To facilitate studies of these proteins, we developed genetically modified mouse lines in which endogenous NPR1 and NPR2 were tagged with the HA epitope. To investigate the role of phosphorylation in regulating NPR1 and NPR2 guanylyl cyclase activity, we developed mouse lines in which regulatory serines and threonines were substituted with glutamates, to mimic the negative charge of the phosphorylated forms (NPR1-8E and NPR2-7E). Here we describe the generation and applications of these mice. We show that the HA-NPR1 and HA-NPR2 mice can be used to characterize the relative expression levels of these proteins in different tissues. We describe studies using the NPR2-7E mice that indicate that dephosphorylation of NPR2 transduces signaling pathways in ovary and bone, and studies using the NPR1-8E mice that indicate that the phosphorylation state of NPR1 is a regulator of heart, testis, and adrenal function.The natriuretic peptide receptors NPR1 and NPR2, also known as guanylyl cyclase A and guanylyl cyclase B, have critical functions in many signaling pathways, but much remains unknown about their localization and function  Natriuretic peptide receptors 1 and 2 (NPR1 and NPR2) are membrane guanylyl cyclases, also known as guanylyl cyclase A and guanylyl cyclase B . Atrial NPR1 and NPR2 are structurally similar proteins, comprised of an extracellular ligand binding domain, a single plasma membrane-spanning region, and an intracellular domain composed of a juxtamembrane kinase homology domain, followed by a dimerization domain and a carboxy-terminal catalytic domain . The kinTryptic phosphopeptide mapping and mass spectrometry studies identified the phosphorylation sites in NPR1 and NPR2 . SubstitMuch has been learned about the physiological functions of NPR1 and NPR2 from studies of natural mutations in humans and genetically modified mice in which these proteins are inactive or overactive , and in in vivo, we generated mice in which the 7 serines and threonines of NPR1 and the 6 serines and threonines in NPR2 that are known to be phosphorylated were changed to glutamates  was added to the extracellular N-termini of the endogenous NPR1 and NPR2 proteins , by CRISThe HA-NPR1 mice were made similarly to the HA-NPR2 mice, except that the Cas9/sgRNA ribonucleoprotein (RNP) complex and ssDNA donor mixture were introduced directly into one-cell C57BL/6J embryos by electroporation rather than microinjection. The positions where the HA sequence and a 3 amino acid spacer were inserted are shown in The mice used for the studies to be described were homozygous for the indicated mutations, and the terms HA-NPR1 and HA-NPR2 refer to homozygotes. The mice used to generate them were obtained originally from The Jackson Laboratory (#000664).Generation and validation of the NPR1-8E and NPR2-7E mice have been previously described . In thesIn brief, the NPR1-8E mice were produced by CRISPR/Cas9 mediated genome editing of C57BL/6J one-cell embryos, to obtain a global knock-in line; they were initially named GC-A-8E . The micHprt-Cre mice /129sv (25%). The mice were maintained on this mixed background. Use of heterozygous breeding pairs to obtain homozygous mutants and wildtype controls ensured that background strain did not affect experimental results (The NPR2-7E mice were generated by homologous recombination in mouse ES cells derived from a F1 (C57BL/6Jx129sv) embryo, to obtain a conditional knock-in line . To remoCre mice . The Hpr results , 2017. I results .2. Frozen organs were weighed and sonicated in an appropriate volume of 50 mM Tris HCl, 150 mM NaCl, 1% SDS; protein concentrations were determined by a BCA assay (Thermo Scientific #23227). Protein samples were loaded on a 12% gel, and separated by SDS-PAGE, allowing the 45 kDa marker to run to the bottom of the gel. Loading of equal amounts of protein per lane was assured by performing BCA assays on all tissues, and confirmed by staining of blots for total protein, using Ponceau S  to stimulate follicle growth. All other tissues were collected from adult male mice (16–20 weeks old). After removal, organs were rinsed in PBS, blotted on a laboratory wipe, and snap frozen in liquid Nonceau S   or rabbit monoclonal (#3724) antibody from Cell Signaling was found to be optimal .These parameters were measured as previously described . In brieTo investigate the utility of the HA-tagged NPR1 and NPR2 mice for comparing expression levels of NPR1 and NPR2 proteins, lysates of several tissues were prepared from homozygous HA-tagged and wild-type mice, and an equal amount of protein from each tissue lysate was separated by SDS-PAGE. Western blots were probed with an HA antibody, and bands present at ∼120–130 kDa in HA-expressing tissues, but not in wild-type tissues, were identified as NPR1 or NPR2 . DoubletOf the tissues tested, NPR1 was most highly expressed in the adrenal gland and testis . In addiOnly a small subset of tissues was examined here, and previous studies indicate that many other tissues  express these proteins as well , 2022. BHA-NPR2 mice and HA antibodies have also been used to analyze the cellular localization of NPR2 within ovarian follicles, by immunofluorescence microscopy . These sIn addition to their use for examining protein expression levels in different tissues, HA-NPR2 mice and HA specific antibodies have been used together with Phostag gel western blots to show that fibroblast growth factor causes NPR2 dephosphorylation in chondrocytes . SimilarImportantly, no obvious morphological or physiological defects have been noted with the HA-NPR1 and HA-NPR2 mice. A previous study demonstrated that addition of an epitope tag of similar size  to the N-terminus of NPR1 had no effect on the ANP concentration required to activate NPR1 guanylyl cyclase activity, or on the kinetics of the activation . The N-tA previous study demonstrated that addition of an HA tag to the N-terminus of NPR2 did not alter physiological processes that depend on NPR2 activity: regulation of meiotic arrest and resumption in ovarian follicles , and depin vitro identified multiple phosphorylation sites in NPR1 and NPR2 that are important for regulation of enzyme activity  cells diffuses through gap junctions to the oocyte, where it prevents meiotic resumption . The midIn chondrocytes of the growth plate in the bones of young mice, cGMP produced by CNP stimulation of NPR2 promotes bone elongation . Bones aOne pathway opposing bone elongation is fibroblast growth factor (FGF) signaling, which dephosphorylates and inactivates NPR2 in chondrocytes , lowerinIn addition to these effects on bone elongation, the bones of NPR2-7E/7E mice have higher mineral density, a larger network of trabeculae, a thicker cortex, and larger diameter compared to wild type . As a ren = 9) remained significantly smaller than hearts from age-matched wild-type male mice (n = 5) .Twelve-week-old male NPR1-8E/8E mice have smaller hearts. This is due to smaller cardiomyocytes, resulting from diminished cardiac extracellular regulated kinase (ERK) activity . ImportaBecause NPR1 is expressed in multiple tissues, and at relatively low levels in the heart itself , the effCorrelating with the high levels of expression of NPR1 in testis and adrenal gland , plasma The discovery 25 years ago that the guanylyl cyclase activity of natriuretic peptide receptors requires receptor phosphorylation ,b, 1999 syee@uchc.edu).Cryopreserved embryos for the mouse lines described here are freely available by request: HA-NPR1 , HA-NPR2 , PA-NPR2 , NPR1-8E , NPR2-7E , and NPR2-7E . Requests should be addressed to S-PY (This animal study was reviewed and approved by Institutional Animal Care and Use Committees at UCONN Health and University of Minnesota.S-PY, KL, and DK designed, generated, and validated the genetically modified mice. JE, TU, and LJ designed, performed, and analyzed the protein expression studies. BW, CH, TO’C, and LP designed, performed, and analyzed the heart measurements. JE, LP, S-PY, and LJ wrote the manuscript. All authors reviewed and approved it.


