Hayashida Kyoko, Kajino Kiichi, Simukoko Humphrey, Simuunza Martin, Ndebe Joseph, Chota Amos, Namangala Boniface, Sugimoto Chihiro
Division of Collaboration and Education, Hokkaido University Research Center for Zoonosis Control, Sapporo, 001-0020, Japan.
Department of Biomedical Sciences, School of Veterinary Medicine, University of Zambia, P.O. Box 32379, Lusaka, Zambia.
Parasit Vectors. 2017 Jan 13;10(1):26. doi: 10.1186/s13071-016-1949-8.
Because of the low sensitivity of conventional rapid diagnostic tests (RDTs) for malaria infections, the actual prevalence of the diseases, especially those caused by non-Plasmodium falciparum (non-Pf) species, in asymptomatic populations remain less defined in countries lacking in well-equipped facilities for accurate diagnoses. Our direct blood dry LAMP system (CZC-LAMP) was applied to the diagnosis of malaria as simple, rapid and highly sensitive method as an alternative for conventional RDTs in malaria endemic areas where laboratory resources are limited.
LAMP primer sets for mitochondria DNAs of Plasmodium falciparum (Pf) and human-infective species other than Pf (non-Pf; P. vivax, P. ovale, P. malariae) were designed and tested by using human blood DNA samples from 74 residents from a malaria endemic area in eastern Zambia. These malaria dry-LAMPs were optimized for field or point-of-care operations, and evaluated in the field at a malaria endemic area in Zambia with 96 human blood samples. To determine the sensitivities and specificities, results obtained by the on-site LAMP diagnosis were compared with those by the nested PCR and nucleotide sequencing of its product. The dry LAMPs showed the sensitivities of 89.7% for Pf and 85.7% for non-Pf, and the specificities of 97.2% for Pf and 100% for non-Pf, with purified blood DNA samples. The direct blood LAMP diagnostic methods, in which 1 μl of anticoagulated blood were used as the template, showed the sensitivities of 98.1% for Pf, 92.1% for non-Pf, and the specificities of 98.1% for Pf, 100% for non-Pf. The prevalences of P. falciparum, P. malariae and P. ovale in the surveyed area were 52.4, 25.3 and 10.6%, respectively, indicating high prevalence of asymptomatic carriers in endemic areas in Zambia.
We have developed new field-applicable malaria diagnostic tests. The malaria CZC-LAMPs showed high sensitivity and specificity to both P. falciparum and non-P. falciparum. These malaria CZC-LAMPs provide new means for rapid, sensitive and reliable point-of-care diagnosis for low-density malaria infections, and are expected to help update current knowledge of malaria epidemiology, and can contribute to the elimination of malaria from endemic areas.
由于传统快速诊断检测（RDTs）对疟疾感染的敏感性较低，在缺乏配备完善的准确诊断设施的国家，无症状人群中该疾病，尤其是由非恶性疟原虫（非Pf）物种引起的疾病的实际流行情况仍不太明确。我们的直接血样干LAMP系统（CZC-LAMP）作为一种简单、快速且高度灵敏的方法，被应用于疟疾诊断，以替代疟疾流行地区实验室资源有限时的传统RDTs。
设计了针对恶性疟原虫（Pf）和除Pf之外的人类感染性疟原虫物种（非Pf；间日疟原虫、卵形疟原虫、三日疟原虫）线粒体DNA的LAMP引物组，并使用赞比亚东部疟疾流行地区74名居民的人类血液DNA样本进行了测试。这些疟疾干LAMP针对现场或即时检测操作进行了优化，并在赞比亚的一个疟疾流行地区使用96份人类血液样本在现场进行了评估。为了确定敏感性和特异性，将现场LAMP诊断获得的结果与其产物的巢式PCR和核苷酸测序结果进行了比较。对于纯化的血液DNA样本，干LAMP对Pf的敏感性为89.7%，对非Pf的敏感性为85.7%，对Pf的特异性为97.2%，对非Pf的特异性为100%。以1μl抗凝血液为模板的直接血样LAMP诊断方法，对Pf的敏感性为98.1%，对非Pf的敏感性为92.1%，对Pf的特异性为98.1%，对非Pf的特异性为100%。在调查区域，恶性疟原虫、三日疟原虫和卵形疟原虫的流行率分别为52.4%、25.3%和10.6%，这表明赞比亚流行地区无症状携带者的比例很高。
我们开发了新的适用于现场的疟疾诊断检测方法。疟疾CZC-LAMP对恶性疟原虫和非恶性疟原虫均显示出高敏感性和特异性。这些疟疾CZC-LAMP为低密度疟疾感染的快速、灵敏和可靠的即时诊断提供了新手段，有望有助于更新当前疟疾流行病学知识，并有助于在流行地区消除疟疾。
