Design of a multi-epitope subunit vaccine candidate for chikungunya virus using computational methodology.

Chikungunya virus (CHIKV) is a neglected tropical pathogen that causes fever and long-lasting severe arthralgia. Despite its high morbidity, there is still no licensed specific therapeutic option for it. This study proposes a multi-epitope subunit vaccine candidate for CHIKV, designed using computational methods. It was based on the E2 spike glycoprotein in CHIKV, from which T- and B-cell epitopes were predicted and then refined. The pan HLA DR-binding epitope (PADRE) was added to this refined construct, then simulated compared with the native protein, where it was predicted to elicit more than twice the number of antibody titers. Thus, this construct is potentially effective against CHIKV, which further experimentation using live models would be able to verify. This study also demonstrates the feasibility of using rational tools in the future to further optimize vaccine design.

Antimicrobial efficacy and activity of ethanolic extract of Piper betle L. on Staphylococcus aureus-infected wound in mice and clinical isolates of multiple drug-resistant bacterial pathogens.

This study aimed to determine the in vivo effectiveness of the ethanolic extract of Piper betle L. leaves against Staphylococcus aureus-infected wounds in mice and its antimicrobial properties on clinical isolates of multiple drug-resistant bacterial pathogens. Twenty mice were divided into four groups. Wounds were created in all mice under anesthesia by excision from the dorsal skin down to the subcutaneous fat and inoculating with S. aureus. After 24 h, the wound of each mouse was treated once daily by application of the respective cream. Group I was treated with mupirocin antibacterial cream; Group II received a cream base containing no active ingredient; Groups III and IV were treated with 2.5% and 5.0% concentrations of P. betle cream, respectively. Further, an in vitro study was performed by adding undiluted, 1:50 and 1:100 dilutions of the four studied creams in normal saline containing 1.5 × 108 CFU/mL of the following bacteria: antimicrobial-susceptible S. aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant S. aureus, extended-spectrum ß-lactamase-producing Escherichia coli, vancomycin-resistant Enterococcus, metallo-ßlactamase-producing P. aeruginosa and carbapenem-resistant Klebsiella pneumoniae. The mice in Groups III and IV had significantly faster wound contraction and significantly shorter reepithelialization time than Group II (p < 0.05), which were not significantly different from Group I (p > 0.05). P. betle creams inhibited all studied bacterial strains at full concentration and at a dilution of 1:50. The inhibitory effect was more significant than Groups I and II (p < 0.05), except on S. aureus. Specifically, S. aureus inhibition was not significantly different for Groups III and IV (p > 0.05) when compared with Group I. Cream formulations derived from P. betle ethanolic extract have great potential as antimicrobial agents for the treatment of wound infection. Further clinical tests are recommended to determine the safety and efficacy of these formulations in other mammalian species.

Antimicrobial efficacy and activity of ethanolic extract of Piper betle L. on Staphylococcus aureus-infected wound in mice and clinical isolates of multiple drug-resistant bacterial pathogens

@#This study aimed to determine the in vivo effectiveness of the ethanolic extract of Piper betle L. leaves against Staphylococcus aureus-infected wounds in mice and its antimicrobial properties on clinical isolates of multiple drug-resistant bacterial pathogens. Twenty mice were divided into four groups. Wounds were created in all mice under anesthesia by excision from the dorsal skin down to the subcutaneous fat and inoculating with S. aureus. After 24 h, the wound of each mouse was treated once daily by application of the respective cream. Group I was treated with mupirocin antibacterial cream; Group II received a cream base containing no active ingredient; Groups III and IV were treated with 2.5% and 5.0% concentrations of P. betle cream, respectively. Further, an in vitro study was performed by adding undiluted, 1:50 and 1:100 dilutions of the four studied creams in normal saline containing 1.5 × 108 CFU/mL of the following bacteria: antimicrobial-susceptible S. aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant S. aureus, extended-spectrum β-lactamase-producing Escherichia coli, vancomycin-resistant Enterococcus, metallo-βlactamase-producing P. aeruginosa and carbapenem-resistant Klebsiella pneumoniae. The mice in Groups III and IV had significantly faster wound contraction and significantly shorter reepithelialization time than Group II (p < 0.05), which were not significantly different from Group I (p > 0.05). P. betle creams inhibited all studied bacterial strains at full concentration and at a dilution of 1:50. The inhibitory effect was more significant than Groups I and II (p < 0.05), except on S. aureus. Specifically, S. aureus inhibition was not significantly different for Groups III and IV (p > 0.05) when compared with Group I. Cream formulations derived from P. betle ethanolic extract have great potential as antimicrobial agents for the treatment of wound infection. Further clinical tests are recommended to determine the safety and efficacy of these formulations in other mammalian species.

Sensitive determination of chromium (VI) in paint samples using a membrane optode coupled to a multisyringe flow injection system.

