Table of Content

30 September 2016, Volume 31 Issue 9

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Orginal Article

Drug resistance of invasive fungi and its mechanisms

ZHAO Hu

2016, 31(9):  735-738.  DOI: 10.3969/j.issn.1673-8640.2016.09.001

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A total of 4 original articles and 1 review about the drug resistance of invasive fungi and its mechanisms are collected for this special topic. The original articles and review describe the drug resistance and mechanisms of Candida albicans,Aspergillus fumigatus and Cryptococcus neoformans. While antifungal agents are widely used in recent years,the drug resistance of fungi increases gradually. It is useful to study the drug resistance mechanism of fungi and the new targets for antifungal agents. It can provide a reference for the research and development of new antifungal agents and the treatment with antifungal agents.

Drug resistance genes of Candida albicans to azoles and their regulation mechanisms

WU Yongqin, YING Chunmei

2016, 31(9):  739-743.  DOI: 10.3969/j.issn.1673-8640.2016.09.002

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Candida albicans is a conditionally pathogenic fungus in human. With azoles being widely applied for a long time,the clinical isolates of Candida albicans against azoles gradually increase. The clinical treatment of Candida albicans has become a thorny issue. The drug resistance genes of Candida albicans and regulation mechanisms are reviewed for finding new therapeutic targets and developing new antifungal agents at the level of transcriptional regulation.

Correlation between C1409A missense mutation in MRR2 gene and fluconazole resistance of Candida albicans

WANG Ying, ZHANG Rong, LIU Jinyan, SHI Ce, LI Wenjing, ZHAO Yue, XIANG Mingjie

2016, 31(9):  744-749.  DOI: 10.3969/j.issn.1673-8640.2016.09.003

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Objective To study the correlation between C1409A missense mutation in MRR2 gene [the gene encoding zinc cluster transcription factor-multidrug resistance regulator 2（Mrr2）] and fluconazole resistance of Candida albicans. Methods The isolates of Candida albicans containing C1409A missense mutation in MRR2 gene were generated by one-step cloning technology and site-directed mutagenesis based on MRR2Δ/Δ mutant,which was recombined from Candida albicans engineering isolate SN152. The mutated MRR2 gene was expressed at ADE2 locus using classical lithium acetate transfection method. Fluconazole susceptibility test and real-time fluorescence quantitation polymerase chain reaction （PCR） were performed to analyze the correlation between C1409A missense mutation in MRR2 gene and fluconazole resistance. Results The recombinant isolates contributed to an almost 3-time increase for CDR1 expression and a 4-time increase for minimal inhibitory concentration（MIC） in fluconazole resistance compared with SN152. Conclusions The C1409A missense mutation in MRR2 gene contributes to fluconazole resistance of Candida albicans with upregulating CDR1 expression.

Candida spectrum and characteristics of antifungal susceptibility in vulvovaginal candidiasis

WANG Dongjiang, GUO Jian, ZHOU Aiping, WEN Donghua, WU Wenjuan

2016, 31(9):  750-754.  DOI: 10.3969/j.issn.1673-8640.2016.09.004

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Objective To investigate the age,Candida distribution and in vitro antifungal susceptibility in patients with vulvovaginal candidiasis（VVC）in Jiading district,and to provide a reference for the diagnosis and treatment of VVC. Methods The vaginal secretions from patients with VVC in the Maternal and Children Healthcare Hospital of Jiading District from July 2015 to October 2015 were collected,and Candida were cultured and identified by CHROMagar Candida color medium and ATB microbiological identification system. All isolates were confirmed by sequencing,using internal transcribed spacer（ITS）1 and ITS4. Drug susceptibility test was performed by Sensititre Yeast One,and minimal inhibitory concentration（MIC） was determined according to the Clinical and Laboratory Standards Institute（CLSI）2012 M27-S4. Results A total of 829 isolates of Candida were isolated,and 82 isolates were collected from recurrent vulvovaginal candidiasis（RVVC） group. Adult women（20-59 years old） accounted for 97.1%. Candida albicans accounted for 77.1%（639/829）,Candida glabrata accounted for 15.1%（125/829）,Candida parapsilosis accounted for 3.4%（28/829）,Candida tropicalis accounted for 2.1%（17/829）,Candida krusei accounted for 1.6%（13/829）,and Saccharomyces cerevisiae accounted for 0.8%（7/829）. The susceptibility rates of Candida albicans in VVC group to fluconazole,itraconazole,voriconazole,flucytosine,caspofungin,anidulafungin and micafungin were 66.1%,81.4%,44.1%,96.6%,100.0%,100.0% and 100.0%,and those in RVVC group were 41.5%,53.7%,26.8%,95.1%,100.0%,100.0% and 100.0%,respectively. Conclusions Candida albicans is the main pathogenic fungus for VVC,and the drug resistance rate to azoles is high. Therefore,antifungal agents should be chosen according to the results of drug susceptibility test to improve the precision of treatment and reduce the incidence of drug resistant isolates.

