Table of Content

30 June 2014, Volume 29 Issue 6

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Application of molecular techniques in clinical microbiological determination

NI Yuxing

2014, 29(6):  581-583.  DOI: 10.3969/j.issn.1673-8640.2014.06.001

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We carefully organized 6 manuscripts, including 1 review and 5 research papers for the molecular microbiology diagnosis special issue, mainly about the diagnosis, treatment(resistance), prevention and control of clinical common bacterium, fungus and mycobacterium infections. The special issue focuses on the application research of molecular biology techniques in the direct detection of clinical specimens, genotyping, resistance gene research, antigen preparation,serological diagnosis and so on. Blood stream infection is a serious infectious disease, but blood cultures have the problem with long turn-around-time and low positive rate. The application of molecular biology techniques can be used to the detection of common or rare pathogenic bacteria, mycobacteria, fastidous bacteria for rapid identification and drug resistance analysis in the blood stream infection, which can shorten the turn-around-time.Clostridium difficile is a kind of important nosocomial infection pathogen which can cause antimicrobial-associated diarrhea. GeneXpert real-time fluorescence quantitation polymerase chain reaction (PCR) can directly detect Clostridium difficile infection and has the advantages of rapid detection, simple operation and so on.Candida glabrata can cause blood stream infection and other infections. It is a common invasive fungus in nosocomial infection with strong drug resistance. Microsatellite polymorphism offers a simple and rapid method for genotyping with resolution, which is higher than multilocus sequence typing (MLST). Therefore, microsatellite polymorphism can be the preferred choice for Candida glabrata genotyping in laboratory.In recent years, Candida albicans azole resistance has increasing trend. The expression of genes related to azole resistance and biofilm in Candida albicans suggests that ERG11 gene over expression for Candida albicans is an important mechanism of azole resistance. High expression of HWP1 genes is associated with Candida albicans biofilm formation.The carbopenem resistance mechanism of Klebsiella pneumoniae is mainly producing plasmid-mediated Klebsiella pneumoniae carbopenem 2 (KPC-2). Homologous recombination knockout bla_KPC-2 gene in clinical isolates of Klebsiella pneumonia establishes lambda red homologous recombination method of knocking out its drug-resistant gene bla_KPC-2 on plasmid contained by clinical Klebsiella pneumoniae drug-resistant strains reliably, which is helpful to further study on the carbopenem resistance mechanism of Klebsiella pneumoniae.On Preparation and serology diagnosis research of recombinant Mycobacterium smegmatis expressing rd ESAT-6 of Mycobacterium tuberculosis, the fusion proteins of Mycobacterium tuberculosis are successfully prepared in this study. The fusion protein has good specificity and sensitivity for the serological diagnosis among tuberculosis patients.

The research progress in the molecular diagnosis of blood stream infection

GUO Jian,WU Wenjuan

2014, 29(6):  584-589.  DOI: 10.3969/j.issn.1673-8640.2014.06.002

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The blood stream infection is a syndrome of serious systemic infection, and blood culture remains being the gold standard for the diagnosis of bacterial blood stream infection, but only 30%-40% blood stream infection pathogens could be found by blood culture. Molecular biology method through analyzing the nucleic acid of microorganisms in blood samples will quickly provide the results of bacterial, fungal or viral infections accurately, and provide a common resistance gene detection results of pathogens. At present, the molecular diagnosis techniques for blood stream infection in clinical laboratoriesmainly include nucleic acid hybridization, nucleic acid amplification and DNA sequence analysis, gene chip, mass spectrometry and so on. The common pathogenic bacteria, mycobacteria, fastidious bacteria, rare pathogenic bacteria use a variety of detection techniques combined with the application of rapid identification and drug resistance analysis, which can provide reliable diagnosis results for clinical in a relatively short period of time, indicate the use of clinical rational drug and improve survival in patients with blood stream infection.

Clinical evaluation of GeneXpert real-time fluorescence quantitation PCR in rapid detection of Clostridium difficile

CHEN Xu, DONG Danfeng, HAN Lizhong, NI Yuxing

2014, 29(6):  590-592.  DOI: 10.3969/j.issn.1673-8640.2014.06.003

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Objective To evaluate the clinical application of GeneXpert real-time fluorescence quantitation polymerase chain reaction (PCR) in rapid detection of Clostridium difficile from stool. Methods Two-head swab was dipped in clinical stool specimens, one-head swab was used for GeneXpert real-time fluorescence quantitation PCR in detecting toxity gene tcdB , and the other was used for anaerobic culture. The results of these 2 methods were compared and analyzed statistically, and the sensitivity, specificity, positive predictive value and negative predictive value of GeneXpert real-time fluorescence quantitation PCR were calculated. Results Totally 141 clinical stool specimens were collected, and GeneXpert real-time fluorescence quantitation PCR revealed that Clostridium difficile was positive in 42 cases, among which Clostridium difficile was cultured in 34 cases. GeneXpert real-time fluorescence quantitation PCR was well consistent with the culture method (Kappa=0.775 0, P<0.01), and the sensitivity, specificity, positive predictive value and negative predictive value of GeneXpert real-time fluorescence quantitation PCR were 87.2%, 92.2%,81.0% and 94.9%, respectively. Conclusions GeneXpert real-time fluorescence quantitation PCR could detect Clostridium difficile from stool specimens directly and accurately, and it has a prospective potential for clinical application.

