Visceral Leishmaniasis and HIV Co-infection in Bihar,India: Long-term Effectiveness and Treatment Outcomes with Liposomal Amphotericin B (AmBisome)

Background

Visceral Leishmaniasis (VL; also known as kala-azar) is an ultimately fatal disease endemic in the Indian state of Bihar, while HIV/AIDS is an emerging disease in this region. A 2011 observational cohort study conducted in Bihar involving 55 VL/HIV co-infected patients treated with 20–25 mg/kg intravenous liposomal amphotericin B (AmBisome) estimated an 85.5% probability of survival and a 26.5% probability of VL relapse within 2 years. Here we report the long-term field outcomes of a larger cohort of co-infected patients treated with this regimen between 2007 and 2012.

Methods and Principal Findings

Intravenous AmBisome (20–25 mg/kg) was administered to 159 VL/HIV co-infected patients (both primary infections and relapses) in four or five doses of 5 mg/kg over 4–10 days. Initial cure of VL at discharge was defined as improved symptoms, cessation of fever, improvement of appetite and recession of spleen enlargement. Test of cure was not routinely performed. Antiretroviral treatment (ART) was initiated in 23 (14.5%), 39 (24.5%) and 61 (38.4%) before, during and after admission respectively. Initial cure was achieved in all discharged patients. A total of 36 patients died during follow-up, including six who died shortly after admission. Death occurred at a median of 11 weeks (IQR 4–51) after starting VL treatment. Estimated mortality risk was 14.3% at six months, 22.4% at two years and 29.7% at four years after treatment. Among the 153 patients discharged from the hospital, 26 cases of VL relapse were diagnosed during follow-up, occurring at a median of 10 months (IQR 7–14) after discharge. After accounting for competing risks, the estimated risk of relapse was 16.1% at one year, 20.4% at two years and 25.9% at four years. Low hemoglobin level and concurrent infection with tuberculosis were independent risk factors for mortality, while ART initiated shortly after admission for VL treatment was associated with a 64–66% reduced risk of mortality and 75% reduced risk of relapse.

Significance

This is the largest cohort of HIV-VL co-infected patients reported from the Indian subcontinent. Even after initial cure following treatment with AmBisome, these patients appear to have much higher rates of VL relapse and mortality than patients not known to be HIV-positive, although relapse rates appear to stabilize after 2 years. These results extend the earlier findings that co-infected patients are at increased risk of death and require a multidisciplinary approach for long-term management.     

Post Kala-Azar Dermal Leishmaniasis following Treatment with 20 mg/kg Liposomal Amphotericin B (Ambisome) for Primary Visceral Leishmaniasis in Bihar,India

Background

The skin disorder Post Kala-Azar Dermal Leishmaniasis (PKDL) occurs in up to 10% of patients treated for visceral leishmaniasis (VL) in India. The pathogenesis of PKDL is not yet fully understood. Cases have been reported in India following therapy with most available treatments, but rarely in those treated with liposomal amphotericin B (Ambisome). Between July 2007 and August 2012 with the support of the Rajendra Memorial Research Institute (RMRI), Médecins Sans Frontières (MSF) supported a VL treatment programme in Bihar, India—an area highly endemic for Leishmania donovani—in which 8749 patients received 20 mg/kg intravenous Ambisome as first-line treatment. This study describes the characteristics of patients who returned to the MSF supported treatment programme with PKDL.

