Detection of Plasmodium Species by High-Resolution Melt Analysis of DNA from Blood Smears Acquired in Southwestern Uganda
Detection of Plasmodium Species by High-Resolution Melt Analysis of DNA from Blood Smears Acquired in Southwestern Uganda
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DOI：
10.1128/jcm.01060-17
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发表时间：
2018-01-01
影响因子：
9.4
通讯作者：
Boum, Yap, II
中科院分区：
文献类型：
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作者：
Kassaza, Kennedy;Operario, Darwin J.;Boum, Yap, II
在资源有限的情况下，使用吉姆萨染色血涂片进行疟疾的显微镜诊断是标准的护理方法。这些涂片是PCR检测确认疟原虫感染或存档样本流行病学研究的潜在DNA来源。因此，我们评估了使用实时PCR检测疟原虫的染色血涂片的DNA提取物的使用。我们从2010年乌干达西南部无症状儿童的存档血涂片和相应的红细胞颗粒中提取DNA。然后，我们进行实时PCR，然后进行高分辨率熔解（HRM），以确定疟原虫的物种，我们比较了我们的结果与显微镜。我们共分析了367例血涂片和相应的红细胞团块，其中185例涂片（50.4%）经显微镜检查呈阳性。与镜检相比，PCR-HRM检测涂片DNA的敏感性为93.0%（95%置信区间[ CI]，88.2 - 96.2%），特异性为96.7%（95% CI，93.0 - 98.8%），沉淀DNA的PCR-HRM分析的灵敏度为100.0%（95% CI，98.0 - 100.0%），特异性为94.0%（95% CI，89.4 - 96.9%）。阳性PCR HRM结果鉴定为恶性疟原虫（92.0%）、卵形疟原虫（5.6%）和三日疟原虫（2.4%）。与显微镜相比，Giemsa染色的厚血涂片或相应的血丸DNA提取物的PCR-HRM分析对疟疾诊断具有高灵敏度和特异性。因此，血涂片可以提供足够的DNA来源，用于确认疟原虫属感染，并可用于回顾性遗传研究。
Microscopic diagnosis of malaria using Giemsa-stained blood smears is the standard of care in resource-limited settings. These smears represent a potential source of DNA for PCR testing to confirm Plasmodium infections or for epidemiological studies of archived samples. Therefore, we assessed the use of DNA extracts from stained blood smears for the detection of Plasmodium species using real-time PCR. We extracted DNA from archived blood smears and corresponding red blood cell pellets collected from asymptomatic children in southwestern Uganda in 2010. We then performed real-time PCR followed by high-resolution melting (HRM) to identify Plasmodium species, and we compared our results to those of microscopy. We analyzed a total of 367 blood smears and corresponding red blood cell pellets, including 185 smears (50.4%) that were positive by microscopy. Compared to microscopy, PCR-HRM analysis of smear DNA had a sensitivity of 93.0% (95% confidence interval [ CI], 88.2 to 96.2%) and a specificity of 96.7% (95% CI, 93.0 to 98.8%), and PCR-HRM analysis of pellet DNA had a sensitivity of 100.0% (95% CI, 98.0 to 100.0%) and a specificity of 94.0% (95% CI, 89.4 to 96.9%). Identification of positive PCR-HRM results to the species level revealed Plasmodium falciparum (92.0%), Plasmodium ovale (5.6%), and Plasmodium malariae (2.4%). PCR-HRM analysis of DNA extracts from Giemsa-stained thick blood smears or corresponding blood pellets had high sensitivity and specificity for malaria diagnosis, compared to microscopy. Therefore, blood smears can provide an adequate source of DNA for confirmation of Plasmodium species infections and can be used for retrospective genetic studies.