Rehabilitation in South Africa (SA) operates independently of major health services and reforms, despite the increasing rehabilitation need. With the introduction of National Health Insurance (NHI), SA is facing another major health reform. Evidence is needed on the current SA rehabilitation situation, regarding shortcomings, opportunities, and priority strategic strengthening actions. We aimed to describe the current rehabilitation capacity in the SA public health sector, which serves the majority and most vulnerable South Africans. A cross-sectional survey was conducted in five provinces, using the World Health Organisation’s Template for Rehabilitation Information Collection (TRIC). Participants were purposively selected for their insights and experiences of rehabilitation in specific government departments, health sectors, organisations, and/or services. TRIC responses were analysed descriptively. Participants explained how timely and effective rehabilitation produced long-term health, social, and economic benefits. Positive initiatives were reported for rehabilitation data collection, service design, and innovation. Challenges included inadequacies in human resources, the integration of rehabilitation at primary care, guidelines, and specialised long-term care facilities. The continuity of care across levels of care was sub-optimal due to inefficient referral systems. Promoting and improving rehabilitation nationally requires concerted, innovative, collaborative, and integrated efforts from multiple stakeholders within, and outside, the health system. Rehabilitation: Rehabilitation is an essential health service that improves the lives of people with a wide range of health conditions—including many people with disabilities—through optimising their day-to-day functioning. Rehabilitation outcomes may include improved mobility, personal care capacity, the ability to communicate, greater independence, as well as joining (or re-joining) the workforce or attending school or university. Rehabilitation is relevant for people of all ages—for instance, premature infants with developmental delay; children with neurological disorders, hearing, or vision loss; or people of any age recovering from illness, trauma, communicable diseases , or dealing with the sequelae of chronic diseases . People with compromised capacity are often unable to study or work, and hence cannot contribute optimally to their families, communities, or the country. Therefore, rehabilitation, more than the medical management of impairments, incorporates a holistic, person-centred approach to enable optimal reintegration  of people with disabilities and impaired functioning into their communities  at universities.”One participant noted the lack of collaborative, multi-level leadership within the rehabilitation discipline with a broad range of stakeholders . Although several committees are available in clinical fields, coordination of a more united approach and action will strengthen rehabilitation:“Rehabilitation is a victim of governance flaws and failures in the system.”Participants acknowledged that rehabilitation governance is complex because rehabilitation itself is complex. It involves coordination within rehabilitation teams, and additional synchrony with the comprehensive care required by the individual. Participants felt that rehabilitation is affected by broader health system failures, for example, inadequate governance and the lack of follow-through of provincial plans or policies nationally. These broader health system factors fall outside the remit of rehabilitation governance, but can significantly impact on rehabilitation service assessment and quality:Participants reflected on strategies that could elevate the visibility of rehabilitation at strategic levels. They noted the dynamic nature of health management, where rehabilitation managers should capitalise on opportunities as they arise. Rehabilitation managers at one predominantly rural province reflected on key challenges, such as litigation related to children with cerebral palsy. Since funding for rehabilitation forms a large proportion of these claims, there are opportunities to strengthen rehabilitation options, which could significantly reduce litigious pay-outs. There were also strong suggestions that the inclusion of rehabilitation in national strategic initiatives for which there are ring-fenced funding , could improve access to, and visibility of, rehabilitation at the national level.A participant from one urban province reported on the formation of a new committee to plan and implement integrated care. Rehabilitation is represented on this committee, which marks a positive step towards integrated care pathways, and a move away from traditional specialised care models such as HIV clinics.All provinces (100%) indicated the existence of an allocated budget for rehabilitation and assistive products. Only one province  could provide the total 2021 expenditure for rehabilitation . In this province, rehabilitation expenses constituted R96 million, about 0.3% of the total provincial health budget. Most participants indicated that they lacked ready access to information about rehabilitation expenditure. Their perceptions were that financing for rehabilitation was not standardised or even monitored across facilities, limiting opportunities to examine funding fluctuations or trends over time. Participants indicated that most rehabilitation expenditure—such as the human resources, consumables, and infrastructure—occurs at facility level, but routine accounting and reporting disaggregated by rehabilitation is mostly not in place, therefore information remains limited.All participants reported the Government  as the major financing mechanism for rehabilitation. The sources mentioned included the Road Accident Fund (RAF) and the Workers’ Compensation Assistance (WCA). The Public Finance Management Act (PFMA) was reported as the regulatory body that ensured the efficient and transparent management of all revenue and expenditure provided by the government. Additional financing mechanisms included the NGOs but was not monitored by the PFMA Act. There were typically no out-of-pocket (OOP) costs incurred for rehabilitation provided at government facilities, while one province (20%) mentioned that this was dependent on patients’ income.The major health financing mechanisms covered all levels of rehabilitation including hospital-based inpatient and outpatient rehabilitation, community-based rehabilitation, and outreach . The training of a few community health workers (CHWs) was covered. No special funding was allocated to rehabilitation that targeted children with developmental delays and disabilities.The quantitative responses indicated notable inefficiencies pertaining to rehabilitation health information. Participants were asked about the routine collection of data on the types of rehabilitation needs that existed in their provinces. Participants mostly reported awareness of the information generated by the Washington Group Short Set, which had been included in the South African Census. No other data appeared to be routinely collected on rehabilitation needs in South Africa.One key finding was the lack of high-level indicators for rehabilitation within national or provincial health monitoring frameworks. The only indicator mentioned was the number of wheelchairs and hearing aids issued during specific time periods, which is included in the national health monitoring and reporting systems. Participants unanimously agreed that these were insufficient, since they failed to provide any indication of service coverage or quality, or how rehabilitation contributed to key performance indicators on population health.Routine data on service delivery  were captured in all five provinces but no data were recorded on actual need, which is estimated to be significantly higher than current service levels. Although a growing number of research projects are being conducted in this area, they are not always conducted in collaboration with clinical services, and moreover, projects are generally focused on clinical rehabilitation topics.The key challenges pertaining to rehabilitation data collection are depicted in Participants commented that they were aware of rehabilitation research projects being conducted in their provinces. However, research funds were limited, and projects were mainly clinically oriented. The key funding bodies mentioned included the state-based South African Medical Research Council (SAMRC) and the National Research Foundation (NRF) and private bursaries available for postgraduate studies. Participants suggested that systems-related rehabilitation research is urgently needed.In one province, an online method had been developed to routinely collect human resource information and service delivery data . The data were verified by on-site visits and regular check-ins with rehabilitation coordinators and managers at a facility level. These data were not integrated into the provincial online systems, although this was a goal going forward.An assessment of workforce capacity highlighted notable limitations in all rehabilitation professions, as the ratio was <1 across provinces. Regarding workforce competence and experience, participants mentioned self-initiated local strategies to support therapist training and competence which included forums, in-service training, CPD courses funded by the skills development fund, and student supervision. Almost all therapists (97%) had undergraduate university degrees (3% had diplomas), but only 6% had a masters or doctoral degree. The participants highlighted the limited career-advancing pathways for therapists and that obtaining a postgraduate degree does not ensure a higher-level post or better salaries.The salaries of entry-level therapists were about one-third of the salary of a medical officer, and slightly less than professional nurses. In addition, participants highlighted that there were no incentives in place to attract or retain therapists . There were once-off allowances for uniform and post-graduate studies.One urban province had initiated special interest groups, chaired by a nominated chair, as acknowledgement of their clinician expertise. This initiative offers affordable and accessible opportunities to maintain or bolster clinical competence.Participants suggested that better collaborative approaches between academic educators, researchers, clinicians, and health managers would yield benefits in terms of rehabilitation capacity.All participants provided perceptions that rehabilitation services were not aligned to the needs of the local population, considering the burden of disease and the workforce shortages.Participants indicated that rehabilitation services were mostly located at tertiary institutions. However, all participants indicated the need to strengthen primary care and community rehabilitation, which hinged on weekly outreach services in three of the provinces see . SpecialRehabilitation services delivered in the community were said to be very limited—existing services were provided by rehabilitation care workers supported by district-based therapists as well as primary care therapists. Non-governmental organisations provided training for carers of people with disabilities and mobility training for people with vision and hearing loss.“Make managers understand what rehab is and how it fits into their programme.” Contextual factors influencing servicesThe need to develop rehabilitation guidelines and integrate rehabilitation into priority programmes and comprehensive care pathways was commonly suggested as a way of improving referral to rehabilitation, and access to services. One rehabilitation manager commented that managers in other services should understand how rehabilitation can contribute to the KPIs of broader health programmes:Socio-economic factors, such as the high crime rates, unemployment, poverty, domestic violence, lack of education, and substance abuse, have a notable effect on the efficiency of rehabilitation services. People requiring rehabilitation have complex needs, and many are in complex domestic and social situations, where they may also require mental health support and rehabilitation. Participants also noted that students were not trained to deal with this level of complexity. Providing appropriate education for rehabilitation recipients and their families/caregivers was reportedly challenging and required high-level skills. For example, teenage pregnancies were common, and these mothers required special support if their children required rehabilitation.Several participants suggested that an increasing number of families and caregivers were unable to take care of patients once they were discharged from hospital, due to less-than-adequate socio-economic circumstances. For example, family members were unable to leave much-needed employment to provide care, or non-working family members were not equipped with skills, or were healthy enough, to act as carers. In addition, the influx of foreign patients (mostly migrants from other African countries) meant that many patients with severe disabilities (in long-term rehabilitation facilities) could not be discharged, as their families were not in South Africa. This presented significant ethical and legal dilemmas since the already-limited number of long-term rehabilitation beds could not be made available for new patients.The high burden on medical and nursing staff limits their ability to educate patients on medical aspects and risk factors related to their condition. Consequently, increased strain is placed on the limited rehabilitation workforce to address this gap; consequently, less time is spent on rehabilitation.The participants indicated that there is often no opportunity to educate patients and their carers due to early discharge. In addition, education is often limited by the lack of available translators in many