Blood culture quality and turnaround time of clinical microbiology laboratories in Chinese Teaching Hospitals: A multicenter study.

PURPOSE: Blood culture (BC) remains the gold standard for the diagnosis of bloodstream infections. Improving the quality of clinical BC samples, optimizing BC performance, and accelerating antimicrobial susceptibility test (AST) results are essential for the early detection of bloodstream infections and specific treatments. METHODS: We conducted a retrospective multicenter study using 450,845 BC specimens from clinical laboratories obtained from 19 teaching hospitals between 1 January 2021 and 31 December 2021. We evaluated key performance indicators (KPIs), turnaround times (TATs), and frequency distributions of processing in BC specimens. We also evaluated the AST results of clinically significant isolates for four different laboratory workflow styles. RESULTS: Across the 10 common bacterial isolates (n = 16,865) and yeast isolates (n = 1011), the overall median (interquartile range) TATs of AST results were 2.67 (2.05-3.31) and 3.73 (2.98-4.64) days, respectively. The specimen collections mainly occurred between 06:00 and 24:00, and specimen reception and loadings mainly between 08:00 and 24:00. Based on the laboratory workflows of the BCs, 16 of the 19 hospitals were divided into four groups. Time to results (TTRs) from specimen collection to the AST reports were 2.35 (1.95-3.06), 2.61 (1.98-3.32), 2.99 (2.60-3.87), and 3.25 (2.80-3.98) days for groups I, II, III, and IV, respectively. CONCLUSION: This study shows the related BC KPIs and workflows in different Chinese hospitals, suggesting that laboratory workflow optimization can play important roles in shortening time to AST reports and initiation of appropriate timely treatment.

Low-Cost Full-Range Detection of C-Reactive Protein in Clinical Samples by Aptamer Hairpin Probes and Coprecipitation of Silver Ions and Gold Nanoparticles.

C-reactive protein (CRP) levels can vary widely related to diverse disease contexts. However, expensive antibodies have impeded the clinical utility of antibody-based full-range CRP assays, especially in developing countries. Herein, we established a low-cost, antibody-free, 96-well plate-based full-range CRP detection method by combining gold nanoparticles (AuNPs), silver iodide (AgI), Eosin Y, and the aptamer hairpin probe (AHP) with Ag+-mediated cytosine-cytosine mismatches, that is, the Au@AgI/Eosin Y-AHP method. After binding the target CRP, the AHP released Ag+, which subsequently induced the aggregation of AuNPs on the surface of AgI colloids, resulting in a significant increase in the adsorption of Eosin Y on the surface of AuNPs. The changes in fluorescence intensity (FI) of Eosin Y in the supernate without and with CRP were proportional to the concentration of the CRP in the wide range of 0.01-40 ng/mL (r = 0.9969), and 96 samples can be detected in 96-well plates simultaneously by a microplate reader within 45 min. Remarkably, the CRP levels of 100 clinical samples achieved with the Au@AgI/Eosin Y-AHP had a good correlation with those obtained with the latex-enhanced immune turbidimetry assay (r = 0.986). Furthermore, the kit based on the Au@AgI/Eosin Y-AHP method costs only $8.1 for 100 tests. Therefore, the new method is beneficial for less developed areas where expensive assays are not affordable.

Characteristics and potential clinical applications of the extracellular vesicles of human pathogenic Fungi.

Extracellular vesicles (EVs) are a heterogeneous group of lipid membrane-enclosed compartments that contain different biomolecules and are released by almost all living cells, including fungal genera. Fungal EVs contain multiple bioactive components that perform various biological functions, such as stimulation of the host immune system, transport of virulence factors, induction of biofilm formation, and mediation of host-pathogen interactions. In this review, we summarize the current knowledge on EVs of human pathogenic fungi, mainly focusing on their biogenesis, composition, and biological effects. We also discuss the potential markers and therapeutic applications of fungal EVs.

High expression of nuclear NRF2 combined with NFE2L2 alterations predicts poor prognosis in esophageal squamous cell carcinoma patients.

