Matrix-assisted laser desorption/ionisation time of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria from agar media. Direct identification from positive blood cultures should decrease the time to obtaining the result. In this study, three different processing methods for the rapid direct identification of bacteria from positive blood culture bottles were compared. In total, 101 positive aerobe BacT/ALERT bottles were included in this study. Aliquots from all bottles were used for three bacterial processing methods, i.e. the commercially available Bruker's MALDI Sepsityper kit, the commercially available Molzym's MolYsis Basic5 kit and a centrifugation/washing method. In addition, the best method was used to evaluate the possibility of MALDI application after a reduced incubation time of 7 h of Staphylococcus aureus- and Escherichia coli-spiked (1,000, 100 and 10 colony-forming units [CFU]) aerobe BacT/ALERT blood cultures. Sixty-six (65%), 51 (50.5%) and 79 (78%) bottles were identified correctly at the species level when the centrifugation/washing method, MolYsis Basic 5 and Sepsityper were used, respectively. Incorrect identification was obtained in 35 (35%), 50 (49.5%) and 22 (22%) bottles, respectively. Gram-positive cocci were correctly identified in 33/52 (64%) of the cases. However, Gram-negative rods showed a correct identification in 45/47 (96%) of all bottles when the Sepsityper kit was used. Seven hours of pre-incubation of S. aureus- and E. coli-spiked aerobe BacT/ALERT blood cultures never resulted in reliable identification with MALDI-TOF MS. Sepsityper is superior for the direct identification of microorganisms from aerobe BacT/ALERT bottles. Gram-negative pathogens show better results compared to Gram-positive bacteria. Reduced incubation followed by MALDI-TOF MS did not result in faster reliable identification.
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Five methods were compared to determine the best technique for accurate identification of coagulase-negative staphylococci (CoNS) (n=142 strains). MALDI-TOF MS showed the best results for rapid and accurate CoNS differentiation (correct identity in 99.3%). An alternative to this approach could be Vitek2 combined with partial tuf gene sequencing.
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Fingerprints are widely used in forensic science for individualization purposes. However, not every fingermark found at a crime scene is suitable for comparison, for instance due to distortion of ridge detail, or when the reference fingerprint is not in the database. To still retrieve information from these fingermarks, several studies have been initiated into the chemical composition of fingermarks, which is believed to be influenced by several donor traits. Yet, it is still unclear what donor information can be retrieved from the composition of one's fingerprint, mainly because of limited sample sizes and the focus on analytical method development. It this paper, we analyzed the chemical composition of 1852 fingerprints, donated by 463 donors during the Dutch music festival Lowlands in 2016. In a targeted approach we compared amino acid and lipid profiles obtained from different types of fingerprints. We found a large inter-variability in both amino acid and lipid content, and significant differences in L-(iso)leucine, L-phenylalanine and palmitoleic acid levels between male and female donors. In an untargeted approach we used full-scan MS data to generate classification models to predict gender (77.9% accuracy) and smoking habit (90.4% accuracy) of fingerprint donors. In the latter, putatively, nicotine and cotinine are used as predictors.
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Traces of condom lubricants in fingerprints can be valuable information in cases of sexual assault. Ideally, not only confirmation of the presence of the condom but also determination of the type of condom brand used can be retrieved. Previous studies have shown to be able to retrieve information about the condom brand and type from fingerprints containing lubricants using various analytical techniques. However, in practice fingerprints often appear latent and need to be detected first, which is often achieved by cyanoacrylate fuming. In this study, we developed a desorption electrospray ionization mass spectrometry (DESI-MS) method which, combined with principal component analysis and linear discriminant analysis (PCA-LDA), allows for high accuracy classification of condom brands and types from fingerprints containing condom lubricant traces. The developed method is compatible with cyanoacrylate (CA) fuming. We collected and analyzed a representative dataset for the Netherlands comprising 32 different condoms. Distinctive lubricant components such as polyethylene glycol (PEG), polydimethylsiloxane (PDMS), octoxynol-9 and nonoxynol-9 were readily detected using the DESI-MS method. Based on the analysis of lubricant spots, a 99.0% classification accuracy was achieved. When analyzing lubricant containing fingerprints, an overall accuracy of 90.9% was obtained. Full chemical images could be generated from fingerprints, showing the distribution of lubricant components such as PEG and PDMS throughout the fingerprint, while still allowing for classification. The developed method shows potential for the development of DESI-MS based analyses of CA treated exogenous compounds from fingerprints for use in forensic science.
