Development of a quadruple qRT-PCR assay for simultaneous identification of hypervirulent and carbapenem-resistant Klebsiella pneumoniae.

IMPORTANCE: Globally, the increasing number of hypervirulent Klebsiella pneumoniae (hvKp) and carbapenem-resistant Kp (CR-Kp) infections poses a huge public health challenge with high morbidity and mortality. Worrisomely, due to the mobility of elements carrying virulence and drug-resistance genes, the increasing prevalence of CR-hvKp has also been found with an overwhelming mortality rate in recent years. However, the current detection methods for hvKp and CR-Kp have many disadvantages, such as long turnaround time, complex operation, low sensitivity, and specificity. Herein, a more sensitive, rapid, single-reaction, and multiplex quantitative real-time PCR was developed and validated to differentiate the circulating lineages of Kp with excellent performance in sensitivity and specificity, providing a useful tool for the differential diagnosis and the surveillance of the circulating Kp.

Early application of awake extracorporeal membrane oxygenation in pneumocystis jirovecii pneumonia complicated with severe acute respiratory distress syndrome: a case report.

Introduction: Patients suffering from severe acute respiratory distress syndrome (ARDS) are usually treated with mechanical ventilation. Extracorporeal membrane oxygenation (ECMO) has traditionally been considered a life-saving therapy and was reserved as a last resort when other treatment options were exhausted. However, this report outlines our successful initial experience with early implementation of awake venovenous extracorporeal membrane oxygenation (VV-ECMO) in a case of pneumocystis jirovecii pneumonia complicated by severe acute respiratory distress syndrome (ARDS), offering a promising new approach for recovery. Case presentation: We present a case report of the effective application of awake VV-ECMO in a 29 years-old man with severe ARDS caused by pneumocystis jirovecii pneumonia. The patient initially received antibiotic treatment and non-invasive ventilation (NIV) for respiratory distress, but these interventions failed to improve the worsening dyspnea that occurred in the patient. Following the combined antifungal therapy, high-flow nasal cannula (HFNC) oxygen therapy, and VV-ECMO for a duration of 7 days, the patient's symptoms improved, showing relief. Conclusion: Awake VV-ECMO proved to be an effective treatment for critically ill patients with ARDS, avoiding the need for invasive mechanical ventilation. However, increased clinical evidence is needed to verify whether awake ECMO could be widely used in severe ARDS caused by other diseases or conditions.

Activation of CD4+ T Cell-Derived Cannabinoid Receptor 2 Signaling Exacerbates Sepsis via Inhibiting IL-10.

The cannabinoid receptor 2 (CB2) is a receptor mainly expressed in immune cells and believed to be immunosuppressive in infective or inflammatory models. However, its role in sepsis has not been fully elucidated. In this study, we delineate the function and mechanism of CB2 in the cecal ligation and puncture-induced septic model in mice. The activation of CB2 signaling with HU308 led to decreased survival rates and more severe lung injury in septic mice, and lower IL-10 levels in peritoneal lavage fluid were observed in the CB2 agonist group. The mice with conditional knockout of CB2-encoding gene CNR2 in CD4+ T cells (CD4 Cre CNR2fl/fl) improved survival, enhanced IL-10 production, and ameliorated pulmonary damage in the sepsis model after CB2 activation. In addition, double-knockout of the CNR2 gene (Lyz2 Cre CD4 Cre CNR2fl/fl) decreased the susceptibility to sepsis compared with Lyz2 Cre CNR2fl/fl mice. Mechanistically, the blockade of IL-10 with the anti-IL-10 Ab abolished its protection in CD4 Cre CNR2fl/fl mice. In accordance with the animal study, in vitro results revealed that the lack of CNR2 in CD4+ cells elevated IL-10 production, and CB2 activation inhibited CD4+ T cell-derived IL-10 production. Furthermore, in the clinical environment, septic patients expressed enhanced CB2 mRNA levels compared with healthy donors in PBMCs, and their CB2 expression was inversely correlated with IL-10. These results suggested that the activation of CD4+ T cell-derived CB2 increased susceptibility to sepsis through inhibiting IL-10 production.

Circular RNA Circ_0008043 promotes the proliferation and metastasis of hepatocellular carcinoma cells by regulating the microRNA (miR)-326/RAB21 axis.

Circular RNAs (circRNAs) are non-coding RNAs with covalently closed structures that modulate the progression of hepatocellular carcinoma (HCC). Here, we explored whether circ_0008043 regulated the biological function of HCC cells. Quantitative real-time polymerase chain reaction (qPCR) was used to detect circ_0008043, microRNA (miR)-326, and RAB21 levels. Expression of E-cadherin, N-cadherin, and vimentin was assessed using qPCR. Cell proliferation, migration, and invasion were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and transwell assays. Xenograft tumors were used to evaluate cell growth in vivo. The interaction between miR-326 and circ_0008043 or RAB21 was assessed using dual-luciferase reporter analysis and RNA pull-down analysis. The data illustrated that circ_0008043 and RAB21 were highly expressed, while miR-326 was expressed at less levels in HCC tissues and cells. Interfering with circ_0008043 suppressed cellular proliferation, migration, invasion, and cell growth. Circ_0008043 was confirmed to be an miR-326 sponge that targets RAB21. Rescue experiments showed that inhibiting miR-326 abrogated the effect induced by knockdown of circ_0008043, and overexpressed RAB21 abolished the effect induced by miR-326 overexpression. In summary, silencing of circ_0008043 impeded HCC progression by regulating the miR-326/RAB21 axis. These data suggest that circ_0008043 may have clinical value in the treatment of HCC.

