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Infection with H274Y-positive influenza A (H1N1) is not associated with a change in nasopharyngeal Streptococcus pneumoniae colonization in patients
International Journal of Infectious Diseases (2009) 13, e321—e322
http://intl.elsevierhealth.com/journals/ijid
LETTER TO THE EDITOR Infection with H274Y-positive influenza A (H1N1) is not associated with a change in nasopharyngeal Streptococcus pneumoniae colonization in patients Animal models have identified influenza virus infection as a contributing factor to nasopharyngeal colonization with Streptococcus pneumoniae.1—3 This colonization is thought to be positively influenced by the activity of viral neuraminidase. Previous studies suggest that H274Y mutants of influenza A isolated before the 2007—2008 respiratory season had a decreased neuraminidase activity compared to wild-type H274 strains.4,5 Thus, it might be postulated that infection with H274Y-positive strains of influenza virus would be associated with a lower S. pneumoniae colonization rate than infection with wild-type H274 strains.4 The purpose of this brief investigation was to determine whether nasopharyngeal specimens from patients infected with H274Y-positive strains of influenza A (H1N1) from the 2007—2008 respiratory season had different levels of S. pneumoniae colonization to specimens from patients infected with H274-positive influenza A. Nasopharyngeal specimens from patients with influenzalike illness were sent to the Ontario Public Health Laboratories. Isolates of influenza A collected from Toronto, Ontario, Canada (estimated population 2.7 million) between November 14th 2007 and February 14th 2008 were screened by reverse transcriptase (RT)-PCR for the H1N1 subtype using previously described primers.6 Typing of influenza isolates was done by Sanger sequencing and sequence alignment. Neuraminidase gene sequencing was undertaken and sequences were aligned using CLUSTALX. Isolate sequences were compared to influenza A virus sequences with described H274Y mutations: GenBank accession nos. EU516123 [A/ Hawaii/28/2007(H1N1)]; CY027037 [A/Kansas/UR06-0104/ 2007(H1N1)]; EU516027 [A/Texas/31/2007(H1N1)].6 Twenty specimens that had yielded H274Y mutant isolates and forty specimens that had yielded H274 wild-type isolates were randomly selected, and tested for the presence of S. pneumoniae. Because of the small number of specimens available for this study, we were unable to match for factors such as patient age, and severity and duration of infection at time of sampling. Specimens were considered to be positive for S. pneumoniae if genes for both pneumolysin7 and autolysin8 were detected. The Fisher’s exact test was carried out using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, California, USA).
Seventeen percent of all influenza A (H1N1) isolates collected during this time period carried an H274Y mutation. All influenza A (H1N1) isolates most closely resembled influenza A/Solomon Islands/03/06 (H1N1). Four of twenty (20%) specimens yielding wild-type H274 isolates and ten of forty (25%) yielding H274Y-positive (H1N1) isolates were positive for S. pneumoniae ( p = 0.8). However, there is a trend towards a lower prevalence of colonization using this assay if only the pneumolysin results are considered (45% infected with wildtype were colonized compared to 30% with the mutant) (Table 1). When all PCR results were analyzed, either pneumolysin or autolysin or both were detected in 9 of 20 (45%) specimens yielding wild-type H274 isolates and 13 of 40 (33%) specimens yielding H274Y-positive isolates ( p = 0.4) (Table 1). There are several caveats to this work. The sensitivity and specificity of this assay might be questionable, as only 14 of 21 specimens that were positive for pneumolysin were also positive for autolysin. These preliminary data suggest that a more comprehensive study of S. pneumoniae colonization using a more sensitive method is warranted. For example, a quantitative method might enable analysis of bacterial burden rather than just bacterial prevalence. Specimens from patients infected with wild-type and mutant strains of influenza were not matched; readers should therefore be aware that these results are preliminary, and that the small sample sizes and the underpowered nature of this study could bias findings. Thus, the lack of a significant difference in S. pneumoniae colonization prevalence between patients should not be exclusively used to rule out the possibility that patients infected with H274Y (mutant strains) of influenza are less likely to be colonized with S. pneumoniae. Multiple factors influence the chances of bacterial superinfection in patients following influenza infection.9 We believe that this study indicates that S. pneumoniae colonization is an important concern in patients infected with both H274-negative and H274Y-positive strains of influenza A (H1N1). This trend is of increasing importance in light of the emergence of H274Y-positive H1N1 influenza A during the 2007—2008 respiratory season10 and is an important complement to understanding the pathogenicity of these strains. One possible interpretation of these data is that strains of H274Y-positive influenza A (H1N1) circulating in 2007—2008 might not have compromised neuraminidase activity and cell culture infectivity compared to wild-type H274 strains.11 However, the caveats discussed above and the general trend towards lower levels of pneumolysin detected in patients
1201-9712/$36.00 . Crown Copyright # 2008 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. All rights reserved. doi:10.1016/j.ijid.2008.10.014
e322
Letter to the Editor
Table 1 The presence of S. pneumoniae genes in H274 and H274Y-positive nasopharyngeal specimens
Autolysin and pneumolysin Autolysin alone Pneumolysin alone No autolysin or pneumolysin
H274Y (n = 20)
H274 (n = 40)
4 0 5 11
10 1 2 27
with H274Y infection suggest that further studies with greater power are required to fully understand the dynamics of influenza and S. pneumoniae co-infections. We look forward to the results of further studies on the pathogenicity of H274Y-positive strains of influenza from the 2007—2008 and 2008—2009 respiratory seasons. Conflict of interest: No conflict of interest to declare.
