaning that one grid cell is capable to detect multiclass objects.With the dataset changed to railway track images, k-means clustering method are assigned to three resolution output with anchor sizes sort descending. The inference time of YOLOv3 slows down because of more predictive quantities. Nevertheless, it could achieve a high enough detection accuracy.The YOLOv2 architecture in detail was displayed in Since these detectors have images as input, their application areas are quite wide. The only difference is that such a model does not guarantee excellent performance in every data set. For a specific data set, the model structure and parameters need to be adjusted and optimized to achieve the best detection effect. This is also one of the focuses of our work.The rail surface images are captured with an image acquisition system that cameras are installed at the bottom of the train. k-means algorithm on normalized labels of training dataset to choose appropriate anchor sizes. According to the clustering results, the average IoU of anchors used in YOLOv2 is 74.22%, in YOLOv3 is 77.91%. As mentioned before, with k increasing, average IoU will increase so the clustering of YOLOv2 has less average IoU than YOLOv3. After training, the weights lead to the highest accuracy on validation set will be evaluated on test set.The experiments are carried out on our lab server . In the training process, loss value gradually decreases as the number of iterations increases. The maximum number of iterations is set to 10,000 to make sure enough training. For each iteration, 64 images are fed as the input to learn. The learning rate is initialized at 0.005 because of the small training volume, and then multiplied by 0.1 at iteration 8000 and 9000, respectively. For fair comparison, the sizes of input images for all three models are raised to 640 × 640. When the widths and heights are changed at pixel levels, interpolation of pixel values is applied to stretch or compress images. The YOLO model directly predicts the coordinates and sizes of bounding boxes, whereas the YOLOv2 and YOLOv3 models employ The detection performance is evaluated using three commonly used metrics: mean average precision (mAP), F1 score, and inference time. The mAP and F1 score are defined as following.r is interpolated by taking the maximum precision measured for a method for which the corresponding recall exceeds r. In a traditional way, mAP values at a single IoU of 0.50  computed over all the three classes . The AP metric considers both the spatial position and the accuracy of categories for the detected objects. By setting thresholds, all the predicted boxes with different class scores (50) values of YOLO and YOLOv2 are both above 90%. Although YOLOv3 has more standard pyramid output architecture, it has no advantage on mAP performance of this component detection. YOLOv3 achieves a similar performance with the other two models in bolt and clip detection, however, for rail, the AP value is lower than the other two models. The precision and recall results when threshold is set at 0.25 are also shown in 75), IoU of 0.95 (AP95) and averaged over multiple IoU values from 0.50 to 0.95 with a step size of 0.05 (AP[0.5:0.95]) are also provided. It’s observed that higher IoU thresholds reward detectors with better localization. Even averaged over IoU, the detection performance of YOLOv2 still outperformed the other two methods. As the IoU threshold increases, the disadvantages of original YOLO model gradually emerge, and the averaged detection results are not as good as YOLOv3.The three one-stage approaches are trained with parameters set in part A. The AP, F1 score, and IPS results of each class for all test samples are summarized in To explain the unexpectedly poor performance of YOLOv3 in our results, two potential reasons are given as following. First, compared with the volume of images used for training, the YOLOv3 with more than 100 layers is overcomplicated so that it gradually showed an overfitting trend during the long training process. Second, the design of FPN in YOLOv3 aims to detect small and dense objects. However, the objects in our railway application is not either small or densely located. In fact, during the training process, the last two output layers with more divided grids usually have “invalid” output with no detection results to calculate recall or precision. The three models with various input sizes including 640 × 640, 448 × 448, 256 × 256, and 128 × 128 are trained for better explanation. The mAP and IPS values on the test data set are plotted in To further investigate the “abnormal” performance of YOLOv3, we tested mAP on different detection layers under different input sizes. Results are displayed in Moreover, to fully understand the effect of anchors on the results, YOLOv2 and YOLOv3 are compared with different anchors. In In this work, one-stage deep learning networks including YOLO, YOLOv2, and YOLOv3 are proposed to detect railway track components including rail, bolt, and clip. The detection performance is evaluated through Hong Kong MTR railway surface images with different sizes and illuminations. The YOLO series models can accurately detect almost all the key components in the railway image. Compared to original YOLO model , the YOLOv2 model improves the performance in both detection accuracy (93% mAP) and inference time (35 IPS) and can detect mostly cropped objects such as clips. In contrast, the YOLOv3 yields the lowest mAP of 89% and takes more detection time than the other two. Besides, all three networks predict detections at different input resolutions. The YOLO and YOLOv2 improve the detection accuracy with larger input sizes but lead to slower inference speed, whereas the YOLOv3 shows slightly better performance at smaller input resolutions and with less anchor boxes. When compared with other one-stage models such as RetinaNet-101 and YOLOv3-PRN, the YOLO series models always show satisfying detection results on both accuracy and speed. To conclude, YOLOv2, with the high accuracy and fastest speed, is the most suitable model for real-time railway inspection and further component detection.Nevertheless, the results suggest some further applications.The parts on both sides of the rail are symmetrical, especially for the images that photographed by the camera directly above the rails. With this, it is hopeful that the missing parts of these components can be detected.With advantages in detection accuracy and speed, YOLOv2 has the potential to be applied to video and finer object detection.YOLOv3 with fine-grained feature maps has advantage of detecting small objects. Therefore, using YOLOv3 for track defects diagnosis is another direction for railway inspection.


