Association between inflammatory cytokine levels and anemia during Plasmodium falciparum and Plasmodium vivax infections in Mangaluru: A Southwestern Coastal Region of India.

BACKGROUND AND OBJECTIVES: Dysregulated production of inflammatory cytokines might play important role in anemia during malaria infection. The objective of this study was to assess the extent of anemia due to malaria, associated complications, and inflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin [IL]-6, and IL-10) across varying anemic intensity during malaria infections.
MATERIALS AND METHODS: A hospital-based cross-sectional study was conducted at District Wenlock hospital in Mangaluru city. Samples from 627 patients and 168 healthy controls (HC) were analyzed for level of hemoglobin (Hb), red blood cells (RBCs), and inflammatory cytokines. The blood cell parameters and inflammatory cytokines levels across varying intensity of anemia were analyzed using Kruskal-Wallis test and pair-wise comparison between two groups were by Mann-Whitney U-test. Correlations were calculated by Pearson's and Spearman rank correlations.
RESULTS: Compared to HC, Hb, and RBC levels were significantly lower in infected patients. On comparison with mild anemia patients (Hb 8-10.9 g/dL), the levels of TNF-α and IL-6 were significantly elevated, whereas IL-10 levels were lower during severe anemia (SA) (Hb <5 g/dL). In this endemic setting, we found a strong negative association between Hb levels and parasitemia, Hb and TNF-α, and positive relationship with IL-10; anemic patients also had significantly high TNF-α/IL-10 ratios. SA was associated with complications such as acute renal failure (16.0%), jaundice (16.0%), metabolic acidosis (24.0%), hypoglycemia (12.0%), hyperparasitemia (4.0%), and hepatic dysfunction (16.0%).
CONCLUSIONS: Contrary to its benign reputation, Plasmodium vivax (Pv) infections can also result in severe malarial anemia (SMA) and its associated severe complications similar to Plasmodium falciparum infections. Dysregulated inflammatory cytokine responses play an important role in the pathogenesis of SMA, especially during Pv infections. Copyright:

INTRODUCTION

Malaria continues to be a major public health crisis in almost all developing countries, especially in the tropical regions across the globe, affecting >40% of the world's population.[1] In 2016, about 216 million cases and 445,000 deaths were reported from 91 countries across the world, in which India accounted for 1,090,724 cases and 331 malarial deaths.[1] In India, malaria is highly endemic and persists mostly in the Northern, Northeastern, and Southwestern regions. In the Southwestern region, Karnataka state is a significant contributor of malarial infections.[2] Mangaluru, the administrative capital of Dakshina Kannada district, is situated on the Arabian seashore of Southern India. The city and its surrounding areas have a warm and humid climate, harboring high vector density leading to high rates of malarial transmission.[3] In 2016, Karnataka state reported a total of 15,816 malarial infections, of which Mangaluru alone accounted for 11,037 (69.7%) cases and the majority were due to Plasmodium vivax (Pv) (9696, 87.9%) and the remaining were Plasmodium falciparum (Pf) (1341,12.1%) infections.[4567]

Anemia is one of the most common complications during malaria, especially in younger children and pregnant women.[8] Malarial anemia, in its milder forms, is an important manifestation responsible for considerable morbidity, whereas severe malarial anemia (SMA) is of major concern due to its high mortality rates, particularly in children below 2 years of age.[910] The pathogenesis of malarial anemia is primarily due to destruction of red blood cells (RBCs), accelerated removal of both parasitized and nonparasitized RBCs, bone marrow dysfunction, and level of parasitemia.[11]

Although the mechanism and pathological basis of malarial anemia are poorly understood, the involvement of imbalanced inflammatory mediators cannot be ruled out.[12] Similar to Pf, Pv infections also leads to specific host immune responses resulting in cytokine release, as well as activation of macrophages, T-cells, natural killer cells, and neutrophils to combat the infections.[13] Various studies in Pf infections have associated predominant T-helper 1 responses characterized by increased tumor necrosis factor alpha (TNF-α) and conversely reduced interleukin-10 (IL-10) levels to be associated with malarial anemia.[1415] TNF-α is implicated in stimulation of nitric oxide levels, increased production of cytokines such as interleukin-6 (IL-6) and inhibition of erythropoiesis.[1617] On the other hand, IL-10 plays a major role in T-helper type 2-like immune responses and has been found to stimulate erythropoiesis in vitro.[18]

