Cutaneous Leishmaniasis Patients Research Articles

Characterization of serum cytokines and circulating microRNAs that are predicted to regulate inflammasome genes in cutaneous leishmaniasis patients

Leishmaniasis is a neglected disease caused by an intracellular protozoan parasite of the genus Leishmania. Infection starts when this protozoan replicates in a phagolysosomal compartment in macrophages, after evading host immune responses. The balance of Th1 and Th2 immune responses is crucial in leishmaniasis because it will determine whether the infection will be under control or if clinical complications will occur. The inflammasome, which is activated during Leishmania infection, involves the action of caspase-1 and release of the proinflammatory cytokines interleukin-1β and interleukin-18. Together, they contribute to the maintenance of an inflammatory response and pyroptosis. Here, we evaluated the serum levels of cytokines and the expression of circulating microRNAs related to inflammasome regulation in twenty-seven patients with cutaneous leishmaniasis in comparison to nine healthy individuals, in the context of the inflammasome activation. Evaluation of serum cytokines activation (IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-17) was performed by flow cytometry using CBA kits (cytometric beads array) while the expression of circulating microRNAs (miR-7, miR-133a, miR-146b, miR-155, miR-223, miR-328, and miR-342) in plasma was measured by quantitative polymerase chain reaction. Our results showed an increase of the expression of miR-7-5p (p < 10−5), miR-133a (p = 0.034), miR-146b (p = 0.003), miR-223–3p (p = 10−5), and miR-328–3p (p = 0.002), and cytokine levels for IL-1β (p = 0.0005), IL-6 (p = 0.001), and IL-17 (p = 0.001) in patients with cutaneous leishmaniasis compared to the controls. These results suggest that microRNAs and cytokines can play an important role in regulating the human immune responses to Leishmania infection. Our findings may contribute to the understanding of the mechanisms of the gene regulation during the cutaneous leishmaniasis and to the identification of possible biomarkers of the infection.

Trypanothione Reductase Gene Mutations in Meglumine Antimoniate Resistant Isolates from Cutaneous Leishmaniasis Patients Using Molecular Dynamics Method.

Background:In this study, mutations in the tripanothion reductase of Leishmania tropica isolated from Iran was investigated using sequencing and simulation of the enzyme by the molecular dynamic method.Methods:Fifteen susceptible and 15 clinical resistant L. tropica specimens were collected from skin lesions from different regions of Iran in 2017. After DNA extraction, trypanothione reductase (TRYR or TPR), gene fragment was amplified using PCR and sequencing methods. In the case of structural mutations, the components were simulated by molecular dynamics using the GROMACS software.Results:Some structural mutations were observed in 9 amino acids surrounding the active site of the TRYR gene of L. tropica with three-dimensional trypanothione reductase alteration.Conclusion:Change in the active site of TRYR of L. tropica, could probably contribute to the development of resistant L. tropica to glucantime. Because of the likely occurrence of mutations in glucantime as well as the ease of development of L. tropica resistant populations, more samples are needed to demonstrate the relationship between mutations in this enzyme and clinical resistance to glucantime. On the other hand, it is recommended that enzymatic studies be performed to confirm the role of mutation in the function and expression of trypanothione reductase in glucantime resistant and susceptible populations.

Estimation of Interferon Gamma and Zinc Concentration in Serum of Cutaneous Leishmaniasis Patients in Tikrit City

