Plasmodium knowlesi occurs throughout Southeast Asia, and is the most common cause of human malaria in Malaysia. Severe disease in humans is characterised by high parasite biomass, reduced red blood cell deformability, endothelial activation and microvascular dysfunction. However, the roles of intravascular haemolysis and nitric oxide (NO)- dependent endothelial dysfunction, important features of severe falciparum malaria, have not been evaluated, nor their role in acute kidney injury (AKI). In hospitalised Malaysian adults with severe (n = 48) and non-severe (n = 154) knowlesi malaria, and in healthy controls (n = 50), we measured cell-free haemoglobin (CFHb) and assessed associations with the endothelial Weibel–Palade body (WPB) constituents, angiopoietin-2 and osteoprotegerin, endothelial and microvascular function, and other markers of disease severity. CFHb was increased in knowlesi malaria in proportion to disease severity, and to a greater extent than previously reported in severe falciparum malaria patients from the same study cohort. In knowlesi malaria, CFHb was associated with parasitaemia, and independently associated with angiopoietin-2 and osteoprotegerin. As with angiopoietin-2, osteoprotegerin was increased in proportion to disease severity, and independently associated with severity markers including creatinine, lactate, interleukin-6, endothelial cell adhesion molecules ICAM-1 and E-selectin, and impaired microvascular reactivity. Osteoprotegerin was also independently associated with NO-dependent endothelial dysfunction. AKI was found in 88% of those with severe knowlesi malaria. Angiopoietin-2 and osteoprotegerin were both independent risk factors for acute kidney injury. Our ﬁndings suggest that haemolysis-mediated endothelial activation and release of WPB constituents is likely a key contributor to end-organ dysfunction, including AKI, in severe knowlesi malaria. 5, 6 1, 7 Introduction falciparum malaria , and fatal cases occur . Features of The monkey parasite Plasmodium knowlesi is an severe knowlesi malaria are similar to those of severe important emerging zoonotic infection in Southeast Asia, falciparum malaria in adults, and include hyperpar- and is now the most common cause of human malaria in asitaemia, jaundice, acute kidney injury (AKI), respiratory 1, 2 3, 4 5, 6 Malaysia and regions of western Indonesia . The risk distress, shock and metabolic acidosis . However, in of severe disease in adults is at least as high as in contrast to P. falciparum, P. knowlesi-attributed coma has not been reported to-date, and endothelial cytoadherence, a key pathogenic feature of severe falciparum malaria, 8, 9 does not appear to occur . Thus, alternative pathogenic Correspondence: Bridget E. Barber (email@example.com) Global and Tropical Health Division, Menzies School of Health Research, mechanisms may play a greater role in severe knowlesi Darwin, NT, Australia malaria. We have recently reported that, as with falci- Infectious Diseases Society Sabah-Menzies School of Health Research Clinical parum malaria, disease severity in knowlesi malaria is Research Unit, Kota Kinabalu, Sabah, Malaysia Full list of author information is available at the end of the article. © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1234567890():,; 1234567890():,; Barber et al. Emerging Microbes & Infections (2018) 7:106 Page 2 of 10 associated with parasite biomass, endothelial activation, controls. Clinical and pathophysiological data from a 10 5, 10 and microvascular dysfunction , as well as reduced red subset of these patients have been previously reported . blood cell (RBC) deformability . However, the roles of Baseline demographic, clinical and laboratory features of intravascular haemolysis and nitric oxide (NO)-dependent patients and controls are shown in Table 1. endothelial dysfunction, important features of severe fal- Among the 48 patients with severe knowlesi malaria, 12–16 ciparum malaria , have not yet been reported. WHO severity criteria included hyperparasitaemia (n = In conditions associated with intravascular haemolysis, 24, 50%), jaundice (n = 21, 44%), respiratory distress (n = such as severe falciparum malaria, the cell-free hae- 14, 29%), severe AKI by WHO criteria (Cr > 265 mmol/L; moglobin (CFHb) released during erythrocyte rupture is n = 11, 23%), shock (n = 11, 23%), metabolic acidosis (n 2+ 3+ readily oxidised from ferrous (Fe ) to ferric (Fe ) hae- = 4, 8%), severe anaemia (n = 5, 10%) and abnormal moglobin. Ferric haemoglobin (methaemoglobin) then bleeding (n = 5, 8%). Nineteen patients (40%) had one releases haem , which due to its hydrophobic nature severity