Numerous studies have documented inconsistent reporting of sexual behaviour by adolescents. The validity and reliability of self-reported data on issues considered sensitive, incriminating or embarrassing, is prone to social-desirability bias. Some studies have found that Audio Computer-Assisted Self Interviewing (ACASI) that removes the personal interaction involved in face-to-face interviews, decreases item non-response and increases reporting of sensitive behaviours, but others have found inconsistent or contradictory results. To reduce social desirability bias in the reporting of sensitive behaviours, face-to-face interviews were combined with ACASI in a cluster randomized trial involving adolescents in Zambia.th time since recruitment. Young, female research assistants who had conducted interviews for the trial were also interviewed for this study.To explore adolescent girls’ experiences and opinions of being interviewed about sexual and reproductive health, we combined Focus Group Discussions with girl participants and individual semi-structured interviews with teachers. This study was done after the participants had been interviewed for the 6Respondents explained often feeling shy, embarrassed or uncomfortable when asked questions about sex, pregnancy and abortion face-to-face. Questions on sexual activity elicited feelings of shame, and teachers, research assistants and girls alike noted that direct questions about sexual activities limit what the participant girls may be willing to share. Responding to more indirect questions in relation to the context of a romantic relationship was slightly easier. Efforts by interviewers to signal that they did not judge the participants for their behavior and increased familiarity with the interviewer reduced discomfort over time. Although some appreciated the opportunity to respond to questions on their own, the privacy offered by ACASI also provided an opportunity to give false answers. Answering on tablets could be challenging, but participants were reluctant to ask for assistance for fear of being judged as not conversant with technology.Strategies to avoid using overly direct language and descriptive words, asking questions within the context of a romantic relationship and a focus on establishing familiarity and trust can reduce reporting bias. For the use of ACASI, considerations must be given to the context and characteristics of the study population. Numerous studies have found that adolescents may not answer truthfully or consistently when questioned about issues such as sexual activity, abortion and pregnancy. Such issues are considered sensitive, incriminating, or embarrassing, and answers may be influenced by a fear of being misjudged or sanctioned. In an attempt to collect more reliable data on sexual behaviour and childbearing, we combined face-to-face interviews with Audio Computer-Assisted Self Interviewing (ACASI) in a research trial involving adolescents in Zambia. In this study we aimed to explore adolescent girls’ experiences and opinions of being interviewed about sexual and reproductive health when participating in the trial. We combined group discussions with girl participants and individual interviews with teachers. Participants said they felt shy, embarrassed or uncomfortable when asked questions about sex, pregnancy and abortion face-to-face. Teachers, girls and interviewers alike noted that very direct questions limit what the participants may be willing to share. Efforts by interviewers to signal that they did not judge the participants for their behaviour, and repeated interviews with the same interviewer reduced discomfort over time. Some liked ACASI whereas some said it made it easier to give false answers, and answering on electronic tablets could be challenging. To reduce embarrassment and increase the likelihood of honest answers we recommend avoiding overly direct language and descriptive words, and to relate questions about sexual activity to a romantic relationship. Interviewers who are able to establish familiarity and trust can make participants more comfortable. Adolescent sexual and reproductive health is a global public health concern which has received growing interest and increased research attention over the last two decades . To addrThe Audio Computer-Assisted Self Interviewing (ACASI) technique has gained some popularity amongst researchers due to the privacy it affords the respondent. When using ACASI, respondents receive and respond to questions using a handheld computer or tablet, which removes the personal interaction involved in face-to-face interviews, and this has been found to reduce socially desirable responses. Encouraging evidence for increased reporting of sexual risk behaviour using ACASI was found in USA in the 90 s and 2000s –12, and In the Research Initiative to Support the Empowerment of Girls (RISE) we combined face-to-face interviews with ACASI to reduce social desirability bias in the reporting of sensitive behaviours. RISE was a cluster randomized controlled trial that aimed to measure the effect of economic support for adolescent girls, sexuality education and community dialogue on childbearing rates amongst adolescent girls in Zambia . ThroughControl Group, (2) Economic support group with cash transfers to girls and their families and payment of school fees or (3) Combined intervention group receiving the same economic support combined with sexuality education and community meetings targeting norms related to education and SRH amongst parents and community members  but the most embarrassing is the sex question, so it is difficult to answer.GirlSo why do you think this particular question is embarrassing? Because you are saying that asking if you have a boyfriend is not difficult to answer.Researcher: :Because they will start thinking of me as a girl who opens her legs (girls laugh) Ah! (FGD 2)GirlQuestions about sexual behaviour were perceived to be the most difficult to answer. Girls in a group elaborated a bit on this:Another group gave several reasons why girls would lie about ever having had sex: “[the girl’s]self-esteem is low”; “Some girls get offended if you ask them that how many men have you ever slept with”; “some fear that their parents may find out at home and tell the Rise teacher at school”; “fear of being laughed at”, “fear she may be reported to people”; “not just understanding the questions”, “maybe they think that when you meet them [RAs] in the streets, you will be looking at them with an eye that you know about them” (FGD 1).: So, are there any questions that make you feel uncomfortable? Maybe even embarrassed?Researcher: (laughs) Those ones whereby she is asking you if you have ever been pregnant, have you ever given birth? They are embarrassing but more embarrassing if you are actually pregnant.Girl 1Researcher 2: Can I ask why they are embarrassing?: If you have been pregnant it is embarrassing or shameful. (FGD 2)Girl 2Questions around pregnancy also made the respondents uncomfortable:“Some people who respond to these questions do not tell the truth. They lie mainly because they feel guilty and ashamed and don’t want to be discovered as a prostitute” . The girls reported that they are continuously reminded and ‘warned’ by parents, teachers and the community that it is wrong and dangerous to have sex before marriage. This may result in an internalization of shame, guilt and negative feelings, which may have direct implications for the way they respond to sex-related questions.: because these girls they know that when they are doing these things [sexual behaviours], they know it’s not right and they are advised not to be doing these things. So when they say that they do it, they feel guilty because they are doing the wrong thing or have been told not to be doing sex before marriage so that they don’t contract STIs and HIV and they don’t become pregnant. So, when they are asked to say ‘Have you ever had sex?’, they are going to deny because they will know now they are not trusted. (Interview with RISE teacher)TeacherQuestions on sexual activity elicited feelings of shame and teachers, RAs and girls alike noted that they limit what the participants may be willing to share: : you said with sex there was guilt and shame, is that the same with having a child? Is there shame?Researcher: sometimes, for having a child they are not shy, they are going to tell you straight away, that one is not very difficult. The most difficult is where you ask them, do you have a boyfriend, have you ever had sex? Are you pregnant? They hide! But if you ask them if they have a child, they don’t usually hide that.TeacherAnother teacher suggested that questions about having a child are less likely to create feelings of shame than questions about sexual behaviour or pregnancy.When FGD participants were asked why some respondents would report having a child in the face-to-face interview but respond ‘no’ to the questions referring to sex in ACASI, they explained that it is easier to say that they have a child because, “it’s like a lighter version” of saying sex. The shame, guilt and perceptions of promiscuity associated with ‘sex’ amongst these adolescent girls, made them want to distance themselves from it.: we have some situations where a girl will say she has a child and then if you ask her later if she’s ever had sex, she’ll say noResearcher: yes, she’ll say no. (laughs). Why? Because of sex! Aha!! Because of sex…. Just that word ‘sex’, it means ‘a man in me’, just that! She will feel shy, unless another term is used. (Interview with RISE teacher)TeacherTeachers and RAs would also prefer to avoid the word ‘sex’ since the word itself is source of embarrassment. It is rarely, if ever, used in the local public sexual and reproductive health discourse. Although the act of sex is implicit when talking about having a child or pregnancy, they considered that using the word itself creates a risk that participants alter their response.: But others are not fine with these questions. Some may end up slapping you over these questions [about miscarriage/stillbirths], it may be that she was once pregnant but had a stillborn, then you are asking her these questions repeatedly. They may not be in the mood or they may respond to you in a way that is not good just to offend you. (FGD 2)GirlQuestions about miscarriages and stillbirth were perceived as offensive and upsetting despite of RAs being trained to approach such sensitive topics with care and empathy. It appeared that this was partly worsened by the structure of the interview which meant that a number of these questions were repeated (they were asked face-to-face as well as in the ACASI portions).: Why do you think these questions [about abortion] are difficult to answer?ResearcherBecause they feel like if they say the truth, someone is going to tell someone and so on. Abortion is not acceptable in society. It’s actually embarrassing to be known as a person who has aborted. (FGD 4)Girl: Reactions to questions relating to induced abortion were particularly strong as there is a lot of shame associated with abortion.There is just one question that I was asked, and I thought to myself ‘what has made this person to ask me this question?’Girl 2: : Which question is that?Researcher: The questions ‘Have you ever aborted?’. I was disappointed because it is not allowed to abort and I thought the person asking me was looking at me as if we do not respect ourselves. (FGD 5)Girl 2Fears around confidentiality and anonymity of answers were frequently cited by RAs and teachers as a reason for participants not responding truthfully to sensitive and/or taboo questions. In contrast, RISE participants explained that the questions about sex and abortion made them feel judged by the interviewer. A girl who has a child and is asked whether she has ever had sex, may answer ‘no’ because she is afraid of being laughed at, according to participants in one of the groups (FGD 3). Questions about unacceptable behaviours made respondents believe they were suspected of engaging in that sort of behaviour, implying that the interviewer had made a negative evaluation of their character.The following section describes the various respondents’ suggestions for overcoming barriers in collecting self-reported SRH data. Four key strategies were mentioned by all three respondent groups.: At first I was uncomfortable and shy because I was not used to being asked these questions by a stranger, a person you don’t know completely, asking you these questionsGirl 1: What made you not be shy afterwards?Researcher: Because I became used to being asked such questions. You can only talk about these things to people you are close to. (FGD 3)Girl 1As outlined above, the sensitive nature of several of the questions meant that respondents required a great deal of confidence in the interviewer if she was to answer honestly. The girls claimed that they felt comfortable being interviewed by the trial RAs and emphasised the importance of ‘getting to know’ and developing a close relationship with them. This familiarity was created in the informal conversations prior to commencing the interview, throughout the course of each interview, and through repeated interviews (conducted every 6 months) by the same person. The respondents became gradually more comfortable with the interview process as well as the RA.: The thing is that we would like to be interviewed by the same person because we are now used to her as opposed to someone we haven’t met. We also feel like she cannot release our information to people and we are not scared of her. (FGD 6)GirlWe were curious whether changing RAs between interview rounds would increase the feeling of anonymity amongst the RISE participants, but this did not appear to be the case. As can be seen in the quote below, when the RAs returned for subsequent interviews, the girls felt they had already heard their secrets.