Haghi Afsaneh Motevalli, Khoramizade Mohammad Reza, Nateghpour Mehdi, Mohebali Mehdi, Edrissian Gholam Hossein, Eshraghian Mohammad Reza, Sepehrizadeh Zargham
Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Korean J Parasitol. 2012 Mar;50(1):15-21. doi: 10.3347/kjp.2012.50.1.15. Epub 2012 Mar 6.
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.
在伊朗，间日疟原虫每年导致超过80%的疟疾病例感染。人类间日疟的控制干预措施主要依赖于已开发的诊断方法。据报道，重组间日疟原虫顶端膜抗原-1（rPvAMA-1）可实现快速、灵敏且特异的分子诊断设计。本研究旨在分离并表达源自生活在疟疾流行地区的伊朗患者的rPvAMA-1。然后，使用间接ELISA方法评估所鉴定的伊朗PvAMA-1的诊断效率。为此，扩增、克隆AMA-1基因的部分区域，并在pET32a质粒中进行表达。纯化重组His标签蛋白并用于包被ELISA板。通过使用rPvAMA-1的间接ELISA评估抗体检测。将该ELISA方法在现场检测抗间日疟原虫抗体的有效性与84例确诊的间日疟患者的光学显微镜检测结果进行比较，并与84例未感染间日疟原虫的个体进行比较。ELISA临界值计算为来自伊朗南部疟疾流行地区人群OD值的平均值+2SD。我们发现OD = 0.311的临界值在间日疟原虫感染确诊血清与健康对照血清之间显示出最佳相关性。在此临界滴度下，灵敏度为81.0%，特异性为84.5%。通过Kappa分析发现，使用rPvAMA-1的ELISA与光学显微镜检测之间存在良好程度的统计学一致性（0.827）。
