nac in malaria

N-acetylcysteine as adjunctive treatment in severe malaria:A randomized, double-blinded placebo-controlled clinical trial*

Prakaykaew Charunwatthana, MD; M. Abul Faiz, MD, PhD; Ronnatrai Ruangveerayut, MD;Richard J. Maude, MRCP; M. Ridwanur Rahman, MD; L. Jackson Roberts II, PhD; Kevin Moore, FRCP, PhD;Emran Bin Yunus, MD; M. Gofranul Hoque, MD; Mahatab Uddin Hasan, MD; Sue J. Lee, PhD;Sasithon Pukrittayakamee, MD, DPhil; Paul N. Newton, MD; Nicholas J. White, DSc, FRCP, FRS;Nicholas P. J. Day, DM, FRCP; Arjen M. Dondorp, MD, PhD

The introduction of the potentand rapidly acting antimalarialdrug artesunate has been asignificant advance in the

treatment of severe malaria, decreasing thecase fatality rate in adult severe malaria by35% compared with quinine (1). By con-trast, of the many other interventions tried,none have been shown to lower mortality.

Severe falciparum malaria has beenassociated with an increase in oxidativestress, in relation with disease severity(2–4). Possible origins of reactive oxygenspecies are malaria parasites or hostwhite cells (3–6). Although reactive oxy-gen species may be important for parasiteclearance (2), they may be harmful to thehost through its damaging effects on var-ious cells and a reduction in erythrocytedeformability (7). Rigidity of erythrocytesreduces their lifespan and is thought tocontribute to impaired microcirculatoryflow, in addition to sequestration of par-asitized erythrocytes (8). Severity of ane-mia correlates with measures of oxidativedamage to the erythrocyte membrane inpediatric severe malaria (9).

N-acetylcysteine (NAC) is a widelyused, safe, and well-tolerated antioxidant.It is the main treatment for acute parac-

etamol poisoning, acting through directscavenging of free radicals and replenish-ment of glutathione and cysteine. It alsoenhances clearance of peroxides throughglutathione peroxidase and related en-zymes. NAC has also been shown to im-prove microcirculatory blood flow inacute liver failure (10). NAC may be ben-eficial in severe malaria through enhanc-ing scavenging of free radicals and reduc-ing expression of endothelial ligands (11).Red cell deformability can be improved byNAC in an in vitro test system (12). A pilotstudy in Western Thailand showed that in-travenous NAC was associated with a sig-nificantly shorter time to normalization ofplasma lactate and Glasgow Coma Score(GCS) in patients with malaria treated withquinine (13). Another study in Thailandconfirmed the safety of NAC as an adjunc-tive treatment to artesunate in severe ma-laria (14). However, NAC was shown topartly antagonize the antimalarial action ofartesunate in vitro (15). The antimalarialeffect of the artemisinin derivatives de-pends on the unique endoperoxide bridge

*See also p. 758.From the Faculty of Tropical Medicine (PC, SJL, SP,

NPJD, NJW, AMD), Mahidol University, Bangkok, Thai-land; Dhaka Medical College (MAF), Dhaka, Bangladesh;Mae Sot General Hospital (RUANGVEERAYUT), Mae Sot, TakProvince, Thailand; Chittagong Medical College Hospital(RAHMAN, EBY, MGH, MUH), Chittagong, Bangladesh; De-partment of Pharmacology (LJR), Vanderbilt University,Nashville, TN; Department of Medicine (KM), Centre forHepatology, Hampstead Campus, University College Lon-don, London, United Kingdom; and Nuffield Department ofClinical Medicine (PNN, RM, SJL, NPJD, NJW, AMD),Centre for Tropical Medicine, John Radcliffe Hospital,University of Oxford, Oxford, United Kingdom.Supported, in part, by The Wellcome Trust of Great

Britain and by grant GM42056 from the National Insti-tutes of Health.The authors have not disclosed any potential con-

flicts of interest.For information regarding this article, E-mail:

[email protected] © 2009 by the Society of Critical Care

Medicine and Lippincott Williams & Wilkins

DOI: 10.1097/CCM.0b013e3181958dfd

Objective: Markers of oxidative stress are reported to be in-creased in severe malaria. It has been suggested that the anti-oxidant N-acetylcysteine (NAC) may be beneficial in treatment.We studied the efficacy and safety of parenteral NAC as anadjunct to artesunate treatment of severe falciparum malaria.

