Utvikling, optimering og felttesting av en filtreringsmetode for påvisning av salmonid alphavirus i sjøvann

The commercial aquaculture is continuously growing with the demand for fish and seafood products. In Norway, Atlantic salmon (Salmo salar L.) constitute 94 % of the total aquaculture production, making it the most important species in Norwegian aquaculture. With the high demand of fish and fish products globally, the farming of salmonids continues to occur intensively. This result in fish being under constant threat of being infected by pathogens and suffer from different diseases. Pancreas disease (PD), caused by salmonid alphavirus (SAV), is a systemic disease and is considered among the most serious virus diseases in sea farmed salmonids with a negative impact on fish welfare. It is also one of the most economically important fish diseases in aquaculture in Europe. This disease was first reported in 1976 in Scotland and in the 1980s in Norway, where it became a notifiable disease (list 3) in 2007. In 2017, the national surveillance program for PD was intensified in Norway, introducing a PD zone for SAV2 and SAV3 (i.e. Western- and Mid-Norway), and two surveillance zones north and south/south-east of the PD zone. This was done as a way to reduce the consequences of the disease, as well as to prevent further spread of SAV within the defined zones. This surveillance program relies on a time-consuming and resourcedemanding approach, involving monthly sampling of at least 20 fish from all SAV-negative marine operative farm sites with salmonid fish, and analyzing heart tissue from each fish by RT-qPCR analysis. For years, significant progress has been made in developing filtration methods for concentrating low amounts of pathogens in water, and surveillance programs for different types of harmful pathogens have already been established based on a filtration technique. Hence, the focus of this study was to develop and optimize a filtration method for the detection of SAV in seawater, making the selective and invasive traditional testing of fish redundant. This study was divided into three steps; initially tested in vitro, followed by evaluation in a challenge model, and assessed under natural field conditions. The in vitro study was performed in order to test five combinations of two different electrocharged filters and four different buffer solutions for concentration and detection of SAV3 in seawater by spiking SAV3 into 1 L of artificial and natural seawater. The SAV3 was quantified by using RT-qPCR and RT-ddPCR in order to compare the SAV3 concentrations measured. In this study, the highest SAV3 recovery and efficiency was made when combining electronegative filter with lysis buffer, by RT-ddPCR and RT-qPCR analysis, with the former performing significantly better at higher dilutions. Following the in vitro study, a SAV3-cohabitant challenge trial using post-smolt Atlantic salmon, was carried out in order to evaluate the five concentration methods further. In this study, an electronegative filter combined with lysis buffer was the most suitable method for recovering SAV3 from seawater by RT-qPCR analysis. In addition, a positive correlation was found between SAV3 detections in cohabitant fish tissue and in water when using this concentration method. Further optimization and field testing of the filtration method for detection of SAV in seawater was with electronegative filter and elution with lysis buffer before sample analysis by RT-qPCR. Under the field conditions, early SAV-detections was made in seawater collected from inside net-pens compared to the monthly screening of fish. Higher SAV-recovery and early SAV detection were made in seawater compared to fish screening. This new method could be a more straightforward, cost-efficient, time-saving, resource-saving, and not the least animal welfare-friendly approach for virus surveillance, with a potential for earlier implementation of disease control measures that may be applied to detect other fish pathogenic viruses than SAV. Moreover, it could