Clinical diagnosis of severe COVID-19: A derivation and validation of a prediction rule.

BACKGROUND: The widespread coronavirus disease 2019 (COVID-19) has led to high morbidity and mortality. Therefore, early risk identification of critically ill patients remains crucial. AIM: To develop predictive rules at the time of admission to identify COVID-19 patients who might require intensive care unit (ICU) care. METHODS: This retrospective study included a total of 361 patients with confirmed COVID-19 by reverse transcription-polymerase chain reaction between January 19, 2020, and March 14, 2020 in Shenzhen Third People's Hospital. Multivariate logistic regression was applied to develop the predictive model. The performance of the predictive model was externally validated and evaluated based on a dataset involving 126 patients from the Wuhan Asia General Hospital between December 2019 and March 2020, by area under the receiver operating curve (AUROC), goodness-of-fit and the performance matrix including the sensitivity, specificity, and precision. A nomogram was also used to visualize the model. RESULTS: Among the patients in the derivation and validation datasets, 38 and 9 participants (10.5% and 2.54%, respectively) developed severe COVID-19, respectively. In univariate analysis, 21 parameters such as age, sex (male), smoker, body mass index (BMI), time from onset to admission (> 5 d), asthenia, dry cough, expectoration, shortness of breath, asthenia, and Rox index < 18 (pulse oxygen saturation, SpO2)/(FiO2 × respiratory rate, RR) showed positive correlations with severe COVID-19. In multivariate logistic regression analysis, only six parameters including BMI [odds ratio (OR) 3.939; 95% confidence interval (CI): 1.409-11.015; P = 0.009], time from onset to admission (≥ 5 d) (OR 7.107; 95%CI: 1.449-34.849; P = 0.016), fever (OR 6.794; 95%CI: 1.401-32.951; P = 0.017), Charlson index (OR 2.917; 95%CI: 1.279-6.654; P = 0.011), PaO2/FiO2 ratio (OR 17.570; 95%CI: 1.117-276.383; P = 0.041), and neutrophil/lymphocyte ratio (OR 3.574; 95%CI: 1.048-12.191; P = 0.042) were found to be independent predictors of COVID-19. These factors were found to be significant risk factors for severe patients confirmed with COVID-19. The AUROC was 0.941 (95%CI: 0.901-0.981) and 0.936 (95%CI: 0.886-0.987) in both datasets. The calibration properties were good. CONCLUSION: The proposed predictive model had great potential in severity prediction of COVID-19 in the ICU. It assisted the ICU clinicians in making timely decisions for the target population.

Monitoring bronchoalveolar lavage with electrical impedance tomography: first experience in a patient with COVID-19.

OBJECTIVE: Patients with the novel coronavirus disease (COVID-19) often have airway secretions that severely compromise ventilation. This study investigates electrical impedance tomography (EIT) monitoring of a therapeutic bronchoalveolar lavage (BAL) in a patient with COVID-19. APPROACH: A patient with COVID-19 developed acute respiratory distress syndrome requiring mechanical ventilation. He received regional BAL to remove mucus in the small airways (20 ml × 5). Regional ventilation changes before BAL, 30 min after and in the following days, were monitored with EIT. MAIN RESULTS: Regional ventilation worsened shortly after BAL and improved in the following days. The improvement of the oxygenation did not exactly match the ventilation improvement, which indicated a possible ventilation/perfusion mismatch. SIGNIFICANCE: Therapeutic BAL might improve regional ventilation for COVID-19 and EIT could be a useful tool at the bedside to monitor the ventilation treatment of COVID-19.

Importance of respiratory airway management as well as psychological and rehabilitative treatments to COVID-19 patients.

The clinical therapy for severe 2019 coronavirus disease (i.e., COVID-19) sufferers is relatively challenging. Herein, the processes involving salvage of a critical COVID-19 patient were retrospectively analyzed. The condition of an obese female critical COVID-19 sufferer progressively worsened in the initial period after admission. According to her symptoms and examination reports, endotracheal intubation and mechanical ventilation were timely conducted and meanwhile high-dose sedatives and analgesics were administrated. In the later therapeutic phase, however, sedative and analgesic dosages were gradually reduced, and psychological and rehabilitative therapies were conducted, concomitantly with enhancement of airway care to facilitate sputum expectoration. Eventually, the endotracheal tube was feasibly removed after intubation for 18 days and subsequently replaced with noninvasive ventilation and a high-flow nasal cannula oxygen therapy. Intensive airway care alongside psychological and rehabilitative therapies can shorten the mechanical ventilation time and improve the prognosis of COVID-19 sufferers.