Nuclear factor erythroid-2 related factor-2 (NFE2L2 or NRF2) is a frequently mutated gene in esophageal squamous cell carcinoma (ESCC). However, the roles of NFE2L2 alterations in ESCC remain elusive. In order to elucidate this issue, 130 ESCC patients who underwent esophagectomy were enrolled. The majority of tumor tissues were positive for NRF2, which was significantly enriched in the nucleus of the primary tumor tissues compared with the noncancerous mucosae. Primary ESCC tumors positive for NRF2 tended to be positive for NAD(P)H quinone oxidoreductase 1 (NQO1) as the downstream target of NRF2. There was a positive correlation between NRF2 and NQO1 expression level in primary tumors. NQO1 staining in primary tumors with NRF2 nuclear expression was significantly stronger than that with NRF2 cytoplasmic expression. In addition, high concordance for the status of NRF2 expression between primary tumors and corresponding metastatic lesions was observed. Next, we found high expression of nuclear NRF2 (the proportion of nuclear NRF2 expression >20% or nuclear NRF2 immunohistochemistry score >20) predicted shorter overall survival in patients with dual-positive expression of NRF2 and NQO1. Captured-based targeted sequencing revealed that NFE2L2 somatic alterations were observed in 52.8% of ESCC patients with dual-positive expression of NRF2 and NQO1. NFE2L2 amplification and mutations within the DLG/ETGE motifs were seen more frequently in ESCC tumors with nuclear or nucleocytoplasmic expression of NRF2 compared with those with cytoplasmic expression of NRF2. We also found high expression of nuclear NRF2 plus the status of NFE2L2 alteration exhibited high performance in predicting prognosis of ESCC patients. Our study demonstrated that high nuclear NRF2 expression and NFE2L2 alterations were associated with poor prognosis of ESCC patients. These findings suggest that NRF2 signaling pathway might play vital roles in ESCC malignancy and the aberrant activation of NRF2 pathway predicts unfavorable prognosis in ESCC.

Epidemiological and clinical characteristics of respiratory viruses in 4403 pediatric patients from multiple hospitals in Guangdong, China.

BACKGROUND: Acute respiratory infections (ARI) cause considerable morbidity and mortality worldwide, especially in children. Unfortunately, there are limited multi-center data on common viral respiratory infections in south China. METHODS: A total of 4403 nasal swabs were collected from children in 10 cities in Guangdong, China in 2019. Seven respiratory viruses, influenza A virus (IFA), influenza B virus (IFB), respiratory syncytial virus (RSV), adenoviruses (ADV) and parainfluenza virus types 1-3 (PIV1, PIV2 and PIV3), were detected by direct immunofluorescence antibody assay. The personal information and clinical characteristics were recorded and analyzed. RESULTS: The results showed that at least one virus was detected in 1099 (24.96 %) samples. The detection rates of RSV, IFA, ADV, PIV3, PIV1 and PIV2 were 7.13 % (314/4403), 5.31 % (234/4403), 4.02 % (177/4403), 3.04 % (134/4403), 1.70 % (75/4403) and 1.16 % (51/4403), respectively. The detection rate of RSV was highest in 0-6-month-old children at 18.18 % (106/583), while the detection rate of IFA was highest in 12-18-year-old children at 20.48 % (17/83). The total detection rates in winter and spring were 35.67 % (219/614) and 34.56 % (403/1166), higher than those in summer, 17.41 % (284/1631), and autumn, 19.46 % (193/992). CONCLUSIONS: RSV and IFA were the main respiratory viruses in children. With increasing age the detection rate of RSV decreased in children, but the trends for the detection rates of IFA and IFB were the opposite. This study provided the viral etiology and epidemiology of pediatric patients with ARI in Guangdong, China.

Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS-CoV-2.

