Abstract
ABSTRACT Visceral Leishmaniasis (VL) causes high morbidity and mortality in low-to-middle-income countries worldwide. In this study, we used Laser Direct-Write (LDW) technology to develop a new Lateral Flow Device (LFD) with double-channel geometry on a low-cost paper platform as a rapid and accurate serodiagnostic assay for human VL. This Duplex VL-LFD was based on a laser-patterned microfluidic device using two recombinant Leishmania proteins, β-tubulin and LiHyp1, as novel diagnostic antigens. The VL-LFD assay was tested with blood/serum samples from patients diagnosed with VL, Tegumentary Leishmaniasis, Leishmaniasis of unknown identity, other parasitic diseases with similar clinical symptoms, i.e. Leprosy Disease and Chagas Disease, and blood from healthy donors, and compared in parallel with commercial rK39 IT-LEISH® Kit. Clinical diagnosis and real-time Polymerase Chain Reaction assay were used as reference standards. VL-LFD Sensitivity (S ± 95% Confidence Intervals (CI)) of 90.9 (78.9-100) and Specificity (Sp ± 95% CI) of 98.7 (96.1-100) outperformed the IT-LEISH® Kit [S = 77.3 (59.8-94.8), Sp = 94.7 (89.6-99.8)]. This is the first study reporting successful development of an LFD assay using the LDW technology and the VL-LFD warrants comparative testing in larger patient cohorts and in areas with endemic VL in order to improve diagnosis and disease management.
Highlights
Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania and is considered endemic in 98 countries, with the highest burden of disease in India, Brazil and other low-to-middle-income countries (LMIC)
The Visceral Leishmaniasis (VL)-Lateral Flow Device (LFD) assay was tested with blood/serum samples from patients diagnosed with VL, Tegumentary Leishmaniasis, Leishmaniasis of unknown identity, other parasitic diseases with similar clinical symptoms, i.e. Leprosy Disease and Chagas Disease, and blood from healthy donors, and compared in parallel with commercial rK39 IT-LEISH® Kit
Samples were collected from patients classified with VL (n = 24), Tegumentary Leishmaniasis (TL, n = 27), Leishmaniasis of unknown identity (L, n = 3), other diseases with similar clinical symptoms, i.e. Leprosy Disease (LD, n = 13) and Chagas Disease (CD, n = 53, with either Chagasic Myocardiopathy (CM) or Indeterminate Form (IF) of CD) or Unknown Infection (UI, n = 49, comprising of patients assessed with diagnosis Not Informed (NI), Anaemia (A) or Lymphoproliferative (LP))
Summary
Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania and is considered endemic in 98 countries, with the highest burden of disease in India, Brazil and other low-to-middle-income countries (LMIC). The mortality rate for VL is believed to approach 100%. Mortality rates are between 5% and 10% and patients can relapse within a year after infection [2]. Molecular assays require specialized equipment and personnel and parasitological diagnosis is affected by variability in detection sensitivity and by the expertise of the pathologist. Conventional serological tests, such as indirect immunofluorescence (RIFI) and ELISA [3], use whole pathogen or soluble extracts as antigens, but these antigen mixtures lead to cross-reactivity with other diseases and decrease specificity, and should be interpreted with caution [4]
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