评价用于检测感染内脏利什曼病患者后白蛉体内杜氏利什曼原虫的分子检测方法。

Evaluation of molecular assays to detect Leishmania donovani in Phlebotomus argentipes fed on post-kala-azar dermal leishmaniasis patients.

机构信息

Institute of Animal Hygiene and Veterinary Public Health, University of Leipzig, An den Tierkliniken 43, 04103, Leipzig, Germany.

Nutrition and Clinical Services Division, International Centre for Diarrheal Disease Research Bangladesh, 1212, Dhaka, Bangladesh.

出版信息

Parasit Vectors. 2021 Sep 9;14(1):465. doi: 10.1186/s13071-021-04961-6.

DOI:10.1186/s13071-021-04961-6

PMID:34503557

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8428120/

Abstract

BACKGROUND

Post-kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani (LD) is a skin disorder that often appears after treatment of visceral leishmaniasis (VL) patients. PKDL patients are potential reservoirs of LD parasites, which can initiate a new epidemic of anthroponotic VL. Therefore, host infectiousness to its sand fly vector is a critical factor for transmission, and its accurate estimation can facilitate control strategies. At present, conventional microscopy serves as the reference method to detect parasites in its vector. However, low sensitivity of microscopy can be a limiting factor.

METHODS

In this study, real-time quantitative PCR (LD-qPCR) and recombinase polymerase amplification (LD-RPA) assays were evaluated against microscopy for the detection of LD DNA extracted from live sand flies five days after controlled feeding on PKDL cases.

RESULTS

The sensitivity of LD-qPCR and LD-RPA assays were found to be 96.43 and 100%, respectively, against microscopy for the selected fed sand flies (n = 28), and an absolute specificity of both molecular tools for apparently unfed sand flies (n = 30). While the proportion of infectious cases among 47 PKDL patients was estimated as 46.81% as defined by microscopic detection of LD in at least one fed sand fly per case, LD-RPA assay evaluation of only the microscopy negative sand flies fed to those 47 PKDL cases estimated an even greater proportion of infectious cases (51.06%). In overall estimation of the infectious cases in retrospective manner, discordance in positivity rate was observed (p < 0.05) between LD-RPA (59.57%) assay and microscopy (46.81%), while LD-RPA had slightly better positivity rate than LD-qPCR (55.32%) as well.

CONCLUSIONS

Considering the sensitivity, cost, detection time, and field applicability, RPA assay can be considered as a promising single molecular detection tool for investigations pertaining to LD infections in sand flies and/or host infectiousness in PKDL, while it can also be useful in confirmation of microscopy negative sand fly samples.

摘要

背景

由利什曼原虫（LD）引起的卡拉-阿扎尔皮肤利什曼病（PKDL）是一种皮肤疾病，常发生在内脏利什曼病（VL）患者治疗后。PKDL 患者是 LD 寄生虫的潜在储主，可能引发新的人间 VL 流行。因此，宿主对其沙蝇媒介的感染性是传播的关键因素，准确估计其感染性有助于控制策略的制定。目前，常规显微镜检查是检测媒介中寄生虫的参考方法。然而，显微镜检查的低灵敏度可能是一个限制因素。

方法

在这项研究中，我们评估了实时定量 PCR（LD-qPCR）和重组酶聚合酶扩增（LD-RPA）检测方法对从 PKDL 病例控制喂养后 5 天的活沙蝇中提取的 LD DNA 的检测效果。

结果

LD-qPCR 和 LD-RPA 检测方法对选定的喂食沙蝇（n=28）的灵敏度分别为 96.43%和 100%，对明显未喂食沙蝇（n=30）的绝对特异性也分别为 96.43%和 100%。当通过显微镜检查在至少一只喂食沙蝇中检测到 LD 来定义 47 例 PKDL 患者中的感染病例比例时，47 例 PKDL 患者的感染病例比例估计为 46.81%，而仅对 LD-RPA 检测方法评估这些 47 例 PKDL 病例中阴性的喂食沙蝇的感染病例比例估计更高（51.06%）。在回顾性的整体感染病例估计中，LD-RPA（59.57%）检测方法与显微镜检查（46.81%）的阳性率存在差异（p<0.05），而 LD-RPA 的阳性率略高于 LD-qPCR（55.32%）。