A Novel Multiplex LAMP Assay for the Rapid and Accurate Diagnosis of Visceral Leishmaniasis Caused by Leishmania infantum from Iran.

IF 2.1 4区 医学 Q3 PUBLIC, ENVIRONMENTAL & OCCUPATIONAL HEALTH Journal of Tropical Medicine Pub Date : 2023-11-18 eCollection Date: 2023-01-01 DOI:10.1155/2023/9326183
Sahar Samsami, Sahar Namavari, Saeed Ataei, Abdolmajid Ghasemian, Ava Yazdanpanah, Neda Sepahi, Gholamreza Hatam, Hossein Faramarzi, Hadi Mirzaei, Razie Ranjbar, Ali Ghanbariasad
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Abstract

Visceral leishmaniosis (VL) is one of the neglected tropical diseases despite being responsible for serious clinical symptoms, some of which lead to fatal outcomes. Thus, there is a need to apply accurate, rapid, and specific diagnostic measurements in order to control the disease and reduce the mortality rate. We aimed to develop and validate a multiplex LAMP assay for the diagnosis of VL caused by Leishmania infantum (L. infantum). Moreover, a thorough assessment was conducted to determine the effectiveness of multiplex LAMP in identifying various Leishmania species, such as Leishmania tropica (L. tropica) and Leishmania major (L. major) in comparison to Leishmania infantum (L. infantum). The diagnostic performance of the multiplex LAMP method for VL was compared to each LAMP assay, real-time polymerase chain reaction (RT-qPCR), and nested PCR technique. Two separated primers were set and used in a multiplex LAMP assay which was designed based on the ITS2 (internal transcribed spacer II) and were selected on the basis of conserved and high copy number region. Multiplex LAMP primers were designed using an online tool available at https://www.primerexplorer.jp/e. The alignment was performed using MEGA5, and the primers were further adjusted utilizing GENE Runner software. All molecular methods were tested on the serial dilution of cloned plasmid containing ITS region from standard strains of L. infantum, L. tropica, and L. major. Moreover, multiplex LAMP assay was evaluated and compared based on both standard strains and 55 clinical samples from humans as well as dogs. Various approaches were applied to interpret the multiplex LAMP reaction which deciphered a higher sensitivity when compared to the RT-qPCR for L. infantum (one copy number of plasmid, equal to 0.85 femtograms (fg) of plasmid concentration, and 0.004 parasite DNA per μL) detection while these three standard strains of Leishmania were confirmed to contain 40 DNA copies using RT-qPCR. Additionally, the multiplex LAMP detection limit was approximately equivalent to RT-qPCR for L. major and L. tropica, which included 0.342 picograms (pg) and 342 femtograms (fg) of plasmid concentration, 4 × 103 and 4 × 102 copy number of plasmid, and 17.1 and 1.71 parasite DNA per μL for L. major and L. tropica, respectively. Nested PCR exhibited a lower detection limit for L. infantum of 4 × 106 plasmid copy number compared to multiplex LAMP and RT-qPCR. Multiplex LAMP has the potential for accurate and rapid detection of infectious disease, successful treatment, and finding and monitoring asymptomatic cases, especially in low-income countries.

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一种快速准确诊断伊朗婴儿利什曼原虫引起内脏利什曼病的新型多重LAMP检测方法。
内脏利什曼病(VL)是一种被忽视的热带病,尽管它会导致严重的临床症状,其中一些会导致致命的后果。因此,有必要采用准确、快速和具体的诊断措施,以控制疾病并降低死亡率。我们旨在开发和验证一种多重LAMP检测方法,用于诊断婴儿利什曼原虫(L. infantum)引起的VL。此外,还进行了全面的评估,以确定多重LAMP识别各种利什曼原虫的有效性,例如热带利什曼原虫(L. tropica)和大利什曼原虫(L. major),并与幼利什曼原虫(L. infantum)进行比较。将多重LAMP法对VL的诊断性能与每个LAMP法、实时聚合酶链反应(RT-qPCR)和巢式PCR技术进行比较。以ITS2 (internal transcriptionspacer II)为设计基础,根据保守区和高拷贝数区选择两个分离引物,进行多重LAMP实验。多重LAMP引物的设计使用可在https://www.primerexplorer.jp/e上获得的在线工具。使用MEGA5进行比对,并使用GENE Runner软件进一步调整引物。所有的分子方法对含有ITS区域的克隆质粒从婴儿乳杆菌、热带乳杆菌和主要乳杆菌的标准菌株进行了连续稀释试验。此外,基于标准菌株和55份人类和狗的临床样本,对多重LAMP法进行了评估和比较。采用多种方法对多重LAMP反应进行解释,与RT-qPCR相比,对婴儿乳杆菌(1个质粒拷贝数,等于0.85个质粒浓度,0.004个寄生虫DNA / μL)的检测具有更高的灵敏度,而对这3个利什曼原虫标准菌株进行RT-qPCR证实含有40个DNA拷贝。多重LAMP检测限与RT-qPCR近似,质粒浓度分别为0.342皮图(pg)和342飞图(fg),质粒拷贝数分别为4 × 103和4 × 102,每μL分别为17.1和1.71个寄生虫DNA。巢式PCR对婴儿乳杆菌的检出限为4 × 106个质粒拷贝数,低于多重LAMP和RT-qPCR。多效LAMP具有准确和快速发现传染病、成功治疗以及发现和监测无症状病例的潜力,特别是在低收入国家。
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来源期刊
Journal of Tropical Medicine
Journal of Tropical Medicine Immunology and Microbiology-Parasitology
CiteScore
3.90
自引率
4.50%
发文量
0
审稿时长
14 weeks
期刊介绍: Journal of Tropical Medicine is a peer-reviewed, Open Access journal that publishes original research articles, review articles, and clinical studies on all aspects of tropical diseases. Articles on the pathology, diagnosis, and treatment of tropical diseases, parasites and their hosts, epidemiology, and public health issues will be considered. Journal of Tropical Medicine aims to facilitate the communication of advances addressing global health and mortality relating to tropical diseases.
期刊最新文献
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