艾滋病患者真菌感染情况及其药敏结果分析

目的 探讨艾滋病患者真菌感染情况及其药敏结果分析,指导临床治疗用药.方法 分别取艾滋病患者的皮屑、血液、骨髓、尿液、痰液、咽拭子等标本进行真菌培养及药敏试验.结果 140例艾滋病患者中有45例检出真菌,共12种类型.其中,白色念珠菌16株,马尔菲青霉菌20株,其他类型23株;混合感染两种以上真菌者有12例.从药敏结果显示,白色念珠菌对两性霉素和氟康唑最为敏感,敏感率为100%,酮康唑、伊曲康唑次之,敏感率为63.16%,57.89%;马尔菲青霉菌对两性霉素和伊曲康唑最为敏感,敏感率为100%,氟康唑次之,敏感率为60.87%;其他类真菌对两性霉素和氟康唑最为敏感,敏感率为100%,伊曲康唑次之,敏感率为76.19%.结论 艾滋病患者真菌感染率较高,选择两性霉素和氟康唑或伊曲康唑作为临床治疗药物可以取得较好效果.     

五种抗真菌药物对马尔尼菲青霉菌的体外抗菌活性观察

目的 了解两性霉素B(AMB)、酮康唑(KET)、氟康唑(FCA)、5-氟胞嘧啶(5-Fc)和伊曲康唑(ICA)5种抗真菌药物对马尔尼菲青霉菌体外抗菌活性,为临床用药提供参考依据.方法 采用浓度梯度法(E-test)测试AMB、KET、FCA、5-Fc、ICA 5种抗真菌药物对52株从不同AIDS患者骨髓、血液、皮肤损害标本中分离的马尔尼菲青霉菌酵母相和菌丝相的体外抗菌活性.数据采用U检验.结暴 AMB、KET、FCA、5-Fc、ICA的90%酵母相马尔尼菲青霉菌的最低抑制浓度(MIC90)分别为0.250、0.160、24.000、4.000和0.006 mg/L,最低抑制浓度(MIC)范围分别为0.004～0.500、00002～0.016、1.000～256.000、0.002～32.000和0.002～0.008 mg/L;对90%菌丝相马尔尼菲青霉菌的MIC90分别为1.500、0.125、256.000、24.000和0.012 mg/L,MIC范围分别为0.064～4.000、0.006～0.940、1.000～256.000、0.125～32.000和0.002～0.064 mg/L.不同抗真菌药对双相马尔尼菲青霉菌的体外抗菌活性不同,以ICA最强,其次为KET.酵母相和菌丝相的马尔尼菲青霉菌对同一药物的MIC比较差异有统计学意义(AMB、KET、FCA、5-Fc和ICA的U值分别为4.221 9、1.912、28.798、6.43、7.21,均P＜0.05).结论 进行马尔尼菲青霉菌体外抗真菌药物敏感实验对临床有重要参考意义.     

马尔尼菲青霉菌溶血磷脂酶基因的识别和序列分析及功能预测

目的 从马尔尼菲青霉菌(PM)酵母相全长cDNA文库中识别溶血磷脂酶基因,预测其结构和功能,为进一步实验研究提供理论依据.方法 利用NCBI在线分析工具对PM酵母相全长cDNA文库进行筛选,识别溶血磷脂酶基因;通过Vector NTI suite 8.0软件包,对其编码蛋白质的各种结构与功能特征进行预测,并构建种系分子进化树.结果 筛选得到的基因长1014 bp,Blastx分析该基因可能是编码PM溶血磷脂酶的全长基因,完整的开放读码框(ORF)共含729 bp,编码243个氨基酸;其编码蛋白的相对分子质量为26 800,预测等电点为5.49,疏水氨基酸占47.7%,亲水氨基酸占26.8%,酸性氨基酸占12.7%,碱性氨基酸占12.8%;该蛋白有潜在的2个酪蛋白激酶Ⅱ磷酸化位点、3个蛋白激酶C磷酸化位点和7个N-肉豆蔻酰位点.结论 该氨基酸序列属于溶血磷脂酶样功能域特征蛋白酶;与球孢子菌亲源关系最近.通过研究,一个PM溶血磷脂酶基因被成功发现,这为进一步探讨该基因的功能提供了条件.

