Liposomal dual delivery systems in visceral leishmaniasis enhance the synergistic effects of combination therapy:A promise for the future

Visceral leishmaniasis(VL)is a neglected tropical disease,and this review has summarized the current treatment scenario and its prospects.It also highlights alternative approaches used by research groups in India and around the world to develop cutting-edge and potent anti-leishmanial treatments.Even though numerous medications could be utilized to treat VL,the limitations of current treatments including their toxicity,cost,route of administration,and duration of doses,have contributed to the emergence of resistance.Combination therapy might be a better option due to its shorter duration,easier route of administration,and ability to extend the lifespan of individual drugs.However,there is a risk of not delivering both the drugs to the target site together,which can be overcome by the liposomal entrapment of those drugs and at a time knock an opportunity to reduce the dosage of amphotericin B if the combination drug provides a synergistic effect with it.Therefore,this review presents a novel strategy to fight against VL by introducing dual drug-loaded liposomes.

Therapeutic response and safety of different treatments for cutaneous leishmaniasis in patients: A retrospective cross-sectional study

Objective:To analyze the therapeutic response and safety of different treatments for cutaneous leishmaniasis,received by patients in the Program for the Study and Control of Tropical Diseases-PECET-Medellín-Colombia.Methods:This is a retrospective cross-sectional study of patients attended at PECET Research Center during 2016-2021.Relevant information regarding sociodemographic characteristics,history of leishmaniasis,characterization of current infection,treatment received,follow-up of therapeutic response and safety was collected from the medical records.Data were analyzed with Pearson's Chi-square association tests and Mann-Whitney U test using statistical software.Results:A total of 486 clinical records of patients were analyzed,and 356 received treatment.Eight different therapeutic alternatives(systemic,local and in combination)were analyzed.The therapeutic response of the different alternatives used(except thermotherapy)was higher than 50%.Most frequent adverse events were myalgias,arthralgias and headache,and vesicles for systemic and local treatment,respectively.Conclusions:Safety profile and performance of local therapeutic alternatives and combined schemes for the treatment of uncomplicated cutaneous leishmaniasis are an interesting option for the management of the disease.

犬利什曼病诊断的回顾性分析

为了总结分析北京地区犬利什曼病的流行特点、临床特征和诊断方法等,本调查收集在中国农业大学教学动物医院确诊的5例利什曼病患犬的病例资料,包括患犬的基本信息、病史、临床症状、病原学检查、实验室检查和影像学检查结果,并对患犬转归情况进行电话追踪访问后汇总。结果显示,3例(3/5)患犬来自北京市门头沟区。所有患犬(5/5)均有不同的皮肤病症状,4例(4/5)患犬体表淋巴结增大;个别患犬症状还包括葡萄膜炎、眼睑炎和不同程度的消瘦等。病原学检查结果显示,所有患犬均呈利氏曼原虫阳性;实验室检查结果显示,患犬表现贫血、高球蛋白血症和肾损伤等;影像学检查结果显示,部分患犬出现肾皮质回声增强。电话回访后得知,4例(4/5)患犬预后良好,1例患犬死亡。结果表明,利什曼病患犬常见临床症状为皮肤病变合并体表淋巴结肿大,但具体临床表现形式多样。通过对皮肤病变组织或淋巴结穿刺物进行利什曼原虫形态学观察和/或PCR特异性核酸检测可确诊该病。利氏曼病为人兽共患病,本调查通过描述患犬不同临床症状表现和诊治方法,为北京市门头沟及其周边区域利氏曼病的诊治提供科学依据,以期提高疾病的诊治效率,保障伴侣动物和人类的健康。

北京再发黑热病利什曼原虫K26基因和ITS-1序列的多态性分析

目的了解北京市再发黑热病病例的病原分子遗传背景。方法使用PCR方法分别扩增其亲水性酰化表面蛋白B(K26)和核糖体DNA内转录间隔区Ⅰ(ITS-1),然后克隆测序,分析其多态性,构建系统发育树以确定患者感染原虫的虫种类型及其遗传关系。结果K26基因扩增出626 bp大小片段,其长度与国内流行株差异明显,其氨基酸序列由14个氨基酸的基序重复排列组成,但个别位置发生氨基酸替代。K26序列的系统进化树分析显示,北京病例虫株与法国和西班牙的婴儿利什曼原虫相近,但与国内新疆、四川、河北等地的婴儿利什曼原虫虫株距离较远;ITS-1序列扩增出314 bp大小的片段,其系统发育分析显示与婴儿利什曼原虫Leishmania infantum属于同一分支。结论该病例感染的为婴儿利什曼原虫L.infantum,且与国内其他流行区的虫株有一定差异。

