Correlates of severe disease in patients with 2009 pandemic influenza (H1N1) virus infection

Background

In the context of 2009 pandemic influenza (H1N1) virus infection (pandemic H1N1 influenza), identifying correlates of the severity of disease is critical to guiding the implementation of antiviral strategies, prioritization of vaccination efforts and planning of health infrastructure. The objective of this study was to identify factors correlated with severity of disease in confirmed cases of pandemic H1N1 influenza.

Methods

This cumulative case–control study included all laboratory-confirmed cases of pandemic H1N1 influenza among residents of the province of Manitoba, Canada, for whom the final location of treatment was known. Severe cases were defined by admission to a provincial intensive care unit (ICU). Factors associated with severe disease necessitating admission to the ICU were determined by comparing ICU cases with two control groups: patients who were admitted to hospital but not to an ICU and those who remained in the community.

Results

As of Sept. 5, 2009, there had been 795 confirmed cases of pandemic H1N1 influenza in Manitoba for which the final treatment location could be determined. The mean age of individuals with laboratory-confirmed infection was 25.3 (standard deviation 18.8) years. More than half of the patients (417 or 52%) were female, and 215 (37%) of 588 confirmed infections for which ethnicity was known occurred in First Nations residents. The proportion of First Nations residents increased with increasing severity of disease (116 [28%] of 410 community cases, 74 [54%] of 136 admitted to hospital and 25 [60%] of 42 admitted to an ICU; p < 0.001), as did the presence of an underlying comorbidity (201 [35%] of 569 community cases, 103 [57%] of 181 admitted to hospital and 34 [76%] of 45 admitted to an ICU; p < 0.001). The median interval from onset of symptoms to initiation of antiviral therapy was 2 days (interquartile range, IQR 1–3) for community cases, 4 days (IQR 2–6) for patients admitted to hospital and 6 days (IQR 4–9) for those admitted to an ICU (p < 0.001). In a multivariable logistic model, the interval from onset of symptoms to initiation of antiviral therapy (odds ratio [OR] 8.24, 95% confidence interval [CI] 2.82–24.1), First Nations ethnicity (OR 6.52, 95% CI 2.04–20.8) and presence of an underlying comorbidity (OR 3.19, 95% CI 1.07–9.52) were associated with increased odds of admission to the ICU (i.e., severe disease) relative to community cases. In an analysis of ICU cases compared with patients admitted to hospital, First Nations ethnicity (OR 3.23, 95% CI 1.04–10.1) was associated with increased severity of disease.

Interpretation

Severe pandemic H1N1 influenza necessitating admission to the ICU was associated with a longer interval from onset of symptoms to treatment with antiviral therapy and with the presence of an underlying comorbidity. First Nations ethnicity appeared to be an independent determinant of severe infection. Despite these associations, the cause and outcomes of pandemic HINI influenza may involve many complex and interrelated factors, all of which require further research and analysis.In April 2009, Canada’s first wave of pandemic influenza (H1N1) virus infections (pandemic H1N1 influenza) began. The highest burden of severe illness in Canada occurred in the province of Manitoba, where 45 Manitobans and 9 out-of-province patients were admitted to an intensive care unit (ICU). In this first wave, ICU staff and equipment were mobilized to expand bed capacity and ventilator capabilities to accommodate clinical need.Although many individuals presented with mild, self-limited symptoms and no sign of pulmonary involvement, some people required admission to an ICU and received maximal life support measures.^1–3 Predicting disease and mitigating hazard in at-risk populations is an important aim of public heath epidemiology, and in preparation for future waves of pandemic H1N1 influenza, determining correlates of the severity of disease may be very important. Initial reports have suggested that, in addition to many of the previously known risk factors for complications of seasonal influenza, obesity⁴ and other underlying comorbidities^3,5 may be risk factors for severe disease. The interval from onset of symptoms to initiation of antiviral therapy or other treatment and supportive care was also associated with adverse outcome in a recent case series.⁶ In a Canadian study of severe pandemic H1N1 influenza, First Nations people were proportionally overrepresented among patients in the ICU.² However, it is unclear if this association was independent of potential confounding factors. The ability to determine correlates of severe pandemic H1N1 disease and subsequent need for ICU resources in at-risk populations would provide opportunities for public and population health analysis and action, public education, strategic prioritization of vaccination efforts, efficient and equitable allocation and use of antiviral drugs, and development of infrastructure within the health system.The objectives of this study were to identify factors that were correlated with severity of disease in confirmed cases of pandemic H1N1 influenza. Our hypothesis, which was based on existing literature, was that obesity, First Nations ethnicity and longer interval from onset of symptoms to treatment would be important determinants of the severity of disease.     