Methods and Principal Findings

Over a 5-year period, Ambisome was administered to 8749 patients with laboratory-confirmed VL (clinical signs, rK39 positive, with/without parasite confirmation) in four intravenous doses of 5 mg/kg to a total of 20 mg/kg, with a high initial-cure rate (99.3%) and low default rate (0.3%). All patients received health education highlighting the possibility and symptoms of developing PKDL, and advice to return to the MSF programme if these symptoms developed. This is an observational retrospective cohort study of the programme outcomes. Of the 8311 patients completing treatment for their first episode of VL, 24 (0.3%) returned passively to the programme complaining of symptoms subsequently confirmed as PKDL, diagnosed from clinical history, appearance consistent with PKDL, and slit-skin smear examination. Of the 24 patients, 89% had macular lesions, with a median time (interquartile range) to development of 1.2 (0.8–2.2) years following treatment. Comparison of the demographic and clinical characteristics of the VL patients treated with Ambisome who later developed PKDL, with those of the remaining cohort did not identify any significant risk factors for PKDL. However, the time to developing PKDL was significantly shorter with Ambisome than in a subset of patients presenting to the programme with PKDL following previous sodium stibogluconate treatment for VL.

Conclusions

In this large cohort of patients with VL in Bihar who were treated with 20 mg/kg Ambisome, PKDL following treatment appears to be infrequent with no predictive risk factors. The shorter median time to developing symptoms of PKDL compared with that after conventional VL treatments should be taken into account when counseling patients treated with regimens including Ambisome.     

Five-Year Field Results and Long-Term Effectiveness of 20 mg/kg Liposomal Amphotericin B (Ambisome) for Visceral Leishmaniasis in Bihar,India

Background

Visceral Leishmaniasis (VL; also known as Kala-azar) is an ultimately fatal disease endemic in Bihar. A 2007 observational cohort study in Bihar of 251 patients with VL treated with 20 mg/Kg intravenous liposomal amphotericin B (Ambisome) demonstrated a 98% cure rate at 6-months. Between July 2007 and August 2012, Médecins Sans Frontières (MSF) and the Rajendra Memorial Research Institute (RMRI) implemented a VL treatment project in Bihar, India—an area highly endemic for Leishmania donovani—using this regimen as first-line treatment.

Methods and Principal Findings

Intravenous Ambisome 20 mg/kg was administered in four doses of 5 mg/kg over 4–10 days, depending on the severity of disease. Initial clinical cure at discharge was defined as improved symptoms, cessation of fever, and recession of spleen enlargement. This observational retrospective cohort study describes 8749 patients with laboratory-confirmed primary VL treated over a 5-year period: 1396 at primary healthcare centers, 7189 at hospital, and 164 at treatment camps. Initial clinical cure was achieved in 99.3% of patients (8692/8749); 0.3% of patients (26/8749) defaulted from treatment and 0.4% (31/8749) died. Overall, 1.8% of patients (161/8749) were co-infected with HIV and 0.6% (51/8749) with tuberculosis. Treatment was discontinued because of severe allergic reactions in 0.1% of patients (7/8749). Overall, 27 patients (0.3%) were readmitted with post Kala-azar dermal leishmaniasis (PKDL). Risk factors for late presentation included female sex, age >15 years and being from a scheduled caste.In 2012, a long-term efficacy survey in the same area of Bihar determined relapse rates of VL after 5 years'' intervention with Ambisome. Of 984 immunocompetent patients discharged between September 2010 and December 2011, 827 (84.0%) were traced in order to determine their long-term outcomes. Of these, 20 patients (2.4%) had relapsed or received further treatment for VL. Of those completing 6, 12, and 15 month follow-up, 0.3% (2/767), 3.7% (14/383), and 2.4% (4/164), respectively, had relapsed. The mean ±SD time-to-relapse was 9.6±3.0 months.

Significance

This is the largest cohort of VL patients treated with 20 mg/kg Ambisome worldwide. The drug has high initial and long-term efficacy, and a low rate of adverse reactions when administered under field conditions in Bihar, India. Although challenging, its use as first line treatment in rural settings in Bihar is safe and feasible.     

Effectiveness Study of Paromomycin IM Injection (PMIM) for the Treatment of Visceral Leishmaniasis (VL) in Bangladesh

Background

This study was conducted in Bangladeshi patients in an outpatient setting to support registration of Paromomycin Intramuscular Injection (PMIM) as a low-cost treatment option in Bangladesh.