Application evaluation of microsatellite polymorphism and multilocus sequence typing in genotyping of Candida glabrata

YAO Dongting, YING Chunmei, ZHENG Bing

2014, 29(6):  593-596.  DOI: 10.3969/j.issn.1673-8640.2014.06.004

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Objective To evaluate the application significance of microsatellite polymorphism in genotyping of Candida glabrata. Methods From January 2011 to December 2012, 59 isolates of Candida glabrata were collected from Renji Hospital and East Hospital. All the 59 isolates were typed by microsatellite polymorphism and multilocus sequence typing (MLST). The results and discrimination of the 2 genotyping methods were compared. Results By MLST, the 59 isolates belonged to 6 clone sequences, including 43 isolates with ST-7, 7 isolates with ST-10, 3 isolates with ST-15, 3 isolates with ST-55, 2 isolates with ST-3 and 1 isolate with ST-43, and the index of discriminatory power (DP) was 0.456. By microsatellite polymorphism, the 59 isolates were classified into 10 genotypes. There were 25 isolates with type A, 10 isolates with type B, 8 isolates with type C, 6 isolates with type D, 3 isolates with type E, 2 isolates with type F, 2 isolates with type G, 1 isolate with type H, 1 isolate with type I and 1 isolate with type J, and the DP was 0.770. Conclusions Microsatellite polymorphism is a simple and rapid method for molecular typing with higher discrimination than MLST. Therefore, microsatellite polymorphism can be the preferred choice in clinical laboratories.

The expression of genes related to azole resistance and biofilm in Candida albicans

HU Lüyin, ZAI Shubei, JIN Xin, CAI Jinfeng, CAO Yushuo, HU Xiangnan, DU Xin, LI Tianming, LI Min

2014, 29(6):  597-602.  DOI: 10.3969/j.issn.1673-8640.2014.06.005

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Objective To investigate the expression of genes related to azole resistance and biofilm formation capability in clinical isolates of Candida albicans. Methods In this study, 104 Candida albicans were isolated from blood, sterile sites and mucosal lesions of 92 infectious disease patients [viral hepatitis, tuberculosis and acquired immune deficiency syndrome (AIDS) in Shanghai Public Health Clinical Center from 2006 to 2011. The minimum inhibitory concentrations (MICs) of fluconazole, itraconazole and voriconazole were determined by ATB FUNGUS3. The gene expression of efflux pump genes (CDR1, CDR2 and MDR1) and azole antifungal target gene ERG11 were determined by semi-quantitative reverse transcription polymerase chain reaction (PCR), and the azole susceptibe and susceptible dose dependent (S-DD) states were analyzed. 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-2H-tetrazolium- 5-carboxanilide (XTT) test was used to screen the high biofilm formation capability isolates (HBPs). The expressions of ALS3 and HWP1 in low biofilm formation capability isolates (LBPs) were analyzed. Results Among 104 isolates of Candida albicans, 16 isolates were tested resistant to at least one azole, 6 isolates were S-DD to at least one azole.There were statistical significance among resistant, S-DD and susceptible isolates in azole antifungal target gene ERG11 (P=0.007 8), and significant difference was found when comparing susceptible isolates with resistant and S-DD ones (P<0.05). No significant difference was found in the expression levels of efflux pump genes, CDR1, CDR2 and MDR1. The expression levels of HWP1 in HBFs were higher than those in LBFswith statistical significance (P=0.007 9), whereas ALS3 showed nostatistical significance. Conclusions The overexpression levels of ERG11 gene may primarily account for resistance to azole in Candida albicans. High expression of hyphal wall protein HWP1 gene is associated to biofilm formation capability in Candida albicans.

Homologous recombination knockout bla_KPC-2 gene in clinical isolates of Klebsiella pneumonia

ZHANG Ying, TIAN Yueru, AI Fuqi, LIU Hong, MA Yimin, WANG Bei, JIANG Xiaofei

2014, 29(6):  603-606.  DOI: 10.3969/j.issn.1673-8640.2014.06.006

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Objective To establish a method of knocking out drug-resistant genes on plasmid contained by clinical Klebsiella pneumonia drug-resistant isolates. Methods Polymerase chain reaction(PCR) was used to amplify the upstream and downstream fragments of target gene bla_KPC-2 of the test isolates and hygromycin resistance gene fragments on plasmid pMQ300, respectively. Gene splicing by overlap extension polymerase chain reaction(SOE-PCR) was used to construct the fusion fragments, and apramycin resistance lambda red plasmid pKOBEG-Apr was used as mediated, homologous recombination knockout bla_KPC-2 gene in clinical Klebsiella pneumonia drug-resistant isolates. LB medium containing hygromycin and apramycin was used to screen recombination, and PCR and reverse transcription-polymerase chain reaction(RT-PCR) amplification were used to detect bla_{KPC - 2} gene and hygromycin resistance gene, and drug sensitive test was used to confirm the recombination. Results The bla_KPC-2 gene was successfully knocked out in 2 clinical Klebsiella pneumonia drug-resistant isolates with different multilocus sequence typing(MLST). Conclusions Lambda red homologous recombination method can be used to knock out clinical Klebsiella pneumonia drug-resistant isolate plasmid gene reliably.

