Nzelu Chukwunonso O, Bahrami Somayeh, Lawyer Phillip G, Peters Nathan C
Department of Microbiology, Immunology, and Infectious Diseases, Cumming School of Medicine, and Faculty of Veterinary Medicine, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada.
Department of Parasitology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
PLoS Negl Trop Dis. 2025 Mar 17;19(3):e0012915. doi: 10.1371/journal.pntd.0012915. eCollection 2025 Mar.
Surveillance of infected insect vectors of vector-transmitted diseases has been recognized for its ability to estimate pathogen prevalence and transmission potential. Classically restricted to microscopic dissection and examination of individual insects, the potential of entomological monitoring has grown due to the advent of rapid molecular DNA detection methods with high specificity and sensitivity. Despite such advancement, a recurring question concerning DNA detection of parasitic pathogens is related to the fact that DNA amplification, by itself, does not differentiate between insects carrying infectious versus dead, non- or poorly-infectious life-cycle stages, thereby limiting it's programmatic usefulness for accurately measuring the transmission potential of infected insects in endemic areas or within experimentally infected populations. Herein, we developed a quantitative real-time PCR with Reverse Transcription (RT-qPCR) based sherp (small hydrophilic endoplasmic reticulum-associated protein) detection assay employing a novel set of sherp-RT-qPCR primers to detect and quantify infectious Leishmania parasites in infected vector sand flies. The sherp RT-qPCR showed significantly increased expression of sherp transcripts in infectious Leishmania metacyclic versus non-metacyclic promastigotes or mammalian-derived amastigotes. The assay displayed detection performance ranging from 106 to 1 parasite and could reliably quantify parasites within infected sand flies without the need for dissection. Sherp transcripts were also successfully amplified from flies stored in ethanol at room temperature, a practical and economical method of sample preservation in resource-limited field settings. Lastly, in conjunction with an established RT-qPCR assay for Leishmania kinetoplast DNA minicircles, we were able to calculate a score for the degree of metacyclogenesis within infected sand flies, a known predictor of transmission potential. These results highlight the potential of the sherp-RT-qPCR assay to identify hotspots of potential transmission, areas of re-emergence, vector competence, and the transmission potential of infected sand fly populations.
媒介传播疾病的受感染昆虫媒介监测因其能够估计病原体流行率和传播潜力而得到认可。传统上，昆虫学监测仅限于对单个昆虫进行显微镜解剖和检查，由于具有高特异性和敏感性的快速分子DNA检测方法的出现，昆虫学监测的潜力得到了提升。尽管有这样的进展，但关于寄生性病原体DNA检测的一个反复出现的问题是，DNA扩增本身无法区分携带传染性与死亡、无感染性或低感染性生命周期阶段的昆虫，从而限制了其在准确测量流行地区或实验感染种群中受感染昆虫传播潜力方面的程序实用性。在此，我们开发了一种基于逆转录定量实时PCR（RT-qPCR）的sherp（小亲水性内质网相关蛋白）检测方法，使用一组新的sherp-RT-qPCR引物来检测和定量感染媒介白蛉中具有传染性的利什曼原虫寄生虫。sherp RT-qPCR显示，与非循环前鞭毛体或哺乳动物来源的无鞭毛体相比，传染性利什曼原虫循环前鞭毛体中sherp转录本的表达显著增加。该检测方法的检测性能范围为106至1个寄生虫，无需解剖即可可靠地定量受感染白蛉体内的寄生虫。sherp转录本也成功地从室温下保存在乙醇中的白蛉中扩增出来，这是资源有限的野外环境中一种实用且经济的样本保存方法。最后，结合已建立的用于利什曼原虫动基体DNA微环的RT-qPCR检测方法，我们能够计算出受感染白蛉体内循环前鞭毛体形成程度的分数，这是传播潜力的一个已知预测指标。这些结果突出了sherp-RT-qPCR检测方法在识别潜在传播热点、再次出现区域、媒介能力以及受感染白蛉种群传播潜力方面的潜力。
