Chronic kidney disease (CKD) is diagnosed using the estimated glomerular filtration rate (eGFR) and the urinary albumin:creatinine ratio (ACR). The eGFR is calculated from serum creatinine levels using the Modification of Diet in Renal Disease (MDRD) or Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations.To compare the performance of one versus two eGFR/ACR measurements, and the impact of equation choice, on CKD diagnosis and classification.Cohort study in primary care in the Thames Valley region of the UK.Data were from 485 participants aged >60 years in the Oxford Renal Cohort Study with at least two eGFR tests. The proportion of study participants diagnosed and classified into different CKD stages using one and two positive tests were compared. Prevalence of CKD diagnosis and classification by CKD stage were compared when eGFR was calculated using MDRD and CKD-EPI equations.2 (95% CI = 1.1 to 1.6) higher using the CKD-EPI equation compared with the MDRD equation. More patients were diagnosed with CKD when using the MDRD equation, compared with the CKD-EPI equation, once  and twice , and 16 individuals, all of who had CKD stages 2 or 3A with MDRD, were reclassified as having a normal urinary ACR when using the CKD-EPI equation.Participants included in the analysis had a mean age of 72.1 (±6.8) years and 57.0% were female. Use of a single screening test overestimated the proportion of people with CKD by around 25% no matter which equation was used, compared with the use of two tests. The mean eGFR was 1.4 ml/min/1.73 mCurrent guidance to use two eGFR measures to diagnose CKD remains appropriate in an older primary care population to avoid overdiagnosis. A change from MDRD to CKD-EPI equation could result in one in 12 patients with a CKD diagnosis with MDRD no longer having a diagnosis of CKD. The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation is the global standard for estimating GFR, although previously the Modification of Diet in Renal Disease (MDRD) equation was most commonly used.92 on two occasions at least 90 days apart.10,10,12CKD is diagnosed if urinary ACR is ≥3 mg/mmol or the estimated glomerular filtration rate (eGFR) is <60 ml/min/1.73 m,Prevalence estimates of CKD can vary widely, depending on whether they are based on a single eGFR  or two tests .13This study reports findings from an analysis of data from the Oxford Renal Cohort Study (OxRen),Diagnosis of chronic kidney disease (CKD) is based on two screening tests; reports have suggested that a single test could overestimate the prevalence of CKD by as much as 50%. This study has found that a single test overestimated CKD by 25% in an older primary care population, suggesting that a second screening test is clinically appropriate. As laboratories move from using the Modification of Diet in Renal Disease (MDRD) equation to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation to estimate glomerular filtration rate, some people will no longer have a CKD diagnosis. This study found that 8% of people with a CKD diagnosis based on use of the MDRD equation will be reclassified as not having CKD if the CKD-EPI equation is used.Data were used from 485 participants in the OxRen study who had two CKD screening tests, which had been conducted between 90 days and 2 years (730 days) apart. The remainder of study participants had either a single screening test or the second test fell outside the time limits. All samples were analysed across two laboratories using identical ACR and isotope dilution mass spectrometry (IDMS)-traceable creatinine assays, and reported the MDRD eGFR. Both samples for each patient were processed in the same laboratory.n = 30, 6.2%) and, as the recruitment area for OxRen has a majority white population, white ethnicity was allocated to 30 patients (6.2%) for whom no ethnicity was recorded. Those for whom no data regarding their sex were recorded  were excluded. People with missing ACR results  were included in the analysis, and the ACR was assumed to be >3 mg/mmol. The majority of participants included in this cohort had two screening tests because the first screening test suggested a positive CKD diagnosis. On this basis the higher ACR was used. The same assumptions were made for the first and second tests. Results were compared with eGFR results calculated directly from serum creatinine levels in a subset of patients for whom this was available. Sensitivity analyses were carried out to explore the impact of these approximations.The MDRD eGFR, patient age (at each screening test), sex, and ethnicity were used to calculate the CKD-EPI eGFR. The mean patient age was used for those for whom age was missing  time between screening visits were tabulated. The mean and SDs of the eGFR calculated using the MDRD and CKD-EPI equations were summarised by using the first measure alone, the second measure alone, and the mean of both measures. Proportions of patients diagnosed with CKD using a single eGFR/ACR screening test and two positive eGFR/ACR screening tests using the MDRD and CKD-EPI equations were tabulated.show how patients were reclassified into different CKD stages using both the MDRD and CKD-EPI equations and two measures, instead of one, were used to calculate eGFRs; andcompare diagnoses and reclassification of CKD stage when the CKD-EPI equation is used instead of the MDRD equation.Using both equations, the numbers of patients classified into each CKD stage using a single test and two eGFR measures  were calculated. Results were tabulated to:Scatter plots were used to compare the first and second eGFR measures for both equations. Bland–Altman plotsParticipants’ mean age was 72.1 (±6.8) years, 57.5% were female, and 98.9% of those with data had white ethnicity. The mean time between screening tests was 153 (±107) days (range 90–728 days) .2  higher than that generated when using the MDRD equation: 68.3 and 66.9ml/min/1.73m2 for CKD-EPI and MDRD respectively  for both equations are presented in The numbers of participants classified into different CKD stages when the eGFR was calculated using the CKD-EPI instead of the MDRD equation are shown in r2 = 0.645) compared with the CKD-EPI equation (r2 = 0.639). Bland–Altman plots showed good agreement between the mean difference in the eGFR between the first and second measurement versus the mean of two measurements for the MDRD and CKD-EPI equations. On average, the first measure was higher than the second measure for each equation (data available from the authors on request). The CKD-EPI eGFR plotted against the MDRD eGFR for the first and second eGFR measures showed overall agreement (data available from the authors on request).Correlation plots between the first and second eGFR measures for both equations are shown in The multinomial logistic regression analysis found a statistically significant association between age and reclassification of CKD stage when moving from the MDRD to the CKD-EPI equation (data not shown). An increase of 1 year in age is associated with a 7% lower risk of being reclassified to a lower CKD stage with the CKD-EPI equation compared with the CKD stage remaining the same , and a 12% higher risk of being reclassified to a higher CKD stage . Multinomial logistic regression directly compared those with an increase in CKD stage with those with a decrease in CKD stage. This confirmed that participants were 20% more likely to be reclassified to a higher stage using the CKD-EPI equation for each increase in year of age than they were to be reclassified to a lower stage, compared with no change in CKD stage between equations.Males were 3.99 times (95% CI = 1.50 to 10.59) more likely to be reclassified to a higher CKD stage than females when the CKI-EPI equation was used instead of the MDRD equation (data not shown).This analysis has found that, in an older primary care population, a single CKD screening test overestimates the number of people diagnosed with CKD by around 25%, compared with two tests. The mean eGFR was higher using the CKD-EPI equation compared with the MDRD equation, indicating that fewer patients will have a diagnosis of CKD when laboratories move to reporting eGFR using the CKD-EPI equation.Most people in the cohort had stage 2 CKD, followed by those with stage 1 CKD, indicating that they had an abnormal ACR result but normal or mildly reduced eGFR. These people would retain their CKD diagnosis regardless of the equation used. Most participants whose first CKD screening test indicated a CKD diagnosis that was not confirmed in the follow-up test were, using the first test alone, initially classified as having stage 1–3A CKD; this suggests that the reclassification stems from variability in the ACR as well as serum creatinine levels. Reclassification was most marked for those with stage 3A CKD using the MDRD equation; in total, 18% of this group were reclassified as having normal urinary ACR using the CKD-EPI equation  — all haTo the authors’ knowledge, this is the largest analysis to compare CKD diagnoses and reclassification, based on single and twice-conducted screening tests in an older population in primary care. The study presented here is unique, as far as the authors are aware, in reporting results from an older primary care population. However, in carrying out this analysis, it was only possible to access eGFRs that had been calculated using the MDRD equation. Using the available data on age and ethnicity, the authors were able to calculate the eGFR using the CKD-EPI equation, but some assumptions had to be made to use the full dataset. The impact of these estimations have been assessed in a sub-sample of patients with creatinine results and in a sensitivity analysis, which showed similar results.A further limitation is that the cohort was mainly of white ethnicity and so may not be generalisable to the general UK population.It is acknowledged that both equations used to calculate the eGFR in this analysis exhibit some bias relative to measuring GFR;18,14,14,Previous studies have suggested that the prevalence of CKD may be overestimated by 30–50% using a single eGFR compared with two positive measures.13,13,21,The finding presented here, that the CKD-EPI equation gave a higher mean eGFR and reduced the prevalence of CKD compared with the MDRD equation, has been reported by other researchers.7,Previous research has found that using the CKD-EPI equation increased CKD prevalence compared with use of the MDRD equation;7,17A recent meta-analysis has shown that the CKD-EPI equation gives a better eGFR than the MDRD equation, particularly in patients with the highest kidney function.7,26The strong association between reclassification of CKD stage in males may be due to the effect of muscle mass on creatinine-based measures,25This study has found that a single eGFR/ACR test would result in an unacceptably high rate of overdiagnosis of CKD, and confirms that current recommendations to base CKD diagnoses on two positive tests — thereby allowing for biological and analytical variability — are appropriate in primary care.As laboratories move from using the MDRD to the CKD-EPI equation, further consideration is needed to determine how those patients who had a CKD diagnosis but fall below the CKD threshold with the new equation should be treated and followed up as there is no guidance on how GPs should respond or how this could be communicated to patients. If these patients retain their CKD diagnoses, despite no longer meeting the diagnostic criteria, they may receive unnecessary monitoring, treatment, and interventions.Fewer than 1% of patients with stage 3A CKD are reported to go on to develop end-stage renal disease during 8 years of follow-upThe time interval between tests may also affect the agreement between the tests. KDIGO defines chronic CKD as a duration >3 months, but this time interval was selected arbitrarily.


