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Objective:To establish a classification model for rapid identification of hypervirulent subtype ST17 clones of Group B Streptococcus (GBS) using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS).Methods:In a retrospective study, 235 strains of GBS strains were selected from multiple centers in China during 2015-2018. For model generation,45 strains of ST17 and 50 strains of non-ST17 (20 ST19, 15 ST12 and 15 ST10 strains) were enrolled as the modeling group. The remaining 90 main ST strains (40 ST17, 16 ST10, 17 ST12 and 17 ST19) were served as validation group. 50 GBS strains classified as other minor ST subtypes were regarded as taxonomic groups. MS spectra were collected by Bruker mass spectrometry, and then loaded for model generation and verification, and screening of differential peptide peaks by genetic algorithm (GA) and model verification on ClinProTools 3.0 software.Results:The recognition rate for ST17-GA model were 99.4% with cross validation value of 96.9%. Among the ten differential peptide peaks for the classification model, the weights of both two main peptide peaks m/z 2 956 and m/z 5 912 were greater than 1, while the weights of the all other eight peptide peaks were less than 0.5. Model validation showed only one of the ST17 was misjudged as non-ST17 strain, resulting in diagnostic accuracy of 98.9%, sensitivity of 97.5% and specificity of 100%, positive predictive value of 100% and negative predictive value of 98.0%, respectively. For other sporadic STs, 42.0% (21/50) of them were misdiagnosed as ST17 subtype.Conclusion:A MALDI-TOF MS classification model for hypervirulent subtype of ST17 GBS strains has been successfully established with good diagnostic efficacy.
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Objective To establish and optimize a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Escherichia coli and its microbial toxin.Methods The LAMP reaction system and reaction conditions were determined by optimizing LAMP reaction,and the optimized LAMP system was used for the detection.Results Primers targeting shiga toxin (stx) gene and O157 antigen gene rfbe were designed.The established and optimized LAMP amplification system contained 1.2 mmol/L dNTPs,10 mmol/L MgSO4,0.4 mol/L betaine,1 μl 10 × Bst DNA polymerase Buffer,8 U Bst DNA polymerase fragment,2 μl DNA template,and the ratio of inner-primer (FIP and BIP) and outerprimer (F3 and B3) were 8∶ 1.Time and temperature for LAMP was 60 min,60 ℃.The sensitivity was 103 times higher than polymerase chain reaction (PCR),reached 5 × 101 CFU/ml.When LAMP was applied to 19 reference strains,102 EHEC strains,the specification was 100％ while identification rate of rfbe,stx1 and stx2 gene reached 100％,95.2％,92.9％.Conclusions The LAMP method showed a promising prospect for the rapid detection of common nosocomial pathogens microbial toxin.
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Objective To explore the sample type and drug resistance characteristics of Streptococcus pneu-monia(Spn)isolated from pediatric patients in Guangzhou district,and their age distribution to offer instruc-tions for prevention and clinical treatment.Methods Spn isolates were cultured and identified according to the national standard procedure for clinical laboratory operation,followed by analysis of sample type and age dis-tribution of pediatric patients with positive isolates of Spn in Guangzhou Women and Children′s Medical Cen-ter from 2013 Jan 1st to 2015 Dec 31st,drug resistance status was determined by MIC test.Results Totally, 1 243 strains of Spn were isolated,which were mainly from pediatric patients under 1 year old(42.80%).Spn isolates were mainly isolated from respiratory tract(72.81%),ear secretions(15.37%),blood(5.63%),cere-brospinal fluid(3.06%)and hydrothorax(2.01%).For all Spn isolates,the resistance rate to erythromycin, tetracycline and sulfamethoxazole was especially high as 94.93%,85.76%,73.53% respectively,with relative high resistance to penicillin G(24.70%),amoxicillin(39.59%),ceftriaxone(24.05%),meropenem(22.85%) and cefotaxime(19.89%),low resistance to quinolone antibiotics(<10.00%),and no resistance to vancomycin and linezolid.Conclusion The major age group of children with Spn infection is infants under one year old in Guangzhou,clinicians should be serious about the high resistant rate of Spn to erythromycin,tetracycline and sulfamethoxazole,the significantly increased resistant rate to penicillin,amoxicillin and ceftriaxone.Clinicians should choose antibiotics rationally according to the characteristics of drug sensitivity for better treatment.
