Sensitive detection assays for influenza RNA do not reveal viremia in US blood donors

Abstract

Background: There have been anecdotal reports of influenza viremia since the 1960s. We present an assessment of the prevalence of seasonal and 2009 H1N1 influenza viremia (via RNA testing) in blood donor populations using multiple sensitive detection assays.

Methods: Several influenza RNA amplification assays, including transcription-mediated amplification (TMA) and 2 reverse-transcription polymerase chain reaction (RT-PCR) assays, were evaluated and used to test donor samples. Retrospective samples from 478 subjects drawn at sites with high influenza activity were tested. Prospective samples were collected from 1004 blood donors who called their donation center within 3 days of donation complaining of influenza-like illness (ILI). The plasma collected on the day of donation for these subjects was tested.

Results: Of the repository samples, 2 of 478 plasma samples were initially reactive but not repeat reactive by influenza TMA. Of blood donors reporting ILI symptoms postdonation, 1 of 1004 samples was TMA initially reactive but not repeat reactive; all samples were nonreactive by RT-PCR testing.

Conclusions: Targeting blood donor populations most likely to have influenza infection, we failed to detect influenza RNA in 1482 donor samples, with most tested by 3 different RNA assays. Seasonal influenza does not appear to pose a significant contamination threat to the blood supply.

Figure 1.

Relative sensitivity of influenza A RNA detection assays. Blood samples spiked with influenza virus RNA were prepared and tested in a blinded fashion using reverse-transcription polymerase chain reaction (RT-PCR) assay (A) 1, (B) 2, or (C) transcription-mediated amplification (TMA). Results for RT-PCR are described as the cycle threshold (C_T) at which signal was detected and for TMA as a signal-to-cutoff (S/CO) ratio. The percentage of replicates that tested positive is shown above each bar. Two replicates were tested in (A) and (C) in 2 separate experiments (1 representative experiment shown), and 6 replicates were tested in (B) in 1 experiment. Line denotes S/CO = 1, error bars represent standard error.

Figure 2.

Detection of influenza viremia in influenza H3N2-infected ferrets. Three ferrets were infected with 10⁶ egg infectious dose (EID₅₀) of influenza A H3N2 virus intranasally and followed up for 6 days, with bleeds prior to infection and at days 1, 2, 4, and 6. RNA was extracted from whole blood samples and was tested for influenza RNA by (A) reverse-transcription polymerase chain reaction (RT-PCR) assay #1 or (B) transcription-mediated amplification (TMA). Line denotes S/CO = 1. The experiments were each performed once.

Figure 3.

Pneumonia and influenza mortality for 122 US cities. Each week, the vital statistics offices of 122 cities reported the total number of death certificates received and the number of those for which pneumonia or influenza was listed as the underlying or contributing cause of death by age group. The percentage of all deaths due to pneumonia and influenza (P&I) were compared with a seasonal baseline and epidemic threshold value calculated for each week. The seasonal baseline of P&I deaths was calculated using a periodic regression model that incorporates a robust regression procedure applied to data from the previous five years. An increase of 1645 standard deviations above the seasonal baseline of P&I deaths is considered the “epidemic threshold,” ie, the point at which the observed proportion of deaths attributed to pneumonia or influenza was significantly higher than would be expected at that time of the year in the absence of substantial influenza-related mortality. Figure from the Centers for Disease Control and Prevention (CDC) website at http://www.cdc.gov/flu/weekly/weeklyarchives2003–2004/03–04summary.htm, accessed 20 September 2011. The gray shaded area represents the time period during which the tested repository samples were collected.

Figure 4.

Comparison of Centers for Disease Control and Prevention (CDC) influenza-like illness (ILI) surveillance data with prospective sample collection dates. A, Percentage of visits for ILI reported by the US Outpatient ILI surveillance Network (ILINet), weekly national summary, 30 September 2007 to 30 July 2011. Figure from the CDC website at http://www.cdc.gov/flu/weekly/weeklyarchives2010–2011/picILI30.htm, accessed 20 September 2011. B, The number of samples collected at Blood Systems, Inc (BSI) sites (early 2008) or American Red Cross (ARC) sites (late 2008 through late 2010). The peaks in sample collection for both organizations corresponded to reported high levels of ILI activity in the community (panel A).