Deciphering microbiome and fungi-bacteria interactions in chronic wound infections using metagenomic sequencing.

PURPOSE: Chronic wounds caused by infections impose a considerable global healthcare burden. The microbial features of these infections and possible correlations between bacteria and fungi may influence wound healing. However, metagenomic next-generation sequencing (mNGS) analyses of these features remain sparse. Therefore, we performed mNGS on chronic wound infection samples to investigate features and correlations between the bacteriome and mycobiome in 66 patients (28: chronic wounds; 38: non-chronic wounds). METHODS: Microbial community characteristics in patients with wound infections, microbiome-systemic inflammation associations, and bacteria-fungi correlations were analyzed. RESULTS: Infections constituted the primary cause of wounds in this study. Nontuberculous mycobacteria (23%) and Mycobacterium tuberculosis (13%) were the most common pathogens associated with chronic wounds, whereas Staphylococcus aureus (15%) was the most prevalent in non-chronic wound infections. Patients with chronic wound infections had a higher abundance of Pseudomonas aeruginosa than those without chronic wounds. Microbes with a high relative abundance in chronic wound infections were less significantly associated with plasma inflammatory factors than those in non-chronic wound infections. Additionally, a positive correlation between Candida glabrata and P. aeruginosa and an association between Malassezia restricta and anaerobic species were detected in patients with chronic wound infections. CONCLUSION: Our results further support the hypothesis that P. aeruginosa is a microbial biomarker of chronic wound infection regardless of the causative pathogens. Moreover, we propose a positive correlation between C. glabrata and P. aeruginosa in chronic wound infections, which advances the current understanding of fungi-bacteria correlations in patients with chronic wound infections.

Evaluation of respiratory samples in etiology diagnosis and microbiome characterization by metagenomic sequencing.

BACKGROUND: The application of clinical mNGS for diagnosing respiratory infections improves etiology diagnosis, however at the same time, it brings new challenges as an unbiased sequencing method informing all identified microbiomes in the specimen. METHODS: Strategy evaluation and metagenomic analysis were performed for the mNGS data generated between March 2017 and October 2019. Diagnostic strengths of four specimen types were assessed to pinpoint the more appropriate type for mNGS diagnosis of respiratory infections. Microbiome complexity was revealed between patient cohorts and infection types. A bioinformatic pipeline resembling diagnosis results was built based upon multiple bioinformatic parameters. RESULTS: The positive predictive values (PPVs) for mNGS diagnosing of non-mycobacterium, Nontuberculous Mycobacteria (NTM), and Aspergillus were obviously higher in bronchoalveolar lavage fluid (BALF) demonstrating the potency of BALF in mNGS diagnosis. Lung tissues and sputum were acceptable for diagnosis of the Mycobacterium tuberculosis (MTB) infections. Interestingly, significant taxonomy differences were identified in sufficient BALF specimens, and unique bacteriome and virome compositions were found in the BALF specimens of tumor patients. Our pipeline showed comparative diagnostic strength with the clinical microbiological diagnosis. CONCLUSIONS: To achieve reliable mNGS diagnosis result, BALF specimens for suspicious common infections, and lung tissues and sputum for doubtful MTB infections are recommended to avoid the false results given by the complexed respiratory microbiomes. Our developed bioinformatic pipeline successful helps mNGS data interpretation and reduces manual corrections for etiology diagnosis.

Microbiological Diagnostic Performance of Metagenomic Next-generation Sequencing When Applied to Clinical Practice.

