Rapid Detection of Haemophilus influenzae and Haemophilus parainfluenzae in Nasopharyngeal Swabs by Multiplex PCR

Objective To establish multiplex PCR-based assays for detecting H.influenzae and H.parainfluenzae. And the PCR-based assays were applied to detect the carriage rates of H.influenzae and H.parainfluenzae in nasopharyngeal swab specimens which were collected from healthy children. Methods Multiplex primers for species-specific PCR were designed by using DNAstar soft based on the sequences of 165 rRNA genes from genus Haemophilus to detect H.influenzae and H.parainfluenzae. Results The sensitivity of the 165 rRNA PCR assay for detecting H.influenzae and H.parainfluenzae was 97.53% and 100% respectively, and the specificity was 95.89% and 96.63% respectively. Youden＇s Index on the ability to detect H.influenzae and H.parainfluenzae was 0.9342 and 0.9663 respectively. 666 nasopharyngeal swab specimens were collected from healthy children. The detection rates of H.influenzae and H.parainfluenzae were 14.11% and 16.07% respectively by using isolation and culture methods. The detection rates of H.influenzae and H.parainfluenzae were 43.54% and 57.96% respectively by 165 rRNA PCR assays. The carriage rates of serotypes a, b, c, d, e, f and non-typeable isolates were 0% （0/666）, 0.15% （1/666）, 1.20% （8/666）, 0.15% （1/666）, 1.20% （8/666）, 1.80% （12/666）, 95.50% （636/666） respectively. Conclusion The multiplex PCR assays were very rapid, reliable and feasible methods for detection of H.influenzae and H.parainfluenzae in pharyngeal swab specimens which were compared to conventional isolation and culture methods. 95.5% of H.influenzae strains in healthy children were nontypeable. The encapsulated or typable strains were mainly three serotypes which was c, e, and f serotype.

Seroprevalence of Bovine Parainfluenza Virus Type 3 (bPI-3V) in Ruminants from Grenada

Respiratory viral infections are known for serious economic losses in ruminants. Bovine parain-fluenza 3 virus (bPI-3V) a member of Respirovirus genus in association with other respiratory viruses causes respiratory disease complex in ruminants. The aim of this study was to estimate the seroprevalence of bPI-3V in non vaccinated cattle, sheep and goats from Grenada. Sera were collected randomly from 60 sheep, 60 goats and 60 cattle from all six parishes of Grenada. Sera were tested for antibodies to bPI-3V using an indirect Enzyme Linked Immunosorbant Assay (ELISA) kit. Antibodies to bPI-3V were detected in 13.4% (Confidence Level (CL): 95%;Confidence Interval (CI): 4.76% to 22.02%) in cattle;16.7% (CL: 95%;CI: 7.26% to 26.14%) in sheep and 11.7% (CL: 95%;CI: 3.57% to 19.83%) in goats. There was statistically no significant difference in prevalence (p > 0.05) of antibodies to bPI-3V in cattle, sheep and goats in Grenada. This is the first report on seroprevalence of bPI-3V in ruminants in Grenada, West Indies.

Virulent endocarditis due to Haemophilus parainfluenzae:A systematic review of the literature

BACKGROUND Haemophilus parainfluenzae(HPI)belongs to the HACEK(Haemophilus spp.,Aggregatibacter spp.,Cardiobacterium spp.,Eikenella spp.,and Kingella spp.)group of organisms.The HACEK group of organisms are a part of the oropharyngeal flora and can cause invasive opportunistic infection such infective endocarditis(IE)in hosts with compromised immunological barriers.AIM To perform a 20-year systematic review of the literature characterizing the clinical presentation,epidemiology and prognosis of HPI IE.METHODS We performed a systematic review of Medline,Pubmed,Scopus and Embase from 2000 to 2022 to identify all cases of HPI IE.RESULTS Thirty-nine adult cases were identified.HPI IE was found to affect males slightly more than females and is common in patients with predisposing risk factors such as underlying valvular abnormalities.It mostly affected the mitral valve and had an indolent course;significantly sized vegetations(>1 cm)developed in most cases.Central nervous system septic embolization was common.It had a favorable prognosis compared to staphylococcal and streptococcal IE.CONCLUSION Clinicians should be attentive to the indolent course of HPI IE and the presence of predisposing risk factors in order to allow for timely management.