criterion, 17 (35%) had two and 12 (25%) had readily intercalates into cell membranes and increases three or more. Using KDIGO criteria to deﬁne AKI (and susceptibility to oxidant-mediate damage . Free haem the MDRD equation to estimate baseline creatinine, see mediates a range of other pathogenic effects, including 'Materials and Methods'), AKI was present on admission increased production of reactive oxygen species and in 40 (83%) patients with severe malaria and 44 (29%) 19, 20 proinﬂammatory cytokines , and upregulation of patients with non-severe malaria. AKI developed during endothelial cell adhesion molecules . CFHb is also able to admission in another two (4%) patients with severe and quench nitric oxide (NO), and in adults with falciparum two (1%) patients with non-severe malaria. malaria is associated with reduced NO-dependent endo- thelial function, and hyperlactatemia, suggesting a role in Intravascular haemolysis impaired tissue perfusion . CFHb was signiﬁcantly higher in patients with severe CFHb has also been shown to stimulate degranulation of and non-severe knowlesi malaria compared to controls endothelial Weibel–Palade bodies (WPBs) via (67,923 ng/mL, 37,568 ng/mL and 15,146 ng/mL, respec- 22, 23 TLR4 signalling . WPBs are storage organelles speciﬁc tively, p < 0.0001 for both comparisons), and higher in to endothelial cells, and upon endothelial activation, fuse those with severe compared to non-severe disease (p < with endothelial cell membranes and release their contents 0.0001) (Table 1 and Fig. 1). CFHb was higher in patients into plasma. Thus, constituents of WPBs, including von with severe knowlesi malaria compared to patients with Willebrand factor (vWF), angiopoietin-2, P-selectin, and severe falciparum malaria from the same cohort (69,923 osteoprotegerin (OPG), are considered key markers and ng/mL vs. 35,322 ng/mL [n = 21; data previously pub- mediators of endothelial activation. Plasma concentrations lished ], p = 0.015). Haptoglobin was lower in patients of vWF have been shown to increase soon after inoculation with severe and non-severe knowlesi malaria compared to of P. falciparum into human volunteers , and both vWF controls (0.11 g/dL, 0.30 g/dL, and 1.44 g/dL, respectively, 25, 26 and OPG increase early in P. berghei-infected mice , p < 0.0001 for both comparisons), and lower in patients suggesting that endothelial activation is an early host with severe compared to non-severe knowlesi malaria (p response in malaria. In both knowlesi and falciparum = 0.004). In patients with severe and non-severe knowlesi malaria, endothelial activation is associated with disease malaria, there was no signiﬁcant difference in CFHb or 10, 27 severity and measures of impaired organ perfusion . haptoglobin in those enrolled prior to, compared to post, In severe knowlesi malaria, high parasitaemias can commencement of antimalarial treatment (Supplemen- develop rapidly, and intravascular haemolysis has been tary Table 1). reported . However, the contribution of this process to disease severity, and the association with endothelial Endothelial and microvascular function activation and dysfunction, has not been evaluated. We Endothelial function, as measured by the reactive- therefore measured CFHb in Malaysian adults with severe hyperaemia index (RHI), was lower in patients with and non-severe knowlesi malaria, and assessed associa- severe knowlesi malaria compared to those with non- tions with the endothelial WPB constituents angiopoietin- severe knowlesi malaria (median RHI 1.47 vs. 1.87, p < 2 and osteoprotegerin, endothelial and microvascular 0.0001; Table 1). However, there was no signiﬁcant dif- function, and other markers of disease severity. ference between controls and patients with non-severe knowlesi malaria. In patients with severe knowlesi Results malaria, endothelial function was at least as low as it was Patients in patients with severe falciparum malaria from the same A total of 202 patients with knowlesi malaria were cohort (1.47 vs. 1.53 [1.26–1.75], n = 17, p = 0.778; pre- enroled, including 154 with non-severe and 48 with severe viously published ). Endothelial function was associated malaria by WHO criteria , in addition to 50 healthy with P. knowlesi parasitaemia (r = −0.20, p = 0.044), and Barber et al. Emerging Microbes & Infections (2018) 7:106 Page 3 of 10 Table 1 Baseline characteristics of patients with knowlesi malaria and healthy controls Variable Controls Non-severe knowlesi Severe knowlesi malaria P value (severe vs. NS (n = 50) malaria (n= 48) knowlesi malaria) (n = 154) Age 35 (22–43) 40 (28–53) 55 (47–62) <0.0001 Male sex, n (%) 34 (68) 