Host diversity and behavior determine patterns of interspecies transmission and geographic diffusion of avian influenza A subtypes among North American wild reservoir species.

Wild birds can carry avian influenza viruses (AIV), including those with pandemic or panzootic potential, long distances. Even though AIV has a broad host range, few studies account for host diversity when estimating AIV spread. We analyzed AIV genomic sequences from North American wild birds, including 303 newly sequenced isolates, to estimate interspecies and geographic viral transition patterns among multiple co-circulating subtypes. Our results show high transition rates within Anseriformes and Charadriiformes, but limited transitions between these orders. Patterns of transition between species were positively associated with breeding habitat range overlap, and negatively associated with host genetic distance. Distance between regions (negative correlation) and summer temperature at origin (positive correlation) were strong predictors of transition between locations. Taken together, this study demonstrates that host diversity and ecology can determine evolutionary processes that underlie AIV natural history and spread. Understanding these processes can provide important insights for effective control of AIV.

Development of a multiplex droplet digital PCR method for detection and monitoring of Mycobacterium tuberculosis and drug-resistant tuberculosis.

BACKGROUND: The prevalence of multidrug-resistant tuberculosis (MDR-TB) among Korean tuberculosis patients is about 4.1%, which is higher than the OECD average of 2.6%. Inadequate drug use and poor patient compliance increase MDR-TB prevalence through selective pressure. Therefore, prompt detection of drug resistance in tuberculosis patients at the time of diagnosis and quantitative monitoring of these resistant strains during treatment are crucial. METHODS: A multiplex droplet digital PCR (ddPCR) assay was developed and assessed using DNA material of nine Mycobacterium tuberculosis strains with known mutation status that were purchased from the Korean National Tuberculosis Association. We collected a total of 18 MDR-TB residual samples referred for PCR analysis. Total DNA was extracted from the samples and subjected to the quadruplex ddPCR assay. Their results were compared to those of known resistance phenotypes. RESULTS: The analytical sensitivity and specificity of the multiplex ddPCR assay for detecting INH, RIF, EMB, FQ, and SM resistance-causing mutations ranged from 71.43 to 100% and 94.12-100%, respectively. Follow-up sample results showed that the quadruplex ddPCR assay was sensitive enough to detect IS6110 and other mutations even after onset of treatment. CONCLUSIONS: We developed a sensitive and accurate multiplex ddPCR assay that can detect the presence of tuberculosis quantitatively and resistance-conveying mutations concurrently. This tool could aid clinicians in the diagnosis and treatment monitoring of tuberculosis.

Fitness costs of Tn1546-type transposons harboring the vanA operon by plasmid type and structural diversity in Enterococcus faecium.

BACKGROUND: This study analyzed the genetic traits and fitness costs of vancomycin-resistant Enterococcus faecium (VREfm) blood isolates carrying Tn1546-type transposons harboring the vanA operon. METHODS: All E. faecium blood isolates were collected from eight general hospitals in South Korea during one-year study period. Antimicrobial susceptibility testing and vanA and vanB PCR were performed. Growth rates of E. faecium isolates were determined. The vanA-positive isolates were subjected to whole genome sequencing and conjugation experiments. RESULTS: Among 308 E. faecium isolates, 132 (42.9%) were positive for vanA. All Tn1546-type transposons harboring the vanA operon located on the plasmids, but on the chromosome in seven isolates. The plasmids harboring the vanA operon were grouped into four types; two types of circular, nonconjugative plasmids (Type A, n = 50; Type B, n = 46), and two types of putative linear, conjugative plasmids (Type C, n = 16; Type D, n = 5). Growth rates of vanA-positive E. faecium isolates were significantly lower than those of vanA-negative isolates (P < 0.001), and reduction in growth rate under vancomycin pressure was significantly larger in isolates harboring putative linear plasmids than in those harboring circular plasmids (P = 0.020). CONCLUSIONS: The possession of vanA operon was costly to bacterial hosts in antimicrobial-free environment, which provide evidence for the importance of reducing vancomycin pressure for prevention of VREfm dissemination. Fitness burden to bacterial hosts was varied by type and size of the vanA operon-harboring plasmid.