In silico genome analysis and drug efficacy test of influenza A virus (H1N1) 2009

The H1N1 2009 virus is pandemic in many countries. The genome of this virus contains eight segments. Among the eight segments maximum numbers of mutation occur at the segment 1 and segment 4 which codes for PB2 subunit and hemagglutinin (HA) and less number of mutations occur in segment 6 which codes for neuraminidase (NA) protein. Neuraminidase (NA) inhibitors (Oseltamivir and Zanamivir) are presently used as an anti-flu drugs. In the present study, the in silico efficacy of different drugs was tested against the swine flu virus. 3D structures of neuraminidase (NA) proteins of H1N1 2009 were generated using Geno3D. The 3D structure of H1N1 1918 was downloaded from PDB. Interaction study was done using Arguslab 4 and PyMol view. Oseltamivir and Zanamivir have good number of interactions with H1N1 2009 virus and the scoring function also support to this result. When compared with the 1918 H1N1 viral protein, 2009 H1N1 NA protein shows more number of interaction and good scoring function. The RMSD value of before and after docking are found to be same at 0.04A° for both the drugs. The force field energy of NA protein 2009 was found to be −15603.529 KJ/mol before docking. The force field energy was found to be decreased after docking at −17620.740 KJ/mol with Tamiflu and −17652.242 KJ/mol with Zanamivir. The number of interaction and scoring function shows that Oseltamivir and Zanamivir will be able to effectively control the present pandemic H1N1 virus 2009.     

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.     

Susceptibility of antiviral drugs against 2009 influenza A (H1N1) virus

The recent outbreak of the novel strain of influenza A (H1N1) virus has raised a global concern of the future risk of a pandemic. To understand at the molecular level how this new H1N1 virus can be inhibited by the current anti-influenza drugs and which of these drugs it is likely to already be resistant to, homology modeling and MD simulations have been applied on the H1N1 neuraminidase complexed with oseltamivir, and the M2-channel with adamantanes bound. The H1N1 virus was predicted to be susceptible to oseltamivir, with all important interactions with the binding residues being well conserved. In contrast, adamantanes are not predicted to be able to inhibit the M2 function and have completely lost their binding with the M2 residues. This is mainly due to the fact that the M2 transmembrane of the new H1N1 strain contains the S31N mutation which is known to confer resistance to adamantanes.     

A metagenomic analysis of pandemic influenza A (2009 H1N1) infection in patients from North America

Although metagenomics has been previously employed for pathogen discovery, its cost and complexity have prevented its use as a practical front-line diagnostic for unknown infectious diseases. Here we demonstrate the utility of two metagenomics-based strategies, a pan-viral microarray (Virochip) and deep sequencing, for the identification and characterization of 2009 pandemic H1N1 influenza A virus. Using nasopharyngeal swabs collected during the earliest stages of the pandemic in Mexico, Canada, and the United States (n = 17), the Virochip was able to detect a novel virus most closely related to swine influenza viruses without a priori information. Deep sequencing yielded reads corresponding to 2009 H1N1 influenza in each sample (percentage of aligned sequences corresponding to 2009 H1N1 ranging from 0.0011% to 10.9%), with up to 97% coverage of the influenza genome in one sample. Detection of 2009 H1N1 by deep sequencing was possible even at titers near the limits of detection for specific RT-PCR, and the percentage of sequence reads was linearly correlated with virus titer. Deep sequencing also provided insights into the upper respiratory microbiota and host gene expression in response to 2009 H1N1 infection. An unbiased analysis combining sequence data from all 17 outbreak samples revealed that 90% of the 2009 H1N1 genome could be assembled de novo without the use of any reference sequence, including assembly of several near full-length genomic segments. These results indicate that a streamlined metagenomics detection strategy can potentially replace the multiple conventional diagnostic tests required to investigate an outbreak of a novel pathogen, and provide a blueprint for comprehensive diagnosis of unexplained acute illnesses or outbreaks in clinical and public health settings.     

