用白念珠菌菌丝相蛋白抗原诊断白念珠菌病的探讨

建立一种特异性强的检测白念珠菌血清抗体方法，协助临床诊断侵袭性念珠菌病。应用超声粉碎和ＣｏｎＡ－Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析法去除细胞甘露糖和糖蛋白后，ＰＡＧＥ电泳显示４７０００部分是主要的胞浆蛋白抗原，用４７０００－２９０００或全菌抗原建立ＥＬＩＳＡ法测血清抗体。     

用噬菌体模拟抗原检测白念珠菌菌丝蛋白抗体的研究

目的 用噬菌体展示文库技术制备白念珠菌菌丝蛋白模拟抗原，建立测定白念珠菌抗体的酶联免疫吸附测定法（ELISA）。方法 用抗白念珠菌菌丝蛋白P47的单克隆抗体从噬菌体随机12肽库中筛选能与之免疫学结合的肽，即模拟抗原表位。将该模拟抗原包被微孔板，用于酶联免疫吸附测定法测定病人血清白念珠菌菌丝蛋白抗体。结果 用噬菌体模拟抗原建立ELISA，其敏感性、特异性及重复性良好，测定了10份确诊白念珠菌侵袭性感染的病人血清，全部阳性，与用纯化的菌丝蛋白抗原P47测定结果一致；服用免疫抑制剂患者血清阳性率33％，健康人群阳性率为2％。结论 用抗白念珠菌菌丝蛋白单克隆抗体从噬菌体展示随机肽库筛到的噬菌体模拟抗原可代替白念珠菌P47抗原进行抗体测定。     

免疫转印法检测白色念珠菌菌丝组分蛋白抗体

目的 建立免疫转印检测方法探讨血清中白色念珠菌菌丝相蛋白组分抗体的实验室诊断意义。方法 应用超声粉碎和Ｃｏｎ Ａ－Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析除去细胞壁甘露糖和甘露糖蛋白后，惧蛋白抗原通过免疫转印技术，检测相应抗原的血清抗体。结果 血库血清４０例，１例阳性：白色念珠菌培养阳性的９０例中５８例阳性；热带念珠菌２阳性１７例，１０例阳性。使用大量抗菌药物和／或免疫抑制剂治疗而培养阴性１４例７例阳性。     

免疫转印法检测白色念珠菌丝组分蛋白抗体

目的：为了探讨血清中白色念珠菌菌丝相蛋白组分抗体的实验室诊断意义。方法 应用超声粉碎和Ｃｏｎ Ａ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析除去细胞壁甘露糖和甘露糖蛋白后，收集蛋白抗原，通过免疫转印技术，检测血清标本中相应抗原的抗体。结果：血库血清４０份中，１例阳性；白色念珠菌培养阳性的９０名患者，抗体阳性５８例；热带念珠菌培养阳性的１７名患者，１０例抗体阳性，使用大量抗菌药物和／或免疫抑制剂治疗而培养阴性的     

白念珠菌果糖二磷酸醛缩酶重组蛋白的制备及鉴定

摘要：目的：制备有免疫原性的白念珠菌果糖二磷酸醛缩酶（fructosebisphosphate aldolase, Fba1)重组蛋白。 方法：以白念珠菌C1标准株基因组DNA为模板,用PCR法扩增Fba1 DNA序列,与克隆载体pMD18-T连接、测序,再与原核表达载体pET28a(+)连接,构建成重组表达质粒pET28a(+)/Fba1,转化大肠埃希菌BL21(DE3),IPTG诱导重组融合蛋白表达,亲和层析柱纯化重组蛋白。用抗His标签的单克隆抗体鉴定融合蛋白,并用确诊为侵袭性白念珠菌病（ICAI）、且抗白念珠菌烯醇化酶抗体阳性的患者血清进行抗原性鉴定。 结果：Fba1 DNA序列与GenBank中的序列一致。在大肠埃希菌中获得白念珠菌Fab1重组蛋白的高效表达,表达产物以可溶性蛋白为主,最终蛋白质得率为7.6 mg/g湿菌。经免疫印迹鉴定,重组蛋白与抗His标签的单克隆抗体和确诊ICAI患者血清呈特异性反应,显示出良好的抗原反应性。 结论：成功制备了具有良好抗原反应性的重组蛋白,为建立特异性诊断ICAI的新方法打下基础。     

