By using a systems biology-based approach, we analyzed the response to viral infection following hospitalization of 19 p2009A(H1N1) critically ill patients admitted to seven Spanish currently intensive care units. Our results indicate that pandemic H1N1 patients with severe respiratory disease and poor outcome are characterized by an impaired activation of those genes participating in the development of the antiviral adaptive response.Materials and methodsStudy design, participants and sample collectionNineteen patients attending the participants’ ICUs with primary viral pneumonia during the acute phase of influenza virus illness with acute respiratory distress and unequivocal alveolar opacification involving two or more lobes with negative respiratory and blood bacterial cultures at admission were recruited from 1 November to 31 December 2009.

Patients older than 65 years and younger than 18 years were excluded from the study to avoid immaturity/aging of the immune system as confusion factor in the analysis. Only those patients with confirmed H1N1 infection by real-time polymerase chain reaction (PCR) were included in the study (n = 19). Serial blood samples for plasma, serum and RNA were collected by using serum, ethylenediaminetetraacetic acid (EDTA) and PaxGene (BD) venous blood vacuum collection following the manufacturer’s instructions at days 1, 3/5 and 7 after admission to the ICU, according to a unified protocol for all the participant centers. A pharyngeal sample was collected in parallel. Fifteen healthy volunteers of similar age to the patients were recruited between workers of the University of Valladolid, Spain.

A standard survey was employed to collect the clinical data, including history and physical examination, oximetric measurement, hematological, biochemical, radiological and microbiological investigation in all the participant centers. Treatment decisions for all patients, including corticosteroid therapy, were not standardized and were decided by the attending physician. Informed consent was obtained directly from each patient or their legal representative and also from the healthy controls before enrollment. Patient and control identification remained anonymous. Approval of the study protocol in both the scientific and the ethical aspects was obtained from the Scientific Committees for Clinical Research of each one of the participant centers.

Samples were stored at -80��C until cytokine, antibody and RNAm profiling. Attending to timing of seroconversion (production of antibodies Drug_discovery against p2009A(H1N1)), day 9 from onset of symptoms was considered as the border between the innate and the adaptive immune response in the patients, establishing two moments in the evolution of the disease: an early phase (from onset of symptoms (day 0) to day 8) and a late phase (from day 9 and above).