Dweik A, Schönian G, Mosleh I M, Karanis P
Medical and Molecular Parasitology Laboratory, Center of Anatomy, Institute II, University of Cologne, Medical School, Joseph-Stelzmann-Strasse 9, Cologne 50931, Germany.
Ann Trop Med Parasitol. 2007 Jul;101(5):399-407. doi: 10.1179/136485907X176436.
It is possible to detect and distinguish Leishmania parasites using PCR-RLFP - a combination of PCR and analysis of the fragment-length polymorphism seen when the amplicons are digested with one or more restriction enzymes. In the present study, clinical samples from 24 Jordanians suspected to have cutaneous leishmaniasis and cultures set up using leishmanial parasites from five Greek dogs were investigated using PCR, in which the internal-transcribed-spacer-1 (ITS1) region of the parasites' ribosomal-RNA gene was amplified, followed by HaeIII digestion of the resulting amplicons. The cultures, which were all maintained in Leibowitz L-15 medium with 20% foetal calf serum, were each investigated as serial dilutions. Using the PCR-RLFP analysis, each culture was identified as L. donovani and each was found positive for this parasite with a mean sensitivity of 66%-100% (depending on the culture dilution tested), a specificity of 100%, a mean positive predictive value of 100%, and a negative predictive value of 74.6%-100%. When simulated clinical samples, created by mixing human blood with known numbers of L. donovani promastigotes, were investigated, the PCR-RFLP gave optimal results (with a value of 100% each for sensitivity, specificity and positive and negative predictive values). When the real clinical samples (25 lesion aspirates and 20 samples of peripheral blood from 24 Jordanian patients) were investigated using the molecular method, 20 (84%) of the patients were found to have lesion aspirates that were PCR-RFLP-positive for L. major (although, by microscopy, only six were found to have amastigote-positive lesion aspirates). None of the blood samples from the Jordanian patients, however, was found PCR-positive.
使用聚合酶链反应-限制性片段长度多态性分析（PCR-RLFP），即聚合酶链反应（PCR）与扩增子用一种或多种限制性酶消化后片段长度多态性分析相结合的方法，能够检测和区分利什曼原虫寄生虫。在本研究中，对24名疑似患有皮肤利什曼病的约旦人的临床样本以及用5只希腊犬的利什曼原虫寄生虫建立的培养物进行了PCR检测，其中扩增了寄生虫核糖体RNA基因的内转录间隔区1（ITS1）区域，随后对所得扩增子进行HaeIII消化。所有培养物均在含有20%胎牛血清的Leibowitz L-15培养基中维持，并对每个培养物进行系列稀释检测。通过PCR-RLFP分析，每个培养物均被鉴定为杜氏利什曼原虫，并且发现每个培养物对该寄生虫呈阳性，平均灵敏度为66%-100%（取决于所检测的培养物稀释度），特异性为100%，平均阳性预测值为100%，阴性预测值为74.6%-100%。当对通过将人血与已知数量的杜氏利什曼原虫前鞭毛体混合制成的模拟临床样本进行检测时，PCR-RFLP给出了最佳结果（灵敏度、特异性以及阳性和阴性预测值均为100%）。当使用分子方法对真实临床样本（24名约旦患者的25份病变抽吸物和20份外周血样本）进行检测时，发现20名（84%）患者的病变抽吸物经PCR-RFLP检测对硕大利什曼原虫呈阳性（尽管通过显微镜检查，仅6名患者的病变抽吸物中发现有无鞭毛体阳性）。然而，约旦患者的血液样本均未检测到PCR阳性。
