The success of liver transplantation has been limited by the unavailability of suitable donor livers. The current organ preservation technique, i.e., static cold storage (SCS), is not suitable for marginal organs. Alternatively, normothermic machine perfusion (NMP) promises to recreate the physiological environment and hence holds promise for the better organ preservation. The objective of this systematic review is to provide an overview of the safety, benefits, and insight into the other potential useful parameters of NMP in the liver preservation.CRD42018086034 for prospective trials comparing the role of NMP device to SCS in liver transplant by searching the PubMed, EMBASE, Cochrane, BIOSIS, Crossref, and Scopus databases and clinical trial registry. We searched the current literature following registration in the International Prospective Register of Systematic Reviews (PROSPERO) with registration number µmol/L (range 8–344 µmol/l) and 30–47.53 µmol/l (range 9–340 µmol/l) in NMP and SCS groups, respectively. A single case of PNF was reported in NMP group in the randomized trial while none of the other preliminary studies reported any in either group. There was intertrial variability in EAD which ranged within 15–56% in NMP group while being within 23–37% in SCS group. Biliary complications observed in NMP group ranged from 0 to 20%. Single device malfunction was reported in randomized controlled trial leading to renouncement of transplant while none of the other trials reported any machine failure, although two user related device errors inadvertent were reported. The literature search identified five prospective clinical trials  comparing 187 donor livers on NMP device to 273 donor livers on SCS. The primary outcome of interest was to assess the safety and graft survival at day 30 after transplant following NMP of the donor liver. Secondary outcomes included were early allograft dysfunction (EAD) in the first seven days; serum measures of liver functions as bilirubin, aspartate aminotransferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), and international normalized ratio (INR) on days 1–7; major complications as defined by a Clavien-Dindo score ≥ 3; and patient and graft survival and biliary complications at six months. The peaked median AST level between days 1 and 7 in the five trials was 417–1252 U/L (range 84–15009 U/L) while on NMP and 839–1474 U/L (range 153–8786 U/L) in SCS group. The median bilirubin level on day 7 ranged within 25–79  This review outlines that NMP not only demonstrated safety and efficacy but also provided the favourable environment of organ preservation, repair, and viability assessment to donor liver prior to the transplantation with low rate of posttransplantation complication as PNF, EAD, and biliary complication; however further studies are needed to broaden our horizon. Liver transplantation is established as the treatment of choice for patients with the end-stage liver disease. While the success of liver transplantation is unquestioned, the scarcity of donor organs limits the delivery of this therapy in a sufficiently timely manner to prevent deaths on the waiting list. Despite the rise in organ donation, the potential requirement of liver transplantation still far exceeds demand, and patients may have compromised outcomes as they end up being transplanted with high model of end-stage liver disease (MELD) scores and in a severely deconditioned state , 2. The The acute termination of oxygenated blood supply during liver procurement initiates a cascade of injury and inflammation, triggered initially by hypoxic anaerobic metabolism, nutrient, cofactor, and adenosine triphosphate (ATP) depletion, with lactic acid accumulation. These injurious processes are further exaggerated during static cold storage (SCS). Disruption of sodium-potassium membrane pumps leads to disruption of electrolyte cell membrane gradients, resulting in cellular edema, influx of free calcium, and subsequent activation of proteolytic enzymes terminating with cell death. Accumulation of xanthine oxide following ATP breakdown generates free radical upon restoration of circulation. These free radicals lead to lipid peroxidation and cellular destruction known as ischemia-reperfusion injury –9.The traditional method of organ preservation involves flushing of cold preservation solution following complete dissection and interruption of blood supply to the donor organ. Cold preservation works on the principle of diminishing cellular metabolism with a decrease in temperature the Q10 effect), which limits need for ATP  effect, –13. The The possibility of avoiding cold ischemic injury altogether in marginal grafts has recently become possible with the introduction of the new technology of ex vivo normothermic liver perfusion (NMP). The concept of normothermic perfusion is simple, in that maintaining an entirely physiological milieu for the liver during transport should diminish risk of ischemic injury within the liver, and ischemia-reperfusion has already occurred on the device, thereby reducing risk of PRS in the recipient . It remaBased upon extensive promising results in preclinical large animal studies in NMP porcine or rodent models –36, at lIn the present systematic review, we provide a detailed analysis of all available human liver NMP studies that assess safety, feasibility, and reliability of this new technology and where possible available evidence reflecting the clinical effectiveness of NMP as an alternative to SCS in patients undergoing liver transplantation is summated. Finally, we explored potential directions for future research and translation of NMP technique into clinical practice.CRD42018086034. An extensive search of all the published literature describing the role with NMP based device in liver transplantation as an alternative to SCS was made on National Library of Medicine Database, EMBASE, Cochrane, BIOSIS, Crossref, and Scopus databases and clinical trial registry on 10 October 2017. The search covered the period from May 2016  to 18 April 2018 and search was last carried out on 18 April 2018 [A comprehensive systematic literature review was performed according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses for Protocols 2015 (PRISMA-P 2015) , followiril 2018 . Our seaOnly studies which systematically and quantitatively assessed the graft safety, functioning, and graft survival on NMP based devices including the OrganOx (Metra), the Organ Assist (Liver Assist), and TransMedics (OCS) in different clinical studies were analyzed. All other publications as editorials, reviews, and letters were excluded. The primary outcome of interest was to assess the safety and graft survival at day 30 after transplant following NMP of the donor liver. Secondary outcomes included were early allograft dysfunction (EAD) on the first seven days; serum measures of liver functions as bilirubin, aspartate aminotransferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), and international normalized ratio (INR) on days 1–7; major complications as defined by a Clavien-Dindo score ≥ 3; and patient and graft survival and biliary complications at six months .Two separate physician reviewers, KJ and IR, employed a two-stage method to conduct study screening independently. At the first stage, titles and abstracts were scrutinized for excluding obviously ineligible studies. At the second stage, the full text was read carefully for further excluding ineligible studies. Disagreements were resolved via consensus and discussion with chief author AMJS. We analyzed literature with empirical studies using a standardized quality assessment tool and prespecified inclusion and exclusion criteria. The present systematic review was performed using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and registered in the PROSPERO, an international database of prospectively registered systematic reviews .The QUADAS-II  based analysis was done to assess the internal validity of prespecified inclusion and exclusion criteria of the various studies. QUADAS-II is an evidence-based bias assessment tool to evaluate the quality of diagnostic accuracy studies in a systematic review. Each study was reviewed comprehensively and data extracted to assess the earlier outlined parameters .Publication bias is formally assessed through funnel plots, but that requires at least ten trials; unfortunately present systematic review involved only five trials, so we could not have assessed publication bias.Our literature search yielded a total of 1299 manuscripts using keywords listed above. After screening titles and abstracts, 5 studies (4 full articles and single poster with limited data) were included in present review analysis, data extraction of four of which involved Metra device and single with OCS (TransMedics) , 42–45 . Two stuThe detailed data related to study characteristics in terms of perioperative and normothermic perfusion, clinical outcomes, safety, adverse events, and survival were summarized in Tables 2–6.P = 0.004) owing to presence of significant steatosis, increasing lactate level, cirrhosis in donor liver, WIT > 30 mins, incidental malignancy (colon and lung cancer), and device related errors [A total of 460 patients were included in the five trials; we have outlined the demographic and clinical data of patients undergoing liver transplantation following organ preservation by NMP or SCS. NMP based organ preservation was done in 187 cases, while the conventional method of cold storage was used in 273 cases. Nasralla et al. reported that 48 donor livers were discarded (16 (11.7%) NMP, 32 (24.1%) SCS; d errors . The medP < 0.001) between study groups [P < 0.000) [The reported median NMP time in trials varied considerably, from 558 to 786 minutes (range 210–1631 minutes) while median CIT on cold storage was 235–634 minutes (range 64–967 minutes). There was no significant difference in organ preservation time in the study by Ravikumar et al., Selzner et al., and Liu et al. , 42, 44.y groups . They fu< 0.000)  Table 2P < 0.001The attributes to assess donor liver functioning while on NMP such as hepatic transaminases, INR, pH, lactate level, bile production, hepatic artery, and portal vein flow were found normal in all the reviewed trials. The perfusate used in all studies was blood based using ABO-blood group O packed red blood cells; however, in the studies by Ravikumar et al., Bral et al., and Nasralla et al., the circuit and liver were additionally primed with gelatin-based plasma expander , whereas in the study by Selzner et al. Steen solution was used instead .The peak median AST level between days 1 and 7 in the five trials was 417–1252 U/L (range 84–15009 U/L) while on NMP and 839–1474 U/L (range 153–8786 U/L) in SCS group.