Design: A randomized, double-blind, placebo-controlled trialon the use of high-dose intravenous NAC as adjunctive treatmentto artesunate.

Setting: A provincial hospital in Western Thailand and a ter-tiary referral hospital in Chittagong, Bangladesh.

Patients: One hundred eight adult patients with severe falci-parum malaria.

Interventions: Patients were randomized to receive NAC orplacebo as an adjunctive treatment to intravenous artesunate.

Measurements and Main Results: A total of 56 patients weretreated with NAC and 52 received placebo. NAC had no significant

effect on mortality, lactate clearance times (p � 0.74), or comarecovery times (p � 0.46). Parasite clearance time was increasedfrom 30 hours (range, 6–144 hours) to 36 hours (range, 6–120hours) (p � 0.03), but this could be explained by differences inadmission parasitemia. Urinary F2-isoprostane metabolites, mea-sured as a marker of oxidative stress, were increased in severemalaria compared with patients with uncomplicated malaria andhealthy volunteers. Admission red cell rigidity correlated withmortality, but did not improve with NAC.

Conclusion: Systemic oxidative stress is increased in severemalaria. Treatment with NAC had no effect on outcome in patientswith severe falciparum malaria in this setting. (Crit Care Med2009; 37:516–522)

KEY WORDS: N-acetylcysteine; severe malaria; adjunctive treat-ment

516 Crit Care Med 2009 Vol. 37, No. 2

within the 1,2,4-trioxane nucleus, and ismediated by the formation of free radicalsupon cleavage of the endoperoxide (16).

We hypothesized that NAC, as an an-tioxidant adjunctive treatment, can de-crease harmful oxidative stress in severemalaria, and can have a beneficial effecton outcome parameters related to prog-nosis. We conducted a randomized, dou-ble-blind, placebo-controlled clinicaltrial, on the effect of NAC as an adjunc-tive treatment to artesunate in adult pa-tients with severe falciparum malaria.Primary end points were the effects onlactate clearance time, coma recoverytime, and parasite clearance time.

PATIENTS AND METHODS

Study Site and Patients. The study wasconducted at Chittagong Medical College Hos-pital, Chittagong, Bangladesh and Mae SotGeneral Hospital, Tak, Thailand, from May2003 to October 2005. Chittagong MedicalCollege Hospital is a 1000-bed teaching hos-pital with facilities for oxygen therapy andblood transfusion. The 250-bed provincial hos-pital in Mae Sot has intensive care unit facil-ities including hemodialysis and mechanicalventilation. Malaria transmission is seasonaland of low intensity in both locations (17, 18).Ethical clearance was obtained from the Min-istries of Health in Thailand and Bangladesh,and from the Oxford Tropical Medicine EthicalCommittee. ISRCTN registration number IS-RCTN20156397.

Adults patients (�16 years old) with slide-confirmed severe malaria according to modi-fied World Health Organization criteria (19)were recruited, provided written informedconsent was obtained from the attending rel-ative. Criteria for severe malaria includedcoma (GCS �11), acute respiratory distresssyndrome, repeated convulsions, severe ane-mia or jaundice (hematocrit �20%, bilirubin�3.0 mg/dL, combined with parasite counts�100,000/uL), renal failure (serum creatinine�3 mg/dL), hypoglycemia (blood glucose �40mg/dL), shock (systolic blood pressure �80mm Hg with cool extremities), hyperpara-sitemia (peripheral asexual stage parasitemia�10%), hyperlactatemia (venous plasma lac-tate �4 mmol/L), or acidemia (venous plasmabicarbonate �15 mmol/L). Pregnancy, breastfeeding, a history suggesting asthma or hyper-sensitivity to NAC, a wheeze on auscultation atadmission, or previous treatment with lactatecontaining intravenous fluids were exclusioncriteria.

Since plasma lactate concentrations have astrong prognostic significance in severe ma-laria (20), we powered the study to demon-strate an increase in the proportion of patientswith venous plasma lactate levels �2 mmol/Lat 24 hours from 20% to 50% with a signifi-cance level (�) of 0.05 and a power (1-�) of

0.80. On the basis of this, we required at least90 patients in the study. The primary endpoint was chosen to inform the decisionwhether to expand to a larger trial with mor-tality as end point.