Abstract：

Objective To identify the gene encoding lysophospholipase from the full length cDNA library of Penicllium marneffei (PM) in yeast phase and predict the structure and function of its deduced protein, to provide with theoretical evidences for further experiments. Methods The PM full-length cDNA library in yeast phase was screened to identify the gene encoding lysophospholipase with the help of NCBI on-line analytical tool. Then, by utilizing the software package of Vector NTI suite 8.0, the protein deduced by the gene was analyzed to predict its corresponding structure and functions, and its molecular cladogram was constructed. Results The gene was composed of 1014 base pairs in the length and was presumed to be the full-length gene encoding lysophospholipase by Blastx, with a complete open reading frame (ORF) comprised of 729 base pairs encoding 243 amino acids with relative molecular weight being 26 800 and the predicted isoelectric point being S.49. The deduced protein included 47.7% hydrophobic amino acids, 26.8% hydrophilic amino acids, 12.7% acid amino acids and 12.8% basic amino acids. The protein had 2 potential casein kinase Ⅱ phosphorylation site, 3 potential protein kinase C phosphorylation site and 7 N-myristoyl site. Conclusions The amino acid sequence belongs to this kind of protease with lysophospholipase-like domain. The protein is most close to Coccidioides posadasii in genetic relationship. A novel gene encoding lysophospholipase is successfully found and the work has made necessary preparations for further research on the gene's function.     

艾滋病患者马尔尼菲青霉菌优势株酵母相全长cDNA文库的构建和初步分析

Objective To construct a full length cDNA library of the dominant strain of Penicillium marneffei (PM) in yeast phase isolated from AIDS patients in Guangdong province and screen UniGenes as well as full-length genes, so as to establish the foundation for the study of PM's functional genes and pathogenic mechanisms. Methods CloneMiner cDNA construction kit was utilized to extract mRNA of the dominant PM strain isolated from AIDS patients in Guangdong province. The mRNA was reversed into cDNA, then cloned into a pDONR222 vector by BP recombination to obtain an Uncut cDNA library, which was homogenized later to construct a normalized cDNA library with the principal of saturation hybridization for DNA genome. 2000 clones were chosen randomly to make a bi-directional sequencing and analyzed with bioinformatics for screening UniGenes and full-length genes. Results The total clone number of the Uncut cDNA library was 1.16 × 107 cfu/mL, with a recombination rate of 95% and an average insertion element being over 1 kb. The total clone number of the normalized cDNA library was 1.18 × 106 cfu/mL, with a recombination rate of 95% and an average insertion element being over 1 kb as well. 1945 genes which DNA length were longer than 1 kb were obtained by sequencing and merged into 1360 UniG enes, of which 632 genes were full-length ones. Conclusions The full-length cDNA library of the dominant strain of PM from AIDS patients in Guangdong province possesses good quality.Meanwhile, the technical routine presents high efficiency in obtaining full-length genes and establishing a gene expression spectrum, which can contentedly meet the needs of future experiments.     

