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Copyright ? 2006 Elsevier Ltd All rights reserved. Influenza vaccination in patients on long-term anticoagulant therapy
A.M. Iorio<sup>a</sup><sup>, </sup><sup>
</sup><sup>, </sup><sup>
</sup>, B. Camilloni<sup>a</sup>, M. Basileo<sup>a</sup>, F. Guercini<sup>b</sup>, S. Conti<sup>b</sup>, F. Ferrante<sup>b</sup>, E. Paccamiccio<sup>c</sup>, S. Iaboni<sup>b</sup>, M. Vecchioli<sup>c</sup> and A. Iorio<sup>b</sup>
<sup>a</sup>Department of Hygiene, University of Perugia, Via del Giochetto, 06100 Perugia, Italy
<sup>b</sup>Department of Internal and Cardiovascular Medicine, University of Perugia, Ospedale Sant? Andrea delle Fratte, Perugia, Italy
<sup>c</sup>ASL 2 of Perugia, Madonna Alta, Perugia, Italy
Available online 8 June 2006.
Abstract
The study evaluates the risk/benefit of influenza vaccination in patients on stable long-term oral anticoagulant therapy (OAT). One hundred and four consecutive patients with indication for influenza vaccination were randomized to receive one dose of 2004/2005 influenza vaccine followed by placebo after 6 weeks, or vice versa, in a cross-over blinded trial. All patients were tested for anticoagulation levels and for hemagglutination inhibiting antibody titres against the influenza vaccine antigens. The highly protective antibody titres induced by influenza vaccination and the absence of statistically relevant interactions between vaccination and OAT suggest that influenza vaccination can be used safely and successfully in elderly patients on OAT.
Keywords: Influenza; Vaccine; Immunogenicity; Oral anticoagulants
Article Outline
<dl><dt>1. Introduction</dt><dt>2. Materials and methods</dt><dl><dt>2.1. Study population</dt><dt>2.2. Study design</dt><dt>2.3. Vaccination</dt><dt>2.4. Blood sampling and laboratory assays</dt><dt>2.5. Statistical analysis</dt></dl><dt>3. Results</dt><dl><dt>3.1. Flow chart of the study and characteristics of the study subjects</dt><dt>3.2. HI antibody response to influenza vaccine</dt><dt>3.3. Persistence of vaccine-induced antibody response</dt><dt>3.4. Coagulation variables and clinical events</dt></dl><dt>4. Discussion</dt><dt>Acknowledgements</dt><dt>References</dt></dl>
1. Introduction
Influenza vaccination is a recommended procedure for subjects at increased risk for complications of influenza, enclosed people with chronic cardio-cerebro-vascular disorders (CCVD) requiring long-term therapy with oral anticoagulants (OAT). In addition to the predictable advantage of influenza vaccination, previous epidemiological data have shown the possible occurrence of multiple interactions between infectious processes and CCVD. Excess deaths from ischemic hearth disease and stroke have been reported during influenza seasons [1], [2] and [3].
The association between influenza virus infection and CCVD was confirmed by studies showing that influenza vaccination may reduce the risk of CCVD-related deaths or hospital admissions [4]. Possible mechanisms of increased risk of CCVD-related events after influenza include alterations in circulating clotting factors, platelet aggregation, fibrinolysis, concentrations of inflammatory response proteins and of cytokines. These changes might enhance thrombotic tendencies, impair vasodilatation, or cause endothelial injury [3].
Although the above observations suggest that influenza might be a serious disease for CCVD patients, a correct appraisal of the risk/benefit ratio of influenza vaccination in CCVD patients requires the consideration of a second issue. In fact, a significant proportion of CCVD patients requires long-term OAT, and some concern has been raised about the potential adverse effect of influenza vaccination on prothrombin time (PT), since previous studies have yielded conflicting results, showing no influence [5] and [6], a moderate reduction [7], or a moderate increase [8].
In order to gain more information about the appropriateness and immunogenicity of influenza vaccination in patients on OAT, a prospective, controlled, randomized, cross-over study was conducted in the winter season 2004/2005.
2. Materials and methods
2.1. Study population
The study was conducted in the 2004/2005 winter season and involved CCVD patients requiring OAT and referring to the ASL 2 Coagulation Monitoring Network, Perugia, Italy. The study protocol was approved by the Ethic Committee of the Umbria Region. Patients participating in the study were all on stable OAT, aged 18 years or more and with indication for influenza vaccination. Stable OAT was defined by two criteria: treatment for more than 6 months and recent history of three consecutive PT values expressed as international normalized ratio (PT-INR) in the specific therapeutic range. Moreover patients were excluded from the study if they met any of the following criteria: sensitivity to vaccine or its components, short expectation of life, previous OAT duration shorter than 6 months, planned surgery within 6 months, refusal to sign informed consent.
2.2. Study design
The study was designed as a randomized, blinded study. To match the ethical need to administer influenza vaccination to all the patients and the investigational need to have a controlled trial, the study was planned as a cross-over study. The volunteers participating to the study were randomized in two groups, and all underwent influenza vaccination at least one month before the expected time of influenza virus circulation. A wash out period of 2 weeks was performed to better discriminate treatment effects.
The randomization was obtained by using a computer generated random series and the method of sealed envelopes. The patients, the physician encharged of OAT monitoring, those encharged of follow up visits and the technicians performing laboratory determinations were all unaware of patients treatment.
2.3. Vaccination
All patients were immunized in the deltoid muscle with one injection of the commercially available trivalent subunit MF-59 adjuvanted 2004/2005 influenza vaccine (FLUAD, Chiron, Siena, Italy; A/Fujian/411/02, H3N2; A/New Caledonia/20/99, H1N1 and B/Shanghai/361/02). Patients of group 1 received influenza vaccine at day 0 (mid of October) followed by an identical appearing placebo at day 42 (end of November). Vice versa, volunteers of group 2 received the placebo at day 0 (mid of October) and the vaccine at day 42 (end of November).
2.4. Blood sampling and laboratory assays
Blood samples were drawn in each phase of the study at baseline and after 1, 2 and 4 weeks from influenza vaccination or placebo administration. Blood was drawn with a clean venipuncture using a 19-gauge needle. Firstly, 10 ml of blood were drawn in dry tubes for serum antibodies determinations; thereafter, blood for coagulation testing was drawn in 0.312 mM trisodium citrate (1:9, vol:vol). Sera were stored in aliquots at −80 ?C until used. Titres of hemagglutination inhibiting (HI) antibody to the three influenza vaccine antigens were determined simultaneously for all serum samples taken from the same subject by a standard microtitre test using 0.5% turkey erythrocytes and egg-grown influenza antigens. Non-specific inhibitors were inactivated by treatment with receptor-destroying enzyme. The results obtained were evaluated as: (a) number of people with HI antibody titres ≥1:40, considered to be associated with protection from influenza; (b) geometric mean titres (GMT) (first dilution for antibody titration was 1:10 and any antibody titre <1:10 was considered 1:5); (c) number of patients with a positive response (seroconversion from <1/10 to ≥1:20 or at least four-fold increase). PT-INR was measured on fresh plasma within 2 h from venipuncture with one-stage prothrombin time method, using Recombiplastin on ACL10000 coagulometer (both from IL, Milan, Italy).
