Merozoite surface protein-3α is a reliable marker for population genetic analysis of Plasmodium vivax

BACKGROUND: The knowledge on population structure of the parasite isolates has contributed greatly to understanding the dynamics of the disease transmission for designing and evaluating malaria vaccines as well as for drug applications. msp-1 and msp-3α genes have been used as a genetic marker in population studies of Plasmodium vivax isolates. In this study, msp-3α was compared and assessed with msp-1 marker in order to find whether msp-3α is a reliable genetic marker for P. vivax population studies. METHODS: This comparative study was designed and carried out as the first assessment of diversity in Pvmsp-3α gene by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in the 50 northern and 94 southern P. vivax isolates from Iran, which had been analysed before for msp-1 gene. RESULTS: Three allele size as, Type A (1.8 kb), Type B (1.5 kb) and Type C (1.2 kb) have been detected among both northern and southern isolates based on PCR results. Type C (70%) and Type A (68.7%) were the predominant fragments among northern and southern parasites, respectively. 99 distinct Pvmsp-3α fragments defined by the size were detected in the 94 southern samples by PCR analysis. However, no mixed genotype infections have been detected among northern isolates. Based on restriction pattern from digestion with Hha I and Alu I 12 and 49 distinct allelic variants have been detected among 50 northern and 94 southern isolates. However, based on msp-1 gene, 30 distinct variants identified in all 146-sequenced Iranian P. vivax isolate. CONCLUSION: The results suggested that PCR-RFLP on msp-3α gene is an adequate, applicable and easily used technique for molecular epidemiology studies of P. vivax isolates without the need for further sequencing analysis

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Abstract Glutathione S-transferases (GSTs) are a major family of detoxification enzymes which possess a wide range of substrate specificities. Interest in insect GSTs has primarily focused on their role in insecticide resistance. In this study, following World Health Organization (WHO) routine susceptibility test, DNA was extracted from specimens of Anopheles stephensi collected from the Kazeroon district in the Fars province as control area and Saravan, Chabahar, Nikshahr districts in Sistan and Baluchistan province representing major malarious areas under insecticide treatment, in Iran. The (Glutathion S-transferase Epsilon class 2) GSTe2 gene including exon I and II and the full sequence of intron I, belonging to An. stephensi specimens were then amplified. The size of the resulting amplicons from the control area and the insecticide treated areas were 492 and 489 bp, respectively. These fragments were purified and then sequenced from both ends. The comparison of total amplified fragments among Kazeroon and Nikshahr and/or other populations of the Sistan and Baluchistan province (Saravan and Chabahar) showed 98% and 97% similarities, including 9-11 nucleotide substitutions, none of which had led to any amino acid change, within these populations. Comparison of the nucleotide sequence of GSTe2 in An. stephensi populations with that of the major world malaria vector, Anopheles gambiae revealed 86% homology, while amino acid similarity between the two species was approximately 90%. However, the main difference between the two susceptible and resistance groups in An. stephensi populations is related to their intron sequence with a distance of 8-9%, while this distance among resistance populations from the Sistan and Baluchistan province varied by approximately 0-4%. The results obtained from this study serve as a first report and baseline data regarding the structure of GSTe2 gene, including exon I, exon II and intron I in susceptible and resistance field specimens of An. stephensi. However, the integration of these data into the malaria control program still remains a challenge in Iran and neighboring countries, especially Afghanistan and Pakistan