also allow assessment of viral transmission and disease dynamics in fish farms. Let’s dive in!Den kommersielle akvakulturen vokser kontinuerlig med etterspørselen etter fisk og sjømatprodukter. I Norge utgjør 94% av det totale havbruket av atlantisk laks (Salmo salar L.), noe som gjør den til den viktigste arten i norsk havbruk. Med den høye etterspørselen etter fisk og fiskeprodukter globalt sett, fortsetter lakseoppdretten på en intensiv måte. Dette fører til at fisk konstant trues av å bli smittet og lide av forskjellige sykdommer. Pankreas sykdom (PD), som er forårsaket av salmonid alphavirus (SAV), er en systemisk sykdom som regnes som en av de mest alvorlige virussykdommene som også påvirker fiskevelferden negativt hos oppdrettslaksen. Det er også en av de mest økonomiskt viktige fiskesykdommene innen havbruk i Europa. Denne sykdommen ble først rapportert i 1976 i Skottland og på 1980-tallet i Norge, hvor den ble en meldepliktig sykdom (liste 3) i 2007. I 2017 ble det nasjonale overvåkingsprogrammet for PD intensivert i Norge, med innføring av en PD-sone for SAV2 og SAV3, og to overvåkingssoner nord og sør/sør-øst om PD-sonen, for å redusere konsekvensene av sykdommen innenfor de definerte sonene, samt for å forhindre videre spredning av SAV. Dette overvåkingsprogrammet er avhengig av en tidkrevende og ressurskrevende tilnærming, som involverer månedlig prøvetaking av minst 20 fisk fra alle SAV-negative operative sjøvannsanlegg for salmonide fisker, og analyse av hjertevev fra hver av disse fisker ved RT-qPCR analyse. Gjennom årene har det blitt gjort noen betydelige fremskritt i utviklingen av filtreringsmetoder for å konsentrere lave mengder patogener i vann, og overvåkingsprogrammer for forskjellige skadelige patogener er allerede etablert basert på denne teknikken. Dermed var fokuset for denne studien å utvikle og optimalisere en filtreringsmetode for påvisning av SAV i sjøvann, noe som gjør selektiv og invasiv tradisjonell testing av fisk overflødig. Denne studien ble delt inn i tre trinn; opprinnelig testet in vitro, etterfulgt av evaluering i en smitteforsøksmodell, og til sist vurdert under feltforhold. In vitro studien ble utført for å teste fem kombinasjoner av to forskjellige elektroladet filtre, og fire forskjellige bufferløsninger for konsentrasjon og påvisning av SAV3 i sjøvann, ved å tilføye SAV3 i 1 liter kunstig og naturlig sjøvann. SAV3 ble kvantifisert ved bruk av RT-qPCR og RT-ddPCR for å sammenligne de målte konsentrasjonene. I denne studien ble de høyeste konsentrasjonene og effektiviteten av SAV3 funne når man kombinerte elektronegativt filter med lyseringsbuffer, ved RT-ddPCR og RT-qPCR-analyse, hvor den førstnevnte presterte betydelig bedre ved høyere fortynninger. Etter in vitro studien ble det utført en SAV3-kohabitant smitteforsøk med post-smolt atlantisk laks for å evaluere de fem konsentrasjonsmetodene. Også i denne studien var elektronegativt filter kombinert med lyseringsbuffer den mest egnede metoden for å påvise SAV3 fra sjøvann ved bruk av RTqPCR. I tillegg ble det funnet en positiv korrelasjon mellom SAV3-påvisninger i kohabitant fiskevev og i vann, ved bruk av denne konsentrasjonsmetoden. Ytterligere optimalisering og feltprøving av filtreringsmetoden for påvisning av SAV i sjøvann var med elektronegativt filter og eluering med lyseringsbuffer, før prøveanalyse ved RT-qPCR. Under feltforhold, ble tidlig SAV-påvisning gjort i sjøvann oppsamlet fra innsiden av merdkanten sammenlignet med månedlig screening av fisk. Det ble også avdekket høyere SAV-konsentrasjoner og effektivitet, og tidligere SAV-påvisning i vann sammenlignet med screeninganalyse av fiskevev. Denne nye metoden kan være en mer rett frem, kostnadseffektiv, tidsbesparende, ressursbesparende og ikke minst dyrevelferdsvennlig tilnærming for virusovervåking, med potensial for tidligere implementering av sykdomskontrolltiltak og med mulighet for å oppdage andre fiskepatogene virus enn SAV. Videre kan det også tillate vurdering av virusoverføring og sykdomsdynamikk i oppdrettsanlegg. La oss hoppe i det

