This cohort study evaluates the association of deceased donor acute kidney injury with recipient graft survival and characterizes recovery and discard practices for kidneys with acute kidney injury by organ procurement organizations. Is deceased donor acute kidney injury (AKI) associated with recipient graft survival after matching on deceased donor AKI propensity?–matched cohort study of deceased donors with and without AKI, deceased donor AKI had no independent association with short-term and long-term recipient graft survival. Recovery and transplantation of AKI kidneys varied by organ procurement organization; most (39 of 58) had high recovery and high discard of AKI kidneys.In this registry-based, propensity scoreThis study’s findings suggest that the transplant community should continue to use deceased donor AKI kidneys and consider research to investigate whether currently discarded AKI kidneys can be used more effectively. The shortage of deceased donor kidneys for transplants is an ongoing concern. Prior studies support transplanting kidneys from deceased donors with acute kidney injury (AKI), but those investigations have been subject to selection bias and small sample sizes. Current allocation practices of AKI kidneys in the United States are not well characterized.To evaluate the association of deceased donor AKI with recipient graft survival and to characterize recovery and discard practices for AKI kidneys by organ procurement organizations.–matched cohort study from January 1, 2010, to December 31, 2013, in the United States. The dates of analysis were March 1 to November 1, 2019. From 2010 to 2013, a total of 6832 deceased donors with AKI and 15 310 deceased donors without AKI had at least 1 kidney transplanted. This study used a 1:1, propensity score–matched analysis to match deceased donors with AKI to deceased donors without AKI and investigated outcomes in their corresponding kidney recipients.Registry-based, propensity scoreDeceased donor AKI, defined as at least 50% or 0.3-mg/dL increase in terminal serum creatinine level from admission.Recipients were assessed for the time to death-censored graft failure and the following secondary outcomes: delayed graft function, primary nonfunction, and the time to all-cause graft failure.P < .001). Few recipients (120 of 25 323 [0.5%]) developed primary nonfunction regardless of deceased donor AKI status. Recovery and transplantation of AKI kidneys varied by organ procurement organization; most (39 of 58) had high recovery and high discard of AKI kidneys.Ninety-eight percent (6722 of 6832) of deceased donors with AKI were matched to deceased donors without AKI. The mean (SD) age of the 13 444 deceased donors was 40.4 (14.4) years, and 63% (8529 of 13 444) were male. A total of 25 323 recipients were analyzed , and their mean (SD) age was 52.0 (14.7) years. Recipients were followed up for a median of 5  years. Deceased donor AKI status had no association with death-censored graft failure  or all-cause graft failure . The results were consistent after examining by AKI stage and adjusting for recipient and transplant characteristics. More recipients of AKI kidneys developed delayed graft function (29% vs 22%, Deceased donor AKI kidneys transplanted in the study period had recipient graft survival comparable to that of non-AKI kidneys. This study’s findings suggest that the transplant community should evaluate whether currently discarded AKI kidneys from donors without substantial comorbidities can be used more effectively. As a result, the use of kidneys with certain risk factors  has increased in an effort to meet the demands of the waiting list.4 initiative was launched with the following 3 primary aims: to reduce the number of patients developing kidney failure, to decrease the number of US individuals receiving dialysis in dialysis centers, and to increase the number of kidneys available for transplant. In particular, the initiative aims to “double the number of kidneys available for transplant by 2030,”4(p3) which will require substantial changes to the organ procurement system.In 2019, the Advancing American Kidney Health5 However, AKI in the setting of deceased donor transplant may not be comparable. A variety of clinical actions can lead to serum creatinine (Scr) level fluctuations that meet criteria for AKI in deceased donors who typically do not have CKD, severe cardiovascular disease, or sepsis. In particular, vasopressin fluctuations during brain death can contribute to fluid losses and a number of hemodynamic changes,6 which do not necessarily lead to the intrinsic tissue injury commonly associated with CKD development.9 Kidneys procured from deceased donors may also have greater repair potential because the donors are typically younger and have fewer comorbidities than patients who experience AKI in settings in which the AKI leading to the CKD paradigm has been developed.12 Literature on repair biomarkers in the deceased donor setting suggests that donor injury can initiate repair processes and ischemic preconditioning, which may be beneficial at the time of transplant in the recipient.13 Taken together, kidneys from deceased donors with AKI could expand the pool of available kidneys for transplants.Kidneys from deceased donors with acute kidney injury (AKI) may help reduce the kidney shortage. In some clinical settings , AKI and chronic kidney disease (CKD) have been hypothesized to be interconnected syndromes.15 A limitation of the KDPI is that it is restricted to the terminal Scr level at the time of organ procurement. A single creatinine level cannot differentiate deceased donor AKI from preexisting CKD. Furthermore, the terminal Scr level obscures much of the heterogeneity of underlying etiologies that contributed to kidney injury.16Current kidney transplant practices are limited in their ability to characterize deceased donor AKI. In 2014, the United Network for Organ Sharing (UNOS) implemented a new Kidney Allocation System (KAS). Through the KAS, deceased donor organ quality is quantified through the Kidney Donor Profile Index (KDPI), which provides greater granularity over prior binary classifications of expanded vs standard criteria donors.26 Despite the overall evidence supporting transplants of AKI kidneys, current allocation practices of AKI kidneys by organ procurement organizations (OPOs) have not been well characterized. Deceased donor AKI has been shown to be strongly associated with kidney discard,18 although that study was limited to only 5 OPOs and does not represent national practices. Furthermore, relevant cohort studies30 are limited by selection bias and confounders. Because a true clinical trial evaluating the role of deceased donor AKI in recipient outcomes is infeasible, we mimicked a clinical trial using registry data from DonorNet and the Organ Procurement and Transplantation Network (OPTN) to match deceased donors with and without AKI and to evaluate the association of deceased donor AKI with recipient graft survival. We then characterized current recovery and discard practices of AKI kidneys across the United States.Evidence from single-center and multicenter cohorts largely supports the use of selected kidneys from deceased donors with AKI.–matched cohort study was performed from January 1, 2010, to December 31, 2013. The dates of analysis were March 1 to November 1, 2019. From 2010 to 2013, a total of 6832 deceased donors with AKI and 15 310 deceased donors without AKI had at least 1 kidney transplanted. We used DonorNet data from the OPTN system, which registers deceased donors and communicates organ offers. The OPTN system includes data submitted by OPTN members on all donors, wait-listed candidates, and transplant recipients in the United States. The Health Resources and Services Administration (HRSA) of the US Department of Health and Human Services provides oversight to the activities of the OPTN contractor. The OPTN data from 2010 to 2013 have previously been validated with medical record–abstracted data for delayed graft function (DGF) and recipient graft survival from 5 OPOs.31 Validation of serial Scr levels from DonorNet with medical record–abstracted Scr levels from the same 5 OPOs is summarized in eTable 1 in the 33 The study was approved by the HRSA and the institutional review boards at participating institutions under a waiver of consent because deidentified data were used. The clinical and research activities reported herein are consistent with the Principles of the Declaration of Istanbul as outlined in the Declaration of Istanbul on Organ Trafficking and Transplant Tourism.This US registry-based, propensity score34 This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.Of the available 32 437 deceased donors in DonorNet from 2010 to 2013, we excluded donors with only 1 Scr level available (n = 735), donors who had both kidneys transplanted to 1 recipient (n = 1238), donors younger than 16 years (n = 1766), donors whose informed consent was not available (n = 308), and donors whose kidneys were recovered for reasons other than transplant (n = 148), for a total analytic data set of 28 242 deceased donors. We then further excluded donors who had both kidneys discarded (n = 6100), for a final analytic sample of 22 142 donors, 6832 of whom had AKI . The enc35  Scr level criteria as follows: stage 1 , stage 2 , and stage 3 , irrespective of urine output or initiation of dialysis because these data were not available.DonorNet provides multiple serial measurements of the Scr level within a given date during hospitalization for each donor. We used the lowest Scr level on the hospital admission date and the highest Scr level on the terminal date (date of nephrectomy). Deceased donor AKI was defined as at least a 50% increase in the terminal Scr level from admission or an absolute increase in the terminal Scr level of at least 0.3 mg/dL , irrespective of urine output (because it was not available in the data set). Stages of AKI were defined by KDIGO2 or less, within the first 90 days after the first week. Six-month eGFR used the Scr level reported on the UNOS 6-month follow-up form31 and was calculated with the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.36 All-cause GF was defined as all-cause mortality, return to dialysis, or retransplantation.The primary outcome of interest was the time to death-censored graft failure (dcGF) from the transplant date. Secondary outcomes of interest were DGF, primary nonfunction (PNF), 6-month renal function, and the time to all-cause graft failure (GF). Delayed graft function was defined as any dialysis in the first week after transplant. Primary nonfunction was defined per UNOS as kidney graft removal or as any dialysis or a creatinine clearance or estimated glomerular filtration rate (eGFR) of 20 mL/min/1.72 m37 We examined standardized differences and created plots to assess the balance of continuous baseline covariates to investigate whether the propensity score was adequate.We completed a propensity score–matched analysis in which the propensity score was composed of deceased donor characteristics of age, sex, race, body mass index, hypertension, diabetes, donation after cardiovascular determination of death, admission Scr level, and hepatitis C seropositivity. Deceased donors with AKI were propensity score matched 1:1 to deceased donors without AKI based on the logit of the estimated propensity score using a greedy matching algorithm. The matching used a caliper of 0.2 of the SD of the logit of the estimated propensity score.39Continuous variables were reported as the median (interquartile range [IQR]) or the mean (SD). Differences in demographic characteristics, clinical characteristics, and recipient outcomes by deceased donor AKI were compared using standardized differences.40 Because a donor may have 1 or 2 recipients, we estimated 95% CIs using robust sandwich estimates to account for the paired nature of the data.41We used Cox proportional hazards regression models to assess the associations of deceased donor AKI with dcGF and all-cause GF. We adjusted Cox proportional hazards regression models for cold ischemia time and the following recipient characteristics: age, sex, black race, diabetes as the cause of recipient ESRD, preemptive transplant , previous kidney transplant, HLA mismatch level, panel reactive antibody (percentage), and body mass index. The proportional hazards assumption was evaluated by the Kolmogorov-type supremum test.43 As a confirmatory analysis, we conducted an unmatched analysis of the entire cohort using inverse probability treatment weighting of the propensity score.44With a sample size of approximately 20 000 recipients of whom 30% received kidneys from deceased donors with AKI, we had more than 90% power to detect a hazard ratio (HR) of 1.15 with an overall event rate of 14%.P < .05. Analyses were conducted in SAS, version 9.4 (SAS Institute Inc) and Stata, version 14 (StataCorp LLC).All inference testing was 2-sided with a significance level of To characterize allocation practices for AKI kidneys at the OPO level, we examined the proportion of AKI kidneys recovered and the proportion of AKI kidneys transplanted. To examine AKI kidney recovery patterns, we restricted the analytic data set of 28 242 deceased donors to those with AKI, for a final analytic sample of 10 275 deceased donors with AKI, yielding 20 550 kidneys available for recovery . We calcTo examine AKI kidney transplant patterns, we looked at the analytic data set of 41 487 kidneys transplanted. We calculated the proportion of AKI kidneys transplanted at both the OPO level and the transplant center level and then mapped the results.https://gis.hrsa.gov/arcgis/rest/).45 All maps were created using ArcGIS Pro .Map files containing donation service area shapefiles were accessed from HRSA map services (We matched 98% (6722 of 6832) of deceased donors with AKI to deceased donors without AKI . The meaMost of the 6772 deceased donors with AKI had stage 1 (4621 [69%]), followed by stage 2 (1409 [21%]) and stage 3 (692 [10%]). Deceased donors without AKI were more likely to have both kidneys transplanted than deceased donors with AKI . Additional deceased donor and recipient characteristics stratified by stages of AKI are listed in eTable 4 in the Follow-up time was similar among recipients regardless of deceased donor AKI status as follows: 4.77  years for no AKI, 4.82  years for stage 1, 4.76  years for stage 2, and 4.77  years for stage 3. Duration of follow-up, recipient sex, black race, and HLA mismatch level were comparable by deceased donor AKI status . RecipieP < .001) (P = .03) but no clear pattern in the proportion of recipients who developed PNF by stages of AKI, with rates of 63 (0.5%), 32 (0.4%), 12 (0.5%), and 13 (1.0%) for no AKI, stage 1, stage 2, and stage 3, respectively. Six-month renal function was statistically significantly different (P < .001) but not meaningfully different across stages of AKI, with mean (SD) 6-month eGFRs of 61 (22), 58 (22), 57 (22), and 62 (22) mL/min/1.73 m2 for no AKI, stage 1, stage 2, and stage 3, respectively.Among recipients of kidneys from deceased donors with AKI, 29% (3643 of 12 513) developed DGF compared with 22% (2779 of 12 810) of recipients of kidneys from deceased donors without AKI  . DelayedP = .69) . When further examined by stages of AKI , there wP = .32) . Further analyzing by AKI stage , there wThe results from inverse probability treatment weighting among the entire cohort are consistent with the results from the propensity score–matched analysis  were recovered during the study period, and 12 711 AKI kidneys were transplanted. As a result, 3030 kidneys from deceased donors with AKI were never procured, and 4757 of 17 468 (27%) AKI kidneys were discarded after recovery. Kidneys with AKI accounted for almost half (4757 of 10 075) of all discarded kidneys during this period. The median proportions of recovery and subsequent discard were 87.7%  and 26.1% , respectively. A scatterplot of all recovery and discard proportions of AKI kidneys by OPOs is shown in eFigure 3 in the Among the 58 OPOs from 2010 to 2013, the lowest tertile of recovery was 77%, and the lowest tertile of discard was 22%. In this registry-based, propensity score–matched study, deceased donor AKI was associated with increased risk for DGF but not with dcGF or all-cause GF. These findings were consistent when further examined by stages of AKI. Despite the evidence in support of transplanting selected AKI kidneys, there is variation in the recovery and transplant of AKI kidneys. The transplant community may need to consider innovative strategies and research regarding the use of kidneys from deceased donors with AKI in settings of additional comorbidities that are typically associated with kidney discard.46 that indicate deceased donor AKI is associated with increased risk for DGF. Differences in risk were observed for PNF, but rates remained 1% or less across all stages of AKI. No differences were observed for dcGF and all-cause GF. Although DGF requires interventions  during initial follow-up,47 the lack of sustained risk suggests that recovery after DGF is comparable between AKI and non-AKI kidneys, without substantial long-term sequelae.Our findings for DGF are consistent with prior studies26 A detailed review of more than 585 AKI kidneys transplanted from 2010 to 2013 across 13 transplant centers showed comparable outcomes, with 1845 kidneys that did not have AKI, even at the highest stages of deceased donor AKI.17 These findings are congruent with the pooled findings in a systematic review and meta-analysis by Zheng et al,46 who found that