Performance of laboratory diagnostics for the detection of influenza A(H1N1)v virus as correlated with the time after symptom onset and viral load

BackgroundTo diagnose influenza A(H1N1)v virus infection, accurate and rapid detection are important. However, there is scanty data on the performance of various laboratory diagnostics.ObjectiveTo compare the performance of rapid antigen test (RAT), viral culture and RT-PCR for the detection of influenza A(H1N1)v virus and to correlate their performance with the time after symptom onset and viral load.Study DesignFrom May 1, 2009 to June 25, 2009, respiratory samples were collected from 5740 individuals suspected of having influenza A(H1N1)v infection. The performance of viral culture and RT-PCR were investigated and correlated with the time after symptom onset. The sensitivity of RAT ESPLINE influenza A & B-N (Fujirebio Inc, Tokyo) was evaluated using a subset of 60 samples from patients diagnosed as having influenza A(H1N1)v infection.ResultsUsing respiratory samples from 587 patients diagnosed with influenza A(H1N1)v infection, comparison of laboratory diagnostics showed viral culture and RT-PCR gave comparable results with overall sensitivity of 93.9% and 98.1%, respectively. For RAT, when testing a subset of 60 samples collected ≤3 days following symptom onset, the sensitivity was 62%.ConclusionsAlthough viral shedding is prolonged and of higher titre in influenza A(H1N1)v infection, RAT showed a low sensitivity of 62% among patients presenting ≤3 days after symptom onset. Viral culture showed comparable performance with RT-PCR and with sensitivity better than that documented for seasonal influenza.     

Characterization of 2009 pandemic influenza A (H1N1) virus specimens with a positive hemagglutinin 1 signal in the Luminex xTAG respiratory viral panel assay

This retrospective review analyzed Luminex xTAG respiratory viral panel (RVP) results for 2009 pandemic influenza A (H1N1) virus specimens. Comparing median fluorescence intensity (MFI) signals for the influenza A virus and hemagglutinin 1 (H1) reactions for specimens with very low positive (MFI < 1,000) or "no-call" H1 results reliably distinguished 2009 H1N1 from seasonal virus.     

儿童甲型H1N1流感和季节性流感患者的病毒载量及相关临床症状研究

目的 分析并比较儿童甲型H1N1流感和季节性流感患者咽拭子标本的病毒载量及相关临床症状.方法 应用荧光PCR方法对采集的咽拭子标本进行检测,并通过建立核酸标准品,绘制标准曲线,测定标本中的病毒载量,同时结合所收集的患者临床症状数据资料应用随机区组方差和卡方检验方法进行统计分析.结果 2009年9月至2010年9月期间收集的1,040份咽拭子标本中,共检出甲型H1N1流感病毒阳性标本120份,甲型H3N2流感病毒阳性标本61份,乙型流感病毒阳性标本99份;对收集的流感阳性标本病毒载量测定结果显示:不同型别,不同发病时间流感患者咽拭子标本的病毒载量差异无统计学意义(P＞0.05);甲型H1N1流感、季节性流感感染者的性别比例差异无统计学意义,甲型H1N1流感感染者出现咳嗽,流涕临床症状者明显高于与乙型流感感染者.结论 甲型H1N1流感患者咳嗽,流涕症状比季节性乙型流感患者多见,而甲型H1N1流感和季节性流感患者咽拭子标本的病毒载量无显著性差异.     

Performance of rapid-test kits for the detection of the pandemic influenza A/H1N1 virus

The early detection of pandemic influenza strains is a key factor for clinicians in treatment decisions and infection control practices. The aims of this study were to determine the analytical sensitivity and clinical performance of the commercially available influenza rapid tests in Taiwan. Four rapid tests for influenza virus (BinaxNow test, QuickVue test, TRU test, and Formosa Rapid test) were evaluated for their detection limit against four influenza viruses (the 2009 pandemic influenza A virus H1N1, seasonal influenza virus H1N1, H3N2, and influenza B virus) circulating in Taiwan. The viral load of these isolates were quantified by rtRT-PCR and then diluted 2-fold serially for the comparison. The lowest detectable viral load of the pandemic influenza A virus H1N1 by the Formosa Rapid test, QuickVue test, TRU test, and Binax Now test was 5.3 × 10⁴, 1.0 × 10⁵, 1.0 × 10⁵, and 4.2 × 10⁵ copies/μL, respectively. Of these four tests, the two most sensitive tests (the QuickVue test and the Formosa Rapid test) were chosen to evaluate 62 nasopharyngeal specimens from patients who were suspected of infection with pandemic influenza A virus H1N1. The positive rate for the Formosa Rapid test and the QuickVue test were 53.2% (33/62) and 45.2% (28/62) (McNemar's test, P = 0.125), respectively. In conclusion, the Formosa Rapid test was the most sensitive test in the present study for the detection of influenza antigens and its clinical performance was similar to that of the QuickVue test (Kappa = 0.776). This suggests that the Formosa Rapid test could be used to aid clinical decision making in primary health care settings during outbreaks of influenza.     