Drug Treatment. Patients were randomlyassigned to either NAC or matching placebo asan adjunctive treatment to parenteral artesu-nate (Guilin No. 2 Pharmaceutical Factory,Guangxi, People’s Republic of China). Com-puterized randomization was balanced inblocks of 40, and allocation codes were kept insealed envelopes. Patients, physicians, andnurses were masked to the treatment as-signed. NAC was obtained as 10 mL ampoulescontaining 20% w/v NAC from Medeva, Cell-tech Group, Berkshire, UK, who also suppliedmatching ampoules containing placebo. NACwas given intravenously in the standard re-gime used in paracetamol intoxication, i.e.,150 mg/kg in 200 mL 5% dextrose water (5%DW) over 15 minutes, followed by 50 mg/kg in500 mL 5% DW over 4 hours, then 100 mg/kgin 1000 mL 5% DW over 16 hours. Since NAChas a distinctive odor, treating doctors werenot involved in the preparation of the infu-sion. The saline placebo was given in 5% DW.The first dose of NAC or placebo was started 2hours after starting intravenous artesunate.The standard dosing regimen for artesunatewas used (1). Supportive treatments were inaccordance with World Health Organization(21) and local hospital guidelines, but avail-ability of renal replacement therapy and me-chanical ventilation was limited in the hospi-tal in Chittagong.

Study Procedures. At admission, a full his-tory and examination were carried out. Ve-nous blood samples were obtained for hemo-globin, hematocrit, parasitemia, plateletcount, white cell count, plasma lactate levels,glucose levels, and full biochemistry. Parasitecounts and lactate levels were assessed every 6hours. Red cell deformability was measured ata shear stress of 1.7 Pa at admission, 6 hoursafter admission, and then daily until dischargeusing a Laser-assisted Optical Rotational CellAnalyzer (8). Lactate concentrations weremeasured from stored plasma samples(�30°C) with YSI 2300 STAT PLUS glucose/L-lactate version 250D. Urine F2-isoprostanemetabolites (F2-isoP-M) levels were used as ameasure of oxidative stress and assayed at ad-mission and daily until discharge or death.Urine was collected from the urinary catheterand stored without preservative at �80°C orin liquid nitrogen. Urinary concentration ofF2-isoP-M was measured using a high-performance liquid chromatography methodcoupled to electrospray ionization–tandemmass spectrometry. The urinary content ofF2-isoprostanes metabolites was corrected forrenal function by calculating the ratio withcreatinine clearance with the formula (22, 23):

�F2-isoP-M�corrected �F2-isoP-M�urine �creatinine�plasma/�creatinine�urine

Excretion of prostanoids (like F2-isoP-M) isdecreased in parallel with creatinine clear-ance, as shown in patients with liver disease(24). Vital signs, oxygen saturation, consciouslevel (GCS), fluid input, and output were re-corded 6 hourly until recovery. Parasite clear-ance time was defined as the interval betweenstart of treatment and the first of two sequen-tial negative thick films. PC50 is the time to50% and PC90 to 90% reduction in parasitedensity. Parasite reduction ratios at 24 hours(PRR24) and 48 hours (PRR48) were definedas the ratio of the parasite count at admissionto that at 24 or 48 hours (25). Fever clearancetime was the time to a tympanic measuredtemperature below 37.5°C. Coma recoverytime was measured from the start of antima-larial treatment to the time the GCS scorereached 15. Time to normal plasma lactatelevel was the time to a decrease in plasmaconcentrations �2 mmol/L (upper limit ofnormal value). Follow-up for all patients wasfrom admission until discharge or death.

Primary outcome measures were lactateclearance time, coma recovery time, and par-asite clearance time. Secondary outcome mea-sures included fever clearance time, mortality,other parasite clearance measures (PC50,PC90, PRR24, and PRR48), change in red celldeformability and measures of oxidative stress,and the frequency of adverse events.