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Objective To understand the molecular epidemiology of penicillin resistance Streptococcus pneumonia (PNSP) isolated from children in Guangzhou area to provide the experimental basis for clinical prevention and control of Streptococcus pneumonia infectious diseases.Methods Specific primers were designed according to Genebank,penicillin binding protein(PBP) genes PBP1A,PBP1B,PBP2A,PBP2B,PBP2X,PBP3 were amplified by PCR.The sequencing analysis was performed.The PCR products were digested by Hinf I,and the restriction fragment length polymorphism (RFLP) was analyzed.Results DNA of PNSP was successfully extracted,the PCR results showed that in 50 strains of PNSP,the positive rates of bacterial strains containing PBP1A,PBP1B,PBP2A,PBP2B,PBP2X and PBP3 were 48.9%,64.4%,71.1%,31.1%,40.0% and 31.1% respectively.The sequencing showed that their homologies with known sequences in GenBank were 99%,98%,100%,97%,95% and 100% respectively.Using RFLP in Hinf I showed that PBP1A,PBP1B,PBP2A and PBP3 only had one kind of genotype,PBP2B and PBP2X had two kinds of genotypes,the positive rates were 71.4%,28.6%,66.7% and 33.3% respectively.Conclusion The gene distribution of PNSP strains among children in Guangzhou is dominated by PBP2A,PBP1B and PBP1A,there are two subtypes in PBP2B,PBP2X when digested by Hinf I,in which the predominant subtype >65%.
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Objective To analyse distribution and antibacterial resistance status of pathogenic bacteria isolated from blood cul‐tures of hospitalized infants ,in order to provide references for rational use of antimicrobial agents in the treatment of bloodstream infection .Methods A total of 299 strains of pathogenic bacteria isolated from positive blood culture specimens from infants(3 or less than 3 months of age) suspected with bloodstream infections in this hospital from January 2011 to May 2015 were collected ,the bacteria identification and drug sensitivity test were carried out by using the VITEK 2 Compact automatic microorganism analyzer . The composition and antibacterial resistance of these isolates were analyzed .Results Among the 299 strains of pathogenic bacteria , there were 169 strains of gram‐positive cocci(accounted for 56 .5% ) ,including 95 strains of coagulase negative Staphylococcus (ac‐counted for 31 .8% ) which was the main isolates ,and followed by 28 strains of Staphylococcus aureus(accounted for 9 .4% );there were 120 strains of gram‐negative bacilli (accounted for 40 .1% ) and mainly were Escherichia coli (53 strains ,accounted for 17 .7% );otherwise ,there were 8 strains of fungi (accounted for 2 .7% ) and 2 strains of gram‐positive bacillus (accounted for 0 .7% ) .The results of drug susceptibility test indicated that the gram‐positive cocci had multiple drug resistance to antibacterial a‐gents except for vancomycin and linezolid;the gram‐negative bacilli shown multiple drug resistance except for amikacin ,imipenem and meropenem .The fungus ,however ,displayed high sensitivty to all antifungal drugs .Conclusion Gram‐positive and gram‐nega‐tive bacteria are the main pathogens of hospitalized infants with bloodstream infection ,and are severely resistant to antibacterial a‐gents .Rational use of antimicrobial agents should be recommend for improving clinical efficacy and prohibiting the emergence of drug‐resistant strains .
