Table of Content

30 March 2016, Volume 31 Issue 3

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Orginal Article

Improving laboratory diagnosis ability of infectious diseases in children using multiple assays

XU Jin.

2016, 31(3):  155-158.  DOI: 10.3969/j.issn.1673-8640.2016.03.001

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Infectious disease is a major problem for children' s health worldwide. Early and accurate identification and diagnosis of pathogens play a very important role for the prevention and control of infectious diseases,especially for the control of antibiotics and anti-viral medicine abuse. In order to improve the laboratory ability for the diagnosis of infectious diseases in children,a variety of clinical and on-site diagnostic systems should be developed for the rapid determination of pathogens or host biomarkers by utilizing multiple techniques and assays,and the molecular epidemiological characteristics of pathogen genotype and its possible relationship with pathogenesis should be deeply investigated.

Research progress of cytomegalovirus envelope glycoprotein B,H and N and their genotypes

DONG Niuniu, XU Jin

2016, 31(3):  159-162.  DOI: 10.3969/j.issn.1673-8640.2016.03.002

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Cytomegalovirus （CMV） infections are very common in human beings. Most of the CMV infections are asymptomatic,however, symptomatic CMV infections in children can cause serious complications. CMV envelope glycoprotein is the main component of the outermost lipid bilayer membrane of CMV and plays an important role in CMV entry and spread between cells. The current situation and prospect of the relationship between gene subtypes and 3 envelope glycoproteins [glycoprotein B（gB）,glycoprotein H（ gH ） and glycoprotein N（ gN ）] are reviewed and summarized in this review,in order to provide new ideas for CMV studies.

Detection of herpes simplex virus ⅠRNA using simultaneous amplification and testing

CAO Lingfeng, SU Liyun, SHI Peng, DONG Niuniu, XU Jin

2016, 31(3):  163-167.  DOI: 10.3969/j.issn.1673-8640.2016.03.003

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Objective To establish a rapid and reliable simultaneous amplification and testing （SAT） for detecting herpes simplex virus Ⅰ（HSV-1）. Methods The specific primers were designed,and the probes were optimized according to RNA sequences in open reading frame Us5 region of HSV-1 from GenBank,and HSV-1 RNA was amplified by M-MLV reverse transcriptase and T7 RNA polymerase. The fluorescence signals were collected and determined by fluorescence quantitation polymerase chain reaction （PCR）. A total of 212 clinical specimens （150 cerebrospinal fluid specimens and 62 plasma specimens） were collected from child patients,who were diagnosed or suspected with viral meningitis,and they were determined. Using nested PCR plus sequencing as reference method,the Kappa consistency analysis was performed on the SAT results. Results The sensitivity of HSV-1 RNA by SAT was 100 copies/μL. A total of 18 specimens were positive by SAT, among which 16 specimens were positive by nested PCR,and 12 specimens were positive by sequencing. The Kappa value between SAT and nested PCR plus sequencing was 0.785,and the sensitivity and specificity were 100.0% and 97.0%,respectively. Conclusions The established SAT is a sensitive,specific,accurate,rapid and reliable method for detecting HSV-1 RNA in cerebrospinal fluid and plasma,and it should be a potential method in rapid diagnosis of herpes simplex encephalitis.

Establishment of nucleic acid sequence-based amplification for detection of respiratory syncytial virus