Statistical Analysis. Analysis was per-formed by SPSS software (version 11.0) (Chi-cago, IL) and STATA (version 9, College Sta-tion, TX). Data were log transformed to obtaina normal distribution, if indicated. Normallydistributed data were compared using Stu-dent’s t test and non-normally distributed datawere compared using Student’s t test for geo-metric means or the Mann-Whitney U test fornonpaired nonparametric data or the Wilcox-on’s signed-rank test for paired nonparametricdata. Categorical data were compared by Pear-son’s chi-squared test and Fisher’s exact test.Multiple groups were compared by analysis ofvariance, and comparisons between individualgroups were corrected for multiple compari-sons (Bonferroni). Univariate correlationswere checked with Pearson’s correlation coef-ficient if the data were normally distributed orcould be transformed to a normal distribution.Kaplan-Meier plots were constructed for time-dependent parameters. For comparison be-tween treatment groups (Wilcoxon-Breslowtest), the lactate clearance times and comarecovery times in patients who died beforeclearance or recovery were set as infinite,since failure to clear lactate or recover fromcoma has strong prognostic significance fordeath. To assess whether treatment had anyeffect on the time to lactate or parasite clear-ance and coma recovery times, Cox regressionanalysis was performed, while adjusting for(log) initial parasitemia and bilirubin, sincethese were significantly different between thetwo groups at baseline. To determine whether

517Crit Care Med 2009 Vol. 37, No. 2

treatment was associated with the risk of dy-ing, a multiple logistic regression analysis wasperformed with mortality as the dependentvariable and allocation to NAC or placebo asthe independent variable. The model was alsoadjusted for presenting severity symptoms.

RESULTS

Hundred eight patients were enrolled inthe study, 23 in Mae Sot General Hospitaland 85 in Chittagong Medical College Hos-pital. Of these, 56 patients (13 at Mae Sot)were randomized to receive NAC and 52 (10at Mae Sot) received placebo.

Baseline Characteristics. Baselinecharacteristics are summarized in Tables1 and 2. Except for differences in admis-sion parasitemia and plasma bilirubin

concentration, the treatment groupswere well matched.

Lactate Clearance Time. Elevated ad-mission plasma lactate concentrations(�2 mmol/L) were present in 99 of 108patients (92%). Median lactate clearancetime was 30 in the NAC treated and 42hours in the placebo group (range, 6–96hours for NAC and 6–144 hours for pla-cebo, excluding expired patients). TheKaplan-Meier plot for lactate clearancetime (Fig. 1a), shows no difference be-tween treatment groups (Wilcoxon-Breslow test, p 0.74), also after ad-justment for �log� admission parasitemiaand bilirubin (Hazard ratio: 0.98; 95%confidence interval �CI�: 0.60–1.62, p 0.95).

Coma Recovery Time. At admission 91of 108 patients (84%) had impaired con-sciousness (GCS �15) of which 78 werein coma (72%) (GCS �11). Median comarecovery time was 72 hours in NAC-treated patients and 96 hours in the pla-cebo group (range 6–120 hours in NACand 6–360 hours in the placebo group,excluding the patients who died). Kaplan-Meier analysis, where the coma recoverytime for the fatal cases was regarded asinfinite, showed no difference betweentreatment groups (Fig. 1b, Wilcoxon-Breslow test, p 0.46). Treatment hadno effect on coma recovery time afteradjustment for �log� admission para-sitemia and bilirubin (Hazard ratio: 1.05;95% CI: 0.58–1.88, p 0.88).

Table 1. Baseline characteristics of 108 patients with severe malaria treated with either N-acetylcysteine or placebo as adjunctive treatment tointravenous artesunate