“We’ve been coming here for a long time—have you ever heard of anyone being ‘told on’ or information being shared?”. Familiarity with the respondent also made it easier to judge the reliability of her answers and the appropriate approach to assume. For example, if the participant was pregnant in the previous interview, the RA could be more attentive when asking questions around childbirth or miscarriage.RAs often used their repeated visits to reinforce the confidentiality of the interviews. Several of them said things like: Teachers and FGD participants suggested that the school was considered the ‘safest’ location to conduct the interviews. The presence of community members at the clinic could create a risk of rumours and curiosity, according to the girls. At their homes, the presence of parents seemed to prevent speaking about sex and reproductive issues. Several of the RAs had experienced that the girls avoided being found in their homes when they arrived.: Another way to make us comfortable so that we can answer truthfully, is to tell the person who interviews us to open up freely. They can say that ‘Even I have slept with a man and know everything that happens between a man and a woman. So whatever girls go through, I have also gone through that, so feel comfortable.’ (FGD 1)GirlWhen RAs were able to normalize sex and pregnancy by giving examples from their own lives, it seemed to be an effective way of making a respondent comfortable enough to answer questions on this topic.RAs occasionally referred, without negative connotations, to a young family member who was pregnant, which was meant to signal to the respondent that they had no negative judgement towards that behaviour.: Ok, if you want to ask a friend if she had sex, how would you ask her?Researcher: I can ask her, ‘Do you have a boyfriend. Or are you dating?’ Then that’s when you ask her if she had slept with him. (FGD 6)Girl“In fact they should be asking these questions like do you have a boyfriend and do you have a child together, so as to avoid the awkwardness or just removing the sex question.” (FGD 2)When reviewing the questionnaire items with respondents, they noted that the sequencing and formulation of the questions may impact on the respondent’s reaction to the interview. The comments of the respondents suggested that in the follow up interviews, sex-related questions were not appropriately contextualised in the frame of a stable and/or romantic relationship. All three respondent groups emphasized the need to present questions around sexual activity with the assumption that it was with a boyfriend.Researcher: Is there some way to make it less direct?Head Teacher: Sometimes you start asking ‘Do you have a boyfriend?’ And from there ‘From the time that you met that boyfriend, have you done anything with him?’ Or maybe ‘What type of stories do you share with your boyfriend, and at what time do you meet your boyfriend?’ Such things. Maybe ‘Do you see your boyfriend in the presence of your mother? Or maybe you have got some places where you meet this boyfriend?’ Just like those minor questions before you hit to the real point of ‘Have you ever slept with your boyfriend?’. (Interview with head teacher)A head teacher also suggested that the interviewed girl should be eased into the question.Ok, the tablet is fine to those who are shy to talk face to face with the interviewer, even though there is a chance they can lie. For instance, if she is pregnant when answering on the tablet, she can say ‘no’. But when asked during face to face, she admits it because you have seen for yourself that she is pregnant. (FGD 3)Girl 1: When the girls shared their opinions about the ACASI interview, the privacy that it provided was often highlighted as a positive feature. However, this privacy also provided an opportunity to give false answers:: Which one do you prefer? The tablet or face to face?ResearcherI prefer being asked live [face to face]Girl 2: : We prefer face to face because we are interacting with a person who can read our expressions and use better judgement. And also if you did not really understand the questions, you can ask if you are not clear. (FGD 3)Girl 3The lack of support and contact with the interviewer was a clear drawback of ACASI.“Sometimes we feel shy because we feel like they will tell someone that we don’t know how to use the tablet” . Indeed, many of the informants—both the girls and RAs—suggested more support and training should be given to those RISE participants who struggled with the ACASI technique.RAs and FGD participants noted that the language and dialect of the audio recordings in ACASI could be challenging, but participants hesitated to ask for assistance for fear of being judged as not conversant with technology: : Ooho! It’s reading sometimes which is the problem. Sometimes you are reading and at the same time you are listening from the headphones, and then you have to press at the same time. You end up making a mistake pressing ‘Yes’ to the question when you meant ‘No’.Girl 1: But the tablet asks which language you understand. It’s just that others do not know or hear the instructions where to press. So, others find it difficult to use.Girl 2: So sometimes you put an answer you didn’t want to put?Researcher: Yes.Girl 2: So, what can we do to make it easier on the tablet?Researcher: Maybe they should come and teach those who find it difficult for some time, not just a short time. (FGD 1)Girl 1When the FGD participants were asked why some girls may prefer the face-to-face interview, they suggested that with the ACASI, being presented with a written text, a voice recording and being asked to select a response at the same time, could be confusing.Several RAs felt that some girls had rushed through the questions in the ACASI portion and gave examples of respondents who completed the ACASI portion of the interview in less than five minutes when on average it took 10 min to complete. FGD participants suspected that some girls may have rushed through the ACASI portion because they were uncomfortable with the method and/or technology. All respondents  emphasised the importance of offering continuous clarification and support throughout the interview even when not requested by the participant herself.This study found that questions about sex, pregnancy and abortion made the female adolescent trial participants uncomfortable and elicited feelings of shame. These reactions were common despite efforts to mitigate them by the use of female research assistants of similar age who were instructed and trained to normalize the questions and topics. Repeated questions about private, sensitive issues such as stillbirths could even elicit feelings of anger. The reasons given for underreporting sexual activity in this study were largely in line with findings from previous studies; namely shyness, embarrassment, social desirability, concerns around confidentiality and fear of penalties and social consequences , 9, 19. Throughout the FGDs and interviews it became apparent that sex-related questions which had been used in the follow-up interviews were perceived to lack any reference or association with love, trust, or intimate and stable relationships. Not surprisingly therefore, all the informants advised the researchers to introduce sexual activity questions in a context which assumed that they were performed with a boyfriend. Similar observations on the need for such contextualising have also been made by interviewed sex-workers in Malawi and South Africa who described their relationship with clients in terms which connoted premarital love and trust; not simply transactional relations , 21.Some participants highlighted that the use of the word ‘sex’ can affect how girls answer certain questions. This may be explained through other studies from Zambia which found concerns that the local terms related to sex emerged as more insulting than the English ones . This suggests that other ways of asking sex-related questions should be considered such as asking about ‘boyfriend’ instead of ‘sex partner’, ‘sleeping with’ instead of ‘having sex’, or ‘dating’ instead of ‘sexual relations’. Although the use of local euphemisms and culturally appropriate terminology may require additional preparation and adjustment of research tools, it can make the interview less awkward for both the researcher and the respondent. Alternative expressions such as ‘slept with’, not only distances the respondent from the act of sex but as found by Langhaug and colleagues (2010), it also avoids the embarrassment of hearing sexual terms being read out loud by the interviewer. Formatting of questions in a vague manner, however, contrast with typical survey design guidelines that recommend clear and unambiguous questions to avoid misunderstandings and to increase the validity of responses , 23.The RISE participants explained that the questions about sex and abortion made them feel that they were judged by the interviewer and suspected of engaging in socially unacceptable behaviour. They seemed most concerned about being associated with promiscuity and/or prostitution. This was also seen in a study of reporting of sexual behaviours amongst South African women, which found that 32% of respondents purposefully under- or overreported certain sensitive behaviors to avoid criticism or to seek praise . As poinThe participating girls stated that the school was the most preferred place for interviews. Having interviewers coming home to them created a risk of embarrassing situations with family members asking why they were talking to strangers about sexuality related issues. They also feared that being seen at the health clinic could cause negative rumours. These findings should be seen in relation to other studies in the region who have reported that adolescents rarely communicate with their parents about sexuality and most often seek to hide any sexual relations or interest in boys. Studies have also suggested that adolescents may avoid health clinics out of fear that fellow community members will suspect them of seeking contraception –30.Discussions around the audio computer assisted (ACASI) section of the interview seemed to confirm previous claims around the increased privacy and anonymity afforded by the technique. The use of ACASI was welcomed by some, but the participants did not believe that adolescent girls would necessarily be more truthful when left to respond to sensitive questions on their own. Some studies in low-income contexts have shown higher rates of self-reported sexual activity using ACASI , 31. Fordoes require in-depth details about adolescents’ sexual activities, careful piloting of alternative wording and combinations of data collection methods may increase the validity of the findings. Comparative studies suggest that culturally specific interactive interviews [Research on adolescent SRH may need more careful consideration of the value, relevance and wording of detailed questions about sexual behaviour. If a study terviews  and in-dterviews  yield hiA strength of this study is that we triangulated the responses from participants, teachers, and the RAs themselves. Their observations and suggestions were similar, thus giving credibility to the findings. Another strength was the use of two interviewers. One was a white, 37-year-old woman from Norway and the other interviewer was a Zambian 25-year-old female. The latter spoke the local languages the girls were comfortable with and her age was close to the participants. As for the former, her Scandinavian background with liberal and open views about SRHR issues may have enabled her to ask more direct questions than what a local person would feel comfortable doing, and it is possible that the presence of an outsider made it possible to talk about things that are normally regarded as too private. However, direct ways of questioning may be intimidating for participants who are less accustomed to talking about SRH. The identification of the researchers with the main study may have caused concern that any negative opinions about the RISE project would have implications for the support that it provided to the girl or the school, but explicit efforts were made to ensure the participants that they could be honest and that their responses would have no bearing on them or their participation in the RISE project.Two FGDs and interviews with RAs were not recorded, which may have resulted in some loss of detail or nuance although detailed notes were made as soon as this was discovered. In addition, the transcriber was not fully conversant with the language used in one of the FGDs and quotes may not have been completely accurate.Since we reached saturation, we believe the findings are transferable to the study population of the RISE project. Due to the special context of the study, the generalizability is uncertain but it is likely that the findings are relevant to other studies on sexual and reproductive health among adolescent girls in sub Saharan Africa.This study found that the female adolescent trial participants felt uncomfortable and embarrassed when asked questions about socially disapproved behaviour such as premarital sexual activity and pregnancy, despite repeated experiences of being asked the same questions. Strategies to avoid using overly direct language and descriptive words, asking questions within the context of a romantic relationship and a focus on establishing familiarity and trust can reduce reporting bias. Considering discrepancies in the research on ACASI, it is premature to make generalised claims about its suitability for research in low-income contexts. The findings of this and previous studies suggest that considerations must be given to the context and characteristics of the study population, as well as the time and resources available to familiarize the respondents with ACASI technology.


Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant tumor with an extremely poor prognosis in digestive tumors. Pyrroline-5-carboxylate reductase 1 (PYCR1) plays an important role in tumor development. Therefore, we aimed to explore the effect of PYCR1 on the growth of PDAC cells.Methods: Tumor tissues and adjacent normal pancreatic tissues were collected from 89 patients with PDAC. And immunohistochemistry (IHC) was used to analyze the expression level of PYCR1 in both. RNA interference was used to inhibit the expression of PYCR1 in PANC- 1 and AsPC-1 cells. After infection, the expression of PYCR1 protein was detected by Western blot. The proliferation and growth of PDAC cells were detected by Celigo analysis, MTT, and clone formation assay. Cell apoptosis was analyzed by flow cytometry. Furthermore, the effect of PYCR1 interference on tumor growth was evaluated in vivo through injecting tumor cells subcutaneously into nude mice.Results: The expression of PYCR1 in pancreatic cancer tissues was significantly higher than in paired adjacent normal pancreatic tissues (P <0.01). In vitro, the downregulation of PYCR1 expression significantly inhibited the cell proliferation and colony formation, and increased apoptosis in PANC-1 cells and AsPC-1 cells compared with the shCtrl group (P <0.01). And in vivo, PYCR1 interference also significantly inhibited tumor growth both in the tumor volume and weight.Conclusion: PYCR1 interference was able to inhibit cell proliferation and promote cell apoptosis of pancreatic cancer. The PYCR1 may serve as a potential therapeutic and prognostic biomarker for the treatment of pancreatic cancer. Pancreatic cancer is a highly malignant tumor with poor diagnosis in the digestive system. Pancreatic cancer is occult without specific symptoms, which means that many patients exhibit locally advanced disease or metastasis at the time of diagnosis Pyrroline-5-carboxylate reductase (PYCR) is mainly located in the mitochondria and plays an important role in the biosynthesis of proline As non-essential amino acids in the human body, proline has a unique secondary amine structure, and enzymes involved in the metabolism of proline play a key role in the control of tumor initiation and progression. According to previous reports, PYCR1, one of the most important enzymes in the process of proline metabolism, is closely associated with tumorigenesis and tumor progression, including prostate cancer, breast cancer, colon cancer, lung cancer, and liver cancer in vitro and in vivo.In this study, we aim to analyze the correlation between PYCR1 and PDAC. The expression level of PYCR1 was evaluated in pancreatic cancer. And we explored the biology behavior of PYCR1 both This study involved 89 tumor tissues and paired adjacent normal pancreatic tissues from patients who underwent curative surgical resection in Zhongshan Hospital, Fudan University from September 2012 to May 2016. Tissues were collected during surgery and frozen in liquid nitrogen. All specimens were pathologically confirmed to be ductal adenocarcinoma of the pancreas. And all the clinical pathological data and detailed follow-up data of the 89 patients were collected. Patients were followed up until May 1, 2020. Furthermore, the tissues were used for the analysis of immunohistochemical (IHC). This study was approved by the Ethics Committee of Zhongshan Hospital, Fudan University. All patients signed informed consent before the operation.All pancreatic tissues were fixed in 10% formalin and embedded in paraffin. The sections were dewaxed with xylene and dehydrated with ethanol. After deparaffinization, the sections were treated with 3% hydrogen peroxide for 10 minutes. For antigen retrieval, the sections were heated in sodium citrate buffer (pH 6.0) for 20 minutes. After that, sections were cooled to room temperature naturally and dipped in distilled water for 10 min. Sections were blocked with 10% fetal bovine serum (FBS) for 30minutes and incubated overnight with primary rabbit polyclonal anti-PYCR1 antibody . Then, the second antibody and Vulcan Fast Red Chromogen kit2 was added. Sections were stained with diaminobenzidine (DAB) and were counterstained with hematoxylin. The results were observed under an optical microscope after dehydration and sealing.The brown staining in the cytoplasm indicated positive staining for PYCR1. The tissues were evaluated by two different pathologists. The expression of PYCR 1 staining was scored according to the following criteria independently.(1) The percentage of positive staining was scored as follows: 0, 0-5% positively stained cells; 1, 6-20% positively stained cells; 2, 21-50% positively stained cells; 3, 51-100% positively stained cells.(2) The intensity of staining was scored as follows: 0, negative; 1, weakly positive; 2, moderately positive; 3, strongly positivity.(3) The histochemistry score  was calculated by multiplying the percentage of positive staining score with the staining intensity score. Then, the tissues were divided into two groups: low expression (score≤3) and high expression (score >3).2.The pancreatic cancer cell lines used in this study include SW1990, PANC-1, AsPC-1, BxPc-3, which were purchased from the Chinese Academy of Sciences Cell Bank . These cells were cultured in DMEM media containing 10% FBS at 37 °C with 5% COSmall-hairpin RNA (shRNA) interfering sequences targeting PYCR1 (shPYCR1: 5'-CAGTTTCTGCTCTCAGGAA-3') and negative shRNA sequences (shCtrl: 5'-TTCTCCGAACGTGTCACGT-3') were designed and synthesized. After that, the single-stranded DNA oligo containing the interference sequence was synthesized and converted to double-stranded DNA by annealing. And GV115 vector  was linearized with Age I and EcoR I restriction enzymes. After digestion, the double-stranded DNA oligo was ligated to the GV115 vector using T4 DNA Ligase (Fermentas) according to the manufacturer's instructions. Then, the products were transformed into the E. coli TOP10 strain . The positive clones were identified and selected by sequencing. According to the manufacturer's instructions of EndoFree Maxi Plasmid Kit , the plasmid was extracted from the previous positive clones for further lentiviral packaging.The AsPC-1 and PANC-1 cells were seeded in a 6-well plate at a density of 2×10^5 per well and infected with recombinant PYCR1 (shPYCR1) or negative control (shCtrl) lentivirus separately (MOI of 20). At 72 hours post infection, the fluorescence signals of green fluorescent protein (GFP) were observed with a fluorescence microscope . Only cells, in a well-growing state and positive for GFP fluorescence over 70%, were collected for further detection. And fluorescence-activated cell sorting  was used to sort for GFP-positive cells.For the examination of PYCR1 expression, total RNA was extracted from the cell lines using TRIzol reagent . And total RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase kit (Promega). The RT-qPCR was performed using SYBR® Premix Ex Taq™  in a volume of 12μL on LightCycler 480 II Real-Time PCR System (Roche). PCR cycling conditions were as follows: 95 °C for 30s, 40 cycles at 95 °C for 5 s, 60 °C for 30s. GAPDH was used as an internal control to calculate the relative expression levels of genes. The relative mRNA expression levels were calculated by the 2-ΔΔCT method. All samples were assayed in triplicate.The primers used in this study were listed as follows.PYCR1-forward (F): 5'-GGCTGCCCACAAGATAATGGC-3';PYCR1-reverse (R): 5'-CAATGGAGCTGATGGTGACGC-3';shPYCR1-F: 5'-TTGGCTGCCCACAAGATAAT-3';shPYCR1-R: 5'-ATCACTGTGCTGCACCGTCT-3';GAPDH-F: 5'-TGACTTCAACAGCGACACCCA-3';GAPDH-R: 5'-CACCCTGTTGCTGTAGCCAAA-3'.The AsPC-1 and PANC-1 cells infected by shPYCR1 or shCtrl lentivirus were seeded in a 96-well plate in triplicate at a density of 1.5×10^3 per well. From the second day after seeding, the cells with GFP fluorescence were counted by the Celigo Imaging Cytometer  for 5 days. And the cell proliferation assay is performed at specific times.Further, MTT assays were performed to examine the effect of PYCR1 on cell viability and proliferation. MTT assay began from the second day after seeding. MTT solution  was added to each well and incubated for 4 hours at 37 °C. After that, the MTT solution was removed. 