Background
Malaria is an infection caused by protozoan parasites of the genus
Plasmodium and transmitted by the bite of infected
Anopheles mosquitoes. Out of the four species that infect humans,
Plasmodium falciparum is the principal cause of severe clinical manifestations [
1]. Cyto-adherence and rosetting are important components of several possible pathogenic mechanisms attributed to the cause of severe infection [
2]. An association between 'O' blood group and lower rosetting capacity has been demonstrated [
3]. However, rosetting capacities of blood group 'A', 'B' or 'AB' have remained controversial [
4‐
7]. On the erythrocyte surface, the A and B antigens are tri-saccharides -A, GalNAcα1-3(Fucα1-2)Gal1β1; and B- Gal1α1-3(Fucα1-2)Galβ1 respectively, that are attached to different glycolipids and glycoproteins [
8]. An enzyme glucotransferase is necessary for the production of A and B antigens. On the other hand, blood group 'O' carries a disaccharide H antigen (Fucα1-2Galβ1) due to the absence of the enzyme glucotransferase [
8]. Variations in gene encoding functional glucotransferase have been associated with protections from severe
P. falciparum malaria [
9] and this observation has been further strengthened by a recent genome wide association study [
10]. Tri-saccharide of 'A' and 'B' blood group is presumed to act as receptors and functions as an important factor for rosetting [
7]. However, RBCs of blood group 'O' do not express tri-saccharide, and rosettes formed by infected 'O' blood group RBCs are smaller and easily disrupted compared to blood groups A, B or AB [
4,
7,
11].
There are limited reports in literature on association of ABO blood group and susceptibility to severe falciparum malaria.. The association of blood group 'AB' and severe malaria has been demonstrated in various populations, viz. Sri Lanka [
12], Mali [
11], and Ethiopia [
1], while a significant association has also been reported between blood group 'A' and severity in Gabon [
13], Ethiopia [
1] and Zimbabwe [
14]. The role of blood group 'B' and severe falciparum infection has not been reported.
Malaria remains a major health problem in India. The National Vector Borne Disease Control Programme (NVBDCP), India, has reported that 1.8 million cases of malaria and 1,000 malaria-related deaths occur annually [
15]. However, the World Health Organization (WHO) estimates that figure to be 20 million cases and 15,000 deaths [
16]. A recent study reported a staggering 1,22,000 deaths due to malaria in India, and Odisha as a major contributor to this mortality [
17]. Although the state is hyper-endemic to
P. falciparum malaria[
18] and contributes 29.8% of deaths related to the infection [
15], no study has been carried out in the local population to assess the association of ABO blood group in severe infection. Therefore, the current study aims to investigate this association and look into the overall role of ABO blood group in risk/resistance to the development of severe malaria by a meta-analysis of results from the current study and earlier published reports.
Methods
Study site and participants
The study was conducted at S.C.B. Medical College, Cuttack, Odisha, India between 2008-2009. Patients (age ≥15 years) admitted to the Department of Medicine with a short history of fever were clinically examined in detail and screened for
P. falciparum infection by Giemsa-stained thick and thin blood smears and immune chromatography test (SD Bio Standard Diagnostics India). Detection of
P. falciparum was also performed by nested polymerase chain reaction (PCR). Individuals infected only with
P. falciparum were included. Clinical categorization was done based on WHO guidelines [
19]. Uncomplicated malaria (UM) was defined as patients with fever and evidence of falciparum infection in the blood. Severe malaria(SM) was categorized into three groups based on distinct clinical features: 1) Cerebral malaria (CM), 2) Non cerebral severe malaria (NCSM) and 3) Multi-organ-dysfuction (MOD). CM was further defined as patients with altered sensorium, GCS (Glasgow Coma Scale) of ≤ 10. NCSM patients had one of the several manifestations of severe malaria without cerebral involvement, namely severe anaemia (haemoglobin <5 g/dl), acute renal failure (serum creatinine >3 mg/dl), jaundice (serum bilirubin >3 mg/dl), acute respiratory distress syndrome (PaO2/FIO2 <200), haemoglobinuria (dark red or black coloured urine positive for haemoglobin) and shock (systolic BP of <80 mm Hg). MOD was diagnosed based on presence of two or more organ involvement like CNS (GCS≤10), respiratory (PaO2/FIO2 <200), renal failure (serum creatinine >3 mg/dl) and hepatic dysfunction (ALT/AST >3 times of normal, prolonged prothrombin time and albuminaemia). 174 healthy controls (HC) of identical ethnicity and hailing from a similar geographical background were enrolled. None of the controls reported history of clinical malaria in the last 5 years. They were essentially healthy and negative for demonstrable
P. falciparum infection. The risk of exposure to malaria was similar for both HC and patients. Criteria for analysis of mortality: Since death occurred in the CM and MOD groups' only patients from these groups were included for analysis. The study was approved by the Institutional Ethics Committee of the Medical College and blood samples were collected after written consent of the patients or accompanying person, depending on the clinical scenario.