Background: Schistosomiasis is a major parasitic disease affecting people living in tropical and sup-tropical areas. Transmission of the parasite has been reported in 78 countries, causing significant morbidity and around 200,000 deaths per year in endemic regions. The disease is currently managed by the mass-administration of praziquantel to populations at risk of infection; however, the reliance on a single drug raises the prospect of parasite resistance to the only treatment widely available. The development of an effective vaccine would be a more powerful method of control, but none currently exists and the identification of new immunogens that can elicit protective immune responses therefore remains a priority. Because of the complex nature of the parasite life cycle, identification of new vaccine candidates has mostly relied on the use of animal models and on a limited set of recombinant proteins.Methods: In this study, we have established an infrastructure for testing a large number of vaccine candidates in mice and used it to screen 96 cell-surface and secreted recombinant proteins fromSchistosoma mansoni. This approach, using standardised immunisation and percutaneous infection protocols, allowed us to compare an extensive set of antigens in a systematic manner.Results: Although some vaccine candidates were associated with a statistically significant reduction in the number of eggs in the initial screens, these observations could not be repeated in subsequent challenges and none of the proteins studied were associated with a strongly protective effect against infection.Conclusions: Although no antigens individually induced reproducible and strongly protective effects using our vaccination regime, we have established the experimental infrastructures to facilitate large-scale systematic subunit vaccine testing for schistosomiasis in a murine infection model. Infections in humans are caused by five main species of platyhelminth parasites of the genusSchistosoma, of whichS. mansoni andS. haematobium are the most prevalent. Infection in humans begins with the penetration of the skin by free-swimming cercariae, which triggers their transformation into schistosomula. After a few days of maturation in the dermis, these enter the bloodstream and progressively develop into male and female worms that pair up in the blood vessels of their host’s liver. Pairs ultimately migrate towards their final place of residence: the mesenteric vessels of the bowel in the case of species causing intestinal schistosomiasis, or the venous plexus of the bladder forSchistosoma haematobium, where female worms start laying eggs four to six weeks after infection. Despite the widespread incidence and debilitating effects of schistosomiasis in chronically infected patients, praziquantel is the only drug routinely used to treat established infections in populations living in endemic areas. The incomplete efficacy of the treatment, its lower efficiency at killing immature schistosomula and the increasing risk of drug resistance3 make the discovery of new therapeutics, and in particular vaccines, even more pressing5. Yet, despite many years of research, very few vaccine candidates have progressed to clinical trials6. TwoS. mansoni antigens  are being tested in humans, while a third antigen, Sm-p80, should soon enter phase I studies7. In the case ofS. haematobium, Sh28-GST has been the only clinically tested candidate against the urogenital form of the disease8.With over 200 million individuals affected every year and a third of all countries considered endemic, schistosomiasis remains one of the most widespread parasitic diseases on the planet. It is one of the world’s major global health problems, being responsible for 200,000 deaths annually9 and advances in transcriptional and proteomics studies18 have revealed many potential candidates whose protective effects are yet to be tested in animal models. Proteins secreted by the parasite or present on its surface are generally considered good vaccine candidates because of their direct accessibility to vaccine-induced antibodies. The surface of the adult schistosome, however, is covered by a thick tegumental double phospholipid bilayer shielding the parasite from host antibodies and capturing host antigens20, allowing it to survive for many years, sometimes decades, in the vasculature of infected individuals10. Targeting the parasite at a different stage of its life cycle, when it could be more vulnerable to the host humoral immune system, has been proposed as a possible approach21.The publication of the parasite’s genome22. In support of this idea, infection of various animal models with irradiated cercariae can elicit immunological protection to reinfection24, suggesting that the parasite is more susceptible to killing by the host in the first few days that follow infection. The use of attenuated parasites as a vaccine is, however, not suitable both in terms of practicability (they can be difficult to prepare and may suffer from batch-to-batch variations) and safety (each batch needs to be thoroughly tested to guarantee the efficacy of attenuation). For these reasons, modern vaccines are often based on chemically defined recombinant proteins, which are much easier to manufacture. Nevertheless, for these proteins to work efficiently as vaccines, they must be in a conformation that will elicit the generation of host antibodies that recognise antigens natively expressed on the surface of the parasite. Although popular heterologous expression systems such as bacteria and yeast have been successfully used for the generation of intracellular proteins, they are not generally regarded as suitable for the systematic production of secreted and cell-surface proteins, which require the formation of disulphide bonds for their correct folding and the addition of glycan structures. Also, the differences in adjuvants, routes of delivery, immunisation regimes and heterologous expression systems used to produce the antigens can lead to contradictory results on the protective effect of certain antigens27. We have recently described a library of secreted and cell-surfaceS. mansoni proteins whose selection was based on published proteomics studies and transcriptional data at the schistosomula stage28. Importantly, these proteins were expressed in human embryonic kidney (HEK)-293 cells, to increase the chances of forming disulphide bonds that are critical for the correct tertiary folding of extracellular proteins. Testing against sera from patients living in a high-endemicity area showed that most of them contain heat-labile conformational epitopes28.Immediately after penetrating the skin, the early schistosomula undergo an extensive surface remodelling, leaving their external surface free of the dense glycocalyx that was covering them at the cercarial stageS. mansoni cercariae.In this study, we have produced and purified 96 antigens from this library for immunisation in mice. By establishing infrastructures for large-scale systematic challenges and standardised infection parameters, we have systematically tested the protective efficacy of these antigens in immunised mice against infection with30. Where expression of the recombinant protein could not be achieved, a rat Cd4d3+4 tag was inserted in the expression construct between the sequence encoding the ectodomain of interest and the biotinylation sequence, which can facilitate expression and secretion of the recombinant protein31. At 6 days after transfection, cell culture supernatants were collected, filtered and supplemented with 20 mM imidazole and 400 mM NaCl before being affinity-purified on HisTrap HP purification columns  using an AKTAPure instrument, according to the manufacturer’s instructions. The purified protein fractions were dialysed in phosphate-buffered saline (PBS) and quantified by optical density measurement at 280 nm. To check for purity and integrity of the purified proteins, a 2-µg sample was resolved by SDS-PAGE on NuPAGE 4–12% Bis-Tris polyacrylamide gels (ThermoFisher) for 50 minutes at 200 V. The gels were then stained with SYPRO Orange (ThermoFisher), destained in 7.5% acetic acid and imaged using a Typhoon 9400 phosphoimager . The remainder of each purified protein was aliquoted and stored at -20°C until the day of immunisation. These gels are shown inUnderlying data32.Antigens  were pro33. Supernatants were collected six days post-transfection, dialysed in HEPES-buffered saline, and their level of expression determined by ELISA as previously described29.To prepare biotinylated antigens for determination of antibody titres, the BirA biotin ligase was co-transfected at the same time as the expression constructs described above into HEK-293-E or -6E cells as previously describedSchistosoma mansoni was maintained by routine infections of BALB/C female mice andBiomphalaria glabrata snails. Infected snails were housed in aerated tanks kept in dark cupboards maintained at 28°C. For collection of cercariae, 20 to 50 infected snails were individually washed in conditioned water, pooled in a small glass beaker, submerged in conditioned water and exposed to bright light34. After 60 minutes, the conditioned water containing live cercariae was collected. To evaluate the concentration of cercariae in solution, 12 5-µL aliquots were sampled, mixed with Lugol (Sigma Aldrich), and cercariae contained in each individual drop were counted under a dissecting microscope. For infection challenges, the cercarial solution was subsequently diluted in conditioned water to a final concentration of 400 cercariae/mL, and immediately used for percutaneous infection.All animal experiments were performed in accordance with UK Home Office regulations under project licenses P77E8A062 and PD3DA8D1F, and following approval from the local Animal Welfare and Ethical Review Body. The life cycle of the NMRI (Puerto Rican) strain ofMice were obtained from the Research Support Facility, Sanger Institute and typically had a mass of 25 g. Mice were housed in GM500 Individually Ventilated Cages or IsoCage N - Biocontainment Systems (Tecniplast) and maintained on individual air handling units kept at between 19 to 23°C and 45–65% humidity; each cage received 60 air volume changes per hour. Mice were given access to food and water ad libitum and maintained on a 12-hour light/dark cycle. Aspen wood chip was used as the base bedding with nestlets and "fun tunnels" for environmental enrichment (Datesand). Mice were housed in groups of no more than 6 adults per cage. Welfare assessments were carried out daily as part of the morning check. This involved moving the cage forward on the rack runners and observing the mice in their micro-environment. Any abnormal signs of behaviour or physical clinical signs of concern were reported. "In hand" weekly health assessments were also performed, and every cage was removed from the rack and each mouse given a full "head to toe" health check. All persons performing welfare checks on animals were trained and assessed as competent by qualified individuals.For each antigen studied and on the day of immunisation, aliquots of purified protein were thawed, diluted in PBS and mixed 50% v/v with Alhydrogel adjuvant 2% (InvivoGen) for 2 hours at room temperature. For each antigen, a group of five 7-week old female BALB/C mice was immunised intraperitoneally initially with 100 µg protein followed by two additional fortnightly immunisations using 20 µg protein. Where the quantity of purified antigen was insufficient to achieve these levels, lower doses of proteins were administered, as described inS. mansoni cercariae for 40 minutes by percutaneous infection through the tail. For this purpose, test tubes containing 5.5 mL conditioned water were prefilled and placed onto a bespoke anaesthesia rig (see below) pre-warmed to a surface temperature of 32°C. Mice were anaesthetised in an induction box using 4% isoflurane; 1 L/min oxygen, and an eye ointment was used to prevent corneal damage. After induction of anaesthesia, the animals’ tails were wiped with a tissue soaked with conditioned water to remove any contaminants from the cage bedding that could inhibit cercarial penetration, immersed once more in conditioned water before carefully transferring the animals onto individual holders on the rigs and inserting their tails into the test tubes. Nose cone positions were adjusted for each