100 μl dimethyl sulfoxide (DMSO) was added to dissolve the formed formazan crystals. The absorbance at 490 nm was measured by a microplate reader . Three independent sets of experiments were performed at least.Cell apoptosis assay was performed by the Annexin V-APC Apoptosis Detection Kit  following the manufacturer's instructions. The PANC-1 and AsPC-1 cells infected by shPYCR1 or shCtrl lentivirus were seeded in 6-well culture plates and cultured for 5 days. The adherent cells were digested with trypsin, washed with PBS, and centrifuged. The cell suspension was mixed with Annexin V-APC and incubated for 10 minutes protected from light. The data were collected and analyzed using a flow cytometer with CellQuest software . Three independent sets of experiments were performed at least.The colony formation assay was performed to assess the effect of PYCR1 on cell survival. The cells infected by the lentivirus were cultured for 5 days and then digested into cell suspension with trypsin. After digestion, the cells were seeded in 6-well culture plates at a density of 2,000 per well. After cultured for 14 days, the cells were fixed with 4% paraformaldehyde and stained with Giemsa solution . The colonies were counted on a fluorescence microscope. Three independent sets of experiments were performed at least.The infected cells were collected, washed twice with PBS, and lysed in lysis buffer. After lysed on ice for 15 min, the cells were broken by sonication. Then, the samples were centrifuged at 10000 g for 15 min at 4 °C to collect the supernatant. The protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit . The proteins were separated by 10% SDS-polyacrylamide gel and electro-transferred onto a polyvinylidene fluoride (PVDF) membrane . The membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature and incubated with primary antibody overnight at 4 °C. And then the samples were incubated with the respective second antibody  for 1.5 hours. The protein signal was visualized using the enhanced chemiluminescence kit . GAPDH was used as an internal control.The antibodies used in this study were as follows: Anti-PYCR1 , Anti-GAPDH , Anti-Mouse IgG , Anti-Rabbit IgG .in vivo. All mice were randomly divided into two groups, normal control group  and knockdown group . AsPC-1 cells (5×10^6 cells) infected with shPYCR1 or shCtrl lentivirus were subcutaneously inoculated into the right flank of the nude mice. Data collection started 1 week after the injection. The mice were weighed and tumor volumes were measured twice per week. The tumor volume was calculated by the formula: Tumor volume = π/6 × Length × Width × Width. After 28 days, all mice were euthanized, and the tumors were removed and weighed. All experimental protocols involving animal experiments were approved by the Ethics Committee of Zhongshan Hospital, Fudan University.Twenty Balb/c nude mice  were purchased from Shanghai Lingchang Biotechnology to assess the effect of PYCR1 In this study, all statistical analyses were performed by the SPSS software version 26.0  and the GraphPad Prism software version 8.0 . The comparison between the means of two groups was analyzed by the F-test to test the homogeneity of variance, followed by paired-samples t-test or Student's t-test when appropriate. A Pearson's chi-square test, Fisher's exact test were used to compare the proportions. Survival analyses were performed by the Kaplan-Meier survival analysis and the overall survival (OS) was calculated from the time of diagnosis until tumor-related death. The log-rank test was used to compare the survival curves. Univariable and multivariable Cox regression analysis were used to identify independent factors.Risk factors are expressed as the hazard ratio . p < 0.05 was considered statistically significant . All experiments were conducted three times and the error bars indicate the standard deviation from the mean of triplicate measurements.To evaluate PYCR1 protein expression in PDAC patients, immunohistochemical staining was performed to measure the expression of PYCR1 in 89 paraffin-embedded PDCA tissues and paired normal adjacent tissues. Our results revealed that PYCR1 expression was higher in tumor tissues compared with that in the adjacent normal tissues . Relative abundances are shown as average ΔCt for low abundance (ΔCt ≥ 16), moderate abundance (12 < ΔCt < 16), and high abundance (ΔCt ≤ 12). The result showed that the mRNA expression abundance of PYCR1 was high in all pancreatic cell lines  and low expression group (n=35) according to the IHC scores of PYCR1 expression. The representative IHC staining of PYCR1 in PDAC tissues and normal pancreatic tissues is shown in Figure And the Kaplan-Meier survival curve was plotted to evaluate the prognostic value of PYCR1. The results revealed that high expression of PYCR1 had an important effect on the OS of PDAC patients. Patients with high PYCR1 levels had significantly longer OS than the low counterparts (Low expression vs High expression = 45.74±5.47 months vs 27.90±3.82 months, P=0.013; Fig. in vitro, we treated the PANC-1 and AsPC-1 cells with shCtrl or shPYCR1 lentivirus. Then the level of mRNA and protein was assayed for PYCR1 using RT-qPCR and Western blot 3 days after the lentivirus infection. Compared with those in the shCtrl group, the mRNA expression of PYCR1 in the shPYCR1 group was significantly inhibited (AsPC-1, 92.6% knockdown, shCtrl vs shPYCR1 = 1.003±0.101 vs 0.074±0.005, P=0.004; PANC-1, 78.6% knockdown, shCtrl vs shPYCR1 = 1.020±0.254 vs 0.214±0.043, P=0.006; Fig. Before proceeding to explore the effect of PYCR1 After infection, the proliferation of PDAC cells was analyzed by Celigo and MTT assays. The results were plotted into line graphs and the results of first day were used as controls. The results showed that the inhibition of cell growth and proliferation was significant in the shPYCR1 group, respectively. (Celigo/Cell count/Day5: AsPC-1, shCtrl vs shPYCR1 = 2757±157 vs 1467±68, P<0.001; PANC-1, shCtrl vs shPYCR1 = 8183±397 vs 1661±162, P<0.001; Fig. in vivo. The AsPC-1 cells were treated with shPYCR1 or shCtrl lentivirus. The average tumor volume in the NC group was significantly larger than that in the KD group (NC vs KD = 748.07±104.83 mm3 vs 578.25±98.43 mm3, P=0.002; Fig. In the final part of the study, the AsPC-1 cells were injected subcutaneously into nude mice to explore the effect of PYCR1 Glutamine is an important energy source for cancer cells and the metabolic disorders of glutamine are closely related to tumorigenesis and cancer progression. Glutamine converses into glutamate in the first step of glutamine metabolism. In particular, glutamate, a key role both in glutamine metabolism and tumorigenesis, is one of the most significant sources for the synthesis of proline in vitro and in vivo. Celigo assay, MTT assay, and colony formation assay showed that the pancreatic cells infected by lentivirus grew slowly. And the results of the flow cytometry showed the percentage of apoptotic cells was decreased in the infected cells. Additionally, PYCR1 interference significantly suppressed tumor growth in nude mice. Taken together, all data demonstrated that PYCR1 plays an essential role in tumorigenesis and progression of PDAC cells.The study found that the expression of PYCR1 in 89 tumor tissues was significantly higher than that in the paired adjacent normal tissues. And the further analysis confirmed that the overexpression of PYCR1 may be associated with pancreatic cancer occurrence and progression. The results showed that the median OS of the high PYCR1 expression group was only 27.9 months, while the median OS of the low PYCR1 expression group was 45.7 months. Multivariate Cox regression analysis in 89 patients revealed that the high PYCR1 expression level was an independent prognostic factor predicting worse OS for PDAC patients. Based on the above study, we hypothesized that there may be a close relationship between PDAC tumorigenesis, progression, and PYCR1 expression. To confirm our discovery, we further investigated the role of PYCR1 both These results corroborate the findings of a great deal of the previous work on the effect of PYCR1 on tumors. A previous study, which collected about 2500 cases from 13 independent microarray datasets, found that PYCR1 was overexpressed in breast cancer. The PYCR1 interference significantly suppressed the breast cancer cell growth and invasion by regulating AKT/ERK signaling pathway. And it enhanced the cytotoxicity of doxorubicin in breast cancer cell lines In summary, according to our preliminary results of the relationship between PYCR1 and PDAC, PYCR1 interference leads to inhibition of proliferation and promotion of apoptosis. It will provide a potential biomarker of diagnosis, prognosis, and therapy for pancreatic cancer. However, more detailed studies are needed to confirm the mechanism of PYCR1 on pancreatic cancer. This remains a goal of our further research.


