The Prion Laboratory Section of the Public Health Agency of Canada supports heath care professionals dealing with patients suspected to have Creutzfeldt-Jakob disease (CJD) by testing cerebrospinal fluid (CSF) for protein markers of CJD. To better serve Canadian diagnostic requirements, a quaking-induced conversion (QuIC)-based assay has been added to the test panel. The QuIC tests exploit the ability of disease-associated prion protein, found in the CSF of a majority of CJD patients, to convert a recombinant prion protein (rPrP) into detectable amounts of a misfolded, aggregated form of rPrP. The rPrP aggregates interact with a specific dye, causing a measurable change in the dye's fluorescence emission spectrum. Optimal test and analysis parameters were empirically determined. Taking both practical and performance considerations into account, an endpoint QuIC (EP-QuIC) configuration was chosen. EP-QuIC uses a thermo-mixer to perform the shaking necessary to produce the quaking-induced conversions. Fluorescence readings are obtained from a microwell fluorescence reader only at the beginning and the end of EP-QuIC reactions. Samples for which the relative fluorescence unit ratio between the initial and final readings represent a ≥4 increase in signal intensity in at least two of the three replicates are classified as positive. A retrospective analysis of 91 CSF samples that included 45 confirmed cases of CJD and 46 non-CJD cases was used to estimate the performance characteristics of the EP-QuIC assay. The diagnostic sensitivity and specificity of the EP-QuIC test of this set of samples were 98 and 91%, respectively. Subacute encephalopathies, including Creutzfeldt-Jakob disease (CJD), constitute a large and heterogeneous group of brain diseases that can be diagnostically challenging . Among tThe diagnostic approach for the most common form of CJD, sporadic CJD, has to date relied upon a standardized clinical presentation and supporting investigations such as the presence of increased amounts of indirect protein markers of disease found in patients' cerebrospinal fluid (CSF) , 4. The 8–in vivo represent a major advance in the diagnoses of sporadic CJD. In quaking-induced conversion (QuIC)   or real-n (QuIC) , the priThe high degree of sensitivity and specificity exhibited by RT-QuIC based tests in a growing number of laboratories strongly supports the inclusion of such a test in the clinical investigation of suspected cases of sporadic CJD and genetic CJD –18. In o–12–This project, involving the use of human specimens of the CJD biobank housed at the National Microbiology Laboratory, was reviewed and approved by the Health Canada and Public Health Agency of Canada's Research Ethics Board (HC-PHAC REB 2014-0033). In total 91 CSF specimens, sent to the Prion Laboratory Section for testing due to suspicion of CJD, were used: 45 pathologically confirmed cases of CJD (43 for sporadic CJD and 2 for genetic CJD) and 46 clinically confirmed non-CJD cases. The final diagnoses of all samples are provided in Table S1 in the supplemental material. All sample handling was performed in a biological safety cabinet inside a biosafety level 3 containment laboratory.The substrate used was Syrian hamster recombinant prion protein , provided by the Bristol Institute for Transfusion Sciences. Previously confirmed negative and positive CSF samples were run on each plate as controls. CSF analyses were performed in triplicate using black 96-well optical-bottom plates with 100-μl reaction volumes. Each well had 15 μl of CSF added to 85 μl of QuIC reaction buffer, resulting in a final composition of 300 mM NaCl, 10 μM EDTA, 10 μM ThT, and 0.1 mg/ml Syrian hamster rPrP in 10 mM phosphate buffer (pH 5.8). All the reagents were made fresh with mass spectrometry-grade water and passed through a 0.22-μm-pore size filter after preparation.Sealing tape was applied and both RT-QuIC and EP-QuIC reaction mixtures were incubated at 42°C with intermittent shaking cycles consisting of 90 s of shaking at 900 rpm and 30 s of rest. Fluorescence readings were taken with an excitation wavelength of 450 nm and emission wavelength of 480 nm by a FLUOstar Omega plate reader. In RT-QuIC reactions the plate remained in the FLUOstar Omega plate reader with continued cycles of double orbital shaking and relative fluorescent unit (RFU) readings every 45 min for the duration of the 90-h experiment. In EP-QuIC, the plates were placed in an Eppendorf Thermomixer C equipped with a Smart Block plate and ThermoTop with shaking cycles consisting of 90 s of circular shaking at 900 rpm and 30 s of rest at 42°C. After 20 min, the plates were removed from the Eppendorf Thermomixer C, and an initial RFU reading was taken on a preheated FLUOstar Omega plate reader using the same excitation and emission spectra used for the RT-QuIC reactions . The plates were then returned to the Eppendorf Thermomixer C, and the intermittent shaking cycles continued for 90 h. An endpoint reading was then taken on the FLOUstar Omega using the excitation and emission spectra described above.At the end of the experiment, the RFU final/RFU initial ratio of each well was calculated. Based on receiver operating characteristic curve analyses, individual wells were scored as positive in RT-QuIC reactions when the RFU final/RFU initial was ≥2.0 . Positive EP-QuIC wells were determined to have a RFU final/RFU initial ratio of ≥4.0 . A sample was considered positive when at least two of the three assay replicates for a sample were positive. In instances where one of the three assay replicates for a sample was positive, the sample was reanalyzed using three different volumes of CSF in the reaction mixture . Samples consistently exhibiting one of the three assay replicate wells as positive were scored as indeterminate. The detailed method is described in in the supplemental material.In the development of any diagnostic test a balance must be struck between its sensitivity and specificity. Too much emphasis on sensitivity increases the odds of false positives. Too much emphasis on specificity increases the odds of false negatives. In this respect EP-QuIC and RT-QuIC were slightly different. As shown in The reliability of the assay was assessed by having 8 CSF samples tested by two analysts on the same day, as well as the reanalysis of 41 samples by EP-QuIC on two separate dates. Both experiments demonstrated 100% agreement between all samples.The results in Indeterminate samples did not return a defined positive or negative result and are therefore excluded from being considered true positive or true negative results. Instead the indeterminate samples known to be CJD positive are included in the false negatives and indeterminate samples known to be non-CJD are included in false positives in the calculation of sensitivity and specificity, as shown in It had been previously determined by an EU Joint Programme–Neurodegenerative Disease Research (JPND) CSF QUIC consortium that the optimal shaking cycles for RT-QuIC reactions consisted of 90 s of shaking at 900 rpm, followed by a 30-s rest . The higTime is obviously another important factor to consider when using such assays in a clinical setting. EP-QuIC throughput could be greatly increased by having multiple thermomixers and a single plate reader. Nonetheless, the 90-h run time discussed is relatively long in comparison to the enzyme-linked immunosorbent assays used to measure 14-3-3 and other surrogate markers of CJD in CSF. Normally, the Prion Laboratory Section runs samples received during the preceding week on Monday and Tuesday, with reports being issued on Wednesday. In order to ensure adherence to this schedule expected by referring physicians, the EP-QuIC reactions are started on Friday and read on Tuesday. Other laboratories are exploring the use of different substrates and run parameters that are reported to reduce the RT-QuIC run time to hours as opposed to days . These aThe lower cost and potentially higher throughput, coupled with sensitivity and specificity comparable to that of established RT-QuIC protocols, strongly support the inclusion of the EP-QuIC protocol described in the clinical investigation of suspected cases of CJD.