μmol/L (range 8–344 μmol/L) and 30–48 μmol/L (range 9–340 μmol/L) in NMP and SCS groups, respectively. Only three trials outlined day 7 median ALP level, 139–245 U/L (range 40–626 U/L) in NMP group while being 147–243 U/L (range 58–743 U/L) in SCS group  and 1.03–1.1 (range 0.90–2.2) in SCS group. The median bilirubin level on day 7 was 25–79 CS group .The PNF was observed in a single recipient in the randomized study done by Nasralla et al. in NMP group while none of the other trials reported such event in either groups. There was intertrial variability in EAD which ranged from 10 to 56% in NMP group while being within 23–30% in SCS group. Nasralla et al. reported 93% less likelihood of developing EAD in DCD liver while on NMP rather than SCS . In the The median intensive care unit (ICU) stay was 3–16 days (range 1–65 days) in NMP group while in SCS group the median was 3-4 days (range 0–41 days). The median hospital stay in NMP and SCS was 12–45 days (range 6–114 days) and 13–25 days (range 7–89 days), respectively. Between 10–22% and 22–37% recipients developed Clavien-Dindo score ≥ 3 in NMP and SCS groups, respectively. Biliary complications 6 months after transplant were observed in NMP group ranging from 0 to 20%. The trial by Bral et al. evidenced lower 6 months' biliary stricture in NMP group (0%) compared to 14.8% in SCS; however a recent randomized study published by Nasralla et al. did not observe any statistical difference in occurrence of nonanastomotic biliary stricture in either groups , 45 Tab.During follow-up, 30 days' graft survival reported by three trials was between 90 and 100% in NMP group and 97.5 and 100% in SCS group , 43, 45.None of the earlier four preliminary trials reported any issues related to device failure; however Nasralla et al. in a recent randomized trial (2018) did report single event of graft loss following device malfunction and two user related device errors . PreviouWith the increasing incidence of the liver disease, the number of transplants required has been outpaced by the number of transplants performed. This disparity between liver transplant candidates and the availability of donor liver has led to an increase in mortality while waiting for transplantation. In order to meet the ever-increasing demand, transplant centres have started exploring the probability for utilization of marginal donor organs. However, the equilibrium has never been achieved owing to the compromised quality of such organs. Recent data from the Organ Procurement and Transplantation Network (OPTN) in the United States reveal that almost 22% of procured livers are discarded before transplantation, and likely many more are never offered as they are considered to have too high risk for cold storage. Currently, SCS technique is the mainstay of organ preservation. However this method works well for livers from healthy donors, achieving acceptable rates of EAD, PNF, and biliary complications , 52. It The efficacy of normothermic perfusion for liver preservation has not been fully described by clinical studies. In the present review, we have compiled all the evidence and shortcomings illustrated in the studies done so far. These trials were primarily done to assess the safety of NMP; however, data related to functioning and viability of organ were also included , 42, 43.The primary aim of this review was to assess the safety of NMP by combining analyses of all available early phase clinical trials. The most frequently cited impediments to broader clinical significance of NMP include the unproven benefit and risk of this new technology, the potential substantial costs associated with complex portable machinery, complex procedures, and additional personnel required to tether and maintain liver stability during ex situ perfusion, risk of microbial contamination, and risk that good livers could be destroyed by exposure to instant warm anoxia if a device were to fail during transportation. Inadvertent incidents as graft loss following single event of unrecognized twist in portal vein or catheter occlusion in hepatic vein and bile duct or other user related errors could be a part learning curve associated with new technology and may get minimized with the greater cumulative team experience , 45, 49.The loss of clinically usable livers remains of potential concern if more centres are to face the learning curve of a more complex technology for liver preservation than the standard cold storage solution box, which is cheap, efficient, and of proven and known quantum over the past 40 years. It is possible that reporting bias may underreport technical errors in early studies.A large body of large animal preclinical and now preliminary clinical data demonstrates that ex situ NMP based liver perfusion is generally safe, in most but not all cases lowers early transaminase levels in the recipient, and can accommodate a much prolonged storage phase than could safely be contemplated with SCS. However, adequately powered larger randomized studies are eagerly awaited to more fully define the risk-benefit balance, and until then the exact technique for this exciting new technology remains to be defined.At present, the viability of any particular donor liver can only be assessed following the transplantation into a recipient, which could potentially prove disastrous when extreme marginal donor livers are transplanted, with elevated risk of PNF, EAD, or other detrimental effects of ischemic-reperfusion injuries, including PRS leading to acute renal injury –60.NMP may help overcome such risks by allowing liver function assessment before implantation of the organ into a recipient. The benefits of NMP have been tested in numerous studies and suggest that the viability of donor organ can be predicted by a combination of hemodynamic, metabolic, and synthetic parameters derived during the ex situ perfusion phase, providing a functional assessment of the donor liver, which heretofore was not possible with SCS , 61. TheCurrently, up to one-third of the total pool of marginal livers are made up of DCD donor livers, even though such livers carry added risk –66. WhilBral et al. did not show significant improvement in opening transaminase levels in the recipients, likely due to their increased proportion of DCD donors and prolonged cold ischemia time while a relatively higher proportion of replaced and accessory hepatic arteries were reconstructed on the back table, and their NMP duration was extended to outer limits often while their small surgical team rested overnight or were engaged in other hepatobiliary surgeries . The priAlthough SCS is considered as the gold standard method for liver preservation, the injurious impacts upon hepatocyte and biliary epithelial survival are well described , 69. TheEarly allograft dysfunction (EAD) reflected by elevated recipient transaminase levels within the first postoperative week also poses increased risk for the recipient –80. In tAnastomotic biliary strictures or more diffuse IC are one of the most feared complications of DCD liver transplantation. The incidence of biliary strictures ranges between 4 and 15% following DBD liver transplantation while being within 30–40% after DCD , 74. InjOp Den Dries et al. and Boehnert et al. reported significantly fewer bile duct related complications following NMP in the porcine model , 69. LiuP = 0.180). However, further research is warranted with NMP and DCD donation to more fully define risk and protection to the biliary epithelium.However, subsequent clinical trials showed varied results as Bral et al. and Selzner et al. did not observe any late biliary complications despite the high number of DCD donors , while Ravikumar et al. did report anastomotic strictures in 4 cases (20%) in the NMP group , 42, 43.P < 0.0001) and EAD  [Currently, the current median liver preservation time in the US is approximately 8 hours . There a= 0.002) , 50. HenSteatotic livers constitute a proportion of ECD grafts but have traditionally been discarded before transplantation due to their known increased risk for PNF , 80. SpiThere are certain limitations to the present systematic review owing to a small number of studies available in the literature which might influence the interpretation of related outcomes. Despite these limitations, this systematic review has outlined the advantages of NMP over SCS in organ preservation and safety issues associated with its usage; however further randomized trials are much warranted to confirm these findings.Over the past 40 years, SCS with refined cold preservation solutions has served the field well and has led to outstanding short and long term outcomes with clinical liver transplantation. As a result, liver transplantation remains a life-saving standard of care for all forms of acute, irreversible, and progressive chronic liver disease. The established success of this therapy and propagation of societal diseases such as hepatitis B, hepatitis C, and now widespread nonalcoholic steatohepatitis (NASH) had placed escalating pressure on transplant lists, driven supply-demand imbalance, and unacceptable rates of waiting list mortality. This situation has warranted a reevaluation of the methodology of storage and transportation of organs, as higher risk livers are used to attempt to match demand. Emerging evidence advocates that NMP may extend the safe utilization of the more marginal spectrum of liver donor grafts, but this remains to be proven in practice. This exciting technology has demonstrated safety and efficacy in preliminary clinical studies, and ongoing trials will continue to explore the full potential of NMP technologies, will determine the need for portability, and will more completely define the cost-benefit balance.