Background: Interferon-gamma is one of the important cytokines, that has a role in the immune system of the body. Also zinc has optimal activity on the immune system and is involved in cell and tissue growth. The aim of this study is to evaluate IFN-γ and Zn concentration in the serum of CL patients and compare to control groups. Patients and Method: A total of 96 persons were included in this study, during the period from January/2018 to the end of June/2018 in the outpatients of Dermatology Department of Tikrit Teaching Hospital. Forty eight patients with CL were included in this study, 43.75% were males and 56.25% were females. Assessment of zinc concentration by using spectrophotometer, so interferon gamma by ELISA. Results: There was low serum zinc in CL patients comparing to control, since mean zinc concentration in patients was 9.6 μmol/I and in control was 11.4 μmol/l. Also this study revealed that there was decrease serum zinc in CL patients in contrast to control groups aged &gt;15 years old, since mean zinc concentration in patients was 9.7 μmol/l while in control was 10.9 μmol/l. Mean zinc concentration in male patients and control were (10.0 and 11.6) μmol/l respectively. Mean zinc concentration in female patients (9.4) μmol/l while in female control is higher (10.8) μmol/l. mean serum interferon gamma is significant increase in serum patients (P&lt;0.01) than control groups aged &lt; 15 old. Since mean of IFN-γ was 0.8 IU/ml in patients and 0.5 IU/ml in control. Also serum IFN-γ in patients and control groups (aged &gt; 15 years old) were (1.0 and 0.6) IU/ml respectively. In addition to that it was found that there is significant increase in IFN-γ concentration (P &lt;0.01) in male patients which was (1.1) IU/ml in comparison with the male control which was (0.6) IU/ml. Also the same thing is noticed in females. Mean IFN-γ in female patients and control were (0.7 l and 0.5) IU/ml Respectively.

Gender differences in the severity and features of lesions among Cutaneous Leishmaniasis patients

Objective: To determine if there is any differences in the severity and features of lesions among patients complaining of cutaneous leishmaniasis in an endemic region . Methods: A cross-sectional, observational, descriptive study was performed ( January 2014 to June 2019 ) in the dermatology clinics of : Rizgary Hospital , Hawler teaching center for Skin Diseases, Shadi Health Center, Hawler Institute of Health Prevention. All the patients were referred from both rural districts of Makhmur and Kalack. The provisional diagnosis was dependent mainly on clinical examination in addition to Giemsa stain. Parasite (amastigote) grading and distribution of number, site, type and size of lesions according to the gender of patients were studied . Results: A total of 1264 cutaneous leishmaniasis cases were diagnosed during the study period . According to stain results, 70.6% of the cases were positive to Giemsa stain. Parasite grading and parasite number /field were higher significantly in males . Features of case severity according to the characters of the lesions (number, size, site, type) were more noted in males than females. Conclusions: Male patients are more prone to more severe infections than females.

Gender differences in the severity and features of lesions among Cutaneous Leishmaniasis patients

Gender differences in the severity and features of lesions among Cutaneous Leishmaniasis patients

Socio-Demographic profile of Cutaneous Leishmaniasis patients in Hambantota District, Sri Lanka

Cutaneous leishmaniasis (CL) is known to Sri Lanka from 1992. The distribution of the disease is increasing and large numbers of CL cases are reported from Hambantota district. This study was focused on selected clinical features and socio-demographic factors of CL positive patients in Hambantota district and this information were collected using a questionnaire from CL suspected patients who attended to the Tangalle Hospital from June, 2016 to January, 2017. For selecting the CL positivity, slit skin smears were obtained from lesions of suspected patients. These smears were stained and examined for Leishmania amastigotes. Smears were positive in 183 patients. Majority of them had one lesion on the body and 117 patients were males (p 50 years. House-wives and students were identified as highly affected occupants. The higher number of CL patients lived in rural areas (p<0.001). Most of the CL patients lived in houses with cemented floors and walls with tiled -roofs meanwhile 117 houses had cracks, either in floors or walls. Further, 95.5% of patients were unaware of sand-fly biting. These findings highlight the socio-demographic profile of the CL patients in Hambantota district which will be important for the disease management strategies.

Effect of Antimonial Therapy on Levels of (TNF-α and IL-1β;) Cytokines in Cutaneous Leishmaniasis Patients in Iraq

Effect of Antimonial Therapy on Levels of (TNF-α and IL-1β;) Cytokines in Cutaneous Leishmaniasis Patients in Iraq

