巨细胞病毒脑内感染小鼠听觉诱发电位的变化

目的 探讨脑内感染小鼠巨细胞病毒（murine cytomegalovirus,MCMV）后脑干听觉诱发电位（auditory brainstem response,ABR）的变化,以明确经脑内感染MCMV后小鼠ABR是否出现异常改变,从而验证经脑内感染MCMV能否建立巨细胞病毒（CMV）感染致听力损伤的动物模型,并在此基础上观察更昔洛韦对CMV感染致听力损伤的干预效果.方法 将24只新生小鼠按窝随机分成正常对照组、模型组、干预组3组,对照组经脑内注射无菌生理盐水10 μl,模型组与干预组经脑内接种TCID50为1×105 U/ml的MCMV病毒悬液10 μl,同时干预组在接种MCMV后第2天开始腹腔注射更昔洛韦60 mg/（kg·d）,连用2周.2周后在小鼠麻醉状态下应用脑干听觉诱发电位仪在电屏蔽装置中检测小鼠的ABR,并记录ABR的波形以及Ⅰ波的潜伏期、波幅.结果 模型组与正常对照组相比潜伏期延长、波幅降低（F=9.151,P=0.011;F=5.095,P=0.043）,更昔洛韦干预组与模型组相比潜伏期缩短、波幅升高（F=13.797,P=0.003;F=14.587,P=0.002）,差异有统计学意义.结论 经脑内感染MCMV可以引起小鼠ABR的异常改变,脑内感染MCMV可建立CMV感染致听力损伤的动物模型,婴幼儿CMV感染后早期应用更昔洛韦对CMV感染致听力损伤的进行性发展有一定的抑制作用.     

全反式维甲酸增强携单纯疱疹病毒胸苷酸激酶基因的超声微泡对SMMC-7721细胞的旁观者效应

目的观察经全反式维甲酸(all-trans retinoic acid,ATRA)诱导后,单纯疱疹病毒胸苷酸激酶/丙氧鸟苷系统(HSV-TK/GCV)对人肝癌细胞SMMC-7721旁观者效应的影响。方法免疫组化法观察经全反式维甲酸诱导前后SMMC-7721细胞连接蛋白Cx32的表达;将含有HSV-TK基因的质粒通过静电吸附在脂质微泡表面上,采用超声辐照转染并用倒置显微荧光镜及RT-PCR观察TK基因的转入及表达;MTT法观察经全反式维甲酸诱导后HSV-TK/GCV对肝癌细胞SMMC-7721的旁观者效应。结果经全反式维甲酸诱导后SMMC-7721细胞膜上Cx32表达明显增加,而胞质内仅少量表达;通过超声辐照后HSV-TK基因可顺利转入SMMC-7721细胞中并稳定表达;与对照组相比,经全反式维甲酸诱导组可明显提高HSV-TK/GCV对肝癌细胞SMMC-7721的旁观者效应(P<0.05)。结论全反式维甲酸可提高SMMC-7721细胞中连接蛋白Cx32的表达且能正确定位于细胞膜上,这可能与其能增强HSV-TK/GCV系统对肝癌细胞SMMC-7721的旁观者效应有关。     

