American Journal of Infection Control 43 (2015) 460-4
Contents lists available at ScienceDirect
American Journal of Infection Control
American Journal of Infection Control
journal homepage: www.ajicjournal.org
Major article
Influenza virus prevalence in asymptomatic and symptomatic subjects during pandemic and postpandemic periods Thaís Boim Melchior MD, Ana Helena Perosa MD *, Clarice Neves Camargo PhD, Celso Granato PhD, Nancy Bellei PhD Clinical Virology Laboratory e Infectious Disease Unit, Medicine Department, Federal University of Sao Paulo, SP, Brazil
Key Words: Infection Caregivers Real-time PCR Respiratory virus
Background: In 2009, Influenza A(H1N1)pdm09 had a major effect on global health, causing thousands of deaths. However, the results of asymptomatic infection in transmission has not been well studied. We analyzed asymptomatic influenza infection in individuals with different risk factors for acquiring influenza in a university hospital. Methods: Respiratory samples from 100 children with respiratory symptoms, their asymptomatic caregivers, 100 asymptomatic HIV-infected patients, and 100 health care workers collected during 2009-2011 were tested for influenza by real time real-time polymerase chain reaction. Results: Influenza infection rate in symptomatic children was 44% and in asymptomatic adults it was 8.3% (P < .01). Children older than age 5 years had a 3.4 times greater chance of being infected during influenza season than younger children. Among the asymptomatic group, influenza was more frequent in caregivers (14%; P ¼ .032) and a higher rate (31.8%) was observed if a child was infected during the 2009 pandemic. Contact with an infected child was an important risk factor for influenza acquisition (odds ratio, 3.8; 95% confidence interval, 1.1-13.2) among caregivers. The mean cluster of differentiation 4 T-cell count of HIV-positive patients infected with influenza was not statistically different from uninfected patients (P ¼ .29). Conclusions: Asymptomatic household contacts of symptomatic children may play an important role in community transmission and a more proactive intervention should be considered during future pandemics. Copyright Ó 2015 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.
The influenza A(H1N1)pdm09 (pH1N1) virus had a major effect on global health in 2009, causing thousands of deaths. In 2009, the virus caused 2,060 deaths and more than 50,000 cases of illness in Brazil, with the highest incidence in children younger than age 2 years and adults aged 20-29 years.1,2 It is known that influenza infection is often asymptomatic, occurring in approximately 30%-50% of cases.3,4 Asymptomatic individuals are not easily identified and isolated, increasing their ability to transmit disease.3,5 Cases of asymptomatic pH1N1
* Address correspondence to Ana Helena Perosa, MD, 781 Pedro de Toledo St, 15th Fl, Vila Clementino, Sao Paulo e SP, CEP 04039-032, Brazil. E-mail address: [email protected] (A.H. Perosa). This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (grant No. 2010/09272-5). TBM, AHP, CG, and NB participated in the data analysis, writing, and review of the manuscript. TBM, AHP, and CNC participated in the laboratory analysis. Conflicts of interest: None to report.
infection in patients and health care workers (HCWs) previously exposed to sick patients have been reported.5-7 Complications resulting from influenza infection are more common in immunocompromised patients.8 There is a lack of knowledge about the effect of pH1n1 in individuals with HIV infection. However, according to the World Health Organization, HIV-positive individuals, particularly those with low cluster of differentiation 4 counts, are at high risk for developing complications from infection by a new viral subtype and should have priority in the prevention and treatment of disease.9,10 Household contact with children seems to be more important in disease transmission than exposure to other patients.11 Exposure to the index patient during the symptomatic phase and longer exposure times can increase the risk of acquiring the infection.12 We analyzed the prevalence of influenza infection in asymptomatic and symptomatic individuals from different populations at a university hospital in Sao Paulo, Brazil, during the pandemic and postpandemic periods. The frequency of influenza infection in
0196-6553/$36.00 - Copyright Ó 2015 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajic.2015.01.032
T.B. Melchior et al. / American Journal of Infection Control 43 (2015) 460-4
461
Fig 1. Distribution of samples collected during the study, by group. HCW, health care worker.