This study aims to assess the efficacy of a universal adhesive  (SB) in total-etch mode, compared to a traditional orthodontic primer  (XT Primer), to perform bonding of orthodontic fixed retainers along with the Transbond XT Light Cure Adhesive Paste (3M ESPE). For the in vitro study, a round section wire (Ortosmail Krugg) was bonded using XT Primer for 20 bovine incisors (Group 1) and SB for other 20 (Group 2). Samples were debonded in a universal testing machine applying a tangential force to specimens (crosshead speed of 1 millimeter per minute). Shear bond strength (SBS) and adhesive remnant index (ARI) scores were calculated. For the in vivo study, 100 patients needing upper and lower canine-to-canine fixed retainers after orthodontic treatment were randomly assigned to two groups of 50 participants each, i.e., group 1 (retainer bonding with XT Primer) and group 2 (retainer bonding with SB). Over two years, examinations were carried out monthly, and detachments were registered by considering the teeth and arches affected. In vitro, no statistically significant differences in SBS and ARI scores were demonstrated between the two groups, both showing a mean bond strength of about 12 MPa and major frequency of ARI “2” (>50% remnant adhesive on the enamel). Conversely, a significantly lower failure rate over 2 years was assessed clinically for group 2 in both arches. Independently of the adhesive and arch, incisors reported a significantly higher failure rate than canines. Scotchbond Universal used in total-etch mode could be a valid alternative to the traditional orthodontic Transbond XT Primer. After obtaining the desired position of the teeth, the final step of an orthodontic therapy consists of a retention procedure that attempts to keep the result achieved, avoiding a certain relapse due to the tendency of teeth to regain their pretreatment position . This phDespite this appliance not necessitating the active cooperation of the patient as required for removable retainers, its main disadvantage remains the risk of detachment ,8. The cDifferent adhesive systems have been developed in the restorative field. Currently, the most recent generation is known as “universal” or “multimode”, and the key point of these adhesives consists of the possibility of being used either with the total-etch mode or with the self-etch one, according to the choice of the operator . In any Until now, several studies have evaluated which composite resin is associated with the lowest incidence of retainers’ failure: Conflicting results have been obtained, as some authors found no statistically significant differences between the forces required to detach the different samples ,16, wherOn the basis of this consideration, it may be hypothesized that the usage of the universal restorative adhesive following the enamel pre-etching could represent a reliable option to avoid retainers’ detachments. Accordingly, the aim of this current in vitro and in vivo study is to analyze even in orthodontics the efficacy of a universal adhesive generally used in restorative dentistry, which is applied here after enamel pre-etching in order to bond fixed retainers to teeth. The research question is whether this last bonding agent is found to be more effective in orthodontic adhesion with respect to a conventional orthodontic primer.The first two null hypotheses investigated in vitro are that there is no significant difference in shear bond strength (SBS) and in the adhesive remnant index (ARI) scores between the two different products tested when used with the same adhesive paste. The third and fourth null hypotheses investigated through a randomized clinical trial are that during a 2-year follow up there are no significant differences in the fixed retainers’ bond failure rates and that there are no significant differences between incisors and canines failure rate.This experiment has been approved by the Unit Internal Review Board. Specimens were prepared from bovine incisors recently extracted. Considering the difficulty in obtaining human teeth for laboratory studies, the bovine ones represent a valid substitute, considering the similarity of the enamel .After the extraction, teeth were kept in complete darkness in a solution of 0.1% (wt/vol) thymol without alcohol . Within Sample size calculation  was performed considering two independent study groups  and a continuous variable. Concerning the primary outcome (bond strength), an expected mean of 24.7 was hypothesized, with a standard deviation of 9.2 ,31. The The root of each element was embedded into cold-curing fast-setting acrylic  inside a plastic cylindrical mold (2 cm height × 2 cm diameter). Teeth were positioned in the