2.5. Statistical analysis
The sample size of the study was calculated considering that the PT-INR value (mean ? S.D.) of the population is 2.61 ? 0.51, and arbitrarily setting as cut off value a clinically significant variation of 0.5 U. Under this assumption, to reach a study power of 80% with an alpha error of 0.05%, 34 patients were needed. Since the standard deviation of the chosen difference is unknown, it was decided to multiply the sample size by five, to allow for withdrawals and to end with a population of at least 100 patients, usually considered adequate also for virological response evaluation. Differences in the frequency of qualitative variables were tested by Student's t-test (comparison of GMT) and χ<sup>2</sup>-test (comparison of number of people with protective antibody titers). The PT-INR data were examined with an ANOVA model for repeated measures coding the entry order in the cross-over design as a covariate. The study was analysed by intention to treat analysis, with the last observation carried forward method to treat missing data in order to avoid loss of power.
3. Results
3.1. Flow chart of the study and characteristics of the study subjects
The flow chart of the study is given in Table 1. Demographic data and clinical indications were obtained from patient at the beginning of the study, while at every study point the patients filled in a questionnaire regarding the ischemic or bleeding side effects or any known adverse reaction expected after vaccination. Weekly dosages of oral anticoagulants were recorded in the computerized program used to manage OAT (PARMA Rel. 5, Web version, IL, Milan, Italy). Table 1.
Study flow chart <table border="1" cellpadding="6" cellspacing="0" frame="hsides" rules="groups"><colgroup span="4"> <col><col><col><col></colgroup><thead valign="top"> <tr> <th align="left" valign="center">
</th><th align="left" valign="center">Blood samples</th><th colspan="2" id="col3" align="left" valign="center">Treatment</th> </tr><tr> <th align="left" valign="center">
</th><th align="left" valign="center">
</th><th align="left" valign="center">Group 1</th><th align="left" valign="center">Group 2</th> </tr></thead> <tbody> <tr> <td class="nowrap" colspan="4" headers="col1" align="left" valign="center">Week</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 0</td><td class="nowrap" align="left" valign="center"><sup>a</sup><sup>,</sup><sup>b</sup></td><td class="nowrap" align="left" valign="center">Vaccine</td><td class="nowrap" align="left" valign="center">Placebo</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 1</td><td class="nowrap" align="left" valign="center"><sup>a</sup></td><td class="nowrap" align="left" valign="center">
</td><td class="nowrap" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 2</td><td class="nowrap" align="left" valign="center"><sup>a</sup></td><td class="nowrap" align="left" valign="center">
</td><td class="nowrap" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 4</td><td class="nowrap" align="left" valign="center"><sup>a</sup><sup>,</sup><sup>b</sup></td><td class="nowrap" align="left" valign="center">
</td><td class="nowrap" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" colspan="4" headers="col1" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" colspan="2" headers="col1" align="left" valign="center">Wash-out</td><td class="nowrap" colspan="2" headers="col3" align="left" valign="center">Cross-over</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 6</td><td class="nowrap" align="left" valign="center"><sup>a</sup><sup>,</sup><sup>b</sup></td><td class="nowrap" align="left" valign="center">Placebo</td><td class="nowrap" align="left" valign="center">Vaccine</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 7</td><td class="nowrap" align="left" valign="center"><sup>a</sup></td><td class="nowrap" align="left" valign="center">
</td><td class="nowrap" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 9</td><td class="nowrap" align="left" valign="center"><sup>a</sup></td><td class="nowrap" align="left" valign="center">
</td><td class="nowrap" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 10</td><td class="nowrap" align="left" valign="center"><sup>a</sup><sup>,</sup><sup>b</sup></td><td class="nowrap" align="left" valign="center">
</td><td class="nowrap" align="left" valign="center">
</td> </tr></tbody> </table> <sup>a</sup> INR determination.
<sup>b</sup> HI antibody titration.
Of the 278 patients referring to the Coagulation Monitoring Network Centre, 217 satisfied all the inclusion criteria. One hundred and thirteen declined to participate (62 refused vaccination; 38 had already been vaccinated elsewhere; 13 refused consent for blood testing) and 104 entered the study giving written informed consent to participate in the trial. The 104 patients enrolled in the study were 57 males, 47 females, mean age 71.3 ? 9.2, mean body mass index 27 ? 3.8. Four patients did not complete the follow-up period because of no compliance. The clinical indication for OAT were the following: (a) atrial fibrillation (54%), (b) prosthetic heart valve (20.1%), (c) dilated cardiomiopathy/heart valve disease (11.5%), (d) recurrent venous thromboembolism (9.6%) and (e) arterial disease (4.8%). The characteristic of the patients entered in the study were overlapping with the whole OAT population of the centre.
Fifty-three patients in OAT were randomized at vaccination followed by placebo (group 1) and 51 at placebo followed by vaccine (group 2). No differences among demographic characteristics, health condition status and indication to OAT were found between the two groups.
3.2. HI antibody response to influenza vaccine
Since pre-vaccination HI antibody values against the three vaccine components were substantially the same in the two groups comparing blood samples collected on day 0 for group 1 and on days 0, 28 and 42 for group 2 (data not shown), vaccine immunogenicity was evaluated comparing HI titres against the three vaccine components in sera collected before and 4 weeks after vaccination. As reported in Table 2, 28 days after vaccination statistically significant increases in the values of GMT and the number of seroprotected patients were found in both groups against the three vaccine antigens. The values found 28 days after vaccination in groups 1 and 2 were similar, except for the GMT value against the A/H3N2 antigen, which was significantly lower in group 2 as compared with group 1 (384 versus 584, Table 2). The results obtained were assessed according to the criteria of European Commission for influenza vaccines in elderly population. The post-vaccination requirements [post-vaccination seroprotection (HI titre ≥1:40) ≥60%, mean fold increase (MFI) of geometric mean titres (GMT) ≥2, seroconversion ≥30%] were always met by recipients of both groups against all three vaccine antigens. The percentages of seroprotected people after vaccination ranged from 92 to 100%, the values of MFI of GMT from 2.7 to 8.9 and percentages of seroconversions from 33 to 82% (Table 2). Table 2.