asymptomatic subjects with different risk factors is described. These subjects included HCWs, HIV-infected patients, and caregivers of children with respiratory symptoms. METHODS Study design A prospective study of 400 samples was established considering the 4 groups evaluated (ie, HCW, HIV, caregivers, and children) so that each was composed of 100 individuals, which was a sample size calculated to be sufficient to determine a prevalence of influenza from 10%-30% with a 90% confidence interval and 0.05 alpha error. The goal of sampling was that we could determine the prevalence for influenza acquisition in 4 different risk groups. Patients Patients were actively recruited in different health care units of a university hospital in Sao Paulo, Brazil, during the 2009 pandemic (June-October), 2010 (March-October), and 2011 (May- October). The inclusion criterion for asymptomatic adults was the absence of respiratory symptoms during the 15 days before sample collection. For symptomatic children (up to age 12 years) inclusion criterion was the diagnosis of acute respiratory infection within a week before sample collection. Samples Three hundred samples were collected from nonhospitalized asymptomatic adults: 100 from HIV-positive outpatients from an HIV ambulatory clinic recruited once a week during a scheduled routine visit, 100 HCWs (physicians and nurses) with direct contact with patient from different hospital wards/clinics (ie, nephrology, pediatric, hematology, infectious diseases, transplant, cardiology, psychiatry, general surgery, laboratory, and intensive care unit), and 100 from caregivers (all household contacts) of symptomatic children. An additional 100 samples were collected from symptomatic children. Children were outpatients recruited on a weekly
basis, twice a week. Asymptomatic caregiver and symptomatic child pairs were interviewed by 1 researcher following referral by a pediatrician. Samples were collected only from children for whom the asymptomatic caregiver agreed to participate. Figure 1 shows the samples collected from these 400 individuals between July 2009 and October 2011. The use of the data, the terms of consent, and all procedures were approved by the Ethics Committee of the Hospital Sao Paulo (protocol No. 0700/10). Laboratory methods Nasopharyngeal swabs were collected, immediately transported to the virology laboratory, and aliquots were frozen at 80 C for further analysis by polymerase chain reaction. Nucleic acid was extracted from 140ul of collected sample using the QIAmp Viral RNA Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The influenza A real-time polymerase chain reaction test was performed according to the Centers for Disease Control and Prevention protocol for pH1N1.13 The influenza B real-time polymerase chain reaction test was performed after standardization in our laboratory, using primers previously described.14 Samples positive for primer INFA and negative for the primers SwINFA and SwH1 in the Centers for Disease Control and Prevention protocol were considered as influenza A nontyped. The reactions were performed with AgPath-ID One Step RT-PCR (Applied Biosystems, Austin, TX) on a model ABI 7500 real-time machine (Applied Biosystems, Austin, TX), and ribonuclease P was used as the internal control. Statistical analysis was performed using c2 or Student t tests to evaluate the variables; a P value 50%, a quarter of household contacts of symptomatic children were also infected with influenza and the potential for transmission cannot be underestimated, even if they remain asymptomatic. These data suggest the use of a more consistent approach, such as prophylaxis or preemptive treatment during future pandemics. A limitation of our study was the recruitment of asymptomatic patients during pandemic periods when the influenza attack rate is higher than during epidemic periods. Another limitation was that HCWs who were concerned about their higher risk of influenza may have protected themselves better than other HCWs, which could decrease the relative risk of influenza infection in this group.
464
T.B. Melchior et al. / American Journal of Infection Control 43 (2015) 460-4
CONCLUSIONS The occurrence of asymptomatic infections and their frequency in different populations highlights the importance of asymptomatic infection in influenza circulation. Family members of children with influenza may have an effect on viral transmission among the general population during pandemic periods. Additional studies should be conducted to better understand the clinical significance of asymptomatic infection by different subtypes of influenza virus during epidemics and pandemics.
