Dermal microdialysis: A method to determine drug levels in the skin of patients with Post kala-azar dermal leishmaniasis (PKDL).

OBJECTIVES: Post-kala-azar-dermal leishmaniasis (PKDL) is an infectious skin disease that occurs as sequela of visceral leishmaniasis (VL) and causes cutaneous lesions on the face and other exposed body parts. While the first-line drug miltefosine is typically used for 28 days to treat VL, 12 weeks of therapy is required for PKDL, highlighting the need to evaluate the extent of drug penetration at the dermal site of infection. In this proof-of-concept study, we demonstrate the use of a minimally invasive sampling technique called microdialysis to measure dermal drug exposure in a PKDL patient, providing a tool for the optimization of treatment regimens. METHODS AND MATERIALS: One PKDL patient receiving treatment with miltefosine (50 mg twice daily for 12 weeks) was recruited to this proof-of-concept study and consented to undergo dermal microdialysis. Briefly, a µDialysis Linear Catheter 66 for skin and muscle, a probe with a semi-permeable membrane, was inserted in the dermis. A perfusate (a drug-free physiological solution) was pumped through the probe at a low flow rate, allowing miltefosine present in the dermis to cross the membrane and be collected in the dialysates over time. Protein-free (dialysates) and total (blood and skin biopsies) drug concentrations were analysed using LC-MS/MS. RESULTS: and conclusions: Using microdialysis, protein-free miltefosine drug concentrations could be detected in the infected dermis over time (Cmax ≈ 450 ng/ml). This clinical proof-of-concept study thus illustrates the potential of dermal microdialysis as a minimally invasive alternative to invasive skin biopsies to quantify drug concentrations directly at the pharmacological site of action in PKDL.

Epidemiology and clinical features of cutaneous leishmaniasis in Khyber Pakhtunkhwa, Pakistan.

Current cross-sectional study was carried out between September 2019 to January 2020 at the Department of Pathology, Mardan Medical Complex (MMC), Mardan, and District Headquarter Hospital North Waziristan, Khyber Pakhtunkhwa (KP), Pakistan. The objectives of the current study were to determine the prevalence of leishmaniasis and its associated risk factors in selected districts of KP province, Pakistan. Altogether, three hundred and seventy-four (n=374) leishmaniosis patients were included in the current study. Skin specimen from the ulcer border were collected. The slides were stained by Giemsa stain and examined for the presence of amastigote. The prevalence of leishmania infected patients in different region of KP were as follows: North Waziristan region 53.7 (n=201) District Mardan 34.7% (n=130); District Nowshera 6.7% (n=25), District Swabi 1.1% (n=4) and other Districts i.e. Dir, Malakand, Buner and Bajawarr were 3.7% (n=14). The frequency of leishmaniasis were more in male and majority of the infected patients were in the age group of <10 years. Among n=374 patients 95.7% (n= 358) had cutaneous leishmaniasis while 3% (n= 11) had mucocutaneous type of infection and 1.3% (n= 5) patients had both cutaneous and mucocutaneous infection. Upon treatment by Sodium stibogluconate (SSG) 97% (n=362) showed clinical signs of complete or partial recovery of their skin lesions. Conclusively, highest incidence of leishmania infection occurred during short study period and majority of the cases showed positive response to treatment.

[Cutaneous leishmaniasis after renal transplantation: Report of 7 cases and review of the literature]. / Leishmaniose cutanée après transplantation rénale : à propos de 7 cas et revue de la littérature.

Leishmaniasis is a zoonosis acquired from the bite of a sandfly that introduces the amastigote forms of leishmania into the bloodstream. It is a frequent infection in the countries of the Mediterranean basin and in Tunisia, where it is rife in an endemo-epidemic mode. However, it is rare after kidney transplantation. It constitutes a challenge due to the diagnostic difficulty, the variability and the polymorphism of the clinical picture in immunocompromised patients. We report seven observations of cutaneous leishmaniasis after kidney transplantation through which we try to identify diagnostic and therapeutic difficulties.

A phase II multicenter randomized study to evaluate the safety and efficacy of combining thermotherapy and a short course of miltefosine for the treatment of uncomplicated cutaneous leishmaniasis in the New World.