Background: Metagenomic next-generation sequencing (mNGS) was suggested to potentially replace traditional microbiological methodology because of its comprehensiveness. However, clinical experience with application of the test is relatively limited. Methods: From April 2017 to December 2017, 511 specimens were collected, and their retrospective diagnoses were classified into infectious disease (347 [67.9%]), noninfectious disease (119 [23.3%]), and unknown cases (45 [8.8%]). The diagnostic performance of pathogens was compared between mNGS and culture. The effect of antibiotic exposure on detection rate was also assessed. Results: The sensitivity and specificity of mNGS for diagnosing infectious disease were 50.7% and 85.7%, respectively, and these values outperformed those of culture, especially for Mycobacterium tuberculosis (odds ratio [OR], 4 [95% confidence interval {CI}, 1.7-10.8]; P < .01), viruses (mNGS only; P < .01), anaerobes (OR, ∞ [95% CI, 1.71-∞]; P < .01) and fungi (OR, 4.0 [95% CI, 1.6-10.3]; P < .01). Importantly, for mNGS-positive cases where the conventional method was inconclusive, 43 (61%) cases led to diagnosis modification, and 41 (58%) cases were not covered by empirical antibiotics. For cases where viruses were identified, broad-spectrum antibiotics were commonly administered (14/27), and 10 of 27 of these cases were suspected to be inappropriate. Interestingly, the sensitivity of mNGS was superior to that of culture (52.5% vs 34.2%; P < .01) in cases with, but not without, antibiotic exposure. Conclusions: mNGS could yield a higher sensitivity for pathogen identification and is less affected by prior antibiotic exposure, thereby emerging as a promising technology for detecting infectious diseases.

Identification of metabolites of the novel anti-tumor drug candidate MDH-7 in rat urine by liquid chromatography coupled with triple quadrupole linear ion trap mass spectrometry.

RATIONALE: Our previous preliminary pharmacokinetic study demonstrated that the novel double pyrimidine tricyclic nucleoside MDH-7 in rats had a very short half-life (<30 min) after oral administration. As a result, the in vivo metabolic profile of MDH-7 should be investigated during early stages of drug development to better select drug candidates. METHODS: In this study, a rapid method was developed to identify the metabolites of MDH-7 in rat urine by means of ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) using a triple quadrupole linear ion trap instrument. MDH-7 and its metabolites were detected and characterized by the combined use of the multiple reaction monitoring-information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode and the precursor scan information-dependent acquisition-enhanced product ion (PREC-IDA-EPI) mode. RESULTS: Ten novel metabolites of MDH-7 were identified and characterized in rat urine by LC/ESI-MS and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) analyses. M1 was identified as 5-fluoro-N(4) -[(pentyloxy)carbonyl]cytosine; M2 and M3 were formed by hydroxylation products of M1. Metabolites M4-M10 were formed by a series of degradation reactions such as: deacetylation, hydroxylation, loss of the defluorocytosine base, oxidative-deamination, loss of the defluorouracil base, N-dealkylation and amide hydrolysis. CONCLUSIONS: Based on the profiles of the metabolites, possible metabolic pathways of MDH-7 in rats were proposed for the first time. This study provides new and available information on the metabolism of MDH-7 which is very useful to further understand its in vivo metabolic fate. Copyright © 2016 John Wiley & Sons, Ltd.

Increased claudin-3, -4 and -18 levels in bronchoalveolar lavage fluid reflect severity of acute lung injury.

BACKGROUND AND OBJECTIVE: Acute lung injury (ALI) is characterized by disruption of lung epithelial and endothelial cells, leading to increased membrane permeability and loss of barrier function. Claudins are key components of tight junctions (TJ) that regulate paracellular permeability, and play an important role in alveolar epithelial barrier function and fluid clearance. However, whether claudin-3, -4, -18 or -5 expression changes in Pseudomonas aeruginosa (PA)-induced ALI and the clinical significance of such change is unknown. METHODS: Rats underwent intratracheal instillation of PA, and samples were collected prior to and 3, 9 and 24 h after instillation. Lung injury was evaluated by bronchoalveolar lavage fluid (BALF) total protein, arterial blood gas analysis, lung injury score, and expression of surfactant protein and von Willebrand factor. Claudin expression in lung was measured with quantitative real-time polymerase chain reaction and western blotting, and in BALF by enzyme-linked immunosorbent assay. The relationship between claudins in BALF and lung injury grade were analysed with Spearman's rank correlation. Alveolar epithelium, endothelium and TJ ultrastructure were observed with electron microscopy. RESULTS: Claudin-4, -18 and -5 mRNA levels increased significantly 24 h after PA instillation in the most severe lung injury cases, whereas there was no significant change in protein levels. Claudin-3, -4 and -18 levels in BALF increased most 24 h after PA instillation; this paralleled alveolar epithelial disruption and lung injury severity. CONCLUSIONS: Claudin-3, -4 and -18 released into the alveolar compartment is highly associated with barrier function loss caused by alveolar epithelial injury.