Methodology

This Phase IIIb, open-label, multi-center, single-arm trial assessed the efficacy and safety of PMIM administered at 11 mg/kg (paromomycin base) intramuscularly once daily for 21 consecutive days to children and adults with VL in a rural outpatient setting in Bangladesh. Patients ≥5 and ≤55 years were eligible if they had signs and symptoms of VL (intermittent fever, weight loss/decreased appetite, and enlarged spleen), positive rK39 test, and were living in VL-endemic areas. Compliance was the percentage of enrolled patients who received 21 daily injections over no more than 22 days. Efficacy was evaluated by initial clinical response, defined as resolution of fever and reduction of splenomegaly at end of treatment, and final clinical response, defined as the absence of new clinical signs and symptoms of VL 6 months after end of treatment. Safety was assessed by evaluation of adverse events.

Principal Findings

A total of 120 subjects (49% pediatric) were enrolled. Treatment compliance was 98.3%. Initial clinical response in the Intent-to-Treat population was 98.3%, and final clinical response 6 months after end of treatment was 94.2%. Of the 119 subjects who received ≥1 dose of PMIM, 28.6% reported at least one adverse event. Injection site pain was the most commonly reported adverse event. Reversible renal impairment and/or hearing loss were reported in 2 subjects.

Conclusions/Significance

PMIM was an effective and safe treatment for VL in Bangladesh. The short treatment duration and lower cost of PMIM compared with other treatment options may make this drug a preferred treatment to be investigated as part of a combination therapy regimen. This study supports the registration of PMIM for use in government health facilities in Bangladesh.

Trial Registration

ClinicalTrials.gov identifier: NCT01328457     

Risk Factors for Visceral Leishmaniasis Relapse in Immunocompetent Patients following Treatment with 20 mg/kg Liposomal Amphotericin B (Ambisome) in Bihar,India

Background

A proportion of all immunocompetent patients treated for visceral leishmaniasis (VL) are known to relapse; however, the risk factors for relapse are not well understood. With the support of the Rajendra Memorial Research Institute (RMRI), Médecins Sans Frontières (MSF) implemented a program in Bihar, India, using intravenous liposomal amphotericin B (Ambisome) as a first-line treatment for VL. The aim of this study was to identify risk factors for VL relapse by examining the characteristics of immunocompetent patients who relapsed following this regimen.

Methods and Principal Findings

This is an observational retrospective cohort study of all VL patients treated by the MSF program from July 2007 to August 2012. Intravenous Ambisome was administered to 8749 patients with VL in four doses of 5 mg/kg (for a total dose of 20 mg/kg) over 4–10 days, depending on the severity of disease. Out of 8588 patients not known to be HIV-positive, 8537 (99.4%) were discharged as initial cures, 24 (0.3%) defaulted, and 27 (0.3%) died during or immediately after treatment. In total, 1.4% (n = 119) of the initial cured patients re-attended the programme with parasitologically confirmed VL relapse, with a median time to relapse of 10.1 months. Male sex, age <5 years and ≥45 years, a decrease in spleen size at time of discharge of ≤0.5 cm/day, and a shorter duration of symptoms prior to seeking treatment were significantly associated with relapse. Spleen size at admission, hemoglobin level, nutritional status, and previous history of relapse were not associated with relapse.

Conclusions

This is the largest cohort of VL patients treated with Ambisome worldwide. The risk factors for relapse included male sex, age <5 and ≥45 years, a smaller decrease in splenomegaly at discharge, and a shorter duration of symptoms prior to seeking treatment. The majority of relapses in this cohort occurred 6–12 months following treatment, suggesting that a 1-year follow-up is appropriate in future studies.     

