Rapid dot sputum and serum assay in pulmonary tuberculosis

A rapid direct sputum (Sp.) and/or antibody assay, based on immunoblotting and enzyme immunoassay is described. The test can detect mycobacterial antigens or antibodies in clinical specimens from pulmonary tuberculosis (TB) patients. In this study, 87 sputa, 87 sera and 40 paired sputa and sera were utilized from smear-positive and smear-negative, culture-positive patients; 59 sputa, 37 sera and 22 paired sputa and sera from nontuberculosis respiratory disease patients and 68 sera from healthy controls. The antigen detection in sputum by dot assay has 86.1% sensitivity on active tuberculosis patients, 92.9% specificity, 91.6% positive predictive value (PPV), 88.2% negative predictive value (NPV) and 10.3% error. The antibody assay has 83.6% sensitivity, 95.4% specificity, 94.4% positive predictive value, 85.6% negative predictive value and 11% error. The test performed on paired sputum and serum (Sr.) samples has a sensitivity of 93.3%, which rose to 96.1% on smear-positive and culture-positive patients, but the specificity decreased to 83% in sputum, whereas in serum it was 92%. The results of the assay, combined with clinical and radiological data, could form the basis for starting an earlier course of treatment for tuberculosis.     

Rapid serodiagnosis of human mycobacteriosis by ELISA using cord factor (trehalose-6,6'-dimycolate) purified from Mycobacterium tuberculosis as antigen

IgG antibodies against purified cord factor (trehalose-6,6'-dimycolate, TDM) in sera of 99 patients infected with mycobacteria (42 patients with tuberculosis excreting tubercle bacilli in the sputum, 11 patients with non-tuberculous mycobacteriosis excreting acid-fast bacilli in the sputum, and 46 patients without bacilli in the sputum but diagnosed as having pulmonary tuberculosis by chest X-ray films and physical examination), five patients with lung cancer, and 100 healthy controls which included subjects positive and negative for the tuberculin test were tested by the ELISA with TDM purified from Mycobacterium tuberculosis H37Rv as the antigen. Of the 99 cases of mycobacteriosis, 83 patients (83.8%) had positive results (48 samples from 53 patients, or 90.5%, with bacilli in the sputum, and 35 samples from 46 patients (76%) with tuberculosis diagnosed clinically). The sera of the five patients with lung cancer and the 100 controls all gave negative results. Thus, the sensitivity and specificity were 83.8% and 100%, respectively. ELISA with TDM as the antigen is simple, reproducible, and useful for the rapid serodiagnosis of general mycobacterial infections including tuberculosis, because it does not involve the cultivation of bacteria.     

Direct and simultaneous identification of Mycobacterium tuberculosis complex (MTBC) and Mycobacterium tuberculosis (MTB) by rapid multiplex nested PCR-ICT assay

The Mycobacterium tuberculosis (MTB) shows different virulence and host infection range from other members of the M. tuberculosis complex (MTBC). Differential identification of MTB from MTBC is thus important in certain occasions. The currently commercially available molecular assays which use either IS6110 or 16S rDNA fragment as identification targets are mainly designed for identifying MTBC but not for MTB. Comparative genomic DNA analysis has provided valuable information on regions of difference (RD) present in MTB but not in other members of the MTBC. RD9 region is further suggested to be a potential target for differential identification of MTB from MTBC. In this study, using IS6110 and Rv3618 (belong to RD9) as the specific identification targets for MTBC and MTB, respectively, we developed and tested a multiplex nested PCR-ICT (immuno-chromatography test) assay for simultaneously and directly detecting not only MTBC but also MTB from 1500 clinical sputum specimens. The results were compared with traditional culture and biochemical identification results together with patients' clinical assessments. This assay showed a 95.5% sensitivity, 97.9% specificity, 2.1% false positive rate and 4.5% false negative rate towards detection of MTBC, and a 93.0% sensitivity, 99.8% specificity, 0.2% false positive rate and 7.0% false negative rate for detection of MTB. This detection system shows great potential in clinical application.     

