用光学显微镜和巢式聚合酶链反应（Nested PCR）方法检测外周血扩展中的疟疾，并通过 18S rRNA 基因调查伊朗东南部的疟原虫种的遗传多样性。

Detection of malaria with light microscopy and Nested polymerase chain reaction (Nested PCR) methods in peripheral blood expansions and investigation of the genetic diversity of Plasmodium species by 18S rRNA gene in Southeast of Iran.

机构信息

Department of Medical Parasitology and Mycology, Kerman University of Medical Sciences, Kerman, Iran.

Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran; Students Research Committee, Kerman University of Medical Sciences, Kerman, Iran.

出版信息

Microb Pathog. 2019 Dec;137:103782. doi: 10.1016/j.micpath.2019.103782. Epub 2019 Oct 7.

DOI:10.1016/j.micpath.2019.103782

PMID:31600540

Abstract

BACKGROUND

Malaria is a public health concern that leads to about a million deaths worldwide every year. Malaria is caused by the genus Plasmodium, which includes P. falciparum, P. vivax, P. malariae, and P. ovale. Molecular phylogeny is essential to better recognition the evolution of the genus Plasmodium genus and detection of the relative degree of Plasmodium species in humans. The aim of this study was to detect malaria with Light Microscopy (LM) and Nested polymerase chain reaction (Nested PCR) methods in peripheral blood expansions and to investigate the genetic diversity of Plasmodium species by 18S rRNA gene in the southeast of Iran.

METHODS

A total of 97 blood smears were collected from patients suspected to malaria in a 6-year period in the southeast of Iran including Hormozgan, Kerman, and Sistan and Baluchestan provinces. Diagnosis of Plasmodium species on blood smears was performed using LM and Nested PCR methods. In addition, 16 Plasmodium-positive samples were chosen for the determination of genetic diversity.

RESULTS

Overall, 97 of 97 (100%) studied cases were positive by LM while 94 of 97 (96.8%) of them were detected as malaria by Nested PCR. Except for seven cases, Nested PCR confirmed the LM results. These samples involved two P. vivax and five P. falciparum in the LM method. Meanwhile, the Nested PCR was detected in all of the cases as a mixed infection with P. vivax and P. falciparum. The results of the phylogenetic analysis revealed two main clades and five different subclades. About 87.5% of the isolates were located in clade I and contained P. vivax. In addition, 12.5% of the studied isolates involved P. falciparum that was in clade II.

CONCLUSION

According to our results, Nested PCR method had higher sensitivity than LM and is suggested as a good approach for malaria detection. Consideration the wide diversity of tested isolates and the importance of vaccine development, which is affected by this diversity, further studies are needed in this regard.

摘要

背景

疟疾是全球每年导致约 100 万人死亡的公共卫生问题。疟疾是由疟原虫属引起的，包括恶性疟原虫、间日疟原虫、三日疟原虫和卵形疟原虫。分子系统发育对于更好地认识疟原虫属的进化和检测人类中疟原虫种的相对程度至关重要。本研究旨在通过外周血涂片的光镜（LM）和巢式聚合酶链反应（Nested PCR）方法检测疟疾，并通过 18S rRNA 基因调查伊朗东南部地区疟原虫种的遗传多样性。

方法

在伊朗东南部的 6 年期间，共收集了来自疑似疟疾患者的 97 份血涂片，包括霍尔木兹甘省、克尔曼省和锡斯坦和俾路支省。使用 LM 和巢式 PCR 方法对血涂片上的疟原虫种进行诊断。此外，选择了 16 个疟原虫阳性样本以确定遗传多样性。

结果

总体而言，97 例研究病例中，LM 检测阳性率为 97/97（100%），Nested PCR 检测阳性率为 94/97（96.8%）。除 7 例外，Nested PCR 均证实了 LM 结果。这些样本包括 LM 法检测到的 2 例间日疟原虫和 5 例恶性疟原虫。同时，Nested PCR 检测到所有病例均为间日疟原虫和恶性疟原虫混合感染。系统发育分析结果显示出两个主要分支和五个不同的亚分支。大约 87.5%的分离株位于 I 分支，包含间日疟原虫。此外，12.5%的研究分离株涉及 II 分支的恶性疟原虫。