Malaria is a mosquito borne protozoan infection caused by Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. In a recent report, World Health Organization (WHO) estimates about198 million cases in 2013 and 1.2 billion subjects are at high riskworldwide (WHO, 2014). Majority of malarial death is a result of P. falciparum infection which leads to severe malaria characterized by cerebral malaria (CM), multi-organ dysfunction (MOD), and non-cerebral severe malaria (NCSM) (Buffet et al., 2011; Panda et al., 2011, 2012; Pattanaik et al., 2012). The pathogenesis of malaria is complex and the severity depends on parasite virulence, transmission dynamics, genetic factors, and host immune response (Buffet et al., 2011; Mangano and Modiano, 2014; White et al., 2014). Malaria infection elicits both innate and adaptive immune response of the host. The innate immune system is composed of diverse pattern-recognizing receptors or soluble pathogen-recognizing molecules (PRMs) (Thiel et al., 2006; Barreiro et al., 2009), which recognize specific molecular motifs on the surfaces of virus, bacteria and parasites. Mannose binding lectin is a liver derived soluble PRMs which play an important role in the innate immune response. MBL binds to sugars on the surface of pathogenic micro-organisms and triggers the complement activation system (Ip et al., 2009). MBL has been shown to bind to parasite infected erythrocytes (Garred et al., 2003b) and children deficient in MBL are prone to severe malaria (Luty et al., 1998), indicating an important role for MBL in protection against P. falciparum malaria.

The human MBL is a 32 KDa protein consisting of 248 amino acids encoded by MBL2 mapped to 10q21.1 (Garred et al., 2006). Common MBL2 genetic variants have been associated with various diseases such as filariasis (Choi et al., 2001; Meyrowitsch et al., 2010), malaria (Boldt et al., 2006; Jha et al., 2014), leishmaniasis (Asgharzadeh et al., 2007), leprosy (de Messias-Reason et al., 2007; Sapkota et al., 2010), tuberculosis (Singla et al., 2012; Chen et al., 2014), trypanosomiasis (Weitzel et al., 2012), HIV infection(Li et al., 2013), systemic lupus erythematosus (Panda et al., 2013), and rheumatoid arthritis (Martiny et al., 2012). The MBL2 gene consists of four exons. Although several single nucleotide polymorphisms (SNPs) have been reported, three SNPs in exon 1 (codons 52: rs5030737, C > T, Arg>Cys; codon 54: rs1800450, G > A, Gly> Asp and codon 57: rs1800451, G >A,Gly>Glu) are of importance since they affect plasma levels of MBL. Variants are denoted as D (codon 52), B (codon 54), and C (codon 57), whereas A is the common wild type allele (Garred et al., 2006). In addition, two other functional polymorphisms at promoter region of the MBL2 gene have been reported (−550: rs11003125,G > C, H/L and−221: rs7096206, C > G, X/Y) which have been shown to affect plasma MBL levels (Garred et al., 2003a). Furthermore, some reports have shown an association of combined exon1 and promoter MBL2 polymorphisms with plasma levels of MBL (Tsutsumi et al., 2001; Panda et al., 2013).

The association between MBL2 polymorphism and P. falciparum malaria in Gabonese children has been reported in several studies but it has shown contradictory results. Luty et al. (1998), showed an association between codon 54 and 57 variants with susceptibility to severe malaria. On the contrary, another study failed to show similar association (Mombo et al., 2003). Interestingly, a novel mutation (−797C > A) has been linked to susceptibility to severe malaria (Boldt et al., 2006).

A recent study in Indian population revealed association between MBL2 LYPA haplotype with protection and MBL2 LXPA haplotype with increased susceptibility to severe malaria (Jha et al., 2014). To the best of our knowledge, there are no studies on the association of plasma MBL and common MBL2 polymorphism with P. falciparum malaria in well-defined clinical phenotypes. We report an association between low plasma MBL and MBL low producer genotypes with multi organ dysfunction in Odisha, India.

In the present case control study, we investigated the role of MBL in P. falciparum malaria in an endemic region of Odisha, India. Our data suggest that low levels of MBL and MBL2 low producer genotypes are associated with multi organ dysfunction. Furthermore, in the current investigation we also confirm a correlation between MBL2 polymorphism with plasma levels of MBL.

