Investigations into human influenza transmission

Limited understanding of influenza transmission has been a frequent obstacle during the development of pandemic influenza infection prevention and mitigation strategies. The science is hotly debated, especially the relative importance of transmission via large droplets or aerosols. Clarification of the relative importance of different modes of transmission is critical for the refinement of evidence-based infection control advice and has been called for by the European Center for Disease Control (ECDC), the World Health Organization (WHO), and the US Institute of Medicine. The primary aims of this thesis were to investigate influenza transmission; i) by obtaining data concerning viral shedding and the presence of influenza virus in the near environment of infected individuals and ii) through the exploration of a human challenge model to study transmission. Two major clinical studies have been performed; • Shedding and environmental deposition of novel A (H1N1) pandemic influenza virus. The primary aims of the study were to correlate the amount of virus detected in a subject’s nose with that recovered from his/her immediate environment (on surfaces and in the air) and with symptom duration and severity. Adults and children, both in hospital and from the community, who had symptoms of influenza infection were enrolled. Information about symptoms was collected and samples were taken including nose swabs, swabs from surfaces and air samples. Forty two subjects infected with influenza A(H1N1)pdm09 were recruited and followed up. The mean duration of nasal viral shedding was 6.2 days (by PCR) and 4.6 days (by culture). Over 25% of cases remained potentially infectious for at least 5 days. Symptom scores and viral shedding were poorly correlated. From surface swabs collected in the vicinity of 40 subjects, 15 (38%) subject locations were contaminated with virus. Overall 36 of 662 (5.4%) surface swabs taken were positive for influenza, two (0.3%) yielded viable virus. Subjects yielding positive surface samples had significantly higher nasal viral loads on illness Day 3 and more prominent respiratory symptom scores. Room air was sampled in the vicinity of 12 subjects and PCR positive samples were obtained from five (42%). Particles small enough to reach the distal lung (≤4µm) were found to contain virus. • Use of a human influenza challenge model to assess person-to-person transmission: Proof-of-concept study. The primary aim of this study was to establish that an experimentally induced influenza infection is transmissible. Healthy subjects deemed sero-susceptible to influenza A/H3N2/Wisconsin/67/2005 were intranasally inoculated (Donors) and when symptoms began, further sero-susceptible subjects (Recipients) were exposed to Donors during an ‘Exposure Event’. Subjects were in close contact, e.g. playing games and eating meals together, for a total of 28 hours during a 2 day period. Samples were collected to confirm infection status. Among 24 healthy adult subjects, nine were randomised to the ‘Donor’ group and 15 to the ‘Recipient’ group. Following inoculation 5 out of 9 Donors (55%) developed illness and 7 out of 9 (78%) were proven to be infected. After exposure, 5 out of 15 Recipients developed symptoms and 3 out of 15 were proven to be infected. Three others were found to be non sero-susceptible prior to exposure. The overall attack rate in Recipients was 20% but was 25% after adjustment for pre-exposure immunity. The contact, droplet and aerosol routes of influenza transmission are all likely to have a role. This thesis shows that transmission of influenza via surfaces may be less important than current infection control policies and public guidance documents imply. Air sampling results add to the accumulating evidence that supports the potential for aerosol transmission of influenza. The human challenge model could be used to investigate routes of influenza transmission further and a study funded by the Centers for Disease Control (CDC) is planned