Preparation and serology diagnosis research of recombinant Mycobacterium smegmatis expressing rdESAT-6 of Mycobacterium tuberculosis

GUO Jian,FAN Qiwen,FAN Xiaoyong, MA Hui, QIAN Xueqin, HU Xiangnan, WU Wenjuan

2014, 29(6):  607-612.  DOI: 10.3969/j.issn.1673-8640.2014.06.007

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Objective To construct the prokaryotic expression vectors containing Mycobacterium tuberculosis(MTB) fusion gene. Meanwhile, the inducible expression of fusion protein recombinant dimer ESAT-6 (rdESAT-6) was performed in the expression systems of Mycobacterium smegmatis. The fusion protein was purified, and the antigenic specificities of fusion protein was analyzed by Western blot. The significance of fusion proteins for the diagnosis of tuberculosis was evaluated. Methods The 2×esat-6 gene was amplified with DNA vaccine HG856A₂. The 2×esat-6 gene was then cloned into plasmid pMF41 for the construction of prokaryotic expression vector pMF41-2×esat-6. The recombinant plasmid pMF41-2×esat-6 was electroporated into Mycobacterium smegmatis. The target gene was induced to express fusion protein rdESAT-6. The antigenic specificities of purified fusion protein was analyzed by Western blot with mouse antiserum against rdESAT-6. The sensitivity and specificity of these fusion proteins for the diagnosis of tuberculosis were assayed by enzyme-linked immunosorbent assay (ELISA) and tuberculosis dot immunogold filtration assay (TB-DOT) test kit for 126 tuberculosis patients and 42 healthy controls. Results The prokaryotic expression vector pMF41-2×esat-6 was constructed successfully, and the related fusion protein was expressed in the form of inclusion body. The fusion protein was purified, and the purity of these proteins were great than 95%. The fusion protein was antigenic with ESAT-6 mouse antiserum. The sensitivities of the 126 tuberculosis patients and negative cases of serological diagnosis were 79.75% (63/79) and 61.70% (29/47), respectively, which were better than those of TB-DOT test kit [62.03% (49/79) and 44.68% (21/47),P<0.05] . The specificity was both 95.24% (40/42). Conclusions The fusion proteins of MTB are successfully prepared in this study with good sensitivity and specificity, which can be used for the diagnosis of tuberculosis.

Study on the utility of thromboelastography in monitoring low molecular weight heparin

SHEN Yun, ZHANG Liwei, LIN Xiaoyi

2014, 29(6):  613-616.  DOI: 10.3969/j.issn.1673-8640.2014.06.009

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Objective To attempt to use thromboelastography (TEG) reaction time (R) to monitor the clinical use of low molecular weight heparin (LMWH). Methods A total of 20 hospitalized patients from nephrology department with nephritic syndrome were enrolled.Blood samples were collected at 4 h after both firstly and secondly receiving dose in order to perform TEG and anti-Xa activity test. TEG R and anti-Xa activity were determined. The associations between TEG R and anti-Xa activity, between the dose of taking drugs at first time and anti-Xa activity, and between the dose of taking drugs at first time and TEG R, were analyzd. The comparative analysis of anti-Xa activity with different doses of taking drugs was performed. The comparative analysis of TEG R with different doses of taking drugs was performed. Results There was no association between TEG R and anti-Xa activity (P=0.54, R=-0.144 9). There was no association between anti-Xa activity and the dose of taking drugs at first time (P=0.26, R=-0.263 6). There was obvious association between TEG R and the dose of taking drugs at first time (P<0.000 1, R=0.875). There was difference in anti-Xa activity between the 2 doses of taking drugs (P=0.000 5, t=4.152). There was significant difference in TEG R between the 2 doses of taking drugs (P<0.000 1, t=15.19). Conclusions TEG R reflects patients' condition of using LMWH much more accurately than anti-Xa activity.

