酶联免疫斑点术和流式细胞术检测γ干扰素对结核性胸膜炎的辅助诊断价值

目的 探讨胸腔积液PEMC中CD+4T淋巴细胞分泌或产生IFN-γ的检测对结核性胸膜炎的辅助诊断价值.方法 分离40例结核性胸腔积液(结核组)和30例恶性胸腔积液患者(疾病对照组)PEMC,经克隆表达的早期分泌抗原靶6(ESAT-6)和培养滤液蛋白10(CFP-10)融合蛋白(简称"E/C")刺激后,用ELISpot检测分泌IFN-γ细胞的斑点形成细胞(SFC)数量和流式细胞术(FCM)结合胞内细胞因子染色检测产生IFN-γ细胞比率,并比较这2项指标在结核组和疾病对照组间的差异,同时评价2种方法检测IFN-γ对结核性胸膜炎的辅助诊断效能.结果 PEMC经E/C刺激后用ELISpot检测结核组SFC数量为205(125 ～450)SFC/5×104PEMC,疾病对照组为5(2～18)SFC/5×104PEMC,两组间的差异有统计学意义(U=20.00,P<0.01);FCM检测结核组CD+4T淋巴细胞产生IFN-γ细胞比率为3.27%(1.81%～7.34%),疾病对照组为0.12%(0.06%～0.46%),两组间的差异有统计学意义(U=45.00,P<0.01).ELISpot检测PEMC经E/C刺激后分泌IFN-γ的敏感度为92.5%(37/40)、特异度为80.0%(24/30)、阳性预测值为0.86、阴性预测值为0.89、阳性似然比为4.63和阴性似然比为0.09,准确性为87.1%;FCM检测经E/C刺激后产生IFN-γ的敏感度为87.5%(35/40)、特异度为90.0%(27/30)、阳性预测值为0.92、阴性预测值为0.84、阳性似然比为8.75和阴性似然比为0.14,准确性为88.6%.结论 经E/C刺激后,用FCM和ELISpot检测CD+4T淋巴细胞产生和分泌IFN-γ细胞的方法对结核性胸膜炎诊断的敏感度和特异度高,且对结核性胸膜炎有辅助诊断价值.

Abstract：

Objective To explore the value of IFN-γ produced or secreted by CD+4 T Lymphocytes from pleural effusion mononuclear cells for the diagnosis of tuberculous pleurisy(plTB).Methods The PEMCs of 40 patients with tuberculous pleural effusion and 30 patients with malignancy pleural effusion were selected as the tuberculosis and disease control groups, then co-cultured with the early secretory antigenic target 6 (ESAT-6) and culture filtered protein 10 (CFP-10) fusion protein (E/C).The numbers of spot forming cells(SFC) secreting IFN-γ were enumerated by ELISpot and the ratios of cells producing IFN-γ were detected by flow cytometry and intracellular cytokine staining.Moreover, the two indicators were compared between tuberculosis and disease control groups to evaluate the 2 methods detecting IFN-γ in the diagnosis of plTB.Results After E/C stimulation, the numbers of SFC were 205(125-450)SFC/5×104 PEMC in tuberculosis group and 5(2-18)SFC/5×104 PEMC in disease control group by ELISpot.The difference between two groups was statistically significant (U= 20.00, P<0.01).The proportion of IFN-γ-secreting CD+4 T lymphocytes was 3.27% (1.81%-7.34%) in tuberculosis group and 0.12% (0.06%-0.46%) in control group detected by FCM. The difference between the two groups was statistically significant (U=45.00, P<0.01).The indicators of ELISpot in detection of IFN-γ which was secreted by PEMC after co-cultured with E/C were as follows: sensitivity 92.5% (37/40), specificity 80.0% (24/30), positive predictive value 0.86, negative predictive value 0.89, positive likelihood ratio 4.63, negative likelihood ratio 0.09 and accuracy 87.1%;and for FCM, they were 87.5% (35/40), 90.0% (27/30), 0.92, 0.84, 8.75 and 0.14, respectively and accuracy 88.6%.Conclusion After E/C stimulation, the assay for IFN-γ-secreting CD+4 T lymphocytes by FCM and ELISpot is highly sensitive and specific for diagnosis of plTB as an auxiliary method.     

实时荧光定量PCR检测VAD1 mRNA评估新型隐球菌性脑膜炎的疗效

Objective To establish a new approach for quantitative detection of VAD1 mRNA in cryptococcus neoformans by RT-FQ-PCR, and evaluate the treatment efficacy of CNM. MethodsThe primers and TaqMan probe were designed according to the published sequence of VAD1 mRNA (GenBank),and RT-FQ-PCR method to detect VAD1 mRNA was established. Cerebrospinal fluid from 25 CNM patients and 30 controls were detected and sensitivity and specificity of the method were evaluated. VAD1 mRNA concentration in cerebrospinal fluid from both acute phase and recovery phase of 25 CNM patients were also detected and significance of CNM treatment efficacy with VAD1 mRNA analysis was evaluated. Results Correlation coefficient of standard curve was - 0. 997 9 in detection of VAD1 mRNA by RT-FQ-PCR, and the detection limit was 101 copies/μl. The intra CV of plasmid standard for high, medium and low concentrations were 0. 65% ,0. 89% and 1.23% respectively, the sensitivity of cryptococcus neoformans detection by RT-FQPCR was 96% (24/25) ,while specificity was 100% (30/30). VAD1 mRNA concentration in acute phase were significant higher than that in recovery phase (3. 042 ±0. 906 vs 2. 187 ±0. 665 ,t =4. 583 ,P ＜0. 01).Conclusions The established RT-FQ-PCR method for the detection of VAD1 mRNA is provided with sound sensitivity, specificity and reproducibility, which might be fit for the detection of VAD1 mRNA. The expression level of VAD1 mRNA is relevant with the treatment efficacy of CNM.     

