Search Results (138)

Background/Objectives: 16S ribosomal RNA sequencing has, for several years, been the main means of identifying bacterial and archaeal species. Low-throughput Sanger sequencing is often used for the detection and identification of microbial species, but this technique has several limitations. The use of high-throughput sequencers may be a good alternative to improve patient identification, especially for polyclonal infections and management. Kraken 2 and KrakenUniq are free, high-throughput tools providing a very rapid and accurate classification for metagenomic analyses. However, Kraken 2 can present false-positive results relative to KrakenUniq, which can be limiting in hospital settings requiring high levels of accuracy. The aim of this study was to establish an alternative next-generation sequencing technique to replace Sanger sequencing and to confirm that KrakenUniq is an excellent analysis tool that does not present false results relative to Kraken 2. Methods: DNA was extracted from reference bacterial samples for Laboratory Quality Controls (QCMDs) and the V2-V3 and V3-V4 regions of the 16S ribosomal gene were amplified. Amplified products were sequenced with the Illumina 16S Metagenomic Sequencing protocol with minor modifications to adapt and sequence an Illumina 16S library with a small 500-cycle nano-flow cell. The raw files (Fastq) were analyzed on a commercial Smartgene platform for comparison with Kraken 2 and KrakenUniq results. KrakenUniq was used with a standard bacterial database and with the 16S-specific Silva138, RDP11.5, and Greengenes 13.5 databases. Results: Seven of the eight (87.5%) QCMDs were correctly sequenced and identified by Sanger sequencing. The remaining QCMD, QCMD6, could not be identified through Sanger sequencing. All QCMDs were correctly sequenced and identified by MiSeq with the commercial Smartgene analysis platform. QCMD6 contained two bacteria, Acinetobacter and Klebsiella. KrakenUniq identification results were identical to those of Smartgene, whereas Kraken 2 yielded 25% false-positive results. Conclusions: If Sanger identification fails, MiSeq with a small nano-flow cell is a very good alternative for the identification of bacterial species. KrakenUniq is a free, fast, and easy-to-use tool for identifying and classifying bacterial infections. Full article

Reliable, timely and economical target organism detection in harbors and ballast water is urgently needed to prevent the spread of aquatic invasive species (AIS) by commercial ships in the North American Great Lakes (NAGL). Inter-Great Lake ships (Lakers) transport large volumes (ca. 52 million metric tons. annually) of untreated lake water between lakes, with over 50% transported against the natural flow from the lower lakes to Western Lake Superior ports. The transport of ballast water is the number one threat of AIS spread throughout the NAGL. A relatively new tool to fight the spread of AIS is the use of eDNA for rapid detection and identification of target organisms. This technology opens doors for advancing control of ballast-mediated AIS through rapid detection. To that end, we have developed species-specific, reliable eDNA primers to target specific detection of four AIS in water samples along with qPCR protocols. Target organisms were selected based on the following criteria: (1) they are known to be invasive in the lower NAGL, (2) they are established in the lower NAGL but not in Superior, (3) they are biodegradable, and (4) they are obtainable, morphologically distinct and have existing DNA sequence information. Working primers, qPCR protocols and detection limits are provided for three invertebrate species and one alga species. These species are Daphnia lumholtzi (a water flea), Cercopagis pengoi (the fishhook water flea), Echinogammarus ishnus (a scud) and Nitellopsis obtusa (Starry Stonewort). Full article

Digital substation technology adhering to the IEC 61850 standard has provided several opportunities and flexibility for the rapid growth and complexity of the present and future electrical grid. The communication infrastructure allows complete interoperability between legacy and modern devices. The emergence of 5G wireless communication and its utilization in substation operation presents significant advantages in terms of cost and scalability, while also introducing challenges. This paper identifies research gaps in the literature and offers valuable insights for future analysis by providing a simulation study using an empirical latency dataset of a 5G network to illustrate three aspects of substation operational challenges: coordination of protection schemes, sequential reception of packet data streams, and time synchronization processes. The findings show a mean latency of 8.5 ms for the 5G network, which is significantly higher than that of a wired Ethernet network. The results also indicate that the high latency and jitter compromise the selectivity of protection schemes. The variability in latency disrupts the sequence of arriving data packets such that the packet buffering and processing delay increases from around 1.5 ms to 11.0 ms and the buffer size would need to increase by 6 to 10 times to handle out-of-sequence packets. Additionally, a time synchronization success rate of 14.3% within a 0.1 ms accuracy range found in this study indicates that the IEEE 1588 protocol is severely affected by the latency fluctuations. Full article

