A recombinant Plasmodium vivax apical membrane antigen-1 to detect human infection in Iran

Abstract

In Iran, Plasmodium vivax is responsible for more than 80% of the infected cases of malaria per year. Control interventions for vivax malaria in humans rely mainly on developed diagnostic methods. Recombinant P. vivax apical membrane antigen-1 (rPvAMA-1) has been reported to achieve designing rapid, sensitive, and specific molecular diagnosis. This study aimed to perform isolation and expression of a rPvAMA-1, derived from Iranian patients residing in an endemic area. Then, the diagnostic efficiency of the characterized Iranian PvAMA-1 was assessed using an indirect ELISA method. For this purpose, a partial region of AMA-1 gene was amplified, cloned, and expressed in pET32a plasmid. The recombinant His-tagged protein was purified and used to coat the ELISA plate. Antibody detection was assessed by indirect ELISA using rPvAMA-1. The validity of the ELISA method for detection of anti-P. vivax antibodies in the field was compared to light microscopy on 84 confirmed P. vivax patients and compared to 84 non-P. vivax infected individuals. The ELISA cut-off value was calculated as the mean+2SD of OD values of the people living in malaria endemic areas from a south part of Iran. We found a cut-off point of OD=0.311 that showed the best correlation between the sera confirmed with P. vivax infection and healthy control sera. A sensitivity of 81.0% and specificity of 84.5% were found at this cut off titer. A good degree of statistical agreement was found between ELISA using rPvAMA-1 and light microscopy (0.827) by Kappa analysis.

Keywords: AMA-1; ELISA; Iran; Plasmodium vivax; microscopy; recombinant antigen.

Fig. 1

SDS-PAGE analysis for rPvAMA-1 production. Lane 1: E. coli BL21(DE 3) bacterial lysate containing pET 32 a plasmid (negative control). Lane 2: E. coli BL21(DE 3) bacterial lysate containing pET 32 a-AMA-1 recombinant plasmid. Lane 3: Protein size marker.

Fig. 2

SDS-PAGE analysis of rPvAMA-1 expression. PSM: protein size marker (kDa). PvAMA-1: His tagged recombinant antigen purified by Ni-NTA affinity column after IPTG induction.