Systematic studies concerning severity of malarial anemia and its associated complications are largely limited to African settings and Northern parts of India where malaria is hyperendemic, and Pf is by far the most prevalent infecting species.[1319] Furthermore, studies concerning the role of inflammatory cytokines and its related pathogenesis have been confined to Pf infections; and hence, there is a paucity of data regarding the involvement of cytokines in malarial anemia during Pv infections.[2021] Overall in Mangaluru, Pv (87.9%) being the major infecting species, the real impact of malarial anemia and its associated clinical pathology on the affected populations in this endemic setting is unknown. In this study, the prevalence of anemia and its intensity, associated severe malarial complications, and measurement of plasma inflammatory cytokine levels across varying degree of malarial anemia were studied.

MATERIALS AND METHODS

Ethics

The study protocol was approved by the Ethical Committee of Kuvempu University, Shivamogga, Karnataka state; the central ethics committee of NITTE University, Mangaluru, and the Institutional Review Board of Pennsylvania State University College of Medicine, Hershey, PA, USA.

Study site

During November 2013 to October 2015, a cross-sectional study was conducted at Government Wenlock Hospital in Mangaluru city. Wenlock hospital is the major government hospital in this region which provides free medical treatment, especially to the socioeconomically poor individuals. The malarial infection in this region is persistent throughout the year, with its peak transmission being during the rainy season (i.e., June–September).

Study design and population

The patients seeking medical attention at malaria outpatient's clinic and inpatient wards and healthy individuals with matching age, sex, and localities, who were attending blood bank for blood donations, were requested to participate in the study. The study participants were recruited irrespective of their socioeconomic status, sex, and ethnicity. The research project was explained to the participants and recruited only after obtaining a signed informed consent from the study participants or their guardians. The study participants were recruited upon their first visit and were not followed up later in the study. Based on the total population, the sample size was estimated as reported by Kwenti et al.[22] Considering a prevalence rate of 28%, a total of 627 patients were analyzed; 310 infected patients were recruited in the 1st year and 317 were recruited in the 2nd year. For comparison, a total of 168 healthy controls (HC) (comprising of 78 in the 1st year and 90 in the 2nd year) were recruited. Trained technicians interviewed the study participants using a structured questionnaire, to obtain information regarding age, sociodemographic profile, economic status, educational level, occupation, and clinical presentation of patients. The inpatients were treated by the attending physicians, evaluated twice daily for severe symptoms and complications as per the WHO guidelines until discharged.[21] Inclusion criteria were adults (≥18 years of age), patients positive for the presence of Plasmodium parasites and axillary temperature ≥37.5°C and HC matching in age, gender, and locality were enrolled in the study. Exclusion criteria were children, pregnant women, and individuals testing positive for dengue, typhoid, hepatitis B and C, HIV, and those who used antipyretics prior to diagnosis.

Malarial diagnosis

The malarial diagnosis was performed by rapid diagnostic kits (RDT) and microscopic examination of Giemsa-stained peripheral blood smears. The malaria Ag Pf/Pv rapid diagnostic test kit (SD Bioline, India) method detects histidine-rich protein II antigen of Pf and lactate dehydrogenase of Pv in human blood.

For Giemsa-stained peripheral blood smears, two thick and thin blood slides were prepared from each study participant, stained with 4% Giemsa stain and observed under the microscope for the presence of Plasmodium and the species type. Parasite densities were quantified as parasites/μl of blood (number of parasites counted/number of white blood cells (WBCs) counted × total number of WBCs per μl of blood) or (number of parasites counted/number of RBCs counted × total number of RBCs per μl of blood). Percentage parasitemia was determined as (number of parasites per μl of blood/number of RBCs per μl of blood) × 100.[23]

Sample collection, hematological and inflammatory cytokine analysis

Upon malarial diagnosis and before any antimalarial medications, about 2–3 ml of venous blood samples were aseptically drawn into heparin-coated vacationers for plasma and to clot activator tubes for serum preparation and kept at 4°C until further use. Within 1 h of collection, blood samples were used for hematological analysis and serum or plasma samples were prepared by centrifugation at room temperature, labeled and stored at −80°C until further use. Hematological analysis of parameters such as hemoglobin (Hb), RBCs, mean corpuscular volume, mean corpuscular Hb, and mean cell Hb concentration were performed by hematology analyzer (Mind Ray-Biomedical, Shenzhen, China). The plasma levels of cytokines such as TNF-α, IL-6, and IL-10 were measured in duplicates by sandwich ELISA, using commercially available kits from R and D Biotech, USA. The serum/plasma samples were screened for comorbid infections such as hepatitis B, C, and HIV using commercially available ELISA kits (J. Mitra and Co, New Delhi).