BACKGROUND: The detection of serum antibodies to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as a new tool for the coronavirus disease 2019 (COVID-19) diagnosis. Since many coronaviruses are sensitive to heat, heating inactivation of samples at 56°C prior to testing is considered a possible method to reduce the risk of transmission, but the effect of heating on the measurement of SARS-CoV-2 antibodies is still unclear. METHODS: By comparing the levels of SARS-CoV-2 antibodies before and after heat inactivation of serum at 56°C for 30 minutes using a quantitative fluorescence immunochromatographic assay RESULTS: We showed that heat inactivation significantly interferes with the levels of antibodies to SARS-CoV-2. The IgM levels of all the 34 serum samples (100%) from COVID-19 patients decreased by an average level of 53.56%. The IgG levels were decreased in 22 of 34 samples (64.71%) by an average level of 49.54%. Similar changes can also be observed in the non-COVID-19 disease group (n = 9). Of note, 44.12% of the detected IgM levels were dropped below the cutoff value after heating, suggesting heat inactivation can lead to false-negative results of these samples. CONCLUSION: Our results indicate that heat inactivation of serum at 56°C for 30 minutes interferes with the immunoanalysis of antibodies to SARS-CoV-2. Heat inactivation prior to immunoanalysis is not recommended, and the possibility of false-negative results should be considered if the sample was pre-inactivated by heating.

Femtomolar Detection of Lipopolysaccharide in Injectables and Serum Samples Using Aptamer-Coupled Reduced Graphene Oxide in a Continuous Injection-Electrostacking Biochip.

A method for microfluidic sample preconcentration to detect femtomolar level of lipopolysaccharide (LPS) is introduced, enabled by 6-carboxyfluorescein (6-FAM) labeled aptamer-LPS binding along with reduced graphene oxide (rGO). The free FAM-aptamers can be adsorbed onto the surface of rGO, resulting in fluorescence quenching of background signals. Conversely, the aptamer-LPS complex cannot be adsorbed by rGO, so the fluorescence is maintained and detected. When an electric field is applied across the microchannel with Nafion membrane in the chip, only the fluorescence of aptamer-LPS complex can be detected and stacked by continuous injection-electrostacking (CI-ES). The method shows a high selectivity (in the presence of pyrophosphate, FAD+, NAD+, AMP, ADP, ATP, phosphatidylcholine, LTA, and ß-d-glucans which respond positively to LAL) to LPS and an extreme sensitivity with the limit of detection (LOD) at 7.9 fM (7.9 × 10-4 EU/mL) and 8.3 fM (8.3 × 10-4 EU/mL) for water sample and serum sample, respectively. As a practical application, this method can detect LPS in injections and serum samples of human and sepsis model mouse and quickly distinguish Gram-negative bacteria Escherichia coli ( E. coli) from Gram-positive bacteria Staphylococcus aureus ( S. aureus) and fungus Candida albicans ( C. albicans). More importantly, by changing the aptamers based on different targets, we can detect different analytes. Therefore, aptamer-coupled rGO in a CI-ES biochip is a universal, sensitive, and specific method. For TOC only.

The Clinical and Pathological Features of Maternal Gastric Adenocarcinoma Metastatic to Placenta.

We describe two mothers with metastatic gastric carcinoma involving the placenta. The first was a 35-year-old woman, who presented with a progressive decline in serum fibrinogen level at 34 + 2gestation week and underwent an emergency cesarean section. The second case was a 30-year-old woman who developed intolerable lumbosacral pain and cesarean section was performed at her 37 + 3gestation week. Gross pathological examinations of both placentas were normal, but microscopy showed poorly differentiated adenocarcinoma infiltration in the intervillous space, later proved to be gastric in origin. The neoplastic cells did not involve the villi, but were associated with massive perivillous fibrin. The two mothers died 180 and 19 days after the delivery. Both babies were free of metastatic carcinoma at 20- and 19-month follow-up.

miRNA-548p suppresses hepatitis B virus X protein associated hepatocellular carcinoma by downregulating oncoprotein hepatitis B x-interacting protein.