Impaired dendritic cell maturation in response to pandemic H1N109 influenza virus

BackgroundInfection with pandemic A/H1N1/2009 influenza virus led to hospitalisation of patients not expected to be at risk of severe disease from seasonal influenza infection.ObjectivesWe sought to establish whether (i) DC maturation was compromised in patients experiencing severe pandemic influenza infection, (ii) the pandemic virus differed from seasonal influenza virus in its ability to induce DC maturation and (iii) there was an associated inability to activate memory B cells or induce antibody.Study designPeripheral blood mononuclear (PBMCs) cells were sampled from individuals with confirmed acute pandemic A/H1N1/2009 influenza infection or from healthy vaccinated controls. DCs were differentiated from the PBMC and tested for their ability to mature following stimulation with pandemic virus, seasonal H3N2 influenza virus or LPS. Serum samples from the patients were used to assess seroconversion to influenza and the levels of influenza specific memory B cells in PBMC were also determined.ResultsDCs obtained from all individuals exhibited negligible maturation marker upregulation when exposed to pandemic A/H1N1/2009 virus but showed a strong response to the seasonal H3N2 virus and LPS. Robust levels of memory B cell were obtained in both groups and patients seroconverted to the virus.ConclusionsOverall, the ability of patient's DC to mature in response to different stimuli was no different to that of control subjects DCs. Importantly, panH1N109 virus failed to induce substantial DC maturation in any individual, contrasting with seasonal virus, but this did not result in failure to mount memory B cell and antibody responses to the virus.     

Pandemic H1N1 influenza virus causes a stronger inflammatory response than seasonal H1N1 influenza virus in ferrets

A 2009 H1N1 influenza virus pandemic, which had its origin in swine, caused severe illness and mortality in humans. Inflammatory responses may be responsible for pathogenesis caused by infection with influenza viruses. To better understand the pathogenic mechanism, clinical signs and inflammatory responses in ferrets infected with the pandemic H1N1 were compared with those caused by seasonal H1N1 influenza virus. Ferrets infected with the 2009 pandemic H1N1 virus displayed higher body temperatures, greater reduction in body weight, and higher viral titers in the tracheae and lungs. Levels of inflammatory cytokines, including interleukin-6, interferon-alpha, and tumor necrosis factor-alpha, were higher in the lungs of ferrets infected with the 2009 pandemic H1N1. The data support the idea that increased pathogenesis caused by the 2009 pandemic H1N1 influenza virus may have been partially mediated by a higher induction of pro-inflammatory cytokines in the lungs of affected humans or animals.     

Tropism and Innate Host Responses of the 2009 Pandemic H1N1 Influenza Virus in ex Vivo and in Vitro Cultures of Human Conjunctiva and Respiratory Tract

