Biophysical and ultrasonographic changes of acute Old World cutaneous leishmaniasis skin lesions in comparison with uninvolved skin: A possible tool for non-invasive early detection and treatment outcome assessment.

Cutaneous leishmaniasis (CL) is a skin disease caused by intracellular protozoa, which is endemic in Iran. The goal of this study was to compare biophysical characteristics in CL lesions with uninvolved skin. Stratum corneum hydration, transepidermal water loss, surface friction, pH, sebum, melanin, erythema, temperature, elasticity parameters (R0, R2, and R5), thickness and echo-density of epidermis and dermis were measured on the active erythematous indurated part of a typical CL lesion in 20 patients, and compared with the same location on the other side of the body as control. Paired t-test was used for statistical analyses and a p < 0.05 was considered significant. Melanin content, R2 and echo-density of dermis were significantly lower, whereas transepidermal water loss, friction index, pH, erythema index, temperature, and the thickness of dermis were significantly higher in CL lesions. There was no significant difference in stratum corneum hydration, sebum, R0, R5, thickness of epidermis, and density of epidermis between CL and normal skin. CL lesions are characterized by certain changes in biophysical and ultrasonographic properties, which are mostly correlated with histological features. These changes are likely to be useful in the non-invasive early detection of CL and also as treatment outcome measures for clinical trials of new treatment modalities for CL in the future.

Immunotherapeutic effects of Glycyrrhiza glabra and Glycyrrhizic Acid on Leishmania major-infection BALB/C mice.

Treatment of cutaneous leishmaniasis (CL) is a public health problem in endemic areas. The objective of the current study was to investigate the immunotherapeutic activities of the hydroalcoholic extract of Glycyrrhiza glabra (HEG) and glycyrrhizic acid (GA) in the treatment of Leishmania major (L. major)-infected BALB/c mice. In this study, the effect of HEG and GA was checked in vitro on growth of L. major promastigote and amastigote using MTT assay and microscopic counting, respectively. For in vivo experiment, the lesion induced by L. major on BALB/c mice were treated intraperitoneally with HEG, GA, meglumine antimoniate or phosphate buffer saline (negative control) for one month. Then, the lesion development and the parasite burden of the lymph node was assessed, the cytokine response (IFN-γ and IL-4) to Leishmania antigens was evaluated using ELISA method. The results showed that HEG and GA significantly inhibited the growth of L. major promastigotes and amastigotes, the lesion development, parasite burden in the lymph nodes, level of IFN-γ and the ratio of IFN-γ/IL-4 in HEG, GA and meglumine antimoniate-treated mice were significantly higher compared with the negative control group, there was no difference between the HEG, GA and meglumine antimoniate group. It is concluded that hydroalcoholic extract of G. glabra and glycyrrhizic acid showed therapeutic and immunomodulatory effects on L. major-infected BALB/c mice.

The in vitro effect of nanoliposomal amphotericin B against two clinically important dermatophytes.

AIMS: The present study aimed to investigate the antifungal activity of amphotericin B-loaded nanoliposomes against Trichophyton interdigitale and Trichophyton rubrum. Moreover, it was attempted to assess the obtained resistance in vitro. METHODS: In total, 29 archived clinical strains, namely, T. interdigitale (n = 16) and T. rubrum (n = 13), were included in this study. These strains were determined using a previous ITS1-ITS2 region sequence. Moreover, a liposomal formulation of amphotericin B was formulated by a thin-film hydration method. Particle size, polydispersity index (PdI), and zeta potential (ZP) were measured by a Zetasizer. Furthermore, physicochemical properties, such as appearance, aggregation of particles, particle size, PdI, and ZP, were determined at 0-, 1-, and 3-month intervals. A scanning electron microscope (SEM) was also used to examine nanoparticles structure. The minimum inhibitory concentration (MIC) of amphotericin B-loaded nanoliposomes, itraconazole, efinaconazole, terbinafine, and ciclopirox was determined according to the protocol of the broth microdilution method of CLSI M38-A2. The morphological changes of T. interdigitale and T. rubrum strains exposed to the amphotericin B-loaded nanoliposomes were observed using SEM. RESULTS: The amphotericin B-loaded nanoliposomes displayed a lower MIC compared to those of the amphotericin B and liposomes when used separately. Based on the results, amphotericin B-loaded nanoliposomes induced no drug resistance in any of the tested strains. CONCLUSION: Accordingly, amphotericin B-loaded nanoliposomes can be a potent antifungal for the topical treatment of onychomycosis. There was no in vitro evidence regarding the resistance of the tested strains to amphotericin B-loaded nanoliposomes. This reflects that amphotericin B-loaded nanoliposomes have a low probability to induce drug resistance in dermatophyte species.