Classification of study participants

Healthy controls

The healthy individuals attending blood bank for blood donations and testing negative for malaria by peripheral blood smear and RDT kits.

Mild malaria

Mild malaria (MM) group was defined as patients, with low-grade clinical symptoms, such as fever, headache, or chills and positive for the presence of Plasmodium parasite by peripheral blood smear and RDT kit, regardless of parasite species with no symptoms or signs of severe malaria or other etiologies.

Severe malaria

Severe malaria (SM) was defined by the presence of Plasmodium parasite at any density plus any one of the following complications as per the WHO guidelines of SM: severe anemia (SA) (Hb <5 g/dl), acute renal failure (serum creatinine ≥3 mg/dl), jaundice/icterus (serum bilirubin ≥3 mg/dl), metabolic acidosis (plasma bicarbonate <15 mmol/l), spontaneous bleeding, hypoglycemia (plasma glucose <40 mg/dl), hyperparasitemia (≥10% parasitemia), acute respiratory distress syndrome, pulmonary edema, and cerebral malaria, thus requiring hospital admission and supportive care.[23]

Classification of study participants based on anemic severity

Based on varying Hb levels (g/dl), the intensity of anemia was classified into four categories as follows:.[2425]

Nonanemic (NA): study participants with Hb ≥11 g/dL

Mild anemia (MA): Hb ranged between 8 and 10.9 g/dL

Moderate anemia (MDA): Hb ranged between 5 and 7.9 g/dL

SA: Hb <5 g/dL.

Statistical analysis

The statistical analysis was performed by entering the data in Microsoft Excel spreadsheet and the data analysis was performed using GraphPad Prism (GraphPad Prism version-6, Inc., San Diego California, USA). Summary statistics were calculated for baseline demographics and quantitative variables. The blood cell parameters and inflammatory cytokines level across varying intensity of anemia were compared between the HC and malaria patients with Pv, Pf, and mixed infections. For robust comparison between various groups, nonparametric Kruskal–Wallis test was used. Pair-wise comparison between two groups was conducted by Mann–Whitney U-test and P values were corrected for multiple comparison error by Bonferroni correction. Correlations between two continuous variables were calculated by Pearson's correlation and Spearman rank correlations. The P < 0.05 was considered to be statistically significant.

RESULTS

Sociodemographic profile

Seven hundred and ninety-five (n = 795) individuals were recruited into the study at Government Wenlock Hospital in Mangaluru city. A total of 627 (78.9%) study participants were found to be infected from malaria, and 168 (21.1%) individuals were enrolled as HC. Among the infected, Pv was the most prevalent species of infection (n = 384, 61.2%) followed by Pf (n = 172, 27.5%) and mixed (n = 71, 11.3%) infections. The mean age of the study participants was 31.4 years (age range, 18–65 years). Malarial infections were found to be higher in males (402, 64.1%) in comparison to females (225, 35.9%). Among the 627 infected patients, 554 (88.4%) patients had MM and were treated on an outpatient basis and 73 (11.6%) patients suffered from SM and thus required hospital admissions for supportive treatment [Table 1].

Table 1

Characteristics of the study participants across various infecting species

	HC	Pv	Pf	Mixed	Overall infected
Number of study participants	168	384 (61.2)	172 (27.5)	71 (11.3)	627 (78.9)
MM	0	351 (63.4)	149 (26.9)	54 (9.7)	554 (88.4)
SM	0	33 (45.2)	23 (31.5)	17 (23.3)	73 (11.6)
Gender
Males	110	246 (61.2)	111 (27.6)	45 (11.2)	402 (64.1)
Females	58	138 (61.3)	61 (27.1)	26 (11.6)	225 (35.9)
Mean age, years (range)	30.1 (18-58)	30.5 (18-65)	32.7 (18-65)	31.7 (18-65)	31.4 (18-65)
18-25	79	175 (67.0)	57 (21.8)	29 (11.1)	261 (41.6)
26-35	45	100 (58.5)	53 (31.0)	18 (10.5)	171 (27.3)
36-45	27	69 (55.2)	41 (32.8)	15 (12.0)	125 (19.9)
>45	17	40 (57.1)	21 (30.0)	9 (12.9)	70 (11.2)