122 (79) 35 (73) NS Fever duration, days NA 5 (4–7) 6 (3–7) NS Time from malaria treatment to NA 5.8 (0–11.8) 6.5 (0–12.1) NS enrolment, h Parasites/µL NA 3781 (979–13,680) 98,974 (24,034–164,304) <0.0001 Haemoglobin, g/dL, mean (SD) NA 12.9 (1.5) 11.9 (2.1) 0.0002 Haemoglobin nadir, g/dL, mean (SD) NA 11.7 (1.5) 9.4 (2.0) <0.0001 Haemoglobin fall, g/dL NA 1.2 (0.5–1.8) 2.2 (1.6–3.2) <0.0001 Cell-free haemoglobin, ng/mL 15,146 (9641–25,256) 37,568 (17,168–55,251) 67,923 (29,292–163,848) <0.0001 Haptoglobin, g/dL 1.44 (1.01–1.72) 0.30 (0.07–1.18) 0.11 (0.04–0.21) 0.004 N = 99 N = 47 Platelets, ×10 /µL NA 51 (36–76) 31 (21–57) 0.0001 Creatinine, µmol/L NA 95 (77–113) 144 (112–207) <0.0001 Bilirubin, µmol/L NA 17 (13–25) 39 (24–88) <0.0001 Aspartate transaminase, IU/L NA 40 (29–52) 58 (39–103) <0.0001 Alanine transaminase, IU/L NA 40 (24–62) 37 (21–57) NS Lactate, mmol/L NA 1.2 (0.9–1.5) 1.5 (1.1–2.3) 0.0001 N = 134 Interleukin-6, pg/mL BDL 27/30 38 (18–83) 182 (56–353) <0.0001 N = 97 N = 47 WBP constituents Angiopoietin-2, pg/mL 1,183 (875–1597) 4,296 (2943–6323) 10,072 (6311–14,072) <0.0001 P-selectin, pg/mL 40 (31–52) 31 (25–39) 39 (30–51) 0.0008 N = 153 N = 46 Osteoprotegerin, pg/mL 986 (625–1463) 2087 (1605–3008) N = 153 4795 (3184–7535) N = 46 <0.0001 vWF, pg/mL 1156 (843–1634) 5328 (3952–6188) 5140 (4555–6336) NS N = 38 N = 47 Adhesion molecules ICAM-1, pg/mL 149 (123–167) 469 (363–621) 563 (430–703) 0.004 E-selectin, pg/mL 19 (13–25) 49 (36–66) 63 (50–90) 0.0003 Microvascular reactivity, units/s 6.62 (5.43–7.25) N = 43 6.1 (5.3–6.9) 3.5 (2.8–5.3) <0.0001 N = 59 N = 41 Endothelial function (RHPAT) 1.97 (1.7–2.27) 1.87 (1.59–2.23) 1.47 (1.33–1.79) <0.0001 N = 64 N = 38 Data are median (IQR) unless otherwise stated NS non-severe, NA not assessed, BDL below detection limit, WPB Weibel–Palade body, vWF von Willebrand factor, ICAM-1 intercellular adhesion molecule-1, RHPAT reactive-hyperaemia peripheral arterial tonometry Barber et al. Emerging Microbes & Infections (2018) 7:106 Page 4 of 10 p<0.0001 A. B. p<0.0001 10 p<0.0001 p<0.0001 p=0.586 p<0.0001 Controls Non-severe Pk Severe Pk Controls Non-severe Pk Severe Pk n=50 n=154 n=48 n=50 n=64 n=38 p<0.0001 C. D. p<0.0001 p<0.0001 p<0.0001 100000 100000 p<0.0001 p<0.0001 Controls Non-severe Pk Severe Pk Controls Non-severe Pk Severe Pk n=50 n=154 n=48 n=50 n=153 n=46 Fig. 1 Cell-free haemoglobin (a), endothelial function (b), angiopoietin-2 (c) and osteoprotegerin (d) in patients with severe and non-severe knowlesi malaria, and healthy controls with lactate (−0.24, p = 0.024), with the latter remaining for parasitaemia (p = 0.001). Using logistic regression and signiﬁcant after controlling for parasitaemia (p = 0.041). controlling for parasitaemia, log CFHb was associated There was no association between endothelial function with risk of AKI by KDIGO criteria on or during admis- and CFHb. sion (OR 1.52 [95% CI: 1.12–2.07], p = 0.008). There was Microvascular function, as measured by NIRS, was no association between CFHb and patient age. reduced in patients with knowlesi malaria in proportion CFHb was correlated with aspartate transaminase (r = to disease severity (Table 1, and as previously reported ). 0.38, p < 0.0001), likely reﬂecting release of this enzyme There was a positive correlation between endothelial and from RBCs . There was no association between CFHb microvascular function as measured by RHI and NIRS, and the other liver aminotransaminase, alanine respectively (r = 0.35, p <0.001), remaining signiﬁcant transaminase. after controlling for parasitaemia and age (r = 0.244, p = 0.020). Cell-free haemoglobin and association with WPB constituents Cell-free haemoglobin and markers of disease severity As CFHb has been shown to stimulate degranulation of 22, 23 CFHb was correlated with parasitaemia (r = 0.24, WPBs , we evaluated plasma concentrations of the p<0.001) (Table 2). CFHb also correlated with lactate WPB constituents angiopoietin-2, OPG, P-selectin, and (r = 0.20, p = 0.006), and with microvascular dysfunction VWF, and their associations with CFHb. Angiopoietin-2 (r = 0.35, p <0.001), with both correlations remaining was increased in patients with knowlesi malaria compared signiﬁcant after controlling for parasitaemia (p = 0.040 to controls, and increased in severe compared to non- and 0.028, respectively). CFHb correlated with creatinine severe disease (Table 1, Fig. 1 and as previously reported (0.31, p < 0.0001), remaining signiﬁcant after controlling in a subset of these patients ). Similarly, OPG was Angiopoietin 2 (pg/ml) Cell free haemoglobin (ng/ml) Osteoprotegerin (pg/ml) Reactivehyperemia index Barber et al. Emerging