Rapid identification of ST17 group B streptococcus using MALDI-TOF MS / 中华检验医学杂志

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.

Comparison of four screening methods for group B streptococcus / 中华检验医学杂志

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.

Comparison of four screening methods for group B streptococcus / 中华检验医学杂志

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.

Clinical analysis of 2 siblings with late-onset meningitis caused by group B streptococcus which were homogenous to the colonization bacteria of their mother / 中华实用儿科临床杂志

Objective To raise awareness of the late-onset meningitis caused by group B streptococcus (GBS) which was homogenous to the maternal colonization.Methods The clinical data of late-onset GBS meningitis in neonates twins whose pathogens were homogenous to their maternal colonization were collected from Department of Neonatology,Guangzhou Women and Children's Medical Center.The general conditions,clinical symptoms,laboratory tests and drug treatment of the twins and their mother were retrospectively analyzed,and the GBS homology during inpatient care was tested.And the progress of the twins' condition was investigated by telephone follow-up.Results The mother had two pregnancies without prenatal GBS screening or intrapartum antimicrobial intervention for GBS,everything was normal during pregnancy and delivery.Twins were born through cesarean section.The elder sister was discharged with Linezolid taken orally after 167 days in hospital without convulsions,shaking or other discomfort.The elder sister was followed up for every 2 weeks,and in the last time of follow-up,cerebrospinal fluid white blood cell counts were 45 × 106/L,protein level was 1.52 g/L and Linezolid was withdrawn.The younger brother was discharged after 58 days in hospital with follow-up for every 2 weeks,and in the last time of follow-up,cerebrospinal fluid white blood cell counts were 30 × 106/L,protein level was 0.66 g/L.During the hospitalization and follow-up without convulsions and irritation,and the cranial magnetic resonance imaging of the twin brother was normal.Test results showed that the GBS bacteria strain for twins and their mother were all serotype Ⅲ.The possibility of the GBS homology was more than 90％.Conclusions The toxicity of serotype Ⅲ GBS strain was strong.More proactive precautions should be considered to apply for the mother whose first birth already had GBS infection.Early identification and intervention of infection risk factors would help optimize the anti-infection treatment program and reduce nerve system damage and other adverse outcomes caused by invasive GBS infection.

Development and evaluation of loop-mediated isothermal amplification assay for the rapid detection of Escherichia coli and its microbial toxin / 中国医师杂志

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.

Clinical analysis and serotypes distribution of neonatal purulent meningitis caused by group B streptococcus / 实用医学杂志

Objective To investigate the clinical characteristics,antibiotic susceptibilities and serotypes of Group B Streptococcus(GBS)isolated from neonatal meningitis to provide references for the prevention and treatment of neonatal GBS meningitis. Methods From June 2013 to June 2016,we surveyed the GBS strains iso-lated from purulent meningitis of < 90 days infants from Guangzhou Women and Children′s Medical Center. The GBS isolates were identified and the minimum inhibitory concentration of the antibiotics was determined by Vitek 2 Compact automatic bacterial identification system.GBS serotyping was performed using Strep-B-Latex?rapid latex agglutination test kit. Results A total of 46 cases of neonatal GBS meningitis,15 cases of early-onset infection and 31 cases of late-onset infection were diagnosed. 78.3% of GBS meningitis with varying degrees of complica-tions.Among 41 survivors with 3~24 months follow-up,50% of the early-onset and 44.8% of the late-onset GBS meningitis with varying degrees of neurological sequelae.Four capsular types were identified among the 46 isolates, serotype-Ⅲwas the most prevalent(73.9%),followed by Ib(19.6%),V(4.3%)and Ia(2.2%).All the isolates were susceptible to penicillins,cephalosporins,linezolid and vancomycin. Conclusion The highly pathogenic serotype-Ⅲ was the predominant serotype among neonatal GBS meningitis in Guangzhou,Therefore,it is neces-sary to strengthen the epidemiological surveillance of GBS invasive infection and the effective implementation of pre-ventive measures.

Interpretation of the guideline or consensus on screening strategy and detection methods for prevention of perinatal group B streptococcal infection in European and American countries / 中华检验医学杂志

Group B streptococcus ( GBS) is the most common cause of neonatal invasive infections in western countries , leading to severe pneumonia , sepsis and meningitis with high mortality and morbidity . Neonatal GBS infections are mainly transmitted from mother to baby .Since the end of 1990 s, prevention strategies of antenatal GBS screening and intrapartum antibiotic prophylaxis ( IAP) have been implemented by European and American area , decreasing the incidence of neonatal GBS disease significantly .The harm of GBS to neonates has been recognized and valued in recent years in China , but the authorized prevention measures are still lacking.Efficacy of IAP-based prevention depends on the accuracy of GBS screening results during pregnancy .Here, the GBS prevention guideline or consensus recommended by European and American CDC, including the screening strategies , detection methods, sample collection, storage and transportation were interpreted .

Penicillin-binding proteins genotyping of penicillin resistance Streptococcus pneumonia isolated from children in Guangzhou area / 国际检验医学杂志

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%.

Clone and expression of APH(3′′)-Ⅰand AAC(2′)-Ⅰ gene of Stenotrophomonas maltophilia / 国际检验医学杂志

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 .

The distribution and drug resistance of respiratory tract infection pathogens isolated from patients of pediatric CICU / 国际检验医学杂志

Objective To analyze the common respiratory tract infection pathogens distribution and their drug resistance in pedi-atric cardiac intensive care unit(CICU),so as to provide reference for clinical rational use of antibiotics.Methods 1 350 cases of sputum specimens from lower respiratory tract infection patients of pediatric CICU in the medical center between January 2011 and December 2012 were cultivated and drug susceptibilities were tested.The results were retrospectively analyzed.Results 490 patho-genic strains were isolated from 1 350 cases of sputum specimens and identified,including Gram negative bacilli 288 strains (58.78%),Gram positive coccus 140 strains(28.57%),fungi 62 strains(12.65%,mainly Candida albicans ).Gram negative bacilli was given priority to with Klebsiella pneumoniae (62 strains,12.65%),followed by Branhamella catarhalis ,Pseudomonas aerugi-nosa and Escherichia coli .The rates of extended-spectrum beta-lactamases(ESBLs)-producing strains among Escherichia coli and Klebsiella pneumoniae were 73.33% and 66.13%,respectively.Gram positive coccus was given priority to with Staphylococcus aureus (65 strains,13.27%),followed by Streptococcus pneumoniae .Methicillin-resistant Staphylococcus aureus (MRSA)accounted for 24.62%.Conclusion Staphylococcus aureus ,Streptococcus pneumoniae and Klebsiella pneumoniae are main pathogens of re-spiratory tract infection in pediatric CICU.And there is multiple drug-resistant bacteria infection.Rational applicattion of antibiot-ics according to the test results of isolation and drug susceptibility is an effective way to control the infection of critical children and reduce the emergence of resistant strains.

Antimicrobial susceptibility surveillance in China: a MOHNARIN program report (2009-2010) / 中华检验医学杂志

Objective To investigate the bacterial resistance in nationwide and understand the distribution of bacterial and resistance trend.MethodsThe 6507 clinical isolates were collected from 19 hospitals in 17 cities.The susceptibility tests were performed using agar dilution method recommended by Clinical and Laboratory Standards Institute (CLSI) in central laboratory.The values of MIC50,MIC90 and MICrange were calculated by SPSS 17.0 and the susceptibilities of isolates to antimicrobial agents were determined by using CLSI (2010) guideline.Of all 6507 isolates,4691 strains were collected from target wards and 1816 were isolated from others wards.ResultsAmong 4691 strains,1156 were Gram-positive (24.6％ ) and 3535 were Gram-negative (75.4％).Based on the minimum inhibitory concentration results,the prevalence of methicillin resistant Stapylococcus aureus and methicillin resistant Stapylococcus epidermidis are 51.6％ ( 325/630 ) and 87.0％ ( 228/262 ) respectively.Staphylococci showing intermediate or full resistance to vancomycin were not observed. Coagulase negative Staphylococci showed 2.5％ (16/642)intermediate rate and 1.6％ ( 10/642 ) full resistance rate to teicoplanin,and showed 0.5％ ( 3/642 )resistance rate to linezolid.Antibiotic resistance rate of Enterococcus faecalis to ampicillin was 17.1％(19/111),while the resistance rate of Enterococcus faecium to ampicillin reached up to 85.0％(164/193).Three Enterococcus faecium were resistant to glycopeptides.The prevalence of penicillin resistance Streptococcus pneumoniae and penicillin intermediate Streptococcus pneumoniae were 41.2％ ( 145/352) and 37.2％ (131/352) respectively based on oral penicillin criterion,while the prevalence were 0.0％ (0/352) and 6.0％(21/352) based on vein to non-meningitis criterion.A vast majority of Enterobacteriaceae maintained high susceptibility to carbapenems,with resistance rate less than 2.0％.In addition,tigecycline,moxalactam,fosfomycin and amikacin displayed desirable antibacterial activity against Enterbacteriaceae,and resistance rates to these drugs were all less than 10.0％.For non-fermenting Gramnegative isolates,resistance rate of Pseudomonas aeruginosa and Acinetobacter baumannii to imipenem were 23.1％ ( 139/601 ) and 53.5％ (419/784) respectively.Resistance rate of Acinetobacter baumannii was much higher than that during the period 2007 - 2008.Colistin,tigecycline,minocycline and fosfomycin demonstrated good antibacterial activity against Acinetobacter baumannii in vitro.Conclusions Compared with MOHNARIN 2007 -2008year surveillance results, significant increase in resistance rate of Acinetobacter baumannii was demonstrated.Resistant strains to linezolid and tigecycline were found.Bacterial resistance has been a widespread problem in our country,which requires much more attention.