The novel pandemic influenza H1N1 (H1N1pdm) virus of swine origin causes mild disease but occasionally leads to acute respiratory distress syndrome and death. It is important to understand the pathogenesis of this new disease in humans. We compared the virus tropism and host-responses elicited by pandemic H1N1pdm and seasonal H1N1 influenza viruses in ex vivo cultures of human conjunctiva, nasopharynx, bronchus, and lung, as well as in vitro cultures of human nasopharyngeal, bronchial, and alveolar epithelial cells. We found comparable replication and host-responses in seasonal and pandemic H1N1 viruses. However, pandemic H1N1pdm virus differs from seasonal H1N1 influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in its receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection with H1N1pdm. A greater viral replication competence in bronchial epithelium at 33°C may also contribute to the slight increase in virulence of the pandemic influenza virus. In contrast with highly pathogenic influenza H5N1 virus, pandemic H1N1pdm does not differ from seasonal influenza virus in its intrinsic capacity for cytokine dysregulation. Collectively, these results suggest that pandemic H1N1pdm virus differs in modest but subtle ways from seasonal H1N1 virus in its intrinsic virulence for humans, which is in accord with the epidemiology of the pandemic to date. These findings are therefore relevant for understanding transmission and therapy.The recent pandemic caused by a novel H1N1 virus (H1N1pdm) arose from the reassortment of three or more viruses of swine origin, including the North American triple reassortant H3N2 and H1N2 viruses, classical swine H1N1, and European swine H1N1/H3N2 viruses.^1,2 Most patients with pandemic H1N1pdm have mild influenza-like illness, but a minority of patients develop a primary viral pneumonia, sometimes leading to acute respiratory distress syndrome and death.^3,4 Many, but not all, patients with severe disease have pregnancy, obesity, or underlying disease states such as asthma, obstructive airways disease, diabetes, and chronic cardiovascular or renal disease. The disease associated with H1N1pdm so far appears to be comparable with that of seasonal influenza and less severe than that seen in the 1918 pandemic or in zoonotic disease caused by highly pathogenic avian influenza (HPAI) H5N1. However, unlike seasonal influenza where morbidity and mortality are mainly seen in the elderly, pandemic H1N1pdm appears to spare this age-group, possibly because of the presence of cross-neutralizing antibody generated by prior repeated seasonal H1N1 infection.⁵ In California, the median age of all cases was 17 years, of hospitalized cases 26 years, and for fatal cases was 45 years.It is therefore important to understand how the pathogenesis and tissue tropism of H1N1pdm virus in humans differs from seasonal influenza viruses. However, there is so far limited information in this regard. The H1N1pdm virus does not possess the genetic motifs of virulence associated with either the HPAI H5N1 or 1918 H1N1 viruses.² In experimentally infected ferrets, macaques, and mice, H1N1pdm causes moderately more severe illness compared with seasonal influenza although being much less virulent than HPAI H5N1 or the 1918 pandemic Spanish flu virus.^6,7,8 In these animal models, H1N1pdm virus was able to infect the alveolar epithelium more readily than seasonal H1N1 virus, but whether this holds true for humans is not known.⁷ Though H1N1pdm was initially reported to have a predominantly α2-6 sialic acid (Sia) receptor binding preference⁸ similar to human seasonal influenza viruses, recent glycan array data indicates that there is binding to both “human” Sia α2-6 and “avian” Sia α2-3.⁹ H1N1pdm virus differs from seasonal influenza viruses in their ability to infect and cause illness in mice without prior adaptation. As the mouse respiratory tract has a predominance of Sia α2-3, rather than Sia α2-6 receptors, these findings support the contention that H1N1pdm viruses have a broader Sia receptor binding profile.⁸ Taken together, these observations suggest that H1N1pdm virus differs in subtle but important ways from seasonal influenza viruses in receptor usage and tissue tropism, and this may be important in its pathogenesis and transmission.Cytokine dysregulation is believed to contribute to the pathogenesis of human disease caused by HPAI H5N1 as well as the 1918 pandemic H1N1 viruses.^{10,11,12,13,14} It is not known whether the H1N1pdm virus differs from seasonal influenza in the induction of proinflammatory host responses in human tissues. The lungs of H1N1pdm-infected mice had a markedly different cytokine profile when compared with seasonal influenza infected animals with elevated levels of interleukin (IL)-4, IL-10, and interferon (IFN)-γ. The lungs of H1N1pdm-infected macaques also had higher levels of chemokines MCP-1, MIP-1α, IL-6, and IL-18.⁶ However, it is not known whether these host responses simply reflect the greater or more extensive replication of the H1N1pdm virus in the lung when compared with seasonal influenza viruses or are attributable to intrinsic differences in the virus itself being able to induce a more potent innate host response as occurs with the highly pathogenic avian influenza H5N1 virus. When primary human cells (macrophages and type I-like pneumocytes) are infected with seasonal and HPAI H5N1 influenza viruses of comparable infectious titers, the HPAI H5N1 viruses differentially hyperinduce a range of proinflammatory responses over a single virus replication cycle.^10,11,14 Thus it is clear that the H5N1 virus has inherent properties that lead to an exaggerated innate immune response. It is relevant to use a similar approach to investigate the host innate immune responses induced by pandemic H1N1pdm compared with that of seasonal influenza H1N1 virus in primary human respiratory epithelium.We have previously used ex vivo cultures of nasopharynx, tonsillar tissue, and lung for investigating virus tropism.¹⁵ We have also established in vitro cultures of polarized primary human respiratory epithelial cells, including type I–like alveolar epithelial cells, nasopharyngeal epithelial cells, and differentiated bronchial epithelial cells for investigating tissue tropism and innate immune host responses elicited by influenza viruses.^10,14,15 These in vitro cultures of bronchial epithelium differentiated at an air–liquid interface (ALI) provide a good representation of the human bronchial epithelium and have a ciliated epithelium as well as mucus producing goblet cells. We have also recently established ex vivo tissue culture models of human conjunctival epithelium. We now use these ex vivo human tissue cultures as well as the primary human respiratory epithelial cell cultures to compare the virus replication competence, cell tropism, and host innate immune responses of the pandemic H1N1pdm virus with that of seasonal influenza H1N1 viruses and, where relevant, avian HPAI H5N1 and H7N7 viruses.We demonstrate that whereas seasonal H1N1 and pandemic H1N1pdm viruses replicate comparably in ex vivo cultures of human nasopharynx and lung tissues, the human conjunctiva is preferentially infected by H1N1pdm rather than seasonal influenza H1N1 or H3N2 viruses. Pandemic H1N1pdm replicates more efficiently than seasonal H1N1 virus in differentiated bronchial epithelial cells in vitro at 33°C, but the two viruses replicate comparably at 37°C. We also demonstrate that the pandemic H1N1pdm virus does not differ from the human seasonal influenza viruses in their ability to induce proinflammatory cytokines and therefore does not appear to have the same potential to induce cytokine dysregulation as that manifested by HPAI H5N1 or the 1918 H1N1 virus.     