Microbes & Infections (2018) 7:106 Page 5 of 10 Table 2 Cell-free haemoglobin and correlations with Table 3 Logistic regression model for predictors of acute markers of severity in knowlesi malaria kidney injury and severe malaria in knowlesi malaria Univariate analysis Controlling for Odds 95% Conﬁdence P value parasitaemia ratio interval Correlation P value Correlation P value Predictors of AKI coefﬁcient coefﬁcient Log angiopoietin-2 4.41 2.02–9.63 <0.0001 Log osteoprotegerin 1.98 1.02–3.82 0.043 Parasite count 0.23 <0.001 Age 1.07 1.04–1.10 <0.0001 Creatinine 0.31 <0.0001 0.23 0.001 Predictor of severe malaria Lactate 0.20 0.006 0.15 0.040 Log angiopoietin-2 5.35 2.01–14.25 0.001 AST 0.37 <0.0001 0.35 <0.0001 Log osteoprotegerin 2.66 1.15–6.14 0.022 ALT 0.09 0.222 NA Log parasite count 1.47 1.15–1.89 0.002 IL-6 0.28 <0.001 0.23 0.001 Microvascular −0.35 <0.001 −0.22 0.028 AKI acute kidney injury as deﬁned by KDIGO. Backward stepwise regression was used, with variables removed if P value was >0.05. Variables included in both reactivity models included: age, angiopoietin-2, osteoprotegerin, parasite count, and cell- free haemoglobin. Patients with hyperparasitaemia as a sole severity criterion Angiopoietin-2 0.33 <0.0001 0.24 0.001 were reclassiﬁed as non-severe for this analysis. Alternative regression models are included in Supplementary Data OPG 0.37 <0.0001 0.34 <0.0001 Age remained an independent risk factor of AKI if included as a binary variable ICAM-1 0.17 0.019 0.12 0.081 of >45 years (OR 6.09 [95% CI: 2.91–12.77], P < 0.0001). For predictors of severe malaria, age was not an independent risk factor, whether included as a E-selectin 0.28 <0.0001 0.18 0.010 continuous variable, or as a binary variable of >45 years Univariate correlations were calculated using Spearman’s correlation coefﬁcient. Partial correlation was used to control for parasitaemia, with all variables log- transformed. Correlations with parasitaemia, OPG, AST and IL-6 all remained remained as independent risk factors for severe malaria signiﬁcant after also controlling for angiopoietin-2. No association was found (with hyperparasitaemia removed as a severity criterion), between cell-free haemoglobin and the Weibel–Palade body (WBP) constituents P-selectin or vWF and for AKI (Table 3; alternative logistic regression AST aspartate transaminase, ALT alanine transaminase, IL interleukin, OPG models shown in Supplementary Tables 2 and 3). vWF osteoprotegerin, ICAM-1 intercellular adhesion molecule, NA not assessed was also associated with risk of AKI on univariate analysis (odds ratio for log-transformed vWF 4.13 [95% CI: increased in severe compared to non-severe knowlesi 1.07–15.98], p = 0.040); however, this did not remain malaria (median 4795 vs. 2087 pg/mL, p < 0.0001), and signiﬁcant after controlling for parasitaemia and age. increased in both groups compared to controls (p < 0.0001 for both comparisons). No increase in P-selectin was seen Osteoprotegerin and correlation with endothelial cell in knowlesi malaria patients overall compared to controls, adhesion molecules and IL-6 and VWF, although increased in knowlesi malaria patients As with angiopoietin-2 (Table 4, and as previously compared to controls, was not increased in severe com- reported ), OPG was also associated with ICAM-1 (r = pared to non-severe disease (Table 1). OPG correlated 0.31, p < 0.0001) and E-selectin (r = 0.34, p < 0.0001), with with angiopoietin-2, after controlling for age and para- both correlations remaining signiﬁcant after controlling sitaemia (r = 0.39, p < 0.0001). for parasitaemia and age (p ≤ 0.0001 for both correla- CFHb correlated with angiopoietin-2 (r = 0.33, p < tions). OPG was also correlated with IL-6 (r = 0.54, p < 0.0001) and OPG (r = 0.37, p < 0.0001), with both corre- 0.0001), remaining signiﬁcant after controlling for para- lations remaining signiﬁcant after controlling for para- sitaemia and age (p < 0.0001). The correlations between sitaemia (p = 0.0005 and p < 0.0001, respectively; Table 2). OPG and E-selectin, ICAM-1 and IL-6 were also inde- The association between CFHb and OPG remained sig- pendent of angiopoietin-2 (Table 4). niﬁcant after also controlling for angiopoietin-2. Both angiopoietin-2 and OPG correlated with age in Osteoprotegerin and markers of disease severity knowlesi malaria patients (r = 0.39, p < 0.0001, and r = In addition to the association with adhesion molecules 0.43, p < 0.0001, respectively), independent of para- and IL-6, after controlling for age and parasitaemia OPG sitaemia. OPG also correlated with age in healthy controls was also independently correlated with all other