Pathogenic bacteria distribution and drug susceptibility in children with acute otitis media in Pearl River Delta / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the pathogenic bacteria distribution and drug susceptibility in children with acute otitis media (AOM) in different age and different season in the Pearl River Delta region.@*METHOD@#Four hundred and forty-two children diagnosed as AOM were divided into three groups by age factor and four groups by season factor. Midge ear pus collecting and culturing were used for bacteria and antimicrobial susceptibility test.@*RESULT@#(1) Strains of bacteria were isolated from 356 children with the positive rate of 80.5%. Streptococcus pneumoniae (39.2%), staphylococcus aureus (25.9%) and haemophilus influenzae (7.4%) were the most frequently isolated pathogens. (2) Streptococcus pneumoniae was the main pathogenic bacteria in 0-1 year group and > 1-3 years group (P 3 years group (P 0.05). Staphylococcus aureus was the main pathogen in January-March group (P < 0.05); (4) Drug sensitivity shown that linezolid and ofloxacin were most sensitive to streptococcus pneumoniae and staphylococcus aureus, and macrolides had a good therapy effect to haemophilus influenzae.@*CONCLUSION@#The pathogens distribution and drug susceptibility in children with AOM were varies in different age and different season. As a result, a treatment should be done based on the climate, environment, and pathogens distribution of a region.

Prevalence of antimicrobial resistance of Streptococcus pneumoniae in Chinese children: four hospitals surveillance / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the nasal carriage of antibiotic-resistant pneumococci in children of < 5 years old in the following four cities, Beijing, Shanghai, Guangzhou and Xi'an.</p><p><b>METHODS</b>A total of 647 pneumococci strains were isolated and detected. Minimal inhibition concentrations (MICs) of antibiotics were determined by E-test. Disk diffusion test was used for the measurement of antimicrobial susceptibility.</p><p><b>RESULTS</b>Prevalence of penicillin non-susceptible Streptococcus pneumoniae in the four cities was 41%, with Guangzhou (60.8%) ranking first, followed by Xi'an (45%), Shanghai (37%) and Beijing (25.9%). The majority of penicillin non-susceptibility isolates (23.9% - 53.8%) had a low level of resistance (MIC 0.64 - 1.5 microg/ml). The most sensitive antimicrobials in terms of percentage of susceptible organisms were amoxicillin-clavulanic acid (99.4%), followed by ceftriaxone (92.1%); cefurxime and cefaclor were slightly more sensitive than penicillin with susceptibility of 74.8% and 77.9%. Erythromycin, tetracycline and TMP-SMZ were highly resistant (83.6%, 82.1% and 76.2% respectively). Among erythromycin resistant isolates, 100% were resistant to azithromycin, 98.6% to clarithromycin, 97.2% to roxithromycin and spiramycin, and 96.6% to clindamycin. 97.2% (141/145) were typical of the macrolides-lincosamides-streptogramons B (MLSB) resistance phenotype, and 2.8% (4/145) were M phenotype. The group of PRSP was with significantly higher rates of non-susceptibility for ceftriaxone (18.4%), cefurxime (58.6%), cefaclor (53.4%), compared with the group of PEN-S (0.5%, 1.8% and 0.2%, respectively) and the rate of multi-drug resistance in the isolates of PRSP group (92.9%) was significantly higher than that of PEN-S group (59.2%).</p><p><b>CONCLUSION</b>The rates of penicillin and multi-drug resistance among isolates of pneumococci carried nasally in are high children and the high prevalence of multi-drug resistance in the Chinese population may be becoming one of the most serious problems in this century.</p>