Ferreira Filho Júlio César Rente, Braz Lucia Maria Almeida, Andrino Marcos Luiz Alves, Yamamoto Lidia, Kanunfre Kelly Aparecida, Okay Thelma Suely
Laboratório de Soroepidemiologia e Imunobiologia, Instituto de Medicina Tropical, Universidade de Sao Paulo, Brazil.
Laboratório de Soroepidemiologia e Imunobiologia, Instituto de Medicina Tropical, Universidade de Sao Paulo, Brazil.
Exp Parasitol. 2019 May;200:13-15. doi: 10.1016/j.exppara.2019.03.007. Epub 2019 Mar 20.
The choice of cost-effective molecular methods for diagnosing and monitoring of Chagas disease before and after treatment is crucial in endemic countries with high patients' demand and limited financial resources. To this end, a kDNA was compared to a satellite real-time quantitative PCR (sat-qPCR), both amplifications using Sybr Green instead of Taqman hydrolysis probes. Non-isogenic Swiss albino mice were infected with a small inoculum of the highly virulent and partially resistant to benznidazole Y strain, belonging to T. cruzi discrete typing unit II (DTU-II) that predominates in Atlantic and Central Brazil. DNA from EDTA-blood samples and 10 organs of mice containing high, moderate and low parasite load levels were extracted by a highly used commercial kit and tested in triplicate, showing no disagreements between the two qPCRs. The melting temperature of positive samples was 79.8 °C ± 1 °C for satellite-DNA and 78.1 °C ± 1 °C for kDNA. DNA from genetically-related parasites such as Leishmania sp. showed no cross-reactions, but the sympatric T. rangeli was detected by both qPCRs, more effectively by kDNA than the satellite system, which required the equivalent of 50 parasites to give a positive result. Samples from infected mice, regardless of the type of biological matrix (blood or organ samples) or the parasite load gave positive results by both qPCRs. The sensitivity of sat-qPCR was 2 × 10 parasite or 240 target copies, and for kDNA, 2 × 10 parasite or 24 target copies. Regarding repeatability and reproducibility, the coefficient of variation (CV) was always < 25% in both assays; linearity of sat-qPCR was 0.991 (±0.002) and 0.991 (±0.008) for kDNA qPCR. In most collection times, the median Ct values found in blood and organs provided by sat-DNA and kDNA qPCRs were similar. In conclusion, although kDNA qPCR achieved a better analytical sensitivity, sat-qPCR gave better specificity results. Nevertheless, further research is intended to test other T. cruzi DTUs and chagasic patients' samples before these cost-effective techniques are incorporated into diagnostic routines.
在患者需求高且财政资源有限的流行国家，选择具有成本效益的分子方法用于恰加斯病治疗前后的诊断和监测至关重要。为此，将一种kDNA与一种卫星实时定量PCR（sat-qPCR）进行了比较，两种扩增均使用SYBR Green而非Taqman水解探针。用少量高毒力且对苯硝唑部分耐药的Y株接种非同源瑞士白化小鼠，该毒株属于克氏锥虫离散分型单元II（DTU-II），在巴西大西洋沿岸和中部地区占主导地位。通过一种常用的商业试剂盒从EDTA血样和小鼠的10个器官中提取DNA，这些器官的寄生虫载量分别为高、中、低水平，并进行了三次重复检测，结果显示两种qPCR之间没有分歧。卫星DNA阳性样本的熔解温度为79.8°C±1°C，kDNA阳性样本的熔解温度为78.1°C±1°C。来自遗传相关寄生虫如利什曼原虫属的DNA未显示交叉反应，但两种qPCR均检测到同域的兰氏锥虫，kDNA比卫星系统检测更有效，卫星系统需要相当于50个寄生虫才能得出阳性结果。来自感染小鼠的样本，无论生物基质类型（血液或器官样本）或寄生虫载量如何，两种qPCR均给出阳性结果。sat-qPCR的灵敏度为2×10个寄生虫或240个靶标拷贝，kDNA的灵敏度为2×10个寄生虫或24个靶标拷贝。关于重复性和再现性，两种检测方法的变异系数（CV）始终<25%；sat-qPCR的线性度为0.991（±0.002），kDNA qPCR的线性度为同样为0.991（±0.008）。在大多数采集时间，sat-DNA和kDNA qPCR在血液和器官中测得的Ct值中位数相似。总之，虽然kDNA qPCR具有更好的分析灵敏度，但sat-qPCR给出了更好的特异性结果。然而，在将这些具有成本效益的技术纳入诊断程序之前，还需要进一步研究以检测其他克氏锥虫DTU和恰加斯病患者样本。