center of the molds and with an inclination, thus allowing a shear force to act on their labial surface.2, calculated by delimiting with a pencil the presence of a 3 mm × 1 mm retainer, was immediately bonded.The 37% orthophosphoric acid  was used to etch buccal enamel for 30 seconds, as generally recommended in dentistry , and theFor this phase, samples were randomly divided into two groups of 20 specimens each. In group 1 , the bonding agent used was Transbond XT Light Cure Adhesive Primer  whereas in group 2  Scotchbond Universal  was used.The assigned bonding material was applied by means of a brush, following the specific procedures listed in 2. In conclusion, light curing of the paste was performed for 10 seconds in an occlusal–apical direction and for 10 seconds in the contrary one.On each sample, a multistranded round section wire , of 3 mm length, was applied along with Transbond XT Light Cure Adhesive Paste (3M ESPE) whose excess was removed with a probe to limit an area of 3 mm2, was used when necessary for all the light curing phases. The distal end of the light guide was placed perpendicular and at contact with the resin to be cured.The LED lamp Starlight Pro , with an output irradiance of 1000 mW/cmThe characteristics of the tested materials and the protocols recommended for their application are shown in All the specimens were then stored in distilled water for 24 h at room temperature before proceeding with the subsequent experimental phase.Each specimen was placed in a universal testing machine . According to a previous study of our group , retaineThe software Bluehill 2  automatically recorded the maximum detachment shear force in Newtons. Results were then transformed into megapascal, with the following formula:After detachment, retainers and teeth surfaces were analyzed under a ×10 optical microscope  , and theTrial designThis study was conceived as a parallel-group, active controlled, and single-center randomized clinical trial (RCT) with a 1:1 allocation ratio. This study has been approved by the Unit Internal Review Board Committee (2016 0113). No variations to the methods occurred during the study. In order to reduce bias, blinding was conceived for patients, the outcome assessor, and data analyst.ParticipantsPatients referred to the affiliation of the authors were engaged from February 2016 to January 2018 and were followed until January 2020. For each patient, the informed consent was achieved . Interventions and data assessment were carried out in the same center.The inclusion criteria were going towards the final step of an orthodontic treatment and the later positioning of both upper and lower fixed retainers. Patients reporting facial trauma, onychophagia, and habitude of biting pencils or pens were excluded from this study. Among the participants selected, three refused to participate. The flow of the participants and the investigation protocol used in the present report are showed in InterventionsAt the end of the orthodontic treatment, participants were randomly subdivided into two groups, in accordance with the product later used for the retainer’s bonding. The materials tested are those previously reported for the in vitro evaluation. Multistranded wires were bonded on upper and lower arches on the palatal/lingual side, involving incisors and canines. In the first group (XT Primer), the adhesion procedure was conducted using the conventional Transbond XT Primer  and in the second group (SB), the universal adhesive Scotchbond Universal (3M). All the other steps, e.g., the use of the Transbond XT Light Cure Adhesive Paste, were the same in the two groups. To guarantee a standardization, interventions were always realized by a same operator who was not involved in the previous in vitro study.OutcomesFrom the date of retainer’s bonding, participants of both groups underwent a 2-year follow up, with periodic visits every month, executed by another operator not taking part in the bonding phase or the in vitro study. Each detachment of retainers was recorded, considering the position (maxillary or mandibular arch) and the respective number of incisors and canines involved. Only the first temporal detachment (or detachments) was been considered, whereas subsequent ones were not further contemplated for the statistics. Participants were instructed to respect the planned appointments and to urgently contact the dentist in case of any suspected detachment.Sample sizeThe established alpha was 0.05 and power was 80%, and considering two independent groups and a continuous primary outcome, the sample size calculation required 100 total participants resulting in 1200 teeth splinted. Concerning the primary outcome (failure