Abstract
Background
Pathogenesis of severe Plasmodium vivax malaria is poorly understood. Endothelial dysfunction and reduced nitric oxide (NO) bioavailability characterize severe falciparum malaria, but have not been assessed in severe vivax malaria.
Methods
In patients with severe vivax malaria (n = 9), patients with nonsevere vivax malaria (n = 58), and healthy controls (n = 79), we measured NO-dependent endothelial function by using reactive hyperemia–peripheral arterial tonometry (RH-PAT) and assessed associations with arginine, asymmetric dimethylarginine (ADMA), and hemolysis.
Results
The L-arginine level and the L-arginine to ADMA ratio (a measure of L-arginine bioavailability) were reduced in patients with severe vivax malaria and those with nonsevere vivax malaria, compared with healthy controls (median L-arginine level, 65, 66, and 98 µmol/mL, respectively [P = .0001]; median L-arginine to ADMA ratio, 115, 125, and 187, respectively [P = .0001]). Endothelial function was impaired in proportion to disease severity (median RH-PAT index, 1.49, 1.73, and 1.97 in patients with severe vivax malaria, those with nonsevere vivax malaria, and healthy controls, respectively; P = .018) and was associated with the L-arginine to ADMA ratio. While the posttreatment fall in hemoglobin level was greater in severe vivax malaria as compared to nonsevere vivax malaria (2.5 vs 1 g/dL; P = .0001), markers of intravascular hemolysis were not higher in severe disease.
Conclusions
Endothelial function is impaired in nonsevere and severe vivax malaria, is associated with reduced L-arginine bioavailability, and may contribute to microvascular pathogenesis. Severe disease appears to be more associated with extravascular hemolysis than with intravascular hemolysis.
Keywords: malaria, Plasmodium vivax, arginine, asymmetric dimethylarginine, endothelial function, nitric oxide, hemolysis
Plasmodium vivax is the most widespread malaria parasite, causing an estimated 13.8 million malaria cases per year [1, 2]. Although previously considered benign, P. vivax is now recognized as a cause of severe and fatal disease [3–8]. Despite this, little is known about the pathogenesis of disease in severe vivax malaria.
In falciparum malaria, a central process in pathogenesis of severe disease is cytoadherence of parasitized erythrocytes to activated endothelium, leading to microvascular sequestration and obstruction with consequent tissue hypoxia and organ dysfunction [9, 10]. Microvascular dysfunction is an additional process associated with tissue hypoxia in severe falciparum malaria [11, 12], with impaired microvascular reactivity associated with both impaired tissue perfusion and death [11]. Recent analyses in falciparum malaria have shown that both microvascular sequestration and endothelial activation are independent predictors of mortality [13], suggesting that both processes contribute to the pathogenesis of severe falciparum malaria. Reduced endothelial nitric oxide (NO) bioavailability contributes to each of these processes, with reduced NO levels shown to be associated with increased parasite cytoadherence [14], increased endothelial activation, and endothelial dysfunction [12, 15, 16]. Several factors have been shown to contribute to the impairment of NO bioavailability in falciparum malaria, including reduced expression of type 2 NO synthase in mononuclear cells [17], reduced concentrations of the NO precursor L-arginine [15, 18], inhibition of NO synthase (NOS) by asymmetric dimethylarginine (ADMA) [19–22], and reduced concentration of the NOS cofactor tetrahydrobiopterin [23, 24]. Intravascular hemolysis has also been shown to be increased in severe falciparum malaria, with NO quenching by cell-free hemoglobin further limiting endothelial NO bioavailability [25].
