血浆(1→3)-β-D-葡聚糖动态检测对评价抗真菌药物疗效的价值

探讨血浆(1→3)-β-D-葡聚糖动态检测对评价深部真菌感染的抗真菌药物疗效的价值.应用MB-80微生物动态快速检测系统及GKT-5M Set动态真菌检测试剂盒定量检测血浆(1→3)-β-D-葡聚糖的含量.在48例深部真菌感染患者的血浆(1→3)-β-D-葡聚糖检测中,有35例患者血浆(1→3)-β-D-葡聚糖水平逐渐...     

血浆(1-3)-β-D-葡聚糖检测对侵袭性肺部真菌感染的诊断价值

为评价(1-3)-β-D-葡聚糖检测(G试验)对侵袭性肺部真菌感染的诊断价值,对肺部感染患者130例行G试验,并将试验结果与病原学诊断结果相比较,计算G试验的灵敏度、特异性、阳性预测值、阴性预测值及准确性.结果表明:G试验对侵袭性肺部真菌感染的灵敏度为76.5%(13/17),特异性为80.7%(88/109),阳性预...     

白血病真菌感染的预防和护理研究

目的 探讨护理干预对预防急性白血病侵袭性真菌感染的影响.方法 取我院50例白血病患者,分别予常规护理和防治真菌护理,通过真菌PCR分子生物学检测和1-3-β-D 葡聚糖检测.结果 常规护理和防治真菌护理两组的临床效果有明显差异(p<0.001).结论 防治真菌感染护理可以有效的降低白血病患者的侵袭性真菌感染率.     

葡聚糖结合蛋白的原核表达及检测深部真菌感染的应用

目的 验证葡聚糖特异性的结合蛋白用于检测深部真菌感染(IFI)患者血清中(1,3)-β-D-葡聚糖的应用价值。方法 通过构建美洲鲎葡聚糖结合蛋白(GBP)252位氨基酸至668位氨基酸活性区域表达质粒——pET30a-GBP252-668,表达并纯化获得GBP252-668重组蛋白。随后通过棋盘法构建双GBP252-668夹心法,并以此检测IFI患者血清中(1,3)-β-D-葡聚糖。同时与G试验(G-Test)进行检测性能的比对。结果 成功纯化获得分子质量约为46 ku的GBP252-668重组蛋白,并确定最佳包被浓度和检测浓度分别为8和16 g/L,以此构建了GBP252-668夹心法。对48名深部真菌感染患者和48名健康人血清进行检测,根据受试者工作特征曲线(ROC)可得其灵敏度为91.67%,特异度为89.58%。其灵敏度与G-Test相当,特异性高于同批次G-Test(79.17%)。结论 本研究构建的双GBP夹心法是一种极具潜力检测侵袭性真菌的方法,其灵敏度与G-Test相当,特异性高于同批次G-Test。     

TGF-β1在糖尿病肾病早期诊断中的应用

目的:探讨转化生长因子-β1(TGF-β1)在糖尿病早期肾病诊断中的临床应用价值。方法:采用放射免疫分析检测糖尿病患者血清及尿中β2-微球蛋白(β2-m),采用酶联免疫分析(ELISA)法检测糖尿病患者血清中TGF-β1。同时检测尿白蛋白(uAlb)及尿微量蛋白,按尿白蛋白的排泄率分为三组。结果:糖尿病患者TGF-β1、血β2-m、尿β2-m与尿Alb具有很好的正相关性。结论:检测血清TGF-β1、血β2-m、尿β2-m是诊断糖尿病早期肾损害的敏感指标,对糖尿病肾病的早期诊断具有一定的临床应用价值。     