Purpose. To investigate whether P. acnes could induce disc degeneration and Modic changes when inoculated into the discs of rabbits. Method. A wild-type strain of P. acnes isolated from a patient associated with Modic change and disc degeneration was inoculated into the intervertebral discs of rabbits. Meanwhile, S. aureus was injected into the discs to establish a model of discitis as the comparison and a standard strain of P. acnes was inoculated as the control. MRI and histological change were observed. Results. Both the P. acnes-inoculated and S. aureus-inoculated rabbits showed hyperintense signals at endplates and hypointense signals at nucleus pulposus on T2WI. However, P. acnes only resulted in moderate disc degeneration and endplates rupture in histological examination, which was different from the pathological change of discitis caused by S. aureus. In addition, higher death rates (2/3 versus 0/5) were observed in S. aureus-inoculated rabbits. Conclusion. Compared to S. aureus, the pathological change caused by P. acnes would be considered as Modic-I change and disc degeneration rather than a discitis. Modic changes are the successive event of disc degeneration , presentOne of the explanations was the bacterial mechanism. It was reported that some low-virulent anaerobic bacteria could be isolated from the intervertebral discs only with degeneration but without pyogenic change , 7. Subs Propionibacterium acnes (P. acnes) was the major and most frequent isolated bacterium, accounting for approximately 85%, so that it ought to be the most possible pathogen for Modic change and low back pain [Among those low-virulent anaerobic bacteria,ack pain , 8. It iack pain . More imack pain . P. acnes was the contamination during tissue harvest rather than the original colonization inside intervertebral discs [Nevertheless, there has been controversies and argumentation against the relationship between bacteria and Modic changes, highlighting that the isolatedal discs . In addial discs . To solval discs , 15. P. acnes isolated from the intervertebral disc of patient associated with Modic changes and disc degeneration has the ability to induce the same pathological change when inoculated into the discs of rabbits. In the meantime, a pyogenic discitis model caused by Staphylococcus aureus (S. aureus) was established to make a comparison and a standard strain of P. acnes was used as the control. To our knowledge, this is the first animal experiment to demonstrate the possible causative relationship between P. acnes and Modic changes, which is very important to reveal the exact role low-virulence anaerobic bacteria are playing in intervertebral discs.Therefore, the primary goal of this investigation was to test whether the wild-type strain ofThe harvest of intervertebral disc from human being was approved by the clinical Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. Animal experiments were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine. A total of eight New Zealand rabbits weighing around 2.0~2.5 kg each were included in this study . They we P. acnes was isolated from a patient who initially took part in the epidemiological study about the relationship between P. acnes and disc degeneration [A wild-type strain ofneration . The pat2-10% CO2-10% H2 mixed gas. When limpid broth turned into cloudy, it was transferred into the Anaerobic Blood Agar  for another three-day culture under the same anaerobic circumstance.For the process of isolation of the bacterium, disc material of L4~L5 was harvested under stringent sterilized situation and some muscle and ligaments around the disc were also obtained as contamination marker. All samples  were enriched in a 9 mL sterilized Tryptone Soya Broth  mixed with 1 mL of bovine serum . The broth was cultured inside an anaerobic Glove Box device  for fourteen days with circumstance of 37°C, 80% N P. acnes was isolated from the intervertebral disc of the patient and no bacterium was found at the muscle and ligament, suggesting that the isolated P. acnes originated from the intervertebral discs rather than from the contamination of the incision. Moreover, this microbe was confirmed as P. acnes by these three identification methods  with adjusting the concentration to 1 × 107 CFU (Colony-Forming Units) per mL using the plate count method. In the meantime, a standard strain of P. acnes was obtained from the Guangdong Microbiology Culture Center  and prepared into the same suspension as the method described above. Additionally, a single colony of S. aureus  harvested from Columbia blood agar plate was suspended in sterilized saline with the same concentration of 1 × 107 CFU per mL.The wild-type strain ofAll rabbits were anesthetized using 2.5% sodium pentobarbital with 1 mL/kg via auricular vein and were then placed in right lateral position. The skin was shaved and sterilized carefully with povidone iodine for three times. Prior to the surgery, the desired vertebrae  and intervertebral discs (L5~L6 and L6~L7) were identified using CT. Meanwhile, the diameters of the desired intervertebral discs on coronal plane were measured using CT to determine the depth of penetration.μL wild-type strain or standard strain of P. acnes suspension was inoculated into the nucleus pulposus at the segment of L6~L7 and 25 μL of sterilized TSB without bovine serum was injected at the segment of L5~L6 as internal control at the same time  and discs (L5~L6 and L6~L7) . Before ame time . The holof L6~L7 . All aniMRI was performed before and every two weeks after the inoculation until the end of follow-up at the eighth week. Briefly, animals were fasted closely on a wooden plate at prone position after deep anaesthetization and scanned with knee coil in a 3.0-T GE HDxt Signa/MRI system . The parameters were as follows: T1W TE/TR, 8.4 ms/540 ms, T2W TE/TR, 48.2 ms/2000 ms, slice thickness, 3 mm, and Field of View, 20 × 20. All MRI images were evaluated by two authors independently (Z. Chen and Z. Zhou). According to Pfirrmann classification system, the degree of intervertebral discs degeneration was evaluated and classed into five grades .μm. Hematoxylin and eosin staining was conducted and digital pictures were captured under the magnification of 100x.Animals were euthanized at the eighth week for histological examination. Briefly, intervertebral discs with endplates were fixed in 4% formaldehyde for 24 hours and then decalcified with EDTA (Ethylenediaminetetraacetic Acid) for three months, processed with routine paraffin embedding, and sectioned at 5  P. acnes-inoculated segment (L6~L7) since the second week, revealing hypointense signals at T1WI and hyperintense signals at T2WI (Figures  P. acnes-inoculated animals.No abnormal signals were found before the surgery Figures . However Figures . Further Figures . By cont Figures . Accordi S. aureus-inoculated intervertebral discs, presenting as hyperintense signals at the endplates and hypointense signals at the nucleus pulposus on T2WI. However, the definition of the endplates and vertebral body was less intact and more severe inflammatory signal changes were found at the S. aureus-inoculated segment (Figures In addition, similar signal changes were also found in the Figures . P. acnes-inoculated animals remained alive until the end of follow-up. However, of the three S. aureus-inoculated rabbits, one died at the fourth week and another died at the fifth week ( S. aureus seemed more fatal than that caused by P. acnes (2/3 versus 0/5, All of the fivefth week . Only on P. acnes-inoculated segment was depicted as endplates rupture and moderate disc degeneration with the disappearance of nucleus pulposus and disorganizing of annulus fibrosus ( S. aureus showed significantly narrowed intervertebral space and annulus fibrosus was completely replaced by cartilage, indicating severe pathological change by discitis ( P. acnes inside intervertebral discs would result in disc degeneration and endplates rupture rather than the severe discitis.In histological examination, TSB-inoculated intervertebral discs showed normal anatomical structure, in which nucleus pulposus was enclosed with normal annulus fibrosus . Howeverfibrosus . Comparadiscitis . Thus, i P. acnes isolated from the patient with disc degeneration and Modic change resulted in the same signal changes when inoculated into the intervertebral discs of rabbits. Associated with histological examination, the signal changes were more reasonable to be considered as Modic change and disc degeneration. Furthermore, compared to the higher death rate and severe pathological change of discitis caused by S. aureus, the inoculation of P. acnes inside intervertebral discs did not result in severe discitis. Therefore, it was strongly indicated that P. acnes was one of the pathogens for Modic change and disc degeneration.The wild-type strain of S. aureus-infected rabbit models, no hyperintense signal changes were observed at intervertebral discs and surrounding soft tissue, which was also similar to this study's result [ P. acnes-inoculated segment and the inflammatory oedema seemed more severe in S. aureus-inoculated segment so that we are inclined to the Modic changes rather than discitis in P. acnes-inoculated segment.Modic change is the abnormal signals at the endplates and subchondral bone on MRI, which is always secondary to the disc degeneration . Howevers result . Here, i P. acnes was one of the pathogens causing Modic changes and disc degeneration. As one of the most important criteria to judge the relationship between a specific microbe organism and a specific disease, “finding abundant microorganism in organisms suffering from the disease” and “isolating and culturing this organism in pure culture medium” were the first and second criteria, and “cultured microorganism could cause disease when introduced into a healthy organism” and “re-isolated from the inoculated, diseased experimental host” were the third and fourth steps [ P. acnes in the nonpyogenic degenerated intervertebral discs [ P. acnes inside animal's intervertebral discs would cause Modic change and disc degeneration. All of these data satisfied the first, second, and third criteria of Koch's Postulates and thus we could extrapolate that P. acnes has a strong connection with Modic change and disc degeneration.Based on Koch's Postulates, we further believed thatth steps . Previoual discs , 8, and al discs . Here, t S. aureus, which was well known as the most frequent pathogen in spondylodiscitis [ P. acnes rarely caused pyogenic discitis. In a thorough review, only twenty-nine cases of P. acnes-induced spondylodiscitis were reported within the last 15 years [ P. acnes in nonpyogenic intervertebral discs was as high as 44% [ P. acnes had different species and some of them are more virulent, but others may be less virulent [ P. acnes they would have more chance to result in the latent low-grade chronic inflammation [Being different fromdiscitis , P. acne15 years . On the h as 44%  and a sih as 44% , 8. One virulent , thus prammation , which i P. acnes gets access into the intervertebral discs [ P. acnes may intrude the intervertebral discs via circulation system [ P. acnes, because the reisolation percentage was as high as 61% after the inoculation of P. acnes directly into the intervertebral discs. More importantly, disc degeneration was also observed on the MRI after P. acnes inoculation, which was consistent with our results [Recently, an illuminating animal paper tried to reveal the routine of howal discs . Althougn system , it prov results . P. acnes induces the Modic changes. One viewpoint was that the propionic acid, the metabolite secreted by P. acnes, resulted in the dissolution of fatty bone marrow and bone, thus represented as Modic changes [ P. acnes [ P. acnes has been proved to have a strong ability to stimulate monocytes to produce proinflammatory factors such as TNF-α and IL-1 β [Unfortunately, it is still unclear how changes . AnotherP. acnes . P. acned IL-1 β , so thatd IL-1 β . Also, id IL-1 β . P. acnes from human being, we followed previous method: muscle and ligaments were extracted as the contamination marker to avoid false-positive culture result [ P. acnes suspension was to maintain bacterial viability. Although macromolecular substances in the TSB may have unpredictable effect, the TSB alone inoculation segment did not show any abnormal changes, suggesting that TSB used as the solvent was a practicable option. Finally, according to previous study, the penetration with 28-gauge needle had minor or no effect on disc degeneration in rabbits [During the isolation ofe result , 16. In  rabbits . coagulase-negative staphylococci, were not tested in this experiment. In addition, observation duration was relatively short in this study. Long-term effect of P. acnes on intervertebral discs and endplates requires further study.However, some limitations still exist in this experiment. First, other low-virulence anaerobic floras, like P. acnes has strong correlation with disc degeneration and Modic changes when inoculated into intervertebral discs, which is definitely different from discitis caused by S. aureus in histological examination and death rate. In clinical practice, one should be more aware of the presence of P. acnes in patients with Modic changes and disc degeneration.Based on Koch's Postulates,


