Direct Identification of Enteroviruses in Cerebrospinal Fluid of Patients with Suspected Meningitis by Nested PCR Amplification

Enteroviruses, the most common human viral pathogens worldwide, have been associated with serous meningitis, encephalitis, syndrome of acute flaccid paralysis, myocarditis and the onset of diabetes type 1. In the future, the rapid identification of the etiological agent would allow to adjust the therapy promptly and thereby improve the course of the disease and prognosis. We developed RT-nested PCR amplification of the genomic region coding viral structural protein VP1 for direct identification of enteroviruses in clinical specimens and compared it with the existing analogs. One-hundred-fifty-nine cerebrospinal fluids (CSF) from patients with suspected meningitis were studied. The amplification of VP1 genomic region using the new method was achieved for 86 (54.1%) patients compared with 75 (47.2%), 53 (33.3%) and 31 (19.5%) achieved with previously published methods. We identified 11 serotypes of the Enterovirus species B in 2012, including relatively rare echovirus 14 (E-14), E-15 and E-32, and eight serotypes of species B and 5 enteroviruses A71 (EV-A71) in 2013. The developed method can be useful for direct identification of enteroviruses in clinical material with the low virus loads such as CSF.     

肠道病毒71型感染手足口病患儿Th1/Th2细胞因子变化

目的探讨肠道病毒71型(EV71型)感染手足口病患儿的常规生化检查及辅助性T细胞(help T cell,Th)1/Th2细胞因子变化。方法选择儋州市人民医院2017年1月-2018年12月住院的EV71型感染手足口病患儿120例作为研究对象,其中普通型67例,重症型53例。另选择医院2017年1月-2018年12月健康体检小儿60名作为对照组。对两组患儿白细胞计数(WBC)、C-反应蛋白(CRP)、白细胞介素-2(IL-2)、IL-4、IL-6、IL-10和干扰素-γ(IFN-γ)水平进行比较。结果感染组WBC计数、CRP、IL-2、IL-4、IL-6、IL-10和IFN-γ含量分别为(12.01±1.24)×10^9/L、(10.83±2.45)mg/L、(10.83±1.24)pg/ml、(11.43±1.46)pg/ml、(34.39±5.46)pg/ml、(10.32±2.41)pg/ml、(4.83±0.97)pg/ml,均高于对照组,差异均具有统计学意义(P<0.05)。重症组WBC计数、CRP、IL-2、IL-4、IL-6、IL-10和IFN-γ含量高于普通组,差异均具有统计学意义(P<0.05)。结论EV71型感染手足口病患儿WBC计数和CRP水平升高,且Th1/Th2细胞因子IL-2、IL-4、IL-6、IL-10和IFN-γ明显升高,且与病情进展密切相关。     

Chimeric enterovirus 71 virus-like particle displaying conserved coxsackievirus A16 epitopes elicits potent immune responses and protects mice against lethal EV71 and CA16 infection

Hand-foot-and-mouth disease (HFMD) is an infectious disease of infants and young children frequently caused by the enterovirus A species, mainly enterovirus 71 (EV71) and coxsackievirus A16 (CA16). In this study, we prepared the EV71 virus-like particle (EV71-VLP) and its chimeras using recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under CMV-IE promoter) proteins in Sf9 cells. EV71-VLP chimera ChiEV71(1E)-VLP or ChiEV71(4E)-VLP displayed single CA16 PEP71 epitope in VP1 or four conserved CA16 neutralizing epitopes (PEP71 in VP1, aa136-150 in VP2, aa176-190 in VP3 and aa48-62 in VP4) by substitution of the corresponding regions of EV71 structure proteins, respectively. In mice, EV71-VLP and its chimeras elicited similar EV71-specific IgG and neutralizing antibody (NAb) titers compared to inactivated EV71. Expectedly, vaccination of ChiEV71(1E)-VLP or ChiEV71(4E)-VLP resulted in significantly increased CA16-specific IgG and NAb production and improved cross-protection against CA16 infection compared to EV71-VLP. Interestingly, the VLPs induced potent cellular immune responses and significantly decreased Th2 type (IL-4 and IL-10) cytokines secretion in the splenocytes of immunized mice compared to inactivated EV71 or inactivated CA16. Neonatal mice born to dams immunized with the chimeric VLPs or neonatal mice passively transferred with sera of immunized mice were completely protected from lethal EV71 challenge and partially protected from lethal CA16 infection. Our study provides a novel bivalent or multivalent vaccine strategy to prevent EV71 and related-enterovirus infections.     

