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Abstract: Pseudomonas syringae (Psy) is a widely distributed bacterial species complex primarily recognized as a foliar pathogen but also inhabits diverse environments, including water habitats, where strains closely related to agricultural pathogens have been identified. The connection between Psy-caused epidemics and its potential presence in nearby irrigation systems remains underexplored. This study comprehensively examined the Psy complex in the Danube-Tisa-Danube Hydrosystem (DTD) in Serbia, assessing its abundance, phylogenetic diversity, and pathogenic potential. To reduce the reliance on the time-consuming steps of isolation and identification, we developed novel high-specific primers and probes for precise detection of strains belonging to phylogroup 2 within Psy complex. Our results demonstrated that dPCR, coupled with highly specific and sensitive primers, outperformed both traditional plating and qPCR in detecting the Psy complex and phylogroup 2 in irrigation waters, making Psy diagnostics more effective. Phylogenetic analysis indicated high strain diversity within the DTD, identifying phylogroups 1, 2, 7, 12, and 13 and haplotypes linked to strains previously encountered in epidemics on sugar beet in Serbia. Notably, 66.67% of the isolates from the DTD were capable of inducing disease. Phylogroup 2 isolates displayed a broad host range, suggesting that the dissemination of Psy from DTD through irrigation, poses a substantial threat to crop health and agricultural productivity.
Keywords: phytopathogen detection, irrigation, qPCR, dPCR
Published in DiRROS: 05.09.2025; Views: 241; Downloads: 94
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Abstract: Background Enteroviruses (EV) cause yearly outbreaks with severe infections, particularly in young children. This study investigates EV circulation, age, and clinical presentations in Europe from 2018 to 2023. Methods Aggregated data were requested from the European Centre for Disease Prevention and Control National Focal Points for Surveillance and European Non-Polio Enterovirus Network. Data included detection month, specimen type, age group, and clinical presentation for the 10 most commonly reported EV types per year. Results Twenty-eight institutions (16 countries) reported 563 654 EV tests during the study period with 33 265 (5.9%) EV positive. Forty-two types were identified (n = 11 605 cases) with echovirus 30 (E30), coxsackievirus A6 (CVA6), EV-D68, E9, E11, CVB5, E18, CVB4, EV-A71, and E6 most frequently reported. E30 declined after 2018/2019, while CVA6, CVB5, E9, E11, and EV-D68 were prevalent both before and after the coronavirus disease 2019 (COVID-19) pandemic, and CVB4 and E18 were prevalent after the pandemic. A shift in seasons (summer to fall) and specimen positivity (feces to respiratory) was observed. Neurological signs predominated among EV-A71, CVB4, CVB5, E6, E9, E11, E18, and E30 (30%–72%). CVB4, CVB5, E9, E11, and E18 were frequently reported among neonates (18%–32%). CVA6 was frequently associated with hand, foot and mouth disease, and EV-D68 with respiratory infections. Paralysis was reported among 22 infections, associated with 10 nonpolio types. Conclusions This study emphasizes the widespread circulation and severity of EV infections in Europe, as well as the (re)emergence of specific types postpandemic. Our findings highlight the need for continuous EV surveillance to monitor variation in circulation, age, and clinical presentations, including paralysis among nonpolio EV infections.