Efficacy and Safety of Amphotericin B Emulsion versus Liposomal Formulation in Indian Patients with Visceral Leishmaniasis: A Randomized,Open-Label Study

Background

India is home to 60% of the total global visceral leishmaniasis (VL) population. Use of long-term oral (e.g. miltefosine) and parenteral drugs, considered the mainstay for treatment of VL, is now faced with increased resistance, decreased efficacy, low compliance and safety issues. The authors evaluated the efficacy and safety of an alternate treatment option, i.e. single infusion of preformed amphotericin B (AmB) lipid emulsion (ABLE) in comparison with that of liposomal formulation (LAmB).

Methods

In this multicentric, open-label study, 500 patients with VL were randomly assigned in a 3∶1 ratio to receive 15 mg/kg single infusion of either ABLE (N = 376) or LAmB (N = 124). Initial cure (Day 30/45), clinical improvement (Day 30) and long term definitive cure (Day 180) were assessed.

Findings

A total of 326 (86.7%) patients in the ABLE group and 122 (98.4%) patients in the LAmB group completed the study. Initial cure was achieved by 95.9% of patients in the ABLE group compared to 100% in the LAmB group (p = 0.028; 95% CI: −0.0663, −0.0150). Clinical improvement was comparable between treatments (ABLE: 98.9% vs. LAmB: 98.4%). Definitive cure was achieved in 85.9% with ABLE compared to 98.4% with LAmB. Infusion-related pyrexia (37.2% vs. 32.3%) and chills (18.4% vs. 18.5%) were comparable between ABLE and LAmB, respectively. Treatment-related serious adverse events were fewer in ABLE (0.3%) compared to LAmB (1.6%). Two deaths occurred in the ABLE group, of which one was probably related to the study drug. Nephrotoxicity and hepatotoxicity was not observed in either group.

Conclusions

ABLE 15 mg/kg single infusion had favorable efficacy and was well tolerated. Considering the demographic profile of the population in this region, a single dose treatment offers advantages in terms of compliance, cost and applicability.

Trial Registration

www.clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00876824","term_id":"NCT00876824"}}NCT00876824     

Surface-Modified Liposomal Formulation of Amphotericin B: <Emphasis Type="Italic">In vitro</Emphasis> Evaluation of Potential Against Visceral Leishmaniasis

Surface modification of liposomes with targeting ligands is known to improve the efficacy with reduced untoward effects in treating infective diseases like visceral leishmaniasis (VL). In the present study, modified ligand (ML), designed by modifying polysaccharide with a long chain lipid was incorporated in liposomes with the objective to target amphotericin B (Amp B) to reticuloendothelial system and macrophages. Conventional liposomes (CL) and surface modified liposomes (SML) were characterized for size, shape, and entrapment efficiency (E.E.). Amp B SML with 3% w/w of ML retained the vesicular nature with particle size of ～205 nm, E.E. of ～95% and good stability. SML showed increased cellular uptake in RAW 264.7 cells which could be attributed to receptor-mediated endocytosis. Compared to Amp B solution, Amp B liposomes exhibited tenfold increased safety in vitro in RAW 264.7 and J774A.1 cell lines. Pharmacokinetics and biodistribution studies revealed high t _1/2, area under the curve (AUC)_0–24, reduced clearance and prolonged retention in liver and spleen with Amp B SML compared to other formulations. In promastigote and amastigote models, Amp B SML showed enhanced performance with low 50% inhibitory concentration (IC₅₀) compared to Amp B solution and Amp B CL. Thus, due to the targeting ability of ML, SML has the potential to achieve enhanced efficacy in treating VL.     