玻璃体内注射联合全身应用更昔洛韦治疗AIDS合并巨细胞病毒视网膜炎的临床疗效

目的　观察玻璃体内注射联合全身应用更昔洛韦治疗AIDS合并巨细胞病毒视网膜炎（cytomegalovirus retinitis,CMVR）的临床疗效。方法　收集2016年1月至2018年1月在广西医科大学第一附属医院收治的AIDS合并CMVR患者7例13眼。患者全身用药为更昔洛韦和膦甲酸钠注射液，更昔洛韦 5.0～7.5 mg·kg^-1，每天2次静脉滴注；成人每次给予膦甲酸钠3 g，每天3次静脉滴注；同时在局部玻璃体内注射更昔洛韦。治疗前后对比的指标有:最佳矫正视力、眼压、眼底照相情况、视觉诱发电位、眼电图和视网膜电图等检查；检测指标包括CD4+T细胞计数，血液、前房水、玻璃体CMV-DNA病毒载量等。通过检查结果辅助判断治疗效果。结果　6例患者治疗后最佳矫正视力为（0.72±0.83）logMAR，较治疗前（1.14±0.83）logMAR提高（P=0.001），其中1例患者经过全身和局部抗病毒治疗后视力不提高。患者治疗前后眼压均在正常范围内，治疗前为（13.62±3.04）mmHg（1 kPa=7.5 mmHg），治疗后为（12.77±2.89）mmHg，差异无统计学意义（P=0.119）。注药后患者眼底病变范围逐渐变小（P<0.05）。治疗1个月后，患者视觉诱发电位N2-P2振幅及眼电图光峰电位较治疗前升高（均为P<0.05），视觉诱发电位P2潜伏期和视网膜电图（明适应）a波及b波潜伏期和振幅治疗前后差异均无统计学意义（均为P>0.05）。3例患者治疗前血液中CMV-DNA病毒载量检测为阴性，1例患者2眼经过治疗后眼内液中CMV-DNA病毒载量检测为阴性。治疗前玻璃体CMV-DNA病毒载量均明显高于前房水，而前房水CMV-DNA病毒载量又明显高于血液（均为P<0.05），说明三个部位的CMV-DNA病毒载量从高到低的排列顺序为玻璃体>前房水>血液。治疗后玻璃体、前房水CMV-DNA病毒载量均低于治疗前，差异均有统计学意义（均为P<0.05）。治疗前后血液CMV-DNA病毒载量差异无统计学意义（P>0.05）。治疗前后CD4+T 细胞计数差异无统计学意义（P>0.05）。治疗前CD4+T细胞计数与眼内液、血液CMV-DNA病毒载量均呈负相关关系（均为P<0.05）；治疗过程中，CD4+T细胞计数与眼内液CMV-DNA病毒载量无相关关系（均为P>0.05），与血液CMV-DNA病毒载量呈负相关关系（P<0.05），但两者之间不存在直线回归关系（P>0.05）。治疗前及治疗过程中玻璃体CMV-DNA病毒载量与前房水CMV-DNA病毒载量均存在正相关关系（回归方程分别为:Y=20 178.973+0.165X，Y=171 849.77+0.168X，均为P<0.05）。所有患者术中和术后均未出现严重并发症。结论　静脉滴注抗病毒药物联合玻璃体内注射小剂量更昔洛韦是治疗AIDS合并CMVR安全有效的治疗方法。     

神经保护因子对巨细胞感染新生儿听力影响的研究

目的 探讨神经保护因子对巨细胞感染新生儿听力的影响。方法 收集78例先天性巨细胞感染的新生儿分为2组,均给予更昔洛韦进行治疗,研究组患者在上述治疗的基础上给予神经保护因子,比较2组患者的治疗效果。结果 2组患儿在出生后3 d听力筛选不通过率差异无统计学意义(P>0.05);患儿听力筛选不通过率在出生后6周研究组(5.1%)明显低于对照组(15.4%,P<0.05)。研究组患儿诱发电位检测V波的阈值在出生后3个月与6个月均明显低于对照组(P<0.05)。结论 在一定程度上神经保护因子能降低先天性巨细胞感染新生儿听力的损伤,对患儿的听觉系统有很好的保护作用,在临床上值得进行推广应用。     

不同启动子指导的HSV1-sr39TK/GCV系统对小鼠淋巴细胞的敏感性研究

目的观察慢病毒载体中不同启动子指导的突变型单纯疱疹病毒1型胸苷激酶(HSV1-sr39TK)基因在小鼠T淋巴细胞内的表达差异,并进一步比较对丙氧鸟苷(ganciclovir,GCV)的敏感性。方法用亚克隆技术将PCR扩增的HSV1-sr39TK基因分别连接至慢病毒载体不同启动子后,然后在HSV1-sr39TK基因后以内部核糖体进入位点(internal ribosome entry sites,IRES)连接绿色荧光蛋白(green fluorescent pro-tein,GFP)报告基因;采用三质粒包装系统包装病毒,将病毒感染刀豆蛋白A(concanavalin,ConA)激活的小鼠淋巴细胞,分别用流式细胞术、RT-PCR法鉴定HSV1-sr39TK和GFP基因在小鼠淋巴细胞的表达,用Cell Counting Kit-8(CCK-8)法观察感染不同病毒的淋巴细胞对前体药物GCV的敏感性。结果不同启动子指导的HSV1-sr39TK及GFP基因均可在小鼠淋巴细胞表达,巨细胞病毒(cytomegalovirus,CMV)启动子驱动表达能力最强,磷酸甘油激酶(phosphoglycerokinase,PGK)启动子最弱。感染含CMV启动子病毒的淋巴细胞对GCV的IC50值最小。结论在小鼠淋巴细胞内CMV启动子驱动的慢病毒载体HSV1-sr39TK基因表达最强,对前体药物GCV敏感性最高。     

Detection of human cytomegalovirus immediate early antigen in leukocytes as a marker of viremia in immunocompromised patients

Peripheral blood polymorphonuclear (PMN) cells from 35 immunocompromised patients (22 heart transplant recipients and 13 AIDS patients) and four normal subjects were tested for the presence of human cytomegalovirus (HCMV) immediate early antigen (IEA) (antigenemia) by indirect immunofluorescence (IFA) and IEA-specific monoclonal antibodies (MAb). PMN samples were tested in parallel for HCMV isolation (viremia) by using MAb to viral early antigens (EA) and the IFA technique 24-48 hr after inoculation onto human fibroblast monolayers. HCMV was isolated from 26 of 83 PMN samples examined: of these, 25 were also positive for HCMV IEA (96% sensitivity). Seven additional PMN samples negative for viral isolation resulted IEA-positive (87.7% specificity). Six of the seven discordant samples were taken from four patients during ganciclovir treatment. The transitory dissociation between positive HCMV antigenemia and negative viremia during antiviral treatment was followed, at the end of the therapy, either by virus clearance and disappearance of IEA-positive PMNs (one patient) or by reappearance of viremia (three patients). Among concordant positive samples, a significant correlation was observed between the number of IEA-positive PMN leukocytes and EA-positive nuclei of infected fibroblasts, when the same number of PMNs were used for both tests.     

