Screening Clinical, Laboratory and Host Markers for Diagnosis of Disease Severity in Plasmodium vivax Clinical Samples

Abstract

Malaria is one of the most infectious disease that affects lives of million people throughout the world. Recently, there are several reports which indicate Plasmodium vivax (P. vivax) causing severe disease in infected patients from different parts of the world. For P. vivax disease severity, the data related to immunological and inflammatory status in human host is very limited. In the present study clinical parameters, cytokine profile and integrin gene were analyzed in P. vivax clinical patients. A total of 169 P. vivax samples were collected and categorized into severe vivax malaria (SVM; n = 106) and non-severe vivax malaria (NSVM; n = 63) according to WHO severity criteria. We measured host biomarker levels of interferon (IFN-γ), superoxide dismutase (SOD-1), interleukins viz. (IL-6, IL-10), and tumor necrosis factor (TNF-α) in patient plasma samples by ELISA for pro- and anti-inflammatory cytokines in severe malaria. Host integrin gene was genotyped using PCR assay. In our study, thrombocytopenia and anemia were major symptoms in severe P. vivax patients. In analyzed SVM and NSVM groups a significant increase in cytokine levels (IL-10, IL-6, and TNF-α) and anti-oxidant enzyme SOD-1 was found. Our study results also showed a higher pro-inflammatory (TNF-α, IL-6 and IFN-γ) to anti-inflammatory (IL-10) cytokine ratio in severe vivax patients. Integrin gene showed no mutation with respect to thrombocytopenic patients among clinically defined groups. It was observed that severe vivax cases had increased cytokine levels irrespective of age and sex of the patients along with thrombocytopenia and other clinical manifestations. The results of current findings could serve as baseline data for evaluating severe malaria parameters during P. vivax infections and will help in developing an effective biomarker for diagnosis.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-024-01324-4.

Introduction

Malaria is one of the major health challenges that the entire world is facing today. According to WHO World Malaria Report 2023, the estimated global malaria in year 2022 was 249 million [1]. The proportion of Plasmodium vivax infection has reduced from 8% in 2000 to 3% in 2022 [2]. About 2% of all malaria cases worldwide were found in South East Asia with India contributing to 66% of these cases. Malaria is not only a consequence of factors related to the Plasmodium parasite but there are also several host molecular and genetic alterations surrounded by environmental factors [3].

Plasmodium falciparum is known to cause severe malaria but since the last decade, P. vivax is also seen to be causing severe malaria in clinical isolates [4]. One of the severe symptoms of P. vivax and P. falciparum malaria, reported commonly in adults and children both is thrombocytopenia. There are several different pathogenetic factors that contribute to malaria thrombocytopenia, like altered bone marrow, oxidative stress, platelet aggregation, splenomegaly and platelet destruction by macrophages [5]. There are few reports that suggest platelets have active role in malaria pathogenesis as platelets produce microparticles into the bloodstream, and these microparticles are linked to acute inflammatory infections [6, 7]. Platelets have five integrins, b3 integrin avb3, aIIbb3, and three b1 integrins that play a role in platelet adhesion to matrix protein collagen, laminin, and fibronectin [8]. Apart from homeostasis platelet contain wide range of angiogenic, inflammatory, and immune modulating factors.

Few studies have suggested important role of cytokines like TNF-α, IL-6, IL-10, and IFN-γ on malaria outcome [9, 10]. Cytokines are small glycoprotein that play a role in host defense as well as in a variety of normal physiological and metabolic processes [11]. Cytokines have been found to possess a pleiotropic effect in acute and chronic inflammatory responses [11]. Certain host-related factors are associated with oxidative stress, such as enzyme haem oxygenase (HO-1) and superoxide dismutase (SOD-1) along with molecules, related to metabolic adaptation to iron overload due to malaria-triggered hemolysis. These types of genes, biomolecules and enzymes are identified as helper biomarkers which can help to distinguish between severe and non-severe malaria patients [12–14]. Earlier P. vivax malaria was known as benign malaria but currently, P. vivax clinical trends have changed, causing severe disease leading to potentially lethal disease in some infections [15, 16]. The unavailability to predict severe P. vivax at an early stage of infection is becoming a point of concern [10]. However, uncontrolled levels of TNF-α, IFN-γ, IL-6, IL-10, IL-1β, and IL-4 have been linked to the development of immunopathology, which is frequently present in severe conditions in malaria infections [17–19]. On the basis of their findings numerous studies have shown that the ratio of pro-inflammatory to anti-inflammatory cytokines serves as an important indicator of disease prognosis [20–22]. There are studies that lack the assessment of thrombocytopenia and disease severity in the clinical isolates of P. vivax and the role of integrins, antioxidant enzymes, inflammatory cytokines, and their ratio in severity of malaria infection. In the present study the association between platelet integrin and different plasma levels of TNF-α, IFN-γ, IL-6, IL-10, and enzyme SOD-1 in clinically defined groups of P. vivax infected patients along with other parameters such as the patient’s age and sex, level of parasitemia in relation to disease severity were analyzed.