African swine fever virus (ASFV) and classical swine fever virus (CSFV) are important transboundary animal diseases (TADs) affecting swine. ASFV is a large DNA virus with a genome size of 170–190+ kilobases (kB) belonging to the family Asfarviridae, genus Asfivirus. CSFV is a single-stranded RNA virus with a genome size of approximately 12 kB, belonging to the family Flaviviridae, genus Pestivirus. Outbreaks involving either one of these viruses result in similar disease syndromes and significant economic impacts from: (i) high morbidity and mortality events; (ii) control measures which include culling and quarantine; and (iii) export restrictions of swine and pork products. Current detection methods during an outbreak provide minimal genetic information on the circulating virus strains/genotypes that are important for tracing and vaccine considerations. The increasing availability and reduced cost of next-generation sequencing (NGS) allow for the establishment of NGS protocols for the rapid identification and complete genetic characterization of outbreak strains during an investigation. NGS data provides a better understanding of viral spread and evolution, facilitating the development of novel and effective control measures. In this study, panels of primers spanning the genomes of ASFV and CSFV were independently developed to generate approximately 10 kB and 6 kB amplicons, respectively. The primer panels consisted of 19 primer pairs for ASFV and 2 primer pairs for CSFV, providing whole genome amplification of each pathogen. These primer pools were further optimized for batch pooling and thermocycling conditions, resulting in a total of 5 primer pools/reactions used for ASFV and 2 primer pairs/reactions for CSFV. The ASFV primer panel was tested on viral DNA extracted from blood collected from pigs experimentally infected with ASFV genotype I and genotype II viruses. The CSFV primer panel was tested on 11 different strains of CSFV representing the three known CSFV genotypes, and 21 clinical samples collected from pigs experimentally infected with two different genotype 1 CSF viruses. ASFV and CSFV amplicons from optimized PCR were subsequently sequenced on the Oxford Nanopore MinION platform. The targeted protocols for these viruses resulted in an average coverage greater than 1,000X for ASFV, with 99% of the genome covered, and 10,000X–20,000X for CSFV, with 97% to 99% of the genomes covered. The ASFV targeted whole genome sequencing protocol has been optimized for genotype II ASF viruses that have been responsible for the more recent outbreaks outside of Africa. The CSFV targeted whole genome sequencing protocol has universal applications for the detection of all CSFV genotypes. Protocols developed and evaluated here will be essential complementary tools for early pathogen detection and differentiation, as well as genetic characterization of these high-consequence swine viruses, globally and within the United States, should an outbreak occur. Full article

International air traffic has contributed to the global spread of SARS-CoV-2 and its variants. In early 2023, wastewater-based epidemiology (WBE) has been implemented at airports as a surveillance tool to detect emerging variants at short notice. This study investigates the feasibility and challenges of applying WBE at Berlin Brandenburg (BER) Airport, including a rapid implementation of wastewater sampling and analysis under unprecedented circumstances. For this purpose, aircraft and airport wastewater was sampled over 13 weeks. Established sampling and analysis protocols for municipal wastewater treatment plants (WWTPs) had to be adapted to the specific conditions of the airport environment. SARS-CoV-2 RNA was quantified and sequenced, revealing SARS-CoV-2 mutations not previously observed in clinical surveillance data in Germany. Despite the logistical and methodological challenges, the study demonstrates that WBE can serve as an early warning system for pathogen introduction. However, our study also underscores the need for realistic timelines for the establishment and validation of WBE monitoring strategies in new contexts. Investments in the establishment of WBE systems, e.g., infrastructure, protocols, trained personnel, and a network of stakeholders at strategic nodes including airports, can act as an effective tool for pandemic preparedness and global health security. Full article