YANG Haiou, YE Xingchen, FU Qihua

2016, 31(3):  168-172.  DOI: 10.3969/j.issn.1673-8640.2016.03.004

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Objective To establish a nucleic acid sequence-based amplification （NASBA） to detect respiratory syncytial virus（RSV）. Methods According to the alignment results from the National Center for Biotechnology Information （NCBI） gene databank,the specific primers for RSV were designed by Primer 5 software. Throat swab samples being positive for RSV by immuno-fluorescence assay were collected,and the corresponding negative samples were as controls. Through optimizing the sample process and amplification system, NASBA for detecting RSV was established and compared to reverse transcription polymerase chain reaction （RT-PCR）. Results Direct freeze-thawing throat swab samples were optimal for the sample process,and the supernatant was as the target for detecting. The specific primers were added with T7 promoter sequence. NASBA could detect the standard RSV RNA at 2.81×10² copies/µL,but RT-PCR could only show blurry products even up to 2.81×10⁴ copies/µL,which indicated that the sensitivity of NASBA assay was 100 times at least higher than that of RT-PCR. NASBA could detect RSV RNA in all of the 7 throat swab samples,while they showed negative in samples,which were positive for other viruses（2 cases of influenza A virus,3 cases of parainfluenza virus and 1 case of adenovirus）and negative RSV samples （3 cases）. These results indicated that NASBA had high specificity. Conclusions The established NASBA is a highly specific,sensitive and time-saving method,overcoming the limitations of current detections for RSV. It is an effective method to detect RSV.

Serum amyloid A and C-reactive protein combined determination for diagnosis of bacterial infection in neonates

SHEN Weihong, YUE Chaoyan, SUN Zhendong, YING Chunmei

2016, 31(3):  173-175.  DOI: 10.3969/j.issn.1673-8640.2016.03.005

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Objective To study the significance of serum amyloid A（SAA）,C-reactive protein （CRP） and white blood cell count determinations for diagnosis of bacterial infection in neonates. Methods SAA,CRP and white blood cell count were determined in 80 neonates with bacterial infection and 58 healthy neonates （non-bacterial infection group）,and the results were analyzed. Results The concentrations of SAA and CRP in infection group [（67.3±38.4）and （19.2±11.2） mg/L] were significantly higher than those in non-bacterial infection group [（8.7±2.1）and （5.6±2.5） mg/L]（P<0.05）. The white blood cell count was >15.00×10⁹/L in 64 neonates of 80 cases of infection group（80%）, which was higher than that in non-bacterial infection group [24 cases （41%）]. Conclusions SAA and CRP could be used as important markers for monitoring bacterial infection in neonates,and SAA is more sensitive than CRP. The combined determination of SAA and CRP could improve the determination rate of bacterial infection,which provides an experimental reference for early diagnosis and monitoring of bacterial infection.

Significance of interferon-gamma release assay in child tuberculosis infection

ZHONG Huaqing, XU Menghua, LU Lijuan, DU Yajuan, ZHANG Jieqiong, XU Jin

2016, 31(3):  176-179.  DOI: 10.3969/j.issn.1673-8640.2016.03.006

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Objective To evaluate the significance of interferon-gamma （IFN-γ） release assay（IGRA） for diagnosis of tuberculosis infection in child. Methods Mycobacterium tuberculosis-specific antigen [early-secreted antigenic target-6 （ESAT-6）and culture filtrate protein-10 （CFP-10）] was used. By IGRA enzyme-linked immunospot assay（ELISPOT）（T-SPOT）,34 active tuberculosis children and 2 200 non-tuberculosis children were enrolled and determined for IFN-γ levels,and the sensitivity and specificity were evaluated. Some of influential factors were analyzed. Results The sensitivity and specificity of T-SPOT for the diagnosis of active tuberculosis were 73.5% and 99.0%,respectively. The results of T-SPOT had no relationship with sex,age,Bacillus Calmette-Guerin vaccination status,the history of exposure to source case,immunity state and infection position. Conclusion T-SPOT is sensitive,specific and helpful for early and rapid diagnosis of active tuberculosis in children.