References 1. Brasil. SVS/MS. Boletim eletrônico epidemiológico: caderno especial influenza, Influenza Pandêmica (H1N1) 2009. Análise da situação epidemiológica e da resposta no ano de 2009 2010;10:1-21. Disponível em, http://portal.saude.gov. br/portal/arquivos/pdf/boletim_eletronico_influenza_25_03.pdf [acesso em maio 2010]. 2. Informe técnico de influenza e Vigilância da Síndrome respiratória aguda grave (SRAG), de síndrome gripal (SG) e de internações por CID J09 a 118. Ministério da Saúde. Secretaria de Vigilância em Saúde. Disponível em, http://portal.saude.gov. br/portal/arquivos/pdf/info_tecn_influenza_31_01_2012_28novo _29.pdf; 2012 [acesso em jan 2012]. 3. Hsu SB, Hsieh YH. On the role of asymptomatic infection in transmission Dynamics of infectious diseases. Bull Math Biol 2008;70:134-55. 4. Lau LL, Cowling BJ, Fang VJ, Chan KH, Lau EH, Lipsitch M, et al. Viral shedding and clinical illness in naturally acquired influenza virus infections. J Infect Dis 2010;201:1509-16. 5. Yang J, Yang F, Huang F, Wang J, Jin Q. Subclinical infection with the novel influenza a (H1N1) virus. Clin Infect Dis 2009;49:1622-3. 6. Cheng VC, Tai JW, Wong LM, Chan JF, Li IW, To KK, et al. Prevention of nosocomial transmission of swine-origin pandemic influenza virus A/H1N1 by infection control bundle. J Hosp Infect 2010;74:271-7. 7. Meyer S, Ilchmann C, Adam M, Sattinger E, Siamkouris D, Arunagirinathan U, et al. Asymptomatic infection with novel influenza A/H1N1 virus in a heart transplant recipient. J Heart Lung Transplant 2010;29:585-6. 8. Wilschut JC, McElhaney JE, Palache AM. Pathogenesis, clinical features and diagnosis. In: Influenza. 2nd ed. Philadelphia: Mosby; 2006. 9. Centers for Disease Control and Prevention. Updated Interim Recommendations d HIV-Infected Adults and Adolescents: Considerations for Clinicians Regarding 2009 H1N1 Influenza. Available from: http://www.cdc.gov/h1n1flu/ guidance_hiv.htm; 2009. Accessed February 8, 2010.
10. World Health Organization. Considerations on influenza A (H1N1) and HIV infection. Available from: http://www.who.int/hiv/mediacentre/influenza_hiv. pdf; 2009. Accessed February 8, 2010. 11. Williams CJ, Schweiger B, Diner G, Gerlach F, Haaman F, Krause G, et al. Seasonal influenza risk in hospital healthcare workers is more strongly associated with household than occupational exposures: results from a prospective cohort study in Berlin, Germany, 2006/07. BMC Infect Dis 2010;10:8. 12. Pang X, Yang P, Li S, Zhang L, Tian L, Li Y, et al. Pandemic (H1N1) 2009 among quarantined close contacts, Beijing, People’s Republic of China. Emerg Infect Dis 2011;17:1824-30. 13. Centers for Disease Control and Prevention. CDC protocol of realtime RTPCR for influenza A (H1N1). Available from: http://www.who.int/csr/resources/ publications/swineflu/CDCRealtimeRTPCR_SwineH1Assay-2009_20090430.pdf; 2009. Accessed January 27, 2010. 14. Selvaraju SB, Selvarangan R. Evaluation of three influenza A and B real-time reverse transcription-PCR assays and a new 2009 H1N1 assay for detection of influenza viruses. J Clin Microbiol 2010;48:3870-5. 15. Glatman-Freedman A, Portelli I, Jacobs SK, Mathew JI, Slutzman JE, Goldfrank LR, et al. Attack rates assessment of the 2009 pandemic H1N1 influenza A in children and their contacts: a systematic review and metaanalysis. PLoS One 2012;7:e50228. 16. Vesikari T, Pellegrini M, Karvonen A, Groth N, Borkowski A, O’Hagan DT, et al. Enhanced immunogenicity of seasonal influenza vaccines in young children using MF59 adjuvant. Pediatr Infect Dis J 2009;28:563-71. 