Compared with falciparum malaria, coma in vivax malaria is very rare [26]. However, other organ dysfunction and hypotension can occur, and severe disease in vivax malaria is well described [7, 8, 27, 28]. Severe vivax malaria occurs at lower levels of peripheral parasitemia than those usually seen in severe falciparum malaria, and the pathogenesis of organ dysfunction in vivax malaria is not well understood [3–8]. We have recently shown evidence in vivax malaria of a hidden biomass underestimated by peripheral parasitemia, with the total P. vivax biomass (as measured by parasite lactate dehydrogenase [pLDH] level) associated with systemic inflammation [27]. While there is little evidence of sequestration of parasitized cells within endothelium-lined vessels [5, 27], P. vivax–parasitized erythrocytes may accumulate in nonendothelial cell–lined vessels and/or extravascular tissues, such as the spleen or bone marrow [4, 27]. We have also shown that in vivax malaria endothelial activation and impaired microvascular reactivity are associated with impaired tissue perfusion and disease severity [27] and are of comparable magnitude in severe vivax malaria and severe falciparum malaria. However, the roles of endothelial function and microvascular NO bioavailability in the pathogenesis of vivax malaria have not been assessed.
In this study we measured endothelial function in patients with vivax malaria and assessed factors that have been shown to correlate with reduced NO bioavailability in severe falciparum malaria—arginine, ADMA, and markers of hemolysis, including cell-free hemoglobin (CFHb). Healthy controls were included for comparison.
METHODS
Ethics Statement
The study was approved by the ethics committees of the Malaysian Ministry of Health (Kota Kinabalu) and the Menzies School of Health Research (Darwin, Australia).
Study Site and Patients
Patients were enrolled as part of a prospective clinical and epidemiological study of all malaria patients admitted to Queen Elizabeth Hospital, an adult tertiary care referral hospital in Sabah, Malaysia [29]. Consecutive patients with polymerase chain reaction (PCR)–confirmed vivax monoinfection were enrolled during September 2010–June 2013 if they were nonpregnant, ≥12 years old, had no major comorbidities or concurrent illness, were within 18 hours of commencing antimalarial treatment, and had not been previously enrolled in the study. Clinical details of these patients have been previously reported [27, 29]. Severe malaria was defined as the presence of ≥1 of the following: unrousable coma (Glasgow coma scale score, <11), multiple (>2) convulsions, respiratory distress (respiratory rate of >30 breaths/minute and oxygen saturation of <94%), hypotension (systolic blood pressure, ≤80 mm Hg), jaundice (bilirubin level of >43 µmol/L plus parasitemia level of >20 000 parasites/µL and/or creatinine level of >132 µmol/L), significant abnormal bleeding, hypoglycemia (blood glucose level, <2.2 mmol/L), metabolic acidosis (bicarbonate level of <15 mmol/L or lactate level of >4 mmol/L), acute kidney injury (AKI; creatinine level, >265 µmol/L), and a hemoglobin level of <7.5 g/dL. Healthy controls were visitors or relatives of patients with malaria who had no history of fever in the past 48 hours and a blood film negative for malaria parasites.
Standardized history and physical examination findings were documented. Hematologic and biochemical findings, acid-base parameters, and lactate level (obtained by bedside blood analysis; iSTAT system) were obtained on enrollment. Parasite counts were determined by microscopy, and parasite species were identified by PCR [30, 31]. Patients were treated according to hospital guidelines, as previously described [29].
Laboratory Assays
Venous blood collected in tubes coated with lithium heparin was centrifuged within 30 minutes of collection, and plasma was stored at −70°C. ADMA and arginine levels were measured using reverse-phase high-performance liquid chromatography with simultaneous fluorescence and UV-visible absorbance detection, as previously described [32]. Plasma concentrations of the endothelial activation markers intracerebral adhesion molecule-1 (ICAM-1) and angiopoietin-2 were measured using an enzyme-linked linked immunosorbent assay (ELISA; R&D Systems), and interleukin 6 (IL-6) and interleukin 10 (IL-10) levels were measured by flow cytometry (BD Cytometric Bead Array). Plasma haptoglobin and CFHb levels were measured by ELISA according to the manufacturers' (ICL Laboratories and Bethyl Laboratories, respectively) instructions. Red blood cell arginase is released during intravascular hemolysis, and plasma arginase activity was measured as an additional measure of hemolysis by using a radiometric assay, as described elsewhere [33]; findings are reported in micromoles per milliliter per hour. P. vivax biomass was estimated on the basis of the pLDH level, measured by ELISA [34].