Fibrillary glomerulonephritis (FGN) is a rare primary glomerular disease that seldom coexists with other diseases. Membranoproliferative glomerulonephritis is a pathologic finding of renal lesions associated with IgM-secreting monoclonal proliferations. We present a case study of a patient with unusual simultaneous FGN and IgM-related renal disorder in nonmalignant monoclonal IgM gammopathy.A 63-year-old male presented with nephrotic syndrome and elevated serum creatinine levels. Laboratory examination revealed elevated levels of serum IgM and low C3 levels. Serum and urine immunofixation electrophoresis showed a monoclonal IgM with a kappa light chain. A bone marrow biopsy revealed less than 5 % bone marrow infiltration by lymphoplasmacytic lymphoma, and a renal biopsy revealed mesangiocapillary glomerulonephritis on light microscopy. Immunofluorescent and immunohistochemical staining indicated granular deposits of immunoglobulin G in the mesangium and granular deposits of immunoglobulin M and κ light chains along the capillary wall. Electron microscopy revealed randomly arranged nonbranching fibrils of approximately 15 nm in diameter in the glomerular mesangium and subendothelial electron-dense deposits. According to these results, we confirmed FGN and membranoproliferative glomerulonephritis, which were attributed to monoclonal IgM deposits.To the best of our knowledge, this is the first report of simultaneous FGN and membranoproliferative glomerulonephritis in nonmalignant IgM monoclonal gammopathy. The term “fibrillary glomerulonephritis” (FGN) was introduced by Alpers et al in 1987 to characterize the glomerular accumulation of nonbranching, randomly arranged fibril, which differ from amyloid deposits in their large size and lack of reactivity to Congo red .IgM monoclonal gammopathies can be categorized into symptomatic, asymptomatic Waldenström’s disease, IgM–related disorders, and IgM monoclonal gammopathy of unknown significance (MGUS) . Renal iWe present a case report of a patient with nonmalignant IgM/κ gammopathy who developed nephrotic syndrome associated with FGN and the renal deposition of IgM and κ light chains.A 63-year-old man presented at our nephrologic outpatient clinic with progressive bilateral leg edema and foamy urine, which he had experienced for 1 month. He was hospitalized for alcoholic pancreatitis in 1999 but not regularly followed up by our hospital after discharge.3 / uL. Urinalysis revealed 2+ occult blood, 3+ protein, and 5-7 red blood cells/high power field; the 24-h protein excretion was 5.7 g/day. Serum immunoglobulin (Ig) and serum complement tests yielded high IgM (498 mg/dL), low C3 and IgG , and normal IgA and C4 levels. Serum and urine immunofixation electrophoresis showed a monoclonal IgM-bearing kappa light chain. The urinary Bence Jones protein was negative. The rheumatoid factor, antinuclear antibody, cryoglobulin, and other autoantibodies were negative. Serum antibodies against HIV, hepatitis B and C were all negative. A bone marrow biopsy revealed hypocellularity with normal maturation of myeloid series, and less than 5 % of the cells had positive immunohistochemical staining of CD138/syndecan-1 plasma cells.Physical examination of the patient revealed a blood pressure of 150/85 mmHg, blood temperature of 36.5 °C, and pulse rate of 78 beats/minute; the grading scale for pitting edema was 3+. The laboratory results were as follows: blood urea nitrogen, 26 mg/dL; serum creatinine, 1.8 mg/dL; albumin, 3.1 g/dL; hemoglobin, 12.4 g/dL and platelets, 212 × 10Renal sonography showed that both kidneys were enlarged. Chest and abdominal computerized tomography ruled out organomegaly and lymphadenopathy. A whole body bone X-ray revealed no lytic bone lesions.Light microscopy of the renal biopsy revealed nodular segmental glomerulosclerosis with mesangial cell proliferation and mesangial matrix expansion Fig.  in 9 of We performed immunofluorescence studies, observing a strong positive staining for IgM with a diffuse, global, granular, and capillary pattern Fig.  ; positiThe patient was treated initially with diuretics, angiotensin receptor blockers, 0.5 mg/kg of prednisolone per day, and 1.5 mg/kg of cyclophosphamide per day. Because steroid-induced diabetes occurred, we halved the steroid dose within the first 3 months. After 6 months, serum creatinine levels were maintained at 1.8 mg/dL and the 24-h protein excretion decreased to 1.8 g/day. 1 year later, the patient experienced herpes zoster; therefore, we discontinued cyclophosphamide treatment. However, the patient progressed to stage 4 chronic kidney disease the second year after diagnosis and advanced to end-stage renal disease (ESRD) without dialysis the third year after diagnosis.FGN, first described by Rosenmann and Eliakim in 1977 , is a raOur patient with FGN exhibited various atypical characteristics, including a strong positive immunofluorescent intensity of IgM along the glomerular capillary wall; the presence of IgM κ, as detected by immunofixation electrophoresis, and hypocomplementemia. In a large single-center series of 66 FGN cases, 47 % of the cases were weakly positive for IgM according to the immunofluorescence stain, and the mean intensity of the positive cases was 1.0+. Of the 11 patients exhibiting a positive M-spike, all were IgG with or without light chains according to immunofixation electrophoresis. Only one patient in this study had hypocomplementemia .At an international workshop on Waldenström’s macroglobulinemia, 4 categories of IgM monoclonal gammopathies were proposed. Patients with bone marrow infiltration by lymphoplasmacytic lymphoma who (1) might or might not (2) have symptoms caused by IgM are categorized as having symptomatic or asymptomatic Waldenström’s disease, respectively. 3) Patients with symptoms attributable to IgM but with no bone marrow infiltration are classified as having IgM-related disorder. (4) Patients with IgM monoclonal gammopathy but no bone marrow infiltration or IgM-related symptoms are classified as having MGUS [ PatientsThe incidence of renal complications of malignant IgM-secreting proliferation is rare, and renal involvement in IgM monoclonal disorders is generally described in patients with Waldenström’s macroglobulinemia . The typOur patient had renal lesions associated with IgM-secreting monoclonal proliferations but did not fulfill the criteria for having Waldenström’s macroglobulinemia. Audard et al. revisited the spectrum of renal lesions occurring in patients with a circulating monoclonal IgM and kidney disease related to B cell proliferation. Of the 14 patients examined, 5 patients had intracapillary IgM monoclonal deposits disease, 3 patients had a neoplastic lymphoplasmacytic infiltration of the interstitium, 3 patients had membranoproliferative glomerulonephritis, 2 patients had amyloidosis, and 1 patient had acute tubular necrosis. According to immunologic data, only 3 patients had low blood complement C3, C4, and CH50 levels; all these patients were diagnosed with Waldenström’s macroglobulinemia . Our pat9/L, and they present with bulky disease, symptomatic hyperviscosity, severe neuropathy, amyloidosis, cryoglobulinemia, cold-agglutinin disease, or evidence of disease transformation [Regarding treatment, although no effective therapy has been established for FGN, some clinicians have treated the disease according to pathologic findings. Cyclosporine has been used successfully to treat patients with a membranous pattern, and rituximab has been used successfully to treat patients with an MPGN pattern , 16. In ormation , 6. SeveIn summary, we report a rare simultaneous occurrence of FGN and membranoproliferative glomerulonephritis attributed to IgM/κ deposits in a patient with nonmalignant IgM monoclonal gammopathy. This patient exhibited a wide spectrum of renal lesions caused by IgM-secreting monoclonal proliferations, and renal prognosis was as poor as that for FGN.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the editor of this journal.


While it is acknowledged that child obesity interventions should cover multiple ecological levels  to maximize their effectiveness, there is a lack of evaluation data to guide the development and implementation of such efforts. To commence addressing this knowledge gap, the present study provides process evaluation data relating to the experiences of groups implementing the EPODE approach to child obesity prevention in various locations around the world. The aim of this exploratory study was to investigate the barriers and facilitators to program implementation in program sites around the world to assist in developing strategies to enhance program outcomes.An online survey that included open-ended questions was distributed to the 25 EPODE programs in operation at the time of the survey (May 2012). The survey items asked respondents to comment on those aspects of program implementation that they found challenging and to suggest areas for future improvement. Eighteen programs representing 14 countries responded to the request to participate in the survey, yielding a 72% response rate. The responses were analyzed via the constant comparative method using NVivo qualitative data analysis software.The main concerns of the various EPODE programs were their ability to secure ongoing funding and their access to evidence-based intervention methods and policy advice relating to relationships with third parties. These issues were in turn impacted by other factors, including (i) access to user-friendly information relating to the range of intervention strategies available and appropriate evaluation measures; (ii) assistance with building and maintaining stakeholder relationships; and (iii) assurance of the quality, independence, and transparency of policies and practices.The findings are facilitating the ongoing refinement of the EPODE approach. In particular, standardized and tailored information packages are being made available to advise program members of (i) the various evaluation methods and tools at their disposal and (ii) methods of acquiring private partner support. Overall, the study results relating to the types of issues encountered by program members are likely to be useful in guiding the future design and implementation of multi-level initiatives seeking to address other complex and intractable health-related problems. The World Health Organization, 2 recogThe prevention and treatment of child obesity is a high health priority because overweight children are more likely than their normal weight counterparts to become overweight adults, 5, 6. ADespite the recognized importance of addressing child obesity, little progress has been made at the population level and prevalence levels remain high, 13. NumGiven the diverse factors that contribute to obesogenic environments, it is recognized that interventions seeking to address child obesity need to operate at multiple ecological levels, 25–28. Most obesity programs to date have been downstream in that they have primarily focused on modifying individuals’ decision making processes through education to achieve desirable weight outcomes, 31. MetMidstream interventions, typically in the form of family-based interventions, have had some success by altering the home environment. Epstein and colleagues demonstrated significant improvements in child obesity over extended time periods, 37. TheUpstream interventions often involve mandatory or voluntary policies that attempt to shape the environment in ways that make healthy choices easier for individuals. Such inChild obesity interventions that cross multiple ecological levels are rare. This situation reflects the high costs, numerous logistical difficulties, and lack of rigorous evaluation data to guide such efforts, 42. It In the context of inadequate evaluation data for comprehensive, multi-level interventions, it has been suggested that the generation of process evaluation data should be an immediate priority to provide vital information for those seeking to implement such interventions. ProcessThe qualitative survey results reported in this article build on an earlier analysis of preliminary EPODE process evaluation data in the form of program-related documents and interviews with implementation staff in three countries. The preDetailed information relating to the origins and philosophy of the EPODE approach is provided elsewhere, 50. Brihttp://www.epode-international-network.com/programmes for descriptions of each program). It is estimated that by 2015, variations of the EPODE approach will have been