Data represented as number of study participants (%). MM: Mild malaria, SM: Severe malaria, HC: Healthy controls, Pv: Plasmodium viva, Pf: Plasmodium falciparum

Hematological changes during malarial anemia

The levels of Hb, RBC, and RBC indices and plasma levels of the cytokines between NA and anemic (A) groups were analyzed and compared across various infecting species. In comparison to NA groups, the mean parasitemia percentage was significantly increased in A groups during Pf (P ≤ 0.0001) and Pv (P ≤ 0.0001) infections. In comparison to NA group, the Hb levels significantly decreased in A group, across all infecting groups. Furthermore, within A groups across various infecting parasite species, the Hb levels were significantly lower in Pv than in mixed and Pf infections [Table 2]. The RBC counts, in comparison to NA groups, were significantly decreased in A groups during Pf and Pv infections, especially during Pv infections [Table 2]. In comparison to NA groups, the inflammatory cytokine levels such as TNF-α and IL-6 were significantly increased and IL-10 levels were significantly decreased in A groups across various infecting species [Table 2]. We also found no significant influence of age and gender on RBC levels, Hb levels and its indices, and inflammatory cytokines analyzed.

Table 2

Changes in the hematological parameters and inflammatory cytokine levels in nonanemic and anemic groups across various infecting species

Variable	HC	Pv		Pf		Mixed		P* (between anemic groups)
		NA	A	NA	A	NA	A	Pv vs. Pf	Pv vs. mixed	Pf vs. mixed
Parasitemia (%)		0.14±0.2	0.58±0.7	0.52±0.6	1.24±1.5	0.66±0.7	0.88±0.8	<0.0001	0.0172	0.4509
Hb (g/dl)	14.1±0.8	13.1±1.5	7.2±2.3	13.1±1.6	8.0±2.5	13.1±1.6	8.2±2.2	0.003	0.0195	0.9986
RBC (×103/µl)	4.99±0.7	4.97±0.7	3.71±1.0	4.87±0.7	4.15±1.0	4.89±0.8	4.69±2.8	0.0015	0.0044	0.4396
HCT (%)	40.88±4.1	41.82±6.2	27.20±9.6	40.99±8.3	31.36±9.3	41.90±10.1	32.80±10.5	0.0001	0.0008	0.32
MCH (pg)	26.83±2.9	25.95±5.1	25.66±2.9	25.77±3.9	24.16±4.4	25.70±3.4	23.60±5.4	0.1053	0.1313	0.3283
MCV (fL)	86.59±6.1	84.69±9.4	82.61±13.7	84.83±10.7	82.30±11.8	86.45±12.0	80.60±16.0	0.8682	0.4829	0.558
MCHC (g/dl)	30.76±1.8	30.93±3.3	29.93±3.6	30.54±3.2	29.73±4.0	30.27±5.4	29.37±5.8	0.2797	0.0386	0.0208
TNF-α (pg/ml)	66.73±29.3	212.16±150.0	328.87±217.7	159.25±160.7	370.17±262.1	183.53±189.8	298.03±174.0	0.4327	0.7092	0.3404
IL-6 (pg/ml)	87.48±54.3	211.11±163.2	269.19±188.5	238.86±170.4	356.22±197.7	261.46±143.8	329.09±236.8	0.0006	0.3226	0.3005
IL-10 (pg/ml)	134.1±103.7	632.83±414.6	549.05±460.5	681.73±433.9	535.09±471.5	710.88±348.3	549.98±441.9	0.0437	0.244	0.5924

Data represented a mean±SD. Comparison of two groups by Mann-Whitney U-test; *P≤0.05 is considered to be significant and are highlighted in bold. Pv: Plasmodium vivax, Pf: Plasmodium falciparum, Hb: Hemoglobin, RBC: Red blood cells, HCT: Hematocrit, MCH: Mean cell hemoglobin, MCV: Mean corpuscular volume, MCHC: Mean corpuscular hemoglobin concentration, TNF-α: Tumor necrosis factor-alpha, IL: Interleukin, NA: Nonanemic, A: Anemic, HC: Healthy controls, SD: Standard deviation