Evaluation of an attenuated vaccine candidate based on the genotype C of bovine parainfluenza virus type 3 in albino guinea pigs

Bovine parainfluenza virus type 3（BPIV3） is considered as one of the most important respiratory tract pathogens of both young and adult cattle, and widespread among cattle in the world. BPIV3 was first reported in China in 2008 and four strains of BPIV3 were isolated from Shandong Province, known as genotype C（BPIV3c）. Pathogen investigations had shown that BPIV3 c infection was very common among cattle in China. To date, BPIV3 can be classified into genotypes A, B and C based on genetic and phylogenetic analysis. Serological survey also demonstrates that BPIV3 infection is widespread in China, however, there is still no available vaccine for BPIV3 prevention in China nowadays. In the present study, the BPIV3 c strain SD0835 was continuously passaged on Madin-Darby bovine kidney（MDBK） cells for hundreds of times, and the pathogenicity of passage 209 was reduced in guinea pigs. The passage 209 of BPIV3 c strain SD0835 was used as a live vaccine candidate to immunize the guinea pigs. The vaccination results revealed that two vaccinations could induce excellent serum neutralizing antibody responses as well as proliferation of T lymphocytes. The vaccinated guinea pigs were well protected against challenge with a low passage of BPIV3 c strain SD0835. Additionally, the percentages of CD4~＋ and CD8~＋ T cell subsets of animals in vaccinated group increased after immunization; T cell subsets on day 2 after challenge in both groups decreased, and the decline of CD4~＋ and CD8~＋ T cell subsets levels of four guinea pigs in vaccinated group was relatively moderate, comparing with that of the control group. These data support further testing of the attenuated virus as an effective candidate vaccine.

Parainfluenza Virus Types 1, 2, and 3 in Pediatric Patients with Acute Respiratory Infections in Beijing During 2004 to 2012

Background： Although human parainfluenza virus （HPIV） has been determined as an important viral cause of acute respiratory infections （ARIs） in infants and young children, data on long-term investigation are still lacking to disclose the infection pattern of HPIV in China. Methods： Nasopharyngeal aspirates were collected from 25,773 hospitalized pediatric patients with ARIs from January 2004 through December 2012 for respiratory virus screen by direct immuno-fluorescence assay. Results： Out of these specimens, 1675 （6.50%, 1675/25,773） showed HPIV positive, including 261 （1.01%, 261/25,773） for HPIVI, 28 （0.11%, 28/25,773） for HPIV2, and 1388 （5.39%, 1388/25373） for HPIV3, 2 of the samples were positive for both HPIV1 and HPIV3, and 36 were co-detected with other viruses. The positive rates of HPIVs were higher in those younger than 3 years old. HPIV3 was detected from all age groups, predominantly from patients under 3 years of age, and the highest frequency was found in those 6 months to 1-year old （352/4077, 8.63%）. HPlV3 was the dominant type in each of the years detected between May and July. HPIV1 showed a peak in every odd year, mainly in August or September. HPIV was detected most frequently from patients with upper respiratory infection （12.49%, 157/1257）, followed by bronchitis （ 11.13%, 176/2479）, asthma （9.31%, 43/462）, bronchiolitis （5.91%, 150/2536）, pneumonia （6.06%, 1034/17,068）, and those with underlying diseases （1.0%, 15/1506）. HPIV3 is the dominant type in these six disease groups referred above, especially in the asthma group. Conclusions： HPIV is one of the important viral causes of ARIs in infants and young children in Beijing based on the data from the hospitalized children covering a 9-year term. HPIV3 is the predominant type in all these years and in most of the disease groups. HPIVs with different types show different seasonality.

