Application of digital PCR technology in the precise molecular diagnosis: opportunities and challenges / 中华检验医学杂志

Digital polymerase chain reaction (dPCR) is an absolute quantitative technique that has been rapidly developed in recent years. This technique assigns the reaction system containing DNA template to a large number of independent reaction units for PCR, and calculates the DNA copy number according to the Poisson distribution and statistical positive signals. In contrast to conventional qPCR, dPCR does not depend on amplification curves, is not affected by amplification efficiency, thus has high accuracy and repeatability, and can achieve the absolute quantification. This article reviews the development history of dPCR and its application in molecular diagnosis, tumor liquid biopsy and prenatal diagnosis of infectious diseases, and looks forward to the application prospect of this technology.

To establish a method of serum detection by Raman spectroscopy for the diagnosis of gastric cancer / 中华检验医学杂志

Objective:To establish a method of serum detection by Raman spectroscopy for the diagnosis of gastric cancer.Methods:Between April and November 2019, 110 patients with gastric cancer [73 males, 37 females, age (57.4±10.3) years] and 74 patients with colorectal cancer [48 males and 26 females, aged (58.3±12.2) years] were collected at the First Affiliated Hospital of Army Military Medical University, along with 100 healthy subjects [59 males and 41 females, aged (55.6±10.61) years] during the same period. Fasting venous blood serum samples were collected from the subjects. A Raman spectrometer XploRA PLUS was used in this experiment, with an excitation light source of 532 nm, a field of view of 100 times, and a spectrum range of 200-2 000 cm -1, etc. The serum samples were detected by nondestructive and non-contact rapid detection, and the Raman spectra of serum samples were collected. Using the Raman spectrum acquisition and processing supporting software LabSpec6 to smooth, baseline, and normalize the obtained Raman spectrum. Multivariate statistical analysis software SIMCA14.1 were applied to import and analyze the obtained Raman spectrum data by principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and other methods for statistical analysis. An operating characteristic curve (ROC) was constructed to evaluate the model analysis effect between serum samples of healthy people and those with gastric cancer. Serum samples from the colorectal cancer group were used to verify the reliability of the model. Results:Six Raman peaks with good repeatability were detected in serum samples in health and gastric cancer group, and peaks were located at 1 001.17, 1 154.63, 1 337.89, 1 446.85, 1 515.33, and 1 658.34 cm -1, respectively. Raman intensities at six Raman peaks were significantly different between healthy and gastric cancer groups. At the displacement of 1 001.17, 1 154.63, and 1 515.33 cm -1, the Raman intensity in the healthy group was higher than that in the gastric cancer group. At 1 337.89, 1 446.85, and 1 658.34 cm -1 displacement, the Raman intensity of the gastric cancer group was higher than that of the healthy group. An OPLS-DA model was constructed to analyze the serum samples of the healthy group and the gastric cancer group. In the model, R 2 is the fitting power, and Q 2 is the predictive ability. The closer the values of R 2 and Q 2 are to 1, the better the performance of the model, and the obtained model's R 2X(cum)=0.809, R 2Y(cum)=0.819, Q 2(cum)=0.758. ROC characteristic curve was drawn based on the OPLS-DA model. The area under the curve (AUC) of the gastric cancer group was 0.998. Six peaks with good repeatability were detected in the serum Raman spectra of gastric cancer stage Ⅰ, Ⅱ, Ⅲ, and Ⅳ, which were located at the displacement of 1 001.85, 1 155.07, 1 338.36, 1 445.75, 1 515.92, and 1 657.68 cm -1, respectively, and at the displacement of 1 155.07 and 1 515.92 cm -1. The Raman intensity of gastric cancer stage Ⅳwas significantly higher than that of gastric cancer stages Ⅰ, Ⅱ, and Ⅲ. Conclusions:According to the model reliability verification, the healthy group, gastric cancer group and colorectal cancer group can also be effectively separated based on OPLS-DA results; it showed a good performance in separating the healthy group from the gastric cancer group. It is possible to detect serum samples from healthy people and gastric cancer patients unlabeled by combining Raman spectroscopy and the OPLS-DA method in multivariate statistics.

The technical development and application prospects of CRISPR/Cas9 in laboratory medicine / 中华检验医学杂志

Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9),a cluster of regularly spaced short palindromic repeats,is a natural immune defense system for bacteria and archaea to identify themselves and exogenous invading DNA fragments,protecting them from viruses.In recent years,CRISPR/Cas9 has become a revolutionary gene editing tool.Its specific targeted spot-cutting ability also plays an important role in nucleic acid detection,bacterial typing,etc.,and has shown great application potential in the field of medical testing.Based on the latest researches,this paper reviews the progress of CRISPR/Cas9 application in the new techniques of nucleic acid detection,pathogen typing and bacterial evolution in laboratory medicine,and also summarizes the application prospect of CRISPR technology in the field of laboratory medicine.