SFTSV Gn-Head mRNA vaccine confers efficient protection against lethal viral challenge.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus, causing thrombocytopenia and hemorrhagic fever, with a fatality rate ranging from 12% to 30%. SFTSV possesses Gn and Gc glycoproteins, which are responsible for host cell receptor attachment and membrane fusion, respectively, to infect host cells. We have previously reported a protein subunit vaccine candidate (sGn-H-FT) of the SFTSV soluble Gn head region (sGn-H) fused with self-assembling ferritin (FT) nanoparticles, displaying strong protective immunogenicity. In this study, we present messenger RNA (mRNA) vaccine candidates encoding sGn-H or sGn-H-FT, both of which exhibit potent in vivo immunogenicity and protection capacity. Mice immunized with either sGn-H or sGn-H-FT mRNA lipid nanoparticle (LNP) vaccine produced strong total antibodies and neutralizing antibodies (NAbs) against sGn-H. Importantly, NAb titers remained high for an extended period. Finally, mice immunized with sGn-H or sGn-H-FT mRNA LNP vaccine were fully protected from a lethal dose of SFTSV challenge, showing no fatality. These findings underscore the promise of sGn-H and sGn-H-FT as vaccine antigen candidates capable of providing protective immunity against SFTSV infection.

Viral codon optimization on SARS-CoV-2 Spike boosts immunity in the development of COVID-19 mRNA vaccines.

Life-long persistent herpesviruses carry "trans-inducers" to overcome the unusual codon usage of their glycoproteins for efficient expression. Strikingly, this "trans-inducibility" can be achieved by simply changing the codon-usage of acute virus glycoproteins to that of persistent herpesvirus glycoproteins with herpesviral trans-inducer. Here, we apply the "persistent viral codon-usage-trans-inducer" principle to SARS-CoV-2 Spike mRNA vaccine platform, in which the codon-usage of Spike is changed to that of Herpes Simplex Virus-1 (HSV-1) glycoprotein B (gB) with its "trans-inducer" ICP27. The HSVgB-ICP27-codon-optimized Spike mRNA vaccine induced markedly high antigen expression and stability, total IgG, neutralizing antibody, and T cell response, ultimately enhancing protection against lethal SARS-CoV-2 challenge. Moreover, the HSVgB- codon-optimized Delta (B.1.617.2) strain Spike mRNA vaccine provided significant enhancements in antigen expression and long-term protection against SARS-CoV-2 challenge. Thus, we report a novel persistent viral codon-usage-trans-inducer mRNA vaccine platform for enhanced antigen expression and long-term protection against lethal viral infection.

Fructose Promotes Leaky Gut, Endotoxemia, and Liver Fibrosis Through Ethanol-Inducible Cytochrome P450-2E1-Mediated Oxidative and Nitrative Stress.

Fructose intake is known to induce obesity, insulin resistance, metabolic syndrome, and nonalcoholic fatty liver disease (NAFLD). We aimed to evaluate the effects of fructose drinking on gut leakiness, endotoxemia, and NAFLD and study the underlying mechanisms in rats, mice, and T84 colon cells. Levels of ileum junctional proteins, oxidative stress markers, and apoptosis-related proteins in rodents, T84 colonic cells, and human ileums were determined by immunoblotting, immunoprecipitation, and immunofluorescence analyses. Fructose drinking caused microbiome change, leaky gut, and hepatic inflammation/fibrosis with increased levels of nitroxidative stress marker proteins cytochrome P450-2E1 (CYP2E1), inducible nitric oxide synthase, and nitrated proteins in small intestine and liver of rodents. Fructose drinking significantly elevated plasma bacterial endotoxin levels, likely resulting from decreased levels of intestinal tight junction (TJ) proteins (zonula occludens 1, occludin, claudin-1, and claudin-4), adherent junction (AJ) proteins (ß-catenin and E-cadherin), and desmosome plakoglobin, along with α-tubulin, in wild-type rodents, but not in fructose-exposed Cyp2e1-null mice. Consistently, decreased intestinal TJ/AJ proteins and increased hepatic inflammation with fibrosis were observed in autopsied obese people compared to lean individuals. Furthermore, histological and biochemical analyses showed markedly elevated hepatic fibrosis marker proteins in fructose-exposed rats compared to controls. Immunoprecipitation followed by immunoblot analyses revealed that intestinal TJ proteins were nitrated and ubiquitinated, leading to their decreased levels in fructose-exposed rats. Conclusion: These results showed that fructose intake causes protein nitration of intestinal TJ and AJ proteins, resulting in increased gut leakiness, endotoxemia, and steatohepatitis with liver fibrosis, at least partly, through a CYP2E1-dependent manner.

