Investigational new drugs for the treatment of leishmaniasis.

INTRODUCTION: Over the past 20 years, significant progress has been made in anti-leishmanial therapy. Three new drugs/formulations are available for the treatment of various forms of leishmaniasis, namely oral miltefosine, paromomycin and liposomal amphotericin B. However, these advances in drug development have added considerable complexity for clinicians including toxicity, emergence of resistance and decreased sensitivity of available drugs. The development of newer drugs with less toxicity and more efficacy is urgently needed. AREAS COVERED: This review comprehensively examines the latest developments and current status of antileishmanial drugs for the treatment of leishmaniasis across the world. Several new investigational drugs that showed anti-leishmanial activity under in vitro or in vivo conditions and either underwent the phase-I/II clinical trials or are on the verge of entering the trials were reviewed. We also delve into the challenges of drug resistance and discuss the emergence of new and effective antileishmanial compounds. EXPERT OPINION: The available treatments for leishmaniasis are limited in number, toxic, expensive, and demand extensive healthcare resources. Every available antileishmanial drug is associated with several disadvantages, such as drug resistance and toxicity or high cost. Miltefosine is potentially teratogenic. New antileishmanial drugs/treatment modalities are sorely needed for expanding future treatment options.

Development and deployment of a solid oral amphotericin B dosage form to treat visceral leishmaniasis within a pediatric population.

Visceral leishmaniasis (VL) is a severe and potentially fatal infection, with over 90% of reported cases occurring in East African countries including Chad, Djibouti, Eritrea, Ethiopia, Kenya, Somalia, South Sudan, Sudan, and Uganda, affecting mainly impoverished individuals, and creating a significant economic burden. Currently, the intravenous single-dose liposomal amphotericin B is the first choice for the treatment of VL. Recently, WHO and DNDi have suggested a combination of intravenous liposomal amphotericin B and oral miltefosine as a potential approach to treat VL. However, miltefosine availability is uncertain, and its side effects frequently cause treatment to be discontinued. Furthermore, due to the difficult route of liposomal amphotericin B administration by intravenous infusion, the lack of formulation's tropical stability, accessibility, injection toxicity, and cost have prevented this injectable formulation of amphotericin B from reaching the most infected populations, particularly the pediatric population. To solve this problem, the development of a solid oral amphotericin B formulation that is cost-effective, safe, tropically stable, and easy to swallow, making it more accessible to children, particularly in rural communities having limited access to medical clinics or trained healthcare professionals is imperative. This viewpoint will discuss the opportunities and challenges of developing an oral amphotericin B formulation for a pediatric population.

Helminth derivative tuftsin-phopshorylcholine to treat autoimmunity.

Autoimmune diseases (AIDs) affect 5 to 10% of the population. There are more than ∼100 different autoimmune diseases. The AIDs are one of the top 10 causes of death in women under 65; 2nd highest cause of chronic illness; top cause of morbidity in women in the US. The NIH estimates annual direct healthcare costs for autoimmune diseases about $100 billion, in comparison, with cancers investment of $57 billion, heart and stroke cost of $200 billion. The current treatments for autoimmune diseases encompasses: steroids, chemotherapy, immunosuppressants, biological drugs, disease specific drugs (like acethylcholine-estherase for myasthenia gravis). The treatments for autooimmune diseases supress the patient immune network, which leads the patients to be more susceptible to infections. Hence, there is a need to develop immunomodulatory small molecules with minimal side effects to treat autoimmune diseases. The helminths developed secreting compounds which modulate the human defense pathways in order to develop tolerance and survive in the host environment. We have imitated the immunomodulatory activity of the helminth by using a derivative of the helminth secretory molecule. A bi-functional small molecule -tuftsin (T)-phosphorylcholine (PC), coined as TPC, was constructed. This chimeric molecule showed its immunomodulatory activity in 4 murine models of autoimmune diseases, attenuating the clinical score and the inflammatory response by immunomodutating the host immune system. Ex-vivo in human peripheral blood mononuclear cells (PBMCs) and biopsies originated from arteries of patients with giant cell arteritis. This paper decipher the mode of action of TPC immunomodulatory activity. Our data propose the potential for this small molecule to be a novel therapy for patients with autoimmune diseases.

