白色念珠菌耐药株CYP51基因突变热点探讨

目的探讨耐唑类药物白色念珠菌CYP51基因突变发生热点. 方法从复发性外阴阴道念珠菌病(RVVC)患者分离出白色念珠菌,采用美国国家临床实验室标准化委员会(NCCLS)公布的酵母菌液基稀释法抗真菌药物敏感试验参考方案(M-27方案)进行体外药敏试验,分离出氟康唑(FLC)及伊曲康唑(ITC)的耐药株;根据PCR-SSCP分析结果确定CYP51突变热点是1 364～1 774 bp之间,随机选择12株耐FCZ(MIC≥64 μg/ml)及ICZ (MIC≥16 μg/ml)的白色念珠菌用D1～D2、E1～E2 2对特异引物进行PCR扩增,将其产物测序,并与NCBI网站提供的白色念珠菌参考株进行比对、分析. 结果 12株耐FLC、ITC的白色念珠菌,扩增CYP51基因片段长度包括突变热点在内的667对碱基,即从1 108～1 774 bp;在12株菌当中,共发现13个位点38个碱基突变,突变频率最高的位点是第1 587位中腺嘌呤(A)被鸟嘌呤(G)取代;37个碱基突变没有引起氨基酸的改变,为无意义突变,只有1 609位的鸟嘌呤(G)被腺嘌呤(A)所取代,导致第488位的缬氨酸被异亮氨酸取代(V488I). 结论 CYP51基因突变是白色念珠菌对唑类抗真菌药物耐药机制之一;CYP51基因突变热点在1 364～1 774 bp之间,但92.3%的碱基突变是无意突变.     

白色假丝酵母菌对氟康唑敏感性及耐药机制研究

目的研究本院临床分离的白色假丝酵母菌对氟康唑的耐药性及其ERG11基因突变情况。方法将临床标本接种至沙堡弱平板,用VITEK 2 Compact微生物分析系统鉴定所得菌株,保存所得白色假丝酵母菌;按照CLSI-M27-A3酵母菌微量肉汤稀释法进行氟康唑敏感性试验;选择部分代表菌株进行ERG11基因的PCR扩增、测序及比对分析。结果共分离得162株白色假丝酵母菌,其中来自痰液标本最多,占68.5%;氟康唑耐药株为11株,占6.8%;剂量依赖敏感株(S-DD)为16株,占9.9%;敏感株为135株,占83.3%;从7株氟康唑耐药和1株氟康唑敏感的白色假丝酵母菌ERG11基因中共发现22个点突变,其中错义突变3个,分别是V51E、D116E、E266D,其余均为同义突变;有2株氟康唑耐药菌株未发现错义突变。结论 ERG11基因的突变是白色假丝酵母菌对氟康唑耐药的重要机制,D116E可能是浙江地区白色假丝酵母菌对氟康唑耐药的主要突变位点;对于白色假丝酵母菌耐氟康唑机制的研究不应局限于ERG11基因的突变,应综合多方面因素。     

白色假丝酵母菌对氟康唑耐药性及Erg11基因突变分析

目的分析白色假丝酵母菌临床分离株对氟康唑的耐药性,阐明耐氟康唑白色假丝酵母菌Erg11基因突变。方法回顾性分析2011年6月-2012年12月医院患者送检各类标本分离的287株假丝酵母菌属,采用美国临床实验室标准化研究所(CLSI)M27-A3推荐的微量稀释法进行体外药敏试验,从177株白色假丝酵母菌中筛选出7株对氟康唑耐药株,对氟康唑靶酶基因Erg11进行PCR扩增、测序,与Genbank上标准序列(X13296)比对分析。结果在7株白色假丝酵母菌氟康唑耐药株和1株敏感株中共发现25个点突变,其中15个同义突变,10个错义突变,10个错义突变分别为V51G、Y53H、C75W、Y79D、V94G、D116E、K119N、K128T、D153E、E266D,其中V51G、Y53H、C75W、Y79D、V94G是新发现的。结论成功分析了白色假丝酵母菌对氟康唑耐药性及Erg11基因突变。     