recipient graft survival of 22 487 kidneys across 14 cohorts was indistinguishable by deceased donor AKI status. The only exception within the meta-analysis was a registry study48 from the United Kingdom, which found modestly inferior recipient outcomes after transplant of stage 3 AKI kidneys. However, the kidney discard rate in the United Kingdom is 150% lower than that in the United States (10% vs 25%),48 suggesting that the elevated risk seen with stage 3 AKI kidneys, if present, is limited to a small subgroup with high severity of disease.In this study, associations of deceased donor AKI with graft outcomes were consistent across stages of AKI, which is largely consistent with prior studies.26 the concerns raised by the registry study48 in the United Kingdom regarding stage 3 AKI kidneys should not deter recovery and transplants of AKI kidneys. Instead, the findings from the United Kingdom should prompt additional safeguards and development of interventions to ensure allograft success. Examples include caution when transplanting AKI kidneys into recipients sensitive to volume overload and risk of heart failure, careful review of procurement biopsy specimens to avoid AKI kidneys with extensive cortical necrosis, and further research into ischemic preconditioning,49 better repair and recovery biomarkers,13 and development of donor-level interventions before nephrectomy and transplant.47 Our results indicate a slightly protective association of stage 3 AKI for all-cause GF, which is most likely explained by increased precautions and closer follow-up. Furthermore, stage 3 accounts for less than 20% of deceased donor AKI in the United States. The transplant community should embrace the use of the 80% of AKI kidneys with less severe injury (stage 1 or stage 2), while advancing transplants of stage 3 AKI kidneys with appropriate safeguards and considerations.In light of the overall evidence supporting the use of AKI kidneys across studies,50 followed by a second cohort of 36 transplant centers,51 to evaluate and increase the use of high-KDPI kidneys. Stewart et al52 recently used SimUNet, a DonorNet offer simulator, to investigate whether biopsy findings substantially and variably alter kidney offer acceptance decisions for deceased donors with and without AKI. Additional SimUNet approaches could be considered to help further understand the consequences of deceased donor AKI. For example, acceptance rates for deceased donor AKI kidneys that provide KDPIs based on admission Scr levels could be compared with current KDPIs that use the terminal Scr level alone.Another important finding of our study is that AKI kidney allocation practices vary by OPO. Although there is no consensus regarding the allocation of AKI kidneys, these kidneys can be carefully incorporated into the transplant system through pilot studies, such as the Collaborative Improvement and Innovation Network (COIIN) project. In 2017, a total of 19 transplant centers participated in the first phase of COINN,48 only 172 deceased donors had stage 3 AKI from 2003 to 2013. In our propensity score–matched analysis, 692 deceased donors from 2010 to 2013 had stage 3 AKI. Second, we confirmed our findings with the entire cohort through inverse probability treatment weighting, which provides reassurance for the generalizability of our findings. Third, DonorNet provides granular information on admission Scr level, which allowed rigorous clinical classification of AKI and its severity that is more consistent with current international guidelines.35Our study has several strengths. First, this is a large, registry-based study encompassing all kidney transplants in the United States over a 3-year period. Prior studies have been limited by small sample sizes, particularly for the number of deceased donors with stage 3 AKI. In the United Kingdom registry,37 Second, our maps are based on DonorNet data from 2010 to 2013. In December 2014, the KAS was introduced,15 and deceased donor kidney transplantation has increased since its implementation. However, the KAS did not substantially alter kidney discard rates, which remained approximately 20%.14 Third, posttransplant kidney function data beyond 6 months were not available. Fourth, our calculations for recovery and discard only capture AKI-associated recovery and discard. The OPO-level decisions are multifactorial and depend on the availability of transplant centers willing to accept the kidneys in question. In fact, a large number of deceased donor kidney offers are declined on behalf of transplant candidates.53 Our analysis does not account for the other factors and regulatory bodies involved in transplant decisions.There are several limitations of our study to consider. First, our results do not provide the true strength of a randomized clinical trial. However, propensity score methods are a popular tool to align baseline characteristics between groups similar to randomization in clinical trials.This study found that the AKI kidneys transplanted in the United States from 2010 to 2013 had comparable rates of recipient graft survival, even among the highest stages of injury. There was OPO-level variation in the allocation practices of AKI kidneys. From our study’s findings, we believe that the transplant community should continue to use deceased donor AKI kidneys and consider research to investigate whether currently discarded AKI kidneys from deceased donors without substantial comorbidities can be used more effectively.


U-test and Kruskal–Wallis test were used for statistical analyzes. EVs isolated from the CSF were more abundant in patients with progressive MS and in those with a clinically isolated syndrome than in all the other groups examined. Furthermore, an important change in the number of EVs and in their surface marker expression occurred during active phases of MS . In particular, the number of CSF-EVs increased in patients affected by MS during clinical relapse; this finding was associated with a decrease in the number of CD19+/CD200+ (naïve B cells) EVs. These markers are expressed by immature and naïve B lymphocytes, and to the best of our knowledge, this double staining has never been associated with MS, but their reduction has been observed in patients with another type of Th1 cell-mediated autoimmune disease. In contrast, the presence of lesions in the brain and spine on gadolinium-enhanced MRI was associated with an increase in the numbers of CCR3+/CCR5+ (subset of CD8 memory T cells), CD4+/CCR3+ (Th2 cells), and CD4+/CCR5+ (Th1 cells) CSF-EVs. Two points are worth emphasizing: (i) the data obtained in this study confirm that CSF-EVs represent a potentially promising tool to identify biomarkers specific for different phases of MS; and (ii) Considering the role of EVs in intercellular communication, our results provide some insights that improve our understanding of the relationships among some of the cell types that are mainly involved in MS pathogenesis .This study was designed based on the hypothesis that changes in both the levels and surface marker expression of extracellular vesicles (EVs) isolated from the cerebrospinal fluid (CSF) may be associated with the clinical form, disease activity, and severity of multiple sclerosis (MS). The analyzes were performed on subjects affected by MS or other neurological disorders. EVs, which were isolated by ultracentrifugation of CSF samples, were characterized by flow cytometry. A panel of fluorescent antibodies was used to identify the EV origin: CD4, CCR3, CCR5, CD19, and CD200, as well as isolectin IB4. The Mann–Whitney  Extracellular vesicles (EVs) are small double lipid membrane vesicles that are released by all cell types. These specialized structures were recently shown to be involved in intercellular communication via both autocrine and paracrine signaling. EVs represent a method for transferring information between cells  located in close proximity or in distant sites through biological fluids . Indeed,Two different types of EVs have been identified based on size and the mechanism of formation: exosomes, which are derived from the multivesicular endosomal cell compartment, and membrane-derived vesicles, which are formed by direct budding from the plasma membrane of the cell . EVs conSeveral studies identified EVs in the CSF and described their roles in both physiological and pathological conditions, such as stroke, inflammatory neurodegenerative diseases and multiple sclerosis (MS) . EVs havMultiple sclerosis is an autoimmune disorder of the central nervous system (CNS) that is associated with demyelination and neurodegeneration . The cliVarious studies conducted on the EVs isolated from both blood and CSF of subjects affected by MS have revealed an important role for these organelles in the pathogenesis of the disease . EVs rel+ EV level was detected in