Influenza A Subtyping: Seasonal H1N1, H3N2, and the Appearance of Novel H1N1

Influenza virus subtyping has emerged as a critical tool in the diagnosis of influenza. Antiviral resistance is present in the majority of seasonal H1N1 influenza A infections, with association of viral strain type and antiviral resistance. Influenza A virus subtypes can be reliably distinguished by examining conserved sequences in the matrix protein gene. We describe our experience with an assay for influenza A subtyping based on matrix gene sequences. Viral RNA was prepared from nasopharyngeal swab samples, and real-time RT-PCR detection of influenza A and B was performed using a laboratory developed analyte-specific reagent-based assay that targets a conserved region of the influenza A matrix protein gene. FluA-positive samples were analyzed using a second RT-PCR assay targeting the matrix protein gene to distinguish seasonal influenza subtypes based on differential melting of fluorescence resonance energy transfer probes. The novel H1N1 influenza strain responsible for the 2009 pandemic showed a melting profile distinct from that of seasonal H1N1 or H3N2 and compatible with the predicted melting temperature based on the published novel H1N1 matrix gene sequence. Validation by comparison with the Centers for Disease Control and Prevention real-time RT-PCR for swine influenza A (novel H1N1) test showed this assay to be both rapid and reliable (>99% sensitive and specific) in the identification of the novel H1N1 influenza A virus strain.The 2009 novel influenza A/H1N1 viral pandemic has presented challenges for hospital laboratories and health care systems seeking to rapidly diagnose, treat, and limit the spread of this virus. As is the case for routine diagnosis of seasonal influenza infections, molecular amplification assays offer the potential for the sensitivity and speed needed to manage an influenza outbreak. However, standardized RT-PCR assays specific for this strain of influenza were not initially available, leaving many laboratories to diagnose this infection through indirect means.Our laboratory has used PCR for rapid detection of influenza A and B for several years, and more recently had implemented a rapid RT-PCR/melt-curve assay designed to differentiate seasonal influenza A subtypes H1N1 and H3N2.¹ This approach was initially developed for viral subtyping to guide clinicians on the appropriate antiviral therapy. Antiviral resistance has risen during recent years, with the majority of seasonal H1N1 strains no longer being sensitive to oseltamivir (Tamiflu), and seasonal H3N2 strains being largely resistant to adamantanes.² Rapid determination of influenza A subtype is essential for determining optimal therapy and for prudent use of antiviral agents. Consequently, this RT-PCR assay has become part of our influenza testing algorithm.The design of the RT-PCR assay exploits minor variations in a relatively conserved sequence within the matrix protein gene. Not surprisingly, the novel H1N1 strain of influenza that appeared in the spring of 2009 had a distinct melting temperature consistent with the published matrix gene sequence and the sequence of the fluorescence resonance energy transfer probes used in this assay designed to differentiate seasonal influenza A subtypes H1N1 and H3N2. As part of our influenza testing algorithm, this assay allowed definitive diagnosis of the 2009 influenza H1N1 from nasopharyngeal swabs within hours after arrival in the clinical laboratory.     

H5N1 influenza virus is changing

Bacterial vaginosis is a synergistic polymicrobial syndrome characterized by depletion of Lactobacillus spp., especially those that produce hydrogen peroxide, and an intense increase in the quantity of commensal vaginal anaerobic bacteria to 100- to 1000-fold above normal levels. While the bacterial spectrum of these organisms has long been known to include Gardnerella vaginalis, Prevotella spp., anaerobic Gram-positive cocci, Mobiluncus spp. and Mycoplasma hominis, innovative use of molecular diagnostics has identified novel species apparently associated with this syndrome, including Atopobium vaginalis. Effecting resolution of bacterial vaginosis is important, in particular for the 8 to 23% of women afflicted with symptomatic disease during their reproductive years. Bacterial vaginosis has been consistently associated with numerous adverse sequelae related to the upper genital tract, including pelvic inflammatory disease and postsurgical infection in the setting of invasive gynecologic procedures, and may increase women’s risk of acquiring HIV infection. Pregnant women with bacterial vaginosis experience a higher rate of preterm delivery and low-birth-weight infants. While antibiotics with activity against anaerobes – typically, metronidazole and clindamycin applied vaginally or taken orally – are the mainstays of therapy, bacterial vaginosis frequently recurs. For these reasons, innovative approaches to therapy are urgently required.     