implemented in the form of 40+ large-scale community-based programs involving more than 400,000,000 people.Variations of the EPODE approach have been adopted in numerous countries, each adapting their implementation to accommodate local cultural, social, and political contexts. Adaptation has also occurred within nations, with different programs being implemented in different areas to reflect the varying needs and characteristics of the relevant populations , a not-for-profit organization, was created April 7, 2011 in Brussels. The objective of EIN is to optimize the effectiveness of the EPODE-based programs around the world by generating global visibility for the EPODE approach, advocating for increased political attention to obesity prevention, encouraging expansion of the scientific evidence base relating to obesity prevention, facilitating information sharing between programs, and fostering links between relevant stakeholders across the public and private sectors. EIN thuThe study protocol received clearance from the Ethics Committee of the Faculty of Medicine, Hasselt University, Belgium #CME2013423). An online survey that included open-ended questions was distributed to the 25 EPODE programs in operation around the world at the time of the survey (May 2012). The link to the online survey was distributed via email by the EIN Coordination team. In line with the World Health Organization’s[3423. An Of the 25 programs that were members of EIN at the time of the survey, 18 responded to the request to participate, yielding a 72% response rate. These 18 programs represented 14 different countries and four different regions. The Western Europe regional group included France, Belgium, the Netherlands, Portugal, and Greece. The Eastern Europe group encompassed Slovakia, Poland, and Romania (two programs). The South America group included Mexico (three programs), Brazil, and Chile, and the Asia Pacific region was represented by Singapore, Australia (two programs), and New Zealand. The non-responding programs were almost all located in South America, with the exception of one program located in the Asia Pacific region. The survey was produced in English, and all responses were received in English. In the findings presented below, respondents’ quotes are attributed to the regions from which they originate to provide anonymity at the program level.The survey responses were imported into NVivo 10  for coding and analysis. An exploratory approach to analysis was adopted that involved using the constant comparative method to identify commonalities and differences in responses across the various programs to generate a thematic analysis. Data weThe themes identified in the data related to those aspects of program delivery that were most salient to the respondents. In accordance with the exploratory approach adopted, the issues prioritized by respondents became the focus of the findings reported below.The respondents commented on their perceptions of the performance of their own programs and discussed those aspects of program delivery that they felt were especially successful or problematic. They also provided a ‘wish list’ of the additional forms of support they felt could assist them improve outcomes in their particular contexts. Of note was the consistency of responses, with few variations evident by region. The sections below outline these findings, with relevant quotes provided from the survey data. Where appropriate, the quotes have been adapted to account for the second-language status of English in many of the participating countries.We are very happy to be part of EIN. We value the opportunity to share our experiences and at the same time learn from all the countries that are participating. It is an opportunity for our program to grow and strengthen. (South America)Overall, the respondents found participation in an international network of organizations undertaking similar activities to be useful in supporting their efforts: There needs to be an explicit statement about the role (of corporate sponsors) in all communications. (Asia Pacific)(We want EIN to) be completely independent from conflict of interests and be able to show it. (Western Europe)Their pride in their association with EIN was reflected in the expectation that the organization will maintain high standards and transparent independence to optimize the short- and long-term success of the network. An important aspect of this independence was perceived to be clear demarcation between industry funding and industry involvement in strategy development and implementation: (We want EIN) to publish reports on the results of the individual programs in international scientific fora. (Western Europe)(We want EIN) to hold the program to high scientific standards so that we deliver definitive new evidence and to disseminate the results widely. (Asia Pacific)Part of the process of maintaining high standards was noted to be an ongoing commitment to publishing results in the scientific literature to provide tangible evidence of the credibility and effectiveness of the comprehensive intervention approach: We have initiated strong, sustainable, long-lasting partnerships with schools, summer day-camps, and local organizations…We have excellent relations with the press and media. (Western Europe)We have had a good response from social, private, and academic sectors. (South America)There is strong political commitment and strong support from schools for our obesity prevention programs. (Asia Pacific)Frequent reference was made to the importance of strong relationships with relevant stakeholders to enhance the functioning of the program. Nominated stakeholders included policy makers, community groups, schools, research institutions, the media, and private sector organizations. While not all respondents could report strong affiliations with all these stakeholders, their ability to progress their objectives was attributed to establishing and maintaining effective working relationships with at least one or more such groups. This reflects the philosophy of EIN, which emphasizes the need to work with a range of collaborators across multiple facets of society to provide a broad-based approach to obesity prevention. All our efforts to obtain sources for financing program from public sources were unsuccessful, even though all political representatives at national  and municipal levels were very impressed by the aim of program and its strategy. (Eastern Europe)We feel as though our actions are limited due to our budget. The development and implementation of large actions could be improved with more resources. (Western Europe)We need assistance with the correct application and interpretation of statistics required. (South America)There is a large gap between the socio-economical levels of our participants, which represents a real challenge in terms of conducting an accurate, significant evaluation of the impact of our actions. (Western Europe)The primary factors that were mentioned as limiting the success of respondents’ programs to date included inadequate financial support from public and/or private sources and the challenges associated with generating the outcome data required for rigorous evaluation. These weaknesses were also the focus of statements relating to desired forms of additional support, as outlined further below. (The database could be used to) share activities, best practices, results (BMI), best practice to involve new financial partners, and best practice in the evaluation of local mobilization…(There could be) a review of the latest news on obesity and overweight prevention in children and a review of methods that have proved their efficiency on the field (Western Europe)Respondents’ experiences with implementing their programs provided them with insights into those areas of high-level administrative support that are important for optimizing fidelity within and across programs. A particularly critical element was considered to be an extensive database that serves as an information resource for participating groups. The primary function of such a database would be to provide comprehensive information about the activities that have been undertaken by the various participating programs and the results of these activities. Inclusion in the database of best practice examples was considered to be potentially very useful in preventing individual programs from having to learn what works through trial and error. Respondents also suggested that such a database could include a repository of scientific information relating to obesity, its causes, and possible solutions. Ideally, this information would be provided in user-friendly formats that could be used in discussions with policy makers and other key stakeholder groups. (It would be good) to standardize the methods for anthropometric measurements in children – weight and height but also abdominal circumference…And to standardize the food and lifestyle questionnaire according to children’s age and adapted to traditional food consumption and life customs for each country. (Eastern Europe)(We need) help to improve the tools that we are using to evaluate our programs. (South America)We could use more accompaniments in the evaluation process. We need evaluation processes which are meaningful and incontestable. (Western Europe)(We need) ways to evaluate that are cost effective and inform policy in a practical way. (Asia Pacific)(It would be good to have a) common evaluation tool - sustainable methodology for implementation, research, and evaluation. (Asia Pacific)Another form of support that was mentioned frequently related to program evaluation measures and procedures. Many respondents expressed a desire for fully specified evaluation criteria and instruments to enable them to produce outcome results that are comparable across programs using methods that would be adequately rigorous for publication in the scientific literature. It was acknowledged that physiological data are especially difficult to access, and assistance in developing appropriate procedures to facilitate the capture of these data was sought. (We would like) assistance with the improvement of the education methodology - new ways of education and spreading the information among the program’s participants. (Eastern Europe)(We would like assistance with an) overall communication strategy, for example including national institutions and organizations related to public health, sport, etc. (Eastern Europe)Similarly, some respondents were desirous of assistance and guidance in developing the communication strategies that are used to disseminate health messages as part of their programs. The specialist expertise required for effective message development and dissemination was not always readily available in local contexts, and it was felt that EIN could assist with these processes at a central level. This concern was most apparent among the Eastern European respondents: We have been totally unsuccessful in obtaining support from the government, municipalities, funds, and private investors. We could operate this project with a minimum 20 people on full time job, but we cannot!!! We would like to meet global representatives of private sector companies who could help us. (Eastern Europe)(We would like EIN) to share best practices in terms of public-private partnership and the methodology behind the success stories. (Eastern Europe)(We would like access to) a list of companies interested in investing and participating with government partnerships. (South America)A final primary area of desired support related to the negotiation and management of relationships with public and private organizations. This was perceived to be a particularly complex issue because of: (i) the need for ongoing financial support to achieve program sustainability and hence the need to develop financial relationships with third parties; (ii) the differing cultural contexts which prevent a unified approach across countries; and (iii) the varying levels of perceived acceptability of food industry support of child obesity interventions in different countries. This complexity appeared to result in considerable stress for some respondents, especially those located in countries where government funding was less likely to be available. (We would like EIN to) build partnerships with the food industry to regulate advertising of their products and encourage involvement in nutrition education strategies for consumers. (South America)(We would like EIN) to provide counselling to stakeholders to improve food marketing and strategies to improve and maintain child general health. (Eastern Europe)A related issue was the desire for expert assistance to influence food advertising practices. It was understood that food advertising is an important determinant of children’s diets, and hence that comprehensive child obesity interventions need to include strategies to address the current emphasis in food advertising