Development, optimization and field testing of a filtration method for detection of salmonid alphavirus in seawater

The commercial aquaculture is continuously growing with the demand for fish and seafood products. In Norway, Atlantic salmon (Salmo salar L.) constitute 94 % of the total aquaculture production, making it the most important species in Norwegian aquaculture. With the high demand of fish and fish products globally, the farming of salmonids continues to occur intensively. This result in fish being under constant threat of being infected by pathogens and suffer from different diseases. Pancreas disease (PD), caused by salmonid alphavirus (SAV), is a systemic disease and is considered among the most serious virus diseases in sea farmed salmonids with a negative impact on fish welfare. It is also one of the most economically important fish diseases in aquaculture in Europe. This disease was first reported in 1976 in Scotland and in the 1980s in Norway, where it became a notifiable disease (list 3) in 2007. In 2017, the national surveillance program for PD was intensified in Norway, introducing a PD zone for SAV2 and SAV3 (i.e. Western- and Mid-Norway), and two surveillance zones north and south/south-east of the PD zone. This was done as a way to reduce the consequences of the disease, as well as to prevent further spread of SAV within the defined zones. This surveillance program relies on a time-consuming and resourcedemanding approach, involving monthly sampling of at least 20 fish from all SAV-negative marine operative farm sites with salmonid fish, and analyzing heart tissue from each fish by RT-qPCR analysis. For years, significant progress has been made in developing filtration methods for concentrating low amounts of pathogens in water, and surveillance programs for different types of harmful pathogens have already been established based on a filtration technique. Hence, the focus of this study was to develop and optimize a filtration method for the detection of SAV in seawater, making the selective and invasive traditional testing of fish redundant. This study was divided into three steps; initially tested in vitro, followed by evaluation in a challenge model, and assessed under natural field conditions. The in vitro study was performed in order to test five combinations of two different electrocharged filters and four different buffer solutions for concentration and detection of SAV3 in seawater by spiking SAV3 into 1 L of artificial and natural seawater. The SAV3 was quantified by using RT-qPCR and RT-ddPCR in order to compare the SAV3 concentrations measured. In this study, the highest SAV3 recovery and efficiency was made when combining electronegative filter with lysis buffer, by RT-ddPCR and RT-qPCR analysis, with the former performing significantly better at higher dilutions. Following the in vitro study, a SAV3-cohabitant challenge trial using post-smolt Atlantic salmon, was carried out in order to evaluate the five concentration methods further. In this study, an electronegative filter combined with lysis buffer was the most suitable method for recovering SAV3 from seawater by RT-qPCR analysis. In addition, a positive correlation was found between SAV3 detections in cohabitant fish tissue and in water when using this concentration method. Further optimization and field testing of the filtration method for detection of SAV in seawater was with electronegative filter and elution with lysis buffer before sample analysis by RT-qPCR. Under the field conditions, early SAV-detections was made in seawater collected from inside net-pens compared to the monthly screening of fish. Higher SAV-recovery and early SAV detection were made in seawater compared to fish screening. This new method could be a more straightforward, cost-efficient, time-saving, resource-saving, and not the least animal welfare-friendly approach for virus surveillance, with a potential for earlier implementation of disease control measures that may be applied to detect other fish pathogenic viruses than SAV. Moreover, it could also allow assessment of viral transmission and disease dynamics in fish farms. Let’s dive in

Development and evaluation of a method for concentration and detection of salmonid alphavirus from seawater

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Sparsentan in patients with IgA nephropathy: a prespecified interim analysis from a randomised, double-blind, active-controlled clinical trial

Background: Sparsentan is a novel, non-immunosuppressive, single-molecule, dual endothelin and angiotensin receptor antagonist being examined in an ongoing phase 3 trial in adults with IgA nephropathy. We report the prespecified interim analysis of the primary proteinuria efficacy endpoint, and safety. Methods: PROTECT is an international, randomised, double-blind, active-controlled study, being conducted in 134 clinical practice sites in 18 countries. The study examines sparsentan versus irbesartan in adults (aged ≥18 years) with biopsy-proven IgA nephropathy and proteinuria of 1·0 g/day or higher despite maximised renin-angiotensin system inhibitor treatment for at least 12 weeks. Participants were randomly assigned in a 1:1 ratio to receive sparsentan 400 mg once daily or irbesartan 300 mg once daily, stratified by estimated glomerular filtration rate at screening (30 to 1·75 g/day). The primary efficacy endpoint was change from baseline to week 36 in urine protein-creatinine ratio based on a 24-h urine sample, assessed using mixed model repeated measures. Treatment-emergent adverse events (TEAEs) were safety endpoints. All endpoints were examined in all participants who received at least one dose of randomised treatment. The study is ongoing and is registered with ClinicalTrials.gov, NCT03762850. Findings: Between Dec 20, 2018, and May 26, 2021, 404 participants were randomly assigned to sparsentan (n=202) or irbesartan (n=202) and received treatment. At week 36, the geometric least squares mean percent change from baseline in urine protein-creatinine ratio was statistically significantly greater in the sparsentan group (-49·8%) than the irbesartan group (-15·1%), resulting in a between-group relative reduction of 41% (least squares mean ratio=0·59; 95% CI 0·51-0·69; p<0·0001). TEAEs with sparsentan were similar to irbesartan. There were no cases of severe oedema, heart failure, hepatotoxicity, or oedema-related discontinuations. Bodyweight changes from baseline were not different between the sparsentan and irbesartan groups. Interpretation: Once-daily treatment with sparsentan produced meaningful reduction in proteinuria compared with irbesartan in adults with IgA nephropathy. Safety of sparsentan was similar to irbesartan. Future analyses after completion of the 2-year double-blind period will show whether these beneficial effects translate into a long-term nephroprotective potential of sparsentan. Funding: Travere Therapeutics