Mechanism of azole resistance in Aspergillus fumigatus with TR34/L98H mutations

WANG Qian, CHEN Wei, WAN Zhe, LI Ruoyu, LIU Wei

2016, 31(9):  755-760.  DOI: 10.3969/j.issn.1673-8640.2016.09.005

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Objective To investigate the mechanism of azole resistance in Aspergillus fumigatus. Methods An isolate of Aspergillus fumigatus was collected from bronchoalveolar lavage fluid（BALF） of a patient with invasive aspergillosis（IA）. By microbroth dilution,the minimal inhibitory concentrations（MIC）/minimal effective concentration（MEC） of itraconazole,voriconazole,posaconazole and amphotericin B and caspofungin were determined according to the Clinical and Laboratory Standards Institute（CLSI） M38-A2. The full-length region of the gene encoding the target enzyme of azoles（cyp51A gene including its promoter） was cloned and sequenced. Microsatellite genotyping was performed by amplifying and determining the isolate's 9 microsatellite loci,and the genetic relationships among isolates with the same genotype were then established by cluster analysis using unweighted pair-group method with arithmetic means（UPGMA）. Results For this isolate,the MIC of itraconazole,voriconazole,posaconazole and amphotericin B were >16,2,0.5 and 2 μg/mL. The MEC of caspofungin was 0.25 μg/mL. The sequence analysis of cyp51A gene showed the presence of 2 copies （-288--322）of a 34 bp sequence in tandem in the promoter of cyp51A gene together with the presence of 3 point mutations at T364A,T960A and T1554A,which were accordingly deduced amino acid substitution including TR34/L98H/S297T/F495I. Microsatellite genotyping indicated that this isolate had a unique genotype being different to those reported previously in Europe and Asia. Conclusions The azole-resistant isolate of Aspergillus fumigatus from BALF harboring amino acid substitution TR34/L98H/S297T/F495I has been isolated with a unique genotype being different to isolates reported before.

The drug susceptibility of Cryptococcus neoformans in East China

ZHU Junhao, HAN Demin, LI Li, ZHANG Qiangqiang

2016, 31(9):  761-764.  DOI: 10.3969/j.issn.1673-8640.2016.09.006

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Objective To study the in vitro drug susceptibility of Cryptococcus neoformans isolated clinically from East China in recent years. Methods The minimal inhibitory concentrations （MIC） of Cryptococcus neoformans to fluconazole,flucytosine,amphotericin B,itraconazole and voriconazole were determined by micro-dilution broth method,according to the Clinical and Laboratory Standards Institute （CLSI）M27-A3. The drug-resistant and susceptible-dose dependent （SDD） isolates were further identified by internal transcribed spacer （ITS） sequencing. Results Among 96 isolates,the MIC₉₀（the range of MIC）were 4（0.5-16）μg/mL for fluconazole,4（0.25-64）μg/mL for flucytosine,0.5（0.03-1）μg/mL for amphotericin B,0.5（0.03-1）μg/mL for itraconazole and 0.25（0.03-0.5）μg/mL for voriconazole. All the drug-resistant and SDD isolates were determined as Cryptococcus neoformans var. grubii. Conclusions Cryptococcus neoformans isolated from East China are susceptible to antifungal agents except itraconazole. The main species of insensitive isolates is Cryptococcus neoformans var. grubii.

