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The effect of regularly dosed paracetamol versus no paracetamol on renal function in Plasmodium knowlesi… Cooper, Daniel J; Plewes, Katherine; Grigg, Matthew J; Rajahram, Giri S; Piera, Kim A; William, Timothy; Chatfield, Mark D; Yeo, Tsin W; Dondorp, Arjen M; Anstey, Nicholas M; Barber, Bridget E Apr 24, 2018

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STUDY PROTOCOL Open AccessThe effect of regularly dosed paracetamolversus no paracetamol on renal function inPlasmodium knowlesi malaria (PACKNOW):study protocol for a randomised controlledtrialDaniel J. Cooper1,2*, Katherine Plewes3,4,5, Matthew J. Grigg1,2, Giri S. Rajahram2,6,7, Kim A. Piera1, Timothy William2,8,Mark D. Chatfield1,9, Tsin Wen Yeo1,2,10, Arjen M. Dondorp3,4, Nicholas M. Anstey1,2†and Bridget E. Barber1,2,9†AbstractBackground: Plasmodium knowlesi is the most common cause of human malaria in Malaysia. Acute kidney injury(AKI) is a frequent complication. AKI of any cause can have long-term consequences, including increased risk ofchronic kidney disease, adverse cardiovascular events and increased mortality. Additional management strategiesare therefore needed to reduce the frequency and severity of AKI in malaria. In falciparum malaria, cell-freehaemoglobin (CFHb)-mediated oxidative damage contributes to AKI. The inexpensive and widely available drugparacetamol inhibits CFHb-induced lipid peroxidation via reduction of ferryl haem to the less toxic Fe3+ state, andhas been shown to reduce oxidative damage and improve renal function in patients with sepsis complicated byhaemolysis as well as in falciparum malaria. This study aims to assess the ability of regularly dosed paracetamol toreduce the incidence and severity of AKI in knowlesi malaria by attenuating haemolysis-induced oxidative damage.Methods: PACKNOW is a two-arm, open-label randomised controlled trial of adjunctive paracetamol versus noparacetamol in patients aged ≥ 5 years with knowlesi malaria, conducted over a 2-year period at four hospital sitesin Sabah, Malaysia. The primary endpoint of change in creatinine from enrolment to 72 h will be evaluated byanalysis of covariance (ANCOVA) using enrolment creatinine as a covariate. Secondary endpoints includelongitudinal changes in markers of oxidative stress (plasma F2-isoprostanes and isofurans) and markers ofendothelial activation/Weibel–Palade body release (angiopoietin-2, von Willebrand Factor, P-selectin,osteoprotegerin) over 72 h, as well as blood and urine biomarkers of AKI. This study will be powered to detect adifference between the two treatment arms in a clinically relevant population including adults and children withknowlesi malaria of any severity.(Continued on next page)* Correspondence: dancooper@doctors.net.uk†Equal contributors1Global and Tropical Health Division, Menzies School of Health Research andCharles Darwin University, Darwin, NT, Australia2Infectious Diseases Society Sabah-Menzies School of Health ResearchClinical Research Unit, Kota Kinabalu, Sabah, MalaysiaFull list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Cooper et al. Trials  (2018) 19:250 https://doi.org/10.1186/s13063-018-2600-0(Continued from previous page)Discussion: Paracetamol is widely available and has an excellent safety profile; if a renoprotective effect isdemonstrated, this trial will support the administration of regularly dosed paracetamol to all patients with knowlesimalaria. The secondary outcomes in this study will provide further insights into the pathophysiology of haemolysis-induced oxidative damage and acute kidney injury in knowlesi malaria and other haemolytic diseases.Trial registration: Clinicaltrials.gov, NCT03056391. Registered on 12 October 2016.Keywords: Malaria, Plasmodium knowlesi, Acute kidney injury, ParacetamolBackgroundThe simian parasite Plasmodium knowlesi, first reportedas a major cause of human malaria in Malaysia in 2004[1], is now known to cause human infections throughoutSoutheast Asia [2]. In Malaysia, P. knowlesi is the mostcommon cause of human malaria, and incidence is in-creasing [3–5]. In Sabah, 1325 cases of knowlesi malariawere notified in 2014, representing a 33% increase on2013 notifications [6]. Recent studies using more specificmolecular diagnostic methods indicate that P. knowlesiis also the most common cause of human malaria inareas of western Indonesia [7, 