Paromomycin-loaded mannosylated chitosan nanoparticles: Synthesis, characterization and targeted drug delivery against leishmaniasis.

Cutaneous leishmaniasis is the most common form of leishmaniasis caused by different species of Leishmania parasites. The emergence of resistance, toxicity, long term treatment, high cost of the current drugs, and intracellular nature of the parasite are the major difficulties for the treatment of leishmaniasis. Although the therapeutic effect of paromomycin (PM) on leishmaniasis has been investigated in different studies, it has a low oral absorption and short half-life, leading to a decreased drug efficacy. Therefore, new and targeted carriers with no such problems are needed. In the present study, PM was loaded into chitosan (CS) nanoparticles accompanied by targeting to macrophages (as the host of Leishmania parasite). PM-loaded into mannosylated CS (MCS) nanoparticles using dextran (PM-MCS-dex-NPs) was prepared by ionic gelation and then characterized. The particle size and zeta potential of PM-MCS-dex-NPs were obtained as 246 nm and +31 mV, respectively. Mannosylation of CS was qualitatively evaluated by Fourier-transform infrared spectroscopy and quantitatively measured by CHNO elemental analysis; also, a mannosylation level of 17% (w) was attained. Encapsulation efficiency (EE), drug release profile, and THP-1 cell uptake potential were determined. A value of 83.5% for EE and a higher release rate in acidic media were achieved. THP-1 cell uptake level of PM-MCS-dex-NPs after 6 h was ˜2.8 and ˜3.9 times of non-mannosylated CS nanoparticles (PM-CS-dex-NPs) and PM aqueous solution, respectively. In vitro cell cytotoxicity and promastigote and amastigote viabilities were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Half-maximal inhibitory concentration values toward the THP-1 cells for PM aqueous solution, Glucantim, PM-CS-dex-NPs, and PM-MCS-dex-NPs after 48 h were obtained as 1846 ±â€¯158, 1234 ±â€¯93, 784 ±â€¯52 and 2714 ±â€¯126 µg mL-1, respectively. Half-maximal inhibitory concentration values toward the promastigotes for PM aqueous solution, Glucantim, PM-CS-dex-NPs, and PM-MCS-dex-NPs after 48 h were obtained as 105.0 ±â€¯14.0, 169.5 ±â€¯9.8, 65.8 ±â€¯7.3 and 17.8 ±â€¯1.0 µg mL-1, respectively. Selectivity (therapeutic) indices for PM aqueous solution, Glucantim, PM-CS-dex-NPs and PM-MCS-dex-NPs after 48 h were obtained as 24.6, 17.5, 3.7 and 214, respectively. The parasite burden in THP-1 cells after 48 h treatment with PM aqueous solution, Glucantim, PM-CS-dex-NPs, and PM-MCS-dex-NPs at a typical concentration of 20 µg mL-1 were 71.78, 69.94, 83.14 and 33.41%, respectively. While the effect of PM-CS-dex-NPs was more salient on amastigotes, PM-MCS-dex-NPs effectively affected both stages of the parasite, especially the amastigote one. This indicated that the mannosylated formulation acts as a targeted delivery system. The findings of this study revealed that this novel targeted formulation represented a strong anti-leishmanial activity.

Paromomycin-loaded mannosylated chitosan nanoparticles: Synthesis, characterization and targeted drug delivery against leishmaniasis.

Cutaneous leishmaniasis is the most common form of leishmaniasis caused by different species of Leishmania parasites. The emergence of resistance, toxicity, long term treatment, high cost of the current drugs, and intracellular nature of the parasite are the major difficulties for the treatment of leishmaniasis. Although the therapeutic effect of paromomycin (PM) on leishmaniasisLeishmania parasite). PM-loaded into mannosylated CS (MCS) nanoparticles using dextran (PM-MCS-dex-NPs) was prepared by ionic gelation and then characterized. The particle size and Zeta potential of PM-MCS-dex-NPs were obtained as 246 nm and + 31 mV, respectively. Mannosylation of CS was qualitatively evaluated by Fourier-transform infrared spectroscopy and quantitatively measured by CHNO elemental analysis; also, a mannosylation level of 17% (w) was attained. Encapsulation efficiency (EE), drug release profile, and THP-1 cell uptake potential were determined. A value of 83.5% for EE and a higher release rate in acidic media were achieved. THP-1 cell uptake level of PM-MCS-dex-NPs after 6 h was ˜2.8 and ˜3.9 times of non-mannosylated CS nanoparticles (PM-CS-dexIn vitroGlucantim, PM-CS-dex-NPs, and PM-MCS-dex-NPs after 48 h were obtained as 1846 ±â€¯158, 1234 ±â€¯93, 784 ±â€¯52 and 2714 ±â€¯126 µg mL-1Glucantim, PM-CS-dex-NPs, and PM-MCS-dex-NPs after 48 h were obtained as 105.0 ±â€¯14.0, 169.5 ±â€¯9.8, 65.8 ±â€¯7.3 and 17.8 ±â€¯1.0 µg mL-1Glucantim, PM-CS-dex-NPs and PM-MCS-dexGlucantim, PM-CS-dex-NPs, and PM-MCS-dex-NPs at a typical concentration of 20 µg mL-1 were 71.78, 69.94, 83.14 and 33.41%, respectively. While the effect of PM-CS-dex-NPs was more salient on amastigotes, PM-MCS-dex-NPs effectively affected both stages of the parasite, especially the amastigote one. This indicated that the mannosylated formulation acts as a targeted delivery system. The findings of this study revealed that this novel targeted formulation represented a strong anti-leishmanial activity.

