Increased Blood Concentrations of Malondialdehyde in Plasmodium Infection: A Systematic Review and Meta-Analysis

Several studies have evaluated the relationship between malondialdehyde (MDA) concentrations and Plasmodium infections; however, the findings remain inconclusive. This study synthesized differences in MDA concentrations among patients with different levels of clinical severity, uninfected controls, and different Plasmodium species. The research protocol was registered in PROSPERO (CRD42023393540). Systematic literature searches for relevant studies were performed using the Embase, MEDLINE, Ovid, ProQuest, PubMed, Scopus, and Google Scholar databases. Qualitative and quantitative syntheses (meta-analyses) of distinct MDA concentrations between the disease groups were performed. Twenty-three studies met the eligibility criteria and were included in the systematic review. Overall, MDA concentrations were significantly elevated in participants with malaria relative to uninfected controls (p < 0.01, Cohen d: 2.51, 95% confidence interval (CI): 1.88–3.14, I2: 96.22%, 14 studies). Increased MDA concentrations in participants with malaria compared with uninfected controls were found in studies that enrolled patients with P. falciparum malaria (p < 0.01, Cohen d: 2.50, 95% CI: 1.90–3.10, I2: 89.7%, 7 studies) and P. vivax malaria (p < 0.01, Cohen d: 3.70, 95% CI: 2.48–4.92, I2: 90.11%, 3 studies). Our findings confirm that MDA concentrations increase during Plasmodium infection, indicating a rise in oxidative stress and lipid peroxidation. Thus, MDA levels can be a valuable biomarker for evaluating these processes in individuals with malaria. However, further research is necessary to fully elucidate the intricate relationship between malaria, antioxidants, oxidative stress, and the specific role of MDA in the progression of malaria.


Introduction
Lipid peroxidation is involved in the pathogenesis of various tissue injuries [1,2].Oxidative stress facilitates lipid peroxidation in the cell membrane, resulting in the formation of harmful substances such as aldehydes, including malondialdehyde (MDA) and 4-hydroxy-2-nonenal, as well as other toxic compounds [3].MDA, a well-known secondary product of lipid peroxidation, can serve as a biomarker for cell membrane damage [4].As a colorless liquid and potent oxidizing agent, MDA is naturally produced in response to oxidative stress [5].Thus, elevated MDA levels indicate increased oxidative stress through the process of lipid peroxidation [6].Increased levels of lipid peroxidation products have been linked to several human illnesses, including malaria [7][8][9].
In humans, malaria is caused by the infection of one or more of the five Plasmodium parasites (predominantly Plasmodium falciparum and P. vivax) through bites from Anopheles spp.mosquitoes [10].Malaria remains one of the leading causes of death among children under the age of 5 years in Africa [11].In response to Plasmodium infection, host cells release reactive oxygen species (ROS), which not only clear the parasite but also damage host cells and tissues, leading to severe pathologies [12].Many studies have reported elevated levels of oxidative stress markers, including MDA, in malaria patients [13][14][15][16][17]. Two basic processes lead to oxidative stress in malaria.First, the parasite structurally damages erythrocytes during its replication cycle, altering characteristics such as stiffness, viscosity, and volume [18,19] and resulting in oxidative stress due to the destruction of host hemoglobin.Second, the host immune system launches a number of defense mechanisms in response to oxidative stress, culminating in the release of free radicals by activated macrophages to combat the parasite [20,21].The production of reactive hydroxyl radicals due to mitochondrial oxidative stress has been linked to liver apoptosis in animals with malaria [22,23].Human erythrocytes infected with malaria parasites show increased levels of oxidative stress [24,25], which impacts disease severity by causing red cell lysis, leading to anemia and a reduction in iron concentration.
The relationship between MDA levels and Plasmodium infections has been investigated in the literature, but the results are inconsistent.Furthermore, MDA concentrations may vary according to the clinical severity of the disease or Plasmodium species.Therefore, this systematic review synthesized differences in MDA concentrations between participants with malaria and uninfected controls.Additionally, we examined variations in MDA levels between patients with severe and nonsevere malaria, as well as between patients with P. falciparum and P. vivax malaria.

