结核抗原特异性IFN-γ ELISPOT检测技术用于诊断HIV感染者人群中结核潜伏感染的研究

目的 探讨结核分枝杆菌抗原特异性IFN-γ酶联免疫斑点(ELISPOT)检测技术在HIV感染者中用于诊断结核潜伏感染的应用价值.方法 运用自行研发的结核分枝杆菌抗原特异性IFN-γ ELISPOT检测方法对110例确诊的HIV感染者及205例HIV阴性健康志愿者的外周血结核特异性IFN-γ释放水平进行检测,并同时进行结核菌素皮肤试验(研).结果 ELISPOT在HIV感染和HIV/AIDS正在接受抗病毒治疗人群中的阳性率分别为24.5%和29.8%,明显高于HIV阴性健康人群7.3%(P＜0.001);对多种不同部位多肽抗原刺激以P8.10的敏感性最高,但与其他抗原的差异无统计学意义(P＞0.05);不同CD4细胞计数水平对ELISPOT检测结果尤影响(P＞0.05);PPD试验在HIV感染人群中的阳性率远远低于ELISPOT检测方法,其差异有统计学意义(P＜0.0001).结论 结核分枝杆菌抗原特异性IFN-γ ELISPOT检测技术在艾滋病合并结核潜伏感染的早期诊断中有很大的应用价值,推荐用于艾滋病合并结核潜伏感染的辅助诊断.     

Rv1813c在结核分枝杆菌潜伏感染诊断中的价值

目的 研究重组结核分枝杆菌潜伏感染蛋白Rv1813c在诊断结核分枝杆菌潜伏感染方面的价值.方法 20例结核潜伏感染者和79例健康志愿者均行胸部X线检查、PPD皮肤试验、结核抗体检测,同时应用ELISA联合重组融合蛋白CFP10-ESAT6和潜伏感染蛋白Rv1813c进行IGRA检测.结果 所有受试者中,PPD皮肤试验和抗体检测的阳性率分别为50％和28％.Rv1813c刺激潜伏感染者后产生IFN-γ的水平显著高于健康对照(P＜0.05),其刺激PPD强阳性组(皮试直径≥15mm)产生的IFN-γ值低于弱阳性组(5mm≤皮试直径＜15mm),但无统计学意义,而CFP10-ESAT6刺激PPD强阳性组后产生的IFN-γ值高于弱阳性组(P＜0.05).CFP10-ESAT6和Rv1813c刺激抗体阴性及阳性组后产生的IFN-γ水平没有显著性差异(P＞0.05).Rv1813c诊断结核感染的ROC曲线下面积为0.834,特异性80％时,灵敏度为85％;特异性为90％时,灵敏度为45％.结论 同时检测Rv1813c及CFP10-ESAT6抗原特异的IFN-γ值有可能筛选出潜伏感染者中的活动感染人群,对于结核分枝杆菌潜伏感染诊断有重要的应用价值.     

潜伏结核感染的分子机制研究进展

潜伏结核感染是活动性结核病的主要来源,也是结核病防控的关键,但是潜伏结核感染的机制尚未完全明确.近年来,在分子水平上对潜伏结核感染所做的大量研究表明,许多重要分子参与了潜伏结核感染的发生过程,进一步阐释了潜伏结核感染的发生机制,为潜伏结核感染诊断、治疗及新疫苗的研发开辟了新方向.现就结核潜伏感染的分子机制研究进展进行综述.     

结核感染干扰素释放试验中的QFT-G试验在肺结核诊断中的应用评价

目的 探讨结核感染干扰素释放试验中的QFT-G试验在肺结核诊断中的应用评价.方法 采用QFT-G检测外周血结核特异性γ-干扰素的含量,同时与FQ-PCR检测痰中结核分枝杆菌核酸结核菌素、纯化蛋白质衍生物(PPD)皮试进行平行比较分析.结果 QFT-G、FQ-PCR和PPD三种方法的敏感度分别为:91.4％、84.2％、78.9％,特异性分别为86.9％、80.8％和54.8％,阳性预测值分别为91.0％、92.2％和71.1％,阴性预测值分别为66.2％、75.4％和46.3％,准确性分别为87.8％、85.0％和59.6％.结论 QFT-G较FQ-PCR和PPD有较高的敏感度、特异性和准确性,是一种诊断肺结核的有价值的检测方法.     