HI antibody titres in group 1 (vaccine/placebo N = 53) and group 2 (placebo/vaccine N = 51) OAT patients before and 28 days after vaccination <table border="1" cellpadding="6" cellspacing="0" frame="hsides" rules="groups"><colgroup span="7"> <col><col><col><col><col><col><col></colgroup><thead valign="top"> <tr> <th align="left" valign="center">Antigen</th><th align="left" valign="center">Group</th><th colspan="2" id="col3" align="left" valign="center">GMT</th><th colspan="2" id="col5" align="left" valign="center">% HI titre ≥40 (N)</th><th align="left" valign="center">% Positive response (N)</th> </tr><tr> <th align="left" valign="center">
</th><th align="left" valign="center">
</th><th align="left" valign="center">Before vaccination</th><th align="left" valign="center">After (MFI) vaccination</th><th align="left" valign="center">Before vaccination</th><th align="left" valign="center">After vaccination</th><th align="left" valign="center">
</th> </tr></thead> <tbody> <tr> <td class="nowrap" align="left" valign="center">A/H3N2</td><td class="nowrap" valign="center">1</td><td class="nowrap" valign="center">78</td><td class="nowrap" valign="center">584 (7.5)<sup>*</sup><sup>,</sup><sup>**</sup></td><td class="nowrap" valign="center">75 (40)</td><td class="nowrap" valign="center">100<sup>**</sup> (53)</td><td class="nowrap" valign="center">74 (39)</td> </tr><tr> <td class="nowrap" headers="col1">
</td><td class="nowrap" valign="center">2</td><td class="nowrap" valign="center">50</td><td class="nowrap" valign="center">384 (7.7)<sup>**</sup></td><td class="nowrap" valign="center">61 (31)</td><td class="nowrap" valign="center">100<sup>**</sup> (51)</td><td class="nowrap" valign="center">82 (42)</td> </tr><tr> <td class="nowrap" colspan="7" headers="col1" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" align="left" valign="center">A/H1N1</td><td class="nowrap" valign="center">1</td><td class="nowrap" valign="center">40</td><td class="nowrap" valign="center">126 (3.2)<sup>**</sup></td><td class="nowrap" valign="center">57 (30)</td><td class="nowrap" valign="center">92<sup>**</sup> (49)</td><td class="nowrap" valign="center">47 (25)</td> </tr><tr> <td class="nowrap" headers="col1">
</td><td class="nowrap" valign="center">2</td><td class="nowrap" valign="center">34</td><td class="nowrap" valign="center">93 (2.7)<sup>**</sup></td><td class="nowrap" valign="center">55 (28)</td><td class="nowrap" valign="center">92<sup>**</sup> (47)</td><td class="nowrap" valign="center">33 (17)</td> </tr><tr> <td class="nowrap" colspan="7" headers="col1" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" align="left" valign="center">B</td><td class="nowrap" valign="center">1</td><td class="nowrap" valign="center">21</td><td class="nowrap" valign="center">186 (8.9)<sup>**</sup></td><td class="nowrap" valign="center">34 (18)</td><td class="nowrap" valign="center">96<sup>**</sup> (51)</td><td class="nowrap" valign="center">81 (43)</td> </tr><tr> <td class="nowrap" headers="col1">
</td><td class="nowrap" valign="center">2</td><td class="nowrap" valign="center">20</td><td class="nowrap" valign="center">118 (5.9)<sup>**</sup></td><td class="nowrap" valign="center">43 (22)</td><td class="nowrap" valign="center">96<sup>**</sup> (49)</td><td class="nowrap" valign="center">68 (35)</td> </tr></tbody> </table> <sup>*</sup> p < 0.05 comparing groups 1 and 2.
<sup>**</sup> p < 0.01 comparing pre and post-vaccination values.
3.3. Persistence of vaccine-induced antibody response
For 50 of the 53 patients of group 1 it was possible to examine antibody persistence after vaccination comparing GMT values determined before, 28, 42 and 70 days after vaccination. As reported in Fig. 1, high level of vaccine-induced antibodies persisted for at least 70 days against all three vaccine antigens. However, as expected, 70 days after vaccination, the values of HI titres were lower than those found 28 days after vaccination, but still statistically higher than those found before vaccine administration. The MFI of GMT on day 28 and on day 70 were 9.3 and 7.8 for A/H3N2, 3.7 and 2.7 A/H1N1 and 8.9 and 6.8 for B Antigen (Fig. 1).
(17K)
Fig. 1. Persistence of GMT HI antibodies values in patients of group 1 at different times after vaccine administration. <sup>*</sup>p < 0.01 comparing pre- and post-vaccination data (Student's t-test). (
) A/H3N2; (
) A/H1N1; (
) B.
3.4. Coagulation variables and clinical events
There was no significant baseline difference in the mean PT-INR values and mean weekly dosage between groups 1 and 2. The average variation of PT-INR value (0.03 ? 0.04 U versus 0.02 ? 0.10 U, active vaccine and placebo, respectively) and the average variation in the dosage of administered oral anticoagulants (1.1 ? 0.4 mg/week versus 0.9 ? 0.7 mg/week active vaccine and placebo, respectively) evaluated at the weeks reported in the study flow chart (Table 1) were similar in both groups. There were no significant differences in the two groups in the number of patients who showed a variation ≥0.5 or ≥1 U of PT-INR, that was on average below 19 and 3%, respectively at all time points. The mean percentage of time spent by patients within the therapeutic range, calculated as proposed by Rosendaal et al. [9], was identical in the groups (71.1%).
A single episode of recurrent venous thromboembolism was observed in a cancer patient with central venous catheter during the long-term follow up period. No major bleeding events were found during follow up, and 11 minor muco-cutaneous hemorrhagic events were recorded, 9 in group 1 (one traumatic and eight spontaneous), and 2 in group 2 (both spontaneous). Only 1 patient out of 11, after placebo injection, had a high value of INR (6.9), while the other 10 patients had PT-INR values in therapeutic range. Seventeen patients (16.3%) complained of local reactions to vaccine administration (itching, tenderness, swelling or cutaneous pain at injection site), while eight patients (7.7%) complained of symptoms after placebo injection. There were no systemic complications.
4. Discussion
This is the first randomized, blinded study of influenza vaccination of patients receiving long-term OAT, evaluating both the immunogenicity and the effect of influenza vaccination on PT-INR values in a well definite CCVD population.