Search Strategy
This research protocol was registered in PROSPERO (CRD42023393540).The study was conducted and reported according to the PRISMA protocol for reporting systematic reviews and meta-analyses [26].Systematic searches of the literature for relevant studies published up to 18 January 2023 were performed using the Embase, MEDLINE, Ovid, ProQuest, PubMed, and Scopus databases.The key terms used in the search strategy were (Propanedial OR Malonyldialdehyde OR Malonaldehyde OR Malonylaldehyde OR "Sodium Malondialdehyde") AND (malaria OR Plasmodium OR "Remittent Fever" OR "Marsh Fever" OR Paludism).For the search in PubMed, the MeSH terms were identified in the search strategy as "(((malaria) OR (malaria [MeSH Terms])) OR (Plasmodium)) OR (Plasmodium [MeSH Terms]) AND (Malonyldialdehyde) OR (Malonyldialdehyde[MeSH Terms])."The search strategy used in different databases differed slightly according to the format of each database (Table S1).The publication date and language were not restricted.The literature searches were also conducted using Google Scholar to ensure that no relevant articles were overlooked and to identify additional relevant articles.

Inclusion and Exclusion Criteria
The eligibility criteria for the review were determined using the patient, intervention, comparison, outcome (PICO) framework [27] as follows: P, participants with malaria; I, none; C, uninfected controls; and O, MDA concentration.The following articles were considered eligible for this review: (i) studies in which malaria cases were diagnosed using a single method (or combination of methods), including microscopy, a rapid diagnostic test, serology, or molecular methods; (ii) studies that evaluated MDA concentration in malaria cases using thiobarbituric acid assay [28]; and (iii) studies that recruited healthy or febrile participants as uninfected controls.Reviews, systematic reviews, meta-analyses, animal studies, in vitro studies, comments, letters to the editor, and case reports were excluded.

Study Selection and Data Extraction
The articles were input into EndNote version 20.0 (Clarivate Analytics, Philadelphia, PA, USA), duplicates were removed, and the remaining articles were checked for eligibility.After removing irrelevant articles, the full texts of the remaining articles were examined to see if they met the requirements.Studies that failed to meet the eligibility requirements were then removed, and a clear explanation was given.Data on the study and participant characteristics and the PICO criteria were extracted from each study, including the name of the first author, publication year, study design and area, the number of participants and age range, MDA concentrations in malaria patients and uninfected controls, Plasmodium identification methods, and MDA concentration methods.Study selection and data extraction were performed independently by two authors (O.M. and M.K.), and any disagreements were discussed with another author (A.M.).

Quality Assessment
The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklists for observational studies were used to assess the quality of the studies [29].These checklists are used to score a maximum of 22 points for each study across all sections of the article: title and abstract, 1; introduction, 2; method, 9; results, 5; discussion, 4; and other information, 1.The quality assessment was performed independently by two authors (O.M. and W.M.), and any disagreements were discussed to reach a final agreement.Studies with a STROBE score percentage of <50%, 50-75%, and >75% were considered low-, moderate-, and high-quality studies, respectively [30] (Table S2).

Data Syntheses
Qualitative synthesis was used to explain qualitative differences in MDA concentrations in the disease groups (malaria vs. uninfected controls, severe vs. nonsevere malaria, and P. falciparum vs. P. vivax infection).Quantitative synthesis (meta-analysis) of distinct MDA concentrations between the disease groups (malaria vs. uninfected controls, severe vs. nonsevere malaria, and P. falciparum vs. P. vivax infection) was performed using the random-effect model [31].I 2 statistics were utilized to assess between-study heterogeneity, with I 2 values <50, 50-75, and >75 categorized as having low, moderate, and high between-study heterogeneity, respectively [30].Meta-regression and subgroup analyses were performed to investigate the potential source(s) of between-study heterogeneity.The funnel plot, Egger test, and trim-and-fill methods were used to determine the publication bias [32].The leave-one-out meta-analysis method was used to determine whether one study affected the overall effect estimate [33].Statistical analyses were performed using STATA 17.0 (StataCorp, College Station, TX, USA).