Milteforan, a promising veterinary commercial product against feline sporotrichosis.

Sporotrichosis, the cutaneous mycosis most commonly reported in Latin America, is caused by the Sporothrix clinical clade species, including Sporothrix brasiliensis and Sporothrix schenckii sensu stricto. Due to its zoonotic transmission in Brazil, S. brasiliensis represents a significant health threat to humans and domestic animals. Itraconazole, terbinafine, and amphotericin B are the most used antifungals for treating sporotrichosis. However, many strains of S. brasiliensis and S. schenckii have shown resistance to these agents, highlighting the importance of finding new therapeutic options. Here, we demonstrate that milteforan, a commercial veterinary product against dog leishmaniasis, whose active principle is miltefosine, is a possible therapeutic alternative for the treatment of sporotrichosis, as observed by its fungicidal activity in vitro against different strains of S. brasiliensis and S. schenckii. Fluorescent miltefosine localizes to the Sporothrix cell membrane and mitochondria and causes cell death through increased permeabilization. Milteforan decreases S. brasiliensis fungal burden in A549 pulmonary cells and bone marrow-derived macrophages and also has an immunomodulatory effect by decreasing TNF-α, IL-6, and IL-10 production. Our results suggest milteforan as a possible alternative to treat feline sporotrichosis. IMPORTANCE: Sporotrichosis is an endemic disease in Latin America caused by different species of Sporothrix. This fungus can infect domestic animals, mainly cats and eventually dogs, as well as humans. Few drugs are available to treat this disease, such as itraconazole, terbinafine, and amphotericin B, but resistance to these agents has risen in the last few years. Alternative new therapeutic options to treat sporotrichosis are essential. Here, we propose milteforan, a commercial veterinary product against dog leishmaniasis, whose active principle is miltefosine, as a possible therapeutic alternative for treating sporotrichosis. Milteforan decreases S. brasiliensis fungal burden in human and mouse cells and has an immunomodulatory effect by decreasing several cytokine production.

The nexus between Leishmania & HIV: Debilitating host immunity and Hastening Comorbid disease burden.

The scintillating association between Leishmania and HIV has contributed exceptionally towards expansion of Visceral Leishmaniasis (VL) with Acquired Immunodeficiency Syndrome (AIDS). The co-infection poses a grievous threat to elimination of VL and containment of Human Immunodeficiency Virus (HIV). When coinfected, Leishmania and HIV complement each other's proliferation and survival by inducing immunesenescence, T cell fatigue and exhaustion. Antigen presentation is lost, co-stimulatory molecules are diminished whereas co-inhibitory molecules such as CTLA-4, TIGIT, LAG-3 etc. are upregulated to ensure a Th2-baised immune environment. As a consequence, Leishmania-HIV coinfection causes poor outcomes, inflates the spread of Leishmania parasites, enhances the severity of side-effects to drugs, as well as escalate the probability of treatment failure and mortality. What makes control extremely strenuous is that there are frequent episodes of VL relapse with no prognostic markers, no standard immunophenotype(s) and appearance of atypical clinical symptoms. Thus, a standard therapeutic regimen has been difficult to develop and treatment is majorly dependent upon a combination of liposomal Amphotericin B and Miltefosine, a therapy that is expensive and capable of causing drastic side-effects in recipients. As World Health Organization is committed to eliminate both VL and HIV in due course of future, the existing therapeutic interventions require advancements to grapple and overcome this hazardous co-infection. In this context, an overview of HIV-VL co-infection, immunopathology of HIV and Leishmania co-inhabitance, available therapeutic options and their limitations in the treatment of co-infection are discussed in-depth.