8].As with falciparum malaria, acute kidney injury (AKI) isa common complication of knowlesi malaria. Severe AKI(serum creatinine > 265 μmol/L) occurred in 24% ofpatients in a prospective tertiary-hospital study of severeP. knowlesi malaria [9] and in 54% of fatal knowlesimalaria cases in Sabah during 2010–2014 [6, 10]. Even inpatients with non-severe knowlesi malaria, mild-moderateAKI is common. In a recent district hospital-based studyinvolving 481 patients with knowlesi malaria of anyseverity, AKI by Kidney Disease: Improving GlobalOutcomes (KDIGO) criteria [11] occurred in 26% ofchildren ≤ 12 years and in 19% of adults [12]. Import-antly, AKI, even if not severe, can have significantlong-term consequences. Regardless of cause, AKI isassociated with increased risk of chronic kidney disease(CKD) [13], progression of existing CKD [14], long-termrisk of end-stage renal disease [15], major adverse cardio-vascular events [16] and mortality [13, 17]. In addition,AKI is associated with an increased duration of hospi-talisation and need for specialised long-term care, thuscontributing to substantial health economic costs [18].Interventions aimed at reducing the incidence and se-verity of AKI are therefore needed, including appropri-ate adjunctive treatments targeting the pathogenicmechanisms underlying AKI.Mechanisms of renal disease in knowlesi malaria arepoorly defined. In falciparum malaria, AKI has beenattributed in part to haemolysis-mediated oxidativedamage [19]. Haemolysis, and subsequent release ofcell-free haemoglobin (CFHb), leads to oxidative stressand lipid peroxidation in the renal tubules via oxida-tion of ferric (Fe3+) to ferryl (Fe4+) haemoglobin [20].Free-radical-induced lipid peroxidation generates F2-isoprostanes (F2-IsoPs) and isofurans (IsoFs), which appearin free form in plasma and subsequently, after glomerularfiltration, in urine. F2-IsoPs and IsoFs are considered ro-bust measures of oxidative stress, and are associated withAKI in haemolytic conditions such as rhabdomyolysis, sep-sis and post-cardiopulmonary bypass [20–24]. Similarly, ina prospective observational study of Bangladeshi adultswith severe falciparum malaria, CFHb and F2-IsoPs/IsoFswere associated with AKI and disease severity, suggestingthat haemolysis-induced oxidative damage contributes tothe pathogenesis of malaria-associated AKI [19].Paracetamol has been shown to inhibit CFHb-inducedlipid peroxidation via reduction of ferryl haem to the lesstoxic Fe3+ state and quenching of globin radicals [20, 25,26]. In a proof-of-concept study in rats, paracetamol sig-nificantly reduced rhabdomyolysis-induced kidney injuryby inhibiting haemoprotein-catalysed lipid peroxidation[20]. In a retrospective study of patients with sepsis andraised CFHb, receiving paracetamol was associated withreduced lipid peroxidation and reduced risk of death [27].In a randomised, placebo-controlled trial, paracetamol wasassociated with reduced plasma F2-IsoPs and improvedrenal function in adults with sepsis and detectable CFHb[28]. Paracetamol was also associated with decreased lipidperoxidation (plasma IsoFs) in adults [21] and children[23] undergoing cardiopulmonary bypass. A recent rando-mised controlled trial in Bangladeshi adults with severeand moderately severe falciparum malaria showed thatparacetamol improved kidney function and reduced oddsof developing AKI, particularly in those with high CFHb atenrolment [29].Paracetamol may also have a potential role in reducingendothelial activation and microvascular dysfunction inmalaria. In previous in vitro studies, CFHb has beenshown to stimulate degranulation of endothelial cellWeibel–Palade bodies (WPBs), leading to release ofmajor WPB constituents von-Willebrand factor (vWF)and P-selectin [30]. In addition, WPBs also store andrelease angiopoietin-2 (Ang-2), an autocrine mediator ofendothelial activation that is associated with mortality andendothelial dysfunction in severe falciparum malaria [31],and with AKI in knowlesi malaria [32]. In both species,CFHb is independently associated with Ang-2 [32–34],Cooper et al. Trials  (2018) 19:250 Page 2 of 11suggesting that CFHb-induced degranulation of WPBsmay also lead to release of Ang-2. The haem-mediated re-lease of WPB constituents has been shown to be mediatedthrough TLR4 signalling, and is dependent on NADPHoxidase (NOX) [30]. Paracetamol has been shown toreduce NADPH isoforms NOX2 and