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Objective To perform the amplification ,sequencing and prokaryotic expression of APH (3′′)‐Ⅰ and AAC (2′)‐Ⅰgenes from the clinically isolated gzch810 strain(SM gzch810)of Stenotrophomonas maltophilia to provide the basic materials for the next step functional test .Methods The SM gzch810 genome chromosome was extracted ,the APH (3′′)‐Ⅰ ,AAC (2′)‐Ⅰ whole genes were amplified by PCR and sequenced after being cloned into pMD18‐T vector .The recombination were subcloned into pGEX‐4T‐1 vector and the expression of the recombinant APH (3′′)‐Ⅰ and AAC (2′)‐Ⅰ were analyzed by SDS‐PAGE .Results The 800bp and 550bp DNA fragments of APH(3′′)‐Ⅰ ,AAC(2′)‐Ⅰ gene were amplified from SM gzch810 by PCR and sequenced ;the sequence comparison analysis showed that DNA and amino acid sequence identities of APH (3′′)‐Ⅰand AAC (2′)‐Ⅰ genes with other strains were 91% and 95% respectively .The sequence of APH (3′′)‐Ⅰand AAC(2′)‐Ⅰ of SM gzch810 were submitted to GenBank(accession number :HQ315852 and HQ315853);two major protein bands corresponding to the expected recombinant GST‐TP fusion proteins (56 × 103 and 46 × 103 respectively) were identified by SDS‐PAGE .Conclusion APH(3′′)‐Ⅰand AAC(2′)‐Ⅰgene of SM gzch810 are successfully cloned and expressed ,which lays a good foundation for further detecting corresponding antibi‐otic resistance and functional evaluation of above two kinds of recombinant E .coli .
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Objective To clone and express Staphylococcus aureus drug resistance adenylyltransferase gene in E .coli BL21 ,and to make the foundation for its function research .Methods Primers were designed on the basis of adenylyltransferase gene in gen‐bank ,PCR was used to amplify adenylyltransferase gene using Staphylococcus aureus genomic DNA as template .The obtained PCR production was attatched with pGEX‐4t‐1(+ ) plasmid ,and transformed into E .coli BL21 (DE3) .The recombinant plasmid was di‐gested by double enzyme digestion and identified by gene sequence .The recombinant protein was induced to expression by IPTG and identified by Western‐blotting .Results Using Staphylococcus aureus genome as a template ,the target fragment about 800 bp was successful amplified .After enzyme‐cutting and DNA‐sequencing ,the target fragment showed that the ORF begin with ATG ,end with TAG ,783 bp in length ,the predicted isoelectric point and molecular weight were 7 .75 and 29 × 103 ,and it was homology 99%homology with the reported sequence gene in genbank .SDS‐PAGE and Western‐blot showed the molecular weight of recombinant fusion protein was about 55 × 103 .Conclusion Adenylyltransferase gene of Staphylococcus aureus was successfully cloned and ex‐pressed in E .coli as a fusion protein ,which makes the foundation for the research of its function .
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Objective To investigate the infection status and drug resistance of imipenem‐resistant Pseudomonas aeruginosa (PAE) isolated from the submitted specimens from children patients in our hospital between October 2012 to September 2013 for mastering the antimicrobial resistance status of Pseudomonas aeruginosa infection among children in Guangzhou and the occurrence situation of imipenem resistant strains .Methods The detection situation of Pseudomonas aeruginosa from infected children during this period was analyzed and the VITEK 2 Compact analyzer produced by the France bioMerieux company was used to identify the bacteria .The minimal inhibitory concentration(MIC) of imipenem resistant Pseudomonas aeruginosa to 11 kinds of antibiotics was detected .Results 161 strains of Pseudomonas aeruginosa were detected from 36 600 specimens ,including 24 strains(14 .9% ) of imipenem resistant Pseudomonas aeruginosa ,the positive strains were mainly originated from phlegmy (50 .3% ) ,the isolation was highest in ICU (27 .4% ) and NICU(21 .8% ) .The drug resistance rate of imipenem‐resistant P .aeruginosa was higher to ceftriax‐one sodium(91 .7% ) ,cefoperazone(29 .0% ) ,ceftazidime(29 .0% ) ,cefoperazone /shubatan(29 .0% ) and aztreonam (25 .0% ) .Con‐clusion Imipenem‐resistant Pseudomonas aeruginosa has the higher detection rate among children in this area and usually has re‐sistance to multiple antibiotics ,which should be paid more attention to and antibiotics should be rationally used .