马尔尼菲青霉菌溶血磷脂酶基因的识别和序列分析及功能预测

Objective To identify the gene encoding lysophospholipase from the full length cDNA library of Penicllium marneffei (PM) in yeast phase and predict the structure and function of its deduced protein, to provide with theoretical evidences for further experiments. Methods The PM full-length cDNA library in yeast phase was screened to identify the gene encoding lysophospholipase with the help of NCBI on-line analytical tool. Then, by utilizing the software package of Vector NTI suite 8.0, the protein deduced by the gene was analyzed to predict its corresponding structure and functions, and its molecular cladogram was constructed. Results The gene was composed of 1014 base pairs in the length and was presumed to be the full-length gene encoding lysophospholipase by Blastx, with a complete open reading frame (ORF) comprised of 729 base pairs encoding 243 amino acids with relative molecular weight being 26 800 and the predicted isoelectric point being S.49. The deduced protein included 47.7% hydrophobic amino acids, 26.8% hydrophilic amino acids, 12.7% acid amino acids and 12.8% basic amino acids. The protein had 2 potential casein kinase Ⅱ phosphorylation site, 3 potential protein kinase C phosphorylation site and 7 N-myristoyl site. Conclusions The amino acid sequence belongs to this kind of protease with lysophospholipase-like domain. The protein is most close to Coccidioides posadasii in genetic relationship. A novel gene encoding lysophospholipase is successfully found and the work has made necessary preparations for further research on the gene's function.     

艾滋病合并皮肤马尔尼菲青霉感染4例

目的探讨艾滋病（AIDS）合并皮肤马尔尼菲青霉感染的临床及实验室特征。方法分析2005年1月至2006年6月本院收治的明确诊断为AIDS合并皮肤马尔尼菲青霉感染的4例患者临床特征;取皮损、血和骨髓分别在25℃和37℃进行真菌培养。观察菌落形态、显微镜下特征;对皮肤活检组织行HE及六胺银染色。观察镜下皮损组织学及马尔尼菲青霉的特征。结果AIDS合并皮肤马尔尼菲青霉感染伴多系统损害。皮损特征：早期表现为淡红色丘疱疹、糜烂性丘疹,继而为坏死性丘疹、传染性软疣样丘疹、皮肤溃疡及血痂。37℃培养呈酵母相,25℃呈菌丝相。皮肤病理活检六胺银染色（＋）。使用二性霉素B、伊曲康唑治疗。3例临床症状缓解、皮疹消退出院。1例死亡。结论AIDS合并皮肤马尔尼菲青霉感染皮损特征：坏死性丘疹、传染性软疣样丘疹。皮损25℃、37℃真菌培养结合皮肤病理活检是确诊的关键。二性霉素B、伊曲康唑是目前治疗AIDS合并皮肤马尔尼菲青霉感染的首选药物。     

固相酶联免疫测定法检测NS1抗原在登革病毒感染早期诊断中的应用

目的探讨固相酶联免疫测定（ELISA）法检测NS1抗原在登革病毒感染早期诊断中的应用价值。方法选取登革病毒感染早期患者血清171份,非登革病毒感染发热患者血清11份,正常人血清10份,采用ELISA法检测全部192份血清的登革病毒NS1抗原和IgM抗体;采用逆转录-聚合酶链反应-限制性内切酶酶切片段长度多态性分析（RT-PCR-RFLP）技术对发病5 d内的125份血清进行扩增和鉴定分型;并采用C6/36细胞微量培养法对发病第1、2天的41份血清进行登革病毒分离培养。结果登革病毒感染患者发病2 d内、3～5 d以及6～10 d血清NS1抗原的检出率分别是92.7%（38/41）、83.3%（70/84）、10.9%（5/46）;IgM抗体的检出率分别是2.4%（1/41）、51.2%（43/84）、97.8%（45/46）;非登革病毒感染的发热患者及正常人血清中,有1例疟疾患者血清登革病毒IgM抗体呈阳性,NS1抗原无一例阳性。RT-PCR在登革病毒感染患者发病第1、2天和3～5天的检出率分别是85.4%（35/41）、83.3%（70/84）;登革病毒感染患者发病第1、2天血清的病毒分离培养阳性率分别是80.0%（16/20）、38.1%（8/21）,总分离率58.5%（24/41）;RT-PCR-RFLP分型鉴定技术及间接免疫荧光法（IFA）均证实2006年广州流行株为登革Ⅰ型病毒。结论ELISA法检测登革病毒NS1抗原操作技术成熟,且具有敏感性高、特异性好的特点,对登革病毒感染的早期诊断和疫情的早期控制具有重要意义,适合于基层医疗机构常规应用。     