Summary Characteristics of the Included Studies
Of the 23 included studies, most (65.2%) were published between 2010 and 2022 and had a cross-sectional design (91.2%).The majority were performed in African countries (41.8%) and Asian countries (41.8%), with Nigeria and India the most representative countries in Africa (63.6%) and Asia (72.7%), respectively.Most studies enrolled patients infected with P. falciparum (47.8%), and most study participants were adults (39.1%).Over half of the studies used microscopy alone for Plasmodium detection (52.2%) (Table 1).

Quality of the Included Studies
Based on the STROBE checklist, seven studies (21.7%) were of high quality, and the remainder (78.3%) were of moderate quality.None of the studies were of low quality (Table S3).To investigate whether the quality of the studies affected the pooled effect estimate, a meta-regression analysis was performed using the study's quality as a covariate.

MDA Concentrations in Severe and Nonsevere Malaria
A qualitative synthesis showed increased MDA concentrations in severe and nonsevere malaria patients in a study by Sakyi et al. [48].Regarding patients with severe malaria with different complications, Villaverde et al. showed no difference in MDA concentrations between children with malaria retinopathy-positive cerebral malaria and children with malaria retinopathy-negative cerebral malaria [51].The analysis of differences in MDA concentrations between patients with severe and nonsevere malaria using the meta-analysis approach could not be performed due to an insufficient number of studies.

MDA Concentrations in Severe and Nonsevere Malaria
A qualitative synthesis showed increased MDA concentrations in seve nonsevere malaria patients in a study by Sakyi et al. [48].Regarding patients with malaria with different complications, Villaverde et al. showed no difference in concentrations between children with malaria retinopathy-positive cerebral mala children with malaria retinopathy-negative cerebral malaria [51].The anal differences in MDA concentrations between patients with severe and nonsevere m using the meta-analysis approach could not be performed due to an insufficient n

MDA in P. falciparum and P. vivax Malaria
In two studies [37,50], a qualitative synthesis showed increased MDA concentrations in P. falciparum malaria compared with P. vivax malaria.Lower MDA concentrations in P. falciparum malaria compared with P. vivax malaria have been reported [41].Differences in MDA concentrations between patients with P. falciparum and P. vivax malaria using the meta-analysis approach could not be determined due to the insufficient number of studies.

Sensitivity Analysis and Publication Bias
In all reruns using the leave-one-out meta-analysis method, participants with malaria had higher MDA concentrations than the uninfected controls (Figure 5).The funnel plot in the comparison analysis of MDA concentrations between participants with malaria and uninfected controls showed asymmetry (Figure 6).The Egger test revealed a statistically significant small-study effect (p < 0.01).The trim-and-fill analysis showed increased MDA concentrations in malaria cases compared with uninfected controls (Cohen d: 1.70, 95% CI: 1.58-1.81).