Clinical significance of serum pancreatic lipase in nonalcoholic fatty liver disease

XIONG Yongrui, MAO Xiaohong, ZHANG Juanwen, ZHAO Ying, WU Jianping

2016, 31(3):  180-184.  DOI: 10.3969/j.issn.1673-8640.2016.03.007

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Objective To investigate the correlation of serum pancreatic lipase（P-LIP） with nonalcoholic fatty liver disease（NAFLD） and the clinical significance for predicting hepatic fibrosis. Methods A total of 700 NAFLD patients diagnosed by abdominal ultrasonography and 310 healthy subjects （healthy control group）were enrolled,and their body parameters and hematological indices were determined,like height,body mass index,systolic blood pressure,diastolic blood pressure,P-LIP,amylase（AMY）,alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,gamma-glutamyltransferase（GGT）,creatine kinase（CK）,triglyceride（TG）,total cholesterol（TC）,high-density lipoprotein cholesterol（HDL-C）,fasting plasma glucose（FPG）, uric acid（UA）,serum iron（Fe）,sialic acid（SA）,high-sensitivity C-reactive protein（hs-CRP） and white blood cell（WBC）. According to the guidelines for the diagnosis of metabolic syndrome （MS） issued by Chinese Diabetes Society of Chinese Medical Association in 2004,the NAFLD patients were classified into 2 groups,MS group and non-MS group. Spearman correlation analysis was used to evaluate the correlation of P-LIP levels with other parameters. Stepwise regression analysis was used to evaluate different P-LIP levels' risk for advanced hepatic fibrosis [fibrosis index based on 4 factors （FIB-4）≥1.3] in NAFLD patients. Results Compared with healthy control group,NAFLD group had significantly low levels of HDL-C and AMY,and the other parameters increased significantly（P<0.05）. The P-LIP levels in MS group [26（4-208）U/L] were significantly lower than those in non-MS group [30（14-298）U/L] and healthy control group [36（5-201）U/L]（P<0.05）. P-LIP was positively correlated with HDL-C and AMY（r = 0.154 and 0.211,P<0.05）,and it was negatively correlated with ALT,GGT,TG,FPG,UA,SA,hs-CRP and WBC（r = -0.171,-0.167,-0.223,-0.155,-0.201,-0.154 and -0.162,P<0.05）. Stepwise regression analysis showed that serum P-LIP was an independent factor for predicting advanced hepatic fibrosis in NAFLD patients[odds ratio（OR）=2.356,95% confidence interval（CI）：1.235-5.331]. Conclusions Serum P-LIP is closely correlated with NAFLD and could be used as an independent factor for predicting hepatic fibrosis in NAFLD patients.

Clinical significance of the determination of thyroglobulin in diagnosis of differentiated thyroid carcinoma

MAO Minjing, YE Tingjun, PENG Yibing, WANG Xuefeng

2016, 31(3):  185-188.  DOI: 10.3969/j.issn.1673-8640.2016.03.008

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Objective To provide a reference for the diagnosis of metastatic differentiated thyroid carcinoma（DTC）,through determining the level of thyroglobulin（Tg） in washout fluid of fine needle aspirate（FNA） for suspicious cervical lymph nodes.Methods A total of 145 FNA washout fluid samples were collected from suspicious cervical lymph nodes of patients with DTC after surgery by B ultrasound,and 16 FNA washout fluid samples of cervical reactive proliferation lymph nodes were used as control group. The FNA-Tg levels were determined by chemiluminescence immunoassay. Combined with pathological analysis,the difference of FNA-Tg levels between negative lymph nodes（27 cases）and metastasis lymph nodes（118 cases）was analyzed retrospectively. Combined with cytological analysis,the diagnosis efficiencies for different FNA-Tg cut-off values were evaluated. Results The FNA-Tg level in metastasis lymph node group [median（P₂₅-P₇₅）] was 4 163（273-13 138）ng/mL,which was higher than that in negative lymph node group [0.1（0.1-0.2）ng/mL]（P<0.01）. The diagnosis sensitivity of cytological analysis was 83.1%,and the specificity was 100%. For cytological analysis,the diagnosis sensitivities were 96.6% and 91.5%,when the cut-off values were 4.7 and 47.1 ng/L,and the specificities were 92.6% and 100%. Combined with cytological analysis,when the cut-off values were 1,5,10,50 and 100 ng/mL,the sensitivities were 97.5%,97.5%,96.6%,96.6% and 96.6%,and the specificities were 88.9%,92.6%,92.6%,100% and 100%. Conclusions FNA-Tg method is an auxiliary method for the determination of lymph node metastasis in the diagnosis of DTC. When the result of cytological analysis is positive,and FNA-Tg cut-off value is > 5 ng/mL,metastasis is from DTC. When the result of cytological analysis is negative,and FNA-Tg cut-off value is >50 ng/mL,lymph node has DTC metastasis.