Clinical significance of serum vascular endothelial growth factor in lung cancer

WANG Kun, HE Naping, WU Qiong, XIANG Manlin, YI Bin

2016, 31(9):  765-769.  DOI: 10.3969/j.issn.1673-8640.2016.09.007

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Objective To investigate the significance of serum vascular endothelial growth factor（VEGF） in lung cancer. Methods The levels of serum VEGF were determined by enzyme-linked immunosorbent assay（ELISA） in 142 patients with lung cancer,30 patients with lung diseases and 25 healthy subjects. Meanwhile,their tumor stages and serum VEGF expression changes before and after chemotherapy and surgery were compared. The receiver operating characteristic（ROC） curve was used to evaluate the significance of serum VEGF in lung cancer. SPSS 13.0 software was used for statistical analysis. Results Serum VEGF level in lung cancer group was 154.09（86.81,260.01） pg/mL,which was higher than that in healthy control group [59.06（27.66,77.81）pg/mL,P<0.01]. Serum VEGF level had no statistical significance for different pathological types of lung cancer group（P=0.493）. Serum VEGF levels before and after chemotherapy had no statistical significance（P>0.05）. Serum VEGF levels with different M stages and tumor stages in non-small cell lung cancer had statistical significance（P<0.05）,and there was no statistical significance between localized and extensive stages（P>0.05）. The area under ROC curve of serum VEGF in the diagnosis of lung cancer was 0.664,and that for non-small cell lung cancer distant metastasis was 0.666. Conclusions The expression of serum VEGF is abnormal in lung cancer group,and it is related to the clinical stages of lung cancer. It is of significance in the diagnosis and metastasis of lung cancer.

Analysis on the results of human papilloma virus determination

XU Hong, WANG Delu, DU Wenjie

2016, 31(9):  770-773.  DOI: 10.3969/j.issn.1673-8640.2016.09.008

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Objective To investigate high-risk human papilloma virus（HPV） in screening of cervical diseases. Methods By polymerase chain reaction（PCR）,23 kinds of HPV subtypes were determined,and pathological analysis was performed by thinprep cytologic test（TCT）. Subjects were classified into 6 groups according to ages. The determination rates were calculated. Results A total of 1 144 specimens were collected. There were 698 cases with HPV infections,and the infection rate was 61.0%. The 23 subtypes had been determined. The subtype with the highest infection rate was high-risk type 52（16.5%）,followed by type 16（14.5%） and type 58（7.1%）. Single subtype infection rate was 36.5%,and multiple infection rate was 24.5%. The determination rates of ≤25,26-35,36-45,46-55 and >55 years old groups were 56.4%,61.8%,59.9%,63.3% and 57.4%,respectively. Conclusions HPV infections in Tongling are mainly single infection and high-risk infection,and the subtype with the highest infection rate is high-risk type 52. The infection rates with different ages are slightly different.

Coagulation function analysis in patients with gestational diabetes mellitus and gestational hypertension during the 3rd trimester pregnancy

HU Peng, CHEN Jingfei

2016, 31(9):  774-777.  DOI: 10.3969/j.issn.1673-8640.2016.09.009

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Objective To analyze plasma coagulation factors and D-dimer（DD） in patients with gestational diabetes mellitus（GDM） and gestational hypertension during the 3rd trimester pregnancy. Methods A total of 108 healthy pregnant women,104 patients with GDM and 52 patients with gestational hypertension were enrolled. Prothrombin time（PT）,activated partial thromboplastin time（APTT）,fibrinogen（FIB） and DD were determined. Results Compared with healthy pregnant women,FIB increased,and PT and APTT decreased in GDM group（P<0.05）. FIB increased,and PT decreased in gestational hypertension group compared with healthy pregnant women（P<0.05）. However,APTT had decreasing tendency without statistical significance（P>0.05）. When excluding 1 patient with severe coagulation disorder,the other 51 patients with gestational hypertension showed decreased APTT compared with healthy pregnant women（P<0.05）. Compared with healthy pregnant women,GDM and gestational hypertension groups showed no statistical significance for DD（P>0.05）. However,there were 3 cases of oligoamnios,fetal distress and venous thrombosis and 1 case of hemolysis,elevated liver enzymes and low platelet（HELLP） syndrome showing remarkable DD increasing. Conclusions Patients with GDM and gestational hypertension trend to be hypercoagulable,and high-level DD is probably associated with adverse pregnancy outcomes.