结核病患者院内念珠菌属感染及药物敏感性

目的了解结核病患者医院内对念珠菌属的二重感染和药物敏感性试验结果。方法对2002年1月~2004年12月3年间共6 280份临床送检的标本进行真菌培养;并对部分阳性念珠菌样品进行5-氟胞嘧啶(5-FC)、氟康唑(FLC)、酮康唑(KET)和伊曲康唑(ITC)4种抗真菌药物的敏感性试验。结果6 280份标本共分离出念珠菌属2 767株,分离率为44.06%;其中白色念珠菌检出2 006株,阳性率占31.94%,其他依此为光滑念珠菌344株(5.48%)、热带念珠菌206株(3.28%)、克柔念珠菌95株(1.51%)和其他念珠菌与真菌116株(1.85%);对4种抗真菌药物的敏感率分别依次为:5-FC 93.79%I、TC 62.76%、FLC 51.03%和KET 47.59%。结论由于结核病需长期用药,因而念珠菌属的二重感染率较高,使患者病情加重,在4种抗真菌药物中,以5-氟胞嘧啶的抗念珠菌活性为最佳。     

ERG5基因在白色念珠菌耐唑类药物中的突变及高表达

目的探讨临床分离白色念珠菌ERG5基因突变、高表达与唑类抗真菌药物耐药的关系。方法对36株临床白色念珠菌进行药物敏感性试验,并对其ERG5基因进行扩增测序,将测序结果与Gen Bank中已发表的序列(Gen Bank序列号:AF069572)进行比对,分析基因突变情况;抽提白色念珠菌临床菌株的总RNA,并反转录合成c DNA,采用实时荧光定量PCR(FQ-RT-PCR)方法检测ERG5基因的表达水平。结果 36株白色念珠菌临床菌株中,共检测出ERG5基因存在1个错义突变位点;氟康唑耐药组有11株发生ERG5高表达,占61.11%,敏感组中5株发生高表达,占31.25%。耐药菌株组ERG5基因的表达量ΔCt值与敏感组比较,差异具有统计学意义(P0.05)。结论白色念珠菌ERG5基因的高表达可能与FCA产生耐药有关,并未发现ERG5耐药菌株在同一位点发生错义突变,白色念珠菌ERG5基因突变、高表达在耐药中的作用还有待于进一步研究证实。     

新疆静脉吸毒者HIV-1原发耐药分析

目的研究新疆伊犁州经静脉吸毒(IDU)感染人类免疫缺陷病毒1型(HIV-1)未经抗病毒治疗者原发耐药株的流行情况。方法于2009年12月—2010年3月收集新疆伊犁州77例IDU HIV-1感染者血样及流行病学信息,提取血浆病毒RNA,逆转录聚合酶链反应(RT-PCR)和巢式PCR方法扩增pol基因区1.3 kb片段并测序;构建系统进化树分析病毒亚型,提交斯坦福大学HIV耐药数据库进行耐药性分析。结果 77例感染者中,76例(98.7%)为CRF07_BC亚型,1例(1.3%)为B亚型;蛋白酶区未检出主要耐药突变,耐药相关次要突变主要出现在第10、58和71位;其中71位氨基酸突变发生频率最高(11/77),其次为第10位(6/77)和第58位(3/77);第71位氨基酸由野生型的A突变为V者7例,突变为T者3例,突变为I者1例;第10位氨基酸由野生型的L突变为I者5例,突变为V者1例;第58位氨基酸由野生型的Q突变为E者3例;1例病例逆转录酶区同时存在Y181C及M184V耐药突变。结论新疆伊犁州未接受抗病毒治疗的IDU感染者中检出原发耐药株,原发耐药率处于较低水平,但应加强该地区IDU感染者的耐药监测,防止耐药株产生和流行。     

大学生口腔念珠菌分离株药物敏感性分析

目的 探讨在校大学生口腔念珠菌对常用抗真菌药物的敏感性.方法 选取云南省某高校在校学生1179名,采集其口腔咽拭子,分离疑似念株菌84株.采用微量液基稀释法,测定念珠菌对氟康唑、酮康唑、氟胞嘧啶和两性霉素B等4种常用抗真菌药物的敏感性,以最低抑菌浓度(MIC值)进行判定.结果 84株念珠菌对上述4种药物敏感性的几何均数分别为3.175,0.070,0.272和0.43 mg/L;耐药率分别为8.3%,9.5%,7.1%和8.3%;非白色念珠菌对酮康唑和两性霉素B的耐药率均高于白色念珠菌(P<0.05);有3株念株菌对2种或2种以上药物同时耐药.结论 在校大学生口腔念珠菌中存在一定程度的耐药株和交叉耐药现象,尤其是非白色念珠菌.     