CD42a was lower in IPF subjects than in controls (p = 0.0153), while CD209, Cd133/1, MCSP, and ROR1 were higher in IPF patients than in controls . Kaplan-Meier survival analysis for IPF patients: for median values and a cut-off of 0.48 for CD25, the two subgroups showed a significant difference in survival rate ; the same was true for CD8 . Conclusion: Our multicenter study showed for the first time the expression of surface epitopes on EVs from IPF patients, providing interesting data on the communication signatures/exosomal profile in serum from IPF patients and new insights into the pathogenesis of the disease and a promising reliability in predicting mid-term survival of IPF patients.Background: Extracellular vesicles (EVs) are secreted by cells from their membrane within circulation and body fluids. Knowledge of the involvement of EVs in pathogenesis of lung diseases is increasing. The present study aimed to evaluate the expression of exosomal surface epitopes in a cohort of idiopathic pulmonary fibrosis (IPF) patients followed in two Italian Referral Centres for Interstitial Lung Diseases, comparing them with a group of healthy volunteers. Materials and Methods: Ninety IPF patients (median age and interquartile range (IQR) 71 (66–75) years; 69 males) were selected retrospectively. Blood samples were obtained from patients before starting antifibrotic therapy. A MACSPlex Exosome Kit, human, , to detect 37 exosomal surface epitopes, was used. Results: CD19, CD69, CD8, and CD86 were significantly higher in IPF patients than in controls ( Extracellular vesicles (EVs) are secreted by the cell membrane into the circulation and body fluids. They contain cytoplasmic components, such as proteins and miRNA. Since EVs express surface proteins linked to their origin, they can adhere and fuse with circulating or distant resident cells. This capturing property of distant cells enables EVs to shuttle functional nucleotides or proteins, providing horizontal transfer of a variety of biological molecules . EVs incIn respiratory medicine, there is increasing evidence of the involvement of EVs in the pathogenesis of lung diseases including lung cancer, chronic obstructive pulmonary disease, and pulmonary fibrosis ,5,6.In the field of lung cancer, studies on EVs are extensively growing, especially about the potential of EVs as diagnostic biomarkers of lung cancer ,7. ExosoA few reports have investigated the role of miRNA in a murine bleomycin-induced lung fibrosis model and in IPF patients. Their results suggest that miRNAs could be useful prognostic indicators of disease .Unlike conventional biomarkers of IPF, such as Krebs von den Lungen-6 (KL-6) ,11,12,13The aim of the present study was to compare the expression of exosomal surface epitopes in a cohort of IPF patients monitored in two Italian referral centres for interstitial lung diseases (ILDs) and in a control group of healthy volunteers.Ninety IPF patients (median age and interquartile range (IQR) 71 (66–75) years; 69 males), monitored at the Siena Regional Referral Centre for Interstitial Lung Diseases and Foggia Regional Referral Centre for Rare Pulmonary Diseases, were selected retrospectively. Patients with clinical evidence of concomitant infections, malignancies, and/or acute exacerbations of the disease were excluded, as well as IPF patients with a radiological pattern of indeterminate usual interstitial pneumonia (UIP). The diagnosis of IPF was confirmed by multidisciplinary discussion according to international guidelines . Only fiBlood samples were obtained from patients before starting antifibrotic therapy ,17. LungDemographic and clinical data, comorbidities, family history, lung function parameters, including diffusion capacity of carbon monoxide (DLCO), and radiological features were obtained from the medical records and entered in an electronic database for statistical analysis.The description of the study design with a flow chart is reported in Healthy controls and patients gave their written informed consent to participate in the study, which was approved by our local ethics committee .g for 10 min. Samples were then frozen at −80 °C.Serum samples were processed as follows: 6 mL blood samples from IPF patients and healthy volunteers were isolated in serum-separating tubes. The blood was spun at 2500× g for 30 min and 10,000× g for 45 min at 4 °C. The pellets were resuspended and filtered through a 0.22 μm membrane. EVs were isolated by ultracentrifuging the supernatant at 100,000× g for 2 h at 4 °C. The EVs were resuspended in 100 μL PBS and stored at −80 °C.According to the manufacturer’s instructions , serum wThe exosome protein concentration was measured by the Bradford protein assay . Equivalent amounts of protein (80 µg) from exosomes were separated by SDS-polyacrylamide 12% gel and transferred to PVDF (Biorad). The membrane was blocked with 5% milk in Tris-buffered saline with 0.1% Tween-20 and incubated overnight at 4 °C with primary antibodies: CD81 , Alix , and β-actin  in blocking solution. After three washes with Tris-buffered saline/0.1% Tween-20, the membrane was incubated for 1 h with HRP-conjugated secondary antibodies. Immunoreactive bands were detected with ECL substrate .The Bradford protein assay was used to measure protein concentrations in BAL fluid samples, as previously reported ,21,22. Tg for 5 min. The supernatant was aspirated and 5 μL MACSPlex Exosome Detection Reagent CD9, CD63, and CD81 were added to each tube followed by incubation for 1 h at room temperature protected from light in an orbital shaker (450 rpm). The washing step was repeated, and 500 μL MACSPlex Buffer was added to each tube followed by incubation for 15 min at room temperature protected from light in an orbital shaker (450 rpm). EV-containing samples were centrifuged and the supernatant aspirated, leaving about 150 μL in the tube. Flow cytometric analysis was carried out on a Facs CantoII flow cytometer followed by Kaluza Analysis 2.1  , which detects 37 exosomal surface epitopes plus two isotype controls . After iCA, USA) a,b. For p value less than 0.05 was considered statistically significant. Statistical analysis was performed by GraphPad Prism 9 software.The results were expressed as means and standard deviations (SD) or medians and quartiles (25th and 75th percentiles) for continuous variables, as appropriate. One-way ANOVA non-parametric test  and Dunn test were performed for multiple comparisons. The Chi-squared test was used for categorical variables. The Kaplan-Meier estimator test was used for survival analysis, and the hazard ratio (Mantel-Haenszel) was calculated. A Clinical, demographic, and immunological data are reported in The purity of serum-derived extracellular vesicle preparations was evaluated by western blot analysis, testing for ALIX and CD81 in three random samples . EVs arep = 0.0023, p = 0.0471, p = 0.0082, and p = 0.0143, respectively) (p = 0.0153), while CD209, Cd133/1, MCSP, and ROR1 were higher in IPF patients than in controls   c. CD42a ctively) c.p = 0.0243, hazard ratio: 0.52 (95%CI 0.29–0.92)  (29–0.92) a; the sa75–2.53) b.The present multicentre study is the first to examine the expression of 37 exosomal surface epitopes in IPF patients. To our knowledge, no data are currently available in the literature on protein expression in EVs from serum of IPF patients. Only one study has investigated miRNA expression and did so in a murine bleomycin-induced lung fibrosis model and in IPF patients: in the murine model, miR-21-5p in EVs was found to be significantly upregulated in acute inflammatory phase and the later fibrotic phase. This increase in miR-21-5p expression in circulating EVs was also observed in IPF patients and showed a significant correlation with disease progression .Our study identified increased expression of many exosomal surface epitopes  in IPF patients with respect to controls, suggesting that an alteration of these markers may be associated with lung fibrosis. From a mechanistic point of view, overexpression of these epitopes in our population is probably related to adaptive immune system dysregulation, which has been described in IPF patients ,25,26,27CD69 is a human transmembrane C-type lectin, initially detected on the surface of activated lymphocytes and known as a very early lymphocyte activation marker antigen . It has Unfortunately, no data are available on NK-CD69-expressing cells in interstitial lung diseases, although increased NK (CD56+) cells were reported in peripheral blood from IPF patients by Esposito et al. . InteresOur study showed high CD42a (known as platelet glycoprotein IX (GP9)) expression in EVs from IPF patients. CD42a is expressed on platelets and megakaryocytes ,41. TranCD209 (known as dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)) was abundantly expressed in our IPF patients. CD209 is a C-type lectin receptor present on the surface of macrophages  and dendOn the other hand, CD133/1 , is a recognized marker of hematopoietic stem cells and committed progenitors  and is aNo data are available in the literature on MCSP  or chondroitin sulphate proteoglycan 4 (CSPG4)) in pulmonary fibrosis. MCSP is a surface type I transmembrane core proteoglycan that is crucially involved in cell survival, migration, and angiogenesis ,59. HoweThe last marker abundantly expressed in EVs of our IPF patients was ROR1 (receptor tyrosine kinase like orphan receptor (1)). This is not surprising since ROR1 and ROR2 are key mediators of WNT5a signalling . WNT5a, Mesenchymal stem-cell-derived EVs have a strong therapeutic effect in cases of organ damage, including acute lung injury . Recent This is a novel and intriguing research study contributing to the definition of EVs’ potential role in the pathogenesis of IPF. Although preliminary, the results of this study were validated in two different cohorts of IPF patients recruited from two Italian Regional Referral Centres for ILD. The main limitations of study include the retrospective design of the research, the lack of data from EVs in relation to IPF treatments and the need to compare our results in different biological fluids.In conclusion, our study shows the expression of surface epitopes on EVs from IPF patients for the first time. It provides interesting data on their communication signatures/exosomal profile in serum from IPF patients, offering new insights into the pathogenesis of the disease. Some of these epitopes proved to reliably predict mid-term survival of IPF patients treated with antifibrotic drugs, suggesting their possible use as prognostic indicators in the management of these patients.