Variable N-acetylcysteine (n 56) Placebo (n 52) p

Age (yrs) 34.2 (30.8–37.8) 34.9 (30.9–38.8) 0.81Male/Female patients 44/12 41/11 0.65Thailand/Bangladesh 13/43 11/41 0.82Days of fever before admission 5.8 (4.6–7.1) 5.6 (4.8–6.3) 0.72Temperature (°C) 38.1 (37.8–38.4) 38.0 (37.6–38.3) 0.64Systolic blood pressure (mm Hg) 111 (107–116) 112 (106–117) 0.96Oxygen saturation (%)a 95 (94–96) 96 (95–97) 0.12Glasgow Coma Scoreb 8 (3–15) 9 (3–15) 0.61Hemoglobin (g/dL) 10.2 (9.5–10.8) 10.1 (9.5–10.8) 0.91Peripheral white blood cell count (cells/mm3) 9555 (8,076–11,034) 9,150 (7,624–10,676) 0.70Platelet count (cells/mm3) 126,026 (102,781–149,272) 139,342 (116,792–161,316) 0.40Parasitemia (�L)a 118,843 (77,535–169,005) 57,178 (33,327–95,531) 0.02Serum creatinine level (mg/dL)a 1.98 (1.54–2.41) 2.04 (1.57–2.78) 0.80Total serum bilirubin level (mg/dL)a 5.04 (3.85–6.60) 3.41 (2.73–4.35) 0.03Serum albumin (g/100 mL) 2.91 (2.77–3.08) 2.70 (2.53–2.85) 0.06Serum aspartate aminotransferase (U/L)a 79 (63–100) 80 (62–98) 0.91Serum alanine aminotransferase (U/L)a 20 (16–25) 23 (17–29) 0.46Serum base excess (mmol/L) �7.9 (�6.7 to �10.0) �7.1 (�4.9 to �9.3) 0.33Plasma lactate level (mmol/L)a 5.3 (4.6–6.7) 4.7 (3.9–5.6) 0.36Red cell deformability, shear stress 1.7 Pa (EI) (n 87) 0.189 (0.179–0.199) 0.194 (0.183–0.205) 0.53Urinary F2-isoP-M, pg/mLa (n 50) 627.4 (395.7–994.7) 496.9 (288.4–856.2) 0.50

F2-isoP-M is F2-isoprostane metabolites values are mean (95% confidence interval), unless indicated otherwise.aGeometric mean (95% confidence interval); bmedian (range).

Table 2. Distribution of baseline presenting severity symptoms in patients with severe malaria according to study site and adjuvant treatment group (NACor placebo)

Thailand(n 23)

Bangladesh(n 85)

NAC(n 56)

Placebo(n 52)

p(NAC vs. Placebo)

Glasgow Coma Score �11 12 (52) 66 (78) 42 (75) 36 (69) 0.50Hematocrit �20% with parasite count �100,000/uL 2 (9) 5 (6) 3 (5) 4 (8) 0.70Bilirubin �3.0 mg/dL with parasite count �100,000/uL 6 (26) 27 (32) 23 (41) 10 (19) 0.01Serum creatinine �3.0 mg/dL 3 (13) 17 (20) 11 (20) 9 (17) 0.75Systolic blood pressure �80 mm Hg and cool extremities 1 (4) 2 (2) 2 (4) 1 (2) 1Parasitemia �10% 7 (30) 20 (24) 16 (29) 11 (21) 0.37Venous lactate �4 mmol/L 10 (43) 55 (65) 34 (61) 31 (60) 0.90Venous bicarbonate �15 mmol/L 3 (13) 34 (40) 17 (30) 20 (38) 0.37

NAC, N-acetylcysteine.Values in the parentheses indicates percentage.


Parasite Clearance Time. UsingKaplan-Meier analysis (Fig. 1c) parasiteclearance was faster in patients receivingplacebo (Wilcoxon-Breslow p 0.03).Parasite clearance time, censoring the fa-tal cases at the time of death, was 36hours (range, 6–120) in the NAC-treatedpatients and 30 hours (range, 6–144) inthe placebo group. However, admissionparasitemias were significantly higher inthe NAC-treated patients (geometricmean: 118,843 parasites/�L �95% CI:77,535–169,005�), compared with 57,178parasites/�L (95% CI: 33,327–95,531) inthe control group (p 0.021), and admis-sion (log) parasitemia correlated with (log)parasite clearance time (r .67, p �0.0005). In the Cox regression analysis, ad-justing for �log� parasitemia and bilirubinlevels at admission, treatment allocation(NAC or placebo) did not significantly affectparasite clearance time (Hazard ratio: 1.17;95% CI: 0.74–1.86, p 0.51).