P47模拟多肽抗原在诊断侵袭性白色念珠菌感染中的应用

目的利用噬菌体肽库来源的P47模拟抗原建立的ELISA法,探讨该模拟抗原在侵袭性白色念珠菌病血清学诊断中的应用价值。方法收集经培养确诊的各类念珠菌侵袭性感染和浅表感染的病人血清130份,用模拟抗原包被ELISA微孔板,测定抗P47抗体,考察该方法能否区分白色念珠菌侵袭性感染和浅表感染,以及与其他念珠菌感染的交叉反应。结果经细菌培养鉴定确证为白色念珠菌侵袭性感染的病人血清35份,抗P47抗体阳性率为100%,浅表感染者阳性率为4.8%(2/42),热带念珠菌侵袭性感染者抗P47抗体阳性率为16.7%(3/18)。光滑念珠菌、近平滑念珠菌以及未能鉴定到种的念珠菌侵袭性和浅表感染者血清,均未检测到抗P47抗体。结论用白色念珠菌菌丝蛋白P47模拟抗原建立ELISA检测抗P47抗体,虽然与热带念珠菌侵袭性感染有部分交叉反应,但仍可用于白色念珠菌侵袭性感染的血清学诊断。     

抗烯醇化酶抗体检测对侵袭性念珠菌病的诊断价值

目的 利用白念珠菌烯醇化酶重组蛋白质为抗原,建立测定人血清中抗白念珠菌烯醇化酶IgG类抗体的ELISA法,评估其在侵袭性念珠菌病(IC)诊断中的应用价值.方法 收集各类确诊IC患者(66例)和白念珠菌定植者(32例)血清,用重组白念珠菌烯醉化酶包被ELISA微孔板,测定血清中的抗烯醇化酶抗体,确定cut off值并对方...     

重组抗原检测抗干燥综合征A 抗原的自身抗体

目的 建立便捷的检测抗干燥综合征（ＳＳ）Ａ抗原（分子量为６００００的多肽成分）的自身抗体（抗ＳＳＡ）的方法,以利用疾病的早期诊断和病程监控。方法 构建基因工程菌,表达ＳＳＡ－６００００融合蛋白。经ＧＳＴ柱层析法纯化后,分别经免疫印迹（ＩＢＴ）法和酶联免疫吸附法（ＥＬＩＳＡ）检测２０份ＳＳ患者血清、６０份系统性红斑狼疮（ＳＬＥ）患者血清和３０份正常人血清。结果 利用重组抗原检测抗ＳＳＡ自身抗体敏感性     

免疫转印法检测白色念珠菌菌丝组分蛋白抗体

目的　为了探讨血清中白色念珠菌菌丝相蛋白组分抗体的实验室诊断意义。方法　应用超声粉碎和ConA Sepharose4B亲和层析除去细胞壁甘露糖和甘露糖蛋白后 ,收集蛋白抗原 ,通过免疫转印技术 ,检测血清标本中相应抗原的抗体。结果　血库血清 40份中 ,1例阳性 ;白色念珠菌培养阳性的 90名患者 ,抗体阳性 5 8例 ;热带念珠菌培养阳性的 17名患者 ,10例抗体阳性。使用大量抗菌药物和 /或免疫抑制剂治疗而培养阴性的 14名患者中 ,抗体阳性 7例。抗体阳性的 75例结果中 ,15例出现 2 9ku和 47ku条带 ,5 1例单独出现 47ku条带 ,9例仅出现 2 9ku条带。结论　免疫转印法检测抗体可作为深部念珠菌感染的参考指标 ,并可提供抗体动态变化模式 ,为深入研究打下基础。     