Influence of military stress on immune function among recruits

AN Liyun, WANG Fukun, JIA Keran, LIU Yanli, TANG Fei, WANG Xianling, LI Fang

2016, 31(3):  189-194.  DOI: 10.3969/j.issn.1673-8640.2016.03.009

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Objective To investigate the changes of cellular immunity and humoral immunity among recruits before and after military training. Methods A total of 60 recruits participated military training in enlisted military training base, and the symptom checklists（SCL-90） were used to scale their psychological symptoms before and after military training, respectively. Their serum immunoglobulin and complement function were determined, and natural killer（NK） cell, B lymphocyte, CD4⁺ T lymphocyte, CD8⁺ T lymphocyte, interleukin 2（IL-2） and interleukin 10（IL-10） levels were determined. Results The SCL-90 total score,somatization, depression and paranoia score after military training increased significantly than those before military training（P<0.05）. The levels of serum IgG, IgA and IgM, B lymphocyte, NK cell ,peripheral blood CD4⁺ T lymphocyte, CD8⁺ T lymphocyte and CD4⁺/CD8⁺ ratio decreased significantly （P<0.05 or P<0.01）. The level of peripheral blood IL-2 decreased after military training（P<0.05）, and the level of peripheral blood IL-10 increased significantly （P<0.05）. The helper T lymphocyte（Th）1/Th2 ratio after military training was significantly higher than that before military training（P<0.05）. ConclusionsMilitary stress can cause body's immune dysfunction among recruits.

Change and significance of peripheral blood lymphocyte immunophenotype in cutaneous T-cell lymphoma

YOU Ran, GONG Yan, QU Chenxue, WANG Yang, YUAN Jiaying, LU Yao, TANG Xiaoling

2016, 31(3):  195-200.  DOI: 10.3969/j.issn.1673-8640.2016.03.010

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Objective To investigate the application significance of peripheral blood lymphocyte immunophenotype in cutaneous T-cell lymphoma. Methods Peripheral blood lymphocyte immunophenotype was analyzed by flow cytometry and morphology in 52 patients diagnosed as skin disorders [31 cases of cutaneous T-cell lymphoma（malignant skin disorder group）and 21 cases of inflammatory skin disorders（benign skin disorder group）]. The characteristics of lymphocyte subset,immunophenotype and morphology between benign and malignant skin disorder groups were compared. Results CD4/CD8 double negative T lymphocyte in malignant group was significantly higher than that in benign group （P < 0.05）,and B lymphocyte percentage in malignant group was significantly lower than that in benign group （P < 0.05）. In malignant group,T lymphocyte percentage,CD4/CD8 ratio and CD4/CD8 double negative T lymphocyte percentage in Sézary syndrome subgroup were higher than those in benign group and other malignant skin disorder subgroups （P < 0.05）,meanwhile B lymphocyte and natural killer（NK） cell percentage decreased （P < 0.05）. There were 14 abnormal cases of malignant group（45.1%） and 5 abnormal cases of benign group（23.8%） for immunophenotype. There were 15 abnormal cases of malignant group（48.4%） and 6 abnormal cases of benign group（28.6%）for morphology. Conclusions Peripheral blood lymphocyte immunophenotype can provide a reference for the diagnosis of cutaneous T-cell lymphoma,and it is helpful with the differential diagnosis.