BACKGROUND: Systemic pentavalent antimonials, mainly meglumine antimoniate, continue to be the first-choice drugs for treatment of cutaneous leishmaniasis (CL) despite their toxicity, difficulty of administration and high cost. In the search for therapeutic alternatives, combining two treatment interventions has emerged as a potential alternative to either reduce the use of antimonials with the associated toxicities, or to increase efficacy. Here, we report the results of a recently completed trial assessing the efficacy and safety of a combination of thermotherapy (TT) plus a short course of miltefosine (MLT) for the treatment of uncomplicated CL in Colombia and Peru. METHODS: A multicenter, randomized, evaluator-blinded, phase II, controled clinical trial was conducted. Adult volunteers with a parasitologically confirmed diagnosis of uncomplicated CL were randomly allocated to receive either a single session of TT or a combination of TT plus a short course of MLT (3 weeks). Therapeutic response outcomes and safety were assessed. RESULTS: 130 subjects were included in the study, of whom 64 were randomly assigned to the TT arm and 66 to the TT + MLT arm. Cure at 3 months' follow-up was achieved in 57.8% (n = 37) and 80.3% (n = 53) in the TT and TT + MLT groups, respectively, in the intention to treat analysis. The TT + MLT regimen was better that TT alone (p = 0.0055). The presence of vesicles at the site of heat application was the most common adverse event reported associated with the use of TT; while vomiting (31.8%) and elevation of liver enzymes (28.8%) were the most frequent adverse events reported associated with the use of MLT. CONCLUSION: The combination of TT plus a short course of MLT was shown to be significantly better than TT alone for the treatment of uncomplicated CL in the New World. TRIAL REGISTRATION: Registered in clinicaltrials.gov NCT02687971.

Transcriptomic landscape of skin lesions in cutaneous leishmaniasis reveals a strong CD8+ T cell immunosenescence signature linked to immunopathology.

The severity of lesions that develop in patients infected by Leishmania braziliensis is mainly associated with a highly cytotoxic and inflammatory cutaneous environment. Recently, we demonstrated that senescent T and NK cells play a role in the establishment and maintenance of this tissue inflammation. Here, we extended those findings using transcriptomic analyses that demonstrate a strong co-induction of senescence and pro-inflammatory gene signatures in cutaneous leishmaniasis (CL) lesions. The senescence-associated signature was characterized by marked expression of key genes such as ATM, Sestrin 2, p16, p21 and p38. The cell type identification from deconvolution of bulk sequencing data showed that the senescence signature was linked with CD8+ effector memory and TEMRA subsets and also senescent NK cells. A key observation was that the senescence markers in the skin lesions are age-independent of patients and were correlated with lesion size. Moreover, a striking expression of the senescence-associated secretory phenotype (SASP), pro-inflammatory cytokine and chemokines genes was found within lesions that were most strongly associated with the senescent CD8 TEMRA subset. Collectively, our results confirm that there is a senescence transcriptomic signature in CL lesions and supports the hypothesis that lesional senescent cells have a major role in mediating immunopathology of the disease.

Impairment of effector molecules response in diabetes induces susceptibility to Leishmania amazonensis infection.

Type 2 Diabetes is a chronic disease resulting from insulin dysfunction that triggers a low-grade inflammatory state and immune impairment. Leishmaniasis is an infectious disease characterized by chronic inflammation resulted from the parasite's immunomodulation ability. Thus, due to the delicate immune balance required in the combat and resistance to Leishmania infection and the chronic deregulation of the inflammatory response observed in type 2 diabetes, we evaluated the response of PBMC from diabetic patients to in vitro Leishmania amazonensis infection. For that, peripheral blood was collected from 25 diabetic patients and 25 healthy controls matched for age for cells extraction and subsequent experimental infection for 2 or 24 h and analyzed for phagocytic and leishmanicidal capacity by optical microscopy, oxidative stress by GSSG generation, labeling of intracellular mediators by enzyme-Linked immunosorbent assay, and cytokines measurement with cytometric beads array technique. We found that the diabetic group had a higher percentage of infected cells and a greater number of amastigotes per cell. Also, even inducing NF-kB phosphorylation and increasing TNF production after infection, cells from diabetic patients were unable to downregulate NRF2 and generate oxidative stress, which may be associated with the exacerbated levels of IL-6 observed. PBMC of diabetic individuals are more susceptible to infection by L. amazonensis and fail to control the infection over time due to the inability to generate effector microbicidal molecules.

Hypoxia-Inducible Factor Signaling in Macrophages Promotes Lymphangiogenesis in Leishmania major Infection.