The 104 participants in the study were elderly CCVD patients on long-term stable OAT considered to be a risk group for which annual influenza immunization is recommended. The patients were randomized in two groups, comparable for demographic and health status characteristics. Since they could not be randomized to placebo for ethical reasons, all of them received one injection of vaccine and one of placebo in a cross-over design. This was possible since, on the basis of laboratory based influenza surveillace data of the previous years (our data not shown), influenza virus activity was generally not found before January in the area where the participants resided (Umbria, Italy). The patients of the first group were vaccinated in mid October and those of the second group at the end of November, all in time for mounting a vaccine induced immune response before influenza virus circulation. The vaccine used in the study was the MF-59 adjuvanted subunit trivalent influenza vaccine (FLUAD, Chiron, Siena, Italy), commercially available for the 2004/2005 winter season.
One of the aims of the study was to investigate on the influenza vaccine immunogenicity and, in accordance with previuos results in elderly [10] and in HIV infected HAART treated volunteers [11], the MF-59 adjuvanted vaccine induced statistically significative increases of antibody titres against all three vaccine components in the elderly patients on OAT (Table 2). The vaccine stimulated HI antibody increases persisted for at least 70 days (Fig. 1). Moreover the HI post-vaccination response quantitatively exceeded the requirements of the European Commission, suggesting that long-term OAT did not influence the antibody response to influenza vaccine (Table 2).
A second aim of this study was to investigate the possibility of interference between influenza vaccination and OAT. Only subjects found to be on stable OAT in the weeks before influenza vaccination were included, since observations made in patients only after influenza vaccination might have the potential bias of attributing long-term effects of previous clinical and biological events to influenza vaccination. All the three main parameters, generally used in OAT interference studies, were considered and no differences were found on comparing the two groups for PT-INR values variation over time, weekly dosages of administered OAT and percentage of time spent within PT-INR value therapeutic range.
These results, obtained by examining a large number of patients and using an accurate controlled study design, support previous studies [5] and [6] showing that influenza vaccination did not have any significant effect on PT-INR values. Evidence for a moderate reduction [7] or a moderate increase [8] in PT-INR values, was less frequently reported and, in general, observed only in limited number of patients studied.
In accordance with previous data [6] a low incidence (16.3%) of local complications was found after intramuscolar injection of influenza vaccine, although intramuscolar injection has the potential to cause muscular hematomas in OAT patients. Clinically overt bleeding events, mainly mucocutaneous, were recorded in 11 patients. Since data from the literature suggest that they might be secondary to transient platelet disorders caused by vaccination or viral infection [12], it would be appropriate to include platelet count or platelet function determination in future studies.
In conclusion our results suggest that CCVD patients who are at risk for influenza-related complications and who are on stable OAT can be safely and successfully immunized with influenza vaccine.
Acknowledgements
We are indebted to Chiron for kindly providing the needed doses of FLUAD and to Instrumentation Laboratory for the reagents used for INR determinations.
References
[1] The Eurowinter Group, Cold exposure and winter mortality from ischemic heart disease, cerebrovascular disease, respiratory disease, and all causes in warm and cold regions of Europe, Lancet 349 (1997), pp. 1341?1343.
[2] M.J.W. Sprenger, R.J.A. Diepersloot, W.E.P. Beyer and N. Masurel, Influenza related excess mortality in The Netherlands, Lancet 336 (1990), pp. 382?383. Abstract + References in Scopus | Cited By in Scopus
[3] F.H.Y. Yap, P.-L. Ho, K.-F. Lam, P.K.S. Chan, Y.-H. Cheng and J.S.M. Peiris, Excess hospital admission for pneumonia, chronic obstructive pulmonary disease, and heart failure during influenza seasons in Hong Kong, J Med. Virol 73 (2004), pp. 617?623. Abstract-EMBASE | Abstract-MEDLINE | Full Text via CrossRef | Abstract + References in Scopus | Cited By in Scopus
[4] K.L. Nichol, J. Nordin, J. Mullooly, R. Lask, K. Fillibrandt and M. Iwane, Influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly, N Engl J Med 348 (2003), pp. 1322?1332. Abstract-MEDLINE | Abstract-EMBASE | Abstract-Elsevier BIOBASE | Full Text via CrossRef | Abstract + References in Scopus | Cited By in Scopus
[5] B.A. Lipsky, R.E. Pecoraro, N.J. Roben, P. de Blaquiere and C.J. Delaney, Influenza vaccination and warfarin anticoagulation, Ann Intern Med 100 (1984), pp. 835?837. Abstract-MEDLINE | Abstract-EMBASE | Abstract + References in Scopus | Cited By in Scopus
[6] G. Raj, R. Kumar and W.P. McKinney, Safety of intramuscolar influenza immunization among patients receiving long-term warfarin anticoagulation therapy, Arch Intern Med 155 (1995), pp. 1529?1533.
[7] D. Poli, L. Chiarugi, M. Capanni, E. Antonucci, R. Abbate and G.F. Gensini et al., Need of more frequent INR monitoring in elderly patients on long-term anticoagulation therapy after influenza vaccination, Blood Coagul Fibrin 13 (2002), pp. 297?300. Abstract-MEDLINE | Abstract-EMBASE | Abstract-Elsevier BIOBASE | Full Text via CrossRef | Abstract + References in Scopus | Cited By in Scopus
[8] P. Kramer, M. Tsuru, C.E. Cook, C.J. McClain and J.L. Holtzman, Effect of influenza vaccine on warfarin anticoagulation, Clin Pharmacol Ther 35 (1984), pp. 416?418. Abstract-EMBASE | Abstract-MEDLINE | Abstract + References in Scopus | Cited By in Scopus
[9] F.R. Rosendaal, S.C. Cannegieter, F.J. van der Meer and E. Briet, A method to determine the optimal intensity of oral anticoagulant therapy, Thromb Haemost 69 (1993), pp. 236?239. Abstract-MEDLINE | Abstract-EMBASE | Abstract + References in Scopus | Cited By in Scopus
[10] A. Podda, The adjuvanted influenza vaccines with novel adjuvants: experience with the MF-59-adjuvanted vaccine, Vaccine 19 (2001), pp. 2673?2680. SummaryPlus | Full Text + Links | PDF (190 K) | Abstract + References in Scopus | Cited By in Scopus
[11] A.M. Iorio, D. Francisci, B. Camilloni, G. Stagni, M. De Martino and D. Toneatto et al., Antibody responses and HIV-1 viral load in HIV-1-seropositive subjects immunised with either the MF59-adjuvanted influenza vaccine or a conventional non-adjuvanted subunit vaccine during highly active antiretroviral therapy, Vaccine 21 (2003), pp. 3629?3637. SummaryPlus | Full Text + Links | PDF (185 K) | Abstract + References in Scopus | Cited By in Scopus
[12] A. Schattner, Consequence or coincidence? The occurrence, pathogenesis and significance of autoimmune manifestations after viral vaccines, Vaccine 23 (2005) (30), pp. 3876?3886. SummaryPlus | Full Text + Links | PDF (140 K) | Abstract + References in Scopus | Cited By in Scopus
<sup></sup>Corresponding author. Tel.: +39 075 5857313; fax: +39 075 5857317.