H5N1 influenza virus and the safety of plasma products

BACKGROUND: The ever-increasing number of human H5N1 influenza virus infections may enable these viruses to acquire the ability to spread effectively among humans and potentially to cause a pandemic. Recently, more systemic virus dissemination was reported during H5N1 virus infection of humans, resulting in significant virus concentrations also in the blood. The observation has raised concerns about the safety of labile blood products for transfusion and consequentially also for plasma derivatives. To confirm the safety margins of plasma products, dedicated virus inactivation processes used during their production were investigated for their effectiveness in inactivating this virus of recent concern. STUDY DESIGN AND METHODS: Virus inactivation by steps commonly used during the manufacture of plasma derivatives, such as pasteurization for human albumin, solvent/detergent treatment for intravenous immunoglobulin (IVIG), vapor heating for factor VIII inhibitor bypassing activity, and incubation at low pH for IVIG, were investigated with a reassortant strain of H5N1 influenza virus. RESULTS: The results show that H5N1 influenza behaves as expected for lipid-enveloped viruses; that is, the virus is effectively inactivated by all the commonly used virus inactivation procedures tested. CONCLUSION: The safety margins of plasma derivatives against the theoretical transmission of H5N1 influenza virus are very substantial.     

Comparison of the efficacies of amantadine treatment of swine-origin influenza virus A H1N1 and seasonal influenza H1N1 and H3N2 in Japan (2008–2009)

Amantadine is not thought to be effective for the treatment of swine-origin influenza virus (S-OIV) based on an analysis of genetic sequences of the M2 protein. However, the actual clinical efficacy of amantadine has not been well documented. Here, we were able to compare the efficacies of amantadine and neuraminidase inhibitors. Subjects consisted of 428 patients, including 144 with seasonal influenza (flu) identified between 2008 and 2009, and 284 with S-OIV identified between July 1 and November 30, 2009. Diagnosis of flu was established using a rapid diagnostic kit obtained commercially in Japan. Body temperature sheets were obtained from 95% of the S-OIV patients. Times required to recover normal body temperature were compared among subjects using different antiviral drugs. Genetic abnormalities in the M2 protein were also investigated in 66 randomly selected subjects from within the patient pool. Overall, the average hours required to recover normal body temperature in S-OIV patients treated with amantadine (160 cases), with oseltamivir (59 cases), or with zanamivir (65 cases) were 33.9 ± 20.7, 31.7 ± 16.0, or 36.3 ± 21.6, respectively. These differences were not statistically significant. The N31S abnormality was found in all 14 samples taken from the H3N2 patients and in all of the 23 samples taken from in S-OIV patients. However, this abnormality was not found in any of the 30 samples taken from seasonal H1N1 patients. Amantadine was found to be equally effective in treating S-OIV patients as neuraminidase inhibitors. The genetic abnormality resulting in S31N amino acid conversion identified in some of the H3N2 and S-OIV patients is thought to alter the function of M2 protein only mildly.     

甲型H1N1流感病毒核酸检测的应用及临床相关性的探讨

实验室诊断季节性流感和世界大流行流感的方法有多种,每种方法都有其优缺点[1].病毒培养被视为"金标准"方法,它敏感度特异度较好,但需要特殊的实验条件,费时耗力,不适用常规检测.血清学方法敏感性高,但特异抗体需在感染后1～3周才可产生,因此抗体检测更适合于回顾性诊断或人群感染状况的流行病调查,不适于早期诊断.快速抗原检测方法虽然在15～30 min可完成,特异性强但敏感性低于核酸检测方法.     