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This report presents the highlights of the 7th European Meeting on Molecular Diagnostics held in Scheveningen, The Hague, The Netherlands, 12-14 October 2011. The areas covered included molecular diagnostics applications in medical microbiology, virology, pathology, hemato-oncology,clinical genetics and forensics. Novel real-time amplification approaches, novel diagnostic applications and new technologies, such as next-generation sequencing, PCR lectrospray-ionization TOF mass spectrometry and techniques based on the detection of proteins or other molecules, were discussed. Furthermore, diagnostic companies presented their future visions for molecular diagnostics in human healthcare.
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Semen traces are considered important pieces of evidence in forensic investigations, especially those involving sexsual offenses. Recently, our research group developed a fluorescence-based technique to accurately determine the age of semen traces. However, the specific compounds resonsible for the fluoresescent behaviour of ageing semens remain unknown. As such, in this exploratory study, the aim is to identify the components associated with the fluorescent behavior of ageing semen traces. In this investigation semen stains and various biofluorophores commonly found in body fluids were left to aged for 0, 2, 4, 7, 14 and 21 days. Subsequently, thin-layer chromatography (TLC) and ultra-performance liquid chromatography (UPLC) mass spectrometry were performed to identify the biofluorophores present in semen. Several contributors to the autofluorescence could be identified in semen stain, these include tryptophan, kynurenine, kynurenic acid, and norharman. The study sheds light on the.
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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.
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Understanding the factors that may impact the transfer, persistence, prevalence and recovery of DNA (DNA-TPPR), and the availability of data to assign probabilities to DNA quantities and profile types being obtained given particular scenarios and circumstances, is paramount when performing, and giving guidance on, evaluations of DNA findings given activity level propositions (activity level evaluations). In late 2018 and early 2019, three major reviews were published on aspects of DNA-TPPR, with each advocating the need for further research and other actions to support the conduct of DNA-related activity level evaluations. Here, we look at how challenges are being met, primarily by providing a synopsis of DNA-TPPR-related articles published since the conduct of these reviews and briefly exploring some of the actions taken by industry stakeholders towards addressing identified gaps. Much has been carried out in recent years, and efforts continue, to meet the challenges to continually improve the capacity of forensic experts to provide the guidance sought by the judiciary with respect to the transfer of DNA.
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Understanding taste is key for optimizing the palatability of seaweeds and other non-animal-based foods rich in protein. The lingual papillae in the mouth hold taste buds with taste receptors for the five gustatory taste qualities. Each taste bud contains three distinct cell types, of which Type II cells carry various G protein-coupled receptors that can detect sweet, bitter, or umami tastants, while type III cells detect sour, and likely salty stimuli. Upon ligand binding, receptor-linked intracellular heterotrimeric G proteins initiate a cascade of downstream events which activate the afferent nerve fibers for taste perception in the brain. The taste of amino acids depends on the hydrophobicity, size, charge, isoelectric point, chirality of the alpha carbon, and the functional groups on their side chains. The principal umami ingredient monosodium l-glutamate, broadly known as MSG, loses umami taste upon acetylation, esterification, or methylation, but is able to form flat configurations that bind well to the umami taste receptor. Ribonucleotides such as guanosine monophosphate and inosine monophosphate strongly enhance umami taste when l-glutamate is present. Ribonucleotides bind to the outer section of the venus flytrap domain of the receptor dimer and stabilize the closed conformation. Concentrations of glutamate, aspartate, arginate, and other compounds in food products may enhance saltiness and overall flavor. Umami ingredients may help to reduce the consumption of salts and fats in the general population and increase food consumption in the elderly.
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