settings, and written information may not be available in all languages. Even if written information is available in local languages, low educational levels of many patients and families, coupled with other cultural factors, mean that patients often cannot read, understand, and/or act on the information. Thus, therapists are often tasked with explaining information to patients, families, and other caregivers, which places additional burdens on already-stretched rehabilitation capacity.Participants reported strategies that complemented available rehabilitation services at community level. These strategies included engaging community health workers and including NGOs and community peer support groups. In one province, orientation and mobility practitioners  were employed to act as conduits between patients and the healthcare system.Most rehabilitation facilities at the tertiary level had the necessary infrastructure for rehabilitation, including access to equipment, gym facilities, and kitchen/bathrooms for the assessment of, and training in, activities of daily living. Most patients require primary care and community rehabilitation but the infrastructure at this level of care is reportedly inadequate and/or inappropriate, particularly to conduct group sessions or manage special groups such as children.The rehabilitation-related medicines that were reported to be mostly available in government services in the country were non-steroidal anti-inflammatory drugs (NSAIDS). Baclofen and corticosteroids were either not available or only available at higher levels of care.The design of new primary care facilities with purpose-built sections for rehabilitation was mentioned by one manager, who suggested that this indicates progress towards strengthening primary care rehabilitation.All five provinces (100%) were not equipped with an essential list of assistive products, and 80% of these did not have a strategy or plan that included assistive products. However, quality regulations, safety and service standards were somewhat available for the procurement and provision of assistive devices in all the provinces.Regulatory bodies for assistive devices were in place to oversee the quality and provision of assistive devices. Participants agreed that most of the basic assistive technologies were available and were procured via national tenders (for those devices that were routinely issued). However, ordering devices that were not available by tender was often constrained by complex procurement processes . These procurement difficulties resulted in delays in the provision of appropriate, specialised, assistive technologies for people with complex disabilities.One urban province has implemented provincial tenders to reduce bureaucracy in ordering assistive technologies that are not on the national tender. This initiative has proven to be feasible and efficient in procuring assistive devices in a timely manner.The participants reported that rehabilitation was poorly integrated in emergency response plans. One cited reason was that rehabilitation workers were not considered to be frontline workers; hence, focus was on medicine and nursing professions. Participants unanimously agreed that rehabilitation capacity was not mapped and known to the Ministry of Health and Health Emergency Operation Centre or National Emergency Management Agency. No contingency plans nor stockpile for assistive products were in place for the continued provision of rehabilitation in the event of a disaster or pandemic. One province mentioned the availability of prefabricated orthoses only. All provinces had no established referral pathways between rehabilitation services in high-risk areas and those in low-risk areas.The COVID-19 pandemic raised awareness regarding the need for strengthening emergency preparedness within rehabilitation, e.g., to accommodate an increased need for assistive devices. Telerehabilitation became increasingly popular as a means of continuing rehabilitation services for non-COVID patients.To our knowledge, this is the first paper to report on a detailed analysis of the public health sector’s rehabilitation capacity in South Africa. The findings provide a broad overview of the current strengths, weaknesses, opportunities, and emerging initiatives/innovations to advocate and enact priority steps for the prioritisation and strengthening of rehabilitation in the health system.The WHO TRIC tool facilitated the collection of comprehensive data on the country’s rehabilitation situation. This validated tool was previously used in LMICs to assess rehabilitation capacity . AlthougThis research identified that South Africa’s rehabilitation capacity has strengths in terms of leadership and governance. Rehabilitation managers are vocal advocates at provincial and national levels and are appropriately positioned at the governance level to drive strategic initiatives, and implement key policies, such as NHI. However, rehabilitation managers reported their limited influential abilities since policy makers do not regard rehabilitation as integral to healthcare, nor as an essential health service. Many factors contribute to the sub-optimal strategic positioning of rehabilitation within the South African health system. One key barrier could be the lack of appropriate and reliable rehabilitation data to drive data-driven advocacy for informed decision-making by policy makers. Although participants in one urban province reported taking significant steps to increase the collection of reliable data, they have not yet crafted an avenue for data to be utilised at a strategic level. Most participants indicated that routine service-related statistics are collected (such as number of people treated); however, this type of data may be inadequate to drive high-level decision-making . In addiSouth Africa, as do many other countries globally, uses mortality and morbidity as standard indicators of population health. Morbidity and multimorbidity levels are high in South Africa due to the quadruple burden of disease . Our parSouth Africa’s envisaged NHI is a major health transformation that presents investment opportunities for strengthening rehabilitation. Our participants suggested that rehabilitation should be better integrated in major health policies and reforms, particularly to facilitate increased investment. Participants reflected on the importance of integrating rehabilitation into major health reforms such as the NHI. It is envisaged that the NHI will necessitate greater data collection which, assuming rehabilitation is included, could be utilised in rehabilitation research. Potentially ring-fenced funding for the NHI could be directed towards impactful research into improving and integrating rehabilitation services, which currently appears to be mostly limited to clinical studies. The roll-out of the NHI will incorporate notable technological transformation, which could support the inclusion of rehabilitation in patient and broader healthcare plans. However, to seize these opportunities, the rehabilitation community should strive to reduce disciplinary boundaries, which were reported by our participants. Unless the therapies are represented by a united voice in the design of major, national health reforms, it could constrain investment and interventions to strengthen rehabilitation in the South African health system.The implementation of the NHI at an operational level involves the design of standardised care packages along care pathways of priority conditions. Our study shows a startling lack of rehabilitation guidance documents available in South Africa, which may hamper the inclusion of rehabilitation along NHI care pathways. To address this challenge in a timely manner, South Africa could leverage on international initiatives in this area, such as the WHO’s Package of Interventions for Rehabilitation (WHO PIR) . The WHOOur participants suggested that the capacity of public sector rehabilitation services is inadequate for the local need, which is based on the notable proportion of South Africans who can benefit from rehabilitation services . HoweverAdvancing rehabilitation nationally and provincially requires efforts from tertiary training institutions. Participants highlighted the need for training to become more focused on team approaches and integrated care models to support the government’s current shift towards integrated care approaches. One urban province mentioned the establishment of an integrated care forum, which can serve as the impetus for educational reform. The educational design of innovative integrated training models that are contextually efficient and feasible for South Africa will ensure that graduates are more open and prepared to work in an integrated manner in local settings. Tertiary institutions play a central role in lobbying for increased funding for health-systems research, which could potentially be focused on strengthening rehabilitation in the health system.Access to healthcare services is enshrined as a basic human right in the Constitution of South Africa . Access The cross-sectional design of this study does not provide information on trends and cause-and-effect relationships. Data collection was mainly conducted online due to the COVID-19 pandemic and face-to-face meetings may have provided further insights through observation. The study was limited to an analysis of the rehabilitation capacity of the public health sector, whereas the TRIC tool should ideally be used to capture information on rehabilitation across other non-health sectors including labour and education; and non-public sectors including non-governmental organisations and the private sector. The study was limited to five provinces and there may be nuances in the other four provinces that were not captured in this analysis. Though our study was valuable in providing an overview of the situation at a national and provincial level, we cannot generalise our findings to the provinces as we were unable to capture the granular differences in rehabilitation services and capacity at district or local levels. We were unable to obtain data on rehabilitation finances from four provinces and could thus not compare this aspect across provinces. This was because it was not possible for participants to identify expenditure, due to its aggregation in other health expenditure, and the non-standardised accounting approaches used across provinces and facilities. A future study should include facility-level stakeholders that may be able to provide accurate data on the financing of rehabilitation.Multiple strategies were used to validate the data, which contributes to the confidence in the findings. A representative sample of five provinces, which was reflective of a range of factors  was used. This assisted in obtaining a comprehensive understanding of the current rehabilitation situation in South Africa.Rehabilitation capacity in South Africa is limited compared with population needs. Rehabilitation faces many challenges, but also opportunities for improvement. Access to quality rehabilitation services to meet increasing need is possible with moderate investment. Political will is critical for increasing investment in rehabilitation to redress key shortcomings, such as human resources as well as primary care rehabilitation services. Rehabilitation coalitions within and across sectors are necessary to drive strategic, priority initiatives to position rehabilitation as a valued health strategy in the South African health system. Future focused, locally centred leadership should prevail as the country’s health sector transforms towards a NHI initiative, in which rehabilitation is a core element.


Accurate measurements of whole blood creatinine can aid in the diagnosis and treatment of renal diseases.GEM Premier ChemSTAT is a new point-of-care system providing rapid creatinine, BUN and tCOHeparinized whole blood samples from different clinical locations were evaluated on the GEM Premier ChemSTAT and results compared to plasma from the same samples on the Beckman AU5800 or whole blood on the GEM Premier 4000. Precision studies were conducted with whole blood and quality control material.ChemSTAT correlated well with plasma samples on the AU5800 (regression slopes (S): 0.957–1.159, correlation coefficients (r)≥0.952) and with whole blood specimens on the GEM Premier 4000 . The repeatability was 0.1%–3.1% and QC precision were within lab and manufacturers’ specifications.ChemSTAT demonstrated strong correlation to the comparative methods and excellent precision. Combining with its continuous quality management, ChemSTAT is suitable for acute care settings to provide rapid, reliable results, which could minimize time-to-treatment and improve patient outcome. •GEM Premier ChemSTAT evaluated against Beckman AU 5800 and GEM Premier 4000.•A multi-use cartridge-based system measures twelve analytes in whole blood sample.•Method comparison and precision meet laboratory and manufacturers' specifications.•Whole blood creatinine assays showed lab quality performance.•Plasma vs serum bias observed for creatinine and urea on lab analyzer. BUNblood urea nitrogenPOCpoint of careeGFRestimated glomerular filtration rateIDMSisotope dilution mass spectrometryMDRDModification of Diet in Renal DiseaseCKD-EPIChronic Kidney Disease Epidemiology CollaborationNKDEPNational Kidney Disease Education ProgramMDLmedical decision level1+), potassium (K+), calcium (Ca++), chloride (Cl−), glucose, creatinine, blood urea nitrogen (BUN), total carbon dioxide (tCO2), lactate, pH and pCO2 in one whole blood specimen could help patient triage, monitoring and treatment. Literature has provided evidence for the relevance of electrolyte abnormality, acute kidney injury (AKI) and chronic kidney disease (CKD) to the severity and outcome of COVID-19 patients [A whole blood point-of-care system that can perform a quick metabolic chemistry panel consisting of sodium . The evaluation protocol was reviewed and approved by the local Institutional Review Board in accordance with the Declaration of Helsinki. Individual participant consent was not required because the study used deidentified remnant blood samples.2.22.2.1The GEM Premier ChemSTAT (hereafter ChemSTAT)  is a new cartridge based POC testing system that has two primary components: the GEM Premier ChemSTAT instrument and a disposable, all-in-one, multi-use cartridge (GEM PAK) that is stored at room temperature and contains all the materials required to perform analytical testing, including sensors, solutions, sampler, tubing and waste bag.