Evaluation of the 3-Minute Diagnostic Confusion Assessment Method for identification of postoperative delirium in older patients

Importance: Delirium is a common postoperative complication in older patients that often goes undetected and might lead to worse outcomes. The 3-Minute Diagnostic Confusion Assessment Method (3D-CAM) might be a practical tool for routine clinical diagnosis of delirium. Objective: To assess the 3D-CAM for detecting postoperative delirium compared with the long-form CAM used for research purposes. Design, Setting, and Participants: This cohort study of older patients enrolled in ongoing clinical trials between 2015 and 2018 was conducted at a single tertiary US hospital. Included participants were aged 60 years or older undergoing major elective surgical procedures that required at least a 2-day hospital stay. Data were analyzed between February and April 2019. Exposures: Surgical procedures of at least 2 hours in length requiring general anesthesia with planned extubation. Main Outcomes and Measures: Patients were concurrently assessed for delirium using the 3D-CAM assessment and the long-form CAM, scored based on a standardized cognitive assessment. Agreement between these 2 methods was tested using Cohen κ with repeated measures, a generalized linear mixed-effects model, and Bland-Altman analysis. Results: Sixteen raters conducted 471 concurrent CAM and 3D-CAM interviews including 299 patients (mean [SD] age, 69 [6.5] years), the majority of whom were men (152 [50.8%]), were White (263 [88.0%]), and had noncardiac operations (211 [70.6%]). Both instruments had good intraclass correlation (0.84 for the CAM and 0.98 for the 3D-CAM). Cohen κ demonstrated good overall agreement between the CAM and 3D-CAM (κ = 0.71; 95% CI, 0.58 to 0.83). According to the mixed-effects model, there was statistically significant disagreement between the 3D-CAM and CAM (estimated difference in fixed effect, -0.68; 95% CI, -1.32 to -0.05; P = .04). Bland-Altman analysis showed the probability of a delirium diagnosis with the 3D-CAM was more than twice the probability of a delirium diagnosis with the CAM (probability ratio, 2.78; 95% CI, 2.44 to 3.23). Conclusions and Relevance: The 3D-CAM instrument demonstrated agreement with the long-form CAM and might provide a pragmatic and sensitive clinical tool for detecting postoperative delirium, with the caveat that the 3D-CAM might overdiagnose delirium

A comparison of alternative assays to measure DNA damage in stallion spermatozoa: TUNEL test versus ‘Nicoletti assay’

The aberrations of sperm DNA may cause various problems and have negative consequences on fertility. These influence embryonic development or might lead to early embryo loss. Sperm Chromatin Structure Assay (SCSA) is the flow cytometric method most often used for the detection of DNA lesions; however, some studies using that method reached confusing conclusions. The aim of this pilot study was to adjust and compare two alternative tests, namely the TUNEL test and the Nicoletti assay. The above-mentioned two flow cytometric methods capable of detecting the fragmented DNA of sperm were tested on 12 frozen-thawed stallion semen samples. The TUNEL test demonstrated much higher DNA fragmentation ratio than the Nicoletti assay (mean ± SD: 30.77 ± 13.03% vs. 1.93 ± 0.89%, respectively). A fluorescent microscopic check of the samples showed that TUNEL labelled the plasma membrane and the mitochondria in a nonspecific way, rather than detecting only the fragmented DNA, thus eventually resulting in a false positive sign. The Nicoletti assay is simpler, quicker and does not detect nonspecific binding; however, further analyses are required to determine its diagnostic value

Attenuated reovirus displays oncolysis with reduced host toxicity

Background: Although the naturally occurring reovirus causes only mild symptoms in humans, it shows considerable potential as an oncolytic agent because of its innate ability to target cancer cells. In immunocompromised hosts, however, wild-type reovirus can target healthy tissues, including heart, liver, pancreas and neural structures. Methods: We characterized an attenuated form of reovirus (AV) derived from a persistently infected cell line through sequence analysis, as well as western blot and in vitro transcription and translation techniques. To examine its pathogenesis and oncolytic potential, AV reovirus was tested on healthy embryonic stem cells, various non-transformed and transformed cell lines, and in severe combined immunodeficiency (SCID) mice with tumour xenografts. Results: Sequence analysis of AV reovirus revealed a premature STOP codon in its sigma 1 attachment protein. Western blot and in vitro translation confirmed the presence of a truncated ?1. In comparison to wild-type reovirus, AV reovirus did not kill healthy stem cells or induce black tail formation in SCID mice. However, it did retain its ability to target cancer cells and reduce tumour size. Conclusion: Despite containing a truncated attachment protein, AV reovirus still preferentially targets cancer cells, and compared with wild-type reovirus it shows reduced toxicity when administered to immunodeficient hosts, suggesting the potential use of AV reovirus in combination cancer therapy

Detection of DNA polymerase activities associated with purified duck hepatitis B virus core particles by using an activity gel assay.