animal, and anaesthesia was maintained at 2% isoflurane; 1L/min oxygen. Individual splash guards were put in place to reduce the possibility of human exposure to infected water. In each test tube, 500 µL of a stock solution containing 400 cercariae/mL was added. After 40 minutes, animals were removed from the anaesthesia rigs, placed back into their cage and monitored until full recovery from the anaesthesia. At 42 days after the challenge, mice were euthanised by intraperitoneal injection of 200 µL of 200 mg/mL pentobarbital supplemented with 100 U/mL heparin; adult worms were recovered after portal vein section and intracardiac perfusion with phenol-red-free DMEM supplemented with 10 U/mL heparin, and whole livers were collected. Worms collected from individual mice were washed three times in PBS and placed in 6-well plates for counting. Liver tissues were weighed and then digested by incubation in 10 mL of 4% potassium hydroxide for 15 hours at 37°C35. The number of eggs per gram of liver was calculated after counting the number of eggs from 12 10-µL aliquots from each liver homogenate.After a 4-week rest period, animals were challenged with 20029.At 8 days after the last immunisation, blood samples (20 to 50 µL) were collected by tail bleed from each animal and left to clot for two hours at room temperature. After centrifugation, sera were collected and stored at -20°C after addition of 2 mM sodium azide. To determine the antibody titre against an antigen of interest, individual sera were serially diluted 1:4 between a 1:2,000 and a 1:8,192,000 dilutions in HBST/2% BSA. Depending on the presence or absence of a Cd4 tag in the antigen studied, these serial dilutions were pre-incubated overnight at room temperature with 1 mg/mL or 100 µg/mL of the rat Cd4d3+4-BLH protein, respectively, to block the fraction of antibodies that were raised against the tag in immunised animals. Serially diluted sera were then transferred to streptavidin-coated ELISA plates on which the biotinylated antigen of interest had been immobilised. To ensure that the antibody fraction directed against the protein tag had been blocked, binding of the lowest dilution of antisera was also tested against the control rat Cd4d3+4-BLH protein immobilised on an ELISA plate to confirm absence of anti-tag reactivity. Detection was performed using an alkaline-phosphatase anti-mouse immunoglobulin secondary antibody followed by incubation with an alkaline phosphatase substrate, as previously described2 scores between the number of eggs per gram of liver and the total number of adults or the total number of mature females were calculated by linear regression analysis. Comparison of average numbers of eggs per gram of liver between groups of immunised animals in a same cohort was performed using one-way ANOVA analysis followed by Bonferroni correction. Where immunisations were repeated on larger cohorts of animals, an unpaired t-test was used to compare average numbers of eggs in immunised and control animals; P-values ≤ 0.05 were considered to be significant.Statistical analysis was performed using GraphPad Prism (version 5) software. Data from serum analysis were analysed using a non-linear regression model to determine half-maximal antibody titres. RS. mansoni infection in mice. These are usually done either by intra-peritoneal injection of cercariae or by exposure of the skin to the parasite (percutaneous infection)34. Because a large fraction of the antigens to be tested correspond to parasite proteins expressed in the early dermal stages of infection, we opted to use the percutaneous method, which is also a natural route of infection. This protocol, however, requires a relatively lengthy exposure of either restrained or anaesthetised animals to infested water. To minimise unnecessary stress to the animal and biological safety risks to the experimentalist, we designed a bespoke anaesthesia apparatus that allowed for prolonged exposure to the parasite by tail immersion into cercariae-containing water while animals were under the influence of a gaseous anaesthetic -293 cells as soluble ectodomains containing a C-terminal rat Cd4d3+4 tag followed by a sequence for enzymatic monobiotinylation and a 6-His tag for purification28. The majority of these proteins were immunoreactive when tested against sera fromS. mansoni-infected patients living in areas of high endemicity, and contained heat-labile epitopes demonstrating the presence of conformational epitopes. We attempted to produce all 103 antigens as soluble 6-His-tagged proteins  and managed to obtain 74 antigens in quantities sufficient to perform immunisations in mice. For another 22 antigens, sufficient expression levels could only be achieved when these proteins were produced as C-terminally Cd4-tagged and 6-His-tagged antigens. Finally, seven proteins  were not expressed at sufficient levels to perform immunisations in either form  and two PBS-immunised controls (n = 6) were compared. In both repeats, the average half-maximal antibody titres were lower than in the primary screen at 1:48,073 for protein 10 and 1:347,980 for protein 58. We did not see any statistical difference between control animals and those immunised with protein 58 in this larger cohort. With the exception of one control animal, which only had a few mature females and therefore had a much lower egg burden, we once again observed a trend towards a reduced number of eggs per gram of liver in animals immunised with protein 10 compared to controls, although this observation did not reach statistical significance  and between the last immunisation and the infection challenge (four weeks); all cercarial preparations were carefully counted and used within one hour of cercarial shedding for infection; the tails of all animals were carefully wiped and immersed in conditioned water before the infection to avoid any inhibitory effect of residual oils present in animal bedding on parasite penetration. Despite these precautions, we still observed significant variability in the efficiency of infection and, in some cases, sex ratio imbalance in the cercarial preparation, which resulted in an excessive number of adult worms compared to the number of eggs produced. One way to better control this imbalance would be to use snails singly infected with male or female miracidia and mix the cercariae produced by these snails in equal quantities.39 or cathepsin B40. Other promising vaccine candidates such as the intracellular protein Sm-p8041 or the multimembrane spanning SmTSP226 were not included in our screen since their subcellular localisation or structure were not compatible with secretion of the recombinant proteins by HEK-293 cells in the culture supernatant. One possible explanation for the lack of protective efficacy observed in our study could be a biased immune response caused by the adjuvant that we used for immunisations. When designing this systematic study, we selected an adjuvant that could induce a good humoral response in mice but could also be transferable to human vaccine studies. Alhydrogel is a mild aluminium-hydroxide-based adjuvant commonly used in human vaccine formulations, which induces a Th2-type immune response mostly skewed towards the production of IgG1 antibodies43. While this antibody isotype results in high-affinity neutralising antibodies in mouse, it is far less efficient in recruiting complement and binding to activating Fcγ receptors44. The use of alternative adjuvants associated with a more balanced Th1/Th2 immune response such as Quil A might therefore be considered. One alternative explanation could be functional redundancy between some of the proteins tested. Our protein library contains multiple members of large protein families  and although these members are immunologically distinct, they could be functionally redundant. Immunisation with mixtures of antigens from the same family of proteins could therefore also be considered.Of the 96 recombinant candidates that were tested, all but 11 were antigenic enough to generate half-maximal titres > 1:10,000 , and only four did not raise any immune response. The lower immunogenicity of some proteins could be attributed to their relatively small sizes (six of them had a molecular mass <50 kDa) or to the lower antigen doses administered during immunisations because of poor expression. Despite overall high reactivities, we could not observe any reproducible protective effect from antibodies raised against the antigens tested. These include proteins that had previously been proposed as vaccine candidates such as Sm2945 and mice immunised with a DNA vaccine encoding the corresponding gene showed a reduced worm burden close to statistical significance15. Interestingly, the proportion of mature adult females recovered after perfusion in our experiment was similar between immunised and control groups , while the average total number of worms recovered was slightly smaller in the group vaccinated with protein 10 (27 worms versus 33 worms in the PBS controls). Individual cohort data for antigens 10 and 58, in addition to repeat analysis on larger groups, are available asUnderlying data32.Of all the candidates that we tested, proteins 10 and 58 showed a statistically significant decrease in the number of eggs per gram of liver in the primary screen when compared to the other two immunised groups from their respective cohort. However, only protein 10 showed a similar trend in the repeat experiment on a larger number of animals, although this did not reach statistical significance. This protein belongs to the Ly6 family of proteins and is also known as SmLy6B/SmCd59b. In previous studies, SmLy6B has been shown to be widely expressed in both male and female wormsS. mansoni as vaccine candidates. Although the vast majority of the 96 proteins tested raised a robust immune response in mice, none of them were associated with a strong protective effect against infection by the parasite and the identification of new vaccine candidates against schistosomiasis remains much needed. We hope nonetheless that these antigens will constitute a useful resource for the scientific community and that their role as vaccine candidates will be further explored in the context of different adjuvants, as components of protein mixtures or in other animal models.In conclusion, we have developed a technical infrastructure to refine the conditions of percutaneous infection in mice and to systematically screen a large number of recombinant cell-surface and secreted antigens fromhttps://doi.org/10.6084/m9.figshare.9862139.v132.Figshare: Data files describing the systemic screening of 96 Schistosoma mansoni cell surface and secreted antigens produced in a mammalian expression system.This project contains the following underlying data:1. Optimisation of the duration of challenge for percutaneous infection.pzf2. Optimisation of the rest period after immunisation.pzf3. S. mansoni proteins gels.pptx4. Half maximal antibody titres.pzf5. Correlation Eggs vs Total Adults or Mature Females.pzf6. Eggs per gram of liver counted across cohorts.xlsx7. Cohort analysis - Individual data for antigens 10 and 58.pzf8. Repeat analysis on larger groups - Proteins 10 and 58.pzfRDS-SPRN-00358.xlsx .Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of the The paper by Crosnier et al. represents a tremendous amount of careful and valuable work – the testing of a large panel of vaccines is clearly not an easy task, especially considering the vagaries of the trial system of vaccination followed by challenge infection with live cercariae. The large variation between the infectivity of the cercariae batches used in this paper shows how difficult it is to standardise schistosome vaccine trials. However, to their credit, the authors went to great lengths in animal husbandry, animal housing (e.g. controls in each cage), methodology in infections (including repeat experiments) and analysis of data to ensure that the results they obtained were reliable. The results of this paper reveal that vaccination with 96 different S. mansoni parasite molecules, selected based on the expression on the surface of early stage schistosomules or secreted by these (i.e. a large complement of the parasite-host interaction), does not induce protection in mice against a challenge of 200 cercariae. The molecules and their recombinant production: As mentioned above, the 96 molecules were rationally selected because of their putative role in the parasite-host interactome – the most likely molecules that vaccine-induced antibodies could access.   