Estimating background rates of Guillain-Barré Syndrome in Ontario in order to respond to safety concerns during pandemic H1N1/09 immunization campaign

Abstract Background The province of Ontario, Canada initiated mass immunization clinics with adjuvanted pandemic H1N1 influenza vaccine in October 2009. Due to the scale of the campaign, temporal associations with Guillain-Barré syndrome (GBS) and vaccination were expected. The objectives of this analysis were to estimate the number of background GBS cases expected to occur in the projected vaccinated population and to estimate the number of additional GBS cases which would be expected if an association with vaccination existed. The number of influenza-associated GBS cases was also determined. Methods Baseline incidence rates of GBS were determined from published Canadian studies and applied to projected vaccine coverage data to estimate the expected number of GBS cases in the vaccinated population. Assuming an association with vaccine existed, the number of additional cases of GBS expected was determined by applying the rates observed during the 1976 Swine Flu and 1992/1994 seasonal influenza campaigns in the United States. The number of influenza-associated GBS cases expected to occur during the vaccination campaign was determined based on risk estimates of GBS after influenza infection and provincial influenza infection rates using a combination of laboratory-confirmed cases and data from a seroprevalence study. Results The overall provincial vaccine coverage was estimated to be between 32% and 38%. Assuming 38% coverage, between 6 and 13 background cases of GBS were expected within this projected vaccinated cohort (assuming 32% coverage yielded between 5-11 background cases). An additional 6 or 42 cases would be expected if an association between GBS and influenza vaccine was observed (assuming 32% coverage yielded 5 or 35 additional cases); while up to 31 influenza-associated GBS cases could be expected to occur. In comparison, during the same period, only 7 cases of GBS were reported among vaccinated persons. Conclusions Our analyses do not suggest an increased number of GBS cases due to the vaccine. Awareness of expected rates of GBS is crucial when assessing adverse events following influenza immunization. Furthermore, since individuals with influenza infection are also at risk of developing GBS, they must be considered in such analyses, particularly if the vaccine campaign and disease are occurring concurrently

Humoral and Cell-Mediated Immunity to Pandemic H1N1 Influenza in a Canadian Cohort One Year Post-Pandemic: Implications for Vaccination

We evaluated a cohort of Canadian donors for T cell and antibody responses against influenza A/California/7/2009 (pH1N1) at 8-10 months after the 2nd pandemic wave by flow cytometry and microneutralization assays. Memory CD8 T cell responses to pH1N1 were detectable in 58% (61/105) of donors. These responses were largely due to cross-reactive CD8 T cell epitopes as, for those donors tested, similar recall responses were obtained to A/California 2009 and A/PR8 1934 H1N1 Hviruses. Longitudinal analysis of a single infected individual showed only a small and transient increase in neutralizing antibody levels, but a robust CD8 T cell response that rose rapidly post symptom onset, peaking at 3 weeks, followed by a gradual decline to the baseline levels seen in a seroprevalence cohort post-pandemic. The magnitude of the influenza-specific CD8 T cell memory response at one year post-pandemic was similar in cases and controls as well as in vaccinated and unvaccinated donors, suggesting that any T cell boosting from infection was transient. Pandemic H1-specific antibodies were only detectable in approximately half of vaccinated donors. However, those who were vaccinated within a few months following infection had the highest persisting antibody titers, suggesting that vaccination shortly after influenza infection can boost or sustain antibody levels. For the most part the circulating influenza-specific T cell and serum antibody levels in the population at one year post-pandemic were not different between cases and controls, suggesting that natural infection does not lead to higher long term T cell and antibody responses in donors with pre-existing immunity to influenza. However, based on the responses of one longitudinal donor, it is possible for a small population of pre-existing cross-reactive memory CD8 T cells to expand rapidly following infection and this response may aid in viral clearance and contribute to a lessening of disease severity

DNA restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolates from HIV-seropositive and HIV-seronegative patients in Kampala, Uganda

