Ghosh Prakash, Sharma Abhijit, Bhattarai Narayan Raj, Abhishek Kumar, Nisansala Thilini, Kumar Amresh, Böhlken-Fascher Susanne, Chowdhury Rajashree, Khan Md Anik Ashfaq, Faisal Khaledul, Hossain Faria, Uddin Md Rasel, Rashid Md Utba, Maruf Shomik, Rai Keshav, Sooriyaarachchi Monica, Abhayarathna Withanage Lakma Kumari, Karki Prahlad, Kumar Shiril, Ranasinghe Shalindra, Khanal Basudha, Routray Satyabrata, Das Pradeep, Mondal Dinesh, Abd El Wahed Ahmed
Nutrition and Clinical Service Division, International Centre for Diarrhoeal Disease Research, Dhaka 1212, Bangladesh.
PATH, 15th Floor, Dr. Gopal Das Bhawan, 28 Barakhamba Road, New Delhi 110001, India.
Microorganisms. 2021 Mar 12;9(3):588. doi: 10.3390/microorganisms9030588.
With the advancement of isothermal nucleic acid amplification techniques, detection of the pathogenic DNA in clinical samples at point-of-need is no longer a dream. The newly developed recombinase polymerase amplification (RPA) assay incorporated in a suitcase laboratory has shown promising diagnostic efficacy over real-time PCR in detection of DNA from clinical samples. For broader application of this point-of-need system, we undertook a current multi-country diagnostic evaluation study towards establishing this technique in different endemic settings which would be beneficial for the ongoing elimination programs for leishmaniasis. For this study purpose, clinical samples from confirmed visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients were subjected to both real-time PCR and RPA assay in Bangladesh, India, and Nepal. Further skin samples from confirmed cutaneous leishmaniasis (CL) patients were also included from Sri Lanka. A total of 450 clinical samples from VL patients, 429 from PKDL patients, 47 from CL patients, and 322 from endemic healthy/healthy controls were under investigation to determine the diagnostic efficacy of RPA assay in comparison to real-time PCR. A comparative sensitivity of both methods was found where real-time PCR and RPA assay showed 96.86% (95% CI: 94.45-98.42) and 88.85% (95% CI: 85.08-91.96) sensitivity respectively in the diagnosis of VL cases. This new isothermal method also exhibited promising diagnostic sensitivity (93.50%) for PKDL cases, when a skin sample was used. Due to variation in the sequence of target amplicons, RPA assay showed comparatively lower sensitivity (55.32%) than that of real-time PCR in Sri Lanka for the diagnosis of CL cases. Except for India, the assay presented absolute specificity in the rest of the sites. Excellent concordance between the two molecular methods towards detection of DNA in clinical samples substantiates the application of RPA assay incorporated in a suitcase laboratory for point-of-need diagnosis of VL and PKDL in low resource endemic settings. However, further improvisation of the method is necessary for diagnosis of CL.
随着等温核酸扩增技术的进步，在临床样本采集现场检测致病DNA已不再是梦想。新开发的整合在手提箱式实验室中的重组酶聚合酶扩增（RPA）检测法，在检测临床样本中的DNA时，与实时PCR相比显示出了良好的诊断效果。为了使这种现场检测系统得到更广泛的应用，我们开展了一项当前的多国诊断评估研究，旨在在不同的地方流行环境中建立该技术，这将有利于正在进行的利什曼病消除计划。出于本研究目的，在孟加拉国、印度和尼泊尔，对确诊的内脏利什曼病（VL）和黑热病后皮肤利什曼病（PKDL）患者的临床样本进行了实时PCR和RPA检测。此外，还纳入了来自斯里兰卡确诊的皮肤利什曼病（CL）患者的皮肤样本。共对450份VL患者的临床样本、429份PKDL患者的临床样本、47份CL患者的临床样本以及322份地方流行区健康/健康对照样本进行了研究，以确定RPA检测法与实时PCR相比的诊断效果。两种方法的比较敏感性研究发现，实时PCR和RPA检测法在诊断VL病例时的敏感性分别为96.86%（95%CI：94.45 - 98.42）和88.85%（95%CI：85.08 - 91.96）。当使用皮肤样本时，这种新的等温方法对PKDL病例也表现出了良好的诊断敏感性（93.50%）。由于目标扩增子序列的差异，在斯里兰卡，RPA检测法在诊断CL病例时的敏感性（55.32%）相对低于实时PCR。除印度外，该检测法在其他地点均表现出绝对特异性。两种分子方法在检测临床样本中的DNA方面具有极好的一致性，这证实了整合在手提箱式实验室中的RPA检测法在资源匮乏的地方流行环境中对VL和PKDL进行现场诊断的应用价值。然而，对于CL的诊断，该方法仍需进一步改进。
