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Pythium insidiosum is a rare fungus-like pathogen that is known to cause pythiosis in mammals with high morbidity and mortality. Identification of the pathogen is essential for timely treatment and rational use of antibiotics. However, Pythium insidiosum is difficult to detect via conventional microbiological tests. The current gold standard is polymerase chain reaction, which is lacking in most hospitals since human pythiosis is rare in China. In this study, we used metagenomic Next-Generation Sequencing and identified Pythium insidiosum in a 56-year-old Chinese male who was hospitalized due to severe edema in the right lower limb with scattered darkening indurations. The patient had a history of cirrhosis and occupational exposure to swamp water. Serological level of immune biomarkers indicated immunodeficiency, and Proteinase 3-Anti-Neutrophil Cytoplasmic Antibody was positive. Surgical incision of the lesions revealed radiating and reticular cutaneous ulcers. Microbial infections were suspected but conventional tests failed to discover the etiology. Empirical use of penicillin, vancomycin, and ceftriaxone had no effect. As a result, the peripheral blood and tissue biopsies were sent for metagenomic Next-Generation Sequencing, which reported Pythium insidiosum. This finding was corroborated by pathological staining, whole-genome sequencing, and internal transcribed spacer sequencing. Notably, antifungal treatment was ineffective, but the patient responded well to oral trimethoprim–sulfamethoxazole, which may be due to the folp gene found in Pythium insidiosum genome. Our study prompts future studies to determine the optimal treatment of skin pythiosis.

Introduction Co-infection pneumonia with Mycobacterium abscessus (M. abscessus) and Pneumocystis jirovecii (P. jirovecii) is rarely reported in previously healthy patients without HIV infection. The diagnosis of pneumonia of M. abscessus and P. jirovecii remains challenging due to its nonspecific clinical presentation and the inadequate performance of conventional diagnostic methods. Case report We report the case of a 44-year-old previously healthy male transferred to our hospital in February 2020 with a 4-month history of productive cough and one month of intermittent fever. At local hospital, the metagenomic next-generation sequencing(mNGS) detected P. jirovecii sequences in blood; with the antifungal therapy (Caspofungin, trimethoprim-sulfamethoxazole [TMP-SMX] and methylprednisolone [MP]), the patient still had hypoxemia, cough and fever. Then he was transferred to our hospital, the mNGS of bronchoalveolar lavage fluid (BALF) detected the sequences of M. abscessus and P. jirovecii. CD4+ T-lymphocytopenia in the peripheral blood cells was presented and HIV serology was negative. Caspofungin, TMP-SMX, clindamycin and MP were used to treat P. jirovecii pneumonia (PJP). Moxifloxacin, imipenem cilastatin and linezolid were used to treat M. abscessus infection. Clinical progress was satisfactory following antifungal combined with anti-M. abscessus therapy. Conclusion Co-infection pneumonia with M. abscessus and P. jirovecii as reported here is exceptionally rare. mNGS is a powerful tool for pathogen detection. M. abscessus infection could be a risk factor for P. jirovecii infection. This case report supports the value of mNGS in diagnosing of M. abscessus and P. jirovecii, and highlights the inadequacies of conventional diagnostic methods.

Current clinical needs require the development and use of rapid and effective diagnostic indicators to accelerate the identification of pneumonia and the process of microbiological diagnosis. MicroRNAs (miRNAs) in extracellular vesicles (EVs) have become attractive candidates for novel biomarkers to evaluate the presence and progress of many diseases. We assessed their performance as biomarkers of pneumonia. Patients were divided into the pneumonia group (with pneumonia) and the control group (without pneumonia). We identified and compared two upregulated miRNAs in EVs derived from bronchoalveolar lavage fluid (BALF-EVs) between the two groups (PmiR–17–5p = 0.009; PmiR–193a–5p = 0.031). Interestingly, in cell-debris pellets and EVs-free supernatants derived from bronchoalveolar lavage fluid (BALF-cell-debris pellets and BALF-EVs-free supernatants), total plasma, and EVs derived from plasma (plasma-EVs), the expression of miR–17–5p and miR–193a–5p showed no difference between pneumonia group and control group. In vitro experiments revealed that miR–17–5p and miR–193a–5p were strikingly upregulated in EVs derived from macrophages stimulated by lipopolysaccharide. MiR–17–5p (area under the curve, AUC: 0.753) and miR–193a–5p (AUC: 0.692) in BALF-EVs are not inferior to procalcitonin (AUC: 0.685) in the diagnosis of pneumonia. Furthermore, miR–17–5p and miR–193a–5p in BALF-EVs had a significantly higher specificity compared to procalcitonin and could be served as a potential diagnostic marker. MiR–17–5p and miR–193a–5p in EVs may be involved in lung inflammation by influencing the forkhead box O (FoxO) signaling pathway and protein processing in endoplasmic reticulum. This study is one of the few studies which focused on the potential diagnostic role of miRNAs in BALF-EVs for pneumonia and the possibility to use them as new biomarkers for a rapid and early diagnosis.