All molecules were produced in HEK-293 cells to ensure correct folding of the proteins. The production of the same molecules (without a His tag) is described in a previous paper by Crosnier (reference 281) who demonstrated by ELISA that the epitopes on the majority of molecules were heat labile – i.e. the purified recombinant bound to antibodies in serum from a pool of infected humans but heat-treated recombinants did not. This does not necessarily prove that the expression system delivers properly folded proteins as heat-treatment can cause precipitation or aggregation of proteins that may affect their binding to ELISA plates. Since the panel of molecules contained a number of proteases and protease inhibitors these could have been tested for functional activity to help validate the expression system. This result leaves myself, and I am sure the authors and members of the schistosome community, a little dazed. Surely, within this wide range of important molecules there should be a least one protective antigen? So how do we explain this result?The vaccine protocol: The protocol used is standard in the field – 2 to 3 immunisations followed by challenge with cercariae. The challenge infection was given 4 weeks after the final vaccination which goes some way in avoiding spurious protective responses that may operate at the level of the lungs in challenge infections given within a week or two after vaccination .  Alhydrogel was chosen as the adjuvant (mixed 50:50 with antigen solution). It was selected because it can be used in humans. Published data suggests it induced Th2-like responses. Given the failure of all molecules to induce protection, it is possible that this adjuvant is not suitable for eliciting the necessary protective responses against schistosomes in mice and therefore, as the authors point out a different adjuvant(s) would be important to test. A small sub-panel of antigens from each group  could be selected for adjuvant studies. The expression system can also add mammalian-like glycans to recombinant proteins. Figure 2 displays an impressive array of purified proteins. Some are well-defined bands while others are smeary and may be glycosylated – it would be useful to know the proportion of proteins that carry mammalian-like glycans.Antibody responses. The vaccinated mice returned with a ‘robust’ immune response to vaccination i.e. they produced high titre total antibodies . It would be informative to know the isotype of antibodies and have some indication of cellular responses to determine if these were similar across all vaccinated groups and, therefore, consisted of a profile of ‘non-protective’ responses .   Whole-mount immunolocalisation studies could determine if immune responses against surface antigens did induce surface-binding antibodies.An interesting finding in this study is that after seven weeks of infection with 200 cercariae (with high variable takes), adult worms numbers correlated with eggs per gram of liver tissue . This could seem obvious – the more females the more eggs! – but in various vaccines studies reported it is not always the case.   It would have been relevant to assess if this correlation held true for intestinal-tissue eggs which are destined to pass on infection to snails, especially since specific immune responses are suggested to aid egg transport through the intestinal wall. The antigen doses administered to the mice seems very high to me. The first vaccine of 100 ug was followed by two boosts of 20 ug. Could this high quantity of vaccine have induced some immune-tolerance, notwithstanding the high antibody response obtained? Generally, two doses of 20 ug would be sufficient to induce memory responses that would re-boosted following challenge. Perhaps dose-ranging some selected antigens would be useful?The mouse model. Does this study suggest mice are simply not a good model for testing recombinant schistosome vaccines? Female Balb/c were used in the studies. It may be worthwhile trying selected antigens in the C57/Bl mice given their propensity to elicit a more Th1-like response.1) most of the antigens are not immunogenic in infected mice at 6 weeks post infection. Although saposin-domain and cathepsin B are immunogenic at this time-point these still did not induce protective immune responses. Most antigens were also reactive with antibodies in pooled serum from infected humans, but we do not know if these antibodies have any protective ability. It seems, immunogenicity/non-immunogenicity in normal infections cannot be used to select vaccines According to Crosnier  or Montanide (used in the Cathepsin B study), these could have been tested as part of the optimisation steps.Schistosoma mansoni cell-surface and secreted antigens does not identify any strongly protective vaccine candidates in a mouse model of infection, using an Alhydrogel adjuvant". The adjuvant, and potential concerns with it, should also be mentioned in the abstract. To be clear about the potential problems with the experimental setup, I suggest the title should be changed to "Systematic screening of 96  In Table 1, where antigens are known by another name  these should be indicated on the table. This would be a useful resource for easily being able to see if a antigen expresses in HEK293 cells, and whether it is immunogenic. In Figure 2, it would be useful for the reader to indicate the expected molecular weight of expressed proteins, as in many cases it is unclear which band is the protein of interest, e.g 14C, 16C, 23C. In Figure 5A, it would increase the readability to have the groups in numerical order of antigen. This would allow the reader to identify a particular antigen of interest to them more easily. The groups could remain colour-coded by cohort so that these could be compared if need be - as data is normalised within each cohort this does not seem critical. Finally, antigen 11 appears to be in its own cohort (no other yellow groups) and also seems to be far below the normalised level of 1 - is this actually showing protection? Interesting also that 10 (which showed a trend for protection), and 11  are both members of the SmLy6 family.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. There's an urgent need to optimize in silico and so-called reverse vaccinology (RV) approaches for parasite vaccines, and bring this to the same status as bacterial vaccines. For example, BEXSERO is now the first licensed bacterial vaccine developed through an RV approach. The study here is an important attempt to apply RV for schistosomiasis. While in this initial attempt no promising antigen was identified, there are opportunities to both enlarge the number of potential candidates and add an extra level of screening for downselection. For example, it would be of interest to modify the approach to include human serologic screening against putatively resistant individuals in endemic areas. This approach was used to develop Sm-TSP-2 now in clinical trials.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Supplemental Digital Content is available in the text. Bronchitic symptoms in children pose a significant clinical and public health burden. Exposures to criteria air pollutants affect bronchitic symptoms, especially in children with asthma. Less is known about near-roadway exposures.Bronchitic symptoms  in the past 12 months were assessed annually with 8 to 9 years of follow-up on 6757 children from the southern California Children’s Health Study. Residential exposure to freeway and non-freeway near-roadway air pollution was estimated using a line-source dispersion model. Mixed-effects logistic regression models were used to relate near-roadway air pollutant exposures to bronchitic symptoms among children with and without asthma.Among children with asthma, a 2 SD increase in non-freeway exposures  and freeway exposures  were significantly associated with increased risk of bronchitic symptoms. Among children without asthma, only non-freeway exposures had a significant association . Associations were strongest among children living in communities with lower regional particulate matter.Near-roadway air pollution was associated with bronchitic symptoms, especially among children with asthma and those living in communities with lower regional particulate matter. Better characterization of traffic pollutants from non-freeway roads is needed since many children live in close proximity to this source. Near-roadway air was associated with an increased risk of bronchitic symptoms in adolescents, especially among those having asthma. These associations with were near-roadway pollution more pronounced in communities that had lower concentrations of regional fine particulate matter, suggesting that even in communities that appear to be “cleaner,” there are some individuals who may be at greater risk than others.2 Several studies have now shown positive associations between regional air pollutants and prevalence of bronchitic symptoms in children.8 Moreover, recent findings from the southern California Children’s Health Study (CHS) suggested that reductions in ambient pollution levels across several cohorts of children were associated with lower prevalence of bronchitic symptoms, further implicating the importance of air pollution in the etiology of this outcome.9Bronchitic symptoms in children, especially among those diagnosed with asthma, represent a significant burden to both clinical and public health that also results in reduced quality of life and considerable economic costs.2) and fine particulate matter (PM2.5) as well as constituents of PM2.5 such as elemental carbon (EC) and organic carbon.4 In southern California, traffic represents the primary source of many of these air pollutants.10 Although reductions in regional air pollution in southern California have taken place over past decades, the number of vehicles and vehicle miles driven have increased.11 Much less is known about whether exposure to air pollutants from near-roadway sources is associated with bronchitic symptoms in children. These near-roadway pollutants include oxides of nitrogen (NOx), ultrafine particles, carbon monoxide (CO), EC, and gaseous and particle phase organic compounds such as polycyclic aromatic hydrocarbons, all of which are corre lated and decay sharply with increasing distance away from a roadway.15 Because exposure to vehicle emissions from freeways and non-freeway roads are likely to be different mixtures because of differences in proportion of heavy-duty trucks, vehicle speed, traffic volumes, and frequency of acceleration, braking, and cold starts,17 we investigated whether the associations varied for exposures from freeways and non-freeway roads. Furthermore, since previous studies have shown that regional air pollution is strongly associated with bronchitic symptoms, and traffic is an important contributor to regional pollution in southern California, we were interested in whether the effect of near-roadway air pollution (NRAP) differed across communities with higher and lower levels of regional pollution.Identifying specific components of the air pollution mixture responsible for observed health effects has been of both academic and regulatory interest. In a previous CHS study, several components of regional pollution were found to be associated with bronchitic symptoms including nitrogen dioxide  in 1993 and 1997  and followed through 18 years of age across 12 communities, while the third cohort was composed of students recruited from kindergarten and first grade classrooms (≈5–6 years of age) in 2003 (hereafter referred to as Cohort E [N = 5600]) and followed through 16 years of age across 13 communities; nine of the communities overlapped across all three cohorts  a daily cough for 3 months in a row, (2) congestion or phlegm other than when accompanied by a cold, or (3) bronchitis. Children from Cohorts C and D were considered to have a history of asthma by 10 years of age if there was a “yes” response to the question, “Has a doctor ever diagnosed this child as having asthma?” on the baseline questionnaire. Because children in Cohort E entered the study at a younger age (5–7 years of age), a child was considered to have a history of asthma by 10 years of age if there was a “yes” response