Frozen/Thawed Samples Can Replace Fresh Samples for Assignment of ISI to Secondary Thromboplastin Standards for Multiple Reagent/Instrument Combinations: Data to Support Possible Revision of WHO Guidelines.

BACKGROUND: Calibration of thromboplastins is required for accurate calculation of the international normalised ratio (INR). Accurate INR results are required for optimal dosing of vitamin K antagonists. Decreases in vitamin K antagonist usage have made the recruitment of sample sets for international sensitivity index (ISI) calibrations more difficult. A possible solution to this would be to allow the use of frozen-thawed samples in place of fresh plasmas in the calibration of secondary standards. OBJECTIVES: We investigated the effect of freezing and thawing samples before usage in ISI calibrations of secondary standards. METHODS: Multiple reagent/instruments were tested to identify the degree of difference between a fresh sample ISI calibration and one performed on frozen-thawed samples. Where possible, the two ISI calibrations were performed on the same sample set. Alternatively, a separate set of samples from different patients was used. RESULTS: The difference in ISI values was <3% for those datasets where the same samples were used, and <6% for those datasets where two sample sets were used. Additionally, other parameters required for a valid ISI calibration showed only minor differences-some calibrations showed fewer outliers in the frozen-thawed datasets. Mean normal prothrombin time for the international reference thromboplastins was <3.5% different across four different calibrations (two for rabbit thromboplastin and two for recombinant human thromboplastin). CONCLUSIONS: This modification to the WHO guidelines would facilitate the recruitment of test plasmas in advance of calibration solving the problem of requiring availability of fresh patient samples with a range of INRs in a 5-h window. TRIAL REGISTRATION: Not a part of any clinical trial.

Defining a metrologically traceable and sustainable calibration hierarchy of international normalized ratio for monitoring of vitamin K antagonist treatment in accordance with International Organization for Standardization (ISO) 17511:2020 standard: communication from the International Federation of Clinical Chemistry and Laboratory Medicine-SSC/ISTH working group on prothrombin time/international normalized ratio standardization.

Calibration of prothrombin time (PT) in terms of international normalized ratio (INR) has been outlined in "Guidelines for thromboplastins and plasmas used to control oral anticoagulant therapy" (World Health Organization, 2013). The international standard ISO 17511:2020 presents requirements for manufacturers of in vitro diagnostic (IVD) medical devices (MDs) for documenting the calibration hierarchy for a measured quantity in human samples using a specified IVD MD. The objective of this article is to define an unequivocal, metrologically traceable calibration hierarchy for the INR measured in plasma as well as in whole blood samples. Calibration of PT and INR for IVD MDs according to World Health Organization guidelines is similar to that in cases where there is a reference measurement procedure that defines the measurand for value assignment as described in ISO 17511:2020. We conclude that, for PT/INR standardization, the optimal calibration hierarchy includes a primary process to prepare an international reference reagent and measurement procedure that defines the measurand by a value assignment protocol conforming to clause 5.3 of ISO 17511:2020. A panel of freshly prepared human plasma samples from healthy adult individuals and patients on vitamin K antagonists is used as a commutable secondary calibrator as described in ISO 17511:2020. A sustainable metrologically traceable calibration hierarchy for INR should be based on an international protocol for value assignment with a single primary reference thromboplastin and the harmonized manual tilt tube technique for clotting time determination. The primary international reference thromboplastin reagent should be used only for calibration of successive batches of the secondary reference thromboplastin reagent.

Evolution of a subtilisin-like protease gene family in the grass endophytic fungus Epichloë festucae.

BACKGROUND: Subtilisin-like proteases (SLPs) form a superfamily of enzymes that act to degrade protein substrates. In fungi, SLPs can play either a general nutritive role, or may play specific roles in cell metabolism, or as pathogenicity or virulence factors. RESULTS: Fifteen different genes encoding SLPs were identified in the genome of the grass endophytic fungus Epichloë festucae. Phylogenetic analysis indicated that these SLPs belong to four different subtilisin families: proteinase K, kexin, pyrolysin and subtilisin. The pattern of intron loss and gain is consistent with this phylogeny. E. festucae is exceptional in that it contains two kexin-like genes. Phylogenetic analysis in Hypocreales fungi revealed an extensive history of gene loss and duplication. CONCLUSION: This study provides new insights into the evolution of the SLP superfamily in filamentous fungi.

Patterns of expression of a lolitrem biosynthetic gene in the Epichloë festucae-perennial ryegrass symbiosis.

