A novel tetra-primer ARMS-PCR for genotyping of the OPRM1 gene rs1799971 variant associated with opioid use disorders.

OBJECTIVES: A SNV is a single nucleotide change that can occur at any point in the genome. SNVs are the most common genetic variants that occur in the human genome, and a number of SNVs have been found to be associated with human traits and disease. Researchers genotype SNVs using TaqMan technology, DNA microarray, MALDI-TOF mass spectrometry, and automated sequencing, which are expensive and time-consuming. The OPRM1 gene rs1799971 (A118G) has been identified for its association with Opioid use disorder (OUD). The present study focused on developing a single step identification test using Tetra-Primer Amplification Refractory Mutation System-PCR (T-ARMS-PCR) to detect the presence of SNV OPRM1 rs1799971 (A118G). This study was performed to optimize the protocol for the designed four primers and validate it using a total of 52 buccal samples from volunteers who are currently under rehabilitation for the drug abuse disorder. RESULTS: Utilizing 52 DNA samples, a novel T-ARMS-PCR assay was successfully developed, tested, and validated. The products of the T-ARMS PCR for rs1799971 contained 395 bp as the control band, 186 bp as G allele (variant) and 257 bp as A allele (wild type), which were observed in the gel image. The genotype frequencies for the OPRM1 gene rs1799971 (A118G) were 44% (22/52) of homozygous variant type (GG), 28.9% (15/52) of homozygous wild type (AA) and 28.9% (15/22) of heterozygous (AG). The G allele frequency was 56.7% and A allele frequency was 43.3%.

One-Pot Reducing Agent-Free Synthesis of Silver Nanoparticles/Nitrocellulose Composite Surface Coating with Antimicrobial and Antibiofilm Activities.

Nitrocellulose with silver nanoparticle (AgNP/NC) composite was prepared in situ using Ag(CH3CO2) and nitrocellulose without any reducing agent. The composite materials synthesized were spray coated onto glass substrates to obtain thin films. The AgNPs/NC composites were characterized by ultraviolet-visible, Fourier transform infrared, X-ray photoelectron spectroscopy, scanning electron microscopy, and transmission electron microscopy. The antimicrobial activity of AgNPs/NC composite was investigated by tube method and time-kill kinetic studies against three microbial species, including Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), and Candida albicans (ATCC 10231). The antibiofilm activities were qualitatively determined against all three organisms. Prepared AgNPs/NC films exhibited good antimicrobial activity and significant inhibition of biofilm development against all three microbial species. The effective dispersion of AgNPs/NC in biofilm was responsible for the significant antibiofilm activity of the prepared material. The reported AgNPs/NC composite can be used as coating additive in bacteriocidal paint which can be applied onto surfaces such as in healthcare environments.

Group B Streptococcus colonisation and their antimicrobial susceptibility among pregnant women attending antenatal clinics in tertiary care hospitals in the Western Province of Sri Lanka.

The proportion of Group B Streptococcus (GBS) colonisation in pregnant women >35 weeks of gestation was 18% and 49% by culture and real-time PCR respectively in selected hospitals from the Western Province of Sri Lanka. A Descriptive cross-sectional study was conducted from January to April 2019. Two low vaginal and rectal swabs were collected from 100 pregnant women. Identification of GBS was done by culture and real-time PCR. GBS isolates were found to be sensitive to penicillin, ampicillin, cefotaxime, vancomycin, while 5 and 4 isolates out of 18 were resistant to erythromycin and clindamycin, respectively. Further, there was a significant association between GBS colonisation and a history of vaginal discharge and unemployment.IMPACT STATEMENTWhat is already known on this subject? Prevalence of GBS colonisation in the vagina and rectum of pregnant women in developing countries ranges from 8.5% to 22%. The Conventional method of culture has been considered the gold standard for diagnosis, however, the culture method does not give positive results for all cases of GBS. Polymerase chain reaction (PCR) has been found to be more sensitive for the detection of GBS than culture. In Sri Lanka, ante-natal screening for GBS is not practiced as the prevalence of GBS is still unlcear due to non-availably of data. Only a few scattered studies have been conducted using culture in Sri Lanka. Thus there is an urgent need to determine the magnitude of the GBS colonisers of ante-natal women in order to set up guidelines for screening and management of GBS.What do the results of this study add? In this study, the overall GBS colonisation rate which was detected using both culture and PCR was 50% in Western Province of Sri Lanka. That was a high figure when compared to the figures which were detected previously in Sri Lanka using only conventional culture methods. The risk factors for GBS colonisation were found to have a significant relationship with the history of abnormal vaginal discharge. Further, it was found that when Candida species coexisted with GBS, the existence of GBS was enhanced. Penicillin remains the antibiotic of choice for GBS.What are the implications of these findings for clinical practice and/or further research? This study emphasises the importance of establishing national policies for screening of pregnant women of >35 weeks of gestation to reduce the risk of neonatal infection. Further, it gives an insight into the options of antibiotics that can be used for treatment of these GBS colonisers from Sri Lanka.

