Dolanchampa Modak

IL-10- and TGF-β-Mediated Susceptibility in Kala-azar and Post-kala-azar Dermal Leishmaniasis: The Significance of Amphotericin B in the Control of Leishmania donovani Infection in India

Visceral leishmaniasis (VL) or kala-azar is known to be associated with a mixed Th1-Th2 response, and effective host defense requires the induction of IFN-γ and IL-12. We address the role of the differential decline of IL-10 and TGF-β in response to sodium antimony gluconate (SAG) and amphotericin B (AmB), the therapeutic success of SAG and AmB in Indian VL, and the significance of IL-10 and TGF-β in the development and progression of post-kazla-azar dermal leishmaniasis (PKDL). In the active disease, PBMC from VL patients showed suppressed Ag-specific lymphoproliferation, IFN-γ and IL-12 production, and elevation of IL-10 and TGF-β. Cure corresponded with an elevation in IFN-γ and IL-12 production and down-regulation of IL-10 and TGF-β. Both CD4+ and CD8+ T cells were involved in IFN-γ and IL-10 production. Interestingly, the retention and maintenance of residual IL-10 and TGF-β in some SAG-treated individuals and the elevation of IL-10 and TGF-β in PKDL, a sequel to kala-azar, probably reflects the role of these cytokines in reactivation of the disease in the form of PKDL. Contrastingly, AmB treatment of VL resulted in negligible TGF-β levels and absolute elimination of IL-10, reflecting the better therapeutic activity of AmB and its probable role in the recent decline in PKDL occurrences in India. Moreover, elucidation of immune responses in Indian PKDL patients revealed a spectral pattern of disease progression where disease severity could be correlated inversely with lymphoproliferation and directly with TGF-β, IL-10, and Ab production. In addition, the enhancement of CD4+CD25+ T cells in active VL, their decline at cure, and reactivation in PKDL suggest their probable immunosuppressive role in these disease forms.

Increased Levels of Interleukin-10 and IgG3 Are Hallmarks of Indian Post-Kala-Azar Dermal Leishmaniasis

BACKGROUND Post-kala-azar dermal leishmaniasis (PKDL), an established sequela of visceral leishmaniasis (VL), is proposed to facilitate anthroponotic transmission of VL, especially during interepidemic periods. Immunopathological mechanisms responsible for Indian PKDL are still poorly defined. METHODS Our study attempted to characterize the immune profiles of patients with PKDL or VL relative to that of healthy control subjects by immunophenotyping, intracellular cytokine staining of peripheral blood mononuclear cells, and enzyme-linked immunosorbent assay for serum cytokines and immunoglobulin G (IgG) subclasses. RESULTS Patients with PKDL had significantly raised percentages of peripheral CD3+CD8+ cells compared with control subjects, a difference that persisted after cure. Patients with PKDL showed an intact response to phytohemagglutinin, with the percentages of lymphocytes expressing interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-10 being comparable to those in control subjects. Patients with VL had decreased IFN-gamma and IL-2 expression, which was restored after cure, and increased IL-10 expression, which persisted after cure. In their response to Leishmania donovani antigen, patients with PKDL showed a 9.6-fold increase in the percentage of IL-10-expressing CD3+CD8+ lymphocytes compared with control subjects, and this percentage decreased with treatment. Patients with PKDL had raised levels of IgG3 and IgG1 (surrogate markers for IL-10), concomitant with increased serum levels of IL-10. CONCLUSIONS IL-10-producing CD3+CD8+ lymphocytes are important protagonists in the immunopathogenesis of Indian PKDL.

Enhanced Lesional Foxp3 Expression and Peripheral Anergic Lymphocytes Indicate a Role for Regulatory T Cells in Indian Post-Kala-Azar Dermal Leishmaniasis

Indian post-kala-azar dermal leishmaniasis (PKDL) is a low-frequency (5-10%) dermal sequela of visceral leishmaniasis (VL) caused by Leishmania donovani; importantly, affected individuals are speculated to be parasite reservoirs. Insight into its immunopathogenesis could translate into rational immunomodulatory therapeutic approaches against leishmaniases. In patients with PKDL (n=21), peripheral lymphocytes were analyzed for surface markers, intracellular cytokines, and lymphoproliferative responses using flow cytometry. In lesional tissue biopsies (n=12), expression of counter-regulatory cytokines (IFN-gamma and IL-10) and the T-regulatory transcription factor forkhead box protein 3 (Foxp3) was analyzed using reverse transcriptase-PCR, along with immunohistochemical detection (n=8) of CD3 and Foxp3 positivity. In patients with PKDL, circulating CD8(+)CD28(-) and antigen-induced IL-10(+)CD3(+) lymphocytes were increased and receded with treatment. CD8(+) lymphocytes showed impaired proliferative responses to L. donovani antigen (LDA) and phytohemagglutinin, which were reinstated after treatment. At presentation, the upregulated lesional IFN-gamma and IL-10 messenger RNA (mRNA), Foxp3 mRNA, and protein were curtailed after treatment. In Indian patients with PKDL, increased frequency of the CD8(+)CD28(-) phenotype, enhanced antigen-specific IL-10 production, and accompanying anergy of circulating lymphocytes suggest their regulatory nature. Furthermore, the concomitantly elevated lesional expression of Foxp3 suggests their possible recruitment into the lesional site, which would sustain disease pathology.