Other parasite clearance measuresalso did not show any difference betweentreatment groups: mean median (range)PRR at 24 hours was 370 (1-infinite) inthe NAC group and 170 (1-infinite) in theplacebo group (p 0.56); PRR at 48hours was 3316 (4-infinite) and 3140 (1-infinite, p 0.94), respectively; the geo-metric mean time (95% CI) to 50% clear-ance of parasites (PC50) was 8.1 hours(6.31–10.42) in the NAC group and 6.6hours (5.13–8.38) in the placebo group(p 0.23); for PC90, times were 19.1hours (15.57–23.53) and 18.6 hours(15.88–21.87, p 0.84), respectively.Thus, there was no evidence that NAC pro-longed parasite clearance by artesunate.

Fever Clearance Time. There was nodifference in fever clearance times be-tween the two treatment groups byKaplan-Meier analysis (Wilcoxon-Breslow,p 0.49). Fever clearance time, censor-ing the fatal cases at the time of death,

was 24 hours for both treatment groups(range, 6–96 hours for the NAC and6–360 hrs in the placebo group) and ad-justment for �log� parasitemia at admis-sion and bilirubin in the Cox model didnot change this result (Hazard ratio:0.81; 95% CI: 0.43–1.53, p 0.52).

Mortality. The overall mortality in thisseverely ill group of patients was 35% (38of 108 patients, Table 3), with 21 fatalcases (38%) in the NAC group vs. 17 inthe placebo group (33%), which was notsignificantly different by survival analysis(Wilcoxon-Breslow, p 0.52). The levelof supportive and intensive care unit carewas higher in Mae Sot General Hospitalin Thailand, and mortality was signifi-cantly lower at this study site (9% vs.42%, p 0.003). Median time to deathwas 16 hours in both the NAC and place-bo-treated groups (range 2–204 hours inthe NAC group and 3–288 in the placebogroup). In a multiple logistic regression

Figure 1. Kaplan-Meier curves for lactate clearance times, defined as the time to a plasma lactate concentration below 2 mmol/L (a, Wilcoxon-Breslow testfor equality of survivor functions, p 0.74), coma recovery times, defined as the time since admission to a Glasgow Coma Score of 15 (b, p 0.46) andparasite clearance times, defined as the time to a negative peripheral blood slide for asexual parasite forms of Plasmodium falciparum (c, p 0.03*). Dottedline represents patients treated with N-acetylcysteine as adjunctive treatment to intravenous artesunate (n 56), solid line patients receiving placebo inaddition to antimalarial treatment with intravenous artesunate (n 52). *After correction for baseline parasitemia and bilirubin the difference in parasiteclearance between treatment groups was not significant (p 0.51).


analysis with mortality as the dependentvariable, allocation to NAC or placebo didnot contribute to the model in addition tothe presenting severity symptoms.

Red Cell Deformability. Admission redcell deformability at a shear stress of 1.7Pa (as mean elongation index, 95% CI)was assessed in 87 patients. Red cell de-formability was lower in fatal cases: 0.182(0.169 – 0.194), compared with 0.198(0.189–0.207) in patients who survived(p 0.03). There was no significantchange in red cell deformability in thefirst 24 hours after admission in eithergroup: mean change (95% CI) 3.1%(�6.5% to 12.8%) in the NAC-treatedgroup (p 0.50) and 3.1% (�6.1% to12.3%) in the placebo group (p 0.49).

Measures of Oxidative Stress. UrineF2-isoP-M concentrations were assessedin 50 of the patients with severe malaria.For comparison, these were also assessedin 15 local volunteers, 13 patients withuncomplicated falciparum malaria, and 4patients with Plasmodium vivax infec-tion. After correction for renal function,geometric mean urine F2-isoP-M weresignificantly higher in patients with se-vere malaria compared with uncompli-cated malaria, P. vivax, and healthy con-trols (Fig. 2); the corrected geometricmean F2-isoP-M concentrations (95% CI,p of difference with value in severe ma-laria) were 556 pg/mL CrCl (392–787) forpatients with severe malaria; for uncom-plicated falciparum malaria 183 pg/mLCrCl (114–293, p 0.002); in vivax ma-laria 139 pg/mL CrCl (23–825, p 0.02),and in healthy controls 176 pg/mL CrCl(89–350, p 0.002). Within the group ofpatients with severe malaria, there wasno difference (p 0.44) in corrected uri-nary F2-isoP-M concentrations at admis-sion between survivors and fatal cases:geometric mean (95% CI) 613 pg/mL

