Simultaneous determination of advanced glycation end products and heterocyclic amines in roast/grilled meat by UPLC-MS/MS.

Advanced glycation end products (AGEs) and heterocyclic amines (HAs) are main harmful Maillard reaction products of meat products. Simultaneous quantification of both with high sensitivity, selectivity and accuracy remains a major challenge due to inconsistencies in their pre-treatment and instrumental methods and the different polarity of AGEs and HAs. We developed a method for the simultaneous determination of AGEs and HAs in roast/grilled meat by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) using dynamic multiple reaction monitoring (D-MRM). The instrument parameters and pre-treatment method were optimized to achieve reasonably good separation and high response for the 11 target analytes within 8 min. From 10 to 200 ng/mL, the limits of detection (LODs) and limits of quantitation (LOQs) ranged from 0.3 to 5.5 µg/L and 0.9 to 6.3 µg/L, respectively, and the correlation coefficient (R2) was >0.99. It was acceptable to recoveries, standard deviations (RSDs), and matrix effects. Six types of roast/grilled meat samples were then tested using the developed method.

Structural and physicochemical characteristics of wheat starch as influenced by freeze-thawed cycles and antifreeze protein from Sabina chinensis (Linn.) Ant. cv. Kaizuca leaves.

The effects of freeze-thawed cycles (FTs) and a new antifreeze protein from Sabina chinensis (Linn.) Ant. cv. Kaizuca leaves (ScAFP) on the structure and physicochemical characteristics of wheat starch were studied. The mechanical breaking exerted by ice crystals on starch granules during FTs gradually deepened, sequentially squeezing the surface (2-6 FTs), amorphous region (8 FTs) and crystalline region (10 FTs) of starch granules. These changes led to reduced thermal stability, increased retrogradation tendency, and weakened gel network structure. The addition of ScAFP retarded the damage of ice crystals on starch granule structure and crystal structure during FTs, and significantly reduced the retrogradation tendency. Compared with native starch, the hardness of freeze-thawed starch without and with added ScAFP after 10 FTs decreased by 17.85% and 9.22%, respectively, indicating ScAFP improved the gel texture properties of freeze-thawed starch. This study provides new strategies for improving the quality of frozen starch-based foods.

A dual-process of targeted and unbiased Nanopore sequencing enables accurate and rapid diagnosis of lower respiratory infections.

BACKGROUND: Nanopore metagenomics has been used for infectious disease diagnosis for bacterial pathogens. However, this technology currently lacks comprehensive performance studies in clinical settings for simultaneous detection of bacteria, fungi, and viruses. METHODS: We developed a dual-process of Nanopore sequencing for one sample, with unbiased metagenomics in Meta process and target enrichment in Panel process (Nanopore Meta-Panel process, NanoMP) and prospectively enrolled 450 respiratory specimens from multiple centers. The filter system of pathogen detection was established with machine learning and receiver operator characteristic (ROC) curve to optimize the detection accuracy based on orthogonal test of 21 species. Antimicrobial resistance (AMR) genes were identified based on the Comprehensive Antibiotic Resistance Database (CARD) and single-nucleotide polymorphism matrix. FINDINGS: Our approach showed high sensitivity in Meta process, with 82.9%, 88.7%, and 75.0% for bacteria, fungi (except Aspergillus), and Mycobacterium tuberculosis groups, respectively. Moreover, target amplification improved the sensitivity of virus (>80.0% vs. 39.4%) and Aspergillus (81.8% vs. 42.3%) groups in Panel process compared with Meta process. Overall, NanoMP achieved 80.2% sensitivity and 98.8% specificity compared with the composite reference standard, and we were able to accurately detect AMR genes including blaKPC-2, blaOXA-23 and mecA and distinguish their parent organisms in patients with mixed infections. INTERPRETATION: We combined metagenomic and enriched Nanopore sequencing for one sample in parallel. Our NanoMP approach simultaneously covered bacteria, viruses and fungi in respiratory specimens and demonstrated good diagnostic performance in real clinical settings. FUNDING: National Key Research and Development Program of China and National Natural Science Foundation of China.