Cost-effectiveness study of therapeutic approaches for mucosal leishmaniasis.

This study aimed to estimate the cost-effectiveness of four therapeutic approaches available for mucosal leishmaniasis in Brazil: miltefosine, meglumine antimoniate, combined with and without pentoxifylline, and liposomal amphotericin B. The perspective adopted was that of the Brazilian Unified National Health System (SUS). The outcome of interest was "cured patient", which was analyzed using a decision tree model. Estimates of direct costs and effectiveness were obtained from the scientific literature. Meglumine antimoniate alone was the base comparator strategy; liposomal amphotericin B showed an incremental cost-effectiveness ratio (ICER) of USD 7,409.13 per cured patient, and the combination of meglumine antimoniate with pentoxifylline presented an ICER of USD 85.13. Miltefosine was absolutely dominated, with higher cost and similar effectiveness when compared to meglumine antimoniate. Sensitivity analyses, varying the cost by ±25%, did not change the results. However, when the cost of miltefosine was estimated at less than USD 171.23, this strategy was dominant over meglumine antimoniate alone. The results confirm that treatment with liposomal amphotericin B remains the option with the highest ICER among the approaches analyzed. Miltefosine may be cost-effective based on the variation in the acquisition price, which deserves attention because it is the only available oral option. The non-accounting of other aspects prevent the use of these results immediately to support decision-making, but they point out the need to negotiate the prices of drugs available for mucosal leishmaniasis and indicates the need of encouraging technology transfer or other actions aimed at expanding the performance of the Brazilian national industrial complex.

Differences in the Cellular Immune Response during and after Treatment of Sudanese Patients with Post-kala-azar Dermal Leishmaniasis, and Possible Implications for Outcome.

BACKGROUND: The host cellular immune response associated with two treatments for post-kala-azar dermal leishmaniasis (PKDL) - paromomycin plus miltefosine (Arm 1), and liposomal amphotericin B plus miltefosine (Arm 2) - was examined in Sudanese patients before treatment (D0), at the end of treatment (D42), and during the post-treatment period (D180). METHODS: Whole blood samples were stimulated with soluble Leishmania antigen for 24 h (whole blood assay [WBA]) and the concentrations of Th1/Th2/Th17-associated cytokines, IP-10, PDL-1 and granzyme B were determined. RESULTS: The Arm 1 treatment (98.2% cure rate) induced a Th1/Th2/Th17 response, while the Arm 2 treatment (80% cure rate) induced a Th1/Th2 response. Five Arm 2 patients relapsed and showed lower IFN-γ, TNF and IL-1ß concentrations at D0 than non-relapsers in this Arm. In patients with low-IFN-γ-production at D0, Arm 1 treatment led to a better host immune response and clinical outcome than Arm 2 treatment. CONCLUSIONS: A Th1/Th2/Th17 response was associated with a higher cure rate. Patients with low IFN-γ, TNF and IL-1ß before treatment are more likely to relapse if they undergo Arm 2-type treatment. Determining IFN-γ, TNF and IL-10 levels prior to treatment could help predict patients at higher risk of relapse/recovery from PKDL. TRIAL REGISTRATION: ClinicalTrials.gov NCT03399955, Registered 17 January 2018, https://clinicaltrials.gov/study/ NCT03399955.

Ophthalmic adverse effects of miltefosine in the treatment of leishmaniasis: a systematic review.