CrCl (384–979) in survivors (n 33) and460 pg/mL CrCl (271–779) in fatal cases(n 17). There was no significant corre-lation with admission hemoglobin, redcell deformability, or lactate concentra-tions. In 32 severe malaria patients, lon-gitudinal data at 24 hours were available.No change in urinary F2-isoP-M concen-trations (corrected for renal function)were observed in the 15 patients treatedwith NAC (F2-isoP-M: mean percentchange, 95% CI 2.7%, �8.7 to 14.2,p 0.62), nor in the 17 patients treatedwith placebo (6.3%, �2.7 to 16.9, p 0.22).

Adverse Events. No adverse events re-lated to NAC were observed, includingallergic reactions. There was no differ-ence in the proportion of patients pre-senting with severe complications uponadmission between the NAC-treatmentgroup and the placebo (Table 3).

DISCUSSION

This study evaluated the effects of NACas an adjuvant to intravenous artesunate

for the treatment of severe falciparummalaria. A smaller pilot study had shownpreviously that clearance of elevatedplasma lactate levels and coma recoverytimes were accelerated by NAC (13). Bothparameters have strong prognostic signif-icance in severe malaria. For both vari-ables, there was no significant differencebetween NAC or placebo-treated patientsin the present study. The main differencewith the earlier pilot study was antima-larial treatment with artesunate ratherthan quinine. Since mortality is lowerand parasite clearance times, and possiblylactate clearance times, shorter in pa-tients treated with artesunate comparedwith quinine, an additional effect of NACmight be more difficult to demonstrate inthe current study. Despite strict random-ization, patients in the NAC groups hadhigher levels of parasitemia and bilirubinat admission. However, the results fromthe Kaplan-Meier analyses were consis-tent with the results from multivariateanalyses, which were controlled for ad-mission parasitemia and bilirubin levels,thereby taking the differences into ac-count.

Mortality, fever clearance time, andcomplications or adverse events also didnot differ between treatment groups. Wehave recently shown that the antiplasmo-dial action of artesunate is slightly antag-onized by the antioxidant NAC (16). Thisinhibitory effect was no longer observedwhen the NAC was added 2 hours afterincubation with artesunate. In the cur-rent study, NAC was started 2 hours afterartesunate administration. Although par-asite clearance times were significantlylonger in the NAC treatment group in theunivariate analysis, when adjusted for ad-mission parasitemia and bilirubin levels,treatment allocation did not have a sig-nificant effect on parasite clearance time.

SM UM PV Healthy10

100

1000

10000p=0.002

p=0.02

p=0.002Ur

ine

F2-is

oP-M

(pg/

ml c

r.cl

)ge

omet

ric

mea

n,95

%C

I

Figure 2. Urine F2-isoprostane metabolites (F2-isoP-M) concentrations corrected for creatinineclearance in patients with severe falciparum ma-laria (SM), compared with uncomplicated falci-parum malaria (UM), vivax malaria (PV), andhealthy local volunteers (“healthy”).

Table 3. Complications according to study site and adjunctive treatment group (NAC or placebo) in 108 patients with severe malaria

Thailand(n 23)

Bangladesh(n 85)

NAC(n 56)

Placebo(n 52)

p(NAC vs. Placebo)

Died 2 (9) 36 (42) 21 (38) 17 (33) 0.60Mechanical ventilation 3 (13) 4 (5) 4 (7) 3 (6) 1Blood transfusion 3 (13) 8 (9) 3 (5) 8 (15) 0.11Vasopressor /inotropic drugs 3 (13) 5 (6) 5 (9) 3 (6) 0.71Dialysis 2 (9) 10 (12) 4 (7) 8 (15) 0.22Acute respiratory distress syndrome 4 (17) 15 (16) 9 (16) 10 (19) 0.66Renal failure 2 (9) 26 (31) 14 (25) 14 (27) 0.82Black water fever 2 (9) 12 (14) 7 (13) 7 (13) 0.88Septicemia 4 (17) 17 (20) 10 (18) 11 (21) 0.66Aspiration pneumonia 3 (13) 22 (26) 14 (25) 11 (21) 0.63

NAC, N-acetylcysteine.Values in the parentheses indicates percentage.


