Development of a sandwich ELISA to detect Leishmania 40S ribosomal protein S12 antigen from blood samples of visceral leishmaniasis patients

Abstract

BackgroundVisceral leishmaniasis (VL), caused by Leishmania donovani complex parasites, is a neglected parasitic disease that is generally fatal if untreated. Despite decades of research to develop a sensitive VL diagnostic test, definitive diagnosis of VL still mainly relies on the visualization of the parasite in aspirates from the spleen, liver or bone marrow, an invasive and dangerous process with variable sensitivity. A sensitive assay that can detect Leishmania antigen from blood samples will help confirm cause, cure or recurrence of VL.MethodsIn this study, rabbit polyclonal antibodies were raised against eight recombinant Leishmania proteins that are highly abundant in Leishmania. The antibodies were purified and labeled with biotin for developing a prototype sandwich enzyme-linked immunosorbent assay (ELISA).ResultsThe ELISA for the Leishmania 40S ribosomal protein S12 detected target antigen with the highest sensitivity and specificity and could detect 1 pg of purified protein or as few as 60 L. donovani parasites. The 40S ribosomal protein S12 sandwich ELISA could detect the target antigen from Peripheral Blood Mononuclear Cell (PBMC) samples in 68% of VL patients and post-kala-azar dermal leishmaniasis (PKDL) patients, providing an estimation of parasitemia ranging from 15 to 80 amastigotes per ml of blood.ConclusionThese results indicate that the 40S ribosomal protein S12 sandwich ELISA warrants further tests with more clinical samples of VL patients and other parasitic diseases. It is hopeful that this ELISA could become a useful tool for confirming VL diagnosis, monitoring treatment progress, disease recurrence and possibly detecting asymptomatic Leishmania infections with a high parasite load.

Evaluation of methods for detection of asymptomatic individuals infected with Leishmania infantum in the state of Piauí, Brazil

“…infantum and false-negative test results underestimate the actual rate. However, ELISA can identify asymptomatic infections as is shown in the present study and also in other studies [6,53,54]. In addition, the ELISA method using crude antigen is generally more sensitive, albeit less specific [11].…”

A case of visceral leishmaniasis found by left oblique hernia: A case report

“…Moreover, clinical manifestations and disease symptoms or signs may produce additional confusion. The absence of the medical history of patients resident in epidemic areas may lead to misdiagnosis of the disease (5). Based on research statistics, the misdiagnosis rate of clinical VL can reach 23.4% (6).…”

Tandem mass tag-based quantitative proteomics analyses of a chicken-original virulent and its attenuated Histomonas meleagridis strain in China

“…In visceral Leishmaniasis, the 60S ribosomal protein L6 was suggested to be potentially associated with its resistance to paromomycin (54). Meanwhile its 40S ribosomal protein S12 has the highest sensitivity and specificity as an ELISA assay to detect the target antigen (55). Based on the lack of researches related to ribosomal proteins in H. meleagridis, it is believed that it has the potential for further research.…”