A Case of Terbinafine-Resistant Tinea Cruris Caused by Trichophyton tonsurans.

A 26-year-old male patient referred to our center with a history of extremely itchy crusted skin lesions in his groins for one year. Moreover, his friend, a 25-year-old male, also developed similar lesions in the groin after using the shared pool, whose condition also did not improve with similar treatment. A regular mycology test (direct and culture test) was performed, as well as molecular examination. The antifungal susceptibility assay to terbinafine, itraconazole, posaconazole, fluconazole, and voriconazole was conducted according to the Clinical and Laboratory Standards Institute M38 third ed. The sequencing study identified T. tonsurans as the causative organism in both patients. The abovementioned organism isolated from both patients displayed resistance against terbinafine and fluconazole (MIC ≥ 4 µg/ml and MIC ≥ 8 µg/ml, respectively). Moreover, the susceptibility of both subjects to posaconazole (0.313 µg/ml), voriconazole (0.25-0.0625 µg/ml), and (1 µg/ml) itraconazole increased. The present report aimed to emphasize the increase in antifungal resistance and a demand for antifungal stewardship, to control this public health threat.

Drug Sensitivity Profile of Fungi Isolated from Onychomycosis Patients and Evaluation of Squalene Epoxidase Mutation in One Terbinafine-Resistant Trichophyton mentagrophytes Species.

Background: The resistance to treatment of onychomycosis is increasingly reported. The present study aimed to assess the antifungal activity of itraconazole, terbinafine, luliconazole, and efinaconazole against dermatophytes, molds, and also yeast isolated from patients with onychomycosis. Furthermore, the mechanism of resistance to terbinafine in resistant Trichophyton mentagrophytes species was evaluated using the squalene epoxidase (SQLE) gene sequence. Methods: A total of 71 fungal isolates were collected from 97 patients with suspected onychomycosis. The identification of fungal species was performed using conventional and molecular approaches. In vitro drug susceptibility for itraconazole, terbinafine, luliconazole, and efinaconazole was carried out using the broth microdilution method according to the CLSI-M60 and CLSI-M38 3rd ed., respectively. The SQLE gene of one terbinafine-resistant T. mentagrophytes was amplified using the specific primers. Results: Efinaconazole and luliconazole demonstrated higher effectiveness against all isolates in the study. One mismatch was detected at position 1177, which showed A → C change associated with Phe397Leu amino acid substitution of the SQLE protein in terbinafine-resistant T. mentagrophytes. Conclusion: The occurrence of resistant strains of organisms causing onychomycosis should be considered and evaluated. Furthermore, the identification of amino acid changes responsible for resistance to antifungals is a useful consideration in drug-target interaction.

Immune Responses in Cutaneous Leishmaniasis: In vitro Thelper1/Thelper2 Cytokine Profiles Using Live Versus Killed Leishmania major.

BACKGROUND: Recovery from cutaneous leishmaniasis (CL) leads to protection against further lesion development. In contrast, vaccination using killed parasites does not induce enough protection; the reason(s) is not currently known but might be related to different immune response induced against live versus killed parasites. In this study, Th1/Th2 cytokine profiles of CL patients were evaluated against live versus killed Leishmania major. METHODS: In this study peripheral blood mononuclear cells (PBMC) of the volunteers with active CL lesion (CL), history of CL (HCL) and healthy volunteers were cultured and stimulated with live or killed Leishmania major, the supernatants were collected and levels of IFN-γ, IL-5 and IL-10 were titrated using ELISA method. RESULTS: The results showed that IFN-γ levels in CL patients (p< 0.001) and HCL volunteers (p< 0.005) are significantly higher when stimulated with live than stimulated with killed L. major. IFN-γ production in PBMC volunteers with CL and HCL stimulated with live or heat-killed L. major was significantly (p< 0.001) higher than in unstimulated ones. The level of IL-5 in CL patients (p< 0.005) and HCL volunteers (p< 0.001) are significantly lower when stimulated with live than killed L. major. There was no significant difference between the levels of IL-10 in PBMC stimulated with either live or killed L. major. CONCLUSION: It is concluded that using live Leishmania induces a stronger Th1 type of immune response which justify using leishmanization as a control measure against CL.