Fibroblast-stimulating lipopeptide-1 as a potential mucosal adjuvant enhances mucosal and systemic immune responses to enterovirus 71 vaccine

To prevent viral infection at the site of entry, mucosal vaccines are potent tools for inducing IgA secretion for defense. Because Toll-like receptor (TLR) ligands serve as strong adjuvants, two ligands that mimic the structure of mycoplasmal and bacterial lipopeptides represent interesting vaccine candidates. Pam3CSK4, a synthetic triacylated lipopeptide, interacts with TLR2/1. Because fibroblast-stimulating lipopeptide-1 (FSL-1), a synthetic diacylated lipopeptide, is recognized by TLR2/6, we targeted the potential immuno-inducibility of Pam3CSK4 and FSL-1 as adjuvants of an enterovirus 71 (EV71) mucosal vaccine. Naïve BALB/c mice were used for intranasal immunization three times over a 3-week interval, with results showing that EV71-specific IgG and IgA in serum, nasal washes, bronchoalveolar lavage fluid, and feces from the EV71 + FSL-1 group were significantly higher than levels observed in mice treated with EV71 + Pam3CSK4, EV71 alone, or the control group treated with phosphate-buffered saline. Furthermore, we observed more EV71-specific IgG and IgA-producing cells in treatments using EV71 formulated with FSL-1. Additionally, T cell-proliferative responses and interferon-γ and interleukin-17 secretion were significantly increased when inactivated EV71 was formulated using FSL-1. Moreover, serum from immunized mice was capable of neutralizing the infectivity of EV71 (C2 genotype) and was able to cross-neutralize the B4 and B5 genotypes of EV71. Our data suggested that FSL-1 could be used as an efficient adjuvant for intranasal EV71-vaccine immunization.     

2013-2014年浙江省奉化市手足口病患者及其密切接触者监测结果分析

目的 了解浙江省奉化市手足口病患者及其密切接触者病原分布情况,分析2013-2014年手足口病疫情和病原学监测结果,为手足口病防控提供科学依据。方法 每月采集手足口病患者5例及其15名密切接触者粪便标本检测肠道病毒,同时开展住院病例病原学监测,对2013-2014年病原学监测结果和手足口病疫情进行描述性流行病学分析。结果 2013-2014年奉化市手足口病发病率分别为225.99/10万和365.07/10万;街道发病率高于乡镇;发病率最高为1岁组。病例肠道病毒A组71型（EV71）、柯萨奇病毒A组16型（Cox A16）和其他肠道病毒阳性率分别为20.94%、20.42%和38.74%;2013年优势病毒为其他肠道病毒（占61.00%）,2014年为Cox A16和EV71;EV71发病年龄大于其他肠道病毒（Z=-3.70,P=0.00）;肠道病毒阳性病例和阴性病例的密切接触者阳性率分别为48.44%和18.75%,差异有统计学意义;不同病毒阳性病例的密切接触者其他肠道病毒阳性率在44.96%～48.15%之间,差异无统计学意义;病例与密切接触者肠道病毒以同型为主（Kappa值0.53）;1～8岁密切接触者阳性率42.08%,12～66岁密切接触者阳性率31.73%,差异无统计学意义。结论 奉化市手足口病处于高流行态势,流动人口密集地区高发。多种病毒流行,优势病毒处于变化中,流行特征与病原体有关,病原监测有助于采取适当的防控措施,必要时应对其他肠道病毒开展进一步分型。密切接触者带病毒率高,应关注密切接触者管理。     

