PLGA nanoparticles loaded with Gallic acid- a constituent of Leea indica against Acanthamoeba triangularis.

Acanthamoeba, a genus that contains at least 24 species of free-living protozoa, is ubiquitous in nature. Successful treatment of Acanthamoeba infections is always very difficult and not always effective. More effective drugs must be developed, and medicinal plants may have a pivotal part in the future of drug discovery. Our research focused on investigating the in vitro anti- acanthamoebic potential of Leea indica and its constituent gallic acid in different concentrations. Water and butanol fractions exhibited significant amoebicidal activity against trophozoites and cysts. Gallic acid (100 µg/mL) revealed 83% inhibition of trophozoites and 69% inhibition of cysts. The butanol fraction induced apoptosis in trophozoites, which was observed using tunnel assay. The cytotoxicity of the fractions and gallic acid was investigated against MRC-5 and no adverse effects were observed. Gallic acid was successfully loaded within poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles with 82.86% encapsulation efficiency, while gallic acid showed 98.24% in vitro release at 48 hours. Moreover, the gallic acid encapsulated in the PLGA nanoparticles exhibited 90% inhibition against trophozoites. In addition, gallic acid encapsulated nanoparticles showed reduced cytotoxicity towards MRC-5 compared to gallic acid, which evidenced that natural product nanoencapsulation in polymeric nanoparticles could play an important role in the delivery of natural products.

Preparation of Poly (dl-Lactide-co-Glycolide) Nanoparticles Encapsulated with Periglaucine A and Betulinic Acid for In Vitro Anti-Acanthamoeba and Cytotoxicity Activities.

Poly (dl-lactide-co-glycolide) (PLGA) microspheres were synthesized as delivery system for the natural anti-parasitic compounds, Periglaucine A (PGA) and Betulinic acid (BA). Periglaucine A and Betulinic acid were encapsulated in PLGA nanoparticles by single emulsion method with an average particle size of approximately 100⁻500 nm. Periglaucine A and Betulinic acid encapsulation efficiency was observed to be 90% and 35% respectively. Anti-Acanthamoeba property of Periglaucine A and Betulinic acid remained intact after encapsulation. PGA-PLGA and BA-PLGA nanoparticles demonstrated inhibition in viability of Acanthamoeba triangularis trophozoites by 74.9%, 59.9%, 49.9% and 71.2%, 52.2%, 88% respectively at concentration of 100 µg/mL, 50 µg/mL and 25 µg/mL. Cytotoxicity of PGA-PLGA and BA-PLGA nanoparticles has been evaluated against lung epithelial cell line and showed dose dependent cytotoxicity value of IC50 2 µg/mL and 20 µg/mL respectively. Futher, increased viability was observed in lung epithelial cell line in higher doses of synthesized polymeric nanoparticles. Results indicate that poly (dl-lactide-co-glycolide) (PLGA) nanoparticles could be exploratory delivery systems for natural products to improve their therapeutic efficacy.

Correction: Pathogenic waterborne free-living amoebae: An update from selected Southeast Asian countries.

[This corrects the article DOI: 10.1371/journal.pone.0169448.].

Pathogenic waterborne free-living amoebae: An update from selected Southeast Asian countries.

Data on the distribution of free-living amoebae is still lacking especially in Southeast Asian region. The aquatic environment revealed a high occurrence of free-living amoebae (FLA) due to its suitable condition and availability of food source, which subsequently causes infection to humans. A total of 94 water samples consisted of both treated and untreated from Laos (31), Myanmar (42), and Singapore (21) were investigated for the presence of pathogenic FLA. Each water sample was filtered and cultured onto non-nutrient agar seeded with live suspension of Escherichia coli and incubated at room temperature. Morphological identification was conducted for both trophozoites and cysts via microscopic stains (Giemsa and immunofluorescence). The presence of Naegleria-like structures was the most frequently encountered in both treated and untreated water samples, followed by Acanthamoeba-like and Vermamoeba-like features. To identify the pathogenic isolates, species-specific primer sets were applied for molecular identification of Acanthamoeba, Naegleria, and Vermamoeba. The pathogenic species of Acanthamoeba lenticulata and A. triangularis were detected from untreated water samples, while Vermamoeba vermiformis was found in both treated and untreated water samples. Our results suggested that poor water quality as well as inadequate maintenance and treatment might be the cause of this alarming problem since chlorine disinfection is ineffective in eradicating these amoebas in treated water samples. Regular monitoring and examination of water qualities are necessary in order to control the growth, hence, further preventing the widespread of FLA infections among the public.

