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Objective@#To evaluate diagnostic performance of Todd-Hewitt (T-H) broth culture method, direct culture method, liquid chromogenic culture method, and loop-mediated isothermal amplification (LAMP) method for screening group B streptococcus (GBS) during late pregnancy.@*Methods@#In the retrospective study, the rectal vaginal secretions samples were collected from pregnant women at 35 to 37 weeks at the obstetrics clinic of Guangzhou Women and Children′s Medical Center affiliated to Guangzhou Medical University during October 2016 to April 2018. For the purposes of clinical evaluation, T-H broth culture was used as the standard reference method, and double-blind trials were used to evaluate diagnostic performance of direct culture method, liquid chromogenic culture method, and LAMP method for screening group B streptococcus during late pregnancy in three research stages. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), coincidence rate and Yoden index for each method were calculated. Also, the level of agreement between each method and T-H broth was assessed using the kappa (k) coefficient.@*Results@#A total of 969 specimens were detected by the T-H enrichment culture method, and 90 were positive (9.3%). The sensitivities from high to low were LAMP method [100% (25/25)], direct culture method [81.5% (22/27), 95%CI:65.8%-97.1%], and liquid color culture method [71.1% (27/38), 95%CI:55.9%-86.2%]. Specificities were direct culture method [100% (282/282)], liquid color culture method [98.1% (455/464), 95%CI:96.8%-99.3%], and LAMP method [94.0% (125/133), 95%CI: 89.9%-98.1%]. The coincidence rates were direct culture method [98.4% (22+282)/309], liquid color culture method [96.0% (27+455)/502], and LAMP method [94.9% (25+125)/158]. The Kappa values of the direct culture method (0.889), LAMP method (0.832) and the enrichment culture method were all ≥0.75, and that of the liquid color culture method was 0.708. The false negative rate of direct culture method was 18.5% (5/27), and no false negative case by LAMP method, but its false positive rate was 6.0% (8/133). The false negative rate and false positive rate of liquid color culture method were 28.9% (11/38) and 1.9% (9/464), respectively.@*Conclusions@#Of the three screening methods compared in this study, only the LAMP method has the advantages in sensitivity, specificity, and coincidence rate compared with T-H enriched culture method, while the others have a certain degree of false negatives rate. The clinical laboratory can introduce these methods based on laboratory facilities and staffing, or refer to the European and American guidelines and combine the recommended antenatal GBS screening method with intrapartum nucleic acid amplification tests to best meet the clinical demands.
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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 evaluate diagnostic performance of Todd-Hewitt (T-H) broth culture method, direct culture method, liquid chromogenic culture method, and loop-mediated isothermal amplification (LAMP) method for screening group B streptococcus (GBS) during late pregnancy.Methods:In the retrospective study, the rectal vaginal secretions samples were collected from pregnant women at 35 to 37 weeks at the obstetrics clinic of Guangzhou Women and Children′s Medical Center affiliated to Guangzhou Medical University during October 2016 to April 2018. For the purposes of clinical evaluation, T-H broth culture was used as the standard reference method, and double-blind trials were used to evaluate diagnostic performance of direct culture method, liquid chromogenic culture method, and LAMP method for screening group B streptococcus during late pregnancy in three research stages. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), coincidence rate and Yoden index for each method were calculated. Also, the level of agreement between each method and T-H broth was assessed using the kappa (k) coefficient.Results:A total of 969 specimens were detected by the T-H enrichment culture method, and 90 were positive (9.3%). The sensitivities from high to low were LAMP method [100% (25/25)], direct culture method [81.5% (22/27), 95 %CI:65.8%-97.1%], and liquid color culture method [71.1% (27/38), 95 %CI:55.9%-86.2%]. Specificities were direct culture method [100% (282/282)], liquid color culture method [98.1% (455/464), 95 %CI:96.8%-99.3%], and LAMP method [94.0% (125/133), 95 %CI: 89.9%-98.1%]. The coincidence rates were direct culture method [98.4% (22+282)/309], liquid color culture method [96.0% (27+455)/502], and LAMP method [94.9% (25+125)/158]. The Kappa values of the direct culture method (0.889), LAMP method (0.832) and the enrichment culture method were all ≥0.75, and that of the liquid color culture method was 0.708. The false negative rate of direct culture method was 18.5% (5/27), and no false negative case by LAMP method, but its false positive rate was 6.0% (8/133). The false negative rate and false positive rate of liquid color culture method were 28.9% (11/38) and 1.9% (9/464), respectively. Conclusions:Of the three screening methods compared in this study, only the LAMP method has the advantages in sensitivity, specificity, and coincidence rate compared with T-H enriched culture method, while the others have a certain degree of false negatives rate. The clinical laboratory can introduce these methods based on laboratory facilities and staffing, or refer to the European and American guidelines and combine the recommended antenatal GBS screening method with intrapartum nucleic acid amplification tests to best meet the clinical demands.
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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 study genotyping and molecular epidemiology distribution of GBS pathogenic strains of GBS positive pregnant women in Guangzhou,for GBS pathogenic strains of rapid molecular diagnosis and epidemiological surveillance pro-vide certain theoretical basis and method.Methods In the Guangzhou area,used multi stage stratified sampling method col-lecting GBS positive pregnant women’s reproductive tract specimens from January to December 2015,drug sensitivity quality control standard strains:Streptococcus pneumoniae (ATCC49619)and Staphylococcus aureus (ATCC25923),took culture of bacterial,strain,identification,DNA extraction,PCR,gene detection method,through the relevant software for data analy-sis,analyzed GBS strains of gene and molecular epidemiology.Results In the study,collected 2 812 samples of secretions,af-ter identification of strains isolated from 178 strains of pathogenic GBS strains,the detection rate was 6.33%.GBS patho-genic strains to linezolid vancomycin,penicillin,nitrfurantion and other antimicrobial drug resistance rate was 0,GBS parho-genic strains to ampicillin,ciprfloxacin moxifloxacin and levofloxacintesistant parts,the restance rates were 1.1%,16.9%, 18.0% and 22.5%,but GBS pathogenic strains to erythromycin,clindamycin tetracydine antibiotics showed a high resistance rate,the resistance rates were 50.6%,47.8%(of which 20 cases of erythromycin induced clindamycin resistance accouted for 23.5%)and 73.0%.Among them,65 strains of GBS detected the mreA gene,56 strains of GBS detected the ermB gene,36 strains of GBS detected the mefA gene,28 strains of GBS detected the mefE gene,5 strains of GBS detected the ermA gene, ermC gene was not detected in the gene.Among them,carried five multidrug resistance gene of 3 strains (1.6 9%)and 4 kinds of resistant gene carried with 15 strains (8.43%),carried three resistance genes of 19 strains (10.67%),2 kinds of resistant gene carrying a 25 strains (14.04%),carried the resistance gene of 5 strains (2.81%),did not carry resistance gene of 1 strain (0.56%).The nucleotide sequences of the five drug resistance genes were 100%,and no gene mutation oc-curred.Conclusion The main GBS disease resistant gene was mreA,ermA,ermB,mrfA,mefE and its nucleotide sequence homology was 100%.The clinical need to strengthen the detection of resistant gene and molecular level and guide clinical more scientific and rational drug use.
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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 .