Anti-HBc IgG Levels: A Predictor of HBsAg Seroclearance in Chronic Hepatitis B Patients with Nucleos(t)ide Analogue-Induced HBeAg Seroclearance.

BACKGROUND/AIMS: We investigated the efficiency of the indirect ratio of anti-HBc IgG at predicting HBsAg seroclearance in patients with nucleos(t)ide analogue (NA)-induced HBeAg seroclearance. METHODS: We performed a retrospective study that included 366 chronic hepatitis B patients (March 2007 to December 2016) at a single tertiary hospital. These patients were HBsAg seropositive, and experienced NA-induced HBeAg seroclearance. The indirect ratio of light absorbance of anti-HBc IgG levels were measured with chemiluminescent microparticle immunoassay using the Architect Anti-HBc assay (Abbott Laboratories, IL, USA) as a qualitative method prior to antiviral therapy. We calculated the cumulative incidences of HBsAg seroclearance based on the anti-HBc IgG levels. RESULTS: After a 10-year follow-up, 48 patients experienced HBsAg seroclearance (13.1%). Thirty-three of 179 patients who had an indirect ratio of light absorbance of anti-HBc IgG < 11 RLU (relative light unit) showed HBsAg seroclearance (18.4%); 15 of 187 patients who had an indirect ratio of light absorbance of anti-HBc IgG ≥ 11 RLU showed HBsAg seroclerance (8.0%) (p = 0.003). In multivariate analysis, age, and ALT at the time of HBeAg seroclearance were predictors of HBsAg seroclearance. Especially, the relative risk of HBsAg seroclearance in patients with baseline anti-HBc IgG levels < 11 RLU was 2.213 (95% CI, 1.220-4.014), compared to that in patients with higher levels of anti-HBc IgG at baseline (p = 0.009). CONCLUSION: Using an indirect method for anti-HBc IgG levels, baseline anti-HBc IgG levels (< 11RLU), age (≥ 50 years), and ALT (≥ 40 IU/L) might be associated with HBsAg seroclearance in patients with NA-induced HBeAg seroclearance.

Hemp-Derived Nanovesicles Protect Leaky Gut and Liver Injury in Dextran Sodium Sulfate-Induced Colitis.

Hemp (Cannabis sativa L.) is used for medicinal purposes owing to its anti-inflammatory and antioxidant activities. We evaluated the protective effect of nanovesicles isolated from hemp plant parts (root, seed, hemp sprout, and leaf) in dextran sulfate sodium (DSS)-induced colitis in mice. The particle sizes of root-derived nanovesicles (RNVs), seed-derived nanovesicles (SNVs), hemp sprout-derived nanovesicles (HSNVs), and leaf-derived nanovesicles (LNVs) were within the range of 100-200 nm as measured by nanoparticle tracking analysis. Acute colitis was induced in C57BL/N mice by 5% DSS in water provided for 7 days. RNVs were administered orally once a day, leading to the recovery of both the small intestine and colon lengths. RNVs, SNVs, and HSNVs restored the tight (ZO-1, claudin-4, occludin) and adherent junctions (E-cadherin and α-tubulin) in DSS-induced small intestine and colon injury. Additionally, RNVs markedly reduced NF-κB activation and oxidative stress proteins in DSS-induced small intestine and colon injury. Tight junction protein expression and epithelial cell permeability were elevated in RNV-, SNV-, and HSNV-treated T84 colon cells exposed to 2% DSS. Interestedly, RNVs, SNVs, HSNVs, and LNVs reduced ALT activity and liver regeneration marker proteins in DSS-induced liver injury. These results showed for the first time that hemp-derived nanovesicles (HNVs) exhibited a protective effect on DSS-induced gut leaky and liver injury through the gut-liver axis by inhibiting oxidative stress marker proteins.