Measurement of Endothelial Function
Endothelial function was measured noninvasively, using peripheral arterial tonometry, by the change in digital pulse wave amplitude in response to reactive hyperemia, giving a reactive hyperemia peripheral arterial tonometry (RH-PAT) index as previously described [15]. The RH-PAT index is at least 50% dependent on endothelial NO production [35] and has been shown to be L-arginine responsive in falciparum malaria [15]. Endothelial function was measured on enrollment and repeated on day 3 in patients who remained hospitalized. Measurement of endothelial function was discontinued in patients with nonsevere malaria, in May 2011.
Statistical Analysis
Statistical analysis was performed with STATA software (version 13.0). For continuous variables, intergroup differences were compared using analysis of variance or Kruskal–Wallis tests, depending on the distribution of data. Student t tests or Mann–Whitney tests were used for pair-wise comparisons. Categorical variables were compared using χ2 or Fisher exact tests. Associations between continuous variables were assessed initially using pair-wise correlation to control for disease severity, with data log transformed to exhibit a normal distribution. The Spearman correlation coefficient was used to assess for association between variables within the severity groups. Multiple linear regression was used to adjust for confounding variables. The Wilcoxon signed rank test was used to compare day 0 and day 3 data.
RESULTS
Patients
A total of 67 patients with vivax malaria were enrolled, including 58 with nonsevere and 9 with severe disease, in addition to 79 healthy controls. The epidemiological, clinical, and biochemical features, including measures of parasite biomass, have been previously reported [27, 29]. Baseline characteristics are shown in Tables 1 and 2. Among the 9 patients with severe vivax malaria, severity criteria included hypotension (n = 6), respiratory distress (n = 1), jaundice (n = 2), metabolic acidosis (n = 1), abnormal bleeding (n = 1), and multiple convulsions (n = 1). Three patients had 2 severity criteria, and 6 had 1 criterion. Cultures of blood specimens obtained before antibiotic treatment yielded negative results for 4 patients, were positive for Streptococcus pneumoniae for 1 [29], and were not done for 4. No deaths occurred. As previously reported [27], the parasite count and pLDH level, a marker of P. vivax biomass, were higher in patients with severe vivax malaria as compared to those with nonsevere vivax malaria.
Table 1.
Baseline Characteristics of Patients With Plasmodium vivax Malaria and Healthy Controls
| Characteristic | Healthy Controls (n = 79) | Patients With Vivax Malaria |
||
|---|---|---|---|---|
| Nonsevere (n = 58) | Severe (n = 9) | P Value | ||
| Age, y | ||||
| Median (IQR) | 35 (23–44) | 24 (18–40) | 39 (31–53) | .171 |
| Range | 14–69 | 13–61 | 14–79 | |
| Male sex, no. (%) | 57 (72) | 37 (73) | 9 (100) | .075 |
| Weight, kg | 58 ± 10 | 56 ± 12 | 58 ± 8 | .584 |
| Current smoker, no. (%) | 33 (42) | 24 (47) | 1 (11) | .045 |
| Fever duration, d, median (IQR) | 5 (3–7) | 4 (3–4) | .135 | |
| Enrollment systolic blood pressure, mm Hg | 123 ± 15 | 114 ± 16 | 119 ± 18 | .400 |
| Enrollment mean arterial blood pressure, mm Hg | 109 ± 13 | 99 ± 13 | 104 ± 16 | .573 |
| Minimum systolic blood pressure, mm Hg | NA | 100 ± 9 | 85 ± 15 | <.0001 |
| Pulse rate, beats/min | 71 ± 12 | 88 ± 20 | 97 ± 14 | .197 |
| Respiratory rate, breaths/min | 20 ± 3 | 24 ± 5 | 26 ± 4 | .210 |
| Temperature, °C | 36.5 ± 0.4 | 37.5 ± 1.2 | 37.0 ± 0.5 | .220 |
| Time from malaria treatment to enrollment, h, median (IQR)a | NA | 4.5 (0–11) | 9.7 (6–15) | .117 |
Data are mean ± SD, unless otherwise indicated, and are from all subjects who had endothelial function measured and/or blood analysis performed.