Replication of hepadnaviruses involves reverse transcription of an intermediate RNA molecule. It is generally accepted that this replication scheme is carried out by a virally encoded, multifunctional polymerase which has DNA-dependent DNA polymerase, reverse transcriptase, and RNase H activities. Biochemical studies of the polymerase protein(s) have been limited by the inability to purify useful quantities of functional enzyme from virus particles and, until recently, to express enzymatically active polymerase proteins in heterologous systems. An activity gel assay which detects in situ catalytic activities of DNA polymerases after electrophoresis in partially denaturing polyacrylamide gels was used by M.R. Bavand and O. Laub (J. Virol. 62:626-628, 1988) to show the presence of DNA- and RNA-dependent DNA polymerase activities associated with hepatitis B virus particles produced in vitro. This assay has provided the only means by which hepadnavirus polymerase proteins have been detected in association with enzymatic activities. Since conventional methods have not allowed purification of useful quantities of enzymatically active polymerase protein(s), we have devised a protocol for purifying large quantities of duck hepatitis B virus (DHBV) core particles to near homogeneity. These immature virus particles contain DNA- and RNA-dependent DNA polymerase activities, as shown in the endogenous DNA polymerase assay. We have used the activity gel assay to detect multiple DNA- and RNA-dependent DNA polymerase proteins associated with these purified DHBV core particles. These enzymatically active proteins appear larger than, approximately the same size as, and smaller than an unmodified DHBV polymerase protein predicted from the polymerase open reading frame. This is the first report of the detection of active hepadnavirus core-associated DNA polymerase proteins derived from a natural host

Detection of an RNase H activity associated with hepadnaviruses.

Replication of the hepadnavirus DNA genome is accomplished via reverse transcription of an intermediate, pregenomic RNA molecule. This process is likely to be carried out by a virally encoded, multifunctional polymerase which possesses DNA- and RNA-dependent DNA polymerase and RNase H activities. However, the nature of the product(s) of the polymerase gene predicted to mediate these functions is unclear. Biochemical studies of the polymerase protein(s) have been limited by its apparent low abundance in virus particles and, until recently, the inability to express active polymerase protein(s) heterologously. We have used activity gel assays to detect DNA- and RNA-dependent DNA polymerase activities associated with highly purified duck hepatitis B virus (DHBV) core particles (S. M. Oberhaus and J. E. Newbold, J. Virol. 67:6558-6566, 1993). Now we report that the same approach identifies a 35-kDa RNase H activity in association with highly purified DHBV core particles and crude preparations of virions from DHBV-infected ducks and woodchuck hepatitis virus-infected woodchucks. This is the first report of the detection of an hepadnavirus-associated RNase H activity. Its apparent size is smaller than any of the DNA polymerase activities that we detected previously and significantly smaller than the full-length protein predicted from the polymerase open reading frame (p85 for DHBV). These data suggest that the viral polymerase and RNase H activities are separable and that these enzymes may coordinate their activities in vivo by forming a complex

Reovirus-induced apoptosis of MDCK cells is not linked to viral yield and is blocked by Bcl-2.

In this study, we investigated the relationship between reovirus-induced apoptosis and viral growth. Madin-Darby canine kidney (MDCK) epithelial cells infected with prototype reovirus strains type 1 Lang (T1L) or type 3 Dearing (T3D) were found to undergo apoptosis, and T3D induced apoptosis of MDCK cells to a substantially greater extent than T1L. By using T1L x T3D reassortant viruses, we found that differences in the capacities of these strains to induce apoptosis are determined by the viral S1 and M2 gene segments. These genes encode viral outer-capsid proteins that play important roles in viral entry into cells. T1L grew significantly better in MDCK cells than T3D, and these differences in growth segregated with the viral L1 and M1 gene segments. The L1 and M1 genes encode viral core proteins involved in viral RNA synthesis. Bcl-2 overexpression in MDCK cells inhibited reovirus-induced apoptosis but did not substantially affect reovirus growth. These findings indicate that differences in the capacities of reovirus strains to induce apoptosis and grow in MDCK cells are determined by different viral genes and that premature cell death by apoptosis does not limit reovirus growth in MDCK cells

Differences in the capacity of reovirus strains to induce apoptosis are determined by the viral attachment protein sigma 1.