Materials and Methods

Ethics Approval and Study Participants

The samples for this study were collected during the years 2016 to 2019 from two different locations viz Safdarjung Hospital, Delhi and Nuh, Primary Health Centre (PHC) Mewat in India. The NIMR ethics committee (NIMR/ECR/EC/2018) granted approval for this research. Based on WHO severity criteria, patients were categorized as severe or non-severe P. vivax malaria (2014). For the collection of their samples, enrolled patients and adults (in the case of minors) provided written consent. Rapid malaria antigen testing (RMAT) and peripheral smear examinations were used to diagnose the Plasmodium infection followed by 18S PCR assay for P.vivax species confirmation in malaria positive samples (Fig. 1) [23].

Fig. 1.

Workflow depicting the P. vivax sample collection and detection methods in study

Collection and Diagnosis of Samples

About 2–3 mL of blood was collected from each P. vivax confirmed patient by vein puncture aseptically. Half volume of collected blood sample was centrifuged to obtain plasma which was aliquoted in vials and remaining volume of samples were used for making filter spots on Whatman No.3 paper strips (GE Healthcare) for further molecular analysis. Thin and thick blood smears of samples were prepared, air-dried, fixed and stained with Giemsa. A total of ~ 10 fields (1000X magnificence) were counted for infected and uninfected erythrocytes and parasitemia level was calculated.

Genomic DNA Isolation and PCR Assay

Parasite DNA of positive samples was extracted from the filter papers containing dried blood spots (DBS) using QIAamp DNA, Blood Mini kit (Qiagen, Germany) as per the manufacturer’s instructions. To confirm the Plasmodium species, the positive samples were amplified using 18S rRNA PCR assay [24]. Samples of P. falciparum, mixed-infections and co-infections were excluded from the study and only P. vivax mono-infections were analyzed further.

Integrin PCR

Genotyping of platelet integrin was done using polymerase chain reaction (PCR) and sequencing was done to analyze the C807T mutation in thrombocytopenic patients [25].

Cytokine Assay

Cytokines IL-10 and TNF-α, IL-6, IFN-γ and SOD-1, were evaluated in plasma samples of vivax patients. Briefly, a flat-bottom 96 microwell plate (Nunc Maxisorb) precoated with capture antibody was used and plasma samples were diluted for IL-10 (1:2), TNF- α (1:2), IL-6 (1:2), IFN-γ (1:2), and SOD-1 (1:5) respectively. The limit of detection (LOD) for IL-10 was (LOD-7.8 pg/ml), TNF-α was (LOD-3.9 pg/ml), IL-6 (LOD-7.8 pg/ml), IFN-γ (LOD-15.63 pg/ml) and SOD-1 was (LOD- 0.005 U/ml), The standards and samples were prepared in triplicates and incubated for two hours. Cytokine concentration in plasma was calculated with the help of standards and mean concentration, standard deviation (SD) values were calculated for patient samples.

Statistical Analysis

Data were keyed in an excel spreadsheet and analysed with graph pad prism (GraphPad Software, San Diego California USA, http://www.graphpad.com) and (SPSS) version 20 for Windows (SPSS, IBM., Chicago, USA). Continuous and categorical variables were presented as mean ± SD and percentages respectively. Mean values were compared for two clinical groups using an unpaired T-test, Chi-square test, and non-parametric Mann–Whitney test. The significance level was set at p-value < 0.05.

Results

Patient Physiognomies

A total of 169 malaria infected patients were enrolled in the study, out of these 106 were severe vivax and 63 non-severe vivax patients. The percentage of male patients was more with 58.4% and 61.9% in comparsion to female patient with 41.5% and 38.1% in both clinical groups. The age group of patients between > 12 ≤ 60 year in both groups (Table 1). The parasitemia was calculated by microscopy for all P. vivax clinical samples which ranged from 0.07 to 1.06%. Our findings revealed no correlation between parasitemia and P. vivax infection disease severity (Fig. 2a).

Table 1.