(1,3)-β-D葡聚糖、半乳甘露聚糖联合降钙素原检测对侵袭性真菌感染的诊断价值

目的 探讨血清中(1,3)-β-D葡聚糖[ (1,3) β-D-glucan,G]和半乳甘露聚糖(galactomannan,GM)联合降钙素原(procalcitonin,PCT)检测对患者侵袭性真菌感染(invasive fungal infections,IFI)的临床诊断价值。方法 选取徐州市中心医院2017年1月至2018年6月收治的具有高危IFI因素的住院患者447例,根据血培养结果分成实验室诊断组和非实验室诊断组。分别采用定量检测、比色法及电化学发光法对血清中G、GM和PCT的含量进行检测,评价三者联合检测对IFI的早期诊断价值。结果 447例患者中实验室诊断组51例,非实验室诊断组396例。51例患者中G试验阳性39例,GM试验阳性14例,PCT阳性41例。G试验、GM试验、PCT检测的敏感性为76.47%、27.45%和80.39%;特异性为76.77%、87.12%和66.67%;阳性预测值为29.77%、21.54%和23.70%;阴性预测值为96.20%、90.31%和96.35%;阳性似然比为3.29、2.13和2.41;阴性似然比为0.31、0.83和0.29;Youden指数为0.53、0.15、0.47。三者联合检测的敏感性为94.12%、特异性为62.88%、阳性预测值为24.62%,阴性预测值为98.81%,阳性似然比为2.54,阴性似然比为0.09,Youden指数为0.57。三者联合检测的敏感性均高于G试验、GM试验、PCT单独检测及G/GM联合试验,差异具有统计学意义(χ2值分别为6.331、47.545、4.320和5.299,P值均<0.05)。三者联合检测的特异性均低于G试验、GM试验单独检测及G/GM联合试验,差异具有统计学意义(χ2值分别为18.127、62.061和16.754,P值均<0.05),但与PCT单独检测相比差异无统计学意义(χ2=1.245,P>0.05)。结论 G、GM以及PCT三者联合检测可显著提高IFI检出的敏感性,并且对IFI的诊断排除有很大的价值,从而降低假阳性率和假阴性率,为IFI的早期诊断提供一定的依据。     

血浆1，3-β-D葡聚糖检测对儿童侵袭性真菌感染诊断价值

目的评价血浆1,3-β-D葡聚糖（BG）检测（G试验）对儿童侵袭性真菌感染（IFI）的诊断价值。方法回顾性分析2008年1月至2011年8月在首都医科大学附属北京儿童医院住院,抗生素应用时间〉10d,并行G试验患儿的临床资料,排除拟诊IFI者。根据IFI诊断标准,将研究对象分为IFI组和非IFI组。G试验测定采用GKT-5MSet动态真菌检测试剂盒,血浆BG浓度≥10pg·mL^-1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。结果研究期间共有525例患儿行G试验,排除拟诊IFI者129例,最终396例患儿纳入分析。IFI组43例,非IFI组353例。IFI组G试验阳性31／43例,阳性率为72．1％;阴性12／43例,假阴性率为27．9％。非IFI组G试验阳性48／353例,假阳性率为13．6％。G试验诊断IFI的敏感度、特异度、阳性预测值和阴性预测值分别为72．1％、86．4％、39．2％和96．2％。根据G试验结果绘制ROC曲线,曲线下面积为0．815（95％CI：0．732—0．898）。结论G试验对儿童IFI具有中等诊断价值。适当提高诊断界值或连续监测可很大程度消除假阳性。     

小鼠Dectin-1胞外区的融合表达及其识别白念珠菌β-葡聚糖的研究

目的 克隆、表达并纯化小鼠腹腔巨噬细胞Dectin-1基因胞外区,探讨其识别并结合真菌β-葡聚糖的能力.方法 应用RT-PCR方法从小鼠腹腔巨噬细胞RNA中扩增Dectin-1基因胞外区,构建原核重组表达载体pET28-CRD,进行融合表达、纯化和复性.将融合蛋白与白念珠菌酵母相共同孵育,检测其识别、结合白念珠菌细胞壁β-葡聚糖的功能.结果 成功克隆并构建原核重组表达质粒pET28-CRD,表达并纯化了融合蛋白,该蛋白能识别、结合白念珠菌酵母细胞壁β-葡聚糖.结论 构建的原核重组表达载体能够在原核细胞内表达,表达产物有识别、结合真菌胞壁β-葡聚糖的功能,为进一步研究相应的真菌检测方法奠定了基础.     