Neoglycoproteins as Carriers for Receptor-Mediated Drug Targeting in the Treatment of Experimental Visceral Leishmaniasis

ABSTRACT. Methotrexate (MTX) coupled to mannosyl bovine serum albumin (BSA) was taken up efficiently through the mannosyl receptors present on macrophages. Binding experiments indicate that conjugation does not decrease the affinity of the neoglycoprotein for its cell surface receptor. The drug conjugate eliminated intracellular amastigotes of Leishmania donovani in mouse peritoneal macrophages about 100 times more efficiently than free drug on the basis of 50% inhibitory dose. Inhibitory effect of the conjugate was directly proportional to the density of sugar on the neoglycoprotein carrier. Colchicine and monensin, inhibitors of receptor-mediated endocytosis, can prevent the leishmanicidal effect of the conjugate. Antileishmanial effect of the conjugate can be competitively inhibited by mannose-BSA and mannan. In a murine model of experimental visceral leishmaniasis the drug conjugate reduced the spleen parasite burden by more than 85% in a 30-day model whereas the same concentration of free drug caused little effect. These results indicate that MTX-neoglycoprotein conjugate binds specifically to macrophages, and is internalized and degraded in lysosomes releasing the active drug to act on Leishmania parasites. These results also represent the potential for a general approach to intracellular targeting of clinical agents for macrophage-associated disorders.     

Visceral Leishmaniasis and Immunocompromise as a Risk Factor for the Development of Visceral Leishmaniasis: A Changing Pattern at The Hospital for Tropical Diseases,London

Visceral leishmaniasis (VL) is a parasitic protozoon infection caused by the Leishmania species and transmitted by sandflies. Patients acquire VL in five main tropical areas and the Mediterranean basin, and clinicians from non-endemic regions regularly see infected patients. We describe the population presenting with VL to the Hospital for Tropical Diseases (HTD), London and identify risk factors for developing VL.

Methods and Principal Findings

A retrospective study of imported VL to the HTD, London including patients diagnosed and/or managed at the HTD between January 1995 and July 2013. We analyse patient demographics, risk factors for developing VL, diagnosis, investigation, management and outcome. Twenty-eight patients were treated for VL at the HTD over an 18 year period. The median age at VL diagnosis was 44 years (range 4–87 years) with a male to female ratio of 2:1. Most patients were British and acquired their infection in the Mediterranean basin. The median time from first symptom to diagnosis was six months with a range of 1–12 months and diagnosis included microscopic visualisation of leishmania amastigotes, positive serological tests (DAT and k39 antibody) or identification of leishmania DNA. Nineteen patients had some form of immunocompromise and this has increased proportionally compared to previously described data. Within the immunocompromised group, the ratio of those with autoimmune disease has increased. Immunocompromised patients had lower cure and higher relapse rates.

Conclusions

The rise of VL in patients with immunocompromise secondary to autoimmune disease on immunomodulatory drugs presents new diagnostic and therapeutic challenges. VL should be a differential diagnosis in immunocompromised patients with pyrexia of unknown origin returning from travel in leishmania endemic areas.     

Immucillins ImmA and ImmH Are Effective and Non-toxic in the Treatment of Experimental Visceral Leishmaniasis

Background

Immucillins ImmA (IA), ImmH (IH) and SerMe-ImmH (SMIH) are synthetic deazapurine nucleoside analogues that inhibit Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis multiplication in vitro without macrophage toxicity. Immucillins are compared to the Glucantime standard drug in the chemotherapy of Leishmania (L.) infantum chagasi infection in mice and hamsters. These agents are tested for toxicity and immune system response.

Methodology/Principal Findings

BALB/c mice were infected with 10⁷ amastigotes, treated with IA, IH, SMIH or Glucantime (2.5mg/kg/day) and monitored for clinical variables, parasite load, antibody levels and splenocyte IFN-γ, TNF-α, and IL-10 expression. Cytokines and CD4+, CD8+ and CD19+ lymphocyte frequencies were assessed in uninfected controls and in response to immucillins. Urea, creatinine, GOT and GPT levels were monitored in sera. Anti-Leishmania-specific IgG1 antibodies (anti-NH36) increased in untreated animals. IgG2a response, high levels of IFN-γ, TNF-α and lower levels of IL-10 were detected in mice treated with the immucillins and Glucantime. Immucillins permitted normal weight gain, prevented hepato-splenomegaly and cleared the parasite infection (85–89%) without renal and hepatic toxicity. Immucillins promoted 35% lower secretion of IFN-γ in uninfected controls than in infected mice. IA and IH increased the CD4+ T and CD19+ B cell frequencies. SMIH increased only the proportion of CD-19 B cells. IA and IH also cured infected hamsters with lower toxicity than Glucantime.