Synthesis, Characterization and Antimicrobial Activity of Garcinol Capped Silver Nanoparticles.

Garcinol, a well-known medicinal phytochemical, was extracted and isolated from the dried fruit rinds of Garcinia quaesita Pierre. In this study, garcinol has successfully used to reduce silver ions to silver in order to synthesize garcinol-capped silver nanoparticles (G-AgNPs). The formation and the structure of G-AgNPs were confirmed by UV-visible spectroscopy, transmission electron microscopy and Fourier transform infrared (FTIR) spectroscopy. The antimicrobial activity of garcinol and G-AgNPs were investigated by well diffusion assays, broth micro-dilution assays and time-kill kinetics studies against five microbial species, including Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), Candida albicans (ATCC 10231) and clinically isolated methicillin-resistant Staphylococcus aureus (MRSA). The formation of G-AgNPs is a promising novel approach to enhancing the biological activeness of silver nanoparticles, and to increase the water solubility of garcinol which creates a broad range of therapeutic applications.

Comparison of Antimicrobial Properties of Silver Nanoparticles Synthesized from Selected Bacteria.

Green silver nanoparticle (AgNP) biosynthesis is facilitated by the enzyme mediated reduction of Ag ions by plants, fungi and bacteria. The antimicrobial activity of green AgNPs is useful to overcome the challenge of antimicrobial resistance. Antimicrobial properties of biosynthesized AgNPs depend on multiple factors including culture conditions and the microbial source. The antimicrobial activity of AgNPs biosynthesized by Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Acinetobacter baumannii (confirmed clinical isolate) were investigated in this study. Biosynthesis conditions (AgNO3 concentration, pH, incubation temperature and incubation time) were optimized to obtain the maximum AgNP yield. Presence of AgNPs was confirmed by observing a characteristic UV-Visible absorbance peak in 420-435 nm range. AgNP biosynthesis was optimal at 0.4 g/L AgNO3 concentration under alkaline conditions at 60-70 °C. The biosynthesized AgNPs showed higher stability compared to chemogenized AgNPs in the presence of electrolytes. AgNPs synthesized by P. aeruginosa were the most stable while NPs of S. aureus were the least stable. AgNPs synthesized by P. aeruginosa and S. aureus showed good antimicrobial potential against E. coli, P. aeruginosa, S. aureus, MRSA and Candida albicans. AgNPs synthesized by S. aureus had greater antimicrobial activity. The antimicrobial activity of NPs may vary depending on the size and the morphology of NPs.

Isolation and characterization of Leptospira interrogans from two patients with leptospirosis in Western Province, Sri Lanka.

Leptospirosis is an endemic infectious disease causing considerable morbidity and mortality in Sri Lanka; however, reports on the isolation of Leptospira from infected patients in Sri Lanka have been largely unavailable since the 1970s. Two isolates were obtained and characterized from 100 blood cultures from leptospirosis-suspected patients. Phylogenic analysis of partial flaB gene sequences identified the isolates as Leptospira interrogans. The patient serum samples from which Leptospira was isolated reacted with the Leptospira serogroups Sejroe and Canicola at a titre of 1 : 200. Exposure to domestic sewage and gutters filled with muddy water was suspected to be the source of infection in these two culture-positive patients. This study reports the successful isolation of pathogenic Leptospira from two patients in Western Province, Sri Lanka.

Enhanced antibacterial activity of TiO2 nanoparticle surface modified with Garcinia zeylanica extract.

BACKGROUND: The antibacterial activity of 21 nm TiO2 nanoparticles (NPs) and particles modified with Garcinia zeylanica (G. zeylanica) against Methicillin resistant Staphylococcus aureus was investigated in the presence and absence of light. RESULTS: Surface modification of TiO2 NPs with the adsorption of G. zeylanica extract, causes to shift the absorption edge of TiO2 NPs to higher wavelength. TiO2 NPs, G. zeylanica pericarp extract showed significant bactericidal activity which was further enhanced in contact with the TiO2 modified G. zeylanica extract. CONCLUSIONS: The antimicrobial activity was enhanced in the presence of TiO2 NPs modified with G. zeylanica and with longer contact time.

Evaluation of bactericidal effect of three antiseptics on bacteria isolated from wounds.