1662. Pushing the Dose: Miltefosine Treatment for a Supersized American with Cutaneous Leishmaniasis

BackgroundMiltefosine (Profounda, FL) is an oral alkylphospholipid drug which is approved by the Food and Drug Administration for the treatment of some species of New World cutaneous leishmaniasis. The maximal daily recommended dosage is 50 mg t.i.d for 28 days; yet there is some evidence that doses <2.5 mg/kg/day may be associated with lesser efficacy. We treated a healthy 30-year-old, 117 kg, 78 inches (low body fat) male with cutaneous Leishmania tropica infection with miltefosine after multiple unsuccessful treatment regimens. This presentation will provide the background considerations and the outcome of our approach of using higher daily dosages of miltefosine.MethodsWe used a miltefosine dose escalation of 50 mg t.i.d (1.28 mg/kg/day) for days 1–5 with fatty food, increasing to 50 mg. q.i.d. (1.71 mg/kg/day) for days 6–15. For days 16–28, the patient received 250 mg daily (2.13 mg/kg/day). Weekly blood testing was done for complete blood count, metabolic panel, and miltefosine pharmacokinetics. Plasma concentrations were assayed using a validated liquid chromatography coupled to tandem mass spectrometry methodology.ResultsThe patient experienced a good clinical result with his two ulcerative lesions on the left leg healing with full epithelialization by day 28. He tolerated miltefosine well until he escalated to 250 mg daily when he noted 2 hours of fatigue and dizziness after the dose, nausea and after the fourth day he developed epididymitis. His serum creatinine was elevated (1.4 mg/dL). The epididymitis resolved after approximately a week, his other symptoms resolved soon after completing the day 28 dose. Serial miltefosine plasma levels accumulated during treatment to 30, 34, 44, and 53 μg/mL on days 7, 14, 21, and 27 after the start of treatment (dropping to 27 μg/mL 8 days post), with an apparent distribution half-life of 7 days.ConclusionMiltefosine yielded healing of recalcitrant L. tropica infection but was associated with adverse effects at the 250 mg daily dose that severely limited the activity of the patient for the final 8 days of therapy; however, they were not dose-limiting. Miltefosine accumulation appeared to be dose-proportional compared with reported concentrations with a median 1.8 mg/kg/day dose in Dutch cutaneous leishmaniasis patients (median weight 85 kg).Disclosures All authors: No reported disclosures.

Genotyping of Causative Agents of Cutaneous Leishmaniasis in Patients Using PCR-RFLP Method in Dasht-e-Azadegan County, Southwest of Iran, in 2016

Background: As a neglected disease, cutaneous leishmaniasis renders considerable incidence rates. Eastern Mediterranean countries such as Iran are endemic for this infection. Objectives: Herein, we determined the species/strains of the causative agents of cutaneous leishmaniasis in Dasht-e-Azadegan, Khuzestan, Iran, using PCR-RFLP in 2016. Methods: We microscopically examined 80 smear slides from suspected patients referring to health centers of the city. After DNA extraction from slide materials, the amplification of the ITS1 fragment was done using the LITSR and L5.8S primer pair for Leishmania molecular detection. Subsequently, HaeIII (species level), TaqI (strain level), DpnI, and HpaII (mutation analysis) digestion was exerted based on the RFLP method. Results: Microscopic examination revealed the amastigote forms of Leishmania in all 80 samples. A 350 bp band was amplified by ITS1-PCR, which confirmed the infection at the molecular level. Following HaeIII digestion, 150 and 200 bp fragments were produced indicating Leishmania major. Also, TaqI digestion rendered 130 and 200 bp bands suggesting the A1 strain. Moreover, no mutations were detected in the genome of the identified L. major A1 strains by DpnI (140 and 200 bp bands) and HpaII (no digestion) digestion. Conclusions: The genotypic heterogeneity of Leishmania species is of utmost importance for better treatment choices, appropriate diagnosis, and preventive measures. Future work in the area should address the parasite strains in alternative vector/reservoir hosts.

