SAG3基因在肾移植患者弓形虫感染诊断中的应用

目的:建立快速、特异、敏感诊断肾移植患者弓形虫感染情况的DNA检测方法,并讨论其临床意义.方法:利用ELISA和PCR方法对68例肾移植受者及20例正常供者同时进行检测.结果:检测肾移植受者68例,PCR和ELISA方法检测弓形虫SAG3基因片段和其抗原,阳性各为5例及4例;其阳性率分别为7.35%和5.91%.作为阴性对照的20名正常者同时进行弓形虫的检测,其结果均为阴性.结论:体外扩增弓形虫SAG3基因的方法具有准确、快速、特异和敏感的优点,可以作为肾移植患者弓形虫感染诊断的有价值的方法之一.     

异基因造血干细胞移植后巨细胞病毒的检测及临床意义

目的 为了降低异基因造血干细胞移植（allo-HSCT）后巨细胞病毒（CMV）感染的相关死亡率，寻求一种更快捷、更特异的诊断CMV感染的分子生物学方法。方法 2001年4月至2003年4月，从79例接受异基因造血干细胞移植的患者中采集了135份外周血标本。采用巢式聚合酶链反应（nested-PCR）方法检测患者外周血中CMVgB DNA，阳性标本做酶切分型，部分行序列测定。结果CMVgB DNA检测中有42例患者（53．9％）的66份标本（48．9％）阳性；对其中阳性患者的45份标本进行了酶切分型，结果 CMV gB1型21例（46．7％），CMV gB2型14例（31．1％），CMV gB3型7例（15．6％），CMV gB4型3例（6．7％）。先后有2种型别的CMV感染者有3例（3．8％）。经分析，CMV gB阳性和CMV gB阴性患者GVHD的发生率分别为81．0％和32．4％（P＜0.01）；CMV gB1、gB2、gB3以及gB4型Ⅱ～Ⅳ度急性GVHD及慢性GVHD的发生率分别为：81．0％、50．0％、42．9％和0。结论 Nested-PCR方法可快捷特异地检测CMV感染。CMVgB1、2型中，重度急性GVHD和慢性GVHD发生率较高。CMV gB基因分型检测可有效地指导临床抗病毒治疗，且对移植后患者的临床转归有一定的预测价值。     

异基因造血干细胞移植后人CMV感染与GVHD相关性的临床研究

目的 探讨异基因造血干细胞移植后人巨细胞病毒(CMV)感染与移植物抗宿主病(GVHD)的关系.方法 对1998年3月至2001年3月进行的63例造血干细胞移植受者的CMV感染情况进行检测,用聚合酶链反应法检测CMV gB分型,并统计受者GVHD的发生情况.结果 40例发生CMV感染,23例未感染,其GVHD发生率分别为82.5％(33/40)和47.8％(11/23),差异有统计学意义(P＜0.01).CMV gB-1、gB-2、gB-3型感染者发生Ⅱ～Ⅳ度急性GVHD的几率分别为58.8％(10/17)、40％(2/5)和66.7％(4/6),三者间差异无统计学意义(P＞0.05).CMV阳性受者中27.5％(11/40)出现移植后继发性造血抑制,CMV阴性的受者13.0％ (3/23)出现,发生率的差异无统计学意义(P＞0.05).结论 CMV感染者GVHD的发生率升高,而CMV感染与GVHD严重程度无关,合理治疗后不会显著抑制移植后的造血重建.     

