Evaluating coverage bias in next-generation sequencing of Escherichia coli

Whole-genome sequencing is essential to many facets of infectious disease research. However, technical limitations such as bias in coverage and tagmentation, and difficulties characterising genomic regions with extreme GC content have created significant obstacles in its use. Illumina has claimed that the recently released DNA Prep library preparation kit, formerly known as Nextera Flex, overcomes some of these limitations. This study aimed to assess bias in coverage, tagmentation, GC content, average fragment size distribution, and de novo assembly quality using both the Nextera XT and DNA Prep kits from Illumina. When performing whole-genome sequencing on Escherichia coli and where coverage bias is the main concern, the DNA Prep kit may provide higher quality results; though de novo assembly quality, tagmentation bias and GC content related bias are unlikely to improve. Based on these results, laboratories with existing workflows based on Nextera XT would see minor benefits in transitioning to the DNA Prep kit if they were primarily studying organisms with neutral GC content

Spectroscopic observations of novae V1065 CEN and V1280 SCO using 45 cm cassegrain telescope at Arthur C Clarke Institute

The spectroscopic observations of two novae namely V1065 CEN and V1280 SCO were made by 45 cm Cassegrain telescope in high resolution ($\lambda/\delta\lambda$=22000) at H$\alpha$ (6563 \r{A}) region. V1065 CEN is He/N-type spectra which characterize a broad (Gaussian FWHM 49 \r{A}), saddle shaped and asymmetric H$\alpha$ emission line without prominent P-Cyg absorption component. Completely different H$\alpha$ profile of V1280 SCO shows prominent P-Cyg absorption and narrow emission line (Gaussian FWHM 26 \r{A}) which can be classified as Fe II type nova. The expansion velocities of these two systems measured from the minima of the P-Cyg profiles are close to 2300 km/s for V1065 CEN, and 716 km/s for V1280 SCO. Based on the photometric analysis, the Nova V1065 CEN can be classified as fast (11$<$t${_2}$$<$25) nova. The derived absolute magnitudes at maximum for nova V1065 CEN to be M$_{o,V}$ = -7.58$\pm$0.18 and M$_{o,B}$= -7.75$\pm$0.25 correspond to a distance 8.51$\pm$0.33 kpc. The parameters t$_{2V}$=12 days and t$_{3V}$=14 days of nova V1280 SCO determine that the nova is in between very fast and fast nova. The mean absolute magnitude at maximum is calculated to be M$_{o,V}$=-8.7$\pm$0.1 and the estimated distance to the nova V1280 SCO is 3.2$\pm$0.2 kpc

Organoids and bioengineered intestinal models: Potential solutions to the Cryptosporidium culturing dilemma

Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes

Influence of culture medium on in-vitro biofilm formation by Candida species

Objectives: Objective of this study was to establish an in vitro biofilm on the 96 well plates and to determine the efficacy of three different culture media on biofilm formation of Candida albicans and C. tropicalis Methods: A 96 well sterile, polystyrene plate was inoculated using 10^6 cell/ml of C. albicans and C. tropicalis suspensions and the growth rate of planktonic cells was determined by measuring the absorbance (OD492) at 2 hour intervals. Adhesion of Candidial cells to initiate the biofilm formation in the presence of three culture media (Yeast Nitrogen Base (YNB) supplemented with 100 mM glucose, Sabouraud Dextrose Broth (SDB) and RPMI1640) was quantified using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Crystal Violet (CV) assay after 90 minutes. Biofilms of C. albicans, C. tropicalis and 1:1 co-biofilms were developed and the growth rates were quantified at 24 hours’ time intervals. Scanning electron microscope (SEM) was performed to assess the architecture. Results: Planktonic cells of both C. albicans and C. tropicalis showed maximum growth with SDB. C. albicans and co-biofilm adhesion were significantly facilitated with RPMI1640 and the best medium for C. tropicalis adhesion was YNB. Biofilms showed the maximum growth rate in RPMI 1640. C. tropicalis exhibited the minimum growth with all three culture media.Conclusions: The maximum growth rate for planktonic C. albicans and C. tropicalis was achieved with SDB. However RPMI 1640 was the best medium for growth of biofilms

Impact of routine laboratory culture media on in-vitro biofilm formation of Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis

Objectives: This study was aimed to determine the efficacy of four routine laboratory culture media onbiofilm formation of Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus feacalis.Methods: A sterile flat bottom 96 well plate was inoculated using 0.5 McFarland equivalent standardcell suspension of P. aeruginosa, S. aureus and E. feacalis and the growth rate of planktonic cells wasquantified by measuring the optical density (OD492) at two hour intervals. Influence of culture mediumon adhesion of bacteria as an initial step of biofilm formation in the presence of four culture media(Nutrient broth (NB), Brain Heart Infusion (BHI) broth, Luria-Bertani (LB) broth and RPMI 1640) wasquantified using MTT (3-[4, 5- dimethylthiazole-2-yl]-2, 5- diphenyltetrazolium bromide) assay after90 minutes adhesion. Biofilms of P. aeruginosa, S. aureus, E. feacalis and their 1:1 mixed biofilmswere developed and the growth was quantified using MTT metabolic activity at 24 hour time intervals.Scanning electron microscopy (SEM) was performed to assess the ultrastructure.Results: On comparing the relative growth of the bacteria in different culture media, the maximumgrowth of all three planktonic cultures was achieved using BHI broth. All mono species and mixedspecies cultures exhibited their maximum adhesion in the presence of RPMI 1640. All biofilm exhibitedthe maximum growth in BHI broth. SEM imaging had shown the enhanced growth of ultrastructure ofthe biofilm with the presence of BHI broth.Conclusions: The maximum planktonic and biofilm growth was achieved with BHI broth. However,bacterial adhesion was enhanced in the presence of RPMI 1640

Preliminary survey of knowledge, attitudes and practices among nurses regarding seasonal influenza and influenza vaccination

Health care workers are  at risk of influenza through occupational exposure. Uptake of influenza vaccine is poor even in countries where it is provided free. We sought to determine the knowledge, attitudes and practices regarding seasonal influenza and barriers for vaccination among nurses in Colombo. A cross sectional survey was carried out from February to March 2020 on 97 randomly selected nurses. Level of knowledge was measured using a scoring system. Only a few (n=7; 7.2%) nurses had been immunized against influenza. Overall knowledge regarding influenza and vaccines was average in most nurses (n=53; 55%). The majority (n=62; 63.9%) believed the vaccine was safe and 79.4% (n=77) were willing to be vaccinated if vaccine is provided free. However, 15 of these 77 (19.5%) were reluctant to be vaccinated annually. Identified barriers for vaccination were the perception that the vaccine was not essential, doubt about its efficacy, fear of vaccines and side effects. Knowledge should be improved, and misconceptions and fears need to be addressed through health education and promotion.</p

An audit of specimens received for superficial fungal studies to the Department of Microbiology, University of Ruhuna

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Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty