Development and Clinical Evaluation of Serum and Urine-Based Lateral Flow Tests for Diagnosis of Human Visceral Leishmaniasis

Abstract

Visceral leishmaniasis (VL), a fatal parasitic infection, is categorized as being neglected among tropical diseases. The use of conventional tissue aspiration for diagnosis is not possible in every setting. The immunochromatography-based lateral flow assay (LFA) has attracted attention for a long time due to its ability to give results within a few minutes, mainly in resource-poor settings. In the present study, we optimized and developed the LFA to detect anti-Leishmania antibodies for VL diagnosis. The performance of the developed test was evaluated with serum and urine samples of Indian VL patients and Brazilian sera. The new test exploits well-studied and highly-sensitive purified antigens, LAg isolated from Leishmania donovani promastigotes and protein G conjugated colloidal-gold as a signal reporter. The intensity of the bands depicting the antigen–antibody complex was optimized under different experimental conditions and quantitatively analyzed by the ImageJ software. For the diagnosis of human VL in India, LFA was found to be 96.49% sensitive and 95% specific with serum, and 95.12% sensitive and 96.36% specific with urine samples, respectively. The sensitivity and specificity of LFA were 88.57% and 94.73%, respectively, for the diagnosis of Brazilian VL using patients’ sera infected with Leishmania infantum. LFA is rapid and simple to apply, suitable for field usage where results can be interpreted visually and particularly sensitive and specific in the diagnosis of human VL. Serum and urine LFA may improve diagnostic outcomes and could be an alternative for VL diagnosis in settings where tissue aspiration is difficult to perform.

Development of Immunological Assays Based on Leishmania donovani Antigen for Diagnosis of Canine Visceral Leishmaniasis and Their Multicenter Evaluation in Brazil and Italy

“…We have earlier reported the diagnostic ability of LAg against both L. donovani-and L. infantum-infected human VL (Ejazi et al, 2019;Ejazi et al, 2021). In a dipstick format, LAg has been validated in eight centers in six countries, including India, Nepal, Sri Lanka, Brazil, Ethiopia, and Spain, and found overall 97.10% sensitivity and 93.44% specificity (Ejazi et al, 2019).…”

“…Identification of several newer antigens of L. infantum in Brazil demonstrated sensitivities between 49% and 97% for CVL diagnosis in comparison with 93% and 68% sensitivity with parasite lysate and rK39 antigen, respectively (Magalhaes et al, 2017). Earlier, LAg in ICT format had been evaluated with Brazilian human sera and had 88.57% and 94.73% sensitivity and specificity, respectively (Ejazi et al, 2021). In this study, we have accessed the performance of LAg-ICT against Brazilian canine sera.…”

The pathogenicity and virulence of Leishmania - interplay of virulence factors with host defenses

“…Using crude antigen, LAg in LFA exhibited sensitivities of 96.49% and 95.12% and specificities of 95% and 96.36% with Indian VL sera and urine, respectively, infected with L. donovani . In a similar study, 88.57% sensitivity and 94.73% specificity were monitored with the Brazilian sera infected with L. infantum [ 290 ]. Among recombinant proteins, rK39-ICT has certainly been the widely used rapid test for VL diagnosis in the last decade.…”

“…However, its performance shows drastic variations among different geographical locations of VL endemicity. Sensitivity rates of rK39-ICT performed in the Indian subcontinent were determined to be 97%, whereas in Brazil and East Africa, sensitivities go down to 85% and 67%, respectively [ 290 , 291 ]. The disparity in rK39-ICT performance initiated the development of numerous proteins for ICT that were isolated from regional L. donovani species, such as rK9, rK26, rK28, rKE16, and rKRP-42.…”

Nutritional Modulation of the Immune Response Mediated by Nucleotides in Canine Leishmaniosis

“…Clinical staging, either following the LeishVet guidelines for the practical management of CanL [7] or the guidelines for the treatment of leishmaniasis in dogs [40], is also a very useful tool, especially during patient monitoring [41], as clinical evolution can have an impact on the prognosis and influence the efficacy of a particular treatment protocol. In an attempt to optimize the detection of infected dogs and to improve the way CanL patients are diagnosed and monitored, several innovative techniques have been developed over the past few years, such as parasite detection and measurement of other immune system biomarkers from saliva samples [42][43][44][45]. Furthermore, it is also expected that in the future, more widespread use of serum measurement of circulating immune complexes will contribute to improving diagnosis, evaluation of disease progression, and monitoring response to treatment [46].…”