Study and clinical application of wild-type blocking probe for improving trace amount mutation detection of BRAF V600E gene / 国际检验医学杂志

Objective To study the screening of trace amount mutation of BRAF V 600E gene for avoiding the appearance of ineffective treatment in cancer patients .Methods The internal competitive amplification fragments were used to improve the inhibition of wild-type blocking (WTB) probe on wile-type BRAF V600E gene to increase the detection efficiency of BRAF V600E genotype of trace amount mutation occurrence .Re-sults When the template DNA concentration was 50 -200 ng/μL ,the constructed trace amount gene muta-tion real time fluorescence quantitative detection method could completely block the amplification of the wild-type BRAF V600E gene .The sensitivity of this assay reached as high as 0 .1％ ,which was in line with the sen-sitivity requirement for the gene trace amount mutation detection technique .In the colorectal biopsy tissues from 50 cases of suspected colorectal cancer ,8 cases (16 .0％ ) of BRAF V600E gene trace amount mutation were detected by using this constructed method ,which had higher detection rate .Conclusion The constructed gene trace amount mutation detection method can make the rapid ,simple and low cost quantitative analysis for BRAF V600E gene trace amount mutation in clinical samples .

Application of terahertz spectroscopy in the detection of cell and tissue / 中华检验医学杂志

As a novel,label-free and non-invasive detection modality,terahertz(THz,1THz=1012 Hz)spectroscopy has been widely used in various areas.For instance,in the biomedical field,it has great potentials to provide real-time scanning of living cells and tissues due to its unique advantages.Significant achievements have been reached in cell detection, related to bacterial identification, cancer cell characterization and blood cell detection.In tissue detection, the THz spectroscopy can be used to provide real-time scanning of living tissues and fast diagnosis.Furthermore, a single system which integrated THz spectroscopy and THz imaging would be able to collect information more sensitive and comprehensive. However,the clinical adaption of THz spectroscopy is still a controversial issue attributed to some intrinsic limitations and technical bottlenecks.In this article, both the application of THz spectroscopy in cell and tissue detection and the existing challenges and strategies to accelerate clinical applications were reviewed comprehensively.

Application of Raman spectroscopy in the rapid detection of pathogenic microorganisms / 中华检验医学杂志

With the rapid development of instrument technology , Raman spectrometer has become one of the fastest growing type of instrument in the molecular spectroscopy . In recent years , Raman spectrometer gradually emerging in the application in the domain of biology and medicine , Raman spectroscopy technology appears new development constantly in the rapid identification and classification of microorganisms because of its rapid , efficient, sensitive, noninvasive, repeatability and other unique advantages.This article describes its application in the rapid detection of bacteria , viruses and other microorganisms , and also prospects for the application of Raman spectroscopy in future clinical work .

Role of TPMT Genetic Polymorphism in the Individualized Treatment of Thiopurine Drug / 现代检验医学杂志

Thiopurine S-methyltransferase (TPMT) is an important and key cytoplasmic enzyme in the metabolism of thiopurine drugs,whose activity can directly determine the amount of thiopurine drugs metabolized to cytotoxic 6-thioguanine nucleotides and consequently influence clinical efficacy and adverse drug reactions of thiopurine drugs.In order to deepen knowledge and role of genetic polymorphism of tpmt in the individualized thiopurine drug treatment,this present review mainly covered the following three frequently concerned aspects,including i) whether or not to determine the activity of TPMT priot to treatment of thiopurine drugs;ii) to genotype or to phenotype;iii) how to choose genotype methods.