Immune response in cutaneous leishmaniasis patients with healing vs. non-healing lesions.

BACKGROUND AND OBJECTIVES: The outcome of Leishmania infection mainly depends upon the Leishmania species which causes the disease and the generation of the type of host immune response, the healing process and protection in leishmaniasis depends upon induction of Th1 response. In this study, the Th1/Th2 cytokine profile in cutaneous leishmaniasis (CL) is evaluated. MATERIALS AND METHODS: This study was carried out in leishmaniasis clinic of CRTSDL, TUMS, during March 2018 to March 2019. Peripheral blood mononuclear cells (PBMC) of volunteers with active healing and non-healing lesion (s) of cutaneous leishmaniasis (CL), volunteers with and without history of CL were cultured and stimulated with Soluble Leishmania antigen (SLA). The supernatants were collected and the levels of IFN-γ, IL-5 and IL-10 were titrated using ELISA method. RESULTS: The results showed a significantly higher levels of IFN-γ in volunteers with active CL healing form (p<0.005), history of CL (p<0.005) than healthy volunteers. A significantly (p<0.005) higher level of IFN-γ was seen in volunteers with active healing form of lesion than non-healing form. There was a significantly (p<0.005) higher level of IL-10 in volunteers with a history of non-healing form and active non-healing form of CL. There was no significant difference in IL-5 production in PBMC of different groups. CONCLUSION: IFN-γ production starts at early stage of cutaneous leishmaniasis and enhance during course of lesion healing, IFN-γ level is significantly higher in all patients compared to healthy volunteers, IFN-γ is significantly higher in patients with healing form than non-healing form of lesion.

Safety Evaluation of Topical Application of Nano-Liposomal Form of Amphotericin B (SinaAmpholeish) on Healthy Volunteers: Phase I Clinical Trial.

BACKGROUND: We aimed to evaluate the safety of SinaAmpholeish in a double-blind, randomized, phase 1 clinical trial in healthy human volunteers. METHODS: The study was carried out in DermaLab of Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran in 2012. A topical Nano-liposomal formulation of 0.4% Amphotericin B was developed against Leishmania under trade name of SinaAmpholeish. In this randomized, double-blind, right-left, comparative, phase I clinical trial, in 2 steps; 7 and 20 healthy volunteers were recruited and applied SinaAmpholeish on the right and its vehicle on the left volar side of forearm, twice a day for one week or 3 times a day for two weeks. Seven biophysical skin parameters were measured in standard conditions before and 2 wk after application. RESULTS: There was no adverse effect when SinaAmpholeish and its vehicle were used twice a day for seven days. However, when were used 3 times a day for two weeks, both SinaAmpholeish and its vehicle induced severe local skin reactions in 2 volunteers leading to discontinuation of application. Mild and temporary local reactions were observed in about half of the application sides and there was no significant difference between SinaAmpholeish and its vehicle. CONCLUSION: The new formulation is safe and worth to be tested in further phase 2 clinical trial and since there was no adverse effect with twice a day application it was decided to use SinaAmpholeish twice a day in phase 2 clinical trial.

Safety Evaluation of Nano-Liposomal Formulation of Amphotericin B (Sina Ampholeish) in Animal Model as a Candidate for Treatment of Cutaneous Leishmaniasis.