In this paper, a case study is presented based on one school, conducted to examine the factors that influenced the process and quality of implementation of the Tier 1 Program of the Project P.A.T.H.S. Through interviews with the school contact person and focus group interviews with the instructors, an integration of the findings showed that several factors related to the program, people, process, policy, and place (5 “P”s) facilitated the implementation process of the Tier 1 Program in the school. Obstacles and difficulties with reference to the 5 “P”s that affected the quality of implementation were also identified. Overall, the quality of program implementation in the school was good, and the program was well received by the program implementers. Implications of the present findings for future program implementation with reference to school administrative arrangements and implementation issues are discussed.


BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (p<0.01), and also higher, albeit not statistically significant, compared with the fusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group . IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome.Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of  BCR-ABL1 fusion gene form a small patient group with a poor prognosis, which has been substantially improved in recent treatment protocols with the addition of imatinib and other tyrosine kinase inhibitors [BCR-ABL1-positive disease and an unfavorable prognosis [BCR-ABL1-like subtype is found in approximately 50% of so-called B-other cases, which are BCP-ALL cases negative for the sentinel cytogenetic lesions BCR-ABL1, ETV6-RUNX1, TCF3-PBX1, rearrangement of MLL, or high hyperdiploidy (51-65 chromosomes). BCR-ABL1-like BCP-ALL shows a high frequency of B-cell development gene aberrations, especially IKZF1 deletions [Children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with the hibitors . Approxirognosis –4. This eletions .BCR-ABL1 fusion gene and expected to be clonal leukemia drivers. Detection of tyrosine kinase fusions could guide targeted treatment with tyrosine kinase inhibitors and improve outcome in a similar way as currently for BCR-ABL1-positive patients.Tyrosine kinase activating lesions are diverse and include tyrosine kinase fusion proteins, cytokine receptor overexpression, or activating point mutations in genes encoding kinases, cytokine receptors, or signaling molecules (recently reviewed in ). In ourin vitro cell models to tyrosine kinase inhibition [Tyrosine kinases known to be involved in fusions include the ABL class kinases ABL1, ABL2, PDGFRB and CSF1R as well as the JAK class kinase JAK2. Each of these kinases has been detected in in-frame fusions with multiple 5′ partner genes resulting in the expression of a constitutively active, oncogenic kinase. The number of patients without sentinel chromosomal abnormalities and the diversity of novel tyrosine kinase fusions poses a challenge to routine fusion gene detection. Previous studies have described the discovery and oncogenic potential of ABL and JAK class fusion genes and the sensitivity of patients’ cells or hibition –7. Selechibition –7. A larhibition . Other shibition –11.We aimed at the detection of recently identified tyrosine kinase fusion genes in 153 B-other cases of a population-based cohort of 574 Dutch/German pediatric BCP-ALL patients at initial diagnosis to address the frequency of tyrosine kinase fusions, their clinical response characteristics, and associated genetic lesions in B-cell development genes.BCR-ABL1-like cases (18%), and none among 76 non-BCR-ABL1-like B-other cases (q34) in their karyotypes (ABL1 is located on 9q34), together with add(10)(q21) in the ZMIZ1-ABL1 case (ZMIZ1 is located on 10q22), and with del(3)(p13) in the FOXP1-ABL1 case (FOXP1 is located on 3p13). Two EBF1-PDGFRB cases showed an interstitial 5q deletion on array-CGH, one case arose by balanced t translocation, and one case showed a more complex copy number alteration, with loss of EBF1 exon 16 and gain of a genomic region encompassing EBF1 exons 7-9 q1 in the -9 Table . EBF1 ex add(10)q1 in the  add(10)q1 in the ns Table . Interesmosome 5 . Finally add(10)q1 in the CRLF2 were found in 16% of BCR-ABL1-like cases, none of which overlapped with ABL/JAK class tyrosine kinase fusion cases, and also in 16% of non-BCR-ABL1-like B-other cases. About 50% of CRLF2-high cases carried a JAK2 mutation [CRFL2 high expression cases with a PAR1 deletion, JAK2 mutation or both was similarly high in BCR-ABL1-like (9/12 cases) and B-other cases , IKZF1 deletions other than exon 4-7 or full deletion, including deletion of exons 1, 1-2, 1-3, 2-3, 2-7, 2-8, 4-8, 5 and 7-8, occurred more frequently in the fusion-positive compared with the fusion-negative BCR-ABL1-like cases  showed an aberrant deletion frequency in tyrosine kinase fusion cases compared with non-BCR-ABL1-like B-other cases  and intrachromosomal amplification of chromosome 21 [Next, we compared the frequency of B-cell development gene lesions between the tyrosine kinase fusion-positive osome 21 , were muBCR-ABL1-like B-other  and also higher, albeit not statistically significant, compared with fusion-negative BCR-ABL1-like cases  and before consolidation (p=0.002) and also, although less significant, compared with fusion-negative BCR-ABL1-like cases  was comparable with the remaining BCR-ABL1-like group (35% ± 6%), and worse than the non-BCR-ABL1-like B-other group , ABL/JAK class tyrosine kinase fusions cases are estimated to represent approximately 2.5% of pediatric BCP-ALL, making this heterogeneous subgroup similar in size to the BCR-ABL1-positive cytogenetic subtype. Although the number of cases evaluated in our study is modest, the frequency of tyrosine kinase fusion cases is similar to those reported in other cohorts: 3% ABL/JAK class fusions in a high-risk pediatric US cohort [We identified ABL/JAK class tyrosine kinase fusion genes in 14/153 9%) of DCOG/COALL B-other cases and these fusions were restricted to the % of DCOGBCR-ABL1-like B-other group, and found them non-overlapping with two chromosomal abnormalities that were described earlier to occur in BCR-ABL1-like cases, iAMP21 and dic [CRLF2 high expression cases are present both in BCR-ABL1-like and non-BCR-ABL1-like B-others, we found that tyrosine kinase fusions and high CRLF2 expression were mutually exclusive. The current study did not aim at discovering novel TK fusions, therefore there is a small probability that a sample with a novel tyrosine kinase gene is included in the tyrosine kinase fusion negative group.The most comprehensive detection of novel tyrosine kinase fusion genes requires transcriptome or whole genome sequencing. These costly methods have not been applied to complete cohorts but rather to subsets of cases, for example cases selected based on Philadelphia-like gene expression or absence of sentinel chromosomal abnormalities, mostly in retrospective studies , 9. Moreic . MoreoveBCR-ABL1-like features, and our earlier finding that tyrosine kinase fusion cases are identified by both the St. Jude and Erasmus MC BCR-ABL1-like expression signatures [Our finding that tyrosine kinase fusions are only found in patients with gnatures , means tgnatures .BCR-ABL1-like B-other ALL cases but a comparable relapse rate as BCR-ABL1-like ALL without tyrosine kinase fusions. In correspondence, a recent Japanese study showed that the event-free survival of tyrosine kinase-activating fusion cases  was unfavorable compared with fusion-negative BCP-ALL [EBF1-PDGFRB-positive patients were MRD positive at the end of induction [Ex vivo leukemic patients’ cells with JAK and ABL class fusions were shown to be sensitive to tyrosine kinase inhibitors [We found that ABL/JAK kinase fusion cases were characterized by poor initial response to induction treatment, high MRD levels, and a higher relapse rate compared with non- BCP-ALL , Schwab nduction , and Robnduction . Recent nduction , 19. Ex hibitors –7, 15. Phibitors , 20–21. BCR-ABL1, MLL rearrangements, ETV6-RUNX1, TCF3-PBX1, and high hyperdiploidy. Among the B-other cases, BCR-ABL1-like cases identified by hierarchical clustering of gene expression data were described previously in our cohort [BCR-ABL1-like cases in the cohort, tyrosine kinase fusion testing was performed   and GermEBF1-PDGFRB, PAX5-JAK2, NUP214-ABL1, RANBP2-ABL1, ETV6-ABL1, RCSD1-ABL1 and STRN3-JAK2 described by Roberts et al. [ABL1, PDGFRB, CSF1R, ABL2, TYK2, and JAK2  [PDGFRB/CSF1R and ABL1 probes (Cytocell) to confirm fusions. The methods applied to each case depended on the type and amount of available patient material and are indicated in Detection of tyrosine kinase fusion genes was performed by RT-PCR followed by Sanger sequencing for seven fusion transcripts s et al. . In addierlands) , 26. We Copy number analysis was performed using Agilent SurePrint G3 Hmn 4×180K arrays  co-hybridized with 1 μg patient DNA labeled with ULS-Cy5 and 1 μg reference genomic DNA male pool  labeled with ULS-Cy3 (Agilent Genomic DNA ULS Labeling Kit). Copy number microarray data were normalized using median log ratio in the CGHcall  version IKZF1 (a3) and the SALSA P202 Multiplex Ligation-dependent Probe Amplification (MLPA) assays (MRC-Holland) were used to identify or confirm genomic lesions on the following genes: IKZF1, CDKN2A, CDKN2B, ETV6, PAX5, RB1, BTG1 and EBF1 as described previously [The SALSA P335 ALL-eviously . In shorMLL, RUNX1, TCF3, and IKZF1, the tyrosine kinase genes ABL1, ABL2, PDGFRB, CSF1R, JAK1, JAK2, JAK3, FLT3, and TYK2, and the cytokine signaling genes CRLF2, EPOR, IL7R, TSLP, SH2B3, and IL2RB [Targeted Locus Amplification (TLA) combined with deep-sequencing was used to detect fusion genes and sequence mutations in regions up to 100 kb around a pre-selected primer pair by crosslinking of physically proximal genomic sequences as described before . Brieflynd IL2RB .PDGFRB and ABL1. The FISH probes for PDGFRB overlap with the neighboring CSF1R locus. At least 100 interphase nuclei were evaluated.FISH was performed on interface nuclei using break apart probes  for 2 and buffer from Applied Biosystems . For primer sequences see cDNA was synthesized from 1 μg total or copy RNA using M-MLV reverse transcriptase and combined oligo-dT and pdN6 priming in 20 μl . PCR was performed on 2.5 μl cDNA using Taq polymerase, MgClCumulative incidence of relapse (CIR) was estimated using a competing risks model. We considered relapse as event, and death as competing event. To test for equality of CIRs, Gray's test has been applied. The CIR probability (pCIR) with standard error was reported. Outcome analyses were performed in R 3.0.1, using the packages cmprsk version 2.2-6 , mstate