malaria (r = 0.30, p = 0.033). Age is a known risk factor for severe severity markers evaluated, including creatinine (r = 0.36, knowlesi malaria . However, in a backward stepwise p < 0.0001), lactate (r = 0.31, p < 0.0001), microvascular logistic regression model controlling for age and para- dysfunction (r = 0.23, 0.024) and endothelial dysfunction sitaemia, both OPG and angiopoietin-2 (but not CFHb) (r = 0.26, p = 0.011) (Table 4). These associations were at Barber et al. Emerging Microbes & Infections (2018) 7:106 Page 6 of 10 Table 4 Comparative correlations between Weibel–Palade body constituents OPG and angiopoietin-2 and biomarkers of severity in knowlesi malaria OPG Angiopoietin-2 Univariate analysis Controlling for parasitaemia Univariate analysis Controlling for parasitaemia and age and age Correlation P value Correlation P value Correlation P value Correlation P value coefﬁcient coefﬁcient coefﬁcient coefﬁcient a a Age 0.43 <0.0001 0.32 <0.0001 0.39 <0.0001 0.30 <0.0001 b b Parasite count 0.45 <0.0001 0.38 <0.0001 0.46 <0.0001 0.32 <0.0001 Creatinine 0.40 <0.0001 0.36 <0.0001 0.54 <0.0001 0.54 <0.0001 Lactate 0.38 <0.0001 0.31 <0.0001 0.34 <0.0001 0.25 0.0009 AST 0.32 <0.0001 0.29 0.0001 0.30 <0.0001 0.29 0.0001 IL-6 0.57 <0.0001 0.34 0.0001 0.45 <0.0001 0.26 0.0002 Microvascular −0.48 <0.0001 −0.23 0.024 −0.46 <0.0001 −0.22 0.029 reactivity Angiopoietin-2 0.52 <0.0001 0.39 <0.0001 VWF 0.26 0.018 0.32 0.004 0.28 0.010 0.28 0.010 P-selectin 0.23 0.0009 0.14 0.039 0.18 0.010 NS ICAM-1 0.31 <0.0001 0.27 0.0001 0.36 <0.0001 0.33 <0.0001 E-selectin 0.34 <0.0001 0.31 <0.0001 0.30 <0.0001 0.26 0.0002 RHPAT −0.39 0.0001 0.26 0.011 −0.23 0.020 NS All biomarkers of severity remained signiﬁcantly correlated with OPG after also controlling for angiopoietin-2, except microvascular reactivity and ICAM-1. In contrast, after controlling for OPG, only creatinine, AST and ICAM-1 remained signiﬁcantly associated with angiopoietin-2 Controlling for parasitaemia only Controlling for age only least as strong as with the well-validated malaria severity cells both contributing. The greater severity of intravas- biomarker angiopoietin-2, and, in the case of lactate, AST, cular haemolysis in severe knowlesi compared to P. fal- creatinine, and endothelial dysfunction, were independent ciparum may reﬂect the 24-h erythrocytic life-cycle of P. of angiopoietin-2 (Table 4). knowlesi, or may reﬂect poor adaption of P. knowlesi to the human host. Massive destruction of RBCs has been Discussion previously reported in P. knowlesi-infected rhesus maca- Intravascular haemolysis (as measured by CFHb) is ques (Macaca mulatta), another unnatural host for this increased in knowlesi malaria in proportion to disease parasite. As well as the red cell agglutination and sludging severity, and to a greater extent than that seen in falci- reported in these early studies , haemolytic phenomena parum malaria. Furthermore, intravascular haemolysis is such as haemoglobinuria, renal tubular acidosis and independently associated with markers of disease severity, haemoglobin casts within tubular lumens were frequently 33, 34 including lactate, microvascular dysfunction, and creati- noted as pre-terminal events . The marked haemolysis nine, suggesting that haemolysis likely contributes to associated with P. knowlesi makes this parasite an ideal impaired tissue perfusion and organ dysfunction in model to study the pathophysiological consequences of knowlesi malaria. With the apparent paucity of P. falci- haemolysis in severe human malaria. parum-like endothelial cytoadherence causing sequestra- The mechanisms by which haemolysis mediates end- tion in knowlesi malaria, our ﬁndings suggest that organ damage in severe malaria are incompletely under- intravascular haemolysis may play a more central role in stood. In severe falciparum malaria, haemolysis is asso- the development of severe disease in knowlesi compared ciated with impaired NO-dependent endothelial to falciparum malaria. dysfunction , and with oxidative damage which con- As with falciparum malaria, the cause of intravascular tributes to AKI . In addition, we now show that in haemolysis in severe knowlesi malaria is likely multi- knowlesi malaria, intravascular haemolysis is indepen- factorial, with lysis of infected and uninfected red blood dently associated with the endothelial cell WPB Barber et al. Emerging Microbes & Infections (2018) 7:106 Page 7 of 10 constituents angiopoietin-2 and OPG, suggesting that production . The association between OPG and endo- endothelial activation is likely a key mediator of thelial dysfunction in severe knowlesi malaria suggests