Virological diagnosis of respiratory virus infection in patients attending an emergency department during the influenza season

To investigate the burden of influenza-like illness (ILI), patients attending an emergency department during the influenza season were tested for several common respiratory viruses, using PCR-based methods. Influenza A viruses were detected in 25 of 103 recruited patients (24%), rhinoviruses in 15%, and respiratory syncytial virus in only one. The data suggest that triage criteria based on ILI case definitions would not contain the spread of the influenza virus during pandemic alerts and could lead to unnecessary isolation of patients with other infections. Application of broader triage criteria followed by timely molecular diagnosis could be effective in preventing new respiratory agent transmission.     

Spontaneous pneumomediastinum complicating pneumonia in children infected with the 2009 pandemic influenza A (HINI) virus

We report two occurrences of spontaneous pneumomediastinum (SPM) complicating pneumonia in Japanese children infected with the novel influenza A (HINI) virus (IV). General practitioners especially should suspect possible SPM when examining and treating children with the novel influenza accompanied by status asthmaticus or wheezing. The presented patients illustrate the specific clinical and radiological signs associated with SPM complicating pneumonia in children infected with A(HINI)v.     

Rhinoviruses delayed the circulation of the pandemic influenza A (H1N1) 2009 virus in France

In contrast to the experience in other European countries, the onset of the A(H1N1)2009 influenza virus epidemic was unexpectedly slow in France during the first part of autumn 2009. Our objective was to test the hypothesis that intense circulation of rhinoviruses might have reduced the probability of infection by A(H1N1)2009 virus at the beginning of autumn 2009. Systematic analysis for the detection of A(H1N1)2009 (H1N1) and human rhinovirus (HRV) was performed by RT-PCR from week 36 to week 48 on respiratory samples sent to the diagnostic laboratory by the paediatric hospital (n = 2121). Retrospective analysis of the obtained data, using 2 × 2 contingency tables with Fisher's exact test, revealed evidence of an inverse relationship between HRV and H1N1 detection. Between weeks 36 and 48 of 2009, both HRV and H1N1 were detected but in different time frames. HRV dispersed widely during early September, peaking at the end of the month, whereas the H1N1 epidemic began during mid-October and was still active at the end of this survey. During the co-circulation period of these two respiratory viruses (weeks 43–46), HRV detection appeared to reduce the likelihood of H1N1 detection in the same sample (OR = 0.08–0.24 p <0.0001). These results support the hypothesis that HRV infections can reduce the probability of A(H1N1) infection. This viral interference between respiratory viruses could have affected the spread of the H1N1 viruses and delayed the influenza pandemic at the beginning of autumn in France.     

The viral etiology of an influenza-like illness during the 2009 pandemic

Many viruses are known to cause influenza-like illness (ILI); however, in nearly 50% of patients, the etiologic agent remains unknown. The distribution of viruses in patients with ILI was investigated during the 2009 A/H1N1 influenza pandemic (A/H1N1p). From June 2009 to January 2010, 660 patients with suspected influenza were questioned and examined, and nasal swabs were collected. All patient samples were tested for influenza virus, and 286 negative nasal swabs were tested further for 18 other respiratory viruses using real-time RT-PCR. Two waves of ILI were observed in the epidemic curve (weeks 35-42 and 42-49). At least eight viruses co-circulated during this period: human rhinovirus (HRV) (58), parainfluenza 1-4 viruses (PIV) (9), human Coronavirus (hCoV) OC43 (9), enterovirus (5), adenovirus (AdV) (4), and human metapneumovirus (hMPV) (2); however, 204 samples remained negative for all viruses tested. ILI symptoms, according to the Centers for Disease Control and Prevention criteria for ILI definition, were reported in 75% of cases. These patients had positive swabs for A/H1N1p, HRV, hCoV-OC43, PIV, AdV, and hMPV without significant difference with non-ILI patients. This study found that many respiratory viruses circulated during this period and that the A/H1N1p did not impact on the kinetics of other respiratory viruses. The proportion of non-documented cases remains high. ILI could not distinguish A/H1N1p infection from that due to other respiratory viruses. However, in multivariate anlaysis, cough, chills, hyperemia, and dyspnea were associated significantly with influenza virus versus other respiratory viruses.     