on unhealthy foods. Some respondents reported that they would value assistance in working with the food industry to address this issue. The qualitative, exploratory approach adopted in this process evaluation study permitted those issues of most importance to respondents to emerge and be integrated into an overall interpretation of the high-level factors affecting program delivery. Ultimately, the primary concern of EIN member programs is their effectiveness in terms of addressing child obesity. As depicted in Figure Issues relating to funding are well recognized in the health intervention literature, 54, andSimilarly, the desire expressed by the study participants for evidence-based strategies reflects best-practice recommendations in the literature, 55, 56.On the basis of the study findings, EIN has commenced the introduction of a range of initiatives to enable them to better support implementation activities across the large and growing number of programs. Table A limitation of this study was the reliance on data from a single representative from each program. It is possible that those in other roles, especially those involved in day-to-day program implementation, may have different perceptions and needs. Future research could access a broader range of program representatives. A further limitation was the use of an online survey to collect qualitative data. This approach facilitated timely and simultaneous data collection from the various programs, but lacked the ability to probe respondents for greater detail or to seek clarification on the issues raised. An alternative approach for future studies could be the use of interviews – either face-to-face or via online videoconferencing systems .The major strength of the study was the high response rate, with the final sample reflecting project coordinators from a wide range of nations. The findings are therefore likely to be representative of the issues faced by the diverse programs affiliated with the EIN, and the initiatives implemented in response should be well-placed to commence addressing these issues. However, given the global prevalence of child obesity, the recognized substantial adverse health consequences of the condition, and the limited resources of EIN as an NGO, higher-level assistance to address the identified issues appears warranted. For example, a best-practice database relating to child obesity prevention strategies and related evaluation measures may be better managed by an entity such as the World Health Organization that has ready access to the required expertise and technical support.Another potential area of support at this higher level would be the development of standards for public-private partnerships in health interventions in general and child obesity prevention in particular. The involvement of private organizations in health promotion can invite criticisms relating to the possibility of interference and suboptimal outcomes. CurrentImplementing an obesity prevention intervention on a global scale brings many challenges. Previous analyses of national health promotion programs have highlighted the difficulties associated with political commitment and resource constraints, 55. Inc


Toxoplasmosis is typically diagnosed by serologic testing. External quality assessment (EQA) of clinical laboratories could ensure the accuracy and reliability of serological tests. We assessed the quality of toxoplasma serological assays in Chinese clinical laboratories by an EQA performed between 2004 and 2013 by the National Center for Clinical Laboratories.EQA panels were prepared and shipped at room temperature to participating laboratories that employed toxoplasma IgG and IgM serological detection. By 2013, 5,384 EQA test reports for toxoplasma-specific IgM and 2,666 reports for toxoplasma-specific IgG were collected. Enzyme-linked immunosorbent (ELISA) and chemical immunofluorescent assays were the most commonly used detection methods. The overall coincidence rates of negative samples were better than those of positive samples. The overall EQA score for toxoplasma-specific IgM detection ranged between 84.3% and 99.6%. The ratio of laboratories that achieved correct IgG detection ranged from 61.1% to 99.3%. However, the inter- and intra-assay variabilities were found to be considerable. The most common problem was failure to detect low titers of antibody.The EQA scheme showed an improvement in toxoplasma serological testing in China. However, further optimization of assay sensitivity to detect challenging samples remains a future challenge. Toxoplasma gondii is a protozoan obligate intracellular parasite that causes the disease toxoplasmosis. It has been estimated that 10% to 70% of the world’s population is infected by T. gondii, and the general infection rate of T. gondii in the northeast and south of China was found to be 12.3% [T. gondii infection in pregnant women by systematic screening have been performed in several countries such as France and Austria [in utero infection, accurate diagnostic tests are essential. Therefore, a number of more sensitive methods, such as a serum IgG avidity test, PCR, and western blotting using serum from mother-baby pairs, have been developed [ Austria . As non-eveloped . Howevereveloped .Quality assurance of serologic testing is important to ensure accurate and reliable screening of susceptible individuals, especially for pregnant women. Therefore, laboratories should participate in external quality assessment (EQA) schemes conducted by independent organizations . ParticiThe toxoplasma EQA scheme consisted of two distributions of human serum specimens for the examination of toxoplasma IgG and IgM antibodies with a request to report qualitative results twice a year (in May and October). Each distribution consisted of two panels of serum, one to test for toxoplasma IgG, another to test for toxoplasma IgM. Each panel consisted of five serum specimens. The panels were majorly prepared using sera pools of blood donors kindly donated by Shenzhen blood center and Liaoning blood center in China or minor sera pools donated by the company Viron/Serion  or minorly purchased from Guangzhou Kang Run biological products development co., LTD  . The NCCL tested the specimens for toxoplasma-specific IgG and IgM antibodies prior to dispatch, using several commercial enzyme-linked immunosorbent assays (ELISA) or chemiluminescence immunoassay (CIA) kit for toxoplasma IgG and IgM. The tests used to produce the EQA panel of sera for each year and precisely detail for positive sera were shown in Tables Hospital laboratories from China that employ toxoplasma IgG and IgM serological detection were invited to participate in this EQA scheme. The participating clinical laboratories tested the EQA panels using their standard protocols and returned the test results to NCCL via a data submission form on the NCCL website. Participants using EIAs were asked to provide signal/cut-off (S/CO) values if possible.The EQA data were scored according to qualitative criteria; every correct positive or negative test result in accordance with the expected results of NCCL of the specimens in each panel was assigned a point of 20, while false negative or false positive results were not scored. The highest possible total EQA score per panel was thus 100 points, and an EQA points <80 was considered unqualified. Data were entered into Microsoft Excel spreadsheets and analyzed using Microsoft Excel and the Prism 6 software  and SPSS 16.0(SataCorp).Chinese clinical hospital laboratories offering toxoplasma IgG/IgM testing were invited to participate in the EQA scheme. At the start of the IgM scheme in 2003, 79 laboratories participated and reported qualitative results. At the start of the IgG scheme in 2008, 94 laboratories were participating. The number of participating laboratories increased steadily over the past years. By September 2013, 843 laboratories had reported 2,666 toxoplasma-specific IgM test results, while 551 laboratories had submitted 5,384 EQA test reports for toxoplasma-specific IgG.All IgM test results, acquired by the different routine detection assays as used by the participating hospital laboratories, were analyzed and scored . The ratIn general, the scores for IgG test results in the EQA scheme were better than those for IgM results . The oveThe diagnostic assays used by participants during the EQA, over ten years in the case of IgM and over six years in the case of IgG, are summarized in Tables As mentioned above, ELISA was the most widely used detection method in the EQA scheme. In 2003, at the start of the IgM scheme, 21 different ELISAs were used for the detection of IgM, of which 17 were Chinese domestic ELISA kits. Currently, 25 different ELISAs are being used of which 16 are Chinese domestic kits. In 2008, 19 ELISA kits were used by participating laboratories for the detection of IgG. By 2013, the number of ELISA kits increased to 23. The assays currently used by at least five participants in the EQA and the accumulative total coincidence rate, for the detection of toxoplasma-specific IgM and IgG, are shown in To determine the inter-assay variability of EIA kits from different manufacturers that are currently used in the EQA by more than five laboratories, the variability of log S/CO values was estimated by determining the standard deviation (SD) of the results of all participating laboratories for all the positive samples distributed in EQA scheme. The mean and SD of log S/CO values for each assay were determined based on the test results of all participants using EIAs, for the IgM-positive  as well The intra-assay variability for kits currently used by more than five participants was analyzed by comparing the assay results for individual positive samples distributed in EQA scheme. Results (mean (log S/CO) ± SD) for the IgM assay kits are given in In this paper, we described the results of a long-term EQA scheme to evaluate the various immunoassays for the detection of toxoplasma-specific IgG and IgM used by the participating laboratories. Conducting such a program is the best method to detect problems with regard to assay sensitivity and accuracy, and to maintain and improve the quality of routine toxoplasmosis diagnosis in clinical laboratories.The primary goal of EQA is to assess the participants’ diagnostic assay performance. A UK National EQA scheme for toxoplasma-specific IgG was established in 1993, followed by a scheme for toxoplasma-specific IgM in 1998. These EAlthough the overall high EQA scores in the current EQA program demonstrated an encouraging improvement in the accuracy of toxoplasma-specific IgG and IgM assays, we found a large variability in the assay results of the participating laboratories. For example, some of the participants reported false-negative results for some samples, while other participating laboratories correctly identified the same samples, using the same assay kit Figs  and 3. OThere is wide variety of assays available for the detection of toxoplasma-specific IgM and IgG. Some analyses based on commercial assay are necessary, because it is a key factor in clinical determination. Tough other techniques, such as Sabin-Feldman dye test(DT), latex agglutination test (LAT)for toxoplasma specific IgG detection, immunosorbent agglutination assay (ISAGA) for toxoplasma specific IgM detection, are mostly reserved for reference laboratories, while ELISA is mostly used in clinical laboratories for routine screening for IgG and IgM. ELISA and CIA are available as commercial kits and on automated platforms . ELISA aMost commercial serological kits use native antigens prepared from tachyzoites grown in mice and/or tissue culture. The preparation methods for these antigens vary in different companies and the produced antigens may contain contaminants from culture media and eukaryotic host cells . The majA detailed comparison of different commercial ELISA kits in the current study showed that some kits did not sufficiently allow for detecting weakly positive samples Figs  and 3. TMost ELISAs can detect IgM for months or years after infection, so IgM detection is not an accurate marker of recent infection . The difIn conclusion, the long-term national EQA toxoplasma serology scheme in China has been beneficial to the participating clinical laboratories; the IgG and IgM assay performance improved and the sensitivity of routine tests as well as the accuracy of assay data analysis increased during the past years, thanks to the educational approach of the EQA scheme. This shows that a continuous monitoring of laboratories by an EQA can improve national performance, underlining the importance of such EQA.S1 Table(DOCX)Click here for additional data file.


Dear Sir:We appreciate the interest of Drs. Benziger and Gilman in the Fogarty International Clinical Research Scholars and Fellows (FICRS-F) Program.We are engaged in a multi-year project to report on the FICRS-F program's outputs and outcomes.