incidence), an expected frequency of 30% was hypothesized. The expected difference between the frequencies was supposed to be 21.8. This margin was supposed by basing on the results of previous reports that evaluated similar topics ,37.A total of 50 patients were requested for each group. Loss to follow-up and incomplete compliance with therapy were excluded. A total sample of 100 patients was finally selected of which 48.5% males (mean age: 25 years and 9 months) and 51.5% females (mean age: 25 years and 3 months). Among the 50 controls, 49% were males (mean age: 27 years and 2 months) and 51% females (mean age: 26 years and 11 months), whereas, among the 50 trials, 48% were males (mean age: 24 years and 2 months) and 52% females (mean age: 23 years and 10 months).Randomization and blindingRandomization sequence was performed by the data analyst using a block randomization table and considering a permuted block randomization with 50 participants for each fixed block. Participants were enrolled by the dental operator who also achieved the allocation concealment by using sequentially numbered and sealed envelopes containing the allocation cards earlier arranged. It has not been technically feasible to blind the operator. On the contrary, participants, the outcome detector, and statistician were blinded throughout the whole trial since they were not aware of the treatment assigned. No visible significant differences could be noticed during the data assessment between the retainers bonded with the two different methods.p < 0.05.The results achieved were analyzed using the R Software . The statistical significance for all the analyses was set at t-test was applied to assess the presence of significant differences in debonding values between the two groups. To reveal significant differences in adhesive remnant index scores, a frequency analysis represented by an χ2 test was carried out.As regards the shear bond strength values, descriptive statistics  were calculated. Data normality was determined through the Kolmogorov–Smirnov test. Homoscedasticity was verified using Levene’s test. The The Fisher exact test was applied to assess differences in the frequencies of the clinical detachments of the groups tested in vivo; moreover, the relative Kaplan–Meier survival curves of the adhesives examined were designed and compared by means of the log-rank test.t-test demonstrated the absence of a significant difference between the two groups (p > 0.05), despite a major SBS assessed for group 2.As reported in 2 test  did not show any significant difference between the two groups (p > 0.05), which both exhibited a higher prevalence of ARI scores “2”, as reported in The χp < 0.05). Considering both arches as well as the upper and lower ones separately, the trial group (group 2) reported significantly lower total detachments. Within group 1, lower teeth had a significantly higher failure percentage (p < 0.05) with respect to the upper ones (27.33% vs. 16.33%), and conversely, within group 2, a higher failure rate was seen for the upper teeth (7.33% vs. 6.00%) but without a statistical significance (p > 0.05).At the end of the follow up, statistically significant differences were found in terms of detachments (failure rate) between the two groups of the study . During the 2-year retention period no wire fractures were found in both groups.Kaplan–Meier survival curves are showed in The first two null hypotheses of the present study were accepted. The results obtained in vitro showed the absence of statistically significant differences between the conventional orthodontic primer and the universal adhesive, for both SBS and ARI. As regards this latter parameter, a major frequency of an ARI score of 2 has been shown for both materials tested, which indicates that a cohesive failure at the wire–composite interface occurs, and more than 50% of the adhesive remains on the teeth, with no failure of the bonding interface (enamel–adhesive), in accordance with the results reported by other authors ,39. ConsAccording to these results, both Scotchbond Universal used in the total-etch mode as well as the conventional orthodontic primer appear to be adequate systems since they showed no bonding failure at the enamel–adhesive interface but only detachments at the composite–wire site (cohesive fracture). To date, we found no studies in literature comparing the average force required for the detachment of splints bonded with Transbond XT Primer and Scotchbond Universal. This prevents us from directly comparing these results with those of analog studies. Transbond XT Primer has been chosen as the only reference of non-universal adhesives because of its wide use in orthodontic bonding.A