Retrospective analysis on chromosome karyotypes among 2 018 fetuses in the 2nd trimester pregnancy

DU Manxing, WANG Weijia, SU Nianhua, LU Jianqiang

2016, 31(9):  778-781.  DOI: 10.3969/j.issn.1673-8640.2016.09.010

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Objective To analyze retrospectively the results of amniotic fluid cell culture and chromosome karyotypes among 2 018 fetuses in the 2nd trimester pregnancy,and to investigate the relationships of abnormal chromosome karyotypes and proportions with the syndromes of prenatal diagnosis and the pregnancy outcomes for fetuses with abnormal mosaic karyotypes. Methods Amniotic fluid samples were extracted from pregnant women according to prenatal diagnosis syndromes,whose gestational ages were 16 to 24 weeks. The samples were determined for amniotic fluid cell culture and chromosome karyotypes. Results A total of 61 cases of abnormal chromosome karyotypes were determined,and the abnormal rate was 3.0%（61/2 018）. The abnormal rates in groups with high risk results of serological screening,high pregnant ages（≥35 years old）,abnormal ultrasound determination results and adverse pregnancy or family history were 2.8%,2.3%,9.2% and 10.6%,respectively. The abnormal rates between the groups with high risk results of serological screening and high pregnant ages showed no statistical significance（P>0.05）. A total of 43 cases of numerical abnormality of chromosome karyotypes were determined,including 23 cases of trisomy 21,9 cases of trisomy 18 and 1 case of trisomy 13 and 10 cases of sex chromosome. There were 12 cases of structural abnormality of chromosome karyotypes,including 7 cases of balanced translocation,2 cases of Robertsonian translocation,1 case of insertion,1 case of deletion and 1 case of inversion. Furthermore,6 cases of mosaic karyotypes were determined,and 2 pregnant women of the 6 cases with low proportion gave birth to their babies,but they showed no abnormal phenotypes after follow-up. Conclusions There are high abnormal rates under different syndromes of prenatal diagnosis. Fetuses with low proportion of mosaic karyotypes may have normal phenotypes. Amniotic fluid chromosome karyotype analysis can provide a reliable reference for the diagnosis of fetal chromosome abnormalities.

Gene mutation types of α-thalassemia and β-thalassemia in Quanzhou

ZHUANG Qianmei, WANG Yuanbai, ZHUANG Jianlong, JIANG Yuying

2016, 31(9):  782-786.  DOI: 10.3969/j.issn.1673-8640.2016.09.011

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Objective To analyze the types and frequencies of gene mutations for α-thalassemia and β-thalassemia in Quanzhou,and to provide a reference for screening and diagnosing thalassemia in Quanzhou. Methods Reverse dot blot（RDB）-polymerase chain reaction （PCR） was used to determine gene mutations of α-thalassemia and β-thalassemia,while gap （Gap）-PCR was used to determine gene deletion of α-thalassemia. Specimens with suspected rare gene mutations were determined for gene sequencing.Results Among 1 121 specimens,195 cases were determined with β-thalassemia,with a positive rate of 17.40%. IVS-2-654（C→T） heterozygous mutation and CD41-42（-TCTT） heterozygous mutation were the most common gene mutations,accounting for 67.18% （131/195）. A total of 325 cases were determined with α-thalassemia,with a positive rate of 28.99%,and --SEA/αα was the most common gene mutation,accounting for 77.23% （251/325）. There was 1 case with rare Thailand type of deletion heterozygous α-thalassemia （-- Thai /αα）,1 case with α-anti4.2 and 1 case with HbG-Chinese in complex with Hb Westmead mutation. Conclusions There is a high carrier rate of thalassemia in Quanzhou,and the gene mutation types are complex. Therefore,the screening and prenatal diagnosis of thalassemia are of significance.