Fasciola gigantica: Immunodiagnosis of fasciolosis by detection of circulating 28.5 kDa tegumental antigen

A monoclonal antibody (MoAb)-based sandwich ELISA was developed for the detection of circulating 28.5 kDa tegumental antigen (28.5 kDa TA) in the sera from mice experimentally infected with Fasciola gigantica. The MoAb was immobilized on a microtiter plate, and the antigen in the serum was captured and detected with biotinylated polyclonal rabbit anti TA antibody. The test could detect 28.5 kDa in the extracts of tegument (TA), whole body (WB) and excretory-secretory (ES) fractions at the concentrations of these crude antigens as low as 600 pg/ml, 16 and 60 ng/ml, respectively. This sandwich ELISA assay could detect the infection from day 1 to 35 post infection and showed that circulating level of 28.5 kDa TA peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using sera from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as healthy mice and hamsters. The sandwich ELISA exhibited a sensitivity and specificity at 94.55% and 100%, respectively, and with a positive predictive value of 100%, a negative predictive value of 97.39%, false positive rate of 0%, false negative rate of 5.50% and an accuracy of 98.2%. Thus, this detection method exhibited high specificity and sensitivity as well as could be used for early diagnosis of fasciolosis by F. gigantica.     

Application of urinary mutagen testing to detect workplace hazardous exposure and bladder cancer

The objectives of this biochemical epidemiologic case-control study were to evaluate urinary mutagen testing for occupational exposure assessment, and for possible screening for bladder cancer in the workplace. Thirty-seven patients (19 bladder cancer cases and 18 controls) completed a questionnaire. Two urine samples, i.e. a work sample taken while at work, and a home sample, were requested from each patient. Twenty-six patients (17 cases and 9 controls) gave a total of 47 24-h urine samples for mutagenicity testing by the Ames test. A positive Ames test was found to be associated significantly with current occupation with hazardous exposure (odds ratio = 3.7, 95%CI 1.1–12.9), and non-significantly with bladder cancer (odds ratio = 1.8, 95%CI 0.5–7.1). Our results show that the urinary Ames test has the potential of being used as a surveillance for current workplace hazardous exposure (sensitivity = 52%, specificity = 77%, positive predictive value = 72%, negative predictive value = 59%, positive likelihood ratio = 2.3), but not as a screening test for bladder cancer cases (sensitivity = 42%, specificity = 71%, positive predictive value = 3%, negative predictive value = 98%, positive likelihood ratio = 1.5).     

结核分枝杆菌可视化抗体检测蛋白芯片的制备

目的：利用克隆表达的7种结核分枝杆菌优势表位抗原,建立可视化抗体检测蛋白芯片,用于结核病辅助诊断。方法：将7种结核分枝杆菌优势表位抗原,即38kD、ESAT-6、CFP10、MPT64、Mtb8、Mtb8.4和Mtb16.3点于修饰的基片上,制备可检测7种结核抗体的多靶点蛋白微阵列,建立免疫金银染色检测系统;使用该芯片对48例临床结核病患者血液样品进行检测,并与“金标准”痰涂片（48例）和痰培养（其中的29例）检测结果进行比较,分析其敏感性;对30名献血员血液样品进行检测,分析其特异性。结果：可视化抗体检测蛋白芯片的敏感性分别为98.5％和96.6％,而痰涂片和痰培养检测方法的敏感性分别为35.4％和48.3％;可视化抗体检测蛋白芯片的特异性为93.3％。结论：建立的结核分枝杆菌可视化抗体检测蛋白芯片检测敏感性显著高于痰涂片和痰培养方法,可用于结核病的临床辅助诊断,提高痰涂片和痰培养假阴性的检出率。     