流式细胞术快速检测白色假丝酵母菌药物敏感性的评价

目的 比较评介流式细胞术快速药敏试验(FCST)和CLSI M27一A2常量液体培养基稀释法、ATB FUN-GUS3半固体培养基法检测白色假丝酵母菌对常用抗真菌药物敏感性符合率.方法 用FCST法和CLSI M27-A2常量液体培养基稀释法、ATB FUNGUS3同时测定46株临床分离的白色假丝酵母菌对氟康唑(FLC)、酮康唑(KET)、伊曲康唑(ITC)、伏立康唑(VRC)等药物的药敏性并进行比较分析.结果 FCST法与M27-A2比较,FLC、KET、ITC和VTC的符合率分别为91.3%、100.0%、80.4%和93.5%; FCST法与ATBF3比较,FLC、ITC和VRC的符合率分别为91.3%、91.3%、和93.5%.结论 FCST法具有快速、重复性好、易于判断MIC值、准确性高等优点;FCST法与M27-A2、ATBF3法测定FLC、KET、ITC和VRC对白色假丝酵母菌的敏感性有较高的符合率,在临床抗真菌药物敏感性检测中具有很大的应用前景.     

白色念珠菌CDR基因表达与氟康唑体外药敏试验的关系研究

目的调查白色念珠菌临床株对氟康唑的耐药情况,研究白色念珠菌临床耐药基因的表达与氟康唑耐药之间的关系。方法采用M27-A2微量肉汤稀释法,对某院微生物实验室2010年分离保存的221株来自就诊患者的痰、中断尿、粪便和咽拭子的白色念珠菌进行氟康唑MIC值测定。以白色念珠菌18S RNA为内参照,采用RT-PCR技术观察比较耐药株和敏感株组在不同氟康唑浓度下CDR1和CDR2基因的转录水平。结果 221株白色念珠菌中,有57.47%（127株）对氟康唑敏感,10.86%（24株）剂量依赖敏感,31.67%（70株）耐药。不同药物浓度作用下,耐药株组CDR1和CDR2的表达量相对要高于敏感株组（P〈0.05）。结论白色念珠球菌对氟康唑耐药率较高,耐药株组CDR1和CDR2表达量高于敏感株组。     

白色念珠菌upc2基因G1927A多态性与唑类药物耐药的关系

目的 探讨白色念珠菌upc2基因C末端单核苷酸多态性以及其对唑类药物耐药的影响。方法 对34株白色念珠菌临床株,纸片法测定5种药物敏感性,DNA测序法检测upc2基因单核苷酸多态性,RT-PCR法测定cdr1基因mRNA转录水平。比较耐药株和敏感株upc2单核苷酸多态性以及cdr1基因mRNA水平的差异。结果 14株白色念珠菌氟康唑耐药株（3株多重耐药,6株双重耐药,5株单一耐药）,20株敏感株对5种药物均敏感。5株耐药白色念珠菌upc2基因存在G1927A型多态性,20株敏感株upc2基因均为G1927野生型,G1927A型upc2基因检出率35.7%,显著高于敏感株（χ2 = 8.37,P ＜0.01）。G1927A型多重耐药株的cdr1基因mRNA水平高于G1927A型双重耐药株,并且二者均高于敏感株。结论 upc2基因G1927A单核苷酸多态性通过上调cdr1基因mRNA转录而引起白色念珠菌对唑类药物产生多重耐药或双重耐药表型。     