2009年甲型H1N1流感研究近况

根据2009年甲型H1N1流感疫情及相关研究资料,回顾近代重大流感疫情的流行病学资料,介绍甲型H1N1流感病毒的结构及抗原特征、甲型H1N1流感流行病学、临床和放射学表现、并发症及目前推荐的诊断治疗策略.     

广州地区无偿献血人群中H1N1和H3N2流感病毒中和抗体的分布及水平

目的 探讨H1N1和H3N2流感病毒中和抗体在广州地区无偿献血人群中的分布及水平,为研究广州地区流感流行病学特征提供参考.方法 于2015年10～11月,采用年龄分层随机抽样法选择广州血液中心无偿献血的498例健康献血者为研究对象.按年龄将其分为18～20岁组(n=150)、21～30岁组(n=210)、31～60岁组(n=138).采用微量病毒中和实验法测定献血者血浆中H1N1和H3N2流感病毒中和抗体滴度,并分别对不同性别及年龄段H1N1和H3N2流感病毒中和抗体阳性率及滴度进行统计学分析.结果 498例健康献血者中,H1N1流感病毒中和抗体阳性率为3.6％ (18/480);H3N2流感病毒中和抗体阳性率为4.6％ (23/498),2种流感病毒中和抗体阳性率比较,差异无统计学意义(x2=0.636,P=0.425).156例男性献血者中,H1N1和H3N2流感病毒中和抗体阳性率分别为3.8％(6/156)和4.5％(7/156);342例女性献血者中H1N1和H3N2流感病毒中和抗体阳性的率分别为3.2％(11/342)和4.4％(15/342).不同性别献血者H1N1和H3N2流感病毒中和抗体阳性率比较,差异均无统计学意义(x2 =0.129、0.003,P＞0.05).18～20岁组、21～30岁组和31～60岁组献血者H1N1流感病毒中和抗体阳性率分别为4.0％、5.2％和0.7％;H3N2流感病毒中和抗体阳性率分别为6.0％、5.2％和2.2％.不同年龄组献血者H1N1和H3N2流感病毒中和抗体阳性率比较,差异均无统计学意义(x2=4.961、2.705,P＞0.05).18～20岁组、21～30岁组和31～60岁献血者H1N1流感病毒中和抗体几何平均滴度(GMT)分别为1∶1.38、1∶1.48和1∶1.05,平均为1∶1.32;H3N2流感病毒中和抗体GMT分别为1∶1.62、1∶1.51和1∶1.19,平均为1∶1.44.3组献血者H1N1和H3N2流感病毒中和抗体滴度比较,差异均无统计学意义(x2 =4.887、2.702,P＞0.05).结论 广州地区无偿献血人群中H1N1和H3N2流感病毒中和抗体水平较低,不同性别和年龄人群对流感病毒普遍易感.     

Que sera,sera: evolution of the swine H1N1 influenza A virus

About 2% of the world’s population is estimated to be chronically infected with hepatitis C virus (HCV). These chronic carriers are at risk of developing liver cirrhosis and its complications. Successful treatment of HCV infection is associated with improved quality of life and increased survival. Antiviral approaches were formerly based on interferon and therefore all patients with a contraindication to interferon were excluded from treatment (e.g., patients with decompensated disease, severe impairment of other organs). Very recently, interferon-free combinations have become available for genotypes 2 and 3. This review focuses on the most recently reported data on the various interferon-free combinations used (namely, sofosbuvir-based combinations, the ABT-450/ombitasvir/dasabuvir/ribavirin combination, the daclatasvir/asunaprevir combination, and the MK-5172/MK-8742 combination). All these combinations yielded amazing results in terms of efficacy (90–100%), tolerability and safety. If the problem of the high cost is overcome, interferon-free therapies will lead to what has long been a chimera, namely, an HCV-free world.     