17. Guatura SB, Watanabe AS, Camargo CN, Passos AM, Parmezan SN, Tomazella TK, et al. Surveillance of influenza A H1N1 2009 among school children during 2009 and 2010 in São Paulo, Brazil. Rev Soc Bras Med Trop 2012;45:563-6. 18. Klein MB, Lu Y, DelBalso L, Coté S, Boivin G. Influenzavirus infection is a Primary Cause of Febrile respiratory illness in HIV-infected adults, despite Vaccination. Clin Infect Dis 2007;45:234-40. 19. Kunisaki KM, Janoff EN. Influenza in immunosuppressed populations: a review of infection frequency, morbidity, mortality, and vaccine responses. Lancet Infect Dis 2009;9:493-504. 20. Sierra JO, Manchón FO, Flecha IC, Quezel-Guerraz NM, Bravo DS. Grupo de Trabajo para el Estudio MARBEGRIP. Asymptomatic infection by influenza AH1N1 virus in healthcare workers: MARBEGRIP study, preliminary results. Rev Esp Salud Publica 2011;85:63-71. 21. Chen MI, Lee VJ, Lim WY, Barr IG, Lin RT, Koh GC, et al. 2009 influenza A(H1N1) seroconversion rates and risk factors among distinct adult cohorts in Singapore. J Am Med Assoc 2010;303:1383-91. 22. Papenburg J, Baz M, Hamelin MÈ, Rhéaume C, Carbonneau J, Ouakki M, et al. Household transmission of the 2009 pandemic A/H1N1 influenza virus: elevated laboratory-confirmed secondary attack rates and evidence of asymptomatic infections. Clin Infect Dis 2010;51:1033-41. 23. Sikora C, Fan S, Golonka R, Sturtevant D, Gratrix J, Lee BE, et al. Transmission of pandemic influenza A (H1N1) 2009 within households: Edmonton, Canada. J Clin Virol 2010;49:90-3.
Contents lists available at ScienceDirect
American Journal of Infection Control
American Journal of Infection Control
journal homepage: www.ajicjournal.org
Major article
Influenza virus prevalence in asymptomatic and symptomatic subjects during pandemic and postpandemic periods Thaís Boim Melchior MD, Ana Helena Perosa MD *, Clarice Neves Camargo PhD, Celso Granato PhD, Nancy Bellei PhD Clinical Virology Laboratory e Infectious Disease Unit, Medicine Department, Federal University of Sao Paulo, SP, Brazil
Key Words: Infection Caregivers Real-time PCR Respiratory virus
Background: In 2009, Influenza A(H1N1)pdm09 had a major effect on global health, causing thousands of deaths. However, the results of asymptomatic infection in transmission has not been well studied. We analyzed asymptomatic influenza infection in individuals with different risk factors for acquiring influenza in a university hospital. Methods: Respiratory samples from 100 children with respiratory symptoms, their asymptomatic caregivers, 100 asymptomatic HIV-infected patients, and 100 health care workers collected during 2009-2011 were tested for influenza by real time real-time polymerase chain reaction. Results: Influenza infection rate in symptomatic children was 44% and in asymptomatic adults it was 8.3% (P < .01). Children older than age 5 years had a 3.4 times greater chance of being infected during influenza season than younger children. Among the asymptomatic group, influenza was more frequent in caregivers (14%; P ¼ .032) and a higher rate (31.8%) was observed if a child was infected during the 2009 pandemic. Contact with an infected child was an important risk factor for influenza acquisition (odds ratio, 3.8; 95% confidence interval, 1.1-13.2) among caregivers. The mean cluster of differentiation 4 T-cell count of HIV-positive patients infected with influenza was not statistically different from uninfected patients (P ¼ .29). Conclusions: Asymptomatic household contacts of symptomatic children may play an important role in community transmission and a more proactive intervention should be considered during future pandemics. Copyright Ó 2015 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.