NOX4, the majorNOX isoforms expressed in the kidney and linked to thepathogenesis of AKI [35]. Thus, prevention of CFHb-induced WPB release may represent an additional reno-protective mechanism of paracetamol.RationaleIn adults with severe knowlesi malaria, CFHb is higherthan in severe falciparum malaria, and AKI at least ascommon [32]. As paracetamol has been shown to reducehaemolysis-induced lipid peroxidation, we hypothesisethat paracetamol may play a similar role in patients withknowlesi malaria [19, 27, 33, 36]. Paracetamol is inex-pensive, safe and widely available. If a renoprotectiverole is demonstrated, this would provide evidence forthe administration of regular paracetamol for all patientswith knowlesi malaria.MethodsWe aim to test whether regularly dosed paracetamolcompared with no paracetamol will reduce kidneydysfunction in patients with P. knowlesi malaria.Trial design and study sitesThis is a two-arm, open-label, randomised controlledtrial conducted at four centres in Sabah, Malaysia,namely Queen Elizabeth Hospital (Kota Kinabalu) andKeningau, Ranau, and Kota Marudu District Hospitals.Queen Elizabeth Hospital and Keningau both have well-equipped intensive care units with facilities for invasiveventilation, haemodynamic support and renal replace-ment therapy. Patients will be followed up at 1, 3 and5 years post-enrolment. A summary of the trial design isshown in Fig. 1.RecruitmentPatients are being identified via referral from hospitalphysicians and/or notification from hospital microsco-pists. Patients are being recruited in Emergency Depart-ments at time of presentation, or in medical wards orFig. 1 PACKNOW trial designCooper et al. Trials  (2018) 19:250 Page 3 of 11outpatient departments. Patient recruitment startedin October 2016 and is expected to be complete bySeptember 2018.ParticipantsInclusion criteria:1. Age ≥ 5 years2. Admitted to hospital with microscopy-diagnosed P.knowlesi infection13. Temperature > 38 ºC on admission or fever duringthe preceding 48 h4. Within 18 h of commencement of antimalarialtreatment5. Written informed consent from patient or attendingrelative/guardianExclusion criteria:1. Contraindication or allergy to paracetamol orartesunate therapy2. Known cirrhosis, or more than six standardalcoholic drinks per day3. Pregnancy2Randomisation and blindingParticipants are randomised to either the paracetamol orthe control arm using computer-generated site-specificblock randomisation by an independent statistician in a 1:1ratio. Allocation tables were uploaded to the ResearchElectronic Data Capture (REDCap) web application hostedat Menzies School of Health Research. REDCap is a secure,web-based application designed to support data capture forresearch studies [40]. Standardised clinical data is entereddirectly into the REDCap electronic case report form(CRF). Enrolment procedures do not delay administrationof standard artemisinin-based treatment. As placebotablets are not used in the no paracetamol arm, it is notpossible to blind clinicians, investigators or patients to thetreatment allocation. However, treatment allocation ismasked from staff performing all laboratory investigations.Treatment arms1) Paracetamol arm: Paracetamol is administeredorally 6-hourly for 72 h by research nurses.Patients unable to swallow receive paracetamolby nasogastric tube. Dosing of paracetamol isweight-based, with patients ≥ 50 kg receiving 1 gand patients < 50 kg receiving 12.5–15 mg/kg. Ifthe patient vomits within 30 min of paracetamoladministration, a further dose is given.2) No paracetamol arm (control): Patients in thecontrol arm do not receive paracetamol; however,paracetamol may be given if temperature remains >39.5 °C for > 30 min despite tepid sponging, or ifdeemed necessary by the treating clinician. There isno specific rescue medication for this trial.The date, time and dose of paracetamol adminis-tered to patients is recorded in the CRF. All patientsreceive standard artemisinin-based treatment for mal-aria. The use of intravenous artesunate and oralartemisinin-combination therapy is at the discretion ofthe treating clinician according to Malaysian Nationalguidelines [41].Day 0, Hour 0 (enrolment) is the time of administra-tion of the first dose of paracetamol administered by theresearch team. Any concomitant medications, or para-cetamol doses prior to enrolment, are recorded on theCRF. There are no medications that