Significance on smear Gram staining microscopy of sputum through nose sampling among children for the early diagnosis of community-acquired pneumonia

KONG Changsheng, CHEN Jun, QI Qiaoli, ZHU Lingna, ZHANG Yi, ZOU Xiaoyan

2016, 31(3):  201-204.  DOI: 10.3969/j.issn.1673-8640.2016.03.011

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Objective To evaluate the significance of smear Gram staining microscopy of sputum through nose sampling among children for the early diagnosis of community-acquired pneumonia（CAP）. Methods From January 2015 to March 2015,596 sputum specimens were collected through nose sampling among children suspected with CAP,and were performed culture and smear Gram staining microscopy. The results of the 2 methods were compared. Results A total of 596 sputum specimens were detected,389 sputum specimens were qualified,and the qualification rate was 65.27%. A total of 368 pathogenic strains were isolated,and the culture positive rate was 61.74% （368/596）. Moraxella catarrhalis,Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureuswere the top 4 species of pathogenic strains. The coincidence rate between the results of the 2 methods was 86.24%（514/596）,and there was no statistical significance between the 2 methods （P=0.845 0）. Conclusions Smear Gram staining microscopy of sputum through nose sampling among children is economical and fast,which could provide important information among children for the early diagnosis and treatment of CAP.

Application significance of S100B in the assessment of blood-brain barrier injury

PAN Zhiguang, QIN Jiaqian, WU Jiong, SONG Binbin, GUO Wei

2016, 31(3):  212-216.  DOI: 10.3969/j.issn.1673-8640.2016.03.014

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Objective To evaluate the performance of serum S100B determination kit based on electro-chemiluminescence immunoassay,and to investigate preliminarily the correlation between serum S100B concentration and the severity of blood-brain barrier injury. Methods The precision was validated according to the Clinical and Laboratory Standards Institute （CLSI） EP-15A protocols. Linearity and accuracy were also validated. A total of 45 patients with blood-brain barrier injury were enrolled. Serum S100B concentrations were determined. The patients were classified into 4 groups according to quotient albumin（QALB）results [<the first quartile（P₂₅）,P₂₅-<the second quartile（P₅₀）,P₅₀-<the third quartile（P₇₅） and ≥P₇₅] ,<P₂₅（low QALB group,11 cases）,P₂₅-<P₅₀ （middle QALB group,11 cases）,P₅₀-<P₇₅ （high QALB group,11 cases） and ≥P₇₅（extremely high QALB group,12 cases）. The correlation between S100B and the severity of blood-brain barrier injury was observed. Results The within-run coefficients of variations（CV） of 3 concentrations （S1 = 0.05 µg/L,S2 = 0.12 µg/L and S3 = 2.75 µg/L）were 5.08%,3.00% and 5.23%,and the between-run CV were 5.07%,3.27% and 5.46%, respectively. Relative biases were 5.26% and 0.41%,respectively. The linear range was 0.01-36.00μg/L, which indicating a satisfied linearity （Y = 0.999X + 0.003,R²= 0.999）. The S100B concentration in low QALB group was significantly lower than that in extremely high QALB group （P = 0.04）,and there was no statistical significance between the other 2 groups（P > 0.05). Conclusions Serum S100B determination kit based on electro-chemiluminescence immunoassay exhibits satisfied clinical performance,which is suitable for current clinical application. Serum S100B concentration could reflect the severity of blood-brain barrier injury,and assist clinicians to evaluate the disease status for patients.