A.M. Iorio<sup>a</sup><sup>, </sup><sup>
</sup><sup>, </sup><sup></sup>, B. Camilloni<sup>a</sup>, M. Basileo<sup>a</sup>, F. Guercini<sup>b</sup>, S. Conti<sup>b</sup>, F. Ferrante<sup>b</sup>, E. Paccamiccio<sup>c</sup>, S. Iaboni<sup>b</sup>, M. Vecchioli<sup>c</sup> and A. Iorio<sup>b</sup> <sup>a</sup>Department of Hygiene, University of Perugia, Via del Giochetto, 06100 Perugia, Italy
<sup>b</sup>Department of Internal and Cardiovascular Medicine, University of Perugia, Ospedale Sant? Andrea delle Fratte, Perugia, Italy
<sup>c</sup>ASL 2 of Perugia, Madonna Alta, Perugia, Italy
Available online 8 June 2006.
Abstract
The study evaluates the risk/benefit of influenza vaccination in patients on stable long-term oral anticoagulant therapy (OAT). One hundred and four consecutive patients with indication for influenza vaccination were randomized to receive one dose of 2004/2005 influenza vaccine followed by placebo after 6 weeks, or vice versa, in a cross-over blinded trial. All patients were tested for anticoagulation levels and for hemagglutination inhibiting antibody titres against the influenza vaccine antigens. The highly protective antibody titres induced by influenza vaccination and the absence of statistically relevant interactions between vaccination and OAT suggest that influenza vaccination can be used safely and successfully in elderly patients on OAT.
Keywords: Influenza; Vaccine; Immunogenicity; Oral anticoagulants
Article Outline
<dl><dt>1. Introduction</dt><dt>2. Materials and methods</dt><dl><dt>2.1. Study population</dt><dt>2.2. Study design</dt><dt>2.3. Vaccination</dt><dt>2.4. Blood sampling and laboratory assays</dt><dt>2.5. Statistical analysis</dt></dl><dt>3. Results</dt><dl><dt>3.1. Flow chart of the study and characteristics of the study subjects</dt><dt>3.2. HI antibody response to influenza vaccine</dt><dt>3.3. Persistence of vaccine-induced antibody response</dt><dt>3.4. Coagulation variables and clinical events</dt></dl><dt>4. Discussion</dt><dt>Acknowledgements</dt><dt>References</dt></dl>
1. Introduction
Influenza vaccination is a recommended procedure for subjects at increased risk for complications of influenza, enclosed people with chronic cardio-cerebro-vascular disorders (CCVD) requiring long-term therapy with oral anticoagulants (OAT). In addition to the predictable advantage of influenza vaccination, previous epidemiological data have shown the possible occurrence of multiple interactions between infectious processes and CCVD. Excess deaths from ischemic hearth disease and stroke have been reported during influenza seasons [1], [2] and [3].
The association between influenza virus infection and CCVD was confirmed by studies showing that influenza vaccination may reduce the risk of CCVD-related deaths or hospital admissions [4]. Possible mechanisms of increased risk of CCVD-related events after influenza include alterations in circulating clotting factors, platelet aggregation, fibrinolysis, concentrations of inflammatory response proteins and of cytokines. These changes might enhance thrombotic tendencies, impair vasodilatation, or cause endothelial injury [3].
Although the above observations suggest that influenza might be a serious disease for CCVD patients, a correct appraisal of the risk/benefit ratio of influenza vaccination in CCVD patients requires the consideration of a second issue. In fact, a significant proportion of CCVD patients requires long-term OAT, and some concern has been raised about the potential adverse effect of influenza vaccination on prothrombin time (PT), since previous studies have yielded conflicting results, showing no influence [5] and [6], a moderate reduction [7], or a moderate increase [8].
In order to gain more information about the appropriateness and immunogenicity of influenza vaccination in patients on OAT, a prospective, controlled, randomized, cross-over study was conducted in the winter season 2004/2005.
2. Materials and methods
2.1. Study population
The study was conducted in the 2004/2005 winter season and involved CCVD patients requiring OAT and referring to the ASL 2 Coagulation Monitoring Network, Perugia, Italy. The study protocol was approved by the Ethic Committee of the Umbria Region. Patients participating in the study were all on stable OAT, aged 18 years or more and with indication for influenza vaccination. Stable OAT was defined by two criteria: treatment for more than 6 months and recent history of three consecutive PT values expressed as international normalized ratio (PT-INR) in the specific therapeutic range. Moreover patients were excluded from the study if they met any of the following criteria: sensitivity to vaccine or its components, short expectation of life, previous OAT duration shorter than 6 months, planned surgery within 6 months, refusal to sign informed consent.
2.2. Study design
The study was designed as a randomized, blinded study. To match the ethical need to administer influenza vaccination to all the patients and the investigational need to have a controlled trial, the study was planned as a cross-over study. The volunteers participating to the study were randomized in two groups, and all underwent influenza vaccination at least one month before the expected time of influenza virus circulation. A wash out period of 2 weeks was performed to better discriminate treatment effects.
The randomization was obtained by using a computer generated random series and the method of sealed envelopes. The patients, the physician encharged of OAT monitoring, those encharged of follow up visits and the technicians performing laboratory determinations were all unaware of patients treatment.
2.3. Vaccination
All patients were immunized in the deltoid muscle with one injection of the commercially available trivalent subunit MF-59 adjuvanted 2004/2005 influenza vaccine (FLUAD, Chiron, Siena, Italy; A/Fujian/411/02, H3N2; A/New Caledonia/20/99, H1N1 and B/Shanghai/361/02). Patients of group 1 received influenza vaccine at day 0 (mid of October) followed by an identical appearing placebo at day 42 (end of November). Vice versa, volunteers of group 2 received the placebo at day 0 (mid of October) and the vaccine at day 42 (end of November).