造血干细胞移植后并发感染121例的临床分析

目的探讨造血干细胞移植(HSCT)后并发感染的临床病原学特点和相关危险因素。方法共有121例造血系统疾病患者接受HSCT,其中51例行异基因外周造血干细胞移植,40例行异基因骨髓移植,4例行异基因外周血造血干细胞和骨髓混合移植,5例行脐带血移植,16例行自体造血干细胞移植,5例行自体CD34~+细胞移植。患者均采用化疗进行预处理。采用短程甲氨蝶呤联合环孢素A预防移植物抗宿主病(GVHD),部分病例加用达利珠单抗或抗淋巴细胞球蛋白。结果121例患者中,116例患者获得造血重建,3例移植失败,2例在移植物植活前因严重感染死亡。移植后60d内,83例(68．6%)共发生感染97次,64例(77．1%)发生感染时中性粒细胞＜0．5×10~9/L,患者均有不同程度的发热。口咽黏膜和呼吸道(上、下呼吸道)是最常见的感染部位,占64．9%。21例患者的23次感染中,共分离出20株细菌和7株真菌,65%为革兰氏阴性杆菌。10例患者并发巨细胞病毒感染。因感染死亡的患者共8例,均为异基因造血干细胞移植患者。发生Ⅱ～Ⅳ度急性GVHD患者的感染发生率(88．9%,24/27)高于0～Ⅰ度急性GVHD患者(60．3%,44/73,P＜0．01)。结论造血干细胞移植后60d内的感染多发生于粒细胞缺乏期；口咽黏膜炎和呼吸道是常见感染部位,下呼吸道感染者的死亡率高,侵袭性真菌感染者的预后不佳；发生Ⅱ～Ⅳ度急性GVHD者有较高的感染发生率。     

性传播性尿道炎的诊断与治疗：附62例报告

为探讨一种对性传播性尿道炎有效的诊断和治疗方法，采用病原体培养和聚合酶链反应（PCR）检测62例性传播性尿道炎，结果病原体培养淋球菌（NG）阳性9例，解脲文原体（UU）阳性30例，两者均阳性5例，均阴性18例；沙眼衣原体（CT）阳性25例，UU阳性17例，两者均阳性20例，其中合并NG阳性17例。病原体培养与PCR显示NG和UU双阳性分别为13例和34例，双明性分别为47例和25例，两种方法不一致考分别为2例和3例。PCR对NG和UU的敏感度分别98．3％和97．2％，特异性分别为97.9％和92．6％。所有患者均口服阿奇霉素500mg，每3d1次，总剂量1．5g。结果治愈50例，显效6例，无效6例，有效率为90．3％。提示PCR对CT、UU及NG感染是一种敏感、特异、快捷和无创伤的检测技术，可作为性传播性尿道炎复查常规检测手段。阿奇霉素对治疗性传播世尿道炎具有疗效好、副作用少、服用方便等优点，值得推广应用。     

血清降钙素原检测对异基因造血干细胞移植术后并发菌血症的诊断价值

目的探讨血清降钙素原(PCT)、C反应蛋白(CRP)和内毒素检测对异基因造血干细胞移植(allo-HSCT)术后并发菌血症患者的诊断价值。方法回顾性分析2013年6月至2016年5月解放军第三〇九医院移植血液科行allo-HSCT术后发热的122例受者,检测血清PCT、CRP和内毒素。以血培养结果作为金标准,计算PCT、CRP、内毒素3种检测方法的灵敏度、特异度、阳性预测值和阴性预测值。绘制受试者工作特征(ROC)曲线,计算ROC曲线下面积。P0.05为差异具有统计学意义。结果根据血培养结果,122例allo-HSCT术后发热患者中有40例为菌血症,其中革兰阴性菌24例,革兰阳性菌16例。PCT和CRP检测ROC曲线下面积分别为0.815、0.727,差异具有统计学意义(P0.05)。当PCT检测截断值为0.845 ng/m L时,灵敏度和特异度分别为72.5%、87.5%,阳性预测值和阴性预测值分别为74.3%、86.7%。当CRP检测截断值为55.4 mg/L,灵敏度和特异度分别为71.8%、66.3%,阳性预测值和阴性预测值分别为50.0%、80.8%。结论血清PCT检测对于allo-HSCT术后合并菌血症患者的的临床诊断价值高于CRP和内毒素,可作为预测allo-HSCT术后发热患者是否并发细菌感染的优选诊断指标。     

