Weigle Kristen A, Labrada Luz Angela, Lozano Caterin, Santrich Cecilia, Barker Douglas C
Department of Epidemiology, University of North Carolina at Chapel Hill, USA.
J Clin Microbiol. 2002 Feb;40(2):601-6. doi: 10.1128/JCM.40.2.601-606.2002.
We evaluated PCR methods for diagnosis of acute and chronic cutaneous leishmaniasis (CL) in an area of Colombia where Leishmania (Viannia) is endemic. The PCR method specifically amplified whole linearized minicircle kinetoplast DNA (kDNA) of the Leishmania subgenus Viannia from biopsy lysates. PCR products were detected in agarose gels. For 255 acute cases, this PCR method had greater sensitivity (75.7%) than each conventional method, i.e., microscopic examination of Giemsa-stained lesion scraping (46.7%), biopsy culture (55.3%), aspirate culture (46.3%), and the conventional methods combined (70.2%). Among 44 cases of chronic CL, amplification of biopsy DNA was more sensitive (45.5%) than the individual (4.5 to 27.7%) and combined (27.3%) conventional methods. The detection of kDNA in biopsies from chronic lesions was enhanced by a chemiluminescent dot blot hybridization, which produced a sensitivity of 65.8% when alone and 90.9% when in combination with DNA extraction of biopsy lysates (P < 0.001). Three biopsies from 84 skin lesions of other etiologies were falsely positive by PCR (specificity, 96.4%). PCR detected kDNA more frequently in biopsies (detection level, 83.9%) than in aspirates (74.7%) from 103 cases of acute CL. Among aspirates from 53 chronic cases of CL, the alternative methods, DNA extraction and hybridization, increased sensitivity from 41.5 to 56.6% (P > 0.05). This enhanced PCR method in chronic biopsies was so much more sensitive than conventional methods that it should be considered the preferred diagnostic method for chronic CL. These findings support the appropriate incorporation of PCR into diagnostic strategies for cutaneous leishmaniasis.
我们在哥伦比亚利什曼原虫（维安尼亚亚属）流行地区评估了用于诊断急性和慢性皮肤利什曼病（CL）的PCR方法。该PCR方法能从活检裂解物中特异性扩增出维安尼亚亚属利什曼原虫的完整线性化微小环动质体DNA（kDNA）。PCR产物在琼脂糖凝胶中进行检测。对于255例急性病例，该PCR方法的敏感性（75.7%）高于每种传统方法，即吉姆萨染色病变刮片显微镜检查（46.7%）、活检培养（55.3%）、抽吸物培养（46.3%）以及传统方法联合使用（70.2%）。在44例慢性CL病例中，活检DNA的扩增比单独的传统方法（4.5%至27.7%）及联合传统方法（27.3%）更敏感（45.5%）。化学发光斑点印迹杂交增强了慢性病变活检中kDNA的检测，单独使用时敏感性为65.8%，与活检裂解物DNA提取联合使用时敏感性为90.9%（P<0.001）。84例其他病因皮肤病变的3份活检经PCR检测为假阳性（特异性为96.4%）。在103例急性CL病例中，PCR在活检中检测到kDNA的频率（检测水平为83.9%）高于抽吸物（74.7%）。在53例慢性CL病例的抽吸物中，DNA提取和杂交等替代方法使敏感性从41.5%提高到56.6%（P>0.05）。这种改进的慢性活检PCR方法比传统方法敏感得多，应被视为慢性CL的首选诊断方法。这些发现支持将PCR适当地纳入皮肤利什曼病的诊断策略中。