Selection and optimization of STR for Shanghai Han population

ZHAO Huijia, GU Zhidong, CHENG Weiwei, TAO Jiong, GAO Jiaqi, HAN Xu

2016, 31(9):  787-791.  DOI: 10.3969/j.issn.1673-8640.2016.09.012

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Objective To select common chromosome short tandem repeat（STR） for Shanghai Han population,and to optimize the site amplification conditions into a determination system. Quantitation fluorescence polymerase chain reaction（QF-PCR） will be used for the rapid prenatal diagnosis of fetal common chromosome aneuploidies. Methods Through the polymorphism analysis of 48 healthy Han population in Shanghai,STR heterozygosities on 13,18,21,X and Y chromosomes were evaluated. STR with high polymorphism was selected and optimized into a determination system,and the sensitivity and specificity were evaluated in the determination system by 36 samples [22 cases of amniotic fluid,9 cases of cord blood and 5 cases of chorionic villus samples（CVS）]. Chromosome karyotype analysis was performed simultaneously,and the effectiveness of the established method was evaluated. Results A total of 14 STR were selected in STR heterozygosity test,and they were D13S258（0.88）,D13S305（0.90）,D13S634（0.92）,D13S742（0.83）,D18S535（0.81）,D18S1002（0.69）,D18S386（0.75）,D18S391（0.77）,D21S1435（0.75）,D21S1412（0.94）,D21S1446（0.58）,D21S1414（0.85）,DXS7132（0.77） and DXY218（0.71）,together with sex chromosome of qualitative and quantitative specific STR of AMXY,SRY and TAF9B into optimizing an amplification system. The system was used to determine 36 samples of amniotic fluid,cord blood and CVS. There were 35 cases with abnormal chromosome numbers,including 15 cases of trisomy 21（Down's）,12 cases of trisomy 18（Edward）,4 cases of trisomy 13（Patau）,2 cases of 45,XO（Turner）,1 case of 47,XXY（Klinefelter） and 1 case of 47,XYY（Super-Man）. The remaining 1 case was 46,XX（healthy female）. The results of QF-PCR were as same as the results of chromosome karyotype analysis. Conclusions Selected 14 STR show high polymorphism in Shanghai Han population,and sex chromosome qualitative and quantitative specific STR constitute a QF-PCR determination system,which can accurately determine 13,18,21,X and Y chromosome aneuploidies. QF-PCR is a rapid,accurate,economic and efficient method for the rapid prenatal diagnosis of fetal common chromosome aneuploidies in Shanghai Han population.

Correlations between serum 25-hydroxyvitamin D and 4 blood lipid items among elderly males in Shanghai

YANG Jing, SONG Binbin, WANG Beili, WU Jiong, ZHANG Chunyan, GUO Wei, PAN Baishen

2016, 31(9):  792-796.  DOI: 10.3969/j.issn.1673-8640.2016.09.013

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Objective To investigate the relationships of serum 25-hydroxyvitamin D [25（OH）D] with triglyceride （TG）,total cholesterol（TC）,high-density lipoprotein cholesterol （HDL-C） and low-density lipoprotein cholesterol （LDL-C） among elderly males in Shanghai. Methods A total of 686 elderly males participating healthy examination were enrolled. Their ages,heights and weights were recorded. Fasting venous blood samples were collected,and the 4 blood lipid levels and 25（OH）D were determined. According to the levels of 25（OH）D ,the classification was performed,G1,G2,G3 and G4 groups. The factors for blood lipid levels between G1 and G4 groups were compared. The correlations of 25（OH）D with blood lipid levels were analyzed regressively. Results Body mass index （BMI） in G1 group was lower than that in G4 group（P=0.001）,and HDL-C in G1 group was higher than that in G4 group（P<0.001）. There was no statistical significance for ages,LDL-C,TC and TG between G1 and G4 groups（P>0.05）. HDL-C was correlated positively with 25（OH）D （R²=0.161 8,P<0.001）. After the adjustment for ages and BMI,HDL-C increased by 0.01 mmol/L with serum 25（OH）D increasing by10 nmol/L. Conclusions HDL-C in elderly males may be affected by 25（OH）D.