A Defective Oxidative Burst and Impaired Antigen Presentation are Hallmarks of Human Visceral Leishmaniasis

PurposeSurvival of the Leishmania parasite within monocytes hinges on its ability to effectively nullify their microbicidal effector mechanisms. Accordingly, this study aimed to delineate this biological niche in patients with visceral leishmaniasis (VL).MethodsIn monocytes, the redox status, antigen presenting capacity, expression of Toll-like receptors (TLRs), co-stimulatory molecules (CD80/86) and generation of intracellular cytokines (IL-8, IL-1β, IL-10 and LAP-TGF-β1) was measured by flow cytometry, levels of circulating cytokines (IL-1β, IL-6, TNF-α, IL-8, IL-4, IL-13, IL-10 and GM-CSF) by ELISA and arginase activity by spectrophotometry.ResultsWithin monocytes, generation of an oxidative burst was markedly attenuated as evident by decreased generation of nitric oxide and reactive oxygen species, concomitant with raised levels of thiols. This was accompanied by lowered frequency of TLR4+ monocytes, but the arginase activity remained unaltered. Pathogen persistence was enhanced by the predominance of anti-inflammatory cytokines within monocytes, notably IL-10. Alongside, development of adaptive immunity was severely attenuated as manifested by a pronounced impairment of antigen presentation and co-stimulation evident by down regulation of CD54, HLA-DR and CD86. Treatment corrected the redox imbalance and reversed the impaired antigen presentation.ConclusionsIn VL, monocyte functions were severely impaired facilitating parasite persistence; anti-leishmanial chemotherapy mediated parasite elimination through modulation of the macrophage microenvironment by restoring its redox status and antigen presenting capacity.

