Effect of Leishmania RNA virus 2 on virulence factors and cytokines gene expression in a human macrophage infected with Leishmania major: A preliminary study.

Cutaneous leishmaniasis (CL) is one of the most important infectious parasitic diseases in the world caused by the Leishmania parasite. In recent decades, the presence of a virus from the Totiviridae family has been proven in some Leishmania species. Although the existence of LRV2 in the Old world Leishmania species has been confirmed, almost no studies have been done to determine the potential impact of LRV2 on the immunopathogenicity of the Leishmania parasite. In this preliminary study, we measured the expression of target genes, including Glycoprotein 63 (gp63), Heat Shock Protein 70 (hsp70), Cysteine Protease b (cpb), Interleukin 1 beta (IL-1ß), IL8 and IL-12 in LRV2 positive Leishmania major strain (LRV2+L. major) and LRV2 negative L. major strain (LRV2-L. major). We exposed THP-1, a human leukemia monocytic cell line, to promastigotes of both strains. After the initial infection, RNA was extracted at different time points, and the relative gene expression was determined using a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Findings showed that the presence of LRV2 in L. major was able to increase the expression of gp63, hsp70, and cpb genes; also, we observed lower levels of expression in cytokine genes of IL-1ß, IL-8, IL-12 in the presence of LRV2+, which are critical factors in the host's immune response against leishmaniasis. These changes could suggest that the presence of LRV2 in L. major parasite may change the outcome of the disease and increase the probability of Leishmania survival; nevertheless, further studies are needed to confirm our results.

Distinct microbiome profiles and biofilms in Leishmania donovani-driven cutaneous leishmaniasis wounds.

The endemic strain of Leishmania donovani in Sri Lanka causes cutaneous leishmaniasis (CL) rather than more common visceral form. We have visualized biofilms and profiled the microbiome of lesions and unaffected skin in thirty-nine CL patients. Twenty-four lesions (61.5%) were biofilm-positive according to fluorescence in situ hybridization. Biopsies of biofilm-positive lesions were dominated by Pseudomonas, class Bacilli and Enterobacteriaceae and distinguished by significantly lower community evenness. Higher relative abundance of a class Bacilli OTU was detected in wound swabs versus contralateral skin. Wound swabs and biopsies had significantly distinct microbiome profiles and lower diversity compared to unaffected skin. Greater abundances of potentially pathogenic organisms were observed in wet ulcers, lesions with high parasite loads and large wounds. In summary, more than half of L. donovani associated CL wounds harboured biofilms and the wounds exhibited a distinct, less diverse, microbiome than unaffected skin.

Resistance Against Leishmania major Infection Depends on Microbiota-Guided Macrophage Activation.

Innate immune cells present a dual role during leishmaniasis: they constitute the first line of host defense but are also the main host cells for the parasite. Response against the infection that results in the control of parasite growth and lesion healing depends on activation of macrophages into a classical activated phenotype. We report an essential role for the microbiota in driving macrophage and monocyte-derived macrophage activation towards a resistance phenotype against Leishmania major infection in mice. Both germ-free and dysbiotic mice showed a higher number of myeloid innate cells in lesions and increased number of infected cells, mainly dermal resident and inflammatory macrophages. Despite developing a Th1 immune response characterized by the same levels of IFN-γ production as the conventional mice, germ-free mice presented reduced numbers of iNOS+ macrophages at the peak of infection. Absence or disturbance of host microbiota impaired the capacity of bone marrow-derived macrophage to be activated for Leishmania killing in vitro, even when stimulated by Th1 cytokines. These cells presented reduced expression of inos mRNA, and diminished production of microbicidal molecules, such as ROS, while presenting a permissive activation status, characterized by increased expression of arginase I and il-10 mRNA and higher arginase activity. Colonization of germ-free mice with complete microbiota from conventional mice rescued their ability to control the infection. This study demonstrates the essential role of host microbiota on innate immune response against L. major infection, driving host macrophages to a resistance phenotype.

Microbiota instruct IL-17A-producing innate lymphoid cells to promote skin inflammation in cutaneous leishmaniasis.