7. Greiner O, Day PJR, Bosshard PP, Imeri F, Altwegg M, Nadal D. Quantitative detection of Streptococcus pneumoniae in nasopharyngeal secretions by real-time PCR. J Clin Microbiol 2001;39:3129—34. 8. McAvin JC, Reilly PA, Roudabush RM, Barnes WJ, Salmen A, Jackson GW, et al. Sensitive and specific method for rapid identification of Streptococcus pneumoniae using real-time fluorescence PCR. J Clin Microbiol 2001;39:3446—51. 9. McCullers JA, Bartmess KC. Role of neuraminidase in lethal synergism between influenza virus and Streptococcus pneumoniae. J Infect Dis 2003;187:1000—9. 10. European Centre for Disease Prevention and Control. Emergence of seasonal influenza viruses type A (H1N1) with oseltamivir resistance in some European Countries at the start of the 2007-2008 influenza season. 2008. http://ecdc.europa.eu/ pdf/080127_os.pdf. 11. Rameix-Welti MA, Enouf V, Cuvelier F, Jeannin P, van der Werf S. Enzymatic properties of the neuraminidase of seasonal H1N1 influenza viruses provide insights for the emergence of natural resistance to oseltamivir. PLoS Pathog 2008;4:e1000103.
AliReza Eshaghia Joanne Blaira Laura Burtona Kam Wing Choia Cedric De Limaa Allison A. McGeerb,c Donald E. Lowa,b,c Tony Mazzullia,b,c Steven J. Drewsa,b,c,*
References 1. Peltola VT, Boyd KL, McAuley JL, Rehg JE, McCullers JA. Bacterial sinusitis and otitis media following influenza virus infection in ferrets. Infect Immun 2006;74:2562—7. 2. Peltola VT, McCullers JA. Respiratory viruses predisposing to bacterial infections: role of neuraminidase. Pediatr Infect Dis J 2004;23:S87—97. 3. Peltola VT, Murti KG, McCullers JA. Influenza virus neuraminidase contributes to secondary bacterial pneumonia. J Infect Dis 2005;192:249—57. 4. Abed Y, Baz M, Boivin G. Impact of neuraminidase mutations conferring influenza resistance to neuraminidase inhibitors in the N1 and N2 genetic backgrounds. Antivir Ther 2006;11:971— 6. 5. Herlocher ML, Truscon R, Elias S, Yen HL, Roberts NA, Ohmit SE, Monto AS. Influenza viruses resistant to the antiviral drug oseltamivir: transmission studies in ferrets. J Infect Dis 2004;190:1627—30. 6. Takao S, Shimazu Y, Fukuda S, Kuwayama M, Miyazaki K. Neuraminidase subtyping of human influenza a viruses by RT-PCR and its application to clinical isolates. Jpn J Infect Dis 2002;55: 204—5.