A significant negative correlation was observed between increasing parasitemia and decreased Hb levels during Pv (r = −0.3925, P ≤ 0.0001), Pf (r = −0.3426, P ≤ 0.0001), and mixed (r = −0.2972, P = 0.019) infections. In all three groups, parasitemia and RBC levels were negatively correlated during Pv (r = −0.2866, P ≤ 0.0001), Pf (r = −0.2158, P = 0.007), and mixed (r = −0.2093, P = 0.025) infections. Decreased Hb and RBC showed a positive relationship across all infecting groups; Pv (r = 0.4684, P ≤ 0.0001), Pf (r = 0.3854, P ≤ 0.0001), and mixed (r = 0.2375, P = 0.046) infections.

Inflammatory cytokine levels across varying intensity of anemia during malaria

Among the 627 patients, overall 238 (38.0%) patients were anemic. In this study, overall among the infected patients, 144 (23.0%) had MA, 51 (8.1%) had MDA, and 43 (6.9%) suffered due to SA. The number of patients with SA in Pv and mixed infections was comparable with Pf infections [Table 3]. The mean plasma cytokine levels across the varying degree of anemia in patients with Pf, Pv, and mixed infections are outlined below:

Table 3

Inflammatory cytokine levels across varying intensity of anemia during Plasmodium viva, Plasmodium falciparum, and mixed infections

Parameter	NA	MA	MDA	SA
	Pv
Number of patients (%)	253 (65.9)	86 (22.4)	23 (6)	22 (5.7)
Hb (g/dl)	12.8 (12-13.7)	10 (9.3-10.6)	6.9 (6.3-7.4)	4.3 (3.9-4.6)
TNF-α (pg/ml)	183.8 (95-297.4)	252 (12.7.7-406.2)	353.7 (200.7-607.6)	455.6 (204.8-794.8)
IL-6 (pg/ml)	177.9 (68.2-297.4)	207.4 (124.1-281.5)	198 (119.2-316.5)	368.4 (211.2-599.2)
IL-10 (pg/ml)	539.9 (384.1-752.4)	643.9 (396.4-907.9)	319.7 (215.8-834.1)	282.1 (113.5-552.4)
Cytokines	Pf
Number of patients (%)	99 (57.6)	40 (23.3)	18 (10.5)	15 (8.7)
Hb (g/dl)	13 (12.2-13.7)	10.2 (9.4-10.6)	7.2 (6.7-7.7)	4.2 (3.5-4.6)
TNF-α (pg/ml)	86 (45.9-233)	195.3 (146.4-310.9)	356.4 (132-591.5)	678.7 (561.5-882.2)
IL-6 (pg/ml)	181.7 (128.7-324.1)	324.1 (198.4-427.1)	262.2 (147.6-576.9)	445.4 (306.7-516.6)
IL-10 (pg/ml)	585.7 (328-934)	487.7 (274.2-1047)	385.2 (186.4-727)	155.4 (101.4-183.1)
Cytokines	Mixed
Number of patients (%)	37 (52.1)	18 (25.4)	10 (14.1)	6 (8.4)
Hb (g/dl)	12.8 (12.1-14)	9.8 (9.3-10.1)	7.3 (6.3-7.5)	4.7 (3.7-4.9)
TNF-α (pg/ml)	93.6 (68.9-169.1)	200.5 (145.5-331)	200 (92.75-481.3)	488.4 (360.5-548.5)
IL-6 (pg/ml)	265.8 (137-369.4)	151.7 (119.1-413.3)	292.3 (128.8-515.9)	501.5 (183.7-867.1)
IL-10 (pg/ml)	657 (422.6-816.4)	577 (298-835.6)	502.5 (273.4-652.2)	226.2 (213.3-311.1)

Data shown as mean (inter quartile range, i.e., 25% and 75%) and were analyzed by one-way nonparametric Kruskal-Wallis test for multiple comparisons and Mann-Whitney U-test for comparison between two groups were corrected for multiple comparison error by Bonferroni corrections. NA: Nonanemic, MA: Mild anemia, MDA: Moderate anemia, SA: Severe anemia, Hb: Hemoglobin, TNF-α: Tumor necrosis factor-alpha, IL: Interleukin, Pv: Plasmodium viva, Pf: Plasmodium falciparum

Tumor necrosis factor alpha

In comparison with HC, irrespective of infecting species, the TNF-α level was found to be significantly increased in NA groups. On comparison with NA groups, the TNF-α level gradually increased with an increase in anemic intensity during Pv, Pf, and mixed infections [Table 3 and Figure 1]. Between SA groups of various infecting species, the TNF-α levels were found to be significantly increased in patients with Pf infections.