AIM: miR-548p is a recently identified and poorly characterized miRNA. However, its role of miR-548p in tumorigenesis and progression remains poorly understood. Here, we aimed to investigate the biofunction of miR-548p in hepatocellular carcinogenesis. METHODS: The expression levels of miR-548p were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The role of miR-548p in hepatocellular carcinoma (HCC) was determined by colony formation, flow cytometry assay and nude mice xenograft experiments. miR-548p target genes were analyzed by miRNA target predication programs and verified by qRT-PCR, western blotting assay and dual-luciferase reporter assay. RESULTS: miR-548p is repressed by hepatitis B virus X protein (HBx) in HCC tumor tissues and hepatoma cells, and inhibited cell growth by inhibiting cell proliferation and promoting cell apoptosis. miR-548p directly downregulated the expression of hepatitis B x-interacting protein (HBXIP) by binding to the 3'-untranslated region of HBXIP mRNA. Further study showed that hepatocyte nuclear factor-4a (HNF4A) promoted the expression of miR-548p and inhibited the transcription of HBXIP. HNF4A is a dominant transcriptional regulator of hepatocyte differentiation and hepatocellular carcinogenesis, and is shown to be repressed by HBx. CONCLUSION: We proposed the model for HBx/HNF4A/miR-548p/HBXIP pathway that controls hepatoma cell growth and tumorigenesis of HCC. miR-548p was identified as a tumor-suppressor in HBx-associated hepatocellular carcinogenesis.

The miR-573/apoM/Bcl2A1-dependent signal transduction pathway is essential for hepatocyte apoptosis and hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with an increasing incidence worldwide. Apolipoprotein M (apoM) is a novel apolipoprotein that is mainly expressed in liver and kidney tissues. However, the anti-tumor properties of apoM remain largely unknown. We evaluated the anti-tumor activities and mechanisms of apoM in HCC both in vivo and in vitro. Bioinformatic analysis and luciferase reporter assay results showed that apoM was a potential target of hsa-miR-573 and was downregulated after transfection with hsa-miR-573 mimics. Overexpression of apoM suppressed migration, invasion, and proliferation of hepatoma cells in vitro. Overexpression of hsa-miR-573 in hepatoma cells reduced apoM expression, leading to promotion of the invasion, migration, and proliferation of hepatoma cells in vitro. In addition, hsa-miR-573 markedly promoted growth of xenograft tumors in nude mice with an accompanying reduction in cell apoptosis. ApoM markedly inhibited growth of xenograft tumors in nude mice and promoted cell apoptosis. Moreover, Bcl2A1 mRNA and protein levels were inhibited by apoM overexpression and an increase in apoptosis rate by apoM was markedly compensated by Bcl2A1 overexpression in HepG2 cells. These results provide evidence that hsa-miR-573 promoted tumor growth by inhibition of hepatocyte apoptosis and this pro-tumor effect might be mediated through Bcl2A1 in an apoM-dependent manner. Therefore, our findings may be useful to improve understanding of the critical effects of hsa-miR-573 and apoM in HCC pathogenesis.

Rapid detection of New Delhi metallo-ß-lactamase gene and variants coding for carbapenemases with different activities by use of a PCR-based in vitro protein expression method.

New Delhi metallo-ß-lactamase (NDM)-producing bacteria are considered potential global health threats. It is necessary to monitor NDM-1 and its variants in clinical isolates in order to understand the NDM-1 epidemic and the impact of its variants on ß-lactam resistance. To reduce the lengthy time needed for cloning and expression of NDM-1 variants, a novel PCR-based in vitro protein expression (PCR-P) method was used to detect blaNDM-1 and its variants coding for carbapenemases with different activities (functional variants). The PCR-P method combined a long-fragment real-time quantitative PCR (LF-qPCR) with in vitro cell-free expression to convert the blaNDM-1 amplicons into NDM for carbapenemase assay. The method could screen for blaNDM-1 within 3 h with a detection limit of 5 copies and identify functional variants within 1 day. Using the PCR-P to analyze 5 recent blaNDM-1 variants, 2 functional variants, blaNDM-4 and blaNDM-5, were revealed. In the initial testing of 23 clinical isolates, the PCR-P assay correctly found 8 isolates containing blaNDM-1. This novel method provides the first integrated approach for rapidly detecting the full-length blaNDM-1 and revealing its functional variants in clinical isolates.