Association between the overall burden of comorbidity and Ct values among the older patients with Omicron infection: Mediated by inflammation.

Objectives: To investigate the associations between the overall burden of comorbidity, inflammatory indicators in plasma and Ct values among the elderly with COVID-19. Methods: We conducted a retrospective observational study. The results of each nucleic acid test of during hospitalization were obtained. Linear regression models assessed the associations between the overall burden of comorbidity, inflammatory indicators in plasma and Ct values among the elderly. A causal mediation analysis was performed to assess the mediation effects of inflammatory indicators on the association between the overall burden of comorbidity and Ct values. Results: A total of 767 COVID-19 patients aged ≥ 60 years were included between April 2022 and May 2022. Patients with a high burden of comorbidity had significantly lower Ct values of the ORF gene than subjects with a low burden of comorbidity (median, 24.81 VS 26.58, P < 0.05). Linear regression models showed that a high burden of comorbidity was significantly associated with higher inflammatory responses, including white blood cell count, neutrophil count and C-reactive protein. Also, white blood cell count, neutrophil count, C-reactive protein and the overall burden of comorbidity assessed by age-adjusted Charlson comorbidity index were independent risk factors for the Ct values. A mediation analysis detected the mediation effect of white blood cells on the association between the burden of comorbidity and Ct values, with the indirect effect estimates of 0.381 (95% CI: 0.166, 0.632, P < 0.001). Similarly, the indirect effect of C-reactive protein was -0.307 (95% CI: -0.645, -0.064, P = 0.034). White blood cells and C-reactive protein significantly mediated the relationship between the burden of comorbidity and Ct values by 29.56% and 18.13% of the total effect size, respectively. Conclusions: Inflammation mediated the association between the overall burden of comorbidity and Ct values among elderly with COVID-19, which suggests that combined immunomodulatory therapies could reduce the Ct values for such patients with a high burden of comorbidity.

[The evaluation of strengthened psychological and behavioral intervention in smoking cessation clinics].

OBJECTIVE: To evaluate the effect of psychological and behavioral intervention combined with varenicline smoking cessation clinics and to analyze predictors of successful quitting. METHODS: Subjects were collected from quitters who went to receive consultation and intervention in "smoking and related diseases" clinic at Zhongshan Hospital, Fudan University from March 2009 to September 2010. Eligible subjects were screened and divided into strengthen follow-up group and control group. The 4 weeks continuous abstinence rate from week 9 through week 12 were observed logistic regression model and used to analyze the predictors of successful quitting. RESULTS: Subjects who are addicted to nicotine received strengthening psychological and behavioral intervention combined with varenicline in smoking cessation clinics. The total continuous cessation rate during the 9(th) - 12(th) week was 52.3%, with 60.9% (28/46) and 46.2% (30/65) of strengthen follow-up group and control group respectively. The most frequent adverse effects were nausea 39.6% (44/111), insomnia and abnormal dreams 17.1% (19/111). Adverse effects were tolerable and withdraw symptoms were few. Preparation and medication time can be used as predictors of successful quitting. CONCLUSION: The quit rate of varenicline therapy combining with strengthen intervention is high and strengthening psychological and behavioral intervention could increase the success rate more obviously, which is a good choice for cessation therapy in smoking cessation clinics. Better preparation and regular adequate treatment can improve quit rate.