两种结核抗原特异性干扰素释放试验试剂用于HIV感染人群中结核潜伏感染诊断的比较

目的 比较评价自行研发及国外进口T-SPOT TB两种结核抗原特异性γ干扰素释放试验在HIV感染人群中诊断结核潜伏感染的价值.方法 102例HIV感染者均来自于深圳市第三人民医院艾滋病门诊,其中男66例,女36例,平均年龄35岁.运用自行研发的结核杆菌抗原特异性γ干扰素ELISpot检测方法及T-SPOT TB试剂盒对102例确诊的HIV感染者的外周血结核特异性γ干扰素释放水平进行检测,并同时进行TST.结果 在HIV感染者中17例患者T-SPOT和ELISpot两种方法检测共同阳性,ELISpot的敏感度、特异度、阳性预测值、阴性预测值和准确性分别为94.4%、94.0%、77.3%、98.8%和94.1%.其中3例患者在TST与T-SPOT两种检验方法检测共同阳性,TST的敏感度、特异度、阳性预测值、阴性预测值和准确性分别为16.7%、98.8%、75.0%、84.7%和84.3%.利用3种抗原对102例患者进行检测,结核分枝杆菌重组抗原(ESAT-6)、PoolA、PoolB刺激所得斑点数的平均值分别为26.89±5.77、18.96±4.75和14.51±3.77,仅ESAT-6与PoolB的差异有统计学意义(H=7.557,P=0.022 9).不同CD4分组ELISpot检测阳性率和T-SPOT TB检测阳性率差异均无统计学意义(x2=0.860 8,P=0.650 2;x2=1.396 4,P=0.497 5).结论 自行研发的结核杆菌抗原特异性γ干扰素ELISpot检测技术在HIV感染人群中用于结核潜伏感染的诊断与T-SPOT TB检测方法类似,两种方法的敏感度与特异度均高于TST,可用于艾滋病合并结核潜伏感染的辅助诊断.

Abstract：

Objective To evaluate the diagnostic value of two tuberculosis-specific IFN-γ release assays in latent tuberculosis infection among HIV-infected individuals. Methods The levels of tuberculosis antigen-specific IFN-γin 102 HIV patients from AIDS Outpatient Clinic of Shenzhen Third People's Hospital were detected by in-house tuberculosis-specific IFN-γ ELISpot assay and commercial T-SPOT TB kit, and tuberculin skin test (TST) were done at the same time. There were 66 males and 36 females,and the average age was 35. Results Seventeen HIV infected patients were positive in both IFN-γ ELISpot and T-SPOT TB methods, the sensitivity, specificity positive predictive value(PPV), negative predictive value(NPV) and compliance rates of ELISpot were 94. 4% ,94. 0% ,77. 3% ,98. 8% and 94. 1% ,respectively. Three patients were positive in both IFN-γELISpot and T-SPOT TB methods, the sensitivity, specificity, PPV, NPV and compliance rates of TST were 16. 7%, 98. 8%, 75.0%, 84. 7% and 84. 3%, respectively. The average number of spots using three kinds of antigen ESAT-6, Pool A,Pool B obtained were 26. 89 ±5. 77,18. 96 ±4. 75 and 14. 51 ± 3.77, respectively. Only ESAT-6 and Pool B have a statistically significant difference (H=7.557,P = 0.022 9), no significant difference was shown between other groups. There was no significant difference between the positive rate and the CD4+ T cellls number(x2 =0. 860 8 ,P =0. 650 2) ,as the same as the T-SPOT TB (x2 = 1. 396 4, P = 0. 497 5 ). Conclusions The performance of this in-house tuberculosis-specific IFN-γ ELISPot assay was comparable to T-SPOT assay in diagnosis of latent tuberculosis infection, and the sensitivity and specificity of both these two assays were all much higher than TST. They canbe recommended in diagnosing latent tuberculosis infection in HIV infected patients.     