Efficacy and safety of sparsentan versus irbesartan in patients with IgA nephropathy (PROTECT): 2-year results from a randomised, active-controlled, phase 3 trial

BackgroundSparsentan, a novel, non-immunosuppressive, single-molecule, dual endothelin angiotensin receptor antagonist, significantly reduced proteinuria versus irbesartan, an angiotensin II receptor blocker, at 36 weeks (primary endpoint) in patients with immunoglobulin A nephropathy in the phase 3 PROTECT trial's previously reported interim analysis. Here, we report kidney function and outcomes over 110 weeks from the double-blind final analysis.MethodsPROTECT, a double-blind, randomised, active-controlled, phase 3 study, was done across 134 clinical practice sites in 18 countries throughout the Americas, Asia, and Europe. Patients aged 18 years or older with biopsy-proven primary IgA nephropathy and proteinuria of at least 1·0 g per day despite maximised renin–angiotensin system inhibition for at least 12 weeks were randomly assigned (1:1) to receive sparsentan (target dose 400 mg oral sparsentan once daily) or irbesartan (target dose 300 mg oral irbesartan once daily) based on a permuted-block randomisation method. The primary endpoint was proteinuria change between treatment groups at 36 weeks. Secondary endpoints included rate of change (slope) of the estimated glomerular filtration rate (eGFR), changes in proteinuria, a composite of kidney failure (confirmed 40% eGFR reduction, end-stage kidney disease, or all-cause mortality), and safety and tolerability up to 110 weeks from randomisation. Secondary efficacy outcomes were assessed in the full analysis set and safety was assessed in the safety set, both of which were defined as all patients who were randomly assigned and received at least one dose of randomly assigned study drug. This trial is registered with ClinicalTrials.gov, NCT03762850.FindingsBetween Dec 20, 2018, and May 26, 2021, 203 patients were randomly assigned to the sparsentan group and 203 to the irbesartan group. One patient from each group did not receive the study drug and was excluded from the efficacy and safety analyses (282 [70%] of 404 included patients were male and 272 [67%] were White) . Patients in the sparsentan group had a slower rate of eGFR decline than those in the irbesartan group. eGFR chronic 2-year slope (weeks 6–110) was −2·7 mL/min per 1·73 m2 per year versus −3·8 mL/min per 1·73 m2 per year (difference 1·1 mL/min per 1·73 m2 per year, 95% CI 0·1 to 2·1; p=0·037); total 2-year slope (day 1–week 110) was −2·9 mL/min per 1·73 m2 per year versus −3·9 mL/min per 1·73 m2 per year (difference 1·0 mL/min per 1·73 m2 per year, 95% CI −0·03 to 1·94; p=0·058). The significant reduction in proteinuria at 36 weeks with sparsentan was maintained throughout the study period; at 110 weeks, proteinuria, as determined by the change from baseline in urine protein-to-creatinine ratio, was 40% lower in the sparsentan group than in the irbesartan group (−42·8%, 95% CI −49·8 to −35·0, with sparsentan versus −4·4%, −15·8 to 8·7, with irbesartan; geometric least-squares mean ratio 0·60, 95% CI 0·50 to 0·72). The composite kidney failure endpoint was reached by 18 (9%) of 202 patients in the sparsentan group versus 26 (13%) of 202 patients in the irbesartan group (relative risk 0·7, 95% CI 0·4 to 1·2). Treatment-emergent adverse events were well balanced between sparsentan and irbesartan, with no new safety signals.InterpretationOver 110 weeks, treatment with sparsentan versus maximally titrated irbesartan in patients with IgA nephropathy resulted in significant reductions in proteinuria and preservation of kidney function