to the same question at any time during follow-up up through age 10 years. Children without asthma were those who never reported having asthma before or during any period of follow-up.Chronic bronchitic symptoms in the prior 12 months were assessed at each annual visit over an 8- to 9-year period (median follow-up: 7 years), as previously described.19 The CALINE4 model used distance to roadways, vehicle counts, vehicle emission rates, and meteorological conditions as inputs. This model accounts for the higher vehicle speeds, fraction of heavy-duty trucks, and traffic volumes on freeways than on non-freeways. It does not account for the enhanced acceleration, braking, and cold starts that occurs on non-freeways compared to freeways. We chose to use NOx as a surrogate for the near-roadway mixture because the emission factor model (EMFAC2014) produces more accurate vehicle emission rates for NOx than other species .21 While NOx was selected as the surrogate to represent this mixture, we will refer to the output from these models as NRAP from freeways and non-freeways. Modeled NRAP, estimated as a time-varying annual average, were lagged a year to temporally align exposures with bronchitic symptoms from the prior 12 months. Total NRAP was computed as the sum of NRAP from freeways and non-freeways.Residential exposure to the NRAP mixture from freeways and other non-freeway roads located within 5 km from their residences was estimated using the California Line Source Dispersion Model (CALINE4).9 We excluded children whose baseline asthma status was unknown or who developed asthma after 10 years of age so that we could focus on the association of near-roadway pollutants with bronchitic symptoms in children with and without asthma rather than attempting to address the issue of whether exposure to near-roadway pollutants is associated with incident asthma and then bronchitic symptoms in these incident cases. Multilevel logistic models were used to examine the associations of NRAP from freeways and non-freeway roads with bronchitic symptoms in these longitudinal analyses. A random intercept for individuals was included to account for serial dependency across observations from the same individual. Models included fixed effects for sex, ethnicity, and age. Models were adjusted for community to focus the associations on within community comparisons and to control for any between community differences . Models were also adjusted for cohort to focus the associations on within-cohort comparisons and to control for any long-term temporal changes in exposure, outcome, and potential confounders. Effect estimates were separately scaled to 2 SD of the total distribution of NRAP from either freeways or non-freeway roads across all individuals. Separate estimates for children with asthma and children without asthma were computed by including an interaction term between asthma status and exposure. Associations were also reported by higher and lower levels of regional PM2.5 and ozone. This was accomplished by taking the median of all available annual concentrations at each community’s regional monitor during the study period and then dichotomizing these communities into “higher” and “lower” PM2.5 or ozone communities . An interaction term between these indicators and exposure to NRAP was then included in the model to obtain separate estimates of NRAP in each of these categories of regional pollutants. Regional NO2 was also considered, but the communities sorted into the same higher and lower categorizations as PM2.5. Several sensitivity analyses were conducted, including adjustment for additional covariates ; use of asthma medication; presence in the home of roaches, dogs, cats, gas stove, mildew, or second-hand smoke; and participant’s overweight/obesity status), restricting data to participants with observations in the first and last years of follow-up, and including participants who developed asthma after the age of 10 years.The primary analyses were restricted to 6757 children with and without asthma who were (1) diagnosed by the age of 10 years and (2) had at least two assessments of bronchitic symptoms, for consistency with a recently published study.All analyses assumed a two-sided alternative hypothesis at a 0.05 level of significance. All models were fitted using SAS version 9.4 .http://links.lww.com/EE/A7. The associations of child ethnicity and parental education with bronchitic symptoms differed by childhood asthma status .Among the 6757 participants who were included in the primary analyses, about half were females . Associations of bronchitic symptoms with total NRAP exposure were similar to that of NRAP from freeways.Exposure to NRAP was associated with bronchitic symptoms Table . Among a2.5. Among all participants living in communities with lower regional PM2.5, a 2 SD increase in total NRAP exposure was associated with a 46% increase in the odds of bronchitic symptoms . Associations of bronchitic symptoms with NRAP from freeways were similar to those of total NRAP exposure in lower and higher PM2.5 communities, but the difference in associations was only marginally significant .Differences in associations between NRAP and bronchitic symptoms were observed by community levels of regional PM2.5 communities among all participants were driven mainly by children with asthma. The prevalence of asthma among participants in communities with lower PM2.5 was 18% compared to 21% of participants in communities with higher PM2.5. Children with asthma living in lower PM2.5 communities were at 89% increased odds of reporting bronchitic symptoms in the past 12 months per 2 SD increase in NRAP from freeways . Associations between non-freeways exposures and bronchitic symptoms were observed among children with asthma in both lower and higher PM2.5 communities, but the difference in magnitude was not statistically different. There was no significant difference in effects of NRAP by higher and lower ozone levels .These differences in NRAP associations with bronchitic symptoms between lower and higher PMhttp://links.lww.com/EE/A7). Stronger associations were observed when analyses were restricted to participants who completed follow-up to the last study visit. Associations were also robust to adjustment for several potential confounders , with increased reports of bronchitic symptoms.4 Moreover, EC, a constituent of PM2.5 and a known marker of diesel emissions, was also associated with bronchitic symptoms.4 Several other studies have shown associations between higher levels of traffic-related air pollutants, including NO2, PM2.5 and EC, with increased frequency of respiratory outcomes such as bronchitis, cough, wheeze, and chest tightness, as well as hospital admission owing to respiratory morbidities.28The near-roadway pollutant mixture is composed of particles and gases. Ambient measures of pollutants include both direct emissions from traffic as well as secondary compounds formed as a result of atmospheric photochemistry and regional transport. In previous CHS studies, we have shown associations between different measures of ambient pollutants  a major non-freeway with primarily passenger vehicles, (2) a limited access freeway, and (3) a heavily traveled non-freeway with a high proportion of truck traffic.16 For example, numerous polycyclic aromatic hydrocarbons, formaldehyde, acetaldehyde, and acrolein had higher concentrations 35 m downwind of the major non-freeway than 40 m downwind of the Interstate freeway that had more than double the traffic volume.13 Cold starts, which are more likely to occur near residences and thus non-freeway roads, result in higher levels of pollutants generated because of catalytic converters having not reached optimal temperature to efficiently reduce emissions.45 Another study found that PM concentrations were higher during delay periods at signalized traffic intersections compared to free-flowing traffic.17 More research is needed to better characterize differences in near-roadway pollutants from freeways and non-freeway roadways and to determine whether differences in composition may explain the difference in observed associations.It is unclear as for the stronger associations of bronchitic symptoms with non-freeway pollutants. CALINE4 estimates of pollutants from non-freeway roads may have been estimated with more error compared to those from freeway roads because of the likelier smaller spatial scale of pollutants from the large network of non-freeway roads and less reliable input data regarding traffic flow on these particular roads. However, the misclassification of non-freeway exposures with respect to bronchitic symptoms is likely to be nondifferential, which would cause a bias of the observed effects toward the null and an underestimation of the true effect. One possible explanation for the difference in associations may be explained by the non-freeway emission mixture being likely different than that from freeways. The fraction of heavy-duty diesel trucks is higher on freeways than on non-freeways in Southern Californian, and a Californian study has shown that the chemical composition of compression-ignition (diesel) engine emissions and spark ignition (gasoline) engine emissions are quite different.46 In the current study, reported associations between near-roadway pollutants and bronchitic symptoms were weakest in communities that historically had higher levels of PM2.5 (and NO2) and strongest in those with lower concentrations. One possible explanation is that the effect of NRAP is masked in communities with higher regional PM2.5 and NO2. In other words, a possible dose–response effect may be occurring where higher regional pollution may have already resulted in an exacerbation of symptoms among children with asthma, so the effect of NRAP is smaller on a relative scale, given a high background risk. Thus, in communities with lower PM2.5 and NO2, the effect of NRAP would be more apparent.In two previously reported studies from the CHS, both regional and NRAP were important determinants of respiratory outcome.47 Another limitation was the use of self-report for case ascertainment of bronchitic symptoms in the past 12 months and for asthma status. Self-report of asthma status has been validated in a subset of participants based on review of medical records, thus, lending credibility to our assessment of asthma status.48 Bronchitic symptoms in this study was defined based on at least one affirmation of either a cough that last for at least 3 months, chronic phlegm, or a report of diagnosis. These are all symptoms that are likely to be remembered well.4 However, misclassification of both bronchitic symptoms and asthma may still have occurred. Any misclassification of bronchitic symptoms or asthma is unlikely to be related to the model-based assignment of exposures, which would result in more conservative effect estimates.A limitation of this study was the use of estimated measures of residential near-roadway exposure based on the CALINE4 model, which may not capture true personal exposures and thus lead to some exposure misclassification. However, this exposure metric has been previously shown to capture adequately the local scale variability of traffic sources in a majority of these study communities in two separate exposure assessment campaigns.This study also has a number of strengths. The longitudinal assessment of bronchitic symptoms among three cohorts of youth across several southern Californian communities with a wide range of traffic and regional air pollutant exposures, spanning a 20-year period, is a major and obvious strength. The use of a population-based sample of participants from a number of schools allows for increased generalizability of findings to other youth in this region. Another strength was the consistent collection of important covariates allowing for adjustment for several factors that could confound the observed relationship.2.5 communities.This is one of the first studies to report associations of near-roadway pollutants with repeated measures of bronchitic symptoms on the same participant. These findings provide further evidence of the potential harm of NRAP, especially among children with asthma. More studies are needed to determine what components of NRAP are likely to induce the observed associations. Additional work is also needed to better understand the variations in effects of NRAP in lower and higher PMgillilan@usc.edu).Fred Lurmann is employed by Sonoma Technology, Inc . All other authors declare they have no actual or potential competing interests beyond grant support from the NIH and the South Coast Air Quality Management District. This work was supported by the National Institutes of Health , the Environmental Protection Agency , the South Coast Air Quality Management District , and the Hastings Foundation. Because of the limitations in the original consent forms and HIPAA requirements, the data from the Southern California Children Health Study (CHS) cannot be freely available in the manuscript, supplemental files, or in a public repository. However, we are committed to sharing the data and results acquired as part of this study. The CHS has a process in place for data sharing that involves approval of proposals by a Data Sharing Committee composed of USC and California ARB selected academic members. Investigators who want access to data will be required to submit a brief research protocol, which will be reviewed by the Children’s Health Study Health Data Release Committee and the USC IRB. Please send requests to access this dataset to the corresponding author, Dr. Frank Gilliland (