+, K+, Ca++, Cl−, glucose, creatinine, BUN (or urea), tCO2, hematocrit, lactate, pH, and pCO2 from a single whole blood specimen. The system uses patented Intelligent Quality Management (iQM) technology. Once the GEM PAK is validated, it uses an active quality process control program (iQM) designed to provide continuous monitoring of the analytical process with automatic error detection, attempts to correct any errors, and documentation of all corrective actions, replacing the use of external quality control. This simplifies use in emergency and other POC critical care settings by non-laboratorians. The creatinine assay on ChemSTAT relies on a differential measurement of both creatinine and creatine electrodes. The creatinine/creatine electrodes are amperometric biosensors involving a three/two-enzyme cascading reaction converting creatinine/creatine to hydrogen peroxide (H2O2). The BUN (urea) assay utilizes a potentiometric sensor consisting of urease enzyme. The bicarbonate sensor relies on a pH selective polymer as a gas permeable outer membrane and an internal bicarbonate buffer. The Na+, K+, Ca++, Cl−, pH and pCO2 assays are based on a potentiometric ion selective sensor, while the glucose and lactate assays are based on an amperometric biosensor, and the hematocrit assay is based on a conductivity sensor. The working principles of the assays were described in detail in literature [The ChemSTAT system provides quantitative measurement of Naterature .GEM Premier 4000 applies the same measurement technology as the ChemSTAT for all the analytes used as comparative methods in this study.2.2.2+, K+ and Cl− electrolyte measurements. Glucose and urea measurements were both colorimetric enzymatic assays. The creatinine measurement was the kinetic colorimetric assay based on the Jaffe method.The reference clinical biochemistry analyzer, Beckman AU5800  uses an indirect potentiometric ion selective electrode method for Na2.3The method comparison study was performed in accordance with Clinical and Laboratory Standards Institute (CLSI) EP09c  with the+, K+, Cl−, and glucose, and also compared against a whole blood POC system, GEM Premier 4000 for Na+, K+, Cl−, Ca++, glucose, lactate, hematocrit, pH and pCO2.Method comparison studies were performed against the AU5800 for creatinine, BUN, NaFive ChemSTAT PAKS were used in this study covering two different sensor and reagent lots. All results except those from quality control material were obtained from a single reagent lot for AU5800 and GEM Premier 4000 systems. Reagents, calibration verification product and QC materials were supplied by the manufacturers.2.4The precision characteristics of ChemSTAT were evaluated according to CLSI EP15-A2  over a pThe within-run repeatability was estimated with a whole blood precision study. Heparinized whole blood specimens were assayed in duplicate on one ChemSTAT system to estimate repeatability with total of thirty-four whole blood specimens.3Analyse-it for Microsoft Excel Method Validation edition (version 5.66), an add-in statistical analysis software for MS Excel  was applied in data analysis and graphic presentation of study results.+, hematocrit, and pH) or Passing-Bablok  regression methods were used to obtain slope, intercept, correlation coefficients and confidence intervals. Deming regression was used for analytes with constant variability (SD) and Passing-Bablok regression was used for analytes with mixed variability  throughout the measuring range. The bias at the medical decision levels (MDL) for the ChemSTAT versus the AU5800 or GEM Premier 4000 was estimated for each evaluated analyte. The estimated mean bias across tested sample ranges for each analyte was obtained from the Bland-Altman analysis to assess the agreement between ChemSTAT and the comparative methods.Deming  per analyte and should be within the Total Error Allowed (TEa). Bland-Altman analysis was performed to evaluate the agreement of the ChemSTAT test results with the comparative methods by evaluating the mean bias, SD of bias and limits of agreement for 95% sample (LoA). The mean bias should be within TEa and the LoA shall be within the range of clinically acceptable limits. For some evaluated analytes, the regression analysis results were not representative due to a narrow range of samples spanning the claimed measuring range. In those cases, the mean bias from Bland-Altman analysis was used to assess the agreement between two methods and the percentage of samples within the total allowable error (TEa) limit was calculated and compared against the proposed acceptance criterion of ≥95%., within-laboratory  mean, standard deviation (SD), and coefficient of variance (%CV) were computed for each analyte of ChemSTAT System Evaluator Levels 1–3 and ChemSTAT Hematocrit Evaluator Levels 1–3, separately. The study results were deemed acceptable if the estimated within-laboratory  SD or %CV were less than or equal to the acceptance criteria set by the Spanish Society of Laboratory Medicine (SEQCML) [For data analysis of the aqueous control precision study(SEQCML) ,8 and th(SEQCML) .For the whole blood repeatability study, the coefficient of variation for each sample was computed and each of these %CV was pooled to determine the within-sample %CV and SD for establishing acceptability. The study results were deemed acceptable if the estimated within-sample (within-run repeatability) SD or %CV were less than or equal to the acceptance criteria set by SEQCML and the manufacturer.44.1vs. AU5800 or GEM Premier 4000. The number of samples, tested sample range, regression slope (95% CI), correlation coefficient (r), and estimated bias at MDL are summarized in +, K+, Cl− and glucose and 0.965 to 1.124 vs. GEM Premier 4000 for electrolytes, glucose, lactate, hematocrit, pH and pCO2. Intercepts ranged from −1.357 to 2.31 vs. AU5800 and -2.936 to 5.973 vs. GEM Premier 4000. Correlation coefficients (r) were 0.952–0.998 and 0.949 to 0.999 vs. AU5800 and GEM Premier 4000 respectively, for all evaluated analytes. The estimated mean bias between ChemSTAT and the comparative methods for all analytes from the Bland-Altman analysis was within TEa and in the range from −0.21 mmol/L for sodium to +1.34 mg/dL for glucose and from +0.004 for pH to +3.4 mg/dL for glucose vs. AU5800  for all the analytes met the recommended criteria of ≥0.95. The slightly elevated regression slope for K+ was likely due to difference between the two assays; consistent K+ results were observed between whole blood assays on ChemSTAT and GEM Premier 4000 which were assayed within 5 min. Despite the elevated slope, Bland-Altman analysis demonstrated good agreement between ChemSTAT and AU5800 K+ results as 98% of the sample biases were within the TEa limit. The method comparison performance for each analyte vs. AU5800 is shown in + for the likely cause discussed above. Good agreement between ChemSTAT WB and AU5800 plasma creatinine was observed; biases at MDL1 (0.6 mg/dL) and MDL2 (1.6 mg/dL) were only −0.001 (0.17%) and 0.013 (0.8%), respectively. For the method comparison analysis vs. GEM Premier 4000 WB for Na+, K+, Cl−, glucose, lactate, hematocrit, pH, and pCO2, the regression slopes were all within the recommended range of 0.90–1.10 except for hematocrit which had a slightly elevated slope of 1.124 and the correlation coefficients (r) for all the analytes met the recommended criteria of higher than 0.95. In this study, the reference hematocrit on GEM Premier 4000 was derived from the measured total hemoglobin by CO-Oximetry while the ChemSTAT hematocrit was measured based on a conductivity method. Difference in measurement methodology and sample inhomogeneity may have contributed to the small deviation of slope from the unity line.For the method comparison analysis During the study, serum samples were also tested on AU5800 for creatinine and urea in parallel and the results were analyzed to compare with those results from plasma for every patient sample to assess variability between the two matrices on a laboratory chemistry analyzer. For AU5800 Crea and Urea assays, small but noticeable positive biases between plasma and serum samples were observed as shown in 4.2For the ChemSTAT, the within-laboratory precision or total precision (SD or CV%) for all the measured parameters were within the specified acceptance criteria in a three level quality control material over the 41 test days. The estimated SD or CV% (highlighted in bold) were compared with the SEQCML criteria and the manufacturer's claim as summarized in 2 at level 1 (p < 0.05). However, the differences of the means among three groups were within ±0.5 mmol/L (4%) at mean recovery of 12.8 mmol/L; therefore, no clinically significant impact is expected.The entire data set was also divided into 20, 30 and 40 three test day groups and the mean for the QC materials was estimated for each group. An analysis of variance (ANOVA) was performed to evaluate significant differences between the three models. The results showed no statistically significant difference (p > 0.05) among three groups for all analytes and all 3 tested levels except for tCOThe repeatability or the within-run sample precision study were carried out for seventeen test days with a total of 68 whole blood samples by multiple medical staff. Sample results were analyzed and the pooled within-sample precision for all analytes are summarized in 52, BUN, and Na+). Before adapting a new POC analyzer such as ChemSTAT it is important to evaluate its analytical performance against the institute's existing test method. Comparison of ChemSTAT against Beckman AU5800 was not available in literature. Many hospitals conduct multiple tests for the same patient on both lab and POC analyzers throughout the patient stay for diagnosis and monitoring purposes. Commutability between POC WB assay and lab results is critical for hospitals in deciding which assay to use depending on the urgency of needing assay results. 2) This study evaluated the ChemSTAT creatinine assay against the Jaffe creatinine assay which is widely used on clinical chemistry analyzers while the previous study compared ChemSTAT against the enzymatic creatinine assay. 3) The current study focused on the patients in nephrology and oncology while the previous study evaluated with ED patients. Both studies provided unique clinical performance of the ChemSTAT to specific patient populations.There has been a published comparison study for ChemSTAT vs. lab analyzer Roche cobas (4) but this study provided unique information in the following aspects: 1) due to the lack of harmonization and commutable reference standards for certain clinical assays, subtle biases exist among assays from different analyzer manufacturers and/or methodology  can be used as criteria. The ChemSTAT reports eGFR values derived from either Modification of Diet in Renal Disease (MDRD) or Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations based on the IDMS traceable whole blood creatinine assay. The small bias (0.011 mg/dL for creatinine up to 2.0 mg/dL with 95% limits of agreement (LoA) from −0.138 to 0.159 mg/dL) and low imprecision (1.8% CV) observed for creatinine in this study made the system capable to predict the change of 0.3 mg/dL in whole blood creatinine. Based on its accuracy and better precision than 0.1 mg/dL, as recommended by the National Kidney Disease Education Program (NKDEP)  the ChemIn this study, when plasma vs. serum analysis was compared on AU5800, it was noticed that systematic biases  were observed between plasma and serum samples for AU5800 creatinine and urea assays. This bias between serum vs. plasma results has also reflected on the ChemSTAT WB vs. AU5800 S as positive biases and wider LoA when correlating to serum samples (for example creatinine bias of 0.031 mg/dL with 95% LoA from −0.141 to 0.204 mg/dL vs. serum as depicted in The 41 day within-laboratory precision study was conducted over a period of four months and utilized five cartridge PAKs. The results of excellent total precision of external quality control material demonstrated the system's capability to maintain its long-term analytical performance consistency through the iQM active quality management program.The ChemSTAT system was easy to use and did not require any troubleshooting or maintenance. The system continuously monitors the analytical process and performs automatic error detection, correction and documentation of all corrective actions, which makes it a suitable system for use in emergency and critical care settings by non-laboratorians.62 and lactate testing could find improved turnaround time from a single whole blood specimen and could support improved patient triage and management.Strong correlations were observed in this study between the ChemSTAT system and the comparative methods with excellent precision in analyzing patient samples. The overall total precision of the ChemSTAT was proven to meet the requirement of clinical laboratory. Implementation of the POC ChemSTAT system for basic metabolic panel as well as pH, pCOAll authors participated in concept, design, analysis, writing, reviewing, editing and approving the manuscript. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.AEG, RB, MM, RF and EC declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article. CX declares employment by Werfen.