Innate lymphoid cells (ILCs) comprise a heterogeneous population of immune cells that maintain barrier function and can initiate a protective or pathological immune response upon infection. Here we show the involvement of IL-17A-producing ILCs in microbiota-driven immunopathology in cutaneous leishmaniasis. IL-17A-producing ILCs were RORγt+ and were enriched in Leishmania major infected skin, and topical colonization with Staphylococcus epidermidis before L. major infection exacerbated the skin inflammatory responses and IL-17A-producing RORγt+ ILC accumulation without impacting type 1 immune responses. IL-17A responses in ILCs were directed by Batf3 dependent CD103+ dendritic cells and IL-23. Moreover, experiments using Rag1-/- mice established that IL-17A+ ILCs were sufficient in driving the inflammatory responses as depletion of ILCs or neutralization of IL-17A diminished the microbiota mediated immunopathology. Taken together, this study indicates that the skin microbiota promotes RORγt+ IL-17A-producing ILCs, which augment the skin inflammation in cutaneous leishmaniasis.

Leishmania major biotin protein ligase forms a unique cross-handshake dimer.

Biotin protein ligase catalyses the post-translational modification of biotin carboxyl carrier protein (BCCP) domains, a modification that is crucial for the function of several carboxylases. It is a two-step process that results in the covalent attachment of biotin to the ϵ-amino group of a conserved lysine of the BCCP domain of a carboxylase in an ATP-dependent manner. In Leishmania, three mitochondrial enzymes, acetyl-CoA carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, depend on biotinylation for activity. In view of the indispensable role of the biotinylating enzyme in the activation of these carboxylases, crystal structures of L. major biotin protein ligase complexed with biotin and with biotinyl-5'-AMP have been solved. L. major biotin protein ligase crystallizes as a unique dimer formed by cross-handshake interactions of the hinge region of the two monomers formed by partial unfolding of the C-terminal domain. Interestingly, the substrate (BCCP domain)-binding site of each monomer is occupied by its own C-terminal domain in the dimer structure. This was observed in all of the crystals that were obtained, suggesting a closed/inactive conformation of the enzyme. Size-exclusion chromatography studies carried out using high protein concentrations (0.5 mM) suggest the formation of a concentration-dependent dimer that exists in equilibrium with the monomer.

Site specific microbiome of Leishmania parasite and its cross-talk with immune milieu.

Microbiota consists of commensal, symbiotic and pathogenic microorganisms found in all multicellular organisms. These micro-organisms are found in or on many parts of the body, including the intestinal tract, skin, mouth, and the reproductive tract. This review focuses on interplay of site specific microbiota, vector microbiota along with immune response and severity of Leishmaniasis. Herein, we have reviewed and summarized the counter effect of microbiome post infection with the Leishmania parasite. We have studied skin microbiome along with the gut microbiome of sand-fly which is the vector for transmission of this disease. Our major focus was to understand the skin and gut microbiome during Leishmania infection,their interaction and effect on immunological responses generated during the infection.Moreover, systems biology approach is envisioned to enumerate bacterial species in skin microbiota and Phlebotmus gut microbiota during Leishmania infection.

Curcumin-loaded self-emulsifying drug delivery system (cu-SEDDS): a promising approach for the control of primary pathogen and secondary bacterial infections in cutaneous leishmaniasis.

Cutaneous leishmaniasis being a neglected tropical disease (NTD) faces several challenges in chemotherapy. If infected with secondary bacterial infections, the treatment regime of cutaneous ulcers in cutaneous leishmaniasis is further complicated which usually require two or more than two chemotherapeutic agents for healing. In the current study, seven curcumin-loaded self-emulsifying drug delivery system (cu-SEDDS) formulations (namely F1-F7) were prepared by mixing different excipients (oils, surfactants, and co-solvents) through stirring (vortex) and sonication. The formulations were characterized regarding their droplet size, polydispersity index (PDI), and zeta potential by zeta sizer. The cu-SEDDS formulations displayed different sizes ranging from 32.4 up to 80.0 nm. The zeta potential of the formulations ranged from - 1.56 up to - 4.8. The antileishmanial activities of the cu-SEDDS formulations in terms of IC50 against Leishmania tropica ranged from 0.19 up to 0.37 µg/ml. The minimum inhibitory concentrations (MICs) of these formulations against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae were in the range of 48-62 µg/ml. The hemolysis caused by formulations was 1-2%. The spreading potential of the formulations (F1 and F5) over damaged skin model was remarkable. These results suggest that cu-SEDDS further enhanced the broad spectrum antileishmanial and antibacterial profile of curcumin and could be used for the treatment of cutaneous leishmaniasis and its associated secondary infections.