Abbreviations: IQR, interquartile range; NA, not applicable.
a A total of 14 of 58 patients with nonsevere vivax malaria and 1 of 9 with severe vivax malaria were enrolled prior to commencing antimalarial treatment.
Table 2.
Laboratory and Physiological Measurements Among Patients With Plasmodium vivax Malaria and Healthy Controls
| Characteristic | Healthy Controls | Patients With Vivax Malaria |
P Value |
||
|---|---|---|---|---|---|
| Nonsevere (n = 58) | Severe (n = 9) | All Groups | Malaria Groups | ||
| Parasite count, parasites/µL | … | 4055 (1599–10 165) | 10 243 (4387–19 520) | .020 | |
| Parasite LDH level,a ng/mL | … | 44.6 (7.8–143) (n = 53) |
307 (258–619) | .016 | |
| Hemoglobin level, g/dL, mean ± SD | |||||
| Overall | … | 12.2 (2.04) | 13.9 (1.22) | .015 | |
| Nadir | … | 11.2 (1.98) | 11.4 (0.8) | .847 | |
| Absolute decrease | … | 1 (0.4–1.5) | 2.5 (1.7–3.4) | .0001 | |
| CFHb level, µM | 15 146 (9641–25 256) (n = 50) |
32 498 (20 068–45 189) (n = 48) |
31 338 (9889–35 824) | .0001 | .161 |
| Plasma LDH level, µL | 213 (174–290) (n = 50) |
311 (254–420) (n = 57) |
295 (272–344) | .0001 | .550 |
| Haptoglobin levela, g/L | 1.44 (1.01–1.72) (n = 60) |
0.27 (0.07–1.09) (n = 53) |
0.28 (0.035–0.530) | .0001 | .846 |
| Plasma arginase clearance rate, µM/mL/h | 0.115 (0.078–0.192) (n = 48) |
0.121 (0.074–0.168) (n = 51) |
0.073 (0.039–0.094) (n = 8) |
.069 | .035 |
| Creatinine level, μmol/L | … | 82 (67–99) (n = 57) |
128 (94–136) | .011 | |
| Bilirubin level, μmol/L | … | 17 (11.6–25.1) (n = 55) |
25.5 (13–41) | .132 | |
| Lactate level, mmol/L | … | 1.14 (0.78–1.47) (n = 51) |
1.28 (1–2.23) | .162 | |
| Angiopoietin-2 level, pg/mL | 1183 (875–1597) (n = 50) |
4557 (3463–6197) (n = 53) |
8857 (6547–9743) | .0001 | .001 |
| ICAM-1 level, pg/mL | 149 (123–167) (n = 50) |
505 (416–639) (n = 53) |
686 (682–832) | .0001 | .077 |
| IL-6 level, pg/mL | Below detection limit in 27/30 patients | 27.8 (9.1–93.6) (n = 46) |
49.5 (36.1–54.0) | .0001 | .369 |
| IL-10 level, pg/mL | Below detection limit in 29/30 patients | 185.5 (41.3–504.1) (n = 46) |
173 (107–319) | .0001 | .733 |
| L-arginine level, µmol/mL | 98.4 (80.1–112.8) (n = 51) |
67.4 (54–82.5) | 65.1 (55.6–71) | .0001 | .520 |
| ADMA level, µM | 0.540 (0.490–0.595) (n = 51) |
0.553 (0.446–0.642) | 0.533 (0.504–0.541) | .792 | .526 |
| L-arginine:ADMA ratio | 187 (153–213) | 125 (109–142) | 115 (103–127) | .0001 | .270 |
| Endothelial functionb | 1.97 (1.64–2.27) (n = 79) |
1.73 (1.46–2.05) (n = 30) |
1.49 (1.37–1.88) (n = 8) |
.018 | .237 |
Data are median value (interquartile range), unless otherwise indicated. Investigations are performed on enrollment, unless otherwise stated. Where a result is below the detection limit, half the lower limit of detection is substituted.