Conclusions/Significance

Immucillins IA, IH and SMIH were effective in treating leishmaniasis in mice. In hamsters, IA and IH were also effective. The highest therapeutic efficacy was obtained with IA, possibly due to its induction of a TH1 immune response. Low immucillin doses were required and showed no toxicity. Our results disclose the potential use of IA and IH in the therapy of visceral leishmaniasis.     

Successful Treatment of Breakthrough Trichosporon asahii Fungemia by the Combination Therapy of Fluconazole and Liposomal Amphotericin B in a Patient with Follicular Lymphoma

Mycopathologia - Invasive trichosporonosis is a rare and lethal fungal infection that occurs in immunocompromised patients. Breakthrough trichosporonosis can occur in patients treated with...     

Isavuconazole and Liposomal Amphotericin B as Successful Combination Therapy of Refractory Invasive Candidiasis in a Liver Transplant Recipient: A Case Report and Literature Review

Invasive fungal infections in liver transplant recipients are associated with elevated morbidity and mortality and pose a challenge to the treating physicians. Despite of lacking clinical data, the use of antifungal combination therapy is often considered to improve response rates in an immunocompromised patient population. We herein report a case of refractory invasive candidiasis in a liver transplant recipient treated successfully with a combination of isavuconazole und high-dose liposomal amphotericin B. The antimycotic combination treatment was able to clear a bloodstream infection with C. glabrata and led to regression of bilomas among tolerable side effects. The use of the above-mentioned antifungal combination therapy in a liver transplant recipient has not been reported previously. This case highlights the efficacy and safety of antifungal combination therapy in immunocompromised patients with refractory invasive candidiasis.

     

A comprehensive analysis of LACK (Leishmania homologue of receptors for activated C kinase) in the context of Visceral Leishmaniasis

The Leishmania homologue of activated C kinase (LACK) a known T cell epitope from soluble Leishmania antigens (SLA) that confers protection against Leishmania challenge. This antigen has been found to be highly conserved among Leishmania strains. LACK has been shown to be protective against L. donovani challenge. A comprehensive analysis of several LACK sequences was completed. The analysis shows a high level of conservation, lower variability and higher antigenicity in specific portions of the LACK protein. This information provides insights for the potential consideration of LACK as a putative candidate in the context of visceral Leishmaniasis vaccine target.     

Use of a Recombinant Cysteine Proteinase from Leishmania (Leishmania) infantum chagasi for the Immunotherapy of Canine Visceral Leishmaniasis

Background

A recombinant cysteine proteinase from Leishmania (Leishmania) infantum chagasi (rLdccys1) was previously shown to induce protective immune responses against murine and canine visceral leishmaniasis. These findings encouraged us to use rLdccys1 in the immunotherapy of naturally infected dogs from Teresina, Piauí, a region of high incidence of visceral leishmaniasis in Brazil.