Program evaluation of this 10 lecture series demonstrated high levels of satisfaction among learners and instructors as well as knowledge gain by learners. An average of 5.5 learners and 2 instructors attended the 10 lectures and completed surveys using a Likert scale to rate statements regarding lecture content, technology, and personal connection. Positive statements about lecture content and the applicability to daily work had 98% or greater agreement as did statements regarding ease of technology and convenience. The learners agreed with feeling connected to the instructors 100% of the time, while instructors had 86.4% agreement with connection related statements. Instructors, joining at their respective work locations, rated convenience statements at 100% agreement. This evaluation also demonstrated effectiveness with an average 10.7% increase in pre- to posttest knowledge scores by learners. As the global health community considers efficiency in time, money, and our environment, telehealth education is a critical method to consider and develop for health worker education. Our pilot program evaluation shows that telehealth may be an effective method of delivering education to frontline health workers in rural Guatemala. While larger studies are needed to quantify the duration and benefits of specific knowledge gains and to perform a cost-effectiveness analysis of the program, our initial pilot results are encouraging and show that a telehealth program between a US-based university and a rural community health program in a low- and middle-income country is both feasible and acceptable.Telehealth education has the potential to serve as an important, low-cost method of expanding healthcare worker education and support, especially in rural settings of low- and middle-income countries. We describe an innovative educational strategy to strengthen a long-term health professional capacity building partnership between Guatemalan and US-based partners. In this pilot evaluation, community health nurses in rural Guatemala received customized, interactive education  Over one billion people globally have limited access to health services, with many living in rural areas in low- and middle-income countries (LMICs) . One impvia teleconference, including in specialties such as obstetrics and gynecology . The anonymous survey data were collected and stored in Google Docs and then further processed in Microsoft Excel©. Two blocks of five lectures were given. Each block was preceded by a pre-test, consisted of one lecture per week for 5 weeks and was followed by a posttest after the last lecture was given. Pre- and posttests assessed knowledge of each of the five topics per block, with 20 points available per topic and an overall 100 points per test for each of the two blocks. Each lecture was followed by a quality and satisfaction evaluation assessing content, technology, and connection between instructors and learners on a 4-point Likert scale. Instructors completed a separate evaluation assessing technology, convenience, and connection. Both evaluations requested details on technology difficulties and general feedback on the teaching. As technical problems can affect the students’ perceptions of quality  with population SD. Percent of “agree” responses (scores 3 and 4) was also calculated per survey question. Survey questions were grouped into assessments of lecture content, technology, and connection among instructors and learners. Knowledge gain was measured by percent improvement per subject and overall pre- to posttest scores. Due to the small number of students, instructors, and lectures, correlation among survey responses per lecture was not performed.n = 4, 1 year post high school) or professional nursing school . All CHNs were female, averaging 25 years of age and 5 years post-completion of nursing school. The lead instructor was a pediatrician at our institution (Kelly A. McConnell). Additional instructors were pediatricians (Maya Bunik and Gretchen J. Domek), a pediatric nurse practitioner (Maureen Lenssen), and a recent medical school graduate from Guatemala (Saskia Bunge Montes). All instructors had been to the site in rural Guatemala and had been involved with the implementation of the community health program.The seven CHN learners in this program had completed either auxiliary nursing school . The amount of time to connect to the system was most frequently 5–10 min but took up to 15 min. Three lectures were rescheduled due to a lack of Internet connection at the site in Guatemala; two were done later in the same day and one was rescheduled to a different day. Overall, the CHNs were extremely satisfied with the lecture delivery via telehealth as well as the direct teaching.There was an average of 5.5 learners present per lecture. Responses from the learners were overall positive, especially regarding lecture content with 98% or greater agreement with each positive statement Table . The tecAn average of two instructors was present for each lecture, one lead instructor and generally one additional team member. Audio quality was again reported to be better than video quality with “I could see the learners well” agreed upon only 59.1% of responses compared to 86.4% for audio  time and travel and accommodation costs. A concept of planetary health linking human health, flourishing civilizations and the environment, described in the Lancet commission on planetary health , must beA major limitation to this study was that a specific cost-effectiveness analysis was not performed as the set-up of equipment, bandwidth, licensing of software, and faculty and CHN salaries were all included free-of-charge to our program. While most economic evaluations of telemedicine have failed to show significant cost savings , there iAnother limitation of our study was that the CHNs completed the surveys and tests anonymously, preventing pairing of pre- and posttest scores or the ability to adjust for lecture attendance. This was, however, done to encourage full participation and to remove concerns about job performance, which we feel aided in more candid survey responses. Furthermore, our dataset of surveys and tests was small, as it was designed as a pilot program evaluation, limiting our ability to calculate further correlation or data beyond central tendency. We do not know from our small pilot study whether the knowledge gain by the CHNs translated into better work performance and for what duration of time the knowledge gain persisted. This will be another important area of future exploration.We are limited in the sustainability of this project which could provide additional quality improvement cycles as well as more detailed data collection, especially as these additional research questions arise. As this program developed in response to an expressed need, our team had several champions for CHN education. This is the main project of a Global Health postdoctoral fellow, who has moved to a different practice location. The program would benefit from an assigned hub of responsibility such as each oncoming fellow, a resident in our global health program or another long-term champion. The project is likely to continue but not necessarily with the same structure or data collection.Several lectures were followed by informal discussions regarding the overall telehealth experience. These discussions informed the following lectures in an unofficial improvement cycle. Throughout our experience with telehealth for education, we observed that the learners were much more engaged if the didactic portion was limited to 30 min and interspersed with interactive questions supplied by the instructor and real patient cases brought by the CHNs. Engagement was not specifically measured by the instructors but was discussed after lectures among team members. Also, limiting the use of video and audio transmission for those instructors not directly teaching improved the clarity and focus of the lead instructor and allowed the lead instructor to see and hear the learners better. This allowed for reengagement, questions, and breaks as necessary, similar to the way a live teacher can respond to a class. The CHNs reported preference for a larger screen for viewing, especially compared to a small laptop screen as was most commonly used. In the future, it will be important to provide a larger screen and speakers to allow for better video conferencing at the rural LMIC site. Flexibility and patience from both the learners and instructors were also important since some lectures had to be delayed or postponed due to poor connectivity, although this was rare. Anecdotally, the instructors noted that experience with telehealth, especially in understanding the delay in videoconferencing, allowed for better interactions and stronger feelings of connection.Our program evaluation shows that telehealth may be an effective and low-cost method of delivering education to frontline health workers, specifically CHNs in rural Guatemala. Post graduate nursing education targeted to current fieldwork was delivered using existing technology systems and easily available resources during work hours with high satisfaction among instructors and learners as well as knowledge improvement among learners from pre- to posttest scores. There are likely to be significant economic benefits to such a telehealth program, including improved fieldwork by CHNs and decreased travel costs by faculty. While larger studies are needed to quantify the duration and benefits of specific knowledge gains and to perform a cost-effectiveness analysis of the program, our initial pilot results are encouraging and show that a telehealth program between an academic university in a high-income country and a rural community health program in a LMIC is both feasible and acceptable. The relationship between the faculty at the US-based Center for Global Health and the frontline health workers at the rural LMIC site in Guatemala is integral to the continued success of this international partnership and community health program, which provides critical health-care access to a rural and impoverished population. The frequent interactions and time and effort given to these educational sessions provide ongoing CHN training in support of the community health program goals and display concern and support for the daily work of the CHNs, contributing to the continuation and longevity of this important partnership.All authors participated in multiple meetings regarding the design of the educational program and the program evaluation. KM was the instructor for all teaching sessions in this program evaluation, with supervision and approval of materials by GD. All authors participated in teaching, primarily MB and ML, prior to this specific program evaluation. LK coordinated educational sessions and analyzed the data with KM. SM, a Guatemala native, assisted with translation and creation of educational materials as well as communication with CHNs. KM did the primary writing, closely working with author GD. All authors reviewed the manuscript and gave final approval.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.