白念珠菌细胞壁不溶性β-葡聚糖升高小鼠血清IL-1β、TNF-α的研究

为探讨白念珠菌β-葡聚糖升高白细胞的机制,通过培养白念珠菌来获取不溶性β-葡聚糖(Candida albicansinsoluble β-glucan,CAIBG),并以rhG-CSF作为阳性对照,计数实验组及对照组动物外周静脉血白细胞总数,用酶联免疫吸附试验(ELISA)检测IL-1β、TNF-α,观测小鼠血清中IL-1β、TNF-α的变化,以及IL-1β、TNF-α与CAIBG诱导的末梢血白细胞计数之间的相关性。结果发现在实验组小鼠外周血白细胞,血清IL-1β、TNF-α较对照组均有明显升高(P<0.05)。结果表明白念珠菌不溶性β-葡聚糖可以通过升高小鼠血清IL-1β、TNF-α来刺激机体白细胞产生增加。     

血浆(1-3)-β-D-葡聚糖对侵袭性真菌感染诊断价值的荟萃分析

目的采用荟萃分析评价血浆(1,3)-β-D-葡聚糖检测对侵袭性真菌感染的诊断价值。方法采用STATA11软件分析纳入研究项目血浆(1,3)-β-D-葡聚糖浓度诊断侵袭性真菌感染总的灵敏度、特异度,阳性似然比和阴性似然比。结果纳入的31组研究数据分析显示总的灵敏度,特异度,阳性似然比和阴性似然比分别为0.79(95%CI,0.73～0.85),0.86(95%CI,0.80～0.90),5.64(95%CI,3.818～8.352),和0.242(95%CI,0.179～0.326)。结论荟萃分析显示血浆(1,3)-β-D-葡聚糖检测对于诊断IFI有较高的临床价值。     

Strategy for the treatment of fungal infections in critically ill surgical patients]

To improve the outcome of invasive Candida infections, earlier empirical therapy before the establishment of the definitive diagnosis is considered to be necessary. However, appropriate use of empirical therapy for suspected candidiasis in febrile non-neutropenic surgical patients has not been defined. According to the guidelines from the Infectious Diseases Society of America, empirical therapy of suspected candidiasis in this setting should be limited to patients with Candida colonization of multiple sites, multiple other risk factors, and absence of any other causes of fever. A corrected colonization index which takes into account both the density and the degree of colonization of Candida spp. was shown to be the independent factors that predict subsequent candidal infection. It may also be appropriate to commence empirical therapy on the basis of a positive serodiagnostic test. Beta-D glucan is a cell-wall constituent of fungi, which is assumed to be a marker of fungal sepsis. However, it has been shown that beta-D-glucan can also be detected in patients without fungal infections, such as those on haemodialysis, and its positive predictive value is relatively low. The mono-utilization of beta-D-glucan for the assessment of fungal infection should therefore be avoided. The combined assessment of beta-D-glucan and extent of colonization with Candida spp. is believed to have the advantage of lessening the likelihood of a false positive reaction of beta-D-glucan.     

Serum glucan levels are not specific for presence of fungal infections in intensive care unit patients

Fungal infections in the critically ill patient are difficult to diagnose and are associated with a high mortality rate. A major obstacle to managing fungal infection is the lack of a reliable clinical assay that will rapidly identify patients with fungal sepsis. Glucans are polymers of glucose that are found in the cell wall of fungi and certain bacteria. Glucans are also released from the fungal cell wall into the extracellular milieu. Several studies have reported that detection of fungal glucan in serum or plasma is useful in the diagnosis of mycoses. However, recent studies have questioned the clinical utility of this assay. In this study, we examined serum glucan levels in intensive care unit (ICU) patients and attempt to correlate serum glucan levels with the presence of fungal infection. Following attainment of informed consent, serum was harvested from 46 ICU patients with confirmed fungal infections, confirmed bacterial infections, or no evidence of infection. Sera from eight healthy volunteers served as control. Serum glucan was assayed with a glucan-specific Limulus assay. Serum glucan levels were increased (69.6 +/- 17 pg/ml; P < 0.001) in ICU patients versus the normal (11.5 +/- 1.3 pg/ml) and noninfected ICU (27.4 +/- 17 pg/ml) controls. However, serum glucan levels were not different in patients with confirmed fungal infections versus those with confirmed bacterial infections. Thus, serum glucan levels did not show a correlation with the presence of fungal infections and do not appear to be specific for fungal infections. However, the assay may be useful as a negative predictor of infection.     