We examined recurrent Buruli ulcer cases following treatment and assumed cure in a large cohort of Australian patients living in an endemic area. We report that while the recurrence rate was low (2.81 cases/year/1000 population), it remained similar to the estimated risk of primary infection within the general population of the endemic area . The majority of recurrent lesions occurred in different regions of the body and were separated by a median time interval of 44 months. Clinical, treatment and epidemiological factors combined with whole genome sequencing of primary and recurrent isolates suggests that in most recurrent cases a re-infection was more likely as opposed to a relapse of the initial infection. Additionally, all cases occurring more than 12 months after commencement of treatment were likely re-infections. Our study provides important prognostic information for patients and their health care providers concerning the nature and risks associated with recurrent cases of Buruli ulcer in Australia. Mycobacterium ulcerans (M. ulcerans) causes a necrotising infection of skin and soft-tissue known as Buruli ulcer. Since the regular use of antibiotics for Buruli ulcer treatment in Australian populations was introduced at the turn of the century, treatment success rates have been very high. However there is no information from the Australian setting on the risk of recurrent disease following treatment and assumed cure, despite this being important prognostic information for patients, their families and health-care providers. Furthermore, it is also not known if recurrent disease represents late relapse of the initial treated infection or a subsequent new infection. In our study we have shown for the first time in Australian patients living in an endemic area that the incidence of recurrent Buruli ulcer following treatment and healing is low, and that this risk is similar to the estimated risk of primary infection within the general population of the endemic area. Furthermore, we have used clinical, treatment and epidemiological data supported by genomic information of M. ulcerans organisms to determine that the majority of recurrent lesions appear to result from re-infection. This suggests that for a proportion of treated patients’ acquired protective immunity against the development of recurrent M. ulcerans disease does not develop from their initial infection. Mycobacterium ulcerans (M. ulcerans) causes a necrotising infection of skin and soft-tissue known as Buruli ulcer.[M. ulcerans infection, as well as ongoing transmission risk in the community. For the first time, whole genome sequencing has recently been used to examine this issue in four cases of recurrent M. ulcerans disease in Benin, Africa, and suggested that three of the cases represented disease relapse and one re-infection.[li ulcer. Since thli ulcer.–4 Diseasli ulcer.,5 Howeveli ulcer. At presenfection.M. ulcerans lesions following treatment and assumed cure in an Australian population and to use whole genome sequencing techniques combined with clinical, treatment and epidemiological data to determine whether recurrent lesions represented late disease relapse or re-infection.The aim of our study was to determine the risk of recurrent M. ulcerans cases managed at Barwon Health, a tertiary referral institution in Victoria, Australia, from 1/1/1998-31/12/2016 were included in the study. A M. ulcerans case was defined as the presence of a lesion clinically suggestive of M. ulcerans plus any of (1) a culture of M. ulcerans from the lesion, (2) a positive PCR from a swab or biopsy of the lesion, or (3) histopathology of an excised lesion showing a necrotic granulomatous ulcer with the presence of acid-fast bacilli (AFB) consistent with acute M. ulcerans infection.All confirmed M. ulcerans lesion appearing after the original lesion had healed that was culture positive for M. ulcerans and occurred ≥ 12 months after initial treatment. Patients were not actively followed up after 12 months from treatment commencement, therefore diagnosis of recurrence relied upon self-presentation or referral to our health service.Recurrence was defined as a new A ‘significant risk’ of relapse following initial treatment was defined as a) those who had surgery without at least 2 weeks of known effective combination antibiotics based on our published risk of relapse of 32% in those who have had surgery alone, and our M. ulcerans Agy99 genome [M. ulcerans isolates obtained from the same endemic region (Bellarine Peninsula) [Where available, whole genome sequencing and single nucleotide polymorphism (SNP) analysis was performed to examine genetic relationships between pairs of isolates from the same patient (two patients did not have paired isolates available). A total of 10 isolates, derived from five patients with recurrent disease, were subjected to whole genome sequencing . Whole g9 genome , includi9 genome  and coreninsula) .Data was collected prospectively using Epi-info 6  and analysed using STATA 12 .This study was approved by the Barwon Health Human Research and Ethics Committee. All previously gathered human medical data were analysed in a de-identified fashion.M. ulcerans were managed at Barwon Health during the study period and included in the analysis. The median age was 57 years (IQR 37–73 years) and 225 (52.8%) were male. Thirty-four (8.0%) patients had diabetes and 35 (8.2%) were immune suppressed. Lesions were classified as World Health Organization (WHO) category one for 79.3%, category two for 10.6% and category three for 10.1% of lesions. The clinical type of lesion was classified as an ulcer for 85.1%, nodule for 6.1%, oedematous for 7.8% and plaque for 0.9%. The median duration of symptoms prior to diagnosis was 42 days (IQR 28–75 days).A total of 426 patients with Of this cohort, seven (1.6%) patients were diagnosed with a recurrent lesion . This waThe recurrent lesions occurred a median 44 months (IQR 16–68 months) after treatment commenced for the initial lesion; 5/7 recurrences occurred at least 3.4 years from the initial lesion. Four (57%) recurrences were on a completely separate limb and side of the body, one was on the same limb but different region of that limb and 2 were on the same limb and in the same region.Treatment of the initial lesion involved surgery alone for 1 patient, antibiotics alone for 2 patients, and antibiotics combined with surgery for 4 patients . AccordiM. ulcerans isolates from the same endemic area. Three of the six isolates  were genetically identical to each other following SNP analysis . In contrast, the isolates from patient #2 –also genetically identical–were only separated by 12 months, and occurred on the same body region. In this case, a late relapse of the initial infection would appear more likely.The whole genome sequence analysis revealed a mix of genetic relationships between isolates. Paired isolates from some patients (#2 and #3) were genetically identical, possibly suggesting either late relapse of the initial infection or re-infection from a genetically homogenous source. In the case of patient #3, the extended time between recurrence (46 months), the fact that the patient received highly effective treatment, and the fact that the lesions were identified in different body areas (right forearm and left ankle), suggests that re-infection from a genetically homogenous source was more likely. While it’s hard to estimate the degree of genetic change that would occur during a latency period M. ulcerans cases in Australia suggests that exposure risk to M. ulcerans is short-term and may not necessarily be from a genetically homogeneous source [M. ulcerans is highly clonal in Australia, with only minor genetic variation [in vivo and thus the cases represent late disease relapse despite a small number of SNP differences. In the case of patients #1 and #5 this latter hypothesis cannot be ruled out, but seems unlikely as in both cases the primary (first) isolate had genetically diverged more from the ‘common’ dominant genotype compared to the second isolate. This is further supported in patient #1 by the long duration between lesions (44 months) and in patient #5 by the recurrent lesion being situated on a completely different body area and the initial treatment being highly effective for curing BU. Combined, these findings suggests that re-infection with a different genotype was the most plausible explanation for the #1 and #5 cases.There were genetic differences between three of the paired isolates  which can be interpreted in two ways. Firstly, it’s possible that they are the result of re-infection from a genetically heterogeneous population. In support of this hypothesis, our previous research examining family clusters of s source . Howeverariation ,16, it iM. ulcerans, putting them at higher risk of relapse [In comparison with the other known study by Eddyani et al. from Africa  that loo relapse , and in  relapse .The two recurrent cases who did not have paired isolates available for WGS (#6 and 7) were classified as re-infections based on a combination of separate body areas (#6 and 7), highly effective treatment (#7) and having a long time interval between cases (#6 and 7). Additionally, our data suggesting that all recurrent cases which occurred more than 12 months after treatment commenced were classified as re-infections, and the only one occurring after 12 months was classified as disease relapse, would support our previous clinical definitions that treatment failure occurs when a recurrent lesion appears within 12 months of commencing treatment.M. ulcerans disease does not develop following an initial infection. Interestingly, the rate of recurrence (2.81 cases/year/1000 population) was similar to the estimated risk of infection in the general population of the Bellarine Peninsula [M. ulcerans disease in those with a prior history of the disease.[If, as suggested by our study, most recurrent cases result from re-infection, then at least for a proportion of treated patients acquired protective immunity against the development of recurrent pulation), suggest disease.M. ulcerans it is likely that any recurrent lesions in patients would have be managed at Barwon Health and therefore we feel the risk of missing recurrent lesions would be small. Additionally, as the incidence of M. ulcerans in the Bellarine Peninsula has fallen in recent years,[A limitation of our study is that we relied on self-presentation or referral to our health service for diagnosis of recurrent lesions more than 12 months after treatment commenced and therefore there is a risk that some recurrent lesions were not captured in our study. However, as we are the only specialised health service in our region managing nt years, if this M. ulcerans re-infection does not develop.There is a low incidence of recurrent Buruli ulcer in treated Australian patients living in endemic regions and the risk is similar to the estimated risk of primary infection within the general population of the endemic area. The majority of recurrent lesions appear to result from re-infection suggesting that for a proportion of treated patients lifelong immunity against


