Therapeutic Efficacy of Arnica in Hamsters with Cutaneous Leishmaniasis Caused by Leishmania braziliensis and L. tropica.

Leishmaniasis may occur in three different clinical forms, namely, visceral, mucocutaneous and cutaneous, which are caused by different species of trypanosomatid protozoans of the genus Leishmania. Pentavalent antimonials are the leading treatment for cutaneous leishmaniasis despite the hepatic, renal, and cardiac toxicity. In addition, the response of some Leishmania species to pentavalent antimonials is increasingly poorer, and therefore new and more potent therapeutic alternatives are needed. Arnica montana L., Asteraceae, is a traditional medicinal plant of Europe and preparations of its flowers are commonly used externally to treat disorders of the musculoskeletal system as well as superficial inflammatory conditions. Previous studies have shown that Arnica tincture (AT), an ethanolic extract prepared from the flowerheads of Arnica montana as well as isolated Arnica sesquiterpene lactones (STLs) have antileishmanial activity in vitro against L. donovani and L. infantum, as well as in vivo against L. braziliensis. In this work, we studied the in vitro cytotoxicity and antileishmanial activity of AT and STLs against both L. braziliensis and L. tropica. The in vivo therapeutic effect of AT was studied in hamsters with cutaneous Leishmaniasis (CL) caused by experimental infection with L. braziliensis and L. tropica. Furthermore, various semisolid Arnica preparations were also evaluated against L. braziliensis. The STLs and the AT possess a very high in vitro activity against both Leishmania species with median effective concentrations (EC50) ranging from 1.9 to 5.9 µg/mL. The AT was not cytotoxic for human tissue macrophages, skin fibroblasts, and hepatic cells. The therapeutic response of hamsters infected with L. braziliensis to the topical treatment with AT was 87.5% at a dose of 19.2 µg STL/2× day/60 d, 72.7% at doses of 19.2 µg STL/1× d/60 d and 67% at a dose of 38.4 µg STL/2× d/60 d. In turn, the therapeutic response in hamsters infected with L. tropica was 100% when treated at a dose of 19.2 µg STL/2× day/60 d and 71% at a dose of 38.4 µg STL/2× d/60 d. On the other hand, the effectiveness of treatment with glucantime administered intralesionally at a dose of 200 mg/every three days for 30 days was 62.5% for L. braziliensis and 37.5% for L. tropica infection. These results are promising and encourage the implementation of clinical trials with AT in CL patients as a first step to using AT as a drug against CL.

Assessment of in vitro and in vivo effect of Quercetin 3-Glucoside, Oxyresveratrol and Quercetin O-Hexoside against Leishmania tropica

Abstract The aim was to scrutinize the in vivo and in vitro activities against Leishmania tropica with compounds of Oxyresveratrol, Quercetin O-Hexoside, and Quercetin 3-Glucoside. The in vitro outcomes against Leishmania were analyzed for 24-48 hours on L. tropica KWH23 promastigotes with compounds materials having 50 - 200 µg/mL concentration with negative control and standard drug Amphotericin B. The compounds were analyzed in L. tropica infected BALB/c mice against Leishmania tropica. The Quercetin 3-Glucoside shows mean inhibition of extracellular promastigotes after 48 hours at 50, 100, 150, 200 µg/mL were 91.02 ± 0.12, 94.50 ± 0.07, 96.15 ± 0.17 and 97.01 ± 0.08 % respectively. In BALB/c mice, the intracellular amastigotes were 91% cured at 200 µg/mL and mean lesion size decreased to 0.41 ± 0.21 mm (p < 0.01). The result shows that Quercetin 3-Glucoside possesses significant anti-leishmanial activity.

Anti-leishmanial activity and identification of chemical constituents of Scrophularia striata essential oil by head space solid phase micro extraction gas chromatography mass spectrometry (HS-SPME GC-MS) / Actividad anti-leishmanial e identificación de componentes químicos del aceite esencial de Scrophularia striata mediante microextracción en fase sólida en el espacio de cabeza seguido de cromatografía gaseosa acoplada a espectrometría de masas (HS-SPME GC-MS)

The aim of the present study was to determine the main constituents of Scrophularia striata essential oil and to evaluate in vitro effect of essential oil on Leishmania tropica and Leishmania major promastigotes and axenic amastigotes. Chemical constituents of the extracted essential oil were separated by headspace solid-phase microextraction (HS-SPME) equipped with a PDMS/DVB fiber. The fiber was injected to gas chromatogram- mass spectroscopy (GC-MS) to determine their identity. Finally, after exposure of parasites to different concentrations of water soluble fraction of essential oil, viability of promastigotes and axenic amastigotes were investigated. Based on the HS-SPME results, 47 compounds representing 95.6% of the total oil, were identified in essential oil. Essential oil analysis showed that nonane (19.7%), α-terpineol (17.4%) and linalool (10.2%) were the most abundant compounds. This study indicates that water soluble fraction of S. striata essential oil has promising anti-leishmanial activity.

El objetivo del presente estudio fue determinar los principales componentes del aceite esencial de Scrophularia striata y evaluar el efecto in vitro del aceite esencial en promastigotes y amastigotes axénicos de Leishmania tropica y Leishmania major. Los componentes químicos del aceite esencial extraído se separaron mediante microextracción de fase sólida en el espacio superior (HS-SPME) equipado con una fibra PDMS/DVB. Para determinar su identidad la fibra se inyectó en un cromatógrafo de gases acoplado un espectrómetro de masas (GC-MS). Finalmente, después de la exposición de los parásitos a diferentes concentraciones de fracción soluble del aceite esencial en agua, se investigó la viabilidad de los promastigotes y los amastigotes axénicos. En base a los resultados de HS-SPME, se identificaron 47 compuestos que representan el 95.6% del aceite total en el aceite esencial. El análisis de aceites esenciales mostró que el nonano (19.7%), el α-terpineol (17.4%) y el linalol (10.2%) fueron los compuestos más abundantes. Este estudio indica que la fracción soluble en agua del aceite esencial de S. striata tiene una actividad antileishmanial prometedora.

Predominance of Leishmania major and rare occurrence of Leishmania tropica with haplotype variability at the center of Iran.

BACKGROUND: Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. METHODS: All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. RESULTS: Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n = 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n = 48, 53.93%) than L. tropica (n = 4, 4.49%) (Mann-Whitney U test: p < 0.05) and two samples (2.25%) remained ambiguous after several sequencing. L. major did not have diversity with two common haplotypes but L. tropica were found to exhibit high diversity with three novel haplotypes. CONCLUSION: L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies.

Predominance of Leishmania major and rare occurrence of Leishmania tropica with haplotype variability at the center of Iran

ABSTRACT Background Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. Methods All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. Results Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n= 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n= 48, 53.93%) than L. tropica (n= 4, 4.49%) (Mann-Whitney U test: p< 0.05) and two samples (2.25%) remained ambiguous after several sequencing. L. major did not have diversity with two common haplotypes but L. tropica were found to exhibit high diversity with three novel haplotypes. Conclusion L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies.

Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing.

Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION™ sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.