RNA-sequencing profiling analysis of pericyte-derived extracellular vesicle-mimetic nanovesicles-regulated genes in primary cultured fibroblasts from normal and Peyronie's disease penile tunica albuginea.

BACKGROUND: Peyronie's disease (PD) is a severe fibrotic disease of the tunica albuginea that causes penis curvature and leads to penile pain, deformity, and erectile dysfunction. The role of pericytes in the pathogenesis of fibrosis has recently been determined. Extracellular vesicle (EV)-mimetic nanovesicles (NVs) have attracted attention regarding intercellular communication between cells in the field of fibrosis. However, the global gene expression of pericyte-derived EV-mimetic NVs (PC-NVs) in regulating fibrosis remains unknown. Here, we used RNA-sequencing technology to investigate the potential target genes regulated by PC-NVs in primary fibroblasts derived from human PD plaque. METHODS: Human primary fibroblasts derived from normal and PD patients was cultured and treated with cavernosum pericytes isolated extracellular vesicle (EV)-mimetic nanovesicles (NVs). A global gene expression RNA-sequencing assay was performed on normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC-NVs. Reverse transcription polymerase chain reaction (RT-PCR) was used for sequencing data validation. RESULTS: A total of 4135 genes showed significantly differential expression in the normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC-NVs. However, only 91 contra-regulated genes were detected among the three libraries. Furthermore, 20 contra-regulated genes were selected and 11 showed consistent changes in the RNA-sequencing assay, which were validated by RT-PCR. CONCLUSION: The gene expression profiling results suggested that these validated genes may be good targets for understanding potential mechanisms and conducting molecular studies into PD.

Mastocytosis-derived extracellular vesicles exhibit a mast cell signature, transfer KIT to stellate cells, and promote their activation.

Extracellular vesicles (EVs) have been implicated in the development and progression of hematological malignancies. We thus examined serum samples from patients with systemic mastocytosis (SM) and found EVs with a mast cell signature including the presence of tryptase, FcεRI, MRGX2, and KIT. The concentration of these EVs correlated with parameters of disease including levels of serum tryptase, IL-6, and alkaline phosphatase and physical findings including hepatosplenomegaly. Given reports that EVs from one cell type may influence another cell's behavior, we asked whether SM-EVs might affect hepatic stellate cells (HSCs), based on the abnormal liver pathology associated with mastocytosis. We found that KIT was transferred from SM-EVs into an HSC line eliciting proliferation, cytokine production, and differentiation, processes that have been associated with liver pathology. These effects were reduced by KIT inhibition or neutralization and recapitulated by enforced expression of KIT or constitutively active D816V-KIT, a gain-of-function variant associated with SM. Furthermore, HSCs in liver from mice injected with SM-EVs had increased expression of α-SMA and human KIT, particularly around portal areas, compared with mice injected with EVs from normal individuals, suggesting that SM-EVs can also initiate HSC activation in vivo. Our data are thus consistent with the conclusion that SM-EVs have the potential to influence cells outside the hematological compartment and that therapeutic approaches for treatment of SM may be effective in part through inhibition of effects of EVs on target tissues, findings important both to understanding complex disease pathology and in developing interventional agents for the treatment of hematologic diseases.

Application of principal component analysis (PCA) to the assessment of parameter correlations in the partial-nitrification process using aerobic granular sludge.

For the first time, principal component analysis (PCA) was used to extract relevant information hidden in the partial-nitrification process using aerobic granular sludge. The objectives of this research are (a) to determine total ammonia nitrogen (TAN), total nitrite nitrogen (NO2-N), nitrate nitrogen (NO3-N), and other water quality parameters; (b) to identify the diversity of nitrification and denitrification bacterial community of wastewater samples during the partial-nitrification process using aerobic granular sludge and; (c) to analyze the correlation of available parameters using PCA. The nitrite accumulation ratio was determined from TAN, NO2-N, and NO3-N. Other water quality parameters were mixed liquor volatile suspended solids (MLVSS), alkalinity, total nitrogen (TN) and sludge volume index (SVI), pH, and dissolved oxygen (DO). The identification of bacterial community was conducted using 16S rRNA gene-based pyrosequencing by GS Junior Sequencing system. The water quality parameters were computed for PCA using software MATLAB. A nitrite accumulation ratio (NAR) between 0.55 and 0.85 was determined while maintaining the aerobic granular sludge's compact and dense structure. The PCA was used to reduce the data dimensionality from the original 8 variables to 2 principal components explaining 75% of the total data variance. Applying PCA to the data analysis in biological wastewater treatment can support detecting data anomalies and separating useful information from unwanted interferences.

