Abstract
BackgroundGlutamate dehydrogenase of malaria parasites (pGDH) is widely used in rapid diagnostic tests for malaria. Variation in the pGDH gene among Korean isolates of Plasmodium vivax was analysed, and a recombinant pGDH protein was evaluated for use as antigens for the serodiagnosis of vivax malaria.MethodsGenomic DNA was purified from blood samples of 20 patients and the pGDH gene of P. vivax was sequenced. Recombinant protein was prepared to determine the antigenicity of pGDH by enzyme-linked immunosorbent assay (ELISA).ResultsPartial sequence analysis of the P. vivax pGDH gene from the 20 Korean isolates showed that an open reading frame (ORF) of 1410 nucleotides encoded a deduced protein of 470 amino acids. The amino acid and nucleotide sequences were conserved among all the Korean isolates. This ORF showed 100% homology with P. vivax strain Sal-I (GenBank accession No. XP_001616617.1). The full ORF (amino acids 39–503), excluding the region before the intron, was cloned from isolate P. vivax Bucheon 3 (KJ726751) and subcloned into the expression vector pET28b for transformation into Escherichia coli BL21(DE3)pLysS. The expressed recombinant protein had a molecular mass of approximately 55 kDa and showed 84.8% sensitivity (39/46 cases) and 97.2% specificity (35/36 cases) in an ELISA. The efficacy of recombinant pGDH protein in seroepidemiological studies was also evaluated by ELISA using serum samples collected from 876 inhabitants of Gyodong-myeon, Ganghwa County, Incheon Metropolitan City. Of these samples, 91 (10.39%) showed a positive reaction with recombinant pGDH protein. Among the antibody-positive individuals, 13 (14.29%) had experienced malaria infection during the last 10 years.ConclusionThe pGDH genes of P. vivax isolates from representative epidemic-prone areas of South Korea are highly conserved. Therefore, pGDH is expected to be a useful antigen in seroepidemiological studies. It was difficult to identify the foci of malaria transmission in Gyodong-myeon based on the patient distribution because of the very low parasitaemia of Korean vivax malaria. However, seroepidemiology with recombinant pGDH protein easily identified regions with the highest incidence of malaria within the study area. Therefore, recombinant pGDH protein may have a useful role in serodiagnosis.
Highlights
Glutamate dehydrogenase of malaria parasites is widely used in rapid diagnostic tests for malaria
Glutamate dehydrogenase (GDH), an enzyme involved in urea synthesis, catalyzes the reversible oxidative deamination of l-glutamate to form α-ketoglutarate and ammonia, using nicotinamide adenine dinucleotide phosphate (NADP(H)) or nicotinamide adenine dinucleotide (NAD(H)) as cofactor [5]
Plasmodium falciparum contains three genes encoding potential parasite glutamate dehydrogenase proteins; two are found on chromosome 14 (PF14_0164 and PF14_0286, encoding pGDHa and pGDHb, respectively) and one is present on chromosome 8 (PF08_0132, encoding pGDHc) [7, 8]. pGDHa and pGDHb are NADP(H) dependent, and the primary sequence of pGDHb suggests that the protein is located in the apicoplast, whereas the localization and cofactor specificity of pGDHc cannot be predicted
Summary
Glutamate dehydrogenase of malaria parasites (pGDH) is widely used in rapid diagnostic tests for malaria. During the last 20 years, the development of alternative diagnostic methods for malaria, such as rapid diagnostic tests (RDTs), has made it possible to extend biological diagnosis to remote areas that lack advanced medical services. RDTs are lateral-flow immunochromatographic tests that detect specific malaria antigens. They are rapid and simple enough to carry out without electricity, specific equipment, or intensive training of personnel [2,3,4]. Plasmodium falciparum contains three genes encoding potential parasite glutamate dehydrogenase (pGDH) proteins; two are found on chromosome 14 (PF14_0164 and PF14_0286, encoding pGDHa and pGDHb, respectively) and one is present on chromosome 8 (PF08_0132, encoding pGDHc) [7, 8]. PGDH has been used as an antigen for malaria detection
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