Counting of Total and Differential White Blood Cells in Cutaneous Leishmaniasis Patients in Nassiryha city Thi-qar Province

The present study was carried out on 50 cutaneous Leishmaniasis patients , without taken treatments and they were having sore skin infection diagnosed by dermatologist as Cutaneous Leishmaniasis and clinical diagnosis and 25 control groups , attended five main hospitals in Thi-Qar province ,there are Imam Hussein Teaching Hospital , Shattra general hospital, Rifya general hospital , Suq Al-Shayokh general hospital and Al-Chibayish general hospital, during the period from the beginning of January 2017 to end in June in the same year. The statistical analysis were P≤ 0.05 considered statistically significant. There was significant decrease at p≤ 0.05 in mean of total WBCs in CL patients(6.36) compared to (controls groups) (7.80), also there was significant differences in mean of basophiles, monocytes , in CL patients(0.75 and 5.65) compared to (controls groups) (1.18 and 6.92) and there was increase and no significant differences were observed in mean of eosinophil’s in CL patients compared to (controls groups)( 2.31and 2.08) .There was significant were observed increase in mean of neutrophils and lymphocytes in CL patients (65.92 and 32.53)compared to controls groups (58.27 and 25.08).

Comparison of Nested-PCR and Loop-Mediated Isothermal Amplification for Cutaneous Leishmaniasis Diagnosis

Background: Leishmaniasis is a protozoan disease caused by Leishmania genus and its most common form is cutaneous leishmaniasis. The number of reported cases of cutaneous leishmaniasis in Khuzestan, southwest Iran, continues to increase. Therefore, early, accurate diagnosis is crucial for successful therapy. Objectives: The Nested PCR is a molecular method with high sensitivity and specificity in detecting leishmaniasis, but this method requires advanced equipment and skilled labor. Therefore, developing a simple yet accurate technique to diagnose leishmaniasis is essential. This study was designed to evaluate the possibility of replacing Nested PCR with loop-mediated isothermal amplification (LAMP) method for diagnosis of cutaneous leishmaniasis. Methods: We obtained 75 clinical samples from cutaneous leishmaniasis patients whose infection had already been confirmed by microscopic examination. The LAMP assay with pre-added malachite green was performed using a set of four primers targeting conserved sequences of the18S ribosomal RNA gene. The nested PCR method was performed using specific kinetoplast minicircle DNA primers. Cultured promastigotes of Leishmania major (MHOM /IR/75/ER), L. tropica (MHOM/IR/02/Mash10), and the virulent RH strain of Toxoplasma gondii were used as controls. Results: Our results showed that LAMP was positive for 100% of microscopically positive samples, which was similar to Nested PCR. The detection of L. major was improved at 104 parasites/ml using the LAMP method. Conclusions: Our findings suggest that considering the simplicity, specificity, and sensitivity of LAMP, it would be a potentially useful method in the diagnosis of leishmaniasis and detection of its various strains/species. This cost and time-effective method can be used as a suitable alternative for surveillance of leishmaniasis, as well as in epidemiological studies.

Immunodiagnosis of human and canine visceral leishmaniasis using recombinant Leishmania infantum Prohibitin protein and a synthetic peptide containing its conformational B-cell epitope

In the present study, Leishmania infantum's Prohibitin was cloned and, alongside a synthetic peptide, evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis (CVL and TL, respectively) in dogs and humans. For TL diagnosis, this study analyzed serum samples from cutaneous (n = 20) or mucosal (n = 39) leishmaniasis patients, and from Chagas disease (CD) patients (n = 8) and non-infected patients (n = 45). For CVL diagnosis, serum samples from asymptomatic (n = 14), symptomatic (n = 71), non-infected (n = 116), and Leish-Tec®-vaccinated (n = 79) dogs were examined, as well as T. cruzi (n = 11) and Ehrlichia canis (n = 10) infected animals. An indirect ELISA method using rProhibitin showed diagnostic sensitivity and specificity values of 91.76% and 89.91%, respectively. L. infantum SLA showed 86.11% and 48.24% of specificity and sensitivity, respectively, for CVL serodiagnosis, and 98.31% and 84.91% sensitivity and specificity, respectively for TL diagnosis. L. braziliensis SLA showed 75.47% and 83.05% of specificity and sensitivity, respectively, for TL diagnosis. The synthetic peptide showed a better result in TL than in CVL diagnosis. In conclusion, preliminary results suggest that the detection of antibodies against the rProhibitin protein and the synthetic peptide improves the serodiagnosis of TL and CVL.