Investigation on the reference ranges of peripheral blood T lymphocyte subsets among healthy adults in Shanghai

DONG Jingyi, NING Xiaoxiao, WANG Lei, TIAN Jianhui

2014, 29(6):  617-622.  DOI: 10.3969/j.issn.1673-8640.2014.06.010

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Objective To establish the reference ranges of peripheral blood T lymphocyte subsets among 333 healthy adults in Shanghai, to analyze and discuss the change characteristics through the establishment of 95% confidence interval, and to provide the reference for immune status analysis and clinical diagnosis and treatment. Methods Multiparameter three-color immunofluorescence was used to stain the peripheral blood T lymphocytes, and BD FACSCalibur flow cytometry was used to acquire and analyze the percentages and absolute counts of T lymphocyte subsets. Results Among females, the CD3⁺ percentage, CD4⁺/CD8⁺ ratio, CD3⁺ CD4⁺ percentage and CD4⁺CD45RA⁺ percentage (72.6%±8.1%), 1.57(0.74-4.70), (44.8%±8.0%) and (1.71%±6.3%) were respectively higher than those among males (70.9%±8.5%), 1.43(0.51-4.12), (40.6%±9.3%) and (15.1%±6.8%). The lymphocyte absolute count and CD8⁺CD28⁺ absolute count in males (2 069.8±474.1) cells/μL and (237.5±95.7) cells/μL were higher than those in females (1 932.8±469.7) cells/μL and (218.1±111.1) cells/μL. The percentage of T lymphocyte subsets increased with the increase of age.There were statistical significances in >50 year-old group with the other groups. The absolute count of 333 healthy adults for peripheral blood T lymphocyte was (1 069.1-2 935.6) cells/μL. The percentages of T lymphocyte subsets were: CD3⁺ 55.3%-87.6%, CD3⁺CD4⁺ 25.2%-57.6%, CD3⁺CD8⁺ 15.0%-42.1%, CD4⁺CD25⁺ 2.5%-10.5%, CD4⁺CD45RA⁺ 5.0%-27.2 % and CD8⁺CD28⁺ 4%-19.3%, and CD4⁺/CD8⁺ ratio was 0.7-2.9. Conclusions There are differences for the percentage distribution of T lymphocyte subsets among healthy adults between sex and age. Since these differences can be ignored, the reference ranges of peripheral blood T lymphocyte subsets among healthy adults could be established in Shanghai.

Clinical significance on the dynamic monitoring of serum lactate dehydrogenase levels in acute leukemia

LIU Hua, ZHENG Qin, ZENG Tingting, YE Yuanxin, LI Guixing, SU Jun

2014, 29(6):  622-626.  DOI: 10.3969/j.issn.1673-8640.2014.06.011

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Objective To monitor dynamically serum lactate dehydrogenase (LDH) levels in acute leukemia (AL) patients,and to investigate the clinical significance on the monitoring of pathogenetic condition and prognosis evaluation. Methods The LDH levels of 2 170 times from 1 184 de novo AL patients were analyzed retrospectively. The relationship of LDH levels with disease progression and AL prognosis was evaluated.The relationships between LDH levels and blood cell counts,the proportion of bone marrow leukemia cells,cytogenetic abnormality, flow cytometry immunophenotyping and the other related diagnostic markers were analyzed. Results LDH levels of AL patients in different stages of the disease were different. The LDH levels of incipient and relapse stages in AL patients were significantly higher than those of remission stage in AL patients and those in healthy controls (P<0.05). The LDH levels of remission stage in AL patients reduced, but were still significantly higher than those in healthy controls (P<0.05). The LDH levels of acute lymphoblastic leukemia (ALL) patients were significantly higher than those of acute myeloid leukemia (AML) patients (P<0.05). Compared with the other diagnostic and prognostic indicators of AL, LDH levels had correlation with the proportion of bone marrow leukemia cells,cytogenetic abnormality and flow cytometry immunophenotyping (P<0.05). Conclusions The serum LDH levels of AL patients in different stages and different subtypes are different significantly. The LDH levels can be used as a supplementary indicator of AL diagnosis, disease progression and therapeutic effect evaluation.

Colonization of group B Streptococcus in late pregnancy by fluorescence quantitation PCR in Nanjing area

JI Xiuqing, LU Gensheng, HU Ping, CHENG Jian, LIU Ye, LIN Ying

2014, 29(6):  628-630.  DOI: 10.3969/j.issn.1673-8640.2014.06.012

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Objective To evaluate the colonization of group B Streptococcus(GBS) in late pregnancy in Nanjing area. Methods The vaginal samples from 9 073 women in late pregnancy(34-37 weeks) were collected. A total of 500 cases of second trimester(20-24 weeks)of healthy pregnant women were chosen as control group. The colonization of GBS was detected by fluorescence quantitation polymerase chain reaction(PCR). The results were analyzed comparatively. Results The results showed that the GBS carrier rate in late pregnancy was 4.17%, the carrier rate in second trimester pregnant women was 5.20%, and there was no statistical significance between the 2 groups(P>0.05). The carrier rate of 30-34 years old group was the highest (4.77%), compared with ≤24 years old group and ≥40 years old group respectively, and the difference was statistically significant (P<0.05), but the difference with the other age groups had no statistical significance (P>0.05). Conclusions The carrier rate of GBS in late pregnancy is within the range of carrier rate of healthy population in Nanjing area. Gestational weeks have little effect on the pregnant women with GBS carrier rate

The diagnosis significance of miR-UL112 level in human peripheral blood mononuclear cells during HCMV infection

