Sato Kimiya, Shinomiya Nariyoshi
Department of Pathology, Japan Self Defense Forces Central Hospital, Setagaya-ku, Tokyo 154-8532.
Rinsho Byori. 2008 Aug;56(8):657-61.
Malaria is one of the most prevalent infectious diseases in the world. Accurate identification of four species of human malaria parasite is essential for appropriate treatment. Here, we developed a simple and rapid method of identifying Plasmodium species using a fast polymerase chain reaction (PCR) assay. Based on the previous literature, we amplified small subunit ribosomal RNA genes of four human malaria parasites. To establish a minimum detection limit, a blood sample with a known number of P. falciparum parasites (parasitemia: 3%) was diluted serially(from 0.03% to 0.000003%). We compared the detection limits between single (one-step) PCR and nested (two-step) PCR. Other clinical blood samples, which were infected with P. falciparum (parasitemia: 2.8%), P. vivax (parasitemia: 0.13%), P. ovale (parasitemia: 0.04%), respectively, were also tested by our PCR system. The PCR findings were compared to those of blood film Giemsa staining and rapid diagnostic tests (RDT). The sensitivity of our method is less than one parasite in 1 microl of blood(estimated parasitemia: 0.000003%) for both single PCR and nested PCR, though an increased number of cycles (40 cycles) was required for single PCR. Using clinical samples, it was proven that amplified products by single PCR could clearly distinguish between P. falciparum, P. vivax, and P. ovale. To detect P. vivax and P. ovale, the PCR system was more sensitive than RDT. The total required time for our method was within three to four hours from DNA extraction to PCR detection. Taken together, our method is easier and faster than the previously reported PCR-based malaria parasite identification systems, and is also useful for cases in which diagnosis by Giemsa staining and RDT is difficult.
疟疾是世界上最普遍的传染病之一。准确识别四种人类疟原虫对于恰当治疗至关重要。在此，我们开发了一种使用快速聚合酶链反应（PCR）检测法来识别疟原虫种类的简单快速方法。基于先前的文献，我们扩增了四种人类疟原虫的小亚基核糖体RNA基因。为确定最低检测限，将含有已知数量恶性疟原虫的血样（疟原虫血症：3%）进行连续稀释（从0.03%至0.000003%）。我们比较了单步（一步法）PCR和巢式（两步法）PCR之间的检测限。其他分别感染了恶性疟原虫（疟原虫血症：2.8%）、间日疟原虫（疟原虫血症：0.13%）、卵形疟原虫（疟原虫血症：0.04%）的临床血样也通过我们构建的PCR系统进行检测。将PCR检测结果与血涂片吉姆萨染色及快速诊断检测（RDT）的结果进行比较。我们所构建方法的灵敏度对于单步PCR和巢式PCR而言，在1微升血液中均低于一个疟原虫（估计疟原虫血症：0.000003%），不过单步PCR需要增加循环数（40个循环）。使用临床样本证明，单步PCR的扩增产物能够清晰区分恶性疟原虫、间日疟原虫和卵形疟原虫。为检测间日疟原虫和卵形疟原虫，该PCR系统比RDT更灵敏。我们所构建方法从DNA提取到PCR检测所需的总时间在三到四个小时以内。综上所述，我们所构建的方法比先前报道的基于PCR的疟原虫识别系统更简便、快速，并且对于吉姆萨染色和RDT诊断困难的病例也很有用。