The clinical value of valve metagenomic next-generation sequencing when applied to the etiological diagnosis of infective endocarditis.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) is widely applied in the etiological diagnosis of infectious diseases. However, the clinical practice of mNGS in infective endocarditis (IE) is relatively less studied. This research aimed to assess the etiological diagnostic value of valve mNGS in IE. METHODS: We retrospectively analyzed 49 IE patients who underwent cardiac valve surgery in Zhongshan Hospital, Fudan University, Shanghai from 1 June 2018 to 30 November 2020. Among these IE patients, 28 were culture positive and 21 were culture negative. The culture results of the culture-positive IE patients were set as gold standard to assess the sensitivity and specificity of valve mNGS in the etiological diagnosis of IE. We studied the positive detection rate of pathogens by valve mNGS among the culture-negative IE patients. During the same period, we also collected the resected valves of 8 patients with non-infective valvular diseases for mNGS as negative controls. RESULTS: The valve mNGS results of the culture-positive IE patients were the exact same as their culture results. Both the sensitivity and specificity of valve mNGS were 100%. The positive detection rate of pathogens by valve mNGS was 100% among the culture-negative IE patients. The stringent mapped reads number of genera (SMRNG), relative abundance of genera, stringent mapped reads number of species (SMRN), relative abundance of species, and coverage rate of valve mNGS results were significantly higher in culture-positive IE participants than in culture-negative IE participants. The valve mNGS results of the 8 participants with non-infective valvular diseases were all negative. CONCLUSIONS: Valve mNGS is a promising technology for the etiological diagnosis of IE, especially culture-negative IE, and it may be used to guide precise antibiotic treatment after surgery.

Micropore-Rich Yolk-Shell N-doped Carbon Spheres: An Ideal Electrode Material for High-Energy Capacitive Energy Storage.

Increasing the energy density of electrochemical double layer capacitors (EDLCs) can broaden their applications in energy storage but remains a formidable challenge. Herein, micropore-rich yolk-shell structured N-doped carbon spheres (YSNCSs) were constructed by a one-pot surfactant-free self-assembly method in aqueous solution. The resultant YSNCSs after activation possessed an ultrahigh surface area of 2536 m2 g-1 , among which 80 % was contributed from micropores. When applied in EDLCs, the activated YSNCSs demonstrated an unprecedentedly high capacitance (270 F g-1 at 1 A g-1 ) in 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF4 ]) ionic liquid, affording an ultrahigh energy density (133 Wh kg-1 at 943 W kg-1 ). The present contribution provides insight into engineering porous carbons for capacitive energy storage.

Evaluation of superinfection, antimicrobial usage, and airway microbiome with metagenomic sequencing in COVID-19 patients: A cohort study in Shanghai.

BACKGROUND: In COVID-19 patients, information regarding superinfection, antimicrobial assessment, and the value of metagenomic sequencing (MS) could help develop antimicrobial stewardship. METHOD: This retrospective study analyzed 323 laboratory-confirmed COVID-19 patients for co-infection rate and antimicrobial usage in the Shanghai Public Health Clinical Center (SPHCC) from January 23rd to March 14th 2020. The microbiota composition was also investigated in patients with critically severe COVID-19. RESULTS: The total population co-infection rate was 17/323 (5.3%) and 0/229 (0), 4/78 (5.1%), and 13/16 (81.3%) for the mild, severe, and critically severe subgroups, respectively. Proven fungal infection was significantly associated with a higher mortality rate (p = 0.029). In critically severe patients, the rate of antimicrobials and carbapenem usage were 16/16 (100%) and 13/16 (81.3%), respectively, in which the preemptive and empiric antimicrobial days accounted for 51.6% and 30.1%, respectively. Targeted therapy only accounted for 18.3%. MS was implemented to detect non-COVID-19 virus co-existence and the semi-quantitative surveillance of bacteremia, with clear clinical benefit seen in cases with MS-based precision antimicrobial management. Airway microbiome analysis suggested that the microbiota compositions in critically severe COVID-19 patients were likely due to intubation and mechanical ventilation. CONCLUSIONS: In the SPHCC cohort, we observed a non-negligible rate of super-infection, especially for the critically ill COVID-19 patients. Fungal co-infection requires intensive attention due to the high risk of mortality, and the clinical benefit of MS in guiding antimicrobial management warrants further investigation.