Emergent 2009 influenza A(H1N1) viruses containing HA D222N mutation associated with severe clinical outcomes in the Americas

Background

During the 2010-2011 influenza season, a small sub-group of 2009 influenza A(H1N1) viruses (hereafter referred to as 2009 A(H1N1)) emerged that was associated with more severe clinical outcomes in Ecuador and North America. Genetically, the haemagglutinin (HA) of this sub-clade was distinct from HAs found in viruses associated with severe outbreaks in 2010 from the United Kingdom and from other global specimens isolated earlier in the season.

Objective

We report the emergence of a novel 2009 A(H1N1) variant possessing a re-emergent HA D222N mutation obtained from patients with severe respiratory illnesses and phylogenetically characterise these D222N mutants with other severe disease-causing variants clustering within a common emerging sub-clade.

Case reports

In early 2011, three cases of 2009 A(H1N1) infection, two from Quito, Ecuador, and one from Washington, DC, USA, were complicated by severe pneumonia requiring mechanical ventilation, resulting in one fatality. These cases were selected due to the reported nature of the acute respiratory distress (ARD) that were captured in Department of Defence (DoD)-sponsored global influenza surveillance nets.

Results

Genetically, the 2009 A(H1N1) strains isolated from two of the three severe cases carried a prominent amino acid change at position 222 (D222N) within the primary HA receptor binding site. Furthermore, these cases represent an emerging sub-clade of viruses defined by amino acid changes within HA: N31D, S162N, A186T and V272I. Phylogenetically, these viruses share a high degree of homology with strains associated with recent fatal cases in Chihuahua, Mexico.

Discussion

Previously, enhanced virulence associated with the change, D222G, has been clinically linked to severe morbidity and mortality. Initial observations of the prevalence of a novel sub-clade of strains in the Americas suggest that viruses with a re-emergent D222N mutation may too correlate with severe clinical manifestations. These findings warrant heightened vigilance for emerging sub-clades of 2009 A(H1N1) and presumptive clinical implications.     

2009甲型H1N1流感流行期间北京急性呼吸道感染儿童常见呼吸道病毒的研究

目的了解2009至2010年甲型H1N1流感大流行期间北京地区急性呼吸道感染患儿中,除流感病毒外的其他几种常见呼吸道病毒感染的流行情况。方法设计并建立检测包括呼吸道合胞病毒（RSV）A/B亚型,副流感病毒（PIV）1、2、3型,腺病毒（ADV）和人博卡病毒（HBoV）在内的多重实时荧光PCR方法,并应用该方法对2009年6月至2010年2月甲型H1N1流感大流行期间,对首都儿科研究所附属儿童医院就诊的急性呼吸道感染患儿中甲型H1N1流感病毒检测阴性的咽拭子标本进行上述呼吸道病毒的核酸检测。结果新建立的多重RT-PCR方法最低可检测的目标基因含量为10～300拷贝数,未见与其他非目标病毒的交叉反应。本研究共检测标本849份,病毒检测总阳性率为39.0%（331份）,5岁以下占87.6%（290/331份）。各病毒的检测阳性率分别为RSV-A亚型1.4%（12份）,RSV-B亚型8.4%（71份）,PIV-1型8.2%（70份）,PIV-2型0.5%（4份）,PIV-3型3.9%（33份）,ADV13.9%（118份）,HBoV2.7%（23份）。RSV检出以B亚型为主（85.5%）,流行高峰在2009年11月至2010年2月。PIV检出以PIV-1型及PIV-3型为主,PIV-1型的流行高峰在2009年7月至2009年10月,PIV-2型及PIV-3型的流行高峰在2009年6～9月。ADV病毒在研究期间均有较高检出率（平均为13.9%）,HBoV的流行高峰在2009年9～12月。结论除流感病毒外,ADV、RSV-B及PIV可能也是2009甲型H1N1流感流行期间引起儿童急性呼吸道感染的重要病原。比较以往的资料可见各病原在2009甲型H1N1流感流行期间的流行季节性及检出率基本未受到新型流感病毒的影响。     