Effective species identification is crucial for the conservation and management of marine mammals, particularly in regions such as the Mediterranean Sea, where several cetacean populations are endangered or vulnerable. In this study, we developed and validated a High-Resolution Melting (HRM) analysis protocol for the rapid, cost-effective, and reliable identification of the four representative marine cetacean species that occur in the Mediterranean Sea: the bottlenose dolphin (Tursiops truncatus), the striped dolphin (Stenella coeruleoalba), the sperm whale (Physeter macrocephalus), and the fin whale (Balaenoptera physalus). Species-specific primers targeting mitochondrial DNA regions (cytochrome b and D-loop) were designed to generate distinct melting profiles. The protocol was tested on both tissue and fecal samples, demonstrating high sensitivity, reproducibility, and discrimination power. The results confirmed the robustness of the method, with melting curve profiles clearly distinguishing the target species and achieving a success rate > 95% in identifying unknown samples. The use of HRM offers several advantages over traditional sequencing methods, including reduced cost, speed, portability, and suitability for degraded samples, such as those from the stranded individuals. This approach provides a valuable tool for non-invasive genetic surveys and real-time species monitoring, contributing to more effective conservation strategies for cetaceans and enforcement of regulations against illegal trade. Full article

Staphylococcus aureus is a leading cause of foodborne intoxication globally, driven by its heat-stable enterotoxins (SEs), which pose significant public health risks. This review critically evaluates modern and traditional methodologies for detecting S. aureus and its enterotoxins in food matrices, emphasizing their principles, applications, and limitations. The review includes a dedicated section on sample preparation and pretreatment methods for diverse food substrates, addressing a critical gap in practical applications. Immunological techniques, including ELISA and lateral flow assays, offer rapid on-site screening but face matrix interference and variable sensitivity challenges. Molecular methods, such as PCR and isothermal amplification, provide high specificity and speed for bacterial and toxin gene detection but cannot confirm functional toxin production. Sequencing-based approaches (e.g., WGS and MLST) deliver unparalleled genetic resolution for outbreak tracing but require advanced infrastructure. Emerging biosensor technologies leverage nanomaterials and biorecognition elements for ultra-sensitive real-time detection, although scalability and matrix effects remain hurdles. Mass spectrometry (MALDI-TOF MS) ensures rapid species identification but depends on pre-isolated colonies. Traditional microbiological methods, while foundational, lack the precision and speed of molecular alternatives. The review underscores the necessity of context-driven method selection, balancing speed, sensitivity, and resource availability. Innovations in multiplexing, automation, AI-based methods, and integration of complementary techniques are highlighted as pivotal for advancing food safety surveillance. Standardized validation protocols and improved reporting of performance metrics are urgently needed to enhance cross-method comparability and reliability in outbreak settings. Full article

Background/Objectives: The recent COVID-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for protocols for rapid development of efficient screening methods to search for the optimal mRNA vaccine structures against mutable viral agents. The unmatched success of mRNA vaccines by Pfizer and Moderna encoding the spike protein of SARS-CoV-2 confirms the potential of lipid nanoparticles for mRNA delivery for an accelerated development of new vaccines. The efficacy of vaccination and the production cost of mRNA-based vaccines largely depend on the composition of mRNA components, since the synthesis of an immunogenic protein requires precise and efficient translation in vivo. The composition of 5′ and 3′ UTR combinations of mRNA has a strong impact on the translation efficiency. The major objective of this study was to increase the probability of producing the immunogenic protein encoded by vaccine mRNA. For this purpose, we proposed to find a new combination of natural UTRs and, in parallel with that, to design and test the system for in vivo selection of translationally active UTRs. Methods: By using Ribo-Seq analysis, sets of candidate short UTRs were generated. These UTRs were tested both in cell cultures and in mice for effective production of secreted nanoluciferase (NLuc) and the S protein of SARS-CoV-2. A combination of the most effective UTRs was used to generate a prototype of an mRNA vaccine capable of inducing neutralizing antibodies against coronavirus. Results: The usefulness of the selected UTRs for vaccine development was tested by implicating the full-length coding sequence of SARS-CoV-2 S protein to produce the main immunogen. As a result, the system for functional screening of UTRs was created by using the NLuc gene. Conclusions: The proposed approach allows non-invasive quantitative assessment of the translational activity of UTRs in the blood serum of mice. By using the full-length sequence of SARS-CoV-2 S protein as a prototype, we demonstrated that the combination of UTRs selected using our luciferase-based reporter assay induces IgG titers and neutralization rates comparable to those obtained by using UTRs from commercial S-protein-based mRNA vaccines. Full article