WU Rong, KONG Qianqian, L Zhiyi, XU Jian, NI Zhenhua, XIANG Fenfen, KANG Xiangdong

2014, 29(6):  631-634.  DOI: 10.3969/j.issn.1673-8640.2014.06.013

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Objective To investigate the levels of micro RNA(miR)-UL112-3p and 5p in human peripheral blood mononuclear cells, and to evaluate the significance of miR-UL112 in the diagnosis of human cytomegalovirus (HCMV) atent and active infections. Methods Human peripheral blood mononuclear cells were isolated from 92 non-cancer patients (57 patients with high-level HCMV specific CD8⁺ T lymphocyte and 35 patients with low-level HCMV specific CD8⁺T lymphocyte) and 30 cancer patients receiving chemotherapy. HCMV DNA, miR-UL112-3p and miR-UL112-5p were determined by real time fluorescence quantitation polymerase chain reaction (PCR). The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic significance of HCMV latent infections. Results In cancer group, the levels of HCMV DNA, miR-UL112-3p and miR-UL112-5p were higher than those in non-cancer group (P<0.05). The levels of HCMV DNA, miR-UL112-3p and miR-UL112-5p in patients with high-level CD8⁺ T lymphocyte were significantly higher than those in patients with low-level CD8⁺ T lymphocyte (P<0.01). As the levels of miR-UL112-3p and miR-UL112-5p in patients with low-level CD8⁺ T lymphocyte for standards, the positive rates in patients with high-level CD8⁺ T lymphocyte were 59.65% and 64.91%. As 1.09 and 1.52 for the cut-off values of miR-UL112-3p and miR-UL112-5p in HCMV high-level latent infection, the sensitivities were 59.65% and 64.91%, and the specificities were 97.1% and 97.1%. In the +s (5.24 and 6.63) standard of miR-UL112-3p and miR-UL112-5p levels of non-cancer group, the positive rates of cancer group were 50.00% and 73.33%. Conclusions There might be diagnostic significance for HCMV latent and active infections by determining the levels of miR-UL112-3p and 5p.

Gene polymorphism of CYP2C9 and VKORC1 and their relationships to the dosage of warfarin

NIU Guoping, WEI Yuanyuan

2014, 29(6):  635-639.  DOI: 10.3969/j.issn.1673-8640.2014.06.014

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Objective To investigate the relationship of cytochrome P450(CYP)2C9 and vitamin K epoxide reductase complex 1(VKORC1)genotypes with warfarin dosage, and to provide reference for the use of warfarin in Xuzhou city, Jiangsu, China. Methods A total of 100 Han-population healthy subjects and 200 patients with warfarin dosage were enrolled. CYP2C9 and VKORC1 genotypes were determined bypolymerase chain reaction and ligase detection reaction (PCR-LDR). Results There were 91 cases of CYP2C9 genotype with *1/*1 and 9 cases of CYP2C9 genotype with *1/*3 in healthy subjects. There were 17 cases of VKORC1 genotype with GA and 83 cases of VKORC1 genotype with AA. VKORC1(1639 G>A) genotype showed homozygote AA(168 cases), heterozygote GA (32 cases) and no GG in patients with warfarin dosageGYP2C9 genotype in 200 patients had 179 cases of *1/*1 and 21 cases of *1/*3, and there was no*2 mutation.Warfarin dosage in VKORC1(1639 G>A) in AA [ (2.59±0.83) mg/d] group was significantly lower than that in GA [(4.51±0.79)mg/d] group.Warfarin dosage in CYR2C9 genotype with *1/*9 [(3.01±1.12) mg/d] was higher than that with *1/*3 [(2.19±0.32)mg/L, P<0.05] . Conclusions CYP2C9 and VKORC1 genotype covariate therapy has the potential significance to improve the safety of warfarin use.