1990—2019年中国利什曼病发病趋势及年龄-时期-队列模型分析

目的分析1990—2019年中国利什曼病发病率的变化趋势,探究年龄、时期及队列因素对利什曼病发病率的影响,为中国利什曼病的防控政策的制定提供参考依据。方法利用2019年全球疾病负担数据库作为数据来源,提取1990—2019年中国利什曼病发病人数、粗发病率、标化发病率等数据。运用Joinpoint回归模型分析1990—2019年中国利什曼病标化发病率的变化趋势,并计算年度变化百分比(annual percentage change,APC)与平均年度变化百分比(average annual percentage change,AAPC)。采用年龄-时期-队列模型分析年龄、时期及队列因素对中国利什曼病发病率的影响。结果1990—2019年中国利什曼病发病数从4487例减至904例,降幅79.85%;粗发病率从0.38/10万降至0.06/10万,降幅84.21%;标化发病率从0.37/10万降至0.08/10万,降幅78.38%。Joinpoint回归模型结果显示,1990—2019年中国利什曼病全人群、男性和女性标化发病率均呈下降趋势,AAPC分别为-5.00%、-5.06%、-4.95%,差异均有统计学意义(t=-90.65、-90.70、-90.82,P均<0.01)。年龄-时期-队列模型结果显示,1990—2019年中国人群利什曼病发病风险随着年龄的增长而降低(RR值从5.24降至0.23),随着时期的推移而降低(RR值从2.14降至0.67),越晚出生的队列其发病风险越小(RR值从7.86降至0.12)。结论1990—2019年我国利什曼病呈低度流行态势,利什曼病防治效果显著,但仍需加强对重点区域传播媒介的干预和高危人群的防护,以降低我国利什曼病疾病负担。

Immune response to inactivated bacterial vector carrying the recombinant K39 antigen of Leishmania infantum in mice

Objective:To evaluate the immunological response elicited by an inactivated bacterial vector carrying the K39 antigen of Leishmania infantum,and a purified antigen.Methods:Mice were subjected to the following treatments:(1)Purified recombinant K39(rK39)protein at a 20μg dose with complete Freund’s adjuvant;(2)Inactivated Escherichia coli(BL21 DE3)carrying the K39 protein at an equivalent total protein content of 200μg;(3)Inactivated bacteria lacking the K39 protein;(4)Non-immunized control animals.Serological monitoring was performed.All groups were challenged by intraperitoneal injection of 10^(7) Leishmania infantum promastigotes.After euthanasia,the liver and spleen were collected to analyze the levels of TNF,IFN-γ,IL-12,IL-4,and IL-10.Results:Mice immunized with purified rK39 or the inactivated bacterial vector carrying the K39 antigen of Leishmania infantum showed a long-lasting immune response with high levels of polyclonal antibodies specifically recognizing the recombinant proteins.The IgG1 subclass was the predominant immunoglobulin;however,the induction of IgG2a and the profile of cytokines produced were indicative of the induction of a mixed-type response.Conclusions:The inactivated bacterial vector carrying the K39 antigen,as well as the purified antigen can induce a long-lasting immune response in immunized mice,predominantly favouring a Th2 profile response.

Molecular Docking Studies on Streptomycin Antileishmanial Activity

Resistance to pentavalent antimonial drugs and the lack of vaccines make it urgent to find novel therapeutic options to treat Leishmaniasis, a tropical disease caused by the Leishmania protozoan parasite. The study reported here is to investigate if Streptomycin, an aminoglycoside, and Amphotericin B, the second-line treatment drug, exhibit antileishmanial activity through a similar mechanism. By using MOE (Molecular Operating Environment), we performed molecular docking studies on these drugs binding to a range of targets including ribosome targets in Leishmania and H. sapiens. Our study shows that the two drugs do not bind to the same pockets in Leishmania targets but to the same pockets in the human ribosome, with some differences in interactions. Moreover, our 2D maps indicated that Amphotericin B binds to the A-site in the human cytoplasmic ribosome, whereas streptomycin does not.