74例无典型皮疹的手足口病脑炎患儿临床特征分析

  目的  分析无典型皮疹的手足口病脑炎患儿临床特征,以指导临床早发现、早诊治,为手足口病的防控提供参考。  方法  收集2018年1月—2019年12月在杭州市儿童医院住院的74例确诊手足口病脑炎但无典型皮疹患儿的临床资料,包括流行病学资料、临床表现、实验室检查结果、病原学检测结果等进行回顾性分析。  结果  74例患儿发病年龄为(6.92±2.72)岁,5岁及以上占82.5%。2018年和2019年各检出42例和32例,2年间检出率比较差异无统计学意义(P>0.05)。检出月份均以6、7月份检出率最高,分别为2018年的35.7%(15/42)和42.9%(18/42),2019年的28.1%(9/32)和37.5%(12/32),与典型手口足病流行时间一致。患儿临床以发热、头痛、呕吐为主要表现,而无典型手、足、臀皮疹和咽峡的疱疹。其皮疹表现为稀疏、细小斑丘疹,未见疱疹,分布不典型以口周、耳颈、躯干、四肢为主,且多为单个部位,达87.8%。外周血常规及脑脊液改变符合手足口病特点,脑脊液肠道病毒通用型检测均阳性,未检出常见肠道病毒EV71和CV-A6、CV-A10、CV-A16。患儿均治愈, 未出现并发症、后遗症。  结论  手足口病流行季节需警惕以年长儿多见,发热、头痛、呕吐为主要表现,而无典型皮疹的手足口病脑炎,避免误诊漏诊。      

糖皮质激素在河南、四川两省重症手足口病中的应用现状及不同剂量激素疗效比较

目的 了解河南、四川两省糖皮质激素在重症手足口病中应用现状;比较不同剂量激素治疗重症手足口病的疗效.方法 采用回顾性多中心研究方法,按照等容量随机抽样法随机抽取河南、四川两省201 1年1月至5月320例重症手足口病例为研究对象,计算糖皮质激素静脉注射使用率.按激素使用情况将病例分为非激素治疗组、小剂量激素治疗组和大剂量激素治疗组,比较3组之间治愈率、发热持续时间、住院时间、神经系统症状持续时间、生命体征及血糖等.结果 320例患者中291例在住院治疗过程中静脉使用糖皮质激素,激素使用率为90.9％.3组之间治愈率差异无统计学意义(P＞0.05),且小剂量激素治疗组、大剂量激素治疗组患儿神经系统症状持续时间、住院时间、发热持续时间比非激素治疗组长,差异均有统计学意义(P均＜0.05);入院第3天,大剂量激素治疗组舒张压明显高于其他2组(P均＜0.05).结论 糖皮质激素在河南、四川两省广泛应用于重症手足口患儿的治疗,在发热、神经系统症状不缓解情况下更倾向于使用大剂量激素治疗.激素治疗重症手足口病患儿并未能提高治愈率,亦不能缩短病程、改善其临床症状,激素对于重症手足口病的疗效有待商榷,且大剂量激素治疗有可能增加患儿药物不良反应.     

2012年江西省儿童急性出血性结膜炎病原学特征分析

目的：了解2012年江西省儿童急性出血性结膜炎(AHC)病原学特征,为其防治提供科学依据。方法采集2012年江西省急性出血性结膜炎患儿44例结膜拭子标本,并运用PCR和RT-PCR方法对这44份结膜拭子标本同时进行肠道病毒70型、柯萨奇病毒24型变异株和腺病毒三种病毒检测。结果2012年江西省儿童流行性出血性结膜炎流行高峰在5-7月份;PCR和RT-PCR检测结果为腺病毒阳性31份。结论2012年江西省儿童急性出血性结膜炎病原体主要为腺病毒。