Vascular remodeling is a phenomenon seen in the cutaneous lesions formed during infection with Leishmania parasites. Within the lesion, Leishmania major infection leads to the infiltration of inflammatory cells, including macrophages, and is associated with hypoxic conditions and lymphangiogenesis in the local site. This low-oxygen environment is concomitant with the expression of hypoxic inducible factors (HIFs), which initiate the expression of vascular endothelial growth factor-A (VEGF-A) in macrophages during the infection. Here, we found that macrophage hypoxia is elevated in the skin, and the HIF target Vegfa is preferentially expressed at the site of infection. Further, transcripts indicative of both HIF-1α and HIF-2α activation were increased at the site of infection. Given that HIF mediates VEGF-A and that VEGF-A/VEGFR-2 signaling induces lymphangiogenesis, we wanted to investigate the link between myeloid HIF activation and lymphangiogenesis during L. major infection. We show that myeloid aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF/VEGF-A signaling promotes lymphangiogenesis (the generation of newly formed vessels within the local lymphatic network), which helps resolve the lesion by draining away inflammatory cells and fluid. Concomitant with impaired lymphangiogenesis, we find the deletion of myeloid ARNT/HIF signaling leads to an exacerbated inflammatory response associated with a heightened CD4+ Th1 immune response following L. major infection. Altogether, our data suggest that VEGF-A-mediated lymphangiogenesis occurs through myeloid ARNT/HIF activation following Leishmania major infection and this process is critical in limiting immunopathology.

Genetic variability of molecules involved in the disease pathogenesis in Leishmania infection.

Macrophages are the primary host cell for Leishmania parasites, by Toll like receptors (TLR-MyD88) that are central components of the innate and adaptive immunity against leishmania infection. The CD40/CD40L interaction has also been shown to be important in resistance to various protozoa. In this context, one of the most important properties of suppressors of cytokine signalling (SOCS) proteins, especially SOCS1 and SOCS3, is the regulation of macrophages cell for Leishmania parasites. In the present study we evaluated variants of molecules involved in activation and modulation of leishmanicidal signaling cascades and the possible associations between polymorphisms present in the TLR2, TLR4, MyD88, CD40, SOCS1, SOCS3 genes with susceptibility/resistent to Leishmania. The results suggest the absence of any association between TLR2 and TLR4 variants and susceptibility to Leishmaniasis. Analysis of the nucleotide sequence encoding the TIR recognition domain of the MyD88 molecule showed that it is highly conserved when compared to the reference sequences. In contrast, heterozygous rs 12953258, which reflects a decrease in the expression of SOCS3, suggesting that it may be involved in the leishmaniasis susceptibility. This study is a first advance in the analysis of polymorphisms of genes involved in the signaling pathway of the macrophage and their relationship with leishmaniases infection and disease progression.

Post kala-azar dermal leishmaniasis: A threat to elimination program.

Leishmaniasis remains a public health concern around the world that primarily affects poor folks of the developing world spanning across 98 countries with mortality of 0.2 million to 0.4 million annually. Post kala-azar dermal leishmaniasis (PKDL) is the late skin manifestation of visceral leishmaniasis (VL). It has been reported that about 2.5% to 20% of patients recovered from VL develop PKDL having stilted macular or nodular lesions with parasites. In the Indian subcontinent (ISC), it manifests a few months after recovery from VL, though in Africa it can occur simultaneously with VL or a little later. New cases of PKDL are also observed without prior VL in the ISC. These individuals with PKDL represent an important but largely neglected reservoir of infection that perpetuates anthroponotic Leishmania donovani transmission in the ISC and can jeopardize the VL elimination program as these cases can infect the sand flies and spread the endemic. Therefore, it becomes imperative to eradicate PKDL as a part of the VL elimination program. With the limited treatment options besides little knowledge on PKDL, this review stands out in focusing on different aspects that should be dealt for sustained VL elimination.

Cutaneous leishmaniasis in male schoolchildren in the upper and lower Dir districts of Khyber Pakhtunkhwa, and a review of previous record in Pakistan.