Anti-encystment and amoebicidal activity of Lonicera japonica Thunb. and its major constituent chlorogenic acid in vitro

OBJECTIVE@#To examine the acanthamoebicidal effects of ethyl acetate, aqueous and butanol fractions of dried flower buds of Lonicera japonica (L. japonica) Thunb. (Flos Lonicerae) in vitro.@*METHODS@#Acanthamoeba triangularis isolates were obtained from environmental water samples and identified by PCR. They were exposed to ethyl acetate, water and butanol fractions of L. japonica Thunb. at concentrations ranging from 0.5 mg/mL to 1.5 mg/mL. The extracts were evaluated for growth inhibition at 24, 48 and 72 h, respectively. Chlorogenic acid at a concentration of 1 mg/mL was examined for inhibition of encystment.@*RESULTS@#Ethyl acetate fraction at a concentration of 1.5 mg/mL evoked a significant reduction of trophozoite viability by 48.9% after 24 h, 49.2% after 48 h and 33.7% after 72 h chlorogenic acid, the major active constituent of L. japonica Thunb. at the concentration of 1 mg/mL reduced the cysts/trophozoite ratio by 100% after 24 h, 84.0% after 48 h and 72.3% after 72 h. This phenolic compound at concentration of 1 mg/mL concurrent with 0.6% hydrogen peroxide inhibited hydrogen peroxide-induced encystment by 92.8% at 72 h.@*CONCLUSIONS@#Results obtained from this study show that ethyl acetate fraction at 1.5 mg/mL is the most potent fraction of L. japonica Thunb. and its major constituent chlorogenic acid showed the remarkable inhibition of encystment at a concentration of 1 mg/mL.

Anti-encystment and amoebicidal activity of Lonicera japonica Thunb. and its major constituent chlorogenic acid in vitro

Objective To examine the acanthamoebicidal effects of ethyl acetate, aqueous and butanol fractions of dried flower buds of Lonicera japonica (L. japonica) Thunb. (Flos Lonicerae) in vitro. Methods Acanthamoeba triangularis isolates were obtained from environmental water samples and identified by PCR. They were exposed to ethyl acetate, water and butanol fractions of L. japonica Thunb. at concentrations ranging from 0.5 mg/mL to 1.5 mg/mL. The extracts were evaluated for growth inhibition at 24, 48 and 72 h, respectively. Chlorogenic acid at a concentration of 1 mg/mL was examined for inhibition of encystment. Results Ethyl acetate fraction at a concentration of 1.5 mg/mL evoked a significant reduction of trophozoite viability by 48.9% after 24 h, 49.2% after 48 h and 33.7% after 72 h chlorogenic acid, the major active constituent of L. japonica Thunb. at the concentration of 1 mg/mL reduced the cysts/trophozoite ratio by 100% after 24 h, 84.0% after 48 h and 72.3% after 72 h. This phenolic compound at concentration of 1 mg/mL concurrent with 0.6% hydrogen peroxide inhibited hydrogen peroxide-induced encystment by 92.8% at 72 h. Conclusions Results obtained from this study show that ethyl acetate fraction at 1.5 mg/mL is the most potent fraction of L. japonica Thunb. and its major constituent chlorogenic acid showed the remarkable inhibition of encystment at a concentration of 1 mg/mL.

A reproducible method for extraction of Plasmodium falciparum DNA by microwave irradiation and its potential for rapid molecular diagnosis

Malaria remains one of the most important communicable diseases. A rapid, simple and accurate method is a crucial part of malaria diagnosis. The aim of this study was to reevaluate the microwave irradiation method to extract DNA from Plasmodium falciparum and compare with six other existing DNA extraction methods such as QIAamp DNA mini kit (Qiagen), FTA elute card, phenol-chloroform, Chelex, Chelex without proteinase-K and Rapid boiling. Two different P. falciparum isolates were used: (i) Laboratory strains with 0.3% parasitemia and (ii) clinical isolate with 0.6% parasitemia. Each DNA extraction method was validated for the presence of P. falciparum by a routine nested and real time PCR. In order to evaluate the sensitivity of the DNA extraction by microwave, double serial dilution of P. falciparum from in vitro culture at parasitemia that ranged from 0.0001 to 0.17% were used to extract the DNA by microwave and the P. falciparum DNA was then detected by nested and real-time PCR. The nested and real-time PCR were able to detect. P. falciparum DNA at the parasitemia level as low as 0.0003% and 0.0001%, respectively. Our results can reproduce the results from earlier studies and reveal microwave as a rapid and simple tool to extract P. falciparum DNA and subsequent molecular diagnosis of malaria.

Molecular identification of blow flies recovered from human cadavers during crime scene investigations in Malaysia

Forensic entomology applies knowledge about insects associated with decedent in crime scene investigation. It is possible to calculate a minimum postmortem interval (PMI) by determining the age and species of the oldest blow fly larvae feeding on decedent. This study was conducted in Malaysia to identify maggot specimens collected during crime scene investigations. The usefulness of the molecular and morphological approach in species identifications was evaluated in 10 morphologically identifi ed blow fl y larvae sampled from 10 different crime scenes in Malaysia. The molecular identifi cation method involved the sequencing of a total length of 2.2 kilo base pairs encompassing the ‘barcode’ fragments of the mitochondrial cytochrome oxidase I (COI), cytochrome oxidase II (COII) and t-RNA leucine genes. Phylogenetic analyses confi rmed the presence of Chrysomya megacephala, Chrysomya rufifacies and Chrysomya nigripes. In addition, one unidentified blow fly species was found based on phylogenetic tree analysis.