IL-10基因单核苷酸多态性与急性淋巴细胞白血病易感性的相关性研究

Objective To observe the relationship of IL-10 gene single nucleotide polymorphism and the susceptibility to ALL. Methods The bone marrow and peripheral blood samples from 115 ALL patients and 323 healthy controls were collected in Peking University First Hospital and Beijing Dao-pei Hospital from January 2007 to December 2009. The DNA were extracted from all samples. The primers of -819C/T and -592A/C in the promoter region of IL-10 gene were designed for the PCR. The restrictive fragment length polymorphism of IL-10 gene was analyzed by using restrictive enzyme Msl Ⅰ and HpyCH4 Ⅲ.Sequencing was done in part of these samples to confirm the results of PCR. The differences of genotypes and allele ratio of -819 and -592 sites were analyzed between the ALL patients and healthy controls. Real-time quantitative PCR was performed to detect the EB virus (EBV) infection and the expression of BCR/ABL fusion gene. The differences of genotypes and allele ratio of -819 and -592 sites were analyzed between the positive and negative group. Results The genotype ratios of -819CC, -819TT, - 819CT, -592AA,- 592CC and - 592AC were 14. 8% ( 17/115 ), 45.2% ( 52/115 ), 40. 0% ( 46/115 ), 43.5% ( 50/115 ),16. 5% ( 19/115 ), 40. 0% ( 46/115 ) in ALL patients, and were 9. 9% ( 32/323 ), 16. 4% ( 53/323 ),73.7% ( 238/323 ), 11.8% ( 38/323 ), 15.5% ( 50/323 ), 72. 8% ( 235/323 ) in the healthy controls,respectively. The genotypes of -819 and -592 sites had statistically significant differences between the two groups(x2 values were 46.000 and 54.550, all P ＜ 0. 05 ). The allele ratio of -819T and -592A were (65.2%, 150/230) and (63.5%, 146/230) in ALL patients, while they were 53.5% (344/646) and 48. 1% (311/646)in the healthy controls. There were statistically significant differences between the two groups (x2 values were 9. 877 and 15.986, all P ＜ 0. 05 ). The EBV DNA were detected in 42 ALL patients,among which 22 were positive and 20 were negative. The genotype ratios of -819CC, -819TT, -819CT,-592AA, - 592CC, - 592AC in EBV positive group were 9. 1% ( 2/22 ), 40. 9% ( 9/22 ), 50. 0%(11/22) ,31.8% ( 7/22 ), 13.6% ( 3/22 ), 54. 5% ( 12/22 ), while they were 35.0% ( 7/20 ), 45.0%(9/20) ,20. 0% (4/20) ,35.0% (7/20) ,45.0% (9/20) ,20. 0% (4/20) in the EBV negative group. The genotypes of -819 and -592 sites showed no statistical differences between the two groups( all P ＞ 0. 05 ).The BCR/ABL fusion gene were detected in 36 ALL patients, among which 20 were positive and 16 were negative. The genotype ratios of - 819CC, - 819TT, - 819CT, - 592AA, - 592CC, - 592AC in BCR/ABL positive group were 0% (0/20) ,45.0% (9/20) ,55.0% ( 11/20), 45. 0% (9/20) ,5.0% (1/20) ,50. 0%( 10/20), while they were 18. 8% ( 3/16 ), 50. 0% ( 8/16), 31.3% ( 5/16 ), 50. 0% ( 8/16 ), 18. 8%(3/16), 31.3 % (5/16)in the BCR/ABL negative group. The genotypes of -819 and -592 sites showed no statistical differences between the two groups ( all P ＞ 0. 05 ). Conclusion The population with - 819TT and - 592AA genotype of IL-10 gene shows susceptibility to ALL.     

焦磷酸测序技术检测结核分枝杆菌四种药物耐药性

目的 利用焦磷酸测序技术检测结核分枝杆菌对利福平、异烟肼、氧氟沙星、阿米卡星的耐药性,并对其临床应用进行评价.方法 收集上海市肺科医院2008-2009年临床确诊的肺结核患者的痰标本培养阳性结核分枝杆菌89株.所有菌株按<结核病诊断实验室检验规程>进行分枝杆菌培养、菌种鉴定.另外10株已知药敏结果的结核分枝杆菌来自上海市肺科医院菌株库.利用焦磷酸测序技术,以10株已知药敏结果和经直接测序已知耐药基因突变情况的结核分枝杆菌为试验菌株,探索焦磷酸测序检测结核分枝杆菌rpoB、katG、gyrA、rrs耐药基因的最佳模式,并用该条件检测89株结核分枝杆菌临床分离株,检测结果与Bactec 960药敏法进行比较.结果 rpoB、gyrA基因检测宜采用焦磷酸测序序列分析模式,katG、rrs基因检测宜采用焦磷酸测序单核苷酸多态性模式.以Bactec 960药敏法测定结果为判断标准,则焦磷酸测序法检测89株结核分枝杆菌临床分离株对利福平、异烟肼、氧氟沙星、阿米卡星耐药性的敏感度分别为98.0%、64.1%、79.5%、78.3%,特异度分别为97.5%、100.0%、90.0%、100.0%,准确性分别为97.8%、77.5%、85.4%、94.4%,该法检测利福平、氧氟沙星、阿米卡星的检测结果与Bactec 960药敏检测结果一致性较好,Kappa值均≥0.7.结论 焦磷酸测序技术检测结核分枝杆菌对利福平、异烟肼、氧氟沙星、阿米卡星耐药性具有快速、准确、高通量的优点,是一种可对结核分枝杆菌多种药物耐药性进行快速检测的方法.