快速鉴定人肺部感染中非结核分枝杆菌的一种简单方法

目的建立一种快速鉴别结核分枝杆菌（tuberculous mycobacteria,TM）和非结核分枝杆菌（non tuberculous mycobacteria,NTM）的简单方法。方法收集165例诊断为肺结核病人的痰标本,经除污染杂菌处理后各取0.1ml的痰液沉淀物,分别加入到含不同浓度对硝基苯甲酸（para-nitro benzoicacid,PNB）的未成熟椰汁液体培养基和罗氏培养基中培养。对在含PNB的培养基中生长的阳性培养物进行烟酸检测。结果所测样本菌株中有18株在含PNB的培养基中生长,其培养物中检测不含烟酸。形态学检查18例未成熟椰汁液体培养基中阳性培养物都不同于典型的结核分枝杆菌。结论含PNB的未成熟椰汁液体培养基,可作为一种快速鉴定人肺部感染中非结核分枝杆菌的简单筛选方法。     

结核分枝杆菌早期培养滤液蛋白的制备和应用

<正>全球结核杆菌感染者高达20亿人,每年新发病例约800万~1000万,其中我国每年新发患者高达150万。结核杆菌流行病学的一个重要特征是结核分枝杆菌的潜伏感染。结核分枝杆菌潜伏感染是一种亚临床状态,其中约有10%的结核分枝杆菌潜伏感染者会发展成为结核病患者[1]。流行病学调查显示,我国受结核杆菌感染人数超过     

结核分枝杆菌抗体检测试剂盒的临床应用

结核分枝杆菌抗体(IgG)是结核病病人血清中重要的标志性抗体之一,检测此抗体对于鉴别活动性结核病和既往结核分枝杆菌感染患者具有辅助诊断价值。本文分别采用酶联免疫吸附法(ELISA)与胶体金法结核分枝杆菌抗体诊断检测试剂盒,对320例病人血清标本进行了结核分枝杆菌抗体(IgG)水平表达检测分析,以评价其在结核病人诊治中的临床应用,现报道如下。     

结核分枝杆菌相关IFN-γ释放试验在结核病诊断及疗效监测中的应用

目的：目的：探讨结核分枝杆菌相关γ-干扰素释放试验（TB-IGRA）在结核病诊断及临床抗结核治疗效果评价中的应用价值。方法：对196例临床确诊结核患者进行相关病史收集,同时进行TB-IGRA、结核菌素皮肤试验、结核杆菌培养、涂片镜检、结核抗体检测,结核患者的治疗过程中按用药疗程（初治患者第2、5、6个月,复治患者第2、5、8个月）对其进行随访,在治疗结束后6个月和1年后各随访一次,比较TB-IGRA与结核菌素皮肤试验在治疗过程中的变化。结果：确诊结核病例196例,其中肺结核158例,肺外结核38例,TB-IGRA在结核诊断中灵敏度为82．1％,特异性为91．7％,阳性预测值为93．1％,阴性预测值为79．3％,与传统实验室检查方法相比有显著性差异（P〈0．01）。在结核患者的抗结核治疗过程中,TB-IGRA阳性率及浓度水平治疗前与治疗后2、6月以及复治后2、5、8月比较显著下降（P〈0．01）,TB—IGRA水平与结核分枝杆菌数量存在较好相关性,与传统的TST方法评估疗效相比有显著性差异（P〈0．01）。结论：TB-IGRA在结核诊断中敏感性和阳性预测值较高,可作为较好的结核诊断的依据,同时TB-IGRA对肺外结核的灵敏度较高,为肺外结核患者的诊断提供了较为可靠的依据,通过治疗过程TB—IGRA检测分析,外周血TB-IGRA的变化随着抗结核治疗的进程下降的趋势明显,与结核涂片镜检结果符合率较高,是临床疗效评估较为理想的指标。     

Mycobacterium tuberculosis infection may protect against allergy in a tuberculosis endemic area