Figure 1

Inflammatory cytokine levels during varying degree of anemia in patients with Pv, Pf, and mixed infections: Plasma levels of TNF-α (a-c), IL-6 (d-f), and IL-10 (g-i) during Pv, Pf, and mixed infections and the cytokine levels TNF (j), IL-6 (k), and IL-10 (l) in severe anemia patients. Data shown as mean ± standard deviation and were analyzed by one-way nonparametric Kruskal–Wallis test for multiple comparisons and Mann–Whitney U-test for comparison between two groups; P ≤ 0.05 were considered to be statistically significant and are highlighted in bold. Pv: Plasmodium vivax; Pf: Plasmodium falciparum; NA: Nonanemic; MA: Mild-anemia; MDA: Moderate anemia; SA: Severe anemia; TNF-α: Tumor necrosis factor-alpha; IL-6: Interleukin-6; IL-10: Interleukin-10

Interleukin-6

The IL-6 levels, in comparison with HC, were significantly increased in NA groups. On comparison with NA groups, the IL-6 levels were found to be significantly increased in MA and SA during Pv and Pf infections. However, the IL-6 levels among SA patients between the various infecting groups did not show any significant change [Table 3 and Figure 1].

Interleukin-10

The levels of anti-inflammatory cytokine IL-10, in comparison with HC, were found to be increased in NA groups. A gradual decrease in IL-10 levels was observed with an increase in anemic severity, particularly in SA patients when compared with NA groups. Between the SA patients across various infecting species, the IL-10 levels were found to be significantly decreased in Pf infections Table 3 and Figure 1]. In this study, irrespective of infecting species, we observed a significantly higher TNF-α/IL-10 ratio in patients with malarial anemia. The comparison within various infecting groups, Pf (P = 0.015) infections showed a significant increase in TNF-α/IL-10 ratios [Figure 2]. This supports the hypothesis that IL-10 is a critical factor in downregulating the TNF-α during the pathogenesis of SA.

Figure 2

The TNF-α and IL-10 ratios among NA (NA–closed gray boxes) and anemic groups (A–closed black boxes) during Pv, Pf, and mixed malarial infections. Data shown as mean ± standard deviation were analyzed by one-way nonparametric Kruskal–Wallis test for multiple comparisons, and Mann–Whitney U-test for comparison between two groups, *** indicate significance value of P < 0.001. Abbreviations used: Pv: Plasmodium vivax; Pf: Plasmodium falciparum; NA: Nonanemic; A: Anemia; TNF-α: Tumor necrosis factor-alpha; IL-10: Interleukin-10

To understand the influence of malarial anemia on increased parasitic burden (i.e., % parasitemia), Hb, and inflammatory cytokines levels, a correlation analysis was performed. There was a significant positive correlation between increased parasitemia percentage and TNF-α levels during Pv (r = 0.3216, P ≤ 0.0001) and Pf (r = 0.3669, P ≤ 0.0001) infections. The decrease in Hb levels also had a significant negative correlation on TNF-α during Pv (r = −0.2513, P ≤ 0.0001), Pf (r = −0.4412, P ≤ 0.0001) and mixed (r = −0.4603, P ≤ 0.0001) infections. A significant inverse relationship was observed between parasitemia and IL-6 levels during Pv (r = −0.1189, P = 0.020) and Pf (r = −0.2892, P ≤ 0.0001) infections. A significant positive correlation was observed between decreased Hb and IL-10 levels during Pv (r = 0.8318, P = 0.001), Pf (r = 0.2338, P = 0.002), and mixed (r = 0.3578, P = 0.002) infections.

Clinical manifestations in patients with severe anemia

In this study, of the total 627 malaria patients, 25 (4%) patients were found to be suffering from SA. Among admitted patients with SA, 2 (8%) patients had SA alone as a complication, 23 (92%) patients suffered from additional complications such as acute renal failure (4, 16.0%), jaundice (4, 16.0%), metabolic acidosis (6, 24.0%), hypoglycemia (3, 12.0%), hyperparasitemia (1, 4.0%), and hepatic dysfunction (4, 16.0%) in various combinations. All SM patients were treated successfully, and there were no death cases [Table 4].