A rare case of adrenal gland abscess due to anaerobes detected by metagenomic next-generation sequencing.

A 50-year-old woman presented with right back pain, low fever, leukocytosis, a high level of C-reactive protein and a high erythrocyte sedimentation rate. Abdominal magnetic resonance imaging (MRI) revealed a hypodense lesion in the right suprarenal region, while PET/CT showed mildly increased metabolic activity. A CT-guided percutaneous puncture was performed, and foul-smelling thick pus was removed, which indicated an anaerobic infection. A smear of the pus showed both gram-positive and gram-negative microorganisms. Traditional culture only detected Escherichia coli, Proteus mirabilis, and Actinomyces turicensis. While surprisingly, metagenomic next-generation sequencing (mNGS) of both the pus and blood showed high reads of multiple pathogens, including anaerobes and the three culture-positive pathogens. Thus, adrenal gland abscess was confirmed, and a combination therapy of catheter drainage and effective antimicrobial treatment was started. Six days later, the patient had clinically improved and mNGS showed dramatically decreased reads of all pathogens. A follow-up lab examination of inflammatory biomarkers was normal, and the adrenal mass was reduced radiographically.

Diagnostic accuracy of metagenomic next-generation sequencing for active tuberculosis in clinical practice at a tertiary general hospital.

BACKGROUND: To evaluate the diagnostic accuracy of metagenomic next-generation sequencing (mNGS) for active tuberculosis (TB). METHODS: We retrospectively collected 820 samples at Zhongshan Hospital, Fudan University in Shanghai, China, between 1 April 2017 and 31 March 2018. They were classified into TB cases (125, 15.2%) and NOT TB cases (695, 84.8%) according to the clinical diagnosis. Specimens were evaluated by a regular clinical microbiological assay and mNGS performed in parallel. RESULTS: Sixty-one confirmed TB cases and 64 clinical TB cases were included. The overall sensitivity of mNGS was 49.6% [95% confidence interval (95% CI), 40.6-58.6%], and the specificity was 98.3% (95% CI, 96.9-99.1%), with peak sensitivities of 88.9% (95% CI, 50.7-99.4%) for lung tissue, 55.0% (95% CI, 32.0-76.2%) for bronchoalveolar lavage fluid (BALF), and 50.0% (95% CI, 32.8-67.2%) for serous fluids. The overall sensitivity of mNGS was superior to that of the culture assay (35.2%, 95% CI, 27.0-44.3), but no superior sensitivity for sputum was observed in mNGS compared with the culture assay (mNGS: 52.3%, 95% CI, 31.1-72.6%; culture: 60.9%, 95% CI, 38.8-79.5%). In clinical TB cases, mNGS detected additional positive results (40.6%, 26/64). mNGS reduced the turnaround time from 2-6 weeks to 32-36 hours. CONCLUSIONS: mNGS may be a promising technology for the early auxiliary diagnosis of active TB, especially sputum-negative pulmonary TB (PTB) and tuberculous serous effusion.

The clinical value of metagenomic next-generation sequencing in the microbiological diagnosis of skin and soft tissue infections.

