Malaria is the most important human parasitic disease. As per WHO Malaria Report 2019, In 2018 an estimated 228 million cases of malaria occurred worldwide, compared with 251 million cases in 2010 and 231 million cases in 2017.Nineteen countries in sub-Saharan Africa and India carried almost 85% of the global malaria burden.  In 2018, there were an estimated 405 000 deaths from malaria globally, compared with 416 000 estimated deaths in 2017, and 585 000 in 2010.As of October 2019, the NVBDCP Indian database suggested that 286091 malaria cases were reported out of which 128544 were falciparum malaria¹.Despite robust anti-malarial programmes, P. vivax is difficult to control because of its harder nature, presence of hypnozoites in its life cycle and availability of fewer drugs to tackle hypnozoites. The end result of this is that the incidence of P. vivax has decreased more slowly than that of P. falciparum specially where the two species coexist. P. vivax may then persist as the principal cause of malaria and pose the main challenge to malaria elimination². Despite microscopic examination being the standard method for malaria diagnosis, it requires skilled personnel, often fails to detect mixed species infections or sub-microscopic parasitemia, and has poor performance in endemic areas. Rapid diagnostic tests (RDTs) offer a quicker, cost-effective alternative by detecting parasite proteins like HRP-II or LDH but face challenges with frozen samples, short shelf life, and operator variability. Existing diagnostic methods, including PCR-based techniques, remain reliable for symptomatic patients, but new approaches are needed for low-density parasitemia detection in high-throughput settings. The role of oxidative stress in malaria remains debated, with some studies suggesting a protective function and others linking it to disease pathology. Malaria infection induces oxidative stress through hydroxyl radicals (OH) in the liver, leading to apoptosis³. Research by Atamna et al. observed higher OH+ and H2O2 production in P. falciparum-infected erythrocytes compared to normal cells, highlighting the impact of oxidative stress in malaria pathophysiology⁴.During malaria, the host's hemoglobin serves as a source of amino acids for the parasite, releasing large amounts of heme that induce intravascular oxidative stress, affecting erythrocytes, endothelial cells, and enabling parasite internalization in tissues like the liver and brain⁵.Oxidative stress in malaria occurs when reactive oxygen species (ROS) levels exceed the body's antioxidant defenses, leading to cellular, tissue, and organ damage^6-8. The study highlights the role of oxidative stress in malaria pathogenesis and its impact on antioxidant defense mechanisms⁹.Comparative serum proteomic analysis of P. falciparum and P. vivax infections reveals alterations in proteins linked to critical physiological pathways¹⁰. Additionally, this research explores the implications of oxidative stress changes on malaria outcomes in Cameroonian pregnant women¹¹.

AIM

To identify and investigate the adult patients infected with Plasmodium vivax and Plasmodium falciparum using clinical, biochemical parameters, and oxidative stress pathway.

This hospital-based comparative cross-sectional study was conducted over a duration of 15 months at the Department of Medicine, S.P. Medical College and P.B.M Hospital, Bikaner. A total of 27 cases meeting the study criteria were included in each group. The participants were selected using a random sampling method.  Inclusion criteria comprised individuals aged 18-65 years of either sex, with P. vivax and P. falciparum infections confirmed through microscopic examination and/or rapid diagnostic test (RDT). Patients who had not received anti-malarial treatment before sample collection and those willing to provide written informed consent and adhere to protocol requirements were also included.  Exclusion criteria consisted of patients with significant systemic diseases such as autoimmune disorders, chronic liver diseases, psychiatric illnesses, and bleeding disorders, as determined by history and physical examination. Additionally, patients with mixed infections of P. vivax and P. falciparum in peripheral smear, as well as those co-infected with dengue fever or leptospirosis (confirmed through serology), were excluded. Subjects unwilling to provide consent were also excluded from the study. 

The study was carried out in two steps. In the first step, malaria-infected patients were identified, and clinical data and serum samples were collected. In the second step, these serum samples were sent to the Indian Institute of Technology, Powai, Mumbai, for oxidative stress pathway analysis, along with appropriate controls, to explore the biochemical parameters and host-pathogen interactions in greater detail.