Table 4

Prevalence of severe malarial complications among patients with severe anemia admitted to the hospital for supportive care

Severe malarial complications	Overall (n=25)	Pv (n=10)	Pf (n=7)	Mixed (n=8)
Acute renal failure	4 (16.0)	2 (20.0)	2 (28.6)	0 (0.0)
Jaundice	4 (16.0)	2 (20.0)	2 (28.6)	0 (0.0)
Metabolic acidosis	6 (24.0)	3 (30.0)	2 (28.6)	1 (14.3)
Hypoglycemia	3 (12.0)	2 (20.0)	1 (14.3)	0 (0.0)
Hyperparasitemia	1 (4.0)	0 (0.0)	1 (14.3)	0 (0.0)
Hepatic dysfunction	4 (16.0)	1 (10.0)	2 (28.6)	1 (14.3)

Data represented as number of patients (%). Pv: Plasmodium viva;, Pf: Plasmodium falciparum

DISCUSSION

Anemia during malaria remains a major public health problem with significant morbidity and mortality.[9] Traditionally, Pf infections have been considered to be resulting in anemia more frequently and with more severe degree than observed during Pv infections. However, in spite of a lower parasitic burden observed during Pv infections as compared to Pf infection the emerging reports across various endemic settings show that Pv malaria may even be associated with higher frequency and more severe degree of anemia than Pf infections.[2627] The aim of the present study was to understand the effect of malarial infection on hematological parameters such as Hb, RBC levels and its indices, anemic severity, associated severe malarial complications, and inflammatory cytokine levels (TNF-α, IL-6, and IL-10) in patients from the endemic region of Mangaluru city and its surrounding regions. The cytokine levels also have been measured across the varying intensity of malarial anemia during Pv, Pf, and mixed (Pv and Pf) infections. In our study site, among the two major infecting species, Pv infections were found to be predominant.[356] Further, higher proportions of males were infected, as most of them were employed as laborers in construction of roads and buildings. These construction sites are known to provide ideal breeding grounds for vector propagation resulting in high vector density and increased spread of infection.[3] Even though anemia is a frequent and important complication of malaria, its pathogenesis is not well understood.[17] It is well known that during Pf infections, higher parasitic load leads to increased hemolysis which may rapidly lead in progression of mild form of malarial anemia into severe form. In contrast to earlier reports observed during Pf infections, in spite of a lower parasitic burden in Pv-infected anemic patients, we observed a significant decrease in Hb and RBC levels and a significant negative relationship with increasing parasitemia and decreasing RBC and Hb levels.[25] The proportion of patients with SA during Pv (5.7%) infections were also comparable with Pf (8.7%) infections.

The pro- and anti-inflammatory cytokines are important mediators of development and outcome of malaria.[27] To explore the influence of inflammatory cytokines, the levels of TNF-α, IL-6, and IL-10 were compared and analyzed in patients across varying degree of anemia during Pv, Pf, and mixed infections. In response to malarial infections, the TNF-α is known to be released by macrophages. Experimental evidence show that TNF-α alone and in combination with various other cytokines and chemokines can be a potent inhibitor of erythropoiesis through the suppression of bone marrow function and increased destruction of RBCs, characteristic to malaria.[2728] In vitro studies proved that TNF suppresses the proliferation of erythroid progenitor cells in human marrow cultures.[29] Murine model studies also have demonstrated that in Plasmodium berghei infected mice, upon administration of recombinant TNF-α, there is an increase in spleen size, reduction of pluripotent stem cells and erythroid progenitor cells in the bone marrow. It is also postulated that TNF-α may not be directly, but indirectly is responsible for dyserythropoiesis and erythrophagocytosis by increased release of reactive oxygen species by leukocytes in the bone marrow.[30] In this study, we observed a significant increase in TNF-α level, particularly during SA, suggesting that increased TNF might result in impaired erythropoiesis not only during Pf but also during Pv infections[3132] A significant positive relationship between increasing parasitic density and TNF-α level were observed in our study as reported earlier.[33] The levels of Hb and TNF-α also showed a negative correlation suggesting TNF-α plays an important role in anemic severity during malaria.[934]