荧光PCR技术在粪便幽门螺杆菌ureA基因检测中的应用

目的 评价荧光PCR技术检测粪便幽门螺杆菌（Helicobacter pylori,HP）urea基因的价值.方法 采用荧光PCR技术检测50例消化内科住院患者的粪便样本,其中23例通过胃黏膜活检组织尿素酶及病理组织染色确诊为HP感染.同时进行HP培养和血清HP尿素酶抗体检测.四格表x2检验比较3种方法的敏感性、特异性、阳性预测值和阴性预测值.结果 荧光PCR技术检测粪便HP感染的敏感性、特异性、阳性预测值和阴性预测值分别为1.00、0.96、96%和100%,而HP培养检测的敏感性、特异性、阳性预测值和阴性预测值分别为0.78、1.00、100%和84%,其中敏感性和阴性预测值与荧光PCR法相比,差异具有统计学意义（X2=5.60和4.44,P值均〈0.05）.血清HP尿素酶抗体检测的敏感性、特异性、阳性预测值和阴性预测值分别为0.96、0.74、76%和95%,其特异性和阳性预测值与荧光PCR法比较,差异具有统计学意义（X2=5.28和4.08,P值均〈0.05）.结论 粪便HPureA基因检测对诊断HP感染具有较高的临床价值.     

非HLA基因单核苷酸多态性对造血干细胞移植后巨细胞病毒感染的影响

目的 探讨非HLA基因单核苷酸多态性(SNP)对造血干细胞移植后巨细胞病毒(CMV)感染的影响.方法 对2008年7月至2011年12月接受异基因造血干细胞移植的受者及其相应供者64例进行研究.采用聚合酶链反应-序列特异性寡核苷酸探针和测序方法检测髓过氧化物酶基因(MPO)、甘露糖结合凝集素基因(MBL)、CD14基因和血管紧张素转换酶(ACE)基因的SNP,结合CMV pp65检测结果,比较CMV阳性组和CMV阴性组ACE、CD14、MPO、MBL基因SNP的差异.结果 ACE 16内含子基因多态性分布:DD型14例(10.9％),ID型72例(56.3％),Ⅱ型42例(38.8％);CD14-159位基因多态性分布:CC型18例(14.1％),CT型81例(63.3％),TT型29例(22.7％);MPO-463位点基因多态性分布:G型100例(78.1％),A型2例(1.6％),GA型26例(20.3％);MBL启动子-550位基因多态性分布:H型28例(21.9％),HL型73例(57.0％),L型27例(21.1％);MBL启动子-221位基因多态性分布:Y型87例(68.0％),YX型38例(29.7％),X型3例(2.3％);MBL外显子1区基因多态性分布:A型94例(73.4％),AB型32例(25.0％),B型2例(1.6％).CMV阳性组和CMV阴性组供、受者ACE、CD14-159位点、MPO-463位点、MBL启动子-221以及外显子1区基因单核苷酸多态性相比较,差异均无统计学意义;但CMV阳性组供者MBL-550位点HL基因频率明显增高,与阴性组比较,差异有统计学意义.结论 供者MBL的单核苷酸多态性对造血干细胞移植后CMV感染有影响.     

造血干细胞移植患儿创伤后应激障碍变化及影响因素分析

目的 调查造血干细胞移植患儿创伤后应激障碍状态及影响因素，为实施针对性干预提供参考。 方法 选取造血干细胞移植患儿148例，应用创伤后应激障碍症状自评量表在造血干细胞移植前（入层流室前1～2 d，T1）、移植后2～3周（T2）、移植后3个月（T3）进行调查，并分析其影响因素。 结果 造血干细胞移植患儿在T1、T2、T3创伤后应激障碍发生率分别为12.8%、69.6%、10.1%。年龄对患儿移植前及移植后3个月创伤后应激障碍产生影响，照顾者与患儿关系对其移植后2~3周产生影响（均P＜0.05）。 结论 患儿移植期间创伤后应激障碍水平呈动态变化，在造血干细胞移植过程中，护理人员应重视年长儿以及母亲照顾者患儿的心理护理。     

多源化巨细胞病毒DNA定量检测在异基因造血干细胞移植后巨细胞病毒肺炎诊断中的应用

目的 研究不同来源[血浆、痰液、支气管肺泡灌洗液(BALF)]标本的巨细胞病毒(CMV)DNA定量检测对异基因造血干细胞移植后CMV肺炎的诊断价值.方法 回顾性分析接受异基因造血干细胞移植的405例受者的临床资料,其中诊断为CMV肺炎的19例受者设为CMV肺炎组,选择同期仅发生CMV血症的229例受者、接受纤维支气管镜...     