the CSF from patients with RRMS than in healthy controls, and the EV concentration was correlated with gadolinium-positive lesions, a sign of active disease  the concentrations of EVs in the CSF from patients with MS who were diagnosed according to 2010 McDonald criteria  with sub the concWe included individuals affected by MS or OND that had a planned lumbar puncture for their diagnostic work-up during the period from February 2012 to September 2015. None of the patients had received any disease-modifying drug treatment 1 month prior to the lumbar puncture. For the purpose of the study, we collected 15–20 ml of CSF according to the international guidelines . SubjectThis research project was approved by the Ethics Committee of Palermo University, and all subjects provided written informed consent.In the 15 days before the lumbar puncture, all patients with MS and CIS underwent MRI of the brain with a 1.5 T scanner Signa HDxt  using standardized procedures. The maximum lag-time of 15 days between MRI and lumbar puncture was planned to minimize the risk that the data obtained from MRI and lumbar puncture represented two different phases of the disease, because the mean lifetime of a gadolinium-enhanced lesion is approximately 30 days . The neug for 15 min at room temperature. The supernatant was centrifuged at 1,730 × g for 15 min at room temperature and then frozen at -80°C until use.Samples of 15–20 ml of CSF were collected in polypropylene tubes. Each sample was centrifuged at 432 × g for 90 min at 4°C. The pellet was resuspended in 10 ml of PBS and the sample was immediately frozen and stored at -80°C until use.The final supernatant was diluted in PBS to a final volume of 40 ml, and EVs were purified by ultracentrifugation at 105,000 × The number of obtained EVs was determined by flow cytometry with a FACSCanto instrument . One microliter of EVs was diluted in a fixed volume of 200 μl of filtered PBS (0.1 μm filter); all samples were analyzed by FACS for 30 s at medium flow rate. Gating was performed as described by Samples were immunostained with a panel of fluorescent dye-conjugated antibodies, and the presence and concentration of specific markers were detected by flow cytometry to identify the cells that released EVs. In particular, we used the following markers in the FACS analysis: FITC-conjugated anti-CD19, PeCy-7-conjugated anti-CD200, Alexa Fluor 488-conjugated anti-CD4, APC-conjugated anti-CCR3, PerCpCY-5-conjugated anti-CCR5, and FITC-conjugated isolectin IB4. Please see Table 2+) and incubated with 2 μl of IB4 for 30 min at 4°C in the dark. EVs were then washed twice, resuspended in 200 μl of filtered PBS with Ca2+ and analyzed by flow cytometry.One microliter of EVs was diluted in a fixed volume (200 μl) of filtered FACS Buffer  and incubated with 1 μl of the different antibodies for 45 min at 4°C in the dark. EVs were then washed by ultracentrifugation, resuspended in 200 μl of filtered PBS and analyzed by flow cytometry. The staining of the selected CDs/CCRs was analyzed in two different experimental sets, according to the fluorochromes used: (1) staining for CCR3, CCR5, and CD4, and (2) staining for CD19 and CD200. For IB4 staining, 1 μl of EVs was diluted in a fixed volume (200 μl) of filtered FACS Buffer , conjugated with the same labeled IgG isotype as the primary antibodies. IgG isotypes were also used as a negative control to determine the fluorescence background. All samples were analyzed by FACS for 30 s at medium flow rate.All antibodies were purchased from eBioscience, and isolectin IB4 was purchased from Life Technologies . The results obtained were analyzed with FlowJo V10.1 software (FlowJo LLC).U-test was used to compare two subgroups of patients, and the Kruskal–Wallis test was used to compare three or more subgroups of patients. SPSS software  was used for statistical analyzes. The two-tailed alpha level was set to p < 0.05 to indicate a significant difference. The data were processed using GraphPad Prism software version 7.04 .Means or medians and the corresponding ranges were used to describe the distributions of all variables. Non-parametric tests were applied for statistical analyzes; in particular, the Mann–Whitney We obtained EVs from the CSF of 39 subjects with MS, 2 subjects with CIS, and 18 subjects with OND. The 39 subjects with MS were classified according to the 2013 Lublin criteria as follows : 35 withWithin the rMS subgroup, 19 patients had experienced at least two clinical attacks, 14 reported one clinical attack but, according to the 2010 McDonald criteria , were diThe yield of EVs at the end of the isolation procedure did not permit us to perform all of the planned characterizations of EVs on the entire sample. Consequently, for each type of analysis, a table with the characteristics of each individual study population will be shown.1). Next, we observed that the majority of EVs analyzed on the NanoSight NS300 instrument under light scatter mode showed a size distribution ranging from 105 to 405 nm   to confirm their identity. Flow cytometry assays showed that CSF-EVs vary in size but are ≤1 μm in diameter  , NTA, elp = 0.49).We assessed EV concentrations (EV/ml) in whole CSF samples from 59 subjects to determine whether patients with MS and patients with OND had different levels of EVs. Figure U-test showed a significant difference between groups, with p = 0.02.Subsequently, we evaluated the concentration of CSF-EVs in the rMS subgroup, according to different clinical and instrumental parameters . Figure We did not observe significant differences among the other subgroups analyzed (data not shown).We proceeded from the hypothesis that EVs present in the CSF of patients diagnosed with different clinical forms of MS and OND displayed dissimilar biomarkers on their surface. This information might be relevant to monitor the progression of the disease and to discriminate between MS and OND. The evaluation of this hypothesis was conducted by monitoring biomarker levels present in circulating CSF-EVs from patients with different diseases.+ EVs in the rMS, pMS, CIS, OIND, and ONIND groups. No statistically significant differences emerged from the comparison among the studied groups (p = 0.83), similar to the comparison among the other selected clinical and instrumental parameters in the rMS subgroups (data not shown). In contrast, after stratifying subjects with rMS according to clinical relapse, we observed a significant increase in the number of myeloid CSF-EVs in patients with stable rMS . For this reason, we determined the concentration of EVs positive for several CDs in the CSF of 25 patients with MS, 2 patients with CIS, and 13 controls using FACS analyzes; the demographic and clinical features are summarized in Table h2 cells .+, CCR3+, CCR5+, CD4+/CCR3+, CD4+/CCR5+, and CCR3+/CCR5+ CSF-EVs in all examined groups. We did not observe statistically significant differences in the investigated markers among the five studied groups. Furthermore, after pooling the rMS subgroup according to some of the selected clinical and instrumental parameters , similar concentrations of all markers were observed (data not shown). Additionally, after stratifying subjects with rMS according to clinical relapse, we did not obtain any statistically significant differences in the levels of these markers , of CCR5+ (p = 0.042), CD4+/CCR3+ (p = 0.018), CD4+/CCR5+ (p = 0.004), and CC3+/CCR5+ (p = 0.004) CSF-EVs  CSF-EVs compared to CD4+/CCR3+ (Th2) CSF-EVs in patients with gadolinium-positive lesions . Instead, we observed a statistically significant reduction in the number of CD19+/CD200+ CSF-EVs in subjects with in the course of a clinical relapse (p = 0.030)  Figure , whereas) Figure –I.The aims of this work were to confirm the presence of circulating EVs in CSF, to identify some of the cells from which they originated, and to investigate the possible use of these CSF-EVs as biomarkers for both the diagnosis and determination of the active status of MS.First, consistent with the literature, our data confirm the presence of EVs in the CSF  on EVs. One of the first examined markers was IB4, an isolectin that labels microglia Figure . Our rese Figure . The dif+ T lymphocytes play a central role in the pathogenesis of MS, predominating in acute lesions . All subjects provided written informed consent in accordance with the Declaration of Helsinki.FG, PR, GSc, GSav, and GSal conceived and designed the experiments. FG, MB, and EA performed flow cytometry analyzes. PR, SR, MM, GV, GSav, and GSal selected patients, performed lumbar puncture and collected and stored cerebrospinal fluid samples. PR, GSav, and GSal analyzed the data. FG, PR, and GSal wrote the manuscript. All authors have read and approved the final version of the manuscript.The 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.