Kaolin application and bunch-zone leaf removal are two cultural practices that contribute to the control of main pests in vineyards, such as leafhoppers and the European grapevine moth. In the context of Integrated Pest Management (IPM), a two-year study on the side effects of these practices on generalist predators was conducted in three vineyards of a grape-growing area in north-eastern Italy. This study provides the first data on the influence of kaolin and bunch-zone leaf removal on spiders and predatory insects occurring in vineyards. It was demonstrated that moderate use of kaolin has a negligible impact on generalist predatory arthropods in vineyards, while no negative effects were associated with bunch-zone leaf removal. Therefore, these practices were compatible with IPM strategies.Lobesia botrana, but their side effects on generalist predators are still poorly understood. In north-eastern Italian vineyards, the impact of kaolin and LR on species and functional diversity of spiders, as well as the abundance of spiders and generalist predatory insects, was assessed in one vineyard for two consecutive years and in two vineyards for one year. The ecological indices of the spider community were never influenced by kaolin and only in one case were they influenced by LR. At the spider family level, kaolin reduced the abundance of Araneidae, Oxypidae and Salticidae, but only in single cases. In single cases, kaolin reduced the amount of Orius sp. anthocorids and increased that of Scymninae coccinellids, whereas LR increased the amount of Aeolothrips sp. The moderate use of kaolin and the application of LR had negligible and inconsistent impacts on generalist predatory arthropods in vineyards and were therefore, compatible with IPM strategies.In vineyards, kaolin application and bunch-zone leaf removal (LR) were effective in the control of leafhoppers and  Plants sprayed with kaolin, a white aluminum silicate, become visually, tactually and chemically unrecognizable to arthropod pests . The treHebata vitis (Göthe) [Empoasca vitis (Göthe)], Zygina rhamni Ferrari, Arboridia kermanshah Dlabola, Erasmoneura vulnerata (Fitch) and Scaphoideus titanus Ball  and functional guilds  ,53. All To evaluate the effects of kaolin and LR on the number of spiders and predatory insects (collected with a drop cloth and yellow sticky traps), for each vineyard and year, an ANOVA test applied to linear mixed-effects models (LMM) followed by Tukey’s post hoc test was used . The samWithin spiders, the relative proportion of hunting-strategy groups (hunters and web-builders) or subgroups (ambushers and active hunters) in each vineyard was compared using a G test of goodness of fit. To compare percentage data, a Fisher’s exact test or Ryan’s test  were useStatistical analyses were performed with SPSS version 20  or R sofSpider populations were more than twice as abundant in Vineyard B (N. 160) compared to the other vineyards . In whole, spiders belonged to nine families  and 28 different taxa . The higp < 0.0001; in 2016: 72%, p < 0.0001) and in Vineyard B , but not in Vineyard C . The proportion of web-builders was significantly higher in Vineyards B and C than in Vineyard A at 0.05 level (Ryan’s test).Based on the hunting strategy, hunters were more represented than web-builders with significant differences at the G test in Vineyard A , with a prevalence of Nuctenea sp. in Vineyard A and Vineyard C, and Mangora acalypha  in Vineyard B. In total, Linyphiidae represented around a third of web-builders.Among web-builders, Araneidae were prevalent in all vineyards , whereas the subgroup “active hunters” was significantly prevalent in Vineyard C . Among ambushers , Xysticus sp. (60%) was the prevalent taxon. Among active hunters, Oxyopidae of the genus Oxyopes (40%) and Salticidae (36%) were the more represented.Among hunters, the subgroup “ambushers”, exclusively represented by Thomisidae, was on average prevalent on the subgroup “active hunters” in Vineyard A and Vineyard B, even if the difference did not reach the significance level at the G test . The number of captured Coccinellidae in Vineyard B was three times higher than in Vineyard A.In both Vineyards A and B, the amount of captured predatory insects belonging to the considered taxa was prevalent on spiders  . Among tTotal spiders were more abundant in Vineyard B than in Vineyard A, but the proportion of web-builders and hunters was the same  . Among hXysticus sp., while Oxyopes sp. was more frequent in the control.During the two years under study (2015 and 2016), spiders collected by the drop cloth method were not affected by kaolin and LR in terms of H’ and FD indices and functional guilds  . The intSignificant differences in spider amount occurred only at the family level, i.e., for active hunters Oxyopidae in 2015 and Salticidae in 2016, and for web-builders Araneidae in 2016 . No signOrius sp. showed lower captures in the kaolin than in the control, but not at a significant level. Aeolothrips sp. showed significantly higher captures in LR than no LR. Spiders captured by yellow sticky traps were not significantly affected by either kaolin or LR. Concerning spider families, Thomisidae showed higher captures in LR than no LR, but not at a significant level.Among predatory insects captured by yellow sticky traps in 2015, kaolin significantly increased Scymninae . On the Xysticus sp., while no LR resulted in a higher occurrence of Oxyopes lineatus Latreille.In neither of the two vineyards were spiders collected by the drop cloth method affected by kaolin in terms of H’ and FD indices and considered functional guilds  . On othep = 0.23; Vineyard C: constant 86%, p = 1.0).The total spiders collected by the drop cloth method in both vineyards were not significantly affected by kaolin and LR, not even when considering hunting-strategy groups or families . The proOrius sp. in the kaolin than in the control were recorded . This occurrence was not confirmed for Vineyard B . Among hunters, a significantly higher percentage of Salticidae was captured with yellow sticky traps than by the drop cloth method .In 2015, in Vineyard A, a significantly higher percentage of web-builders than hunters was associated with yellow sticky traps compared to the drop cloth method  , recorded in the present study, has been reported feeding on H. vitis nymphs in French vineyards, although its contribution to leafhopper control has not been considered important [H. vitis dead adults were found on spider webs [Oxyopes lineatus (Oxyopidae), whose individuals were observed preying on H. vitis adults during the present study, has been reported feeding on Cicadellidae [H. vitis, as the species Bathyphantes pallidus (Banks) was found to be a valuable predator of the leafhopper Empoasca fabae (Harris) in a field of alfalfa in Kentucky (USA) [Erythroneura spp. leafhoppers [Anagrus spp. egg parasitoids, seem to be more important than spider abundance [Cheiracanthium sp. (Miturgidae), also found in two vineyards in the present study, are widespread predators of Erythroneura spp. leafhoppers in Californian vineyards [The same taxa of spiders found in the vineyards of the present study are reported as predators of  studies ,63,64,65mportant . In Portdellidae . Even Liky (USA) . Moreovefhoppers , even ifbundance ,33,70. Fineyards ,36,38,71Araniella sp. (Araneidae) did not protect its owner from insecticide spraying [Among web-builders, the prevalence of Araneidae in the present study is in agreement with other studies ,72. Theispraying . Neverthspraying .The comparison of yellow sticky traps and drop cloth to monitor spiders in vineyards was performed in the present study and in that of Nobre et al. . Both stAeolothrips sp., Scymninae, Coccinellidae non-Scymninae, Orius sp. and C. carnea s.l.) are reported feeding on grapevine pests. Aeolothrips sp. are predators of phytophagous thrips in vineyards [Chrysoperla carnea s.l. is reported as a predator of L. botrana larvae [H. vitis and Z. rhamni [Orius spp. are effective control agents of tetranychid and eriophyid mites in vineyards [H. vitis [H. vitis and Orius niger (Wolff) population densities was observed using yellow sticky traps, even if the predatory activity could not be confirmed visually [Orius spp. and coccinellids .Araniella cucurbitina (Clerck) was observed under laboratory conditions after the ingestion of great amounts of silk covered with kaolin [In this study, kaolin affected the spider communities in terms of abundance only in three cases recorded in Vineyard A, i.e., the Oxyopidae and Salticidae active hunters, and the Araneidae web-builders. The prevalence of cases with negative kaolin effects on hunters compared to web-builders agrees with studies conducted in apple orchards ,47. Conch kaolin , it is bh kaolin . This feH. vitis and Z. rhamni populations in plots sprayed with kaolin [In the present study, the relatively reduced negative effects of kaolin on spiders can be explained by the low number (two or three) of kaolin applications (20 kg/ha per application). In the literature, negative effects on spiders were sometimes observed with a higher number of kaolin applications than in the present study  ,44,46,47h kaolin ,17 couldh kaolin ,84,85,86Orius sp. decreased and Scymininae increased. With the same number of kaolin applications, a negative effect on Orius sp. was also reported in olive orchards [Anthocoris nemoralis (Fabricius) (Anthocoridae), negative effects of kaolin were found under laboratory and field conditions [Orius sp. could also be an indirect effect of the reduction of leafhopper populations by kaolin [H. vitis control [Two kaolin applications interfered with the abundance of predatory insects only in two cases: orchards . For anonditions ,87, but nditions . In the y kaolin , as anth control ,82. In V control ,90.In the present study, kaolin did not affect Coccinellidae non-Scymninae in agreement with other studies ,43,46, eC. carnea s.l. was not negatively influenced by kaolin, although the data reported in the literature did not always confirm the absence of negative effects [C. carnea and Chrysoperla externa (Hagen) [Aeolothrips sp., whose individuals spend most of their life feeding on small arthropods and pollen occurring in herbaceous vegetation of the inter-row.Additionally, in the present study,  effects ,92. Howe (Hagen) ,88,93. KAeolothrips sp. that were more abundant in LR of Vineyard A and spiders that showed lower species diversity and functional evenness in Vineyard C. The increase in captures of Aeolothrips can be explained by the preference of Thysanoptera for yellow sticky traps exposed to sunlight [Bunch-zone leaf removal influenced captures of spiders and predatory insects only in two cases: sunlight ,95 as ocRegarding the effect of LR on the spider community, which significantly reduced spider diversity in one of the three vineyards, opposing suggestions were reported in the literature. The study of Pennington et al. , in whicIn vineyards, moderate use of kaolin and the application of LR have shown minor impact on spiders and generalist predatory insects; therefore, they are suitable cultural practices in the context of IPM strategies. The negative effects are also negligible after two consecutive years of kaolin applications on the same plots. Even the few cases of a decrease in hunter spiders could be a consequence of the negative effects of kaolin on leafhoppers, which are the most frequent prey of spiders inhabiting grapevine canopy. Regardless, considering the positive effects of kaolin and LR on the control of grapevine key pests, the overall outcome of their adoption is positive. The use of these cultural practices in the context of IPM reduces the risk that pests exceed their economic thresholds and, thus, the need for insecticide applications in vineyard.