Viral load and epidemiological profile of patients infected by pandemic influenza a (H1N1) 2009 and seasonal influenza a virus in Southern Brazil

Correlation between virologic profile and clinical features of patients infected by influenza virus provides important information for epidemiological control and clinical management of future disease outbreaks. Samples from patients in Southern Brazil, from June to December 2009, were examined and the viral load was correlated with epidemiological data. All samples were analyzed by qRT-PCR for detection of the 2009-pandemic Influenza A (H1N1). Relative viral loads were assessed based on the 2(-ΔCT) method and epidemiological data were obtained for each patient, following ethical policies. A total of 933 samples were positive for pH1N1 (2009) influenza; 172 were positive for seasonal influenza A; 13 were undetermined; 1992 samples were negative for influenza A. Combined molecular and epidemiological data were available for 38 seasonal and 198 pandemic samples. The median viral load was higher in pandemic than in seasonal influenza samples; in patients infected with pH1N1 (2009), viral load associated positively with chills, myalgia and rhinorrhea, and negatively with dyspnea, but no association was observed with other symptoms, nor with clinical conditions such as pregnancy, smoking, immunodepression and co-morbidities. Regarding patients infected with seasonal influenza, viral loads did not show statistically significant association with any of the symptoms. This is the first study in Brazil that examines epidemiological and molecular data from the 2009 influenza pandemic. The results may serve as a basis for developing strategies to control human-to-human infection and viral dissemination, and for implementing effective measures and public health policies against future novel disease outbreaks.     

Histopathological Evaluation of the Diversity of Cells Susceptible to H5N1 Virulent Avian Influenza Virus

