A one-step multiplex qPCR assay for simultaneous identification and quantification of Leishmania martiniquensis and Leishmania orientalis/Leishmania chancei and detection and quantification of trypanosomatids in clinical samples.

IF 2.4 2区医学 Q2 PARASITOLOGY

Parasite Pub Date : 2025-01-01 Epub Date: 2025-06-24 DOI:10.1051/parasite/2025030

Chonlada Mano, Piyapha Hirunpatrawong, Patiya Prasertsilp, Saowalak Kaewmee, Parinda Limprasert, Padet Siriyasatien, Adisak Tantiworawit, Derek Gatherer, Michael D Urbaniak, Paul A Bates, Narissara Jariyapan

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Abstract

Leishmaniasis is one of the most important zoonotic diseases. Recently, Leishmania (Mundinia) martiniquensis, Leishmania (Mundinia) orientalis, and Leishmania (Mundinia) chancei have been reported as new human pathogens. Trypanosomatids, apart from Leishmania spp., such as Crithidia spp., are also occasionally capable of infecting humans. Here, a one-step multiplex qPCR assay for the simultaneous identification and quantification of L. martiniquensis and L. orientalis/L. chancei and detection and quantification of trypanosomatids was developed using ITS1 as the molecular target and human RNase P as the internal control gene. The assay was evaluated using 44 positive residual DNA samples from leishmaniasis patients and 25 negative DNA samples. Results revealed that the limits of detection (LOD) of the assay for L. martiniquensis, L. orientalis, and Crithidia sp. (CLA-KP1) were 1.699 (0.0255), 1.717 (0.0292), and 1.763 (0.0882) fg/reaction (parasite equivalents/reaction), respectively. The assay had high analytical specificity. The mean Cq values of the intra-assays and inter-assays differed by less than 1, indicating the reliability of the assay. Evaluation results revealed that the assay could identify L. martiniquensis and L. orientalis in clinical samples with 100% sensitivity and 100% specificity. In conclusion, the ITS1/human RNase P multiplex qPCR assay offers a rapid and reliable diagnostic tool for identifying and quantifying L. martiniquensis and L. orientalis/L. chancei and detecting and quantifying trypanosomatids in clinical samples within a single reaction. This assay provides an advancement in the diagnostic capabilities for leishmaniasis and trypanosomatid infections, potentially improving patient management and surveillance efforts.

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马尼提克利什曼原虫和东方利什曼原虫/机会利什曼原虫的一步多重qPCR同时鉴定和定量以及临床样品中锥虫的检测和定量

利什曼病是最重要的人畜共患疾病之一。最近，马提尼克利什曼原虫、东方利什曼原虫和机会利什曼原虫被报道为新的人类病原体。锥虫，除了利什曼原虫，如克里蒂亚原虫，偶尔也能感染人类。本研究采用一步多重qPCR方法同时鉴定和定量马提尼库马和东方蓟。以ITS1为分子靶点，以人RNase P为内控基因，建立了锥虫的检测与定量方法。使用来自利什曼病患者的44份阳性残留DNA样本和25份阴性DNA样本对该检测方法进行了评估。结果表明，该方法对马提尼松、东方松和Crithidia sp. （CLA-KP1）的检出限（LOD）分别为1.699（0.0255）、1.717（0.0292）和1.763 (0.0882)fg/reaction（寄生虫当量/reaction）。该方法具有较高的分析特异性。测定内测定和测定间测定的平均Cq值相差小于1，表明测定的可靠性。评价结果表明，该方法能以100%的灵敏度和100%的特异性鉴别临床样品中的马提尼克松菌和东方松菌。综上所述，ITS1/human RNase P多重qPCR方法可为马提尼库氏乳杆菌和东方乳杆菌的鉴定和定量提供快速可靠的诊断工具。单次反应中临床样品中锥虫的形成、检测和定量。这种检测方法提高了利什曼病和锥虫病感染的诊断能力，有可能改善患者管理和监测工作。

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来源期刊

Parasite 医学-寄生虫学

CiteScore

5.50

自引率

6.90%

发文量

审稿时长

3 months

期刊介绍： Parasite is an international open-access, peer-reviewed, online journal publishing high quality papers on all aspects of human and animal parasitology. Reviews, articles and short notes may be submitted. Fields include, but are not limited to: general, medical and veterinary parasitology; morphology, including ultrastructure; parasite systematics, including entomology, acarology, helminthology and protistology, and molecular analyses; molecular biology and biochemistry; immunology of parasitic diseases; host-parasite relationships; ecology and life history of parasites; epidemiology; therapeutics; new diagnostic tools. All papers in Parasite are published in English. Manuscripts should have a broad interest and must not have been published or submitted elsewhere. No limit is imposed on the length of manuscripts, but they should be concisely written. Papers of limited interest such as case reports, epidemiological studies in punctual areas, isolated new geographical records, and systematic descriptions of single species will generally not be accepted, but might be considered if the authors succeed in demonstrating their interest.