Associated-risk determinants for anthroponotic cutaneous leishmaniasis treated with meglumine antimoniate: A cohort study in Iran.

BACKGROUND: The control of cutaneous leishmaniasis (CL) is facilitated by knowledge of factors associated with the treatment failures in endemic countries. The aim of this evaluation was to identify the potential risk determinants which might affect the significance of demographic and clinical characteristics for the patients with anthroponotic CL (ACL) and the outcome of meglumine antimoniate (MA) (Glucantime) treatment. METHODOLOGY/PRINCIPAL FINDINGS: This current was executed as a cohort spanning over a period of 5 years which centered in southeastern part of Iran. Altogether, 2,422 participants were evaluated and 1,391 eligible volunteer patients with ACL caused by Leishmania tropica were included. Overall, 1,116 (80.2%) patients received MA intraleisionally (IL), once a week for 12 weeks along with biweekly cryotherapy, while 275 (19.8%) patients received MA alone (20 mg/kg/day for 3 weeks) (intramuscular, IM). The treatment failure rate in ACL patients was 11% using IL combined with cryotherapy plus IM alone, whilst 9% and 18.5% by IL along with cryotherapy or IM alone, respectively. Multivariate logistic regression model predicted 5 major associated-risk determinants including male (odds ratio (OR) = 1.54, confidence interval (CI) = 1.079-2.22, p = 0.018), lesion on face (OR = 1.574, CI = 1.075-2.303, p = 0.02), multiple lesions (OR = 1.446, CI = 1.008-2.075, p = 0.045), poor treatment adherence (OR = 2.041, CI = 1.204-3.46, p = 0.008) and disease duration > 4 months (OR = 2.739, CI = 1.906-3.936, p≤0.001). CONCLUSIONS/SIGNIFICANCE: The present study is the original and largest cohort of ACL patients who treated with MA. A comprehensive intervention and coordinated action by the health authorities and policy-makers are crucial to make sure that patients strictly follow medical instructions. Early detection and effective therapy < 4 months following the onset of the lesion is critical for successful treatment of the patients. Since a significant number of patients are still refractory to MA, reducing man-vector exposure and development of new effective alternative drugs are essential measures against ACL due to L. tropica.

Study of the efficacy of N-methyl glucamine antimoniate (SbV) associated with photodynamic therapy using liposomal chloroaluminium phthalocyanine in the treatment of cutaneous leishmaniasis caused by Leishmania (L.) amazonensis in C57BL6 mice.

BACKGROUND: Pentavalent antimonials remain first-line drugs in the treatment of cutaneous leishmaniasis (CL); however, adverse effects and drug resistance have led to the search for less toxic and more effective treatments. As an alternative, topical phthalocyanine has been studied and its efficacy and low toxicity demonstrated. We aimed to study the in vivo efficacy of N-methyl glucamine antimoniate (NMG) associated with photodynamic therapy (PDT) with topical liposomal chloroaluminium phthalocyanine (AlClPC) in the treatment of experimental CL by L. amazonensis. METHODS: Experimental study with 54 C57BL6 isogenic mice divided into 9 groups including uninfected control, untreated control, PDT with AlClPC + NMG at doses of 10 and 20 mgSbV/Kg/day. The criteria to evaluate the treatment efficacy were: paw diameter, amastigote count, culture, viability test and parasite counts using MTT (3-bromo-4,5-dimethylthiazol-2,5-diphenyl-tetrazolium bromide). RESULTS: Treatment of CL with the association of NMG20 + PDT with AlClPC showed significant reduction of paw diameter, amastigote count, cultures, viability test and parasite counts. Parasite reduction occurred at the 10th and 20th days of treatment and 60 days after treatment ended, indicating that parasites did not multiply again. The NMG10 + PDT group with AlClPC presented results equivalent to gold-standard treatment (20 mgSbV/kg/day). Biochemical and histopathological evaluation showed minor changes. CONCLUSION: Treatment of CL caused by L. amazonensis with NMG20 mgSbV/kg/day + PDT with AlClPC was more effective than the traditional NMG20 mgSbV/kg/day.

