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Application of Quantitative PCR in the Diagnosis and Evaluating Treatment Efficacy of Leishmaniasis
Frontiers in Cellular and Infection Microbiology ( IF 4.6 ) Pub Date : 2020-09-11 , DOI: 10.3389/fcimb.2020.581639
Yun Wu 1, 2 , Xiaojun Tian 1, 2 , Nan Song 1, 2 , Minjun Huang 1, 2 , Zhaoyong Wu 1, 2 , Shaogang Li 1, 2 , Nicholas R Waterfield 3 , Bin Zhan 4 , Lei Wang 1, 2 , Guowei Yang 1, 2
Affiliation  

Leishmaniasis is still a serious neglected tropical disease that may cause death in infected individuals. At present, the clinical diagnosis and treatment monitoring still rely on parasitological culture and microscopy that needs experienced technicians. The low sensitivity and inconvenience of microscopic examination could cause misdiagnosis and relapse of leishmaniasis. There is an urgent need for developing a sensitive and easily operated diagnostic method for the diagnosis and disease management of leishmaniasis. Thus, a quantitative real-time PCR (qPCR) based on the conversed regions of kinetoplast minicircle DNA (mkDNA) of Leishmania spp. was developed to detect different species of Leishmania. The designed mkDNA-based qPCR was able to detect as low as one copy of Leishmania mkDNA or DNA from single parasite. It also detected Pan-Leishmania protozoa including Leishmania donovani, Leishmania infantum and Leishmania major without cross-reaction with other pathogen DNAs available in our lab. This method was clinically applied to quantitatively detect skin lesion samples from 20 cutaneous leishmaniasis (CL) and bone marrow and/or PBMC samples from 30 current and cured visceral leishmaniasis (VL) patients, and blood samples from 11 patients with other infections and 5 normal donors as well. Total 20 skin lesion samples from current CL patients and 20 bone marrow and/or PBMC samples from current VL patients were all detected as positive with qPCR without cross-reaction with samples from patients with malaria, brucellosis and dengue or normal donors. Two VL patients with parasite converted to microscopically negative after treatment were detected positive with qPCR. The patients with bigger skin lesion in CL and higher level of immunoglobulin or splenomegaly in VL, had the higher parasite load detected by qPCR. The parasite load was significantly reduced after treatment. In conclusion, the mkDNA-based qPCR assay that we developed in this study can be used not only for diagnosis of both cutaneous and visceral leishmaniasis with high sensitivity and specificity, but also for evaluating the severity and treatment efficacy of this disease, presenting a rapid and accurate tool for clinical surveillance, treatment monitoring and the end point determination of leishmaniasis.



中文翻译:


定量PCR在利什曼病诊断及疗效评价中的应用



利什曼病仍然是一种严重被忽视的热带疾病,可能导致感染者死亡。目前,临床诊断和治疗监测仍然依赖于寄生虫培养和显微镜检查,需要经验丰富的技术人员。镜检灵敏度低、不便,容易导致利什曼病的误诊和复发。迫切需要开发一种灵敏且易于操作的诊断方法来诊断和治疗利什曼病。因此,基于利什曼原虫动质体小环 DNA (mkDNA) 转换区域的定量实时 PCR (qPCR)。开发用于检测不同种类的利什曼原虫。设计的基于 mkDNA 的 qPCR 能够检测低至一份利什曼原虫 mkDNA 或来自单一寄生虫的 DNA。它还检测了泛利什曼原虫,包括杜氏利什曼原虫、婴儿利什曼原虫和大型利什曼原虫,且不与我们实验室提供的其他病原体 DNA 发生交叉反应。该方法应用于临床定量检测20例皮肤利什曼病(CL)的皮损样本和30例当前和治愈的内脏利什曼病(VL)患者的骨髓和/或PBMC样本,以及11例其他感染患者和5例正常人的血液样本捐助者也是如此。当前 CL 患者的总共 20 份皮肤病变样本以及当前 VL 患者的 20 份骨髓和/或 PBMC 样本均经 qPCR 检测为阳性,与疟疾、布鲁氏菌病和登革热患者或正常供体的样本没有交叉反应。两名患有寄生虫的 VL 患者在治疗后转为镜检阴性,但 qPCR 检测呈阳性。 CL 中皮损较大、VL 中免疫球蛋白水平较高或脾肿大的患者,qPCR 检测到的寄生虫载量较高。治疗后寄生虫负荷显着减少。总之,我们在本研究中开发的基于 mkDNA 的 qPCR 检测不仅可以用于诊断皮肤和内脏利什曼病,具有高敏感性和特异性,而且可以用于评估该疾病的严重程度和治疗效果,提供快速的诊断方法。用于利什曼病临床监测、治疗监测和终点确定的准确工具。

更新日期:2020-10-07
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