High prevalence of unusual genotypes of Toxoplasma gondii infection in pork meat samples from Erechim, Southern Brazil

Toxoplasmosis is the most common cause of infectious uveitis in Brazil, with a higher frequency in the South of the country. We have collected samples from porcine tongue and diaphragm obtained in both large and small abattoirs and used molecular biological technique to determine the prevalence of infection and RFLP analysis to type the parasites. Seventeen out of 50 (34%) samples from the diaphragm and 33 out of 50 (66%) samples from the tongue demonstrated a positive PCR reaction for T. gondii and restriction analysis of four of the positive samples revealed that all had a type I genotype at SAG2. However, when other unlinked loci were analyzed, these strains had a type III genotype at markers BTUB, SAG3, and GRA6. One of the strains (8T) had a type II allele at SAG3, indicating it has a combination of alleles normally seen in the clonal lineages. Our sampling indicates a high prevalence of infection and suggests that unusual genotypes of T. gondii are found in Brazil even among domesticated pigs.     

Detection of Aspergillus galactomannan by enzyme immunoabsorbent assay in recipients of allogeneic hematopoietic stem cell transplantation: a prospective study

BACKGROUND: Invasive aspergillosis (IA) has become the leading infectious cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This is partially because of the lack of a sensitive, specific, and noninvasive diagnostic test. New diagnostic tests for IA, such as the detection of Aspergillus galactomannan antigen (AGA) by sandwich enzyme-linked immunoabsorbent assay (ELISA), have recently been described. This study validates the usefulness of this diagnostic tool in the allo-HSCT setting. METHODS: From January 1999 to January 2001, all consecutive adult patients undergoing allo-HSCT were prospectively studied with a galactomannan antigenemia assay (ELISA test) twice weekly from admission until death or discharge, and weekly afterward if the patient received immunosuppressive therapy. Proven, probable, and possible IA were defined according to the European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. RESULTS: During the 2 years of study, 74 patients underwent an allo-HSCT. A total of 832 serum samples were collected. According to the European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria, it was ascertained that 66 patients did not fulfill any criteria of IA, 2 patients were classified with possible IA, 5 patients were classified with probable IA, and 1 patient was classified with proven IA. Fourteen samples were positive for AGA, all from patients with IA. The sensitivity and specificity of the test were 75% and 100%, respectively. The positive predictive and negative predictive values were 100% and 97%, respectively. CONCLUSIONS: In this study, AGA detection was clearly related to IA. Although the ELISA test did not have any role in the anticipation of the diagnosis, it clarifies the diagnosis of IA in allo-HSCT.     

HLA-I类PCR-SSP基因分型在异基因造血干细胞移植中的应用

目的：提高异基因造血干细胞移植供、受者HLA－I类配型的精确度,有效防止移植物抗宿主病（GVHD）的发生。方法 采用准确、简便的DNA、RNA提取方法和HLA－I类PCR－SSP基因分型方法。建立HLA－A33、A68、B40、B13 PCR－SSP分型方法以及HLA基因序列测序分析。结果：对300例中清学难以判断结果的30例临床标本进行基因水平的研究发现,DNA及RNA提取方法可以满足此项研究对样本的要求。HLA－I类PCR－SSP分型方法具有良好的稳定性、可靠性和特异性;重复率达100％。同时,HLA－A33、A68、B40、B13结果与HLA－I类PCR－SSP分型结果一致。HLA基因测序分析进一步肯定了HLA－I类PCR－SSP分型的特异性。HLA－I类PCR－SSP不受某些血清学影响因素的干扰。整个实验过程仅耗时2．5h。结论：HLA－I类PCR－SSP基因分型方法准确、特异、重复性好,可作为临床骨髓移植配型和正常人群无关供者筛选的常规方法,对GVHD的发生必将起到重要的预防作用。     

