Distribution and transmission characteristics of thirty-five dual genotype vancomycin-resistant Enterococcus faecium carrying both vanA and vanM in an intensive care unit / 中华传染病杂志

Objective To investigate the distribution and transmission characteristics of vancomycin -resistant Enterococcus faecium (VREF) carrying both vanA and vanM in the intensive care unit.Methods VREF strains were isolated from patients in the intensive care unit of Jinshan Hospital , Fudan University in Shanghai from 2013 to 2017.Antimicrobial susceptibilities of the VREF strains to nine antibiotics , including vancomycin, teicoplanin, linezolid and chloromycetin , were tested by broth microdilution method.Multiple polymerase chain reaction (PCR) was used for van genotyping and pulsed field gel electrophoresis (PFGE) was used for homology analysis.Results Thirty-five strains were mainly isolated from urine (16 strains), blood (11 strains), feces ( five strains ), bile ( two strains ) and pleural effusion ( one strain ).All the strains (100.00%) were resistant to vancomycin , ampicillin and levofloxacin , but only 40.00% were resistant to teicoplanin.All the strains were sensitive to linezolid.The results of van genotyping showed that 33 (94.3%) strains belonged to vanA and vanM dual genotype VREF, and the other two were vanA type VREF.PFGE results showed that 35 strains could be divided into 14 PFGE patterns, and seven out of 10 strains isolated in 2014 were identical and the other three belonged to three different PFGE patterns.Conclusions A dual genotype VREF carrying both vanA and vanM has been emerging and spreading in the intensive care unit of Jinshan Hospital , Fudan University in Shanghai.

Resistance mechanism and clinical feature of linezolid-resistant Staphylococcus capitis isolated from blood samples / 中国感染与化疗杂志

Objective To understand the resistance mechanism and clinical feature of linezolid-resistant S.capitis isolated from blood samples.Methods Antimicrobial susceptibility testing was carried out to determine the susceptibility of clinical strains.PCR and sequencing analysis were used to analyze cfr gene and 23S rRNA mutation,which were associated with linezolid resistance.Patterns of pulsed-field gel electrophoresis (PFGE) were analyzed in combination with clinical data to understand the clinical feature of S.capitis strains.Results Five linezolid-resistant S.capitis strains were isolated from blood samples of 3 patients.These strains were resistant not only to linezolid,but also to most of the commonly used antimicrobial agents except glycopeptides,rifampin,and trimethoprim-sulfamethoxazole.Mutation was identified in 23S rRNA genes of all the five strains and cfr gene was found in four of the five strains.PFGE typing showed the same type,which supported the homology of the 5 strains.Three patients had deep vein indwelling catheter and two of them were treated with linezolid.Conclusions Linezolid-resistant S.capitis isolates showed the phenotype of resistance to multiple antimicrobial agents.Linezolid resistance may be mediated by cfr gene and 23S rRNA mutations in S.capitis.Long-term use of deep vein indwelling catheter and linezolid treatment may increase the risk of linezolid-resistant S.capitis infection.

The efficiency of serum lncRNA GAS5/miR-21 as biomarkers in patients with diabetes and diabetic nephropathy / 中华肾脏病杂志