Demographic details of clinical samples

	Parameters
P. vivax clinical samples (n = 169)	Sex (%)		Age (years) (%)
	Male	Female	≥ 1 ≤ 12	> 12 ≤ 60
Severe (n = 106)	62 (58.4)	44 (41.5)	43 (40.5)	63 (59.4)
Non-severe (n = 63)	39 (61.9)	24 (38.1)	7 (11.1)	56 (88.8)

Fig. 2.

(a Parasitemia was analyzed in SVM and NSVM group and results were evaluated statistically with an unpaired t-test where the p-value was set at a significant value (p-value < 0.05). b Comparison of thrombocytopenic conditions with respect to normal platelet count in P. vivax patients. One-way ANOVA (non-parametric test) was used to compare mean values between SVM and NSVM where the p-value was set at less than 0.05

Thrombocytopenia in Clinical Samples

Severe anemia was the most prevalent laboratory parameter with 69.8% among SVM patients. Similarly mild anemia with 31.7% the most prevalent laboratory parameter among NSVM patients. Severe thrombocytopenia was observed in 73.5% of SVM patients and in NSVM patients, 19.04% mild and 26.9% moderate thrombocytopenia was seen. Severe anemia and severe thrombocytopenia were significantly greater in SVM cases in comparison to NSVM cases in the study (p-value < 0.0001) (Table 2).

Table 2.

Laboratory parameters of SVM and NSMV patients

Laboratory parameters
Clinical groups	Normal platelet count	Thrombocytopenia			Normal Hb count	Anemia
		Mild	Moderate	Severe		Mild	Moderate	Severe
Severe, n = 106 (%)	2(1.88)	7(6.60)	19(17.92)	78(73.5)	7(6.6)	6(5.6)	19(17.9)	74(69.8)
Non-severe, n = 63 (%)	30(47.6)	12(19.04)	17(26.98)	4(6.34)	32(50.7)	20(31.7)	11(17.4)	0
Total no of pateints, n = 169 (%)	32(18.93)	19(11.24)	36(21.30)	82(48.52)	39(23.07)	26(15.38)	30(17.75)	74(43.7)
Chi square (df)	53.8(1)	6.132(1)	1.93(1)	71.5(1)	43.4(1)	20.6(1)	0.006(1)	78.2(1)
p-value	< 0.0001	0.013	0.164	< 0.0001	< 0.0001	< 0.0001	0.93	< 0.0001

Normal Hb =  > 11–16 g/dl; severe anemia: Hb < 5 g/dl (children); Hb < 7 g/dl (adult); mild anemia (Hb > 9–11 g/dl); moderate anemia (Hb 7–9 g/dl); normal platelet = 150,000–400,000 platelets/μl; moderate thrombocytopenia = 50,000–100000 platelets/µl, mild thrombocytopenia = 100,000–150000 platelets/µl; severe thrombocytopenia < 50,000platelets/µl

The majority of P. vivax infected patients had severe thrombocytopenia (48.52%) followed by moderate thrombocytopenia (21.30%), normal platelet count (18.93%), and mild thrombocytopenia (11.24%). The different thrombocytopenic conditions were compared with normal platelet count in all P. vivax patients, which showed a highly significant difference among the thrombocytopenic conditions (p-value < 0.0001) (Fig. 2b).

Integrin Analysis

Integrin alpha2 C807T genotype (single nucleotide polymorphism) distribution was genotyped in clinically defined groups. No mutation in integrin alpha2 with respect to thrombocytopenia using allele CC as a reference i.e. (CT and TT) (supplementary Fig. 1) was seen. The neighbor-joining phylogenetic tree was constructed between reference and clinical groups, but no pattern of relatedness was seen between wild type sequences of phylogenetic cladogram in severe vivax and non-severe vivax isolates (supplementary Fig. 2).

Cytokine Analysis

In the present study, we observed a significantly higher level of TNF-α (42.22 ± 8.91, p-value < 0.0001), IL-6 (148.0 ± 47.90, p-value = 0.0024), IL-10 (20.55 ± 4.75, p-value < 0.0001), and SOD-1 (1.21 ± 0.29, p-value < 0.0001) in SVM, whereas IFN-γ (-40.04 ± 111.7, p-value = ns) showed no significant difference between SVM and NSVM (Fig. 3). Also, log levels of SOD-1, TNF-α, IL-10, IL-6, and IFN- γ did not correlate with log percent parasitemia (p-value = ns) (Supplementary Fig. 3 and Supplementary Table 1).

Fig. 3.