Abstract：

Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikacin by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequencing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates,when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid,ofloxacin and amikacin is 98.0%, 64. 1%, 79.5%, 78. 3% respectively, the specificity is 97.5%,100. 0%, 90. 0%, 100. 0% respectively, the accuracy is 97.8%, 77. 5%, 85.4%, 94. 4% respectively,tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa ≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for detecting Mycobacterium tuberculosis resistance to these four drugs.     

携IgG单抗靶向超声造影剂的构建及其靶向效能的体外评价

Objective To construct targeted ultrasound contrast agent carried goat anti-mouse IgG antibody (UCA-IgG) and evaluate the effectiveness of its targeted adhesion using parallel plate flow chamber. Methods The ultrasound contrast agent targeted to mouse IgG was designed by conjugating monoclonal antibodies against mouse lgG to the lipid monolayer shell of the agent using biotin-streptavidin. The binding of IgG antibodies to the ultrasound contrast agent were identified by fluorescence in vitro. The attachment and detachment of UCA-IgG to mouse IgG immobilized on a culture dish were assessed in a parallel-plate flow chamber. While the plate lacked mouse IgG,or blocked with large number of goat anti-mouse IgG were served as two control groups. Results UCA-IgG issued a bright green fluorescence, while the contral lipid ultrasound contrast agent didn't show fluorescence. The number of UCA-IgG bound to mouse IgG of experimental group was greater than two control groups,increased with increasing coverslips surface antibody concentrations (P<0. 05),and there was significant positive correlation between the number of UCA-IgG bound to mouse IgG and time of combination (P<0.05). The adhesion rate of experimental group increased with shear stress before 0. 5×10-5 N/cm2 (P<0.05) and then decreased (P<0. 05). There was limited adherence of control groups to the UCA-IgG. The stess of half-maximal detachment was increased with increasing coverslips surface antibody concentrations (P<0.05). Conclusions UCA-IgG could adhere to mouse IgG in the physical conditions. It may provide strong supports for studying other targeted ultrasound contrast agent preliminary and fatherly in vitro.     

尿流式有形成分及干化学分析在尿路感染诊断中的应用评价

Objective To evaluate the clinical application of automated urine formed elements analyzer and/or urine dipstick analyzer for examination of urinary formed elements in screening urinary tract infection (UTI). Methods 148 fresh midstream clear-catch urine samples from the UTI patients and 284 fresh midstream clear-catch urine samples from non-UTI subjects were selected. Bacteria culture was performed for bacterial colony counting and identification. Bacteria counts ( BACT), yeast-like fungus and WBC were performed by UF-looOi automated urine formed elements analyzer. Leukocyte esterase test (LEU) and nitrite test (NIT) were performed by URISYS 2400 urine dipstick analyzer. We evaluated data obtained from urine dipstick analyzer, UF-1000i and combination of UF-1000i with urine dipstick analyzer and the results was compared with those obtained from quantitative bacterial culture. Then we evaluated the sensibility, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy. Results Among the 148 patients with UTI, the positive rate of the quantitative bacterial culture was 73.6% (109/148), the positive rate of LEU and NIT detected by dipstick test 26. 4% (39/148).There was significantly statistical difference between bacterial culture and strip test(χ2 = 55.68 ,P < 0. 05 ). The positive rate of urine flow cytometry by UF-1000i with either positive of BACT and WBC was 91.2%(135/148), which was higher than the positive rate of the quantitative bacterial culture. There was significant difference between two methods (χ2 = 14. 70, P < 0. 05 ). The positive rate of anyone positive among BACT, WBC, LEU and NIT was 94. 6% (140/148) when detected with combination of dipstick test and UF-1000i, which was higher than the positive rate of the quantitative bacterial culture. And there was significant difference between two methods (χ2 = 20. 45, P < 0. 05 ). The sensitivity of dipstick test was low (26. 4% ,39/148 ), and specificity was high ( 99. 3%, 282/284 ) . The sensitivity, specificity, positive predictive value, negative predictive value of BACT detected by UF-1000i in diagnosing urinary tract infection were 92. 6% ( 137/148 ), 39. 8% ( 113/284 ). 44. 5% ( 137/308 ) and 91.1% ( 113/124 ), respectively. If the dipstick test was combined with UF-1000i, the sensitivity, negative predictive value, specificity, positive predictive value and accuracy were 98.0% ( 145/148 ), 97.1% ( 100/103 ). 35.2% (100/284) ,44. 1% (145/329) and 56. 7% (245/432), respectively. Conclusions The combination of urine dipstick test and automated urine formed elements analyzer UF-1000i plays an important role in early diagnosis of UTI. And it has significant value in diagnosis of UTI, especially for the patients with negative bacterial cultures of urine sample.     