Early and appropriate antibiotic treatment reduces morbidity and mortality of pneumonia. However, early prediction of pulmonary infection remains a challenge for clinicians. Current clinical needs require the development and use of rapid and effective diagnostic indicators to accelerate the identification of pneumonia and process of microbiological diagnosis. MicroRNAs (miRNAs) in exosomes (EXOs) have become attractive candidates for novel biomarkers to evaluate the presence and progress of many diseases. We assessed their performance as biomarkers of pneumonia. Patients were divided into pneumonia group (with pneumonia) and control group (without pneumonia). We identified and compared two upregulated miRNAs in EXOs derived from bronchoalveolar lavage fluid (BALF) between the pneumonia and control groups (miR-17-5p, p=0.009; miR-193a-5p, p=0.031). Interestingly, miR-17-5p and miR-193a-5p in BALF-cell-debris pellets, BALF-EXO-free supernatants, total plasma, and plasma-EXOs did not differ significantly between both groups. In vitro experiments revealed that miR-17-5p and miR-193a-5p were strikingly upregulated in EXOs derived from macrophages stimulated by LPS. Receiver operator characteristic (ROC) curve analysis indicated that exosomal miR-17-5p (area under the curve, AUC: 0.753) and exosomal miR-193a-5p (AUC: 0.629) have acceptable diagnostic value. This study is one of the few studies on BALF-EXO-miRNAs in patients with pneumonia, providing potential diagnostic biomarkers and therapeutic targets for pneumonia.

Background: The diagnosis of T. marneffei infection remains challenging due to its non-specific clinical presentations and the inadequate performance of conventional diagnostic methods. Meanwhile, there is no good indicator to monitor when induction treatment should switch to consolidation treatment.Case summary: A 53-year-old male patient presented with a one-week history of high fever on March 21, 2019, 10 years after a liver transplant. A chest CT showed multiple pulmonary nodules. Blood culture revealed T. marneffei. After two weeks of intravenous treatment of voriconazole and caspofungin, the patient was discharged without fever and was given oral voriconazole on April 8. Two days later, the patient had a fever again. The patient was fever-free soon after intravenous voriconazole. However, a high fever occurred three days later. Cultures of blood and sputum were negative. Metagenomics next-generation sequencing (mNGS) detected T. marneffei sequences in blood and Enterococcus faecium sequences in sputum. Despite the use of caspofungin and linezolid, the patient maintained a daily fever of 38.5°C. Chest CT revealed that pulmonary nodules were growing in size and number. After administering Amphotericin B, the temperature quickly returned to normal. Liposomal amphotericin B was used due to increased creatinine. When the sequence of T. marneffei turned negative in the blood, the patient was discharged on May 24 and received 14-week oral voriconazole without relapse. Conclusion: mNGS can directly detect the sequence of T. marneffei in the blood. Consequently, mNGS is a powerful technique in precise diagnosis and a good monitoring technique for treating disseminated T. marneffei.

MiR30a plays diverse roles in inflammatory diseases, including autoimmune hepatitis (AIH). Klf14 is associated with the inflammation in AIH. We investigated whether miR30a exerts its regulatory function via Klf14. Concanavalin A (Con A)-induced AIH mice were infected with a miR30a agomir or antagomir. MiR30a expression was quantified using qRT-PCR. TargetScan and luciferase reporter assays were used to predict the relationship between miR30a and Klf14. Liver inflammation was evaluated by measuring serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, performing histology, and measuring mRNA expressions of inflammatory cytokines and Klf14 by qRT-PCR, protein of Klf14 by western blotting, and Tregs by FACS. MiR30a was downregulated in the hepatocytes (HCs) of AIH mice, which was negatively associated with the liver inflammation. MiR30a overexpression alleviated the inflammation, whereas downregulation of endogenous miR30a aggravated it. The mRNA and protein level of Klf14 were inversely correlated with the miR30a expression. The luciferase reporter assay validated the relationship between Klf14 and miR30a. Moreover, the frequency of Tregs was consistently correlated with the expression of miR30a. MiR30a may play an essential role in AIH, and its ability to regulate the inflammatory responses may, at least partially, be mediated by targeting Klf14 to modulate Tregs.