FUS-100尿沉渣分析仪在住院患者尿路感染筛检中的应用

目的 探讨FUS-100全自动尿沉渣分析仪(简称FUS-100)中的细菌和酵母定量计数在筛查住院患者尿路感染时的价值.方法 用定量细菌培养法和FUS-100检测505例疑似泌尿系统感染患者清洁中段尿标本.结果 505份尿标本培养阳性192份,阳性率为38.0％,其中36份标本有两种细菌生长,共分离出228株菌,革兰阳性球菌30株(13.2％),革兰阴性杆菌108株(47.4％),真菌90株(39.4％).以尿细菌计数≥23.72/μL、酵母计数≥0.65/μL为感染阳性标准,与中段尿培养相比较,FUS-100细菌定量计数的敏感性为85.5％,特异性为89.5％,阳性似然比为8.14,阴性似然比为0.16;酵母定量计数的敏感性为80.4％,特异性为97.2％,阳性似然比为28.71,阴性似然比为0.20.结论 FUS-100定量计数尿细菌和酵母可用于住院患者尿路感染的快速筛查.     

Serodiagnosis of extrapulmonary tuberculosis by enzyme-linked immunosorbent assay (ELISA)

Antibodies against Mycobacterium tuberculosis antigenic glycolipids were determined by enzyme-linked immunosorbent assay (ELISA). The 720 sera were collected from adult patients under investigation, suspected with extrapulmonary tuberculosis. The test performance was estimated according to definitive diagnosis in terms of specificity, sensitivity, positive predictive value and negative predictive value. These parameters calculated on 142 sera from patients with extrapulmonary tuberculosis and on 578 sera from patients with different nontuberculosis diseases were 92%, 81.6%, 70.9% and 95.1%, respectively. The specificity decreased to 85% when tuberculosis was associated with cancer or hepatic cirrhosis. In reactivated tuberculosis the sensitivity and the positive predictive value were 86.9% and 83.3%, respectively. Our results showed that ELISA was conclusive for patients with active tuberculosis, before the initiation of the treatment. The sensitivity decreased to 30% in inactive forms. It was demonstrated that ELISA was positive in cases with negative microscopy genitourinary tuberculosis. ELISA could be used as a supporting test in the laboratory diagnosis of active extrapulmonary tuberculosis in adults, disregarding the site involved.     

C反应蛋白检测在感染性和非感染性发热鉴别中的作用

目的：探讨C反应蛋白（C reactive protein,CRP）在感染性和非感染性发热鉴别中的作用。方法：对758例以发热为主要症状的患者进行各项检查,并在入院24小时内采集静脉血用免疫比浊法检测CRP,计算特异度、灵敏度、准确度、阳性预测值、阴性预测值、阳性似然比、阴性似然比。结果：758例患者中,确诊为感染性发热的患者616例,占81.27%,非感染性发热患者142例,占18.73%。感染性发热患者的CRP值显著高于非感染性发热患者,差异具有统计学意义（P〈0.05）。CRP检验的特异度、灵敏度、准确度、阳性预测值、阴性预测值、阳性似然比和阴性似然比的值分别为87.32%、97.89%、95.91%、97.10%、90.51%、7.72和0.02。结论：CRP的检测能有效鉴别感染性发热与非感染性发热。     

Diagnostic Accuracy and Turnaround Time of the Xpert MTB/RIF Assay in Routine Clinical Practice

The Xpert MTB/RIF assay was introduced for timely and accurate detection of tuberculosis (TB). The aim of this study was to determine the diagnostic accuracy and turnaround time (TAT) of Xpert MTB/RIF assay in clinical practice in South Korea. We retrospectively reviewed the medical records of patients in whom Xpert MTB/RIF assay using sputum were requested. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the diagnosis of pulmonary tuberculosis (PTB) and detection of rifampicin resistance were calculated. In addition, TAT of Xpert MTB/RIF assay was compared with those of other tests. Total 681 patients in whom Xpert MTB/RIF assay was requested were included in the analysis. The sensitivity, specificity, PPV and NPV of Xpert MTB/RIF assay for diagnosis of PTB were 79.5% (124/156), 100.0% (505/505), 100.0% (124/124) and 94.0% (505/537), respectively. Those for the detection of rifampicin resistance were 57.1% (8/14), 100.0% (113/113), 100.0% (8/8) and 94.9% (113/119), respectively. The median TAT of Xpert MTB/RIF assay to the report of results and results confirmed by physicians in outpatient settings were 0 (0–1) and 6 (3–7) days, respectively. Median time to treatment after initial evaluation was 7 (4–9) days in patients with Xpert MTB/RIF assay, but was 21 (7–33.5) days in patients without Xpert MTB/RIF assay. Xpert MTB/RIF assay showed acceptable sensitivity and excellent specificity for the diagnosis of PTB and detection of rifampicin resistance in areas with intermediate TB burden. Additionally, the assay decreased time to the initiation of anti-TB drugs through shorter TAT.     

