First molecular epidemiological study of cutaneous leishmaniasis in Libya.

BACKGROUND: Cutaneous leishmaniasis (CL) is a major public health problem in Libya. The objective of this study was to investigate, for the first time, epidemiological features of CL outbreaks in Libya including molecular identification of parasites, the geographical distribution of cases and possible scenarios of parasite transmission. METHODOLOGY/PRINCIPAL FINDINGS: We studied 450 patients that came from 49 areas distributed in 12 districts in north-west Libya. The patients' ages ranged from 9 months to 87 years (median age 25 years); 54% of the cases were males. Skin scrapings spotted on glass slides were collected for molecular identification of causative agent. The ribosomal internal transcribed spacer 1 (ITS1) was amplified and subsequently characterized by restriction fragment length polymorphism (RFLP) analysis. In total, 195 samples were successfully identified of which 148 (75.9%) were Leishmania major, and 47 (24.1%) Leishmania tropica. CL cases infected with L. major were found in all CL areas whereas L. tropica cases came mainly from Al Jabal Al Gharbi (46.4%), Misrata (17.8%) and Tarhuna districts (10.7%). A trend of seasonality was noticed for the infections with L. major which showed a clear peak between November and January, but was less pronounced for infections by L. tropica. CONCLUSION: The first molecular study on CL in Libya revealed that the disease is caused by L. major and L. tropica and the epidemiological patterns in the different foci were the same as in other Mediterranean foci of CL.

SDS-coated atovaquone nanosuspensions show improved therapeutic efficacy against experimental acquired and reactivated toxoplasmosis by improving passage of gastrointestinal and blood-brain barriers.

Toxoplasmic encephalitis (TE) is the most common clinical manifestation of reactivated infection with Toxoplasma gondii in immunocompromised patients that is lethal if untreated. The combination of pyrimethamine plus sulfadiazine or clindamycin is the standard therapy for the treatment of TE, but these combinations are associated with hematologic toxicity and/or life-threatening allergic reactions. Therefore, alternative treatment options are needed. Atovaquone is safe and highly effective against T. gondii in vitro, but the oral micronized solution shows poor bioavailability. We synthesized atovaquone nanosuspensions (ANSs) coated with poloxamer 188 (P188) and sodium dodecyl sulfate (SDS) to improve oral bioavailability and passage through the blood-brain barrier (BBB). Coating of ANSs with SDS resulted in enhanced oral bioavailability and enhanced brain uptake of atovaquone compared to Wellvone(®) in murine models of acute and reactivated toxoplasmosis as measured by high performance liquid chromatography (HPLC). Parasite loads and inflammatory changes in brains of mice treated with SDS-coated ANS were significantly reduced compared to untreated controls and to Wellvone(®)-treated mice. In conclusion, nanosuspensions coated with SDS may ultimately lead to improvements in the treatment of TE and other cerebral diseases.

The role of apolipoprotein E in uptake of atovaquone into the brain in murine acute and reactivated toxoplasmosis.

We investigated whether coating of atovaquone nanosuspensions (ANSs) with apolipoprotein E (apoE) peptides improves the uptake of atovaquone into the brain. The passage across the blood-brain barrier (BBB) of ANSs stabilized by polysorbate 80 (Tween 80), poloxamer 184 (P184), or poloxamer 338 (P338) and the same formulations coated with apoE peptides were analyzed in vitro and in vivo. Passage through a rat coculture model of the BBB did not differ between individual atovaquone formulations, and the addition of apoE peptides did not enhance the transport. Following the induction of toxoplasmic encephalitis (TE) in mice, treatment with all atovaquone formulations reduced the number of parasites and inflammatory foci compared with untreated mice. Uptake of atovaquone into the brain did not depend on coating with apoE. Finally, incubation of apoE peptide-coated ANSs with brain endothelial cells for 30 min did result in the accumulation of nanoparticles on the cell surface but not in their uptake into the cells. In conclusion, ANSs coated with Tween 80 or poloxamers showed therapeutic efficacy in murine toxoplasmosis. ApoE- and apoE-derived peptides do not induce the uptake of ANSs into the brain. Alternative mechanisms seem to be in operation, thereby mediating the passage of atovaquone across the BBB.

A new combined flow-cytometry-based assay reveals excellent activity against Toxoplasma gondii and low toxicity of new bisphosphonates in vitro and in vivo.

OBJECTIVES: The aim of this study was to investigate the antiparasitic activity and toxicity of bisphosphonates using a new combined flow cytometry assay. METHODS: Using Toxoplasma gondii tachyzoites carrying the green-fluorescent protein (GFP), we established a new flow cytometry assay combining testing of in vitro and in vivo activity plus toxicity of newly synthesized bisphosphonates against T. gondii. Toxicity as determined by this assay was compared with toxicity as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. RESULTS: In vivo, therapeutic efficacy was 100% for bisphosphonates 2F, 3B, 18A, 22A and 30B at 490, 1000, 512, 44.05 and 47.6 microM concentrations, respectively. Toxicity at 100% inhibitory concentrations was 20% for 2F and 3B, 60% for 22A and 30B, and 75% for 18A. In vitro, 6 (91A, 203A, 200C, 210A, 204A and 282A) of 15 newly synthesized bisphosphonates (12 nitrogen-containing and 3 n-alkyl) inhibited parasite replication by >50% at a concentration of 100 microM. Whereas substances 91A and 282A (high efficacy) showed moderate and low toxicity (cell viability between 70% and 100%), respectively, toxicities of 203A, 200C, 210A and 204A were 70%, 65%, 80% and 70%, respectively, as determined by flow cytometry. Compounds 290A, 218A, 214A, 266A and 219A inhibited parasite replication by between 20% and 50% at a concentration of 100 microM. CONCLUSIONS: Newly synthesized bisphosphonates 2F, 3B, 91A and 282A showed excellent therapeutic activity and low toxicity. These antiparasitic drugs may therefore be promising compounds for use in patients with acute and reactivated toxoplasmosis. The new flow cytometry assay allowed simultaneous determination of therapeutic efficacy and toxicity.