Easy Test for Visceral Leishmaniasis and Post–Kala-azar Dermal Leishmaniasis

To the Editor: Diagnosis of visceral leishmaniasis (VL), fatal if untreated, is complex because the symptoms are the same for many fever-associated ailments. Despite limitations, diagnosis remains based on finding Leishmania amastigotes in spleen and/or bone marrow aspirates (1). Sophisticated laboratory methods, although sensitive, are costly. The immunochromatographic strip test that uses recombinant K39 antigen (rK39), although satisfactory in India, is less sensitive in Africa, Latin America, and Mediterranean regions (2). Post–kala-azar dermal leishmaniasis (PKDL), a sequel to VL in India and Africa, is often confused with other skin diseases (3,4). Diagnosis of VL in dogs in Latin America and Mediterranean countries remains confusing because of rampant asymptomatic infections and elevated antibodies against Leishmania spp (5). Earlier we reported the diagnostic potential of L. donovani (MHOM/IN/83/AG83) promastigote membrane antigens (LAg) (3,6). Here we report applicability of LAg-based ELISA and dipstick systems even at primary health centers. Using randomized sampling, we tested samples from 122 kala-azar patients from India, 20 PKDL patients from India, and 40 VL patients from Brazil. VL was confirmed by finding parasites in aspirates. Serum samples were collected before chemotherapy was given. PKDL was diagnosed as described (3). Control samples were collected from 24 healthy persons from non–disease-endemic areas in India; 15 healthy persons from disease-endemic areas in India; 20 healthy persons from disease-endemic areas in Brazil; and 21 persons with Hansen disease, 7 with filariasis, 4 with tuberculosis, 1 with lymphoma, 1 with leukemia, 2 with virus-induced fever, and 5 with malaria. Consent was obtained from all human donors. This study was approved by Ethical Committee on Human Subjects at Indian Institute of Chemical Biology and the Ethical Board for Human Subjects and Animal Experimentation of the Federal University of Piaui. We developed a diagnostic ELISA with modifications of our previous method (6). Microtiter plates were coated with 2.5 μg LAg at pH 7.5 (100 μL/well) and kept at 4°C overnight, after which they were blocked with 1% bovine serum albumin, dried, and stored at 4°C as precoated plates. The assay performed at room temperature took ≈2.5 h. Test and control serum samples (1:1,000 dilution, 100 μL/well) were applied to the plates for 45 min and shaken occasionally. Horseradish peroxidase–conjugated goat anti-human immunoglobulin (Ig) G (Genei, Bangalore, India) was applied at 1:5,000 (100 μL/well) for 45 min. Color development with ortho-phenylenediamine (Sigma-Aldrich, St. Louis, MO, USA) was allowed for 5–10 min. Positive results were determined by comparing colors with those on a card previously prepared for positive and negative wells. ELISA, performed for the VL and PKDL patients from India, was 100% sensitive (percentage of patients with confirmed disease and positive test results) and 96.3% specific (percentage of negative controls with negative test results) (Figure, panel A); sensitivity and specificity were higher than that reported earlier (6) and by other studies that used crude leishmanial antigens (2). One sample from each of filariasis, lymphoma, and disease-endemic area controls was marginally false positive. Seropositivity was diagnosed for 1 patient who had a negative spleen aspirate but clinical signs of VL and for 1 patient who refused spleen or bone marrow aspiration. Figure Representative results of ELISA and dipstick testing. A) Samples underwent ELISA in duplicate. Upper panel, positive samples in duplicate (1–2 and 3–4) in wells A–H, except A1–A2 (blank), and G3–G4 and H3–H4 ... To avoid any visible cross-reaction in the dipstick assay, we optimized LAg concentration, test serum dilution, and control serum dilution. Optimum concentration for human studies is 500 μg/mL LAg, 1:2,000 serum dilutions, 1:2,000 horseradish peroxidase–conjugated goat anti-human IgG, and 15 mg 3,3′-diaminobenzidine (Sigma-Aldrich) as substrate in 30 mL Tris-buffered saline. LAg was bound to a long nitrocellulose piece at the test line (line on which LAg is coated). Goat anti-human IgG (Genei) at 1:25 was coated as an internal control line. Free sites were blocked with 2% bovine serum albumin containing 0.01% NaN3 and were air dried. LAg-coated membranes were affixed to the end of a plastic support (with a free end as handle) with double-adhesive tape, cut into 4-mm–wide sticks, and stored at room temperature. During the testing process at room temperature, dipsticks were incubated in diluted serum for 30 min, washed 2×, incubated for 30 min with secondary antibody, washed 3×, and incubated in substrate solution for 1 min. Finally, dipsticks were washed in water, dried on tissue paper, and examined for specific reaction. When stored at room temperature without desiccation, dipsticks performed consistently for 12 months. Dipsticks appeared equally sensitive and specific (100%) for VL from India and Brazil and for PKDL. Because internal control lines remained positive, analyses were considered valid (Figure, panel B). LAg dipsticks are more sensitive for diagnosing VL in Brazil than rK39 (7) and cost ≈70× less (8). Although further validation with a larger sample size and healthy controls from disease-endemic areas and controls for other diseases is warranted, these easy, simple, and low-cost methods could emerge as efficient tools for diagnosis of VL and PKDL.

Severe skin rash with lamivudine in HIV infected patients: Some unusual case reports

Lamivudine (3TC) is a nucleoside reverse transcriptase inhibitor (NRTI) licensed for as a first line drug in Human immunodeficiency virus (HIV) infection and also in the treatment of hepatitis B. It is relatively nontoxic in nature and potentiates the antiviral effects of other NRTIs like zidovudine. Although lamivudine is well-tolerated, certain dermatological side effects like severe skin rash may appear. We report a case series of severe skin rashes in four HIV-infected patients, probably due to lamivudine.

Effectiveness of targeted viral load strategy in a public health HIV/AIDS programme

Methods Data of patients referred to School of Tropical Medicine, Kolkata for suspected treatment failure (TF) was analyzed for the period of December2008 to November2013 to ascertain the positive predictive value (PPV) of immunologic and/or clinical parameter for confirming virologic failure (VF) and to observe the short term outcome of secondline treatment. Paired ttest was used for data analysis.

Symmetrical peripheral gangrene: A rare complication of dengue fever

Symmetric peripheral gangrene is associated with a variety of infective and non-infective etiologies. SPG is always presented with disseminated intravascular coagulation (DIC) and carries a higher mortality. Herein, we describe a 42-year-old female with dengue fever and rash developed bilateral symmetric dry gangrene of 2 nd and 3 rd toes. There was no history of taking B-blockers, ergot etc. All the peripheral pulses of the affected limbs were palpable. Color Doppler of lower limb vessels was done, which indicated normal flow. Blood was positive for Fibrin degradation products and D dimers. Patient was managed with IV fluids, LMWH, FFP etc. Her general condition improved within 72 hours with no further progression of gangrene.