IL-8 and IL-23 Levels in Peripheral Blood Mononuclear Cells of Patients with Cutaneous Leishmaniasis Caused by Leishmania major: A Case-Control Study

Background: Several cytokines contribute to cutaneous leishmaniasis outcomes. IL-23 is a key cytokine in inflammation and a central cytokine in controlling Th17. IL-8, also known as a neutrophil chemotactic factor in the lesion site of cutaneous leishmaniasis, indicates disease promotion. Objectives: This study aimed to evaluate the relationship of Leishmania major with IL-8 and IL-23 expression. Methods: This case-control study was performed on 25 healthy individuals and 25 cutaneous leishmaniasis patients who lived in Southwestern Iran, in 2017. Peripheral blood mononuclear cells (PBMCs) were first isolated. Total RNA was extracted and reverse-transcribed into cDNA. The expression levels of IL-8 and IL-23 were measured by the real-time PCR. The data were statistically analyzed using SPSS version 16. Results: Our findings indicated that the expression of IL-8 was significantly higher in the leishmaniasis group than in the control group with the median (IQR) of 0.39 (0.92) and 0.03 (0.11), respectively (P = 0.02). The expression of IL-23 was higher in the leishmaniasis group than in the control group with the median (IQR) of 0.13 (0.43) and 0.11 (0.61), respectively (P = 0.48). The increased IL-8 expression accompanied the location sites in face and hand and the increased number of skin lesions. Conclusions: The expression of IL-8 in patients with cutaneous leishmaniasis is an index of increased activity of local neutrophils that can contribute to leishmaniasis survival or inflammation increase. However, IL-23 appears to be less important in the inflammatory reaction than IL-8.

A comparative morphometrics of amastigote forms of &lt;em&gt;Leishmania donovani&lt;/em&gt; found in cutaneous leishmaniasis patients in Sri Lanka: evidence for the presence of promastigote-like structures

Objective: Investigations have not yet been carried out to identify the different morphological forms of amastigotes present in patients with cutaneous leishmaniasis (CL) in Sri Lanka. Thus, this paper describes the existence of different amastigote forms in cutaneous lesions for the first time in Sri Lanka. Methods: This was a retrospective study. One hundred and thirty skin smears were investigated to identify the different morphometric forms of L. donovani. In addition, demographic data (age, gender, occupation, household characteristics, and geographic area) were analyzed using department records. Results: Of the 130 samples, 84 (60.83%) samples had amastigote forms. Three (2.31%) samples had amastigotes in intracellular locations while 43 (33.08%) had amastigotes extracellularly. Nineteen (14.62%) samples had amastigotes in intracellular and extracellular locations simultaneously. Promastigote-like structures (PLS) were found in 65 (50%) samples. Of the 65 samples, 19 (14.62%) had both PLS and amastigote forms. PLS alone (no association with amastigote forms) were found in 46 (35.38%) samples. Amastigotes were found predominantly in lesions Conclusion: Microscopic examinations of skin smears revealed the presence of promastigote-like structures for the first time in patients with CL in Sri Lanka. Therefore, we suggest that different morphometric features of amastigotes should not be ignored as they may be useful in diagnosis of CL in clinically suspected patients.

Alterations in monocyte subsets and cytokine production after TLR activation in American Cutaneous Leishmaniasis.

Phenotypic and functional aspects of monocytes from Localized Cutaneous Leishmaniasis (LCL) patients were evaluated. The frequencies of monocyte subsets and TLR2/TLR4 expression were evaluated in fresh peripheral blood whereas cytokine production was evaluated in whole blood cell cultures stimulated with TLR agonists or Leishmania braziliensis antigen (Ag). CD16+ monocytes frequency was increased in patients compared with controls. A TLR4 agonist (LPS) induced expression of TNF and IL-10 in monocyte subsets of patients and controls. The CD14+ CD16+ monocytes expressed higher levels of these cytokines than CD14+ CD16- cells. The levels of secreted TNF were higher in whole blood cell cultures from patients than controls after LPS/TLR4 or Ag stimulation. Whereas in controls there was a positive correlation between TNF and IL-10 levels, this was not observed in stimulated cell cultures from patients. The high levels of LPS-induced TNF were associated with the number of lesions and the percentages of CD14hi CD16+ monocytes. The levels of TLR2-induced TNF were also associated with number of lesions. All monocyte subsets from patients expressed higher levels of TLR2 and TLR4 than controls. Data suggest that systemically activated monocytes contribute for an imbalance in pro- and anti-inflammatory cytokine production during LCL, participating in the immunopathogenesis of the disease.