AIM To assess whether elderly patients are prioritized under the emergency triage system in Guangzhou, China. METHODS This was a cross-sectional survey of clinical data from adult visitors to the emergency department of the Third Affiliated Hospital of Sun Yat-sen University between 1 August 2015 and 31 December 2017. The primary end-point was receiving the first medical service within the target waiting time, which varied according to the triage level of the patient. Multivariate logistic regression was used to determine whether age was an independent predictor of a shorter waiting time. RESULTS Data from 262 282 emergency patients were analyzed. The mean age of patients was 35.97 years, and 7.5% were aged ≥65 years. In total, 88.3% of patients received medical service within the target waiting time, and 87.4% of elderly patients received medical service within the target waiting time. Multivariate logistic regression analysis showed that advanced age was independently associated with receiving medical service within the target waiting time (adjusted odds ratio 1.258, 95% confidence interval 1.198-1.321; P

BACKGROUND: Mechanical ventilation (MV) is an important lifesaving method in intensive care unit (ICU). Prolonged MV is associated with ventilator associated pneumonia (VAP) and other complications. However, premature weaning from MV may lead to higher risk of reintubation or mortality. Therefore, timely and safe weaning from MV is important. In addition, identification of the right patient and performing a suitable weaning process is necessary. Although several guidelines about weaning have been reported, compliance with these guidelines is unknown. Therefore, the aim of this study is to explore the variation of weaning in China, associations between initial MV reason and clinical outcomes, and factors associated with weaning strategies using a multicenter cohort. METHODS: This multicenter retrospective cohort study will be conducted at 17 adult ICUs in China, that included patients who were admitted in this 17 ICUs between October 2020 and February 2021. Patients under 18 years of age and patients without the possibility for weaning will be excluded. The questionnaire information will be registered by a specific clinician in each center who has been evaluated and qualified to carry out the study. DISCUSSION: In a previous observational study of weaning in 17 ICUs in China, weaning practices varies nationally. Therefore, a multicenter retrospective cohort study is necessary to be conducted to explore the present weaning methods used in China. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR) (No. ChiCTR2100044634).

Pneumocystis jirovecii (P. jirovecii) pneumonia (PJP) is an opportunistic fungal infection after renal transplantation, which is always severe, difficult to diagnose, combined with multiple complications and have poor prognosis. We retrospectively analyzed clinical data, including risk factors, diagnosis, treatment and complications of seven clinical cases suffered with severe PJP after renal transplantation in our department in 2019. All the seven recipients were routinely prescribed with PJP prophylaxis after renal transplantation, and six of them suffered acute graft rejection before the infection. P. jirovecii sequence was identified in blood or broncho-alveolar lavage fluid (BALF) by the metagenomic next-generation sequencing (mNGS) in all patients. All the patients were improved with the therapy trimethoprim-sulfamethoxazole (TMP-SMX) combined with caspofungin for the PJP treatment, but suffered with complications including renal insufficiency, leukopenia, thrombocytopenia, gastrointestinal bleeding, mediastinalemphysema, pulmonary hemorrhage, and hemophagocytic syndrome and other severe infections. Taken together, mNGS is a powerful tool that could be used to diagnose PJP in renal transplantation recipients. And PJP prophylaxis should be prescribed during and after treatment for acute rejection. TMP-SMX is the first-line and effective drug for PJP treatment, but the complications are always life-threatening and lead to poor prognosis. We should pay attention to these life-threatening complications.