2.4. Blood sampling and laboratory assays
Blood samples were drawn in each phase of the study at baseline and after 1, 2 and 4 weeks from influenza vaccination or placebo administration. Blood was drawn with a clean venipuncture using a 19-gauge needle. Firstly, 10 ml of blood were drawn in dry tubes for serum antibodies determinations; thereafter, blood for coagulation testing was drawn in 0.312 mM trisodium citrate (1:9, vol:vol). Sera were stored in aliquots at −80 ?C until used. Titres of hemagglutination inhibiting (HI) antibody to the three influenza vaccine antigens were determined simultaneously for all serum samples taken from the same subject by a standard microtitre test using 0.5% turkey erythrocytes and egg-grown influenza antigens. Non-specific inhibitors were inactivated by treatment with receptor-destroying enzyme. The results obtained were evaluated as: (a) number of people with HI antibody titres ≥1:40, considered to be associated with protection from influenza; (b) geometric mean titres (GMT) (first dilution for antibody titration was 1:10 and any antibody titre <1:10 was considered 1:5); (c) number of patients with a positive response (seroconversion from <1/10 to ≥1:20 or at least four-fold increase). PT-INR was measured on fresh plasma within 2 h from venipuncture with one-stage prothrombin time method, using Recombiplastin on ACL10000 coagulometer (both from IL, Milan, Italy).
2.5. Statistical analysis
The sample size of the study was calculated considering that the PT-INR value (mean ? S.D.) of the population is 2.61 ? 0.51, and arbitrarily setting as cut off value a clinically significant variation of 0.5 U. Under this assumption, to reach a study power of 80% with an alpha error of 0.05%, 34 patients were needed. Since the standard deviation of the chosen difference is unknown, it was decided to multiply the sample size by five, to allow for withdrawals and to end with a population of at least 100 patients, usually considered adequate also for virological response evaluation. Differences in the frequency of qualitative variables were tested by Student's t-test (comparison of GMT) and χ<sup>2</sup>-test (comparison of number of people with protective antibody titers). The PT-INR data were examined with an ANOVA model for repeated measures coding the entry order in the cross-over design as a covariate. The study was analysed by intention to treat analysis, with the last observation carried forward method to treat missing data in order to avoid loss of power.
3. Results
3.1. Flow chart of the study and characteristics of the study subjects
The flow chart of the study is given in Table 1. Demographic data and clinical indications were obtained from patient at the beginning of the study, while at every study point the patients filled in a questionnaire regarding the ischemic or bleeding side effects or any known adverse reaction expected after vaccination. Weekly dosages of oral anticoagulants were recorded in the computerized program used to manage OAT (PARMA Rel. 5, Web version, IL, Milan, Italy).
Table 1. Study flow chart <table border="1" cellpadding="6" cellspacing="0" frame="hsides" rules="groups"><colgroup span="4"> <col><col><col><col></colgroup><thead valign="top"> <tr> <th align="left" valign="center">
</th><th align="left" valign="center">Blood samples</th><th colspan="2" id="col3" align="left" valign="center">Treatment</th> </tr><tr> <th align="left" valign="center">
</th><th align="left" valign="center">
</th><th align="left" valign="center">Group 1</th><th align="left" valign="center">Group 2</th> </tr></thead> <tbody> <tr> <td class="nowrap" colspan="4" headers="col1" align="left" valign="center">Week</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 0</td><td class="nowrap" align="left" valign="center"><sup>a</sup><sup>,</sup><sup>b</sup></td><td class="nowrap" align="left" valign="center">Vaccine</td><td class="nowrap" align="left" valign="center">Placebo</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 1</td><td class="nowrap" align="left" valign="center"><sup>a</sup></td><td class="nowrap" align="left" valign="center">
</td><td class="nowrap" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 2</td><td class="nowrap" align="left" valign="center"><sup>a</sup></td><td class="nowrap" align="left" valign="center">
</td><td class="nowrap" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 4</td><td class="nowrap" align="left" valign="center"><sup>a</sup><sup>,</sup><sup>b</sup></td><td class="nowrap" align="left" valign="center">
</td><td class="nowrap" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" colspan="4" headers="col1" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" colspan="2" headers="col1" align="left" valign="center">Wash-out</td><td class="nowrap" colspan="2" headers="col3" align="left" valign="center">Cross-over</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 6</td><td class="nowrap" align="left" valign="center"><sup>a</sup><sup>,</sup><sup>b</sup></td><td class="nowrap" align="left" valign="center">Placebo</td><td class="nowrap" align="left" valign="center">Vaccine</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 7</td><td class="nowrap" align="left" valign="center"><sup>a</sup></td><td class="nowrap" align="left" valign="center">
</td><td class="nowrap" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 9</td><td class="nowrap" align="left" valign="center"><sup>a</sup></td><td class="nowrap" align="left" valign="center">
</td><td class="nowrap" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" align="left" valign="center"> 10</td><td class="nowrap" align="left" valign="center"><sup>a</sup><sup>,</sup><sup>b</sup></td><td class="nowrap" align="left" valign="center">
</td><td class="nowrap" align="left" valign="center">
</td> </tr></tbody> </table> <sup>a</sup> INR determination.
<sup>b</sup> HI antibody titration.
Fifty-three patients in OAT were randomized at vaccination followed by placebo (group 1) and 51 at placebo followed by vaccine (group 2). No differences among demographic characteristics, health condition status and indication to OAT were found between the two groups.
3.2. HI antibody response to influenza vaccine
Since pre-vaccination HI antibody values against the three vaccine components were substantially the same in the two groups comparing blood samples collected on day 0 for group 1 and on days 0, 28 and 42 for group 2 (data not shown), vaccine immunogenicity was evaluated comparing HI titres against the three vaccine components in sera collected before and 4 weeks after vaccination. As reported in Table 2, 28 days after vaccination statistically significant increases in the values of GMT and the number of seroprotected patients were found in both groups against the three vaccine antigens. The values found 28 days after vaccination in groups 1 and 2 were similar, except for the GMT value against the A/H3N2 antigen, which was significantly lower in group 2 as compared with group 1 (384 versus 584, Table 2). The results obtained were assessed according to the criteria of European Commission for influenza vaccines in elderly population. The post-vaccination requirements [post-vaccination seroprotection (HI titre ≥1:40) ≥60%, mean fold increase (MFI) of geometric mean titres (GMT) ≥2, seroconversion ≥30%] were always met by recipients of both groups against all three vaccine antigens. The percentages of seroprotected people after vaccination ranged from 92 to 100%, the values of MFI of GMT from 2.7 to 8.9 and percentages of seroconversions from 33 to 82% (Table 2).