haemolysis-induced end-organ damage. An association OPG likely exacerbates endothelial NO deﬁciency, con- between CFHb and angiopoietin-2 has been previously tributing to severe disease. OPG inhibition of eNOS and 12 13 demonstrated in adults and children with falciparum endothelial NO production is reversed in vitro by malaria; however, we now extend these ﬁndings by RANKL . Taken together, these ﬁndings raise the pos- demonstrating an association in P. knowlesi between sibility that RANKL may be a candidate adjunctive CFHb and both angiopoietin-2 and OPG that is inde- treatment to improve NO bioavailability in conditions pendent of parasite biomass. Our ﬁndings are consistent associated with elevated OPG and endothelial dysfunction with previous in vitro and murine reports demonstrating such as severe malaria. 22, 23 that CFHb stimulates degranulation of WPBs . In the In addition to its release from endothelial cells, OPG is current study, the lack of independent associations with also expressed in immune cells, including dendritic cells CFHb and markers of end-organ damage, after controlling and macrophages, and may modulate inﬂammatory for angiopoietin-2 and/or OPG, further supports the role responses through inhibition of RANKL/RANK signal- of endothelial activation in mediating pathophysiological ling . As RANKL has been shown to reduce macrophage consequences of intravascular haemolysis. production of proinﬂammatory cytokines , inhibition of While increased angiopoietin-2 is well documented in RANKL by OPG may be expected to increase inﬂamma- severe malaria, and known to be a key marker of disease tory responses. In keeping with this, in our study, OPG 10, 27 severity , a notable ﬁnding of our study was the was independently associated with IL-6. This is also marked elevation of the other key WPB constituent, OPG, consistent with a murine study, in which inﬂammatory in severe knowlesi malaria. OPG is a member of the cytokines, including IL-6, TNF, IL-1B and MCP-1, were tumour necrosis factor (TNF) receptor superfamily, and is reduced in OPG knockout mice and in WT mice infused a soluble decoy receptor for the receptor activator of NF- with RANKL . кB ligand (RANKL), thus modulating the interaction In this study we found that OPG and angiopoietin-2 between RANKL and its receptor RANK . OPG has a were both independently associated with AKI, suggesting widespread tissue distribution, including in vascular and that haemolysis-induced endothelial activation is an immune tissues. In vascular tissues, release of OPG from important mechanism of malaria-associated AKI. AKI is endothelial cells is upregulated by cytokines including common in knowlesi malaria, occurring in 44% of all 36, 37 TNF, IL-1a and IL-1b patients in this study, and in 88% of those with severe . OPG has been shown to sti- mulate endothelial cell migration , to increase leukocyte disease. AKI is now recognised to have signiﬁcant long- adhesion to endothelial cells both in vitro and in vivo , term consequences, including increased risk of chronic and to upregulate endothelial cell adhesion molecules in kidney disease, cardiovascular disease and death (reviewed 40 48 the presence of TNF . This latter effect of OPG is con- in ref. ), and new treatment strategies to prevent sistent with the results of our current study, with OPG malaria-associated AKI are needed. In falciparum malaria, independently associated with endothelial adhesion haemolysis has been linked to AKI from oxidative stress molecules ICAM-1 and E-selectin. Upregulation of and lipid peroxidation , and further studies are war- adhesion molecules by OPG may also explain in part the ranted to determine if haemolysis-induced oxidative stress association between OPG and mortality in a recent study also contributes to AKI in knowlesi malaria. The patho- of African children with cerebral malaria . genic pathways of CFHb may present targets for adjunc- We also found an independent association of OPG with tive treatments to protect against AKI in both falciparum 50, 51 endothelial and microvascular dysfunction, as measured and knowlesi malaria . by RHPAT and NIRS, respectively. This is consistent with Our study is associated with several limitations. First, other studies which have demonstrated an association although our ﬁndings suggest that haemolysis-induced between OPG and endothelial dysfunction in other con- endothelial activation and WPB release may be key ditions, including hyperuricemia , Hashimoto’s thyr- pathogenic mechanisms in severe malaria, it is possible 42 43 oiditis and type 1 diabetes mellitus . In addition, OPG that release of WPB constituents may also