The impact of hepatitis B virus x protein and microRNAs in hepatocellular carcinoma: a comprehensive analysis.

microRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs, approximately 22 nucleotides (nts) in length, widely found in animals, plants, and viruses. Mature miRNAs control gene expression at a post-transcriptional level through blocking protein translation or inducing mRNA degradation. Many recent studies have shown that hepatitis B virus x protein (HBx), a viral protein with a crucial role in hepatogenesis, is associated with the regulation of miRNAs. This interaction impacts fundamental tumor processes, such as cell proliferation, differentiation, and apoptosis. In this review, we summarized the recent literature on the roles of HBx-regulated miRNAs in the pathogenesis of hepatocellular carcinoma.

[Morphologic features suggestive of submucosal invasive adenocarcinoma in colorectal mucosal biopsy specimens].

OBJECTIVE: To observe and summarize the morphologic features that may suggest submucosal invasive adenocarcinoma in colorectal mucosa biopsy specimens. METHODS: The study cohort included 432 colorectal biopsy specimens were obtained from 2006 to 2012. All the cases had radical surgery. Basing on the pathologic diagnoses, the cases were divided into 366 invasive adenocarcinoma (IAC) and 66 high-grade intraepithelial neoplasms (HGIN). These two groups were compared. RESULTS: In the IAC group, the percentage of tumor forming cribriform structures, acute angle-shaped glands, diffuse carcinoma cell proliferation was 61.2% (224/366) , 33.8% (124/366) and 7.4% (27/366) , respectively. In the HGIN group, cribriform gland structures appeared in 6.0% (4/66) of the cases, while no acute angle-shaped gland or diffuse carcinoma cell proliferation was detected. The difference of these three characteristics in the two group was statistically significant (all P < 0.01). Glandular branching was detected in 89.9% (329/366) of IAC cases and 66.7% (44/66) of HGIN cases; this difference was not significant. There was no difference in cellular atypia between the two groups. Interstitial fibrosis was detected more frequently in the IAC group (85.5%, 313/366 in IAC versus 0 in HGIN, P < 0.01). In biopsy specimens of IAC, a few cases showed neoplastic glands in close contact with large lymphatics, adipose tissue, and ganglion. CONCLUSIONS: In colorectal biopsy specimen, the five features that suggest submucosal invasion of the neoplastic glands including the formation of cribriform structure, angular gland, diffuse carcinoma cells, interstitial fibrosis and neoplastic glands in close contact with the thick-walled vessels.

DNA methylation profiling identifies epigenetic signatures of early gastric cancer.

Research on the DNA methylation status of gastric cancer (GC) has primarily focused on identifying invasive GC to develop biomarkers for diagnostic. However, DNA methylation in noninvasive GC remains unclear. We conducted a comprehensive DNA methylation profiling study of differentiated-type intramucosal GCs (IMCs). Illumina 850K microarrays were utilized to assess the DNA methylation profiles of formalin-fixed paraffin-embedded tissues from eight patients who were Epstein-Barr virus-negative and DNA mismatch repair proficient, including IMCs and paired adjacent nontumor mucosa. Gene expression profiling microarray data from the GEO database were analyzed via bioinformatics to identify candidate methylation genes. The final validation was conducted using quantitative real-time PCR, the TCGA methylation database, and single-sample gene set enrichment analysis (GSEA). Genome-wide DNA methylation profiling revealed a global decrease in methylation in IMCs compared with nontumor tissues. Differential methylation analysis between IMCs and nontumor tissues identified 449 differentially methylated probes, with a majority of sites showing hypomethylation in IMCs compared with nontumor tissues (66.1% vs 33.9%). Integrating two RNA-seq microarray datasets, we found one hypomethylation-upregulated gene: eEF1A2, overlapped with our DNA methylation data. The mRNA expression of eEF1A2 was higher in twenty-four IMC tissues than in their paired adjacent nontumor tissues. GSEA indicated that the functions of eEF1A2 were associated with the development of IMCs. Furthermore, TCGA data indicated that eEF1A2 is hypomethylated in advanced GC. Our study illustrates the implications of DNA methylation alterations in IMCs and suggests that aberrant hypomethylation and high mRNA expression of eEF1A2 might play a role in IMCs development.