Investigations into human influenza transmission

Limited understanding of influenza transmission has been a frequent obstacle during the development of pandemic influenza infection prevention and mitigation strategies. The science is hotly debated, especially the relative importance of transmission via large droplets or aerosols. Clarification of the relative importance of different modes of transmission is critical for the refinement of evidence-based infection control advice and has been called for by the European Center for Disease Control (ECDC), the World Health Organization (WHO), and the US Institute of Medicine. The primary aims of this thesis were to investigate influenza transmission; i) by obtaining data concerning viral shedding and the presence of influenza virus in the near environment of infected individuals and ii) through the exploration of a human challenge model to study transmission. Two major clinical studies have been performed; • Shedding and environmental deposition of novel A (H1N1) pandemic influenza virus. The primary aims of the study were to correlate the amount of virus detected in a subject’s nose with that recovered from his/her immediate environment (on surfaces and in the air) and with symptom duration and severity. Adults and children, both in hospital and from the community, who had symptoms of influenza infection were enrolled. Information about symptoms was collected and samples were taken including nose swabs, swabs from surfaces and air samples. Forty two subjects infected with influenza A(H1N1)pdm09 were recruited and followed up. The mean duration of nasal viral shedding was 6.2 days (by PCR) and 4.6 days (by culture). Over 25% of cases remained potentially infectious for at least 5 days. Symptom scores and viral shedding were poorly correlated. From surface swabs collected in the vicinity of 40 subjects, 15 (38%) subject locations were contaminated with virus. Overall 36 of 662 (5.4%) surface swabs taken were positive for influenza, two (0.3%) yielded viable virus. Subjects yielding positive surface samples had significantly higher nasal viral loads on illness Day 3 and more prominent respiratory symptom scores. Room air was sampled in the vicinity of 12 subjects and PCR positive samples were obtained from five (42%). Particles small enough to reach the distal lung (≤4µm) were found to contain virus. • Use of a human influenza challenge model to assess person-to-person transmission: Proof-of-concept study. The primary aim of this study was to establish that an experimentally induced influenza infection is transmissible. Healthy subjects deemed sero-susceptible to influenza A/H3N2/Wisconsin/67/2005 were intranasally inoculated (Donors) and when symptoms began, further sero-susceptible subjects (Recipients) were exposed to Donors during an ‘Exposure Event’. Subjects were in close contact, e.g. playing games and eating meals together, for a total of 28 hours during a 2 day period. Samples were collected to confirm infection status. Among 24 healthy adult subjects, nine were randomised to the ‘Donor’ group and 15 to the ‘Recipient’ group. Following inoculation 5 out of 9 Donors (55%) developed illness and 7 out of 9 (78%) were proven to be infected. After exposure, 5 out of 15 Recipients developed symptoms and 3 out of 15 were proven to be infected. Three others were found to be non sero-susceptible prior to exposure. The overall attack rate in Recipients was 20% but was 25% after adjustment for pre-exposure immunity. The contact, droplet and aerosol routes of influenza transmission are all likely to have a role. This thesis shows that transmission of influenza via surfaces may be less important than current infection control policies and public guidance documents imply. Air sampling results add to the accumulating evidence that supports the potential for aerosol transmission of influenza. The human challenge model could be used to investigate routes of influenza transmission further and a study funded by the Centers for Disease Control (CDC) is planned

The effect of influenza virus on the human oropharyngeal microbiome

© The Author(s) 2018. Background. Secondary bacterial infections are an important cause of morbidity and mortality associated with influenza infections. As bacterial disease can be caused by a disturbance of the host microbiome, we examined the impact of influenza on the upper respiratory tract microbiome in a human challenge study. Methods. The dynamics and ecology of the throat microbiome were examined following an experimental influenza challenge of 52 previously-healthy adult volunteers with influenza A/Wisconsin/67/2005 (H3N2) by intranasal inoculation; 35 healthy control subjects were not subjected to the viral challenge. Serial oropharyngeal samples were taken over a 30-day period, and the V1-V3 region of the bacterial 16S ribosomal RNA sequences were amplified and sequenced to determine the composition of the microbiome. The carriage of pathogens was also detected. Results. Of the 52 challenged individuals, 43 developed proven influenza infections, 33 of whom became symptomatic. None of the controls developed influenza, although 22% reported symptoms. The diversity of bacterial communities remained remarkably stable following the acquisition of influenza, with no significant differences over time between individuals with influenza and those in the control group. Influenza infection was not associated with perturbation of the microbiome at the level of phylum or genus. There was no change in colonization rates with Streptococcus pneumoniae or Neisseria meningitidis. Conclusions. The throat microbiota is resilient to influenza infection, indicating the robustness of the upper-airway microbiome

Influenza A (H3) illness and viral aerosol shedding from symptomatic naturally infected and experimentally infected cases

BackgroundIt has long been known that nasal inoculation with influenza A virus produces asymptomatic to febrile infections. Uncertainty persists about whether these infections are sufficiently similar to natural infections for studying human‐to‐human transmission.MethodsWe compared influenza A viral aerosol shedding from volunteers nasally inoculated with A/Wisconsin/2005 (H3N2) and college community adults naturally infected with influenza A/H3N2 (2012‐2013), selected for influenza‐like illness with objectively measured fever or a positive Quidel QuickVue A&B test. Propensity scores were used to control for differences in symptom presentation observed between experimentally and naturally infected groups.ResultsEleven (28%) experimental and 71 (86%) natural cases shed into fine particle aerosols (P [less than] .001). The geometric mean (geometric standard deviation) for viral positive fine aerosol samples from experimental and natural cases was 5.1E + 3 (4.72) and 3.9E + 4 (15.12) RNA copies/half hour, respectively. The 95th percentile shedding rate was 2.4 log10 greater for naturally infected cases (1.4E + 07 vs 7.4E + 04). Certain influenza‐like illness‐related symptoms were associated with viral aerosol shedding. The almost complete lack of symptom severity distributional overlap between groups did not support propensity score–adjusted shedding comparisons. ConclusionsDue to selection bias, the natural and experimental infections had limited symptom severity distributional overlap precluding valid, propensity score–adjusted comparison. Relative to the symptomatic naturally infected cases, where high aerosol shedders were found, experimental cases did not produce high aerosol shedders. Studying the frequency of aerosol shedding at the highest observed levels in natural infections without selection on symptoms or fever would support helpful comparisons