Sumoylation of Human Parainfluenza Virus Type 3 Phosphoprotein Correlates with A Reduction in Viral Replication

Human parainfluenza virus type 3(HPIV3), a member of the Paramyxoviridae family, can cause lower respiratory disease in infants and young children. The phosphoprotein(P) of HPIV3 is an essential cofactor of the viral RNA-dependent RNA polymerase large protein(L). P connects nucleocapsid protein(N) with L to initiate genome transcription and replication.Sumoylation influences many important pathways of the target proteins, and many viral proteins are also themselves sumoylated. In this study, we found that the P of HPIV3 could be sumoylated, and mutation of K492 and K532 to arginine(PK492 R/K532 R) failed to be sumoylated within P, which enhances HPIV3 minigenome activity. Biochemical studies showed that PK492 R/K532 Rhad no effect on its interactions with N, formation of homo-tetramers and formation of inclusion bodies.Finally, we found that incorporation of K492 R/K532 R into a recombinant HPIV3(rHPIV3-PK492 R/K532 R) increased viral production in culture cells, suggesting that sumoylation attenuates functions of P and down-regulates viral replication.

2022年白银市某院呼吸道感染住院患儿的病毒检出情况分析

目的分析2022年白银市某院呼吸道感染住院患儿的病毒检出情况。方法选取2022年1月至12月白银市第一人民医院儿科收治的908例因呼吸道感染住院治疗的患儿为研究对象,采用间接免疫荧光法检测5种主要呼吸道病毒的免疫球蛋白M抗体阳性情况,包括副流感病毒(PIV)、乙型流感病毒(IFVB)、甲型流感病毒(IFVA)、腺病毒(ADV)、呼吸道合胞病毒(RSV)。结果有180例呼吸道病毒检出阳性,阳性率达19.8%。男551例,病毒检出95例(17.2%);女357例,病毒检出85例(23.8%)。不同性别病毒总检出率、ADV检出率比较,差异有统计学意义(P<0.05)。2~3岁患儿411例,病毒检出96例(23.4%)。上呼吸道感染性疾病278例,病毒检出50例(18.0%);支气管感染疾病120例,病毒检出34例(28.3%)。PIV、RSV、IFVB、ADV的检出率分别为10.1%、3.0%、4.0%、2.8%,PIV检出数量在7~8月达到高峰,IFVB检出数量在2、9月达到高峰。结论2022年白银市某院呼吸道感染住院患儿中,病毒检出阳性率为19.8%,主要病原体为PIV,感染高发季节在夏季,支气管感染疾病的病毒检出率最高,不同性别、年龄段的病毒检出率存在差异。

胸部放射学严重程度指数对人副流感病毒相关下呼吸道感染患儿的短期预后价值

目的 探讨胸部放射学严重程度指数(RSI)对人副流感病毒(HPIV)相关下呼吸道感染(LRTI)患儿的短期预后价值。方法 回顾性收集HPIV相关LRTI患儿80例,按照短期(7天)预后分为预后不良组(n=21)和预后良好组(n=59)。比较两组C反应蛋白(CRP)、中性粒细胞/淋巴细胞比值(NLR)、RSI。分析患儿预后不良的影响因素,以及CRP、NLR和RSI对预后不良的预测效力。结果 与预后良好组比较,预后不良组CRP升高,NLR降低,RSI得分升高(P<0.05)。相关性分析结果表明,RSI得分与CRP呈正相关,与NLR呈负相关(P<0.001)。CRP、NLR和RSI是患儿短期预后不良的独立影响因素(P<0.05)。ROC分析结果显示,RSI、CRP、NLR对患儿短期预后均具有良好的预测价值,3指标联合时预测效能最高(P<0.05)。结论 RSI对于HPIV相关LRTI患儿短期预后有一定预测效能,将其与血液学指标联用可提高其预测的应用价值。

一起肉牛副流感病毒3型、巴氏杆菌、牛支原体混合感染的诊治

牛呼吸道综合征往往是多种病原体混合感染引起,致病机制复杂。云南省昆明市某肉牛养殖场部分犊牛出现了以咳嗽、气短、流鼻涕、腹泻等呼吸道症状为主要表现的呼吸道疾病,随后多头相继死亡,剖检主要病变为气管、胃黏膜、小肠出血,肺脏肝变,肝脏出血肿大,心脏出血,心包黏连等;经采病料用常见呼吸道疾病病原(牛病毒性腹泻病毒,牛冠状病毒,牛副流感病毒3型,牛呼吸道合胞体病毒,牛疱疹病毒1型,多杀性巴氏杆菌,溶血性曼氏杆菌,牛支原体)8联PCR检测试剂盒检测,结果为副流感病毒3型、多杀性巴氏杆菌、牛支原体混合感染。经用加米霉素、10%替米考星和20%氟苯尼考等大环内酯类药物治疗,结合加强饲养管理,多数病牛恢复了健康。