艾滋病合并马尔尼菲青霉病的临床分析

 [目的]探讨艾滋病合并马尔尼菲青霉病的临床及实验室特征。[方法]回顾分析2002年11月至2005年12月本院收治明确诊断为艾滋病合并马尔尼菲青霉病的53例临床及实验室资料。[结果]艾滋病合并马尔尼菲青霉病以发热、消瘦、咳嗽、皮疹、贫血等为主要临床特点,皮损主要表现为坏死性丘疹、脐凹状丘疹、溃疡、结节、血痂;外周血CD4^＋细胞显著减少;在沙氏琼脂培养基中马尔尼菲青霉呈酵母相（37℃）或菌丝相（25℃）;药敏结果显示伊曲康唑、酮康唑对马尔尼菲青霉的MIC值最低,两性霉素B、5-氟胞嘧啶次之,氟康唑最高;病理组织六胺银染色见圆形、椭圆形或腊肠样病原体,部分有横隔。[结论]艾滋病合并马尔尼菲青霉病临床表现复杂,主要发生于CD4^＋计数少于50细胞∕μL的患者,真菌培养鉴定结合组织病理检查是确诊的关键,治疗上建议使用伊曲康唑、两性霉素B。     

马尔尼菲青霉菌溶血磷脂酶基因的克隆、表达及纯化

目的 研究马尔尼菲青霉菌(PM)溶血磷脂酶(LysoPLs)基因的克隆、表达和纯化,为研究其致病机制奠定基础.方法 采用生物信息学方法,从PM酵母相全长 cDNA 文库中识别出PMLysoPLs基因的同源序列及其全长编码区.通过PCR方法 扩增PMLysoPLs基因的编码区序列,构建原核表达载体pET 30a(+)-PMLysoPLs,经DNA 序列测定鉴定其序列,在大肠埃希菌BL-21/DE3中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,重组产物采用His-镍蛋白纯化柱进行纯化.结果 PMLysoPLs基因长度为991 bp,其全长编码序列长度为732 bp,编码243个氨基酸,其编码蛋白理论分子量为26.8 kDa.重组表达载体经序列测定及酶切鉴定与理论推测结果 相符.经IPTG诱导,该基因在大肠埃希菌BL-21/DE3中得到高效的可溶性上清表达,纯化后的重组蛋白分子量为25~35 kDa,其单一蛋白纯度达95%以上.结论 本研究成功构建了PMLysoPLs基因的pET 30a(+)原核重组质粒,获得的可溶性重组蛋白表达效率高,可用于进一步研究PM溶血磷脂酶的功能.     

艾滋病患者马尔尼菲青霉菌优势株酵母相全长cDNA文库的构建和初步分析

目的 构建广东地区艾滋病患者马尔尼菲青霉菌(PM)优势株酵母相全长cDNA文库,筛选UniGene和全长基因,为PM的功能基因组学研究和致病机制的探讨奠定基础.方法 应用CloneMiner cDNA construction kit提取广东地区艾滋病患者PM优势株酵母相mRNA,反转录成cDNA后通过BP重组方法克隆进pDONR222载体制备成Uncut型cDNA文库,然后利用基因组DNA饱和杂交原理,对Uncut型cDNA文库进行均一化处理,构建均一化cDNA文库.针对均一化文库,随机挑取2000个克隆子进行双向测序,并进行生物信息学分析,筛选UniGene和全长基因.结果 Uncut型cDNA文库总克隆数为1.16×107 cfu/mL,重组率95%,平均插入片段长度＞1 kb;均一化文库总克隆数为1.18×106 cfu/mL,重组率95%,平均插入片段长度＞1 kb.测序获得1945条基因片段长度＞1 kb的序列,归并获得1360个UniGene,632个全长基因.结论 所构建的艾滋病患者PM优势株全长cDNA文库质量较好,采用的技术路线获取全长基因建立基因表达谱的效率较高,可以很好满足下一步实验需要.