Performance verification of Addcare Elisa800 automatic enzyme immunoassay workstation and its clinical application

FU Jie, SU Peng, JIANG Xingyu, PU Xiaoyun

2016, 31(9):  797-800.  DOI: 10.3969/j.issn.1673-8640.2016.09.014

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Objective To verify the performance of Addcare Elisa800 automatic enzyme immunoassay workstation,and to evaluate its clinical application. Methods Electronic balance was used to determine the sample capacity and residue capacity of washing machine,thermometer was used to determine temperature of each incubation model,standard filter was used to determine the performance of enzyme-mark analyzer,carry-over rate was calculated,and whether the performance met technical requirements or not was evaluated,which was compared with that of Tecan Freedom Evolyzer automatic enzyme immunoassay system. The significance of Addcare Elisa800 automatic enzyme immunoassay workstation was verified. Results Sample capacities,residue capacities of washing machines,the temperatures of incubation models,the performance of enzyme-mark analyzers and carry-over rates were all in line with technical requirements. Compared with Tecan Freedom Evolyzer automatic enzyme immunoassay system,the coincidence rate was 100%,without statistical significance（P>0.05）. Conclusions Addcare Elisa800 automatic enzyme immunoassay workstation can provide high quality of determination results,and it can provide scientific reference for clinical diagnosis and treatment.

The determination of D-dimer by dual particle size latex immunoturbidimetry

FANG Qing

2016, 31(9):  801-806.  DOI: 10.3969/j.issn.1673-8640.2016.09.015

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Objective To improve the analytical sensitivity and linear range of latex immunological reagent,and to compare the sensitivities and linear ranges of dual and single particle size latex immunoturbidimetry in the determination of D-dimer（DD）. Methods Carboxylic latex particles with diameters of 49 and 150 nm were prepared,and a covalent coupling method was used to link DD monoclonal antibody to the particles. The 2 sensitized latex particles were mixed at various proportions,and the sensitivity and linear range were determined,which were compared with those of imported reagents. Results If 4 parts of 49 nm sensitized latex particles were mixed with 1 part of 150 nm sensitized latex particles,the optimal linearity of calibration curve was obtained,with the sensitivity of 0.03 and linear range of 61.88 μg/mL,and it correlated well with imported reagents. Conclusions Dual particle size latex immunoturbidimetry is better than traditional DD latex diagnostic assay,and is of potential for clinical application.

Application of SYSMEX XE-5000 automatic blood analyzer for the routine determination of cerebrospinal fluid among children

SUN Kuixia, YANG Wenshuang, JIANG Shiju

2016, 31(9):  807-809.  DOI: 10.3969/j.issn.1673-8640.2016.09.016

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Objective To investigate the significance of SYSMEX XE-5000 automatic blood analyzer for the routine determination of cerebrospinal fluid among children. Methods A total of 194 cerebrospinal fluid samples were collected and determined by XE-5000 and manual microscopy. The consistency was evaluated. Results When white blood cell（WBC） count was ≥30 cells/μL,there was no statistical significance between the 2 methods（P>0.05）. However,when WBC count was <30 cells/μL,there was a statistical significance between the 2 methods（P<0.05）. When WBC count was ≥10 cells/μL,there were good correlations in counting mononuclear cells and multinuclear cells between the 2 methods （P<0.01）. When red blood cell（RBC） count was >1 000 cells/μL,there was a good correlation between the 2 methods（r=0.98,P<0.01）. Conclusions XE-5000 can be used in the determination of cerebrospinal fluid among children,when WBC count is ≥30 cells/μL.

BIOMED-2 standardized immunoglobulin gene rearrangement detection in paraffin-embedded tissues for diffuse large B cell lymphoma