A protective role for complement C3 protein during pandemic 2009 H1N1 and H5N1 influenza A virus infection

Highly pathogenic H5N1 influenza infections are associated with enhanced inflammatory and cytokine responses, severe lung damage, and an overall dysregulation of innate immunity. C3, a member of the complement system of serum proteins, is a major component of the innate immune and inflammatory responses. However, the role of this protein in the pathogenesis of H5N1 infection is unknown. Here we demonstrate that H5N1 influenza virus infected mice had increased levels of C5a and C3 activation byproducts as compared to mice infected with either seasonal or pandemic 2009 H1N1 influenza viruses. We hypothesized that the increased complement was associated with the enhanced disease associated with the H5N1 infection. However, studies in knockout mice demonstrated that C3 was required for protection from influenza infection, proper viral clearance, and associated with changes in cellular infiltration. These studies suggest that although the levels of complement activation may differ depending on the influenza virus subtype, complement is an important host defense mechanism.     

Human avian influenza A (H5N1) virus infection in China

Highly pathogenic influenza A (H5N1) virus causes a widespread poultry deaths worldwide. The first human H5N1 infected case was reported in Hong Kong Special Administrative Region of China in 1997. Since then, the virus re-emerged in 2003 and continues to infect people worldwide. Currently, over 400 human infections have been reported in more than 15 countries and mortality rate is greater than 60%. H5N1 viruses still pose a potential pandemic threat in the future because of the continuing global spread and evolution. Here, we summarize the epidemiological, clinical and virological characteristics of human H5N1 infection in China monitored and identified by our national surveillance systems. Chinese Nature Science Foundation Key Project (Grant No. 30599433), Chinese Basic Science Research Program (973)Key Project (Grant No. 2005CB523006)     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

The seroprevalence of pandemic influenza H1N1 (2009) virus in China

Background

Mainland China experienced pandemic influenza H1N1 (2009) virus (pH1N1) with peak activity during November-December 2009. To understand the geographic extent, risk factors, and attack rate of pH1N1 infection in China we conducted a nationwide serological survey to determine the prevalence of antibodies to pH1N1.

Methodology/Principal Findings

Stored serum samples (n = 2,379) collected during 2006-2008 were used to estimate baseline serum reactogenicity to pH1N1. In January 2010, we used a multistage-stratified random sampling method to select 50,111 subjects who met eligibility criteria and collected serum samples and administered a standardized questionnaire. Antibody response to pH1N1 was measured using haemagglutination inhibition (HI) assay and the weighted seroprevalence was calculated using the Taylor series linearization method. Multivariable logistic regression analyses were used to examine risk factors for pH1N1 seropositivity. Baseline seroprevalence of pH1N1 antibody (HI titer ≥40) was 1.2%. The weighted seroprevalence of pH1N1 among the Chinese population was 21.5%(vaccinated: 62.0%; unvaccinated: 17.1%). Among unvaccinated participants, those aged 6-15 years (32.9%) and 16-24 years (30.3%) had higher seroprevalence compared with participants aged 25–59 years (10.7%) and ≥60 years (9.9%, P<0.0001). Children in kindergarten and students had higher odds of seropositivity than children in family care (OR: 1.36 and 2.05, respectively). We estimated that 207.7 million individuals (15.9%) experienced pH1N1 infection in China.

Conclusions/Significance

The Chinese population had low pre-existing immunity to pH1N1 and experienced a relatively high attack rate in 2009 of this virus. We recommend routine control measures such as vaccination to reduce transmission and spread of seasonal and pandemic influenza viruses.     

Risk factors for severe outcomes following 2009 influenza A (H1N1) infection: a global pooled analysis

Background

Since the start of the 2009 influenza A pandemic (H1N1pdm), the World Health Organization and its member states have gathered information to characterize the clinical severity of H1N1pdm infection and to assist policy makers to determine risk groups for targeted control measures.

Methods and Findings

Data were collected on approximately 70,000 laboratory-confirmed hospitalized H1N1pdm patients, 9,700 patients admitted to intensive care units (ICUs), and 2,500 deaths reported between 1 April 2009 and 1 January 2010 from 19 countries or administrative regions—Argentina, Australia, Canada, Chile, China, France, Germany, Hong Kong SAR, Japan, Madagascar, Mexico, the Netherlands, New Zealand, Singapore, South Africa, Spain, Thailand, the United States, and the United Kingdom—to characterize and compare the distribution of risk factors among H1N1pdm patients at three levels of severity: hospitalizations, ICU admissions, and deaths. The median age of patients increased with severity of disease. The highest per capita risk of hospitalization was among patients <5 y and 5–14 y (relative risk [RR] = 3.3 and 3.2, respectively, compared to the general population), whereas the highest risk of death per capita was in the age groups 50–64 y and ≥65 y (RR = 1.5 and 1.6, respectively, compared to the general population). Similarly, the ratio of H1N1pdm deaths to hospitalizations increased with age and was the highest in the ≥65-y-old age group, indicating that while infection rates have been observed to be very low in the oldest age group, risk of death in those over the age of 64 y who became infected was higher than in younger groups. The proportion of H1N1pdm patients with one or more reported chronic conditions increased with severity (median = 31.1%, 52.3%, and 61.8% of hospitalized, ICU-admitted, and fatal H1N1pdm cases, respectively). With the exception of the risk factors asthma, pregnancy, and obesity, the proportion of patients with each risk factor increased with severity level. For all levels of severity, pregnant women in their third trimester consistently accounted for the majority of the total of pregnant women. Our findings suggest that morbid obesity might be a risk factor for ICU admission and fatal outcome (RR = 36.3).