The influenza A(H1N1)pdm09 (pH1N1) virus had a major effect on global health in 2009, causing thousands of deaths. In 2009, the virus caused 2,060 deaths and more than 50,000 cases of illness in Brazil, with the highest incidence in children younger than age 2 years and adults aged 20-29 years.1,2 It is known that influenza infection is often asymptomatic, occurring in approximately 30%-50% of cases.3,4 Asymptomatic individuals are not easily identified and isolated, increasing their ability to transmit disease.3,5 Cases of asymptomatic pH1N1
* Address correspondence to Ana Helena Perosa, MD, 781 Pedro de Toledo St, 15th Fl, Vila Clementino, Sao Paulo e SP, CEP 04039-032, Brazil. E-mail address: [email protected] (A.H. Perosa). This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (grant No. 2010/09272-5). TBM, AHP, CG, and NB participated in the data analysis, writing, and review of the manuscript. TBM, AHP, and CNC participated in the laboratory analysis. Conflicts of interest: None to report.
infection in patients and health care workers (HCWs) previously exposed to sick patients have been reported.5-7 Complications resulting from influenza infection are more common in immunocompromised patients.8 There is a lack of knowledge about the effect of pH1n1 in individuals with HIV infection. However, according to the World Health Organization, HIV-positive individuals, particularly those with low cluster of differentiation 4 counts, are at high risk for developing complications from infection by a new viral subtype and should have priority in the prevention and treatment of disease.9,10 Household contact with children seems to be more important in disease transmission than exposure to other patients.11 Exposure to the index patient during the symptomatic phase and longer exposure times can increase the risk of acquiring the infection.12 We analyzed the prevalence of influenza infection in asymptomatic and symptomatic individuals from different populations at a university hospital in Sao Paulo, Brazil, during the pandemic and postpandemic periods. The frequency of influenza infection in
0196-6553/$36.00 - Copyright Ó 2015 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajic.2015.01.032
T.B. Melchior et al. / American Journal of Infection Control 43 (2015) 460-4
461
Fig 1. Distribution of samples collected during the study, by group. HCW, health care worker.
asymptomatic subjects with different risk factors is described. These subjects included HCWs, HIV-infected patients, and caregivers of children with respiratory symptoms. METHODS Study design A prospective study of 400 samples was established considering the 4 groups evaluated (ie, HCW, HIV, caregivers, and children) so that each was composed of 100 individuals, which was a sample size calculated to be sufficient to determine a prevalence of influenza from 10%-30% with a 90% confidence interval and 0.05 alpha error. The goal of sampling was that we could determine the prevalence for influenza acquisition in 4 different risk groups. Patients Patients were actively recruited in different health care units of a university hospital in Sao Paulo, Brazil, during the 2009 pandemic (June-October), 2010 (March-October), and 2011 (May- October). The inclusion criterion for asymptomatic adults was the absence of respiratory symptoms during the 15 days before sample collection. For symptomatic children (up to age 12 years) inclusion criterion was the diagnosis of acute respiratory infection within a week before sample collection. Samples Three hundred samples were collected from nonhospitalized asymptomatic adults: 100 from HIV-positive outpatients from an HIV ambulatory clinic recruited once a week during a scheduled routine visit, 100 HCWs (physicians and nurses) with direct contact with patient from different hospital wards/clinics (ie, nephrology, pediatric, hematology, infectious diseases, transplant, cardiology, psychiatry, general surgery, laboratory, and intensive care unit), and 100 from caregivers (all household contacts) of symptomatic children. An additional 100 samples were collected from symptomatic children. Children were outpatients recruited on a weekly
