Glycoproteins in circulating immune complexes are biomarkers of patients with Indian PKDL: A study from endemic districts of West Bengal, India

Abstract

BackgroundPost Kala Azar Dermal Leishmaniasis (PKDL) occurs as dermal consequence of previous Visceral Leishmaniasis (VL) infection and serves as an important reservoir for transmission of VL. Diagnosis of PKDL is often challenging for its symptomatic resemblance to other co-endemic diseases like Leprosy or Vitiligo. Parasitological examination by slit-skin smear and culture are the standard methods but lack high sensitivity. Thus, for efficient control of VL, reliable diagnostic and prognostic assay of PKDL are required.ObjectivePreviously, glycoproteins (9-OAcSA) have been reported as promising biomarkers of Indian VL patients. However, till date, the status of glycans in Indian PKDL patients remains unexplored. Accordingly, in this study, the glyco-profile of PKDL Circulating Immune Complexes (CICs) as compared to other cross diseases like Vitiligo and Leprosyhas been investigated. Further, a novel Glyco CIC assay has been developed for efficient Indian PKDL patient diagnosis.Methods/principal findingIn the present study, 90 PKDL patients were enrolled from 3 VL endemic districts of West Bengal during 2015–16. Glycosylation profile of isolated CICs from sera of PKDL patients were initially analyzed through gradient SDS gel electrophoresis followed by PAS silver double staining, which revealed the presence of several glycan rich PKDL specific proteins of varying molecular weights. To further characterize the glyco-profile of acid dissociated affinity purified immuno-reactive antigens present in the CICs, glycosylation was demonstrated in these purified CIC antigens by DIG glycan differentiation kit with or without glycosidase as well as neuraminidase treatment. Diagnostic evaluation of the newly developed colorimetric Glyco CIC assay through Receiver Operating Characteristic (ROC) curve analysis revealed excellent (0.99) AUC value as compared to other conventional serodiagnostic assays like PEG CIC, Parasite ELISA (IgG and IgM). Additionally, longitudinal monitoring of 18 PKDL patients further revealed its good prognostic utility.ConclusionThese results highlight the glycosylation status of CICs among Indian PKDL patients present in all the studied endemic districts of West Bengal. These PKDL biomarkers were completely absent in cross diseases like Vitiligo and Leprosy. Further, the newly developed Glyco CIC assay had an improved sensitivity of 95.6%, specificity of 99.3%, NPV of 97.1% and PPV of 98.9%.

High levels of serum glycans monovalent IgG immune complexes detected by dissociative ELISA in experimental visceral leishmaniasis

“…This type of assay limits the interaction of antibodies to proteins and glycoproteins, minimizing the presence of other specific antibodies, especially anti‐glycans. Protein or glycoprotein ICs (> 10 000 MW) are generally detected at low levels in VL or other leishmaniasis, such as post‐Kala‐azar dermal leishmaniasis …”

“…Protein or glycoprotein ICs (> 10 000 MW) are generally detected at low levels in VL 18 or other leishmaniasis, such as post-Kala-azar dermal leishmaniasis. 22,23 Structural composition of antigens complexed to IgG anti-leishmania antibodies is an important tool for the understanding of trigger mechanisms, permanence and elimination of ICs. Our search of low-molecular-weight antigens in PSE by sequential molecular exclusion chromatography revealed glycans (950-1800 MW) probably with 6-13 sugar residues by MALDI-TOF.…”

Clinical Proteomics Profiling for Biomarker Identification Among Patients Suffering With Indian Post Kala Azar Dermal Leishmaniasis

“…Previous reports have repeatedly suggested association of parasite infections with high levels of CICs which directly leads to tissue damage (Moulds et al, 1992). Previous studies from our lab have reported the presence of high levels of CICs among PKDL patients (Datta et al, 2015;Jaiswal et al, 2018). Further highthroughput studies are required to identify the specific peptide fragments from PKDL patients' CICs, representing specific proteins, which could act as glycated CICs proteome biomarker for both diagnostic and prognostic care of PKDL patients after treatment completion.…”

“…These studies highlight the role of proteomics in identifying disease specific protein markers that can help in detection of VL, when parasite load is scanty. Previous reports from our lab have reported altered CICs glycosylation status of PKDL patients (Datta et al, 2015;Jaiswal et al, 2018). High throughput proteomic analysis of PKDL patients' glycated CICs will assist in the identification of major PKDL disease specific protein biomarkers among Indian (MAC and POLY) PKDL population.…”

“…Albumin depleted samples were treated with Glycine-HCL buffer as described by Jaiswal et al (2018), to dissociate the antigen-antibody complex (Jaiswal et al, 2018). Next the acid dissociated CICs were affinity purified using equilibrated Protein A Sepharose 4B column (Invitrogen, USA), at room temperature (RT) for 45 minutes.…”