Documented reports are limited, showing cutaneous leishmaniasis (CL) as a severe threat to schoolchildren in Pakistan. The present study aimed to investigate the clinico-epidemiology and associated risk factors of CL in local and Afghan male schoolchildren between 6 and 16 years of age. The experimental strategy involved a questionnaire for the collection of information and clinical diagnosis (microscopy and semi-nested PCR) of 113 CL symptomatic schoolchildren out of 8,833 schoolchildren (7,175 local and 1,658 Afghan refugees) studying in nineteen schools of the Upper and Lower Dir Districts, Khyber Pakhtunkhwa, Pakistan. Previous records of CL in Pakistan was studied, and spatial analysis was performed on elevation and agro-ecological maps using Arc-GIS v10.3.1. Active lesions were found predominant (n=113, 1.2%: cutaneous lesions, 97, 86%, and lesions with mucosal involvement, 16, 14%) than scars (20, 0.25%). Active lesions of both local (100, 88%) and Afghan refugees (13, 12%), and infected age groups were found significantly different. Majority of the lesions were dry crusted (98, 86.7%), single (83, 73%), and frequently infecting facial region (59, 52%). Avoiding bed nets, living in mud houses and animal shelters were highly associated with CL infection. Temergara (30, 26.5%) and Rabath (14, 12.3%) were hyperendemic CL foci. Microscopically, 71 (63%) cases were positive, while the PCR assay revealed Leishmania tropica in 110 (97.3%) cases. Previous record revealed that L. tropica is dominant throughout Pakistan, and dry mountains and plateaus of northwestern and southwestern regions are spatially at high-risk. Measures should be taken to reduce CL infection by eliminating the associated risk factors, promoting PCR-based diagnosis and basic medical facilities.

Advanced case of PKDL due to delayed treatment: A rare case report.

Post-kala-azar dermal leishmaniasis (PKDL) is clinical outcome of visceral leishmaniasis (VL) and is thought to be the potential reservoir of parasite. Miltefosine (MF) is the only oral drug existing for treatment of post-kala-azar dermal leishmaniasis (PKDL). Increased miltefosine tolerance in clinical isolates of Leishmania donovani has been reported and is one of the major concerns in the treatment of PKDL. Here, we report a highly ulcerated PKDL case that was successfully cured after miltefosine treatment.

Changing clinico-epidemiology of post-kala-azar dermal leishmaniasis (PKDL) in India: Results of a survey in four endemic states.

BACKGROUND & OBJECTIVES: Detection and treatment of post-kala-azar dermal leishmaniasis (PKDL) cases is considered important for kala-azar elimination. The objective of our study was to find out the proportion of different forms of lesions, interruption of treatment and rate of treatment completion, cure rates of PKDL, risk factors for developing severe forms of PKDL and utilization of services offered by the kala-azar elimination program. METHODS: A cross-sectional survey of PKDL patients registered for treatment at all levels of care during 2015 and 2016 was done. RESULTS: 576 PKDL patients who had started treatment in 2015 and 2016 were studied. Three-fourths of all patients were found to be clinically cured after a year of follow-up. Around 90% lesions were of macular type. Interruption of treatment was observed in one-fourth of PKDL patients. Median duration between kala-azar treatment and development of PKDL was 4.5 years. Around 79% patients had past history of kala-azar treatment. Discontinuation of treatment during earlier kala-azar episode was significantly associated with the development of papular and nodular forms of lesion. 43% of patients had received the incentive of INR 2000 after completion of treatment. Around three-fourths women in the reproductive age group were found not to use any contraceptive method during PKDL treatment. INTERPRETATION & CONCLUSION: PKDL treatment interruption should be reduced through ensuring drug supply and timely retrieval of patients. Directly observed treatment should be implemented and combination regimen should be explored to improve final cure rate. Delivery of financial incentive to PKDL patients and counselling and contraception to women of reproductive age group should be improved.

Not-knowing and the proliferation of assumptions: local explanations of Cutaneous Leishmaniasis in Suriname.

Why do patients and others confronted with cutaneous leishmaniasis (CL) - a parasitic skin disease - in the hinterland of Suriname, South America, provide a dazzling variety of aetiological explanations for one single illness? And how do these explanations reflect local knowledge of and interest in the origin of illness? In this article, we explore these questions using the concept of 'not-knowing', as introduced by Murray Last in 1981. One of Last's conclusions is that 'don't knows' or 'don't cares' reflect people's disinterest in medicine. The aim of this article, however, is to draw attention to another aspect of not-knowing: it may lead to a proliferation of explanatory assumptions, unhindered by precise knowledge. In other words, multiple explanations mask not-knowing, which is from a methodological point of view a rarely observed element in social science research and constitutes an important addition to Murray Last's well known argument. The paper describes findings based on anthropological fieldwork carried out between September 2009 and December 2010 at the Dermatology Service in Suriname's capital Paramaribo and among 205 CL patients and 321 inhabitants in various communities in the hinterland. As this article shows, both knowing and not-knowing are rooted in the various contexts of people's daily lives and reflect their historical, socio-cultural, occupational, educational, biological, environmental, and public health-related conditions. Public health authorities should explore not-knowing more seriously in their efforts to prevent illness, since knowing about not-knowing is valuable in the design of health education and prevention programmes.