Diagnostic imaging is fundamental in dentistry for disease detection, treatment planning, and outcome assessment. Traditional radiographic methods, such as periapical and panoramic radiographs, along with cone beam computed tomography (CBCT), utilize ionizing radiation and primarily focus on visualizing bony structures. Magnetic resonance imaging (MRI) is emerging as a non-ionizing alternative that offers superior soft tissue contrast. However, standard MRI sequences face challenges visualizing mineralized tissues due to their short transverse relaxation times (T2), which results in rapid signal decay. Recent advancements exploring short T2 sequences, including Ultrashort Echo Time (UTE), Zero Echo Time (ZTE), and Sweep Imaging with Fourier Transformation (SWIFT), allow direct visualization of dental hard tissues. UTE captures signals from short T2 tissues using rapid pulse sequences, while ZTE employs encoding gradients before radiofrequency pulses to reduce signal loss. SWIFT enables near-simultaneous excitation and acquisition, improving ultrashort T2 detection. Additionally, customized intraoral and extraoral surface coils enhance the image resolution and signal-to-noise ratio (SNR), increasing MRI’s relevance in dentistry. Research highlights the potential of these short T2 sequences for early caries detection, pulp vitality assessment, and diagnosing jaw osseous pathology. While high-field MRI (3 T–7 T) improves resolution and increases susceptibility artifacts, low-field systems with specialized coils and short sequences offer promising alternatives. Despite obstacles such as cost and hardware constraints, ongoing studies refine protocols to enhance clinical applicability. Incorporating MRI in dentistry promises a safer, more comprehensive imaging methodology, potentially transforming diagnostics. This review emphasizes three types of short T2 sequences that have potential applications in the maxillofacial region. Full article

Importance: Genomic sequencing enables the rapid identification of a breadth of genetic variants. For clinical purposes, sequencing for small genetic variations is considered a solved problem, while challenges remain for structural variants, given the lower sensitivity and specificity. Interest has recently risen among governing bodies in developing protocols for population-wide genetic screening. However, usefulness is constrained when the probability of being affected by a rare disease remains low, despite a positive genetic test. This is a common scenario in neurodegenerative disorders. The problem is recognised among statisticians and statistical geneticists but is less well-understood by clinicians and researchers who will act on these results, and by the general public who might access screening services directly without the appropriate support for interpretation. Observations: We explore the probability of subsequent disease following genetic screening of several variants, both single nucleotide variants (SNVs) and larger repeat expansions, for two neurological conditions, Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS), comparing these results with screening for phenylketonuria, which is well-established. The risk following a positive screening test was 0.5% for C9orf72 in ALS and 0.4% for HTT in HD when testing repeat expansions, for which the test had sub-optimal performance (sensitivity = 99% and specificity = 90%), and 12.7% for phenylketonuria and 10.9% for ALS SOD1 when testing pathogenic SNVs (sensitivity = 99.96% and specificity = 99.95%). Subsequent screening confirmation via PCR for C9orf72 led to a 2% risk of developing ALS as a result of the reduced penetrance (44%). Conclusions and Relevance: We show that risk following a positive screening test result can be strikingly low for rare neurological diseases, even for fully penetrant variants such as HTT, if the test has sub-optimal performance. Accordingly, to maximise the utility of screening, it is vital to prioritise protocols with very high sensitivity and specificity, and a careful selection of markers for screening, giving regard to clinical interpretability, actionability, high penetrance, and secondary testing to confirm positive findings. Full article