Objective To elucidate the efficiency lncRNA GAS5 and miR-21 as biomarkers in diabetes mellitus and diabetic nephropathy.Methods The patients were divided into three groups,diabetic nephropathy group (DN group proven by renal biopsy,n=25,14 males and 11 females),diabetes group (DM group,with normal urine albumin creatinine ratio,n=10,4 males and 6 females),and normal control group (NC group,n=9,4 males and 5 females).The expressions of lncRNA GAS5 and miR-21 in serum samples were detected by real-time quantitative PCR.The correlation between serum lncRNA GAS5 and miR-21 expressions and the clinical parameters was analyzed by T-test,Pearson,Spearman test and multivariate linear regression analysis.Differences of lncRNA GAS5 and miR-21 in different groups were analyzed by one-way analysis of variance.The ROC curve was used to analyze the diagnostic efficacy of lncRNA GAS5 and miR-21 in diabetes and diabetic nephropathy.All data were analyzed by SPSS 20.0 and GraphPad software,with P ＜ 0.05 as considered statistically significant.Results (1) The expression of serum lncRNA GAS5 was significantly down-regulated and serum miR-21 was significantly up-regulated in both diabetes mellitus and diabetic nephropathy patients compared to the NC group all (P ＜ 0.05).(2) In DN patients,the expression of serum lncRNA GAS5 was gradually up-regulated along with the increment of 24 h urinary protein.The expression of serum miR-21 was gradually up-regulated along with renal biopsy stage Ⅱb-Ⅲ of DN (P ＜ 0.05).(3)FBG and HbA1c were all negatively correlated with serum lncRNA GAS5 (P ＜ 0.05),and FBG was independently correlated with serum lncRNA GAS5 (P ＜ 0.05).Urine microalbumin,Total cholesterol (TC),Scr,Urea and SBP were all positively correlated with serum miR-21(P ＜ 0.05).Albumin (ALB)and estimated GFR (eGFR) were negatively correlated with serum miR-21(P ＜ 0.05),and ALB was independently correlated with serum miR-21 (P ＜ 0.05).(4) The diagnostic efficiency of serum lncRNA GAS5,miR-21 and lncRNA GAS5/miR-21 as "diagnostic signature" for DM were was good (P ＜ 0.05).(5) The diagnostic efficiency of serum miR-21 and lncRNA GAS5/miR-21 as "diagnostic signature" for DN were was good (P ＜ 0.05).Conclusions (1) Serum lncRNA GAS5 had good diagnostic efficiency in diabetes mellitus.The sensitivity of lncRNA GAS5/miR-21 for diagnosis of diabetes was 85.71％,and specificity was 88.89％.(2) The level of serum miR-21 can be used as a noninvasive diagnostic marker for diabetic nephropathy.

Rapid detection of high-level vancomycin-resistant genes, vanA, vanB, vanD and vanM, in enterococci / 中华传染病杂志

Objective To develop a multiple polymerase chain reaction (PCR) technique based assay for rapid detection of vanA, vanB, vanD and vanM in high-level vancomycin-resistant enterococci.Methods After analyzing the uncleotide sequence divergence among D-Ala∶D-Lac ligase genes, an multiplex PCR assay for vanA, vanB, vanD and vanM genes in high-level vancomycin-resistant enterococci were designed.By using recombination plasmids containing vanA, vanB, vanD and vanM genes as positive control, and non-vancomycin resistant enterococci (non-VRE) common pathogenic bacterial DNA as negative control, the sensitivity and specificity of the assay were evaluated.Fifty vancomycin-resistant enterococci (VRE) isolates were detected by the assay.Fifty clinical strains of VRE were isolated from 9 hospitals in Shanghai from January 2006 to December 2014.The results were compared with the conventional PCR and sequencing methods.Results The identity of the D-Ala∶D-Lac ligase genes were 60.8%-71.3% of vanA, vanB, vanD and vanM genes.The multiplex PCR assay could identify the genotypes of the positive control samples accurately.No false positive results were found in negative control samples.Among fifty VRE strains detected by the assay, 18 were vanA genotype and 32 were vanM genotype.Comparison of the multiplex PCR assay and sequencing methods revealed sensitivity and specificity of 100%.The detection limit of the assay was 2×10 copies/PCR reaction.The experiment could be done within 3.5 h.Conclusions A multiplex PCR assay is developed to rapid identify the genotype of the high-level vancomycin-resistant enterococci, which can be used for the molecular epidemiology research and detection of VRE.

Fosfomycin resistance and f os gene detection in clinical St ap hy lococcus aureus strains / 中国感染与化疗杂志

Objective To understand fosfomycin resistance and prevalence of fos gene in clinical Staphylococcus aureus strains . Methods A total of 109 clinical strains of S .aureus were isolated from the patients in Huashan Hospital from January to March in 2014 .Antimicrobial susceptibility testing was performed by agar dilution method .The genes related to fosfomycin resistance including fosA ,fosB and fosC were detected by PCR .The flanking sequences of fos gene were determined by primer walking sequencing .The multilocus sequence typing (MLST) was carried out for fos gene positive strains .Results Forty‐four strains were resistant to fosfomycin (MIC> 32 mg/L) ,including 13 positive for fosB gene .Thirteen of the 109 (11 .9% ) strains carried fosB gene .However ,no fosA or fosC gene was identified .ST1 was a dominant MLST type in the strains carrying fosB gene .The three strains positive for fosB gene and associated with high level fosfomycin resistance (MIC> 512 mg/L) belonged to three different ST types . Walking sequencing showed that the fosB gene located on a transferable element containing a transposase gene .Conclusions High prevalence of f osB gene in fosfomycin‐resistant S . aureus strains indicates that f osB gene may mediate or contribute to fosfomycin resistance .