Patients infected with highly pathogenic avian influenza A H5N1 viruses (H5N1 HPAIV) show diffuse alveolar damage. However, the temporal progression of tissue damage and repair after viral infection remains poorly defined. Therefore, we assessed the sequential histopathological characteristics of mouse lung after intranasal infection with H5N1 HPAIV or H1N1 2009 pandemic influenza virus (H1N1 pdm). We determined the amount and localization of virus in the lung through IHC staining and in situ hybridization. IHC used antibodies raised against the virus protein and antibodies specific for macrophages, type II pneumocytes, or proliferating cell nuclear antigen. In situ hybridization used RNA probes against both viral RNA and mRNA encoding the nucleoprotein and the hemagglutinin protein. H5N1 HPAIV infection and replication were observed in multiple lung cell types and might result in rapid progression of lung injury. Both type II pneumocytes and macrophages proliferated after H5N1 HPAIV infection. However, the abundant macrophages failed to block the viral attack, and proliferation of type II pneumocytes failed to restore the damaged alveoli. In contrast, mice infected with H1N1 pdm exhibited modest proliferation of type II pneumocytes and macrophages and slight alveolar damage. These results suggest that the virulence of H5N1 HPAIV results from the wide range of cell tropism of the virus, excessive virus replication, and rapid development of diffuse alveolar damage.Seasonal, pandemic, and zoonotic influenza A virus infections show substantial morbidity and mortality in humans. Seasonal influenza A virus infections in humans are usually mild and cause pneumonia only in a few infected individuals. Pandemic influenza virus infections vary in their disease outcome. Zoonotic influenza virus infections in humans vary from self-limiting conjunctivitis to severe, often fatal, pneumonia. Highly pathogenic avian influenza H5N1 virus (H5N1 HPAIV), implicated in poultry outbreaks,^1,2 can be transmitted zoonotically to humans, as has been observed in areas of Asia and Africa.^3–5 Fatal outcomes have been reported at approximately 60% in the sporadic transmission of this avian influenza H5N1 virus to humans.^5–7 There is no evidence that the avian influenza virus has become efficiently transmissible among humans, a change that could result in a new pandemic.⁸The outcome after infection with influenza virus can range from slight to severe illness, depending on the kinds of cells that are affected during lung tissue infection.^9–11 Events occurring early in infection determine the extent of damage, which can range from bronchitis to pneumonia. In the most severe cases, diffuse alveolar damage (DAD) may be induced during the early stages, and healing and/or scarring may ensue, depending on the persistence of disease. Occasionally, bacterial infection also may occur, with associated effects expressed mainly in the later stages of the disease. Pathological damage caused by influenza viruses in humans and in animal models depends on the virulence of the infective agent and on the host response. All influenza viruses infect the respiratory tract epithelium from the nasal passages to the bronchioles; however, highly virulent viruses (eg, H1N1 1918 and H5N1 HPAIV) tend to infect pneumocytes and resident macrophages in the alveoli. In susceptible individuals, inflammation of the alveolar walls results in DAD. In contrast, low-virulence viruses (seasonal H1N1) primarily cause inflammation, congestion, and epithelial necrosis of the trachea, bronchi, and bronchioles. Tissue tropism is an important factor, and depends largely on the ability of the virus to attach to the host cell.^12–14 We investigated virus replication and histopathological progression of lung tissue in mice infected with H5N1 HPAIV, particularly focusing on the lower respiratory tract and alveoli, with direct comparison to the histopathological characteristics of mice infected with H1N1 pandemic (pdm) influenza virus 2009 virus.     

Simultaneous and complete genome sequencing of influenza A and B with high coverage by Illumina MiSeq Platform

Active global surveillance and characterization of influenza viruses are essential for better preparation against possible pandemic events. Obtaining comprehensive information about the influenza genome can improve our understanding of the evolution of influenza viruses and emergence of new strains, and improve the accuracy when designing preventive vaccines. This study investigated the use of deep sequencing by the next-generation sequencing (NGS) Illumina MiSeq Platform to obtain complete genome sequence information from influenza virus isolates. The influenza virus isolates were cultured from 6 respiratory acute clinical specimens collected in Thailand and Nepal. DNA libraries obtained from each viral isolate were mixed and all were sequenced simultaneously. Total information of 2.6 Gbases was obtained from a 455 ± 14 K/mm² density with 95.76% (8,571,655/8,950,724 clusters) of the clusters passing quality control (QC) filters. Approximately 93.7% of all sequences from Read1 and 83.5% from Read2 contained high quality sequences that were ≥Q30, a base calling QC score standard. Alignments analysis identified three seasonal influenza A H3N2 strains, one 2009 pandemic influenza A H1N1 strain and two influenza B strains. The nearly entire genomes of all six virus isolates yielded equal or greater than 600-fold sequence coverage depth. MiSeq Platform identified seasonal influenza A H3N2, 2009 pandemic influenza A H1N1and influenza B in the DNA library mixtures efficiently.     