一类具有饱和型发病率的双时滞利什曼病模型的全局稳定性分析

利什曼病或称为黑热病,它通过受感染的白蛉叮咬而得以传播.该病最常见的两种临床表现分别为内脏利什曼病和皮肤利什曼病.为了有效控制该疾病的传播,该文提出了一类具有饱和型发病率的双时滞利什曼病模型.首先分析平衡点的存在性,并确定了基本再生数;接着通过构造适当的李雅普诺夫函数,并利用LaSalle不变性原理,研究该系统平衡点的全局稳定性;最后通过数值模拟来验证结果的可行性并给出相应的结论.

Worldwide risk factors in leishmaniasis

Recently, Vector-borne parasitic diseases such as leishmaniasis have been emerged or reemerged in many geographical areas and resulted in global health and economic concerns that involve humans, domestic animals and wild life. The ecology and epidemiology of leishmaniasis are affected by the between host, reservoir and vector(human, animal and sandfly) and the environment. Important drivers for the emergence and spread of leishmaniasis include environmental factors such as alterations in temperature and water storage, irrigation habits, deforestation, climate changes, immunosuppression by HIV or organ transplant, development of drug resistance, increase traveling to endemic regions and dog importation. War, poor socio-economic status and low level household are also major contributors to the spread of this disease. Health education via the public media and training should be implemented by international organizations and governmental agencies in collaboration with research institutions. During transmission season, fully protection can be mentioned by using bednets and insecticides and reservoirs' control in the planning. Based on the findings of the recent studies and high prevalence of leishmaniasis, it is concluded that serious public health monitoring should be considered.

Forecasting the number of zoonotic cutaneous leishmaniasis cases in south of Fars province, Iran using seasonal ARIMA time series method

Objective: To predict the trend of cutaneous leishmaniasis and assess the relationship between the disease trend and weather variables in south of Fars province using Seasonal Autoregressive Integrated Moving Average(SARIMA) model,Methods: The trend of cutaneous leishmaniasis was predicted using Mini tab software and SARIMA model,Besides,information about the disease and weather conditions was collected monthly based on time series design during January 2010 to March 2016,Moreover,various SARIMA models were assessed and the best one was selected,Then,the model's fitness was evaluated based on normality of the residuals' distribution,correspondence between the fitted and real amounts,and calculation of Akaike Information Criteria(AIC) and Bayesian Information Criteria(BIC),Results: The study results indicated that SARIMA model(4,1,4)(0,1,0)(12) in general and SARIMA model(4,1,4)(0,1,1)(12) in below and above 15 years age groups could appropriately predict the disease trend in the study area,Moreover,temperature with a three-month delay(lag3) increased the disease trend,rainfall with a four-month delay(lag4) decreased the disease trend,and rainfall with a nine-month delay(lag9) increased the disease trend,Conclusions: Based on the results,leishmaniasis follows a descending trend in the study area in case drought condition continues,SARIMA models can suitably measure the disease trend,and the disease follows a seasonal trend.

Leishmaniasis:Current status of available drugs and new potential drug targets

The control of Leishmania infection relies primarily on chemotherapy till date.Resistance to pentavalent antimonials,which have been the recommended drugs to treat cutaneous and visceral leishmaniasis,is now widespread in Indian subcontinents.New drug formulations like amphotericin B,its lipid formulations,and miltefosine have shown great efGcacy to treat leishmaniasis but their high cost and therapeutic complications limit their usefulness.In addition, irregular and inappropriate uses of these second line drugs in endemic regions like state of Bihar, India threaten resistance development in the parasite.In context to the limited drug options and unavailability of either preventive or prophylactic candidates,there is a pressing need to develop true antileishmanial drugs to reduce the disease burden of this debilitating endemic disease.Notwithstanding significant progress of leishmanial research during last few decades, identification and characterization of novel drugs and drug targets are far from satisfactory.This review will initially describe current drug regimens and later will provide an overview on few important biochemical and enzymatic machineries that could be utilized as putative drug targets for generation of true antileishmanial drugs.