Analysis of distribution of 15249 strains of nosocomial infection pathogens and drug resistance / 国际检验医学杂志

Objective To monitor the distribution of pathogens and the drug resistance of inpatients in Southwest Hospital in ChongQing ,and to analyze the prevalence of pathogens in various departments .Methods A total of 15249 pathogens cultured from clinical specimens in our hospital in 2016 and the antimicrobial susceptibility testing results were retrospectively analyzed .The anti-microbial susceptibility testing was carried out by using the paper diffusion method (K-B) or the automated instrument method (MIC) .The data were analyzed by WHONET5 .5 software according to the standard of CLSI2017-M100 .Results Compared with 2015 ,a total of 15249 pathogenic microorganisms were isolated from the hospital in 2016 ,among which 9742 were Gram-negative bacteria ,down 11 .45% and 4188 were Gram-positive bacteria ,up 15 .34% and 1319 were fungi ,down 11 .48% .Top five depart-ments which collected most microbiological culture specimen were Burn ,Pediatric ,ICU ,Hepatobiliary surgery ,Neurosurgery .The main specimen type of culture was sputum ,accounting for 27 .28% ,followed by blood ,wound secretion ,urine ,abdominal fluid .The first five pathogens were Klebsiella pneumoniae ,Acinetobacter baumannii ,Escherichia coli ,Staphylococcus aureus ,Pseudomonas aeruginosa .Conclusion Gram-negative bacteria are mainly infected organisms in our hospital .However ,Gram-positive bacteria are also important pathogens .Antibiotics should be selected according to the results of antimicrobial susceptibility testing .The distribu-tion of pathogens and the changes of drug sensitivity should be emphasized ,which can provide an effective theoretical basis for treat-ment .

The exploration and practice of practice teaching for laboratory medicine students / 中华医学教育探索杂志

With the goal of practice teaching for laboratory medicine students, we focus on culti-vating students' clinical thinking and scientific thinking through setting up reasonable teaching task and designing the scientific graduation thesis, and try to develop a novel practice teaching mode for laboratory medicine students. Therefore, we do exploration and practice in the following aspects: orientation training, clinical practice, medical ethics education, clinical communication ability, and scientific research design, hoping to improve students' research thinking ability and innovation ability. Through actual operation and graduation thesis writing, we try to help students to establish and improve their clinical thinking ability and innovation ability.

Exploration of the course construction of clinical biochemistry under the new medical model / 中华医学教育探索杂志

Clinical biochemical test course construction in the new medical model, first of all, requires a combination of subject characteristics of the course, and proceeds with the improvement from the teaching methods and evaluation system. Secondly, we should cultivate students' doctor-patient communication skills and humanities quality in the teaching process. Finally, we should establish the effective clinical biochemical test teaching mode to achieve both teaching and learning.

New areas of laboratory medicine in the 21st century:terahertz laboratory medicine / 中华检验医学杂志

The terahertz laboratory medicine (THz-LabMed) is an intergrated cross-frontier field which involves multi-disciplinary including medicine,biology,biomedical engineering,physics,optics,computer science,information and materials.Using THz technologies for label-free detection and analysis of biomedical macromolecules,cell and tissues,the THz-LabMed is also the core components of terahertz biomedine (THz-BioMed) which focuses on the biomedical application of THz technologies.THz label-free detection is being paid more and more attention because of its unique advantages worldwide and becomes a hot spot for the application of THz wave technology and methods in life science.Application of THz technology in biomedicine involves many fields globally,including disease diagnosis,recognition of protein status,label-free DNA sequencing,mechanism for absorption differences of biological tissue to THz wave,and radiation influence on biological samples and biological process.THz-LabMed is the global synchronized research in THz-BioMed field in China.It is important to seize the opportunities to develop new disciplines of laboratory medicine.

Analysis of glycosylation hemoglobin by capillary electrophoresis / 国际检验医学杂志

Objective To discuss the application value of the capillary electrophoresis in detecting the glycosylation hemoglobin (HbA1c)level.Methods By utilizing the uncoated fused silica capillary column(30 cm in length and 25 μm in diameter)and the specific adsorption peak,the HbA1c levels in the healthy people,patients with diabetes mellitus(DM)and high risk people were de-tected by the photodiode array(PDA)method at the wavelength of 415 nm.During the research process,the precision and accuracy were perfomred the accuracy analysis.Results HbA1c could be effectively separated from non-HbA1c,with a sharp peak.And the relative quantity of HbA1c could be determined.Conclusion The capillary electrophoresis is characterized by specificity,high precision,and high accuracy.Therefore,it should be widely applied in clinic.