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Objective To investigate the changes of pathogens distribution and antimicrobial resistance in children with urinary tract infection (UTI) in 10 years. Methods The results of urine culture and drug sensitivity in children with UTI from January 2001 to December 2003, and from January 2011 to December 2013 were retrospectively analyzed.Results In recent 10 years, there was no obvious change in the ratio of gram-negative bacteria to gram-positive bacteria. Escherichia coli was still the main bacteria causing UTI in children. The detection rate of enterococcus was signiifcantly increased from 18.3%in 2011-2013 to 7.5%in 2001-2003 (P<0.05) and it had become the second pathogenic bacteria. The isolation rate of ESBLs producing strains was signiifcantly higher in 2011-2013 than in 2001-2003 (P<0.05). The rate of Escherichia coli sensitive to imipenem re-mained at 100%and it is also sensitive to enzyme inhibitors antibiotics and nitrofuranto. Sensitivities to antibiotics were changed in different species of enterococcus. Conclusions The distribution of pathogens and antimicrobial resistance in children with UTI are constantly changing. The clinician should pay close attention to changes of epidemiology in the region and hospital and rational use of antimicrobial drugs.
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Objective To explore the main pathogenic bacteria and antibiotic resistance patterns in children with bacterial diar‐rhea from Guangzhou region .Methods Regular bacterial culture of stool samples from children with suspicious bacterial diarrhea was performed to isolate the pathogen during 2011 to 2012 ,followed by the analysis of its composition and serum type ,ward distri‐bution characteristics and drug resistance to 12 antimicrobacterial drugs .Results 416 strains of pathogenic bacteria were isolated from diarrhea children during 2011-2012 ,in which salmonella ,enteropathogenic E .coli ,Campylobacter jejuni and Candida albicans isolates accounted for 53 .61% ,37 .98% ,5 .29% and 1 .68% respectively .Drug resistance rate of the main strains to 12 antimicrobi‐al agents was 85 .25% to ampicillin ,54 .28% to compound sulfamethoxazole ,44 .70% to cefotaxime ,42 .53% to ceftriaxone , 40 .66% to chloramphenicol ,23 .55% to ceftazidime ,23 .36% to aztreonam ,14 .88% to ciprofloxacin ,8 .07% to cefepime ,7 .99% to cefperazone/sulbactam ,7 .42% to piperacillin/tazobactam respectively ,and no resistance to imipenem was detected .Conclusion The pathogenic bacteria causing diarrhea mainly includes salmonella ,pathogenic e .coli ,campylobacter jejuni in children from guang‐zhou region ,the top five sensitive antimicrobial reagents for the main strains includes imipenem ,piperacillin/tazobactam ,cefpera‐zone/sulbactam ,cefepime and ciprofloxacin .