Discussion
MDA is a well-known marker of oxidative stress in several diseases [7][8][9].It is generated by free radicals that cause membrane lipid peroxidation and has been shown to deplete antioxidant levels, increase proinflammatory cytokines, and increase oxidative stress [56].The present study confirmed previous reports of increased MDA levels in participants with malaria compared with uninfected controls [7,[34][35][36][37][38][39][40][41][42][43][44][45][47][48][49][50][53][54][55].It is possible that lipid peroxidation on erythrocyte membranes-which are vulnerable to oxidative damage-is the cause of the elevated MDA levels found in malaria patients.The lipid peroxides released into the bloodstream by erythrocyte membranes that have suffered oxidative damage are degraded, resulting in increased MDA concentrations [57].These findings suggest that Plasmodium infection in humans results in the host's release of ROS, which aid in parasite clearance [12].However, high levels of these ROS can harm host cells and tissues, predisposing them to severe disease outcomes [12].The primary cause may be the malaria parasite's dependence on hemoglobin as a source of vital amino acids necessary for growth and maintenance during the erythrocytic stage of its life cycle [58].As a result, the extent of hemoglobin degradation depends on malaria severity.A low hemoglobin level suggests increased oxidative stress, which is reflected by an increased level of MDA but a decreased level of antioxidants [59].Thus, MDA levels can be used to measure the disease severity in malaria patients along with other evaluations.
The subgroup meta-analysis revealed elevated MDA concentrations in cases of malaria caused by P. falciparum or P. vivax.Due to the limited number of studies investigating MDA levels in both of these Plasmodium species, the results exhibit significant heterogeneity and lack clarity.Specifically, only two studies [37,50] reported higher MDA concentrations in P. falciparum than in P. vivax infections.This disparity may be attributed to the more severe nature of P. falciparum infection, which induces greater oxidative stress in the host cells.However, another study found lower MDA concentrations in P. falciparum malaria than in P. vivax malaria [41].This discrepancy may be explained by the increased susceptibility of patients with P. vivax malaria to oxidative stress, potentially due to lower levels of ascorbic acid [53].Overall, the findings of this study are consistent with previous research indicating similar MDA concentrations in malaria caused by both Plasmodium species, regardless of parasitemia [60].The subgroup meta-analysis demonstrated increased MDA concentrations in malaria cases, regardless of whether the quality of the included studies was high or moderate.Furthermore, when combined with the sensitivity analysis, the results strongly suggest that malaria leads to an excessive accumulation of MDA, which is indicative of oxidative stress.This conclusion is supported by robust findings in high-quality studies, affirming that Plasmodium infections induce oxidative stress-related MDA buildup.
The presence of MDA in malaria-infected individuals indicates the occurrence of lipid peroxidation and oxidative stress.Notably, MDA is one of many markers used to assess oxidative stress and lipid peroxidation, and its measurement alone may not provide a comprehensive understanding of the overall oxidative status in malaria.Furthermore, the association between MDA and the antioxidant system is an important aspect to consider in malaria.The antioxidant system is crucial to maintain the balance between ROS production and elimination [61].It consists of enzymatic and nonenzymatic antioxidants that work together to neutralize and scavenge ROS, thereby protecting cells and tissues from oxidative damage [61,62].Several studies investigating the relationship between MDA and the antioxidant system in the context of malaria showed that the activities of various antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase, and catalase, were decreased during malaria [17,63,64].A longitudinal birth cohort study reported that several polymorphisms in antioxidant enzymes, including glutathione reductase, glutamylcysteine synthetase, glutathione S-transferase P1, haem oxygenase 1, and SOD2, were associated with the oxidative stress status of children [65].Since these enzymes are important for eliminating ROS and maintaining redox homeostasis, their decreased activity during malaria can impair the antioxidant defense system, leading to increased oxidative stress and lipid peroxidation, which contribute to MDA formation.Furthermore, the depletion of nonenzymatic antioxidants, including reduced glutathione, vitamin C, and vitamin E, during malaria has been reported [66,67].These antioxidants directly scavenge ROS and help to regenerate enzymatic antioxidant activity.Reductions in their levels during malaria can impair the overall antioxidant capacity of the system, leading to increased lipid peroxidation and MDA formation.
Our systematic review and meta-analysis had some limitations.First, there was heterogeneity in MDA levels between the studies included in the meta-analysis.This heterogeneity might potentially be influenced by the very wide range of MDA levels due to differences in the quality of the included studies, as demonstrated by the subgroup analysis.Furthermore, MDA levels could be potentially influenced by characteristics of the diseased cohorts, such as the age of participants, the timing, and disease severity.Second, publication bias was observed in the meta-analysis of MDA concentrations between malaria patients and uninfected controls due to the small number of studies.