Synergistic Activity of Imipenem in Combination with Ceftazidime/Avibactam or Avibactam against Non-MBL-Producing Extensively Drug-Resistant Pseudomonas aeruginosa.

Extensively drug-resistant Pseudomonas aeruginosa (XDRPA) infection is a significant public health threat due to a lack of effective therapeutic options. New ß-lactam-ß-lactamase inhibitor combinations, including ceftazidime-avibactam (CZA), have shown a high resistance rate to XDRPA. This study was therefore conducted to describe the underlying genomic mechanism of resistance for CZA nonsusceptible XDRPA strains that are non-metallo-ß-lactamase (MBL) producers as well as to examine synergism of CZA and other antipseudomonal agents. Furthermore, the synergistic antibacterial activity of the most effective antimicrobial combination against non-MBL-producing XDRPA was evaluated through in vitro experiments. The resistance profiles of 15 CZA-resistant XDRPA strains isolated from clinical specimens in China-Japan Friendship Hospital between January 2017 to December 2020 were obtained by whole-genome sequencing (WGS) analysis. MBL genes blaIMP-1 and blaIMP-45 were found in 2 isolates (2/15, 13.3%); the other underlying CZA-resistance mechanisms involved the decreased OprD porin (13/13), blaAmpC overexpression (8/13) or mutation (13/13), and upregulated efflux pumps (13/13). CZA-imipenem (CZA-IPM) combination was identified to be the most effective against non-MBL-producing XDRPA according to the results of WGS analysis and combined antimicrobial susceptibility tests, with an approximately 16.62-fold reduction in MICs compared to CZA alone. Furthermore, the results of checkerboard analysis and growth curve displayed the synergistic antimicrobial activity of CZA and IPM against non-MBL-producing XDRPA. Electron microscopy also revealed that CZA-IPM combination might lead to more cellular structural alterations than CZA or IPM alone. This study suggested that the CZA-IPM combination has potential for non-MBL-producing XDRPA with blaAmpC overexpression or mutation, decreased OprD porin, and upregulated efflux pumps. IMPORTANCE Handling the infections by extensively drug-resistant Pseudomonas aeruginosa (XDRPA) strains is challenging due to their complicated antibiotic resistance mechanisms in immunosuppressed patients with pulmonary diseases (e.g., cystic fibrosis, chronic obstructive pulmonary disease, and lung transplant), ventilator-associated pneumonia, and bloodstream infections. The current study suggested the potentiality of the ceftazidime-avibactam-imipenem combination against XDRPA with blaAmpC overexpression or mutation, decreased OprD porin, and/or upregulated efflux pumps. Our findings indicate the necessity of combined drug sensitivity tests against XDRPA and also lay a foundation for the development of prevention, control, and treatment strategies in XDRPA infections.

Characterization of an NDM-5-producing hypervirulent Klebsiella pneumoniae sequence type 65 clone from a lung transplant recipient.

The emergence of New Delhi Metallo-ß-lactamase (NDM)-producing Klebsiella pneumoniae has aroused critical concern worldwide. Herein, we reported the first emergence of NDM-5-producing K. pneumoniae isolates in a 68-year-old lung transplant recipient, who died of septic shock 13 days after surgery. The K. pneumoniae strain KP22937 isolated from the bloodstream of the patient was analyzed for phenotypes and genotypes. KP22937 belonged to sequence type (ST) 65 and capsule serotype K2, contained iucABCDiutA and iroBCDN virulence clusters, showed high virulence to mice, and was therefore considered a hypervirulent K. pneumoniae. The blaNDM-5 gene was located on a genomic island region of the IncX3-type plasmid pNDM22937, which was successfully transferred to Escherichia coli EC600 with insignificant fitness costs. The transconjugant demonstrated similar antimicrobial susceptibility and growth kinetics to the recipient E. coli EC600. The plasmid pNDM22937 was almost identical to the blaNDM-5-carrying IncX3 plasmids previously reported in K. pneumoniae strains with different ST types and in other species. Our findings raise concerns about the horizontal spread of blaNDM-5 gene mediated by IncX3 plasmid, where hypervirulent K. pneumoniae strains are also involved. Stricter control measures are needed to prevent the dissemination of the novel clone in hospital settings.