山羊副流感病毒3型的研究进展

山羊群中普遍存在山羊副流感病毒3型(CPIV3)病毒的感染,严重危害养羊业的健康发展。该文简要介绍了CPIV3的临床表现、流行病学、病原学、致病性、诊断技术、疫苗研发及防控技术等内容。研究表明,研制出安全有效的疫苗是控制山羊副流感病毒3型的方法,对预防由CPIV3引起的呼吸道疾病具有重要意义。

IFN-α对山羊副流感病毒3型的抗病毒活性

旨在探讨原核表达纯化的山羊α干扰素(IFN-α)对山羊副流感病毒3型(CPIV3)的抗病毒活性。通过分析山羊IFN-α的序列特点,比对不同种属IFN-α的同源性,进而构建山羊IFN-α成熟蛋白编码基因(去除信号肽基因序列)的原核表达载体pET-30a-gIFN-α,将其转化至感受态细胞Rosetta(DE3),IPTG诱导后镍柱及亲和纯化获得山羊IFN-α。利用RT-qPCR测定山羊IFN-α作用于牛肾细胞(Madin-Darby bovine kidney cell,MDBK cell)后6种干扰素刺激基因(interferon-stimulated genes,ISGs)的相对表达水平;同时,利用TCID50及Western blot测定其对CPIV3的抗病毒活性。结果表明,原核表达的山羊IFN-α蛋白含量为0.20 mg·mL^(-1),Western blot结果表明表达产物相对分子质量约为20 ku,与预期结果相符。RT-qPCR结果显示,山羊IFN-α孵育MDBK细胞后,可显著刺激RSAD2、STAT1及ISG15等6种ISGs基因的转录上调。与对照组细胞相比,山羊IFN-α显著下调MDBK细胞中CPIV3的病毒滴度,Western blot结果显示,山羊IFN-α孵育显著下调CPIV3非结构蛋白C的表达水平。山羊IFN-α对CPIV3在MDBK细胞中的增殖具有显著抑制作用。本研究扩展了I型干扰素的研究范围,为CPIV3等反刍动物疫病的防控提供新的思路。

金振口服液对流感病毒H1N1/FM1株和副流感病毒感染致小鼠肺炎的药理作用

目的考察金振口服液对流感病毒H1N1/FM1和副流感病毒感染所致的小鼠肺炎药理作用,为金振口服液的临床应用提供一定的参考。方法采用流感病毒H1N1/FM1和副流感病毒滴鼻感染小鼠造模,考察金振口服液高、中、低剂量组(生药8、4、2 g·kg^(-1)·d^(-1))对小鼠的肺指数、肺组织病理变化和死亡保护作用,并设西药磷酸奥司他韦颗粒、利巴韦林颗粒和中成药小儿热速清口服液作为阳性对照药。结果金振口服液3个剂量组均可不同程度的降低小鼠肺指数、减轻肺组织病理损伤,并对小鼠具有死亡保护作用。结论金振口服液对流感病毒H1N1/FM1和副流感病毒感染所致的肺炎小鼠具有治疗作用。

牛副流感病毒3型和牛支原体混合感染引起牛呼吸道综合征的分析

为查明广西某牛场一起以呼吸道症状为主的病因,试验采用流行病学调查法、临床观察法、病理解剖观察法、病原分离培养和RT-PCR/PCR扩增法进行诊断,并对分离株进行药物敏感性试验。牛副流感病毒3型和牛支原体为阳性,牛冠状病毒、牛呼吸道合胞体病毒等为阴性。该起病例由牛副流感病毒3型和牛支原体混合感染引起的,导致本场牛大批发病死亡的原因是由外购牛传染引起。