Furthermore, other measures of parasiteclearance, which are less dependent onthe admission parasitemia, such as thePRR, were not different between treat-ment groups. Fever clearance times werealso not different between the studygroups. This argues against a major an-tagonistic effect of NAC on the antima-larial activity of artesunate in vivo.

An earlier study in children with se-vere malaria described an association be-tween an increase in reactive oxygen in-termediate production measured byluminol-enhanced chemiluminescence,and severity of disease, especially severeanemia (2). Severe malarial anemia hasbeen linked to oxidative damage of thered cell membrane (9). In our study,urine concentrations of F2-isoP-M weresignificantly increased in patients withsevere malaria, compared with patientswith uncomplicated malaria, vivax ma-laria, and healthy controls from the sameethnic background. Treatment with anartemisinin, which has oxidative prop-erties, could have contributed to theurinary reactive oxygen intermediatesin patients with severe malaria and un-complicated falciparum malaria, in con-trast to those with vivax malaria, whowere treated with chloroquine, or healthycontrols. We did not observe a correlationwith severity of anemia. Recently, it hasbeen shown that plasma L-arginine levelsare reduced in severe malaria and thatthis is associated with decreased nitricoxide production by nitric oxide synthase(26). With low L-arginine levels, nitricoxide synthase generates superoxiderather than nitric oxide, which can add tothe increase in oxidative stress in severemalaria. Measurement of F2-isoprostanesmetabolites in the urine has emerged asa reliable noninvasive method to deter-mine systemic levels of oxidative stressin vivo (27).

Although our study affirms that oxida-tive stress is increased in severe malaria,intervention with high-dose NAC did notshow any measurable effect. Labile pros-taglandin H2-like endoperoxides (H2-isoprostanes) are intermediates in theformation of F2-isoprostanes. H2-isopros-tanes are reduced to form F2-isopros-tanes, but can undergo rearrangement invivo to form E-ring and D-ring isopros-tanes. Since reduction to F2-isoprostanesis catalyzed by thiols (28), NAC couldincrease the ratio between F2-isopros-tanes and E2/D2 isoprostanes, obscuringits antioxidative effect.

The current study reinforces the prog-nostic significance of red cell rigidity, aspreviously shown in both adult and pedi-atric severe malaria (8, 29). Oxidativedamage to the red cell membrane hasbeen proposed as a cause for decreasedred cell deformability (7), but in ourstudy deformability did not correlate withurinary F2-isoprostanes concentrationsand was not restored by NAC.

In addition to its antioxidative proper-ties (30), additional beneficial effects ofNAC have been described in septic shock,such as a vasodilatory effect (10, 31–33),associated with a reduction in serum lac-tate concentrations (34, 35), as well as areduction in nuclear factor kappa-B (36),tumor necrosis factor (34), and interleu-kin-8 levels (36, 37) with reduced expres-sion of endothelial receptors vascular celladhesion molecule-1 and endothelial leu-kocyte adhesion molecule. However,some of these findings were not repro-duced in other studies (38, 39). Vasodila-tory effects might be less relevant in se-vere malaria, where microcirculatoryhypoperfusion is mainly determined bythe mechanical obstruction resultingfrom sequestration of parasitized redblood cells (8). In patients with severemalaria, no changes in plasma concentra-tions of tumor necrosis factor-�, inter-leukin-6, interleukin-8, or interferon-gamma could be demonstrated withhigh-dose NAC treatment (13). In themouse model, antioxidants other thanNAC have been shown to reduce mortal-ity (40), and different antioxidant sub-stances vary in their effect on immunefunction (41). It cannot, therefore, be ex-cluded that other antioxidants mighthave a beneficial effect in human pa-tients. However, the pathogenesis of ce-rebral malaria in mice is quite distinctfrom the disease in humans.

In conclusion, this study showed thatalthough measures of oxidative stresswere increased in severe malaria, no ben-eficial effects from the use of high-doseNAC as an adjunctive treatment to intra-venous artesunate could be observed inthe setting of the study.

ACKNOWLEDGMENTS

We thank the staff and nurses fromMae Sot General Hospital in Thailand andChittagong Medical College Hospital inBangladesh and Dr. Samuel Douthwaitefor their dedication and help.

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