Self-care is key to the daily management of chronic heart failure (HF). After discharge from hospital, patients may struggle to recognize and respond to worsening HF symptoms. Failure to monitor and respond to HF symptoms may lead to unnecessary hospitalizations.This study aimed to (1) determine the feasibility of lung impedance measurements and a symptom diary to monitor HF symptoms daily at home for 30 days following hospital discharge and (2) determine daily changes in HF symptoms of pulmonary edema, lung impedance measurements, and if self-care behavior improves over time when patients use these self-care monitoring tools.This study used a prospective longitudinal design including patients from cardiology wards in 2 university hospitals—one in Norway and one in Lithuania. Data on HF symptoms and pulmonary edema were collected from 10 participants  male) with severe HF (New York Heart Association classes III and IV) who were discharged home after being hospitalized for an HF condition. HF symptoms were self-reported using the Memorial Symptom Assessment Scale for Heart Failure. Pulmonary edema was measured by participants using a noninvasive lung impedance monitor, the CardioSet Edema Guard Monitor. Informal caregivers aided the participants with the noninvasive measurements.P=.007) from a median of 56 (IQR range 22-75) at discharge to a median of 81 (IQR range 72-98) 30 days later.The prevalence and burden of shortness of breath varied from participants experiencing them daily to never, whereas lung impedance measurements varied for individual participants and the group participants, as a whole. Self-care behavior score improved significantly (Noninvasive measurement of lung impedance daily and the use of a symptom diary were feasible at home for 30 days in HF patients. Self-care behavior significantly improved after 30 days of using a symptom diary and measuring lung impedance at home. Further research is needed to determine if daily self-care monitoring of HF signs and symptoms, combined with daily lung impedance measurements, may reduce hospital readmissions. Self-care is recognized as an important aspect of daily management of heart failure (HF) and recommended in international guidelines -3. Self-The 5 most characteristic HF symptoms of fluid retention are shortness of breath, shortness of breath when supine, shortness of breath that awakens the patient during sleep, feeling tired, and ankle swelling . MoreoveAnother promising method for self-monitoring is using noninvasive lung impedance devices to measure pulmonary congestion before HF symptoms are recognized by the patient ,20. As pKnowledge is lacking for self-care approaches that combine the use of a self-reported symptom diary with noninvasive lung impedance measurements. The aim of this study was, therefore, to assess patients’ HF symptoms, lung impedance, and self-care behavior at home for 30 days after hospital discharge. This study sought answers to the following research questions:1. How feasible is it for patients with HF and their caregivers to measure symptoms and signs daily at home using a diary and a noninvasive device for measuring lung impedance during a 30-day period after discharge from hospital?2. How do daily HF symptoms and lung impedance change during the 30-day assessment period?3. How does self-care behavior change when patients use a symptom diary and a noninvasive device to measure lung impedance during the assessment period?This longitudinal observational design study was conducted from May 2017 to November 2017 using eligible patients discharged from cardiology wards in 2 university hospitals in Norway and Lithuania. Norway is a high-income country ranked as number 28 by the World Bank, and Lithuania is ranked as number 84 . The heaPatients were eligible if they were hospitalized with a primary diagnosis of HF, aged older than 18 years, fluent in Norwegian or Lithuanian, and possessed sufficient cognitive abilities to understand and complete the study protocol. Cognitive abilities were judged by the nurses or cardiologists at the hospital ward. Only patients with New York Heart Association (NYHA) Functional Classification III and IV were included in the study.2 [Patients with severe HF in need of surgical intervention, advanced chronic kidney disease defined as estimated glomerular filtration rate less than 25 mL per min per 1.73 m2 , a body The symptom diary comprised 3 components  and was Candidate participants were identified by cardiologists at the hospital wards. Before discharge, the included participants were instructed on how to use the printed symptom diary, which consisted of spaces to rate symptoms and to write down lung impedance measurements and on-demand medications. Participants and informal caregivers were trained at their home on how to perform lung impedance measurements and about sending the measurements by SMS text messages to a study mobile phone used by the HF study nurse. The HF study nurse was contacted if the participant had problems with bad electrode connections or other technical problems . Study participants and informal caregivers were instructed on how to recognize HF symptoms and signs through the use of a paper handout with textual explanations, color-coded information related to HF condition, and prominent contact information.presence or absence of each listed symptom and then scored the symptom frequency on a Likert scale from 1 to 5 . Symptom severity was also scored on a Likert scale from 1 to 5 (from mild to extremely), and symptom distress was scored on a Likert scale from 1 to 5 (from a little bit to extremely). The total score is the sum of all the symptoms present, with a minimum score of 0 and a maximum score of 32. Symptom burden scores are determined by calculating the mean frequency, severity, and distress of each symptom, with a possible maximum score of 5 for each experienced symptom. The MSAS-HF was used with permission from the developers [Participants rated their symptoms using an adapted version of the Memorial Symptom Assessment Scale-Heart Failure (MSAS-HF) , which wvelopers ,29. To ovelopers . A totalvelopers ,29.dry lung impedance of each participant as a reference value. This baseline value was based on each participant’s age, gender, height, weight, NYHA class, and lung impedance measurements [The participants and informal caregivers measured lung impedance at home daily using a CardioSet Edema Guard Monitor , a noninvasive impedance monitor developed to measure pulmonary congestion or edema in HF patients ,31. The urements . Lung imurements . In thisurements ,20. FiguOn-demand medication was prescribed, for example, additional diuretics for some of the included participants to be used at home if necessary. Every day, these HF patients recorded their use of any of these on-demand medications in their diary.The European Heart Failure Self-care Behavior Scale (EHFScBS) is a self-rating instrument that measures HF-related self-care. It comprises 9 items that are self-scored on a 5-point Likert scale. A standardized score ranges from 0 to 100 results, with higher scores indicating better HF self-care -34. The A standard clinical examination was performed at hospital discharge and at the outpatient clinic 30 days later  by a carP value <.05 was considered statistically significant [EpiData Entry  was used for entering and managing data to optimize data accuracy and entry across the 2 countries. This step is in line with recommendation from the data protection managers at both university hospital sites. Raw data were converted from the EpiData Entry format to the SPSS format for analysis. Variables were either categorical or continuous and were presented as counts, percentages, means and SDs, or median and IQRs. Missing data from the symptom diary were not included in the analysis  and reponificant . Data weThe study was conducted in accordance with the Declaration of Helsinki. Ethical approval was obtained from Regional Committees for Medical and Health Research Ethics in Norway (REC number: 2014/1890) and Vilnius Regional Ethics Committee in Lithuania . All participants received verbal and written information about the purpose of the study, signed a written informed consent form before participation, and were free to withdraw from the study at any time. The daily lung impedance measurements were securely sent to the HF study nurse in each country using dedicated study mobile phones. Exchanged study data between the 2 university hospital study sites were deidentified with files encrypted in an email, which required a separate SMS code to be opened. This privacy assurance of research subject data and identity is in accordance with requirements of the data protection officer at the university hospital study sites.The Edema Guard Monitor lung impedance measuring devices were purchased from the company CardioSet Medical Ltd, Matan, Israel, and researchers and the company signed a written contract with no obligations to the company.A total of 10 participants with HF in NYHA classes III and IV at inclusion (5 from each country) were recruited. The participants’ mean age was 64.5 years, 8 participants had comorbidities, 1 participant was female, and most of the participants lived with their spouse or children . None ofParticipants used the symptom diary daily, and with support from their caregivers, they measured lung impedance daily. A total of 7 participants were able to provide data for the full 30-day assessment period. For impedance data, 262 of 300 (87%) recorded measurements were successfully made and sent. For the symptom diary, 1332 of 1500 (89%) entries were successfully made. Missing data were mainly from 2 participants who were classified as NYHA class IV. These participants provided diary and lung impedance data for 19 of 30 days (57%) and 22 of 30 days (63%), respectively. These 2 participants had a few missing lung impedance measurements because of issues with poor signal quality presumably related to suboptimal electrode connections, despite performing more than 3 required measurements and receiving advice from the HF study nurse. A third participant did not have home caregiver support for measuring lung impedance after day 26; thus, participation was terminated on day 26 for that participant. Moreover, 2 participants lived alone, but 1 of them moved in with a family member during the 30-day home assessment period. The other participant received support from an HF study nurse every day to apply the electrodes, although the participant performed the measurement.Prevalence and burden assessment of the 5 symptoms selected from the MSAS-HF varied among the participants . Out of shortness of breath and lacking energy was higher than the total burden score of difficulty breathing lying flat; wake up breathless; or swelling of hands, legs, or feet. Moreover, 1 participant did not experience any of the 5 HF symptoms or signs during the 30-day period. Details of HF symptom prevalence and total burden scores are presented in The total burden score  of shortness of breath for 5 to 26 days and lack of energy for 1 to 28 days.A total of 9 participants experienced symptoms indicating fluid accumulation for 1 or more days. They reported feeling At hospital discharge, individual ΔLIRs ranged from −20.9% to −37.7%. Thus, on day 1 of the study, all participants may have presence of pulmonary edema. The participants’ LIRs over time are presented in The number of days the patients participated in the self-assessment varied because of technical or health-related reasons. Patient number 2 had 26 days of data, patient number 3 had 19 days of data, and patient number 4 had 23 days of data . PatientP=.007) than that at hospital discharge. The subscale score on consulting behavior also significantly improved (P=.049) after 30 days. At the item level, the most frequently performed self-care behavior (score 5) was taking medicationas prescribed, and the least frequently performed self-care behavior (score 1) was to exercise regularly. Details of the self-care behavior results are presented in Self-care behavior improved from the time they were discharged to the end of the 30-day assessment period. The total score on the EHFScBS at the end was significantly better (Our main finding was that HF patients were able to successfully use a symptom diary and measure aided noninvasive lung impedance at home to self-monitor and report HF symptoms during the 