三种方法诊断不孕症患者子宫内膜息肉的临床价值研究

目的:探讨单用阴道超声(TVS)、子宫输卵管造影(HSG)、超声子宫水造影(SIS)以及三种方法联合诊断不孕症患者子宫内膜息肉(EP)的临床价值。方法:以206例行宫腔镜联合诊刮或病检的不孕症患者为研究对象,回顾性分析各种检查方法对EP的筛查结果,评价各种检查方法的真实性、可靠性以及预测值。结果:206例不孕症中,共确诊EP患者60例,阳性率29.1%。三种检查方法中,TVS的灵敏度最高(70.0%),特异度最低(73.3%),漏诊率最低(30.0%),误诊率最高(26.7%),正确诊断指数最高(43.3%),阴性似然比最小(0.409),阴性预测值最高(85.6%);SIS检查的灵敏度最低(38.7%),漏诊率最高(61.3%),但是特异性最高(93.3%),误诊率最低(6.7%),阳性似然比最大(4.284),阳性预测值最大(66.6%),正确诊断指数最低(32.0%);HSG检查的上述各项评价指标均介于TVS和SIS之间。TVS和SIS与金标准的符合率低,Kappa值均小于0.4;HSG符合率最高(86.2%),Kappa值0.647。三种检查联合诊断的灵敏度89.3%,漏诊率10.7%,特异度91.4%,误诊率8.6%,正确诊断指数80.7%,阳性似然比10.384,阴性似然比0.117,符合率89.3%,Kappa值0.792,阳性预测值83.3%,阳性预测值94.6%。结论:对于宫腔可能存在内膜息肉的不孕症患者,单一采用阴道超声检查、子宫输卵管造影或超声子宫水造影方法的灵敏度均较低,漏诊率高,与金标准的一致性较差,而三种方法联合用于诊断不孕症患者EP的真实性、可靠性及预测值均较好。     

Accuracy of touch imprint cytology in diagnosing lung cancer.

A 3-year study assessed the diagnostic accuracy of touch imprint smears in the diagnosis of lung cancer. Touch imprint smears were prepared from 90 computerized tomographic-guided core needle lung biopsies. Cytological diagnosis of touch imprint smears were correlated with the histological diagnosis of the corresponding core needle biopsy specimen, which was taken as the gold standard. The sensitivity, specificity, positive predictive value and negative predictive value of imprint smear results were 89%, 100%, 100% and 68%, respectively. There were no false positives, and all patients with small cell lung cancer were correctly diagnosed with this technique. Imprint cytology can be used to provide a rapid, preliminary diagnosis of lung cancer.     