BACKGROUND: Epidemiological studies have shown an inverse relation of mycobacterial infection and the frequency of allergic diseases and asthma. Recent evidence suggests that allergic inflammation may be inhibited in the presence of chronic and persistent infections, such as that by Mycobacterium tuberculosis (MTB). The relation of tuberculin skin test (TST) size, an accepted marker of MTB infection and the frequency of allergic disease symptoms has not been reported from an area where MTB infection is endemic. OBJECTIVE: To investigate the association of TST and allergic disease symptoms, in children living in a tuberculosis (TB) endemic area. METHODS: In this cross-sectional study, 841 children aged 6-14 years from randomly selected household addresses in two poor communities of Cape Town, South Africa, were investigated with TST and standardized International Study on Asthma and Allergies in Childhood-based questionnaire on allergic disease symptoms. RESULTS: Children with positive TST (> or =10 mm) were significantly less likely to have allergic disease symptoms, in particular allergic rhinitis (AR) (adjusted odds ratio 0.43; 95% confidence interval 0.24-0.79) than those with negative TST. This association remained significant after adjusting for possible confounders and correcting for the effect of clustering (>1 child per household address) in the sample. There was a significant inverse linear trend in the relation of TST size in millimetre and the frequency of allergic disease symptoms, in particular AR (P<0.001). CONCLUSIONS: These results of inverse association of strong TST reaction and allergic disease symptoms in children from a TB endemic area are in support of the hypotheses that allergic inflammation may be inhibited by chronic infections, such as MTB.     

Detection of Mycobacterium tuberculosis in mixed broth cultures using DNA probes

Objective: To investigate the ability of a commercial acridinium ester-labeled DNA probe (AccuProbe, Gen-Probe Inc., USA) to detect the presence of Mycobacterium tuberculosis complex in a mixed culture with Mycobacterium avium complex.
Methods: The density of organisms required to produce a positive result for Mycobacterium tuberculosis complex alone in broth culture was compared with the density required to produce a positive result in the presence of Mycobacterium avium complex.
Results: A threshold density of 1.5 × 10⁶ CFU/mL was required for detection of Mycobacterium tuberculosis and this threshold remained unaltered in the presence of Mycobacterium avium complex. The presence of Mycobacterium avium complex had no effect on detection of Mycobacterium tuberculosis in a mixed broth culture incubated and probed over a 21-day period.
Conclusions: The findings of the study suggest that the presence of Mycobacterium avium complex has no effect on the detection of Mycobacterium tuberculosis and that the Accuprobe test is potentially capable of detecting a dual infection with organisms of both complexes.     

Inverse association between Mycobacterium tuberculosis infection and atopic rhinitis in children

BACKGROUND: The association between Mycobacterium tuberculosis (MTB) infection and atopy remains controversial. AIM: To investigate the association between MTB infection and atopic rhinitis in children living in a high TB incidence area. METHODS: In this cross-sectional study 418 children aged 6-14 years from an established epidemiological research-site in a poor urban community were invited to participate. They were assessed for allergic rhinitis (ISAAC questionnaire) and skin responses to tuberculin and eight environmental allergens. The presence of a BCG scar was documented, intestinal parasites and total and Ascaris lumbricoides-specific IgE levels were measured. Atopic rhinitis was defined, using the new World Allergy Organization (WAO) definition, as reported allergic rhinitis and a positive skin prick test (SPT > or =3 mm) to any allergen. RESULTS: Among the 337 children enrolled 10.4% had allergic rhinitis, 17.5% a positive SPT and 53% a positive tuberculin skin test (TST > or =10 mm). Children with a positive TST were significantly less likely to have recent atopic rhinitis (OR(adjusted) 0.06; 95% CI 0.007-0.5) than those with a negative TST. SPTs were significantly more common in children with negative TST who had recent allergic rhinitis (OR(adj) 34.0; 95% CI 7.6-152.6), but not in children with positive TST and recent allergic rhinitis (OR(adj) 0.6; 95% CI 0.07-5.2). CONCLUSIONS: MTB infection seems to reduce the prevalence of atopic rhinitis, and influences SPT reactivity in children with allergic rhinitis from a high TB incidence area.     