However, the viability of using μEMA to capture behaviors and states in a large-scale longitudinal study has not yet been demonstrated.Ecological momentary assessment (EMA) uses mobile technology to enable in situ self-report data collection on behaviors and states. In a typical EMA study, participants are prompted several times a day to answer sets of multiple-choice questions. Although the repeated nature of EMA reduces recall bias, it may induce participation burden. There is a need to explore complementary approaches to collecting in situ self-report data that are less burdensome yet provide comprehensive information on an individual’s behaviors and states. A new approach, microinteraction EMA (μEMA), restricts EMA items to This paper describes the μEMA protocol currently used in the Temporal Influences on Movement & Exercise (TIME) Study conducted with young adults, the interface of the μEMA app used to gather self-report responses on a smartwatch, qualitative feedback from participants after a pilot study of the μEMA app, changes made to the main TIME Study μEMA protocol and app based on the pilot feedback, and preliminary μEMA results from a subset of active participants in the TIME Study.The TIME Study involves data collection on behaviors and states from 246 individuals; measurements include passive sensing from a smartwatch and smartphone and intensive smartphone-based hourly EMA, with 4-day EMA bursts every 2 weeks. Every day, participants also answer a nightly EMA survey. On non–EMA burst days, participants answer μEMA questions on the smartwatch, assessing momentary states such as physical activity, sedentary behavior, and affect. At the end of the study, participants describe their experience with EMA and μEMA in a semistructured interview. A pilot study was used to test and refine the μEMA protocol before the main study.single-question selection method and smartwatch vibrotactile prompting. We also added sensor-triggered questions for physical activity and sedentary behavior. As of June 2021, a total of 81 participants had completed at least 6 months of data collection in the main study. For 662,397 μEMA questions delivered, the compliance rate was 67.6% (SD 24.4%) and the completion rate was 79% (SD 22.2%).Changes made to the μEMA study protocol based on pilot feedback included adjusting the temporally dense intensive longitudinal self-report data in a sustainable manner. Data suggest that μEMA may be valuable for understanding behaviors and states at the individual level, thus possibly supporting future longitudinal interventions that require within-day, temporally dense self-report data as people go about their lives.The TIME Study provides opportunities to explore a novel approach for collecting  Mobile technologies create new opportunities to design personalized health interventions in 2 broad ways. First, mobile technologies such as smartphones and smartwatches can be used to improve the assessment of behavioral, psychological, and contextual states, reducing reliance on known-to-be-problematic retrospective recall surveys that may poorly capture within-day variations -3. Seconwe currently need self-reportinput to measure many subjective experiences  that play an important role in predicting health behavior changes  there is a survey, I answer the question and I am done...The watch questions are actually less burdensome than the phone questions. Watch questions are pretty convenient because you can just touch [tap] on the watch and you’re done basically.F, aged 24 yearsHowever, several participants felt that the μEMA question did not stay available for long enough. A participant stated as follows:I like how it was really simple, it wasn’t like a series of questions. They were just “yes” or “no.” What made it difficult was the timing...it seems like it only lasts for five seconds and it disappears. Sometimes I may be doing something like resting and then survey comes up. Then I have to stick my arm out of the blanket to answer but then it goes away before I could.F, aged 22 years.” Although the smartwatch vibration creates a quiet buzzing sound when on the arm, if the smartwatch is sitting on a surface , the resulting surface vibration can make a surprisingly loud sound that is difficult to ignore.Initially, the prompt only remained active for 15 seconds, which we later updated during the pilot to 20 seconds. Participants generally noticed the smartwatch vibration on their wrists with a delay, despite a relatively aggressive vibration pattern. In fact, pilot participants described the μEMA vibration pattern  as “too loudParticipants reported difficulty answering μEMA questions when both hands were occupied, especially while driving. However, they were instructed to only answer μEMA questions when it was safe to do so, and they were given the specific example of driving as a situation in which they should ignore the prompts. Participants also reported difficulty answering μEMA questions while doing other cognitively demanding activities such as writing. In fact, a participant mentioned missing prompts while being in an examination room, stating as follows:Sometimes if I was taking a quiz or test in class, I didn’t want to be caught using my watch, so I didn’t touch  it.M, aged 19 yearsParticipants found the ability to undo their responses useful, especially when there were accidental taps on the smartwatch. A participant stated as follows:Yes [Undo] was useful because sometimes on the watch...maybe because of the clothes I’m wearing...but [if] an option would be chosen so I’d be able to go “undo” and choose the right answer.F, aged 24 yearsSort of answer option helpful in instances when they were not sure of their experience at the moment and would have had difficulty entering a more limited Yes or No response. A participant stated as follows:Participants also found the Sort of” made me think about my answer a little more rather than an outright yes or no, which is how I kind of determine how I’m going to answer yes or no...if you’re feeling such and such, [then] I do like the “sort of” option because you’re not feeling fully something. And you are not “not” feeling it completely either. So [“Sort of”] is a pretty easy option for people who are feeling like in the middle. “middle answer such as sort of.In response to such observations with the goal of achieving cognitive simplicity to support microinteraction, we designed all the questions to have a In the pilot study, each question presented was randomly sampled from the pool of questions. This resulted in questions either being overrepresented or repeated too consecutively for some individuals, adding monotony, which some participants noted. A participant stated as follows:I think there were questions based on time or place. Questions like how was your day?...It would ask multiple times a day. I don’t know if it was intentional, but it feels like I’m answering the same question over and over again. Same thing for places. It would ask me multiple times “is this your home?” or something like this.M, aged 18 yearsIn the pilot study, each μEMA prompt selected a question at random. As a result, the app had less control over preventing overrepresentation of questions within the day. On the basis of the interviews, we learned that participants found this repetitiveness of the questions monotonous. Thus, for the main study we developed a new question-selection algorithm that (1) guarantees that a given question is never asked consecutively and (2) ensures that a question is not answered more than a predetermined number of times.only when it was contextually relevant.In the pilot study, we randomly sampled questions on PA, SB, and the participant’s current location. However, these questions also felt repetitive to the pilot participants. Thus, in the main study protocol, these questions were asked when the passive sensors (using accelerometer and GPS) detected a relevant event, gathering information validation questions to check whether the participants are paying attention to the μEMA prompts. Second, we added questions related to the slowly changing characteristics of the participants; these questions add variety to the question pool while also gathering useful information. Finally, on the μEMA undo screen, we added more variety in the thank-you messages that participants receive after answering a μEMA prompt. As the main study would last for 12 months per participant, we deemed reducing burden and increasing engagement to be important considerations.We added 2 additional types of questions for μEMA. First, we included We increased the time duration that a μEMA question stays visible on the smartwatch face from 15 seconds to 20 seconds. To address participant concerns about the loud vibration, we changed the pattern from an 11-second-long vibration to a 6-second pattern of 2-second vibrations separated by 1-second pauses.The protocol described here is based on changes made to the μEMA app after the pilot study feedback.The TIME Study uses a nested design with multiple measurement bursts of smartphone-based EMA across a 12-month study period . Each meHere we describe the self-report measures, question-selection algorithms, and simulation results to validate the filtering algorithm to select questions for μEMA.Core-construct questions measure different behaviors, momentary states, and contexts that cannot be objectively captured using sensors and are expected to change throughout the day. The questions are selected to capture reflective processes, reactive processes, and internal and external processes that can predict or explain behavior adoption and maintenance . Examplesurprising participants with approximately 4 novel questions a day. These questions follow the wording requirements of other μEMA questions . The system uses a question bank of >280 questions so that they will not repeat for approximately 3½ months (assuming 60 μEMA prompts per day).In addition to the core-construct questions, 8% (5/62) of the scheduled prompts during waking hours are used to present questions about person-level characteristics. These are questions that gather data on attributes such as whether a participant owns a car, cares for a pet, self-describes as a morning person, or has other specific personality characteristics. The questions serve two purposes: gathering information that changes relatively slowly and breaking monotony  by 1 + 3 = 5?—Yes or No), trivial trivia questions (Sun rises in the east?—Yes or No), intentionally silly questions (Do pigs fly?—Yes or No), and simple attention-assessment questions . To break the question monotony and enhance engagement, validation questions rarely repeat. Our item bank has >300 validation questions for an estimated 7½ months of question prompting without repetition.The remaining 2% (1/62) of the waking-day prompts are used to present validation questions designed to assess whether participants are paying attention to the μEMA questions and answering them carefully . The validation questions are like other μEMA questions—cognitively simple single questions with 2-3 answers. However, these questions always have an unambiguously correct answer to verify response accuracy. Validation questions include simple math problems . In real time, we compute the area under the curve (AUC) of the high-passed raw accelerometer signal for 10-second epochs . The AUConly when a specific sensor event of interest occurs that suggests contextual relevance. The questions are Physically active [Δ] min ago? and Sedentary [Δ] min ago?, with answer options Yes, Sort of, and No, where Δ is the time difference from the current time to the end of the activity plus 25% of the activity bout length . This additional 25% of bout length is used to ensure that the question is asked at approximately the moment in time when the behavior was likely taking place but not at the behavioral transition.Sensor-triggered questions are prompted At each scheduled μEMA prompt time, if there is an eligible sensor-triggered question, the system will trigger the question only 50% of the time. This is done to avoid continuous and monotonous sensor-triggered prompting for prolonged sedentary and physical activities. When the sensor-triggered questions are eligible to be prompted, they take priority over the core-construct, validation, and engagement questions. For instance, if the next scheduled prompt has a core-construct question queued but the app has identified a PA event, then 50% of the time, that core-construct question is replaced by the PA confirmation question. If both PA and SB are identified, the question about the most recent event is presented.A way to gather self-report data on a comprehensive set of behaviors using μEMA is to randomly select a question to ask the user—as was done in our pilot study. However, it may be useful to measure internal factors  more frequently within a day rather than external factors . The app uses a