血浆(1,3)-β-D葡聚糖检测对器官移植患者侵袭性真菌感染诊断价值

目的 探讨血浆(1,3)-β-D葡聚糖(BG)检测(G试验)在诊断器官移植术后合并侵袭性真菌感染(IFI)的诊断价值.方法 回顾性分析2011年1月～2012年12月130例在本院肝、肾移植中心住院疑似IFI的患者的血浆标本,进行G实验检测.其中64例最终确诊或临床诊断为IFI患者,设为IFI组,余66例为非IFI组.应用MB-80微生物动态快速检测系统和GKT-5M Set动态真菌检测试剂盒,血浆BG浓度≥10 pg/mL判定G试验阳性.采用四格表计算G试验诊断IFI的敏感度、特异度、阳性预测值和阴性预测值.对G试验结果进行受试者特征工作曲线(ROC曲线)分析,并计算曲线下面积.结果 130例患者中,IFI组中G试验阳性57/64,阳性率89.1％;阴性7例,假阴性率10.9％.非IFI组G试验阳性15/66,假阳性率22.7％.G试验阳性诊断IFI的敏感度、特异度、阳性预值和阴性预测值分别为89.1％,77.3％,79.2％和87.9％.根据G试验结果绘制ROC曲线,曲线下面积为0.875(95％ CI:0.813～0.937).结论 G试验对器官移植患者IFI具有中等诊断价值.适当提高诊断界值及重复检测可较大程度地消除假阳性.     

A rapid detection of multidrug-resistant Mycobacterium tuberculosis by a nitrate reductase assay on blood agar

The susceptibility of 49 Mycobacterium tuberculosis clinical isolates to isoniazid (INH) and rifampisin (RIF) (28 multi-drug resistant-tuberculosis samples) was determined by a nitrate reductase assay (NRA) on blood agar. Agreement between the NRA and other testing methods was found to be 93.8% for both INH and RIF. The sensitivity, specificity, positive predictive value and negative predictive value for INH were 92.8%, 94.2%, 86.6% and 97%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for RIF were 90.4%, 96.4%, 95% and 93.1%. In conclusion, we show here that blood agar can be used effectively for the NRA test.     

Evaluation of a rapid molecular screening approach for the detection of toxigenic Clostridium difficile in general and subsequent identification of the tcdC Δ117 mutation in human stools

We have developed and validated a rapid molecular screening protocol for toxigenic Clostridium difficile, that also enables the identification of the hypervirulent epidemic 027/NAP1 strain. We describe a multiplex real-time PCR assay, which detects the presence of the tcdA and tcdB genes directly in stool samples. In case of positive PCR results, a separate multiplex real-time PCR typing assay was performed targeting the tcdC gene frame shift mutation at position 117. We prospectively compared the results of the screening PCR with those of a cytotoxicity assay (CTA), and a rapid immuno-enzyme assay for 161 stool samples with a specific request for diagnosis of C. difficile infection (CDI). A total of 16 stool samples were positive by CTA. The screening PCR assay confirmed all 16 samples, and gave a PCR positive signal in eight additional samples. The typing PCR assay detected the tcdC Δ117 mutation in 2/24 samples suggesting the presence of the epidemic strain in these samples. This was confirmed by PCR ribotyping and sequencing of the tcdC gene. Using CTA as the “gold standard”, the sensitivity, specificity, positive predictive value, and negative predictive value, for the screening PCR were 100%, 94.4%, 66.7%, and 100%, respectively. In conclusion, PCR may serve as a rapid negative screening assay for patients suspected of having CDI, although the low PPV hamper the use of PCR as a standalone test. However, PCR results may provide valuable information for patient management and minimising the spread of the epidemic 027/NAP1 strain.     

Diagnostic Value of the Amplicor PCR Assay for Initial Diagnosis and Assessment of Treatment Response for Pulmonary Tuberculosis

We evaluated the Amplicor PCR assay as an initial diagnostic tool on the basis of clinical diagnosis, and assessed this assay as a follow-up test for patients with pulmonary tuberculosis during chemotherapy. Of the 208 specimens from 155 patients who were bacteriologically and/or clinically diagnosed with active tuberculosis before chemotherapy, 144 were Amplicor PCR-positive (sensitivity, 69.2%), which was equal to the results of culturing. Among 89 specimens which showed positive results by smear and culturing, the Amplicor PCR assay detected 87 (97.8%), whereas among 55 specimens which showed smear-negative but culture-positive results, the Amplicor PCR assay detected 46 (83.6%)(P = 0.003). No false positive results were found in the two systems (specificity, 100%, 120/120). The Amplicor PCR assay was also evaluated as a follow-up test using 926 specimens from 207 patients receiving active tuberculosis chemotherapy. Among 433 specimens which showed Amplicor-PCR positive, 222 (51.3%) were culture-negative. On the other hand, among 233 culture-positive specimens, only 12 (5.2%) were Amplicor PCR-negative. Therefore, this assay is useful for the rapid diagnosis of tuberculosis. The duration of Amplicor PCR-positive after culture-negative conversion was significantly associated with the presence of cavitary lesion, smear-positive specimens before treatment, and smear-positive specimens with negative cultures during chemotherapy.     