Evaluating educational programs can improve the quality of education. The present study evaluated the undergraduate occupational health program at the Semnan University of Medical Sciences in Semnan, Iran, with a focus on the associations between alumni perceptions of the learning environment and the outcomes of the occupational health program.A cross-sectional questionnaire survey was conducted among alumni of the undergraduate occupational health program. We asked alumni to rate their perceptions of the items using a 4-point Likert scale. The associations between alumni perceptions of the educational program and curriculum, faculty, institutional resources, and learning outcomes were modeled and described using structural equation modeling procedures.A descriptive analysis of alumni perceptions indicated low evaluations for the administrative system, practical and research-based courses, and the number of faculty members. We found that a structural model of the evaluation variables of curriculum, faculty qualifications, and institutional resources significantly predicted undergraduate educational outcomes. The curriculum had direct and indirect effects on learning outcomes, mediated by faculty.The findings of our study highlight the usefulness of the structural equation modeling approach for examining links between variables related to the learning process and learning outcomes. Surveys of alumni can provide data for reassessing the learning environment in the light of the professional competencies needed for occupational health graduates. In recent years, the academic literature and many national reports have recommended paying greater attention to the evaluation of educational programs . The thePrevious studies have suggested that curriculum design may enhance students’ motivation for learning ,8. The fIn Iran, the occupational health departments of medical sciences universities are responsible for the design and delivery of undergraduate occupational health educational and training programs. This study examined the associations between occupational health program alumni’s perceptions of the learning environment and their learning outcomes. We performed a comprehensive test of the associations among context, process, and outcome variables that were used for educational evaluation, based on responses from Iranian alumni engaged in occupational health activities. A cross-sectional survey was performed from September 2015 to September 2016.The target subjects comprised alumni who graduated from a 4-year undergraduate occupational health degree program at the Semnan University of Medical Sciences between 1998 and 2014. The inclusion criteria were enrollment in the occupational health program at the Semnan University of Medical Sciences and professional engagement in occupational health activities. After obtaining the addresses of alumni from the educational affairs unit database, the questionnaires along with instructions were distributed to all alumni by mail and email.Faculty members with experience in the occupational health program contributed to developing the alumni survey instrument. The study evaluated the perceived opinions of alumni on a variety of important topics to assess the undergraduate program in occupational health. The survey included descriptive items for alumni self-reporting on the quality of the undergraduate program, as well as items on the demographic characteristics of alumni, such as their involvement in postgraduate studies and employment status.The first draft of our questionnaire consisted of 35 items distributed across 4 scales. That version was completed by 97 subjects to perform exploratory factor analysis. We deleted items with factor loadings less than 0.4, resulting in the elimination of 7 items. Of the study population, 126 alumni completed and returned the second version of the questionnaire. We performed confirmatory factor analysis to validate the structure of the survey instrument.The final version of the questionnaire consisted of 28 items in 4 scales. The outcomes assessed the degree to which respondents perceived enhancements of their cognitive knowledge, skills, and abilities after their course of study. Five items assessed the program outcomes, with a focus on curriculum, learning sessions, and administration in the undergraduate occupational health program. Twelve items assessed the occupational health curriculum and educational program. The faculty construct measured the quality of faculty in terms of education and training. Six items assessed the faculty. The category of institutional resources described the equipment and facilities of the university, as measured by 5 items. Responses were given on a 4-point Likert frequency scale. Alumni perceptions of the undergraduate program were analyzed according to gender using multiple analysis of variance and comparison of means.A structural equation model was used to conduct confirmatory factor analysis and to evaluate the research model in the LISREL software program . Our meaConfirmatory factor analysis was carried out to confirm the validity of the instrument and the measurement model . For allThe Cronbach alpha coefficient was used to measure the internal consistency and reliability of our survey constructs. The explanatory associations of the model were assessed by estimating the coefficient of determination of the endogenous dependent learning outcomes construct. In order to assess the extent to which the individual constructs were discriminated from each other by the survey instrument, we estimated the square root of average variance extracted for each construct and inter-construct correlations .In our conceptual model, the curriculum and program, resources, and faculty variables were assumed to affect program outcomes. We estimated the goodness of fit of the proposed model by the chi-square test, goodness-of-fit index, root mean square error of approximation, and standardized root mean square residual, as suggested by Kline .Ethical approval was obtained from the Ethics Committee Review Board of the research center of Semnan University of Medical Sciences (Reference code: IR.SEMUMS.REC.1395.233). We considered completed and returned questionnaires to indicate the provision of informed consent.Data from a total of 126 alumni who answered and completed the survey were included in the study, resulting in an approximate response rate of 37%. Raw data were available from Our survey measured alumni perceptions of the level of cognitive and behavioral knowledge they achieved by participating in the undergraduate occupational health program. Forty-eight percent of the participants agreed or strongly agreed with the item assessing learning outcomes. The item relating to their understanding of occupational health concepts received the highest score. Furthermore, alumni evaluated the overall structure of the curriculum and program as relatively appropriate. Seventy-six percent of the alumni reported strong agreement regarding clear announcements of the goals of the program, curriculum content and its availability, and educational materials.However, they identified several areas requiring modifications and improvements. For the items related to administration system, laboratory courses, research and project internships, and the number of faculty members specialized in occupational health, over 40% of the responses indicated disagreement or strong disagreement. The majority of alumni reported agreement or strong agreement for the item on library facilities. However, the respondents presented poor evaluations of other items describing laboratory equipment and the number of technical staff.Fifty-four percent of the alumni reported encouraging opinions on the items evaluating whether faculty members dedicated enough time to student instruction and had appropriate qualifications and experience in teaching and research, except for the items “faculty workload allowed them to fully carry out their role” and “faculty members provided students with various learning methods,” which received responses of disagree or strongly disagree by 43% of the alumni.Our results indicate that for all educational program evaluation constructs, the variance in participants’ scores was on average moderate. Learning outcomes had the largest variance of all 5 constructs. The least variance was found for the educational curriculum and program.Male alumni evaluated learning outcomes more favorably than female alumni (P< 0.05). However, female perceptions were more favorable toward faculty members and the curriculum (P< 0.05).The associations of alumni perceptions of the curriculum and program, faculty, resources, and learning experiences with the outcome variables were analyzed by means of structural equation modeling. The fit indices of the hypothesized initial and final models showed a relatively adequate fit to the data , implyinOur analysis found that faculty perceptions had the largest positive effect on learning outcomes (β= 0.65). Therefore, the more positively alumni perceived the level of quality and activities of the faculty members, the more positively they perceived their learning achievements.Perceptions of the curriculum were associated significantly and positively with learning outcomes. However, the curriculum had a direct effect of 0.32 and an indirect effect of 0.13 through the faculty variable, and thus a total effect of 0.51 on alumni learning outcomes. This means that the more positively alumni perceived the curriculum and program, the more they were satisfied with their learning experiences and the outcomes of their undergraduate program.Finally, the institutional resources variable had a direct effect of 0.26 and an indirect effect of 0.11 on learning achievements through the curriculum and program. Therefore, the total effect of institutional resources on the learning outcomes was considerable. This implies that as alumni perceive and evaluate institutional resources more positively, they are likely to have a favorable evaluation of their learning outcomes.The measurement of variance associated with the dependent learning outcomes variable was used to assess the explanatory influence of the proposed model. The coefficient of determination showed that 72% of the variation in the alumni perception of learning outcomes could be explained by their perceptions of the curriculum and program, faculty, and resources.The purpose of this study was to present and analyze how alumni evaluated an undergraduate program in occupational health. We applied an experimental methodology capable of analyzing the degree of influence of curriculum and program, faculty performance, and institutional resources on program outcomes.The current study found that faculty had a