Conclusions
Our study confirms that MDA concentrations increase in cases of Plasmodium infection and are independent of the Plasmodium species (P.falciparum and P. vivax), at least for the limited studies included in this meta-analysis.The measurement of MDA levels can serve as a useful biomarker to evaluate oxidative stress and lipid peroxidation in individuals with malaria.These findings suggest that MDA concentrations can be suitably tracked as a potential indicator of Plasmodium infection.However, further research is necessary to fully understand the relationship between malaria, oxidative stress, and the role of MDA in the disease.

Figure 2 .
Figure 2. Forest plot showing differences in MDA concentration between participants with m and uninfected controls.Blue square, MDA concentration; green diamond, pooled Cohen d

Figure 3 .
Figure 3. Forest plot of differences in MDA concentration between participants with malaria an uninfected controls (stratified by Plasmodium species)[36,38,40,42,43,[47][48][49]53,54].Blue squar MDA concentration; green diamond, pooled Cohen d; crimson diamond, pooled Cohen d in eac subgroup; gray line, no difference in MDA concentration between the two groups; red line, poole Cohen d.Abbreviations: N, number of participants; mean, mean MDA concentrations (using th Cohen d as an effect estimate, any unit of mean MDA concentrations can be used in the met analysis); SD, standard deviation.

Figure 3 .
Figure 3. Forest plot of differences in MDA concentration between participants with malaria and uninfected controls (stratified by Plasmodium species)[36,38,40,42,43,[47][48][49]53,54].Blue square, MDA concentration; green diamond, pooled Cohen d; crimson diamond, pooled Cohen d in each subgroup; gray line, no difference in MDA concentration between the two groups; red line, pooled Cohen d.Abbreviations: N, number of participants; mean, mean MDA concentrations (using the Cohen d as an effect estimate, any unit of mean MDA concentrations can be used in the meta-analysis); SD, standard deviation.

Figure 4 .
Figure 4. Forest plot of differences in MDA concentration between participants with mal uninfected controls (stratified by the study's quality) [34,36-38,40,42-44,47-50,53,54].Blue MDA concentration; green diamond, pooled Cohen d; crimson diamond, pooled Coh subgroups; gray line, no difference in MDA concentration between the two groups; red line Cohen d.Abbreviations: N, number of participants; mean, mean MDA concentrations (u Cohen d as an effect estimate, any unit of mean MDA concentrations can be used in th analysis); SD, standard deviation; CI, confidence interval.

Figure 4 .
Figure 4. Forest plot of differences in MDA concentration between participants with malaria and uninfected controls (stratified by the study's quality) [34,36-38,40,42-44,47-50,53,54].Blue square, MDA concentration; green diamond, pooled Cohen d; crimson diamond, pooled Cohen d of subgroups; gray line, no difference in MDA concentration between the two groups; red line, pooled Cohen d.Abbreviations: N, number of participants; mean, mean MDA concentrations (using the Cohen d as an effect estimate, any unit of mean MDA concentrations can be used in the meta-analysis); SD, standard deviation; CI, confidence interval.

Figure 6 .
Figure 6.Funnel plot presenting the Cohen d of individual studies (blue do sides of the pooled Cohen d (red line).

Figure 6 .
Figure 6.Funnel plot presenting the Cohen d of individual studies (blue dot) on the left and right sides of the pooled Cohen d (red line).

Table 1 .
Summary characteristics of the included studies.

Table 2 .
Qualitative data of MDA concentrations in malaria and uninfected controls.
[38]increase in MDA levels in P. falciparum malaria patients was much more than in P. vivax malaria patients.6Dasetal., 1990[38]India 1-12 years Plasma MDA levels were significantly higher in malaria patients than in control subjects (p < 0.001).