are not permittedduring the study; however, it is recommended to avoidnon-steroidal anti-inflammatory drugs to avoid exacer-bating any renal dysfunction.Paracetamol 500 mg tablets administered in this studyare produced locally in a Good Manufacturing Practice-qualified facility (Paracil™, SM Pharmaceuticals, Malaysia).Sample sizeOur sample size for ANCOVA was calculated using theStata command sampsi, with α = 0.05; 1 − β (power) = 0.9;log-transformed estimated mean 72 h creatinine in controlgroup = 4.38 μmol/L (estimated SD 0.36); log-transformedestimated mean 72 h creatinine in treatment group = 4.275 μmol/L (estimated SD 0.36); correlation betweenbaseline and 72 h creatinine (data log-transformed) = 0.59.The mean 72 h creatinine in the control group, the correl-ation between baseline and 72 h creatinine, and the stand-ard deviation of the creatinine was estimated usingexisting data from prospectively enrolled patients withnon-severe knowlesi malaria at a tertiary hospital in Sabah[9], with non-severe malaria patients used to more accur-ately reflect the patients at the district hospital sites. Themean 72 h creatinine in the treatment group was esti-mated using an estimated effect size of 10%, consistentwith data from a pilot randomised trial of regularlydosed paracetamol, versus no paracetamol, in patientswith severe and moderately severe falciparum malariain Bangladeshi adults [29]. We estimated that this effectsize would be similar in patients with knowlesi malariaof any severity, given the greater haemolysis that occursin severe knowlesi malaria compared to severe falcip-arum malaria, and in non-severe knowlesi malaria com-pared to non-severe falciparum malaria [32].Using the above calculation, a minimum sample sizeof 324 will be required. Allowing for a loss to follow-up of 10% (primarily due to patients discharged fromCooper et al. Trials  (2018) 19:250 Page 4 of 11hospital < 72 h), we will require a minimum of 360patients enrolled in the study, 180 in each arm.Data collection methodsVenous blood and urine will be collected on enrolment.On venous blood, standard haematology and biochemistry(electrolytes, creatinine, bicarbonate, pH, glucose, lactateand liver transaminases) will be performed through thehospital laboratories. Plasma will be separated within30 min of collection. Both plasma and urine will be storedat –70 °C until transport for analysis.Serum creatinine will be measured on enrolment (day 0)then 12-hourly until 72 h, then at 1, 2 and 4 weeks.Follow-up creatinine is measured at 1, 3 and 5 years(Architect c4000 Clinical Chemistry Analyzer, AbbotLaboratories, Illinois, USA). Plasma CFHb concentrationswill be measured at enrolment, at 12 h and then daily for72 h. CFHb will be measured by enzyme linked immuno-sorbent assay (ELISA) using commercially available kits(Bethyl Laboratories Inc., Montgomery, Texas, USA).Biomarkers of oxidative stress (plasma F2-IsoPs andIsoFs) and endothelial activation/WPB release (Ang-2,vWF, P-selectin, osteoprotegerin (OPG)) will be assessedat admission and then daily for 72 h, with markers ofendothelial activation also measured at day 28 and at1 year. Plasma F2-IsoPs and IsoFs will be quantifiedusing gas chromatography-mass spectrometry [42, 43].Plasma CFHb, Ang-2, vWF, P-selectin and OPG will bemeasured by ELISA (RnD Systems Inc., Minneapolis,USA). Laboratory assessment and biomarkers of AKI, in-cluding microscopy, urine haemoglobin and urine andplasma neutrophil gelatinase-associated lipocalin (RnDSystems Inc., Minneapolis, USA), will be measured dailyfor 72 h. Urine albumin:creatinine ratio will be per-formed at 0 and 72 h, then at 1, 2 and 4 weeks and 1, 3and 5 years. Urinalysis will be performed 12-hourly for72 h. Urinary glycocalyx degradation products will bemeasured daily for 72 h [44]. Urine will be collectedfrom a voided specimen or urinary catheter andstored without preservative. Polymerase chain reaction(PCR) for Plasmodium spp. will be performed at theSabah State Reference Laboratory [45, 46] or othervalidated PCR. Paracetamol levels on enrolment andthen 6-hourly for 72 h will be quantified using a vali-dated liquid chromatography-tandem mass spectrom-etry method [47, 48].Microscopic peripheral blood parasitaemia will beassessed by research microscopists on admission and 6-hourly until two consecutive negative smears are ob-tained. Asexual parasite counts will be calculated onthick