Conclusions

Our results demonstrate that risk factors for severe H1N1pdm infection are similar to those for seasonal influenza, with some notable differences, such as younger age groups and obesity, and reinforce the need to identify and protect groups at highest risk of severe outcomes. Please see later in the article for the Editors'' Summary     

Prevalence of 2009 pandemic influenza A (H1N1) virus antibodies, Tampa Bay Florida--November-December, 2009

Background

In 2009, a novel influenza virus (2009 pandemic influenza A (H1N1) virus (pH1N1)) caused significant disease in the United States. Most states, including Florida, experienced a large fall wave of disease from September through November, after which disease activity decreased substantially. We determined the prevalence of antibodies due to the pH1N1 virus in Florida after influenza activity had peaked and estimated the proportion of the population infected with pH1N1 virus during the pandemic.

Methods

During November-December 2009, we collected leftover serum from a blood bank, a pediatric children''s hospital and a pediatric outpatient clinic in Tampa Bay Florida. Serum was tested for pH1N1 virus antibodies using the hemagglutination-inhibition (HI) assay. HI titers ≥40 were considered seropositive. We adjusted seroprevalence results to account for previously established HI assay specificity and sensitivity and employed a simple statistical model to estimate the proportion of seropositivity due to pH1N1 virus infection and vaccination.

Results

During the study time period, the overall seroprevalence in Tampa Bay, Florida was 25%, increasing to 30% after adjusting for HI assay sensitivity and specificity. We estimated that 5.9% of the population had vaccine-induced seropositivity while 25% had seropositivity secondary to pH1N1 virus infection. The highest cumulative incidence of pH1N1 virus infection was among children aged 5–17 years (53%) and young adults aged 18–24 years (47%), while adults aged ≥50 years had the lowest cumulative incidence (11–13%) of pH1N1 virus infection.

Conclusions

After the peak of the fall wave of the pandemic, an estimated one quarter of the Tampa Bay population had been infected with the pH1N1 virus. Consistent with epidemiologic trends observed during the pandemic, the highest burdens of disease were among school-aged children and young adults.     

Antibody dynamics of 2009 influenza A (H1N1) virus in infected patients and vaccinated people in China

Background

To evaluate the risk of the recurrence and the efficiency of the vaccination, we followed-up antibody responses in patients with the 2009 pandemic H1N1 influenza and persons who received the pandemic H1N1 vaccine in Guangzhou China.

Methods

We collected serum samples from 129 patients and 86 vaccinated persons at day 0, 15, 30, 180 after the disease onset or the vaccination, respectively. Antibody titers in these serum samples were determined by haemagglutination inhibition (HI) assay using a local isolated virus strain A/Guangdong Liwan/SWL1538/2009(H1N1).

Results

HI antibody positive rate of the patients increased significantly from 0% to 60% at day 15 (χ² = 78, P<0.001) and 100% at day 30 (χ² = 23, P<0.001), but decreased significantly to 52% at day 180 (χ² = 38, P<0.001), while that of vaccinated subjects increased from 0% to 78% at day 15 (χ² = 110, P<0.001) and 81% at day 30 (χ² = 0.32, P = 0.57), but decreased significantly to 34% at day 180 (χ² = 39, P<0.001). Geometric mean titers (GMT) of HI antibodies in positive samples from the patients did not change significantly between day 15 and day 30 (T = 0.92, P = 0.36), but it decreased significantly from 80 at day 30 to 52 at day 180 (T = 4.5, P<0.001). GMT of vaccinated persons increased significantly from 100 at day 15 to 193 at day 30 (T = 4.5, P<0.001), but deceased significantly to 74 at day 180 (T = 5.1, P<0.001). Compared to the patients, the vaccinated subjects showed lower seroconversion rate (χ² = 11, P<0.001; χ² = 5.9, P = 0.015), but higher GMT (T = 6.0, P<0.001; T = 3.6, P = 0.001) at day 30 and day 180, respectively.