Risk Factors for Mortality of Inpatients with Pseudomonas aeruginosa Bacteremia in China: Impact of Resistance Profile in the Mortality.

PURPOSE: Pseudomonas aeruginosa bacteremia presents a severe challenge to hospitalized patients. However, to date, the risk factors for mortality among inpatients with P. aeruginosa bacteremia in China remain unclear. PATIENTS AND METHODS: This retrospective multicenter study was performed to analyze 215 patients with culture-confirmed P. aeruginosa bacteremia in five healthcare centers in China during the years 2012-2019. RESULTS: Of 215 patients with P. aeruginosa bacteremia, 61 (28.4%) died during the study period. Logistic multivariable analysis revealed that cardiovascular disease (OR=3.978, P=0.001), blood transfusion (OR=5.855, P<0.001) and carbapenem-resistant P. aeruginosa (CRPA) phenotype (OR=4.485, P=0.038) constituted the independent risk factors of mortality. Furthermore, both CRPA and multidrug-resistant P. aeruginosa (MDRPA) phenotypes were found to be significantly associated with 5-day mortality (Log-rank, P<0.05). CONCLUSION: This study revealed a high mortality rate amongst hospitalized patients with P. aeruginosa bacteremia, and those with cardiovascular diseases, CRPA and MDRPA phenotypes, should be highlighted and given appropriate management in China.

Severity of influenza virus and respiratory syncytial virus coinfections in hospitalized adult patients.

BACKGROUND: With the introduction of molecular diagnostic techniques over the past decades, different kinds of viral pathogens in the same sample are detected simultaneously more frequently. Nevertheless, influenza virus (Flu) and respiratory syncytial virus (RSV) coinfection in adults was reported only occasionally. Moreover, the clinical implications of Flu/RSV coinfection in the respiratory tract of adults remain unclear. METHODS: This retrospective study analyzed adult patients with acute respiratory infection from January 2017 to June 2019 in China-Japan Friendship Hospital. RESULTS: A total of 574, 235 and 113 patients were positive for influenza A-only (FA-only), influenza B-only (FB-only) and RSV-only in influenza seasons (from Nov 2017 to Mar 2018 and from Nov 2018 to Mar 2019), respectively. Of these, 19 cases were coinfected by Flu and RSV and admitted to this hospital. Compared with 809 Flu-only infected patients and 113 RSV-only infected patients, both the rates of intensive care unit(ICU) admission and use of invasive mechanical ventilation in Flu/RSV coinfected patients were higher (ICU admission: 47.4% vs. 20.1%, P=0.004; 47.4% vs. 22.1%, P=0.020; invasive mechanical ventilation: 47.4% vs.13.2%, P<0.001; 47.4% vs. 17.7%, P=0.004). Furthermore, 60-day all-cause mortality attributed to Flu/RSV coinfections was significantly greater than that for Flu and RSV mono-infected patients (36.8% vs. 8.0%,P<0.001; 36.8% vs. 11.5%, P=0.004. CONCLUSION: The findings of this study suggest that coinfection of Flu/RSV in adults is associated with a high adverse outcome. Thus, Flu/RSV coinfections should be increasingly appreciated and given appropriate management.

Severity and mortality of respiratory syncytial virus vs influenza A infection in hospitalized adults in China.