Data Collection

This study was undertaken with the approval of the Institutional Ethics Committee of S.P. Medical College and PBM Hospital, Bikaner, and written informed consent was obtained from each participant prior to participation in the study and the sample collection process. Blood samples were collected from patients (n=27) suffering from vivax malaria diagnosed on the basis of clinical examination and confirmed by microscopy/RDT. Blood samples were collected in the defervescent stage from malaria patients with 2–5 days of fever. Blood specimens were collected from age- and sex-matched falciparum malaria (FM) patients (n=27) as febrile disease controls, and healthy subjects (n=27), to perform comparative proteomic analysis. Patients with uncomplicated, non-severe diagnosed by microscopic examination and confirmed through rapid diagnostic testing (RDT) were enrolled for this study.

Table 1. Distribution of cases according to age group in relation to type of malaria

Table 1 shows that out of 27 cases, 2 were PF (1 each aged 21–30 and 31–40 years) and 25 were PV (15 aged 21–30, 6 aged 31–40, and 4 aged >40 years), with mean ages of 33.50±4.95 years (PF) and 31.48±12.65 years (PV), showing no statistically significant difference (p>0.05).

Table 2. Distribution of cases according to presenting complaints in relation type of malaria

Presenting complaints showed fever in all cases, chills (1 PF, 17 PV), rigor (1 PV), red urine (2 PV), rash (1 PV), weakness (3 PV), loss of appetite (2 PV), vomiting (1 PF, 1 PV), headache (1 PF, 5 PV), and body ache (1 PV), with vomiting being the only statistically significant symptom (p<0.05).

Table 3. Distribution of cases according to USG findings in relation to type of malaria

USG findings showed hepatomegaly in 5 cases, splenomegaly in 11 cases (1 PF, 10 PV), hepatosplenomegaly in 2 cases (1 each PF and PV), 2 pregnant cases (both PV), and 7 PV cases with normal USG findings, with no statistically significant difference (p>0.05).

Table 4. Distribution of cases according to hemoglobin (mg/dl) in relation to type of malaria

Among 27 cases, all severe (Hb <7 g/dl) and moderate anaemia (Hb 7.1–9.0 g/dl) cases were in the PV group, with PF cases showing no severe anaemia; mean Hb levels between PF (12.85±2.90 g/dl) and PV (10.56±2.52 g/dl) groups were not significantly different (p>0.05).

Table5.Distribution of cases according to urine examination (pus cells/HPF) in relation to type of malaria

According to above table, both PF cases had their urine examination within normal range (0-5) while out of total 25 PV cases only 1 case had >10 pus cell while 24 case were within normal range (0-5 pus cells) (p>0.05).

Table 6. Distribution of cases according to serum bilirubin(mg/dl) total parameters in relation to type of malaria

Out of 27 cases, all 10 with normal serum bilirubin (0–1) and 15 of 17 with elevated bilirubin (1.1–3.0) belonged to the PV group, with mean serum bilirubin levels showing no significant difference between PF (1.20±0.00) and PV (1.18±0.42) groups (p>0.05).

Graph Distribution of cases according to SGOT (U/L) in relation to type of malaria

Out of 27 cases, all 7 with SGOT <40 and 9 with SGPT <56 belonged to the PV group, while 20 cases with SGOT 41–5UNL and 18 with SGPT 57–5UNL included 2 PF and 18 PV cases for SGOT, and 2 PF and 16 PV cases for SGPT. The mean SGOT (128.00±5.66 vs. 74.08±42.87) and SGPT (90.50±10.61 vs. 93.32±58.80) levels showed no statistically significant difference (p>0.05) between PF and PV groups.

Table 7: Proteins Found in Oxidative Stress

Uncomplicated malaria, characterized by hot stage, cold stage and sweating stage, is in most instances uneventful01. Complicated malaria on the other hand has mortality and morbidity associated with it. Systemic involvement including cerebral malaria, ARDS, liver and kidney dysfunction, splenic involvement, hematological alteration, endocrinological alteration, as well as fetal death and low birth weight in pregnant patient encompasses the spectrum of presentation of complicated malaria. This involvement was quite characteristic of P. falciparum (which resulted in it being labeled malignant malaria) and resulted in great mortality and morbidity.

Out of total 27 cases, 2 cases were of P. falciparum malaria and 25 cases were of P. vivax malaria. This result was in accordance to the study by Kochar et al¹² (2007) which showed that P. vivax is the predominant cause of malaria in north-west Rajasthan.