OBJECTIVE: Miltefosine stands as the sole oral medication approved for the treatment of leishmaniasis. The appearance of severe ophthalmic toxicities induced by miltefosine in the context of leishmaniasis treatment is a matter of significant concern. The main objective of this study is to present a comprehensive summary of the ophthalmic adverse effects associated with miltefosine when used in the treatment of leishmaniasis. METHODS: A systematic search was performed on PubMed, ScienceDirect, Embase, Scopus, and Google Scholar, covering articles from inception up to June 2023, without language restrictions, to identify relevant studies documenting ocular toxicity following miltefosine treatment for leishmaniasis. RESULTS: A total of eight studies involving 31 leishmaniasis patients who developed ocular toxicities while undergoing miltefosine treatment were included in the analysis. These studies were conducted in various regions, with five originating from India, two from Bangladesh, and one from Nepal. Patients presented a spectrum of ophthalmic complications, including uveitis, keratitis, scleritis, and Mooren's ulcer. Commonly reported symptoms included pain, redness, excessive tearing, partial vision impairment, permanent blindness, light sensitivity, and the appearance of white spots on the eye. On average, patients received miltefosine treatment for a duration of 47 days before experiencing the onset of ocular problems. It is important to note that the risk of ocular toxicities increases with prolonged use of miltefosine. CONCLUSIONS: Therefore, to mitigate the potential for irreversible damage to the eyes, it is imperative that all individuals undergoing miltefosine therapy undergo regular eye examinations.

Efficacy assessment of miltefosine and curcumin against Clonorchis sinensis infection.

Praziquantel (PZQ) is currently the only approved drug for treating clonorchiasis, but its poor efficacy against Clonorchis sinensis larvae has highlighted the need to develop newer drugs. In this study, to address this challenge, we investigated the anti-parasitic efficacy of miltefosine (MLT), curcumin (CUR), and PZQ against C. sinensis metacercariae (CsMC), newly excysted juvenile worms (CsNEJs), and adults. Larvicidal effects of MLT and CUR surpassed those elicited by PZQ in vitro. These two drugs exerted their effect against both CsMC and CsNEJs in a dose- and time-dependent manner. To confirm the effect of these drugs in vivo, Syrian golden hamsters were orally infected with 100 CsMC and subsequently treated with MLT, CUR, or PZQ at 1 and 4 weeks post-infection (wpi). MLT and CUR reduced the worm recoveries at 1 and 4 wpi, indicating that these drugs were efficacious against both larvae and adult C. sinensis. PZQ was only efficacious against adult worms. Interestingly, both MLT and CUR showed lower levels of C. sinensis-specific IgG responses than the infection control group, implying that worm burden and bile IgG responses could be correlated. These results indicate that MLT and CUR are efficacious against both larval and adult stages of C. sinensis, thereby highlighting their potential for further development as alternative therapeutic options for clonorchiasis.

Photodynamic therapy offers a novel approach to managing miltefosine-resistant cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a neglected disease caused by Leishmania parasites. The oral drug miltefosine is effective, but there is a growing problem of drug resistance, which has led to increasing treatment failure rates and relapse of infections. Photodynamic therapy (PDT) combines a light source and a photoactive drug to promote cell death by oxidative stress. Although PDT is effective against several pathogens, its use against drug-resistant Leishmania parasites remains unexplored. Herein, we investigated the potential of organic light-emitting diodes (OLEDs) as wearable light sources, which would enable at-home use or ambulatory treatment of CL. We also assessed its impact on combating miltefosine resistance in Leishmania amazonensis-induced CL in mice. The in vitro activity of OLEDs combined with 1,9-dimethyl-methylene blue (DMMB) (OLED-PDT) was evaluated against wild-type and miltefosine-resistant L. amazonensis strains in promastigote (EC50 = 0.034 µM for both strains) and amastigote forms (EC50 = 0.052 µM and 0.077 µM, respectively). Cytotoxicity in macrophages and fibroblasts was also evaluated. In vivo, we investigated the potential of OLED-PDT in combination with miltefosine using different protocols. Our results demonstrate that OLED-PDT is effective in killing both strains of L. amazonensis by increasing reactive oxygen species and stimulating nitric oxide production. Moreover, OLED-PDT showed great antileishmanial activity in vivo, allowing the reduction of miltefosine dose by half in infected mice using a light dose of 7.8 J/cm2 and 15 µM DMMB concentration. In conclusion, OLED-PDT emerges as a new avenue for at-home care and allows a combination therapy to overcome drug resistance in cutaneous leishmaniasis.