BACKGROUND: Metagenomic next-generation sequencing (mNGS), with its comprehensiveness, is widely applied in microbiological diagnosis. Etiological diagnosis is of paramount clinical importance in patients with skin and soft tissue infections (SSTIs). However, the clinical application of mNGS in SSTIs is relatively less studied. MATERIALS AND METHODS: From April 1, 2017 to December 31, 2019, 96 SSTI cases were collected. The positive rates of pathogens detected by mNGS and culture were compared by analyzing tissue samples, pus, swabs, and/or interstitial fluids obtained from the infected parts. Modification of the antibiotic treatment strategy due to mNGS was also assessed. RESULTS: The sensitivity of mNGS for detecting pathogens in SSTI cases was superior to that of culture testing (67.7% vs 35.4%; p < 0.01). Significantly higher identification rates for viruses (10.4% vs 0.0%; p < 0.01) and anaerobes (11.5% vs 1.0%; p < 0.01) were obtained with mNGS compared to culture. Of note, rare pathogens such as Vibrio vulnificus and Bartonella henselae were also detected by mNGS. Importantly, the proportion of multi-pathogen SSTIs detected by mNGS was higher than that of multi-pathogen SSTIs detected by culture (16.7% vs 6.3%; p = 0.035). The rate of targeted antibiotic treatment was significantly higher in mNGS-positive cases than in mNGS-negative cases (41.7% vs 3.8%; p < 0.01). In culture-negative and mNGS-positive cases, the improvement rate was higher than that in mNGS-negative cases, but this was not statistically significant (75.0% vs 73.1%; p = 0.864). CONCLUSIONS: mNGS is a promising tool for the etiological diagnosis of SSTIs, particularly in identifying viruses, anaerobes, and multi-pathogen infections. The application of mNGS testing in clinical practice could change antibiotic treatment strategies and partly benefit clinical outcomes.

Progranulin is a novel biomarker for predicting an acute exacerbation of chronic obstructive pulmonary disease.

BACKGROUND: Progranulin is a pleiotropic glycosylated protein precursor that plays an important role in inflammation. Limited data exist regarding the role of progranulin in the acute exacerbation of chronic obstructive pulmonary disease (AECOPD). OBJECTIVES: The study is to assess the efficiency of progranulin as a circulating biomarker of AECOPD. METHODS: The plasma progranulin levels were measured and compared in patients with AECOPD (n = 52), patients with stable COPD (n = 56), and healthy controls (n = 36). In patients with AECOPD, plasma progranulin levels were measured repeatedly on the last day of hospitalization. Demographical data, pulmonary function, and laboratory parameters were recorded. RESULTS: Patients with AECOPD had higher plasma progranulin levels than both stable COPD patients and healthy controls (158.77 ± 48.17, 109.00 ± 25.05, 93.67 ± 14.71 ng/mL, respectively; P < .001). In patients with AECOPD, the plasma progranulin levels significantly decreased on the last day of hospitalization compared with those on the first day of hospitalization (138.51 ± 44.68 vs. 158.77 ± 48.17 ng/mL, P = .042). The progranulin levels were negatively correlated to FEV1%pred but positively correlated to neutrophil-to-lymphocyte ratio and C-reactive protein in all patients with COPD. Multivariate logistic regression and ROC analysis revealed progranulin (odds ratio 1.05, 95% confidence interval 1.03-1.08, P < .001) as an independent predictor of AECOPD, with an area under the curve of 0.82. CONCLUSIONS: Progranulin may be a valuable blood biomarker of AECOPD and progranulin may be involved in the pathogenesis of AECOPD by disturbing inflammatory responses.

Multiclass analysis of 25 veterinary drugs in milk by ultra-high performance liquid chromatography-tandem mass spectrometry.

The aim of this work was to develop a simple and rapid analytical method for the simultaneous determination of a wide range of drug residues in milk by UHPLC-MS/MS. A total of 25 typical veterinary drugs investigated belong to six families including ß-lactams, quinolones, ß-agonists, phenicols, glucocorticoids and nitrofurans. The samples were extracted by acetonitrile and defatted with n-hexane twice. Electrospray ionization and positive/negative polarity switching were utilized for the analysis of 25 veterinary drugs in a single chromatographic run. The linearity, recovery, precision and matrix effects of the method were fully validated. The intra- and inter-precision were in the range of 1.7-11.1% and 2.5-10.4%, respectively. The average recoveries ranged from 65.9% to 123.5% with RSD less than 10.8% at three concentration levels. The proposed method was demonstrated to be simple, economical and reliable for the fast monitoring of these drug residues in milk samples.