Methodology/Principal Findings

Thirty naturally infected mongrel dogs displaying clinical signs of visceral leishmaniasis were randomly divided in three groups: one group received three doses of rLdccys1 in combination with the adjuvant Propionibacterium acnes at one month interval between each dose; a second group received three doses of P. acnes alone; a third group received saline. The main findings were: 1) dogs that received rLdccys1 with P. acnes did not display increase of the following clinical signs: weight loss, alopecia, onychogryphosis, cachexia, anorexia, apathy, skin lesions, hyperkeratosis, ocular secretion, and enlarged lymph nodes; they also exhibited a significant reduction in the spleen parasite load in comparison to the control dogs; 2) rLdccys1-treated dogs exhibited a significant delayed type cutaneous hypersensitivity elicited by the recombinant antigen, as well as high IgG2 serum titers and low IgG1 serum titers; sera from rLdccys1-treated dogs also contained high IFN-γ and low IL-10 concentrations; 3) control dogs exhibited all of the clinical signs of visceral leishmaniasis and had low serum IgG2 and IFN-γ levels and high concentrations of IgG1 and IL-10; 4) all of the dogs treated with rLdccys1 were alive 12 months after treatment, whereas dogs which received either saline or P. acnes alone died within 3 to 7 months.

Conclusions/Significance

These findings illustrate the potential use of rLdccys1 as an additional tool for the immunotherapy of canine visceral leishmaniasis and support further studies designed to improve the efficacy of this recombinant antigen for the treatment of this neglected disease.     

Evaluation of Amphotericin B and Chloramphenicol as Alternative Drugs for Treatment of Chytridiomycosis and Their Impacts on Innate Skin Defenses

Chytridiomycosis, an amphibian skin disease caused by the emerging fungal pathogen Batrachochytrium dendrobatidis, has been implicated in catastrophic global amphibian declines. The result is an alarming decrease in amphibian diversity that is a great concern for the scientific community. Clinical trials testing potential antifungal drugs are needed to identify alternative treatments for amphibians infected with this pathogen. In this study, we quantified the MICs of chloramphenicol (800 μg/ml), amphotericin B (0.8 to 1.6 μg/ml), and itraconazole (Sporanox) (20 ng/ml) against B. dendrobatidis. Both chloramphenicol and amphotericin B significantly reduced B. dendrobatidis infection in naturally infected southern leopard frogs (Rana [Lithobates] sphenocephala), although neither drug was capable of complete fungal clearance. Long-term exposure of R. sphenocephala to these drugs did not inhibit antimicrobial peptide (AMP) synthesis, indicating that neither drug is detrimental to this important innate skin defense. However, we observed that chloramphenicol, but not amphotericin B or itraconazole, inhibited the growth of multiple R. sphenocephala skin bacterial isolates in vitro at concentrations below the MIC against B. dendrobatidis. These results indicate that treatment with chloramphenicol might dramatically alter the protective natural skin microbiome when used as an antifungal agent. This study represents the first examination of the effects of alternative antifungal drug treatments on amphibian innate skin defenses, a crucial step to validating these treatments for practical applications.     

Association of Pro-Inflammatory Cytokines and Iron Regulatory Protein 2 (IRP2) with Leishmania Burden in Canine Visceral Leishmaniasis

Leishmania infantum infection in humans and dogs can evolve with a wide range of clinical presentations, varying from asymptomatic infections to visceral leishmaniasis. We hypothesized that the immune response elicited by L. infantum infection could modulate whether the host will remain asymptomatic or progress to disease. A total of 44 dogs naturally infected with L. infantum were studied. Leishmania burden was estimated in the blood and spleen by qPCR. The expression of IFN-γ, TNF-α, IL-10 and Iron Regulatory Protein 2 (IRP2) were determined in the spleen by quantitative PCR. Sera cytokines were evaluated by ELISA. Dogs were grouped in quartiles according parasite burden. Increased expression of IFN-γ and TNF-α was associated with reduced Leishmania burden, whereas increased IL-10 and IRP2 expressions were associated with higher Leishmania load. Increased plasma albumin and IFN-γ expression explained 22.8% of the decrease in parasite burden in the spleen. These data confirm that lower IFN-γ response and higher IL-10 correlated with increased parasite load and severity of the visceral leishmaniasis in dogs. The balance between the branches of immune response and the intracellular iron availability could determine, in part, the course of Leishmania infection.