MBL is an important component of the innate immune system. It plays a significant role in the defense against various infections. It recognizes carbohydrate domain on the microbial surface, opsonizes the pathogen and/or activates lectin pathways (Turner, 1996). Role of MBL in malaria has been demonstrated earlier (Luty et al., 1998; Juliger et al., 2002; Thevenon et al., 2009). In the present study, we observed a significantly higher level of plasma MBL in healthy controls from the endemic areas compared to uncomplicated and severe malaria patients, indicating a possible role for plasma MBL in the defense against P. falciparum infection. MBL binds to infected erythrocytes (Garred et al., 2003b) and possibly assists in the clearance of infected cells. Individuals with lower plasma MBL levels may have an increased susceptibility to malarial infection due to lack of this mechanism, and the infection may progress to develop severe disease. However, previous reports from the African continent indicate that normal healthy controls have lower MBL levels compared to patients with severe malaria (Luty et al., 1998) and P. falciparum infected cases (Juliger et al., 2002). Our study deals with adult patients with clinically distinct clinical phenotypes of severe malaria from an endemic area that has seasonal transmission. The earlier studies were from geographically different areas with distinct population base and transmission dynamics (Luty et al., 1998; Juliger et al., 2002). Children are mostly susceptible to severe malaria in Africa and they have a different clinical profile while adults do not suffer from severe disease. The discordance of observation in our study and the earlier reported data could be due to the difference in the population studied. Among the various categories of severe malaria patients, MOD patients had lower plasma MBL levels compared to CM and NCSM. MOD has high mortality among those with severe malaria. Serum MBL levels appear to modulate severity of disease by mechanisms which are not clearly defined.

Although large numbers of SNPs in MBL2 gene have been reported, only limited SNPs have functional relevance. Variants at amino acids coding region may lead to deformed proteins which degrade leading to its lower levels. Mutation at promoter regions may similarly affect binding of transcription factors leading to diminished production of transcripts which ultimately translate to lesser protein expression. Our study revealed an association of BB and AB genotypes with lower plasma MBL similar to studies reported earlier in Danish (Madsen et al., 1995; Steffensen et al., 2000), Australian (Minchinton et al., 2002), and Indian population (Panda et al., 2013). We also observed association between MBL2 Y-221X polymorphism with plasma MBL levels similar to our earlier report (Panda et al., 2013). However, MBL2 combined genotype and H-550L polymorphism did not correlate with plasma levels of MBL. This could be a result of small sample size available for plasma MBL estimation.

We observed a significant association between MBL2 codon 54 and promoter Y-221X polymorphism with susceptibility to multi organ dysfunction. Subjects with heterozygote genotype for codon 54 (AB) and Y-221X (YX) polymorphism had 2.34 and 2.06 fold higher chance of developing MOD. Furthermore, combined analysis of codon and promoter polymorphism revealed significant association of low producer genotype (LXA/LYB, LYB/LYB, LXB/LXB) with susceptibility to MODs (OR = 4.03). Earlier studies reported the role of MBL2 polymorphism in severe malaria. MBL2 codon 54 and 57 variants in Gabonese children, LXPA haplotype in Indian population (Jha et al., 2014) and a novel mutation (−797 C>A) (Boldt et al., 2006) have been linked with susceptibility to severe malaria. The present study also showed similar susceptibility of MBL2 codon 54 and Y-221X variants to severe malaria but did not reach significance after Bonferoni correction.

In the present investigation, distribution of MBL codon 54 (A/B) and promoter (H-550L) polymorphism deviated from HWE in line with our earlier observation (Panda et al., 2013). Disparity of genotype distribution from HWE has been attributed to population stratifications and/or selection pressure (Hosking et al., 2004). In the current study, patients and controls were enrolled from a similar geographical area, and the possible reason for deviation of HWE could be selection pressure. Malaria exerts a strong selection pressure on the human genome and beneficial allele are positively selected and become more prevalent in endemic areas. The studied population is also endemic to filariasis, and a previous study by our group (unpublished results), and others (Choi et al., 2001; Meyrowitsch et al., 2010) had shown an association between MBL2 variants with susceptibility to lymphatic filariasis. Infectious diseases exert selection pressure on human population (Miller, 1999; Ghosh, 2008; Fumagalli et al., 2009) and parasite endemic areas are significantly affected.

Our study clearly defines the association between low plasma levels of MBL and the low producer MBL genotype with severe disease, notably, MOD. Although it is known that MBL functions as an important protective component of the innate immune system, the mechanism(s) that modulates severity in P. falciparum remains to be clearly defined.

AP was involved in design, performing experiments, analysis, interpretation, performed statistics, and writing the first draft of the manuscript. BD made a contribution in the design, data interpretation, work supervision and critically revising the manuscript. All authors read and approved the manuscript.