Mixed viral infections of the respiratory tract; an epidemiological study during consecutive winter seasons

The current study aimed to describe the molecular epidemiology of mixed respiratory viral infections during consecutive winter seasons in a tertiary care hospital. Patients with symptoms of respiratory tract infection were evaluated during the 2009‐2011 and 2013‐15 winter seasons. A clinical microarray technique was used for viral detection. Clinical and epidemiological data were correlated with mixed viral detection and the need for hospitalization. In 332 out of 604 (54.4%) evaluated patients (17.6% children) a respiratory virus was identified. Mixed viral infections were diagnosed in 68/332 (20.5%) patients with virus detection (66.2% mixed Influenza‐RSV infections). Mixed viral infections were more commonly detected in children (OR 3.7; 95%CI 1.9‐5.6, P < 0.01) and patients with comorbidities. In logistic regression analyses, mixed viral infections were associated with younger age (mean age 30.4 years vs. 41.8 years, P ≤ 0.001) and increased rates of fever (OR: 2.7; 95%CI 1.04‐7.2, P < 0.05) but no adverse outcomes or increased rates of hospitalization. High rates of mixed viral infections were noted during all winter seasons (especially Influenza and RSV) and were more common in younger patients. The clinical significance of mixed respiratory viral infection needs further elucidation.

     

Pathologic study of pandemic influenza A (H1N1) 2009 cases from India

The pandemic influenza A (H1N1) 2009 originated in Mexico and rapidly spread to the United States and many other countries. India reported the first pandemic influenza case in May 2009. Autopsy studies describing the pathology of pandemic influenza infection in humans have appeared in the literature and most of these were from Western countries. We present the clinicopathologic features in 46 fatal cases with confirmed pandemic influenza A (H1N1) 2009 virus infection during August 2009 to October 2010. Postmortem needle biopsy tissues were examined for histopathological changes and distribution of virus antigen by immunohistochemistry. The results are comparable with previous autopsy studies. Diffuse alveolar damage was the consistent finding in the lung tissues. However, underlying medical conditions were not noted in the cases from present study. Consistent presence of viral antigen was noted in the bronchiolar epithelium without any reference to the duration of illness. This study also emphasizes the use of the postmortem needle biopsy technique whenever an autopsy is not possible.     

Sero‐immunity and serologic response to pandemic influenza A (H1N1) 2009 virus in Hong Kong

To study the serologic response to the new pandemic influenza A (H1N1) 2009 virus in Hong Kong, the level of immunity was measured before and after the occurrence of the outbreak, and the titer of antibody to the pandemic influenza A (H1N1) 2009 virus in serum samples of laboratory confirmed cases. The presence of pre‐outbreak pandemic influenza A (H1N1) 2009 virus antibodies in 37% of individuals older than >65 years suggested previous exposures to heterologous virus strains may have elicited cross‐reacting antibody. Following large outbreaks of pandemic influenza A 2009 virus that peaked in September 2009, there is a change in immunity level in various age groups consistent with the attack rates among population in Hong Kong. Among individuals with mild clinical presentation, the antibody response to pandemic influenza A (H1N1) 2009 virus was stronger in those individuals aged ≤24 years but took more time to reach a titer of 40 when compared with those aged >24 years; however, the antibody level declined slower among individuals aged ≤24 years. Regardless of age, the antibody response rose rapidly and reached much higher titer among individuals with severe clinical presentation. Further study is required to collect additional data on antibody persistence and determine how much protection is conferred by previous exposure to seasonal influenza A (H1N1) viruses. J. Med. Virol. 82:1809–1815, 2010. © 2010 Wiley‐Liss, Inc.     