Clinical survey of 88 cases of candidemia / 中国感染与化疗杂志

Objective To understand the clinical features of candidemia.Methods A retrospective analysis was performed based on the data of 88 candidemia cases treated in Huashan Hospital during the period from 2007 to 2012.The clinical data were re-viewed in terms of species distribution,underlying diseases,clinical manifestations,treatment and outcomes.The prognostic factors were analyzed by chi-square test or Fisher exact probability test.Multivariate analysis was conducted by multiple Logis-tic regression.Results Candida albicans (40/88,45.5%)was the most common pathogen isolated from these candidemia ca-ses,followed by Candida tropicalis (20/88,22.7%),Candida parapsilosis (17/88,19.3%),Candida glabrata (10/88, 11 .4%),and Candida krusei (1/88,1 .1 %).Solid malignancy,diabetes,and surgical procedure were the most frequently identified underlying diseases.Fatal or deteriorative outcome was reported in 28 cases.The attributable mortality was 18.2%. Multivariate prognostic analysis indicated that presence of central venous catheter (OR:6.322,95% CI :1 .055-37.891 ,P =0.044)was independently correlated to increased mortality.Appropriate antifungal therapy was an independent predictor of de-creased overall mortality (OR:0.137,95% CI :0.039-0.480,P =0.002).Conclusions The pathogen distribution of candi-demia has changed slightly.Appropriate antifungal therapy plays a key role in the treatment of candidemia.

Meta-analysis of flucytosine or fluconazole plus amphotericin B for the treatment of acquired immunodeficiency syndrome-associated cryptococcal meningitis / 中华传染病杂志

Objective To evaluate the survival benefit of amphotericin B (AmB) plus flucytosine or fluconazole for treatment of patients with acquired immunodeficiency syndrome (AIDS)-associated cryptococcal meningitis.Methods The following database were searched from the beginning to October 2013,including Cochrane library,PubMed,OVID,Embase,Wanfang Date,CNKI and Chinese Biomedical Database,and the references of eligible studies were manually screened.Reference lists of relevant articles were screened according to selection and extraction criteria.Meta-analysis was performed using RevMan 5.2.Results Four prospective controlled studies with a total of 399 patients with cryptococcal meningitis were identified,including 386 patients with AIDS-associated cryptococcal meningitis and 13 human immunodeficiency virus (HIV)-negative patients.Two hundred and twentyseven patients were treated with AmB and flucytosine combination therapy,including 217 patients with AIDS-associated cryptococcal meningitis and 10 HIV-negative patients.One hundred and seventy-two patients were treated with AmB and fluconazole combination therapy,including 169 patients with AIDS-associated cryptococcal meningitis and 3 HIV-negative patients.The Meta-analysis revealed that the mortality rate in AmB plus flucytosine combination therapy group was 6.6％ (95％ CI:18.5％-31.6 ％) at two weeks point,which was significantly lower than that in AmB plus fluconazole combination group (19.7％,95％CI:-23.6％-62.9％; OR=0.51,95％CI:0.27-0.93,P＜0.05).But at 10 weeks point,the mortality rate in flucytosine combination group was 12.9％ (95％oo CI:-22.2％-48.0％),which was lower than that in fluconazole combination group (31.4％,95％ CI:-23.1％-85.9 ％).However,there was no statistically significant difference between these two groups at 10 weeks point (OR=0.70,95％CI:0.44-1.13,P=0.15).Conclusion Administration of AmB plus flucytosine at early stage can reduce the mortality rate in patients with AIDS-associated cryptococcal meningitis.