Background: Nusinersen is an antisense-oligonucleotide (ASO) approved for treatment of 5q-spinal muscular atrophy (SMA). Since the drug cannot cross the blood-brain barrier (BBB), it must be administered into the cerebrospinal fluid (CSF) space repeatedly by lumbar puncture. However, little is known whether ASOs have an impact on CSF routine parameters that may yield information on CSF flow and/or intrathecal inflammation. The objective of this study was to examine CSF routine parameters in SMA patients treated with nusinersen.Methods: Routine CSF parameters  of 60 SMA patients  were retrospectively analyzed.Results: White cells ranged from 0 to 4/μL in CSF; a singular case of pleocytosis (8/μL) was observed in a patient in parallel with a systemic infection. Total protein and Qalb showed a mild increase from baseline to the following lumbar punctures . Lactate levels revealed a stable course. In one patient, positive OCB in CSF were transiently observed. The slight change in total CSF protein and Qalb may be caused by repeated lumbar puncture and/or intrathecal administration of the drug.Conclusion: Our data suggest that a regular examination of routine CSF parameters in patients in which intrathecal ASOs are administered is important to obtain information on possible side effects and to gain further insights into intrathecal processes. SMN) 1 gene on chromosome 5 leading to muscle weakness and muscle atrophy  is a rare neuromuscular disease caused by homozygous deletion or point mutation in the survival of motoneuron  and all patients or their relatives  gave informed written consent to participate in the study.Patient samples were collected from June 2017 to April 2019 at the following centers of the MND-NET (German Network for Motoneuron Diseases): Department of Neurology, Ulm University Hospital (Germany), Department of Neurology, Technische Universität Dresden (Germany), Department of Neurology, University Medical Center Göttingen (Germany) and Department of Neurology, Klinikum rechts der Isar der Technischen Universität München .SMN1 gene). Patients history and clinical data were gathered before therapy started. Motor scores .Although there was a statistical difference between the white cell count of T1 and T2 . In the remaining 48 patients, total protein levels were detected below the reverence boarder of 450 mg/L in baseline CSF.p = 0.0004), T1–T3 (p < 0.0001), T1–T4 (p = 0.0012), T1–T5 (p < 0.0001), T1–T6 (p = 0.0001), T1–T7 (p = 0.0010), and T1–T8 (p = 0.0195)]. While a mild increase in total protein could be observed from baseline (T1) to treatment days T2, T3, T5, T6, T7, and T8, median total protein levels were slightly lower at T4 compared to baseline protein levels.CSF total protein differed significantly from baseline (T1) to all following lumbar punctures ; white cell count in this patient was within the normal range, lactate levels minimal elevated at T4 (2.2 mmol/L) and OCB were not determined. Apart from a pronounced scoliosis, there were no further anamnestic or clinical abnormalities in this patient, neither before nor during therapy. The highest total protein (715 mg/L) along with the highest (age-related) Qalb level (11.4 × 103) in baseline CSF (T1) was detected in a 15-year-old SMA type 3 patient, in whom positive OCB in CSF (type 2) in baseline and follow-up samples were observed. In this patient, total protein slightly increased to 913 mg/L in the second lumbar puncture (T2), but showed a stable course in the following lumbar punctures . A medical history for inflammatory CNS diseases was not reported by the patient and no clinical features beyond the flaccid amyotrophic proximal tetraparesis were noticed. Because white cell counts and lactate levels were normal, we classify the findings in this patient as immunological scar due to a former infection.The highest total protein levels of all CSF samples  were detected in a 46-year-old SMA type 3 patient with baseline total protein of 710 mg/L (T1), who showed the highest (age-related) Qalb levels of all samples in accordance . At T8 median Qalb levels were lower compared to baseline (T1) (p = 0.0469), however, only 8 patient samples could be evaluated at T8.Qalb was elevated in 14 SMA patients at baseline (T1). Qalb increased from baseline (T1) to the following six lumbar punctures .Qalb did not differ significantly from baseline (T1) to the following lumbar punctures in this subgroup . Our data suggest that a systematic examination of routine CSF parameters in patients in which intrathecal ASOs are administered is important to obtain information on possible side effects and to gain further insights into intrathecal processes.In summary, the examination of CSF routine parameters in 60 SMA patients  treated with nusinersen revealed that repeated intrathecal administration of the drug seems to be in general safe and well-tolerated. This is in line with previous but limited results from clinical phase 1 studies on intrathecal delivery of ASOs in human –42. NeveThe datasets generated for this study will not be made publicly available as it concerns patient data.The studies involving human participants were reviewed and approved by University of Ulm approval number 19/12; 2012. Written informed consent to participate in this study was provided by the patients and/or patients' legal guardian/next of kin.CW and RG designed the study. CW and JD performed statistical analysis. CW wrote the manuscript. All authors, were involved in the data collection process analyzed global study data and revised and approved the final version of the manuscript.The authors declare the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: CW has received honoraria from Biogen as advisory board member and for lectures and as consultant from Hoffmann-La Roche. She also received travel expenses from Biogen. JK has received financial research support from TEVA Pharmaceuticals and honoraria as speaker/consultant for AbbVie, Allergan, Biogen, Ipsen, and AveXis/Novartis. MO received honoraria as consultant for Biogen, Axon, and Fujirebio. ZU has received honoraria from Biogen as consultant. BW has received honoraria from Biogen for a lecture. PW has received honoraria from Biogen and Desitin for lectures and as a consultant from Hoffmann-La Roche and Abbvie. AL received financial research support from AB Science, Biogen Idec, Cytokinetics, GSK, Orion Pharma, Novartis, TauRx Therapeutics Ltd. and TEVA Pharmaceuticals. He also has received honoraria as consultant from Mitsubishi, Orion Pharma, Novartis, Teva and as advisory board member of Biogen, Treeway, and Hoffmann-La Roche. MD received a travel grant from Biogen and speaker honoraria from Desitin and Genzyme. PL has received financial research support from TEVA Pharmaceuticals and honoraria as speaker/consultant for AbbVie, BIAL, Desitin,Licher MT, Medtronic, Novartis. HT has received research support and/or honoraria as speaker/consultant/advisory board member from Bayer, Biogen, Celgene, Fresenius, Genzyme Sanofi, Merck, Mylan, Novartis, Roche Diagnostics, Teva. AH has received honoraria from Biogen and Desitin as advisory board member. RG has received honoraria from Biogen as advisory board member. 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.


Diarrhea and acute respiratory infections (ARI) account for 30% of deaths among children displaced due to humanitarian emergencies. A wealth of evidence demonstrates that handwashing with soap prevents both diarrhea and ARI. While socially- and emotionally-driven factors are proven motivators to handwashing in non-emergency situations, little is known about determinants of handwashing behavior in emergency settings.We conducted a qualitative investigation from June to August 2015 in a camp for internally displaced persons with a population of 6360 in the war-torn eastern region of the Democratic Republic of Congo. We held key informant interviews with 9 non-governmental organizations and camp officials, in-depth interviews and rating exercises with 18 mothers of children < 5 years, and discussions with 4 groups of camp residents and hygiene promoters to identify motivators and barriers to handwashing.At the time of the study, hygiene promotion activities lacked adequate resources, cultural acceptability, innovation, and adaptation for sustained behavioral change. Lack of ongoing provision of hygiene materials was a major barrier to handwashing behavior. When hygiene materials were available, camp residents reported that the primary motivator to handwashing was to prevent illness, particularly diarrheal disease, with many mentioning an increased need to wash hands during diarrhea outbreaks. Emotionally- and socially-related motivators such as “maintaining a good image” and social pressure to follow recommended camp hygiene practices were also reported to motivate handwashing with soap. Residents who engaged in day labor outside the camp had limited exposure to hygiene messages and handwashing facilities. Interviewees indicated that the harsh living conditions forced residents to prioritize obtaining basic survival needs over good hygiene.Hygiene promotion in camp settings must involve preparedness of adequate resources and supplies and ongoing provision of hygiene materials so that vulnerable populations affected by emergencies can apply good hygiene behaviors for the duration of the camp’s existence. Compared to non-emergency contexts, illness-based messages may be more effective in emergency settings where disease poses a current and ongoing threat. However, failure to use emotive and social drivers that motivate handwashing may present missed opportunities to improve handwashing in camps. Diarrhea and acute respiratory infections (ARI) account for about 30% of deaths among children under five years living in refugee camps . In acutIn the non-emergency context, socially- and emotionally-driven factors, such as nurture, disgust, cleanliness, and affiliation, are important motivators to individual handwashing behavior , 12, andThe Integrated Behavioral Model for Water, Sanitation and Hygiene (IBM-WASH) employs three intersecting dimensions, including the contextual, psychosocial and technological dimensions, that operate on five levels  to influence WASH behaviors . This coIn a camp of internally displaced persons (IDP) in the Democratic Republic of Congo (DRC), we conducted a qualitative investigation to identify motivators and barriers to handwashing from an emic perspective. We also aimed to examine hygiene promotion activities carried out in the camp, availability of handwashing technologies and materials and perceptions regarding their appropriateness, hygiene and handwashing knowledge and behaviors prior to entering the camp, and how the camp conditions and sociocultural environment affected handwashing behaviors. Findings were used to inform trials of handwashing interventions in the camp environment .The study was conducted from June to August 2015 in Camp Kishusha, an IDP camp in Rubaya, North Kivu Province, in the war-torn eastern region of the DRC. The camp is situated on steep mountainous terrain where rain falls from August to May. Residents entered Kishusha in three waves perpetuated by escalations in hostilities, starting in July 2012 through October 2013. The camp was densely populated with people from different ethnic groups who had formally been in conflict sharing neighborhoods and camp facilities. Languages spoken were Kinyarwanda, Kihunde, Kiswahili, and French.At the time of the study, the camp hosted 