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<p><b>OBJECTIVE</b>To evaluate the sensitivity and specificity of fully automated immunohistochemistry (IHC), with comparison to FISH, in the detection of EML4-ALK rearrangement in lung adenocarcinoma (ADC); and the use of IHC as a pre-screening tool.</p><p><b>METHODS</b>A total of 404 paraffin-embedded NSCLC samples from surgical resections were tested by IHC with Ventana anti-ALK rabbit monoclonal antibody (D5F3) and ultrasensitive detection kit. ALK rearrangement was further confirmed by FISH.</p><p><b>RESULTS</b>Twenty-nine of 404 lung ADCs (7.2%) were positive for ALK by IHC. ALK positive tumor cells demonstrated strong and diffused granular cytoplasmic staining. All the ALK IHC-positive cases were confirmed to harbor ALK rearrangement by FISH. None of the ALK IHC-negative cases was FISH-positive.</p><p><b>CONCLUSIONS</b>IHC can effectively detect ALK rearrangement in lung cancer. It may provide a reliable and cost-effective diagnostic approach in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy.</p>
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Adenocarcinoma , Genetics , Metabolism , Carcinoma, Non-Small-Cell Lung , Genetics , Metabolism , Gene Rearrangement , Immunohistochemistry , In Situ Hybridization, Fluorescence , Lung Neoplasms , Genetics , Metabolism , Oncogene Proteins, Fusion , Genetics , Metabolism , Sensitivity and SpecificityABSTRACT
Objective To understand and analysis of the pathogens distribution and drug resistance of nosocomial infection in children′s hospital,so as to provide reliable scientific basis for the prevention and control of hospital infection.Methods 396 cases of upper respiratory tract specimens were collected from pediatric patients with nosocomial infection.These specimens were detected by sputum specimens conventional methods of microorganism cultivation,and K-B method was used to determine the bacteria sensi-tivities to clinical common drug.Results There were 225 cases of specimens were pathogen positive among all the 396 specimens, and 234 strains of bacteria were isolated in all.The positive isolated rate was 56.8%(225/396).Among the 234 isolated strains, Gram negative bacteria accounted for 72.6%(170/234),and Klebsiella occupied the first place[49.4%(84/170)].Gram positive bacteria accounted for 23.5%(55/234),and Staphylococcus had the highest isolated rate in Gram positive bacteria[58.2% (32/55)].In all the 9 kinds of clinical common antimicrobial agents,imipenem had high drug sensitivity to the 234 isolated strains,and the aminoglycosides came next.Conclusion It is necessary for the pediatric patients with nosocomial infection to collect upper re-spiratory tract specimens for bacteriologic studies and drug sensitivity tests.
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Objective To assess the role of promyelocytic leukemia protein (PML)and P53 in the progression of esophageal squamous cell carcinoma(ESCC)and its precursor lesions. Methods Different expression patterns of PML and P53 of 241 cases of ESCC combined with adjacent precursors were analyzed by tissue array and immunohistochemistry and correlated with clinicopathological parameters. Results In ESCC and its precursor lesions, PML and P53 displayed positive or strong positive, while in normal esophageal epithelia, these proteins showednegative or stained positive only in parabasal cell layer. The expression level of PML was correlated with the depth of invasion of esophageal carcinomas (X2=29.461,P<0.001),lymph metastasis status(X2=15.226,P<0.001)and pTNMs(x2=26.956,P
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<p><b>OBJECTIVE</b>To study the clinical, pathological and immunophenotypic characteristics of the primary breast lymphoma (PBL).</p><p><b>METHODS</b>Analyses of clinical history, preoperative findings, histological and immunohistochemical features of eight patients with PBL were performed.</p><p><b>RESULTS</b>Malignant lymphoma was difficult to diagnose preoperatively. All patients were women. The age range was from 34 approximately 65 years (mean 46.4 years). The right breast was involved initially in three patients, the left in four. One patient presented bilateral involvement. Seven patients were assessed at stage IE, one with ipsolateral axillary lymph nodes involvement at stage IIE. According to the WHO classification, five patients were diagnosed as diffuse large B-cell lymphoma (4/5 centroblast, 1/5 immunoblast); the other three patients as MALT lymphoma, all with lymphoepithelial lesions. The paraffin-embedded tissues of all cases showed immunoreactivity for B-cell markers CD20, CD45RA. CD5 and CD10 were negative in all cases. Follow-up data were obtained in six patients, none recurred or died within 8 to 108 months after diagnosis.</p><p><b>CONCLUSIONS</b>This study indicates that most PBL are diffuse large B-cell lymphoma and MALT lymphoma and have a better prognosis after comprehensive therapy.</p>