blood smear using the formula: parasite count perμL blood = parasites × total white blood cell count (fromthe matched daily automated white blood cell count)/200 white blood cells counted.G6PD genotyping will be performed on all enrolledpatients, as G6PD status may influence oxidative stressand may thus affect the beneficial effects of paracetamol.Primary outcomes The change in log-transformed creatinine fromenrolment (hour 0) to 72 h. The change in log-transformed creatinine fromenrolment (hour 0) to 72 h stratified by the presenceof haemolysis, as measured by CFHb (defined byusing a receiver-operating curve analysis todetermine the cut-off CFHb that providesmaximal sensitivity/specificity for predicting AKIby KDIGO criteria).Secondary outcomes Longitudinal change in creatinine over 72 h, asmeasured by the area under the creatinine-timecurve, and the effect of enrolment CFHb on thelongitudinal change in creatinine. Longitudinal change in creatinine over 72 h, asmeasured by the area under the creatinine-timecurve, and the effect of enrolment CFHb on thelongitudinal change in creatinine in patients withsevere knowlesi malaria and in those with AKI (seeTable 1: Criteria forsevere P. knowlesi malaria). Development of AKI (as defined by KDIGO criteria)at 72 h. Duration of AKI: Length of time elapsed until serumcreatinine returns to measured baseline or ifTable 1 Criteria for severe Plasmodium knowlesi malariaUnrousable comaa Glasgow coma scale < 11Respiratory distress Oxygen saturation < 92% with respiratoryrate > 30 breaths/minShock Systolic blood pressure < 80 mmHg withcool peripheries or impaired capillary refillJaundice Bilirubin > 50 μmol/L, withparasitaemia > 20,000/μL and/orcreatinine > 132 μmol/LSevere anaemia Haemoglobin < 7.0 g/dL (adults)Haemoglobin < 5.0 g/dL (children)aSignificant abnormalbleedingIncluding recurrent or prolonged bleeding(from the nose, gums or venipuncture sites),haematemesis or melenaHypoglycaemia Blood glucose < 2.2 mmol/LMetabolic acidosis Bicarbonate < 15 mmol/L orlactate > 5 mmol/LAcute kidney injury Creatinine > 265 μmol/LHyperparasitaemia Parasite count > 100,000/μL (or > 2%infected red blood cells)aNot reported to date in P. knowlesi malariaCooper et al. Trials  (2018) 19:250 Page 5 of 11unavailable, presumed basline (estimated by back-calculation of creatinine using the Modification ofDiet in Renal Disease equation) in the absence of renalreplacement therapy. Longitudinal changes in CFHb and markers ofoxidative stress (plasma F2-IsoP and IsoFs) over72 h. Longitudinal changes in markers of endothelialactivation/WPB release (Ang-2, vWF, P-selectin,OPG) over 72 h. Longitudinal changes in markers of endothelialdysfunction as measured by degradation productsof the endothelial glycocalyx over 72 h. Longitudinal changes in blood and urinebiomarkers of AKI over 72 h, includingneutrophil gelatinase-associated lipocalin andurine albumin:creatinine ratio. Fever clearance time (FCT): time taken for auraltemperature to fall below 37.5 °C (FCT-A) and timetaken for temperature to fall below 37.5 °C andremain there for at least 24 h (FCT-B). Parasite clearance time: Time from commencementof antimalarial treatment to the first of twoconsecutive negative blood films, with blood filmsassessed by microscopy every 6 h, and parasitehalf-life [49]. Safety of paracetamol in severe and uncomplicatedknowlesi malaria, as assessed by the number ofpatients with adverse events or severe adverse events(SAEs)Participant timelineSee Fig. 2 – Participant timeline (SPIRIT figure).Statistical analysis planPrimary endpointThe primary endpoint will be analysed using ANCOVAwith enrolment creatinine (hour 0) as a covariate. Thesame analysis will be performed in patients stratifiedby the presence or absence of significant haemolysis.Clinically significant haemolysis will be defined byusing a receiver-operating curve analysis to determinethe cut-off CFHb that provides maximal sensitivity/specificity for predicting AKI (KDIGO criteria). Ana-lysis of the primary end-point will be by modifiedintention-to-treat, with patients’ data excluded fromthe primary analysis if enrolment (hour 0) or 72 hcreatinine is unavailable. Patients receiving haemodi-alysis will be categorised as having had a creatininerise of 132.6 μmol/L [50]. Based on existing local datain Sabah, this is only expected to be a small propor-tion of patients (< 5%).Patients will be