Reoviruses are important models for studies of viral pathogenesis; however, the mechanisms by which these viruses produce cytopathic effects in infected cells have not been defined. In this report, we show that murine L929 (L) cells infected with prototype reovirus strains type 1 Lang (TIL) and type 3 Dearing (T3D) undergo apoptosis and that T3D induces apoptosis to a substantially greater extent than T1L. Using T1L x T3D reassortant viruses, we found that differences in the capacity of T1L and T3D to induce apoptosis are determined by the viral S1 gene segment, which encodes the viral attachment protein sigma 1 and the non-virion-associated protein sigma 1s. Apoptosis was induced by UV-inactivated, replication-incompetent reovirus virions, which do not contain sigma 1s and do not mediate its synthesis in infected cells. Additionally, T3D-induced apoptosis was inhibited by anti-reovirus monoclonal antibodies that inhibit T3D cell attachment and disassembly. These results indicate that sigma 1, rather than sigma 1s, is required for induction of apoptosis by the reovirus and suggest that interaction of virions with cell surface receptors is an essential step in this mechanism of cell killing

Effect of Sampling Method on Detection of the Equine Uterine Microbiome during Estrus.

Bacterial endometritis is among the most common causes of subfertility in mares. It has a major economic impact on the equine breeding industry. The sensitivity of detecting uterine microbes using culture-based methods, irrespective of the sample collection method, double-guarded endometrial swab, endometrial biopsy, or uterine low-volume lavage (LVL), is low. Therefore, equine bacterial endometritis often goes undiagnosed. Sixteen individual mares were enrolled, and an endometrial sample was obtained using each method from all mares. After trimming, quality control and decontamination, 3824 amplicon sequence variants were detected in the dataset. We found using 16S rRNA sequencing that the equine uterus harbors a distinct resident microbiome during estrus. All three sampling methods used yielded similar results in composition as well as relative abundance at phyla (Proteobacteria, Firmicutes, and Bacteroidota) and genus (Klebsiella, Mycoplasma, and Aeromonas) levels. A significant difference was found in alpha diversity (Chao1) between LVL and endometrial biopsy, suggesting that LVL is superior at detecting the low-abundant (rare) taxa. These new data could pave the way for innovative treatment methods for endometrial disease and subfertility in mares. This, in turn, could lead to more judicious antimicrobial use in the equine breeding industry

Effect of Sampling Method on Detection of the Equine Uterine Microbiome during Estrus

Bacterial endometritis is among the most common causes of subfertility in mares. It has a major economic impact on the equine breeding industry. The sensitivity of detecting uterine microbes using culture-based methods, irrespective of the sample collection method, double-guarded endometrial swab, endometrial biopsy, or uterine low-volume lavage (LVL), is low. Therefore, equine bacterial endometritis often goes undiagnosed. Sixteen individual mares were enrolled, and an endometrial sample was obtained using each method from all mares. After trimming, quality control and decontamination, 3824 amplicon sequence variants were detected in the dataset. We found using 16S rRNA sequencing that the equine uterus harbors a distinct resident microbiome during estrus. All three sampling methods used yielded similar results in composition as well as relative abundance at phyla (Proteobacteria, Firmicutes, and Bacteroidota) and genus (Klebsiella, Mycoplasma, and Aeromonas) levels. A significant difference was found in alpha diversity (Chao1) between LVL and endometrial biopsy, suggesting that LVL is superior at detecting the low-abundant (rare) taxa. These new data could pave the way for innovative treatment methods for endometrial disease and subfertility in mares. This, in turn, could lead to more judicious antimicrobial use in the equine breeding industry

Metagenomic characterization of the equine endometrial microbiome during anestrus

The equine uterus is highly interrogated during estrus prior to breeding and establishing pregnancy. Many studies in mares have been performed during estrus under the influence of high estrogen concentrations, including the equine estrual microbiome. To date, it is unknown how the uterine microbiome of the mare is influenced by cyclicity; while, the equine vaginal microbiome is stable throughout the estrous cycle. We hypothesized that differences would exist between the equine endometrial microbiome of mares in estrus and anestrus. The aim of this study was two-fold: to characterize the resident endometrial microbiome of healthy mares during anestrus and to compare this with estrus. Double-guarded endometrial swabs were taken from healthy mares during estrus (n = 16) and in the following non-breeding season during anestrus (n = 8). Microbial population was identified using 16S rRNA sequencing. Our results suggest that the equine uterine microbiome in estrus has a low diversity and low richness, while during anestrus, a higher diversity and higher richness were seen compared to estrus. Despite this difference, both the estrus and anestrus endometrial microbiome were dominated by Proteobacteria, Firmicutes, and Bacteroidota. The composition of the microbial community between anestrus and estrus was significantly different. This may be explained by the difference in the composition of the endometrial immune milieu based on the stage of the cycle. Further research investigating the function of the equine endometrial microbiome and dynamics changes within the uterine environment is required