利福霉素耐药结核分枝杆菌rpoB突变与利福布丁耐药水平的关系

目的研究利福霉素耐药结核分枝杆菌 rpoB基因突变与利福布丁耐药水平的相关性。方法倍比稀释法测定64株利福霉素耐药及6株敏感菌株对利福布丁的最低抑菌浓度(MIC),并分析其对异烟肼的耐药情况。同时对rpoB全基因扩增后测序,分析rpoB突变位点和突变性质与利福布丁MICs高低及多重耐药的关系。结果6株敏感株rpoB未突变,MICs为 0.25～0.50 mg/L。64株耐药株rpoB突变率为100％。37株利福布丁高度耐药(MICs≥4 mg/L)株中,S531L突变27株,H526R突变和Y389C突变各2株,S531W、H526Y、Q513K、V176F、D516Y联合Q253R突变与D516G联合L511P突变各1株。17株中度耐药 (MICs 2～4 mg/L) 株中,S531L突变16株,D516G联合L511P和S509R突变1株。10株低度耐药（MICs 0.25～1 mg/L）株中,L533P、H526L、H526S、D516V、D516Y单点突变各2株。93.75％(60/64)的利福霉素耐药株对异烟肼耐药。结论检测rpoB突变即可初步筛选多重耐药结核分枝杆菌;中、高水平利福布丁耐药株以S531L突变占绝对优势,rpoB突变位点及突变类型与利福布丁耐药水平有一定相关性。     

基因多态性在肺癌发生中交互作用

目的探讨代谢酶基因与修复酶基因多态性在肺癌发生中的交互作用。方法采用1：1配对病例一对照研究，收集原发性肺癌患者227例和相应非肿瘤对照227例，对CYP1B1、CYP2C19、CYP2D6、CYP2E1、GSTM1、GSTT1、GSTP1、mEH、NQ01、XRCC1、XRCC3、hOGG1、NAT2、XPD基因多态性进行检测，应用Logistic回归对基因一基因交互作用进行分析。结果CYP2C19突变基因型与NQ01突变基因型。mEH-exon3突变基因型与NQ01突变基因型之间对肺癌的发生存在交互作用，可导致肺癌易感性的增高。未见其他代谢酶基因与修复酶基因在汉族人群肺癌发生中存在交互作用。结论对基因多态性的联合检测更有利于筛选易感人群。今后应加大样本含量，进行多基因之间的联合作用分析。     

长沙市4例输入新型冠状病毒Omicron变异株全基因组序列特征分析

目的 分析长沙市境外输入新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)的全基因组序列特征以及遗传变异情况。方法 采用高通量测序方法对2021年12月的SARS-CoV-2进行全基因组序测序,序列进行比对以及进化分析。结果 本研究获得4株SARS-CoV-2全基因组序列,长度为29 685 bp。毒株核苷酸序列与Wuhan-Hu-1参考株(EPI＿ISL＿402125)相比,同源性为99.6%。与Omicron变异株(EPI＿ISL＿8890653)相比,同源性为99.9%。进化树分析表明毒株序列位于BA.1.1分支,与SARS-CoV-2 Omicron变异株位于同一分支。氨基酸序列位点分析发现毒株具有典型的Omicron序列突变位点。ORF1ab区域发现G5494S,K4346R,T5035I和E6945D突变。S蛋白抗体结合区域发生R346K突变。结论 长沙市输入的Omicron变异株携带已报道可明确导致病毒传播和致病力发生变化的突变位点,应继续加强疫情应对措施。     

The effect of lycopene on cytochrome P450 isoenzymes and P-glycoprotein by using human liver microsomes and Caco-2 cell monolayer model

Lycopene is widely used as a dietary supplement. However, the effects of lycopene on cytochrome P450 (CYP) enzymes or P-glycoprotein (P-gp) are not comprehensive. The present study was performed to investigate the effects of lycopene on the CYP enzymes and P-gp activity. A cocktail method was used to evaluate the activities of CYP3A4, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. Caco-2 cell monolayer model was carried out to assay lycopene on P-gp activity. The results indicated that lycopene had a moderate inhibitory effect on CYP2E1, with IC50 value of 43.65?μM, whereas no inhibitory effects on CYP3A4, CYP2C19, CYP2D6 and CYP2E1, with IC50 values all over 100?μM. In addition, lycopene showed almost no inhibitory effect on rhodamine-123 efflux and uptake (p?>?.05), indicated no effects on P-gp activity. In conclusion, there should be required attention when lycopene are coadministered with other drugs that are metabolised by CYP2E1.     

