Development, Characterization, and In Vitro Biological Performance of Amphotericin B and Terbinafine Microemulsions Against Leishmania major.

This study evaluated the effect of microemulsion (ME) containing Amphotericin B (AmB) alone and associated with Terbinafine (Tbf) on Leishmania major (L. major) using in vitro models. The ME formulations of these drugs were formulated and described. After evaluating their cytotoxicity on the J774A1 macrophage (MΦ), their potency against promastigotes and intracellular amastigotes models was evaluated using an in vitro MTT assay and Giemsa stain, respectively. Based on pseudo ternary phase diagrams, unloaded ME, Tbf-loaded ME (ME-Tbf), and, AmB-loaded ME (ME-AmB) with mean droplet sizes 3.4 ± 0.81, 10.05 ± 0.21, and 8.21 ± 0.46 were successfully prepared, respectively. Concerning toxicity, ME-AmB and ME-Tbf indicated lower toxicity on MΦs compared to the free drugs. The ME formulations showed considerably inhibitory effects compared to the free drug forms when the IC50 was examined. The IC50 values of AmB (59.19 ± 1.74 and 36.4 ± 3.2 µg/mL), ME-AmB (7.5 ± 0.9 and 0.8 ± 0.05 µg/mL), Tbf (234.5 ± 9 and 128.8 ± 0.28 µg/mL), ME-Tbf (26.27 ± 0.2 and 11.97 ± 0.6 µg/mL), AmB + Tbf (30.18 and 24.93 µg/mL), and ME-AmB + ME-Tbf (4.79 and 0.37 µg/mL) were estimated after 48 and 72 h, respectively.

Evaluation of the Efficacy of Amphotericin B and Terbinafine Microemulsions and Their Combination on Cutaneous Leishmaniasis and Comparison with the Conventional Drug Form in BALB/c Mice.

Intracellular parasitic protozoa of Leishmania sp. causes leishmaniasis. The restricted access of the drugs to affected cells in the treatment of intracellular infections such as leishmaniasis is frequently hampered. Furthermore, most of today's drugs have limited uses due to some containing toxic compounds, and drug resistance is on the rise. In the present investigation, Amphotericin B (AmB) and Terbinafine (Tbf) were loaded in microemulsion (ME) in combination and alone, and the in vivo efficacy was considered in BALB/c mice infected with Leishmania major (L. major). The wound size at the base of the mouse tail was measured, and real-time PCR was performed to quantify the parasite load after the infection challenge. The study demonstrated that the ME-AmB and ME-Tbf formulations are safe and effective compounds for the treatment of cutaneous leishmaniasis by enhancing the effectiveness of AmB and Tbf in reducing the parasite burden.

Nephrotoxicity of Albendazole and Albendazole Loaded Solid Lipid Nanoparticles in Mice with Experimental Hydatidosis.

Purpose: Cystic echinococcosis (CE) is a serious contemporary public health problem. Different CE treatment methods are of considerable importance, with albendazole (ABZ) being one of the most preferred drugs for CE treatment and prophylaxis. In this study, we evaluated the nephrotoxicity caused by ABZ and ABZ-loaded solid lipid nanoparticles (SLNs) in mice with experimental hydatid cyst. Methods: ABZ-loaded SLNs were produced by micro-emulsification and a high shear homogenization technique. Thereafter, we evaluated the physicochemical characterization of the product. Live protoscolices were injected into mice to induce experimental hydatidosis. Mice were then treated with ABZ and ABZ-loaded SLNs. The nephrotoxicity effects were evaluated by biochemical and histopathological surveys. Results: Significantly different blood urea nitrogen (BUN) levels were observed between the two infected groups (ABZ treatment and ABZ-loaded SLN treatment) and the control group. The kidney malondialdehyde (MDA) and glutathione (GSH) levels of the infected groups were not significantly different from those of the control group. The histopathological study revealed nephropathic and pathologic changes in the ABZ and ABZ-loaded SLN groups. Conclusion: ABZ formulated for ABZ-loaded SLNs had a more prominent chemoprophylactic efficacy on CE and fewer side effects than ABZ alone. Neither ABZ nor ABZ-loaded SLNs caused significant biochemical and histopathological defects on the kidney, and all functional biochemical markers stayed within the normal range. Therefore, ABZ-loaded SLNs could be a potential new product for CE treatment.

Comparison of survival time of Echinococcus granulosus protoscolices in different culture media and temperatures and evaluation of their ability to generate cysts in mice.

Hydatidosis is a disease caused by the larval stage of Echinococcus granulosus, which has great importance in medicine and veterinary medicine. Protoscolices (PSCs) of fertile hydatid cysts play a critical role in secondary echinococcosis after surgery. Fertile cysts were acquired from infected sheep at the local municipal abattoir in Ahvaz, southwest of Iran. PSCs were obtained aseptically and transferred to 10 different culture media and kept at 4°C and 37°C to determine the duration of PSCs' survival. Then, 2000 live PSCs from each of the culture media were injected into the peritoneal cavity of BALB/c mice. After five months, the mice were evaluated in terms of cyst number, size, and weight. The highest PSCs survival time at 4°C was related to RPMI-1640 medium and cyst fluid for 50 and 45 days, respectively. Also, at 37°C, the longest survival time of PSCs was related to cyst fluid and RPMI-1640 media for 30 and 29 days, respectively. The highest level of infection and median cyst number was observed in mice received PSCs from the RPMI-1640 medium at 4°C, and the highest level of infection in mice at 37°C was related to the DMEM low glucose (L) medium. The current study indicated that 4°C was a more suitable temperature for in vitro storage of live PSCs. The maximum amount of infection was observed in mice received PSCs from the RPMI-1640 medium at 4°C. The present study is the first attempt to compare the ability of PSCs to generate hydatid cysts in mice after being cultured in different media and at various temperatures.

