In vitro evaluation of albendazole-loaded nanostructured lipid carriers on Echinococcus granulosus microcysts and their prophylactic efficacy on experimental secondary hydatidosis.

To enhance the therapeutic effects of albendazole (ABZ) on Echinococcus granulosus protoscoleces and metacestodes, ABZ-loaded nanostructured lipid carriers (ABZ-NLCs) are prepared by the hot high-speed homogenization method. Protoscoleces and microcysts were treated in vitro with free ABZ and ABZ-NLCs (concentrations of 1, 5, and 10 µg/ml), and the corresponding effects were monitored by methylene blue exclusion test and scanning and transmission electron microscopy. Chemoprophylactic treatment was performed on Balb/C mice 1 day before intraperitoneal injection of viable protoscoleces. The drugs were administered daily by intragastric inoculation for a period of 30 days. The prophylactic efficacy was assessed based on the number and weight of cysts developed in treated mice. The ultrastructural alterations in cysts were examined by transmission electron microscopy. After 18 days, all the protoscoleces incubated with 10 µg/ml ABZ-NLCs were killed, while 51.25 ± 4.03% of the protoscoleces incubated with 10 µg/ml free ABZ were still viable. Microcysts treated with ABZ-NLCs underwent degenerative alterations in a shorter time than when free ABZ was applied. The mean weight of the cysts recovered from mice of ABZ-NLCs group was significantly lower than that of the free ABZ group (P < 0.05), yielding prophylactic efficacy of 92.45% and 38.53%, respectively. The cysts treated with ABZ-NLCs showed marked ultrastructural changes in the germinal layer. This study demonstrated that both in vitro and in vivo treatments with ABZ-NLCs are significantly more efficient than treatment with free ABZ against E. granulosus protoscoleces, metacestodes, and prevention of cyst development in mice.

Efficiency of flubendazole-loaded mPEG-PCL nanoparticles: A promising formulation against the protoscoleces and cysts of Echinococcus granulosus.

None of the existing drugs can effectively treat the human cystic echinococcosis. This study aimed to improve the efficacy of flubendazole (FLBZ) against the protoscoleces and cysts of Echinococcus granulosus by preparing polymeric FLBZ-loaded methoxy polyethylene glycol-polycaprolactone (mPEG-PCL) nanoparticles. The protoscoleces and microcysts were treated with FLBZ-loaded mPEG-PCL nanoparticles (FLBZ-loaded nanoparticles) and free FLBZ at the final concentrations of 1, 5, and 10 µg/mL for 27 and 14 days, respectively. The chemoprophylactic efficacy of the drugs was evaluated in experimentally infected mice. The nanoparticles were stable for 1 month, with an average size of 101.41 ± 5.14 nm and a zeta potential of -19.13 ± 2.56 mV. The drug-loading and entrapment efficiency of the FLBZ-loaded nanoparticles were calculated to be 3.08 ± 0.15% and 89.16 ± 2.93%, respectively. The incubation of the protoscoleces with the 10 µg/mL nano-formulation for 15 days resulted in 100% mortality, while after incubation with the 10 µg/mL free FLBZ, the viability rate of the protoscoleces was only 44.0% ± 5.22%. Destruction of the microcysts was observed after 7 days' exposure to the FLBZ-loaded nanoparticles at a concentration of 10 µg/mL. The in vivo challenge showed a significant reduction in the weight and number of the cysts (P < 0.05) in the mice treated with the FLBZ-loaded nanoparticles, yielding efficacy rates of 94.64% and 70.21%, correspondingly. Transmission electron microscopy revealed extensive ultrastructural damage to the cysts treated with the FLBZ-loaded nanoparticles. The results indicated that the FLBZ-loaded nanoparticles were more effective than the free FLBZ against the protoscoleces and cysts of E. granulosus both in vitro and in vivo.

PCR-based Diagnosis of Toxoplasma Parasite in Ocular Infections Having Clinical Indications of Toxoplasmosis.

BACKGROUND: The diagnosis of ocular toxoplasmosis is mainly based on clinical features. However, ocular fluid testing by PCR may be very helpful for approval or rejection of this etiology. In this study, we utilized a nested-PCR technique, targeting the B1 partial sequence to analyze the aqueous and vitreous samples for evaluating the presence of the Toxoplasma DNA. METHODS: Fifty aqueous or vitreous humor samples were obtained from patients with clinical features of ocular toxoplasmosis admitted to ophthalmology hospitals and clinics in Iran, within 2014. The samples were subsequently subjected to DNA extraction and purification. For nested amplification of the Toxoplasma B1 gene, two primer pairs were used. The outer and inner primers are expected to produce a 193 bp and a 96 bp fragments, respectively. RESULTS: The first-round PCR resulted in the detection of T. gondii in 58% of samples by amplification of the expected 193bp DNA fragment. The nested-PCR using the inner primers, detected 15 additional samples from those with negative amplicons in the first round PCR (overall positivity of 88%). In addition, vitreous samples showed relatively more positive cases than aqueous humor in detection of the infection. CONCLUSION: The nested-PCR protocol using the B1 gene, with the high detection power, could be a useful complimentary method to clinical diagnose of ocular toxoplasmosis.

Genetic characterization of livestock and human hydatid cyst isolates from northwest Iran, using the mitochondrial cox1 gene sequence.

Cystic echinococcosis (CE), caused by larval stages of the tapeworm Echinococcus granulosus, is one of the most important zoonoses distributed worldwide. Genotype analysis of the parasite isolates from various hosts is required to better understand the host specificity and transmission routes. The aim of this study was to identify the genotypes of E. granulosus isolated from humans and domestic animals from northwest of Iran (Zanjan Province) using the mitochondrial cox1 gene sequence. A total of 86 hydatid cysts including 49 sheep and 28 cattle isolates from the slaughterhouse and nine human isolates from surgical wards of local hospitals were collected. The isolates were subjected to DNA extraction, PCR amplification, and sequence. Eighty-two (95.35 %) isolates, including 47 sheep, 26 cattle, and all nine human isolates, were determined as G1 genotype, and the remaining four (4.65 %), including two sheep and two cattle isolates, were identified as G3 genotype. From the cox1 sequence data, 13 different haplotypes (10 G1s and three G3s) were detected and named as EGH1-EGH13 (GenBank accession numbers, KP859559-KP859571). EGH1 was the major variant among the haplotypes, and it was identified in 46 (53.49 %) isolates (31 sheep, 14 cattle, and one human). Alignment of the partial cox1 sequences showed 12 point mutations including seven (58.3 %) synonymous and five (41.7 %) non-synonymous substitutions. Based on the results, G1 was the major genotype of E. granulosus in northwest of Iran affecting sheep, cattle, and humans. In addition, a minor group of G3 genotype was found to be circulating in this region.