Impact of vanA-Positive Enterococcus faecium Exhibiting Diverse Susceptibility Phenotypes to Glycopeptides on 30-Day Mortality of Patients with a Bloodstream Infection.

This study was performed to evaluate the impacts of vanA positivity of Enterococcus faecium exhibiting diverse susceptibility phenotypes to glycopeptides on clinical outcomes in patients with a bloodstream infection (BSI) through a prospective, multicenter, observational study. A total of 509 patients with E. faecium BSI from eight sentinel hospitals in South Korea during a 2-year period were enrolled in this study. Risk factors of the hosts and causative E. faecium isolates were assessed to determine associations with the 30-day mortality of E. faecium BSI patients via multivariable logistic regression analyses. The vanA gene was detected in 35.2% (179/509) of E. faecium isolates; 131 E. faecium isolates exhibited typical VanA phenotypes (group vanA-VanA), while the remaining 48 E. faecium isolates exhibited atypical phenotypes (group vanA-atypical), which included VanD (n = 43) and vancomycin-variable phenotypes (n = 5). A multivariable logistic regression indicated that vanA positivity of causative pathogens was independently associated with the increased 30-day mortality rate in the patients with E. faecium BSI; however, there was no significant difference in survival rates between the patients of the vanA-VanA and vanA-atypical groups (log rank test, P = 0.904). A high 30-day mortality rate was observed in patients with vanA-positive E. faecium BSIs, and vanA positivity of causative E. faecium isolates was an independent risk factor for early mortality irrespective of the susceptibility phenotypes to glycopeptides; thus, intensified antimicrobial stewardship is needed to improve the clinical outcomes of patients with vanA-positive E. faecium BSI.

Oncogenic D816V-KIT signaling in mast cells causes persistent IL-6 production.

Persistent dysregulation of IL-6 production and signaling have been implicated in the pathology of various cancers. In systemic mastocytosis, increased serum levels of IL-6 associate with disease severity and progression, although the mechanisms involved are not well understood. Since systemic mastocytosis often associates with the presence in hematopoietic cells of a somatic gain-of-function variant in KIT, D816V-KIT, we examined its potential role in IL-6 upregulation. Bone marrow mononuclear cultures from patients with greater D816V allelic burden released increased amounts of IL-6 which correlated with the percentage of mast cells in the cultures. Intracellular IL-6 staining by flow cytometry and immunofluorescence was primarily associated with mast cells and suggested a higher percentage of IL-6 positive mast cells in patients with higher D816V allelic burden. Furthermore, mast cell lines expressing D816V-KIT, but not those expressing normal KIT or other KIT variants, produced constitutively high IL-6 amounts at the message and protein levels. We further demonstrate that aberrant KIT activity and signaling are critical for the induction of IL-6 and involve STAT5 and PI3K pathways but not STAT3 or STAT4. Activation of STAT5A and STAT5B downstream of D816V-KIT was mediated by JAK2 but also by MEK/ERK1/2, which not only promoted STAT5 phosphorylation but also its long-term transcription. Our study thus supports a role for mast cells and D816V-KIT activity in IL-6 dysregulation in mastocytosis and provides insights into the intracellular mechanisms. The findings contribute to a better understanding of the physiopathology of mastocytosis and suggest the importance of therapeutic targeting of these pathways.

Pericyte-Derived Extracellular Vesicle-Mimetic Nanovesicles Restore Erectile Function by Enhancing Neurovascular Regeneration in a Mouse Model of Cavernous Nerve Injury.

