Kipanga Purity N, Omondi David, Mireji Paul O, Sawa Patrick, Masiga Daniel K, Villinger Jandouwe
Martin Lüscher Emerging Infectious Diseases (ML-EID) Laboratory, Molecular Biology and Bioinformatics Unit, International Centre of Insect Physiology and Ecology (icipe), P,O, Box 30772, Nairobi 00100, Kenya.
Malar J. 2014 Nov 15;13:429. doi: 10.1186/1475-2875-13-429.
Microscopy and rapid diagnostic tests (RDTs) are common tools for diagnosing malaria, but are deficient in detecting low Plasmodium parasitaemia. A novel molecular diagnostic tool (nPCR-HRM) that combines the sensitivity and specificity of nested PCR (nPCR) and direct PCR-high resolution melting analysis (dPCR-HRM) was developed. To evaluate patterns of anti-malarial drug administration when no parasites are detected, nPCR-HRM was employed to screen blood samples for low parasitaemia from febrile patients without microscopically detectable Plasmodium infections in a rural malaria-endemic setting.
Blood samples (n = 197) were collected in two islands of Lake Victoria, Kenya, from febrile patients without Plasmodium detectable by microscopy or RDTs. 18S rRNA gene sequences were amplified from extracted DNA by nPCR-HRM, nPCR, and dPCR-HRM to detect and differentiate Plasmodium parasites. The limits of detection (LoD) were compared using serial dilutions of the WHO International Standard for P. falciparum DNA. Data on administration of anti-malarials were collected to estimate prescription of anti-malarial drugs to patients with and without low parasitaemia Plasmodium infections.
The coupled nPCR-HRM assay detected Plasmodium parasites with greater sensitivity (LoD = 236 parasites/mL) than either nPCR (LoD = 4,700 parasites/mL) or dPCR-HRM (LoD = 1,490 parasites/mL). Moreover, nPCR-HRM detected and differentiated low-parasitaemia infections in significantly greater proportions of patients than did either nPCR or dPCR-HRM (p-value <0.001). Among these low-parasitaemia infections, 67.7% of patients were treated with anti-malarials, whereas 81.5% of patients not infected with Plasmodium parasites were treated with anti-malarials.
The enhanced sensitivity of nPCR-HRM demonstrates limitations of differential febrile illness diagnostics in rural malaria endemic settings that confound epidemiological estimates of malaria, and lead to inadvertent misadministration of anti-malarial drugs. This is the first study that employs low-parasitaemia Plasmodium diagnostics to quantify the prescription of anti-malarial drugs to both non-malaria febrile patients and patients with low-parasitaemia Plasmodium infections. nPCR-HRM enhances low-parasitaemia malaria diagnosis and can potentially surmount the deficiencies of microscopy and RDT-based results in determining low-parasitaemia Plasmodium infection rates for evaluating malaria elimination efforts. The findings highlight the need for improved differential diagnostics of febrile illness in remote malaria endemic regions.
显微镜检查和快速诊断检测（RDTs）是诊断疟疾的常用工具，但在检测低疟原虫血症方面存在不足。一种结合巢式PCR（nPCR）的敏感性和特异性以及直接PCR-高分辨率熔解分析（dPCR-HRM）的新型分子诊断工具（nPCR-HRM）被开发出来。为了评估在未检测到寄生虫时抗疟药物的使用模式，在一个农村疟疾流行地区，采用nPCR-HRM对发热患者的血样进行低疟原虫血症筛查，这些患者经显微镜检查未检测到疟原虫感染。
在肯尼亚维多利亚湖的两个岛屿上，从发热患者中采集血样（n = 197），这些患者经显微镜或RDTs检测未发现疟原虫。通过nPCR-HRM、nPCR和dPCR-HRM从提取的DNA中扩增18S rRNA基因序列，以检测和区分疟原虫。使用世界卫生组织恶性疟原虫DNA国际标准的系列稀释液比较检测限（LoD）。收集抗疟药物使用数据，以估计对有和没有低疟原虫血症疟原虫感染的患者使用抗疟药物的处方情况。
与nPCR（LoD = 4700个寄生虫/毫升）或dPCR-HRM（LoD = 1490个寄生虫/毫升）相比，联合nPCR-HRM检测疟原虫的敏感性更高（LoD = 236个寄生虫/毫升）。此外，nPCR-HRM检测和区分低疟原虫血症感染的患者比例显著高于nPCR或dPCR-HRM（p值<0.001）。在这些低疟原虫血症感染患者中，67.7%的患者接受了抗疟药物治疗，而81.5%未感染疟原虫的患者接受了抗疟药物治疗。
nPCR-HRM增强的敏感性表明，在农村疟疾流行地区，鉴别发热疾病诊断存在局限性，这混淆了疟疾的流行病学估计，并导致抗疟药物的不当误用。这是第一项采用低疟原虫血症疟原虫诊断方法来量化对非疟疾发热患者和低疟原虫血症疟原虫感染患者使用抗疟药物处方的研究。nPCR-HRM增强了低疟原虫血症疟疾的诊断，并且有可能克服显微镜检查和基于RDTs的结果在确定低疟原虫血症疟原虫感染率以评估疟疾消除工作方面的不足。这些发现凸显了在偏远疟疾流行地区改进发热疾病鉴别诊断的必要性。