<p>Abstract</p> <p>Background</p> <p>The identification and differentiation of strains of <it>Mycobacterium tuberculosis </it>by DNA fingerprinting has provided a better understanding of the epidemiology and tracing the transmission of tuberculosis. We set out to determine if there was a relationship between the risk of belonging to a group of tuberculosis patients with identical mycobacterial DNA fingerprint patterns and the HIV sero-status of the individuals in a high TB incidence peri-urban setting of Kampala, Uganda.</p> <p>Methods</p> <p>One hundred eighty three isolates of <it>Mycobacterium tuberculosis </it>from 80 HIV seropositive and 103 HIV seronegative patients were fingerprinted by standard IS<it>6110</it>-RFLP. Using the BioNumerics software, strains were considered to be clustered if at least one other patient had an isolate with identical RFLP pattern.</p> <p>Results</p> <p>One hundred and eighteen different fingerprint patterns were obtained from the 183 isolates. There were 34 clusters containing 54% (99/183) of the patients (average cluster size of 2.9), and a majority (96.2%) of the strains possessed a high copy number (≥ 5 copies) of the IS<it>6110 </it>element. When strains with <5 bands were excluded from the analysis, 50.3% (92/183) were clustered, and there was no difference in the level of diversity of DNA fingerprints observed in the two sero-groups (adjusted odds ratio [aOR] 0.85, 95%CI 0.46–1.56, <it>P </it>= 0.615), patients aged <40 years (aOR 0.53, 95%CI 0.25–1.12, <it>P </it>= 0.100), and sex (aOR 1.12, 95%CI 0.60–2.06, <it>P </it>= 0.715).</p> <p>Conclusion</p> <p>The sample showed evidence of a high prevalence of recent transmission with a high average cluster size, but infection with an isolate with a fingerprint found to be part of a cluster was not associated with any demographic or clinical characteristics, including HIV status.</p

T cell analysis in the Toronto seroprevalence and case/control cohorts.

<p>(A) Bin separation of IFNγ responses in CD8 and CD4 T cells specific to pH1N1 stimulation. Frequencies have been corrected for background IFNγ production in LCMV and unstimulated control cultures. (B) Spearman correlation between pH1N1-responding CD8 T cells and donor age. (C) Combinations of effector molecule expression of IFNγ⁺ CD8 T cells from the responder subset. P values above the bars indicate the level of statistical significance compared to all other bars as determined by ANOVA and Tukey test. (D) Spearman correlation between the CD8 T cell response to pH1N1 and the frequency of responding cells with multiple effector functions. (E) CD8 T cell response in case and control subjects. Groups were compared using a nonparametic Mann-Whitney test. (F) Spearman correlation for pH1N1 response and frequency of CD8 T cells with multiple effector functions in cases and controls.</p

Infection followed by vaccination boosts antibody but not T cell responses to pandemic H1N1.

<p>(A) Antibody titers against pH1N1 for vaccinated and unvaccinated donors in the entire cohort 8-10 months post-pandemic. Vaccinations were self-reported from October 2009 to January 2010. A non-parametric Mann-Whitney test was used for statistical significance. (B) CD8 and CD4 responses to pH1N1 for vaccinated and unvaccinated donors in the total Toronto cohort, measured 8-10 months post-pandemic. Groups were compared using a Mann-Whitney test. (C) IFNγ⁺ CD8 T cell responses in donors with both antibody and CD8 T cell responses, T cell responses only, antibodies only, or no antibody or T cell response to pH1N1. Data has been normalized using log transformation to represent Gaussian distribution; groups were compared using ANOVA and Tukey test. (D) Normalized CD8 T cell response in cases and controls with differing vaccination history for pH1N1. Groups were compared by ANOVA and Tukey test. PCR-confirmed infections were reported from April-November 2009; vaccination was self-reported from October 2009-January 2010. (E) Pandemic-specific antibody responses as measured by microneutralization in the case/control cohort, separated by self-reported vaccination history for the monovalent pH1N1 vaccine. PCR-confirmed infections were reported from April-November 2009; vaccination was self-reported from October 2009-January 2010. Nonparametric Kruskal-Wallis and Mann-Whitney tests were performed to determine statistical significance.</p

Detection of influenza-responsive CD8 T cells by multicolour flow cytometry.

<p>Total PBMC were stimulated for 18 hours with pH1N1 influenza, or as a control, with LCMV Armstrong, or left unstimulated and then assessed for IFNγ production by intracellular cytokine staining and flow cytometry. Gates are based on fluorescence minus one controls. (A) Representative gating used to identify IFNγ⁺ CD8 T cells from total PBMC. (B) Sample non-responder, weak responder, and strong responder to pH1N1 identified in the Toronto cohort 8-10 months post-pandemic; positive versus non-responder is defined in the results. A representative “weak” responder was arbitrarily chosen from the bottom third of positive responses whereas the “strong” responder was from the top third of responders.</p

Acute and persisting antibody and memory T cell responses to pandemic H1N1 infection in one PCR case-confirmed donor.

<p>Longitudinal samples of unfractionated PBMC were challenged with influenza virus or controls for 18h. (A) Frequency of pandemic H1N1-responsive CD8 T cells out of total CD8 T cells as measured by IFNγ staining. IFNγ responsive CD8 T cells were also sub-divided by expression of other effector markers, granzyme B and CD107a. (B) Memory phenotypes of influenza-responsive CD8 T cells at various times post-onset of influenza symptoms. (C) Frequency and phenotypes of IFNγ⁺ CD4 T cells after pandemic H1N1 challenge. (D) Antibody titers in serum as detected by microneutralization (MN), hemagglutination inhibition (HAI), and a pandemic H1-specific ELISA assay. BLD  =  below the limits of detection.</p