IL-6 also could play a role in inflammatory response and pathophysiology of SMA.[35] It is well known that IL-6 production is upregulated by TNF-α and acts together with other inflammatory mediators to reduce the parasitic burden. In this study, in comparison to HC, we observed increased IL-6 levels among infected patients and a reciprocal relationship was also observed between increasing parasitemia and IL-6 levels. Further, the IL-6 levels were found to be significantly increased during SA. A study in Malawian children has shown a positive association between the increased IL-6 levels and acute iron deficiency.[36] Research studies also suggest that increased IL-6 levels have a strong association with increase in transferrin receptor density, and decreased transferrin synthesis which play a significant role in anemic severity.[3536] Our observation of a negative correlation between decreased Hb levels and increased IL-6 suggests that higher IL-6 production which may contribute to the pathogenesis of SMA.[25]

IL-10 is a monocyte/lymphocyte-derived cytokine-mediated by the T-helper type 2-like response.[373839] The higher level of this anti-inflammatory cytokine is known to inhibit the overproduction of TNF-α and IL-6, thus excreting protective effects against SA.[17] Studies in IL-10 knockout mice infected with Plasmodium chabaudi resulted in increased severity and mortality, in comparison to wild-type mice suggesting a significant role of IL-10 in the inhibition of pro-inflammatory cytokines such as interferon gamma.[40] Various in vivo studies also suggested that lower IL-10 levels influence the bioavailability of nitric oxide which can affect erythropoiesis and thus plays an important role in pathogenesis of malarial anemia.[1441] Administration of recombinant IL-10 in humans during a double-blind, placebo-controlled study showed a decrease in Hb levels, suggesting an important role of IL-10 in the development of anemia.[42] Recent studies have proven IL-10 to be a regulator of hepcidin production (master regulator of iron homeostasis) by STAT3 phosphorylation, especially in macrophages.[43] The lower IL-10 levels were observed in African children with SA suggesting that insufficient IL-10 response to high TNF-α concentrations may have a central role in anemic severity.[12] In this study, as observed during Pf infections, we found a significant decrease in IL-10 levels during Pv and mixed infections also.[1439] Irrespective of type of infection, a significant positive correlation between decreased Hb and IL-10 levels was observed. In this study, the lower levels of IL-10 among anemic patients might have resulted in decreased circulatory levels of hepcidin, resulting in SA.

Our study agrees with the earlier reports of high TNF-α/IL-10 ratio during malarial anemia, in comparison to NA groups across various infecting species. The high TNF-α/IL-10 ratio reflects an insufficient production of IL-10 in patients with SA to counteract the inhibition of erythropoiesis and increased erythrophagocytosis due to increased TNF-α level.[33944] SA is found to be associated with several life-threatening complications, especially during severe Pf infections.[4546474849] In this study, SA patients experienced a range of malarial complications such as acute renal failure, jaundice, metabolic acidosis, hypoglycemia, and hepatic dysfunction in various combinations. Interestingly, in the endemic settings of Mangaluru city, the proportion of SA patients and the intensity of accompanying SM complications among these patients were comparable across Pv, Pf, and mixed infections. However, it is to be noted that all the SA patients recovered well, and there were no reported cases of death.

Limitations

Although the study is extensive, there are some limitations such as (i) only adults were recruited into the study excluding children and (ii) the malarial diagnosis was performed by microscopic examination of Giemsa-stained blood smears and no molecular methods such as polymerase chain reaction were used.

CONCLUSIONS

The present findings of our study in this region, for the first time, give an insight into the severity of malarial anemia and inflammatory cytokine profile and its associated complications during malarial anemia. We observed a significant reduction in Hb levels and increased levels of inflammatory cytokine in patients suffering from malarial anemia. The increased levels of TNF-α, IL-6, and decreased IL-10 levels suggest a critical role of these cytokines in the pathogenesis of malarial anemia, especially during Pv infections. Further detailed studies are needed to understand the risk factors, burden of malarial anemia among children, and to understand the role of inflammatory cytokines and its association with severe malarial complications of anemia, especially during Pv infections.

Financial support and sponsorship

This work was supported by the Grant D43 TW008268 from the Fogarty International Center of the National Institutes of Health, USA, under the Global Infectious Diseases Program.

Conflicts of interest

There are no conflicts of interest.