Rougemont Mathieu, Van Saanen Madeleine, Sahli Roland, Hinrikson Hans Peter, Bille Jacques, Jaton Katia
Institute of Microbiology, University Hospital of Lausanne, Switzerland.
J Clin Microbiol. 2004 Dec;42(12):5636-43. doi: 10.1128/JCM.42.12.5636-5643.2004.
There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the "gold standard" in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.
有报告称，移民和旅行者中疟疾病例数量不断增加。尽管血液涂片显微镜检查仍是诊断的“金标准”，但该方法灵敏度不足，且需要相当专业的知识。为改善诊断，开发了一种多重实时聚合酶链反应（PCR）。设计了一组针对疟原虫属18S核糖体RNA（rRNA）基因高度保守区域的通用引物；该引物组内部具有足够的多态性，可设计针对恶性疟原虫、间日疟原虫、三日疟原虫和卵形疟原虫的四种物种特异性探针。使用物种特异性探针的实时PCR可特异性检测到恶性疟原虫、间日疟原虫、三日疟原虫和卵形疟原虫的一个质粒拷贝。使用18S筛选探针的实时PCR对所有物种均实现了相同的灵敏度。对97份血样进行了研究。其中66份（60名患者）的血样，比较了显微镜检查和实时PCR结果，疟原虫检测的粗略一致性为86%。对不一致的结果结合临床、分子和测序数据进行重新评估以解决问题。18S筛选PCR与显微镜检查之间的所有9处不一致均以分子方法为准得到解决，两种方法均呈阳性的31份样本中，物种特异性PCR在物种水平上的9处不一致中有8处以分子方法为准得到解决。对另外31份血样进行检测以监测7名患者的抗疟疾治疗情况。7名患者中有6名患者通过实时PCR检测到的疟原虫数量与显微镜下测定的疟原虫血症同时迅速下降。这表明定量PCR在监测接受抗疟疾治疗的患者中具有作用。