strategic sampling algorithm to maximize the value of data collected about some constructs by limiting presentation of some questions, ensure that questions differ within an hour, and guarantee that each day includes questions from each of four broad construct categories .Each μEMA day, from our bank of 30 μEMA questions of core constructs, the app selects 4 (13%) questions related to internal factors, 2 (7%) to external factors, 2 (7%) to reflective processes, and 2 (7%) to reactive processes. More internal factors than other categories are selected because they are expected to vary throughout the day . Rather Feeling stressed?) than questions that assess more stable constructs . If the number of answers received for a question reaches the question’s limit, that question is no longer presented that day.Each μEMA question is assigned a maximum number of presentations per day . This enFeeling stressed? is answered, it is not asked again for at least one hour. However, if the question is not answered, it could be presented at the next prompt, within the hour.Once a particular μEMA question has been answered, it will not be presented again until at least 60 minutes have elapsed. For instance, if the question μEMA_prompting_day, line 1), the app first generates a day-level subset  of 10 questions from the pool of 30 μEMA questions . Next, at each μEMA prompt, day-level questions that have reached their limit  or have been asked recently  are filtered. From the remaining list, a question is selected at random . If the question prompted is answered, the question’s prompt count is updated , as is the last-answered time . If the question is not answered, then it is available for selection in the next scheduled μEMA prompt.The app uses the following algorithm to select μEMA questions . For eacAlgorithm used to select μEMA questionsFor each μEMA_prompting_day:day_level_subset=subset For each μEMA prompt:filtered_questions=remove_maxed_out_questions (day_level_subset)filtered_questions=remove_questions_answered_within_one_hour (filtered_questions)question_to_ask=select_question_at_random (filtered_questions)if question_to_ask is answered:update_prompt_count (question_to_ask)update_answer_time (question_to_ask)To verify our filtering algorithm , we simuWe simulated question selection for 365 days for 300 users, where 261 days were reserved for μEMA prompting, resulting in 78,300 total μEMA days. We assumed 8 hours of sleep and 16 hours of wake time per day, resulting in 15½ hours available for μEMA prompting. Targeting 4 prompts an hour results in 62 μEMA prompting opportunities. Of these, 90% (55/62) were available for core constructs, resulting in 55 slots for core-construct questions each day. We assumed that 75% (46/62) of the scheduled prompts would be answered .Compared with random sampling , the filParticipants were eligible for the TIME Study main trial if they (1) owned an Android smartphone running Android version ≥6.0 as their only personal mobile phone with no intention to switch to a non-Android smartphone, (2) did not wear a smartwatch already, (3) were aged 18-29 years and living in the United States, (4) were currently engaged in recommended levels of PA (or intended to within the next 12 months) , (5) spoBecause of COVID-19 restrictions on in-person recruitment for human subjects research, all participant recruitment and onboarding was conducted remotely. After screening, research staff individually met with each participant remotely (through Zoom) to obtain informed consent. Staff then guided the participants through the TIME app installation on their personal smartphones. Researchers then observed EMA burst compliance for the first 4-day EMA burst  during the next 2 weeks. If the compliance for the run-in period was <8 surveys per day on all days, the participants were withdrawn from the study. Otherwise, they could continue in the study and were mailed a smartwatch (Fossil Gen 4 or Gen 5 model). Once the participants received the smartwatch, a staff member scheduled a second remote orientation session with the participant to guide them through the TIME smartwatch setup. Expectations for smartwatch wear (including wearing it during sleep), charging it, and answering μEMA questions were explained. Participants were instructed that to remain compliant they should wear the smartwatch for 23 hours a day and charge it every day. They were also instructed to wear the smartwatch while sleeping. For μEMA prompting, participants were instructed as follows:“You will be prompted on the smartwatch with single questions of the type Are you walking right now? Yes | Sort of | No. These questions will take only 2-4 seconds to respond to—just like checking time on the smartwatch. There may be up to six such question prompts in an hour, on average, and the frequency of question prompts may vary at different times of your waking day. All the smartwatch questions will be outside of the 4-day smartphone survey burst periods. Each watch prompt will wait for up to 20 seconds for you to respond; if the question is not answered within 20 seconds, the question may disappear from the watch. Unlike phone surveys, watch questions are not re-prompted.”After the smartwatch orientation, participants could use the smartwatch as they deemed fit , while answering μEMA questions when they appeared. Participants receive US $20 per month for wearing the smartwatch for 23 hours on at least 24 days of the month. Participants could earn up to an additional US $80 per month for EMA burst compliance . In addition, if at the end of the study, participants have >50% compliance with μEMA, they were able to keep the smartwatch as their personal device. However, no monthly or regular compensation was provided for μEMA compliance. Participants do not receive feedback on their μEMA compliance, and their withdrawal from the study does not depend on μEMA compliance.We measure compliance rate, completion rate, undo rate, and validation rate to characterize participant response behavior when answering μEMA questions.Compliance rate is measured as the percentage of μEMA questions answered out of all the scheduled questions, including those not prompted because of the device being turned off or in DND mode.Completion rate is measured as the percentage of μEMA questions answered out of all the delivered questions, excluding those not prompted because of the device being turned off or in DND mode.Undo? option was selected after providing an initial answer.Undo rate is measured as the total number of μEMA questions answered when the Validation rate is measured as the percentage of μEMA validation questions answered correctly out of all the validation questions answered.Overall, the compliance rate (n=81 at ≥6 months) for μEMA was 67.7% (SD 13.7%) and the completion rate was 80.23% (SD 13.3%). The μEMA validation rate for the participants (n=81) was 92.9% (SD 23.7%)—indicating that they were paying attention to the μEMA question content and not carelessly tapping to dismiss them.Table 4 presents a summary of the response behavior. We also verified the filter-based question-selection algorithm’s performance against the real-world data from 2% (2/81) of the study participants who completed 6 months in the study . We founOur work with μEMA demonstrates that the technique may enable temporally dense ILD collection with manageable burden to support longitudinal studies or interventions, although some limitations of this work will require further investigation.Traditional mobile phone–based EMA can lead to interruption and response burden. To prevent this burden, researchers often compromise on the temporal density of prompts  and reduce the number of questions or constructs being measured. Complementary forms of EMA are needed where both the researchers’ needs for comprehensive understanding of health behaviors and users’ concern for burden are taken into account. μEMA provides such an opportunity, where the quick microinteractions not only make it less burdensome for the users to answer questions on the smartwatch, but also enable data gathering at a higher temporal density.The TIME μEMA substudy combines smartphone-based EMA with low-burden μEMA on a smartwatch to gather real-time self-report data from naturalistic settings using the personal smartphones of participants. Participants are encouraged to use their smartphone and the loaned smartwatch normally with only a few limitations . Participants are incentivized to wear the smartwatch, but they can pause smartwatch survey prompting as needed. Participants are not directly compensated for high μEMA compliance, although they are told that they can keep the smartwatch if they have >50% μEMA compliance at the end of 1 year. EMA compliance, by contrast, is reinforced with an explicit and compliance-contingent reward provided at regular monthly intervals.Our preliminary results from the pilot and ongoing TIME μEMA substudy show that despite a high interruption rate, μEMA could be sustainable for gathering self-report data longitudinally. Controlled filtering of μEMA questions enables measurement of a comprehensive set of behaviors repeatedly during waking hours. The validation questions make it possible to identify careless responding on both μEMA and smartphone-based EMA.The TIME μEMA substudy can be used to assess the utility and sustainability of μEMA. Comparing response rates with the qualitative data from the exit interviews, for example, suggests that μEMA can be used to gather temporally dense self-report information. In addition, passively collected data on wrist motion (using accelerometers) and location (using GPS) may help explore contextual factors that affect μEMA compliance. The filtering-based μEMA question-selection algorithm may be used to capture diurnal patterns of different constructs during waking hours. These diurnal patterns could be used to explore (1) different clusters of individuals who have similar diurnal patterns for variables of interest  and (2) Overall, the purpose of μEMA is not to replace traditional EMA but to complement it. The TIME Study’s μEMA substudy protocol provides an opportunity to explore how small amounts of information gathered at high frequency can help us learn about health behavior change and maintenance, even as constructs related to behavior change deviate by the hour. Beyond observation studies, TIME Study data could enable exploration of the viability of μEMA for longitudinal interventions , where self-report on many different behaviors may be required. For instance, when optimizing mobile health interventions as part of microrandomized trials , μEMA maThere will be several limitations of the TIME μEMA substudy that will provide opportunities for future research. Findings about the μEMA methodology may not generalize to other population age groups because the main protocol is designed to study health behavior adoption and maintenance in young adults (age 18-29 years). There has been an increased interest in exploring the acceptance of wearable technologies in older adults -62, youtWe had to restrict our data collection to Android users because our research app used advanced sensor capabilities not programmatically available on iOS devices; thus, the TIME μEMA substudy excludes iOS users. Nevertheless, we observed more demographic diversity in the Android users who participated in our screening surveys  versus the iOS users . We have no reason to believe that the platform itself would affect μEMA response patterns.Yes, Sort of, and No) that may be less sensitive to construct variance than mobile phone–based EMAs with more response options. Yet, with a median of ≥4 measurements per construct within a day .The TIME μEMA substudy provides an opportunity to explore a new method of collecting temporally dense self-report data. Although EMA provides information on temporal dynamics of behavior, the burden of accessing the mobile phone, unlocking it, and then answering multiple complex questions compromises engagement. In this formative research, we explored the feasibility of using μEMA to measure multiple constructs in an ILD study. When deploying μEMA, each prompt is a cognitively simple single question that can be answered with a quick glanceable microinteraction with an always-accessible smartwatch. Because of this simplicity, μEMA permits a higher interruption rate than EMA without a corresponding increase in perceived burden. The TIME Study is the first ILD study to deploy μEMA for an entire year; thus, it could provide insights on methodological properties of μEMA—especially the ability to sustain participant engagement. Data from the study could be used to study what influences μEMA compliance, what can be learned about an individual’s behavior using μEMA, and how μEMA might be used along with EMA and passive sensors to richly characterize human behavior, especially how it may change throughout the day in response to rapidly changing contexts.