To evaluate whether patients aged over 70 years old is an independent predictor for prolonging the waiting time, and to know whether the population can be prioritized in the emergency grading system.A retrospective analysis was conducted. All the clinical data of adult visitors to the emergency department of the Third Affiliated Hospital of Sun Yat-sen University from January 1st in 2016 to December 31st in 2017 were collected. All the patients received four emergency first aid triages and treated according to the emergency classification results. The relevant data of selected patients were retrieved from the hospital information system (HIS) including gender, age, triage level, triage department, outpatient service and initial diagnosis time. According to the conclusion of pre-phase analysis that the patients over 65 years old was not associated with waiting time, all subjects were divided into ≥ 70 years old group and70 years old group for further analysis. The waiting time was defined as the difference in registration time and the initial diagnosis time, and the patients were divided into two groups according to whether it exceeded the target waiting time (TWT). TWT was considered as follows according to each emergency triage level: level I instantly, level II 10 minutes, level III 30 minutes, level IV 60 minutes (subacute patients) or 2-4 hours (non-urgent patients). The multivariable Logistic regression analysis was conducted to determine whether the factor that aged over 70 years old was an independent predictor for prolonging waiting time over TWT.There were 272 989 adult visits recorded totally, of which obstetrics and gynecology department visits, incomplete personnel information, invalid diagnosis (or no diagnosis), triage level I, abnormal waiting time (the waiting time exceeded 99% of the average waiting time of all samples) records were excluded. Finally, 219 299 emergency patients were enrolled, with 102 218 male and 117 081 female, which showed an average age of (35.86±15.81) years, 208 082 patients (94.9%) in70 years old group, and 11 217 (5.1%) in ≥ 70 years old group. Totally 191 588 patients (87.4%) were diagnosed within the TWT, and the number was 86.5% (9 701/11 217) in the ≥ 70 years old group. Multivariate Logistic regression analysis indicated that age under 70 years old was independently associated with an exceeded TWT [adjusted odds ratio (OR) = 1.106, 95% confidence interval (95%CI) = 1.041-1.176, P = 0.001]. Meanwhile, visits during the peak period or low triage level was also a risk factor of exceeded TWT. According to the Logistic regression analysis of different departments and removing other factors, in the emergency internal medicine with the most elderly patients, the probability of exceeded TWT in ≥ 70 years old group was lessened (adjusted OR = 1.203, 95%CI = 1.121-1.291, P0.001).In the emergency pre-grading system, the elderly aged (≥ 70 years old) was more likely to see a doctor in TWT than those under 70 years old, so they were easy to get priority treatment.

Human CD4+ T cells play an important role in the immune response to Mycobacterium tuberculosis (MTB). However, little is known about the spectratyping characteristics of the CD4+ T-cell receptor (TCR) α- and β-chains CDR3 region in tuberculosis (TB) patients. We sorted MTB peptide E7-bound CD4+ T cells by using E7/HLA-DR tetramers constructed with different HLA-DRB1 alleles and extracted the CDR3 amino-acid sequences of TCR α- and β-chains. The results showed that the CDR3 sequences of E7-bound CD4+ T cells were completely or partially identical in a single patient. The sequences of MTB peptide C5-bound CD4+ T cells shared another, and non-peptide bound CD4+ T cells, as well as unbound CD4+ T cells with tetramers were different from each other. Specifically, diverse CDR3 sequences of E7-bound CD4+ T cells displayed similar protein tertiary structure in one TB patient. In summary, the TCR α- and β-chains of CDR3 lineage of CD4+ T cells in TB patients apparently drifted, and the predominant CDR3 sequences of TCR α- and β-chains that recognized the MTB antigen exhibited peptide specificity, and certain HLA-DR restriction was also established. This study elucidates the possible causes and mechanisms of peptide-specific CD4+ T-cell-related presentation against MTB.

Purpose The relationship between arterial hyperoxia exposure and clinical outcome is under increasing scrutiny. We therefore performed an update meta-analysis to evaluate the effect of arterial hyperoxia on hospital mortality in critically ill adults. Methods We searched relevant articles for trials that investigated the relationship between arterial hyperoxia and mortality in critically ill adults. The end-point was hospital mortality of critically ill patients. Results Three RCTs and 26 cohort studies involving 257,223 patients were identified. Hyperoxia exposure was associated with increased mortality in critically ill patients (crude OR 1.42, 95% CI 1.26–1.61; adjusted OR 1.20, 95% CI 1.09–1.32). There was no change in significance for outcome in meta-analysis of RCTs (OR 1.36; 95% CI 1.04–1.77) and sensitivity analysis of the included prospective studies (OR 1.32; 95% CI 1.04–1.67). This association was also established in patients admitted to critical care units following cardiac arrest (adjusted OR 1.32; 95% CI 1.12–1.56), ischemic stroke (crude OR 1.31; 95% CI 1.03–1.65) and intracerebral hemorrhage (crude OR 1.47; 95% CI 1.19–1.81). Conclusions The results of current meta-analysis suggest that arterial hyperoxia may be associated with increased hospital mortality in critically ill patients.

A total of 10 non-repetitive multi-drug-resistant Acinetobacter strains were collected. With reference to A. calcoaceticus (ATCC23055), A. baumannii (ATCC19606), A. lwoffii (ATCC17986), and A. junii (NCTC5866),DNA fingerprint technique, amplified ribosomal DNA restriction analysis (ARDRA), and random amplified polymorphism DNA (RAPD) were carried out to identify the genomic species of Acinetobacter spp. The distances between them were calculated by the unweighted pair group method with arithmetic (UPGMA). Genotypes of Acinetobacter spp. were effectively classified and an A. junii together with nine A. baumannii isolates was genomically identified. The combination of ARDRA and RAPD DNA-fingerprint technique shows high complementarity, and could be a useful tool in Acinetobacter genomic species identification.