Risk factors of diabetic foot in China: a Meta analysis

ZHAO Jingjing,WANG Weiling,ZHENG Peili

2014, 29(6):  640-645.  DOI: 10.3969/j.issn.1673-8640.2014.06.015

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Objective To use Meta analysis to evaluate the risk factors of diabetic foot in China. Methods The following databases such as China National Knowledge Infrastructure (CNKI), Chinese Weipu Database, Pubmed,EMbase, Chinese Biological Medicine Database(CBM),WANFANG Database and handsearched relevant literature were searched to collect case-control studies on the related risk factors of diabetic foot in China.The retrieval time reached from inception to December, 2013. Two reviewers independently screened the literature according to the inclusion and exclusion criteria, extracted the data and assessed the quality.The Meta analysis was performed by RevMan5.0 software. The various risk factors were analyzed by fixed effect model or random effect model pooled odds ratio(OR)and 95% confidence interval (CI), with stability evaluated by sensitivity analysis.Moreover, the funnel plot was used to assess the published bias of articles. Results A total of 15 articles were included, the key risk factors such as age(OR=7.51, 95%CI: 6.74-8.28), course of disease(OR=3.34, 95%CI: 2.96-3.72), body mass index(BMI)(OR=-0.53, 95%CI:-0.80-0.26), systolic blood pressure(OR=9.84, 95%CI: 8.08-11.60), diastolic blood pressure(OR=1.03, 95%CI: 0.03-2.03), fasting blood glucose(FPG)(OR=1.48, 95%CI: 1.19-1.77), 2 h post prandial blood glucose(2 hPG)(OR=2.15, 95% CI: 1.71-2.59), glycosylated hemoglobin A_1c(HbA_1c)(OR=1.46, 95%CI: 1.03-1.88), total cholesterol(TC)(OR=-0.31, 95%CI: -0.42-0.19) and high-density lipoprotein-cholesterol(HDL-C)(OR=-0.07, 95%CI: -0.10-0.03) (P<0.01)affected the happening of diabetic foot, but triglyceride (TG) (OR=-0.06, 95%CI: -0.16-0.04), low-density lipoprotein-cholesterol(LDL-C)(OR=-0.05, 95%CI: -0.21-0.11)did not statistically influence on diabetic foot(P>0.05). Conclusions Age, course of disease, BMI, systolic blood pressure, diastolic blood pressure, FPG, 2 hPG, HbA_1c, TC and HDL-C are primarily determined as the important risk factors of diabetic foot, which can provide the reference for the early prevention of diabetic foot occurred in clinic.

Research on the drug resistance mechanisms of meropenem and ciprofloxacin in clinical isolates of Pseudomonas aeruginosa

ZHANG Guodong, WANG Ying, ZHU Hongsheng

2014, 29(6):  646-650.  DOI: 10.3969/j.issn.1673-8640.2014.06.016

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Objective To investigate the drug resistance mechanisms of both meropenem and ciprofloxacin in clinical isolates of Pseudomonas aeruginosa. Methods A total of 30 isolates of Pseudomonas aeruginosa which had been tested resistance to both meropenem and ciprofloxacin by VITEK-2 Compact were collected. Agar dilution method was used to reexamine the minimal inhibitory concentrations (MIC) of meropenem and ciprofloxacin. Carbapenemases were detected by modified three-dimensional test. The resistance genes were analyzed by polymerase chain reaction (PCR) amplification. The expression of efflux systems was analyzed by reverse transcription PCR. Results The same results were showed between agar dilution method and VITEK-2 Compact. Carbapenemases were not detected by modified three-dimensional test. The overexpression ofmexX and mexC genes were detected by reverse transcription PCR. The sequencing of regulatory genes showed that there were 4 isolates which the Gly(GGC) of nfxB was substitute for Val(GTC) and 6 isolates which the Val(CTG)of mexZ was substitute for Gly (CAG). Conclusions The overexpression of the MexXY-OprM and MexCD-OprJ efflux system, due to the mutation of regulatory genes of efflux system, is contributed to the drug resistance to both meropenem and ciprofloxacin of Pseudomonas aeruginosa.

Comparison on the consistency between ROCHE URYSIS-2400 and AIKELAI AX-4030 urine dry-chemistry analyzers

WU Hui, WANG Beili, GUO Wei, PAN Baishen

2014, 29(6):  651-655.  DOI: 10.3969/j.issn.1673-8640.2014.06.017

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Objective To compare the consistency of ROCHE URYSIS-2400 and AIKELAI AX-4030 urine dry-chemistry analyzers. Methods A total of 500 urine specimens were detected by the 2 kinds of urine chemistry analyzers in parallel, and the results were analyzed statistically by positive detection rates and accordance rates. A total of 250 cases were randomly selected for reviewing erythrocytes (ERY) and leulcocytes (LEU) by urine sediment microscopy. Experiments by adding vitamin C were used to evaluate URYSIS-2400 and AX-4030 anti-interference abilities against vitamin C. Results The mean difference percentage of specific gravity (SG) between the 2 analyzers was <1%, and all the general consistency rates of the remaining 9 projects were> 90%. The consistency rates of URYSIS-2400 and AX-4030 were 82.4% and 84.4% with urine sediment microscopy on ERY, and 76.0% and 83.2% on LEU. URYSIS-2400 and AX-4030 anti-interference abilities against vitamin C were up to 50mg/dL on glucose (GLU) and ERY. Conclusions There is a good consistency between the results of URYSIS-2400 and AX-4030, but urine chemistry analyzer is used only for screening,and the results should be reviewed by urine sediment microscopy in order to meet clinical needs.

The fit of four parameter logistic calibration of DXI 800 chemiluminescence analyzer by Origin software

WANG Qiang, MA Xiaohong, WENG Xiumei

2014, 29(6):  656-658.  DOI: 10.3969/j.issn.1673-8640.2014.06.018

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Objective To fit 4 parameter logistic calibration of DXI 800 chemiluminescence analyzer by Origin software and compare the datum difference between Origin and DXI 800, and to investigate the calculation possibility of unknown results of DXI 800 by Origin software. Methods The calibration results of testosterone, total thyroxine, estradiol, troponin I, thyrotropic hormone, carcino-embryonic antigen and progesterone were collected and fit by Origin software, and the calculation results with the original results of DXI 800 system were analyzed comparatively. Results The calculation results of Origin software were very close to the original results of DXI 800, and mean bias was from -0.85% to 1.68%. Conclusions Origin software is suitable for the calculation of unknown results of DXI 800 chemiluminescence analyzer.