Differential diagnosis of pandemic (H1N1) 2009 infection by detection of haemagglutinin with an enzyme-linked immunoassay

A sensitive and convenient immunoassay that can directly differentiate pandemic (H1N1) 2009 (pH1N1) virus from seasonal influenza virus can play an important role in the clinic. In the presented study, a double-sandwich ELISA (pH1N1 ELISA), based on two monoclonal antibodies against haemagglutinin (HA) of the pH1N1 virus, was developed. After laboratory determination of the sensitivity and specificity characteristics, the performance of this assay was evaluated in a cohort of 904 patients with influenza-like illness. All seven strains of pH1N1 virus tested were positive by pH1N1 ELISA, with an average lower detection limit of 10^{3.0 ± 0.4} tissue culture infective dose (TCID)₅₀/mL (or 0.009 ± 0.005 HA titre). Cross-reaction of the assay with seasonal influenza virus and other common respiratory pathogens was rare. In pH1N1-infected patients, the sensitivity of the pH1N1 ELISA was 92.3% (84/91, 95% CI 84.8–96.9%), which is significantly higher than that of the BD Directigen EZ Flu A + B test (70.3%, p <0.01). The specificity of pH1N1 ELISA in seasonal influenza A patients was 100.0% (171/171, 95% CI 97.9–100.0%), similar to that in non-influenza A patients (640/642, 99.7%, 95% CI 98.9–100.0%). The positive predictive value for pH1N1 ELISA was 97.7% and the negative predictive value was 99.1% in this study population with a pH1N1 prevalence of 10.1%. In conclusion, detection of HA of pH1N1 virus by immunoassay appears to be a convenient and reliable method for the differential diagnosis of pH1N1 from other respiratory pathogens, including seasonal influenza virus.     

Evaluation of direct immunofluorescence test with PCR for detection of novel influenza A (H1N1) virus during 2009 pandemic

During the 2009 novel influenza (H1N1) pandemic, the sensitivity of direct immunofluorescence assay (DFA) for H1N1 infection was 62% (266/429) of that of the polymerase chain reaction (PCR) test. The sensitivity of the DFA differed significantly with the age of patients: the sensitivity was the highest (71.8%) for patients aged <10 years and the lowest for patients aged ≥30 years. The sensitivity of DFA in patients aged ≥30 years was 40.7%. Furthermore, the sensitivity (67.3%, 171/254) of DFA was higher for patients who had a high temperature at admission. An increase in the incidence of H1N1 infection did not influence the sensitivity of DFA (62.1% vs. 62%; p=0.984) test, but resulted in a decrease in the negative predictive value, from 92.4% (700/757) to 69.6% (247/355). PCR may be useful as the initial test for diagnosing H1N1 infection in patients aged ≥30 years with a normal temperature at presentation.     

Changes in epidemiology,clinical features and severity of influenza A (H1N1) 2009 pneumonia in the first post-pandemic influenza season

Although the influenza A (H1N1) 2009 virus is expected to circulate as a seasonal virus for some years after the pandemic period, its behaviour cannot be predicted. We analysed a prospective cohort study of hospitalized adults with influenza A (H1N1) 2009 pneumonia at 14 teaching hospitals in Spain to compare the epidemiology, clinical features and outcomes of influenza A (H1N1) 2009 pneumonia between the pandemic period and the first post-pandemic influenza season. A total of 348 patients were included: 234 during the pandemic period and 114 during the first post-pandemic influenza season. Patients during the post-pandemic period were older and more likely to have chronic obstructive pulmonary disease, chronic kidney disease and cancer than the others. Septic shock, altered mental status and respiratory failure on arrival at hospital were significantly more common during the post-pandemic period. Time from illness onset to receipt of antiviral therapy was also longer during this period. Early antiviral therapy was less frequently administered to patients during the post-pandemic period (22.9% versus 10.9%; p 0.009). In addition, length of stay was longer, and need for mechanical ventilation and intensive-care unit admission were significantly higher during the post-pandemic period. In-hospital mortality (5.1% versus 21.2%; p <0.001) was also greater during this period. In conclusion, significant epidemiological changes and an increased severity of influenza A (H1N1) 2009 pneumonia were found in the first post-pandemic influenza season. Physicians should consider influenza A (H1N1) 2009 when selecting microbiological testing and treatment in patients with pneumonia in the upcoming influenza season.     