direct influence on the learning experience and outcomes, with the strongest association in the total structural model 0.65). This influence was around twice as strong as the influence of the curriculum and administrative procedures. This implies that faculty quality is important for explaining alumni perceptions of the undergraduate educational process and learning outcomes. In other words, experiencing more mentor support in the academic environment is highly likely to improve alumni perceptions of learning outcomes. This is consistent with previous research reporting that mentorship could enhance the learning process [5. This iOur study revealed that curriculum and program administration both directly and indirectly influenced the outcomes variable through the faculty variable. A previous study recommended that evaluations of academic programs should be focused not only on curricula, but also on the activity of the faculty . ResearcThe present study clearly showed that faculty can play a crucial role in predicting educational outcomes. Therefore, academic institutions and departments should be careful in recruiting and evaluating faculty members. Academic departments offering an occupational health program at the undergraduate level should encourage faculty members to consider their interactions with learners, qualifications, expertise, and effective learning methods. In academic settings, quality enhancement practices may be introduced to improve the skills of faculty members. Meanwhile, previous reports have emphasized that departments must provide support for faculty members to incorporate research and project-based learning into their teaching approach. This will assist students to learn how to deal with real situations that they may encounter in their future careers ,11.The quality of the curriculum can be considered a decisive factor in improving learning outcomes. Alumni who evaluated the content of the curriculum as suitable more often reported improvements in their learning ability, teamwork, and field knowledge. An earlier study indicated that curricular design had a positive effect on the judgements of graduates concerning the usefulness of the educational program for their current jobs . The typIn our study, alumni believed that little attention had been paid to incorporating research and projects in the undergraduate occupational health program. We suggest that occupational health departments integrate problem-solving and project-based learning into the curriculum and incorporate practice-based research into their professional educational program. However, to achieve this successfully, faculty members interested in engaging in research activities should be recruited .In conclusion, the network of associations in the proposed structural model revealed that learning outcomes were statistically related to the curriculum and program, institutional resources, and faculty. The structural equation model is valuable for program evaluation and obtaining information from alumni, because it allowed us to obtain useful data for internal program planning and for assessing the fit of the educational environment and occupational health discipline curriculum with alumni’s professional opportunities. However, a limitation of the present study is that we analyzed data from a single academic institution with a focus on occupational health education, and the variables described might be peculiar to this area of study. It should be emphasized that because the data were collected through a cross-sectional design, our analysis was limited to identifying associations between measured latent variables, rather than causality.


Yersinia pestis by fluorescence microscopy. In response, for each of the 17 affected communities, village-wide IRS was conducted to control rodent-associated fleas within homes, and community sensitization was conducted to raise awareness of plague signs and prevention strategies. No additional presumptive Y. pestis-positive carcasses were detected in these villages within the 2-month expected duration of residual activity for the insecticide used in IRS. Despite comparatively high historic case counts, no human plague cases were reported from villages participating in the surveillance program; five cases were reported from elsewhere in the districts. We evaluate community participation and timeliness of response, report the frequency of human plague cases in participating and surrounding villages, and evaluate whether a program such as this could provide a sustainable model for plague prevention in endemic areas.Plague, primarily a disease of rodents, is most frequently transmitted by fleas and causes potentially fatal infections in humans. In Uganda, plague is endemic to the West Nile region. Primary prevention for plague includes control of rodent hosts or flea vectors, but targeting these efforts is difficult given the sporadic nature of plague epizootics in the region and limited resource availability. Here, we present a community-based strategy to detect and report rodent deaths , an early sign of epizootics. Laboratory testing of rodent carcasses is used to trigger primary and secondary prevention measures: indoor residual spraying (IRS) and community-based plague education, respectively. During the first 3 years of the program, individuals from 142 villages reported 580 small mammal deaths; 24 of these tested presumptive positive for  Yersinia pestis. The plague bacterium has a nearly global distribution; however, in recent decades most plague cases have been reported from East Africa and Madagascar.2 In Uganda, plague is endemic in the highlands of the far northwest, which are known as the West Nile region.6 Here, Y. pestis is maintained in enzootic cycles among sylvatic and peridomestic rodents and their fleas, with Arvicanthis niloticus and Crocidura sp. likely playing important roles in plague epizootics.7 During plague epizootics, infections are assumed to spill over into Rattus rattus, which is commonly encountered within households in the West Nile region, is highly susceptible to plague infection, and harbors efficient Y. pestis vectors (Xenopsylla cheopis and Xenopsylla brasiliensis). Based on these findings, humans are presumed to be exposed to plague bacteria most commonly in and around homes when rats die, forcing their potentially infectious fleas to find an alternative host, including humans.8 Most plague cases occur between the months of September and December in the West Nile region.9 However, the number of cases occurring per year is highly variable; interannual variation in suspect plague cases has been correlated with seasonal rainfall patterns.9Plague is a life-threatening, flea-borne, rodent-associated zoonosis caused by 10 Recent studies from the West Nile region have shown that indoor residual spraying (IRS) and insecticide delivery tubes effectively reduce flea loads on rodents in the home environment where most exposures are believed to occur.12 While most households in a 2013 study self-reported using some form of rodent control, such as lethal trapping, application of rodenticide, or cat ownership;13 thus far, rodent control, specifically the use of poisons, or traps, have not proven to be effective given the massive resources required to sustain suppression of populations.14 Secondary prevention of plague aims to reduce case fatality rates through education campaigns that emphasize recognition of signs of plague and urging persons with symptoms consistent with plague to seek care without delay.10 Early diagnosis followed by an appropriate antibiotic therapy significantly improves outcomes of patients with plague.15Primary prevention of human plague typically focuses on vector control or rodent reduction within limited areas affected by plague epizootics.16 previous environmental investigations of human plague cases in the West Nile region have noted that villagers commonly report seeing a larger than usual number of rat carcasses  before the onset of human plague cases.7 We wanted to know if we could capitalize on these observations, using them to inform timely, targeted interventions to prevent human cases.Although the factors that trigger epizootics remain poorly defined,Y. pestis-positive results trigger community education and target implementation of vector control (IRS) to prevent human plague cases. Our specific objectives are to evaluate community participation and timelines of response under the RFS program, and to report frequency of human plague cases in participating and surrounding villages. More broadly, we aim to describe the success and limitations of this community-based plague prevention program and evaluate whether RFS might be applied successfully within resource-limited endemic areas.Here, we describe an animal-based surveillance and early response program  that engages members of the community, volunteer village health teams (VHTs), subcounty environmental health officers, and local leaders. Through the program, rat falls are reported, carcasses are collected and tested, and 6 were added to the surveillance network in September 2014. During the evaluation period, two villages were elected to split into four; these four villages were treated separately during analysis.This program was implemented and evaluated in the Arua and Zombo districts of the West Nile region, Uganda, from July 1, 2013 to June 30, 2016. A total of 83 villages, representing a local estimate of approximately 37,000 persons, were selected for participation among 563 local villages with a history of plague and included many of those reporting the greatest number of confirmed or suspect human plague cases between 1999 and 2011  . To expa13 Within each village, one VHT member, a volunteer elected by local leaders and under the direction of subcounty environmental health officers to support the goals of the Uganda Ministry of Health, was invited to participate in the program.The 83 selected villages were typical of those situated throughout the two districts where subsistence farming is the dominant occupation type, and most families live in huts of traditional construction .A schematic overview of the RFS and response program is shown in 17 Carcasses were then necropsied and touch-preparation slides of the liver and spleen tissues were tested for the presence of Y. pestis F1 antigen using a direct fluorescent antibody (DFA) assay described elsewhere.18 Carcasses were considered presumptive positive for Y. pestis and actionable if the presence of the Y. pestis F1 antigen was detected in one or both tissues. For the purpose of brevity, we later refer to carcasses testing positive or equivocal by DFA as Y. pestis-positive.Processing, testing, and storage of potentially infectious carcasses were