To accelerate differentiation between Staphylococcus aureus and Coagulase Negative Staphylococci (CNS), this study aimed to compare six different DNA extraction methods from 2 commonly used blood culture materials, i.e. BACTEC and Bact/ALERT. Furthermore, we analyzed the effect of reduced blood culture times for detection of Staphylococci directly from blood culture material. A real-time PCR duplex assay was used to compare 6 different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with MRSA. Bacterial DNA was isolated with: automated extractor EasyMAG (3 protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus, and a combination between MolYsis Plus and the EasyMAG. The most optimal isolation method was used to evaluate reduced bacterial culture times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the EasyMAG resulted in the most sensitive detection of S.aureus, with a detection limit of 10 CFU/ml, in Bact/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S.aureus load of 1 CFU/ml blood can be detected after 5 hours of culture in Bact/ALERT3D by combining the sensitive isolation method and the tuf LightCycler assay.
Considering activity level propositions in the evaluation of forensic biology findings is becoming more common place. There are increasing numbers of publications demonstrating different transfer mechanisms that can occur under a variety of circumstances. Some of these publications have shown the possibility of DNA transfer from site to site on an exhibit, for instance as a result of packaging and transport. If such a possibility exists, and the case circumstances are such that the area on an exhibit where DNA is present or absent is an observation that is an important diagnostic characteristic given the propositions, then site to site transfer should be taken into account during the evaluation of observations. In this work we demonstrate the ways in which site to site transfer can be built into Bayesian networks when carrying out activity level evaluations of forensic biology findings. We explore the effects of considering qualitative vs quantitative categorisation of DNA results. We also show the importance of taking into account multiple individual’s DNA being transferred (such as unknown or wearer DNA), even if the main focus of the evaluation is the activity of one individual.
Mobile Rapid-DNA devices have recently become available on the market. These devices can perform DNA analyses within 90 min with an easy ‘sample in–answer out’ system, with the option of performing comparisons with a DNA database or reference profile. However, these fast mobile systems cannot yet compete with the sensitivity of the standard laboratory analysis. For the future this implies that Scene of Crime Officers (SoCOs) need to decide on whether to analyse a crime sample with a Rapid-DNA device and to get results within 2 h or to secure and analyse the sample at the laboratory with a much longer throughput time but with higher sensitivity. This study provides SoCOs with evidence-based information on DNA success rates, which can improve their decisions at the crime scene on whether or not to use a Rapid-DNA device. Crime samples with a high success rate in the laboratory will also have the highest potential for Rapid-DNA analysis. These include samples from e.g. headwear, cigarette ends, articles of clothing, bloodstains, and drinking items.
Due to the existing pressure for a more rational use of the water, many public managers and industries have to re-think/adapt their processes towards a more circular approach. Such pressure is even more critical in the Rio Doce region, Minas Gerais, due to the large environmental accident occurred in 2015. Cenibra (pulp mill) is an example of such industries due to the fact that it is situated in the river basin and that it has a water demanding process. The current proposal is meant as an academic and engineering study to propose possible solutions to decrease the total water consumption of the mill and, thus, decrease the total stress on the Rio Doce basin. The work will be divided in three working packages, namely: (i) evaluation (modelling) of the mill process and water balance (ii) application and operation of a pilot scale wastewater treatment plant (iii) analysis of the impacts caused by the improvement of the process. The second work package will also be conducted (in parallel) with a lab scale setup in The Netherlands to allow fast adjustments and broaden evaluation of the setup/process performance. The actions will focus on reducing the mill total water consumption in 20%.
Environmental nano- and micro-plastics (NMPs) are highly diverse [2]. Accounting for this diversity is one of the main challenges to develop a comprehensive understanding of NMPs detection, quantification, fate, and risks [3]. Two major issues currently limit progresses within this field: (a) validation and broadening the current analytical tools (b) uncertainty with respect to NMPs occurrence and behaviour at small scales (< 20 micron). Tracking NMPs in environmental systems is currently limited to micron size plastics due to the size detection limit of the available analytical techniques. There are currently many uncertainties regarding detecting nanoplastics in real environmental systems, e.g. the inexistence of commercially available NMPs and incompatibility between them and those generated from plastic fragments degradation in the environment. Trying to tackle these problems some research groups synthesized NMPs dopped with metals inside [16]. However, even though elemental analysis techniques (ICP-MS) are rather sensitive, the low volume of these metals encapsulated in the nanoparticles make their detection rather challenging. At the same time, due to Sars-Cov-19 pandemic, nucleic acid identification technologies (LAMP, PCR) experienced a fast evolution and are able to provide detection at very low levels with very compact and reliable equipment. Nuclepar proposes the use of Electrohydrodynamic Atomization (EHDA) to generate NMPs coated with nucleic acids of different polymer types, sizes, and shapes, which can be used as support for detection of such particles using PCR-LAMP technology. If proven possible, Nuclepar might become a first step towards an easy NMPs detection tool. This knowledge will certainly impact current risk assessment tools, efficient interventions to limit emissions and adequate regulations related to NMPs.
Routine neuropathology diagnostic methods are limited to histological staining techniques or directed PCR for pathogen detection and microbial cultures of brain abscesses are negative in one-third of the cases. Fortunately, due to improvements in technology, metagenomic sequencing of a conserved bacterial gene could provide an alternative diagnostic method. For histopathological work up, formalin-fixed paraffin-embedded (FFPE) tissue with highly degraded nucleic acids is the only material being available. Innovative amplicon-specific next-generation sequencing (NGS) technology has the capability to identify pathogens based on the degraded DNA within a few hours. This approach significantly accelerates diagnostics and is particularly valuable to identify challenging pathogens. This ensures optimal treatment for the patient, minimizing unnecessary health damage. Within this project, highly conserved primers in a universal PCR will be used, followed by determining the nucleotide sequence. Based on the obtained data, it is then precisely determined which microorganism(s) is/are responsible for the infection, even in cases of co-infection with multiple pathogens. This project will focus to answer the following research question; how can a new form of rapid molecular diagnostics contribute to the identification of microbial pathogens in CNS infections? The SME partner Molecular Biology Systems B.V. (MBS) develops and sells equipment for extremely rapid execution of the commonly used PCR. In this project, the lectorate Analysis Techniques in the Life Sciences (Avans) will, in collaboration with MBS, Westerdijk Institute (WI-KNAW) and the Institute of Neuropathology (Münster, DE) establish a new molecular approach for fast diagnosis within CNS infections using this MBS technology. This enables the monitoring of infectious diseases in a fast and user-friendly manner, resulting in an improved treatment plan.