尿流式有形成分及干化学分析在尿路感染诊断中的应用评价

Objective To evaluate the clinical application of automated urine formed elements analyzer and/or urine dipstick analyzer for examination of urinary formed elements in screening urinary tract infection (UTI). Methods 148 fresh midstream clear-catch urine samples from the UTI patients and 284 fresh midstream clear-catch urine samples from non-UTI subjects were selected. Bacteria culture was performed for bacterial colony counting and identification. Bacteria counts ( BACT), yeast-like fungus and WBC were performed by UF-looOi automated urine formed elements analyzer. Leukocyte esterase test (LEU) and nitrite test (NIT) were performed by URISYS 2400 urine dipstick analyzer. We evaluated data obtained from urine dipstick analyzer, UF-1000i and combination of UF-1000i with urine dipstick analyzer and the results was compared with those obtained from quantitative bacterial culture. Then we evaluated the sensibility, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy. Results Among the 148 patients with UTI, the positive rate of the quantitative bacterial culture was 73.6% (109/148), the positive rate of LEU and NIT detected by dipstick test 26. 4% (39/148).There was significantly statistical difference between bacterial culture and strip test(χ2 = 55.68 ,P < 0. 05 ). The positive rate of urine flow cytometry by UF-1000i with either positive of BACT and WBC was 91.2%(135/148), which was higher than the positive rate of the quantitative bacterial culture. There was significant difference between two methods (χ2 = 14. 70, P < 0. 05 ). The positive rate of anyone positive among BACT, WBC, LEU and NIT was 94. 6% (140/148) when detected with combination of dipstick test and UF-1000i, which was higher than the positive rate of the quantitative bacterial culture. And there was significant difference between two methods (χ2 = 20. 45, P < 0. 05 ). The sensitivity of dipstick test was low (26. 4% ,39/148 ), and specificity was high ( 99. 3%, 282/284 ) . The sensitivity, specificity, positive predictive value, negative predictive value of BACT detected by UF-1000i in diagnosing urinary tract infection were 92. 6% ( 137/148 ), 39. 8% ( 113/284 ). 44. 5% ( 137/308 ) and 91.1% ( 113/124 ), respectively. If the dipstick test was combined with UF-1000i, the sensitivity, negative predictive value, specificity, positive predictive value and accuracy were 98.0% ( 145/148 ), 97.1% ( 100/103 ). 35.2% (100/284) ,44. 1% (145/329) and 56. 7% (245/432), respectively. Conclusions The combination of urine dipstick test and automated urine formed elements analyzer UF-1000i plays an important role in early diagnosis of UTI. And it has significant value in diagnosis of UTI, especially for the patients with negative bacterial cultures of urine sample.     

尿流式有形成分及干化学分析在尿路感染诊断中的应用评价

Objective To evaluate the clinical application of automated urine formed elements analyzer and/or urine dipstick analyzer for examination of urinary formed elements in screening urinary tract infection (UTI). Methods 148 fresh midstream clear-catch urine samples from the UTI patients and 284 fresh midstream clear-catch urine samples from non-UTI subjects were selected. Bacteria culture was performed for bacterial colony counting and identification. Bacteria counts ( BACT), yeast-like fungus and WBC were performed by UF-looOi automated urine formed elements analyzer. Leukocyte esterase test (LEU) and nitrite test (NIT) were performed by URISYS 2400 urine dipstick analyzer. We evaluated data obtained from urine dipstick analyzer, UF-1000i and combination of UF-1000i with urine dipstick analyzer and the results was compared with those obtained from quantitative bacterial culture. Then we evaluated the sensibility, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy. Results Among the 148 patients with UTI, the positive rate of the quantitative bacterial culture was 73.6% (109/148), the positive rate of LEU and NIT detected by dipstick test 26. 4% (39/148).There was significantly statistical difference between bacterial culture and strip test(χ2 = 55.68 ,P < 0. 05 ). The positive rate of urine flow cytometry by UF-1000i with either positive of BACT and WBC was 91.2%(135/148), which was higher than the positive rate of the quantitative bacterial culture. There was significant difference between two methods (χ2 = 14. 70, P < 0. 05 ). The positive rate of anyone positive among BACT, WBC, LEU and NIT was 94. 6% (140/148) when detected with combination of dipstick test and UF-1000i, which was higher than the positive rate of the quantitative bacterial culture. And there was significant difference between two methods (χ2 = 20. 45, P < 0. 05 ). The sensitivity of dipstick test was low (26. 4% ,39/148 ), and specificity was high ( 99. 3%, 282/284 ) . The sensitivity, specificity, positive predictive value, negative predictive value of BACT detected by UF-1000i in diagnosing urinary tract infection were 92. 6% ( 137/148 ), 39. 8% ( 113/284 ). 44. 5% ( 137/308 ) and 91.1% ( 113/124 ), respectively. If the dipstick test was combined with UF-1000i, the sensitivity, negative predictive value, specificity, positive predictive value and accuracy were 98.0% ( 145/148 ), 97.1% ( 100/103 ). 35.2% (100/284) ,44. 1% (145/329) and 56. 7% (245/432), respectively. Conclusions The combination of urine dipstick test and automated urine formed elements analyzer UF-1000i plays an important role in early diagnosis of UTI. And it has significant value in diagnosis of UTI, especially for the patients with negative bacterial cultures of urine sample.     

