Bivalirudin Monitoring in Pediatric Ventricular Assist Device and Extracorporeal Membrane Oxygenation: Analysis of Single-Center Retrospective Cohort Data 2018-2022.

OBJECTIVES: The activated partial thromboplastin time (aPTT) is the most frequently used monitoring assay for bivalirudin in children and young adults on mechanical circulatory support including ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO). However, intrinsic variability of the aPTT complicates management and risks bleeding or thrombotic complications. We evaluated the utility and reliability of a bivalirudin-calibrated dilute thrombin time (Bival dTT) assay for bivalirudin monitoring in this population. DESIGN: Retrospective analysis of clinical data (including aPTT, dilute thrombin time [dTT]) and results of residual plasma samples from VAD patients were assessed in two drug-calibrated experimental assays. One assay (Bival dTT) was validated for clinical use in VAD patients, and subsequently used by clinicians in ECMO patients. Pearson correlation and simple linear regression were used to determine R2 correlation coefficients between the different laboratory parameters using Statistical Package for Social Sciences (Armonk, NY). SETTING: ICUs at Cincinnati Children's Hospital Medical Center. SUBJECTS: Children on VAD or ECMO support anticoagulated with bivalirudin. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: One hundred fifteen plasma samples from 11 VAD patients were analyzed. Both drug-calibrated experimental assays (anti-IIa and Bival dTT) showed excellent correlation with each other ( R2 = 0.94) and with the dTT ( R2 = 0.87), but poor correlation with aPTT ( R2 = 0.1). Bival dTT was selected for validation in VAD patients. Subsequently, clinically ordered results (105) from 11 ECMO patients demonstrated excellent correlation between the Bival dTT and the standard dTT ( R2 = 0.86) but very poor correlation with aPTT ( R2 = 0.004). CONCLUSIONS: APTT is unreliable and correlates poorly with bivalirudin's anticoagulant effect in ECMO and VAD patients. A drug-calibrated Bival dTT offers superior reliability and opportunity to standardize results across institutions. Additional studies are needed to determine an appropriate therapeutic range and correlation with clinical outcomes.

Development of a Diagnostic Assay for Race Differentiation of Podosphaera macularis.

Hop powdery mildew (caused by Podosphaera macularis) was confirmed in the Pacific Northwest in 1996. Before 2012, the most common race of P. macularis was able to infect plants that possessed powdery mildew resistance based on the R-genes Rb, R3, and R5. After 2012, two additional races of P. macularis were discovered that can overcome the resistance gene R6 and the partial resistance found in the cultivar Cascade. These three races now occur throughout the region, which can complicate management and research efforts because of uncertainty on which race(s) may be present in the region and able to infect susceptible hop genotypes. Current methods for determining the races of P. macularis are labor intensive, costly, and typically require more than 14 days to obtain results. We sought to develop a molecular assay to differentiate races of the fungus possessing virulence on plants with R6, referred to as V6-virulent, from other races. The transcriptomes of 46 isolates of P. macularis were sequenced to identify loci and variants unique to V6 isolates. Fourteen primer pairs were designed for 10 candidate loci that contained single nucleotide polymorphisms (SNP) and short insertion-deletion polymorphisms. Two differentially labeled locked nucleic acid probes were designed for a contig that contained a conserved SNP associated with V6-virulence. The resulting duplexed real-time PCR assay was validated against 46 V6 and 54 non-V6 P. macularis isolates collected from the United States and Europe. The assay had perfect discrimination of V6-virulence among isolates of P. macularis originating from the western U.S. but failed to predict V6-virulence in three isolates collected from Europe. The specificity of the assay was tested with different species of powdery mildew fungi and other microorganisms associated with hop. Weak nonspecific amplification occurred with powdery mildew fungi collected from Vitis vinifera, Fragaria sp., and Zinnia sp.; however, nonspecification amplification is not a concern when differentiating pathogen race from colonies on hop. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.

Transcriptome-Derived Amplicon Sequencing Markers Elucidate the U.S. Podosphaera macularis Population Structure Across Feral and Commercial Plantings of Humulus lupulus.