Xenobiotic reductase B (XenB) catalyzes the reduction of the aromatic ring or nitro groups of nitroaromatic compounds with methyl, amino or hydroxyl radicals. This reaction is of biotechnological interest for bioremediation, the reuse of industrial waste or the activation of prodrugs. However, the structural factors that explain the binding of XenB to different substrates are unknown. Molecular dynamics simulations and quantum mechanical calculations were performed to identify the residues involved in the formation and stabilization of the enzyme/substrate complex and to explain the use of different substrates by this enzyme. Our results show that Tyr65 and Tyr335 residues stabilize the ligands through hydrophobic interactions mediated by the aromatic rings of these aminoacids. The higher XenB activity determined with the substrates 1,3,5-trinitrobenzene and 2,4,6-trinitrotoluene is consistent with the lower energy of the highest occupied molecular orbital (LUMO) orbitals and a lower energy of the homo orbital (LUMO), which favors electrophile and nucleophilic activity, respectively. The electrostatic potential maps of these compounds suggest that the bonding requires a large hydrophobic region in the aromatic ring, which is promoted by substituents in ortho and para positions. These results are consistent with experimental data and could be used to propose point mutations that allow this enzyme to process new molecules of biotechnological interest. The enzyme xenobiotic reductase B (XenB) catalyzes the transfer of a hydride ion from the cofactor FMN to various substrates of clinical, industrial or biotechnological interest . The stuThe ability of an enzyme to process different substrates has been termed promiscuity and is a property identified in several enzymes . It is aIn general—and probably valid for XenB—it has been proposed that the substrate promiscuity of enzymes is determined by the structural properties of the active site of the protein, analogous to substrate specificity. The structural mechanisms underlying this enzymatic capacity can be explained on the basis of the conformational diversity of the enzyme active site, different protonation states, different subsites within the active site and cofactor ambiguity . EnzymesXenB can reduce the nitro groups or the aromatic ring of 2,4,6-trinitrotoluene (TNT) or 2,4,6-trinitrophenol (TNF). It can act on aromatic and aliphatic substrates, with different functional groups: TNT, TNF, nitroglycerin, trinitroxylene (TNX), hexahydro−1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), 2,6-dinitrotoluene, 2,4-dinitrobenzene, 1,3, 5-trinitrobenzene, 4-amino-2, 6-dinitrotoluene, cyclohexen-2-one (CHE), N-ethylmaleimide (NEM), 5-[bis-2(chloroethyl)-amino]-2,4-dintrobenzamide (SN23862) or 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) ,13,14. TThe structural factors that determine the substrate promiscuity of XenB, allowing it to process compounds of different structure or to discriminate between chemically similar nitro compounds, are unknown. Some structural information on the interaction of XenB with TNT, TNF, nitroglycerin or biomimetic analogues of NADPH has been obtained through molecular dynamics simulations ,16. In aConsidering the environmental impact of XenB-processed substrates and their potential application in prodrug design, in the present work, we evaluated the structural factors determining the interaction of XenB with TNT, DN4, DN6, 4AD, 2AD, TNB and NBZ.In previous works, it has been experimentally demonstrated that XenB presents enzymatic activity with the benzene compounds TNT, DN6, 4AD and TNB but not in the presence of DN4, 2AD or NBZ . All of To evaluate the participation of specific aminoacidic residues in the formation of the enzyme/ligand complex, the non-covalent interactions between different ligands and XenB were characterized by quantum mechanics (QM) calculations. The general reactivity of each compound and its charge distribution  can be used to explain the experimental activities determined for XenB in the presence of the different substrates. However, it is possible that a difference in the stability of the aromatic ring contributes to the lower XenB activity determined with some compounds. To evaluate this factor, the current intensity of the ring was calculated by QM, since a higher current is associated with a higher aromaticity. According to these quantum calculations, we can identify differences in charge distribution, the feasibility of receiving electrons, or ring stability and, based on these characteristics, explain the differences in XenB activity observed with these compounds.According to reports by other researchers, XenB can act on TNT, DN6, 4AD and TNB but not on DN4, 2AD or NBZ . To idenIt has been shown that the activation energy of the hydride transfer from the FMN cofactor to TNT increases with the distance between the hydride acceptor and the donor group (HAD distance). In accordance with this observation, higher enzyme activity should be correlated with smaller HAD distances. As shown in Molecular dynamics simulations of 200 ns were performed, recording 50 frames every 1 ns . According to the simulations, the XenB/ligand complex adopts a stable conformation that can be identified by cluster analysis . Using tThe enzymatic reaction catalyzed by XenB requires the binding of the substrate to the substrate binding site and the generation of a stable complex. As observed in quantum mechanical calculations, the ligands are stabilized at the substrate binding site by non-covalent interactions. These Van der Waals bonds hold the ligands at distances that allow the transfer of the hydride ion from the cofactor to the substrate. However, this distance  or confoFor the model studied, it is observed that ligand binding is necessary but not sufficient for XenB activity, as observed for XenB/2AD, which remains bound in the simulation but does not show activity. Another factor that could determine the activity of XenB on a substrate is the reactivity of each ligand. Thus, for example, the higher activity of XenB on TNB than for TNT could be associated with a different reactivity.−), electroacceptance (ω+) and net electrophilicity (Δω±) for the seven nitroaromatic compounds  for which we have experimental data of enzymatic activity , global hardness (η) electrophilicity (ω), electrodonation (ωactivity  21]. In. In−), ep-boundary orbitals provide information about the energies of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), which can act as electron donors and acceptors, respectively [The ectively . The bou2), which are favored by electrophilic attacks. In the case of 4AD, 2AD, DN4, DN6 and NBZ, the aromatic rings present a slight green zone, which can facilitate aromatic weak interactions. It is worth mentioning that, in the case of TNB and TNT, the oxygen atom of the nitro group attracts electrons and generates electronic deficiency in the aromatic ring, favoring nucleophilic attack.To show the electrostatic representation of the molecules, electrostatic potential maps (EPM) were developed . These a−1 and −4.95 nAT−1, respectively [−1 and verified its aromaticity because of the positive sign. The RCS value obtained with the same level of theory shows the aromatic character of nitrobenzene, which is weaker than for benzene, a fact that is deduced from its more positive paratropic ring currents. Therefore, we can compare them and predict a greater aromatic character of 2AD and NBZ with net RCS values close to 12.0 nAT−1 with respect to the TNB, TNT and DN6 compound whose net RCS obtained were close to 9.0 nAT−1. This similar aromaticity of TNB, TNT and DN6 shows that the aromatic ring of these compounds presents similar reactivity. Compound 4AD has the lowest net RCS value (close to 8.0 nAT−1), which makes the ring of this compound more reactive, explaining the catalytic capacity shown by XenB for this compound in relation to 2AD.Using the ring current strength (RCS) and vector diagram visualization , we can ectively . Moreoveectively ,25,26,272 group attached to carbon atom 6 were eliminated. For 4AD, on the other hand, the N and O atoms of the NO2 group attached to carbon 4 were removed, and an NH2 group was added at that position. This methodology maintains the distances between the cofactor and substrate functional groups at a similar magnitude to that observed for the enzyme complex with TNT.The crystal structure of the P. putida XenB enzyme in complex with TNT and the cofactor FMN was obtained from the Protein Data Bank (PDB) with PDB ID: 4AEO at a resolution of 1.8 Å. The XenB/TNT enzyme model was generated without considering water molecules. For the models of XenB in complex with the other compounds, the Avogadro program was used to modify the coordinates by removing or adding functional groups . For exa2) for protein and ligand; (2) full structure minimization considering 1500 steps of steepest minimization and 500 steps of conjugate gradient minimization; (3) the minimized systems were progressively heated to 300 K with harmonic constraints of 10 kcal/(mol∙Å2) on the carbon skeleton and ligand for 0.5 ns; (4) the system was then equilibrated for 0.5 ns maintaining the constraints and then for 5 ns without constraints at 300 K in a canonical ensemble (NVT); and (5) for production, molecular dynamics simulations were run for 200 ns with a 2 fs time step, unconstrained, in an isothermal–isobaric (NPT) ensemble at 300 K. In the MD simulation, temperature was controlled with a Langevin thermostat with a collision frequency of 1 ps−1 (NVT) and pressure with a Berendsen barostat (NPT), with 2 ps of relaxion time [The polypeptide chain of XenB was modeled considering the protonation states of ionizable residues at pH 7.0, determined with the H++ web server . The proion time ,31,32. Iion time . All MD ion time .Statistical methodology groups similar data and separates them from data sets with different properties. To perform the clustering, the density-based spatial method of applications with noise (DBSCAN), which is implemented in the CPPTRAJ tool, was used ,35. This−) and electroaccepting (ω+) powers and net electrophilicity (Δω±), were calculated to evaluate the reactivity of the molecules [Seven nitroaromatic compounds by xenobiotic reductase XenB: 2,4,6 trinitrotolueno (TNT), 2,4, dinitrotolueno (DN6), 2,6 dinitrotolueno (DN4), 4-amino-2,6-dinitrotolueno (4AD), 2-amino-4,6-dinitrotolueno (2AD), 1,3,5-trinitrobenzeno (TNB) and nitrobenzeno (NBZ), were drawn using Discovery Studio 3.1 , and geometry optimizations and vibrational frequency calculations were carried out using the Gaussian16 program , using tolecules ,41.The current densities were computed using the GIMIC program , which epro), calculated as the sum of all atomic contributions. The NCI is based on the