occur through is elevated in other conditions associated with endothelial alternate mechanisms, such as direct effect of parasite dysfunction, such as cardiovascular disease, and in products, or cytokines induced at schizogony . Parasite patients with diabetes mellitus is associated with adverse products may directly stimulate endothelial cells and have cardiovascular outcomes and mortality . Endothelial been implicated in WBP exocytosis in falciparum dysfunction is a key feature of severe malaria, resulting malaria . Interestingly, the related parasite Cryptospor- from reduced NO bioavailability . OPG is known to idium has been shown to upregulate OPG mRNA in block RANKL-induced activation of the intracellular intestinal epithelial cells, with the increase in OPG serving eNOS pathway in vitro, and to reduce endothelial NO as an anti-apoptotic and parasite survival strategy . Barber et al. Emerging Microbes & Infections (2018) 7:106 Page 8 of 10 Nevertheless, in our study the association between CFHb equation , with an assumed eGFR of 100 mL/min per 1.73 and OPG was independent of both parasitaemia and IL-6, m . Healthy controls were visitors or relatives of malaria consistent with a direct role of CFHb in WPB release. patients, with no history of fever in the past 48 h and with Second, although we have demonstrated an increase in blood ﬁlm negative for malaria parasites. OPG in severe knowlesi malaria and hypothesise that this Standardised history and physical examination were is a result of WBP exocytosis, OPG is also expressed in documented. Haematology, biochemistry, acid–base other tissues (such as vascular smooth muscle cells and parameters and lactate (by bedside blood analysis; iSTAT macrophages). Thus, we cannot conﬁrm that endothelial system) were obtained on enrolment. Parasite counts were cells are the source of the increased plasma OPG. How- determined by microscopy, and parasite species identiﬁed 57, 58 ever, the consistent ﬁnding of increased endothelial acti- by PCR . Patients with severe disease were treated 10, 27, 54, 55 vation in severe malaria , the very early increases with intravenous artesunate, while those with non-severe 24, 25 in plasma OPG observed in other studies , and the disease received oral artemisinin combination treatment, concurrent elevation of and correlation with endothelial as previously described . cell-speciﬁc marker angiopoietin-2, suggest that endo- thelial cells are a likely source of such markedly elevated Laboratory assays levels of OPG. Venous blood collected in lithium heparin and citrate In conclusion, we have demonstrated that intravascular tubes was centrifuged (including a second high-spin speed haemolysis is increased in severe knowlesi malaria, and to for the citrate tube) within 30 min of collection and a greater extent than falciparum malaria. Furthermore, we plasma stored at −70 °C. Plasma CFHb and vWF were demonstrate that CFHb is independently associated with measured on the citrated platelet-free plasma by ELISA angiopoietin-2 and OPG, and that OPG is associated with (Bethyl Laboratories and Biomedica Diagnostics, respec- endothelial cell adhesion molecules and microvascular tively). Haptoglobin was measured on lithium heparin and endothelial dysfunction, as well as with clinical bio- plasma by ELISA (ICL Laboratories). Plasma concentra- markers of severity, including lactate and AKI. These tions of angiopoietin-2, P-selectin and adhesion molecules ﬁndings suggest that haemolysis-mediated endothelial ICAM-1 and E-selectin were measured on lithium heparin activation and release of WPB constituents, including plasma using quantikine ELISA kits from RnD. OPG was OPG, is likely a key contributor to end-organ dysfunction measured on lithium heparin plasma using a duoset in severe knowlesi malaria. ELISA from RnD. IL-6 was measured by ﬂow cytometry (BD cytometric bead array, Becton Dickinson). Materials and methods Ethics statement Measurement of endothelial and microvascular function The study was approved by the Ethics Committees of Endothelial function was measured non-invasively on the Malaysian Ministry of Health and Menzies School of enrolment using peripheral arterial tonometry (EndoPAT) Health Research. Informed written consent was provided by the change in digital pulse wave amplitude in response by all participating adults, and by the parent or guardian to reactive hyperaemia, giving a reactive hyperaemia of any participant aged <18 years. peripheral arterial tonometry (RHPAT) index, as pre- viously described . The RHPAT index is at least 50% Study site and patients dependent on endothelial NO production and has been Patients