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Optimized protocol for culturing and extracting DNA from fungal isolates associated with brown spot needle blight in pine trees

by Temitope Ruth Folorunso, Gabriel Silva, Marilis E. Girón, Tess Lindow, Micah Persyn, Lori Eckhardt, Janna R. Willoughby

Effective culturing and DNA extraction protocols are essential for advancing research on fungal pathogens of brown spot needle blight (BSNB) that infect loblolly pine (Pinus taeda) and other Pinus species. We evaluated the performance of four widely used fungal media, including Sabouraud dextrose, malt extract, potato dextrose, and yeast extract peptone dextrose, in both solid (agar) and liquid (broth) formats, quantifying fungal growth through colony diameter and biomass accumulation over a three-week period. Sabouraud dextrose agar and broth consistently supported the most rapid and extensive growth in both formats, while potato dextrose underperformed across these metrics. To identify an optimal protocol for downstream molecular applications, we also compared four DNA extraction methods, three of which were modified variants of the CTAB (cetyl-trimethyl-ammonium bromide) chemistry as well as the Qiagen DNeasy kit following the yeast DNA extraction protocol. DNA yield, quantified by fluorometry, was highest for the high-salt CTAB polyvinylpyrrolidone (PVP) protocol and DNA purity (assessed by 260/280 absorbance ratio) was optimal for both PVP and Qiagen extractions. From these comparisons, we suggest that Sabouraud dextrose culturing combined with CTAB PVP extraction for use as a robust and accessible pipeline for generating high-quality fungal DNA.

Heart failure in Southern Sweden (HISS): a cross-sectional analysis of primary care patients characteristics and physicians adherence to guideline-directed medical therapy

Por: Svensson Söderberg · K. · Kronestedt Lundevall · L. · Lindow · T. · Ö Braun · O. · Smith · J. G. · Sundquist · K. · Milos Nymberg · V.
Objectives

We aimed to describe clinical and diagnostic characteristics of primary care patients with heart failure and physicians’ adherence to guideline-directed medical therapy (GDMT) for treating chronic heart failure.

Design

Cross-sectional study based on baseline data from the prospective primary care-based study Heart failure in Southern Sweden (HISS).

Setting

Patients with heart failure were included from 20 primary healthcare centres in the southernmost region of Sweden (Skåne).

Participants

Between 2020 and 2023, patients were included in HISS, resulting in a total of 587 participants. Of these, 558 patients (95% of the HISS participants) had available data on left ventricular ejection fraction and were included in this study. Adult patients aged 18 years or older diagnosed with heart failure (International Classification of Diseases, 10th Revision codes I50, I11.0, I42, I43) were considered eligible for inclusion in HISS. Community-dwelling patients with assisted care were excluded.

Primary and secondary outcomes

The primary outcome measures were distribution of heart failure subtypes and prescribed medications. The secondary outcomes were temporal trends in GDMT and the association between physicians’ adherence to GDMT and clinical characteristics of patients, using logistic regression models.

Results

Heart failure with preserved ejection fraction (HFpEF) was the most prevalent subtype (42%), followed by mildly reduced (30%) and reduced ejection fraction (HFrEF, 28%). Among patients with HFrEF, 20% were prescribed the recommended GDMT according to the European Society of Cardiology (ESC) 2021 guidelines, which consisted of a renin-angiotensin system inhibitor, a beta-blocker, a mineralocorticoid receptor antagonist and a sodium-glucose 2 inhibitor. We observed no significant change in the prescribing trends for the quadruple therapy in patients with HFrEF when comparing the 2 years before and after the publication of the ESC 2021 guidelines. Similarly, we observed no association between patient characteristics and the prescription of GDMT according to ESC 2021 for patients with HFrEF.

Conclusion

HFpEF was the most prevalent subtype, with conclusive and recent echocardiography data among two-thirds of the cohort. Temporal trends in prescription patterns showed no appreciable increase in the use of GDMT for HFrEF during the two years following guideline publication compared with the two preceding years. These findings indicate a need for inclusion of primary care patients as a basis for intensified medical recommendations and implementation strategies.

Trial registration number

NCT04129658.

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