SUN Juan, AI Xiaofei, LI Qinghua, CAO Zeng

2016, 31(9):  810-813.  DOI: 10.3969/j.issn.1673-8640.2016.09.017

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Objective To investigate the feasibility of detecting diffuse large B cell lymphoma（DLBCL） with the application of BIOMED-2 standardized immunoglobulin（IG） gene rearrangement detection in paraffin-embedded tissues. Methods DNA was extracted from 45 paraffin-embedded tissues. Multiple polymerase chain reaction（PCR） amplification was performed,and clonality analysis for IG gene rearrangement was performed by BIOMED-2 system primer. Results When DNA concentration was diluted to 50-100 ng/μL from 100-500 ng/μL,the proportion of the longest amplification fragment（300-400 bp） of DNA was improved from 10.0% to 90.0%. In 45 cases of DLBCL,the positive rate of immunoglobulin heavy chain（IGH）+ immunoglobulin kappa chain（IGK） was 84.4%. For IG/T-cell receptor（TCR） clonality analysis,no clonal rearrangement was found in 15 cases of reactive lymphoid tissue hyperplasia. Conclusions Dilution of DNA is a method to improve not only the proportion of the longest amplification fragment but also the detection rate of clonality. BIOMED-2 standardized immunoglobulin gene rearrangement detection is an efficient and reliable method for the diagnosis of DLBCL,with important clinical significance.

Comparative study of determining phenylalanine on dry blood spot by tandem mass spectrometry and fluorescence assay

TIAN Guoli, WANG Yanmin, XU Hongping, GUO Jing, ZHOU Zhuo, YAO Jing

2016, 31(9):  814-819.  DOI: 10.3969/j.issn.1673-8640.2016.09.018

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Objective To compare the distribution and differences of phenylalanine（Phe） concentration on dry blood spot by fluorescence assay and tandem mass spectrometry,and to provide a methodological reference for newborn hyperphenylalaninemia（HPA）screening program. Methods A total of 62 510 dry blood spot samples were collected for metabolic disease screening,and the Phe concentration was determined by fluorescence assay and tandem mass spectrometry. Tandem mass spectrometry was used to determine tyrosine（Tyr） concentration. The differences and correlation between the 2 methods were analyzed by paired Wilcoxon test and rank correlation analysis,and the consistency was compared by Bland-Altman analysis. Results Both fluorescence assay and tandem mass spectrometry determined 3 cases of HPA in 62 510 dry blood spot samples,and the sensitivities were 100%. The positive predictive value of fluorescence assay was 33.3%. The positive predictive value of tandem mass spectrometry was 18.8%,and it can be up to 100% combined with Phe/Tyr ratio. The distribution of Phe concentrations of normal newborn samples was skewed,and the results of fluorescence assay and tandem mass spectrometry as <60 μmol/L accounted for 96.31% and 95.08%,respectively,and the results as ≥120 μmol/Laccounted for only 0.01% and 0.03%. The concentration by fluorescence assay was lower than that by tandem mass spectrometry. To the 97% percentile （Phe≈63 μmol/L）,the concentration went to flat,then gradually was higher than that by tandem mass spectrometry. The 2 methods were related positively （r=0.43,P<0.01）. For Bland-Altman analysis,the higher the concentration of Phe was,the smaller the bias of the 2 methods was. When Phe was >120 μmol/L,19 sample points all fell within the 95% limit of consistency,the 2 methods were in good consistency,and the results can be replaceable. Conclusions There are differences by fluorescence assay and tandem mass spectrometry to determine Phe with low concentrations,while the 2 methods are in good consistency for that with high concentrations. The 2 methods had no effect on clinical judgment for HPA,and they can be used in newborn screening. Tandem mass spectrometry can simultaneously determine Phe and Tyr concentrations,and the positive predictive value for HPA can be up to 100% combined with Phe/Tyr ratio.