一种国产HBsAg确认试剂的临床应用评价

Objective To carry out the clinical validation of a domestic HBsAg kit to evaluate its application value. Methods 543 serum samples with HBsAg ELISA values of S/CO ≥ 0. 7 were tested by HBsAg confirmatory test. Specific anti-HBs reagent and control reagent were added separately into duplicate wells of HBsAg ELISA plate, in which test sample was also added. After incubation at 37℃, HBsAg was detected by routine ELISA, and the inhibition rate was calculated using absorbanee (A) result of anti-HBs reagent well and control reagent well according to the provided formula. The sample was confirmed as HBsAg positive when the inhibition rate was≥50%. For HBsAg weakly positive samples, "prolonged confirmatory test" (conjugate reaction time was prolonged to 120 rain) was applied to increase the sensitivity. 39 samples were randomized selected for testing and comparison with Abbott Murex confirmatory test. Results 543 serum samples in total were tested by the confirmatory test. Among the 504 cases which showed positive reaction in screening HBsAg ELISA, 89 ( 17. 7% ) were confirmed as negative. According to their S/CO value of the screening HBsAg test, the ratio of negative cases / tested eases in the group were:S/CO≤<5.0, 87/143 (60. 8% ) ;5.0 < S/CO ≤ 10. 0,0/25 (0) ;10. 0 < S/CO ≤ 15.0, 1/21 (4. 8% ) ;15.0 < S/CO ≤ 20. 0, 1/23 (4. 4% ) ;S/CO 20. 0, 0/292(0). Among 39 cases with negative HBsAg (0. ≤相似文献   

Utility of a combination of RD1 and RD2 antigens as a diagnostic marker for tuberculosis

We evaluated the diagnostic potential of a cocktail of 4 antigens encoded by regions of difference (RD) 1 and 2 of Mycobacterium tuberculosis, that is, early secretory antigenic target-6, culture filtrate protein-10 (CFP-10), CFP-21, and mycobacterial protein from species tuberculosis-64 (MPT-64) on the basis of antigen and antibody detection by enzyme-linked immunosorbent assay. Parallel detection of antigens and antibodies in the serum samples of pulmonary tuberculosis (PTB) patients resulted in higher sensitivity as compared to either of the single tests in both smear-positive (90%) and smear-negative (60%) PTB patients. In addition, combined detection of antigens and antibodies in the fluids of extrapulmonary tuberculosis (EPTB) patients could detect >90% of the patients with high specificity. These results demonstrate the ability of the combination of antigen and antibody detection assays based on the cocktail of RD antigens to diagnose a substantial number of PTB and EPTB cases with high specificity.     

Circulating immune complexes in human tuberculosis sera: demonstration of specific antibodies against Mycobacterium tuberculosis glycolipid (DAT,PGLTb1, LOS) antigens in isolated circulating immune complexes

An enzyme-linked immunosorbent assay (ELISA) for IgG using three glycolipid antigens from Mycobacterium tuberculosis in 65 tuberculosis (TB) patients and 50 healthy control subjects was performed. The circulating immune complexes (CICs) were isolated by precipitation with polyethylene glycol 6000 (PEG). This method associated to ELISA measured the specific antibodies present in these CICs. PEG [optical density (OD) 280] was shown to be significantly elevated (P < 0.001) in tuberculous samples. The concentrations of IgG antibodies complexed to the three glycolipid antigens were shown to be higher in patient with tuberculosis than in normal control subjects (P < 0.001). No correlation was observed between levels of free and CIC-bound antibodies. These antibodies isolated from CICs were responsible for almost all of the false-negative serological results. However, great heterogeneity was noticed depending on the antigen used, showing a more positive ELISAs against DAT (77%) than against LOS (71%) or PGLTb1 (18.5%). No correlation was established between the presence of specific CIC-complexed IgG and the bacteriological load or the tuberculosis localization (pulmonary vs. extrapulmonary). The sensitivity of ELISA for CIC-complexed IgG to DAT and LOS was lower in HIV-infected TB patients. From these results, we conclude that detection of complexed IgG DAT and LOS glycolipid antigen will be useful as a complementary technique for the serodiagnosis of tuberculosis.     

γ干扰素释放试验在结核病诊断中的应用价值

目的分析γ干扰素释放试验(IGRA)在结核病诊断中的应用价值。方法收集2017年11月-2019年1月广州南方医院796例进行IGRA检测的住院患者资料,分析IGRA对结核病的诊断价值,并将其与痰涂片抗酸染色镜检、结核抗体胶体金法、结核分枝杆菌DNA荧光定量PCR法、结核菌素皮肤试验(TST)、结核分枝杆菌感染T细胞斑点试验(T-SPOT.TB)进行比较。结果 IGRA对肺结核和肺外结核检出阳性率分别为80.2%和84.1%,差异无统计学意义(P>0.05);肺结核组与非结核组阳性率差异有显著统计学意义(P<0.01)。IGRA对结核诊断的灵敏度、特异度、阳性预测值和阴性预测值分别为 81.9%、81.3%、60.1%和92.9%。痰涂片抗酸染色镜检、结核抗体胶体金法、结核分枝杆菌DNA荧光定量PCR法、TST、T-SPOT.TB的灵敏度分别为3.4%、21.1%、15.2%、66.7%、82.9%。结论 IGRA灵敏度和阴性预测值较好,对结核辅助诊断具有较高临床应用价值。     