Staphylococcus aureus (MSSA) is the most frequent organism, and the majority are endogenous. Decolonisation reduces PJIs but there is a paucity of evidence comparing treatments. Aims; compare 3 nasal decolonisation treatments at (1) achieving MSSA decolonisation, (2) preventing PJI.Periprosthetic joint infection (PJI) causes significant morbidity. Methicillin sensitive Our hospital prospectively collected data on our MSSA decolonisation programme since 2013, including; all MSSA carriers, treatment received, MSSA status at time of surgery and all PJIs. Prior to 2017 MSSA carriers received nasal mupirocin or neomycin, from August 2017 until August 2019 nasal octenidine was used.P < 0.0001). There was no difference in PJI rates (P = 0.452).During the study period 15,958 primary hip and knee replacements were performed. 3200 (20.1%) were MSSA positive at preoperative screening and received decolonisation treatment, 698 mupirocin, 1210 neomycin and 1221 octenidine. Mupirocin (89.1%) and neomycin (90.9%) were more effective at decolonisation than octenidine  and PJI rates. Further research is needed to compare alternative MSSA decolonisation treatments. Hip and knee arthroplasty offers excellent outcomes for end-stage osteoarthritis and other painful conditions . PeriproStaphylococcus aureus (MSSA) is the most common organism isolated in early postoperative PJI [Methicillin sensitive tive PJI , accounttive PJI . A link tive PJI . DNA anative PJI , 10, 11.tive PJI , 11, 12.S. aureus decolonisation is effective at reducing PJIs [There is strong evidence that nasal ing PJIs , 14. Howing PJIs . There iing PJIs .Octenidine HCl is a topical antiseptic with activity against both gram-positive and gram-negative bacteria. As an antiseptic nasal gel octenidine can be supplied by the surgical pre-assessment team without prescription, streamlining the process, and reducing cost. Although it has less potential for inducing resistance than mupirocin , there iNeomycin is an aminoglycoside antibiotic active against both gram-positive and gram-negative bacteria. There is limited research into the efficacy of neomycin ointment for nasal MSSA decolonisation. Leigh et al. showed neomycin achieved nasal decolonisation in 61% of cases compared to 95% with mupirocin at 8 days after treatment . ResistaThis study aims to compare the efficacy of nasal mupirocin, neomycin or octenidine, as part of an MSSA eradication bundle with octenidine skin wash, at both achieving MSSA decolonisation and reducing rates of MSSA PJI in primary hip or knee replacement.S. aureus media  without pre-enrichment. Plates were read at 18 and 24 h, further identification was performed using the VITEK MS  and sensitivity testing using the VITEK 2. Clinical and Laboratory Standards Institute (CLSI) breakpoint standards were used to define methicillin sensitivity. Screening was performed around 14 days prior to surgery, if surgery was delayed beyond 28 days then screening was repeated. Patients testing positive for MRSA were excluded from this study.In 2010 our organisation introduced an MSSA screening and decolonisation programme for patients undergoing elective hip and knee joint arthroplasty. Patients underwent seperate groin and nose swabs for detection of MSSA during their preoperative assessment. Liquid swabs  were originally used until a change to flocked swabs  in June 2018. Swabs were directly streaked onto Colorex Patients testing positive for MSSA carriage were given nasal decolonisation treatment to use 5 days prior to surgery and 5 days after . Group one, from 2010 until August 2017, used nasal mupirocin 2% . Group two, from August 2017, used an octenidine HCl 0.1% nasal decolonisation agent . Group three used neomycin 0.5% and Chlorhexidine 0.1% nasal cream , this was used as an alternative to mupirocin due to periods of national supply shortage. All patients received an octenidine HCl body wash  for daily use 5 days prior to surgery.Nasal cavity cleared if needed.Cream introduced into the anterior nasal cavity with a finger or cotton bud for 2 min.Cream spread further by pressing the sides of the nose together.Any excess cream removed.Patients received verbal and written instructions on how to apply both the nasal treatment and the body wash. Octenisan body wash was applied, once per day, to wet hair and body using a wash cloth, after 1 min skin contact this was washed off. Hair was only included on alternate days (days two and four). Mupirocin and octenidine were both applied twice daily, neomycin was applied four times per day. The steps for nasal treatment are:Primary outcome was decolonisation efficacy, measured by MSSA positive culture on the day of surgery. The secondary outcome was MSSA PJI.Data was collected prospectively by the SSI surveillance team locally on all MSSA carriers from September 2013. This data has been retrospectively analysed for this study. Four groups of interest were identified from this database. The first three groups were positive at initial screening and treated as per protocol at the time, receiving nasal mupirocin, octenidine or neomycin. The fourth were negative at initial screening but positive when swabbed on the day of surgery  criteria . Cases oStatistical analysis was performed using Statistical Package for the Social Sciences (SPSS Version 26). All results quoted to two significant figures. Categorical data was analysed using chi squared test.Data for cases from September 2013 to August 2019 was analysed. Groups one and two covers a period of 47 months, group three covers 24 months . Significantly more patients remained colonised with MSSA after treatment with octenidine (50.0%) than with mupirocin  or with neomycin . There was no statistical difference between mupirocin and neomycin (P = 0.393).Microbiology records were reviewed, in total 1776 patients (56.8%) were swabbed for MSSA on day of surgery , MSSA was isolated from deep tissue samples in ten cases (0.23% MSSA PJI rate). There was no statistical difference between the four groups . This is likely to represent both false negatives at screening and intermittent carriers.Staphylococcus aureus is recognised as the most common causative organism in early postoperative PJI [tive PJI . Since ttive PJI , 21, 22,tive PJI , 24). Sitive PJI . Decolontive PJI .The topical antibiotic mupirocin is the only treatment that currently has good study data. The majority of existing research focuses on decolonisation as a method to reduce SSI and nosocomial infections i.e. an active treatment is compared to a non-active control. There are very few studies comparing different treatment options. In the UK, National Institute for Clinical Excellence (NICE) guidance states mupirocin should be considered whenever MSSA is a likely cause of SSI .Mupirocin is a topical antibiotic working through inhibition of bacterial isoleucyl-tRNA synthetase . WidesprS. aureus infections, nasal decolonisation rates were 95% for mupirocin and 61% for neomycin 8 days after treatment [In our cohort of 3129 patients, mupirocin and neomycin were significantly more effective at MSSA decolonisation (89% and 91% of patients decolonised respectively) than octenidine (50% decolonised). On the day of surgery patients were 5.1 times more likely to be positive on swab culture when treated with octenidine. In the largest study of nasal decolonisation Perl et al. compared mupirocin to placebo in over 4000 patients . Mupirocreatment . The autreatment , 33. OurDespite the significant difference in decolonisation efficacy, in our study, this did not translate into a difference in MSSA PJI rate. There was no statistical difference in MSSA PJI rate between the three treatment groups or with group 4. The authors acknowledge that the sample size is too small to draw firm conclusions on PJI.Interestingly, all three infected cases in group 3 (octenidine) were caused by MSSA. There were no PJI caused by other organisms. Octenidine has a broad spectrum of activity , it may A meta-analysis in 2020 showed that mupirocin nasal decolonisation reduced PJI compared to control groups (placebo or no treatment) . Using aS. aureus SSI. Povidone-iodine is of particular interest as it is applied as a single treatment on the day of surgery potentially reducing issues of compliance. MSSA decolonisation is dependent on the total number of doses received [Phillips et al. compared mupirocin to a povidone-iodine solution for nasal decolonisation in a cohort undergoing arthroplasty or spinal fusion . Similarreceived . Our insThe European Medicines Agency (EMA) has recommended that, based on a presumed impact on clinical end points, microbiological outcomes can used when evaluating nasal decolonisation for orthopaedic or cardiac surgery . Our resth group also had no statistical difference in deep infection rates. Bacterial density clearly plays a significant role that is not fully understood but could explain why intermittent carriers and incomplete eradication may not increase the risk of PJI. Around one third of patients with MSSA carriage appear to have intermittent carriage which can be missed on screening. This supports the concept of universal decolonisation for all patients undergoing high risk surgery without screening, or the use of more sensitive detection techniques such as PCR. Previous cost analysis published from our instituition showed screening costs of £8 per patient compared to £7.73 for decolonisation treatment. Universal decolonisation without screening is cheaper (£8 saving for carriers and £0.27 for non-carriers) and avoids missed carriers. The potential downside is the development for increased resistance with increased use of mupirocin. This further highlights the need for research to compare alternative agents.MSSA carriers represented 20% of our cohort, with a further 9.1% newly positive on day of surgery, possibly representing intermittent carriers. Despite commencing treatment peri-operatively, this 4P = 0.108). This reduction could have impacted on the MSSA PJI rates, concealing a higher PJI rate with octenidine. The changes referred to would not be expected to impact on the efficacy of the MSSA decolonisation regime used at the time. As a result of this analysis our organisation returned to using mupirocin as the primary agent. This change was again audited to ensure its effectiveness, 6/72 (8.3%) were positive on admission for surgery. This is consistent with the original decolonisation efficacy suggesting there are no confounding factors that changed over the time period of the study. Over the course of the study the swabs used to acquire samples were changed. Regular internal validation was performed to ensure there was no change in the sensitivity of the techniques and equipment used.The main limitation of this study is the lack of randomisation. The data covers 6 years, during which multiple changes were made to standard practice and surgical techniques to improve patient care including reducing infections. The PJI rate did decrease markedly over this time, this is evident in the difference in overall PJI rate between the two treatment groups in Table Over time the proportion of patients re-swabbed on admission increased, as such there is a statistically significant difference in the proportion between the groups. This is a potential source of bias. The research team discussed this with the nursing staff responsible for admitting patients, it appears the decision was down to the member of staff’s interpretation of the protocol and not any patient factors.Due to the low incidence, this study was likely to be underpowered to detect a difference in the secondary outcome, MSSA PJI. A Post-hoc power analysis was performed for our study, for the group 2 versus group 3 MSSA PJI analysis we calculated the power to be 0.27. A sample size of 14,094 would be needed to achieve a power of 0.8.There is now good evidence to support MSSA decolonisation therapy prior to major joint arthroplasty and other high risk surgeries. Mupirocin is currently the only agent with strong clinical evidence but mupirocin, neomycin and octenidine are in common use. There is a significant lack of evidence to compare different decolonisation agents. In our non-randomised cohort of 3129 patients we have studied the efficacy of mupirocin, neomycin and octenidine, and found no statistical difference in efficacy between mupirocin and neomycin. However, they were both significantly more effective at MSSA decolonisation than octenidine, with 5.1 times more patients remaining MSSA positive after treatment with octenidine. Any possible differences in the agents and decolonisation did not translate to a difference in deep infection rate.With the threat of drug resistance further randomised research is needed to establish effective alternatives to mupirocin. The importance of bacterial load must be better understood to develop improved screening and treatment protocols.