Pandemic and seasonal human influenza virus infections in domestic cats: prevalence, association with respiratory disease, and seasonality patterns

Domestic cats have several features that make them ideal vehicles for interspecies transmission of influenza viruses; however, they have been largely overlooked as potential reservoirs or bridging hosts. In this study, we conducted serological surveillance to assess the prevalence of novel pandemic H1N1 as well as seasonal human influenza virus infections in domestic cats in Ohio. Four hundred serum samples collected from domestic cats (September 2009 to September 2010) were tested using a hemagglutination inhibition (HI) test. The seroprevalences of pandemic H1N1, seasonal H1N1, and H3N2 were 22.5%, 33%, and 43.5%, respectively. In addition, a significant association between clinical feline respiratory disease and influenza virus infection was documented. In this sample of cats, the prevalence of pandemic H1N1 did not follow the seasonality pattern of seasonal H1N1 or H3N2 influenza, similar to observations in humans. Pandemic H1N1 seroprevalence did not vary in relation to ambient temperature changes, while the seroprevalence of seasonal H3N2 and H1N1 influenza viruses increased with the decline of ambient temperature. Our results highlight the high prevalence of influenza virus infection in domestic cats, a seasonality pattern of influenza virus infection comparable to that in humans, and an association of infection with clinical respiratory disease.     

Duration of viral shedding in patients admitted to hospital with pandemic influenza A/H1N1 2009 infection

Little is known about the kinetics of viral shedding of pandemic influenza A/H1N1 2009 virus. Influenza RNA, as a surrogate for viral clearance, was therefore measured on days 1, 5, 7, and 10 or more in patients admitted to hospital with pandemic influenza A/H1N1 2009 infection. A total of 72 patients who were admitted to hospital with confirmed pandemic influenza A/H1N1 2009 at a tertiary care hospital, Seoul, South Korea, between 1 September and 11 November 2009 were evaluated. The median duration of viral shedding, as assessed by RT-PCR, was 9 days, as determined by the Kaplan-Meier method. Patients who were positive by RT-PCR at their last assay, but who were discharged before the next RT-PCR test due to symptom improvement, were censored from the analysis. If such patients were included, with the assumption that they had negative viral status at discharge, the median duration of viral shedding was 5 days (interquartile range, 2-8 days). These calculations thus suggest that the true median duration of viral shedding is between 5 and 9 days. Univariate analysis showed that delayed administration of antiviral therapy and comorbidity were associated with slower viral clearance. Multivariate analysis showed that oseltamivir started after the first day of symptoms (OR 2.7, 95% CI 1.2-5.7) was associated independently with slower viral clearance. These findings indicate that, in about 50% of patients admitted to hospital with pandemic influenza A/H1N1 2009, virus can be positive as tested by RT-PCR on the eighth day after developing symptoms of influenza. The present findings also indicate that starting antiviral therapy within 24 hr of the onset of symptoms is associated with more rapid viral clearance.     

H1N1 viral proteome peptide microarray predicts individuals at risk for H1N1 infection and segregates infection versus Pandemrix® vaccination

A high content peptide microarray containing the entire influenza A virus [A/California/08/2009(H1N1)] proteome and haemagglutinin proteins from 12 other influenza A subtypes, including the haemagglutinin from the [A/South Carolina/1/1918(H1N1)] strain, was used to gauge serum IgG epitope signatures before and after Pandemrix^® vaccination or H1N1 infection in a Swedish cohort during the pandemic influenza season 2009. A very narrow pattern of pandemic flu‐specific IgG epitope recognition was observed in the serum from individuals who later contracted H1N1 infection. Moreover, the pandemic influenza infection generated IgG reactivity to two adjacent epitopes of the neuraminidase protein. The differential serum IgG recognition was focused on haemagglutinin 1 (H1) and restricted to classical antigenic sites (Cb) in both the vaccinated controls and individuals with flu infections. We further identified a novel epitope VEPGDKITFEATGNL on the Ca antigenic site (251–265) of the pandemic flu haemagglutinin, which was exclusively recognized in serum from individuals with previous vaccinations and never in serum from individuals with H1N1 infection (confirmed by RNA PCR analysis from nasal swabs). This epitope was mapped to the receptor‐binding domain of the influenza haemagglutinin and could serve as a correlate of immune protection in the context of pandemic flu. The study shows that unbiased epitope mapping using peptide microarray technology leads to the identification of biologically and clinically relevant target structures. Most significantly an H1N1 infection induced a different footprint of IgG epitope recognition patterns compared with the pandemic H1N1 vaccine.     