Genotyping the causative agents of cutaneous leishmaniasis in Ahvaz southwest of Iran.

With sand flies as the main vectors, Leishmania species cause ancient zoonotic diseases called leishmaniasis. Iran is an endemic country regarding cutaneous leishmaniasis. A number of 100 smear slides were collected from cutaneous lesions referred to Ahvaz health centers. The DNA was extracted and ITS1-PCR using LITSR and L5.8S primer pair was performed to detect the genus Leishmania. Then, enzymatic digestion of PCR products was done by HaeIII (species detection), TaqI (strain detection), DpnI and HpaII (mutation assessment). Furthermore, 50 samples were sent for sequencing. Microscopic examination showed amastigote form in all 100 slides. Also, molecular identification confirmed the infection of all cases to Leishmania genus, representing a 350 bp band. HaeIII digestion yielded 150 and 200 bp bands, indicating Leishmania major, while 130 and 200 bp fragments following TaqI digestion suggested A1 strain of the parasite. Moreover, no likely mutations was detected in ITS1 fragment of obtained parasites using DpnI (140 and 200 bp digestion) and HpaII (without digestion). The sequencing result also was consistent with our findings, having 100% homology to A1 strain sequence (AY550178). Leishmania major A1 strain was the predominant species in clinical samples of Ahvaz. Nevertheless, future researches should address the parasite strains in other foci and hosts of epidemiological significance.

Evaluation of the Protoscolicidal Effects of Albendazole and Albendazole Loaded Solid Lipid Nanoparticles.

BACKGROUND: Protoscolex plays an important role in the development of hydatid cyst. Albendazole is one of the most effectual protoscolicidal agents for averting the reappearance of this disease, nonetheless, its low solubility and its low intestinal absorption necessitates the presence of a drug carrier to enhance its efficacy. In this study, the effect of albendazole and its nano-form on protoscolices in cultured media and in vivo was evaluated. METHODS: Microemulsion method was used to prepare the Solid lipid nanoparticles (SLNs) containing albendazole. Infected livers were collected from the Slaughterhouse of Ahvaz, Khuzestan in 2017. The protoscolices were stored in RPMI 1640 for one week, and their survival under the influence of albendazole and nano-albendazole on days 3 and 7 at concentrations of 250 and 500µg / ml was investigated. The live protoscolices exposed on day 3 at a concentration of 250 µg/ml were injected to mice for evaluation of pathogenicity. Three months later, after autopsy of the mice, the pathogenicity was evaluated. RESULTS: Protoscolicidal efficacy was highest in both concentrations on day 7 for albendazole and on day 5 for nano-albendazole. Following the autopsy of the mice, cyst growth was reported in all mice, and only two mice from the albandazole loaded SLNs group did not have any cyst. CONCLUSION: Albendazole loaded SLNs showed a higher protoscolicidal property than the free form of this drug; therefore, the use of nano-formulation of this drug is recommended to prevent the onset of this disease.

Molecular genotyping of Giardia duodenalis in children from Behbahan, southwestern Iran.

Giardia duodenalis is an intestinal flagellated protozoan that infects humans and several animal species. Giardiasis causing more than 200 million symptomatic infections globally is one of the most common causes of diarrhea in developing countries. Based on molecular studies mainly targeting the small-subunit (SSU) rRNA gene locus of the parasite, eight assemblages (A to H) have been identified in humans and other animal species. The aim of the current study was to evaluate the frequency and molecular diversity of G. duodenalis in children from rural and urban day care centers from Behbahan, southwestern Iran. This cross-sectional study was based on a concentration method for the microscopic detection of G. duodenalis in stool samples of 450 children, aged 1-7 years, in Behbahan, southwestern Iran. The survey was conducted from December 2015 to May 2016. PCR methods targeting the SSU rRNA and triose phosphate isomerase (TPI) genes of G. duodenalis were used for the identification and genotyping of the parasite isolates. Based on sucrose flotation and microscopy techniques, 2.7% (12/450) of children were infected with G. duodenalis, of which six (50.0%) were males and the other six (50.0%) were females. Overall, 91.7% (11/12) of the infections were detected in children from rural areas. The SSU rRNA and TPI genes were amplified successfully in nine and eight, respectively, of the Giardia-positive samples at microscopy. Among the eight TPI sequences, assemblage A, sub-assemblage AII, was identified in five of the isolates. The sequences of the three remaining samples were untypable. Although no significantly statistical difference between genotype and clinical symptoms was found, five out of the eight isolates identified as assemblage A were obtained in asymptomatic children. Giardia duodenalis infections were more prevalent in children from rural day care schools, and the predominant assemblage was A, sub-assemblage AII. The higher prevalence of giardiasis in rural areas might be related to differences in personal hygiene habits, parents' education level, source of drinking water, and inadequate hygienic toilet facilities in rural areas.