Abbreviations: ADMA, asymmetric dimethylarginine; CFHb, cell-free hemoglobin; ICAM-1, intracerebral adhesion molecule-1; IL-6, interleukin 6; IL-10, interleukin 10; LDH, lactate dehydrogenase.
a Levels were below the detection limit in 1 of 60 controls, 11 of 53 patients with nonsevere vivax malaria, and 3 of 9 patients with severe vivax malaria.
b Determined on the basis of the reactive hyperemia peripheral arterial tonometry index.
Levels of Plasma Arginine and ADMA and Ratio of Arginine to ADMA
Plasma arginine levels were reduced in the severe and nonsevere vivax malaria groups as compared to the control group (median level, 65, 66, and 98 µmol/mL, respectively; P = .0001; Table 1 and Figure 1), although there was no difference between malaria severity groups. There was no difference in plasma ADMA levels on enrollment between patients with vivax malaria and healthy controls. The arginine to ADMA ratio (reflecting arginine bioavailability) was lower in patients with severe or nonsevere vivax malaria, compared with controls (median ratio, 115, 125, and 187, respectively; P = .0001; Figure 1).
Figure 1.
Plasma concentrations of arginine and asymmetric dimethylarginine (ADMA), the arginine to ADMA ratio (a measure of arginine bioavailability), and endothelial function (determined on the basis of the reactive hyperemia peripheral arterial tonometry [RH-PAT] index) in patients with severe Plasmodium vivax malaria, those with nonsevere P. vivax malaria, and healthy controls.
The arginine level was strongly correlated with the ADMA level in all patients with vivax malaria (r = 0.55, P < .0001), with this relationship remaining significant after adjustment for disease severity (Table 3). The ADMA level was inversely correlated with the IL-10 level in all patients with vivax malaria, after adjustment for disease severity (r = −0.37, P = .006). The ADMA level was positively associated with the ICAM-1 level (r = 0.48, P = .0001) but not with the angiopoietin-2 level in all patients with vivax malaria, after adjustment for disease severity, and in both severity groups. In a multivariate model including disease severity, ICAM-1, IL-6, IL-10, and arginine levels, ADMA levels remained significantly associated with the arginine level and inversely associated with the IL-10 level. There was no association between ADMA and CFHb levels in patients with vivax malaria.
Table 3.
Correlations With Endothelial Function and Asymmetric Dimethylarginine (ADMA)–Associated Values in Patients With Nonsevere or Severe Plasmodium vivax Malaria
| Correlation | Patients With Vivax Malaria, by Severity |
|||||
|---|---|---|---|---|---|---|
| Overalla |
Nonsevere |
Severe |
||||
| Rb | P Value | Rb | P Value | Rb | P Value | |
| Between RH-PAT indexc and | ||||||
| Arginine level | 0.407 | .011 | 0.438 | .015 | 0.017 | .968 |
| ADMA level | − 0.106 | .527 | ||||
| Arginine:ADMA ratio | 0.505 | .001 | 0.568 | .001 | −0.217 | .606 |
| CFHb level | 0.396 | .017 | 0.435 | .018 | 0.237 | .572 |
| Angiopoietin-2 level | −0.203 | .221 | ||||
| ICAM-1 level | −0.004 | .979 | ||||
| Between ADMA leveld and | ||||||
| Arginine level | 0.551 | <.0001 | 0.555 | <.0001 | 0.489 | .182 |
| CFHb level | 0.048 | .722 | ||||
| IL-6 level | −0.234 | .085 | −0.267 | .073 | 0.149 | .702 |
| IL-10 level | −0.365 | .006 | −0.421 | .004 | 0.194 | .617 |
| ICAM-1 level | 0.479 | .0001 | 0.430 | .001 | 0.853 | .004 |
| Angiopoietin-2 level | 0.204 | .112 | ||||
Abbreviations: ADMA, asymmetric dimethylarginine; CFHb, cell-free hemoglobin; ICAM-1, intracerebral adhesion molecule-1; IL-6, interleukin 6; IL-10, interleukin 10; RH-PAT, reactive hyperemia peripheral arterial tonometry index.