SOD-1, TNF-α, IL-10, IL-6 and IFN-γ levels were evaluated in SVM and NSVM groups. a SOD-1 b TNF-α c IL-10 d IL-6 show significant p-value whereas e IFN-γ no significant (ns) difference between SVM and NSVM. Non-parametric Mann–Whitney test was used to compare mean values between the two groups where the p-value was set at less than 0.05

Cytokine Level vs Thrombocytopenia

The platelet count was compared among different clinical groups and a significant difference was seen between the two groups and among different thrombocytopenic categories. Our results showed significant difference between cytokines and enzymes with respect to platelet count i.e. TNF-α (p-value ≤ 0.0001), IL-6 (p-value ≤ 0.0001), IL-10 (p-value ≤ 0.0001), IFN-γ (p-value ≤ 0.0001) and SOD-1 (p-value ≤ 0.0001) (Fig. 4). Majority of SVM had severe thrombocytopenia, followed by moderate and mild thrombocytopenia. In NSVM samples mostly normal platelet counts was seen followed by mild, moderate thrombocytopenia and few cases of severe thrombocytopenia.

Fig. 4.

Platelet counts were compared among SVM and NSVM clinical groups and statistical analysis was done using Two-way ANOVA where the p-value was set at less than 0.05. Thrombocytopenia, classified as moderate (50,000–1,00,000), mild (1,00,000–150,000/mm³) and severe (< 50,000/mm³). Normal platelet count is classified as platelet counts > 150,000/mm³. Normal platelet count (red hexagon), moderate thrombocytopenia (green circle), mild thrombocytopenia (blue box), and severe thrombocytopenia (yellow triangle)

Plasma Cytokine Ratio

In the current study we explored the pro- and anti-inflammatory cytokine ratio (IL-6/IL-10, TNF-α/IL-10, and IFN-γ/IL-10) and ratios of cytokine level with respect to antioxidant enzyme (IL-6/SOD-1, TNF-α/SOD-1, IFN-γ/SOD-1, and IL-10/SOD-1) level in patient groups by Mann–Whitney test. The mean ratios of pro-inflammatory and anti-inflammatory cytokines, IL-6/IL-10 (p-value = 0.032), TNF-α/IL-10 (p-value = 0.040), and IFN-γ/IL-10 (p-value = 0.020), were significantly different in our findings (Fig. 5a). TNF-α/SOD-1 mean ratios in SVM samples were significantly higher in comparison to NSVM (p-value = 0.046), whereas IL-6/SOD-1, IFN-γ/SOD-1, and IL-10/SOD-1 mean ratios among two clinical groups were not significant (Fig. 5b).

Fig. 5.

Log ratios compared for different cytokines and enzymes and statistically analyzed using the Mann–Whitney test. Significance difference seen between the mean ratios of A (i) IL-6/IL-10 (p-value = 0.032), (ii) TNF-α/IL-10 (p-value = 0.040), (iii) IFN-γ/IL-10 (p-value = 0.020) and B (i) IL-6/SOD-1 (p-value = ns), (ii) TNF-α/SOD-1 (p-value = 0.046), (iii) IFN-γ/SOD-1 (p-value = ns) and (iv) IL-10/SOD-1 (p-value = ns) between two clinical groups where the p-value was set at less than 0.05

Discussion

Recently several studies indicate the shift in P. vivax pathogenesis towards increased severity risk and mortality [4, 26]. While severe malaria can manifest with low parasite counts, high parasite counts are linked to an increased risk of worsening and subsequent treatment failure, even in the absence of any severity-related signs or symptoms [27]. In our report no correlation between the parasitemia in peripheral blood with disease severity was also observed in other studies [28].

In our study severe anemia (69.8%; Hb < 7 g/dl) and severe thrombocytopenia (73.5%; < 50,000platelets/µl) were the most important complications in severe vivax patients as seen in other studies too from different endemic regions [29, 30]. Similarly, another study from Northwest regions in India found high prevalence of severe thrombocytopenia in P. vivax [31, 32]. The possible reason for severe anemia in P. vivax infected patients is preference of P. vivax parasites for the reticulocytes [30, 33, 34]. The cause for thrombocytopenia in vivax can be attributed to immunological reactions, lytic effect, sequestration, and oxidative stress during the disease pathogenesis [35–37].

In our study we did not find any polymorphism in integrin gene for thrombocytopenic patients. Campos and his colleagues showed integrin polymorphism to be associated with P. vivax induced severe thrombocytopenia [35, 38]. More thorough research for polymorphisms/haplotypes of various integrins is needed to decipher the function of integrin gene in malaria infections.