excluded if PCR confirms any Plasmo-dium infection other than P. knowlesi. In addition,patient data will be excluded from the primary analysis ifeither 0 or 72 h creatinine measures are not available.For patients with no 72 h creatinine result excluded inthe primary analysis, any data collected may still be usedin secondary analyses. Patients may voluntarily withdrawfrom the study at any point for any reason (see below);however, data collected to the point of withdrawal maybe used in the relevant analyses.Secondary endpointsThe secondary area under the curve endpoints will becompared by Student’s t test or Wilcoxon rank-sumtest, depending on distribution. Longitudinal data willbe analysed using mixed-effects modelling. The param-eters of primary interest will be the fixed effect inter-action terms between treatment group and times, andtreatment group and CFHb at enrolment, to describewhether change in secondary outcomes differs betweenthe two groups across the observation period, andwhether the treatment effect depends on enrolmentCFHb, respectively. Time-to-event analyses will be usedto calculate duration of AKI, fever clearance time andparasite clearance time.No interim analysis will be performed.Additional analyses1. The effect of paracetamol on the change increatinine at 72 h and longitudinal change increatinine over 72 h in patients with CKD,compared to patients without CKD.2. Change in creatinine at 72 h and longitudinalchange in creatinine over 72 h in patients withtherapeutic concentrations of paracetamol,compared to patients with absent or lowconcentrations of paracetamol.3. Population pharmacokinetics properties ofparacetamol, including (1) peak plasmaconcentration (Cmax), (2) time to peak plasmaconcentration (Tmax) and (3) area under theplasma drug concentration-time curve (area underthe curve).4. Pharmacodynamic effects of paracetamol oncreatinine, fever and parasitemia.5. To assess the effect of intravascular haemolysis(CFHb) on AKI via specific pathophysiologicalpathways (oxidative stress, endothelial activation), amediation analysis will be performed. Usingregression modelling, the effect of CFHb on AKImediated by each specific pathway will be assessedusing the product of direct effect of CFHb onpathophysiological outcome (oxidative stress,Cooper et al. Trials  (2018) 19:250 Page 6 of 11Fig. 2 (See legend on next page.)Cooper et al. Trials  (2018) 19:250 Page 7 of 11endothelial activation), and direct effect of thesepathophysiological outcomes on AKI.Other data to be reported6. A description of all patients screened and reasonsfor non-inclusion.7. Clinical, epidemiological and laboratory features ofall patients included in the study.8. The proportion of all patients lost to follow-up orwithdrawn, with reasons for withdrawal.9. The number of patients with de novo CKD,hypertension and proteinuria at 1, 3 and 5 yearsafter enrolment.Additional considerationsHepatotoxicityPrevious studies of malaria patients treated with stand-ard dosing of paracetamol have shown no evidence ofclinically significant paracetamol-related hepatotoxicity[19, 29, 47, 51]. The standard dosing of paracetamoladministered in this study is below that known to beassociated with significant hepatotoxicity, and is in ac-cordance with the Malaysian National Drug Formulary.All patients receiving paracetamol will be monitoreddaily for right upper quadrant pain and tender hepato-megaly, and aspartate transaminase (AST) levels will bemeasured on admission, at 72 h and on recovery. Add-itional alanine aminotransferase (ALT) measurementsmay be collected at the discretion of the treating clin-ician. If symptoms or signs of hepatotoxicity are present,or there is a rising AST, comprehensive investigationsincluding total bilirubin, International Normalized Ratioand creatinine will be performed. If there is evidence ofsevere hepatic toxicity (peak AST or ALT levels > 10times the upper limit of normal), paracetamol adminis-tration will be stopped and the patient referred to thegastroenterology consultation service for considerationof N-acetylcysteine therapy.Adverse events/termination of the trialAll adverse and SAEs will be recorded as per standardreporting guidelines. SAEs will be reported within 1 dayof awareness by the site investigator to the chair of theSafety Monitoring Committee. The Principal Investiga-tor will report the SAE to Malaysian and Menzies ethicscommittee