30-day postassessment. This is the first study in which HF patients would use the Edema Guard Monitor to measure their lung impedance on a daily basis for 30 days following hospital discharge to home. Previously, the Edema Guard Monitor had been used by health care professionals once a month at an outpatient clinic with HF patients . Lung imThe age range of our participants was 37 to 85 years, and our findings are in line with some reports showing that older patients accept and manage well when using new technology . All parTypically, after discharge from hospital, HF patients are referred to their general practitioner in the community health care services for follow-up and medication adjustments. In Norway, follow-up at an HF outpatient clinic requires a referral from the hospital ward or general practitioner, currently occurring only 21% of the time . In LithAnother finding was that participants’ self-reported ratings on the 5 key HF symptoms and on the lung impedance measurements varied greatly. The variability in HF symptoms over the course of the assessment period stresses that constant attention is needed to achieve symptom relief. Some authors also warn that HF patients acclimate to symptoms and adapt their activities and lifestyle to reduce the HF symptoms; thus, they might not recognize these symptoms as warning signs -49. TakeHF decompensation is the worsening of HF symptoms and signs and is characterized by a gradual decrease in lung impedance measurements in patients at high risk for volume overload . In thisWe observed that self-care behavior of the study participants significantly improved after using the daily symptom diary and taking daily lung impedance measurements. At discharge, our participants had inadequate self-care. According to the EHFScBS, a score of 70 or higher reflects adequate self-care behavior , which iOne limitation is that only 10 patients participated in this study. Nonetheless, we were able to obtain a substantial amount of data from these participants, and these data provided us with an in-depth understanding of the trajectory of HF symptoms during the first month at home after hospitalization. The variability of the data for HF symptoms and LIR, in addition to our small sample, made it not advisable to perform an analysis for possible associations among these variables. Another limitation is the use of an unvalidated version of the MSAS-HF for the purpose of daily self-monitoring for 30 days at home. However, the unvalidated version did include 5 HF symptoms of fluid overload that are also recommended in guidelines [This is the first study showing that discharged HF patients can successfully use a symptom diary combined with measurement of their noninvasive lung impedance at home to self-monitor on a daily basis, with support from informal caregivers. We found a significant improvement in the participants’ self-care behavior in the period between hospital discharge and end of the 30-day home assessment. These self-care tools can be used by nurses in clinical practice to educate HF patients and their informal caregivers before discharge to home, which could also reduce readmissions. For future research, a larger study population is needed to determine whether self-monitoring of particular HF symptoms at home and LIR are predictive of HF deterioration. Early detection of worsening HF at home will aid health care professionals in providing better HF care.


Regular physical activity (PA) is a cornerstone in the management of complications associated with type 1 diabetes (T1D). Most national guidelines advocate for regular PA for persons living with T1D, however the evidence to support these recommendations has not be reviewed recently. Additionally, in an era of patient-centered care and patient oriented research, the role of patient partners in the area of PA and T1D interventions has never been explored. The purpose of this narrative review is to overcome these two gaps in the literature. Here we review selected epidemiological evidence and identify gaps in research that would add important information to guide practitioners and future guidelines. We also provide an overview of patient-oriented research projects co-developed with persons living with T1D. Significant gaps in the field include: (1) a lack of adequately powered prospective cohort studies using serial measures of PA and hard chronic disease end-points; (2) no multi-centered, highly powered, randomized controlled trials of PA, and long-term health outcomes; (3) little data on the role of new technologies to support PA-related behavior change, and (4) no trials that involved patients in the design and execution of PA-based clinical trials. This review provides a template for scientists and patient partners to develop future research priorities and agendas in the field. Rates of type 1 diabetes (T1D) have increased globally over the past 2 decades  and it cEvidence for most PA guidelines are derived largely from prospective observational cohort studies , 21. Recp < 0.05) to develop macro-vascular disease, compared to those that did. In a follow-up study, the authors found that participation in competitive sports as an adolescent was associated with a reduced odds of nephropathy, neuropathy and macro-vascular disease, relative to those that did not participate in competitive sports during high school. This association was not observed in women and it is unclear to what extent the analyses controlled for confounders.Observational studies clearly demonstrate that regular PA is associated with several health benefits for patients with T1D , includiMore recent studies of larger cohorts  of persons living with T1D in Europe and Scandinavia suggest that the association between PA and long-term cardiovascular disease (CVD) risk is modest and may be modified by other lifestyle factors that cluster with activity levels, particularly smoking. The FinnDiane study has published several analyses of self-reported PA on all-cause and CVD-specific mortality , 50. FinThese associations provide promising evidence for a protective association of PA on CVD and mortality among persons living with T1D, however they are far from conclusive. The findings presented above could be significantly strengthened by (1) conducting a rigorous systematic review of observational studies; (2) conducting larger cohort studies or a meta-analysis of individual level data with longer follow-up using objective measures of PA; (3) creating cohort studies with serial measures of PA over time to determine the association between changes in PA and long term CVD risk and finally, (4) creating cohort studies that conduct more robust analyses using propensity score matching or instrumental variable analysis to rigorously control for measured and unmeasured confounding. Finally, as PA is a behavior that tracks from adolescence through to adulthood, there is a major gap in our understanding of PA behavior in adolescence and health outcomes in adulthood. As recently argued , there ip = 0.6). However, structured exercise lowered daily insulin requirements  and body weight MD: −2.20 kg, 95% CI: −3.79, −0.61; p = 0.007) and increased peak oxygen uptake . The authors attempted to discern a dose effect of PA on health outcomes as others have done (n = 51) of adolescents with T1D  observed an overall effect size of 0.16 (95% CI: 0.0–0.3), suggesting modest improvements in glycemic control with behavioral interventions. This effect was nearly twice as large if the intervention was grounded in an established theoretical model . An updated review with recently published trials has not been conducted and therefore the efficacy of behavioral-based interventions to increase PA among persons with T1D remains unclear.Randomized trials of structured, supervised exercise provide insight into the physiological adaptations associated with increased PA. In contrast, behavioral trials provide insight into how best to motivate individuals to adopt a more active lifestyle . The mosIn a recent behavioral pilot trial in the UK , 58 younAn excellent example of a clinically relevant behavioral intervention to support increased PA was recently published by the FLEX study group . An 18 mThe major take-away from these systematic reviews and recently published trials is that there is a glaring lack of adequately powered clinical trials of PA on health-related outcomes in persons living with T1D  significantly increases glucose disposal into skeletal muscle  effectively normalized nocturnal blood glucose levels and decreased the time spent in hypoglycemia (≤ 3.9 mmol/L) by >70%  inclusive research processes; (2) collaborating respectfully with stakeholder populations; (3) recognizing the value of patient experiences with conditions and; (4) conducting research informed by the needs of the patients . As therPatient engagement in PA and T1D research programs is in its infancy, relative to other areas of endocrine research. The two main goals for engaging patients in PA-based research are to improve health outcomes during interventions and optimizing the patient experience of health care . In termThe first step in the process of patient engagement for PA-related research for persons living with T1D is to set priorities from patients, caregivers and providers. Several recent examples for persons living with type 1 and type 2 diabetes serve as models for future research programs. One example is a large multinational survey study published in 2013 collected data from 16,000 individuals with a significant connection to type 2 diabetes  . The surIn 2012, data from a piloted intervention became available that tested the effectiveness of an iPhone application named “bant” to increase the frequency of blood glucose testing in youth . The appPerhaps one of the largest examples of patient engagement in T1D research is the D1 Now study in Ireland , 135. ThThe James Lind Alliance published the results from their T1D partnership in 2012 . This paA starting point for working with patient partners in PA and health outcomes research for persons living with T1D, would be to ensure that future studies include patient reported outcome measures and patient reported experiences, as they pertain to PA. With regards to patient reported outcome measures, simple tools including quality of life, diabetes-related quality of life and measures related to self-management are standard in other clinical trials. As PA can be viewed as burdensome to patients, potentially increasing the challenging of achieving target glucose control, these measures become even more important for providers and caregivers.The patient experience is a key element of quality of care and health systems improvement. The experience of a provider's confidence prescribing activity, ability to tailor management to meet the needs of individuals active lifestyle and ability to share in the decision making process around including PA in a person's self-management plan are examples of patient experiences that could limit engagement in regular daily PA. Our group intends to conduct an extensive scoping review of clinical trials published to date, to identify if any trials have been conducted that included patient-reported outcome measures, patient experiences or evidence of patient engagement in research related to PA. To the best of our knowledge, these experiences have yet to be captured within PA-based trials, therefore best practices for enhancing the experience of providing PA consultation to persons living with T1D remains unclear.We recognize that the studies described about were not systematically selected and therefore are at risk of a selection bias. However, we attempted to focus on the most robust, representative and clinically relevant studies published to date, in an effort to identify gaps in the most rigorously designed studies to date. Despite this limitation, among the studies described, we identified that the area of PA and health among persons living with T1D is rife with gaps that limit the uptake of this work into clinical practice . Across NK and JM designed the concept for the manuscript. NK, AM, and JM all contributed to writing and revising the manuscript. NK and AM reviewed abstracts and extracted data for the systematic review. All authors read and approved the final version.JM has received speaker fees from Medtronic Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.