大鼠器官移植术后的弓形虫感染及特异性抗体的检测

目的 观察大鼠器官移植术后弓形虫感染发病情况及环抱素A(CsA)对感染的影响，并探讨该病的诊断方法。方法 以Wistar大鼠为供者，SD大鼠为受者，按供、受者术前弓形虫的感染情况分为供者隐性感染组(20只)和受者隐性感染组(20只)，两个组均接受异位心脏移植，术后每组各有10只接受CsA治疗，另10只不用。术后观察动物的存活情况、发病表现，监测弓形虫特异性抗体IgM、IgG滴度和弓形虫循环抗原(CAg)的变化。结果移植术后供者隐性感染组的弓形虫感染死亡率、CAg阳性率、弓形虫感染发病率均高于受者隐性感染组；使用CsA可使弓形虫感染危险性增高；弓形虫特异性抗体IgG、IgM的测定结果有时受CsA的影响，而CAg不受CsA影响，且较抗体出现早。结论 CsA使用可使大鼠心脏移植后的弓形虫感染发病率升高；弓形虫特异性抗体IgM、IgG和循环抗原(CAg)的测定，并结合临床表现，对于弓形虫感染的早期诊断有帮助。     

Combined treatment of neurotrophin-3 gene and neural stem cells is propitious to functional recovery after spinal cord injury

We present an insight of the effects of combination therapy with neurotrophin-3 and neural stem cell on functional recovery after spinal cord injury (SCI). Total RNA was extracted from neural stem cell line C17.2 and reversed transcribed into cDNA. Neurotrophin-3 (NT-3) gene was amplified by PCR and subcloned into plasmid to construct an expression vector pNT-3. A positive clone containing pNT-3, named SHN2, was obtained and used for transplantation. Thirty adult mice received mechanical injury at the T8 vertebra level. Cell survival, NT-3 gene expression, and functional recovery were observed through X-Gal staining, RT-PCR, and open field locomotion, respectively. The results show that NT-3 gene comprising 777 bp nucleotides was cloned and a more than twofold expression was detected when transfected into neural stem cell line C17.2. Quantitative analysis of cellular density revealed a significant increase in SHN2 compared to the control cells (p < 0.01). Thirty days after transplantation, SHN2 showed significant increase near the lesion site. Furthermore, the functional recovery indicated an active effect by detecting Basso-Beattie-Bresnahan (BBB) locomotor rating scale (p < 0.01). In conclusion, combined treatment of neural stem cells and NT-3 gene can facilitate functional recovery. It offers an effective approach to treat SCI.     

聚合酶链反应在器官移植受者人类疱疹病毒6型感染检测中的应用

目的 探讨器官移植受者人类疱疹病毒６型（ＨＨＶ－６）感染的诊断方法。方法 应用聚合酶链反应（ＰＣＲ）技术,检测３１例器官移植受者术前１周、术后１６天及１０例移植术后发热时的外周血单个核细胞（ＰＢＭＣｓ）中的ＨＨＶ－６ＤＮＡ。结果 术前７例和术后１９例的ＰＢＭＣｓ中检测出ＨＨＶ－６ＤＮＡ,１０例发热患者中８例ＨＨＶ－６ＤＮＡ阳性。对１０例发热患者用阿昔洛韦治疗,８例症状得到控制,２例死亡。结论 应用     

The role of PCR in detection of cytomegalovirus in blood and urine of transplanted patients

Rapid and reliable laboratory diagnosis of cytomegalovirus (CMV) infection in transplanted patients becomes more significant considering possible prevention or moderation of CMV disease. The aim of this study was comparison of CMV isolation in cell culture and CMV detection by PCR in blood and urine samples of transplanted patients. The study comprised 21 patients undergoing immunosuppressive therapy after renal or bone marrow transplantation. MRC-5 cell culture was used for viral isolation. Inoculated cultures were maintained for 4 weeks or until appearance of specific cytopathic effect (CPE). Primers used in PCR were specific for the sequence within egzon 4 of immediate-early 1 (IE-1) gene. Presence of anti-CMV IgG and IgM antibodies was determined by ELISA. PCR was more sensitive for blood (4/21) and urine samples (3/19) than cell culture method--blood (0/21) urine (2/19). Statistically significant correlation was not shown between presence of CMV(determined by PCR) in blood and urine of the same patient.     