Molecular testing of respiratory swabs aids early recognition of meningococcal disease in children

Early meningococcal disease (MD) diagnosis is difficult. We assessed rapid molecular testing of respiratory specimens. We performed genotyping of respiratory swabs, blood, and cerebrospinal fluid from children with suspected disease and nasal swabs (NSs) from matched controls. Thirty-nine of 104 suspected cases had confirmed disease. Four controls were carriers. Throat swab ctrA and porA testing for detection of disease gave a sensitivity of 81% (17/21), specificity of 100% (44/44), positive predictive value (PPV) of 100% (17/17), negative predictive value (NPV) of 92% (44/48), and relative risk of 12. NS ctrA and porA testing gave a sensitivity of 51% (20/39), specificity of 95% (62/65), PPV of 87% (20/23), NPV of 77% (62/81), and relative risk of 4. Including only the 86 NSs taken within 48 h of presentation, the results were sensitivity of 60% (18/30), specificity of 96% (54/56), PPV of 90% (18/20), NPV of 82% (54/66), and relative risk of 5. Swab type agreement was excellent (kappa 0.80, P < 0.001). There was exact phylogenetic agreement from different specimen sites for individuals. Carried genosubtypes were P1.7 and P1.21-7. Prehospital rapid molecular testing of easily obtained respiratory specimens could accelerate diagnosis of MD.     

Loop-mediated isothermal amplification (LAMP) in the respiratory specimens for the diagnosis of pediatric pulmonary tuberculosis: A pilot study

BackgroundLoop-mediated isothermal amplification (LAMP) assay is a novel molecular diagnostic technique that can be used for the diagnosis of tuberculosis (TB). It is most suited for developing countries as it is rapid, inexpensive, highly sensitive, requiring minimal infrastructure, training and manpower. Studies in pediatric TB are lacking. We evaluated LAMP in the diagnosis of pediatric pulmonary TB.MethodologyThis was a cross-sectional analytical study conducted at a tertiary care teaching hospital in North India from July 2014 to June 2015 involving 60 children with suspected pulmonary TB. Respiratory specimens (sputum, gastric lavage, bronchoalveolar lavage and/or endotracheal aspirates) were collected and subjected to BACTEC MGIT 960 culture, IS6110 PCR, and LAMP assay targeting IS6110 gene of Mycobacterium tuberculosis.ResultsThirty seven children had confirmed and probable TB according to the composite reference standard (CRS). Among all the 3 tests used for diagnosis of Pulmonary TB, LAMP had highest sensitivity (37.8%) followed by PCR (27%), and culture (21.6%) when compared against the predefined CRS. Culture had maximum specificity of 100%; and PCR, and LAMP had specificity of 95–96%. The sensitivity, specificity, PPV, and NPV of LAMP against culture as reference standard were 75%, 72.4%, 42.9%, and 91.3% respectively. Similarly sensitivity, specificity, PPV, and NPV of PCR against culture as reference standard were 75%, 86.2%, 60%, and 86.2% respectively. On combining LAMP with culture, sensitivity increased to 45.7% (7.8% increase, p = 0.04).ConclusionWe noted that LAMP had highest sensitivity when compared to culture and PCR and comparable specificity.     

肺炎嗜衣原体三种不同抗原的临床诊断价值评价

目的 研究肺炎嗜衣原体3种重组抗原Cpn0146、Cpn0147及Cpn0308应用于肺炎嗜衣原体感染血清学诊断中的价值.方法 构建pGEX6p-2/Cpn0146、Cpn0147、Cpn0308重组质粒,并诱导表达重组蛋白,采用间接ELISA法和免疫印迹法(Western-blot)分析其免疫原性及其免疫反应性.建立分别以3种重组蛋白为包被抗原的间接ELISA法,与肺炎嗜衣原体IgG商品诊断试剂盒平行检测183份呼吸道感染患者的血清标本及32份沙眼衣原阳性患者血清标本,比较各方法检出准确性及阳性率.结果 GST-Cpn0146、Cpn0147、Cpn0308免疫兔血清中特异性IgG抗体效价滴度分别达1:6 400、1:12 800和1:12 800以上;以3种重组蛋白为包被抗原的ELISA法准确性分别为92.3%、94.5%、96.7%.各方法检测出阳性率结果显示,Cpn0146组的阳性率为38.8%(71/183)、与肺炎嗜衣原体IgG试剂盒的阳性率比较差异有统计学意义(x~2=12.07,P<0.05);Cpn0147阳性率40.9%(75/183),与肺炎嗜衣原体IgG试剂盒的阳性率比较差异有统计学意义(x~2=8.10,P<0.05);Cpn0308阳性率44.8%(75/183),与肺炎嗜衣原体IgG试剂盒的阳性率比较差异无统计学意义(x~2=0.57,P>0.05).结论 Cpn0308在肺炎嗜衣原体感染血清学诊断实验中表现出良好的准确性(96.2%)和较高的阳性率(44.8%),可望用于进一步研制肺炎嗜衣原体诊断试剂盒.     