白念珠菌烯醇化酶双抗体夹心ELISA检测方法的建立

摘要：目的：建立检测白念珠菌烯醇化酶（enolase, Eno）的双抗体夹心ELISA法,并试用于几种真菌培养上清液的检测。 方法：将抗白念珠菌Eno单抗作为包被抗体,辣根过氧化物酶标记的羊抗Eno抗体作为检测抗体,用方阵滴定法确定包被抗体和酶标抗体浓度,建立检测Eno的ELISA法,对方法的精密度、特异性、最低检测限等指标进行评价。用该法检测白念珠菌、热带念珠菌、光滑念珠菌、近平滑念珠菌、季也蒙念珠菌、新生隐球菌和酿酒酵母等不同真菌培养上清中Eno水平。 结果：Eno浓度为20 ng/mL和5 ng/mL时,批内和批间变异系数(CV)分别为6.61%、9.19%和6.98%、13.81%;最低检测限为1.25 ng/mL。本法可从白念珠菌37 ℃培养24 h后的上清中检出Eno,Eno含量与白念珠菌菌丝含量呈正相关。本法对近平滑念珠菌有微弱交叉反应,与热带念珠菌、光滑念珠菌、季也蒙念珠菌、新生隐球菌和酿酒酵母均无交叉反应。 结论：建立的检测白念珠菌Eno的双抗体夹心ELISA方法有较好的特异性,可用于评价Eno检测在侵袭性白念珠菌感染中的诊断价值。     

Effects of caspofungin against Candida guilliermondii and Candida parapsilosis

The in vitro activity of caspofungin (CAS) was investigated against 28 yeast isolates belonging to Candida albicans (n = 5), Candida guilliermondii (n = 10), and Candida parapsilosis (n = 13). CAS MICs obtained by broth dilution and Etest methods clearly showed a rank order of susceptibility to the echinocandin compound with C. albicans > C. parapsilosis > C. guilliermondii. Similarly, time-kill assays performed on selected isolates showed that CAS was fungistatic against C. albicans and C. parapsilosis, while it did not exert any activity against C. guilliermondii. In a murine model of systemic candidiasis, CAS given at doses as low as 1 mg/kg of body weight/day was effective at reducing the kidney burden of mice infected with either C. albicans or C. guilliermondii isolates. Depending on the isolate tested, mice infected with C. parapsilosis responded to CAS given at 1 and/or 5 mg/kg/day. However, the overall CFU reduction for C. guilliermondii and C. parapsilosis was approximately 100-fold less than that for C. albicans. Our study shows that CAS was active in experimental systemic candidiasis due to C. guilliermondii and C. parapsilosis, but this activity required relatively high drug dosages.     

Sustained gastrointestinal colonization and systemic dissemination by Candida albicans, Candida tropicalis and Candida parapsilosis in adult mice

The ability of nine clinical isolates of Candida species (three C. albicans, three C. tropicalis and three C. parapsilosis) to colonize and invade the gastrointestinal (GI) tract of adult male CD-1 (ICR) mice was determined. The effect of dietary tetracycline plus glucose supplementation on colonization was evaluated. Strains were intragastrically inoculated. Tetracycline and glucose altered substantially aerobic flora, especially streptococci (average fall 1.1 +/-0.3 log(10) CFU/g, p<0.01 by the Student's t test). At two weeks after oral challenge, sustained and high colonization of GI tract by Candida (mean 5,28 +/- 0.18 log(10) CFU/g, p<0.01) was achieved in all mice receiving glucose-tetracycline supplementation, excepting in animals inoculated with one of C. tropicalis isolates. Histologic sections of the stomachs revealed multiple intraepithelial micro-abscesses associated with hyphae in the region of the cardial-atrium fold. Under immunosuppression, systemic spread of C. albicans and C. tropicalis was observed in 62% and 24% of animals receiving dietary supplementation respectively. Dissemination was not noted for C. parapsilosis isolates. We have developed a simple and inexpensive murine model of sustained colonization of GI tract. This model could be useful for analyzing prophylaxis, treatment and diagnosis of systemic Candida infections and for evaluating virulence of strains.     

