Echinoderm mass mortality events shape marine ecosystems by altering the dynamics among major benthic groups. The sea urchin Diadema antillarum, virtually extirpated in the Caribbean in the early 1980s by an unknown cause, recently experienced another mass mortality beginning in January 2022. We investigated the cause of this mass mortality event through combined molecular biological and veterinary pathologic approaches comparing grossly normal and abnormal animals collected from 23 sites, representing locations that were either affected or unaffected at the time of sampling. Here, we report that a scuticociliate most similar to Philaster apodigitiformis was consistently associated with abnormal urchins at affected sites but was absent from unaffected sites. Experimentally challenging naïve urchins with a Philaster culture isolated from an abnormal, field-collected specimen resulted in gross signs consistent with those of the mortality event. The same ciliate was recovered from treated specimens postmortem, thus fulfilling Koch’s postulates for this microorganism. We term this condition D. antillarum scuticociliatosis.
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Targeted mutagenesis of CCoAOMT1 in Petunia using CRISPR/Cas 9 Technology
Standard SARS-CoV-2 testing protocols using nasopharyngeal/throat (NP/T) swabs are invasive and require trained medical staff for reliable sampling. In addition, it has been shown that PCR is more sensitive as compared to antigen-based tests. Here we describe the analytical and clinical evaluation of our in-house RNA extraction-free saliva-based molecular assay for the detection of SARS-CoV-2. Analytical sensitivity of the test was equal to the sensitivity obtained in other Dutch diagnostic laboratories that process NP/T swabs. In this study, 955 individuals participated and provided NP/T swabs for routine molecular analysis (with RNA extraction) and saliva for comparison. Our RT-qPCR resulted in a sensitivity of 82,86% and a specificity of 98,94% compared to the gold standard. A false-negative ratio of 1,9% was found. The SARS-CoV-2 detection workflow described here enables easy, economical, and reliable saliva processing, useful for repeated testing of individuals.
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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.
Bijna alle klinische samples bevatten genetisch materiaal van het infectieus agentia, waardoor sequencing een aantrekkelijke benadering is voor de detectie en identificatie van pathogenen. Deze nieuwe vorm van moleculaire diagnostiek omzeilt het lange proces van traditionele, op cultuur gebaseerde analyses. Innovatieve amplicon specifieke next generation sequencing (NGS) technologie heeft de mogelijkheid de ziekteverwekkers op basis van DNA binnen enkele uren te identificeren. Deze aanpak versnelt daarmee de diagnostiek enorm en is met name een waardevolle aanvulling bij niet- of moeilijk kweekbare pathogenen. Dit zorgt ervoor dat de patiënt optimaal behandeld kan worden en er geen onnodige gezondheidsschade optreedt. Binnen dit project zal gebruik worden gemaakt van sterk geconserveerde primers in een universele PCR, gevolgd door het bepalen van de nucleotiden volgorde. Aan de hand van de verkregen data wordt vervolgens exact bepaald welk(e) micro-organisme(n) verantwoordelijk is/zijn voor de infectie, zelfs wanneer er co-infectie van meerdere pathogenen is opgetreden. MKB partner Molecular Biology Systems B.V. (MBS) ontwikkelen en verkopen apparatuur waarmee de veelgebruikte PCR extreem snel kan worden uitgevoerd. In samenwerking met MBS, MUMC+ en het lectoraat Analyse Technieken in de Life Sciences wordt binnen dit project een nieuwe moleculaire aanpak opgezet middels de MBS technologie, waarbij op een snelle en gebruiksvriendelijke manier infectieziekten gemonitord kunnen worden wat resulteert in een verbeterd behandelplan. Alle partners kunnen direct profiteren van de resultaten van dit onderzoek. Zo is er directe vertaling van resultaten naar een nieuwe werkwijze voor MUMC+, zal Avans de resultaten direct integreren in haar onderwijs en zal MBS deze snelle innovatieve werkmethode verder door ontwikkelen in de medische diagnostiek.