in accordance with local requirements. Anyserious safety concerns identified by the Safety Monitor-ing Committee may result in modification or termin-ation of the study as necessary.Removal of patients from trialEach participant has the right to withdraw from thestudy at any time. Additionally, any patient found tohave an ALT/AST of 10 times the upper limit of normalwill be withdrawn from the study.The site investigator may also withdraw a participantif the treating clinician determines that further adminis-tration of paracetamol is contraindicated for any medicalreason. Withdrawn patients will continue to receivestandard care from the hospital clinician. The reason forwithdrawal will be recorded in the CRF, and any dataobtained from the participant up to the time of with-drawal from the study will be included in the analysis.Withdrawn subjects will be replaced.Data managementClinical, biochemical and parasitological information willbe collected and managed using REDCap electronic datacapture tools hosted at Menzies School of HealthResearch, Darwin [40]. Analysis will be conducted usingSTATA, V.14 (StataCorp Ltd., Texas, USA). Data collec-tion will be performed by the study nurses and studyphysician. An onsite data manager will oversee accuracyand completeness of data entry. The REDCap CRF willinclude all parameters necessary to assess endpoints.Reimbursement for transportParticipants in the study will be reimbursed for theirtransport to attend all follow-up visits to the study sites.Patients will be paid MYR45 for each day of travelling.No other gifts or payments will be made.ConfidentialityAll patient information will remain confidential and beshared only by the study team. Unique identifiers areused for computer-based data entry and blood samples.In all cases, the Principal Investigator will ensure thatscreening forms, CRFs and the completed identificationcode list are kept in locked files. Subject confidentialitywill be protected in any dissemination of results.(See figure on previous page.)Fig. 2 Participant timeline (SPIRIT figure). (x) = Patients without parasite clearance. * Intensive paracetamol level sampling will be performed on asubset of patients at 0.5, 1.5, 2.5, 4.0, 72.5, 73.5, 74.5 and 76.0 h, in addition to 6-hourly as above. AE adverse event, BUSE blood urea andserum electrolytes, LFTs liver function tests (including bilirubin), CFHb cell-free haemoglobin, FBC full blood count, HCT haematocrit, F2-IsoP F2-isoprostanes, F2-IsoF F2-isofurans, WPB Weibel–Palade bodies, Ang-2 angiopoietin-2, vWF von-Willebrand factor, OPG osteoprotegerin,ACR albumin:creatinine ratio, NGAL neutrophil gelatinase-associated lipocalinCooper et al. Trials  (2018) 19:250 Page 8 of 11DiscussionThis study will be the largest clinical trial to date in know-lesi malaria, and is powered to detect a difference betweenthe two treatment arms in a clinically relevant populationincluding adults and children with knowlesi malaria of anyseverity. Paracetamol is widely available and has an excel-lent safety profile; if a renoprotective effect is demon-strated, this trial will support the administration ofregularly dosed paracetamol to all patients with knowlesimalaria. The secondary outcomes in this study will pro-vide further insights into the pathophysiology ofhaemolysis-induced oxidative damage and acute kidneyinjury in knowlesi malaria, and other haemolytic diseases.The lack of blinding of study arm randomisation topatients and the treating team is not expected to influ-ence the primary endpoint of change in creatinine, asthis is entirely objective. The laboratory staff measuringand reporting creatinine are blinded to treatment alloca-tion. All primary and secondary endpoints listed aboveare laboratory-based parameters, and therefore not ableto be influenced by subjective factors.All patients will receive standard artemisinin-basedtherapy as per Malaysian Ministry of Health guidelinesand treating clinicians are independent of the study(Additional file 1).Trial statusThe trial began recruitment on October 18, 2016 and isexpected to complete in September 2018.Endnotes1P. knowlesi now accounts for over 90% of all reportedmalaria cases in Malaysia [4, 6, 37, 38]; however, micros-copy is unreliable for distinguishing P. knowlesi from P.falciparum and P. vivax [39]. For blood films reported asP. falciparum, a Pf-HRP2-based rapid diagnostic test (FirstResponse™; Premier Medical Corp. Ltd., India) is per-formed, and patients with a positive result are excluded.Patients with nominal P. vivax on microscopy are en-rolled. Final species confirmation by PCR is performed onall patients; any patient with malaria due to a Plasmodiumspecies other than P. knowlesi (including mixed P. know-lesi infections) will be excluded from all analyses.2Pregnant women are excluded due to uncertainty re-garding potential adverse effects of untreated fever inpregnancy.Additional fileAdditional file 1: Populated SPIRIT checklist. (DOC 121 kb)AbbreviationsAKI: Acute kidney injury; ALT: Alanine aminotransferase; Ang-2: Angiopoietin-2; AST: Aspartate transaminase; CFHb: Cell-free haemoglobin; CKD: Chronickidney disease; CRF: Case report form; F2-IsoPs: F2-Isoprostanes; FCT: Feverclearance time; IsoFs: Isofurans; KDIGO: Kidney Disease: Improving GlobalOutcomes; NOX: NADPH oxidase; OPG: Osteoprotegerin; PCR: Polymerasechain reaction; SAE: Serious adverse event; SPIRIT: Standard Protocol Items:Recommendations For Interventional Trials; vWF: von-Willebrand factor;WPB: Weibel–Palade bodiesFundingThis trial is funded by peer-reviewed grants from the National Health andMedical Research Council of Australia (Program Grant #1037304), ‘ImprovingHealth Outcomes in the Tropical North: A multidisciplinary collaboration[HOT NORTH] #113193, and Fellowships to NMA [#1042072], BEB [#1088738]and MJG [#1138860]. This work is solely the responsibility of the authors anddoes not reflect the views of the NHMRC. DJC is supported by AustralianGovernment UPRS & PIRTS scholarships.Availability of data and materialsNot applicable.Dissemination of resultsResults will be disseminated through peer-reviewed publications, academicpublications and key local stakeholder and community forums.Authors’ contributionsDJC, K Plewes, MJG, GSR, TW, TWY, AD, NMA and BEB conceived anddesigned the study. DJC, K Plewes, MJG, AD, NMA and BEB wrote the studyprotocol. KP assisted with the plan for the laboratory methods. MDCprovided statistical advice. All authors read and approved the finalmanuscript.Ethics approval and consent to participateThe study has been reviewed, and approved, by the Malaysian ResearchEthics Committee (protocol number NMRR-16-356-29,088) and the EthicsCommittee of Menzies School of Health Research, Darwin, Australia(reference number 2016-2544). The study is registered under www.clini-caltrials.gov (NCT03056391). First registered October 12, 2016; https://clin-icaltrials.gov/ct2/show/NCT03056391.Consent for publicationConsent will be obtained by trained study staff nurses in Bahasa Malaysia orEnglish. Parents or guardians of participants < 18 years old will be asked toprovide consent, and assent will be sought from participants between 12and 17 years old as per Malaysian Research Ethics Committee guidelines. Ifthe patient is illiterate, a thumbprint will be obtained on the consent form inaccordance with Malaysian GCP guidelines. Informed consent may bewithdrawn at any time and will have no effect on the patient’s clinicalmanagement at the study site.Competing interestsThe authors declare that they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1Global and Tropical Health Division, Menzies School of Health Research andCharles Darwin University, Darwin, NT, Australia. 2Infectious Diseases SocietySabah-Menzies School of Health Research Clinical Research Unit, KotaKinabalu, Sabah, Malaysia. 3Mahidol Oxford Tropical Medicine Research Unit,Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. 4Centrefor Tropical Medicine and Global Health, Nuffield Department of Medicine,University of Oxford, Oxford, UK. 5Division of Infectious Diseases, Faculty ofMedicine, University of British Columbia, Vancouver, Canada. 6InfectiousDiseases Unit, Clinical Research Centre, Queen Elizabeth Hospital, KotaKinabalu, Sabah, Malaysia. 7Sabah Department of Health, Kota Kinabalu,Sabah, Malaysia. 8Jesselton Medical Centre, Kota Kinabalu, Sabah, Malaysia.9Queensland Institute of Medical Research, Brisbane, Australia. 10Lee KongChian School of Medicine, Nanyang Technological University, Singapore,Singapore.Cooper et al. 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Trans R Soc Trop Med Hyg. 1995;89(5):507–9.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Cooper et al. Trials  (2018) 19:250 Page 11 of 11

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