Table 2. HI antibody titres in group 1 (vaccine/placebo N = 53) and group 2 (placebo/vaccine N = 51) OAT patients before and 28 days after vaccination <table border="1" cellpadding="6" cellspacing="0" frame="hsides" rules="groups"><colgroup span="7"> <col><col><col><col><col><col><col></colgroup><thead valign="top"> <tr> <th align="left" valign="center">Antigen</th><th align="left" valign="center">Group</th><th colspan="2" id="col3" align="left" valign="center">GMT</th><th colspan="2" id="col5" align="left" valign="center">% HI titre ≥40 (N)</th><th align="left" valign="center">% Positive response (N)</th> </tr><tr> <th align="left" valign="center">
</th><th align="left" valign="center">
</th><th align="left" valign="center">Before vaccination</th><th align="left" valign="center">After (MFI) vaccination</th><th align="left" valign="center">Before vaccination</th><th align="left" valign="center">After vaccination</th><th align="left" valign="center">
</th> </tr></thead> <tbody> <tr> <td class="nowrap" align="left" valign="center">A/H3N2</td><td class="nowrap" valign="center">1</td><td class="nowrap" valign="center">78</td><td class="nowrap" valign="center">584 (7.5)<sup>*</sup><sup>,</sup><sup>**</sup></td><td class="nowrap" valign="center">75 (40)</td><td class="nowrap" valign="center">100<sup>**</sup> (53)</td><td class="nowrap" valign="center">74 (39)</td> </tr><tr> <td class="nowrap" headers="col1">
</td><td class="nowrap" valign="center">2</td><td class="nowrap" valign="center">50</td><td class="nowrap" valign="center">384 (7.7)<sup>**</sup></td><td class="nowrap" valign="center">61 (31)</td><td class="nowrap" valign="center">100<sup>**</sup> (51)</td><td class="nowrap" valign="center">82 (42)</td> </tr><tr> <td class="nowrap" colspan="7" headers="col1" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" align="left" valign="center">A/H1N1</td><td class="nowrap" valign="center">1</td><td class="nowrap" valign="center">40</td><td class="nowrap" valign="center">126 (3.2)<sup>**</sup></td><td class="nowrap" valign="center">57 (30)</td><td class="nowrap" valign="center">92<sup>**</sup> (49)</td><td class="nowrap" valign="center">47 (25)</td> </tr><tr> <td class="nowrap" headers="col1">
</td><td class="nowrap" valign="center">2</td><td class="nowrap" valign="center">34</td><td class="nowrap" valign="center">93 (2.7)<sup>**</sup></td><td class="nowrap" valign="center">55 (28)</td><td class="nowrap" valign="center">92<sup>**</sup> (47)</td><td class="nowrap" valign="center">33 (17)</td> </tr><tr> <td class="nowrap" colspan="7" headers="col1" align="left" valign="center">
</td> </tr><tr> <td class="nowrap" align="left" valign="center">B</td><td class="nowrap" valign="center">1</td><td class="nowrap" valign="center">21</td><td class="nowrap" valign="center">186 (8.9)<sup>**</sup></td><td class="nowrap" valign="center">34 (18)</td><td class="nowrap" valign="center">96<sup>**</sup> (51)</td><td class="nowrap" valign="center">81 (43)</td> </tr><tr> <td class="nowrap" headers="col1">
</td><td class="nowrap" valign="center">2</td><td class="nowrap" valign="center">20</td><td class="nowrap" valign="center">118 (5.9)<sup>**</sup></td><td class="nowrap" valign="center">43 (22)</td><td class="nowrap" valign="center">96<sup>**</sup> (49)</td><td class="nowrap" valign="center">68 (35)</td> </tr></tbody> </table> <sup>*</sup> p < 0.05 comparing groups 1 and 2.
<sup>**</sup> p < 0.01 comparing pre and post-vaccination values.
For 50 of the 53 patients of group 1 it was possible to examine antibody persistence after vaccination comparing GMT values determined before, 28, 42 and 70 days after vaccination. As reported in Fig. 1, high level of vaccine-induced antibodies persisted for at least 70 days against all three vaccine antigens. However, as expected, 70 days after vaccination, the values of HI titres were lower than those found 28 days after vaccination, but still statistically higher than those found before vaccine administration. The MFI of GMT on day 28 and on day 70 were 9.3 and 7.8 for A/H3N2, 3.7 and 2.7 A/H1N1 and 8.9 and 6.8 for B Antigen (Fig. 1).
Fig. 1. Persistence of GMT HI antibodies values in patients of group 1 at different times after vaccine administration. <sup>*</sup>p < 0.01 comparing pre- and post-vaccination data (Student's t-test). (
There was no significant baseline difference in the mean PT-INR values and mean weekly dosage between groups 1 and 2. The average variation of PT-INR value (0.03 ? 0.04 U versus 0.02 ? 0.10 U, active vaccine and placebo, respectively) and the average variation in the dosage of administered oral anticoagulants (1.1 ? 0.4 mg/week versus 0.9 ? 0.7 mg/week active vaccine and placebo, respectively) evaluated at the weeks reported in the study flow chart (Table 1) were similar in both groups. There were no significant differences in the two groups in the number of patients who showed a variation ≥0.5 or ≥1 U of PT-INR, that was on average below 19 and 3%, respectively at all time points. The mean percentage of time spent by patients within the therapeutic range, calculated as proposed by Rosendaal et al. [9], was identical in the groups (71.1%).
A single episode of recurrent venous thromboembolism was observed in a cancer patient with central venous catheter during the long-term follow up period. No major bleeding events were found during follow up, and 11 minor muco-cutaneous hemorrhagic events were recorded, 9 in group 1 (one traumatic and eight spontaneous), and 2 in group 2 (both spontaneous). Only 1 patient out of 11, after placebo injection, had a high value of INR (6.9), while the other 10 patients had PT-INR values in therapeutic range. Seventeen patients (16.3%) complained of local reactions to vaccine administration (itching, tenderness, swelling or cutaneous pain at injection site), while eight patients (7.7%) complained of symptoms after placebo injection. There were no systemic complications.
4. Discussion
This is the first randomized, blinded study of influenza vaccination of patients receiving long-term OAT, evaluating both the immunogenicity and the effect of influenza vaccination on PT-INR values in a well definite CCVD population.
The 104 participants in the study were elderly CCVD patients on long-term stable OAT considered to be a risk group for which annual influenza immunization is recommended. The patients were randomized in two groups, comparable for demographic and health status characteristics. Since they could not be randomized to placebo for ethical reasons, all of them received one injection of vaccine and one of placebo in a cross-over design. This was possible since, on the basis of laboratory based influenza surveillace data of the previous years (our data not shown), influenza virus activity was generally not found before January in the area where the participants resided (Umbria, Italy). The patients of the first group were vaccinated in mid October and those of the second group at the end of November, all in time for mounting a vaccine induced immune response before influenza virus circulation. The vaccine used in the study was the MF-59 adjuvanted subunit trivalent influenza vaccine (FLUAD, Chiron, Siena, Italy), commercially available for the 2004/2005 winter season.