limitation of this in vitro study might be represented by the fact that it was conducted on bovine teeth and not on human ones, due to the difficulty in obtaining the latter for research purposes. As stated in the systematic review with a meta-analysis conducted by Soares et al.,  despite Considering that clinical studies should follow to confirm the in vitro results, a randomized clinical trial was conducted to test the same materials. The third null hypothesis was partially rejected. In both arches overall as well as in the upper and lower ones considered separately, significantly lower failure rates were assessed during a 2-year follow up for retainers bonded with the Scotchbond Universal adhesive after enamel pre-etching. Since the same orthodontic composite resin was used in this report, the two different bonding agents tested had a significant influence on the different number of failures assessed under clinical conditions. All the detachments assessed in vivo occurred at the enamel–adhesive interface, which appears as the most fragile (resin debonding); on the contrary, no detachment of the retainers from the adhesive (wire debonding) was disclosed. This is in accordance with a previous in vitro study, which showed that lingual appliances bonded on etched enamel almost left no adhesive after detachment; conversely, enamel pretreatment by means of sandblasting, before etching, resulted in more remnant adhesive on the tooth surface .Within group 1, a significant difference between failures of the upper and lower arch was reported only for the lower teeth splinted with the traditional orthodontic primer which had significantly higher failure rates than the upper ones in the same condition (27.33% vs. 16.33%). Conversely, within group 2, a higher failure rate of the multistrand wires was assessed for the upper arch rather than for the lower one (7.33% vs. 6.00%), in accordance with other studies that attribute this outcome to occlusal factors . HoweverThe fourth null hypothesis was rejected. When comparing the clinical bonding failure on incisors and canines, the rates were significantly higher for the former, regardless of the adhesive and of the splinted arch. As reported in the review of Iliadi et al. , bonding®  was equal to 88%, considerably higher than the one reported by glass fiber reinforced bonded orthodontic retainers (49%).Finally, the Kaplan–Meier survival curves clearly show a statistically higher survival rate for Scotchbond Universal (93.33%) following a 24-month follow up, rather than for the conventional Transbond XT Primer (78.17%). In any case, both these percentages are in accordance with the study of Tacken et al. , showingThe results obtained with the clinical trial disagree with those assessed in vitro but a direct comparison is not possible and, conversely, it would be misleading due to the different experimental conditions between the two studies. The rationale of the in vitro shear bond strength test is that of conducting an initial exploratory evaluation of the behavior of the two adhesives, limited to an ideal and short-term storage. A complete laboratory simulation is not always possible in dentistry because of the high variability occurring under in vivo conditions, which justify the need for having recourse to clinical trials . In any Considering that both products tested in this study were applied on pre-etched enamel, the better outcome assessed in vivo for Schotchbond Universal should be ascribed to the chemical bond formation between 10-MDP and VP-copolymer of Schotchbond Universal with the apatite structure of acid-etched enamel that is missing from the Transbond XT Primer-etched enamel interface, the latter solely based on micromechanical retention.The follow up here considered might be relatively short since patients should keep their bonded fixed retainer as long as possible in order to avoid orthodontic relapse. However, studies on this topic, as those considered in the systematic review of Al-Moghrabi et al. , generalIt is well accepted that results of in-vitro studies might not replicate clinical applications ,50; as aFuture studies should be conducted to confirm the results here obtained and to verify whether the same outcomes referred to the adhesives here tested would be obtained even when bonding other type of wires, like fiber-reinforced composites retainers.Despite the limitations of the two studies conducted, the universal adhesive Schotchbond Universal used in total etch bonding strategy could be a valid alternative to the conventional Transbond XT Primer for orthodontic retainers’ adhesion. Accordingly, the use of this product could be extended from restorative dentistry even to orthodontics.