BACKGROUND: Extracellular vesicle (EV)-mimetic nanovesicles (NVs) from embryonic stem cells have been observed to stimulate neurovascular regeneration in the streptozotocin-induced diabetic mouse. Pericytes play important roles in maintaining penile erection, yet no previous studies have explored the effects of pericyte-derived NVs (PC-NVs) in neurovascular regeneration in the context of erectile dysfunction. AIM: To investigate the potential effect of PC-NVs in neurovascular regeneration. METHODS: PC-NVs were isolated from mouse cavernous pericytes, and neurovascular regeneration was evaluated in an in vitro study. Twelve-week-old C57BL/6J mice were used to prepare cavernous nerve injury model. Erectile function evaluation, histologic examination of the penis, and Western blots were assessed 2 weeks after model creation and PC-NVs treatment. OUTCOMES: The main outcomes of this study are PC-NVs characterization, intracavernous pressure, neurovascular regeneration in the penis, and in vitro functional evaluation. RESULTS: The PC-NVs were extracted and characterized by cryotransmission electron microscopy and EV-positive (Alix, TSG101, CD81) and EV-negative (GM130) markers. In the in vivo studies, PC-NVs successfully improved erectile function in cavernous nerve injury mice (∼82% of control values). Immunofluorescence staining showed significant increases in pericytes, endothelial cell, and neuronal contents. In the in vitro studies, PC-NVs significantly increased mouse cavernous endothelial cells tube formation, Schwann cell migration, and dorsal root ganglion and major pelvic ganglion neurite sprouting. Finally, Western blot analysis revealed that PC-NVs upregulated cell survival signaling (Akt and eNOS) and induced the expression of neurotrophic factors (brain-derived neurotrophic factor, neurotrophin-3, and nerve growth factor). CLINICAL IMPLICATIONS: PC-NVs may be used as a strategy to treat erectile dysfunction after radical prostatectomy or in men with neurovascular diseases. STRENGTHS & LIMITATIONS: We evaluated the effect of PC-NVs in vitro and in a mouse nerve injury model, cavernous nerve injury. Additional studies are necessary to determine the detailed mechanisms of neurovascular improvement. Further study is needed to test whether PC-NVs are also effective when given weeks or months after nerve injury. CONCLUSION: PC-NVs significantly improved erectile function by enhancing neurovascular regeneration. Local treatment with PC-NVs may represent a promising therapeutic strategy for the treatment of neurovascular diseases. Yin GN, Park S-H, Ock J, et al. Pericyte-Derived Extracellular Vesicle-Mimetic Nanovesicles Restore Erectile Function by Enhancing Neurovascular Regeneration in a Mouse Model of Cavernous Nerve Injury. J Sex Med 2020;17:2118-2128.

Performance evaluation of a new matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, ASTA MicroIDSys system, in bacterial identification against clinical isolates of anaerobic bacteria.

INTRODUCTION: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced for bacterial identification. The ASTA MicroIDSys system (ASTA, Suwon, Korea) is a new MALDI-TOF MS system developed for species identification of microorganisms. We evaluated the performance of MicroIDSys against clinical isolates of anaerobic bacteria. MATERIAL AND METHODS: A total of 370 non-duplicated clinical isolates of anaerobic bacteria were tested in this study. Bacterial identification with MicroIDSys was performed with a direct smear method, and measured spectra were analyzed using respective software. The results of MicroIDSys were compared with the results of Bruker Biotyper and 16S rRNA sequencing. RESULTS: The overall agreement rates for the 370 clinical isolates (34 genera and 99 species) were 95.4% (353/370) at the genus level and 91.6% (n = 340) at the species level. Only 17 isolates were incorrectly identified at the genus level: five misidentifications and 12 unidentifications. The MicroIDSys system exhibited excellent performance in the identification of clinically relevant bacterial species. Most of the Bacteroides isolates (98.0%, 99/101) and all of the Clostridium difficile (100%, n = 11), Clostridium perfringens (100%, n = 10), Finegoldia magna (100%, n = 11), and Parvimonas micra (100%, n = 10) isolates were correctly identified at the species level. CONCLUSION: The MicroIDSys system proved useful in the identification of anaerobic bacteria, especially clinically relevant species. This system could be of use in clinical microbiology laboratories as a primary tool for identifying anaerobic bacteria.