危重症甲型H1N1流行性感冒病毒肺炎的临床特征——附17例报告

目的:探讨危重症甲型H1N1流行性感冒病毒(甲流)肺炎患者的主要临床特点,分析各临床检验指标与病情严重程度的关系。方法:收集入住呼吸重症监护病房(RICU)的17例危重症甲流肺炎患者的临床资料,归纳分析其临床特征。结果:17例患者以发热、咽痛、咳嗽、肌肉酸痛为主要症状。双肺多发实变影12例(71%)。入院后白细胞为(3.4～25.1)×109/L,中性粒细胞(2.72～21.84)×109/L,中性粒细胞比例58.6%～96.4%,淋巴细胞5.1%～21.6%。CRP 34.6～381.9 mg/L,乳酸脱氢酶(LDH)310～820 U/L,白蛋白<30 g/L10例(59%),ALT升高6例(35%),血清肌酐升高5例(29%),脑钠肽升高4例(24%)。氧合指数<300 mm Hg 13例(76%)。均予奥司他韦治疗,气管插管机械通气支持治疗3例(18%)。死亡2例(12%),均为男性,均为气管插管机械通气者。结论:LDH、CRP升高、炎性细胞增多提示危重症甲流肺炎患者肺部损害加重;机械通气支持治疗对于部分肺内广泛实变者效果不理想,且相关的并发症是部分患者病情加重或死亡的促发因素。     

甲型H1N1流感病毒与非甲型H1N1流感病毒患者外周血象的对比分析

目的:对航天中心医院就诊的甲型H1N1流感病毒感染者与非甲型H1N1流感病毒感染者、正常对照者的外周血象进行对比分析,以期为临床的诊断、治疗以及病情监测提供有利的工具.方法:采用RT-PCR的方法对患者是否患甲型H1N1流感进行确认.采用流式细胞技术的方法,利用全血细胞计数仪对甲型H1N1组、非甲型H1N1组患者以及正常对照组外周血象进行对比分析.利用免疫比浊的方法对3组患者外周血中C反应蛋白(CRP)浓度进行比较分析.结果:甲型H1N1组患者中单核细胞百分数阳性百分率占77.1%,非甲型H1N1组患者单核细胞百分数阳性百分率占7.8%.正常对照组单核细胞百分数阳性百分率占6.7%.甲型H1N1组白细胞总数、淋巴细胞百分比以及嗜酸细胞百分比与非甲型H1N1组相比明显降低,但甲型H1N1组中性粒细胞百分比与正常对照组相比明显升高,而单核细胞百分比在甲型H1N1组中显著升高.甲型H1N1组血小板总数、血小板压积与非甲型H1N1组相比降低,而血小板分布宽度相比非甲型H1N1组数值升高,但与正常对照组相比,血小板总数以及3项参数未有明显差异.甲型H1N1组中CRP浓度与非甲型H1N1组相比差异无显著性,但与正常对照组相比明显升高.结论:甲型H1N1感染患者外周血象与一般流感存在相似之处,但它有其独特特点,在诊断过程中不应该以一般流感的外周血象以及CRP浓度特点来排除甲型H1N1流感病毒的感染.     

H6亚型禽流感病毒概述

2013年5月,台湾报道了首例人感染H6N1禽流感病毒病例,该病例也是人感染H6亚型禽流感的直接证据。研究发现,该病毒由在台湾家禽中流行的不同基因谱系的H6N1病毒重配而成,HA蛋白受体结合位点的一些重要氨基酸发生了突变,这些突变可能会增加病毒与人源受体的结合能力并增强该病毒对哺乳动物的致病性。本研究从病原学、流行病学、受体结合蛋白结构等方面对H6亚型禽流感病毒的特征进行了总结和分析,对于H6亚型禽流感的预警和防控工作具有一定的参考意义。     