BACKGROUND: Respiratory syncytial virus (RSV) is an important cause of medically attended acute respiratory illnesses in older adults but awareness of the relevance of RSV in older people remains lower than that of influenza, which exhibits similar clinical characteristics to those of RSV. OBJECTIVES: This study was performed to assess the clinical significance of RSV in respiratory samples from hospitalized adults. METHODS: Characteristics and outcomes in adults (≥18 years) hospitalized for RSV infection (n = 51) were compared with a cohort hospitalized for influenza A infection (n = 279) in a single-center retrospective cohort study in Beijing, China. RESULTS: Respiratory syncytial virus patients were slightly older, with no significant differences in underlying chronic conditions. Lower respiratory tract infection and cardiovascular complications were more frequent (P < .05) in RSV patients. Rates of mortality in the RSV cohorts were significantly higher within 30 days (13.7% vs 5.0%, P = .019) and 60 days (17.6% vs 7.5%, P = .021). Bacterial co-infection in respiratory samples was associated with reduced survival among RSV patients (log rank, P = .013). CONCLUSIONS: Respiratory syncytial virus is a common cause of serious illness among hospitalized adults in China with greater mortality than influenza A. Increased awareness and the availability of antiviral agents might increase the scope for successful management.

Comparison of the Cepheid Xpert Xpress Flu/RSV assay and commercial real-time PCR for the detection of influenza A and influenza B in a prospective cohort from China.

BACKGROUND: The Xpert Xpress Flu/RSV assay is released by FDA for rapid detection of influenza A (FluA), influenza B (FluB), and respiratory syncytial virus (RSV). This study aimed to evaluate its clinical performance in comparison to that of the RT-PCR assay cleared by China FDA (CFDA-PCR). METHODS: Nasopharyngeal specimens were collected from patients and tested by the two assays side by side. Discordant results were tested with a laboratory-developed real-time PCR for resolution. Viral load in the sample was quantified with a droplet digital PCR. RESULTS: A total of 658 specimens were involved and gave 94.7%-99.1% agreement between the two assays. The Xpert assay showed higher sensitivity for FluA (100% vs. 89.8%) and FluB detection (100% vs. 95.3%), and also higher accuracy (98.9% vs. 95.7%) for FluA than the CDFA-PCR. The positive and negative predictive values (NPV) for the three viruses ranged from 90.5% to 100% in the two assays, with higher NPV for FluA and FluB in Xpert assay. Moreover, the Xpert Ct values showed a linear correlation with virus titer in specimens tested. CONCLUSION: Overall, the Xpert assay is a reliable and sensitive tool for the detection of FluA, FluB and RSV in our clinical settings.

Oral Delivery of Pingyangmycin by Layer-by-Layer (LbL) Self-Assembly Polyelectrolyte-Grafted Nano Graphene Oxide.

With the increasing development in scientific technology, building a nanocarrier system for cancer drugs has become a bliss for cancer patients. To allow for the oral administration of hydrophilic drugs, a nanocarrier that was based on negatively charged graphene oxide (GO) and prepared through the Layer-by-Layer (LbL) self-assembly of poly(acrylic acid)-cysteine (PAA-cys) and poly(allylamine hydrochloride) (PAH) was established. In the present study, we demonstrated the excellent biological properties of GO, the biological adhesiveness of PAA-cys, and the protection and controlled release profiles of polyelectrolyte. Pingyangmycin (PYM) was loaded onto the nanocarrier through non-covalent interactions. In vitro drug release studies of the prepared PAA-cys-PAH-GO-PYM were pH-sensitive and showed sustained release effects for over 8 h, before they were completely expelled by gastrointestinal peristalsis. Furthermore, cell viability experiments using A549 lung adenocarcinoma cells revealed that the IC50 of PAA-cys-PAH-GO-PYM, free drug, and GO-PYM were 159.241 µM, 134.960 µM, and 129.815 µM respectively, indicating the higher retention and cytotoxicity of PYM in vitro. When comparing the oral bioavailability of PYM with free drug, in vivo pharmacokinetics studies showed a 1.03-fold and 1.74-fold increase after GO loading and double-layer polyelectrolyte coating, respectively. Thus, PAA-cys-PAH-GO was successfully developed for oral delivery of PYM as anti-cancer therapy, and may provide further insight for oral administration of GO-based nanomaterials.