One of the aims of the study was to investigate on the influenza vaccine immunogenicity and, in accordance with previuos results in elderly [10] and in HIV infected HAART treated volunteers [11], the MF-59 adjuvanted vaccine induced statistically significative increases of antibody titres against all three vaccine components in the elderly patients on OAT (Table 2). The vaccine stimulated HI antibody increases persisted for at least 70 days (Fig. 1). Moreover the HI post-vaccination response quantitatively exceeded the requirements of the European Commission, suggesting that long-term OAT did not influence the antibody response to influenza vaccine (Table 2).
A second aim of this study was to investigate the possibility of interference between influenza vaccination and OAT. Only subjects found to be on stable OAT in the weeks before influenza vaccination were included, since observations made in patients only after influenza vaccination might have the potential bias of attributing long-term effects of previous clinical and biological events to influenza vaccination. All the three main parameters, generally used in OAT interference studies, were considered and no differences were found on comparing the two groups for PT-INR values variation over time, weekly dosages of administered OAT and percentage of time spent within PT-INR value therapeutic range.
These results, obtained by examining a large number of patients and using an accurate controlled study design, support previous studies [5] and [6] showing that influenza vaccination did not have any significant effect on PT-INR values. Evidence for a moderate reduction [7] or a moderate increase [8] in PT-INR values, was less frequently reported and, in general, observed only in limited number of patients studied.
In accordance with previous data [6] a low incidence (16.3%) of local complications was found after intramuscolar injection of influenza vaccine, although intramuscolar injection has the potential to cause muscular hematomas in OAT patients. Clinically overt bleeding events, mainly mucocutaneous, were recorded in 11 patients. Since data from the literature suggest that they might be secondary to transient platelet disorders caused by vaccination or viral infection [12], it would be appropriate to include platelet count or platelet function determination in future studies.
In conclusion our results suggest that CCVD patients who are at risk for influenza-related complications and who are on stable OAT can be safely and successfully immunized with influenza vaccine.
Acknowledgements
We are indebted to Chiron for kindly providing the needed doses of FLUAD and to Instrumentation Laboratory for the reagents used for INR determinations.
References
[1] The Eurowinter Group, Cold exposure and winter mortality from ischemic heart disease, cerebrovascular disease, respiratory disease, and all causes in warm and cold regions of Europe, Lancet 349 (1997), pp. 1341?1343.
[2] M.J.W. Sprenger, R.J.A. Diepersloot, W.E.P. Beyer and N. Masurel, Influenza related excess mortality in The Netherlands, Lancet 336 (1990), pp. 382?383. Abstract + References in Scopus | Cited By in Scopus
[3] F.H.Y. Yap, P.-L. Ho, K.-F. Lam, P.K.S. Chan, Y.-H. Cheng and J.S.M. Peiris, Excess hospital admission for pneumonia, chronic obstructive pulmonary disease, and heart failure during influenza seasons in Hong Kong, J Med. Virol 73 (2004), pp. 617?623. Abstract-EMBASE | Abstract-MEDLINE | Full Text via CrossRef | Abstract + References in Scopus | Cited By in Scopus
[4] K.L. Nichol, J. Nordin, J. Mullooly, R. Lask, K. Fillibrandt and M. Iwane, Influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly, N Engl J Med 348 (2003), pp. 1322?1332. Abstract-MEDLINE | Abstract-EMBASE | Abstract-Elsevier BIOBASE | Full Text via CrossRef | Abstract + References in Scopus | Cited By in Scopus
[5] B.A. Lipsky, R.E. Pecoraro, N.J. Roben, P. de Blaquiere and C.J. Delaney, Influenza vaccination and warfarin anticoagulation, Ann Intern Med 100 (1984), pp. 835?837. Abstract-MEDLINE | Abstract-EMBASE | Abstract + References in Scopus | Cited By in Scopus
[6] G. Raj, R. Kumar and W.P. McKinney, Safety of intramuscolar influenza immunization among patients receiving long-term warfarin anticoagulation therapy, Arch Intern Med 155 (1995), pp. 1529?1533.
[7] D. Poli, L. Chiarugi, M. Capanni, E. Antonucci, R. Abbate and G.F. Gensini et al., Need of more frequent INR monitoring in elderly patients on long-term anticoagulation therapy after influenza vaccination, Blood Coagul Fibrin 13 (2002), pp. 297?300. Abstract-MEDLINE | Abstract-EMBASE | Abstract-Elsevier BIOBASE | Full Text via CrossRef | Abstract + References in Scopus | Cited By in Scopus
[8] P. Kramer, M. Tsuru, C.E. Cook, C.J. McClain and J.L. Holtzman, Effect of influenza vaccine on warfarin anticoagulation, Clin Pharmacol Ther 35 (1984), pp. 416?418. Abstract-EMBASE | Abstract-MEDLINE | Abstract + References in Scopus | Cited By in Scopus
[9] F.R. Rosendaal, S.C. Cannegieter, F.J. van der Meer and E. Briet, A method to determine the optimal intensity of oral anticoagulant therapy, Thromb Haemost 69 (1993), pp. 236?239. Abstract-MEDLINE | Abstract-EMBASE | Abstract + References in Scopus | Cited By in Scopus
[10] A. Podda, The adjuvanted influenza vaccines with novel adjuvants: experience with the MF-59-adjuvanted vaccine, Vaccine 19 (2001), pp. 2673?2680. SummaryPlus | Full Text + Links | PDF (190 K) | Abstract + References in Scopus | Cited By in Scopus
[11] A.M. Iorio, D. Francisci, B. Camilloni, G. Stagni, M. De Martino and D. Toneatto et al., Antibody responses and HIV-1 viral load in HIV-1-seropositive subjects immunised with either the MF59-adjuvanted influenza vaccine or a conventional non-adjuvanted subunit vaccine during highly active antiretroviral therapy, Vaccine 21 (2003), pp. 3629?3637. SummaryPlus | Full Text + Links | PDF (185 K) | Abstract + References in Scopus | Cited By in Scopus
[12] A. Schattner, Consequence or coincidence? The occurrence, pathogenesis and significance of autoimmune manifestations after viral vaccines, Vaccine 23 (2005) (30), pp. 3876?3886. SummaryPlus | Full Text + Links | PDF (140 K) | Abstract + References in Scopus | Cited By in Scopus
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