Case report: Oseltamivir‐induced resistant pandemic influenza A (H1N1) Virus infection in a patient with AIDS and Pneumocystis jirovecii pneumonia

Pneumocystis jirovecii pneumonia is the main cause of severe respiratory failure in patients with advanced HIV disease who do not receive P. jirovecii prophylaxis. Other aetiological agents may contribute to the respiratory failure in these patients, which is highlighted by the case described below: A patient with advanced HIV disease was treated for a dual‐infection with pandemic influenza A (H1N1) and P. jirovecii. Initially, his condition improved, but deteriorated after the emergence of oseltamivir‐resistant influenza virus. This is the first documented case of emergence of drug‐resistant influenza virus in a patient infected with HIV with a pandemic influenza A (H1N1) and P. jirovecii double infection. J. Med. Virol. 85: 941–943, 2013. © 2013 Wiley Periodicals, Inc.     

Influenza virus A(H1N1)pdm09 hemagglutinin polymorphism and associated disease in southern Germany during the 2010/11 influenza season

A novel influenza A virus emerged in early 2009 to cause the first influenza pandemic of the 21^st century. Understanding the evolution of influenza virus is crucial to determine pathogenesis, vaccine efficacy, and resistance to antiviral drugs. In this study, we investigated the molecular evolution of influenza virus A(H1N1)pdm09 in the 2010/11 influenza season in southern Germany by sequence analysis of the influenza virus hemagglutinin gene from 25 patients with mild, moderate, and severe disease. Phylogenetic analysis revealed co-circulation of different genetic groups. The D222G mutation, which had previously been observed in severe cases, was not detected. Immunocompromised patients were not affected more severely than non-immunocompromised patients (p>0.05), although longer shedding was observed in some of them. Interestingly, additional mutations and potential glycosylation sites were detected in samples from the lower respiratory tract in two patients, but not in the corresponding upper respiratory tract specimens. The H275Y mutation in the influenza virus neuraminidase gene, known to confer resistance to the neuraminidase inhibitor oseltamivir, was detected in one patient.     

2009 Pandemic Influenza A (H1N1) : Pathology and Pathogenesis of 100 Fatal Cases in the United States

In the spring of 2009, a novel influenza A (H1N1) virus emerged in North America and spread worldwide to cause the first influenza pandemic since 1968. During the first 4 months, over 500 deaths in the United States had been associated with confirmed 2009 pandemic influenza A (H1N1) [2009 H1N1] virus infection. Pathological evaluation of respiratory specimens from initial influenza-associated deaths suggested marked differences in viral tropism and tissue damage compared with seasonal influenza and prompted further investigation. Available autopsy tissue samples were obtained from 100 US deaths with laboratory-confirmed 2009 H1N1 virus infection. Demographic and clinical data of these case-patients were collected, and the tissues were evaluated by multiple laboratory methods, including histopathological evaluation, special stains, molecular and immunohistochemical assays, viral culture, and electron microscopy. The most prominent histopathological feature observed was diffuse alveolar damage in the lung in all case-patients examined. Alveolar lining cells, including type I and type II pneumocytes, were the primary infected cells. Bacterial co-infections were identified in >25% of the case-patients. Viral pneumonia and immunolocalization of viral antigen in association with diffuse alveolar damage are prominent features of infection with 2009 pandemic influenza A (H1N1) virus. Underlying medical conditions and bacterial co-infections contributed to the fatal outcome of this infection. More studies are needed to understand the multifactorial pathogenesis of this infection.In April 2009, novel influenza A (H1N1) virus infection was first reported in two US children,¹ followed by identification of cases with acute respiratory illness caused by the identical virus infection in Mexico.² Global transmission of 2009 influenza A (H1N1) [2009 H1N1] virus led to the first influenza pandemic since 1968. Although most case-patients of 2009 H1N1 had mild-to-moderate illness, severe and fatal disease has been reported,^3,4,5 with an estimated mortality of 0.048% among symptomatic US cases.⁶In previous influenza pandemics, studies of autopsy specimens revealed histopathological findings of bronchitis, thrombosis, interstitial inflammation, hyaline membrane formation, and various degrees of intra-alveolar edema, hemorrhage, and inflammation.^7,8,9,10 In contrast, studies of fatal seasonal influenza cases illustrated that viral localization is primarily in major airways, with rare involvement of alveolar cells and lung parenchyma.^11,12 Several studies of previous pandemics concluded that the majority of deaths were likely due to viral infection concurrent with bacterial pneumonia.^10,13 A few histopathological and immunohistologic studies of fatal 2009 H1N1 cases have been recently described.^14,15,16,17 In this report, we describe some of the unique clinicopathologic and epidemiological aspects of a large number of US deaths associated with 2009 H1N1 virus infection. We performed histopathological, immunohistochemical (IHC), and other laboratory methods to study viral distribution and cellular localization and to provide further insights into the pathogenesis of this disease.     

