Because of limitations in study design and because they represented unexpected findings, we interpreted the results of this outbreak investigation as a paradoxical signal of possible concern—thought‐provoking but inconclusive and warranting further evaluation. Canadian investigators thus embarked on a series of confirmatory studies using more rigorous methods and laboratory‐confirmed outcomes through the summer of 2009, each of which corroborated findings from this initial outbreak investigation. In combination, these showed 1.4–2.5‐fold increased risk of medically attended, laboratory‐confirmed pH1N1 illness among prior 2008–2009 TIV recipients. An additional Canadian study using the linked Manitoba immunization registry and administrative databases has also shown similar findings of increased risk (Dr Carole Beaudoin, Public Health Agency of Canada, personal communication). Thus, in Canada, 6 observational studies based on different methods and settings, including the current outbreak investigation, consistently showed increased risk of pH1N1 illness during the spring and summer of 2009 associated with prior receipt of the 2008–2009 TIV. Conversely, studies conducted outside of Canada have provided inconsistent results: 3 studies (from the United States and Australia) reported null effects, 4 (from the United States and Mexico) reported protective effects, and 1 other outbreak investigation (from the Untied States) reported increased risk.
Findings of pH1N1 risk associated with TIV—consistent in Canada but conflicting elsewhere—may have been due to methodological differences and/or unrecognized flaws, differences in immunization programs or population immunity, or a specific mechanistic effect of Canadian TIV. High rates of immunization and the use of a single domestic manufacturer to supply >75% of the TIV in Canada may have enhanced the power within Canada to detect a vaccine‐specific effect. Given the changed immunologic landscape following the first spring‐summer pandemic wave and the mass pH1N1 vaccination campaign during the fall 2009, it may not be possible to further resolve this issue epidemiologically. Studies using animal models, banked serum samples, or other in vitro experiments are needed to further assess this association.
Procedures for Respiratory Virus Detection and HI and MN Serologic Assays
Respiratory virus detection.Influenza detection in nasal/nasopharyngeal specimens was by a screening RT‐PCR assay targeting the matrix (M) gene of influenza A. Subtyping for seasonal human influenza A (A/H1 or A/H3) was performed by real‐time RT‐PCR targeting the HA gene. Subtyping for pH1N1 employed a conventional (end‐point) RT‐PCR assay targeting the HA gene (National Microbiology Laboratory [NML] RT‐PCR protocol distributed 24 April 2009). During the outbreak period, respiratory specimens were also tested by Luminex RVP Assay which detects a wide range of respiratory viruses including influenza A and B.
Serologic survey.Nurses collected 3‐mL blood in serum separation tubes from consenting individuals. Serum samples were separated within 8 h after blood collection, and stored at 2°C–4°C for up to 3 days until shipped to the BC Centre for Disease Control laboratory, where serum was stored at −20°C until testing. All serum samples were assayed in duplicate, and the geometric mean titer (GMT) of the duplicate results was reported as the individual result.
For the HI assay, antigen was produced in embryonated hens’ eggs inoculated with a BC isolate of A/California/07/2009‐like strain and used in its second passage. The hemagglutinin gene of this virus was sequenced and was shown to be identical at antigenic sites to that of A/California/07/2009 at the nucleotide. Serum was treated with Receptor Destroying Enzyme (RDE) to remove nonspecific agglutinins then heated at 56°C for 60 min to inactivate the RDE. Serum was tested for residual nonspecific agglutinins by reaction with guinea pig erythrocytes and those from which nonspecific agglutinins were not totally removed were further reacted with a 50% preparation of erythrocytes. Serum was then serially diluted from 1:10 to 1:1280 with PBS and 25 μL were reacted with 4 HA units (25 μL) of pH1N1 antigen for 45 minutes. To each mixture 50 μL of 0.7% guinea pig erythrocytes were added, and after mixing, the preparations were incubated for 1 h. The HI titre was designated as the inverse of the highest dilution to still show hemagglutination inhibition.
For the MN assay, the same virus was propagated in Madin‐Darby Canine Kidney (MDCK) cell cultures and titrated by median tissue culture infective dose (TCID50). The serum was subjected to 2‐fold serial dilution starting at a dilution of 1:10 and to each dilution 100 infectious units of virus were added. After incubation at 37°C for 2 h the mixtures were added to respective MDCK cell monolayers in 96‐well microtitre culture plates. After 3 h of further incubation the medium was aspirated from the monolayers and replaced with fresh Megavir serum‐free medium (Hyclone) containing L‐1‐tosylamido‐2‐phenylethyl chloromethyl ketone (TPCK)–treated trypsin. The monolayers, incubated at 37°C, were examined for the appearance of cytopathic effects (CPE) recorded on days 3, 4, and 5. The MN titer was defined as the inverse of the serum dilution in the well immediately preceding that manifesting CPE.