basis, twice a week. Asymptomatic caregiver and symptomatic child pairs were interviewed by 1 researcher following referral by a pediatrician. Samples were collected only from children for whom the asymptomatic caregiver agreed to participate. Figure 1 shows the samples collected from these 400 individuals between July 2009 and October 2011. The use of the data, the terms of consent, and all procedures were approved by the Ethics Committee of the Hospital Sao Paulo (protocol No. 0700/10). Laboratory methods Nasopharyngeal swabs were collected, immediately transported to the virology laboratory, and aliquots were frozen at 80 C for further analysis by polymerase chain reaction. Nucleic acid was extracted from 140ul of collected sample using the QIAmp Viral RNA Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The influenza A real-time polymerase chain reaction test was performed according to the Centers for Disease Control and Prevention protocol for pH1N1.13 The influenza B real-time polymerase chain reaction test was performed after standardization in our laboratory, using primers previously described.14 Samples positive for primer INFA and negative for the primers SwINFA and SwH1 in the Centers for Disease Control and Prevention protocol were considered as influenza A nontyped. The reactions were performed with AgPath-ID One Step RT-PCR (Applied Biosystems, Austin, TX) on a model ABI 7500 real-time machine (Applied Biosystems, Austin, TX), and ribonuclease P was used as the internal control. Statistical analysis was performed using c2 or Student t tests to evaluate the variables; a P value 50%, a quarter of household contacts of symptomatic children were also infected with influenza and the potential for transmission cannot be underestimated, even if they remain asymptomatic. These data suggest the use of a more consistent approach, such as prophylaxis or preemptive treatment during future pandemics. A limitation of our study was the recruitment of asymptomatic patients during pandemic periods when the influenza attack rate is higher than during epidemic periods. Another limitation was that HCWs who were concerned about their higher risk of influenza may have protected themselves better than other HCWs, which could decrease the relative risk of influenza infection in this group.
464
T.B. Melchior et al. / American Journal of Infection Control 43 (2015) 460-4
CONCLUSIONS The occurrence of asymptomatic infections and their frequency in different populations highlights the importance of asymptomatic infection in influenza circulation. Family members of children with influenza may have an effect on viral transmission among the general population during pandemic periods. Additional studies should be conducted to better understand the clinical significance of asymptomatic infection by different subtypes of influenza virus during epidemics and pandemics.
References 1. Brasil. SVS/MS. Boletim eletrônico epidemiológico: caderno especial influenza, Influenza Pandêmica (H1N1) 2009. Análise da situação epidemiológica e da resposta no ano de 2009 2010;10:1-21. Disponível em, http://portal.saude.gov. br/portal/arquivos/pdf/boletim_eletronico_influenza_25_03.pdf [acesso em maio 2010]. 2. Informe técnico de influenza e Vigilância da Síndrome respiratória aguda grave (SRAG), de síndrome gripal (SG) e de internações por CID J09 a 118. Ministério da Saúde. Secretaria de Vigilância em Saúde. Disponível em, http://portal.saude.gov. br/portal/arquivos/pdf/info_tecn_influenza_31_01_2012_28novo _29.pdf; 2012 [acesso em jan 2012]. 3. Hsu SB, Hsieh YH. On the role of asymptomatic infection in transmission Dynamics of infectious diseases. Bull Math Biol 2008;70:134-55. 4. Lau LL, Cowling BJ, Fang VJ, Chan KH, Lau EH, Lipsitch M, et al. Viral shedding and clinical illness in naturally acquired influenza virus infections. J Infect Dis 2010;201:1509-16. 5. Yang J, Yang F, Huang F, Wang J, Jin Q. Subclinical infection with the novel influenza a (H1N1) virus. Clin Infect Dis 2009;49:1622-3. 6. Cheng VC, Tai JW, Wong LM, Chan JF, Li IW, To KK, et al. Prevention of nosocomial transmission of swine-origin pandemic influenza virus A/H1N1 by infection control bundle. J Hosp Infect 2010;74:271-7. 7. Meyer S, Ilchmann C, Adam M, Sattinger E, Siamkouris D, Arunagirinathan U, et al. Asymptomatic infection with novel influenza A/H1N1 virus in a heart transplant recipient. J Heart Lung Transplant 2010;29:585-6. 8. Wilschut JC, McElhaney JE, Palache AM. Pathogenesis, clinical features and diagnosis. In: Influenza. 2nd ed. Philadelphia: Mosby; 2006. 9. Centers for Disease Control and Prevention. Updated Interim Recommendations d HIV-Infected Adults and Adolescents: Considerations for Clinicians Regarding 2009 H1N1 Influenza. Available from: http://www.cdc.gov/h1n1flu/ guidance_hiv.htm; 2009. Accessed February 8, 2010.