a Bivariate model adjusting for disease severity.
b Pair-wise correlation coefficient, with data for all variables log transformed to normality.
c The reactive hyperemia peripheral arterial tonometry (RH-PAT) index was determined as a measure of endothelial function on 30 patients with nonsevere vivax malaria and 8 patients with severe vivax malaria. The index remained significantly correlated with the arginine to ADMA ratio (P = .019) in a multivariate model including disease severity and CFHb.
d The ADMA level was measured in 58 patients with nonsevere vivax malaria and 9 patients with severe vivax malaria. The level remained significantly associated with the arginine level (P = .049) and inversely associated with the IL-10 level (P = .005) in a multivariate model including disease severity, IL-6 level, and ICAM-1 level.
Endothelial and Microvascular Function
Endothelial function, as measured by the RH-PAT index, was lower in patients with severe or nonsevere vivax malaria, compared with healthy controls (median RH-PAT index, 1.49, 1.73, and 1.97, respectively; P = .018; Figure 1). Endothelial function was inversely correlated with the arginine level and the arginine to ADMA ratio in all patients with vivax malaria, after adjustment for disease severity (Table 3), with the association with the arginine to ADMA ratio remaining significant on multivariate analysis. There was no correlation between endothelial function and levels of endothelial activation markers or lactate.
Measures of Intravascular Hemolysis
CFHb and plasma LDH levels were higher and the haptoglobin level lower in patients with severe or nonsevere vivax malaria, compared with controls (Table 2). However, there was no evidence that intravascular hemolysis increased with the severity of vivax malaria, with no difference in CFHb, haptoglobin, or LDH levels between severity groups. There was no difference in levels of plasma arginase (released from hemolyzed red blood cells) between healthy controls (0.115 µM/mL/hour) and patients with nonsevere vivax malaria (0.121 µM/mL/hour), and levels were unexpectedly lower in patients with severe disease (0.073 µM/mL/hour), compared with healthy controls (P = .026) and patients with nonsevere disease (P = .035). Arginase can also be released from alternatively activated (M2) monocytes or macrophages [36]. The M2 phenotype has previously been shown to be associated with IL-10 [36], and in the current study arginase level was correlated with the IL-10 level in patients with nonsevere (r = 0.29, P = .05) but not in those with severe (r = −0.35, P = ns) vivax malaria. The hemoglobin level on admission was higher in patients with severe vivax malaria as compared to those with nonsevere vivax malaria (13.9 vs 12.2 g/dL, P = .015), but the fall in hemoglobin level to the nadir level was greater in severe as compared to nonsevere disease (2.5 vs 1 g/dL; P = .0001). The CFHb level fell following treatment in patients with severe or nonsevere vivax malaria, with the decrease similar between both groups (Table 4).
Table 4.
Longitudinal Results in Patients With Severe or Nonsevere Plasmodium vivax Malaria
| Vivax Malaria Severity, Factor | Day 0 | Days 2–4 | P Value |
|---|---|---|---|
| Severe | |||
| Arginine level, µmol/L (n = 5) | 65 (60–68) | 127 (122–128) | .043 |
| ADMA level, µM (n = 5) | 0.506 (0.504–0.533) | 0.653 (0.576–0.823) | .043 |
| Arginine:ADMA ratio | 118 (105–127) | 156 (147–186) | .043 |
| RH-PAT index (n = 6) | 1.44 (1.31–1.54) | 2.04 (1.76–2.24) | .046 |
| CFHb level, µM (n = 7) | 35 434 (18 672–40 986) | 23 656 (17 281–36 700) | .128 |
| Nonsevere | |||
| Arginine level, µmol/L (n = 16) | 76 (61–94) | 127 (122–128) | .026 |
| ADMA level, µM (n = 16) | 0.610 (0.529–0.664) | 0.717 (0.638–0.864) | .0009 |
| Arginine:ADMA ratio | 131 (110–168) | 165 (135–194) | .179 |
| RH-PAT index (n = 14) | 1.84 (1.50–2.22) | 1.95 (1.67–2.00) | .975 |
| CFHb level, µM (n = 24) | 36 130 (33 551–57 546) | 27 918 (19 960–46 522) | .092 |
Data are median value (interquartile range).