Obligately biotrophic plant pathogens pose challenges in population genetic studies due to their genomic complexities and elaborate culturing requirements with limited biomass. Hop powdery mildew (Podosphaera macularis) is an obligately biotrophic ascomycete that threatens sustainable hop production. P. macularis populations of the Pacific Northwest (PNW) United States differ from those of the Midwest and Northeastern United States, lacking one of two mating types needed for sexual recombination and harboring two strains that are differentially aggressive on the cultivar Cascade and able to overcome the Humulus lupulus R-gene R6 (V6), respectively. To develop a high-throughput marker platform for tracking the flow of genotypes across the United States and internationally, we used an existing transcriptome of diverse P. macularis isolates to design a multiplex of 54 amplicon sequencing markers, validated across a panel of 391 U.S. samples and 123 international samples. The results suggest that P. macularis from U.S. commercial hop yards form one population closely related to P. macularis of the United Kingdom, while P. macularis from U.S. feral hop locations grouped with P. macularis of Eastern Europe. Included in this multiplex was a marker that successfully tracked V6-virulence in 65 of 66 samples with a confirmed V6-phenotype. A new qPCR assay for high-throughput genotyping of P. macularis mating type generated the highest resolution distribution map of P. macularis mating type to date. Together, these genotyping strategies enable the high-throughput and inexpensive tracking of pathogen spread among geographical regions from single-colony samples and provide a roadmap to develop markers for other obligate biotrophs.

High Levels of Insensitivity to Phosphonate Fungicides in Pseudoperonospora humuli.

Phosphonate (phosphite; HPO3-2) is fungicidal against oomycetes and certain other organisms. The Fungicide Resistance Action Committee has deemed phosphonate to be at low risk of resistance development, and reduced sensitivity to phosphonate has been reported only occasionally in plant pathogens. Reduced sensitivity to the fungicide fosetyl-Al was documented in the hop downy mildew pathogen, Pseudoperonospora humuli, in the early 2000s, but disease caused by insensitive isolates could still be managed commercially if the fungicide rate was doubled from 2.24 to 4.48 kg/ha. In this research, we document the occurrence of isolates of P. humuli in Oregon that possess even higher levels of insensitivity to fosetyl-Al and other phosphonate fungicides. The median estimated effective concentration required to reduce infection by 50% (EC50) for isolates collected from two farms reporting disease control failures was 2.7% (vol/vol) phosphonate (range = 1.6 to 164.2), which was 1.6 times (range = 0.9 to 96.0) the maximum labeled rate of the phosphonate fungicide utilized. In contrast, the median EC50 for isolates obtained from experimental plots that have received only a single application of a phosphonate fungicide was 0.6% (vol/vol) phosphonate (range = 0.11 to 2.3) or 0.3 times the maximum allowable rate. Sensitivity of isolates to a phosphorous acid fungicide, fosetyl-Al, and a plant nutrient product containing an unspecified level of phosphorous acid were linearly related. Insensitivity to the maximum allowable rate of a phosphorous acid fungicide was widespread within and among hop farms in Oregon. Among 54 isolates assayed for phosphonate insensitivity, 96% had EC50 values that exceeded the maximum allow rate of the fungicide used in the assays. Field studies conducted in 2 years further demonstrated that a phosphorous fungicide, a nutrient product containing phosphorous acid, and fosetyl-Al failed to provide commercially acceptable suppression of downy mildew when applied at the maximum allowable rates and even double these rates, whereas fungicides with different modes of action provided 91% or greater disease control. The whole of this research indicates that P. humuli has been selected to tolerate fosetyl-Al and other phosphonate fungicides at rates four times greater than those used earlier to obtain satisfactory suppression of downy mildew. This finding has implications for management of the disease not only in Oregon but also, in other production regions should insensitive isolates be introduced on infected planting material.

Definitive Identification of Laribacter hongkongensis Acquired in the United States.

Laribacter hongkongensis is a potential emerging pathogen associated with community-acquired gastroenteritis and traveler's diarrhea. We report the isolation of L. hongkongensis from the stool of a patient who had no history of travel outside the United States. The organism was identified by phenotypic tests, mass spectrometry, and gene sequencing.