Establishment and Clinical Applications of a Portable System for Capturing Influenza Viruses Released through Coughing

Coughing plays an important role in influenza transmission; however, there is insufficient information regarding the viral load in cough because of the lack of convenient and reliable collection methods. We developed a portable airborne particlecollection system to measure the viral load; it is equipped with an air sampler to draw air and pass it through a gelatin membrane filter connected to a cone-shaped, megaphone-like device to guide the cough airflow to the membrane. The membrane was dissolved in a medium, and the viral load was measured using quantitative real-time reverse transcriptasepolymerase chain reaction and a plaque assay. The approximate viral recovery rate of this system was 10% in simulation experiments to collect and quantify the viral particles aerosolized by a nebulizer. Using this system, cough samples were collected from 56 influenza A patients. The total viral detection rate was 41% (23/56), and the viral loads varied significantly (from <10, less than the detection limit, to 2240 viral gene copies/cough). Viable viruses were detected from 3 samples with ?18 plaque forming units per cough sample. The virus detection rates were similar among different groups of patients infected with different viral subtypes and during different influenza seasons. Among patients who did not receive antiviral treatment, viruses were detected in one of six cases in the vaccinated group and four of six cases in the unvaccinated group. We found cases with high viral titers in throat swabs or oral secretions but very low or undetectable in coughs and vice versa suggesting other possible anatomical sites where the viruses might be mixed into the cough. Our system is easy to operate, appropriate for bedside use, and is useful for comparing the viral load in cough samples from influenza patients under various conditions and settings. However, further large-scale studies are warranted to validate our results

Viral loads in coughs of unvaccinated influenza patients.

<p>Data from days 2 and 3 of symptom onset were plotted together in respect to the status of receiving antiviral treatment, including treatment time before cough collection. The open circle, closed circle, and closed triangle represent subjects untreated, and treated with oseltamivir or zanamivir, respectively. Viruses isolated in oseltamivir-treated cases were all subtype A(H1), which is considered to be oseltamivir-resistant.</p

Viral loads of coughs compared to those in throat or oral samples in individual patients.

<p>The combined data of viral loads of the cough and throat swab, or cough and oral secretion samples of individual patients were plotted on two-dimensional graphs. The open and closed circles represent cases with and without symptom of coughing, respectively, and daggers indicate cases in which active virus was detected in cough samples using the plaque assay.</p

A schematic diagram of simulation experiments to collect biomist particles.

<p>Settings for the experiments to simulate biomist collection from coughs to estimate the viral recovery rate of this system. Control experiments with mist particles generated from a nebulizer were drawn directly into the gelatin filter membrane, with or without a hollow paper tube to guide the mist to the membrane (A), and the collection of the particles through the cone-shaped device to guide particles expelled through a cough (B).</p

Viral loads in coughs of influenza patients.

<p>Cough samples were collected from individual patients and subjected to viral quantification. The viral load/cough was plotted on the ordinate along with days of illness during sample collection on the abscissa. The onset day of any influenza symptom was defined as day 1. Viral subtypes are presented using different symbols: open circle, closed circle, and open triangle represent infections with A(H1), A(H3), and A(H1N1)pdm09 virus, respectively. Daggers indicate cases in which the active virus was detected using a conventional plaque assay.</p

Viral loads in coughs of vaccinated and unvaccinated influenza patients.

<p>Viral loads were plotted against vaccination status. Data from days 2 and 3 of cases infected with A(H1) or A(H3) virus are shown together. Viral loads in coughs of antiviral-untreated influenza patients (A) and those of all cases including antiviral-treated cases (B). Open and closed circles represent unvaccinated and vaccinated cases, respectively.</p

Viral loads of samples from patients with or without cough symptom.

<p>Viral loads in cough and throat swab samples from cases with and without a symptom of coughing were compared. The viral load in the throat swab was calculated from the viral load of the transport medium in which the swab fluid was eluted and the volume of the eluted swab fluid, which was tentatively estimated by the increase of the weight of the cotton swab used for sample collection. Circle and triangle represent cases with and without symptoms of coughing. Closed circles indicate cases with higher viral loads among cough or throat swab samples, and identical numbers associated with the closed circles indicate that the data were from the same patient.</p

Viral recovery rate of collection and quantification system.

a<p>The experiments were performed in triplicate for the high amount experiment controls and the number is the average and standard error (SE) of the results.</p>b<p>The low dose experiment was performed once to confirm the reproducibility of the recovery rate, as determined by the high dose experiment.</p>c<p>Average copy number of the influenza A M1 gene in the gelatin membrane.</p>d<p>Percentage of viral load on the gelatin membrane to that of collection when the nebulizer was set close to the membrane.</p>e<p>The nebulizer was set 10 cm from the gelatin membrane filter through which the viral mist was suctioned.</p>f<p>The cone-shaped, megaphone-like device of 50 cm in length was connected with the gelatin membrane filter and the nebulizer was set at the entrance of the device.</p>g<p>Percentage of viral load in the product of each step to those at the start of each step.</p>h<p>Viral load in the viral fluid poured into the gelatin membrane solution.</p>i<p>Viral load in the solution of the gelatin membrane filter dissolved in 10 mL of MEM.</p>j<p>The gelatin solution containing the virus was digested with 10 µg/mL of collagenase.</p>k<p>The digested fluid was ultra-centrifuged at 125,000 g for 100 min and the viral RNA was extracted from the precipitate and concentrated by freeze-drying and vacuum centrifugation.</p