conducted in an access-controlled laboratory under BSL-2 conditions. When possible, carcasses were identified to species using a published key.Y. pestis, UVRI staff notified the District Health Officer, Uganda Ministry of Health to recommend timely IRS treatment of all the huts in the reporting village to reduce the numbers of hut-associated, potentially infectious fleas. For the duration of the program reported here, a wettable powder formulation of bendiocarb  was applied to interior walls and floors after a modified application method described previously.12 The residual activity of Ficam-VC against rodent-associated fleas was unknown; therefore, the minimum expected duration of activity against malaria vectors on mud surfaces, or 2 months, was assumed.20When a carcass tested positive for Y. pestis-positive carcass results. Immediately after notification of the test result, the VHT reported the finding directly to the household where the carcass was found. UVRI staff, together with village leadership, hosted meetings at places of worship, schools, markets, and other community spaces to raise awareness of plague at the village level and to share messages of primary and secondary plague prevention. Finally, the UVRI staff notified the health care facilities within the catchment area of the affected village, as well as local traditional healers to alert them to the increased potential for human plague cases.In addition to IRS, a number of community sensitization efforts were initiated in response to Y. pestis was not detected in a carcass, the laboratory staff directly notified the submitting VHT who then gave the result to the individual(s) who reported the carcass. After notification of the person(s) reporting the small mammal death, no further response actions were initiated.When To improve the surveillance and response program and address specific programmatic issues, UVRI and Centers for Disease Control and Prevention (CDC) leadership elicited feedback from VHTs, district- and sub-county-level representatives, environmental health officers, field and laboratory staff, and others annually at refresher training workshops and through organized periodic meetings with key stakeholders. To identify where disposable supplies were needed, what equipment types were useful or problematic, and which areas of the communication or response program could be improved, the UVRI staff also contacted VHTs by phone on a monthly basis and responses were summarized.Y. pestis-positive and Y. pestis-negative carcasses. The overall metric used to evaluate the timeliness of the program was the mean or median number of days between the initial report of a small mammal carcass and the completion of the response.Village-level reporting data, notification, and test result dates were used to evaluate the timeliness of the RFS program for both Statistical comparisons of various response times were made for all carcasses between years 1, 2, and 3 using analysis of variance among them, assuming unequal variance, and if any statistically significant difference was found; a Tukey’s multiple comparison of means test was used to identify where the difference lay. Difference of medians were compared using the Wilcoxon–Mann–Whitney exact test. The Mann–Kendall test was used to evaluate whether a trend existed in IRS response times, whereas a linear model with periodic covariates was used to evaluate carcass submissions by month.21 whereas figures and comparisons were produced using R statistical software.22Proportions were compared using a mid-p exact test for two proportions and a Fisher’s exact test for three proportions. For all statistical comparisons, significance was declared at the alpha = 0.05 level. Data summarization was performed using the JMP software suite,Before the initiation of the program, all protocols were reviewed and approved by the UVRI Research Ethics Committee, Uganda National Council for Science and Technology, and the Uganda President’s Office. Review by the CDC, the National Center for Emerging and Zoonotic Infectious Diseases Human Subjects Coordinator determined the protocols for this program to be nonhuman subjects’ research, thus a full committee review was not required.Of the 83 participating VHTs, 68 (80%) reported and submitted at least one small mammal carcass through the program during the 3 years of evaluation. In addition, small mammal deaths were reported through spontaneous channels of communication by community members from another 74 villages throughout the region that were not initially selected for participation in the program . During N = 484; 92.5%) were made to report the observation of a single carcass, whereas the remainder  were made to report multiple carcasses on a single day. Small mammal deaths were reported to the surveillance program in every month of the year (P < 0.0001). The most commonly reported species was R. rattus (N = 432), which represented 74.6% of the carcasses submitted, followed by A. niloticus , and Mastomys sp. ; the remaining nine other species combined (N = 27) represented 4.7% of total submissions. A small number of carcasses were not identifiable because of poor condition of the carcass . Species belonging to the genera Mastomys and Crocidura are difficult to distinguish morphologically; therefore, these identifications were made at the genus level. Based on earlier molecular identifications from the same region, however, small mammals identified as Mastomys spp. were likely either Mastomys natalensis or Mastomys erythroleucus and Crocidura spp. were most likely Crocidura olivieri.7 Of the carcasses for which location of collection data were available (N = 567), 69.1% were found indoors (N = 392), 30.5% were found near the home (N = 173) and 0.4% were found more than 30 m from the home (N = 2).Between July 1, 2013 and June 30, 2016, a total of 580 small mammal carcasses were submitted through the RFS program. Of the 523 calls received over the 3-year surveillance period, most  tested negative for Y. pestis, and 1 (< 1%) was too desiccated to test. All carcasses that tested Y. pestis-positive were reported between the months of September and April , whereas A. niloticus and Mastomys sp. comprised 16.7% and 4.2% of the total, respectively. There was not a statistically significant difference in infection prevalence among R. rattus (4.4% positive of 433 submitted), A. niloticus (4.4% of 90), and Mastomys sp. (6.3% of 16) (P = 0.72). Although less commonly reported, small mammal carcasses submitted in groups (> 1 per day from the same village), were significantly more likely to test positive for Y. pestis (10.3% of 39 carcass groups) than carcasses submitted singly (2.5% of 484 carcasses) . There was no significant difference in the percent of carcasses testing positive for Y. pestis submitted from participating and “out of network” villages .Of 580 carcasses submitted, 24 (4.1%) tested positive by DFA for nd April . Most caY. pestis-positive carcass, including one village that was treated twice, once in November 2013 and again in November 2014 . For all comparisons, the final report of test results back to the community member(s) who observed the carcass was conducted by the VHTs; dates of this final response step were not recorded and could therefore not be evaluated.The average number of days between critical communication and action steps are shown in Y. pestis-positive carcass from a village and completion of the two main response efforts, IRS and community sensitization, are shown in P = 0.01).The number of days between the detection of the first Y. pestis-positive carcasses were evaluated to identify delays. Test results were communicated from the laboratory through the UVRI epidemiology coordinator, and the recommendation to use IRS was made to district staff within a median of 1 day (range: 0–5) and mean of 1.1 days (95% CI: 0.3–1.9). District-level coordination to implement IRS initially added an additional 26.9 days on average to the total response time . Input from key stakeholders revealed that delayed mobilization of supplies, lack of funding for sprayer stipends, and poor motivation of IRS spray operators were, in part, the source of district-level delays to IRS intervention efforts. Key stakeholders from all levels of the program met to resolve these issues, and subcounty and district leadership resolved to provide timely reimbursement to IRS spray operators and to decentralize spray equipment. As a result, IRS response time decreased significantly after the meeting, from a median of 30 days to 7 days  (Intermediate communication steps after the detection of < 0.001) .n = ∼480) reported approximately 19 confirmed and 1,260 suspect and probable human plague cases during the same time period. During the surveillance period , five laboratory-confirmed human cases of plague were reported from three villages outside the surveillance network, whereas no laboratory-confirmed plague cases were reported from the 83 participating villages. We chose not to compare case counts between villages inside and outside the network primarily because carcasses were accepted from all villages in the region regardless of how they learned about the program. Moreover, we responded to all positive carcasses and any human cases in the area regardless of their role in this study. Therefore, we did not have a valid comparison group to assess the impact of the program on plague case counts.In the years leading up to RFS, from 1999 to 2012, there were 59 laboratory-confirmed and 1,149 suspect and probable cases of human plague among the 83 villages participating in the RFS program. Other villages from Arua and Zombo districts with historical plague activity . Single or multiple challenges were raised on 812 calls and included problems with program-critical supplies such as cell phones or solar chargers (N = 460) and accessory supplies, such as GPS units and batteries, insulated cold-storage boxes, or bicycles (N = 239), requests for disposable supplies, such as plastic bags, gloves, or insect repellent (N = 205), issues with the program administration or key stakeholders (N = 57), or other concerns (N = 33). Although cell phone coverage was available across the study area, service outages, and phone calls made to persons outside of service range were common, and resulted in 844 of the total 1,039 (81%) failed communication attempts.UVRI staff attempted a total of 2,541 monthly phone calls to enrolled plague monitors to obtain feedback on the program and supplies. Of these calls, approximately half were received , recognition of plague symptoms, and the importance of seeking care early in the course of infection to improve outcomes of infection.15 Although we did not statistically evaluate whether the program