electron density (ρ), its derivatives and the reduced density gradients (s). The reduced density gradient is given by:The non-covalent interaction index (NCI) was calculated for each representative conformation (200 ns simulation) obtained from the cluster analysis. Non-covalent interactions such as: hydrogen bonds, steric repulsion and van der Waals interactions, were identified and mapped using the promolecular densities ρ. The surfaces are colored on a blue–green–red scale according to the strength and type of interaction. Blue indicates strong attractive interactions; green indicates weak van der Waals interactions, and red indicates strong unbound superposition. All calculations were performed with NCIPlot software .2 or NH3) at the ortho or para position; however, this binding is not sufficient for the enzyme to process the substrate. As observed in molecular dynamics simulations, the Tyr65 residue may be critical for ligand binding to the XenB substrate binding site for all ligands studied and the Tyr335 residue for DN4 and TNB. Electrostatic potentials show that compounds that bind XenB have a large positively charged region surrounding the aromatic ring and including the N atoms of the substituents. The aromatic nitrocompounds TNT (14.7 ev.) and TNB (15.6 ev.) against which XenB shows the highest relative activity show the highest net electrophilicity. All compounds tested and processed by XenB show similar overall hardness (between 2.2 and 2.8 ev.), which does not correlate with the different activity of XenB by these compounds. The more aromatic compounds, with ring current densities on the order of 12.0 nAT−1, such as 2AD and NBZ, show high stability, which is consistent with the inactivity of XenB by these compounds. From these results, it is possible to explain the activity of XenB by the tested compounds, which could be used to estimate its activity by other nitro compounds in future studies.The binding of nitro compounds to XenB requires deactivating groups (NO


The quality of provided health care may be an important source of variation in rehabilitation outcomes, increasing the interest in associations between quality indicators (QIs) and improved patient outcomes. Therefore, we examined the associations between the quality of rehabilitation processes and subsequent clinical outcomes among patients with rheumatic and musculoskeletal diseases (RMDs).In this multicentre prospective cohort study, adults with RMDs undergoing multidisciplinary rehabilitation at eight participating centres reported the quality of rehabilitation after 2 months and outcomes after 2, 7, and 12 months. We measured perceived quality of rehabilitation by 11 process indicators that cover the domains of initial assessments, patient participation and individual goal-setting, and individual follow-up and coordination across levels of health care. The patients responded “yes” or “no” to each indicator. Scores were calculated as pass rates (PRs) from 0 to 100% (best score). Clinical outcomes were goal attainment , physical function (30 s sit-to-stand test), and health-related quality of life (EuroQoL 5D-5L). Associations between patient-reported quality of care and each outcome measure at 7 months was analysed by linear mixed models.A total of 293 patients were enrolled in this study . Primary diagnoses were inflammatory rheumatic disease (64%), fibromyalgia syndrome (18%), unspecific neck, shoulder, or low back pain (8%), connective tissue disease (6%), and osteoarthritis (4%). The overall median PR for the process indicators was 73% (range 11–100%). The PR was lowest (median 40%) for individual follow-up and coordination across levels of care. The mixed model analyses showed that higher PRs for the process indicators were not associated with improved goal attainment or improved physical function or improved health-related quality of life.The quality of rehabilitation processes was not associated with important clinical outcomes. An implication of this is that measuring only the outcome dimension of quality may result in incomplete evaluation and monitoring of the quality of care, and we suggest using information from both the structure, process, and outcome dimensions to draw inferences about the quality, and plan future quality initiatives in the field of complex rehabilitation.NCT03102814).The study is part of the larger BRIDGE trial (ClinicalTrials.gov  Rheumatic and musculoskeletal diseases (RMDs) are major contributors to the overall need for rehabilitation services worldwide . In the Rehabilitation is frequently described as a patient-centred process, reflecting how patients and health professionals engage with each other and collaborate towards the best possible function for the patients in interaction with their environments , 6. A geProgress towards more optimal delivery of rehabilitation may be aided by quality indicators (QIs), as these measures are designed to compare actual patient care to norms or ideal criteria . SeveralIn the field of RMDs, the relationship between process and outcome is inconsistent –27, and This study was part of a large multi-centre study, the BRIDGE trial, which aimed to improve the quality, continuity, and coordination of rehabilitation for patients with RMDs . In the In the present study, we analysed the patient sample as one cohort regardless of group allocation. This approach was considered to be the most suitable design for our study because it provided a larger variety of responses to the process indicators, as reported by the participants in the BRIDGE trial.Eligible patients were ≥ 18 years old and admitted to 2–4 weeks of multidisciplinary rehabilitation care  due to inflammatory rheumatic diseases, systemic connective tissue diseases, osteoarthritis, fibromyalgia syndrome or chronic widespread pain, osteoporosis, or unspecific neck, shoulder, or low back pain (persistent for > 3 months). Further inclusion criteria were the ability to read and understand questionnaires in Norwegian and access to a smartphone or equivalent device for digital data collection, including a personal electronic credential for secure identification online. Exclusion criteria were fracture(s), cognitive impairment, or severe psychiatric disorders. Health professionals at eight rehabilitation centres in different regions of Norway performed the eligibility screening and inclusion procedures.All included patients received verbal and written information about the study and provided written informed consent. The study was approved by the Norwegian Regional Committee for Medical Research Ethics . Two patient research partners were members of the trial steering committee and involved in all stages of the trial.1) and discharge (T2) from the rehabilitation stay, and at home 2, 7, and 12 months after admission . The patients answered the QI questionnaire only at T3. This time point was chosen to capture the patient perspective of the rehabilitation process in fair proximity to the rehabilitation stay, as well as in proximity to the first month of the follow-up period.Patients were included from August 2017 to August 2018 and followed for 1 year. They used an online solution for self-reported health care assessments at admission , civil status (living with partner [yes/no]), education level (yes ≥ tertiary education), paid employment (yes = part- or full-time), comorbidities , weekly physical training , and smoking .We collected patients’ background characteristics at TSupported by the Norwegian Health Directorate, a QI set for use in rehabilitation for RMDs has been developed by an expert panel comprising clinicians, researchers, and patient research partners . This ex, as experienced by the individual patient.In Table We calculated the results as pass rates (PRs). The PR for a single indicator was “the total number of patients who answered yes for this particular indicator” divided by “the total number of patients who answered yes or no for the same indicator”. In addition, we calculated a summary PR score for each patient as the total of “yes” answers from the patient divided by the eligible QI items for the same patient. The minimum number of eligible items was 8 due to mandatory responses to P01 and P03–09 ; Group B, Patient participation and individual goal-setting through the rehabilitation process (P03-P06); and Group C, Patient participation, individual follow-up, and coordination across levels of health care (P07-P11). For each patient, we calculated a summary PR score for each group of indicators. The PR score for Group A was the total “yes” answers to P01 and P02 divided by the eligible QI items in Group A for that patient. The PR score for Group B was the total “yes” answers to P03 - P06 divided by 4, because eligible QI items in Group B is always 4. Finally, the PR score for Group C was the total “yes” answers to P07 – P11 divided by the eligible QI items in Group C for that patient. In the statistical analyses, we used the term “PR variables” for the summary PR scores and PRs for Group A, B, and C.For statistical analyses, we aimed to record the overall influence of the perceived quality of the rehabilitation process as reflected by the summary PR score. In addition, to distinguish between essential components of the rehabilitation process, we grouped the single indicators into three categories reflecting the main themes of the rehabilitation stay and follow-up-period as presented in Table The primary outcome in the BRIDGE trial was goal attainment after 7 months, as measured by the Patient-Specific Functional Scale (PSFS) , 32. TheThe worst health you can imagine” and 100 indicating “The best health you can imagine” [The secondary outcomes were physical function and HRQoL. We used the 30-s sit-to-stand test (30secSTS) –35 to asimagine” , 36. Allimagine” .We investigated the mean performance score for all reported goals for each patient, termed PSFS. In addition, we examined the first reported goal separately, termed PSFSA1, because the first goal set by the patient is reported to be the most reliable in terms of test-retest stability .1 was the time point for baseline values for the 30secSTS, EQ5D-index, and EQ5D-vas, T2 was the baseline time point for PSFS.Furthermore, based on clinical experience and research , we knewquality of care, which was defined by the Institute of Medicine [rehabilitation is understood as a planned and coordinated process that reaches across levels of care, is tailored to the patient’s experiences and goals, and assists the individuals in their own efforts to achieve their best possible functioning and coping [According to Donabedian’s model for evaluating the quality of care, a good structure should increase the likelihood of a good process, and a good process, in turn, should increase the likelihood of good outcomes . High-quMedicine  as “the