The first genetic characterization of a D4 measles virus strain derived from a patient with subacute sclerosing panencephalitis

Measles virus (MV) strains derived from patients with subacute sclerosing panencephalitis (SSPE), SSPE strains, possess numerous mutations when compared to viruses belonging to the same genotype and circulating in similar time period. Although many SSPE strains have been extensively characterized, none of them belongs to D4 genotype which currently predominates in Europe where it has caused a number of recent outbreaks/epidemics.We sequenced an MV derived from a patient with long-term SSPE; the virus was named MVs/Zagreb.CRO/30.06[D4] (SSPE). Initial genetic analysis showed that it belongs to D4 genotype. The sequences of genes encoding matrix and fusion proteins indicate premature protein terminations. Putative hemagglutin (H) protein is lengthened for 20 amino acids, which is the longest H protein elongation so far found in SSPE viruses.Nucleotides 1421 A, 1422 G, 1507 C and 1542 C in nucleoprotein gene open reading frame seem to be specific for this D4 strain, differentiating it from other D4 non-SSPE strains. Besides, a unique mutation at position 543 of H protein was found, histidine instead of tyrosine.As persistent MV infections are initially established by “normal” wild-type MV strains, the presented comparative analyses describe alterations that could be involved in the maintenance of persistent infection, disease development and progression.     

Vitamin D and Diseases of Mineral Homeostasis: A Cyp24a1 R396W Humanized Preclinical Model of Infantile Hypercalcemia Type 1

Infantile hypercalcemia type 1 (HCINF1), previously known as idiopathic infantile hypercalcemia, is caused by mutations in the 25-hydroxyvitamin D 24-hydroxylase gene, CYP24A1. The R396W loss-of-function mutation in CYP24A1 is the second most frequent mutated allele observed in affected HCINF1 patients. We have introduced the site-specific R396W mutation within the murine Cyp24a1 gene in knock-in mice to generate a humanized model of HCINF1. On the C57Bl6 inbred background, homozygous mutant mice exhibited high perinatal lethality with 17% survival past weaning. This was corrected by crossbreeding to the CD1 outbred background. Mutant animals had hypercalcemia in the first week of life, developed nephrolithiasis, and had a very high 25(OH)D₃ to 24,25(OH)₂D₃ ratio which is a diagnostic hallmark of the HCINF1 condition. Expression of the mutant Cyp24a1 allele was highly elevated while Cyp27b1 expression was abrogated. Impaired bone fracture healing was detected in CD1-R396^w/w mutant animals. The augmented lethality of the C57Bl6-R396W strain suggests an influence of distinct genetic backgrounds. Our data point to the utility of unique knock-in mice to probe the physiological ramifications of CYP24A1 variants in isolation from other biological and environmental factors.     

Azole resistant Aspergillus fumigatus: An emerging problem

Azole resistance has appeared recently in Aspergillus fumigatus and increased dangerously in the last decade. The main resistance mechanism is a point mutation of CYP51A, the gene encoding 14α-sterol demethylase, the target enzyme of azole antifungal drugs. This mutation can induce resistance to itraconazole alone or multi-azole resistance. CYP51A mutation can occur in two cases. The first usually concerns patients receiving long-term azole therapy, most of the time for chronic aspergillosis, and involves a wide range of mutations. The second is due to the use of azole fungicides in agriculture. The latter favors a single mutagenesis event: a substitution of leucine for histidine at codon 98 and the tandem repeat of a 34-base pair tandem sequence in the CYP51A gene promoter region. This confers cross-resistance to all azole antifungal drugs. This emerging and environmentally linked issue is of growing concern for the management of antifungal therapy. This mechanism of resistance was first described in the Netherlands and is now reported worldwide. It may have become the leading mechanism of azole resistance in A. fumigatus. Azoles are major agents for the treatment of aspergillosis, and the only oral antifungals. Infection with antifungal-resistant strains is correlated with treatment failure. This emerging phenomenon stresses the urgent need for new preventive strategies (controlled use of antifungals and azole prophylaxis), new diagnostic strategies (early detection of resistance), and new therapeutic strategies in the management of A. fumigatus infections.     