significantly reduced human plague case occurrence, it is noteworthy that despite participating villages having a history of elevated case counts and evidence of plague activity in small mammals during the surveillance period, no human plague cases were reported from any of these villages. By contrast, five laboratory-confirmed human cases of plague were reported from three villages outside the surveillance network.IRS is intended to reduce flea loads on hut-dwelling rats and human contact with 12 More cost-effective methods of flea control, such as topical insecticide rodent tubes11 may provide an alternate strategy. Given sufficient residual activity, this or other similar low-cost interventions could be deployed before the start of the plague season to reduce the risk of infectious flea bites or within the current program in response to rodent die offs. Alternately, community-level sensitization in response to reports of small mammal carcasses in the absence of IRS would be comparatively inexpensive and could reduce human plague cases and fatality rates through encouragement of plague prevention strategies and early care-seeking behavior. While the efficacy of this type of community awareness-only approach has not been evaluated for RFS, an ongoing plague detection and referral program in West Nile, may provide evidence that such a program can succeed.23The absence of human plague cases after IRS and community sensitization are suggestive that a combined response to detection of rat falls is an effective plague prevention strategy. Although IRS is effective against on-host fleas, the costs of such interventions can be significant when supplies, training, and transportation are included.Natural fluctuations in the rodent and flea populations could likewise have contributed to the reduction in cases within participating villages, although given the proximity of these villages to those that reported plague cases, it seems unlikely that they would have experienced unique ecological shifts. It is difficult to draw conclusions, however because the scope of our evaluation did not include systematic live trapping efforts.Y. pestis, to direct limited resources toward intervention efforts. Confirmation of DFA results can be achieved by culture isolation and specific bacteriophage lysis.18 However, the timely sourcing and shelf life of these specialized supplies proved problematic in this setting. Further, culture and phage lysis steps are time-consuming, and add days to the turnaround times for incoming samples.24 Therefore, for this program, DFA was chosen because the assay can detect the presence of Y. pestis in tissues of infected animals for days or weeks after death24 and because results can be obtained rapidly, to allow for rapid response when Y. pestis transmission is suspected. In the future, the integration of field-based diagnostic platforms, such as lateral flow (dipstick)25 or loop-mediated isothermal amplification assays,26 could improve sustainability of this type of program, given adequate sensitivity and specificity for field-collected carcass samples.One limitation of this program was the use of DFA, a presumptive test for Y. pestis-positive carcasses were submitted from any of the 17 affected villages within the 2-month range of expected residual activity after IRS. However, 7 of the 17 village-based sprays were enacted late, from 22 to 113 days after the submission of a Y. pestis-positive carcass; in these villages, there were no Y. pestis-positive carcasses submitted during the interim between detection of the first positive carcass and IRS. Therefore, it is difficult to determine if the absence of additional Y. pestis-positive submissions from these villages was solely due to IRS, whether enzootic activity halted independently of the treatment, whether subsequent rodent deaths were occurring but were not detected by homeowners, or whether presumptive testing for Y. pestis from the carcass produced a false positive result. In future evaluations, control of host-associated fleas by the specific chemical to be applied by IRS could be measured and should be conducted in advance of scheduled changes to chemical types.Although the RFS program was not structured to answer questions about IRS effectiveness and chemical residual activity against rodent-associated fleas, we nonetheless evaluated submission records to explore the idea that enzootic transmission was interrupted in villages after IRS treatment. We observed that no additional Y. pestis and prevent human exposure to plague bacteria.10 Therefore, the timeliness of communications and response efforts were key indicators of program success. Rapid reporting, collection, submission, testing, and relaying of test result meant that at the conclusion of the third year of the program, village residents were informed of Y. pestis test results within 2 days of detecting small mammal deaths in their community. Communities experiencing small mammal deaths attributable to transmission of Y. pestis received timely information on the signs, symptoms, and treatment options for plague infection; throughout the duration of the 3-year period of evaluation, community sensitization efforts were consistently conducted within 4 days and never more than 9 days after the initial discovery of small mammal deaths. Issues with reimbursement of IRS technical staff and distribution of supplies initially slowed the deployment of village-based IRS; however, decisions made between key stakeholders during the first year of the program decreased response times significantly, from more than 40 days to fewer than 10. Overall, the time from a community member discovering a carcass to village-wide IRS decreased significantly over the 3 years of evaluation, meaning that affected communities were treated against infectious fleas sooner, thus reducing the likelihood of human cases.Rapid plague control interventions are important to halt enzootic transmission of Rattus rattus, the most common species submitted through the surveillance program, is highly susceptible to plague infection and maintains a close association with human habitations in the West Nile region.27 Because of this combination of characteristics, R. rattus in particular is a good sentinel for plague activity because increased mortality in and around huts may be more easily detected by village residents compared with mortality in sylvatic rodents and insectivores.28 Indeed, most small mammal carcasses reported through the program were found either indoors or in peridomestic areas. On the other hand, much of the mortality that village residents reported was not attributable to Y. pestis infection, resulting in a large number of samples tested yielding few plague-positive results.Y. pestis. However, the harms associated with failure to apply appropriate interventions in the event of an active epizootic are high, and in the 3 years of evaluation, use of this adapted submission criteria would have resulted in treatment and sensitization of only 3 of the 17 (17.6%) affected villages. Another strategy to reduce program costs associated with testing a large number of negative samples might be to limit the number of months per year that carcasses can be submitted because most human cases in the region are reported during the months of September through December.9 However, during the 3 years of RFS evaluation, if we assumed a submission window of July through December to allow lead time in recognition of epizootics before human cases, only 15 of the 24 Y. pestis-positive carcasses (62.5%) would have been detected and would have resulted in failure to apply interventions 7 out of 17 times (41.2%). Therefore, using a limited time frame for submissions could not only introduce confusion over when to report carcasses, but could ultimately result in failure of the system to detect and respond to epizootic events when they occur outside a defined temporal window. Indeed, while most human cases in the region are reported from September through December, suspect human plague cases have been reported in every month of the year.9 Finally, because numbers of carcass submissions by month were highest during the “plague season,” testing on nearly 400 Y. pestis-negative carcasses would have been conducted during an annual July through December timeframe, reducing testing effort during the 3 years by only 204 samples (∼35%).To reduce the number and associated cost of reporting, retrieval, and testing of negative carcasses submitted through the RFS program, we explored limiting the definition of “rat fall” from the observation of one or more dead rodents, to include only the observation of more than one dead rodent in a single day because carcasses submitted in groups (> 1 per day) were more likely to test positive for 30 may diminish the need for program-supplied cell phones in the future. Furthermore, local participation in and acceptability of the surveillance program was high during the 3 years of evaluation, as evidenced by spontaneous reporting from “out of network” villages, repeated submissions from participating villages, and IRS acceptance rates. The potential expansion of cell-phone use in the area paired with continued local interest may support the long-term continuation of this early-warning plague surveillance program in West Nile.Other challenges to long-term sustainability of the program involve costs associated with material supplies provided to the VHTs. Participating VHTs reported problems with solar charging panels and cell phones, supplies that were critical to reporting rodent carcasses, in approximately 20% of monthly feedback calls. Personal use of these study supplies was encouraged, but reduced the longevity of these devices, and regular replacements were made at a cost to the program. The rapid expansion of mobile phone ownership throughout the sub-Saharan region and specifically in UgandaY. pestis-positive carcasses were submitted, and no human cases were reported. Given these successes, we consider this RFS program to be a useful means of plague prevention in the West Nile region of Uganda, where plague is endemic but difficult to predict using broad-scale geographic and climate indicators.In summary, implementation of a surveillance program for plague successfully identified rat falls, an early warning sign of local transmission, in 17 villages of the West Nile region of Uganda during 3 years of evaluation. Community engagement and participant input were key for making improvements to the program so that in the final year of evaluation, IRS and community sensitization were initiated within 10 days of notification. Among the villages where plague activity was detected and IRS was conducted, within 2 months of IRS application, no additional