用液相芯片方法检测禽流感病毒H5N1亚型的研究

目的基于液相芯片（MASA）技术建立一种对H5N1亚型禽流感病毒进行快速检测的新方法。方法对GenBank上已有H5和N1核酸片段进行比对分析,设计简并引物和探针,将合成的探针与荧光编码微球进行偶联,建立检测方法。利用该方法检测H5N1亚型禽流感病毒和其他常见亚型（H1N1、H2N2、H3N2、H9N2）的标本。结果利用该方法能够特异性地检测出H5N1标本,检测信号具有较高荧光强度,对其他亚型标本的检测则为阴性结果。该方法对H5NI亚型RNA的最低检出量为10pg。结论利用液相芯片技术研制的H5N1亚型禽流感病毒检测方法能够用于禽流感病毒H5N1亚型的快速、灵敏、特异性检测及鉴定。     

免疫层析法用于甲型H1N1流感病毒抗原筛查的初步评价

目的 评估免疫层析法检测甲型H1N1流感病毒抗原的准确性,推广免疫层析法在甲流H1N1抗原检测中的临床应用.方法 收集2009年8至12月广东省中医院356例甲型H1N1流感疑似患者的鼻拭子标本,采用免疫层析法进行甲型H1N1流感病毒抗原的快速检测,同时将患者咽拭子送往广州市疾病预防控制中心(CDC)进行甲型H1N1流...     

Thrombotic thrombocytopenic purpura triggered by influenza A virus subtype H1N1 infection

We report a case of acquired thrombotic thrombocytopenic purpura (TTP) triggered by influenza A virus subtype H1N1 infection. In December 2010, a 27-year-old man was diagnosed with pneumonia from influenza A virus infection at a local clinic. Two days later, he was admitted to our hospital because of a worsening condition and unexplained thrombocytopenia. The influenza A virus subtype H1N1 real-time polymerase chain reaction test was positive. The patient had typical clinical signs of TTP, thus he was diagnosed with TTP. He received treatment with oseltamivir and high dose methylprednisolone. Plasma exchange therapy was started daily at a 1.5 dose volume of his whole blood. After the 17th plasma exchange therapy, the symptoms and abnormal laboratory results had recovered to normal. Finally, 47 days after admission, the patient had recovered completely and was discharged. This case suggests that the influenza A virus subtype H1N1 infection may have triggered acquired TTP.     

危重症甲型H1N1流感病毒肺炎的多层螺旋CT表现

目的：探讨危重症甲型H1N1流感病毒肺炎患者的多层螺旋CT的影像学特点。方法：回顾性分析21例确诊的危重症甲型H1N1流感患者的64层螺旋CT影像资料。结果：21例患者中,①所有病变均表现为双侧分布,且主要为弥漫性分布,多灶性病变以中下肺叶为主。其中累及右肺上叶9例,右肺中叶14例,右肺下叶19例,左肺上叶9例,左肺下叶19例。②病变形态表现为：支气管血管束增粗（21/21,100%）,小叶中心结节（12/21,57.1%）,小叶间隔增厚（9/21,42.9%）,网状结节（3/21,14.3%）,磨玻璃样密度影（14/21,66.7%）,斑片融合影（7/21,33.3%）,大片实变密度影（9/21,42.9%）,支气管气象（5/21,23.8%）。③胸膜腔积液（8/21,38.1%,其中5例单侧,3例双侧）;淋巴结肿大（9/21,42.9%,其中5例腋窝淋巴结肿大,3例纵隔淋巴结肿大,2例腋窝、纵隔均淋巴结肿大）;胸膜肥厚（12/21,57.1%）;心包积液（1/21,4.76%）。④9例复查危重症甲型H1N1流感患者中3例死亡者首诊MSCT见双肺中下叶斑片及实变密度影、磨玻璃密度影。复查见病变进展迅速,双肺实变明显,死于呼吸衰竭。6例好转者肺内病变明显吸收,仅遗留少许索条或小斑片状影。结论：危重症甲型H1N1流感病毒肺炎64层螺旋CT主要表现为双肺内弥漫分布的磨玻璃密度影及多灶性实变影,多同时累及肺实质及肺间质,可伴有胸膜腔积液、淋巴结肿大、胸膜肥厚等。病程进展以磨玻璃密度影及实变影范围扩大为主要标志。