Avian colibacillosis caused by avian pathogenic Escherichia coli (APEC) strains is a bacterial disease responsible for enormous economic losses in the poultry industry, due to high mortality rates in farms, antibiotic therapy costs, and seizures at slaughterhouses. The aim of this study was to characterize the serogroups and molecular features of extended spectrum β-lactamase (ESBL)-producing APEC isolates recovered from 248 liver samples of 215 broilers and 33 turkeys with colibacillosis lesions in northeast Algeria. For this, microbiological tests were carried out, according to the recommended standards: E. coli isolates were recovered using standard microbiological protocols, and identification was carried out by MALDI-TOF MS. Serogrouping was performed using a rapid agglutination slide and the antisera of three O somatic groups (O1, O2, O78). Antimicrobial susceptibility was determined by the disk diffusion method. PCR assays and sequencing were used to detect antimicrobial resistance genes, integrons, phylogrouping, and MLST. Conjugation experiments were also conducted to determine the transferability of the retrieved ESBL-encoding genes. Overall, 211 (85.1%) APEC isolates were collected (one per positive sample), and 164 (77.7%) of them were typable. The O2 and O1 serogroups were the most detected (46.1% in broiler typable isolates and 61.5% in turkey typable isolates). Seventeen APEC isolates were ESBL-producers and harbored the following genes (number of isolates): bla_CTX-M-1 (14), bla_CTX-M-15 (2), and bla_SHV-12 (1). They belonged to phylogroups D (10 isolates), B1 (6 isolates), and B2 (1 isolate). The MLST of 13 ESBL producers revealed seven STs: ST23, ST38, ST48, ST117, ST131, ST1146, and ST5087. The ESBL-encoding genes were transferred by conjugation among 15 ESBL-producing isolates, and transconjugants acquired either the IncK or IncI1 plasmids. Concerted efforts from all poultry actors are needed to establish surveillance monitoring strategies to mitigate the spread of ESBL-producing isolates implicated in avian colibacillosis. Full article

In this study, we developed, validated and applied an NGS-based SNP genotyping protocol for the molecular identification of Vitis vinifera varieties, demonstrating a reliable and efficient approach for distinguishing grapevine cultivars. By utilizing a small but highly informative set of SNP loci, this method provides effective molecular genotyping while capturing the genetic diversity needed for accurate identification. This straightforward and accessible approach allows for the rapid generation of genetic profiles, which can be compared with the profiles in existing databases to precisely identify grapevine varieties, even in cases where traditional morphological methods fall short due to environmental variability or developmental differences. The process is designed to be both time-efficient and cost-effective, making it a practical tool for routine use in vineyard management, breeding programs, and conservation efforts. Furthermore, the workflow minimizes the need for whole-genome sequencing or other resource-intensive techniques, making molecular profiling accessible to a wider range of researchers, growers, and industry professionals. Analysis of the molecular profiles of known varieties validated the accuracy of the protocol. Moreover, 14 autochthonous Greek grapevine varieties that have not been previously identified were also genotyped and the data were compared with those of all Greek varieties in the Vitis International Variety Catalogue, revealing no matching multilocus genotypes across Greece. Full article

Introduction: The differential diagnosis of splenomegaly is a complex process that encompasses a wide variety of diseases. Moreover, it is not always standardized and lacks a definitive consensus on which tests should be performed and in what order. Gaucher disease (GD) and acid sphingomyelinase deficiency (ASMD) are lysosomal diseases (LD) that present with splenomegaly, the diagnosis of which requires a high index of suspicion and specific biochemical and genetic techniques. The aim of the project for the education and diagnosis of Gaucher disease and acid sphingomyelinase deficiency (PREDIGA) was to conduct educational training alongside an observational, multicenter, ambispective, cross-sectional, single-cohort study among patients having an enlarged spleen or undergone splenectomy to further assess these subjects to exclude two lysosomal diseases, namely GD and ASMD. Methods: Using dried blood spot (DBS) testing, we identified patients with abnormally low values of the enzymes glucocerebrosidase and acid sphingomyelinase, who then underwent sequencing of the GBA1 and SPMD1 genes, respectively. The study involved 34 hospitals and 52 medical specialists. Results: We identified 220 patients (208 adults and 12 children under 18 years) with cryptogenic splenomegaly or who had undergone splenectomy (12 patients) without having reached a diagnosis. The median age was 11 years (interquartile range [IQR] 3–16) in the pediatric population and 51 years (IQR 38–65) in the adult population. Lower-than-normal enzyme values were detected in 19 DBSs, confirming eight positive cases, which corresponded to six patients with GD and two with ASMD. The rest of the DBSs with low enzyme activity were not genetically confirmed (58%). We determined that lysosomal diseases accounted for 3.6% of cryptogenic splenomegaly/splenectomy cases in our setting: 2.7% were GD and 0.9% ASMD, in a ratio of 1 ASMD patient to every 3 GD patients. Lyso-GL1 values in patients with GD were elevated in all but one individual, corresponding to a child diagnosed at 4 months old. The variants detected in the GBA1 gene were consistent with the most frequent variants found in Spain. Discussion/Conclusion: The development and implementation of this protocol for the education and diagnosis of cryptogenic splenomegaly/splenectomy, even in asymptomatic patients, constitutes a comprehensive, simple, rapid, and effective screening method for the diagnosis of GD and ASMD. Full article