A phase II, non-comparative randomised trial of two treatments involving liposomal amphotericin B and miltefosine for post-kala-azar dermal leishmaniasis in India and Bangladesh.

BACKGROUND: In Southeast Asia, treatment is recommended for all patients with post-kala-azar dermal leishmaniasis (PKDL). Adherence to the first-line regimen, twelve weeks of miltefosine (MF), is low and ocular toxicity has been observed with this exposure period. We assessed the safety and efficacy of two shorter-course treatments: liposomal amphotericin B (LAmB) alone and combined with MF. METHODOLOGY/PRINCIPAL FINDINGS: An open-label, phase II, randomized, parallel-arm, non-comparative trial was conducted in patients with parasitologically confirmed PKDL, 6 to ≤60 years. Patients were assigned to 20 mg/kg LAmB (total dose, in five injections over 15 days) alone or combined with allometric MF (3 weeks). The primary endpoint was definitive cure at 12 months, defined as complete resolution of papular and nodular lesions and >80% re-pigmentation of macular lesions. Definitive cure at 24 months was a secondary efficacy endpoint. 118/126 patients completed the trial. Definitive cure at 12 months was observed in 29% (18/63) patients receiving LAmB and 30% (19/63) receiving LAmB/MF (mITT), increasing to 58% and 66%, respectively, at 24 months. Most lesions had resolved/improved at 12 and 24 months for patients receiving LAmB (90%, 83%) and LAmB/MF (85%, 88%) by qualitative assessment. One death, unrelated to study drugs, was reported; no study drug-related serious adverse events were observed. The most frequent adverse drug reactions were MF-related vomiting and nausea, and LAmB-related hypokalaemia and infusion reactions. Most adverse events were mild; no ocular adverse events occurred. CONCLUSIONS/SIGNIFICANCE: Both regimens are suitably safe and efficacious alternatives to long-course MF for PKDL in South Asia. TRIAL REGISTRATION: CTRI/2017/04/008421.

[Complex cutaneous leishmaniasis with bone involvement]. / Komplexe kutane Leishmaniose mit ossärer Beteiligung.

We report an immunocompromised patient with a complex cutaneous leishmaniasis (CL) who suffered from singular bone involvement of the little left toe due to Leishmania (L.) infantum infection. The diagnosis was confirmed by positive polymerase chain reaction (PCR) testing in skin and bone tissue. The patient was successfully treated with miltefosine. In immunocompromised patients with CL, extracutaneous manifestations should always be ruled out. This is the first case report describing osseous involvement in CL due to Leishmania infantum.

Skin pharmacokinetics of miltefosine in the treatment of post-kala-azar dermal leishmaniasis in South Asia.

INTRODUCTION: Post-kala-azar dermal leishmaniasis (PKDL) arises as a dermal complication following a visceral leishmaniasis (VL) infection. Current treatment options for PKDL are unsatisfactory, and there is a knowledge gap regarding the distribution of antileishmanial compounds within human skin. The present study investigated the skin distribution of miltefosine in PKDL patients, with the aim to improve the understanding of the pharmacokinetics at the skin target site in PKDL. METHODS: Fifty-two PKDL patients underwent treatment with liposomal amphotericin B (20 mg/kg) plus miltefosine (allometric dosing) for 21 days. Plasma concentrations of miltefosine were measured on study days 8, 15, 22 and 30, while a punch skin biopsy was taken on day 22. A physiologically based pharmacokinetic (PBPK) model was developed to evaluate the distribution of miltefosine into the skin. RESULTS: Following the allometric weight-based dosing regimen, median miltefosine concentrations on day 22 were 43.73 µg/g (IQR: 21.94-60.65 µg/g) in skin and 33.29 µg/mL (IQR: 25.9-42.58 µg/mL) in plasma. The median individual concentration ratio of skin to plasma was 1.19 (IQR: 0.79-1.9). In 87% (45/52) of patients, skin exposure was above the suggested EC90 PK target of 10.6 mg/L associated with in vitro susceptibility. Simulations indicated that the residence time of miltefosine in the skin would be more than 2-fold longer than in plasma, estimated by a mean residence time of 604 versus 266 hours, respectively. CONCLUSION: This study provides the first accurate measurements of miltefosine penetration into the skin, demonstrating substantial exposure and prolonged retention of miltefosine within the skin. These findings support the use of miltefosine in cutaneous manifestations of leishmaniasis. In combination with parasitological and clinical data, these results are critical for the future optimization of combination therapies with miltefosine in the treatment of PKDL.