Cutaneous leishmaniasis in Iran: Results from an epidemiological study in urban and rural provinces

Objective: To examine the prevalence and clinical manifestations of cutaneous leishmaniasis(CL) in Iran.Methods: This study was conducted in Iran between 2011 and 2013. Sampling, preparing, developing, and fixing of suspicious skin lesions were completed in healthcare centers in 31 Iranian provinces as well as in the Academic Reference Laboratory and the National Reference Laboratory. The information was then analyzed at the Ministry of Health's Information Management Center of Contagious Diseases.Results: Over a three-year period, the number of people identified with CL was 56 546.The highest incidence was reported in 2011(27.5 per 100 000). Wet CL accounted for 43.7% of cases while 43.3% resulted from sporotrichoid leishmaniasis. The results showed that there was a higher incidence of CL due to Leishmania major(50.2%) than to Leishmania tropica. The results of this study found that the highest incidence of CL had happened respectively in Ilam, Fars and, Khorasan Razavi Provinces between 2011 and 2013.Conclusions: Although the incidence of the disease is declining, CL is still a public health concern and disease control protocols need to be established. Therefore, further studies are needed to identify the vectors, reservoirs, and disease species as well as to develop appropriate disease control strategies.

Phlebotomus papatasi and Meriones libycus as the vector and reservoir host of cutaneous leishmaniasis in Qomrood District,Qom Province,central Iran

Objective:To determine the sand flies species responsible for most transmission of Leishmania major(L major) to human,as well as to determine the main reservoir hosts of the disease. Methods:Sand flies were collected using sticky papers and mounted in Puri’s medium for species identification.Rodents were trapped by live Sherman traps.Both sand flies and rodents were subjected to molecular methods for detection of leishmanial parasite.Results:Phlebotomus papatasi(P.papatasi) was the common species in outdoor and indoor resting places.Employing PCR technique only three specimens of 150 P.papatasi(2%) were found naturally infected by parasites with a band of 350 bp which is equal to the L major parasite.Forty six rodents were captured by Sherman traps and identified.Microscopic investigation on blood smear of the animals for amastigote parasites revealed 1(3.22%) infected Meriones libycus(M.libycus). Infection of this animal to L.major was confirmed by PCR against rDNA loci of the parasite. Conclusions:This is the first molecular report of parasite infection of both vector(P.papatasi) and reservoir(M.libycus) to L major in the region.The results indicated that P.papatasi was the primary vector of the disease and circulating the parasite between human and reservoirs and M. libycus was the most important host reservoir for maintenance of the parasite source in the area.

In vitro activity and in vivo efficacy of a combination therapy of diminazene and chloroquine against murine visceral leishmaniasis

The present study evaluated the in vitro activity and in vivo efficacy of diminazene combined with chloroquine as a potential drug against Leishmania donovani. Amphotericin B was used as a positive control drug. In vitro activity involved incubation of various drug concentrations with promastigotes or vero cells in culture before determination of parasite growth inhibition or cell death while in vivo evaluations involved infection of various mice groups with virulent L. donovani parasites and treatment with test drug compounds following disease establishment. Weight changes in experimental mice were also evaluated before infection and throughout the experiment. The results indicated that the diminazene-chloroquine combination was at least nine times more efficacious than individual drugs in killing promastigotes in culture. The diminazene-chloroquine combination was safer （Ld50=0.03±0.04） than Amphotericin B （Ld50=0.02±0.01）. Body weight in infected mice increased significantly （P=0.0007） from day 7 to day 37 following infection （P=0.026）. However, body weight remained comparable in all mice groups during treatment （P=0.16）. The diminazene-chloroquine combination significantly reduced splenic parasite numbers as compared to individual drug therapies （P=0.0001） although Amphotericin B was still more efficacious than any other treatment （P=0.0001）. Amongst the test compounds, the diminazene-chloroquine combination showed the lowest level of IgG antibody responses with results indicating significant negative correlation between antileishmanial antibody responses and protection against disease. These findings demonstrate the positive advantage and the potential use of a combined therapy of diminazene-chloroquine over the constituent drugs. Further evaluation is recommended to determine the most efficacious combination ratio of the two compounds.

