Diagnosis of visceral leishmaniasis (VL) is often hindered by cross-reactions with antigens from other related parasite infections. This study aimed to develop an immunochromatographic test (ICT) which can detect the antigen present in circulating immune complexes (CICs) of VL patients using B-cell epitope-specific antibodies. MS analysis of six immunoreactive 2DE spots revealed two epitopes i.e. RFFVQGDGIGQHSLQEALERR (P) and RRVAVLVLLDRL (P) (From a hypothetical protein [Acc No: XP_003861458.1]). The epitope conservancy analysis suggested that the linear epitope (PP) is 97-100% conserved among Leishmania species and diverged from Homo sapiens (61% query coverage and 80% identity). Further, immunoinformatics analysis of hydrophilicity and flexibility confirmed the antigenicity of the peptide fragment. The linear epitope (PP) was synthesized (98% purity) and the purity was confirmed by high-performance liquid chromatography and MS. The indirect Enzyme linked immunosorbent assay results confirmed the presence of the corresponding antibody in VL patient's sera but not in those of healthy and other diseases. The result demonstrated a sensitivity 90%; Se Cl95% (82.16-96.27)% and specificity 100%; Sp Cl95% (84.56-100)% which indicated the possibility to be used as a diagnostic tool. Sensitivity, specificity, and diagnostic efficiency of colloidal gold conjugated anti-PP antibody ICT strip was 100, 95.2, and 96.7%, respectively, which is slightly better as compared to other ICT for VL. Though, our result indicated the utility of anti-PP antibody to detect CICs epitopes, a large-scale inspection in endemic and non-endemic area and in different ethnic population is needed for its validation and authentication.
Visceral leishmaniasis (VL) is one of the most fatal
and neglected
tropical diseases caused by Leishmania donovani (L. donovani). The applications of currently available
chemotherapy (amphotericin B, miltefosine, and others) in VL treatment
have been limited due to their poor bioavailability, unfavorable toxicity
profile, and prolonged parenteral dosing. Quercetin (QT), a potent
natural antioxidant, is a prominent target when conducting investigations
on alternative therapies against L. donovani infections. However, the therapeutic applications of QT have been
restricted due to its low solubility and bioavailability. In the present
study, we developed and evaluated the antileishmanial activity (ALA)
of quercetin-loaded nanoemulsion (QTNE) against L.
donovani clinical strains. In vitro anti-promastigote assay results demonstrated that QTNE (IC50 6.6 μM, 48 h) significantly inhibited the growth of parasites
more efficiently than the pure QT suspension in a dose- and time-dependent
manner. Results of the anti-amastigote assay revealed that the infected
macrophages (%) of QTNE were significantly more than those of the
pure QT suspension at all concentrations (6.6, 26.4, and 52.8 μM; p < 0.05, p < 0.01 compared to the
control). Moreover, the results of in vitro and ex vivo studies assisted in determining the mechanistic
insights associated with the ALA of QTNE. The overall findings suggested
that QTNE exhibited potential ALA by enhancing the intracellular ROS
and nitric oxide levels, inducing distortion of membrane integrity
and phosphatidylserine release (AV/PI), rupturing the parasite DNA
(late apoptosis/necrosis process), and upregulating the immunomodulatory
effects (IFN-γ and IL-10 levels). Additionally, QTNE showed
superior biocompatibility against all of the treated healthy cells
(PBMCs, PECs, and BMCs) as compared to the control. In conclusion,
QTNE acts as a potential antileishmanial agent targeting both promastigote
and intracellular amastigote forms of L. donovani, which thus opens a new avenue for the use of QTNE in VL therapy.
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