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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.

Tests for visceral leishmaniasis (VL) are not uniformly effective for all endemic regions. In a serological assay, a novel antigen, otubain cysteine peptidase, compared with rK39, showed comparable sensitivity with Indian VL serum samples and prominently increased sensitivity with Brazilian samples, as well as improved monitoring of the treatment response.

Correction to: Scientific Reports https://doi.org/10.1038/s41598-019-46283-9, published online 09 July 2019 The original version of this Article contained an error. The information in the “Dipstick development and assay” section was incomplete. Therefore, the original text, “The assay comprises of incubation of the dipstick with diluted serum samples (1:2000) for 30 min which is followed by washing with TBST (twice). Subsequently, it is incubated with HRP conjugated anti-human IgG (1: 2000) for 30 min. Finally, after two washes in TBST and one in TBS, the strips are dipped in a freshly prepared substrate composed of 0.05% 3, 3′-diaminobenzidine tetrahydrochloride (DAB, Sigma, USA) containing 0.05% of H2O2 in 100 mM TBS. The reaction is stopped by dipping in distilled water. The appearance of dark brown coloured bands at both the test and control line indicates VL positivity and a single band at the control line is indicative of VL negativity.” now reads: “The general assay consists of incubation of the dipsticks with diluted serum (1:2000) samples for 30 min, followed by washing with TBST (twice). Subsequently, it is incubated with HRP conjugated anti-human IgG (1:2000) for 30 min. Finally, after two washes in TBST and one in TBS, the strips are dipped in a freshly prepared substrate composed of 0.05% 3, 3′-diaminobenzidine tetrahydrochloride (DAB, Sigma, USA), containing 0.05% of H2O2 in 100 mM TBS. The reaction is stopped by dipping in distilled water, and the strips dried at RT. In Spain, there were several modifications to the assay; the dipsticks are incubated in diluted serum (1:100), and HRP conjugated anti-human IgG, for 60 min, followed by a 10 min incubation in substrate composed of 0.07% of DAB and 0.2% of H2O2 in 60 mM TBS, before being dried at RT. The appearance of dark brown coloured bands at both the test and control line indicates VL positivity and a single band at the control line is indicative of VL negativity. Whenever this signal was hard to assess, we classified it as not clear, and therefore grouped it together with those displaying no bands. Dipsticks that did not present with a reactive band, or a control band, were excluded from further consideration.” These changes do not affect the overall conclusions of the Article. This has now been corrected in the PDF and HTML versions of the Article.

Author Correction: A multicentric evaluation of dipstick test for serodiagnosis of visceral leishmaniasis in India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain. Scientifc Reports. 2021; 11:3967. DOI: 10.1038/s41598-021-83332-8. PMID: 33574485 Visceral leishmaniasis (VL) is one of the leading infectious diseases affecting developing countries. Colloidal gold-based diagnostic tests are rapid tools to detect blood/serum antibodies for VL diagnosis. Lack of uniformity in the performance of these tests in different endemic regions is a hurdle in early disease diagnosis. This study is designed to validate a serum-based dipstick test in eight centres of six countries, India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain with archived and fresh sera from 1003 subjects. The dipstick detects antibodies against Leishmania donovani membrane antigens (LAg). The overall sensitivity and specificity of the test with 95% confidence intervals were found to be 97.10% and 93.44%, respectively. The test showed good sensitivity and specificity in the Indian subcontinent (>95%). In Brazil, Ethiopia, and Spain the sensitivity and specificity of the dipstick test (83.78-100% and 79.06-100%) were better as compared to the earlier reports of the performance of rK39 rapid test in these regions. Interestingly, less cross-reactivity was found with the cutaneous form of the disease in Spain, Brazil, and Sri Lanka demonstrating 91.58% specificity. This dipstick test can therefore be a useful tool for diagnosing VL from other symptomatically similar diseases and against cutaneous form of leishmaniasis. Sí

Visceral leishmaniasis (VL), a potentially fatal disease is an outcome of infection caused by the parasite Leishmania donovani. The clinical diagnostic tests for this disease are still related to invasive tissue aspiration or serological immunochromatography. Advancements in immunoproteomics such as two-dimensional gel electrophoresis, mass spectrometry, B cell epitope prediction, and peptide synthesis have enabled researchers to discover newer biomarkers for disease diagnosis. In this study, we have screened several urine-reactive leishmanial membrane proteins as potential biomarker candidates. In the immunoblot assay, three proteins 51, 55 and 63 kDa showed 100% reactivity to the urine of 47 VL patients and nonreactive to 18 healthy and other diseases. Mass spectrometry revealed the identity of 51, 55 and 63 kDa proteins as elongation factor 1α (EF1-α), α-tubulin, and glycoprotein 63, respectively. B cell reactive epitopes of these proteins were mapped through bioinformatic tools and one epitope from each protein that had the highest score were synthesized. All the three native electroeluted proteins and their corresponding synthetic peptides were tested through ELISA for reactivity with VL and control urine samples. While all three demonstrated good reactivity, the diagnostic performance of EF1-α was the best. Our findings illustrate the use of urine-based proteomic approach for biomarker discovery in non-invasive clinical diagnosis of VL.

Cerebral ischemia-reperfusion is a classic example of reactive oxygen species (ROS) mediated acute damage to brain. Post-ischemic reperfusion induced oxygen free radicals production causes damage to brain cell mitochondria. Antioxidants like quercetin (Qc) have potentials to manage oxidative stress related pathophysiology. However low oral bioavailability and poor cell membrane permeability restrict its therapeutic efficacy. To overcome these hurdles mitochondria specific delivery of Qc nanocapsules was designed to efficiently counteract cerebral ischemia-reperfusion induced cell death and neurodegeneration in young and aged rats. The orally deliverable quercetin loaded polymeric nanocapsules (N1QC) were made mitochondria specific by using triphenylphosphonium cation as one of the matrix components. N1QC demonstrated higher brain uptake and remarkable mitochondrial localization post cerebral ischemia-reperfusion. This unique controlled mitochondrial delivery of quercetin ameliorated histopathological severity by preserving mitochondrial structural and functional integrity through sequestering ROS thus modulating mitochondrial ROS mediated apoptotic cell death in young and aged rats.

The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8+ cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4+ and CD8+ T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4+ and CD8+ T cells. Furthermore, lymphoid CD8+ T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8+ T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8+ T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

Somsubhra Thakur Choudhury,1 Nirmalendu Das,2 Swarupa Ghosh,2 Debasree Ghosh,2 Somsuta Chakraborty,2 Nahid Ali1 1Infectious Diseases and Immunology, 2Drug Development, Diagnostics and Biotechnology, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India Abstract: The liver plays a vital role in biotransforming and extricating xenobiotics and is thus prone to their toxicities. Short-term administration of carbon tetrachloride (CCl4) causes hepatic inflammation by enhancing cellular reactive oxygen species (ROS) level, promoting mitochondrial dysfunction, and inducing cellular apoptosis. Curcumin is well accepted for its antioxidative and anti-inflammatory properties and can be considered as an effective therapeutic agent against hepatotoxicity. However, its therapeutic efficacy is compromised due to its insolubility in water. Vesicular delivery of curcumin can address this limitation and thereby enhance its effectiveness. In this study, it was observed that both liposomal and nanoparticulated formulations of curcumin could increase its efficacy significantly against hepatotoxicity by preventing cellular oxidative stress. However, the best protection could be obtained through the polymeric nanoparticle-mediated delivery of curcumin. Mitochondria have a pivotal role in ROS homeostasis and cell survivability. Along with the maintenance of cellular ROS levels, nanoparticulated curcumin also significantly (P