Sc). Recent studies applied the RT-QuIC methodology to cerebrospinal fluid (CSF) for diagnosing human prion diseases. However, to date, there has not been a formal multi-centre assessment of the reproducibility, validity and stability of RT-QuIC in this context, an indispensable step for establishment as a diagnostic test in clinical practice. In the present study, we analysed CSF from 110 prion disease patients and 400 control patients using the RT-QuIC method under various conditions. In addition, “blinded” ring trials between different participating sites were performed to estimate reproducibility. Using the previously established cut-off of 10,000 relative fluorescence units (rfu), we obtained a sensitivity of 85 % and a specificity of 99 %. The multi-centre inter-laboratory reproducibility of RT-QuIC revealed a Fleiss’ kappa value of 0.83 (95 % CI: 0.40–1.00) indicating an almost perfect agreement. Moreover, we investigated the impact of short-term CSF storage at different temperatures, long-term storage, repeated freezing and thawing cycles and the contamination of CSF with blood on the RT-QuIC seeding response. Our data indicated that the PrPSc seed in CSF is stable to any type of storage condition but sensitive to contaminations with blood (>1250 erythrocytes/μL), which results in a false negative RT-QuIC response. Fresh blood-contaminated samples (3 days) can be rescued by removal of erythrocytes. The present study underlines the reproducibility and high stability of RT-QuIC across various CSF storage conditions with a remarkable sensitivity and specificity, suggesting RT-QuIC as an innovative and robust diagnostic method.Real-time quaking-induced conversion (RT-QuIC) allows the amplification of miniscule amounts of scrapie prion protein (PrPThe online version of this article (doi:10.1007/s12035-015-9133-2) contains supplementary material, which is available to authorized users. Sc) in brain tissue. They can occur spontaneously (sporadic) but also be due to familial and iatrogenic causes. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common prion disease in human, followed by genetic CJD (gCJD) and iatrogenic CJD (iCJD). Different molecular sCJD subtypes, which show unique clinicopathological phenotypes and transmission characteristics [PRNP) as well as the type of PrPSc, have been reported [Transmissible spongiform encephalopathies or prion diseases are characterized by the aggregation and accumulation of misfolded scrapie prion protein  was an innovation for the pre-mortem diagnosis. RT-QuIC analysis uses recombinant prion protein (recPrP) as a substrate to amplify very small amounts of PrPSc seed in human CSF to detectable levels. Time course of conversion can be monitored in real-time by a fluorescent dye using a fluorescent reader. Four previous studies demonstrated the diagnostic potential of RT-QuIC through amplification of PrPSc from human CSF, derived from sCJD and gCJD patients [The adaptation of in vitro amplification systems for the detection of PrPpatients –7.However, despite these initial reports, establishing RT-QuIC as a routine diagnostic test in clinical practice requires a comprehensive validation and standardization. To date, there is a lack of studies dealing with inter-laboratory reproducibility and standardization, assessment of short- and long-term stability and contamination of CSF samples with potential inhibitory blood cells of the RT-QuIC seeding response in human CSF of CJD patients. All of these parameters may lead to an artificial decrease or increase of PrP seeding activity in CSF and to false positive or negative results.The aim of the present study was to further analyse the applicability of RT-QuIC for routine diagnostic purposes. Analysis of 110 CSF samples from prion disease patients and 400 from a control panel consisting of 200 retrospectively tested patients  and 200 prospectively randomly selected patients was undertaken to obtain diagnostic data more aligned to a routine clinical setting. Also, two ring trials were performed to generate data about the reproducibility of the RT-QuIC assay. Finally, the influence of different CSF storage conditions (long- and short-term storage or repeated freezing and thawing cycles) and the influence of blood contamination on the RT-QuIC seeding response were examined.CSF samples were collected through routine activities of the German National Prion Disease Surveillance Center associated with the University Medical Center Göttingen. Samples were derived from 110 prion disease patients, consisting of 64 sCJD, 39 gCJD  and 7 FFI (D178N mutation), as well as 400 control patients. Initial data on the cohort have been published elsewhere [The 200 retrospectively tested control patients represent those with either a clinically or pathologically defined alternative diagnosis . This control group consisted of AD patients , patients with depression, Dementia with Lewy bodies, PD, psychosis, bipolar disorder, multiple sclerosis, epilepsy, schizophrenia, inflammatory CNS disease and others. The 200 prospective samples were enrolled from the Neurochemistry Laboratory at the Department of Neurology in the framework of quality assurance and were tested blinded for clinical and personal data. Gender- and age-dependent effects on the RT-QuIC seeding response were excluded before [CSF samples were stored at −80 °C prior to analysis. Haemorrhagic CSF samples were excluded from the study.The study was conformed to the Code of Ethics of the World Medical Association and informed consent was given by all study participants or their legal next of kin with the study being approved by the local ethics committee in Goettingen (No. 24/8/12). All samples were analysed blinded for at least personal data.The RT-QuIC was performed as described previously . Briefly154 Q171 polymorphic haplotype as described before [All RT-QuIC experiments were performed using the chimeric recPrP composed of the Syrian hamster residues 14 to 128 followed by sheep residues 141 to 234 of the Rd before .To investigate the reproducibility of the RT-QuIC assay, we conducted two ring trial studies between different laboratories. Fifty-four CSF samples obtained from sCJD patients and 32 non-prion disease controls were examined in two laboratories . In a second ring trial, one sCJD and five controls were investigated in four laboratories .To calculate specificity, sensitivity and predictive values of the RT-QuIC assay, we analysed CSF samples from 110 prion disease patients, as well as from 400 control patients.n = 12) and control samples (n = 6) were incubated under short- and long-term storage conditions.To determine the stability of RT-QuIC, sCJD CSF samples (n = 6–8) and control samples (n = 6–8) with defined amounts of blood cells from control patients.To assess the impact of blood contamination of CSF samples on the RT-QuIC response, we spiked CSF from sCJD (n = 8) and control samples (n = 8) with a defined amount of blood cells from control patients and incubated the CSF samples for 0, 1, 3 and 8 days at room temperature.To investigate the impact of transportation on blood-contaminated samples, we spiked CSF from sCJD  analysis was performed on this dataset, and the best cut-off value was estimated to be 10,000 rfu based on the Youden index. We thereby confirmed the analysis of McGuire et al.  in an int tests as appropriate. For graphical illustration, time series plots are presented at each step of the analysis, displaying means of seeding activity for each point in time (each measurement) in the respective groups. Thus, these illustrations show aggregated data for each point in time, but information about means of AUCs or individual maximums cannot be read out of these plots [For the analysis of the stability of RT-QuIC, a two-step approach was performed. First, positivity rates were calculated  and were compared between groups using ANOVAs. Agreement levels and Fleiss’ kappas were calculated in order to investigate the stability of positivity under different circumstances. Then, measurements of seeding efficiency were calculated for each individual sample defined by relative area under the curve (rel. AUC) and maximal ThT signal (maximum), and groups were compared using paired and unpaired se plots .Sensitivity, specificity and predictive values of RT-QuIC were calculated for the entire dataset as well as for distinct subgroups. All analyses were conducted using Stata 12 software  and R 2.15.3 software.Levels of 14-3-3 protein in CSF were determined by using the CircuLex 14-3-3 Gamma ELISA Kit  according to a previously established protocol .CSF levels of total tau protein were measured using a commercially available ELISA kit . For the determination of tau levels, we followed the manufacturer’s instructions.Sc amplification via RT-QuIC assay, we initiated two ring trial studies and included comparison to other biomarker proteins (tau and 14-3-3). Results of inter-laboratory agreement were indicated as Fleiss’ kappa, which was calculated in a first ring trial with two partners as 0.75 (95 % CI: 0.40–1.00) revealing a substantial agreement, comparable to 14-3-3 and tau proteins  and control (n = 6) samples, which were stored for 2–3, 5–6 or 8–9 years in a −80 °C freezer. Additionally, we subjected 12 sCJD and six control CSF samples to up to 16 repeated freezing and thawing cycles.CSF samples derived from 12 sCJD and six control patients were stored in polypropylene tubes at room temperature or 4 °C for up to 8 days. To estimate the impact of long-term storage on the stability of RT-QuIC seeding response, we analysed sCJD  in 80 h , which is important for CJD diagnostic. Our data indicated that the PrPles Fig. . Under sSc was not significantly influenced, neither under short-term nor under long-term storage conditions or after repeated freezing and thawing cycles of CSF samples . Again, we observed that the seeding efficiency of PrPles Fig. .Fig. 3TiControl samples without prion disease showed negative RT-QuIC responses  under these conditions (data not shown).Sc seeding activity in the RT-QuIC assay, we spiked CSF derived from sCJD patients (n = 6–8) and non-CJD controls (n = 6–8) with different amounts of sonicated red blood cells from control donors .Since erythrocytes and platelets in human blood contamination of CSF may influence PrPOur results indicated that an erythrocyte concentration higher than 1250 cells/μL showed a false-negative RT-QuIC response. Blood contaminations significantly influenced the number of positive replicates Fig.  as well In contrast, erythrocyte concentrations below 1250 cells/μL are tolerable for diagnostics because they did not show any significant impact on the number of positive replicates and the intensity of the RT-QuIC signal Fig. . CSF fron = 8) and control samples (n = 8) with a defined amount of blood cells (5000 cells/μL) from control patients. After 0, 1, 3 and 8 days, we centrifuged the samples to remove the blood cells. The haemolysis and release of inhibitory proteins, which influence the number of positive replicates and the intensity of the RT-QuIC signal significantly, started after 3 days resulting in a false-negative RT-QuIC seeding response in sCJD patients , enhanced quaking induced conversion (eQuIC) or RT-QuIC, exploit the self-propagating replication of the abnormally folded PrPuman CSF –7.The use of RT-QuIC in routine CSF diagnostics for prion diseases requires standardization of sample treatments, definitions of cut-off values by analyzing a high number of samples and assessments of reproducibility and resistance of the RT-QuIC response to short- and long-term storage conditions; until now, only scarce information on these topics is available in the literature.To evaluate the inter-laboratory reproducibility of the RT-QuIC assay, we initiated ring trials between different laboratories. A first RT-QuIC ring trial between two different partners using a high number of samples (54 sCJD and 32 controls) indicated substantial agreement between raters (Fleiss’ kappa = 0.75 (95 % CI: 0.40–1.00)).Even though the inter-laboratory agreement was relatively high, we intended to establish optimal conditions and standard operating procedures for the RT-QuIC method. The assay precision depends on various factors including (1) inter-laboratory variations in staff, protocols and substrates; (2) sensitivity of the assay to different CSF sample storage and shipping temperatures and (3) different fluorescent readers. By minimizing such variations, we achieved in a second ring trial an almost perfect agreement (Fleiss’ kappa = 0.83 (95 % CI: 0.40–1.00)) showing a high reproducibility