Prevalence of Sickle Cell Anemia in Association of Plasmodium falciparum from Rajnandgaon, India

Special Article - Anemia

Ann Nutr Disord & Ther. 2021; 8(1): 1067.

Prevalence of Sickle Cell Anemia in Association of Plasmodium falciparum from Rajnandgaon, India

Tripathi S¹, Mishra N² and Kumar A²*

¹Department of Science and Technology, Women Scientist-A, India

²Department of Biotechnology, Government VYT PG Autonomous College, India

*Corresponding author: Anil Kumar, Department of Biotechnology, Government VYT PG Autonomous College, Durg-491001, Chhattisgarh, India

Received: December 15, 2020; Accepted: January 07, 2021; Published: January 14, 2021


Sickle cell anemia is a blood disorder resulting from the inheritance of abnormal genes from parents. It is caused due to mutation in the β-globulin gene. Sickle cell anemia is widespread across the world and in Central India. The present study was undertaken to study the prevalence of the disorder in the Rajnandgaon district of Central India. A random sampling of 6088 people was done to test the sickle cell anemia problem by slide test method and a total of 249 (4.09%) people were found sickled positive. Further electrophoresis test was performed for all 249 of which 67 were found homozygous (HbSS) and 182 were found heterozygous (HbAS) positive.

Besides the above analysis, chloroquine prophylaxis associated with a high prevalence of Plasmodium falciparum Pfcrt K76T mutation in people (n=26) with sickle cell anemia was also analyzed. The genotype of the subject was screened using the hemoglobin electrophoresis system and the P. falciparum Pfcrt genotyping was carried out using PCR-Restriction Fragment Length Polymorphism (RFLP). The prevalence rate of Pfcrt K76T mutant gene was proportionately found higher in the hemoglobin SS (n=40, m=32, r=0.67) genotype individuals than the hemoglobin AS (n=52, m= 27, r=0.519) and AA (n=182, m= 68, r= 0.37).

Keywords: Sickle cell anemia; P. falciparum; Chloroquine; Pfcrt K76T, PCR-RFLP


Sickle Cell Hemoglobin (HbS) is the first molecular disease known to man [1]. It is a structural variant of hemoglobin in which glutamic acid (an amino acid), at position no.6 of a β-globin chain of hemoglobin is replaced by valine. This happens due to the change of nucleotide, adenine to thymine (GAG/GTG) of codon 6 of a β-globin gene, located on the short arm of chromosome 11. This substitution of amino acid changes the net charge of hemoglobin, oxygen affinity, and three-dimensional structure of hemoglobin, thus rendering it unstable hemoglobin. Sickle hemoglobin gets polymerized at low oxygen tension and deforms the red cell from discoid shape to sicklelike form causing a lot of pathogenicities [2]. Due to its genetic nature of origin, the disease is inheritable following the Mendelian Principle. In malaria-infested areas, the high frequency of hemoglobinopathies, such as Sickle-Cell Disease (SCD), exhibits their protective role against P. falciparum malaria [3]. However, in homozygous sickle hemoglobin (SS) cases, the tenacity of P. falciparum could set off acute hemolytic events [4] and/or recurrence of Vaso-Occlusive Crises (VOCs).

The exact mechanism by which sickle-cell trait (AS) condition imparts resistance to malaria is not known. Various factors are likely to be involved that contribute to varying degrees of defense against malaria [5]. Erythrocytes of sickle cell trait, infected with the P. falciparum parasite are deformed since the parasite drastically decreases the oxygen tension within the red cells as it carries out its metabolism also. This deformation of sickle trait (AS) erythrocytes makes these cells abnormal and obvious targets for phagocytosis [6].

Plasmodium genus is approximated to have evolved around 150 million years ago, even before the evolution of Homo sapiens [7]. So, humans have evolved in the presence of malaria, and this co-evolution has shaped the human genome [8]. Various polymorphisms are protective against severe forms of malaria. The worldwide distributions of Hemoglobinopathies like sickle cell anemia, thalassemias, Glucose-6-phosphate dehydrogenase (G6PD) deficiency, and blood group polymorphisms suggests the prevalence of malaria, indicating that malaria has been a selective force for such mutations [9][7]. Also, genetic variations in human P450 genes (CYP2C8, CYP3A4, and CYP2A6) results in differential metabolism of anti-malarial drugs in humans and have important imputations in both anti-malarial drug efficacy and bearability [10,11].

Chloroquine is a 4-amino-quinoline anti-malarial drug that interferes with the sequestration of toxic heme, which is produced when hemoglobin is digested by an intra-blood parasite to obtain essential amino acids. The parasite crystallizes heme into hemozoin in its acidic digestive vacuoles.

Chloroquine (and other similar drugs) binds to heme and prevents the detoxification process [12]. Parasite resistance is believed to be achieved by the decreased accumulation of chloroquine in the digestive vacuole of the parasite.

In 2000, the P. falciparum chloroquine resistance transporter (pfcrt), the key gene involved in this resistance, was discovered [13]. CQ resistance is associated with a T76 mutation of the P. falciparum chloroquine resistance transporter gene (Pfcrt) [13] while a multidrug resistance analog (Pfmdr1) Y86 variation may modulate its degree [14]. The gene is present on chromosome 7 and encodes a drug and metabolite transporter protein (PfCRT) located on the membrane of the digestive vacuole. A unique mutation pfcrt K76T, was found in connection with other compensatory amino acid residue changes at loci PfCRT 72-76, where PfCRT 72-76 CVMNK is the sensitive haplotype and CVIET and SVMNT are considered the most common resistant haplotypes [15].

Chloroquine resistance depends upon the genetic background of the parasite line i.e., pfcrt K76T enhances the tolerance to chloroquine so that resurgence is likely to occur, but it does not always result in clinical dereliction [12].

According to a hypothesis regarding the resistance is the protonation of chloroquine in the acidic environment of the digestive vacuole. Efflux of the chloroquine (CQ2+) is reduced by the charged lysine (PfCRT K76) amino acid residue in the Chloroquine sensitive strain. When lysine is replaced by neutral amino acid residue threonine (PfCRT 76T) then CQ2+ can egresses down its concentration gradient through PfCRT, eliminating the drug from its target site. Another drug, Verapamil, which is a calcium (Ca2+) channel blocker, is can reverse this resistance by competing with chloroquine for binding at PfCRT, thereby blocking the efflux of the drug from the digestive vacuole.

For a very long time, it is supposed that the malarial parasite has tempted RBC for various mutations for protection. Probably sickling is one of them. A lot of Epidemiological and biochemical evidence is available related to the concept. Chhattisgarh state (India) is one of the major centers of malaria epidemics, especially for Plasmodium falciparum. We have noticed the development of malaria among the sickled population against the previous concept.

So banking on the prevalence of both malaria epidemics and sickling in the society of Chhattisgarh state (India), we have undertaken the present study to detect mutation in Plasmodium falciparum in an association of Heterozygous Sickling (HbAS) and Homozygous Sickling (HbSS) with a correlation of normal hemoglobin.

Material and Method

For the study, seven villages of Rajnandgaon District of Chhattisgarh state (India) within a radius of 60-70kms from district headquarters were selected. Two milliliters of intravenous blood was collected from each person by paramedical staff following ICMR and Institutional Ethics Committee norms. From the first village total, 6088 samples were collected from all seven villages (Table 1). During sample collection, it was taken into note that some of the donors were suffering from malaria and had taken chloroquine as medicine.