Next-generation sequencing technology for diagnosis and efficacy evaluation of a patient with visceral leishmaniasis: A case report

BACKGROUND Visceral leishmaniasis(VL)is a parasitic disease caused by Leishmania and transmitted by infected sand flies.VL has a low incidence in China,and its clinical presentation is complex and atypical.This disease is easily misdiagnosed and can become life-threatening within a short period of time.Therefore,early,rapid and accurate diagnosis and treatment of the disease are essential.CASE SUMMARY A 25-year-old male patient presented with the clinical manifestations of irregular fever,hepatosplenomegaly,increased polyclonal globulin,and pancytopenia.The first bone marrow puncture biopsy did not provide a clear diagnosis.In order to relieve the pressure and discomfort of the organs caused by the enlarged spleen and to confirm the diagnosis,splenectomy was performed,and hemophagocytic syndrome was diagnosed by pathological examination of the spleen biopsy.Following bone marrow and spleen pathological re-diagnosis and metagenomic next-generation sequencing(mNGS)technology detection,the patient was finally diagnosed with VL.After treatment with liposomal amphotericin B,the body temperature quickly returned to normal and the hemocytes recovered gradually.Post-treatment re-examination of the bone marrow puncture and mNGS data showed that Leishmania was not detected.CONCLUSION As a fast and accurate detection method,mNGS can diagnose and evaluate the efficacy of treatment in suspicious cases of leishmaniasis.

Climate change and potential distribution of zoonotic cutaneous leishmaniasis in Central Iran: Horizon 2030 and 2050

Objective: To investigate and predict the effects of climate change on the potential distribution of the main vector and reservoir hosts of the disease in Yazd province in the future.Methods: Distribution data for vector and reservoir hosts of zoonotic cutaneous leishmaniasis in Yazd province were obtained from earlier studies conducted in the area.MaxEnt ecological niche modeling was used to predict environmental suitability.BCC-CSM1-1(m) model and two climate change scenarios, RCP 4.5 and RCP 8.5 were used for horizons 2030 and 2050 climate projections.Future projections were based on data of a regional climate change model.Results: With both scenarios in 2030 and 2050, the results of jackknife test indicated that the mean temperature of wettest quarter and temperature annual range had the greatest effect on the model for the vector and the reservoir hosts, respectively.Conclusions: The climate conditions are the major determinants of zoonotic cutaneous leishmaniasis incidence rate in Yazd Province.These climate conditions provide favorable habitats for ease transmission of zoonotic cutaneous leishmaniasis in this endemic area.Habitats suitability for the vector and reservoir will be expanding in the coming years compared with the current conditions, such that, in horizon 2030 & 2050, the probability of the presence of the vector and reservoir within 38 580 and 37 949 km^2, respectively, from Yazd province is above 60%.Moreover, an increase is predicted in the presence of the vector in the western parts and the reservoir in the northern and central parts of the province in the future.Understanding the role of environmental and bioclimatic factors in zoonotic cutaneous leishmaniasis occurrence can provide a guide for policy-makers in the creation and implementation of more effective policies for prevention and control.

Predicting the number of visceral leishmaniasis cases in Kashgar, Xinjiang, China using the ARIMA-EGARCH model

Objective:To forecast the visceral leishmaniasis cases using autoregress integrated moving average(ARIMA)and hybrid ARIMAEGARCH model,which offers a scientific basis to control visceral leishmaniasis spread in Kashgar Prefecture of Xinjiang,China.Methods:The data used in this paper are monthly visceral leishmaniasis cases in the Kashgar Prefecture of Xinjiang from 2004 to 2016.The sample data between 2004 and 2015 were used for the estimation to choose the best model and the sample data in 2016 were used for the forecast.Time series of visceral leishmaniasis started on 1 January 2004 and ended on 31 December 2016,consisting of 1790 observations reported in Kashgar Prefecture.Results:For Xinjiang,the total number of reported cases were 2187,the male-to-female ratio of cases was 1:1.42.Patients aged between 0 and 10 years accounted for 82.72%of all reported cases and the largest percentage of visceral leishmaniasis cases was detected among scattered children who accounted for 68.82%.The monthly incidences fitted by ARIMA(2,1,2)(1,1,1)12 model were consistent with the real data collected from 2004 to 2015.However,the predicted cases failed to comply with the observed case number;we then attempted to establish a hybrid ARIMA-EGARCH model to fit visceral leishmaniasis.Finally,the ARIMA(2,1,2)(1,1,1)12-EGARCH(1,1)model showed a good estimation when dealing with volatility clustering in the data series.Conclusions:The combined model has been determined as the best prediction model with the root-mean-square error(RMSE)of 7.23%in the validation phase,which means that this model has high validity and rationality and can be used for short-term prediction of visceral leishmaniasis and could be applied to the prevention and control of the disease.