Isolation and identification of pathogenic free-living amoeba from surface and tap water of Shiraz City using morphological and molecular methods.

Free-living amoebae (FLA) are the most abundant and widely distributed protozoa in the environment. An investigation was conducted to determine the presence of free-living amoebae (FLA), Acanthamoeba and Vermamoeba in waterfronts of parks and squares and tap water of Shiraz City, Iran. FLA are considered pathogenic for human. These ubiquitous organisms have been isolated from different environments such as water, soil, and air. Eighty-two water samples were collected from different places of Shiraz City during the summer of 2013. All samples were processed in Dept. of Parasitology and Mycology, Shiraz University of Medical Sciences, Fars, Iran. Samples were screened for FLA and identified by morphological characters in the cultures, PCR amplification targeting specific genes for each genus and sequencing determined frequent species and genotypes base on NCBI database. Overall, 48 samples were positive for Acanthamoeba and Vermamoeba in non-nutrient agar culture based on morphological characteristics. The PCR examination was done successfully. Sequencing results were revealed T4 (62.96 %) genotypes as the most common genotype of Acanthamoeba in the Shiraz water sources. In addition, T5 (33.33 %) and T15 (3.71 %) were isolated from water supplies. Vermamoeba vermiformis was known the dominant species from this genus. The high frequency of Acanthamoeba spp. and Vermamoeba in different environmental water sources of Shiraz is an alert for the public health related to water sources. The result highlights a need for taking more attention to water supplies in order to prevent illnesses related to free-living amoebae.

Genotyping of Giardia lamblia isolates from human in southern Iran.

Giardia lamblia cysts isolated from human faeces in South of Iran were analyzed with PCR-restriction fragment length polymorphism (RFLP) assay, based on the detection of glutamate dehydrogenase (gdh) genes. Among 205 faecal samples from microscopically diagnosed giardiasis patients, the gdh gene was amplified from 172 cases with a semi nested PCR assay and typed by RFLP analysis. Of the 172 positive samples, 128 (74.41%) were typed as assemblage AII, 30 (17.44%) assemblage BIII, 6 (3.49%) assemblage BIV and in 8 (4.66%) isolates, mixed assemblages AII and BIV were detected. Clinical features were available for 52 successfully typed cases and the possible correlation of Giardia assemblages and clinical symptoms was evaluated. Both assemblages caused similar illness, but assemblage AII was significantly more frequently associated with abdominal pain, nausea and vomiting. Since these isolates, A and B, are of human origin, anthroponotic transmission of Giardia can be suggested for the route of infection in this region.

First microscopical and molecular-based characterization of Leishmania major within naturally infected Phlebotomus salehi (Diptera; Psychodidae) in Fars province, southern Iran.

Zoonotoc cutaneous leishmaniasis is endemic in several parts of Iran. Jahrom district is one of the most important endemic foci of leishmaniasis located in Fars province, southern Iran. To identify the vectors of leishmaniasis in this area, a total of 349 sandflies were collected during May to August 2009. They were caught from outdoors in five regions of Jahrom district including villages of Mousavieh, Ghotb-Abad, Heydar-Abad, Fath-Abad and Jahrom County. Eleven species of Phlebotomine (three Phlebotomus spp. and eight Sergentomyia spp.) were detected. To determine the sandflies naturally infected by Leishmania spp., 122 female sandflies were dissected and evaluated microscopically using Giemsa-stained slides. Natural infection of 2 out of 38 (5.26%) P. papatasi and 1 out of 8 (12.5%) P. salehi to Leishmania major was confirmed in the region. Sequencing and nested polymerase chain reaction-based detection of Leishmania were carried out to confirm the microscopic findings. Five (13.16%) P. papatasi and two (25%) P. salehi were positive in nested polymerase chain reaction assay. All positive samples were shown 72-76% similarity with L. major Friedlin. On the basis of our knowledge, this is the first molecular detection of L. major within naturally infected P. salehi in this region in southern Iran.