Access to breast cancer genetic counseling is suboptimal for patients with limited health literacy or a migrant background due to ineffective communication and lack of healthcare professionals’ recommendation. This study examines the effect of a blended training program  for healthcare professionals on their awareness, knowledge, and self-efficacy towards communication about genetic counseling with patients with limited health literacy or a migrant background. In total, 59 breast surgeons and specialized nurses from 16 Dutch hospitals completed an online module and group training. Knowledge, self-assessed awareness, and self-efficacy were assessed before the training and 33 participants also completed a posttest questionnaire 6 months after the training program. We also assessed the perceived applicability and relevance of the training program from healthcare professionals’ perspectives. We found a significant increase in self-assessed awareness of the prevalence and impact of limited health literacy and in healthcare professionals’ self-efficacy to recognize limited health literacy and to communicate effectively with patients with limited health literacy or a migrant background. We did not find an increase in knowledge score. Almost all healthcare professionals reported that they use the techniques learned in the training, such as the teach-back method and plain language, and felt more confident discussing breast cancer genetic counseling. Our results suggest that a blended training program for healthcare professionals has potential to improve their ability to communicate effectively about breast cancer genetic counseling with patients with limited health literacy or a migrant background and offers a promising way to increase the referral rate for these groups of patients. Breast cancer, the most commonly diagnosed type of cancer, affects about 12% of women worldwide  based on healthcare professionals’ and patients’ needs and preferences, to increase healthcare professionals’ knowledge, awareness, and self-efficacy when communicating about breast cancer genetic counseling with communication-vulnerable patients, i.e., patients with limited health literacy or a migrant background  and a group training in one of the participating hospitals (2 h). The online module included information about the prevalence of low literacy and limited health literacy in the Netherlands, the relevance of health literacy in understanding and appraising information from healthcare professionals, and the way health literacy relates to socio-economic and background characteristics of patients. It also included information about how to communicate effectively with patients with a migrant background. In the group training, the teach-back method—a methodology used by healthcare providers to check whether a patient understands what has been discussed—was used as a technique to identify patients with limited health literacy and role-play was used as a strategy to acquire required communication skills and to practice the use of plain language  to get access to the online knowledge module. Within 2 weeks, they were invited to attend a group training on location. We used a pre-/posttest design to evaluate the effect of the Erfo4all intervention on knowledge, self-assessed awareness, and self-efficacy related to communication about breast cancer genetic counseling with patients with limited health literacy or a migrant background. Six months after the group training, the healthcare professionals were asked to fill out a second questionnaire (T1), again to assess knowledge, awareness, and self-efficacy. At T1, we added self-report questions concerning the applicability and relevance of the training program as well as healthcare professionals’ perceived awareness regarding the problem of health literacy in general and their confidence to adapt their communication effectively. Only healthcare professionals who completed the whole program and filled out the T0 and T1 questionnaire were considered in the pre- and post-intervention analysis. For the additional questions, all healthcare professionals who filled in the T1 questionnaire were included.Outcome variables were assessed with self-constructed online questionnaires, before (T0) and 6 months after the training (T1). The items in these questionnaires were based on the intervention mapping approach and the matrix of change used for the development of our training program  literacy among people with a migrant background, the prevalence of adults with limited health literacy in the Netherlands, the level of education in relation to the level of health literacy, and option of using of a professional interpreter (self-reported). Each item was rated as correct (1) or incorrect (0) and a total knowledge score was computed as the number of correct answers.Awareness was assessed by three items: prevalence and impact of health literacy in the Netherlands, impact of health literacy on medical communication, and the importance of taking into account cultural factors when communicating with patients with a migrant background.Items were scored on a 5-point Likert scale ranging from (1) hardly aware to (5) very highly aware.Self-efficacy was assessed by five statements on having confidence in recognizing limited health literacy in patients, communicating effectively about breast cancer genetic counseling with patients with limited health literacy, understanding which customs and habits from patients with a migrant background might influence communication, coping with cultural factors in communication with patients with a migrant background, and coping with a language barrier.Items were scored on a 5-point Likert scale ranging from (1) totally disagree to (5) totally agree.At T1, we also assessed healthcare professionals’ perceived awareness regarding the problem of limited health literacy in general and their confidence to tailor communication about breast cancer genetic counseling to the needs of communication-vulnerable patients. These additional items were scored on a 5-point Likert scale ranging from (1) totally disagree to (5) totally agree. The relevance of the training was assessed by asking whether the trained healthcare professionals shared information about the training with colleagues in multidisciplinary oncology meetings. Application of skills, such as the use of the teach-back method and plain language, was assessed with a 5-point Likert scale ranging from (1) never to (5) very regularly.Univariate analysis was used to describe the background characteristics of healthcare professionals.