Knowledge,attitude,and practices related to cutaneous leishmaniasis in an endemic focus of cutaneous leishmaniasis,Southern Iran

Objective:To assess knowledge,attitudes,and practices(KAP)of inhabitants of an endemic area it)Fars province toward cutaneous leishmaniasis(CI).Methods:the study was carried out in Lapui district in Fars province,south of Iran,one of the most important foci of CL in this province.Sample size(237 residents)was calculated based on population.House-to-house survey was done to collect the data regarding knowledge,attitudes,and practices of the inhabitants.The head of each household was interviewed by a trained staff to assess his/her KAP related to CL.A semi-structured KAP questionnaire was used for data collection.Results:Mean age of participants was 39 and more titan half of the respondents were in the age group of 31-40.Males constituted 172(72.5%)of subjects.Most of the respondents(84.3%)were literate.The majority of the study poulation(83%)had heard about SalaJc(local name for CL)and most of these respondents(91%)were aware that CL is presented with a cutaneous lesion.Nearly two-third of the participants(63.5%)stated the bite of mosquito(not specifically sandflies)for CL transmission.The respondents'attitude regarding the treatment of CL was not satisfactory since Only 48%believed that CL can be treated by medicine.A noticeable proportion of respondents(21%)believed in indigenous medicine for the treatment of CL.A small proportion of respondents(14%)stated that traditional healers are good at treating this disease.More than two-third(69%)of respondents believed that the disease is preventable although most of interviewees did not know about preventive measures.Conclusions:In this study,insufficient knowledge of community about infection nature,vector,transmission mode and preventive measures of CL,highlights the needs for a health education initiative to enhance the awareness of people about CL.This would improve inhabitants'contribution in control program of CL in this area.

Leishmania donovani whole cell antigen delivered with adjuvants protects against visceral leishmaniasis in vervet monkeys (Chlorocebus aethiops)

In a previous immunogenicity and efficacy study in mice, montanide ISA 720 （MISA） was indicated to be a better adjuvant than bacillus calmette guerin vaccine （BCG） for a Leishmania vaccine. In the present study, we report the safety, immunogenicity and efficacy of Leishmania donovani （L. donovani） sonicated antigen delivered with alum-BCG （A1BCG）, MISA or monophosphoryl lipid A （MPLA） in vervet monkeys following intradermal inoculums. Vaccinated and control animals were challenged with virulent L. donovani parasites and the parasitic burden was determined. Only animals vaccinated with alum-BCG adversely reacted to the inoculum by produc- ing ulcerative erythematous skin indurations. Non-parametric ANOVA followed by a post test showed signifi- cantly higher IgG antibodies, and revealed the presence of lymphoproliferative and interferon gamma responses in both AIBCG＋Ag and MISA＋Ag as compared to the MPLA＋Ag or other groups （P 〈 0.001）. We conclude that L. donovani sonicated antigen containing MISA is safe and is associated with protective immune response against Leishmania donovani infection in the vervet monkey model.

Disseminated cutaneous leishmaniasis due to Leishmania(Leishmania) amazonensis in human immunodeficiency virus(HIV)-infected patients: A report of two cases

Rationale: Co-infection of human immunodeficiency virus(HIV) and Leishmania spp. has impact on clinical and therapeutic outcomes of leishmaniases. Most studies do not present the identification of Leishmania species causing American tegumentary leishmaniasis in co-infections. In the Americas, Leishmania(L.) Viannia(V.) braziliensis and L.(V.) guyanensis have been identified. Patient concerns: In this study, two cases of American tegumentary leishmaniasis in patients infected with HIV are described. Patients presented several lesions with rapid dissemination and mucosal involvement. Diagnosis: Disseminated cutaneous leishmaniasis caused by L. amazonensis was identified by molecular test. Interventions: The patients were treated with conventional therapies for HIV infection and American tegumentary leishmaniasis. Outcomes: In co-infection, the clinical manifestations are atypical and the treatment response can be impaired. Lessons: These cases show that HIV infection impacts L. amazonensis infection and point to the relevance of identifying Leishmania species, which can lead to a better patient management.