2020—2022年华中地区犬副流感病毒检测及其F、HN基因系统发育分析

为探究华中地区犬副流感病毒(CPIV)的流行及遗传进化情况,利用RT-PCR方法对华中地区2020年9月—2022年1月收集的418份病料样品进行了CPIV检测,并利用生物信息学软件对分离的CPIV毒株的F、HN基因进行同源性、氨基酸位点分析及系统发育分析。结果显示:CPIV阳性率为15.8%,扩增获得9株CPIV的F、HN基因的序列,并成功分离培养9株CPIV毒株;同时,将分离株的F、HN基因与19株副流感病毒参考毒株进行序列比对分析,结果表明,F基因核苷酸同源性为95.1%~100%,氨基酸同源性为94.6%~99.8%,HN基因核苷酸同源性为95.9%~99.9%,氨基酸同源性为95.8%~99.6%;CPIV的F及HN基因与参考毒株相比均有部分氨基酸发生了突变;系统发育分析结果显示,本研究获得的8株分离株与美国株CPI-和CPI+处于同一分支上,亲缘关系较近,另一株分离株CS-324与中国株He N0718和韩国株D277位于同一分支上。本研究丰富了华中地区CPIV的流行病学调查资料,并为该病的综合防控提供了理论依据。

26例儿童重症人副流感病毒感染的临床特点及危险因素分析

目的:分析儿童重症人副流感病毒(HPIV)感染的临床特点和危险因素。方法:选择2019年9月—2021年8月深圳市儿童医院HPIV感染的住院患儿627例,回顾性研究其中重症HPIV感染临床特点及危险因素。结果:(1)重症HPIV感染有26例(4.15%),冬季是高发季节;(2)单因素分析示:重症HPIV感染与非重症HPIV感染相比,在气促、发绀、血小板计数、基础疾病4项指标的差异有统计学意义(P<0.05),但年龄差异无统计学意义(P>0.05);(3)多因素Logistic回归分析示:气促、发绀、基础疾病与重症HPIV感染正相关,血小板与重症HPIV感染负相关,是其保护因素。结论:重症HPIV感染在住院患儿中占有一定比例,冬季为高发季节。气促、发绀、基础疾病是重症HPIV感染的危险因素,血小板升高(≥100×10~9/L)是其保护因素。

综合护理对小儿急性上呼吸道感染副流感病毒感染的影响和家长满意度评价

研究综合护理对小儿急性上呼吸道感染副流感病毒感染的影响和家长满意度的影响。选取铜仁市妇幼保健院2023年1月—2023年5月收治的80例小儿急性上呼吸道感染副流感病毒感染患儿为研究对象,随机将其分为对照组和观察组,每组40例。对照组使用常规护理,观察组使用综合护理,对两组患儿临床症状消失时间、患儿家属护理满意度、病情复发率、患儿家属疾病知识掌握率、生活质量进行观察。研究发现,在小儿急性上呼吸道感染副流感病毒感染患儿中采用综合护理,可以快速改善其临床症状,减少病情复发,提升患儿生活质量,且患儿家属的护理满意度较高,对疾病知识的掌握程度较高,值得临床推广。

猴副流感病毒RT-PCR检测方法的建立及初步应用

为对易感动物以及相关生物材料和生物制品进行猴副流感病毒(SV5)的快速病原检测,根据SV5病毒SH基因序列,设计了1对引物,建立了检测SV5的RT-PCR方法,通过优化确定了RT-PCR体系的最佳工作条件。核酸序列分析显示,所扩增片段与GenBank数据库中报道的SV5病毒株WR-21005序列同源性达到97%,证明该方法特异。敏感性分析显示,该方法可检测的最小RNA浓度为2.1 ng/μL,能检出的最小病毒滴度为3.98CCID50/0.1 mL。初步将该方法运用于猴、地鼠、常用传代细胞和猴源生物制品的检测,在所检测的样品中均未检出猴副流感病毒核酸序列。结果表明,所建立的RT-PCR方法可用于猴副流感病毒(SV5)的诊断和分子流行病学调查。