First Report and In Silico Analysis of Leishmania virus (LRV2) identified in an autochthonous Leishmania major isolate in Turkey.

Leishmania virus (LRV) has previously been identified in different Leishmania species. Host-LRV interaction is associated with exacerbated clinical manifestations of cutaneous leishmaniasis (CL) and may cause poor therapeutic response. CL cases due to L. major with large skin lesions resistant to routine therapy were recently identified in Turkey. Here, we report the first autochthonous case of cutaneous leishmaniasis caused by LRV-positive Leishmania major, using conventional PCR targeting the viral capsid protein of LRV. The lesion of the case was 6 months old, relatively large (4 cm), and did not recover despite three consecutive intralesional applications of glucantime. Assessment of LRV's influence on prognosis and clinical outcomes of leishmaniasis, based on additional studies, is required.

Schistosoma mansoni rSm29 Antigen Induces a Regulatory Phenotype on Dendritic Cells and Lymphocytes From Patients With Cutaneous Leishmaniasis.

The immune response induced by Schistosma mansoni antigens is able to prevent immune-mediated diseases. Conversely, the inflammatory response in cutaneous leishmaniasis (CL), although responsible for controlling the infection, is also associated with the pathogenesis of disease. The aim of this study was to evaluate the potential of the S. mansoni Sm29 antigen to change certain aspects of the profiles of monocyte derived dendritic cells (MoDCs) and lymphocytes from subjects with CL in vitro. Expression of surface molecules and intracellular cytokines in the MoDCs and lymphocytes as well as the proliferation of Leishmania braziliensis were evaluated by flow cytometry. Levels of cytokines were evaluated in culture supernatants by ELISA. It was observed that stimulation by rSm29 increased the frequency of expression of CD83, CD80, CD86, and IL-10R in MoDCs compared to non-stimulated cultures. Additionally rSm29 decreased the frequency CD4+ and CD8+ T cells expressing CD28 and increased the frequency of CD4+CD25hi and CD4+CTLA-4+ T lymphocytes. Addition of rSm29 to cultures increased IL-10 levels and decreased levels of IL-12p40 and IFN-γ, while not altering TNF levels compared to non-stimulated cultures. This study showed that rSm29 induced a regulatory profile in MoDCs and lymphocytes and thereby regulated the exaggerated inflammation observed in CL. Considering that there are few therapeutic options for leishmaniasis, the use of rSm29 may be an alternative to current treatment and may be an important strategy to reduce the healing time of lesions in patients with CL.

Molecular identification of Leishmania spp. isolates causes cutaneous leishmaniasis (CL) in Sanliurfa Province, Turkey, where CL is highly endemic.

Cutaneous leishmaniasis (CL) is an important public health problem in Turkey. CL has been most frequently seen in Sanliurfa. There is an expectation of increase in the population of leishmaniasis cases with the influence of Syrian refugees arriving in Turkey. In this study we aimed to diagnosis of CL and identifying of parasite from Leishmania isolates by using ITS 1 PCR RFLP. Samples were collected from 135 CL patients in Sanliurfa. After the specimens were inoculated in medium NNN, the ones which were cultures positive were cultivated in RPMI 1640 followed by PCR-RFLP. Genomic DNA was extracted phenol-chloroform procedure. Samples were examined by using ITS 1 PCR followed by RFLP analysis. Our results indicated that two species, L. tropica (132 samples) and L. major (3 samples), are responsible for cutaneous leishmaniasis in Sanliurfa. Our study is the first scientific study in which it is reported molecular analyses of cutaneous leishmaniasis cases caused by L. major in Sanliurfa in Southestern Anatolia Region. Because CL cases caused by L.major are detected in our study, it is considered that genotyping is important for diagnosis of Leishmania and following change of epidemiology.

Long-term cell culture isolated from lesions of mice infected with Leishmania amazonensis: a new approach to study mononuclear phagocyte subpopulations during the infection.