Comparative evaluation of the microplate nitrate reductase assay and the rezasurin microtitre assay for the rapid detection of multidrug resistant Mycobacterium tuberculosis clinical isolates

The microplate nitrate reductase assay (MNRA) and the rezasurin microtitre assay (REMA) were used for the susceptibility testing of 73 clinical isolates and the results were compared with those that were obtained using the Bactec 460 TB and Bactec MGIT 960 systems. The REMA and the MNRA were performed in 96-well plates. For the REMA, the concentrations of isoniazid (INH) and rifampicin (RIF) ranged from 1.0-0.01 μg/mL and 2.0-0.03 μg/mL, respectively. For the MNRA, the INH concentration was between 1.0-0.03 μg/mL and the RIF concentration was between 2.0-0.06 μg/mL. For the MNRA, the sensitivity, specificity, positive predictive value, negative predictive value and INH/RIF agreement were 100/95.6, 97.6/100, 96.8/100, 100/98 and 98.6/98.6, respectively, and for the REMA, they were 100/91.3, 90.4/100, 88.5/100, 100/96.1 and 94.5/97.2, respectively. Our data suggest that these two rapid, low-cost methods may be inexpensive, alternative assays for the rapid detection of multidrug resistant tuberculosis in low-income countries.     

Dominant TNF-α+ Mycobacterium tuberculosis-specific CD4+ T cell responses discriminate between latent infection and active disease

Rapid diagnosis of active Mycobacterium tuberculosis (Mtb) infection remains a clinical and laboratory challenge. We have analyzed the cytokine profile (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2)) of Mtb-specific T cells by polychromatic flow cytometry. We studied Mtb-specific CD4+ T cell responses in subjects with latent Mtb infection and active tuberculosis disease. The results showed substantial increase in the proportion of single-positive TNF-α Mtb-specific CD4+ T cells in subjects with active disease, and this parameter was the strongest predictor of diagnosis of active disease versus latent infection. We validated the use of this parameter in a cohort of 101 subjects with tuberculosis diagnosis unknown to the investigator. The sensitivity and specificity of the flow cytometry-based assay were 67% and 92%, respectively, the positive predictive value was 80% and the negative predictive value was 92.4%. Therefore, the proportion of single-positive TNF-α Mtb-specific CD4+ T cells is a new tool for the rapid diagnosis of active tuberculosis disease.     

急诊脑卒中识别评分量表在院前急救中筛选价值的探讨

目的:探讨急诊脑卒中识别评分量表(ROSIER)在院前急救筛选中的应用价值。方法:收集2013年1月至2014年1月期间,我院急诊科收治的可疑脑卒中病例114例,在院前急救中应用ROSIER量表筛查,并以辛辛那提院前脑卒中识别评分量表(CPSS)作为对照,以头颅CT或MRI检查、神经专科医师意见作为最终诊断,比较ROSIER与CPSS的对于脑卒中的筛选价值。结果:ROSIER对脑卒中的特异度、阳性似然比依次为83.67%、4.80,均显著高于CPSS的67.35%、2.36(P0.05);假阳性为16.33%,显著低于CPSS的32.65%;ROSIER的Kappa值为0.621,显著高于CPSS的0.462,差异具有统计学意义(P0.05);两组的敏感度、假阴性率及阴性拟然比无明显差异(P0.05)。结论:ROSIER应用于脑卒中筛查具有较高的敏感度和特异度,对于脑卒中的院前筛查以及院前急救具有重要指导意义。