Frequency of respiratory virus infections and next-generation analysis of influenza A/H1N1pdm09 dynamics in the lower respiratory tract of patients admitted to the ICU

Abstract

Recent molecular diagnostic methods have significantly improved the diagnosis of viral pneumonia in intensive care units (ICUs). It has been observed that 222G/N changes in the HA gene of H1N1pdm09 are associated with increased lower respiratory tract (LRT) replication and worse clinical outcome. In the present study, the frequency of respiratory viruses was assessed in respiratory samples from 88 patients admitted to 16 ICUs during the 2014-2015 winter-spring season in Lombardy. Sixty-nine out of 88 (78.4%) patients were positive for a respiratory viral infection at admission. Of these, 57/69 (82.6%) were positive for influenza A (41 A/H1N1pdm09 and 15 A/H3N2), 8/69 (11.6%) for HRV, 2/69 (2.9%) for RSV and 2/69 (2.9%) for influenza B. Phylogenetic analysis of influenza A/H1N1pdm09 strains from 28/41 ICU-patients and 21 patients with mild respiratory syndrome not requiring hospitalization, showed the clear predominance of subgroup 6B strains. The median influenza A load in LRT samples of ICU patients was higher than that observed in the upper respiratory tract (URT) (p<0.05). Overall, a greater number of H1N1pdm09 virus variants were observed using next generation sequencing on partial HA sequences (codons 180-286) in clinical samples from the LRT as compared to URT. In addition, 222G/N/A mutations were observed in 30% of LRT samples from ICU patients. Finally, intra-host evolution analysis showed the presence of different dynamics of viral population in LRT of patients hospitalized in ICU with a severe influenza infection.

Fig 1. Frequency distribution of respiratory viruses.

The absolute number of positive patients is reported in brackets.

Fig 2. Phylogenetic comparison of influenza A(H1N1)pdm09 HA gene sequences.

Reference sequences of strains circulating in 2009–2015 are presented in italics (GISAID numbers are provided). Influenza strains of patients with severe infection are reported with a black circle and strains from patients with a mild infection are reported with a white circle. Bootstrap values are given at nodes, and the scale bar is given in numbers of substitutions per site.

Fig 3. Distribution of the number of variants in both URT and LRT samples of ICU patients and URT samples of patients with mild respiratory syndrome.

Fig 4. Frequencies of 222 polymorphisms are displayed as a stacked histogram for four patients with sequential lower respiratory tract samples.

The number of variants observed and the corresponding viral load are reported above each histogram, while the time after admission and the antiviral treatment period are reported below each histogram. NA, not available.