Plasma superoxide dismutase-1 as a surrogate marker of vivax malaria severity

Abstract

Background: Severe outcomes have been described for both Plasmodium falciparum and P. vivax infections. The identification of sensitive and reliable markers of disease severity is fundamental to improving patient care. An intense pro-inflammatory response with oxidative stress and production of reactive oxygen species is present in malaria. Inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and antioxidant agents such as superoxide dismutase-1 (SOD-1) are likely candidate biomarkers for disease severity. Here we tested whether plasma levels of SOD-1 could serve as a biomarker of severe vivax malaria.

Methodology/principal findings: Plasma samples were obtained from residents of the Brazilian Amazon with a high risk for P. vivax transmission. Malaria diagnosis was made by both microscopy and nested PCR. A total of 219 individuals were enrolled: non-infected volunteers (n = 90) and individuals with vivax malaria: asymptomatic (n = 60), mild (n = 50) and severe infection (n = 19). SOD-1 was directly associated with parasitaemia, plasma creatinine and alanine amino-transaminase levels, while TNF-alpha correlated only with the later enzyme. The predictive power of SOD-1 and TNF-alpha levels was compared. SOD-1 protein levels were more effective at predicting vivax malaria severity than TNF-alpha. For discrimination of mild infection, elevated SOD-1 levels showed greater sensitivity than TNF-alpha (76% vs. 30% respectively; p<0.0001), with higher specificity (100% vs. 97%; p<0.0001). In predicting severe vivax malaria, SOD-1 levels exhibited higher sensitivity than TNF-alpha (80% vs. 56%, respectively; p<0.0001; likelihood ratio: 7.45 vs. 3.14; p<0.0001). Neither SOD-1 nor TNF-alpha could discriminate P. vivax infections from those caused by P. falciparum.

Conclusion: SOD-1 is a powerful predictor of disease severity in individuals with different clinical presentations of vivax malaria.

Figure 1. Plasma SOD-1 and TNF-alpha as markers of severe vivax malaria.

A, SOD-1 protein and TNF-alpha plasma levels according to vivax malaria clinical severity. NI, non-infected volunteers (n = 90); Asy, asymptomatic infection (n = 60); Sympt, symptomatic infection (n = 69). Differences among the groups were calculated using the Kruskal Wallis analysis of variance with Dunn's multiple comparisons test. B, Plasma levels of SOD-1 and TNF-alpha in individuals with mild P. vivax infection (n = 50) compared to those with severe vivax malaria (Sev; n = 19). Boxes represent median and interquartile interval; whiskers represent maximum and minimum values. Differences were estimated using Mann-Whitney test. Lines represent median values. P values are shown in each graph.

Figure 2. Correlations between plasma SOD-1 or TNF-alpha concentrations and laboratory parameters of malaria severity.

Correlation of SOD-1 or TNF-alpha with several laboratory parameters in symptomatic vivax malaria patients (n = 69). Column at left (A, C and E): Correlations of SOD-1 with parasitaemia (A), plasma creatinine (C) and alanine amino-transaminase (ALT; E). Column at right: Correlations of TNF-alpha to parasitaemia (B), plasma creatinine (D) and ALT (F). Correlation between TNF-alpha and SOD-1 plasma protein levels is shown in G. The statistical significances were calculated using the Spearman test. The values of p and r are illustrated in each graph.

Figure 3. Effect of anti-malaria treatment on plasma concentrations of SOD-1 and TNF-alpha in individuals with severe vivax malaria.

Plasma levels of SOD-1 protein (red boxes, left Y axis) and TNF-alpha (blue boxes, right Y axis) were estimated before treatment (at admission to the Hospital) and after seven days of in-hospital treatment with intravenous quinine and hemodynamic support in individuals with severe vivax infection who successful recovered (n = 13). Boxes represent median and interquartile interval; whiskers represent maximum and minimum values. Wilcoxon matched pairs test was performed to calculate the statistical significance. P values are plotted in each graph.

Figure 4. Effectiveness of plasma concentrations of SOD-1 and TNF-alpha measurements as markers of vivax malaria severity.

A, ROC curves of SOD-1 (dashed line) and TNF-alpha (solid line) plasma levels for discriminating asymptomatic infection from mild P. vivax malaria cases. B, ROC curves of SOD-1 (dashed line) and TNF-alpha levels (solid line) for discriminating severe from mild P. vivax malaria cases. C-statistics are illustrated in the tables and were used to verify the validation of the ROC curves and the predictive power of each biomarker. AUC, area under the curves; CI, confidence interval.

Figure 5. Plasma concentrations of SOD-1 and TNF-alpha during P. vivax and P. falciparum infections.

Plasma levels of SOD-1 (red boxes) and TNF-alpha (blue boxes) were measured in patients with mild P. vivax (n = 50) or mild P. falciparum (n = 16) malaria. Boxes represent median and interquartile interval; whiskers represent maximum and minimum values. The differences between P. vivax and P. falciparum infections were not significant when compared by the Mann-Whitney test (p>0.05).