Severe post-kala-azar dermal leishmaniasis successfully treated with miltefosine in an Ethiopian HIV patient.

Post-kala-azar dermal leishmaniasis (PKDL) is a neglected tropical disease characterized by a dermatosis which often appears after successful treatment of visceral leishmaniasis caused by Leishmania donovani. PKDL treatment options are few and have severe limitations. In East-Africa, the standard treatment of PKDL is with daily painful potentially toxic sodium stibogluconate injections, administered for a prolonged duration of 30-60 days. In the Indian subcontinent, PKDL is mainly treated with miltefosine, a safer orally administered drug. However, in East-Africa, there is very limited experience in the use of miltefosine for treatment of severe PKDL, with only one published case report. Here we report a severe PKDL case in an Ethiopian HIV patient successfully treated with oral miltefosine (100mg/day for 28 days). Miltefosine was efficacious, safe and well tolerated, suggesting that it can play an important role in the treatment of severe PKDL also in East-African patients. Further research is warranted.

The safety and efficacy of miltefosine in the long-term treatment of post-kala-azar dermal leishmaniasis in South Asia - A review and meta-analysis.

BACKGROUND: Miltefosine (MF) is the only oral drug available for treatment of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). Although the drug is effective and well tolerated in treatment of VL, the efficacy and safety of MF for longer treatment durations (>28 days) in PKDL remains unclear. This study provides an overview of the current knowledge about safety and efficacy of long treatment courses with MF in PKDL, as a strategy in the VL elimination in South Asia. METHODOLOGY/PRINCIPAL FINDINGS: Literature was searched systematically for articles investigating MF treatment in PKDL. A meta-analysis included eight studies (total 324 PKDL patients) to estimate the efficacy of MF in longer treatment regimens (range 6-16 weeks). We found a per-protocol (PP) initial cure rate of 95.2% (95%CI 89.6-100.8) and a PP definite cure rate of 90% (95%CI 81.6-96.3). Descriptive analysis showed that 20% of patients experienced adverse events, which mostly had an onset in the first week of treatment and were likely to get more severe after four weeks of treatment. Gastrointestinal (GI) side effects such as vomiting, nausea, diarrhoea, and abdominal pain were most common. CONCLUSIONS/SIGNIFICANCE: Longer treatment regimens with MF are effective in PKDL patients in India, however with the caveat that the efficacy has recently been observed to decline. GI side effects are frequent, although mostly mild or moderate. However, on the basis of limited data, we cannot conclude that longer MF treatment regimens are safe. Moreover, VL and PKDL pharmacovigilance studies indicate a risk for serious adverse events, questioning the safety of MF. The provision of safer treatment regimens for PKDL patients are therefore recommended. Until these regimens are identified, it should be considered to halt the use of MF monotherapy for PKDL in order to preserve the drug's efficacy.

Cutaneous leishmaniasis: Spatial distribution and environmental risk factors in the state of Pará, Brazilian Eastern Amazon.

INTRODUCTION: Cutaneous leishmaniasis is an infectious disease transmitted by phlebotomine sandflies and is considered a great environmental and public health problem. Thus, this work presents initial results of the analyses about the relationship between the spatial distribution of this disease and its environmental risk factors in three municipalities, in the state of Pará, Brazil, from 2012 to 2016. METHODOLOGY: It was used data from the Ministry of Health, the National Institute for Space Research and the Brazilian Institute of Geography and Statistics. The statistical and spacial analysis of the variables were done using G-test goodness-of-fit, kernel interpolation technique and the Bivariate Global Moran Index (I). RESULTS: The analyses showed that the most affected individuals were males, adults, low schooling, residents in rural areas and small farmers. The disease spatial distribution was not homogeneous in the municipalities and it was associated to different relationships between the land use and occupation and the notificated cases density, with direct spatial autocorrelation. CONCLUSIONS: The deforestation was the most significant risk factor linked to the cases occurrence in all the studied area. We emphasize the need of intensification of epidemiological and environmental surveillance actions in the studied areas.