Multilaboratory testing of two-drug combinations of antifungals against Candida albicans, Candida glabrata, and Candida parapsilosis

There are few multilaboratory studies of antifungal combination testing to suggest a format for use in clinical laboratories. In the present study, eight laboratories tested quality control (QC) strain Candida parapsilosis ATCC 22019 and clinical isolates Candida albicans 20533.043, C. albicans 20464.007, Candida glabrata 20205.075, and C. parapsilosis 20580.070. The clinical isolates had relatively high azole and echinocandin MICs. A modified CLSI M27-A3 protocol was used, with 96-well custom-made plates containing checkerboard pairwise combinations of amphotericin B (AMB), anidulafungin (AND), caspofungin (CSP), micafungin (MCF), posaconazole (PSC), and voriconazole (VRC). The endpoints were scored visually and on a spectrophotometer or enzyme-linked immunosorbent assay (ELISA) reader for 50% growth reduction (50% inhibitory concentration [IC(50)]). Combination IC(50)s were used to calculate summation fractional inhibitory concentration indices (FICIs) (ΣFIC) based on the Lowe additivity formula. The results revealed that the IC(50)s of all drug combinations were lower or equal to the IC(50) of individual drugs in the combination. A majority of the ΣFIC values were indifferent (ΣFIC = 0.51 to 2.0), but no antagonism was observed (ΣFIC ≥ 4). Synergistic combinations (ΣFIC ≤ 0.5) were found for AMB-PSC against C. glabrata and for AMB-AND and AMB-CSP against C. parapsilosis by both visual and spectrophotometric readings. Additional synergistic interactions were revealed by either of the two endpoints for AMB-AND, AMB-CSP, AMB-MCF, AMB-PSC, AMB-VRC, AND-PSC, CSP-MCF, and CSP-PSC. The percent agreements among participating laboratories ranged from 37.5% (lowest) for AND-CSP and POS-VOR to 87.5% (highest) for AMB-MCF and AND-CSP. Median ΣFIC values showed a wide dispersion, and interlaboratory agreements were less than 85% in most instances. Additional studies are needed to improve the interlaboratory reproducibility of antifungal combination testing.     

含光滑念珠菌与白色念珠菌白带的比较分析

目的分析女性含白色念珠菌和光滑念珠菌白带的性状及镜下特征,以便认识并区分这两类念珠菌所引起的妇科感染。方法连续选取2000例做白带检查的妇科门诊患者,询问患者的典型症状并观察单纯含念珠菌妇科涂片的镜下特征,用显色培养基对白带内的真菌进行鉴定,比较分析白色念珠菌组与光滑念珠菌组的差异。结果共检出476例含念珠菌标本,其中白色念珠菌393例,光滑念珠菌60例,其他念珠菌23例。白色念珠菌与光滑念珠菌阴道感染患者均表现出较高的性交痛症状。与白色念珠菌比较,光滑念珠菌组白带无豆渣样改变,患者少见外阴瘙痒;高倍视野下白细胞数量较少,上皮细胞、乳酸杆菌及真菌孢子数量较多,清洁度较好。结论含光滑念珠菌白带与白色念珠菌白带比较具有较为明显的差异,光滑念珠菌感染后患者临床症状及白带镜下特征均无白色念珠菌典型。     

Candida peritonitis

PURPOSE OF REVIEW: The review highlights current insights in the epidemiology, diagnosis and therapy of Candida peritonitis, focusing on complicated secondary and tertiary peritonitis. RECENT FINDINGS: Candida peritonitis is still associated with poor prognosis. Antifungal prophylaxis is therefore recommended in patients with an overt risk profile for invasive candidiasis (immunodeficiency and prior antibiotic exposure). The clinical and microbiological diagnosis of Candida peritonitis remains problematic. It is still unclear which peritonitis patients may benefit from antifungal treatment. Antifungal therapy can be suggested in critically ill patients with nosocomial peritonitis where Candida is diagnosed based on perioperatively sampled peritoneal fluid. Patients with prior exposure to fluconazole are at risk for Candida nonalbicans spp. involvement with possible reduced susceptibility. SUMMARY: The main challenge in Candida peritonitis remains the interpretation of Candida cultured from the peritoneal cavity. Future research should focus on more conclusive diagnosis and on factors potentially confounding outcome, such as site of the perforation and failure of surgical source control. While awaiting progress to discriminate Candida colonization from invasive infection, antifungal therapy is recommended in high-risk critically ill surgical patients. Rapid detection of Candida might be beneficial in this regard. Besides antifungal therapy, adequate source control is of key importance.