of the RT-QuIC assay with improved standardization and supporting the potential of the method for routine diagnostic purposes.We analysed CSF samples from a patient cohort consisting of 110 patients with prion disease  and 400 controls. When setting the cut-off at 10,000 rfu, we obtained a specificity of 99 % and a sensitivity of 85 % for all prion diseases. Among different prion diseases, the sensitivity varied from 100 % for gCJD, 80 % for sCJD and 57 % for FFI cases. Our data are in line with two previous studies, which reported a specificity of almost 100 % and a sensitivity between 80 and 90 % . DiscrepA general problem of biomarker-based diagnostics is the sensitivity of biomarker proteins to storage and shipment conditions. During shipment, CSF is subjected for a defined period of time (between 1 and 8 days) to different temperatures, which are between 20 and 25 °C. After arrival, CSF is usually stored at 4 °C before analysis. Samples, which are used for research, may undergo numerous cycles of freezing and thawing or will be stored for years at −80 °C.Sc seed in CSF of sCJD patients resisted the variations posed with across the analysed conditions, and we did not obtain any loss of sensitivity or specificity or in relation to the intensity of the RT-QuIC responses.For simulation of these conditions, we explored the effect of short-term storage (up to 8 days at room temperature or at 4 °C), long-term storage (up to 8–9 years at −80 °C), as well as the influence of 16 repeated freezing and thawing cycles on the RT-QuIC seeding response. Our data demonstrated that the PrPCompared to other biomarker proteins such as tau or Aβ42, which become degraded in particular under higher temperatures during storage –13, the Sc seeding activity which resulted in a false-negative RT-QuIC response in prion disease patients. Our findings may explain the variable sensitivity of the RT-QuIC assay (between 80 and 90 %) among different laboratories [Contamination with blood cells, such as erythrocytes, may influence the RT-QuIC response. In routine diagnostic laboratory practice, between 5 and 10 % of the CSF samples are contaminated with blood cells during the lumbar puncture. Therefore, there is a need to define clear guidelines for pre-analytical sample handling. Our data revealed an artificial inhibition of the PrPratories , 5. The In conclusion, our data emphasize that RT-QuIC analysis of CSF is a robust and very useful tool for prion diagnosis and should become part of the clinical workup in rapidly progressive dementia cases.Supplement 1Reproducibility of RT-QuIC in comparison to 14-3-3 and tau proteins. CSF from sCJD patients and control donors were analysed in two ring trials. Results of inter-laboratory agreement were shown as Fleiss’ kappa. In a first ring trial with two partners RT-QuIC assay revealed a substantial agreement, which is comparable to 14-3-3 and tau. A second ring trial with more standardized condition showed an almost perfect agreement (Fleiss’ kappa =0.83). (GIF 43 kb)High resolution image (TIFF 3756 kb)Supplement 2Time course of prion seeding activity of two control samples showing a potential false positive RT-QuIC response. Patient 1 showed no abnormalities, the second patient exhibited an elevated tau and 14-3-3 level as well as a CJD typical MRI which are indices for a prion disease. (GIF 403 kb)High resolution image (TIFF 2144 kb)Below is the link to the electronic supplementary material.


A bespoke cross-sectional questionnaire measured pre-season risk perceptions, outcome expectancies, task self-efficacy, facilitators, barriers, and FIFA 11+ implementation intention. Most coaches (90.0%) and players (80.0%) expected the program to reduce injury risk but reported limited intention to use it. Player data demonstrated an acceptable fit to the hypothesized model  = 0.08; root mean square of error of approximation (RMSEA) = 0.06 (0.047–0.080); comparative fit index (CFI) = 0.93; Tucker Lewis index (TLI) = 0.91) Task self-efficacy  and outcome expectancies (β = 0.13 p ≤ 0.05) were positively associated with intention, but risk perceptions were not (β = −0.02). The findings suggest that the HAPA model is appropriate for use in this context, and highlight the need to target task self-efficacy and outcome expectancies in FIFA 11+ implementation strategies.The Fédération Internationale de Football (FIFA) 11+ warm-up program is efficacious at preventing lower limb injury in youth soccer; however, there has been poor adoption of the program in the community. The purpose of this study was to determine the utility of the Health Action Process Approach (HAPA) behavior change model in predicting intention to use the FIFA 11+ in a sample of 12 youth soccer teams (coaches  The Fédération Internationale de Football (FIFA) estimates that there are over 22 million youth soccer players globally , and theExercise-based prevention programs are efficacious at reducing sport injury risk ,14,15,16A number of barriers to FIFA 11+ use have been identified amongst youth soccer players and their coaches, including low expectation that prevention strategies are effective and having insufficient time or resources to implement the program . NotablyPrevention program delivery must also be considered when evaluating adherence. In both research and practical settings, emphasis has been placed on providing injury prevention content to coaches who can then disseminate information to players. This has been moderately successful ,19, but Behavior change theory has seen limited application in sport injury research ,25. In aThe Health Action Process Approach (HAPA) model is a theory of health behavior change that is particularly suited to describing the adoption of preventive behaviors . It compAlthough other theories of behavior change have been explored in injury prevention ,25, manyTherefore, the purpose of this study was to explore the utility of the HAPA model in predicting intention to adopt the FIFA 11+ amongst youth soccer coaches and players, as an initial proof of concept to inform the design of larger prospective studies. The primary objective was to describe perceived task self-efficacy, outcome expectancies, and risk perceptions in this setting. The secondary objectives were to determine whether these factors predicted intention to use the FIFA 11+ at the start of the soccer season and to assess the fit of the HAPA model as an appropriate behavior change theory to apply in this context. This was a cross-sectional study conducted at the start of the 2013 outdoor soccer season (May–August) in Calgary, Canada. This study was approved by the Conjoint Health Research Ethics Board at the University of Calgary (No. 24985).a priori sample size was calculated. Coaches were eligible to participate if they were the head or assistant coach of a female soccer team competing in Tier 1 or 2 of the U14 or U16 age group at the start of the season, and were excluded if they had any previous experience using the FIFA 11+. Players were eligible if they belonged to a female soccer team competing in the U14 or U16 age group at the start of the season and their team coach was participating in the study (as coach permission was required to approach players for recruitment). Exclusion criteria for players included a recent (within six weeks) history of injury requiring medical attention or the inability to participate in soccer for at least one day and preventing full participation at the start of the 2013 season, or a history of systemic disease or neurological disorder preventing full participation at the start of the 2013 season. Teams were randomly approached for recruitment by club once teams were formed prior to the start of the season. All coaches provided written informed consent, and players provided written informed parental consent and personal assent to take part in the study.The study population consisted of coaches and youth female soccer players (aged 12–16 years) competing in the Calgary Minor Soccer Association. Due to the exploratory nature of the study, no formal The FIFA 11+ program is a 20-min warm-up that consists of running exercises, dynamic stretching, plyometrics, and agility, strengthening, and balance exercises . It was Prior to the start of the season, coaches attended a two-hour FIFA 11+ instructional workshop, delivered by the lead author (who has experience playing and coaching soccer at the level targeted in this study). Each exercise in the program was demonstrated, with clear directions on identifying correct and incorrect technique, and coaches were given copies of the program resources. The emphasis of the workshop was on the program content, with only a brief rationale for its use  presented for context. Coaches were clearly told that choosing to use the FIFA 11+ in the following season would be entirely voluntary, and the purpose of the study was to better understand why teams might or might not decide to use the program. After this familiarization, coaches completed a questionnaire to assess demographic characteristics, perceptions about soccer injury, and the HAPA constructs. Player questionnaires were administered by study personnel in a supervised team setting at a training session prior to the start of the season. Coaches explained the FIFA 11+ to the players, and the questionnaires were answered based on the players’ understanding of the program at that time.A bespoke questionnaire was developed for this study, with separate versions for coaches and players see . Sectionnot at all serious to very serious)  see . The quep = 0.05 and, due to the exploratory nature of the study, no corrections were made for multiple comparisons. These analyses were conducted using STATA v.13 .Descriptive analyses ) were used to assess demographic characteristics and questionnaire responses. Due to the low number of coaches in the sample, data were checked for normal distribution and Pearson correlations were subsequently used to assess the association between HAPA variables and intent to use the FIFA 11+. The threshold for statistical significance was set at To assess the fit of the HAPA model for predicting player intention, structural equation modeling was employed  and a twOf 40 teams approached for recruitment, 12 consented to participate in the study. One team was excluded because the coach had experience with the FIFA 11+, 22 responded that they were not interested in engaging in research, and five were unable to schedule a data collection session prior to the start of the season and so declined participation. Two consenting coaches did not attend the pre-season workshop, leaving a final sample of 10 coaches. Of the 203 eligible players, all were successfully recruited but only 200 provided complete questionnaire responses and were retained for analysis. Participant characteristics are presented in Baseline injury risk perceptions and prevention expectancies are presented in Overall, 44.0% of players believed injuries were ‘quite’ or ‘definitely’ preventable, whereas coaches generally believed they were only ‘slightly’ preventable. The majority of coaches (90.0%) and players (80.0%) expected that the FIFA 11+ would reduce injury risk if used at every training and game session. Perceived self-efficacy is presented in The most commonly-reported facilitators for program use amongst coaches were access to FIFA 11+ materials  (endorsed by 100% of coaches), having the proper equipment (40.0%), and having enough space to accommodate the exercises (20.0%). The most common barriers were not having enough time (40.0%), poor player buy-in/lack of player cooperation (40.0%), and coaching staff absences (20.0%). For players, the most common facilitators were personal motivation/willingness to put in effort (35.5%), having enough time (31.5%), making the program a routine (29.0%), and someone taking responsibility for organizing and leading the warm-up (29.0%). Player barriers included poor team buy-in to the program (52.0%), finding the program too difficult/tiring (35.5%), and low personal motivation (29.0%). A complete list of barriers and motivators can be found in the Coaches reported limited intention to make injury prevention a priority in the upcoming season  or to use the FIFA 11+ with their team . Similarly, players reported no distinct intention to use the FIFA 11+ at every session during the season , or to perform the exercises with full effort if they did use the program . r = 0.84, p ≤ 0.01) and self-efficacy for using the program  were the only variables significantly associated with intention.The associations between HAPA predictor variables and intention