A non-controlled, multicenter open-label study to evaluate the safety, tolerability and efficacy of caspofungin in the treatment of invasive candidiasis and esophageal candidiasis / 中国感染与化疗杂志

Objective This study was designed to evaluate the safety ,tolerability and efficacy of intravenous caspofungin for treatment of invasive candidiasis and esophageal candidiasis in Chinese adults .Methods This was a non-controlled ,multicenter ,candidiasis .All the 63 patients were included in the safety set (SS) and the full analysis set (FAS) .In the SS ,19 SAEs occurred in 14 patients .All these SAEs were unrelated to caspofungin .There were 73 caspofungin-related non-serious AEs in 31 patients (49 .2% ) .Five patients (7 .9% ) had both clinical AEs and laboratory abnormalities .Eight patients (12 .7% ) had clinical AEs (mainly rashes) ,and 27 patients (42 .9% ) had laboratory abnormalities ,mainly increases in liver enzymes alanine transaminase and aspartate transaminase and reduction in blood potassium .About 91 .7% of the clinical AEs were mild to moderate .Treatment was discontinued in 1 patient (1 .6% ,1/63) due to AEs .The overall efficacy was 58 .1% (36/62) in the FAS and 70 .0% (35/70) in the per-protocol set (PPS) .In the FAS ,the therapeutic efficacy was 57 .6% (34/59) for invasive candidiasis and 66 .7% (2/3) for esophageal candidiasis .In the PPS , the therapeutic efficacy was 68 .8% (33/48 ) for invasive candidiasis and 100% (3/3 ) for esophageal candidiasis .Conclusions The AEs of caspofungin were mostly mild to moderate in the treatment of invasive candidiasis and esophageal candidiasis in Chinese adults .Only one patient terminated therapy due to drug-related AE .Caspofungin is safe and effective for the treatment of invasive candidiasis and esophageal candidiasis in Chinese adults .

Screening for fosfomycin resistance genes in Enterococcus faecium via high-throughput sequencing / 中华传染病杂志

Objective To screen fosfomycin-resistant genes in the clinical isolates of Enterococcus faecium Efm-HS0661 and verify their functions. MethodsAntimicrobial susceptibility and conjugation experiments were carried out to determine if the antimicrobial resistance in clinical strain was transferable.By Solexa high-throughput sequencing,the genes conferring fosfomycin resistance were screened. The function of resistance gene was identified by cloning.ResultsThe clinical isolates of Enterococcus faecium Efm-HS0661 were resistant to glycopeptide antibiotics and fosfomycin, and the fosfomycin resistance was found to be transferred by conjugation. Within the 2414 bp nucleotide sequence obtained by high-throughput sequencing, fosB, a plasmid-mediated fosfomycin resistance gene was found. The fosB gene was 420 bp in length, which shared 99. 8％ amino acid identity with other fosB from Staphylococcus spp. The minimal inhibitory concentration (MIC) of DH5α transformant containing fosB gene against fosfomycin was higher than that of DHSa transformant without fosB gene. ConclusionsThe high-throughput sequencing can be used to screen unknown resistance genes in clinical isolates. The plasmidmediated resistance gene fosB can confer fosfomycin resistance in Enterococcus faecium.

Clinical and pathological analysis of 2380 patients with bacterial infectious diarrhea / 中华传染病杂志

Objective To investigate the clinical and pathological characteristics of bacterial infectious diarrhea.Methods The clinical and pathological characteristics of 2380 cases of bacterial infectious diarrhea in Jinshan Hospital,Fudan University from 1998 to 2007 were analyzed retrospectively.Enumeration data were analyzed by X~2 test.Results Among the 20 169 patients who went to hospital because of diarrhea in 10 years,2380 cases fecal bacterial culture were positive,including Vibrio parahaemolyticus(2247 cases,94.4%),Shigella(99 cases,4.2%),Salmonella (29 cases,1.2%),Vibrio alginolyticus(3 cases),pathogenic Escherichia coli(2 cases).Patients with diarrhea were common from June to 0ctober in each year.The main manifestations of Vibrio parahaemolyticus infection were abdominal pain,diarrhea,nausea,vomit or dehydration.The main manifestations of Shigella infection were fever,abdominal pain and diarrhea.Conclusions The bacterial culture positive rate of stool samples from patients with bacterial infectious diarrhea is not high in Jinshan district.Shanghai.The major pathogens are Wbrio parahaemolyticus and Shigella.