Identification of Streptococcus G and its drug resistance

HE Lihua, NI Lijun, LIU Hong, WANG Bei, TIAN Yueru, GUO Jian, WU Wenjuan

2016, 31(9):  820-824.  DOI: 10.3969/j.issn.1673-8640.2016.09.019

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Objective To investigate the significance of 16S rDNA sequencing method in identifying beta-hemolytic Streptococcus G and determine the drug resistance of beta-hemolytic Streptococcus G to antibiotics,like penicillin,erythromycin and clindamycin,by in vitro drug susceptibility test,in order to determine the drug resistance phenotypes and genotypes of erythromycin-resistant isolates. Methods A total of 50 beta-hemolytic Streptococcus G isolates were collected from Fudan University Huashan Hospital,and they were identified by API 20 Strep Streptococcus identification system,Lancefield's streptococcal grouping test and 16S rDNA sequencing method. The in vitro drug susceptibility test was performed by agar dilution method. Erythromycin-resistant phenotypes were determined by D test. The drug resistance genes,ermB and mefA,were determined by polymerase chain reaction（PCR）. Results The 50 beta-hemolytic Streptococcus G isolates were identified by API 20 Strep Streptococcus identification system asStreptococcus dysgalactiae. For 16S rDNA sequencing method,there were 42 isolates of Streptococcus dysgalactiae and 8 isolates of Streptococcus anginosus,and the identification rate was >97%. 16S rDNA sequencing method was used as standard,and the identification accuracy of API 20 Strep Streptococcus identification system was 84%. The results of Lancefield's streptococcal grouping test were all group G. The results of drug susceptibility test showed that there was no levofloxacin-,ceftriaxone-,vancomycin- and penicillin-resistant isolates. Erythromycin resistance was found in 28 isolates（56%）,among them,there were 26 isolates（52%） with cMLs,2 isolates（4%） with Ms and no isolate with iMLs. Furthermore,4 isolates （8%） were intermediately resistant to erythromycin. A total of 31 isolates（62%） were clindamycin-resistant,among them,3 isolates（6%） were sensitive to erythromycin and resistant to clindamycin,and 2 isolates（4%） were intermediately resistant to erythromycin and resistant to clindamycin. All cMLs-resistant isolates carried ermB,and 2 Ms-resistant isolates carried mefA. There were 2 isolates of 4 isolates with intermediate resistance to erythromycin carried ermB and mefA,respectively. Conclusions 16S rDNA sequencing method could identify Streptococcus G. The drug resistance rates to erythromycin and clindamycin increase. However,all the isolates are sensitive to penicillin. The beta-lactam antibiotic may be the first choice for the treatment of Streptococcus G infection.

Prokaryotic expression of recombinant human carcinoembryonic antigen and its application in quality control materials

LI Jiangfeng, SONG Xiaoli, QIAN Wei, JIAO Liyuan, ZHAO Xiaoni, WANG Jihua, CAI Lei

2016, 31(9):  825-829.  DOI: 10.3969/j.issn.1673-8640.2016.09.020

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Objective To use prokaryotic cell to express human carcinoembryonic antigen（CEA）,and use it in the development of quality control materials. Methods Gene engineering methods were used to construct prokaryotic expression vector pCold1-cea,and the vector was transferred into Escherichia coli BL21（DE3） to express recombinant human carcinoembryonic antigen（rhCEA）. Results The inclusion bodies of rhCEA were formed in Escherichia coli BL21（DE3） after purification and refolding,which was used into the preparation of quality control materials. The results of quality control materials on Abbott platform were 235.35 ng/mL for high level,68.04 ng/mL for middle level and 11.97 ng/mL for low level. The results on Roche platform were 32.27 ng/mL,7.99 ng/mL and 1.35 ng/mL for high,middle and low levels,respectively. The results on Wondfo platform were 5 ng/mL for high level,and the results for middle and low levels had not been determined. Conclusions Quality control materials prepared by rhCEA have been analyzed in different analysis systems with obvious determination results. The quality control materials are more suitable for Abbott platform than the other 2 platforms.

Application of urine albumin for the diagnosis and treatment of kidney disorders in adults

CHEN Yanwen, HU Yao

2016, 31(9):  830-834.  DOI: 10.3969/j.issn.1673-8640.2016.09.021

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The determination of urine albumin （Alb） is common in the diagnosis and treatment of kidney disorders. However,until now,its uncertainties and controversies regarding the diagnosis and treatment of acute and chronic kidney disorders still exist. The reference ranges for urine Alb have changed recently. This article reviews the latest clinical guidelines and summarizes the application of urine Alb determination in the diagnosis and treatment of acute kidney injury,acute and chronic kidney disorders in adults. The international organization Kidney Disease：Improving Global Outcomes（KDIGO） attempted to resolve these controversies by updating original consensus,staging systems and proper laboratory evaluation for acute and chronic kidney disorders. KDIGO also provided recommendations for the screening and confirmation test of these diseases.