for coaches are presented in p ≤ 0.01). Outcome expectancies had a small but positive significant effect on intention (β = 0.13 p ≤ 0.05), but risk perceptions demonstrated no association (β = −0.02). Player data demonstrated good fit to the hypothesized HAPA prediction model ; CFI = 0.93; TLI = 0.91) . OverallConsistent with previous studies in this population ,23, coacA larger proportion of coaches and players believed that injuries might be preventable in the present study than in previous studies in the same population . FurtherSelf-efficacy for using the program, however, was low amongst coaches and players. Bandura  posits tProgram length, lack of time and space, and limited exercise variety have been reported as barriers to prevention program uptake in several sports ,39. Partr = 0.55). This could point to a meaningful contribution of specific risk perceptions to FIFA 11+ adoption, but should be interpreted with caution given the small sample size. Moreover, although no direct relationship emerged between risk perceptions and intention in the full player model, previous studies suggest that low risk perceptions are related to limited intention to engage in injury prevention behaviors [Although the coach sample was too small to conduct a factor analysis, item-wise correlations suggest that only task self-efficacy was significantly associated with intention. For players, outcome expectancies and task self-efficacy were related to intention, while risk perceptions were not. The relative strengths of these relationships are consistent with the HAPA literature, with self-efficacy being the strongest predictor of intention and risk perception contributing only a small amount to the overall model ,27,28,29ehaviors ,42,43. TBased on the fit indices of the player model, it appears that HAPA is a suitable framework in this context. Additional research will be needed to better quantify the strength of the relationships between predictor constructs and intention, and the volitional stages of the model must be investigated to determine whether it can be used to predict behavior. Yet, the results of the present study suggest that FIFA 11+ promotion strategies that focus on injury risk education may be largely ineffective . This maA number of limitations to this study must be acknowledged. First, the questionnaires have not undergone a rigorous validation process. Although they performed reasonably well for exploratory purposes, it is possible that the associations under investigation were under- or over-estimated due to imprecision in the measurement. Furthermore, the small sample of coaches prevented analysis of the model fit for this group. Future studies should, therefore, refine the questionnaires and seek to validate them in larger samples. This study also used intention as an end point, and there is a widely acknowledged gap between intention and behavior in prevention program implementation . While tFinally, in youth team sports, coach intentions likely influence team behaviors more than those of individual athletes. Therefore, better understanding how coach perceptions match with the perceptions of their players, or how information is communicated between coaches and players, may help to inform FIFA 11+ delivery strategies and provide greater insight into the appropriate content and audience for instructional workshops. Developing workshop content based on the HAPA constructs  will also be important moving forward.This is the first study to examine the HAPA model in predicting injury prevention intention in female youth soccer. The findings suggest that the model is indeed appropriate for use in this context, and highlights the strong relationship between task self-efficacy and intention to use the FIFA 11+ warm up program. This study has also demonstrated that perceived facilitators and barriers to program adoption differ between coaches and players, providing possible avenues for improving program delivery in the community.


Injuries are a leading cause of childhood death and disability. Many injuries are a result of youths taking risks and not avoiding risky situations. An educational program to reduce adolescent injury risk (Prevent Alcohol and Risk-Related Trauma in Youth) has operated out of the Misericordia Hospital in Edmonton Canada since 1992. This reality-based program was evaluated to see if it was impacting program participants. An increase in correct answers for some knowledge, behavior, and attitude questions were found at one week and one month following this 1-day reality-based program. This program was thus considered as having some relevancy in educating grade-9 youths. Although a longitudinal study is needed to determine if this relevancy is long term, this study highlights the importance of reality-based public health programs. Slide and video presentations, role-playing, and discussions are featured. The discussions are led by injury victims, emergency room nurses, police officers, and paramedics. All talk frankly about their roles, experiences, and frustrations over needless accidents. Issues arising from alcohol and drug consumption, and peer pressure are highlighted throughout the day.Injury survivors also speak about what it is like to live with brain, spinal cord, and other permanent disabling injuries. The program is designed so students can engage in candid discussions with injury survivors and see firsthand some of the daily challenges of living with brain, spinal cord, and other serious injuries. Students have the opportunity to use a wheelchair and eat lunch as if they were a quadriplegic. Through this reality-based education, youths are encouraged to consider the possible consequences before engaging in risky actions and not avoiding risky situations. This program also emphasizes personal choice, as children at this age are becoming increasingly independent from their parents and teachers . They tySome evaluations of programs designed to reduce substance abuse and accidents among youths have been conducted, with these published articles available to influence the design of a program evaluation research study. The findings from these program evaluations are also helpful for being optimistic about educational programs. A systematic review of educational programs aimed at youth alcohol reduction, conducted by et al.’s (2011) 18-session prison education program aimed at reducing unprotected sex when alcohol or drugs were used was revealed as effective when participants were tested nine months after discharge. A study of young women who were assigned to a control or brief intervention program that was similarly aimed at reducing unprotected sex also revealed considerable value from this educational program . After this development, the contact person at all schools who were planning to have their grade-9 students attend the program in the fall of 2009 were contacted and told about the study. Specifically, these contacts were told that the hospital wanted to evaluate the program and that this voluntary study involved the same questionnaire administered three times; the first on the morning immediately prior to the program starting, the second one week after the program, and the third one month after the program. The research ethics committee would not allow students to be named or numbered or tracked, and with voluntary participation, this requirement meant that the research team could not track an individual’s scores from the first through third questionnaire, nor determine if the same individual completed all three questionnaires or not. Group data instead were thus compared from the first to second questionnaire to determine for improved knowledge, attitudes, and reported behaviors following the program. Group data were also compared from the first to third questionnaire to determine if these improvements were retained over a one month period.Three school representatives agreed to ensure that all of their attending students and their parents received an information letter and consent form. This form needed to be signed by one or both parents before their child could take part in this study, and research participation was not mandatory for the students taking the program. As expected some students (numbers outlined later) from the three schools completed it immediately prior to the program, one week later, and one month after the program. As indicated, a pre-post analysis of data from all 385 questionnaires was done to see if knowledge, attitudes, and reported behaviors about risk-related trauma increased and remained improved for one month. The SPSS (version 17) program was used to analyze data, with the consent forms locked away and only accessible to the research team. The questionnaire did not ask for the student’s name, and so this data was not entered onto the computer spreadsheet. Simple descriptive statistics, chi-square analyses, and Spearman correlations were used to summarize the data and assess for differences in responses.A total of 385 surveys were returned; 140 pre-program surveys, 123 1-week surveys, and 122 1-month surveys. The return rate for the first survey was 64%, with an attrition of 12-13% after. Because no identifying information about the students could be collected, it is not known whether students completed more than one survey.Six questions assessed risk-related behaviors. As shown in Eight questions assessed risk-related attitudes. As shown in This study revealed a number of improvements in knowledge, attitudes, and reported behaviors; a major reason for continuing this program and possibly expanding it to other schools offering grade-9 education or education to youths somewhat younger or older. The hospital setting, timing , content, and chosen teaching/learning methods could have all contributed to these improvements. Among these factors, the hospital may have been key for providing a realistic setting or environment for learning. Few students at this age and their siblings or parents are likely to have been hospitalized in the past, as less than 10% of Canadians are hospitalized each year now . Having It was notable that the risk-related knowledge of students increased and was sustained over one month. Many of the students, however, had relatively high initial knowledge. This high initial knowledge indicates students pay attention to risk information from their parents, peers, schools, communities, or other sources. Improvements in some reported risk-related behaviors were also found, with these improvements suggesting students assumed more responsibility for being safe. The students were not presented with an exhaustive list of behaviors to report on; so it is hopeful that if one improvement was made, such as seatbelt buckling up, other risk-reduction behavior improvements could also be occurring.The low response rate and answers to some behavior questions is of concern, however. It is possible that some questions were not relevant to all or many of these students, as not all would be with older persons who could be potentially drinking and driving. It is also possible that students are not comfortable revealing the truth about their behaviors. Regardless, the responses of students to attitude questions showed a number of significant immediate and sustained improvements. These improvements are of great interest; as attitudes impact behaviors, and attitudes can be highly resistant to change. Caution must be taken, however, in putting too much confidence in the findings of this study and generalizing the findings to other students of this age or to other risk-reduction programs. The Hawthorne effect, a temporary change in behavior in response to research conditions, could have impacted student responses. In addition, long-term follow-up studies are needed to see if life-long attitudes, knowledge, and behaviors that reduce risk are gained through targeted programs such as the P.A.R.T.Y program.In conclusion, the findings of this study indicate a reality- and hospital-based P.A.R.T.Y. program has positive impacts on the risk-related knowledge, attitudes, and behaviors of grade-9 students. These are most welcome findings as these students will shortly enter the prime age (15-19) for injuries and deaths due to alcohol and substance abuse. This study suggests the importance of targeted community-based programs to equip youths with the information and attitudinal or behavior tools to minimize or prevent harm to themselves and others. A carefully designed, concentrated reality-based program that is held in a hospital may be key, however, for impacting youth risk-related knowledge, behaviors, and attitudes.