2425例小儿呼吸道感染7种常见病毒检出情况分析

目的分析急性下呼吸道感染患儿7种常见病毒[副流感病毒Ⅰ(PIVⅠ)、副流感病毒Ⅱ(PIVⅡ)、副流感病毒Ⅲ(PIVⅢ)、呼吸道合胞病毒(RSV)、流感病毒A(IVA)、流感病毒B(IVB)、腺病毒(ADV)]的感染情况。方法收集2 425例急性呼吸道感染的住院患儿鼻咽深部分泌物标本,进行呼吸道病毒7项指标检测,从患儿年龄[婴幼儿(≤3岁)1 526例、学龄前儿童(4～7岁)650例、儿童(≥8岁)250例]、病毒分布情况、季节因素等方面进行临床流行病学特点分析。结果 2 425例患儿中有552例7种呼吸道病毒阳性,总阳性率为22.76%;其中单一病毒感染360例(65.22%)、混合病毒感染192例(34.78%);在各年龄组中婴幼儿组感染者377例(68.29%),其中单病毒感染242例(43.84%)、混合病毒感染135例(24.46%)。7种病毒中PIVⅢ感染最多,为196例(35.51%),其次为RSV[183例(33.15%)]。从季节分布来看,春、夏、秋、冬四季的检出率分别为17.79%、19.73%、22.37%、31.01%,以冬季阳性率为最高。结论 PIVⅢ感染是本地区小儿呼吸道病毒感染的主要病毒;≤3岁患儿呼吸道病毒感染率最高;以冬季为呼吸道病毒感染高发期。

小儿肺炎的病毒性病原学研究

目的:探讨小儿肺炎的病毒性病原学特点.方法:应用直接免疫荧光法,连续3年对13 642例住院肺炎患儿呼吸道分泌物中呼吸道合胞病毒(RSV),副流感病毒(PIV) 1、2、3型,流感病毒(IV)A型、B型和腺病毒(ADV)抗原进行检测.结果:病毒性肺炎占34.3%,其中RSV占25.8%,PIV4.7%,IV A 2.4%,IV B 0.2%,ADV 1.3%.RSV在≤1岁组检出率 33.1%,>1～3岁组19.7%,>3岁组5.1%(趋势χ2=763.4,P<0.001).ADV肺炎,>1～3岁组与>3岁组分别为2.3%及2.5%,均高于≤1岁组的0.7%(P<0.01).>1～3岁组IV A检出率3.4%,高于≤1岁组(χ2=18.2,P<0.01).>1～3岁组PIV 1肺炎占1.2%,较其它年龄组高(P<0.05).PIV 3,≤1岁组检出率 4.7%,>1～3岁组3.2%,>3岁组1.4%(趋势χ2=52.4,P<0.01).RSV感染率11月开始明显增高,持续到次年的3～4月份,但每年仍有差别.肺炎中RSV月感染率最高62.8%,IV A达15.7%.结论:小儿肺炎的病毒性病原,感染率高低依次为RSV、PIV、IV和ADV.RSV、PIV 3,年龄越大检出率越低.≤1岁婴儿ADV感染较少.幼儿较婴儿易致IV A感染.RSV流行见于冬春季,但存在变化.

催化材料对病毒的吸附和灭活作用及对哺乳动物细胞的毒性

提出了以吸附和催化原理灭活病毒的设想 ,旨在开发出对病毒有过滤、吸附及灭活作用的高效非特异性催化材料 ,应用于各种防护设施 ,有效控制非典型肺炎 (SARS)的传播 .采用与SARS病毒相似的副流感病毒作为模拟对象 ,进行了吸附及灭活该病毒的催化材料研究 ,并考察了催化材料对哺乳动物细胞的毒性 .结果表明 ,病毒气溶胶的阻留及吸附结果与基于DNA吸附的色谱分析结果相一致 ;部分材料可以强烈地吸附病毒 ( 10 0 % ) ,甚至在强烈振荡下并洗脱至第 3次 ,病毒也不能脱附 ;一些材料不仅可以吸附病毒 ,而且强烈振荡后的洗脱液虽然表现出一定的血凝效价 ,但接种鸡胚后 ,病毒并不增殖 ,说明材料具有明显的催化病毒灭活性能 ;对细胞毒性极低的材料可以用在与人体接触的防护材料和设施中 .筛选出的性能优异的催化材料 。