Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Salta, Argentina.
Cátedra de Química Biológica, Facultad de Ciencias Naturales, Universidad Nacional de Salta, Salta, Argentina.
Am J Trop Med Hyg. 2018 Nov;99(5):1156-1161. doi: 10.4269/ajtmh.18-0223.
Cutaneous leishmaniasis is endemic in Salta province, which belongs to the northwest of Argentina. spp. DNA from Giemsa-stained slides of up to 12 years in storage of patients from Salta was characterized through polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP). One hundred smears positive for microscopy, classified in a semiquantitative scale for amastigote density, were analyzed. Also, Leishmanin skin test (LST) results were included. DNA extraction was carried out applying lysis buffer with proteinase K, and then DNA was amplified with ribosomal internal transcribed spacer 1 primers. PCR products were digested with HaeIII enzyme. All PCR-positive smears (74/100) belonged to subgenus. A statistically significant, directly proportional relationship between semiquantitative microscopy and PCR results was detected. All patients had LST-positive results (induration ≥ 5 mm), and the smears of those with smaller induration (LST < 19 mm) gave a higher proportion of positive PCR results. This study determined that smear age did not affect PCR positivity, which allows retrospective analyzes and suggests smears might be useful for molecular complementary diagnosis. Because () is the main circulating species in the study area, determining subgenus in all analyzed samples confirms previous findings. PCR positivity showed statistically significant differences according to semiquantitative microscopy, highlighting the importance of parasite burden in the diagnostic sensitivity of the method. Considering that smears of patients with smaller LST induration were more positive in PCR, a negative smear from patients with positive LST response, but < 19 mm, could actually represent a false-negative result.
皮肤利什曼病在属于阿根廷西北部的萨尔塔省流行。通过聚合酶链反应（PCR）限制性片段长度多态性（RFLP）对来自萨尔塔的长达 12 年储存的患者吉姆萨染色载玻片上的 spp. DNA 进行了特征描述。对 100 张显微镜检查阳性、按阿米巴原虫密度半定量量表分类的涂片进行了分析。此外，还包括利什曼菌素皮肤试验（LST）结果。应用含蛋白酶 K 的裂解缓冲液进行 DNA 提取，然后用核糖体内部转录间隔区 1 引物扩增 DNA。用 HaeIII 酶消化 PCR 产物。所有 PCR 阳性涂片（74/100）均属于 亚属。检测到半定量显微镜检查和 PCR 结果之间存在统计学上显著的直接比例关系。所有患者的 LST 结果均为阳性（硬结≥5 毫米），硬结较小（LST<19 毫米）的患者的涂片得出更高比例的 PCR 阳性结果。本研究确定涂片年龄不影响 PCR 阳性率，这允许进行回顾性分析，并表明涂片可能对分子补充诊断有用。由于 () 是研究区域中主要的循环种，确定所有分析样本中的 亚属证实了先前的发现。PCR 阳性率根据半定量显微镜检查有统计学显著差异，突出了寄生虫负荷在该方法诊断敏感性中的重要性。考虑到 LST 硬结较小的患者的涂片在 PCR 中更呈阳性，因此对于 LST 反应阳性但<19 毫米的患者，阴性涂片实际上可能代表假阴性结果。