Interpretation,pitfalls of biomarkers in diagnosis of invasive fungal diseases

BackgroundInvasive Fungal Diseases (IFD), account for high morbidity and mortality in immunocompromised and seriously ill patients worldwide. Early, faster and accurate diagnosis with timely and appropriate patient management is critical for improved patient outcome and antifungal stewardship. Clinical/radiological presentations in IFD are non-specific and microscopy/culture based tests have low sensitivity and long turnaround time. Biomarkers have clinical utility for diagnosing IFD but their interpretation is not straight forward.ObjectivesThis review discusses the salient characteristics, clinical usefulness and limitations of common biomarkers such as Galactomannan (GM), 1–3, β D glucan (βDG), mannan, anti-mannan antibody (Mn/antiMn), Cryptococcal antigen test (CrAg), Nucleic Acid Amplification (NAA) tests and next generation sequencing for diagnosing IFD.ContentsFungal biomarkers are useful adjuncts as screening and diagnostic tools for IFD and are much more suited for ‘ruling out’ rather than ‘ruling in’ disease. GM, NAA tests are promising biomarkers for screening of invasive Aspergillosis in high risk asymptomatic patients who are not on antifungal therapy and for diagnosis of breakthrough infections in symptomatic patients. 1–3, β D glucan has limitations both as a ‘rule in’ and ‘rule out’ test and is useful in only specific clinical settings. Two consecutive positive 1-3-βDG tests or combined positivity with GM increases its specificity. Mn/antiMn, T2Candida nano diagnostic panel are promising candidates for diagnosing invasive candidiasis. Combining two or more biomarkers improves the sensitivity for prompt initiation of antifungal therapy and the negative predictive value for suspension of empirical treatment.Serum CrAg test is a good ‘rule in’ rather than a ‘rule out’ test in immunocompetent patients but has good diagnostic accuracy in immunocompromised patients. Detection of single nucleotide polymorphisms by next generation sequencing is useful for fungal characterization and identification of host determinants responsible for increased susceptibility to fungal infections but is still in experimental stages.     

Serum Glucan Levels Are Not Specific for Presence of Fungal Infections in Intensive Care Unit Patients

Fungal infections in the critically ill patient are difficult to diagnose and are associated with a high mortality rate. A major obstacle to managing fungal infection is the lack of a reliable clinical assay that will rapidly identify patients with fungal sepsis. Glucans are polymers of glucose that are found in the cell wall of fungi and certain bacteria. Glucans are also released from the fungal cell wall into the extracellular milieu. Several studies have reported that detection of fungal glucan in serum or plasma is useful in the diagnosis of mycoses. However, recent studies have questioned the clinical utility of this assay. In this study, we examined serum glucan levels in intensive care unit (ICU) patients and attempt to correlate serum glucan levels with the presence of fungal infection. Following attainment of informed consent, serum was harvested from 46 ICU patients with confirmed fungal infections, confirmed bacterial infections, or no evidence of infection. Sera from eight healthy volunteers served as control. Serum glucan was assayed with a glucan-specific Limulus assay. Serum glucan levels were increased (69.6 ± 17 pg/ml; P < 0.001) in ICU patients versus the normal (11.5 ± 1.3 pg/ml) and noninfected ICU (27.4 ± 17 pg/ml) controls. However, serum glucan levels were not different in patients with confirmed fungal infections versus those with confirmed bacterial infections. Thus, serum glucan levels did not show a correlation with the presence of fungal infections and do not appear to be specific for fungal infections. However, the assay may be useful as a negative predictor of infection.     