α = 0.05. Statistical analyses were performed using SPSS version 24.0 .Categorical data, number of healthcare professionals, discipline, sex, and clinical setting are presented in numbers and percentages. Continuous data, like age and work experience, are presented as means and standard deviations. To analyze T0 and T1 data, we performed paired analysis or repeated measurement, since multiple responses from the same subject cannot be regarded as independent from each other. In this analysis, subjects were included only when data were available from both time points (T0 and T1). To check for potential selective drop-out, we compared the group that only completed the T0 (T0 only) with the group who completed both questionnaires (T0+T1) and looked for differences in demographics and outcome variables. To analyze pre-post differences in the outcome measures’ knowledge, awareness, and self-efficacy, we used the Wilcoxon signed-rank for related samples. Tests for statistical significance were two-sided with n = 59) and of those who completed both questionnaires (n = 33). Based on background characteristics, no statistical differences were found between both groups, indicating that drop-out between T0 and T1 was not selective.The baseline questionnaire (T0) was completed by 65 participants of whom 59 participants from 16 hospitals completed the whole training program (online module and group training). In total, 37 participants filled out the T1 questionnaire, of whom 33 participants from 14 hospitals filled out both questionnaires (T0 and T1). Table n = 33). At the posttest (after the training), there was a significant positive change on six outcome measures compared to the pretest (before the training). The largest increase was observed in participants’ self-efficacy. Understanding the customs and habits of patients with a migrant background, the ability to recognize limited health literacy, to communicate effectively about breast cancer genetic counseling, and to cope with cultural factors or a language barrier significantly increased from baseline to T1. Moreover, awareness of the prevalence and impact of limited health literacy in the Netherlands increased significantly. The total knowledge score did not increase over time.Table n = 37). More than 80% (n = 30) of the healthcare professionals reported having used plain language to explain genetic testing; of these, almost 41% (n = 12) reported using it (very) regularly. Even more, 92% (n = 34) reported applying the teach-back method to discover whether a patient understood information and to identify limited health literacy, while 62% (n = 23) reported using the teach-back method (very) regularly. More than 97% (n = 36) of the respondents felt more aware of health literacy problems and assessed their ability to recognize patients with limited health literacy higher and 86% (n = 32) reported that their ability to communicate effectively with these groups of patients had improved. Most healthcare professionals expected to benefit from a booster training session after 1 year and more than 41% (n = 15) reported sharing their experience with the training program in multidisciplinary oncology meetings with colleagues.In our prior study, we assessed acceptability and usefulness of the training program directly after the group training  aimed at increasing awareness, knowledge, and self-efficacy regarding limited health literacy and communication with patients with limited health literacy or a migrant background leads to significant improvement in awareness of the prevalence and impact of limited health literacy, and self-efficacy in communicating about breast cancer genetic counseling with these groups of patients. No significant differences were found in pre- and posttest knowledge scores, on awareness of the impact of health literacy on medical communication and the importance of taking into account cultural factors when communicating with patients with a migrant background. Almost half (41%) of the breast surgeons and specialized nurses who participated in the training reported to share their experience with colleagues and almost all reported to apply the techniques taught in the training in daily practice .Healthcare professionals experience several problems in discussing genetic counseling and testing with patients with limited health literacy or a migrant background. Low awareness of the problem of limited health literacy, difficulties in recognizing limited health literacy, coping with cultural factors and a language barrier in communication with patients with a migrant background, and a lack of the skills needed to discuss referral effectively with these patients are the main problems. This is often compounded by patients’ limited health literacy which has been shown to negatively affect their ability to play an active role in their own health care, by asking questions, participating in shared decision-making, and taking initiative  health literacy knowledge   apply certain communication skills. Although it is unknown whether the increased scores on awareness and self-efficacy in this case indeed led to sustainable changes in communication behavior in daily practice, feelings of self-efficacy have been linked previously with behavioral change (Mata et al. In conclusion, our study shows improvements in relevant outcome measures among a diverse group of healthcare professionals involved in surgical breast cancer care in different regions in the Netherlands. It is promising that the skills learned during the training seem applicable in daily practice, even in the long term, and that healthcare professionals reported gains in awareness and self-efficacy. We implemented the Erfo4all training program in three regions in the Netherlands in different clinical settings (academic and non-academic hospitals and among healthcare professionals from different disciplines. Our previous study showed that the acceptance and perceived usability of the program was high (van der Giessen et al.