Reports suggest that even with low parasitemia P. vivax infection is capable of eliciting the inflammatory cytokine response that is sufficient to generate pyrogenic responses and other complications related to it [39]. Our study results showed an increased level of inflammatory cytokines TNF-α, IL-6, IL-10, and anti-oxidant enzyme SOD-1 in severe group as also seen in another study [12, 40]. An article by Prakash D and his colleague has shown that cerebral malaria (CM) has a high level of TNF-α, IL-1b, TGF-b, and IL-10 followed by severe malaria group (SM) and mild malaria group (MM) in P. falciparum infected patients [41]. Also, higher levels of IL-10 have a role to play in malaria pathogenesis as reported and could serve as a useful diagnostic marker for malaria [42]. A direct correlation between hemolytic activity and SOD-1 which causes defects in the regulatory responses leading to disease severity has been observed [43]. Several studies from Sri Lanka, Gambia, Mali, Madagascar, Gulf of Guinea, and Vietnam reported higher levels of TNF-α, IL-10, IL-6, and IFN-γ in complicated malaria cases [42, 44–49]. Our results showed resemblance with another study where excessive production of pro-inflammatory cytokines such as TNF-α, and IL-6 was responsible for pathogenesis of severe malaria [44].

A study reported no correlation between TNF-α levels and parasitemia in UM (uncomplicated malaria) or SM (severe malaria) patients, which is in accordance with our findings too [44, 50]. The high levels of TNF-α and IL-10 in severely thrombocytopenic patients have been reported by various studies as similar to our study results [51–54]. Perera et al. [44] has found significantly higher levels of TNF-α, IL-6, and IL-10 in P. falciparum-infected patients with severe malaria complications when compared to uncomplicated malaria.

An association of pro-inflammatory to anti-inflammatory cytokine was studied to define the cytokine imbalance and host pathogenesis that could be used as severity biomarker. A significant higher ratio of TNF-α/IL-10, IL-6/IL-10, and IFN-γ/IL-10 severe vivax was seen in our study as also reported by others [44]. Significantly high ratios of TNF-α/IL-10 were seen in children with respiratory distress, a symptom underlying metabolic acidosis which is a major risk factor for mortality in severe malaria in comparison to those without respiratory distress [22]. In our research, there were significant differences in mean pro-inflammatory to anti-inflammatory cytokine ratios of IL-6/IL-10, TNF- α/IL-10, and IFN-γ/IL-10. The mean ratios of TNF- α /SOD-1 in SVM samples were significantly greater than in NSVM groups. The observed imbalance between pro- and anti-inflammatory cytokines is responsible for severe symptoms during the P.vivax infection [55].

The importance of this study lies in the fact that host biomarkers like SOD-1, TNF- α, IL-10, IL-6 could serve as markers for analyzing the disease severity in clinical isolates. The limitation of this study was that sample analysis was done at a single time point only and there was paucity of sample volume. It is important to carry out further studies in this direction with inclusion of more host biomarkers for validation and correlation with disease severity in large sample size.

Conclusion

The results generated by this study are of most importance to improve the current knowledge about the P.vivax immunopathological concepts of this widespread malaria disease. In our study, an increased level of cytokines (TNF-α, IL-6, IL-10, and SOD-1) and an imbalance in the ratio of pro- to anti-inflammatory cytokine (IL-6/IL-10, TNF/IL-10, and IFN/IL-10), in severe vivax patients were seen which may have a role to play in the pathogenesis of SVM. More studies are required to understand the role of host factors such as inflammatory cytokines and the integrin gene during P. vivax malaria infection.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

WHO

World Health Organization

SVM

Severe vivax malaria

NSVM

Non-severe vivax malaria

P. falciparum

Plasmodium falciparum

P. vivax

Plasmodium vivax

SOD-1

Superoxide Dismutase-1

TNF-α

Tumor necrosis factor

IL-10

Interleukine-10

IL-6

Interleukine-6

IFN-γ

Interferon—γ

RMAT

Rapid malaria antigen test

DBS

Dried blood spots

PCR

Polymerase chain reaction

LOD

Limit of detection

SD

Standard deviation

Author contributions

VS conceived the idea of the paper. AA, SC, KY, ST carried out the lab work, methodology, Formal analysis, Investigation Software. SSM, MM provided samples, AA drafted the first version of manuscript with KY, ST and SC. VS revised the manuscript for important intellectual content. VS supervised the work at all stages. VP, SSM, MM carried out manuscript writing-review. All authors read and approved final version of the paper before submission.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability

All manuscript related data is available in the tables provided and in supplementary material. If any further information is needed, the corresponding author may be contacted for the same.

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Ethical approval

The NIMR ethics committee (NIMR/ECR/EC/2018) granted approval for this research.