Keywords: enterovirus, laboratory detection, surveillance, Europe, epidemiology
Published in DiRROS: 27.06.2025; Views: 360; Downloads: 208
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Abstract: In the beginning of 2020, the outbreak of the COVID-19 pandemic led to a crisis in which diagnostic methods for the genome detection of SARS-CoV-2 were urgently needed. Based on the very early publication of the basic principles for a diagnostic test for the genome detection of SARS-CoV-2, the first noncommercial laboratory-developed tests (LDTs) and commercial tests were introduced. As there was considerable uncertainty about the reliability and performance of different tests and different laboratories, INSTAND established external quality assessment (EQA) schemes for the detection of SARS-CoV-2 starting in April 2020. In close partnership in a scientific network, the EQA schemes were enhanced, especially the April, June and November 2020 terms. The enhancement included: (i) immediate provision of suitable virus including variants of concern at the beginning of the pandemic outbreak, (ii) short frequency of EQA schemes, (iii) concentration dependency of the testing and sensitivity check, achieved by using SARS-CoV-2-positive samples from a 10-fold dilution series of the same starting material, (iv) specificity check of the testing, achieved by using SARS-CoV-2-negative samples containing human coronaviruses or MERS CoV, (v) revealed samples for orientation on test performance during an ongoing or at the start of an EQA scheme using a pre-quantified SARS-CoV-2-positive EQA sample with a low viral RNA load of only 1 570 copies/mL assigned by digital PCR (dPCR) in June 2020 and (vi) quantified reference materials based on the experiences of the first two EQA schemes with dPCR-assigned values in copies/mL beginning in November 2020 for self-evaluation of the applied test system. This manuscript summarizes the results of a total of 13 EQA schemes for the detection of SARS-CoV-2 between April 2020 and June 2023 in which a total of 1 413 laboratories from 49 countries participated. The qualitative results for the detection of SARS-CoV-2-positive samples were between 95.8 % and 99.7 % correct positive, excluding extremely low concentration samples. For all SARS-CoV-2-negative EQA samples, the qualitative success rates ranged from 95.1 % to 99.4 % correct negative results. The widely varying values for the cycle threshold (Ct)/crossing point (Cq) reported for the different target genes and test systems were striking. A few laboratories reported quantitative results in copies/mL for several VOCs with an acceptable rate of over 93 % correct positive results in the majority of cases. The description of the enhanced EQA schemes for SARS-CoV-2 detection in terms of timing and scope can serve as a blueprint for the rapid development of a quality assessment of diagnostics for an emerging pathogen.
Keywords: COVID-19 pandemic, SARS-CoV-2, virus genome detection tests, reference materials, external quality assessment, laboratory medicine, epidemiology
Published in DiRROS: 18.06.2025; Views: 421; Downloads: 405
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Abstract: In July 2021, during an official survey for Tomato brown rugose fruit virus (ToBRFV), a sample composed of leaves and fruit was taken from three tomato (Solanum lycopersicum cv. Factor F1) plants growing in a greenhouse producing fresh tomatoes in central Slovenia. The sampled plants were slightly dwarfed and showed deformations such as leaf curling, narrowing and small leaves (Figure 1), while no virus symptoms were observed on the fruit.
Keywords: detection, identification, plant virus disease
Published in DiRROS: 26.02.2025; Views: 636; Downloads: 541
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Abstract: Rapid and accurate identification of bacterial pathogens is crucial for effective treatment and infection control, particularly in hospital settings. Conventional methods like culture techniques and MALDI-TOF mass spectrometry are often time-consuming and less sensitive. This study addresses the need for faster and more precise diagnostic methods by developing novel digital PCR (dPCR) assays for the rapid quantification of biomarkers from three Gram-negative bacteria: Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Utilizing publicly available genomes and the rapid identification of PCR primers for unique core sequences or RUCS algorithm, we designed highly specific dPCR assays. These assays were validated using synthetic DNA, bacterial genomic DNA, and DNA extracted from clinical samples. The developed dPCR methods demonstrated wide linearity, a low limit of detection (approx. 30 copies per reaction), and robust analytical performance with measurement uncertainty below 25 %. The assays showed high repeatability and intermediate precision, with no cross-reactivity observed. Comparison with MALDI-TOF mass spectrometry revealed substantial concordance, highlighting the methods’ suitability for clinical diagnostics. This study underscores the potential of dPCR for rapid and precise quantification of Gram-negative bacterial biomarkers. The developed methods offer significant improvements over existing techniques, providing faster, more accurate, and SI-traceable measurements. These advancements could enhance clinical diagnostics and infection control practices.
Keywords: digital PCR (dPCR), Gram-negative bacteria, pathogen detection, respiratory infections, biomarkers, RUCS algorithm
Published in DiRROS: 05.11.2024; Views: 713; Downloads: 664
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