Rapid detections of OprD2 gene in Pseudomoas aeruginosa using loop-mediated isothermal amplification / 中华检验医学杂志

Objective To establish a rapid detection approach by visual interpretation directly for OprD2 resistance gene of Pseudomonas aeruginosa based on the Loop-mediated isothermal amplification (LAMP),and provide a quick and effective method for clinical monitoring of Pseudomonas aeruginosa strains.Methods Totally 47 strains of Pseudomonas aeruginosa collected from December 2011 to June 2012 in Southwest Hospital of microorganisms were prospectively studied.Four LAMP primers (two inner,two outer) were designed according to the six zones of the OprD2 gene of Pseudomonas aeruginosa.A positive reaction is indicated by the color change after adding an intercalating dye (hydroxy naphthol blue) to the reaction solution.This method was used to detect and analyze the distribution of OprD2 resistance gene in 47 strains of Pseudomonas aeruginosa and its coirelation with antibiotic resistance.Results The LAMP assays showed 100％ specificity for the OprD2 gene,and the sensitivity (with the lowest detection limits of 17.414 μg/L) was 10-fold higher than that of conventional PCR assays.The OprD2 gene was negative in 23 strains by both conventional PCR and LAMP.In OprD2 negative strains,the resistance rate of cefotaxime,levofloxacin,aztreonam,piperacillin,imipenem and meropenem was 100％ (23/23),57％ (13/23),48％ (11/23),48％ (11/23),48％ (11/23) and 43％ (10/23).Compared with the OprD2 positive strains,statistical analysis showed that the resistance rate of imipenem,levofloxacin and meropenem in OprD2 negative strains increased significantly (chisquare value is 9.155,4.846,4.037,P value was 0.002,0.028,0.045,and so there was significant difference).Conclusions The established LAMP approach in this study enables rapid,sensitive and specific detection of OprD2 gene in Pseudomonas aeruginosa by visual interpretation.Deficiency of OprD2 gene confers Pseudomonas aeruginosa a basal level of resistance to carbapenems especially to imipenem.The identification of OprD2 gene distribution in Pseudomonas aeruginosa is helpful to the selection of antimicrobial agents in the infection treatment.

The improvement of cardiopulmonary resuscitation (CPR) efficiency by rescue team through the clinical access to pre-hospital care / 中华急诊医学杂志

Objective To explore the improvement of cardiopulmonary resuscitation (CPR) efficiency by rescue team through the clinical access to pre-hospital care.Methods Mter establishment of clinical approaches to cardiac arrest,the training program of first line personnel of rescue teams in the Hangzhou Emergency Center was carried out with practice on simulated patients and scenario.A total of 45 eligible teams were randomly enrolled for study by observing the performance of some essential resuscitation techniques before and after training.Result The efficiency of resuscitation performed by rescue team for cardiac arrest was generally not good enough before training evidenced by the shortage of application of ECG monitoring,endotracheal intubations and establishing intravenous line which were only 8 (17.8％),5(11.1％),6 (13.3 ％),respectively,and the interruption time of chest compression during the first three minutes was (102.13 ± 13.68) seconds and the successfully artificial respiration ratios by assistant members was (0.37 ± 0.09),and ratios of ECG forensics and written inform consent were 8 (17.8％) and 6 (13.3％) respectively,CPR and forensics done simultaneously was only 2 (4.4％).The efficiency of rescue for cardiac arrest was obviously improved after training by the clinical approaches proved by the increase in application of ECG monitoring,endotracheal intubations,intravenous line set up reached to 45 (100％),43 (95.6％),43 (95.6％),respectively,and the interruption time of chest compression during the first three minutes was shorten to (69.7 ± 7.7) seconds and the successfully artificial respiration ratios done by assistant members was (0.57 ±0.12) after training.The ratios of on-site ECG forensics and written inform consent were 40 (88.9％) and 43 (95.6％),respectively,and CPR and evidence obtained simultaneously was up to 36 (80.0％).The efficiency of work done by teams was obviously improved and the risk of miserable events was controlled.Conclusions The clinical approaches to cardiac arrest in prehosptial care is the efficient strategy to rescue the patient with cardiac arrest and it is worthy to popularize at present.

Teaching reform on laboratory diagnostics for eight-year-program clinical medicine majors at undergraduate level / 中华医学教育探索杂志

Supported by Center of Laboratory Medicine of PLA,the Third Military Medical University launched teaching reform on laboratory diagnostics by integrating and optimizing teaching contents,adopting case centered teaching method,introducing thinking modes of evidence-based laboratory medicine and strengthening bilingual teaching.Abilities of students to determine the clinical stage and to evaluate the effect of treatment as well as prognosis of diseases were promoted.

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.