Conclusion

Vaccination of 2009 influenza A (H1N1) was effective. However, about half or more recovered patients and vaccinated persons might have lost sufficient immunity against the recurrence of the viral infection after half a year. Vaccination or re-vaccination may be necessary for prevention of the recurrence.     

Dose-response time modelling for highly pathogenic avian influenza A (H5N1) virus infection

Aims: To develop time‐dependent dose–response models for highly pathogenic avian influenza A (HPAI) of the H5N1 subtype virus. Methods and Results: A total of four candidate time‐dependent dose–response models were fitted to four survival data sets for animals (mice or ferrets) exposed to graded doses of HPAI H5N1 virus using the maximum‐likelihood estimation. A beta‐Poisson dose–response model with the N₅₀ parameter modified by an exponential‐inverse‐power time dependency or an exponential dose–response model with the k parameter modified by an exponential‐inverse time dependency provided a statistically adequate fit to the observed survival data. Conclusions: We have successfully developed the time‐dependent dose–response models to describe the mortality of animals exposed to an HPAI H5N1 virus. The developed model describes the mortality over time and represents observed experimental responses accurately. Significance and Impact of the Study: This is the first study describing time‐dependent dose–response models for HPAI H5N1 virus. The developed models will be a useful tool for estimating the mortality of HPAI H5N1 virus, which may depend on time postexposure, for the preparation of a future influenza pandemic caused by this lethal virus.     

Genetical features of influenza virus A (H1N1) strain that caused the 2009 pandemic

Genetical features of the A(H1N1) influenza virus strain that caused the 2009 pandemic are analyzed in the review. Mutations typical for this strain, unique and similar to influenza viruses of swine, avian and seasonal types, and phenotypic (pathologic) features associated with them, that are experimentally confirmed, are described. A possibility of reassortation of avian and swine influenza viruses and possible epidemiologic consequences are discussed.     

Male predominance of pneumonia and hospitalization in pandemic influenza A (H1N1) 2009 infection

Background

Based on our clinical experience, the H-reflex amplitude asymmetry might be an earlier sign of nerve root involvement than latency in patients with S1 radiculopathy. However, no data to support this assumption are available. The purpose of this study was to review and report the electrophysiological changes in H-reflex amplitude and latency in patients with radiculopathy in order to determine if there is any evidence to support the assumption that H-reflex amplitude is an earlier sign of nerve root involvement than latency.

Results

Patients with radiculopathy showed significant amplitude asymmetry when compared with healthy controls. However, latency was not always significantly different between patients and healthy controls. These findings suggest nerve root axonal compromise that reduced reflex amplitude earlier than the latency parameter (demyelination) during the pathologic processes.

Conclusion

Contrary to current clinical thought, H-reflex amplitude asymmetry is an earlier sign/parameter of nerve root involvement in patients with radiculopathy compared with latency.     

Inside the outbreak of the 2009 influenza A (H1N1)v virus in Mexico

Background

Influenza viruses pose a threat to human health because of their potential to cause global disease. Between mid March and mid April a pandemic influenza A virus emerged in Mexico. This report details 202 cases of infection of humans with the 2009 influenza A virus (H1N1)v which occurred in Mexico City as well as the spread of the virus throughout the entire country.

Methodology and Findings

From May 1st to May 5th nasopharyngeal swabs, derived from 751 patients, were collected at 220 outpatient clinics and 28 hospitals distributed throughout Mexico City. Analysis of samples using real time RT-PCR revealed that 202 patients out of the 751 subjects (26.9%) were confirmed to be infected with the new virus. All confirmed cases of human infection with the strain influenza (H1N1)v suffered respiratory symptoms. The greatest number of confirmed cases during the outbreak of the 2009 influenza A (H1N1)v were seen in neighbourhoods on the northeast side of Mexico City including Iztapalapa, Gustavo A. Madero, Iztacalco, and Tlahuac which are the most populated areas in Mexico City. Using these data, together with data reported by the Mexican Secretariat of Health (MSH) to date, we plot the course of influenza (H1N1)v activity throughout Mexico.

Conclusions

Our data, which is backed up by MSH data, show that the greatest numbers of the 2009 influenza A (H1N1) cases were seen in the most populated areas. We speculate on conditions in Mexico which may have sparked this flu pandemic, the first in 41 years. We accept the hypothesis that high population density and a mass gathering which took in Iztapalapa contributed to the rapid spread of the disease which developed in three peaks of activity throughout the Country.