a
Ontario Public Health Laboratories, Toronto, ON, Canada b Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada c Mount Sinai Hospital, Toronto, ON, Canada *
Corresponding author. Tel.: +416 235 5703 E-mail address: [email protected] (S.J Drews) Corresponding Editor: William Cameron 28 August 2008
http://intl.elsevierhealth.com/journals/ijid
LETTER TO THE EDITOR Infection with H274Y-positive influenza A (H1N1) is not associated with a change in nasopharyngeal Streptococcus pneumoniae colonization in patients Animal models have identified influenza virus infection as a contributing factor to nasopharyngeal colonization with Streptococcus pneumoniae.1—3 This colonization is thought to be positively influenced by the activity of viral neuraminidase. Previous studies suggest that H274Y mutants of influenza A isolated before the 2007—2008 respiratory season had a decreased neuraminidase activity compared to wild-type H274 strains.4,5 Thus, it might be postulated that infection with H274Y-positive strains of influenza virus would be associated with a lower S. pneumoniae colonization rate than infection with wild-type H274 strains.4 The purpose of this brief investigation was to determine whether nasopharyngeal specimens from patients infected with H274Y-positive strains of influenza A (H1N1) from the 2007—2008 respiratory season had different levels of S. pneumoniae colonization to specimens from patients infected with H274-positive influenza A. Nasopharyngeal specimens from patients with influenzalike illness were sent to the Ontario Public Health Laboratories. Isolates of influenza A collected from Toronto, Ontario, Canada (estimated population 2.7 million) between November 14th 2007 and February 14th 2008 were screened by reverse transcriptase (RT)-PCR for the H1N1 subtype using previously described primers.6 Typing of influenza isolates was done by Sanger sequencing and sequence alignment. Neuraminidase gene sequencing was undertaken and sequences were aligned using CLUSTALX. Isolate sequences were compared to influenza A virus sequences with described H274Y mutations: GenBank accession nos. EU516123 [A/ Hawaii/28/2007(H1N1)]; CY027037 [A/Kansas/UR06-0104/ 2007(H1N1)]; EU516027 [A/Texas/31/2007(H1N1)].6 Twenty specimens that had yielded H274Y mutant isolates and forty specimens that had yielded H274 wild-type isolates were randomly selected, and tested for the presence of S. pneumoniae. Because of the small number of specimens available for this study, we were unable to match for factors such as patient age, and severity and duration of infection at time of sampling. Specimens were considered to be positive for S. pneumoniae if genes for both pneumolysin7 and autolysin8 were detected. The Fisher’s exact test was carried out using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, California, USA).
Seventeen percent of all influenza A (H1N1) isolates collected during this time period carried an H274Y mutation. All influenza A (H1N1) isolates most closely resembled influenza A/Solomon Islands/03/06 (H1N1). Four of twenty (20%) specimens yielding wild-type H274 isolates and ten of forty (25%) yielding H274Y-positive (H1N1) isolates were positive for S. pneumoniae ( p = 0.8). However, there is a trend towards a lower prevalence of colonization using this assay if only the pneumolysin results are considered (45% infected with wildtype were colonized compared to 30% with the mutant) (Table 1). When all PCR results were analyzed, either pneumolysin or autolysin or both were detected in 9 of 20 (45%) specimens yielding wild-type H274 isolates and 13 of 40 (33%) specimens yielding H274Y-positive isolates ( p = 0.4) (Table 1). There are several caveats to this work. The sensitivity and specificity of this assay might be questionable, as only 14 of 21 specimens that were positive for pneumolysin were also positive for autolysin. These preliminary data suggest that a more comprehensive study of S. pneumoniae colonization using a more sensitive method is warranted. For example, a quantitative method might enable analysis of bacterial burden rather than just bacterial prevalence. Specimens from patients infected with wild-type and mutant strains of influenza were not matched; readers should therefore be aware that these results are preliminary, and that the small sample sizes and the underpowered nature of this study could bias findings. Thus, the lack of a significant difference in S. pneumoniae colonization prevalence between patients should not be exclusively used to rule out the possibility that patients infected with H274Y (mutant strains) of influenza are less likely to be colonized with S. pneumoniae. Multiple factors influence the chances of bacterial superinfection in patients following influenza infection.9 We believe that this study indicates that S. pneumoniae colonization is an important concern in patients infected with both H274-negative and H274Y-positive strains of influenza A (H1N1). This trend is of increasing importance in light of the emergence of H274Y-positive H1N1 influenza A during the 2007—2008 respiratory season10 and is an important complement to understanding the pathogenicity of these strains. One possible interpretation of these data is that strains of H274Y-positive influenza A (H1N1) circulating in 2007—2008 might not have compromised neuraminidase activity and cell culture infectivity compared to wild-type H274 strains.11 However, the caveats discussed above and the general trend towards lower levels of pneumolysin detected in patients
1201-9712/$36.00 . Crown Copyright # 2008 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. All rights reserved. doi:10.1016/j.ijid.2008.10.014
e322
Letter to the Editor
Table 1 The presence of S. pneumoniae genes in H274 and H274Y-positive nasopharyngeal specimens
Autolysin and pneumolysin Autolysin alone Pneumolysin alone No autolysin or pneumolysin
H274Y (n = 20)
H274 (n = 40)
4 0 5 11
10 1 2 27
with H274Y infection suggest that further studies with greater power are required to fully understand the dynamics of influenza and S. pneumoniae co-infections. We look forward to the results of further studies on the pathogenicity of H274Y-positive strains of influenza from the 2007—2008 and 2008—2009 respiratory seasons. Conflict of interest: No conflict of interest to declare.