Isolation and Molecular Identification of Leishmania spp. in Patients With Cutaneous Leishmaniasis in Golestan Province, Iran

Background and aims: Cutaneous leishmaniasis is a major health issue in many parts of Iran. Diagnosis of cutaneous leishmaniasis in a non-endemic area is not as simple as that in endemic foci. The management and treatment of this disease are global dilemmas. The purpose of this study was to identify Leishmania species isolated from human cutaneous leishmaniasis lesions in patients referred to health centers of Golestan province, Iran. Methods: Cutaneous leishmaniasis patients with suspected lesions were clinically examined. History of journey to the endemic areas of zoonotic cutaneous leishmaniasis and/or anthroponotic cutaneous leishmaniasis and the characteristics of their lesion(s) were recorded. Diagnosis of the lesion was done by using direct smear microscopy and conventional polymerase chain reaction. Results: Out of 360 patients, 202 (57.4 %) were male and 158 (42.8 %) were female. A total of 360 samples were selected from different cities. The number of infected samples in the cities is as follows: Azad Shahr [3 (1.5%)], Aq Qala [11 (5.7%)], Ali Abad [4 (2.1%)], Bandar Turkmen [3 (1.5%)], Bandar Gaz [2 (1%)], Ramian [3 (1.5%)], Kalaleh [23 (12.1%)], Kord koy [1 (0.5%)], Galikesh [7 (3.7 %)], Gomishan [12 (3.6 %)], Gorgan [13 (6.8 %)], Gonbad Qabus [99 (52.1 %)], Marave Tappe [7 (3.6%)] and Minoodasht [2 (1%)]. In the molecular investigations, 186 (96.8%) samples were observed to acquire rural cutaneous leishmaniasis, 4 (2.1%) to acquire urban cutaneous leishmaniasis and 170 (47.2%) to be uninfected. Conclusion: Although history of visiting endemic areas is an important factor to be considered in diagnosis, parasitological confirmation is necessary for the initiation of treatment.

Evaluation of clinical and demographic characteristics of Turkish and Syrian pediatric cutaneous leishmaniasis patients from Hatay, Turkey after the Syrian civil war.

IntroductionCutaneous leishmaniasis (CL) is a skin disease affecting all ages but especially children. Cutaneous leishmaniasis exists in Turkey, and, especially together with emigration from Syria to Turkey in recent years due to the civil war in Syria, the incidence of the disease has increased.AimTo investigate Syrian patients (SP) and Turkish patients (TP) in the pediatric age group who were diagnosed with CL and to compare the age, gender, clinical type, number of lesions, and lesion localizations of these patients.Material and methodsWe included CL patients aged 0-18 who were admitted to the outpatient clinic in the period 2015–2017 and in the first half of 2018. A total of 121 patients (SP, n = 87; TP, n = 34) were included in the study.ResultsThe mean ages of TP and SP were 12.06 ±4.47 and 8.68 ±5.18 years and the disease durations were 6.25 ±3.86 and 4.73 ±3.39 months respectively (p = 0.049). The total number of lesions was 247. The mean lesion number per child was 2.35 ±2.28 in SP, and 1.23 ±0.55 in TP (p = 0.002). Two and multiple lesions were significantly higher in SP (p = 0.005). It was found that the lesions were most frequently located in the head/neck (HN) region (76.9%) and 44.1% of patients with HN localization belonged to the 7–12 age group. We also found that 57.1% of the HN lesions were of the papule type in the patients.ConclusionsWe obtained similar results as in other studies in terms of age, gender, localization, and duration of lesions in general. When SP and TP were compared, we found that the number of lesions was higher, the disease duration was shorter, and the mean age was younger in SP.