DIRMC: a database of immunotherapy-related molecular characteristics.

Cancer immunotherapy has brought about a revolutionary breakthrough in the field of cancer treatment. Immunotherapy has changed the treatment landscape for a variety of solid and hematologic malignancies. To assist researchers in efficiently uncovering valuable information related to cancer immunotherapy, we have presented a manually curated comprehensive database called DIRMC, which focuses on molecular features involved in cancer immunotherapy. All the content was collected manually from published literature, authoritative clinical trial data submitted by clinicians, some databases for drug target prediction such as DrugBank, and some experimentally confirmed high-throughput data sets for the characterization of immune-related molecular interactions in cancer, such as a curated database of T-cell receptor sequences with known antigen specificity (VDJdb), a pathology-associated TCR database (McPAS-TCR) et al. By constructing a fully connected functional network, ranging from cancer-related gene mutations to target genes to translated target proteins to protein regions or sites that may specifically affect protein function, we aim to comprehensively characterize molecular features related to cancer immunotherapy. We have developed the scoring criteria to assess the reliability of each MHC-peptide-T-cell receptor (TCR) interaction item to provide a reference for users. The database provides a user-friendly interface to browse and retrieve data by genes, target proteins, diseases and more. DIRMC also provides a download and submission page for researchers to access data of interest for further investigation or submit new interactions related to cancer immunotherapy targets. Furthermore, DIRMC provides a graphical interface to help users predict the binding affinity between their own peptide of interest and MHC or TCR. This database will provide researchers with a one-stop resource to understand cancer immunotherapy-related targets as well as data on MHC-peptide-TCR interactions. It aims to offer reliable molecular characteristics support for both the analysis of the current status of cancer immunotherapy and the development of new immunotherapy. DIRMC is available at http://www.dirmc.tech/. Database URL: http://www.dirmc.tech/.

Rapid, Ultrasensitive, and Visual Detection of Pathogens Based on Cation Dye-Triggered Gold Nanoparticle Electrokinetic Agglutination Analysis.

Rapid prescribing of the right antibiotic is the key to treat infectious diseases and decelerate the challenge of bacterial antibiotic resistance. Herein, by targeting the 16S rRNA of bacteria, we developed a cation dye-triggered electrokinetic gold nanoparticle (AuNP) agglutination (CD-TEAA) method, which is rapid, visual, ultrasensitive, culture-independent, and low in cost. The limit of detection (LOD) is as low as 1 CFU mL-1 Escherichia coli. The infection identifications of aseptic fluid samples (n = 11) and urine samples with a clinically suspected urinary tract infection (UTI, n = 78) were accomplished within 50 and 30 min for each sample, respectively. The antimicrobial susceptibility testing (AST) of UTI urine samples was achieved within 2.5 h. In ROC analysis of urine, the sensitivity and specificity were 100 and 96% for infection identification, and 100 and 98% for AST, respectively. Moreover, the overall cost of materials for each test is about US$0.69. Therefore, the CD-TEAA method is a superior approach to existing, time-consuming, and expensive methods, especially in less developed areas.

High-resolution melting curve analysis for rapid detection of rifampin resistance in Mycobacterium tuberculosis: a meta-analysis.