Latent tuberculosis infection among new recruits to the army in Beijing, China in 2009

China is a country with high latent tuberculosis infection (LTI) and has a policy of routine BCG vaccination. To monitor tuberculosis (TB) infection, a total of 907 new recruits to the army were interviewed and routinely examined by chest radiographs. They were intradermally injected with purified protein derivative (PPD) and detected with enzyme-linked immunospot (ELISPOT) assay with recombinant CFP-10/ESAT-6 (rCFP-10/ESAT-6) fusion protein, as a stimulus, from December 2009 to February 2010. The prevalence of LTI among new recruits was estimated as 50.2% and 30.7% by PPD skin test and ELISPOT assay, respectively. Of 452 PPD-negative and 455 PPD-positive volunteers, 132 (29.2%) and 146 (32.1%) were ELISPOT positive, respectively. The overall consistency between these two tests was 51.4% (466/907). Although 65.6% of PPD-positive volunteers and 27.8% of ELISPOT-positive volunteers (spot-forming cells; SFC 12.2 ± 21.2) could find the vaccination scars on their arms, 21.3% of PPD-positive volunteers and 35.9% of ELISPOT-positive volunteers (SFC 18.3 ± 34.6) could not. Thus, the TB infection rate in army recruits in China was not as high as previously reported. The results suggest that the ELISPOT technique, we presented, is an accurate method for screening TB infection in China.     

Infection due to a novel mycobacterium,mimicking multidrug-resistant Mycobacterium tuberculosis

The treatment of multidrug-resistant tuberculosis (TB) requires the use, for long periods, of drugs liable to cause significant side effects. In the case of misdiagnosis of multidrug-resistant TB, the patient is exposed to toxic substances without any benefit. In low-income countries, where the microbiological diagnosis of TB relies on microscopy only, the misdiagnosis of multidrug-resistant TB is very frequent in patients persistently smear-positive despite anti-TB treatment, with the possibility of an infection due to non-tuberculous mycobacteria (NTM) being neglected. The isolation of a mycobacterium from the sputum of a Somali patient apparently confirmed the previous diagnosis of cavitary pulmonary disease. Preliminary investigations led, at first, to the strain being identified as multidrug-resistant Mycobacterium tuberculosis, with findings fully in agreement with the patient's history, which was characterized by repeated interruptions of anti-TB treatment. Thorough phenotypic and genotypic analyses led subsequently to the recognition that the strain was a previously unreported non-tuberculous mycobacterium. The patient, who was unresponsive to the anti-TB treatment, dramatically improved once a drug combination active against NTM was used. A major objective of this article is to alert the medical community to the risk, present also in settings in which sophisticated diagnostic techniques are used, that a cavitary infection due to NTM, and consequently not responding to the anti-TB standard regimen, will be mistaken for multidrug-resistant TB.     

Impacts of interleukin-12 on multiplication of Mycobacterium tuberculosis and Mycobacterium avium complex in mice

Objective: To evaluate the potential impacts of exogenous administration of murine recombinant interleukin-12 (IL-12) on multiplication of Mycobacterium tuberculosis and M. avium complex (MAC) in murine models.
Methods: Swiss or beige mice were infected intravenously with M. tuberculosis H37Rv or MAC respectively, and were treated by subcutaneous injection with various doses of IL-12, either alone or in combination with chemotherapy. Effectiveness of treatment was assessed by the enumeration of CFUs in the spleens and lungs, together with other indicators.
Results: Multiplication of M. tuberculosis was reduced by IL-12 in a dose-dependent manner if the treatment began at day 1, whereas no statistically significant suppression was observed if the treatment began at day 14. Combination with IL-12 did not enhance the bactericidal activity of antituberculosis chemotherapy. The growth curves of MAC in IL-12-treated mice were almost identical to those of untreated controls, indicating that IL-12 did not affect the multiplication of MAC in beige mice. In both experiments, the dosing of IL-12 approached levels of severe toxicity for the mouse strains used.
Conclusions: IL-12 had a positive affect on early multiplication of M. tuberculosis . It had no effect on early multiplication of M. avium complex.     