Fungal infections in the immunocompromised host

Invasive fungal infections cause significant morbidity and mortality in patients with impaired immune defences. Defects in neutrophil function and neutropenia predispose to disseminated Candida, Aspergillus and Mucoraceae infections while altered T-lymphocyte mononuclear phagocyte function predisposes to infection with C. neoformans, Histoplasma and Coccidioides. Fungal infections in the immunocompromised host are difficult to diagnose and difficult to treat successfully. The diagnosis is often missed or delayed because of the non-specific clinical features, the failure to isolate or difficulty in interpreting the presence of the fungus from routine microbiological cultures, and the limited usefulness of available serological tests. The assay for cryptococcal antigen is the only currently available reliable serological test used to diagnose an invasive fungal infection. Definitive diagnosis is made by histopathological demonstration of the fungus in tissue or a positive culture from a sterile body site. Invasive procedures are often necessary to obtain adequate tissue for histology and culture. The treatment of invasive fungal infection in the immunocompromised host is amphotericin B with or without 5FC. The usual recommended dose is 1.5 to 3 g total amphotericin B over 6 to 12 weeks. The optimal dose and duration of therapy for each infection is not known. Treatment failures and relapses are common in patients who do not achieve remission of their underlying disease. Ketoconazole, a new broad-spectrum oral antifungal medication, does not appear to be effective therapy for invasive fungal infection in the immunocompromised patient based on results of small clinical trials. New diagnostic methods and therapeutic approaches are necessary to improve the outcome of these infections. Areas of current research include serological assays for fungal antigens and metabolites which may allow earlier diagnosis, treatment with combinations of antifungal agents, and the development of new antifungal agents.     

Efficacy study of early presumptive therapy (EPT) for deep fungal infection]

We retrospectively studied the efficacy of early presumptive therapy (EPT). SUBJECTS AND METHOD: Of the critically ill patients admitted from January 1998 to the end of December 2000 to Kyorin University Trauma Burn and Intensive Care Center, 77 cases were diagnosed with suspected deep fungal infection, and EPT was administered. The diagnosis of suspected deep fungal infection was made by definition. EPT (FLCZ 200 to 400 mg/day x 14 days) was started as soon as the diagnosis was made and continued for two weeks. Its efficacy was retrospectively studied by analyzing the clinical findings, changes in local organisms, and hematological tests. RESULTS: After treatment, 62% of the patients showed improvement in clinical signs of infection, elimination of locally detected fungus, and improvement in the serum diagnosis test. Post-EPT detection levels of the fungus had decreased to 21%. The mean pre-EPT body temperature was 38.7 degrees C +/- 0.6 degrees C, but the mean post-EPT temperature was 36.7 degrees C +/- 0.6 degrees C. The mean level of blood 1,3-beta-D-glucan was 35 plus minus 13 pg/ml at the time the diagnosis was made, but returned to normal levels after treatment had concluded. No patients died as a direct result of the fungal infection. CONCLUSION: This study of early presumptive therapy in critically ill patients in the emergency and intensive care medicine fields showed the therapy in these, and in high risk patients to be efficacious.     

Histologic examination of bone marrow core biopsy specimens has limited value in the diagnosis of mycobacterial and fungal infections in patients with the acquired immunodeficiency syndrome

Bone marrow cultures and biopsy specimens are commonly obtained to rule out disseminated infections, especially in persons with the acquired immunodeficiency syndrome (AIDS) and cytopenias. Using culture as the gold standard, we reviewed 130 consecutive bone marrow cores obtained from 114 AIDS patients along with results of concurrent blood and/or bone marrow aspirate cultures to determine the usefulness of histologic examination for diagnosis of mycobacterial and fungal infections. We also compared the ability of Ziehl-Neelsen, auramine-rhodamine (AR), polyclonal antibody to Mycobacterium bovis (Ab), and Gomori's methenamine silver staining to detect infections. Twenty-seven patients had mycobacterial infection (25 Mycobacterium avium-intracellulare complex cases and two Mycobacterium tuberculosis cases) detected by blood and/or bone marrow cultures. The maximum sensitivity of histology was 50% when the auramine-rhodamine stain and the polyclonal antibody to M bovis were used in combination. The single best stain was auramine-rhodamine, with a sensitivity of 44%, followed by the polyclonal antibody to M bovis (35%). Granulomas were observed in nine cases of mycobacterial infection and did not correlate with the presence of stainable organisms. Of seven patients with positive fungal cultures of bone marrow, four had granulomas and a positive Gomori's methenamine silver stain, one had only a positive stain, and two had neither granulomas nor a diagnostic stain. Overall, granulomas were not sensitive for the detection of infections when culture-proven mycobacterial and fungal cases were evaluated together. We conclude that bone marrow examination has a limited value in the routine evaluation of common opportunistic infections in AIDS patients and recommend that less-invasive tests, such as blood cultures, be obtained initially in most circumstances.