Risk of viral transmission via bone marrow progenitor cells versus umbilical cord blood hematopoietic stem cells in bone marrow transplantation

Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for children and certain adults with malignant and nonmalignant hematologic disease. Since viral infections are the major problem, this study examined those that might potentially be transmitted to HSCT recipients via bone marrow (BM) versus umbilical cord blood (UCB). BM progenitor cells, peripheral blood leukocytes, and plasma samples were collected from 30 allogenic BM donors. Umbilical cord blood hematopoietic stem cells and plasma samples were also collected from 34 UCB donors. Viral DNA extracted and purified from collected specimens was processed using nested polymerase chain reactions (PCR) to detect human parvovirus B19 (HPV B19), human herpesvirus-6 (HHV-6), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV). The prevalences of HCMV DNA in collected BM progenitor cells versus UCB hematopoietic stem cells were 73% versus 23%, respectively. Conversely, HHV-6 DNA was not detected in any collected specimen by simple PCR. Distribution of the other investigated virus DNAs except EBV DNA was similar in specimens collected from both groups. EBV DNA was not determined in UCB hematopoietic stem cells. The results indicate that the risk of viral transmission to BM transplant recipients via UCB hematopoietic stem cells is less than that with BM progenitor cells.     

荧光原位杂交法检测造血干细胞移植后骨髓嵌合状态和微量残留病灶

目的 探讨荧光原位杂交（FISH）法在异基因造血干细胞移植（allo-HSCT）后检测供、受者骨髓嵌合状态和微量残留病灶（MRD）的作用。方法 2001年2月至2005年6月对83例患者进行allo-HSCT。对供者为异性的64例受者实施骨髓细胞性染色体着丝粒探针FISH法检测，评价供、受者骨髓嵌合状态及其动态改变；对供、受者性别相同而有特殊染色体异常的19例受者，应用相应的基因探针（BCR／ABL、AMIL1／ETO和MLL）对骨髓细胞行FISH法检测，评价微量残留病灶的发生。结果 64例异性allo-HSCT的受者中，50例供、受者骨髓嵌合度在99％以上；7例早期嵌合度偏低（96．2％～98．7％），在随访过程中逐渐上升至99％以上，移植后均未复发原病；另外7例嵌合度在随访过程中呈进行性下降，其中3例微量残留病灶在10％以上，均同时出现血液学复发；4例患者微量残留病灶在2％～5％之间，其中2例经快速停用免疫抑制剂后出现严重的移植物抗宿主病（GVHD）；1例在免疫抑制剂快速减量后骨髓嵌合度逐步上升至99．9％，目前仍处于完全缓解（CR）状态中；1例持续处于CR状态。19例供、受者性别相同的allo-HSCT受者中，16例移植后未检测到原来的异常核型；1例检测到10％的微量残留病灶，经免疫抑制剂减量4个月后降为1％，移植后1年仍在完全缓解中；2例分别在移植后1个月和4个月时出现原来的染色体异常。复查骨髓为原病复发，再次化疗未缓解。结论 应用FISH法检测allo-HSCT后骨髓嵌合状态和微量残留病灶，对判断植入、复发和指导早期干预性免疫治疗有重要意义。     

肾移植受者HLA特异性抗体的监测及临床应用

目的：探讨人类白细胞抗原（HLA）群体反应性抗体（PRA）对肾脏移植效果的影响。方法：采用酶联免疫吸附法（ELISA）和微量补体依赖性细胞毒试验（CDC）对897例例次肾移植受者的PRA进行动态监测。结果856例初次肾移植受者中术前PRA阳性者121例,占11％,而33例次2次移植和8次3次移植受者术前PRA阳性分别为58％和87％,与初次肾移植受者组比较,PRA阳性受者比例均显著增高（P＜0．001）。PRA阳性组受者肾移植术后排斥发生率为40％,而阴性组受者仅17％,两组比较有显著性（P＜0．01）。PRA阳性组受者的移植物存活率则明显低于阴性组（P＜0．001）,尤其是PRA＞40％的高效敏受者的1、3、5年和7年多移植物存活率与阴性组比较分别下降了24％、38％、57％和56％,均为P＜0．001。ELISA法与CDC法的双盲试验发现,319份受者血清中CDC－PRA的假阳性和假阴性率分别为3．8％和3．1％。结论PRA是预测受者致敏状态的敏感指标,对移植后排斥反应和移植物存活率均有明显影响。