Anil Kumar Gupta

Leishmania donovani in blood smears of asymptomatic persons.

Leishmania amastigotes have been detected in the peripheral blood smears of Indian kala-azar patients mostly during night. It was, therefore, thought worthwhile to find out whether such amastigotes could be shown in healthy subjects who did not have any symptoms by history or signs on clinical examination. Leishmania-stained blood smears of 450 asymptomatic healthy individuals residing in an endemic village in Bihar (India) were examined by oil-immersion microscopy for the detection of amastigote, six people (1.3%) showed the presence of Leishmania amastigotes. Given the low sensitivity of a single smear examination it is likely that a much greater proportion of asymptomatic persons had parasitemia than the observed 1.3%. This finding is important from the point of view of transmission of disease, as also for modifying the current control measures.

Identification of B-cell Epitope of Leishmania donovani and its application in diagnosis of visceral leishmaniasis

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 (P1) and RRVAVLVLLDRL (P2) (From a hypothetical protein [Acc No: XP_003861458.1]). The epitope conservancy analysis suggested that the linear epitope (P1P2) 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 (P1P2) 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-P1P2 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-P1P2 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.

Leishmania donovani resistant to Ambisome or Miltefosine exacerbates CD58 expression on NK cells and promotes trans-membrane migration in association with CD2

HighlightsHigher expression of CD58 but not of CD2 was observed on CD56+ cells during Visceral Leishmaniasis.Expression of CD58 on CD56+ cells were further exacerbated in Ambisome or Miltefosine relapsed VL.Ratio of CD56+CD58+IFN‐&ggr;+/CD56+CD58+IL‐10+ cells reduces after stimulation with Ld.Ambisome or Miltefosine resistance Ld significantly increase CD58 expression on CD56+ cells.Antagonist to CD58 or CD2, down‐regulates CD56+ cell recruitment at Ld infection site. Abstract Visceral leishmaniasis (VL) is a disease that is associated with compromised immunity and drug un‐responsiveness as well as with the emergence of drug resistance in Leishmania donovani (Ld). Ld down‐modulates cellular immunity by manipulating signaling agents, including a higher expression of the adhesion molecule CD58. The expression of CD58 and CD2 on natural killer (NK) cells facilitates intercellular adhesion and signaling. The influence of drug‐resistant Ld on the expression of CD58 and CD2 was addressed in this study. The mean florescence intensity (MFI) of CD58 but not of CD2 was twofold higher on CD56+ cells during VL, but was down‐regulated after treatment. In addition, MFI of CD58 on CD56+ cells was further exacerbated in VL subjects who had relapsed after Ambisome or Miltefosine treatment. The same pattern of CD58 expression was also obtained upon stimulation of healthy peripheral blood mononuclear cells with Miltefosine‐ or Ambisome‐resistant Ld. The ratio of CD56+CD58+IFN‐&ggr;+/CD56+CD58+IL‐10+ cells was reduced by 6.98‐fold after stimulation with Ld. Further, an antagonist to CD58 or its counter‐receptor CD2 down‐regulated CD56+ NK cell recruitment across a polycarbonate trans‐membrane at Ld infection sites. This study reports that factors associated with drug resistance in Ld probably promote higher expression of CD58 on CD56+ cells and their migration to the infection site in association with CD2.

Degree of anemia correlates with increased utilization of heme by Leishmania donovani parasites in visceral leishmaniasis.

Anemia in kala-azar patients is a serious problem. The present study has addressed this problem with the hypothesis that as the Leishmania parasite is completely devoid of heme biosynthetic pathway, therefore the excessive use of heme by the parasites in the human patients can be one of the possible reason of anemia. We investigated that whether, the inability of Leishmania donovani to synthesize heme, can enforce Leishmania parasite to utilize heme derived from host sources in Indian KA patients. Patients had higher tendency of their macrophages to bind with Hb which was pronounced after sensitization with drug resistant Leishmania strain compared to sensitive.

Agar barrier method for removal of yeast and other fungal contamination from Leishmania promastigotes culture

Abstract The present study explains a novel method of Leishmania promastigotes culture decontamination. The method is based on motility of Leishmania promastigotes across agar barrier which facilitates decontamination from yeast and other non motile contamination. This is inexpensive, easy, rapid and reliable physical method and is able to save valuable isolates in culture.

Energy band gap and spectroscopic studies in Mn1−xCuxWO4 (0 ≤ x ≤ 0.125)

A study on the effect of nonmagnetic Cu2+ substitution at Mn2+ site on the structural and energy band gap of the MnWO4 is reported. Convenient solid state reaction route has been adopted for the synthesis of Mn1-xCuxWO4. X-ray diffraction (XRD) pattern showed high crystalline quality of the prepared samples. Raman spectroscopic studies were carried out to understand the structural aspects of the doping. 15 Raman active modes were identified out of 18, predicted for wolframite type monoclinic structure of MnWO4. UV-visible diffuse reflectance spectra were recorded and analyzed to get energy band gap of the studied system and are found in the range of 2.5 eV to 2.04 eV with a systematic decrease with the increase in Cu2+ concentration. Energy band gap values are verified by Density Functional Theory calculations based on projector augmented wave (PAW) method. The calculated values are in good agreement with the experimental data.