A rapid, simple, accurate, and affordable method for the detection of drug-resistant tuberculosis is very critical for the selection of antimicrobial therapy and management of patient treatment. High-resolution melting curve analysis has been used for the detection of rifampin resistance in Mycobacterium tuberculosis and has shown promise. We did a systematic review and meta-analysis of published studies to evaluate the accuracy of high-resolution melting curve analysis for the detection of rifampin resistance in clinical M. tuberculosis isolates. We searched the PubMed, BIOSIS Previews, and Web of Science databases to identify studies and included them according to predetermined criteria. We used the DerSimonian-Laird random-effects model to calculate pooled measures and applied Moses' constant for linear models to fit the summary receiver operating characteristic curve. According to the selection criteria, most of the identified studies were excluded, and only seven studies were included in the final analysis. The overall sensitivity of the high-resolution melting curve analysis was 94% (95% confidence interval [CI], 92% to 96%), and the overall specificity was very high at 99% (95% CI, 98% to 100%). The values for the pooled positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were 63.39 (95% CI, 30.21 to 133.00), 0.06 (95% CI, 0.04 to 0.09), and 892.70 (95% CI, 385.50 to 2,067.24), respectively. There was no significant heterogeneity across all included studies for the measurements we evaluated. The summary receiver operating characteristic curve for the same data shows an area of 0.99 and a Q* value of 0.97. High-resolution melting curve analysis has high sensitivity and specificity for the detection of rifampin resistance in clinical M. tuberculosis isolates. This method might be a good alternative to conventional drug susceptibility tests in clinical practice.

A high throughput multiplex PCR assay for simultaneous detection of seven aminoglycoside-resistance genes in Enterobacteriaceae.

BACKGROUND: The aminoglycoside-resistance genes encoding aminoglycoside modifying enzymes and 16S rRNA methyltransferases are main factors contributing to increasing resistance to aminoglycosides. Characterization and distribution of antimicrobial resistance gene profiles provide important information on the potential difficulty of treatment of bacteria. Several molecular methods have been developed to investigate the prevalence of aminoglycoside-resistance genes. These existing methods are time-consuming, labor-intensive, expensive or limited sensitivity in the epidemiological investigation. Therefore, it is necessary to develop a rapid, less-costly and high throughput and sensitive method to investigate the distribution of antimicrobial resistance gene in clinical isolates. RESULTS: In this study, we developed a GeXP analyzer-based multiplex PCR assay to simultaneously detect seven aminoglycoside-resistance genes, including aac(3)-II, aac(6')-Ib, aac(6')-II, ant(3″)-I,aph(3')-VI,armA and rmtB, and to analyze the distribution of these genes in clinical Enterobacteriaceae isolates. Under optimized conditions, this assay achieved a limit-of-detection as low as 10 copies of each of the seven genes. The presented method was applied to analyze the distribution of aminoglycoside-resistance genes in 56 clinical Enterobacteriaceae isolates, and the results were compared with that of the conventional single PCR assay. Kappa values of the two methods for detecting each of the seven resistance genes were 0.831, 0.846, 0.810, 0.909, 0.887, 0.810 and 0.825, respectively. CONCLUSION: This GeXP assay is demonstrated to be a rapid, cost-effective and high throughput method with high sensitivity and specificity for simultaneously detecting seven common aminoglycoside-resistance genes.

Poliovirus receptor (PVR) mediates carboplatin-induced PD-L1 expression in non-small-cell lung cancer cells.

Programmed death-ligand-1 (PD-L1) is an immune suppressor that inhibits T cell based immunity. Anti-PD-L1/PD-1 immunotherapy benefits those patients receiving platinum-based combinational chemotherapy. However, the underlying mechanism is still largely unknown. In this study, we found that carboplatin could induce PD-L1 expression in NSCLC H292, A549 and H1299 cells in a dose-dependent manner. mRNA sequencing and the subsequent validation assays found that carboplatin significantly induced PVR expression, which is considered as an immuno-adhesion molecule. Mechanistically, PVR knockdown significantly abrogated carboplatin-induced PD-L1 expression. Functionally, knockdown of PVR significantly reversed the CD3+ T cells proliferation inhibition caused by carboplatin increased PD-L1. Moreover, the carboplatin-induced PVR and subsequent up-regulation of PD-L1 might be mediated via the EGFR, PI3K/AKT, and ERK signaling pathways. Immunohistochemical staining results showed that the PD-L1 expression was positively associated with PVR expression in clinical NSCLC samples. Our study reveals a novel regulatory mechanism of PD-L1 expression, provides evidence that carboplatin inhibits tumor immune response by up-regulating PD-L1 expression and explains the rationale for combining platinum-based chemotherapy with PD-L1/PD-1 inhibitors.