Lolitrem B is synthesized by Epichloë festucae in associations with Pooid grasses. A complex cluster of at least 10 genes (ltm genes) is required for its synthesis. An early step in this pathway is catalyzed by ltmM, a symbiosis-expressed gene. PltmM-gusA reporter gene analysis was used to monitor ltmM gene expression patterns in planta. The minimum promoter length required for high-level gusA expression in infected seedlings is in the range of 480 to 782 bp. gusA was expressed by the endophyte in all infected vegetative plant tissues and in epiphyllous hyphae. Spikelets from reproductive tillers were analyzed at different developmental stages. During pre-anthesis, gusA expression was observed in all infected floral organs except the immature gynoecium. In post-anthesis florets, gene expression occurred almost exclusively in the gynoecium. Expression of gusA by the endophyte was observed in germinating seeds 24 h postimbibition and seedlings older than 6 days postimbibition in hyphae from the mesocotyl to the tip of the emerging first leaf. This work provides a detailed analysis of the spatial and temporal expression patterns of a symbiosis-expressed gene in planta.

Eimeria species (Apicomplexa: Eimeriidae) from arctic ground squirrels (Spermophilus parryii) and red squirrels (Tamiasciurus hudsonicus) in Alaska and in Siberia, Russia.

Fecal samples from arctic ground squirrels (Spermophilus parryii) collected in Alaska (n = 90) and Russia (n = 46) and from red squirrels (Tamiasciurus hudsonicus) in Alaska (n = 35) were examined for the presence of Eimeria spp. (Apicomplexa: Eimeriidae). Four species were recovered from arctic ground squirrels, including Eimeria callospermophili (prevalence = 18%), Eimeria cynomysis (23.5%), Eimeria lateralis (19%), and Eimeria morainensis (77%). A single species, Eimeria tamiasciuri (91%), was recovered from red squirrels. Eimerians recovered from arctic ground squirrels represent new host records, and the single species from red squirrels is a new geographic record. Alaskan arctic ground squirrel prevalence was higher for E. callospermophili (Alaska = 22% vs. Russia = 9%), E. cynomysis (34% vs. 2%), and E. lateralis (27% vs. 4%), but not E. morainensis (78% vs. 76%).

Effects of knowledge and internal locus of control in groups of health care workers judging likelihood of pathogen transfer.

OBJECTIVE: A study was conducted to measure the effects of attitudes and beliefs on the risk judgments of health care workers. BACKGROUND: Lack of hand hygiene compliance is a worldwide issue in health care, contributing to infections, fatalities, and increased health care costs. Human factors methods are a promising solution to the problem of compliance, although thus far, the concentration has been on process and engineering methods, such as the design of no-touch sinks. Factors internal to the health care worker, such as their attitudes and beliefs about hand hygiene, have received less attention. METHOD: For this study, three groups of health care workers completed measures of attitudes, control beliefs, and hand hygiene knowledge. They then provided risk judgments of touching various surfaces via a factorial survey. RESULTS: Attitudes, knowledge, control beliefs, and surface type all predicted the risk judgments of the sample of health care workers, with differences between professional groups. CONCLUSION: Health care workers perceive less risk when touching surfaces,which may explain historically low rates of hand hygiene compliance after surface contact. APPLICATION: Although more research is needed to directly connect risk judgments to failures of hand hygiene, the current results can inform interventions targeting the internal attitudes and beliefs of health care workers.

Functional analysis of a beta-1,6-glucanase gene from the grass endophytic fungus Epichloë festucae.

beta-1,6-glucanases degrade the polysaccharide beta-1,6-glucan, a cell wall component in some filamentous fungi. A single copy of a beta-1,6-glucanase gene, designated gcnA, was identified in each of the grass endophytic fungi Neotyphodium lolii and Epichloë festucae. Phylogenetic analysis indicates that the GcnA protein is a member of glycosyl hydrolase family 5, and is closely related to fungal beta-1,6-glucanases implicated in mycoparasitism. The E. festucae gcnA gene was expressed in mycelium grown in culture and in both vegetative and reproductive tissues of perennial ryegrass. A gcnA replacement mutant had reduced beta-1,6-glucanase activity when grown in media containing pustulan as the major carbon source. beta-1,6-glucanase activity was restored in the replacement mutant by introducing multiple copies of the gcnA gene. Growth of DeltagcnA and gcnA-overexpressing strains in vegetative grass tissues was indistinguishable from wild type strains.