d coping , 40.3. We analysed data in STATA IC, version 16, and set the statistical significance level to 0.05.In the statistical analyses, we included all participants from the BRIDGE trial who completed the QIs at T4 minus the score for the same outcome at baseline. For the actual process indicators, there was no former established PR cut-off for high-quality care. Therefore, we used quartiles  for the quality variable when we examined changes in outcomes by summary PR score for the process indicators.We performed descriptive analyses to report demographic data, quantify the quality of the received rehabilitation process, and describe the observed changes for each clinical outcome, calculated as the outcome score at TAs a preparatory analysis, we performed two regressions treating the summary PR for the quality variable as the response variable. In the first analysis, we regressed R on study centre alone. In the second regression, PR was regressed on the baseline predictors , in addition to study centre.4 was treated as the response, and the fixed effects were its baseline value, the PR variable, and a variable capturing elapsed time since study start. To account for centre level clustering, we included centre as a random effect in the basic model. In the fully adjusted model, we included a wider range of baseline predictors: age, sex, BMI, weekly training, comorbidity, paid employment, education level, civil status, and smoking. In a separate analysis, the primary independent variable was replaced by the three summary PR values for the single indicators grouped into categories (Group A-C). We used the same basic and fully adjusted models as described above.We used a linear mixed model approach to assess the association between the process dimension of the quality of rehabilitation (the PR variable) and the study outcomes . First, our primary independent variable was the summary PR for the process variables. For each outcome, its value at Tnull model), one with the summary PR , and one with PRs for Groups A to C . Subsequently, the association between the quality PR and the outcome was assessed by the likelihood ratio test, comparing each of the latter two models to the first. In other words, we used the likelihood ratio test to examine whether the alternative model  provided significant improvement (better fit) over the null model.For each outcome, three models were fit: one without PR variable(s)  for PSFS, and not. MCID for PSFS is 2 or more points  and ther3 and were included in the current analysis. The participants were mostly female (76%), with a mean age of 52 (±12.3) years. They were referred to multidisciplinary rehabilitation most frequently due to inflammatory rheumatic disease (64%) or fibromyalgia syndrome (18%). Fifty percent of the study cohort had other chronic diseases (≥ 1 comorbidity) in addition to their primary diagnosis. Median disease duration for the primary diagnosis was 17 years. All of the baseline characteristics are presented in Table A total of 293 (78%) of the 374 BRIDGE trial participants completed the QI questionnaire at TThe response rate for the patient-reported QI questionnaire was 100% (no missing items). The median summary PR score of the 11 process indicators was 73% (range 11–100%). For single indicators, the PRs ranged from 9 to 99%. We found the highest PR score for the indicator reflecting patient participation in tailored goal-setting (indicator P04), whereas the lowest PR scores were found for indicators regarding attendance in rehabilitation meetings for family or next of kin , or important others/external professionals  are presented in Table 4  and case-mix (adjusted R-squared for the baseline predictors were 0.10).4 and baseline did not differ much when we examined these changes visually as PR scores for each quartile. Thus, from these initial descriptive analyses, we assumed that patients who reported higher fulfilment of the specified processes of rehabilitation (higher PRs) had only slightly or no better outcomes than patients who received less of the content addressed by the QIs  may have challenged the recommended proximity of the outcomes to the received process of care [4 with data collected at T3 (2 months after admission). However, we did not find any associations between the process PRs and any of the outcome variables.During the observation period of this study, the support from health professionals was intended to decrease as the degree of patients’ self-management increased. The choice of T of care . EspeciaIn this study, we did not find any associations between the quality of provided rehabilitation processes and subsequent clinical outcomes of multidisciplinary rehabilitation for adults with RMDs. The PR values for the process indicators were not associated with improvements in either patient-specific goal attainment, physical function, or HRQoL measured 7 months after the initiated rehabilitation process in the BRIDGE trial.Regarding PRs, we found lower values for QIs within the domain of follow-up and coordination compared to PR values for indicators regarding initial assessments and tailored goal-setting. These findings may indicate a suboptimal transition between the rehabilitation process introduced in secondary care and the expected continuation in a community-based setting. As highlighted by others , improve4 compared to patients who reported a lesser quality process. One reason may be found in the relationship between patients’ outcome expectations and their agreements with clinicians regarding appropriate goal-setting. As the mean RMD duration in our sample was more than 15 years, some participants may have been striving towards maintenance of function as opposed to expectations of clinical improvement. Therefore, future quality initiatives and research should address both maintenance and improvement as desired outcomes [In contrast to what we hypothesized, patients who reported receiving higher quality rehabilitation did not report better outcomes at Toutcomes . In addioutcomes , 47. Sucoutcomes , 49. Clioutcomes . In our outcomes , 48, 49.outcomes .Taken together, weak associations between the process quality and outcomes do not necessarily devalue the importance of a high-quality rehabilitation process. Methodological challenges in identifying associations between the process and patient outcomes have been reported by others , 47–50. self-efficacy, readiness for change, and health literacy, could have been added in the analysis to better address potential self-management-related factors influencing the outcome [3), patients may not accurately remember the process they underwent before discharge. Nevertheless, at this first time point at home, their memory was probably helped by re-scoring the clinical outcomes, by a mandatory follow-up phone call from the rehabilitation centre between discharge and T3, and, optimally, the beginning of the extended care from the community. Lastly, limitations due to the yes/no format of the QI questionnaire yield information restricted to confirmed/unconfirmed for the content addressed by each indicator. Consequently, we did not know patients’ opinions on whether the goals were appropriately ambitious, whether plans for self-management and follow-up were feasible in their context at home, or whether potential barriers were identified and planned for. In future research, we will address these questions.The strengths of this study include a large study cohort, a statistical methodology accounting for the hierarchical data structure, and a > 76% response rate 7 months after baseline. Furthermore, we evaluated QIs and outcomes from the same perspective (the patient perspective). This study also has some limitations. First, a small difference in quality when looking at PRs for Group A and Group B may reduce the potential to explain variations in outcome(s) at 7 months. Second, confounders, such as  outcome . Third,  outcome . There mThis study is a first step to exploring associations between rehabilitation processes and the subsequent clinical outcomes using the process indicators from a QI set for rehabilitation in patients with RMDs. Our results support the need to promote the process indicators as a useful tool to be aware of, recognize, and deliver all aspects of best rehabilitation practice. Using the process indicators, we revealed that the quality of rehabilitation is still suboptimal regarding coordination between care levels and sufficient support for the patients’ self-management strategies after discharge. However, in rehabilitation, it can be difficult to relate the outcomes directly to the quality of the delivered rehabilitation process due to the additional influences of environmental factors and non-medical events arising along the highly personalized rehabilitation process. However, we consider information about clinical outcomes to be valuable and meaningful in evaluating and monitoring rehabilitation quality, but preferably in combination with information about the process dimension of quality.For clinicians, improving the quality of rehabilitation processes may be difficult if the present structural conditions are disadvantageous. Therefore, quality initiatives from policymakers and leaders need to address structural factors aimed at increasing the likelihood of good processes, such as sufficient competence and resources in all care levels, written procedures, and establishment of good structures for mutual information flow and efficient referral routines. This broader perspective, including all dimensions of quality, applies well in complex rehabilitation, in which the health system, providers, and patients are mutually accountable for the clinical outcomes.Results from this work may inform decisions on expected standards of rehabilitation services, such as stakeholders’ efforts to identify and reduce unwarranted variance in quality. Moreover, providers’ and receivers’ input on how quality initiatives apply and work in different contexts, will be essential in future work for further developing of national plans and indicators for quality improvement in rehabilitation.To the best of our knowledge, this study is the first examination of associations between the quality of rehabilitation processes and clinical outcomes based on the process indicators from a QI set for use in rehabilitation for adults with RMDs. We conclude that the quality of rehabilitation processes is not associated with subsequent clinical outcomes. An implication of this is that measuring only one dimension of quality may result in incomplete evaluation and monitoring of the quality of care, and we suggest using information from both the structure, process, and outcome dimensions to draw inferences about the quality and plan future quality initiatives in the field of complex rehabilitation.