10. World Health Organization. Considerations on influenza A (H1N1) and HIV infection. Available from: http://www.who.int/hiv/mediacentre/influenza_hiv. pdf; 2009. Accessed February 8, 2010. 11. Williams CJ, Schweiger B, Diner G, Gerlach F, Haaman F, Krause G, et al. Seasonal influenza risk in hospital healthcare workers is more strongly associated with household than occupational exposures: results from a prospective cohort study in Berlin, Germany, 2006/07. BMC Infect Dis 2010;10:8. 12. Pang X, Yang P, Li S, Zhang L, Tian L, Li Y, et al. Pandemic (H1N1) 2009 among quarantined close contacts, Beijing, People’s Republic of China. Emerg Infect Dis 2011;17:1824-30. 13. Centers for Disease Control and Prevention. CDC protocol of realtime RTPCR for influenza A (H1N1). Available from: http://www.who.int/csr/resources/ publications/swineflu/CDCRealtimeRTPCR_SwineH1Assay-2009_20090430.pdf; 2009. Accessed January 27, 2010. 14. Selvaraju SB, Selvarangan R. Evaluation of three influenza A and B real-time reverse transcription-PCR assays and a new 2009 H1N1 assay for detection of influenza viruses. J Clin Microbiol 2010;48:3870-5. 15. Glatman-Freedman A, Portelli I, Jacobs SK, Mathew JI, Slutzman JE, Goldfrank LR, et al. Attack rates assessment of the 2009 pandemic H1N1 influenza A in children and their contacts: a systematic review and metaanalysis. PLoS One 2012;7:e50228. 16. Vesikari T, Pellegrini M, Karvonen A, Groth N, Borkowski A, O’Hagan DT, et al. Enhanced immunogenicity of seasonal influenza vaccines in young children using MF59 adjuvant. Pediatr Infect Dis J 2009;28:563-71. 17. Guatura SB, Watanabe AS, Camargo CN, Passos AM, Parmezan SN, Tomazella TK, et al. Surveillance of influenza A H1N1 2009 among school children during 2009 and 2010 in São Paulo, Brazil. Rev Soc Bras Med Trop 2012;45:563-6. 18. Klein MB, Lu Y, DelBalso L, Coté S, Boivin G. Influenzavirus infection is a Primary Cause of Febrile respiratory illness in HIV-infected adults, despite Vaccination. Clin Infect Dis 2007;45:234-40. 19. Kunisaki KM, Janoff EN. Influenza in immunosuppressed populations: a review of infection frequency, morbidity, mortality, and vaccine responses. Lancet Infect Dis 2009;9:493-504. 20. Sierra JO, Manchón FO, Flecha IC, Quezel-Guerraz NM, Bravo DS. Grupo de Trabajo para el Estudio MARBEGRIP. Asymptomatic infection by influenza AH1N1 virus in healthcare workers: MARBEGRIP study, preliminary results. Rev Esp Salud Publica 2011;85:63-71. 21. Chen MI, Lee VJ, Lim WY, Barr IG, Lin RT, Koh GC, et al. 2009 influenza A(H1N1) seroconversion rates and risk factors among distinct adult cohorts in Singapore. J Am Med Assoc 2010;303:1383-91. 22. Papenburg J, Baz M, Hamelin MÈ, Rhéaume C, Carbonneau J, Ouakki M, et al. Household transmission of the 2009 pandemic A/H1N1 influenza virus: elevated laboratory-confirmed secondary attack rates and evidence of asymptomatic infections. Clin Infect Dis 2010;51:1033-41. 23. Sikora C, Fan S, Golonka R, Sturtevant D, Gratrix J, Lee BE, et al. Transmission of pandemic influenza A (H1N1) 2009 within households: Edmonton, Canada. J Clin Virol 2010;49:90-3.