Toxic Shock Syndrome Toxin 1-Producing Methicillin-Resistant Staphylococcus aureus of Clonal Complex 5, the New York/Japan Epidemic Clone, Causing a High Early-Mortality Rate in Patients with Bloodstream Infections.

This study was performed to evaluate the clinical impacts of putative risk factors in patients with Staphylococcus aureus bloodstream infections (BSIs) through a prospective, multicenter, observational study. All 567 patients with S. aureus BSIs that occurred during a 1-year period in six general hospitals were included in this study. Host- and pathogen-related variables were investigated to determine risk factors for the early mortality of patients with S. aureus BSIs. The all-cause mortality rate was 15.0% (85/567) during the 4-week follow-up period from the initial blood culture, and 76.5% (65/85) of the mortality cases occurred within the first 2 weeks. One-quarter (26.8%, 152/567) of the S. aureus blood isolates carried the tst-1 gene, and most (86.2%, 131/152) of them were identified to be clonal complex 5 agr type 2 methicillin-resistant S. aureus (MRSA) strains harboring staphylococcal cassette chromosome mec type II, belonging to the New York/Japan epidemic clone. A multivariable logistic regression showed that the tst-1 positivity of the causative S. aureus isolates was associated with an increased 2-week mortality rate both in patients with S. aureus BSIs (adjusted odds ratio [aOR], 1.62; 95% confidence interval [CI], 0.90 to 2.88) and in patients with MRSA BSIs (aOR, 2.61; 95% CI, 1.19 to 6.03). Two host-related factors, an increased Pitt bacteremia score and advanced age, as well as a pathogen-related factor, carriage of tst-1 by causative MRSA isolates, were risk factors for 2-week mortality in patients with BSIs. Careful management of patients with BSIs caused by the New York/Japan epidemic clone is needed to improve clinical outcomes.

Prospective Observational Study of the Clinical Prognoses of Patients with Bloodstream Infections Caused by Ampicillin-Susceptible but Penicillin-Resistant Enterococcus faecalis.

The purpose of this study was to evaluate the clinical impacts of ampicillin-susceptible but penicillin-resistant (ASPR) phenotypes of Enterococcus faecalis on clinical outcomes in patients with bloodstream infection (BSI). A total of 295 patients with an E. faecalis BSI from six sentinel hospitals during a 2-year period (from May 2016 to April 2018) were enrolled in this study. Putative risk factors, including host-, treatment-, and pathogen-related variables, were assessed to determine the associations with the 30-day mortality rate of patients with an E. faecalis BSI. The proportion of ASPR E. faecalis isolates was 22.7% (67/295). ASPR isolates (adjusted odds ratio, 2.27; 95% confidence interval, 1.01 to 5.02) exhibited a significant association with an increased 30-day mortality rate, and a significant difference in survival was identified in a group of patients treated with ampicillin- and/or piperacillin-based regimens who were stratified according to the penicillin susceptibility of the causative pathogen (P = 0.011 by a log rank test). ASPR E. faecalis BSIs resulted in a >2-fold-higher 30-day mortality rate (26.9%; 18/67) than for the BSIs caused by penicillin-susceptible strains (12.3%; 28/228). The differences in mortality rates of patients stratified by penicillin susceptibility were likely due to the treatment failures of ampicillin and/or piperacillin in patients with an ASPR E. faecalis BSI.

Mortality dynamics of Pseudomonas aeruginosa bloodstream infections and the influence of defective OprD on mortality: prospective observational study.