Polymerase chain reaction
Genomic DNA was extracted by GenElute™ Blood Genomic DNA Kit (SIGMA) from whole blood according to the manufacturer's instructions. Polymerase chain reaction (PCR), has been used to detect up to species level - falciparum, vivax, ovale and malariae [
20,
21]. In the population under study,
P. falciparum is the major cause of malaria (>80%) followed by
P. vivax (10-15%) [
22]. With slight modification, genus and species specific nested PCR technique was used to detect
P. falcipaurm and/or
P. vivax infections [
20]. In brief, primary PCR (to detect
Plasmodium genus) was performed in a 20 μl PCR reaction containing 3 μL genomic DNA, 1× Taq buffer containg MgCl
2 (SIGMA), 250 μM dNTP (SIGMA)), 200 nM of two genus-specific primer rPLU1 (5'-TCA AAG ATT AAG CCA TGA AAG TGA-3') and rPLU5 (5'-CCT GTT GTT GCC TTA AAC TCC-3') (Integrated DNA technologies) and 1 U of DNA polymerase (SIGMA). The cycle conditions were as follows: an initial de-naturation step at 95°C for 7 minutes, followed by 38 cycles of 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, and a final extension at 72°C for 7 minutes. For secondary PCR (to detect falciparum and/or vivax species), 3 ul of PCR product of previous reaction was used as template and species-specific primers set were used {rFAL1(5'-TTA AAC TGG TTT GGG AAA ACC AAA ACC AAATAT ATT)/rFAL2(5'-ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC-3') and rVIV1(5'-CGC TTC TAG CTT AAT CCA CAT AAC TGA TAC-3')/rVIV2(5'-ACT TCC AAG CCG AAG CAA AGA AAG TCC TTA-3')} in different tubes. Reaction mixture and thermo-cycler condition was similar to primary PCR. PCR products were analysed in agarose gel electrophoresis.
ABO blood group typing
Blood samples of both falciparum-infected individuals and healthy controls were typed by commercial haemagglutination kit (Tulip Diagnostics, Goa, India). In brief, about 20 ul of whole blood was taken on a clean slide and 20 ul of antisera A, B and Rhesus blood group applied, mixed by means of an applicator stick and results were noted.
Identification of studies for meta-analysis
An extensive PubMed search was performed for articles published on the association of ABO blood group in severe P. falciparum malaria. Cross-references were checked including studies not located in PubMed. The following key words and subject terms were searched: ABO blood group, Plasmodium falciparum, severe malaria and uncomplicated malaria.
Statistical analysis
Statistical analyses were performed by using GraphPad Prism (version 5.01). Difference between means was analysed by ANOVA. The Fisher exact test was used to analyse the association between blood groups and severe malaria or different clinical manifestation. Odds ratio (OR) were calculated with 95% confidence interval. The 'O' blood group was taken as a reference to compare the prevalence of other blood groups, 'A', 'B', 'AB' and non 'O', defined as A+B+AB combined, in severe and uncomplicated malaria. A probability value of ≤ 0.01 (0.05/4) was considered statistically significant after Bonferroni correction. Meta-analysis and all related statistics were obtained by Comprehensive Meta-analysis V2 software. Variation within and among different study (heterogeneity) was assessed by Q-test with the null hypothesis that all studies have the same effects. The random effects model was used for meta-analysis if the Q-statistic was significant (P < 0.05) which indicate heterogeneity across studies. On the other hand, lineage of all studies to null hypothesis, the fixed effect model, was used for necessary meta-analysis.