Antimony susceptible Leishmania donovani: evidence from in vitro drug susceptibility of parasites isolated from patients of post-kala-azar dermal leishmaniasis in pre- and post-miltefosine era.

Post-kala-azar dermal leishmaniasis (PKDL) patients are a key source of Leishmania donovani parasites, hindering the goal of eliminating visceral leishmaniasis (VL). Monitoring treatment response and parasite susceptibility is essential due to increasing drug resistance. We assessed the drug susceptibility of PKDL isolates (n = 18) from pre-miltefosine (MIL) era (1997-2004) with isolates (n = 16) from the post-miltefosine era (2010-2019) and post-miltefosine treatment relapse isolates (n = 5) towards miltefosine and amphotericin B (AmB) at promastigote stage and towards sodium antimony gluconate (SAG) at amastigote stage. PKDL isolates were examined for mutation in gene-encoding AQP1 transporter, C26882T mutation on chromosome 24, and miltefosine-transporter (MT). PKDL isolates from the post-miltefosine era were significantly more susceptible to SAG than SAG-resistant isolates from the pre-miltefosine era (P = 0.0002). There was no significant difference in the susceptibility of parasites to miltefosine between pre- and post-miltefosine era isolates. The susceptibility of PKDL isolates towards AmB remained unchanged between the pre- and post-miltefosine era. However, the post-miltefosine era isolates had a higher IC50 value towards AmB compared with PKDL relapse isolates. We did not find any association between AQP1 gene sequence variation and susceptibility to SAG, or between miltefosine susceptibility and single nucleotide polymorphisms (SNPs in the MT gene. This study demonstrates that recent isolates of Leishmania have resumed susceptibility to antimonials in vitro. The study also offers significant insights into the intrinsic drug susceptibility of Leishmania parasites over the past two decades, covering the period before the introduction of miltefosine and after its extensive use. IMPORTANCE: Post-kala-azar dermal leishmaniasis (PKDL) patients, a key source of Leishmania donovani parasites, hinder eliminating visceral-leishmaniasis. Assessment of the susceptibility of PKDL isolates to antimony, miltefosine (MIL), and amphotericin-B indicated that recent isolates remain susceptible to antimony, enabling its use with other drugs for treating PKDL.

Successful treatment of recurrent visceral leishmaniasis relapse in an immunocompetent adult female with functional hypopituitarism in Bangladesh.

BACKGROUND: Currently available treatment options are mostly effective in achieving long-term cure in visceral leishmaniasis (VL) patients. However, there have been reports of recurrence of this illness in both immunosuppressed and immunocompetent patients. CASE PRESENTATION: We report the first case of recurrent VL relapse in a 19-year-old immunocompetent female with functional hypopituitarism (hypogonadotropic hypogonadism with central hypothyroidism) from Bangladesh, who has been treated three times previously with optimal dosage and duration- liposomal amphotericin B (LAmB) alone and in combination with miltefosine. We treated the patient successfully with a modified treatment regimen of 10 mg/kg body weight LAmB for two consecutive days along with oral miltefosine for seven days as loading dose. For secondary prophylaxis, the patient received 3 mg/kg body weight LAmB along with oral miltefosine for seven days monthly for five doses followed by hormonal replacement. The patient remained relapse free after 12 months of her treatment completion. CONCLUSION: In the absence of protective vaccines against Leishmania species and standard treatment regimen, this modified treatment regimen could help the management of recurrent relapse cases.

