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Table of Contents
RESEARCH ARTICLE
Year : 2019  |  Volume : 56  |  Issue : 2  |  Page : 122-126

C-reactive protein as a prognostic marker of Plasmodiumfalciparum malaria severity


1 ICMR-National Institute of Malaria Research, New Delhi; Department of Biotechnology, Kumaun University, Bhimtal, Uttarakhand, India
2 ICMR-National Institute of Malaria Research, New Delhi, India
3 Department of Biotechnology, Kumaun University, Bhimtal, Uttarakhand, India

Date of Submission26-Mar-2019
Date of Acceptance14-Jun-2019
Date of Web Publication31-Jul-2019

Correspondence Address:
Veena Pande
Head, Department of Biotechnology, Kumaun University, Bhimtal-263 136, District Nainital, Uttarakhand
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.263727

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  Abstract 

Background & objectives: Plasmodiumfalciparum malaria causes wide variety of clinical symptoms ranging from a mild febrile illness to life-threatening complications. For prevention of the severity and early diagnosis, evaluation of potential biomarkers is much needed. C-reactive protein (CRP) is an acute phase protein and well-recognized marker of inflammation in the body. It is synthesized by liver in response to pro-inflammatory responses and has correlation with complications associated with malaria. The study was aimed to assess, if it could serve as a predictive marker for malaria disease severity.
Methods: In the present study, 74 P. falciparum patients and 22 healthy controls were enrolled. Turbidimetric immunoassay was used to measure the CRP in serum samples of all the study participants. Mann-Whitney U-test for continuous data and chi-square test for categorical data were used to compare all malaria cases vs. healthy control group and uncomplicated vs. severe malaria groups. Using receiver operating characteristic (ROC) analysis, best threshold value was determined for CRP in severe malaria patients.
Results: CRP level was significantly elevated in all malaria case groups (1.6 mg/dl IQ 1-2.6) as compared to healthy controls (0.10 mg/dl IQ 0.1-0.20), with p-value <0.0001. Further, CRP level was significantly higher in the severe malaria group (2 mg/dl IQ 1.8-3.9) as compared to the uncomplicated malaria group (1.4 mg/dl IQ 1-2.47) and healthy control group (0.10 mg/dl IQ 0.10-0.20), with p-value <0.05.
Interpretation & conclusion: The present study findings suggest that CRP level can be used to differentiate severe malaria from uncomplicated malaria. Elevated CRP level could be helpful in early prediction of the disease severity in patients infected with P. falciparum and may play an important role in diagnosis of falciparum malaria where improper initial test and clinical manifestations like fever may be absent even with a high load of parasite.

Keywords: Biomarker; C-reactive protein; malaria; Plasmodium falciparum; severe malaria; uncomplicated malaria


How to cite this article:
Bhardwaj N, Ahmed MZ, Sharma S, Nayak A, Anvikar AR, Pande V. C-reactive protein as a prognostic marker of Plasmodiumfalciparum malaria severity. J Vector Borne Dis 2019;56:122-6

How to cite this URL:
Bhardwaj N, Ahmed MZ, Sharma S, Nayak A, Anvikar AR, Pande V. C-reactive protein as a prognostic marker of Plasmodiumfalciparum malaria severity. J Vector Borne Dis [serial online] 2019 [cited 2019 Nov 17];56:122-6. Available from: http://www.jvbd.org/text.asp?2019/56/2/122/263727




  Introduction Top


Malaria is a significant public health problem in India and Plasmodiumfalciparum infections contribute significantly in terms of morbidity and mortality. The subsidiary burden of malaria like malnutrition and anaemia may increase the risk of complications and severity[1],[2]. Plasmodiumfalciparum cases are potentially severe and life-threatening, especially when managed inappropriately. A major reason for progression from mild through complicated to severe disease is missed or delayed diagnosis[3]. Neither parasite density nor parasitaemia constantly define malaria severity[4]. Hyper-parasitaemia does not essentially have primary prognostic significance in semi-immune individuals, as they often tolerate high parasitaemia burden without any physiological signs of disease or severe effects. Hence, prognostic biomarker of severe malaria for detection at an early stage to avoid complications and severity, is the need of the hour.

When P. falciparum enters the host it produces various immunological stress and proteomic modification, leading to serious complications and severity; still a reliable predictive marker for the severe form of malaria is unrecognized[5]. Several blood biomarkers like total leukocyte count, liver function test and glucose test are available to assess the malaria complications, but these are not always capable of defining disease severity. Some studies have identified few markers like angiopoietin 1 and 2 as a biomarker of severity; however, reliability and application of such biomarkers in the routine practice are challenging[6].