BACKGROUND: To assess the mortality dynamics of patients with Pseudomonas aeruginosa bloodstream infections (BSIs) and the influence of OprD deficiencies of the microorganism on early mortality. METHODS: A prospective multicentre observational study was conducted with 120 patients with P. aeruginosa BSIs occurring between May 2016 and April 2017 in six general hospitals in South Korea. PCR and sequencing were carried out to identify the alterations in oprD and the presence of virulence factors. Cox regression was used to estimate the risk factors for mortality at each timepoint and Kaplan-Meier survival analyses were performed to determine the mortality dynamics. RESULTS: During the 6 week follow-up, 10.8% (13/120) of the patients with P. aeruginosa BSIs died in 2 weeks, 14.2% (17/120) in 4 weeks and 20.0% (24/120) in 6 weeks, revealing a steep decrease in cumulative survival between the fourth and sixth weeks. ICU admission and SOFA score were risk factors for mortality in any weeks after BSI onset and causative OprD-defective P. aeruginosa had a risk tendency for mortality within 6 weeks. Among the 120 P. aeruginosa blood isolates, 14 were XDR, nine produced either IMP-6 or VIM-2 MBL, and 21 had OprD deficiency. CONCLUSIONS: BSIs caused by OprD-defective P. aeruginosa resulted in a 2-fold higher 6 week mortality rate (33.3%) than that of BSIs caused by OprD-intact P. aeruginosa (17.2%), likely due to the decreased susceptibility to carbapenems and bacterial persistence in clinical settings.

Antimicrobial resistance and virulence factors of Klebsiella pneumoniae affecting 30 day mortality in patients with bloodstream infection.

Objectives: To investigate the risk factors of patients with Klebsiella pneumoniae (KP) bloodstream infection (BSI) with a focus on antimicrobial resistance and virulence factors. Methods: All KP BSI patients (n = 579) from six general hospitals during a 1 year period were included in this study. The risk factors of hosts and causative KP isolates were assessed to determine associations with the 30 day mortality of KP BSI patients by multivariate Cox hazards modelling. Results: The 30 day mortality rate of KP BSI patients was 16.9% (98/579). Among the host-associated factors, increased SOFA score and leucopenia status exhibited strong associations with increased 30 day mortality. Among the pathogenic factors, carriage of the pks gene cluster (adjusted HR 1.80; 95% CI 1.16-2.79) was a risk factor, especially when accompanied by MDR. In this regard, KP isolates of the wzi50 capsular type (n = 22) frequently harboured pks (63.6%, 14/22) and ybtA (68.2%, n = 15) and mostly exhibited MDR (63.6%, n = 14), resulting in increased 30 day mortality. In contrast, hypermucoviscous KP isolates showed an inverse association with 30 day mortality (adjusted HR 0.55; 95% CI 0.33-0.90). Conclusions: Despite the reported virulence of hypermucoviscous KP strains, they were associated with good prognoses in KP BSI patients. Importantly, carriage of the pks gene cluster, which is responsible for the synthesis of colibactin, was a relevant marker of early mortality.

Analytical and clinical performance of newly developed immunoassay for detecting thyroid-stimulating immunoglobulin, the Immulite TSI assay.

Graves' disease (GD) is caused by autoantibodies against the thyrotropin receptor (TRAb), and among the three types of TRAbs, only the stimulating type (TSI) is known to be associated with GD. In this study, we evaluated the analytical performance of a new fully automated chemiluminescent TSI immunoassay, namely, the Immulite TSI assay, and compared the diagnostic efficacy of the assay with the Elecsys Anti-TSH receptor (TSHR) assay. Precision was evaluated using two levels of quality control reagents, and linearity was evaluated across the expected analytical measurement range (0.18-37.35 IU/L) at five levels using clinical samples. A comparative evaluation between the two assays was performed using 187 clinical samples, and the concordance of qualitative results was also assessed. The repeatability and total imprecision (% coefficient of variation) of the Immulite TSI assay were 3.19% and 3.46% at 0.93 IU/L, and 3.76% and 5.42% at 19.3 IU/L, respectively. The linearity of this assay ranged from 0.16 to 6.17 IU/L. A high degree of correlation was observed between quantitative values from each assay (correlation coefficient = 0.819). Moderate agreement between methods was observed with an overall qualitative agreement of 93.0%. Among 13 cases with discordant qualitative results, the Immulite TSI assay generated more favorable results consistent with clinical diagnoses of patients than the Elecsys Anti-TSHR assay. The Immulite TSI assay showed reliable analytical performance and good correlation with the Elecsys Anti-TSHR assay and we expect this method will be helpful for clinicians to evaluate patients with hyperthyroidism.