Contemporary seasonal influenza A (H1N1) virus infection primes for a more robust response to split inactivated pandemic influenza A (H1N1) Virus vaccination in ferrets

Human influenza pandemics occur when influenza viruses to which the population has little or no immunity emerge and acquire the ability to achieve human-to-human transmission. In April 2009, cases of a novel H1N1 influenza virus in children in the southwestern United States were reported. It was retrospectively shown that these cases represented the spread of this virus from an ongoing outbreak in Mexico. The emergence of the pandemic led to a number of national vaccination programs. Surprisingly, early human clinical trial data have shown that a single dose of nonadjuvanted pandemic influenza A (H1N1) 2009 monovalent inactivated vaccine (pMIV) has led to a seroprotective response in a majority of individuals, despite earlier studies showing a lack of cross-reactivity between seasonal and pandemic H1N1 viruses. Here we show that previous exposure to a contemporary seasonal H1N1 influenza virus and to a lesser degree a seasonal influenza virus trivalent inactivated vaccine is able to prime for a higher antibody response after a subsequent dose of pMIV in ferrets. The more protective response was partially dependent on the presence of CD8(+) cells. Two doses of pMIV were also able to induce a detectable antibody response that provided protection from subsequent challenge. These data show that previous infection with seasonal H1N1 influenza viruses likely explains the requirement for only a single dose of pMIV in adults and that vaccination campaigns with the current pandemic influenza vaccines should reduce viral burden and disease severity in humans.     

A novel H1N1 virus causes the first pandemic of the 21st century

A novel H1N1 virus of swine origin (H1N1v ) is currently spreading in humans, giving rise to the first pandemic in 40 years. The disease is of moderate severity but has notable differences from seasonal influenza. In contrast to seasonal influenza, those over 60 years are relatively spared, a likely consequence of the presence of H1N1v cross‐neutralizing antibody in this age group. Most patients appear to have mild influenza‐like illness and many of the complications leading to hospitalization and mortality occur in those with underlying disease conditions or pregnancy. Studies in animal models suggest that the novel H1N1v pandemic virus causes a more severe illness and appears to have a greater predilection for the alveolar epithelium than seasonal influenza viruses. As there are as yet little data on the pathogenesis and immunology of H1N1v infection in humans, we have reviewed relevant data from past pandemics, from seasonal influenza and avian influenza H5N1 to highlight key issues pertaining to pathogenesis and immunology.     

Seasonal H1N1 2007 influenza virus infection is associated with elevated pre‐exposure antibody titers to the 2009 pandemic influenza A (H1N1) virus

The new influenza strain detected in humans in April 2009 has caused the first influenza pandemic of the 21st century. A cross‐reactive antibody response, in which antibodies against seasonal H1N1 viruses neutralized the 2009 pandemic influenza A (H1N1) virus (2009 pH1N1), was detected among individuals aged >60 years. However, factors other than age associated with such a cross‐reactive antibody response are poorly documented. Our objective was to examine factors potentially associated with elevated pre‐exposure viro‐neutralization and hemagglutination‐inhibition antibody titers against the 2009 pH1N1. We also studied factors associated with antibody titers against the 2007 seasonal H1N1 virus. One hundred subjects participating in an influenza cohort were selected. Sera collected in 2008 were analysed using hemagglutination inhibition and viro‐neutralization assays for the 2009 pH1N1 virus and the 2007 seasonal H1N1 virus. Viro‐neutralization results were explored using a linear mixed‐effect model and hemagglutination‐inhibition results using linear‐regression models for interval‐censored data. Elevated antibody titers against 2009 pH1N1 were associated with seasonal 2007 H1N1 infection (viro‐neutralization, p 0.006; hemagglutination‐inhibition, p 0.018). Elevated antibody titers were also associated with age in the viro‐neutralization assay (p <0.0001). Seasonal 2007 H1N1 infection is an independent predictor of elevated pre‐exposure antibody titers against 2009 pH1N1 and may have contributed to lowering the burden of the 2009 pH1N1 pandemic.