Journal Article

Development of nucleic acid lateral flow immunoassay for duplex detection of Leishmania martiniquensis and Leishmania orientalis in asymptomatic patients with HIV

Ruang-areerate, Toon, Mungthin, Mathirut, Leelayoova, Saovanee, Nawattanapaibool, Namfon, Piyaraj, Phunlerd, Siripattanapipong, Suradej

2024

Overview

Leishmaniasis, a neglected tropical disease caused by parasitic protozoa of the Leishmania genus, remains a global health concern with significant morbidity and mortality. In Thailand, the rising incidence of autochthonous leishmaniasis cases involving Leishmania ( Mundinia ) martiniquensis and novel Leishmania ( Mundinia ) orientalis underscores the critical need for accurate diagnosis and effective control strategies. This study presents a sensitive and specific nucleic acid lateral flow immunoassay (NALFIA) that integrates a duplex PCR assay with a lateral flow device (LFD) strip format. Targeting the internal transcribed spacer 1 (ITS1) region, known for its unique combination of conserved and variable sequences, this assay employs primers labeled with biotin, digoxigenin, and fluorescein isothiocyanate (FITC) markers, enabling precise species identification and differentiation of these two Leishmania species. Remarkably, the assay achieves a sensitivity that surpasses agarose gel electrophoresis, detecting as few as 10 −2 parasite/μL for L . martiniquensis and 10 −4 parasite/μL for L . orientalis . Notably, the assay exhibited reliable specificity, revealing no cross-amplification with other major viscerotropic Leishmania species or reference organisms. Evaluation using 62 clinical samples further confirms the effectiveness of the PCR-LFD assay, with a sensitivity of 100% for L . martiniquensis and 83.3% for L . orientalis , and an excellent agreement ( κ value = 0.948) with nested PCR. This integrated assay represents a promising advancement in diagnostic tools, offering rapid and accurate results that can significantly contribute to effective disease management and control. Given the increasing relevance of these Leishmania species in current public health scenarios, this assay serves as a valuable tool for both diagnostic and research applications.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Acquired immune deficiency syndrome / AIDS / Analysis / Biology and Life Sciences / Biotin / Care and treatment / Diagnosis / Diagnostic software / Digoxigenin / Disease control / DNA, Protozoan - analysis / DNA, Protozoan - genetics / Effectiveness / Electrophoresis / Ethylenediaminetetraacetic acid / Evaluation / Fluorescein isothiocyanate / Global health / Health aspects / HIV / HIV (Viruses) / HIV Infections / HIV patients / Human immunodeficiency virus / Humans / Immunoassay / Immunoassay - methods / Israel / Leishmania / Leishmania - genetics / Leishmania - isolation & purification / Leishmaniasis / Leishmaniasis - diagnosis / Leishmaniasis - parasitology / Leishmania (Mundinia) / Medicine and Health Sciences / Morbidity / Mortality / Nucleic acids / Parasites / Parasitic diseases / Patient outcomes / Polymerase chain reaction / Polymerase Chain Reaction - methods / Protozoa / Public health / Research and Analysis Methods / Sensitivity analysis / Sensitivity and Specificity / Thailand / Thailand - epidemiology / Vector-borne diseases