Abbreviations: ADMA, asymmetric dimethylarginine; CFHb, cell-free hemoglobin; RH-PAT, reactive hyperemia peripheral arterial tonometry.
The median CFHb level was higher in the 3 patients with severe vivax malaria without hypotension, compared with those with hypotension (35 824 µM and 14 372 µM, respectively); however, this was not statistically significant, and there was no correlation between CFHb level and mean arterial pressure in patients with vivax malaria. There was also no correlation between the CFHb level and levels of creatinine, lactate, or markers of endothelial activation (ICAM-1 and angiopoietin-2).
Longitudinal Course of Arginine Level, ADMA Level, Arginine to ADMA Ratio, and Endothelial Function
Arginine levels increased significantly from baseline to days 2–4 in patients with severe or nonsevere vivax malaria (Table 4 and Figure 2). In both patient groups, ADMA levels also increased, with day 3 levels being above those of healthy controls. Despite the increase in ADMA levels, the arginine to ADMA ratio increased in all patient groups from day 0 to days 2–4.
Figure 2.
Plasma concentrations of arginine and asymmetric dimethylarginine (ADMA), the arginine to ADMA ratio (a measure of arginine bioavailability), and endothelial function (determined on the basis of the reactive hyperemia peripheral arterial tonometry [RH-PAT] index) in patients with severe Plasmodium vivax malaria.
In patients with vivax malaria who had the RH-PAT index measured on day 3, median endothelial function improved, significantly so in patients with severe malaria (from 1.49 to 2.04 [n = 6]; P = .046) but not in those with nonsevere malaria (from 1.73 to 1.95 [n = 14]; P = .975). In all patients with vivax malaria, the improvement in endothelial function from baseline to day 3 was correlated with an increase in the arginine to ADMA ratio over the same period (r = 0.64, P = .035 [n = 11]).
DISCUSSION
In this study, we show that in vivax malaria arginine bioavailability (as measured by the arginine to ADMA ratio) is reduced and is associated with NO-dependent endothelial dysfunction. The degree of impairment of endothelial function in severe vivax malaria is comparable to that in patients with severe falciparum malaria from the same study cohort (median RHPAT index, 1.58 [IQR, 1.26–1.96] [21]. As has been demonstrated in falciparum malaria, endothelial function improved with recovery from vivax malaria, with improvement in endothelial function correlating with an increase in the arginine to ADMA ratio. Taken together, these results suggest that impaired L-arginine bioavailability contributes to endothelial dysfunction in vivax malaria.
In severe falciparum malaria, reduced endothelial NO bioavailability is partly accounted for by a reduced concentration of the NO precursor L-arginine [15]. In this study, the concentration of L-arginine was also low in patients with vivax malaria; however, arginine levels were similar between patients with severe disease and those with nonsevere disease. This is consistent with previous studies of Indonesian [15] and Malaysian [21] adults with falciparum malaria, in which arginine levels were also similar between patients with severe or nonsevere falciparum malaria. Nevertheless, arginine bioavailability (measured as the arginine to ADMA ratio) was lowest in severe falciparum malaria [19, 21] and thought contributory to the greater endothelial dysfunction found in those with severe disease [15]. In this study, although ADMA levels did not differ between patients with vivax malaria and healthy controls, the reduction in arginine bioavailability (ie, the arginine to ADMA ratio) in both uncomplicated and severe vivax malaria suggests that, as with falciparum malaria, ADMA levels are inappropriately high. Also, consistent with previous studies in falciparum malaria [21, 22], the ADMA level was strongly correlated with the arginine level. This association has been demonstrated in other acute inflammatory conditions [34, 37] and may reflect arginine-mediated inhibition of dimethylarginine-dimethylaminohydrolase [38] or competitive transport through cationic amino acid transporters [39]. As with falciparum malaria [21], in vivax malaria the ADMA level was inversely correlated with the IL-10 level, suggesting a role of inflammatory cytokines in ADMA regulation and consequent NO bioavailability.