Discussion
Results of the current study suggest that patients with blood group 'B' have a four-fold increased risk of developing severe infection. In addition, it also reiterates the observation that 'O' blood group was significantly associated with a decreased risk of severe malaria. Other blood groups ('A' and 'AB') did not show any association.
The role of ABO blood group in malaria has been investigated in various populations, but robust data is limited [
24]. This study was an attempt to analyse the association of ABO blood group in large number of adult patients and healthy controls in Odisha, a state, highly endemic for falciparum infection [
18]. Higher prevalence of blood group 'O' was observed in uncomplicated cases, an indication of its possible protective property against severity as indicated in previous reports [
1,
11‐
13]. The mechanism of protection is not clearly understood. It is postulated that the phenomenon of rosetting is one of the mechanisms that contributes to disease severity [
2]. This rosetting capacity varies among different blood groups [
3]. Lowest rosette formation is observed in blood group 'O' individuals. This study also clearly highlights the association of severe malaria with blood group 'B'. Interestingly, this association was valid across all grades of severity. Although the number of patients who died (n = 20) was small, frequency of blood group 'B' was higher in these subjects (80%) in comparison to survivors (54%). Previously, studies on patients from Zimbabwe [
14], Gabon [
13] and Ethiopia [
1] showed a significant association of 'A' blood group with severe malaria. Blood group 'AB' has also been reported to be associated with severity in Sri Lanka [
12], Mali [
11] and Ethiopian populations [
1]. There are no reports implicating blood group 'B' with severity. The variability of observations made with regard to different blood groups may be attributable to different rosetting capacity, heterogenous population groups and varied infective strains [
3]. Blood group 'A' in Uganda and Gambia [
4,
7], 'B' group in Thailand [
4] and 'AB' group in Kenya [
6] have been associated with increased rosetting phenomenon.
It is presumed that the prevalence of blood group 'O' would be higher in malaria endemic areas due to its capacity to confer protection. An analysis of blood group in healthy controls revealed a distribution of 'O' (42%), which was much higher compared to 'A' (21%), 'B' (29%) and 'AB' (8%). A community-based study in a tribal population of Odisha, where malaria is endemic, also showed higher prevalence of blood group 'O' [
25]. Significantly, a lower prevalence 'O' blood group has been observed in other malaria non-endemic states like Maharashtra [
26,
27] and Uttar Pradesh [
28] in India, indicating a selective advantage of this blood group in endemic localities. This hypothesis is further supported by higher prevalence of 'O' blood group worldwide where malaria infection is prevalent [
29].
Meta-analysis combines results of several similar studies to produce a single estimate of the major effect with enhanced precision [
30]. Current analysis revealed significant association of blood group 'O' with protection against severe malaria (OR = 0.45) In contrast, blood groups 'A' and 'AB' were associated with susceptibility to severity: Blood group 'A' and 'AB' conferred 1.27- and 1.74- fold higher risks respectively. Although in the current study, 'B' blood group was significantly associated with severe malaria on meta-analysis the association was insignificant. This variation may be a population specific phenomenon.
There are however limitations in the present study. Several RBC polymorphisms, including those linked to glucose-6-phosphate dehydrogenase, pyruvate kinase, complement receptor-1 and haemoglobinopathies, have a role in the clinical outcome of malaria [
31], but were not included for analysis in the present study. Such polymorphisms have only been reported from the Western belt of Odisha among tribal communities [
32,
33] and not in areas from which patients and HC in the current study were enrolled.
Acknowledgements
The authors are grateful to the patients and healthy controls who participated in this study. Senior Research fellowship to AKP and SKP was provided by Council of Scientific and Industrial Research and Indian Council of Medical Research, respectively. The authors are also thankful Mr Subrat Kumar Mohanty and Mr Paritosh Nath for typing of blood group in malaria patients and healthy controls. Institute of Life Sciences is funded by Department of Biotechnology, Government of India.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AKP was involved in detection of malaria by PCR, analysis, interpretation, performed statistics and writing the first draft of the manuscript. SKP was also involved in species-specific PCR and analysis. ANS, RT, BR and BKD made a contribution in the design, data interpretation, work supervision and critically revising the manuscript. All authors read and approved the manuscript.