泛耐药鲍氏不动杆菌碳青霉烯酶基因型研究

目的 研究医院泛耐药鲍氏不动杆菌(PDRAB)不同克隆的耐药性及碳青霉烯酶基因型特征,为有效治疗和控制PDRAB医院感染提供参考资料.方法 应用脉冲场凝胶电泳(PFGE)技术对PDRAB进行分子分型,E试验法检测PDRAB各克隆最低抑菌浓度(MIC)值,聚合酶链反应(PCR)及核酸测序确定PDRAB各克隆碳青霉烯酶基因型. 结果 90株PDRAB分子分型出现A、B、C、D、E 5种克隆,A、C克隆出现亚型,A、C、E克隆为主要流行株,分别占 70.0％、7.8％、l4.4％；各型克隆均对多黏菌素B敏感,部分流行克隆对米诺环素、阿米卡星敏感,其余抗菌药物均表现高水平耐药；A、C及C亚型克隆携带blaOXA-23、bla OXA-51和blaPER-1基因,A亚型、D克隆携带blaOXA-23和blaPER-1基因,B克隆携带blaOXA-51和blaPER-1基因,E克隆携带blaOXA-23、blaOXA-51和blaGIM-1基因,未发现携带blaOXA-24、blaOXA-58、blaIMP、blaVIM及blaSPM-1基因的PDRAB克隆.结论 医院PDRAB流行克隆存在多样性,各克隆耐药表型及碳青霉烯酶基因型有所不同,产生OXA-23酶和PER-1金属酶是该地区PDRAB耐药的主要机制.     

2005年广西H1N1亚型流感病毒基因特性研究

目的阐明2005年广西流行的H1N1亚型流感病毒血凝素基因变异情况。方法挑选3株2005年广西流感病毒分离株,进行血凝素基因HA1区核苷酸序列测定并推导出其氨基酸序列,然后使用DNA—STAR分析软件进行进化特性分析。结果HA1区核苷酸序列和氨基酸序列分析表明,与2005年的国际疫苗推荐株A／New Caledonia／20／1999（H1N1）比较,三株分离株的HA1蛋白均含有7个糖基化位点。分离株A／Gx／566／2005发生以下氨基酸位点替换：82T〉K、94Y〉R、145R〉K、165V〉A,208R〉K、251W〉R、266T〉N,其中165位点位于HA1的抗原决定簇上。分离株A／GX／8／2005及分离株A／GX／13／2005发生了以下位点变异：165V〉A、194E〉G、251W〉R、252Y〉F、314V〉A,其中165及194位于HA1抗原决定簇上。结论2005年在广西同时流行着三系不同的H1N1病毒。一系接近国际疫苗推荐株A／New Caledonia／20／1999（H1N1）东部江苏的2000—2002年毒株,一系接近江苏2005—2006年毒株。尤其需要密切关注与疫苗株HA1蛋白发生较大变异一系H1N1,避免此系H1N1病毒的流行和暴发。     

Correlation of phylogenetic clade diversification and in vitro infectivity differences among Cosmopolitan genotype strains of Chikungunya virus

Cosmopolitan genotypes of Chikungunya virus caused the large-scale febrile disease outbreaks in the last decade in Asian and African continents. Molecular analyses of these strains had revealed significant genetic diversification and occurrence of novel mosquito-adaptive mutations. In the present study we looked into whether the genetic diversification has implications in the infectivity phenotype. A detailed sequence and phylogenetic analyses of these virus strains of Indian Ocean lineage from Kerala, South India from the years 2008 to 2013 identified three distinct genetic clades (I, II and III), which had presence of clade-specific amino acid changes. The E2 envelope protein of the strains from the years 2012 to 2013 had a K252Q or a novel K252H change. This site is reported to affect mosquito cell infectivity. Most of these strains also had the E2 G82R mutation, a mutation previously identified to increase mammalian cell infectivity, and a novel mutation E2 N72S. Positive selection was identified in four sites in the envelope proteins (E1 K211E, A226V and V291I; E2 K252Q/H). In infectivity analysis, we found that strains from clade III had enhanced cytopathogenicity in HEK293 and Vero cells than by strains representing other two clades. These two strains formed smaller sized plaques and had distinctly higher viral protein expression, infectious virus production and apoptosis induction in HEK293 cells. They had novel mutations R171Q in the nsP1; I539S in nsP2; N409T in nsP3; and N72S in E2. Our study identifies a correlation between phylogenetic clade diversification and differences in mammalian cell infectivity phenotype among Cosmopolitan genotype CHIKV strains.