Cytomegalovirus cultures during maintenance DHPG therapy for cytomegalovirus (CMV) retinitis in acquired immunodeficiency syndrome (AIDS)

Nine patients with acquired immunodeficiency syndrome (AIDS) and cytomegalovirus (CMV) retinitis on maintenance therapy with ganciclovir: 9(1,3-dihydroxy-2-propoxymethyl) guanine (DHPG) at high dose (30 mg/kg/week) or low dose (20 mg/kg/week) were tested every 1-2 weeks for CMV isolation from blood, saliva, and urine. Duration of therapy ranged from 1.5 to 12 months (average 5.3 months). During pretreatment and low-dose and high-dose maintenance therapy, CMV was isolated from 48/59 (81%), 90/211 (43%), and 40/290 (14%) of specimens, respectively. Three patients with progressive retinitis had viraemia more frequently than did six patients with stable retinitis, CMV being isolated from 29/47 (62%) and 17/121 (14%) of blood samples, respectively.     

The Role of Antiviral Prophylaxis for the Prevention of Epstein–Barr Virus–Associated Posttransplant Lymphoproliferative Disease in Solid Organ Transplant Recipients: A Systematic Review

The role of antiviral prophylaxis for the prevention of posttransplant lymphoproliferative disease (PTLD) remains controversial for solid organ transplantation (SOT) recipients who are seronegative for Epstein–Barr virus (EBV) but who received organs from seropositive donors. We performed a systematic review and meta‐analysis to address this issue. Two independent assessors extracted data from studies after determining patient eligibility and completing quality assessments. Overall, 31 studies were identified and included in the quantitative synthesis. Nine studies were included in the direct comparisons (total 2366 participants), and 22 were included in the indirect analysis. There was no significant difference in the rate of EBV‐associated PTLD in SOT recipients among those who received prophylaxis (acyclovir, valacyclovir, ganciclovir, valganciclovir) compared with those who did not receive prophylaxis (nine studies; risk ratio 0.95, 95% confidence interval 0.58–1.54). No significant differences were noted across all types of organ transplants, age groups, or antiviral use as prophylaxis or preemptive therapy. There was no significant heterogeneity in the effect of antiviral prophylaxis on the incidence of PTLD. In conclusion, the use of antiviral prophylaxis in high‐risk EBV‐naive patients has no effect on the incidence of PTLD in SOT recipients.     

Preventive Strategies Against Cytomegalovirus and Incidence of α‐Herpesvirus Infections in Solid Organ Transplant Recipients: A Nationwide Cohort Study

We assessed the impact of antiviral preventive strategies on the incidence of herpes simplex virus (HSV) and varicella‐zoster virus (VZV) infections in a nationwide cohort of transplant recipients. Risk factors for the development of HSV or VZV infection were assessed by Cox proportional hazards regression. We included 2781 patients (56% kidney, 20% liver, 10% lung, 7.3% heart, 6.7% others). Overall, 1264 (45%) patients received antiviral prophylaxis (ganciclovir or valganciclovir, n = 1145; acyclovir or valacyclovir, n = 138). Incidence of HSV and VZV infections was 28.9 and 12.1 cases, respectively, per 1000 person‐years. Incidence of HSV and VZV infections at 1 year after transplant was 4.6% (95% confidence interval [CI] 3.5–5.8) in patients receiving antiviral prophylaxis versus 12.3% (95% CI 10.7–14) in patients without prophylaxis; this was observed particularly for HSV infections (3% [95% CI 2.2–4] versus 9.8% [95% CI 8.4–11.4], respectively). A lower rate of HSV and VZV infections was also seen in donor or recipient cytomegalovirus‐positive patients receiving ganciclovir or valganciclovir prophylaxis compared with a preemptive approach. Female sex (hazard ratio [HR] 1.663, p = 0.001), HSV seropositivity (HR 5.198, p < 0.001), previous episodes of rejection (HR 1.95, p = 0.004), and use of a preemptive approach (HR 2.841, p = 0.017) were significantly associated with a higher risk of HSV infection. Although HSV and VZV infections were common after transplantation, antiviral prophylaxis significantly reduced symptomatic HSV infections.