In present study, maximum 16 cases (59.3%) belonged to age group 21-30 years followed by 7 cases (25.9%) in 31-40 age group. Mean age in PF group was 33.50±4.95 years while mean age in PV group was 31.48±12.65 years and this difference was found statistically insignificant (p>0.05). The results are comparable to studies by Gupta et al¹³ and Naha et al¹⁴ where mean age was observed to be 32.26 and 33.17 respectively. This age group is more affected probably due to the greater mobility and greater risk of exposure due to more outdoor activities.

During the study we attempted to identify the spectrum of manifestations of patients presenting with malaria. It was observed that fever was the most common complaint present in all individuals. This is associated with chills (66.7%) and headache (22.2%). Other complaints noted were weakness (11.1%); loss of appetite, vomiting and red colour urine (7.4% each); rigor, rash and bodyache (3.7% each). Similar observations were noted by Quispe et al¹⁵ and Gupta et al¹³ where fever was the most common complaint followed by chills and other constitutional symptoms.

In the present study, 4 cases had severe and 2 cases and moderate anaemia, all the 6 cases belonged to P. vivax group. Maximum 14 cases (51.9%) had normal haemoglobin, out of which 13 had P. vivax malaria and 1 had P. falciparum malaria. Mean Hb in PF group was 12.85±2.90 gm/dl while in PV group mean haemoglobin was 10.56±2.52gm/dl and this difference was found statistically insignificant (p>0.05). Previous studies have presented with a wide range of anaemia (<11 g/dL) from 30%107 to 86.0%113. Anaemia is a frequent finding in malaria cases, particularly in developing nations. The pathogenesis of aanemia is multifactorial and includes hemolysis of infected RBCs, accelerated destruction of parasitized and non parasitized RBCs, bone marrow dyserythropoiesis, splenic pooling. This often occurs on a background of chronic anaemia in the developed nations where intestinal parasites and malnutrition prevail particularly in women.

In present study, we found that hepatometaly was present in 5 cases, splenomegaly was present in 11 cases and out of them 1 case PF positive, hepatosplenomegaly was present in 2 case and 1 was PF positive. These results were in accordance to Zha et al¹⁶ found and splenomegaly is associated with the most of the cases of malaria.

Acute renal failure in malaria is usually oliguric or anuric but urine output may be normal or increased, making daily measurement of serum creatinine to be the most important investigation¹⁷.

In the present study, it was observed that the liver enzyme levels were elevated in both falciparum and vivax malaria patients. 63% cases had serum bilirubin levels between 1.1- 3.0, 74.1% cases had AST between 41- 5×UNL and 66.7% cases had ALT between 57- 5×UNL. None of the cases had values of liver enzyme in the range of severe malaria. The finding of the present study correlated with finding of previous studies of Oluwole et al¹⁸ and ELbadawi et al¹⁹ who reported that most of the malaria patients show elevation in liver enzymes indicating liver damage.

In response to Plasmodium infection, the host defense mechanism activates phagocytes, leading to the production of ROS and RNS that induce oxidative stress. This study investigated oxidative stress markers and found that SOD-1, ceruloplasmin (CP), HBB, and PRDX2 play significant roles in malaria pathogenesis. SOD-1 was upregulated, indicating its role in converting superoxide anions to hydrogen peroxide, with findings supported by Andrade et al.,²⁰ showing its correlation with disease severity. CP levels were also elevated, aligning with Das B.S.'s²¹ observations that ceruloplasmin increases with malaria severity. HBB, associated with protective hemoglobin variants like HbS and HbC, was upregulated, highlighting its role in malaria susceptibility. Conversely, PRDX2, an antioxidant enzyme that protects cells from oxidative damage, was downregulated, suggesting impaired peroxide signaling in malaria patients.

In our study we found that SOD-1 (Superoxide Dismutase-1), CP (Ceruloplasmin) and HBB (Hgb-β-globin) was up-regulated in malaria patient sera, whereas PRDX2 (Peroxiredoxins) was found to be down regulated in malaria patients’ sera as compared to healthy individual sera. The resulting oxidative stress has been suggested to be one of the major mediators of erythrocyte damage, anemia, hepatic and renal dysfunction in malaria

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