C-reactive protein is an inflammatory marker which is found in blood and considered as a predictor of severity in malaria. Its level in blood has shown strong correlation with parasite density even in patients without clinical symptoms[7]. It is synthesized by liver in response to pro-inflammatory responses, induced by the infection, stress and tissue damage[8]. While circulating, CRP stimulates the classical complement pathway and is a key player of numerous essential innate immune processes such as phagocytosis and inflammatory responses[9]. Thus, CRP level measurement could help in understanding the manifestation, severity and pathogenicity of the P. falciparum malaria. This study was aimed to find a correlation between CRP level and disease severity due to P. falciparum infection and whether it can be used as disease marker.


  Material & Methods Top


This study was carried out at District Hospital, Dha-lai, Tripura, India during September 2016–October 2017. Blood samples were collected from a total of 96 individuals, which included 74 malaria patients and 22 healthy controls. All the patients were tested for P. falciparum infection by microscopy and those who were positive for malaria were treated according to the national drug policy. Parasite densities were confirmed by Giemsa- stained thick and thin blood smears in conventional light microscopy. Parasites were counted as number of asexual parasite/μl and calculated as per formulae:

Parasitaemia = [No. of parasites per 1000 RBCs on thin smear x haematocrit (Hct) x 125.6]

Parasitaemia = [No. of parasites per 500 WBCs on thick smear x 8000 WBCs count]

Ethical statement

The study was approved by the Institutional Ethics Committee of ICMR-National Institute of Malaria Research, New Delhi (Approval No. ECR/NIMR/ EC/2016/278).

Study procedures

Participants were screened by physicians for infection or diseases other than malaria at the time of enrolment in the present study. Out of 74 malaria cases only seven patients fulfilled one or more than one criteria of severe malaria according to the WHO[10] severe malaria guidelines. Patient with parasitaemia >100,000/μl along with multiple convulsion and respiratory distress (n = 1), hyperparasitaemia >5% with multiple convulsion and respiratory distress (n = 4) and hyperparasitaemia >5% with respiratory distress (n = 2) were selected.

Sample collection

Blood samples were collected in plain red top vac- utainer (BD Vacutainer®, India) from all the study participants. These were stored upright at room temperature for 30 min and then were spun at 1300 x g for 15 min at 4 °C. Supernatant (upper layer serum) was collected and stored at –80 °C till use.

C-reactive protein measurement

C-reactive protein level was measured by turbidi- metric immunoassay using a commercially available compatible TURBILYTE® CRP kit in a Coralyzer 200 instrument (Tulip Diagnostics Pvt. Ltd, Goa, India).

Statistical analyses

C-reactive protein level was compared between case group vs. healthy control group and also within the case group using Mann-Whitney U/-test. Median values along with interquartile ranges were recorded for each group. Chi-square test was used to compare the male/female ratio within malaria cases and malaria case vs healthy control group. Linear regression analysis was performed to assess the prediction of malaria severity. Using receiver operating characteristic (ROC) analysis, best threshold value was determined for CRP in severe malaria patients. A p-value of <0.05 was considered significant. Analyses were performed using SPSS software, version 17.


  Results Top


Baseline characteristics

C-reactive protein was measured in P. falciparum infected patients as a prognostic marker of severe malaria. The demographic characteristics like age was comparable in malaria case group [18 yr (13–29.25)] vs healthy controls [28 yr (25.75–40.75)] (p <0.05), and uncomplicated malaria group [18 yr (14–30)] vs severe malaria group [12 yr (10–25)] (p < 0.05); while male and female ratio in malaria case group vs healthy control group and uncomplicated malaria vs severe malaria group was not statistically significant. Pulse and respiratory rates showed significant change in the malaria case group in comparison to healthy control group; median pulse was 96 vs 82.5 bpm (p ≤ 0.010) and median respiratory rate was 26/min vs 20/ min (p <0.0001), respectively.

Similarly, in the severe malaria group respiratory rate was higher (35/min) than uncomplicated malaria group (25/min) (p ≤ 0.0001), while pulse was not significantly altered in severe malaria group (93 bpm) than uncomplicated malaria group (96 bpm) (p = 0.536). The median value (interquartile range) of parasitaemia, i.e. parasite/ μl was 58027.5 (11,840–103,243) in all malaria cases group; 48,796 (10,816–92,944) in uncomplicated malaria group and 253,210 (212,892-375,418) in severe malaria group.

Levels of C-reactive protein in all case groups and control

C-reactive protein level was significantly elevated in all the malaria cases [1.6 mg/dl (1–2.60)] as compared to the healthy control group [0.10 mg/dl (0.10–0.20)]; p <0.0001 (Mann-Whitney U-test was used to calculate the p-value). Furthermore, CRP level was significantly higher in severe malaria group [2 mg/dl (1.8-3.9)] as compared to uncomplicated malaria group [1.4 mg/dl (1–2.47)]; p = 0.013 (on the basis of Mann-Whitney U-test).