The construction and expression of AFP-enhanced green fluorescent protein recombinant and the analysis of measurement characteristics

ZHANG Jian, GE Danhong, WANG Xueliang

2014, 29(6):  659-663.  DOI: 10.3969/j.issn.1673-8640.2014.06.019

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Objective To construct the fusion protein of alpha fetoprotein(AFP)-enhanced green fluorescent protein(eGFP) recombinant, and to analyze the stability and measurement characteristics. Methods The sequence of eGFP was amplified from pcDNA3.0 plasmid and inserted PET28a plasmid to construct expression plasmid PET28a-eGFP. The coding sequence of AFP was synthetized according to the coding sequence of AFP from the National Center for Biotechnology Information (NCBI) database, added and linked to eGFP sequence by a linker sequence to construct expression plasmid PET28a-eGFP-AFP. Recombinant protein eGFP and eGFP-AFP were purified, and the measurement characteristics and stability were analyzed. Results The expression plasmid of recombinant protein eGFP and eGFP-AFP were constructed, and the proteins were purified. Recombinant protein eGFP and eGFP-AFP had the same excitation spectrum and emission spectrum, which were 450 and 509 nm optimally, and its fluorescence could be stable over 12 months. eGFP-AFP could be tested for the level of AFP by routine AFP immunoassay. Conclusions Recombinant protein eGFP and eGFP-AFP have the same fluorescence characteristics and could react with routine immunoassay which establishes the foundation for the next research using eGFP-AFP as calibration and quality control materials.

Study on bactericidal and bacteriostatic effects of Schisandra chinensis fruit against Acinetobacter baumannii in vitro

ZHANG Jinghao, LI Yanhong, ZHAO Hu

2014, 29(6):  664-667.  DOI: 10.3969/j.issn.1673-8640.2014.06.020

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Objective To observe the bactericidal and bacteriostatic effects of Schisandra chinensis fruit against Acinetobacter baumannii which was resistant to cefoperazone-sulbactam or non-resistant to cefoperazone-sulbactam. Methods The extract of Schisandra chinensis fruit was prepared, bacteriostatic and bactericidal test in vitro by tube double dilution method was performed to observe their minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) to Acinetobacter baumannii. Results The MIC50 of Schisandra chinensis fruit against Acinetobacter baumannii resistant to cefoperazone-sulbactam or non-resistant to cefoperazone-sulbactam were 15.6 and 7.8 mg/mL, and on this drug concentration, the inhibitory rate against non-resistant to cefoperazone-sulbactam Acinetobacter baumannii was higher than that against resistant to cefoperazone-sulbactam (χ²=4.26 and 3.98, P<0.05). When the drug concentration reached to 31.3mg/mL, the inhibitory rates were all 100%. The MBC50 of Schisandra chinensis fruit against Acinetobacter baumannii which was resistant tocefoperazone-sulbactam or non-resistant to cefoperazone-sulbactam were 31.3 mg/mL, and the bactericidal rate had no statistical significance (χ²=0.74, P>0.05). Conclusions The Schisandra chinensis fruit has certain bactericidal and bacteriostatic effects on Acinetobacter baumannii which was resistant to cefoperazone-sulbactam or non-resistant to cefoperazone-sulbactam in vitro, and the MIC against Acinetobacter baumannii is a little higher. It suggests that Schisandra chinensis fruit could be used for the clinical treatment of Acinetobacter baumannii.

The investigation on the internal quality control of HBV DNA determination by fluorescence quantitation PCR

JIANG Lingli, WANG Xueliang, XIAO Yanqun, WANG Hualiang

2014, 29(6):  668-670.  DOI: 10.3969/j.issn.1673-8640.2014.06.021

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Objective To investigate the internal quality control of hepatitis B virus (HBV) DNA determination by real time fluorescence quantitation polymerase chain reaction (PCR). Methods The coefficients of variation (CV) of previous 20 internal quality control data of HBV DNA determination under routine condition from PCR laboratories in Shanghai region were analyzed, and the control chart was drawn by the mean(±s).1_3s/2_2s multi-rule quality control method and Levey-Jennings single-rule quality control method were used to determine internal quality control data from laboratory A,B and C whose CV were ≥10%,5%-<10% and 1%-<5%, respectively. Results When the concentrations of the internal quality control materials were 5×10⁴ and 5×10⁶ IU/mL, there were no error data detected in laboratory A whose CV were 14.96% and 12.15% by 1_3s/2_2s multi-rule quality control method and Levey-Jennings single-rule quality control method, respectively. There were both 2 random error data detected in laboratory B whose CV were 6.49% and 5.00% by1_3s/2_2s multi-rule quality control method and Levey-Jennings single-rule quality control method, respectively. The CV of laboratory C were 4.36% and 2.43%. There were 3 systematic error data detected by 13s/2_2s multi-rule quality control method, and there were no error data detected by Levey-Jennings single-rule quality control method. Conclusions When the CV of the previous 20 internal quality control data were ≥ 10%, the error detections are all low both by the multi-rule quality control method and single-rule quality control method. The laboratory should set the suitable CV and use the multi-rule quality control method to improve the systematic error detection.