1328 families and 6360 residents and was demarcated into 49 blocks, with 25–30 households per block. Families lived in small tarpaulin-made tents spaced a few feet apart. Each block was supposed to have banks of functioning pit latrines shared by multiple families, with communal handwashing stations next to latrines. There were 57 functional communal pit latrine banks (each consisting of 2–4 latrine doors) throughout the camp, resulting in an average of one latrine bank per 112 residents. Several water pumps were available in the camp. Since the inception of the camp, different non-governmental organizations (NGOs) had provided residents with foodstuffs and basic needs, including materials for handwashing such as bar soap and water receptacles. The camp had experienced periodic cholera epidemics with the most recent occurring about three months prior to our study.We employed complementary qualitative data collection methods as described below.Key informant interviews included NGO representatives implementing WASH activities, camp residents participating in camp governing and hygiene committees, and hygiene promoters overseeing routine hygiene activities. Eligible NGOs involved those promoting hygiene, as well as funding or coordinating organizations working at regional or country levels on WASH. Potential categories of NGO respondents included hygiene promotion managers and senior WASH staff coordinating activities with extensive experience in camp settings. Initial informants were selected purposively, and we subsequently used snowball sampling to identify additional informants.With key informants we examined hand hygiene practices in the camp, handwashing promotion strategies, monitoring and evaluation of handwashing promotion activities, and coordination of handwashing activities with other organizations working in emergency settings. Aspects of handwashing promotion that were examined included factors that guided the development and evolution of the handwashing behavior change strategy, selection, distribution and acceptability of the handwashing hardware, perceived comprehensibility and persuasiveness of the communication messages, and challenges related to the implementation of handwashing activities.Using a freelisting approach, at the end of each key informant interview we asked informants to list motivators or factors that stimulate handwashing behaviors. Specifically, we explained that we were interested in learning what motivates camp residents to wash their hands, with research assistants presenting examples of potential handwashing motivators to ensure that informants understood the exercise. Research assistants introduced the topic by stating, ‘We know that people wash their hands for different reasons, such as to feel good, feel clean or get rid of a bad odor, can you tell us why people in the camp wash their hands?’ Once an initial list of motivators was generated, we used probing techniques to elicit additional motivational drivers of handwashing until the list was exhaustive. After lists were generated by all of the key informants, we assessed the frequency by which each motivator was listed. A final list of seventeen of the most frequently listed motivators to handwashing was combined with three emotional drivers including ‘disgust,’ ‘attract other people’ and ‘habit’ proven effective in non-emergency contexts , 14. SubIn-depth interviews were conducted with female caregivers of young children under five years old to explore knowledge of handwashing, past and current handwashing practices, motivators and barriers to handwashing, and perceptions of the handwashing promotion strategy in the camp. We purposively targeted females due to their role as primary child caregivers and responsibilities related to food preparation and maintaining household hygiene. We selected four blocks geographically separated in the camp. Interviewers were requested to find a central location in the block and spin a bottle to identify a household where they inquired whether a caregiver meeting our criteria was available to participate. If the approached household did not have a person eligible or willing to participate, the interviewer moved to the closest household to the left. When a potential respondent was identified, verbal informed consent was obtained and an interview scheduled. After the interview, the researcher approached another household five households to the left. This process continued until five mother-respondents in each block were interviewed. We anticipated administering 20 in-depth interviews or until reaching data saturation.After in-depth interviews, rating exercises to assess perceptions of handwashing motivators were conducted with the same women. The structured rating task has been used successfully in communities with high rates of illiteracy . Prior tGroup discussions were carried out with separate groups of female caregivers of children under five years of age, male heads of households, and camp residents overseeing hygiene promotion activities. Main topics explored included motivators and barriers to handwashing and perceptions of the handwashing promotion strategy in the camp. Another purpose was to share preliminary findings from the key informants and mother-respondents and elicit recommendations to improve behavioral change approaches. Participants in groups of female caregivers and male household heads were selected in the four blocks where in-depth interviews were conducted with assistance from camp hygiene committee members and promoters and block leaders. We purposively selected communicative inhabitants who were available during the day to participate, with the target of conducting six group discussions.Data collection was conducted by two experienced male Congolese qualitative researchers not previously affiliated with camp activities with assistance from a female international investigator. Both Congolese researchers held Master’s degrees and the international researcher had a doctoral degree. Prior to data collection, a three-day training was led by the international investigator to introduce the study protocol, methods, and ethical procedures and to test and modify the instruments.Initial data collection involved key informant interviews, which were carried out by the two Congolese researchers and the international investigator in a workplace where privacy could be maintained. Key informant interviews lasted approximately 1 h and continued for the duration of the study. During early interviews, we used a guide to address topics related to handwashing promotion, obtained information on the physical layout and sociocultural composition of the camp, and generated lists of motivators to handwashing. Subsequent interviews focused on clarification and interpretation of information gathered through other research methods. An iterative process involving the review of completed interviews and additional questioning was carried out by the two qualitative researchers until no new information emerged.Each researcher was assigned two blocks where they conducted in-depth interviews and group discussions. Using an interview guide developed by the research team, efforts were made to administer in-depth interviews in a private setting, either in the household or an outdoor location next to the household, with each interview limited to 1 h 15 min duration. Techniques were used to probe for additional information according to respondents’ responses. If the researcher was unable to address all interview topics, a follow-up session was scheduled.Group discussions were comprised of 8–12 people with similar background characteristics and held in a relatively private space, such as a church or a school classroom, with sessions limited to 1 h 30 min. One of the researchers moderated the discussions and another researcher took notes to facilitate data transcription. A study guide developed by the study team reflecting research themes and preliminary results procured through key informant and in-depth interviews was used to lead discussions, with adjustments in questioning made based on the content of discussions.Key informant interviews were conducted in French or respondent’s preferred local language, and in-depth interviews and group discussions were administered in Kiswahili or Kinyarwanda with assistance from an interpreter. Interviews and group discussions were audio-recorded; interviewers took handwritten notes that provided additional insights into the data.Audio-recorded interviews were transcribed in Microsoft Word. Separate coding systems were developed for key informant interviews, in-depth interviews, and group discussions. Coding categories were derived from initial research themes and questions and key concepts that emerged during data collection. Coding of interview transcripts was done by one of the researchers and the international investigator on ATLAS.ti, a text-organizing software . ContentKey informants included four NGO representatives, with three informants overseeing implementation and monitoring of WASH activities in emergency camps and the fourth informant working on community outreach. Key informants responsible for managing WASH activities had worked in emergency settings on average for over 10 years, while the community outreach worker had three years’ experience. These key informants had spent an average of 9 months delivering WASH services in the Kishusha camp. Two of these key informants were based in the provincial capital 63 km by dirt road from the camp and three participated in the cluster or working group in charge of coordinating WASH activities. We also conducted key informant interviews with the presidents of the camp administrative and hygiene committees and three hygiene promoters. Table We carried out 20 in-depth interviews with child caregivers. Unfortunately, two interviews were not audio recorded properly and thus information from 18 interviews was analyzed. The average age of respondents was 29 years  of the latrine banks. Throughout the research period, we observed several potential hazards, including non-functional latrines which were not properly closed, and deep holes that had been created for latrines but were never used, which discussion group participants claimed had caused the demise of several residents who had fallen into the holes.NGO key informants reported that block presidents and hygiene promoters were oriented on good hygiene and handwashing practices and sensitization activities were initiated shortly after the first wave of IDPs entered the camp. All three hygiene promoter key informants reported never receiving formal training on hygiene, but rather were only given work directives from hygiene committee members. Representatives from the lead WASH NGO reported conducting group discussions to assess baseline hygiene knowledge and behaviors and adapt handwashing messages to coincide with residents’ understandings. Mother-respondents stated that during the early phase, NGO representatives promulgated information on what constitutes dirty substances, the importance of handwashing with soap to protect against germs and illness, critical times for handwashing, how best to wash hands with soap, and the dangers of exposing others to illness if soap is not used. The F diagram, which describes transmission pathways for fecal pathogens, and other health-related messages were primarily employed to promote handwashing practices . SubsequIn addition to awareness raising, two hygiene promoters stationed in each