The prediction result of CRP level was significant for severe malaria vs uncomplicated malaria (p = 0.027; Odds ratio = 2.13) using univariate binary logistic regression model. Even in the multivariate binary logistic regression model analysis (adjusting the confounding factor like age), CRP level showed higher significance (p = 0.019, Odds ratio = 2.57) [Table 1].
Table 1: Logistic regression analysis of CRP in uncomplicated malaria (UM) vs severe malaria (SM)

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The diagnostic performance and accuracy of CRP was assessed by area under receiver operating characteristic (AUROC) analysis at 95% confidence intervals (CI). C-reactive protein level showed significant AUROC in case group vs healthy control group [area under curve (AUC): 98.9%)] [Figure 1]a. Remarkably, the C-reactive protein level showed good discriminated values (cut-off=1.85, sensitivity = 71.4, and specificity = 68.7) between uncomplicated malaria vs severe malaria group with good AU- ROC (AUC: 78.6%) [Figure 1]b. The box plot analysis of CRP in uncomplicated malaria, severe malaria and healthy control groups suggested similar pattern [Figure 2].
Figure 1: Receiver operating characteristics curve analysis of CRP level in: (a) All malaria cases group vs healthy control group, area under curve-98.9%; and (b) Severe malaria vs uncomplicated malaria group, area under curve-78.6%.

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Figure 2: Box plot graphical representation of C-reactive protein level showing median (interquartile ranges) value of uncomplicated malaria, severe malaria and healthy control groups.

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  Discussion Top


Severe malaria can cause life-threatening conditions like renal failure, hypotension, hypoglycaemia, etc. in pa- tients[5]. Hence, it is essential to develop biomarkers which can predict the severity of P. falciparum malaria for avoiding complications of targeted therapy. In the present study, CRP— A first acute phase protein of inflam-mation and a known sensitive marker of inflammation and tissue damage[11]; was measured in terms of its level in blood in P. falciparum malaria patients as a prognostic marker of severity.

Interleukin 1-ß (IL-1ß) and interleukin 6 (IL-6) are well-known inflammatory markers, and play important role during induction of CRP expression levels in human hepatocytes[12]. The CRP stimulation in hepatocytes is controlled at transcriptional level by IL-6, a regulation that is enhanced by IL-1ß[13]. The early elevation in CRP levels during disease progression and rapid kinetics makes it a widely used inflammatory protein marker. Activation of platelet and complement pathways are triggered by the CRP which results in initiation of effector pathway, leading to complications. Therefore, CRP level measurement could be helpful in understanding the pathogenesis of severe cases of malaria[2].

In this study, CRP levels were significantly elevated in case group compared to healthy controls (p < 0.0001); the elevated levels of CRP may help in the early detection of disease even in asymptomatic malaria patients[7]. This protein has pathophysiological role in the recognition, clearance and destruction of ailing red blood cells (RBCs). In a typical malaria case; anaemia may be the reason of unhealthy erythrocytes lysis, as it effectively binds differentially with infected erythrocyte to clear them from circula-tion[14]. Appropriately, in the present study, CRP levels were significantly elevated in severe malaria as compared to the uncomplicated malaria (p = 0.013). So it might have role in evaluating the P. falciparum malaria severity as well as in response to the antimalarial treat-ment. The present study findings suggest that elevated CRP level is subtle to predict severe malaria from the uncomplicated malaria. However, altered level of CRP was also reported in sepsis disease[15] which causes inflammation in the body. The expression of CRP was found to be elevated in cardiovascular and kidney diseases[16]. In a study by Stauga et al[17], it was stated that the CRP level (cut-off value as 10.8 mg/l) can discriminate the malaria cases from healthy controls. Similarly, in this study the cut-off value of 1.85 mg/dl (equivalent to 18.5 mg/l) for CRP level with sensitivity 71.4% and specificity 68.7%, indicates its adequacy in discriminating severe malaria from uncomplicated malaria (with 78.6% area under curve). Thus, CRP can be used as a prognostic biomarker of severe malaria.

Limitations: The present study has lower study sample size including very few severe malaria cases, and the CRP level differences between uncomplicated and severe malaria were not very much significant. Though, CRP levels cannot be considered as a major biomarker for P. falciparum malaria severity, it could serve as an important early prediction marker for P. falciparum malaria severity. Further study is required to establish its role in predicting the P. falciparum malaria severity with more severe sample.


  Conclusion Top


Elevated CRP levels could be helpful in predicting the disease severity in patients infected with P. falciparum malaria and may play an important role in diagnosis of P. falciparum where improper initial test and clinical manifestations like fever may be absent even with a high load of parasite.

Conflict of interest: None.


  Acknowledgements Top


The author NB would like to express his gratitude to the ICMR–National Institute of Malaria Research, New Delhi for infrastructure, funding and overall support and the Kumaun University for Ph.D registration. MZA is thankful to DBT for fellowship.



 
  References Top

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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1]


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