Alternaria is a genus that contains several important plant pathogens affecting nearly 400 plant species worldwide, including economically important crops such as grapes, citrus, and ornamental plants. Rapid, scalable, and efficient methods of pathogen detection are crucial for managing plant diseases and ensuring agricultural productivity. Current amplicon sequencing protocols for Alternaria detection often require the enzymatic barcoding of amplicons, increasing hands-on time, cost, and contamination risk. We present a proof-of-concept study using custom barcoded primers, combining universal primers targeting ITS1 and ITS2 regions (600 bp) coupled with Oxford Nanopore Technologies (ONT) barcode sequences. Sequencing was performed on infected grapevine, mandarin orange, thuja, and maple tree samples. In total, we analyzed 38 samples using qPCR; 8 tested positive for Alternaria, which were sequenced using a newly developed protocol. As a result, we could identify Alternaria in every positive sample, and besides the pathogen of interest, we could identify the associated mycobiome. This protocol reduces hands-on time and cost, making a significant advancement over current sequencing methods. Future work will focus on optimizing our approach for high-throughput sequencing of up to 96 samples and determining the method’s applicability for large-scale mycobiome analysis. Full article

Background: A rapid intervention team is a broad category of special teams used by police and emergency respondents to cover various needs. It is essential to ensure the safety and well-being of people in emergencies, minimising the risk of harm and maximising the chances of survival. Objective: This study aimed (i) to identify the fitness profiles and levels of POs from the EIR of the Lisbon Metropolitan Command (COMETLIS, PSP, Portugal), considering age classes; (ii) to directly compare the observed fitness profiles to previous research and normative data; and (iii) to compare the fitness profile of POs from the EIR with cadets from the Police Academy. Methods: This cross-sectional observational study included the participation of 121 male POs from the EIR of the Lisbon Metropolitan Command (Portugal) and 92 male cadets from the Police Academy (Lisbon, Portugal). The assessment protocol sequence involved the collection of biosocial data (age classes: ≤29 years; 30–39 years; 40–49 years), a body size assessment, and a fitness assessment (horizontal jump, handgrip strength, 60 s sit-ups and 20 m shuttle run). Results: (i) In the ≤29 years age class, POs performed better in all fitness tests (highlighting that the age class had a statistically significant effect on performance in the horizontal jump, sit-ups, 20 m shuttle run, and predicted VO₂max), and they showed significantly better performance than cadets in handgrip (left, right, and sum), and significantly worse performance in sit-ups and predicted VO₂max. (ii) In the 30–39 years age class, POs had significantly worse performance than cadets in the horizontal jump, sit-ups, 20 m shuttle run, and predicted VO₂max, even after controlling for age. Conclusions: (i) The fitness performance decreased as the age class became older; (ii) the handgrip strength and cardiovascular capacity attributes were between the standard and excellent levels according to the ACSM guidelines for the general population; (iii) POs from the EIR were stronger than cadets in terms of handgrip strength but weaker in terms of lower limb power, abdominal muscular endurance, and aerobic capacity; and (iv) the differences observed between POs from the EIR and cadets in the 30–39 years age class emphasise the importance of physical training after the training period and throughout professional life. Full article