Leishmanioses are neglected diseases and the parasite Leishmania survives and proliferates within mononuclear phagocytes, particularly macrophages. In vitro studies of the immunology and cell biology of leishmaniosis are performed in murine peritoneum and bone marrow macrophages and immortalized cell lines despite the normal and injured tissue-specific heterogeneity of macrophages. In this work, we established an ex vivo methodology to culture lesional cells from BALB/c mice infected with Leishmania amazonensis. The cells were successfully isolated from footpad skin lesions and those exhibiting macrophage morphology were maintained in long-term culture (12 days), while the small number of lymphocytes, polymorphonuclear and unidentified cells died after 1 day of culture. The frequency of infected cells decreased over 2 days. Most lesional cells cultivated ex vivo were myeloid CD11b+ CD14+ F4/80+ CD68+ cells. Low levels of IFN-γ and IL-4, IL-10 production and low arginase and phagocytic activities were detected in ex vivo lesional cell cultures. The ex vivo model developed in this study open perspectives for studying the biology of leishmanial lesions in cellular subpopulations and at the single-cell level.

Effect of secondary infection on epithelialisation and total healing of cutaneous leishmaniasis lesions.

BACKGROUND: Cutaneous leishmaniasis (CL) generally presents with a single or several localised cutaneous ulcers without involvement of mucous membranes. Ulcerated lesions are susceptible to secondary contamination that may slow the healing process. OBJECTIVE: This study verified the influence of non-parasitic wound infection on wound closure (epithelialisation) and total healing. METHODS: Twenty-five patients with a confirmed diagnosis of CL and ulcerated lesions underwent biopsy of ulcer borders. One direct microbial parameter (germ identification in cultures) and four indirect clinical parameters (secretion, pain, burning sensation, pruritus) were analysed. FINDINGS Biopsies of ten lesions showed secondary infection by one or two microorganisms (Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Streptococcus pyogenes and Candida parapsilosis). "Secretion" and "burning sensation" influenced epithelialisation time but not total healing time. Positive detection of germs in the ulcer border and "pain" and "pruritus" revealed no influence on wound closure. CONCLUSIONS: Our borderline proof of clinical CL ulcer infection inhibiting CL wound healing supports the need to follow antimicrobial stewardship in CL ulcer management, which was recently proposed for all chronic wounds.

Effect of secondary infection on epithelialisation and total healing of cutaneous leishmaniasis lesions

BACKGROUND Cutaneous leishmaniasis (CL) generally presents with a single or several localised cutaneous ulcers without involvement of mucous membranes. Ulcerated lesions are susceptible to secondary contamination that may slow the healing process. OBJECTIVE This study verified the influence of non-parasitic wound infection on wound closure (epithelialisation) and total healing. METHODS Twenty-five patients with a confirmed diagnosis of CL and ulcerated lesions underwent biopsy of ulcer borders. One direct microbial parameter (germ identification in cultures) and four indirect clinical parameters (secretion, pain, burning sensation, pruritus) were analysed. FINDINGS Biopsies of ten lesions showed secondary infection by one or two microorganisms (Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Streptococcus pyogenes and Candida parapsilosis). "Secretion" and "burning sensation" influenced epithelialisation time but not total healing time. Positive detection of germs in the ulcer border and "pain" and "pruritus" revealed no influence on wound closure. CONCLUSIONS Our borderline proof of clinical CL ulcer infection inhibiting CL wound healing supports the need to follow antimicrobial stewardship in CL ulcer management, which was recently proposed for all chronic wounds.

Spatial distribution, Leishmania species and clinical traits of Cutaneous Leishmaniasis cases in the Colombian army.

In Colombia, the cutaneous leishmaniasis (CL) is the most common manifestation across the army personnel. Hence, it is mandatory to determine the species associated with the disease as well as the association with the clinical traits. A total of 273 samples of male patients with CL were included in the study and clinical data of the patients was studied. PCR and sequencing analyses (Cytb and HSP70 genes) were performed to identify the species and the intra-specific genetic variability. A georeferenced database was constructed to identify the spatial distribution of Leishmania species isolated. The identification of five species of Leishmania that circulate in the areas where army personnel are deployed is described. Predominant infecting Leishmania species corresponds to L. braziliensis (61.1%), followed by Leishmania panamensis (33.5%), with a high distribution of both species at geographical and municipal level. The species L. guyanensis, L. mexicana and L. lainsoni were also detected at lower frequency. We also showed the identification of different genotypes within L. braziliensis and L. panamensis. In conclusion, we identified the Leishmania species circulating in the areas where Colombian army personnel are deployed, as well as the high intra-specific genetic variability of L. braziliensis and L. panamensis and how these genotypes are distributed at the geographic level.