Risk factors, symptom reporting, healthcare-seeking behaviour and adherence to public health guidance: protocol for Virus Watch, a prospective community cohort study

IntroductionThe coronavirus (COVID-19) pandemic has caused significant global mortality and impacted lives around the world. Virus Watch aims to provide evidence on which public health approaches are most likely to be effective in reducing transmission and impact of the virus, and will investigate community incidence, symptom profiles and transmission of COVID-19 in relation to population movement and behaviours.Methods and analysisVirus Watch is a household community cohort study of acute respiratory infections in England and Wales and will run from June 2020 to August 2021. The study aims to recruit 50 000 people, including 12 500 from minority ethnic backgrounds, for an online survey cohort and monthly antibody testing using home fingerprick test kits. Nested within this larger study will be a subcohort of 10 000 individuals, including 3000 people from minority ethnic backgrounds. This cohort of 10 000 people will have full blood serology taken between October 2020 and January 2021 and repeat serology between May 2021 and August 2021. Participants will also post self-administered nasal swabs for PCR assays of SARS-CoV-2 and will follow one of three different PCR testing schedules based on symptoms.Ethics and disseminationThis study has been approved by the Hampstead National Health Service (NHS) Health Research Authority Ethics Committee (ethics approval number 20/HRA/2320). We are monitoring participant queries and using these to refine methodology where necessary, and are providing summaries and policy briefings of our preliminary findings to inform public health action by working through our partnerships with our study advisory group, Public Health England, NHS and government scientific advisory panels.</jats:sec

Pasteurella multocida meningoencephalitis in an immunocompetent adult with multiple cat scratches

We present the case of a 49-year-old woman admitted to our Acute Medical Unit with a 2-day history of fever, vomiting and confusion. The patient was alcohol dependent and had sustained several scratches from her pet cat, which her pet dog had licked. She deteriorated in the Emergency Department-developing high fever, worsening confusion and meningism. Blood cultures were taken and broad spectrum antibiotics commenced prior to CT scanning and diagnostic lumbar puncture. Blood cultures and CSF 16S ribosomal PCR confirmed a diagnosis of Pasteurella multocida bacteraemia and meningoencephalitis. The patient was successfully treated with 14 days of intravenous antibiotics. P multocida is a Gram-negative coccobacillus which frequently colonises the nasopharynx of animals; it is a recognised but very rare cause of meningoencephalitis in immunocompetent adults. This case highlights the need to consider P multocida infection in patients with prior animal contact, regardless of their immune status

Minimal transmission in an influenza A (H3N2) human challenge-transmission model within a controlled exposure environment

Uncertainty about the importance of influenza transmission by airborne droplet nuclei generates controversy for infection control. Human challenge-transmission studies have been supported as the most promising approach to fill this knowledge gap. Healthy, seronegative volunteer ‘Donors’ (n = 52) were randomly selected for intranasal challenge with influenza A/Wisconsin/67/2005 (H3N2). ‘Recipients’ randomized to Intervention (IR, n = 40) or Control (CR, n = 35) groups were exposed to Donors for four days. IRs wore face shields and hand sanitized frequently to limit large droplet and contact transmission. One transmitted infection was confirmed by serology in a CR, yielding a secondary attack rate of 2.9% among CR, 0% in IR (p = 0.47 for group difference), and 1.3% overall, significantly less than 16% (p<0.001) expected based on a proof-of-concept study secondary attack rate and considering that there were twice as many Donors and days of exposure. The main difference between these studies was mechanical building ventilation in the follow-on study, suggesting a possible role for aerosols

Viral emissions into the air and environment after SARS-CoV-2 human challenge: a phase 1, open label, first-in-human study