block were responsible for ensuring that water and a cleansing agent were available at communal handwashing stations, cleaning latrines and handwashing stations, and monitoring hygiene practices at communal stations. Hygiene promoters also conducted household visits to inspect for cleanliness and remind residents when and how to wash their hands. Work-related challenges mentioned by hygiene promoters at the time of the study included that the camp no longer provided soap for handwashing, the unwillingness of some inhabitants to subscribe to camp mandated hygiene and handwashing practices, and that hygiene maintenance equipment was old and not being replaced.The diamond (previous handwashing promotion involving provision of soap and messaging about handwashing) has become a simple mineral (referring to the deterioration in handwashing promotion activities). . . Before it was good; now people are discouraged, awareness raising regarding good hygiene is not like before.Mother-respondents lamented the decline in essential handwashing promotion activities, underlining that the NGO overseeing hygiene promotion at the time of the study discontinued provision of soap, communication activities had become sporadic or discontinued, and diarrhea once again posed a threat. One mother-respondent (P18) describing the decline in hygiene promotion commented,Those overseeing hygiene have succumbed to a state of lethargy due to lack of motivation. They have become negligent in their work.Several mother-respondents suggested that reductions in food distribution and the discontinuation of soap had negatively impacted activities of the hygiene promoters and committee members, with one (P3) stating,Hygiene promoters and committee representatives continued to monitor hygiene practices and fine residents who did not adhere to camp rules.These materials are helpful, they are all we have, we are poor. Flight from our villages did not allow us to bring these items. . . I like these materials, it made me feel good when they gave it to me because I felt they were looking out for me. We had nothing when we arrived here.Key informants reported that initially hygiene kits including jerry cans, plastic buckets, brooms, rakes, squeegees, shovels, water taps and soap were distributed to hygiene promoters for latrine maintenance. At the time of the study, maintenance materials had not been distributed for over six months, and recently constructed latrines did not have handwashing stations. NGOs had also distributed basic household materials such as water containers, dishware and utensils, and a tarpaulin just after IDPs entered the camp, as well as a basin, jerry can, goblets, buckets, and bar soap for handwashing, which were generally appreciated by mother-respondents. One mother-respondent (P18) said,However, many mothers reported that hygiene materials had been stolen when they were outside the camp or they had not received hardware for over a year and what was previously distributed was old and no longer functional, or in the case of bar soap, had been used long ago.Of the 12 hygiene committee members, there are only 2 who live in a block where stations are functional. You can ask them. They come and give us messages about practices they themselves cannot follow. Who can follow the messages they give us?Most mother-respondents valued communal handwashing stations, highlighting the convenience and fact that they serve as a reminder of handwashing after latrine use and enable hygiene promoters to monitor handwashing activities. They preferred removing “dirty substances” immediately after latrine use to avoid running the risk of having to greet people on the path home with unwashed hands or forgetting to wash their hands and contaminating others. Minimal space for handwashing at home and the desire not to deplete home water supplies for handwashing also favored communal stations. However, in one study block the communal station had stopped functioning  three months prior to our study, forcing residents to carry water to the latrine or to wash their hands in their homes or adjacent blocks, triggering conflict with block inhabitants. In another block, the latrines were no longer used because they were full, causing handwashing stations to become obsolete. Generally, mother-respondents and group discussion participants complained that communal station hardware was in poor condition or non-existent, and that since the NGO changeover, maintenance of stations was less rigorous, diminishing handwashing practices. One female group discussion participant said,Although mother-respondents living in blocks where stations were functional stated that water is generally available, ash was often not. Many maintained that only soap is effective in removing dirty and hard to remove substances like oil or dirt and eliminating “microbes” that cause illness, particularly diarrhea. Mother-respondents explained that washing without soap is equivalent to not washing, making them vulnerable to illness. Soap was also considered preferable due to its multi-faceted uses, including washing the body, clothes and dishes, as well as it being manufactured, which signified high quality and special ingredients. Many mothers indicated that only soap motivates handwashing.It is difficult to say that ash prevents illness the same way as soap, because ash does not have a chemical composition that guarantees the same effect. Ash is natural. It is impossible to wash clothes with ash. Only soap is effective in ridding clothes of dirt. This leads people to have more confidence in soap than ash.Mother-respondents and group discussion participants reported that ash does not remove all dirty substances associated with illness or have cleansing capabilities, with many never previously using ash for handwashing. Other disadvantages cited were that ash leaves a white substance on the hands, does not make the hands feel good, makes handwashing hardware dirty and less attractive, is not valued, and is only used for handwashing after using the toilet, but not during other events or for other purposes. A male group discussion participant said,Group discussion participants reported a perceived increase in diarrhea and cholera since the discontinuation of soap.When NGOs distribute food in the camp, they give to some but not others. When our neighbor does not eat, he will not be in good health. Health is not only about providing water and handwashing.NGOs overseeing hygiene were also involved in food distribution. At the time of the study, NGO key informants reported that less than 25% of households met donor criteria related to food insecurity and camp inhabitants were encouraged to seek employment to provide for their families. Mother-respondents and group discussion participants cited inequities in receipt of food, which they reported affected morale, caused conflict, decreased NGO credibility, and served as a disincentive to subscribe to good handwashing practices. One female group discussion participant said,Several mother-respondents indicated that feeding family members is of greatest concern, and without sufficient food, hygiene and handwashing initiatives lose importance.I could eat without washing my hands because people in the village were few, but here in the camp there are many people. For me many people bring uncleanliness. That is why I started to wash my hands.Study participants indicated that their natal villages were in isolated areas where hygiene information was limited or non-existent, with some suggesting that the sparse populations rendered handwashing unnecessary. One hygiene committee member said,Because I lived in a secluded area, I had no idea about hygiene. After farming, it was sufficient to dip our hands in water and then we ate. We put the water in a receptacle and we washed with it until it got dirty. Then we threw it away.Mother-respondents reported using only water for handwashing, explaining that soap was unaffordable and they had been unaware that ash cleansed. Most indicated that family members washed their hands in a common receptacle, with men sometimes provided a separate basin. Mentioned times for handwashing included after waking from sleep, after farming, before eating and when they bathed. One mother-respondent (P2) said,Several mothers maintained that it was important to wash hands before church or when receiving guests. Other barriers to handwashing included distances and time required to obtain water and the need to eat quickly when sharing a communal plate with family members.We were taught the rules of hygiene and the number of illnesses diminished. If we cannot follow these rules, it is due to our habits from the village. We can see that following the handwashing messages has helped us. If we do not, we pay the consequences (by getting sick).Mother-respondents reported that initially residents ridiculed handwashing messages because they were unaware of the importance of hand hygiene. However, they claimed that exposure to the same communications from different sources and experiences involving residents either complying or not complying with handwashing promotion strategies in the camp where disease was perceived as a persistent threat, confirmed their veracity and led to the general acceptance by mothers of promotional messages. These respondents consistently maintained that following good handwashing practices protects their health, particularly against diarrheal disease. Several made reference to a diarrhea outbreak which killed camp residents, raising fear and validating that handwashing saves lives. One mother-respondent (P5) stated,When we walk around the camp we greet many people, we don’t know what they have touched. When we arrive home, we must wash our hands.Pouring water over the hands was described as the best handwashing approach. Reported critical times to wash hands included before food preparation or consumption, before breastfeeding, after household chores or work outside the camp, when the hands are visibly dirty, and after getting up from bed, with mother-respondents stressing heightened risk of contamination after shaking hands, using the latrine or washing a child who has defecated. A mother (P1) stated,Illness is caused by contact, and if you have eaten without washing your hands, you have eaten dirty substances or microbes that leads to illness.All mothers reported that failure to wash hands before food preparation and consumption can lead to ingestion of dirty substances causing diarrhea, vomiting or other illness, with some mentioning exposing family members who eat from the same receptacle. Mother-respondent (P1) claimed,Here, if someone is exposed to germs it is a danger for everybody. That is why it is important to remind others to wash their hands.Most mother-respondents reported washing their hands more often than other household members due to their involvement in food preparation, exposure to dirty items while cleaning the household, caring for animals and providing childcare, and ongoing access to water. The majority claimed to remind others, particularly children, to wash hands. This mother (P16) said,Now it is ash, since the departure of X (previous NGO), soap is not available. Maybe it is for this reason many people are no longer interested in washing their hands each time (they use the latrine). Lack of soap discourages people from washing their hands.At the time of the study, mothers reported washing their hands at public stations or at home. Many indicated that handwashing had become a habit when soap was available, but the majority refused to use ash. One mother-respondent (P13) said,When people leave the toilet, they don’t find soap by the handwashing station, they only see ash and say, ‘I will wash at home.’ There is the risk of forgetting.A hygiene committee member said,Most respondents reported handwashing with plain water, which they recognized to be ineffective in removing dirty substances.The most commonly cited motivator for handwashing by mother-respondents was to rid the hands of dirty substances or “microbes” and thus protect against illness, with diarrhea mentioned most frequently. The camp was described as a very dirty place, raising fears about contracting illness and necessitating that residents wash their hands to maintain health, especially during diarrhea outbreaks, with some underscoring that as caregivers they must stay healthy. Limiting illness transmission to family members, particularly young children who they considered highly susceptible, was also mentioned as a key motivator. Results from the rating exercise presented in Table Soap was reported to encourage handwashing, with some asserting that the lather eliminates dirty substances and conveys cleanliness. Other attributes included that soap makes hands smell good, feel light, smooth, and soft, and look clean and pretty, making the user feel good, at ease and confident. Some mothers suggested that clean hands engender pride and boosts self-esteem, and eliminating offensive odors that cause embarrassment enhances their image Table . MaintaiThe most common reason for failing to wash hands related to preoccupations with other pressing issues, particularly that family members eat sufficiently Table . Some moOther explanations for neglecting to wash hands included that people working outside the camp are too busy, handwashing stations are non-functional, soap is no longer provided in the camp and unaffordable, or some people refuse to modify habits. Group discussion participants mentioned that people engaged in wage labor, who are mostly men, have less exposure to handwashing hardware and camp-based messaging, while female residents have greater contact with hygiene promoters.In the DRC camp, hygiene promotion was introduced shortly after the camp was formed. However, study results show that two years after initiation of WASH activities, hygiene promotion was hampered by inadequate resources, and lack of cultural acceptability, innovation, and adaptation for sustained behavioral change. Camp residents reported that handwashing was primarily motivated by the desire to protect against illness, particularly diarrhea disease, reflecting camp-based messaging which appeared to garner acceptance in the congested camp where ongoing disease threats prevailed. Emotionally- and socially-related motivators were also reported to stimulate handwashing with soap. The change from providing soap to promoting ash and failure to replace old handwashing technology prevented residents from handwashing with highly valued soap and made them opt to wash with plain water. Results give special insights into the challenges of hygiene promotion in an IDP camp where harsh living conditions and rampant poverty forced residents to prioritize basic survival needs over following good hand hygiene behavior.We found that communication strategies lacked clear objectives, innovation and a long-term vision, thus undermining sustained and widespread behavioral change. Hygiene knowledge of study participants mirrored a hybrid of cultural beliefs and customs held prior to entering the camp and knowledge and practices influenced by the camp context, hygiene messages and rules. A prevailing concept supported by study respondents was that handwashing with soap is effective in protecting against illness, reflecting the disease transmission and prevention focus of camp messages. Although NGOs reported conducting formative research, there was no indication that hygiene sensitization was adapted to residents’ beliefs and practices or the evolving camp context. Rather, the same standard messages were disseminated from the camp inception, with modifications occurring only during diarrhea outbreaks. Still, the ongoing perceived threat of diarrhea disease in the densely populated camp environment appears to have influenced acceptability of illness-based messages. One theory is that when disease symptoms are clear-cut, the disease threat is pervasive, and effective measures are available, cause and effect problem solving is heightened . In thisWhile motivators to handwashing were primarily related to illness prevention, residents cited other emotionally- and socially-related motivators such as “feeling clean” or “being proud” and “maintaining a good image” that stimulated handwashing with soap. The mix of research methods provided a rich combination of data which allowed us to triangulate and validate the results related to handwashing motivators. Findings show that it was particularly important for mother-respondents to maintain a clean appearance and fresh smelling hands during public events such as Sunday church, one of the rare opportunities women had to socialize and project their status in the community. In the DRC, an emphasis to look good prevails, particularly on Sunday, when community interactions are heightened and Congolese try to convey an image of well-being and prosperity, which involves being clean, smelling good and dressing well . Our respondents also unscored social pressure to follow camp hygiene regulations designed to diminish disease transmission to fellow residents. Social science literature has illuminated the importance of social rather than just individual change, since the former better ensures widespread acceptability and long-term impact of healthy behaviors . A sociaStudy results revealed the failure to maintain latrines and communal handwashing stations and distribute materials needed to apply good hand hygiene practices. While residents believed in the effectiveness of soap in preventing illness, they were unable to continue handwashing with soap due to the poor state of the public handwashing stations, discontinuation of soap distribution, and fact that soap was unaffordable to purchase. A quantitative study showed handwashing at communal stations to be far less common when residents were provided ash compared to when they were provided soap . IronicaThe transfer to ash also coincided with the changeover from one highly respected NGO to an organization which reportedly did minimal work, leading to a sharp decline in camp hygiene activities. While the hygiene committee continued functioning, their work seemed in part to be galvanized by the desire to collect fines instituted when residents failed to follow camp rules. Study results point to inadequate oversight and commitment by NGO implementers and the WASH cluster regarding maintenance of effective hygiene promotion activities and a safe living environment. NGO representatives providing assistance often lived far from the camp, permitting only sporadic visits, which likely did not allow them to appreciate the intricacies of camp life and residents’ needs. A camp setting like Kishusha, where inhabitants representing different ethnic backgrounds had previously been in conflict, can be complex, thus requiring a regular presence to understand the social dynamics needed to develop effective handwashing approaches. In addition, security restrictions prevented donors from making firsthand situational assessments and informed decisions about funding needs. The short funding mandates allotted by donors and internal NGO policies restricting time commitments in a camp setting appeared to limit NGO capacity to implement relevant and sustainable strategies. Findings also suggest that NGO implementers had limited understanding of effective handwashing communication approaches and behavioral change theory. WASH experts have reported that inadequate expertise, failure to contextualize communication approaches, and lack of a formal behavioral change strategy undermines the success of hygiene promotion in camp settings . These fResults highlight how the precarious nature of a camp setting can impede handwashing promotion activities, illustrating that basic survival needs take precedence, rendering hygiene practices secondary. NGO-led food distribution targeted a small percentage of families considered food insecure, causing resentment and potentially affecting willingness to adhere to other NGO recommendations regarding hygiene and handwashing practices. IDPs were expected to seek wage labor to eke out enough money to feed family members. When away from their homes, they fell victim to crime, including stealing of hygiene hardware. Those working outside the camp constituted a hard to reach group whose absence during the day limited exposure to handwashing messages and technology. Handwashing promotion should take into consideration the daily patterns of camp residents and modify approaches to better ensure all residents are reached through strategies.A limitation to this study was that we only carried out in-depth interviews with women. While group discussions included male household heads, because discussions were held during the day time we did not include males who routinely work outside of the camp. Failure to collect information from hard to reach males regarding their exposure to and perceptions of the handwashing promotion activities and motivators and barriers to handwashing behavior was a missed opportunity. Another limitation is that some respondents were at times reticent to openly share information. We suspect this relates to the history of camp residents, who had fled conflict zones prior to entering the camp, and historical ethnic tensions of groups residing in the camp, raising suspicion amongst residents. While the researchers were highly skilled, they were not from the study area and therefore did not speak the local languages.Motivating and sustaining optimal hand hygiene practices among emergency-affected populations is multifaceted and requires a long-term vision and strategy in prolonged emergency situations. In Kishusha, assistance given during the emergency phase appeared to be extensive and well received, but over time a deficit in hygiene promotion resources prevented residents from applying the handwashing knowledge acquired and practices encouraged in the camp where disease threats persisted.Illness-based messages may be more effective in camp settings where overcrowding and suboptimal WASH conditions heighten the risk of infectious disease transmission compared to non-emergency contexts, likely altering perceptions of disease threats. However, lack of exploration and use of emotive and social drivers that stimulate handwashing may present missed opportunities to incorporate approaches proven effective in improving handwashing in development settings. The findings underscore the need to design contextually relevant approaches that follow sound hygiene practices and to modify handwashing strategies as camp conditions and resident’s knowledge base change. The poor state of WASH and other services uncovered during the study emphasize the need to provide acceptable and quality assistance that meets basic standards for the duration of the camp’s existence. Additional research to assess whether health-related or psychosocial motivators are more effective can help to prioritize messages related to hygiene promotion during emergencies.