Asymptomatic human carriers of Leishmania infantum: possible reservoirs for Mediterranean visceral leishmaniasis in southern Iran.

Over the last decade, the incidence of visceral leishmaniasis (VL) has increased in many districts of the province of Fars, in southern Iran. Recent epidemiological reports indicate that asymptomatic human infections with Leishmania infantum (the causative agent of VL throughout the Mediterranean basin) occur more frequently in Iran than was previously believed. Between 2004 and 2006, blood samples were collected from 802 apparently healthy subjects from communities, in the north-west and south-east of Fars province, where VL cases had been recorded. Each of these samples was tested for anti-Leishmania antibodies, in direct agglutination tests (DAT), and for L. infantum kinetoplast DNA, in PCR-based assays. Of the 426 subjects from north-western Fars, eight (1.9%) were found seropositive and 68 (16.0%) PCR-positive. The corresponding values for the 376 subjects from south-eastern Fars were lower, with five (1.3%) seropositive and 32 (8.5%) PCR-positive. Of the 100 PCR-positive subjects, 18 (18.0%) each lived in a household in which there had been a case of VL, and six (6.0%) had had VL themselves (in each case, more than a year before the blood sampling for the present study). Although 21 of the PCR-positives have now been followed-up for at least 18 months, none has developed symptomatic VL. Since positivity in the PCR-based assay probably indicated the presence of L. infantum amastigotes in the peripheral blood of 12.5% of the subjects, it is clear that asymptomatic human carriers of L. infantum are quite common in the study areas and probably act as reservoirs in the transmission of the parasite, to humans and to dogs, by sandflies.

First detection of Leishmania infantum in Phlebotomus (Larroussius) major (Diptera: Psychodidae) from Iran.

Ghir-Karzin district is one of the most important endemic foci of visceral leishmaniaisis (VL) in Fars province of southern Iran. To identify the vector(s) of Leishmania in this focus, a total of 2,539 sand flies were collected during June to September 2005. Eleven species (six Phlebotomus and five Sergentomyia) were identified, of which Phlebotomus papatasi Scopoli and Phlebotomus major Annandale were the first (37.4%) and third (11.2%) most common species, respectively. Natural leptomonad infections were observed in two P. papatasi (4.25%) and three P. major (6.65%) specimens on dissection and microscopic examination. Using a Leishmania genus-specific standard polymerase chain reaction (PCR; primers RV1-RV2) and a species-specific nested-PCR (primers LINR4, LIN17, and LIN 19), Leishmania infantum and Leishmania major kinetoplast minicircle DNA was detected in 6 of 72 P. major (8.3%) and 4 of 65 P. papatasi (6.1%), respectively. This is the first detection of L. infantum in P. major, implicating this sand fly as a probable vector of VL in Iran.

A search for the rodent hosts of Leishmania major in the Larestan region of southern Iran: demonstration of the parasite in Tatera indica and Gerbillus sp., by microscopy, culture and PCR.

Zoonotic cutaneous leishmaniasis (CL) caused by Leishmania major occurs widely in Iran, where several species of rodent serve as the parasite's 'reservoir' hosts. In an attempt to identify the rodent hosts in the Larestan region, which lies in the Fars province of southern Iran (where the incidence of human CL has been rising), 32 rodents (20 Tatera indica, eight Meriones crassus, four Gerbillus sp.) were caught and checked for leishmanial infection. Using two detection methods (the microscopical examination of stained tissue smears and the culture of tissue samples) and a PCR to identify any leishmanial parasites detected, L. major was identified in six of the rodents caught: two male T. indica from Alamarvdasht, two female T. indica from Lamerd, and two females of the genus Gerbillus (one caught in Lamerd and one in Lar). Although the samples were too small to prove that M. crassus is not a significant host of L. major in Larestan, they were large enough to indicate that T. indica and members of the genus Gerbillus serve as reservoir hosts of L. major in the region. Tatera indica appears to be an important host of L. major across much of Iran but this appears to be the first time that the genus Gerbillus has been found to be involved in the epidemiology of CL in the country.