Analysis of the human serological immune response to a variable region of the attachment (G) protein of respiratory syncytial virus during primary infection

The serum antibody responses of babies to the variable carboxy-terminal region of the attachment (G) protein of respiratory syncytial virus (RSV) have been analysed using paired acute and convalescent sera from infants experiencing their first RSV infection with viruses of known genotype. The variable 84–85 carboxy-terminal amino acids of the G protein of six recent isolates of group A RSV were expressed in Escherichia coli as fusion proteins with glutathione S-transferase. About half the infants developed antibodies which recognised these fusion proteins. The patterns of response obtained in enzyme linked immunosorbant assays and immunoblotting assays were closely related to the infecting genotype. © 1996 Wiley-Liss, Inc.     

2009甲型H1N1流感大流行期间北京儿童的流感监测

目的 了解2009年甲型H1N1流感大流行期间北京地区儿童中流感流行的情况.方法 采用WHO推荐的实时荧光定量RT-PCR和国家流感中心推荐的分型方法,对2009年甲型H1N1流感大流行期间因流感样症状来首都儿科研究所附属儿童医院就诊患儿的咽拭子标本进行流感病毒核酸检测.结果 2009年6月1日至2010年2月28日期间共检测了4363份咽拭子标本,其中623例为甲型H1N1阳性,阳性率为14.3%,657例为其他甲型流感病毒阳性(15.1%),所有甲型流感病毒的总阳性率为29.3%.623例中有23例为危重症病例(占阳性患者的3.7%),其中5例死亡.618例信息完整的甲型H1N1病例中,患儿年龄为14天～16岁,性别比例为男比女为1.3:1.1～3岁儿童占25.2%,3～6岁学龄前儿童和6～12岁学龄儿童所占比例相近,各约占30%.在监测期间,仅呈现了一个甲型H1N1的流行波.2009年11月达到最高峰,随后减弱,2010年2月快速下降至2.7%.对监测期间每周20～30份临床标本同时进行季节性流感的监测显示,季节性H3N2、甲型H1N1和乙型流感交替流行.呼吸道合胞病毒(RSV)在甲型H1N1流行趋势减缓后逐渐流行成为流行优势株.结论 2009年6月至2010年2月北京地区儿童中出现甲型H1N1的流行,主要累及学龄前和学龄儿童.季节性流感和RSV与甲型H1N1交替流行.     

Comparison of multiplex PCR assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness

The performances of four multiplex PCR (m-PCR) were compared to direct immunofluorescence assay (DFA) and HuH7 cell culture for the detection of viruses in 263 children admitted to hospital with an acute respiratory illness. One hundred fifty (57.6%) nasal aspirates were found DFA-positive; 188 (72.3%) were found positive by both DFA and HuH7 cell culture, and 242 (92%) were PCR-positive. The m-PCR detected 124 viruses which were not found by conventional methods: 68 rhinovirus, 17 human metapneumovirus, 15 respiratory syncytial virus (RSV), 8 parainfluenza virus (PIV), 5 coronavirus 229E, 3 OC43 and 3 NL63, 4 enterovirus, 2 influenza virus B and C virus. The m-PCR were more sensitive, had the advantages of a shorter delay in specific diagnosis, and a lower cost than DFA and culture. Using these m-PCR, the prevalence of each virus was compared between in-patient and out-patient groups of children attending the emergency unit of the hospital. Nasal aspirates from 411 (91.5%) children were found positive by the PCRs. RSV, rhinovirus, and influenza virus were the most frequent viruses detected in this population, representing 43.6%, 31.8%, and 8.8% of the virus found, respectively, followed by human metapneumovirus (4.4%), coronavirus (3.4%), parainfluenza virus (3.2%), adenovirus (2.3%), and enterovirus (2.1%). RSVs were detected more significantly in the in-patient group than in the out-patient group, and influenza viruses were detected more frequently in the out-patient group than in the in-patient group. Moreover, the use of m-PCR pointed out the frequency of rhinovirus and mixed viral detections in these patients. In conclusion, according to the requirements of speed and low cost of the methods, and to achieve the highest rate of detection of respiratory viruses, the combined use of DFA and m-PCR is today likely to be the best way to improve diagnosis of respiratory illnesses in children.