BACKGROUND: Development of a topical treatment for cutaneous leishmaniasis (CL) is an important step in the improvement of lesion management. Amphotericin B (AmB) is effective against Leishmania species but it is toxic, a Nano-liposomal form of AmB with a size of about 100nm (Lip-AmB) was developed and showed to be effective against Leishmania major, and Leishmania tropica in vitro and against L. major in vivo in animal model. This study was designed to check the irritancy Draize test in rabbits and was completed in the Center for Research and Training in Skin Diseases and Leprosy, TUMS, in 2012. METHODS: Twenty rabbits in 3 steps were housed individually with artificial lighting (12/12h light/dark). SinaAmpholeish cream or empty liposomes (prepared under GMP condition at Minoo Company, Tehran, Iran), was applied on a gauze patch and the patches were placed on the designated sites of the skin in the back of the rabbits. At 48 and 72h later, the erythema and oedema were checked, scored and recorded. RESULTS: The erythema score in rabbits was 0.83+0.41 for the SinaAmpholeish and 0.5+0.55 for empty liposomes (P= 0.16). The average score for oedema was 0.67+0.52 for SinaAmpholeish and 0.33+0.52 for empty liposomes (P= 0.16). CONCLUSION: Based on skin irritancy reactions the topical formulation of SinaAmpholeish is safe and could be further checked in human trials.

Lived Experiences of Patients Suffering from Acute Old World Cutaneous Leishmaniasis: A Qualitative Content Analysis Study from Iran.

BACKGROUND: The aim of this study was to explore the experiences of patients who suffer from acute cutaneous leishmaniasis in Iran, focusing on quality of life. METHODS: The study was conducted at two different sites in Iran in 2010-2011. Individual in-depth interviews were conducted with six men and six women parasitologically confirmed acute cutaneous leishmaniasis. Interviews were recorded, transcribed verbatim, and translated into English. Qualitative content analysis was used for data analysis. RESULTS: The participants, aged 23 to 63yr, had mild to severe disease. Based on the analysis four main themes were developed. "Fearing an agonizing disease" reflects patients' experiences of disease development resulting in sadness and depression, "struggling to cope" and "taking on the blame" both illustrate how patients experience living with the disease, which included both felt and enacted stigma as major social concerns. "Longing for being seen and heard" refers to patients' experiences with healthcare as well as their expectations and demands from communities and healthcare to be involved in closing the knowledge and awareness gap. CONCLUSION: Mental and social dimensions of cutaneous leishmaniasis were complex and adversely affected patients' lives by causing psychological burden and limiting their social interactions. Health authorities have to plan programs to increase the disease awareness to prevent the existing stigma to improve patients' social condition and medical care.

The efficacy of hydro alcoholic extract of Seidlitzia rosmarinus on experimental zoonotic cutaneous leishmaniasis lesions in murine model.

OBJECTIVE: Leishmaniasis is one of the most important parasitic infectious diseases in the world. Since last century, many efforts have been made to control and treat the disease, but appropriate vaccines, pesticides and medicines are not available or even eligible. The purpose of this study was to evaluate the effect of hydro-alcoholic extract of Seidlitzia rosmarinus on the lesions of experimental Cutaneous Leishmaniasis (CL) in Balb/c mice. MATERIALS AND METHODS: The population study was 60 Ballb/c mice which divided to 6 groups, all infected with Leishmania major [MRHO/75/IR]. Soon after the ulcer started to appear in the early stage, a dose of provided herbal extract with 5, 10 and 15% concentration applied on each lesion. The surface area of the lesions measured during an interval of 10 days. Direct Giemsa stained smears prepared two and four weeks after treatment. RESULTS: Increasing the mean size of the lesions was statistically significant compared to those in control group (p>0.001). Visceral Leishmaniasis (VL) developed in all of the mice including the control group that received Eucerine alone. Survival rate in group receiving 15% S. rosmarinus extracts showed significantly higher compared to mice in control group (p<0.001). CONCLUSION: Hydro-alcoholic extracts of S.rosmarinus with concentrations below15% did not show a therapeutic effect on experimental CL ulcers of Balb/c mice. Further studies with higher concentrations or nano particles are recommended.

CCR7+ central and CCR7- effector memory CD4+ T cells in human cutaneous leishmaniasis.