Unmasking the Mechanism behind Miltefosine: Revealing the Disruption of Intracellular Ca2+ Homeostasis as a Rational Therapeutic Target in Leishmaniasis and Chagas Disease.

Originally developed as a chemotherapeutic agent, miltefosine (hexadecylphosphocholine) is an inhibitor of phosphatidylcholine synthesis with proven antiparasitic effects. It is the only oral drug approved for the treatment of Leishmaniasis and American Trypanosomiasis (Chagas disease). Although its precise mechanisms are not yet fully understood, miltefosine exhibits broad-spectrum anti-parasitic effects primarily by disrupting the intracellular Ca2+ homeostasis of the parasites while sparing the human hosts. In addition to its inhibitory effects on phosphatidylcholine synthesis and cytochrome c oxidase, miltefosine has been found to affect the unique giant mitochondria and the acidocalcisomes of parasites. Both of these crucial organelles are involved in Ca2+ regulation. Furthermore, miltefosine has the ability to activate a specific parasite Ca2+ channel that responds to sphingosine, which is different to its L-type VGCC human ortholog. Here, we aimed to provide an overview of recent advancements of the anti-parasitic mechanisms of miltefosine. We also explored its multiple molecular targets and investigated how its pleiotropic effects translate into a rational therapeutic approach for patients afflicted by Leishmaniasis and American Trypanosomiasis. Notably, miltefosine's therapeutic effect extends beyond its impact on the parasite to also positively affect the host's immune system. These findings enhance our understanding on its multi-targeted mechanism of action. Overall, this review sheds light on the intricate molecular actions of miltefosine, highlighting its potential as a promising therapeutic option against these debilitating parasitic diseases.

Therapeutic success and failure in using miltefosine to treat dogs naturally infected with Leishmania infantum.

In urban environments, domestic dogs (Canis familiaris) are a major reservoir for the parasite Leishmania infantum. Miltefosine has been used as the standard treatment for canine visceral leishmaniasis in Brazil. However, therapeutic failures have been reported. In the present study, two dogs (CG03 and CG06) with a diagnosis of infection by L. infantum underwent two cycles of treatment with miltefosine (Milteforan™ - Virbac®). Analyses showed increases in the parasite load of both CG03 and CG06, even after treatment. The clinical score of CG03 dropped from 1 to 0 (after one round of treatment), such that this dog became asymptomatic. CG06 showed clinical worsening, such that its score increased from 1 to 2. After the second therapeutic round, the parasite load in CG03 was found to have decreased, but it was still higher than before drug treatment even though this dog was physically asymptomatic. There was no decrease in the parasite load in CG06 and there was clinical worsening. The clinical response of these dogs to the treatment differed, but the parasite load remained high in both cases, which poses a risk to public health, making it essential take measures to prevent the sandfly vector from accessing the dog.

The status of combination therapy for visceral leishmaniasis: an updated review.

For the past 15 years, trials of combination therapy options for visceral leishmaniasis have been conducted with the aim of identifying effective, and safe treatment regimens that were shorter than existing monotherapy regimens and could also prevent or delay the emergence of drug resistance. Although first-line treatment currently relies on combination therapy in east Africa, this is not true in Latin America owing to disappointing trial results, with lower than expected efficacy seen for the combination treatment group. By contrast, several effective combination therapy regimens have been identified through trials on the Indian subcontinent; yet, first-line therapy is still AmBisome monotherapy as the drug is part of a free donation programme and is highly effective in this region. Achieving a short all-oral combination treatment will require new chemical entities, several of which are currently under evaluation. Future studies should systematically include pharmacological substudies to ensure optimal dosing for all patient groups. To achieve maximal impact of new combination treatments, mechanisms to ensure drug availability and access after trials should be established. Enhancing the longevity of current and novel treatments will require effective systems for early detection of emerging drug resistance.