Efficacy and safety of sparsentan versus irbesartan in patients with IgA nephropathy (PROTECT): 2-year results from a randomised, active-controlled, phase 3 trial

Background Sparsentan, a novel, non-immunosuppressive, single-molecule, dual endothelin angiotensin receptor antagonist, significantly reduced proteinuria versus irbesartan, an angiotensin II receptor blocker, at 36 weeks (primary endpoint) in patients with immunoglobulin A nephropathy in the phase 3 PROTECT trial's previously reported interim analysis. Here, we report kidney function and outcomes over 110 weeks from the double-blind final analysis. Methods PROTECT, a double-blind, randomised, active-controlled, phase 3 study, was done across 134 clinical practice sites in 18 countries throughout the Americas, Asia, and Europe. Patients aged 18 years or older with biopsy-proven primary IgA nephropathy and proteinuria of at least 1·0 g per day despite maximised renin–angiotensin system inhibition for at least 12 weeks were randomly assigned (1:1) to receive sparsentan (target dose 400 mg oral sparsentan once daily) or irbesartan (target dose 300 mg oral irbesartan once daily) based on a permuted-block randomisation method. The primary endpoint was proteinuria change between treatment groups at 36 weeks. Secondary endpoints included rate of change (slope) of the estimated glomerular filtration rate (eGFR), changes in proteinuria, a composite of kidney failure (confirmed 40% eGFR reduction, end-stage kidney disease, or all-cause mortality), and safety and tolerability up to 110 weeks from randomisation. Secondary efficacy outcomes were assessed in the full analysis set and safety was assessed in the safety set, both of which were defined as all patients who were randomly assigned and received at least one dose of randomly assigned study drug. This trial is registered with ClinicalTrials.gov, NCT03762850. Findings Between Dec 20, 2018, and May 26, 2021, 203 patients were randomly assigned to the sparsentan group and 203 to the irbesartan group. One patient from each group did not receive the study drug and was excluded from the efficacy and safety analyses (282 [70%] of 404 included patients were male and 272 [67%] were White) . Patients in the sparsentan group had a slower rate of eGFR decline than those in the irbesartan group. eGFR chronic 2-year slope (weeks 6–110) was −2·7 mL/min per 1·73 m2 per year versus −3·8 mL/min per 1·73 m2 per year (difference 1·1 mL/min per 1·73 m2 per year, 95% CI 0·1 to 2·1; p=0·037); total 2-year slope (day 1–week 110) was −2·9 mL/min per 1·73 m2 per year versus −3·9 mL/min per 1·73 m2 per year (difference 1·0 mL/min per 1·73 m2 per year, 95% CI −0·03 to 1·94; p=0·058). The significant reduction in proteinuria at 36 weeks with sparsentan was maintained throughout the study period; at 110 weeks, proteinuria, as determined by the change from baseline in urine protein-to-creatinine ratio, was 40% lower in the sparsentan group than in the irbesartan group (−42·8%, 95% CI −49·8 to −35·0, with sparsentan versus −4·4%, −15·8 to 8·7, with irbesartan; geometric least-squares mean ratio 0·60, 95% CI 0·50 to 0·72). The composite kidney failure endpoint was reached by 18 (9%) of 202 patients in the sparsentan group versus 26 (13%) of 202 patients in the irbesartan group (relative risk 0·7, 95% CI 0·4 to 1·2). Treatment-emergent adverse events were well balanced between sparsentan and irbesartan, with no new safety signals. Interpretation Over 110 weeks, treatment with sparsentan versus maximally titrated irbesartan in patients with IgA nephropathy resulted in significant reductions in proteinuria and preservation of kidney function.</p