Diagnosis of Mycobacterium tuberculosis infection using ESAT-6 and intracellular cytokine cytometry

Diagnosis of infection with Mycobacterium tuberculosis (MTB) using tuberculin skin testing (TST) is often hampered by prior Bacille Calmette-Guérin (BCG) vaccination. ESAT-6 is a protein that is expressed by MTB but absent in BCG. It has been postulated that it might be useful in distinguishing MTB-specific immune responses. This study measured CD4 T cell responder frequencies specific for ESAT-6 and the TST reagent purified protein derivative (PPD) in patients with tuberculosis (n = 16), controls with non-tuberculous pneumonia (n = 8) and normal subjects (n = 7). Responses were identified using the intracellular cytokine staining technique and flow cytometry on whole blood samples, and performed blinded to the patient condition. Antigen-specific CD4 cells were defined by CD69 positivity and one or more cytokine [interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma] and/or CD40L positivity. With ESAT-6 stimulation it was found that TB patients had significantly higher frequencies of IFN-gamma and CD40L-positive CD4 T cells compared to the normal group, while no significant differences were measured with PPD stimulation. A responder frequency of 0.01% or higher for at least one of the measured cytokines/CD40L was defined as a positive response. Using this criterion to compare the two patient groups, PPD had 100% sensitivity but 0% specificity while ESAT-6 had 100% sensitivity and 88% specificity. Use of MTB-specific proteins such as ESAT-6 in combination with intracellular cytokine staining and flow cytometry has the potential to identify individuals with MTB infection.     

Pattern and diversity of cytokine production differentiates between Mycobacterium tuberculosis infection and disease

Tuberculosis (TB) remains a global health problem. The solution involves development of an effective vaccine, but has been limited by incomplete understanding of what constitutes protective immunity during natural infection with Mycobacterium tuberculosis. In this study, M. tuberculosis‐specific responses following an overnight whole‐blood assay were assessed by intracellular cytokine staining and luminex, and compared between TB cases and exposed household contacts. TB cases had significantly higher levels of IFN‐γ⁺TNF‐α⁺IL‐2⁺CD4⁺T cells compared with contacts. TB cases also had a significantly higher proportion of cells single‐positive for TNF‐α, but lower proportion of cells producing IL‐2 alone and these differences were seen for both CD4⁺and CD8⁺ T cells. Cytokine profiles from culture supernatants were significantly biased toward a Th1 phenotype (IFN‐γ and IL‐12(p40)) together with a complete abrogation of IL‐17 secretion in TB cases. Our data indicate that despite a robust response to TB antigens in active TB disease, changes in the pattern of cytokine production between TB infection and disease clearly contribute to disease progression.     

结核分枝杆菌PhoPR双组分系统与不同毒力结核分枝杆菌致病性的相关性研究

目的：通过比较分析卡介苗株（BCG）、结核分枝杆菌国际标准无毒株（H37Ra）、结核分枝杆菌国际标准强毒株（ H37Rv）、新疆地区流行的优势强毒株结核分枝杆菌临床分离株（ XJ-MTB）的PhoP基因和PhoR基因的表达水平差异,探讨研究结核分枝杆菌PhoPR双组分系统与不同毒力结核分枝杆菌的致病性是否具有相关性。方法：首先提取上述四种不同毒力结核分枝杆菌菌株的总RNA,并进行完整性鉴定;再运用SYBR Green I实时荧光定量PCR技术检测各组菌株中PhoP基因和PhoR基因的表达水平;最后比较不同毒力结核分枝杆菌PhoP基因和PhoR基因的表达水平差异。结果：四种不同毒力菌株PhoP基因的相对表达量,由高到低依次为XJ-MTB（9.05）、H37Rv（1.00）、H37Ra（0.25）、BCG（0.08）,且四种菌株中PhoP基因的表达有显著的统计学差异（P＜0.05）;同时四种不同毒力菌株PhoR基因的相对表达量,由高到低依次为XJ-MTB（5.72）、H37Rv（1.00）、H37Ra（0.18）、BCG（0.07）,且四种菌株中PhoR基因的表达有显著的统计学差异（P＜0.05）。其中XJ-MTB菌株PhoP基因和PhoR基因的表达水平与BCG、H37Rv、H37Ra相比存在显著的统计学差异（P＜0.05）;H37Rv菌株PhoP基因和PhoR基因的表达与BCG、H37Ra相比存在显著的统计学差异（P＜0.05）;而BCG菌株PhoP基因和PhoR基因的表达与H37Ra相比统计学差异不显著（ P＞0.05）。结论：结核分枝杆菌PhoPR双组分系统中PhoP基因和PhoR基因在不同毒力结核分枝杆菌中的表达存在差异,并且该系统与不同毒力结核分枝杆菌的致病性存在相关性。