Analysis of the combined application of different syphilis serological tests

ZHONG Xiaofen, MAO Lingzhe

2016, 31(3):  217-219.  DOI: 10.3969/j.issn.1673-8640.2016.03.015

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Objective To evaluate the significance of the combined application of different syphilis serological tests. Methods A total of 285 patients with syphilis were enrolled as syphilis group,and 150 patients without syphilis at the same period in Department of Surgery were enrolled as control group. Their serum samples were determined by treponema pallidum-enzyme-linked immunosorbent assay（TP-ELISA）,rapid plasma reagin（RPR）test and treponema pallidum particle assay（TPPA）. The significance of 3 single determinations and combined determination was compared. Results For primary syphilis,secondary syphilis,latent syphilis and control groups,the positive rates of these 3 methods had statistical significance（P < 0.05）. The sensitivities of TP-ELISA,RPR test and TPPA were 93.68%,82.46% and 97.54%. The specificities were 99.33%,98.00% and 100.00%. The accuracies were 95.63%,87.82% and 98.39%. The combined determination improved the sensitivity and accuracy,and the combined determination of TP-ELISA,RPR test and TPPA had the highest sensitivity and accuracy（98.95% and 99.31%）. Conclusions The combined determination of different syphilis serological tests can improve the accuracy in syphilis determination. For ensuring the accuracy of clinical diagnosis with no misdiagnosis and missed diagnosis,it is advisable to screen specimens by TP-ELISA in clinic and retest the suspected specimens by the combined determination of RPR test and TPPA.

Correlation of AS160 phosphorylation with tumor cell proliferative activity in primary breast cancer

JIANG Xiaohua, WEI Yu, SUN Jianwen, REN Konghua, CUI Beinian, WANG Wei, CHENG Yi

2016, 31(3):  220-223.  DOI: 10.3969/j.issn.1673-8640.2016.03.016

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Objective To investigate the correlation of phosphorylation AS160 （Thr642） regulation with tumor cell proliferative activity in breast cancer tissue. Methods The clinical data of 152 patients with breast cancer in 85 Hospital of People's Liberation Army from January 2003 to January 2015 were analyzed retrospectively. Immunohistochemical method was used to determine phosphorylation AS160（Thr642） and proliferation Ki-67（MIB-1） levels. Mitotic index was evaluated,and the expression characteristics of phosphorylation AS160（Thr642） was investigated in breast cancer cell line MDA-MB-231. The correlations of phosphorylation AS160（Thr642） with mitotic index and Ki-67（MIB-1） were analyzed by Spearman nonparametric test. Results The phosphorylation AS160 （Thr642） was positively correlated with mitotic index （r=0.493,P<0.001） and Ki-67 （MIB-1） （r=0.472,P<0.001）. The phosphorylation AS160（Thr642） in mitotic tumor cells was strongly positive in cell line and breast cancer tissue. Conclusions The phosphorylation AS160 is correlated with tumor cell proliferative activity and might be useful as a marker and a potential treatment target for breast cancer.

Decreased beta-lactam antibiotic susceptibility caused by outer membrane protein 38 deletion in Acinetobacter baumannii

WANG Dairong, LI Xi, ZHU Hong, WANG Aihua

2016, 31(3):  224-227.  DOI: 10.3969/j.issn.1673-8640.2016.03.017

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Objective To investigate the influence of outer membrane protein 38（Omp38） on the susceptibility of beta-lactam antibiotic in Acinetobacter baumannii. Methods The isolate with Omp38 deletion was induced by in vitro experiment. The contribution of mutation to beta-lactam antibiotic susceptibility was validated by complementation experiment. The cell membrane potential change of isolates was determined by flow cytometry. The growth rates were determined in order to evaluate mutant gene fitness cost. Results The Omp38 was confirmed to be correlated with the decreased beta-lactam antibiotic susceptibility. The Omp38 decreased cell membrane potential,and the fitness cost of Omp38 was approximately 3% through measuring growth rates（P< 0.05）. Conclusions Omp38 is deemed to play an important role in beta-lactam antibiotic resistance in Acinetobacter baumannii. The minor fitness cost of Omp38 may be a significant reason for the development of beta-lactam antibiotic resistance in Acinetobacter baumannii.

Clinical application of serum folate and red blood cell folate determinations

CHEN Pu, PAN Baishen

2016, 31(3):  232-236.  DOI: 10.3969/j.issn.1673-8640.2016.03.019

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Folate deficiency is closely related to neural tube defect in newborns and megaloblastic anemia. The determinations of serum folate and red blood cell folate could be primary evidence for folate deficiency. However,the determination methods have not yet been standardized. This article will describe the biological characteristics of folate,the causes of folate deficiency,its epidemiological study,clinical manifestation,diagnostic cut-off values and the clinical application of folate determinations.