BackgroundEffectively implementing strategies to curb SARS-CoV-2 transmission requires understanding who is contagious and when. Although viral load on upper respiratory swabs has commonly been used to infer contagiousness, measuring viral emissions might be more accurate to indicate the chance of onward transmission and identify likely routes. We aimed to correlate viral emissions, viral load in the upper respiratory tract, and symptoms, longitudinally, in participants who were experimentally infected with SARS-CoV-2.MethodsIn this phase 1, open label, first-in-human SARS-CoV-2 experimental infection study at quarantine unit at the Royal Free London NHS Foundation Trust, London, UK, healthy adults aged 18-30 years who were unvaccinated for SARS-CoV-2, not previously known to have been infected with SARS-CoV-2, and seronegative at screening were recruited. Participants were inoculated with 10 50% tissue culture infectious dose of pre-alpha wild-type SARS-CoV-2 (Asp614Gly) by intranasal drops and remained in individual negative pressure rooms for a minimum of 14 days. Nose and throat swabs were collected daily. Emissions were collected daily from the air (using a Coriolis μ air sampler and directly into facemasks) and the surrounding environment (via surface and hand swabs). All samples were collected by researchers, and tested by using PCR, plaque assay, or lateral flow antigen test. Symptom scores were collected using self-reported symptom diaries three times daily. The study is registered with ClinicalTrials.gov, NCT04865237.FindingsBetween March 6 and July 8, 2021, 36 participants (ten female and 26 male) were recruited and 18 (53%) of 34 participants became infected, resulting in protracted high viral loads in the nose and throat following a short incubation period, with mild-to-moderate symptoms. Two participants were excluded from the per-protocol analysis owing to seroconversion between screening and inoculation, identified post hoc. Viral RNA was detected in 63 (25%) of 252 Coriolis air samples from 16 participants, 109 (43%) of 252 mask samples from 17 participants, 67 (27%) of 252 hand swabs from 16 participants, and 371 (29%) of 1260 surface swabs from 18 participants. Viable SARS-CoV-2 was collected from breath captured in 16 masks and from 13 surfaces, including four small frequently touched surfaces and nine larger surfaces where airborne virus could deposit. Viral emissions correlated more strongly with viral load in nasal swabs than throat swabs. Two individuals emitted 86% of airborne virus, and the majority of airborne virus collected was released on 3 days. Individuals who reported the highest total symptom scores were not those who emitted most virus. Very few emissions occurred before the first reported symptom (7%) and hardly any before the first positive lateral flow antigen test (2%).InterpretationAfter controlled experimental inoculation, the timing, extent, and routes of viral emissions was heterogeneous. We observed that a minority of participants were high airborne virus emitters, giving support to the notion of superspreading individuals or events. Our data implicates the nose as the most important source of emissions. Frequent self-testing coupled with isolation upon awareness of first symptoms could reduce onward transmissions.FundingUK Vaccine Taskforce of the Department for Business, Energy and Industrial Strategy of Her Majesty's Government

Minimal transmission in an influenza A (H3N2) human challenge-transmission model within a controlled exposure environment.

Uncertainty about the importance of influenza transmission by airborne droplet nuclei generates controversy for infection control. Human challenge-transmission studies have been supported as the most promising approach to fill this knowledge gap. Healthy, seronegative volunteer 'Donors' (n = 52) were randomly selected for intranasal challenge with influenza A/Wisconsin/67/2005 (H3N2). 'Recipients' randomized to Intervention (IR, n = 40) or Control (CR, n = 35) groups were exposed to Donors for four days. IRs wore face shields and hand sanitized frequently to limit large droplet and contact transmission. One transmitted infection was confirmed by serology in a CR, yielding a secondary attack rate of 2.9% among CR, 0% in IR (p = 0.47 for group difference), and 1.3% overall, significantly less than 16% (p&lt;0.001) expected based on a proof-of-concept study secondary attack rate and considering that there were twice as many Donors and days of exposure. The main difference between these studies was mechanical building ventilation in the follow-on study, suggesting a possible role for aerosols

Safety, tolerability and viral kinetics during SARS-CoV-2 human challenge

To establish a novel SARS-CoV-2 human challenge model, 36 volunteers aged 18-29 years without evidence of previous infection or vaccination were inoculated with 10 TCID 50 of a wild-type virus (SARS-CoV-2/human/GBR/484861/2020) intranasally. Two participants were excluded from per protocol analysis due to seroconversion between screening and inoculation. Eighteen (~53%) became infected, with viral load (VL) rising steeply and peaking at ~5 days post-inoculation. Virus was first detected in the throat but rose to significantly higher levels in the nose, peaking at ~8.87 log 10 copies/ml (median, 95% CI [8.41,9.53). Viable virus was recoverable from the nose up to ~10 days post-inoculation, on average. There were no serious adverse events. Mild-to-moderate symptoms were reported by 16 (89%) infected individuals, beginning 2-4 days post-inoculation. Anosmia/dysosmia developed more gradually in 12 (67%) participants. No quantitative correlation was noted between VL and symptoms, with high VLs even in asymptomatic infection, followed by the development of serum spike-specific and neutralising antibodies. However, lateral flow results were strongly associated with viable virus and modelling showed that twice-weekly rapid tests could diagnose infection before 70-80% of viable virus had been generated. Thus, in this first SARS-CoV-2 human challenge study, no serious safety signals were detected and the detailed characteristics of early infection and their public health implications were shown. ClinicalTrials.gov identifier: NCT04865237