In this work, the potential of a membrane optode coupled to a multisyringe flow injection system (MSFIA) was assessed for determining the Cr(VI) concentration in paint samples. The detection is based on the color obtained from the reaction of Cr(VI) with 1,5-diphenylcarbazide in the presence of sulfuric acid (H(2)SO(4)). The redox product was immobilized on a poly(styrene-divinylbenzene) (SDB-XC) membrane optode. The analyte in the sample was then directly quantified at the surface of the disk by measuring the intensity of reflected incident light using a bifurcated optical fiber at 540 nm. Experimental parameters (concentration of reagents, sample volume, flow rate of sample solutions, eluent concentration, and effect of diverse ions) were studied in detail. The overall time required for the complete procedure was 4 min and only required 0.2 mL of the sample volume. The dynamic working response of Cr(VI) was found within the concentration range of 2.4-1000 µg L(-1) with a limit of detection (LOD) of 0.7 µg L(-1), while the relative standard deviation (RSD) for 400 µg L(-1) Cr(VI) was lower than 2% (n=6). This developed method was used to determine Cr(VI) concentrations in the paint samples, for which an alkaline extraction procedure was proposed. The extraction procedure was based on the use of a 7.5% Na(2)CO(3)/5% NaOH solution at 90 °C for 30 min. Under optimal conditions, the recoveries ranged from 99% to 101%. The complete method was validated using a certified reference material (ERA-QC540, soil sample) and by comparing the results with those obtained using atomic absorption spectrometry (AAS).

Identification of the 18S-ribosomal-DNA genotypes of Acanthamoeba isolates from the Philippines.

Cyst morphology has been commonly used to identify the free-living amoeba Acanthamoeba to subgenus level. A more accurate and consistent method, based on the sequence analysis of the gene coding for the amoeba's small-subunit ribosomal RNA (Rns), has, however, been developed. There have been no attempts to identify the Acanthamoeba genotypes circulating in the Philippines. In this study, therefore, the ASA.S1 region of the Rns gene from 17 Acanthamoeba isolates, collected from soil, water and contact-lens storage cases in different regions of the Philippines, was sequenced. After the isolates were genotyped, using the BLAST program, their phylogenetic positions relative to known Acanthamoeba isolates were determined. For this, the model-based (GTR + Gamma) neighbour-joining, maximum-likelihood and Bayesian-inference analyses and the non-model-based maximum-parsimony analysis were used. All but two of the isolates were identified as the T5 or T4 genotypes, which are probably common in soil, water and contact-lens cases across the Philippines. The only other genotypes identified were T15 (as a single isolate from a contact-lens case) and T3 (as a single soil isolate).

Detection of Entamoeba dispar DNA in macaque feces by polymerase chain reaction.

We used the polymerase chain reaction (PCR) to determine the prevalence of Entamoeba histolytica and E. dispar in the wild population of macaque monkeys (Macaca fuscata) in Mt. Takasaki, Oita Prefecture, Japan. Of the 101 samples collected, 41 (42.57%) were found to be positive for E. dispar. However, no E. histolytica was detected from the collected samples. The results of this survey demonstrate the high prevalence of E. dispar in macaque monkeys in the study area. Moreover, they provide additional baseline information on naturally acquired infectious agents of macaque monkeys and offer an accurate tool for detection of E. histolytica and E. dispar, which are needed for biomedical research using nonhuman primate models.

Field study on the distribution of Entamoeba histolytica and Entamoeba dispar in the northern Philippines as detected by the polymerase chain reaction.

We used the polymerase chain reaction (PCR) to study the distribution of Entamoeba histolytica and E. dispar in 1,872 individuals in 14 communities in the northern Philippines. Here we report a field study using a DNA extraction protocol from formalin-fixed stool specimens as previously reported. This assay detected 137 stools (7.318%) containing E. dispar and 18 stools (0.961%) containing E. histolytica. The most affected age group for E. histolytica/E. dispar infections were those 5-14 years of age. There was no significant difference in the sex distribution of E. histolytica, while in the case of E. dispar, a higher prevalence was observed in females (9.186%) than in males (5.731%) (P < 0.01). An apparent clustering of stool-positive cases of E. histolytica and E. dispar was also observed in the northern part of the study area. The results of this survey demonstrate that E. dispar is highly prevalent in the communities studied. Moreover, it offers promise for the PCR using DNA extracted from formalin-fixed stools as a sensitive epidemiologic tool for detecting E. histolytica and E. dispar infections.

Application of the polymerase chain reaction (PCR) in the epidemiology of Entamoeba histolytica and Entamoeba dispar infections.

In this paper, we briefly summarize the latest information on the polymerase chain reaction (PCR) as an epidemiologic tool for Entamoeba histolytica and Entamoeba dispar infections. This method which employs DNA template directly extracted from formalin fixed stool specimens offers a good promise for an accurate and reliable epidemiology of the two species. The assay is, sensitive enough to detect as few as five cysts in the stool sample, rapid and selectively differentiates E. histolytica from E. dispar DNA from stool specimens without the need for prior cultivation.

Differentiation of Entamoeba histolytica and E. dispar DNA from cysts present in stool specimens by polymerase chain reaction: its field application in the Philippines.

It has been established that two distinct species exist within what was originally known as Entamoeba histolytica. These are E. dispar and E. histolytica, for the nonpathogenic and pathogenic forms, respectively. Differentiation of these two organisms is of great clinical importance since they are morphologically indistinguishable and both forms can infect the human intestinal cavity to different degrees. A simple and rapid DNA-extraction method that can be used directly on formalin-fixed stool specimens has been developed. The extracted DNA was used for the identification of the species existing in the stools by polymerase chain reaction (PCR). A total of 72 randomly collected stool samples from the Philippines were analyzed. In all, 19 samples reacted with E. dispar primers, resulting in the expected 101-bp PCR products; however, none reacted with E. histolytica primers. Furthermore, sensitivity assay suggests that genomic DNA from as few as five cysts can be used as a template for PCR. These observations imply that the use of genomic DNA directly extracted from formalin-fixed stool specimens for PCR amplification is a useful tool for obtaining a sensitive and accurate diagnosis that can be applied even in epidemiology studies.