7. Greiner O, Day PJR, Bosshard PP, Imeri F, Altwegg M, Nadal D. Quantitative detection of Streptococcus pneumoniae in nasopharyngeal secretions by real-time PCR. J Clin Microbiol 2001;39:3129—34. 8. McAvin JC, Reilly PA, Roudabush RM, Barnes WJ, Salmen A, Jackson GW, et al. Sensitive and specific method for rapid identification of Streptococcus pneumoniae using real-time fluorescence PCR. J Clin Microbiol 2001;39:3446—51. 9. McCullers JA, Bartmess KC. Role of neuraminidase in lethal synergism between influenza virus and Streptococcus pneumoniae. J Infect Dis 2003;187:1000—9. 10. European Centre for Disease Prevention and Control. Emergence of seasonal influenza viruses type A (H1N1) with oseltamivir resistance in some European Countries at the start of the 2007-2008 influenza season. 2008. http://ecdc.europa.eu/ pdf/080127_os.pdf. 11. Rameix-Welti MA, Enouf V, Cuvelier F, Jeannin P, van der Werf S. Enzymatic properties of the neuraminidase of seasonal H1N1 influenza viruses provide insights for the emergence of natural resistance to oseltamivir. PLoS Pathog 2008;4:e1000103.
AliReza Eshaghia Joanne Blaira Laura Burtona Kam Wing Choia Cedric De Limaa Allison A. McGeerb,c Donald E. Lowa,b,c Tony Mazzullia,b,c Steven J. Drewsa,b,c,*
References 1. Peltola VT, Boyd KL, McAuley JL, Rehg JE, McCullers JA. Bacterial sinusitis and otitis media following influenza virus infection in ferrets. Infect Immun 2006;74:2562—7. 2. Peltola VT, McCullers JA. Respiratory viruses predisposing to bacterial infections: role of neuraminidase. Pediatr Infect Dis J 2004;23:S87—97. 3. Peltola VT, Murti KG, McCullers JA. Influenza virus neuraminidase contributes to secondary bacterial pneumonia. J Infect Dis 2005;192:249—57. 4. Abed Y, Baz M, Boivin G. Impact of neuraminidase mutations conferring influenza resistance to neuraminidase inhibitors in the N1 and N2 genetic backgrounds. Antivir Ther 2006;11:971— 6. 5. Herlocher ML, Truscon R, Elias S, Yen HL, Roberts NA, Ohmit SE, Monto AS. Influenza viruses resistant to the antiviral drug oseltamivir: transmission studies in ferrets. J Infect Dis 2004;190:1627—30. 6. Takao S, Shimazu Y, Fukuda S, Kuwayama M, Miyazaki K. Neuraminidase subtyping of human influenza a viruses by RT-PCR and its application to clinical isolates. Jpn J Infect Dis 2002;55: 204—5.
a
Ontario Public Health Laboratories, Toronto, ON, Canada b Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada c Mount Sinai Hospital, Toronto, ON, Canada *
Corresponding author. Tel.: +416 235 5703 E-mail address: [email protected] (S.J Drews) Corresponding Editor: William Cameron 28 August 2008