In severe falciparum, reduced NO bioavailability also occurs as a result of increased intravascular hemolysis, leading to NO quenching from CFHb, degradation of arginine by erythrocyte-derived arginase, and inhibition of NOS by erythrocyte-derived ADMA [10]. In contrast, in the current study we did not find evidence that intravascular hemolysis was increased in severe as compared to nonsevere vivax malaria or that it was associated with the endothelial dysfunction in vivax malaria. Measures of intravascular hemolysis were not increased in severe compared to nonsevere vivax malaria, despite the higher circulating parasitemia level and higher total parasite biomass seen in patients with severe relative to those with nonsevere vivax malaria. Indeed, the level of plasma arginase, released from red cells during intravascular hemolysis, was lower in patients with severe malaria, compared with that in patients with nonsevere vivax malaria. Importantly, however, the fall in hemoglobin level was significantly greater during severe as compared to nonsevere vivax malaria, despite a posttreatment reduction in CFHb level in both groups. Taken together, these findings suggest that in severe vivax malaria there is a greater proportion of extravascular infected red cell loss, compared with nonsevere disease [40]. This notion is also consistent with our previous findings suggesting a greater accumulation of infected red blood cells in non–endothelial-lined and/or extravascular tissues in severe vivax malaria than in nonsevere vivax malaria, possibly in the spleen and bone marrow [27]. This is also further supported by the recent finding that P. vivax merozoites preferentially infect immature reticulocytes [41]. The lack of association of hemolysis with disease severity also suggests that heme-mediated toxicity may not be an important contributor to mechanisms of severe disease in vivax malaria.
In vivax malaria, hypotension occurs more commonly than in falciparum malaria [3, 28] and, in the current study, occurred in 6 of 9 patients. It is possible that this increased frequency of hypotension is due in part to a lesser degree of intravascular hemolysis and vascular NO quenching in severe vivax malaria than in severe falciparum malaria, potentially allowing greater vasodilation. Although we did not detect an association between the CFHb level and enrollment mean arterial pressure (MAP) in this study, the level of CFHb was nonsignificantly higher in the 3 patients without shock as compared to the 6 with shock. Larger series will be required to further evaluate the association between the CFHb level and MAP/systemic vascular resistance in severe vivax malaria.
A limitation of this study was the small number of patients with severe vivax malaria, which limited our ability to assess for associations with disease severity and other variables. In addition, a proportion of patients were enrolled following commencement of antimalarial treatment, which may have affected baseline measurements of ADMA levels, arginine levels, and/or endothelial function.
In conclusion, our study demonstrated that, as with falciparum malaria, NO-dependent endothelial function is impaired in vivax malaria, is related to arginine bioavailability, and improves following commencement of antimalarial treatment. In the absence of significant endothelial cytoadherence of P. vivax–infected red blood cells, these processes may contribute to impaired microvascular perfusion and disease pathogenesis. In contrast to falciparum malaria, however, intravascular hemolysis was not increased in severe disease, suggesting that this is not a significant cause of impaired NO bioavailability in severe vivax malaria. Furthermore, the greater fall in hemoglobin level during severe disease in the absence of greater intravascular hemolysis suggests that extravascular hemolysis may be a more important contributor to loss of infected and uninfected red cells in severe vivax malaria.
Notes
Acknowledgments. We thank all the patients enrolled in the prospective study at Queen Elizabeth Hospital and the clinical staff involved in their care; Uma Paramaswaran, Rita Wong, Beatrice Wong, Ann Wee, and Kelly Nestor, for assistance with clinical and laboratory study procedures; Sarah Auburn and Jutta Marfurt, for supervising the PCR assays; the Clinical Research Centre, Sabah, for logistical support; and the Director General of Health, Malaysia, for permission to publish this study.
Financial support. This work was supported by the Australian National Health and Medical Research Council (Program Grants 496600 and 1037304, project grant 1045156, and fellowships to N. M. A., T. W. Y., and B. E. B. and scholarship to M. J. G.).
Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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