In Vitro and In Vivo Control of Secondary Bacterial Infection Caused by Leishmania major.

Bacterial infections of cutaneous leishmaniasis cause skin ulcers on mice, resulting in increased tissue deterioration, and these infections can be controlled with liquid allicin. To isolate and identify the incidences of real secondary bacterial infections in mice, we performed the current study by injecting mice (n = 50) with Leishmania major. L. major infections were initiated by an intramuscular injection of 0.1 mL Roswell Park Memorial Institute (RPMI 1640 media/mouse (107 promastigote/mL)). Scarring appeared 2-6 weeks after injection, and the bacteria were isolated from the skin ulcer tissues. Allicin (50 µL/mL) and ciprofloxacin (5 µg; Cip 5) were used for controlling L. major and bacteria. One hundred samples from skin ulcers of mice were examined, and 200 bacterial colonies were isolated. Forty-eight different genera and species were obtained and identified by Gram staining and physiological and biochemical characterization using identification kits. All samples were positive for secondary bacterial infections. Of the isolates, 79.16% were identified as Gram-negative bacteria, and 28.84% were identified as Gram-positive bacteria; only one yeast species was found. Interestingly, pure allicin liquid at a concentration 50 µL/mL exhibited antibacterial activity against a wide range of Gram-negative and some Gram-positive bacteria, in addition to yeast, and was 71.43% effective. Antimicrobial resistance patterns of all genera and species were determined using 15 different antibiotics. Allicin (50 µL/mL) and Cip 5 were the most effective against L. major and 92.30% of isolated bacteria. Stenotrophomonas maltophilia was the most resistant bacterium to the tested antibiotics with a survival rate of 73.33%, and it exhibited resistance to allicin.

Skin Dysbiosis Goes "Off-Leish".

There is increasing interest in the contribution of microbes to skin disease. In this issue of Cell Host & Microbe, Gimblet et al. (2017) demonstrate that cutaneous leishmaniasis alters the human skin microbiota. In mice, this dysbiosis is transferable to naive animals, where it augments skin inflammation and disease severity.

Cutaneous Leishmaniasis Induces a Transmissible Dysbiotic Skin Microbiota that Promotes Skin Inflammation.

Skin microbiota can impact allergic and autoimmune responses, wound healing, and anti-microbial defense. We investigated the role of skin microbiota in cutaneous leishmaniasis and found that human patients infected with Leishmania braziliensis develop dysbiotic skin microbiota, characterized by increases in the abundance of Staphylococcus and/or Streptococcus. Mice infected with L. major exhibit similar changes depending upon disease severity. Importantly, this dysbiosis is not limited to the lesion site, but is transmissible to normal skin distant from the infection site and to skin from co-housed naive mice. This observation allowed us to test whether a pre-existing dysbiotic skin microbiota influences disease, and we found that challenging dysbiotic naive mice with L. major or testing for contact hypersensitivity results in exacerbated skin inflammatory responses. These findings demonstrate that a dysbiotic skin microbiota is not only a consequence of tissue stress, but also enhances inflammation, which has implications for many inflammatory cutaneous diseases.

A label-free, PCR-free and signal-on electrochemical DNA biosensor for Leishmania major based on gold nanoleaves.

Detection of leishmaniasis is important in clinical diagnoses. In the present study, identification of Leishmania parasites was performed by a label-free, PCR-free and signal-on ultrasensitive electrochemical DNA biosensor. Gold nanoleaves were firstly electrodeposited by an electrodeposition method using spermidine as a shape directing agent. The biosensor was fabricated by immobilization of a Leishmania major specific DNA probe onto gold nanoleaves, and methylene blue was employed as a marker. Hybridization of the complementary single stranded DNA sequence with the biosensor under the selected conditions was then investigated. The biosensor could detect a synthetic DNA target in a range of 1.0×10-10 to 1.0×10-19molL-1 with a limit of detection of 1.8×10-20molL-1, and genomic DNA in a range of 0.5-20ngµL-1 with a limit of detection of 0.07ngµL-1. The biosensor could distinguish Leishmania major from a non-complementary-sequence oligonucleotide and the tropica species with a high selectivity. The biosensor was applicable to detect Leishmania major in patient samples.