Summary analysis on accreditation on-site review nonconformity items of 18 medical laboratories in Shanghai

HUANG Weigang, WAN Haiying, FAN Xiaoxia, CAI Feng, HUANG Sheng, XIANG Mingjie

2014, 29(6):  672-675.  DOI: 10.3969/j.issn.1673-8640.2014.06.022

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Objective To perform summary analysis on ISO15189 accreditation on-site review nonconformity items of 18 medical laboratories in Shanghai, to give the correct methods and pathways for the nonconformity items, and to provide reference for other medical laboratories. Methods ISO15189 accreditation on-site review nonconformity items of 18 medical laboratories in Shanghai were collected, screened and analyzed. According to the nonconformity description, cause analysis, corrective action, correct implementation and correct verification, the correct methods and pathways for common problems are put forward. Results A total of 297 nonconformity items were collected, including 76 items about management (25.6%) and 221 items about technique (74.4%). There were 61 items for the quality insurance of laboratory procedure, accounting for 20.5%(61/297), with 6 classes of 47 forms. Conclusions Through the summary and rearrangement analysis of nonconformity items, the causes of nonconformity items are cleaned up, and the qualities of medical laboratory and laboratory accreditation are improved with reference effects.

Investigation on two cases of amikacin application in validation for EDTA-dependent pseudo thrombocytopenia

ZHANG Jingquan, CHEN Liting, SHI Hourong, WANG Jianbiao

2014, 29(6):  676-678.  DOI: 10.3969/j.issn.1673-8640.2014.06.023

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Objective To investigate 2 cases of amikacin application in validation for ethylene diamine tetracetic acid (EDTA)-dependent pseudo thrombocytopenia. Methods Two cases of EDTA-dependent pseudo thrombocytopenia were collected, and their EDTA-K₂ anticoagulant blood were collected and added amikacin at different time periods.The blood samples were determined for hematology analysis and blood smears. Results For the 2 patients with or without amikacin, the platelet count increased gradually with time, and the histogram disappeared with time. Adding amikacin would obtain more reliable results. Conclusions Amikacin can not immediately dissociate the platelet aggregation in the 2 cases, however, with time, the slow dissociation of platelet achieves a relatively stable state for 4 h. Amikacin is added within 1 h is preferable. The relatively common EDTA anticoagulation can effectively accelerate the dissociation process, in order to obtain the desired results.

Mutation analysis of iron metabolism regulated genes in a patient of hemochromatosis

GUAN Yu, AN Peng, ZHANG Xiaofeng

2014, 29(6):  679-684.  DOI: 10.3969/j.issn.1673-8640.2014.06.024

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Objective To detect the mutation of hemochromatosis-related gene in a patient suspected with hereditary hemochromatosis and his family members. Methods The peripheral blood samples were collected from the patient and his family members after recording patient's clinical data. The indices of iron metabolism including serum iron (SI), total iron binding capacity (TIBC), serum ferritin (SF)and transferrin saturation (TS) were determined. The genomic DNA from peripheral blood was isolated, and polymerase chain reaction (PCR) was used to amplify exon and intron splice junctions, and the 5' and 3' untranslated regions (UTRs) of HFE,HJV,HAMP,TFR2 and SLC40A1 genes. After agarose gel electrophoresis and purification, the PCR products were submitted to bidirectional sequence analysis for determining mutation. Results SF, SI and TS increased significantly in the patient. Mutation analysis in the patient revealed c.224C>T (p.Ala75Val) and c.714C>G (p.Ile238Met) heterozygous mutations respectively located in exon2 and exon5 of TFR2. Additional synonymous mutation was also found in exon6 of SLC40A1 (c.663T>C, p.Val221Val).There was no mutation being found in HFE, HAMP and HJV in the patient. SLC40A1 V221V mutation was all detected in the patient's family members, but it did not happen to the aforementioned TFR2 mutations. Conclusions TFR2 A75V and I238M mutations may be the genetic basis of hereditary hemochromatosis in this patient, but its pathogenic mechanism remains to be further studied.

The research advance of biofilm in the mechanisms of azole resistance to Candida albicans

SHI Ce, LIU Jinyan, WEI Bing, XIANG Mingjie

2014, 29(6):  692-694.  DOI: 10.3969/j.issn.1673-8640.2014.06.028

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Candida albicans is the most important opportunistic fungus. As the first choice to treat the infection, azole is widely used, but the resistance to azole is more and more common. In the lately years, researchers have focused on the mechanism of azole resistance and biofilm formation. This review summarizes the research advance of biofilm.