Three hundred and ninety-five eases of nosocomial bloodstream infection / 中华传染病杂志

Objective To characterize clinical feature, frequency of isolation and antimicrobial susceptibility of pathogens isolated from patients with nosocomial bloodstream infections in Huashan Hospital, Fudan University from 1995 to 2004. Methods The clinical data of all patients who were diagnosed with nosocomial bloodstream infections based on national diagnostic criteria of nosocomial bloodstream infections were retrospectively analyzed. The pathogens were routinely isolated and identified. Susceptibilities against antimicrobial agents were determined by Kirby-Bauer methods and analyzed by WHONET 5.0 software. Results During the 10-year study period, a total of 395 patients were diagnosed with nosocomid bloodstream infection with 435 strains isolated from blood specimen.Gram positive bacteria, Gram negative bacilli and fungi, accounted for 47.4%, 45.1 % and 7.6%,respectively. Coagulase-negative Staphylococci (21.4%), S. aures (17.9%), E.coli (13.6%), K. pneumoniae (10.8%), Candidaspp (7.4%), Enterococci (6.0%), Pseudomonasspp (6.0%) and Acinetobacter spp (3.7%) were frequently identified isolates. S. aures and coagulase-negative Staphylococci resistant to methicillin were 62.8% and 87.1%, respectively. The susceptibilities of cefotaxime and ceftazidime against E. coli and K. preumonine were 46%-78% and 27.7%-40.4%, respectively. Conclusions The Gram positive cocci are slightly more prevalent than Gram negative bacilli in nosocomial bloodstream infections and resistance to the first line antibiotics is common among all pathogens isolated. Candida spp is the fifth leading cause of nosocomial bloodstream infections.

Rapid detection of clinical pathogenic bacteria and antimicrobial-resistant genes using DNA chip / 中华传染病杂志

Objective To detect the clinical pathogenic bacteria and antimicrobial-resistant genes quickly and sensitively using DNA chip.Methods Based on the analysis of 23S rRNA gene se- quences and other genes sequences associated with antimicrobial resistance(SHV＜CTX_M),oligo nucleotide microarray was designed according to different bacteria and antimicrobial-resistant genes. The DNA fragments were amplified by labeling Cy5 fluorescence and detect clinical pathogenic bacte- rias and antimicrobial-resistant genes by hybridization.Results The result of detection(10~3-10~6 bac- teria/ml)was consistent with that of some documents in domestic and overseas under ideal circum- stances of detecting bacteria genomic DNA by the Reagent Box.And it was specific and reproducible when the detection system were evaluated with some clinical isolates and drug-resistant standard strain.DNA chip could identify 16 species and 7 generics including common diverse clinical pathogenic bacteria,and could detect the drug-resistant of extended spectrum?lactamase gene simultaneously. Conclusions The methods that we have established DNA chip is a sensitive,specific and reproducable tool for supplying routine methods.

Partial sequence analysis of 23S rRNA gene in common bacteria and its application in the diagnosis of bacterial infection / 中华传染病杂志

Objective To analyze the 23S rRNA gene partial sequences of common bacteria, and establish molecular biologic techniques to identify bacteria by the difference of gene sequences. Methods Analyzing the sequences of variable region of bacterial 23S rRNA genes, primers and oligonucleotide probes were designed accordingly. Thereafter, bacteria were identified by PCR gel electrophoresis and PCR reverse hybridization. Results There exists significant sequence difference between Gram negative bacteria and Gram positive bacteria and it could be used to differentiate these 2 kinds of bacteria quickly with PCR gel electrophoresis. Meanwhile, sequence variety in different species of bacteria was also observed and PCR reverse hybridization could be used to identify different bacterial species further.Conclusions There exist significant sequence differences among 23S rRNA genes in different common bacteria. By the sequence differences, a specific, sensitive and rapid molecular biologic techniques could be established to quickly identify the pathogens of bacterial infections.