PURPOSE: The profile of central (=T(CM)) and effector (=T(EM)) memory CD4(+) T cell subsets and the possible role as surrogate markers of protection is studied in the volunteers with history of cutaneous leishmaniasis (HCL). METHODS: Profile of T cell subsets based on CCR7/CD45RA expressions and phenotypic changes after soluble Leishmania antigen (SLA) stimulation were analyzed. Then, sorted CD4(+)CD45RO(-)CD45RA(+) naïve T, CD4(+)CD45RO(+)CD45RA(-)CCR7(-) T(EM,) CD4(+)CD45RO(+)CD45RA(-)CCR7(+) T(CM) subsets were cultured with SLA for proliferation, cytokine production and intracellular cytokine assays. RESULTS: In the HCL and control volunteers, the mean frequencies of CD4(+)CD45RA(+)CCR7(+) naïve T cells and CD4(+)CD45RA(-)CCR7(-) T(EM) cells were higher than the other subsets before culture. Frequency of naïve T cells and CD4(+)CD45RA(-)CCR7(+) T(CM) cells was significantly decreased (P=0.01 for naïve T and P<0.05 for T(CM) cells) and frequency of T(EM) cells was significantly increased after SLA stimulation compared to before culture (P<0.001). By CFSE labeling, CD4(+)CD45RO(+)CD45RA(-)CCR7(+) T(CM) cells showed more proliferation potential than CD4(+)CD45RO(+)CD45RA(-)CCR7(-) T(EM) cells. Stimulation of the T(EM) cells in HCL volunteers induced a significantly higher IFN-γ production (P=0.04) with higher number of intracellular IFN-γ positive cells (P=0.032) than the same cells from controls. A significantly higher number of T(CM) cells produced IL-2 in HCL volunteers compared with controls (P<0.05). Most of the intracellular IFN-γ positive T(EM) cells were proliferating CFSE-dim populations (P<0.05). CONCLUSIONS: A combination of Leishmania-reactive IFN-γ producing CD4(+)CD45RO(+)CD45RA(-)CCR7(-) T(EM) and Leishmania-reactive IL-2 producing CD4(+)CD45RO(+)CD45RA(-)CCR7(+) T(CM) are identified in individuals with history of CL which might play a role in protective recall immune response against Leishmania infection.

Evaluation of a Possible Synergistic Effect of Meglumine Antimoniate with Paromomycin, Miltefosine or Allopurinol on in Vitro Susceptibility of Leishmania tropica Resistant Isolate.

BACKGROUND: Pentavalent antimonials are still the first choice treatment for leishmaniasis, but with low efficacy and resistance is emerging. In the present study, the effect of meglumine antimoniate (MA, Glucantime) combined with paromomycin, miltefosine or allopurinol on in vitro susceptibility of Leishmania tropica resistant isolate was evaluated. METHOD: The drugs were obtained from commercial sources and diluents of each drug in medium were prepared on the day of experiment. J774 A.1 murine macrophage cell lines were attached to the cultured on slide and incubated at 37 °C with 5% CO2 for 24 h. Then the stationary phase promastigotes were added to the cells and after 4 hrs of incubation different concentrations of MA, paromomycin, miltefosine or allopurinol were added and incubated for an additional of 72 h. Then the slides were dried and fixed with methanol, stained by Giemsa and studied under a light microscope. Drug activity was evaluated by assessing the macrophage infection rate and the number of amastigotes per infected macrophage was done by examining 100 macrophages. The experiment was done in triplicates. RESULT: Various concentrations of MA along with paromomycin, miltefosine or allopurinol significantly inhibited (P<0.01) the proliferation of L. tropica amastigote stage in the macrophage cell line as compared with MA alone or positive control. CONCLUSION: Combination of Glucantime with paromomycin, miltefosine or allopurinol showed a synergistic effect on the clinical isolate of L. tropica in vitro. Use of combination therapy is a new hope and a logical basis for therapy of the patients with cutaneous leishmaniasis. Further investigations are needed to evaluate the therapeutic effects of these drugs on the CL patients.

Altered serum pro-inflammatory cytokines in children with Down's syndrome.