Loss of heterozygosity on multiple chromosomes in patients with esophageal cancer and its clinical significance / 中华检验医学杂志

Objective To investigate the loss of heterozygosity at 17 microsatellites of 10 chromosome arms in 68 resected specimens of esophageal cancer, and the relationship to the clinicopathological phenotypes of patients. Methods 68 tumor specimens (20 well-differentiated squamous carcinomas, 30 moderately differentiated carcinomas and 18 poorly differentiated carcinomas) and their matched blood samples were analyzed for LOH at 17 microsatellites by using PCR and fluorescence-based DNA sequencing technology, and the association of LOH with the clinicopathological phenotypes of patients was compared statistically. Results The lowest detection frequency of LOH in our subjects was observed at D8S261 with 33. 3%, and the highest frequency was at D9S125 with 85. 2%. There were 12 markers with the frequency of LOH higher than 50.0%, and 3 markers (D3S1597, D3S1285 and D9S125) with the frequency higher than 75. 0%. There was a significant difference in the frequency of LOH at D9S111 and D13S153 between tumors with different histological grades. LOH at D9S111 was observed in 2 of 12 tumors with well differentiation in 14 of 20 tumors with moderate differentiation, and in 14 of 16 tumors with poor differentiation. LOH at DI3S153 was observed in 2 of 8 tumors with well differentiation, in 12 of 28 tumors with moderate differentiation, and in 11 of 12 tumors with poor differentiation. There was a significant difference in the frequency of LOH at D8S261 between tumors with lymph node metastasis and without lymph node metastasis. LOH at D8S261 was found in 1 of 14 tumors with lymph node metastasis, and in 12 of 22 tumors without lymph node metastasis. Conclusions The widespread and frequent loss of heterozygosity may exist in esophageal cancer, and the candidate genes located in the site of frequent LOH may be involved in the development of this cancer; LOH at D9S11 and D13S153 are more commonly observed in the patients with higher histological grades, the tumors with LOH at D8S261 may have a low tendency to lymph node involvement.

Detecting Micro-luminescence of 330 Codon in emb gene of Mycobacterium tuberculosis by Molecular Beacon Hybridization / 中华医院感染学杂志

OBJECTIVE To design molecular beacon detecting embB330 codon of ethambutol-resistant Mycobacterium tuberculosis(MTB),meanwhile,and try to detect fluorescence of mutation site of embB330 codon in liquid by fluorescence microscope by compareing the mutation strains and standard strains.METHODS The software,Beacon designer,was used to design molecular beacon detecting embB330codon and detecting fluorescence signal from hybridization between the amplified product and probe by fluorescence microscope,and to confer to the sequencing results.RESULTS The difference between PCR products from standard strain and ethambutol-resistant one was obvious in detecting the fluorescent light by use of fluorescence microscope.We detected fluorescent light signal between the 33 ethambutol-resistant strains and 10 H37RV standard strains.The rate of ethambutol-resistant strains was about 3%,and the rate of sequencing was about 3%.CONCLUSIONS The technology of molecular beacon effectually can detect mutation single base site of embB330codon.Fluorescence microscope owns characteristics such as high sensitiveness to detect the fluorescent light.

Surveillance of Antimicrobial Resistance and Classification in Non-fermenting Bacteria Isolated from Lower Respiratory Tract for 3 Consecutive Years / 中华医院感染学杂志

OBJECTIVE To investigate the distribution and resistance of non-fermenting bacteria isolated from our hospital and offer the basis for the treatment of bacterial infection.METHODS Totally 2177 strains of non-fermenting bacteria were isolated from clinical sputum samples between Jan 2006 and Dec 2008.All of the isolated bacteria were identified with API identified test(API Inc,France) and Kirby-Bauer(K-B) test used for the antibiotics susceptive test.RESULTS From them the most common bacteria were Pseudomonas auruginosa(34.9%),followed by Acinetobacter baumannii(28.4%) and Stenotrophomonas maltophilia(11.4%).These bacteria had various resistances to the tested antibiotics.CONCLUSIONS Non-fermenting bacteria have high isolate rate and multi-antibiotic resistance,so antibiotics should be correctly used under the guidance of antibiotic susceptibility test.

Distribution and Drug-resistance of ESBLs Produing Gram-negative Bacteria in Blood Culture / 中华医院感染学杂志

OBJECTIVE To study the constituent ratio of the Gram-negative bacteria of blood culture in recent 36 months and their resistance.METHODS Blood culture of patients in our hospital was performed by BacT/Alert 3D and the isolated bacteria were identified by API identified tests(API Inc.France) and additional antibiotics sensitivity test by Kirby-Bauer(K-B).RESULTS Of the 13766 specimens,1468(10.6%) were positive,including 734 Gram-negative bacteria strains(50.0%),476 Gram-positive bacteria strains(32.4%),233 fungi strains(15.9%),and 25 others strains(1.7%).CONCLUSIONS It is important and necessary to monitor the circumstance of the Gram-negative bacteria of blood culture.