were enrolled as part of a prospective observa- shown to be L-arginine responsive in falciparum tional study of all malaria patients admitted to Queen malaria . Measurement of endothelial function was dis- Elizabeth Hospital, an adult tertiary-referral hospital in continued on patients with non-severe malaria in July 5, 10 Sabah, Malaysia . For the current study, patients enroled 2011. Microvascular function was assessed on enrolment between September 2010 and December 2012 were inclu- as previously described , using near infra-red spectro- ded if they had PCR-conﬁrmed P. knowlesi monoinfection, scopy (InSpectra 650, Hutchinson Technology, Hutch- were non-pregnant, ≥12 years old, had no major comor- inson, MN) as previously reported . bidities or concurrent illness and were within 18 h of commencing antimalarial treatment. Severe malaria was Statistics deﬁned according to modiﬁed WHO criteria, as previously Statistical analysis was performed with STATA software described . Renal function was further assessed using the (version 14). For continuous variables, intergroup differ- kidney disease: Improving Global Outcomes (KDIGO) ences were compared using analysis of variance or criteria for AKI. Using this deﬁnition, AKI is deﬁned as an Kruskal–Wallis tests depending on distribution. Student’s increase in serum creatinine of ≥26.5 µmol/L within 48 h, t-test or Wilcoxon–Mann–Whitney tests were used for or to ≥1.5× baseline . Baseline creatinine was estimated two-group comparisons. Categorical variables were com- using modiﬁcation of diet in renal disease (MDRD) pared using χ or Fisher’s exact tests. Associations between Barber et al. Emerging Microbes & Infections (2018) 7:106 Page 9 of 10 continuous variables were assessed using Spearman’s cor- 3. Herdiana, H. et al. Malaria risk factor assessment using active and passive surveillance data from Aceh Besar, Indonesia, a low endemic, malaria elim- relation coefﬁcient. Partial correlation was used to evaluate ination setting with Plasmodium knowlesi, Plasmodium vivax,and Plasmodium associations between variables after adjusting for para- falciparum. Malar. J. 15, 468 (2016). sitaemia, with non-normally distributed variables log- 4. Lubis, I. N. et al. Contribution of Plasmodium knowlesi to multispecies human malaria infections in North Sumatera, Indonesia. J. Infect. Dis. 215,1148–1155 transformed to normality. Backward stepwise regression (2017). was used to evaluate predictors of severe malaria and AKI, 5. Barber,B.E.etal. A prospective comparativestudy of knowlesi,falciparumand with variables removed at a signiﬁcance level of >0.05. For vivax malaria in Sabah, Malaysia: high proportion with severe disease from Plasmodium knowlesi and P. vivax but no mortality with early referral and this analysis, patients with hyperparasitaemia as a sole artesunate therapy. Clin. Infect. Dis. 56,383–397 (2013). severity criterion were reclassiﬁed as having non-severe 6. Grigg, M. J. et al. Age-related clinical spectrum of Plasmodium knowlesi malaria malaria. For comparison of intravascular haemolysis in P. and predictors of severity. Clin. Infect. Dis. (2018). In Press 7. Rajahram,G.S. et al. Deaths due to Plasmodium knowlesi malaria in Sabah, knowlesi vs. P. falciparum malaria, median plasma CFHb in Malaysia: association with reporting as P. malariae and delayed parenteral patients with severe knowlesi malaria was compared to artesunate. Malar. J. 11, 284 (2012). previously published CFHb measurements from patients 8. Cox-Singh, J. et al. Severe malaria-a case of fatal Plasmodium knowlesi infection with post-mortem ﬁndings. Malar. J. 9, 10 (2010). with severe falciparum malaria enroled contemporaneously 9. Govindasamy, G. et al. Retinal changes in uncomplicated and severe Plas- in the same study cohort . modium knowlesi malaria. J. Infect. 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Alterations in systemic extracellular heme and hemo- Research Council (Programme Grants 496600 and 1037304, Project Grant pexin are associated with adverse clinical outcomes in Ugandan children with 1045156 and fellowships to B.E.B., M.G., T.W.Y. and N.M.A.). A.M.D. was severe malaria. J. Infect. Dis. 214,1268–1275 (2016). supported by the Wellcome Trust of Great Britain. K.P. was supported by the 14. Dalko, E. et al. Multifaceted role of heme during severe Plasmodium falciparum Clinician Investigator Programme at the University of British Columbia, Canada. infections in India. Infect. Immun. 83,3793–3799 (2015). We thank all the patients enroled in the prospective study at Queen Elizabeth 15. Plewes, K. et al. 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