OBJECTIVE: Antiseptics are widely used in wound management to prevent or treat wound infections due to their proven wound healing properties regardless of their cytotoxicity. The objective of this study was to determine the bactericidal effects of three antiseptics on pathogens known to cause wound infections. METHOD: The study was carried out at a tertiary care hospital and a university microbiology laboratory in Sri Lanka in 2013. The three acids (acetic acid, ascorbic acid and boric acid) in increasing concentration (0.5%, 0.75% and 1%) were tested against bacterial suspensions equivalent to 0.5 McFarland standard. The Bacteria isolates used were isolated from wound and standard strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. RESULTS: There were 33 (68.8%) Coliforms, 10 (20.8%) Pseudomonas species, and 5 (10.4%) strains of Staphylococcus aureus. Acetic acid at concentration of 0.5% inhibited growth of 37 (77%) and 42 (87.5%) of tested isolates when exposed for 30 and 60 minutes, respectively. However 100% inhibition was achieved at four hours. At a concentration of 0.75%, 40 (83.3%) and 44 (91.7%) were inhibited when exposed for 30 and 60 minutes, respectively, with 100% inhibition at 4 hours. At concentration of 1%, 46 (95.8%) inhibition was seen at 30 minutes and 100% inhibition at 60 minutes. Ascorbic acid, at 0.5% and 0.75 % concentrations, inhibited growth of 45(93.7%) and 47(97.9%) of isolates respectively when exposed for 30 minutes. At these two concentrations, 100% inhibition was achieved when exposed for one hour. At 1% concentration, 100% inhibition was achieved at 30 minutes. Boric acid did not show bactericidal effect at concentrations of 0.5%, 0.75 % and 1%. Pseudomonas species were inhibited at 30 minutes by 0.5% acetic acid. Bactericidal effect against all the standard strains was seen with three acids at each concentration tested from 30 minutes onwards CONCLUSION: Ascorbic acid was bactericidal for all organisms tested within the shortest exposure time at the lowest concentration compared to other two acids. Despite promising bactericidal effects, further studies warrant, as ongoing debates on toxicity of acids on tissue epithelialisation. Application of antiseptics for a shorter duration could overcome this problem without losing bactericidal activity. DECLARATION OF INTEREST: The authors have no conflict of interest and no funding was received for this study.

Proportion of lower limb fungal foot infections in patients with type 2 diabetes at a tertiary care hospital in Sri Lanka.

BACKGROUND: Superficial fungal foot infection (SFFI) in diabetic patients increases the risk of developing diabetic foot syndrome. Sixteen percent of urban population is suffering from diabetes in Sri Lanka. As the diabetes patients are more prone to get fungal foot infections, early intervention is advisable owing to the progressive nature of the infection. There is no data on the prevalence of SFFIs in diabetic patients in Sri Lanka. OBJECTIVE: To determine the etiological agents causing SFFI in patients with type 2 diabetes. MATERIALS AND METHODS: Three hundred eighty five diabetic patients were included. Nail clippings and swabs were collected from the infected sites using the standard protocol. Laboratory identification was done and pathogens were identified to the species level by morpho physiological methods. RESULTS: Clinically 295 patients showed SFFI, of which 255 (86%) were mycologically confirmed for infection. Out of 236 direct microscopy (KOH) positives, 227 (96%) were culture positive. Two hundred and fifty one patients (98%) with SFFI had diabetes for more than 10 years. Of the patients with SFFIs 92% had >100 mg/dl FBS and 81% had >140 mg/dl PPBS levels and 80% had both elevated FBS and PPBS. Non-dermatophyte fungal species were the commonest pathogens followed by yeast and dermatophytes. CONCLUSION: Aspergillus niger was the commonest pathogen followed by Candida albicans. SFFIs were seen significantly with the increasing age, gender, duration of diabetes and with less controlled glycaemic level.

Residual bioburden in reprocessed side-view endoscopes used for endoscopic retrograde cholangiopancreatography (ERCP).

BACKGROUND AND STUDY AIM: Worldwide some endoscopy units routinely continue to use manual reprocessing techniques for disinfection of side-view endoscopes. The aim of this study was to evaluate the outcome quality of manual reprocessing techniques for removal and inactivation of the bioburden from side-view endoscopes used for endoscopic retrograde cholangiopancreatography (ERCP) in a tertiary referral endotherapy unit in Sri Lanka. METHODS: 102 samples obtained from two different flexible side-view endoscopes (Olympus TJF Q 180V and Olympus TJF 160 R) were tested for microbial growth. Three samples were collected each time; one swab from the tip before and another after manual reprocessing. The third sample was collected by flushing the working channel with sterile normal saline after manual reprocessing. Microorganisms were identified by culturing the samples. RESULT: : After reprocessing, culture-positive rates were 20 % and 9 % for the samples obtained from the tip and the working channel of the side-view endoscopes, respectively. Klebsiella spp. and Candida spp. were found to be the commonest microorganisms in the samples from the tips and from the working channels, respectively, of the reprocessed side-view endoscopes. CONCLUSION: There is a high culture-positive rate after reprocessing of the side-view endoscopes using the manual reprocessing procedure, despite strict adherence to the protocol for reprocessing.