There are reports showing that pro-inflammatory cytokines are dysregulated in patients with Down's syndrome (DS). However, most of these reports concern adults. We analyzed cytokine levels in serum samples from children with DS, and compared them with samples from intellectually disabled (ID), and healthy, control children. Blood samples were collected from 24 DS, 24 age-/sex-matched ID, and 24 age-/sex-matched healthy, control children. Serum levels of the cytokines IL-5, IL-10, IL-13, IFN-γ, and TNF-α were measured using a sandwich ELISA method, . The age range of the children was 1-15 years, with a mean ± SD of 5.75 ± 4.36 years. TNF-α levels were significantly higher in the DS and ID groups compared with those found in healthy, control children (P<0.05). The DS and ID groups had significantly higher IFN-γ levels compared with healthy, control children (P = 0.0002 and P<0.01, respectively), with significant higher levels in the DS than the ID group (P<0.05). Serum from the ID group showed significantly higher IL-10 levels compared with those from the DS group (P<0.05), but not the healthy, control group. Significant correlations were found between the differences in TNF-α and IFN-γ levels, in both ID (rs = 0.558; P = 0.005) and DS children (rs = 0.405; P<0.05). There were no significant differences found in serum levels of IL-13 between the groups, and IL-5 was not detectable in any of the serum samples. Levels of TNF-α and IFN-γ were increased, and IL-10 decreased in serum from children with DS. It may be that these differences contribute to the clinical symptoms seen in DS: consequently, these pro-inflammatory cytokines might be useful as early biomarkers of the disorders associated with DS.

Phenotyping of circulating CD8⁺ T cell subsets in human cutaneous leishmaniasis.

Recovery from CL is usually accompanied with long-lasting protection and induction of strong immune response. The phenotypes, generation and maintenance of central (=T(CM)) and effector (=T(EM)) memory T cell subsets in human leishmaniasis are not well known. Profile of T cell subsets were analyzed on peripheral CD8⁺ T cells from volunteers with history of cutaneous leishmaniasis (HCL). In HCL and control groups, mean frequencies of CCR7⁺CD45RA⁺CD8⁺ naïve and CCR7⁻CD45RA⁻CD8⁺ T(EM) cells were higher than other subsets before culture, but after stimulation with soluble Leishmania antigen, the frequency of naïve T cells was significantly decreased and the frequency of T(EM) cells was significantly increased. T(EM) phenotype composed the highest portion of proliferating Carboxy Fluorescein diacetate Succinimidyl Ester (CFSE)-dim population which was significantly higher in HCL volunteers than in control group. Stimulation of isolated CD8⁺ memory T cells, but not naïve T cells, from HCL volunteers induced a significantly higher IFN-γ production compared with that of healthy controls. Intracellular IFN-γ staining provided the same result. Memory population is shown to be responsible for Leishmania-induced IFN-γ production. Leishmania-reactive proliferating T(EM) cells were identified as the most frequent subset which may play a role in recall immune response and protection against Leishmania infection.

Role of Mycobacterium vaccae in the protection induced by first generation Leishmania vaccine against murine model of leishmaniasis.

Various Leishmania antigens showed to induce protection when used with IL-12 as an adjuvant in an animal model of leishmaniasis. Limitations in using IL-12 justify searching for an appropriate adjuvant to accelerate induction of a Th1-type immune response and protection. In this study, the role of Mycobacterium vaccae as an adjuvant mixed with either autoclaved Leishmania major (ALM) or freeze-thawed-killed L. major (KLM) in increasing protection in susceptible and resistant mice was studied. Nineteen groups of BALB/c and 19 groups of C57BL/6 mice, ten mice per group, were immunized three times in 45 days interval with different doses of either KLM or ALM alone or mixed with either BCG or different doses of M. vaccae. Immunized groups of mice and PBS-injected control group were challenged with 2 x 10(6) promastigotes of L. major at the base of the tail. The evolution of the lesion was monitored, and the size of the lesion was measured and recorded weekly. Anti-Leishmania total IgG Ab was titrated before and after challenge. The results showed that immunization of either susceptible or resistant mice with KLM or ALM mixed with low dose of M. vaccae increased protection defined by significantly smaller ulcer size in immunized mice compared with the PBS-injected control group.