Anti-tumour necrosis factor-induced cutaneous leishmaniasis in patient with psoriatic arthritis and uveitis

Anti-tumour necrosis factor-induced cutaneous leishmaniasis in patient with psoriatic arthritis and uveitis

Genomic insights into virulence mechanisms of Leishmania donovani: evidence from an atypical strain

BackgroundLeishmaniasis is a neglected tropical disease with diverse clinical phenotypes, determined by parasite, host and vector interactions. Despite the advances in molecular biology and the availability of more Leishmania genome references in recent years, the association between parasite species and distinct clinical phenotypes remains poorly understood. We present a genomic comparison of an atypical variant of Leishmania donovani from a South Asian focus, where it mostly causes cutaneous form of leishmaniasis.ResultsClinical isolates from six cutaneous leishmaniasis patients (CL-SL); 2 of whom were poor responders to antimony (CL-PR), and two visceral leishmaniasis patients (VL-SL) were sequenced on an Illumina MiSeq platform. Chromosome aneuploidy was observed in both groups but was more frequent in CL-SL. 248 genes differed by 2 fold or more in copy number among the two groups. Genes involved in amino acid use (LdBPK_271940) and energy metabolism (LdBPK_271950), predominated the VL-SL group with the same distribution pattern reflected in gene tandem arrays. Genes encoding amastins were present in higher copy numbers in VL-SL and CL-PR as well as being among predicted pseudogenes in CL-SL. Both chromosome and SNP profiles showed CL-SL and VL-SL to form two distinct groups. While expected heterozygosity was much higher in VL-SL, SNP allele frequency patterns did not suggest potential recent recombination breakpoints. The SNP/indel profile obtained using the more recently generated PacBio sequence did not vary markedly from that based on the standard LdBPK282A1 reference. Several genes previously associated with resistance to antimonials were observed in higher copy numbers in the analysis of CL-PR. H-locus amplification was seen in one cutaneous isolate which however did not belong to the CL-PR group.ConclusionsThe data presented suggests that intra species variations at chromosome and gene level are more likely to influence differences in tropism as well as response to treatment, and contributes to greater understanding of parasite molecular mechanisms underpinning these differences. These findings should be substantiated with a larger sample number and expression/functional studies.

Ligand-Capped Ultrapure Metal Nanoparticle Sensors for the Detection of Cutaneous Leishmaniasis Disease in Exhaled Breath

Human cutaneous leishmaniasis, although designated as one of the most neglected tropical diseases, remains underestimated due to its misdiagnosis. The diagnosis is mainly based on the microscopic detection of amastigote forms, isolation of the parasite, or the detection of Leishmania DNA, in addition to its differential clinical characterization; these tools are not always available in routine daily practice, and they are expensive and time-consuming. Here, we present a simple-to-use, noninvasive approach for human cutaneous leishmaniasis diagnosis, which is based on the analysis of volatile organic compounds in exhaled breath with an array of specifically designed chemical gas sensors. The study was realized on a group of n = 28 volunteers diagnosed with human cutaneous leishmaniasis and a group of n = 32 healthy controls, recruited in various sites from Tunisia, an endemic country of the disease. The classification success rate of human cutaneous leishmaniasis patients achieved by our sensors test was 98.2% accuracy, 96.4% sensitivity, and 100% specificity. Remarkably, one of the sensors, based on CuNPs functionalized with 2-mercaptobenzoxazole, yielded 100% accuracy, 100% sensitivity, and 100% specificity for human cutaneous leishmaniasis discrimination. While AuNPs have been the most extensively used in metal nanoparticle-ligand sensing films for breath sensing, our results demonstrate that chemical sensors based on ligand-capped CuNPs also hold great potential for breath volatile organic compounds detection. Additionally, the chemical analysis of the breath samples with gas chromatography coupled to mass spectrometry identified nine putative breath biomarkers for human cutaneous leishmaniasis.