荧光定量聚合酶链反应检测在肺结核诊断中的应用价值

目的分析荧光定量聚合酶链反应(PCR)检测支气管肺泡灌洗液(BALF)中结核分枝杆菌脱氧核糖核酸(TbDNA)在诊断肺结核中的应用价值。方法选择2018年1月-2020年1月湖南省儿童医院收治的21例肺结核患儿作为肺结核组,另外选择同时期21例排除肺结核感染的肺部疾病患儿作为非肺结核组。全部患儿均行痰涂片抗酸染色镜检、纤维支气管镜(纤支镜)痰涂片抗酸染色镜检和BALF中TbDNA荧光定量PCR检测。比较3种检测方法对肺结核诊断的特异度、敏感度、阳性预测值(PPV)和阴性预测值(NPV)。结果痰涂片抗酸染色镜检、纤支镜痰涂片抗酸染色镜检和荧光定量PCR的敏感度分别为19.05%、14.29%、76.19%,特异度分别为100.00%、100.00%、95.24%,PPV分别为100.00%、100.00%、94.12%,NPV分别为55.26%、53.85%、80.00%。荧光定量PCR检测BALF中TbDNA的敏感度明显高于痰涂片抗酸染色镜检和纤支镜痰涂片抗酸染色镜检(76.19%比19.05%、14.29%),差异有统计学意义(P<0.05);3种检测方法的特异度比较差异无统计学意义(P>0.05)。结论通过荧光定量PCR检测BALF中的TbDNA,具有快速、敏感度高、特异度高的特点,利于早期诊断肺结核,尤其是对于痰涂片检测呈阴性及无典型影像学特征的患儿,可以明显提高确诊率,其结果能够作为诊断肺结核的主要指标。     

乳腺病灶超声全容积成像临床应用价值

目的 探讨常规二维超声、弹性成像、自动乳腺全容积成像(ABVS)对乳腺良恶性病灶鉴别诊断价值.方法 应用二维超声、弹性成像、ABVS对84例患者经手术病理证实的98个乳腺病灶进行超声检查,并将结果进行比较.结果 36例良性患者共46个病灶,48例恶性患者共52个病灶,二维超声诊断乳腺恶性病灶的敏感度为78.9%(41/52),特异度为76.1%(35/46),准确性为77.6%(76/98),阳性预测值78.8%,阴性预测值76.1%;弹性成像诊断乳腺恶性病灶敏感度为82.7%(43/52),特异度为95.6%(44/46),准确性为88.8%(87/98),阳性预测值95.6%,阴性预测值83.0%;ABVS 诊断乳腺恶性病灶的敏感度为84.6%(44/52),特异度为73.9%(34/46),准确性为79.6%(78/98),阳性预测值78.6%,阴性预测值81.0%.结论 ABVS在乳腺病灶定性诊断上有一定优势,与二维超声诊断敏感度、特异度、准确性、阳性预测值、阴性预测值无明显差异;ABVS较弹性成像诊断特异度、准确性、阳性预测值、阴性预测值稍低.     

Comparative study of three different mycobacterial antigens with a novel lipopolysaccharide antigen for the serodiagnosis of tuberculosis

Demonstration of Mycobacterium tuberculosis in a smear or culture is the most reliable method for diagnosing tuberculosis (TB). In the last 10 years, several enzyme-linked immunosorbent assays (ELISAs) based on mycobacterial antigens (such as antigen 60, 38 kDa antigen, and antigen Kp90) have been used for the rapid diagnosis of TB. In this study, we report the isolation of an immunodominant lipopolysaccharide (LPS) antigen from M. tuberculosis H(37)Rv, which can be used for the serodiagnosis of TB. The LPS antigen was compared with three commercially available mycobacterium-specific antigens for the detection of TB. The antigens were evaluated using serum samples obtained from 59 Indian patients (19 patients with active pulmonary TB, 20 with extrapulmonary TB, and 20 with nontuberculous pulmonary disease) and 20 healthy adults. Antigen 60 IgG (sensitivity 89%, specificity 97%) and LPS (sensitivity 84%, specificity 97%) were more sensitive and specific than 38 kDa antigen IgG (sensitivity 79%, specificity 97%) and Kp90 IgA (sensitivity 82%, specificity 40%). These results indicate that the LPS antigen can be used as a sensitive tool for the serodiagnosis of TB and could be utilized to develop an ELISA for the screening of patients for TB.     

结核菌脂多糖和酚提取蛋白在临床血清诊断中的应用

以结核菌脂多糖和酚提取蛋白为抗原，用ＥＬＩＳＡ方法对肺结核痰菌阳性患者，肺癌和老慢支患者、多蓖型麻风患者和正常人进行了ＩｇＧ抗体检测。抗脂多糖抗体和抗酚提取蛋白抗体测试的敏感度都是７４．７％，特异性分别为９７％和９５％。两种测试的相关系数ｒ＝０。５５７，两者并联测定敏感度为８５．３％，特异性为９４％，如果将阳性判断标准设为两种测试全为阳性，或其中一项强阳性，则敏感度为７２．６％，特异性达９９％，因