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The virologist and the flu
Journal of Clinical Virology 69 (2015) 200–202
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Case report
The virologist and the flu Sabine Wicker a , Holger F. Rabenau b,∗ a b
Occupational Health Service, University Hospital Frankfurt, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany Institute of Medical Virology, University Hospital Frankfurt, Goethe University, Paul-Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany
a r t i c l e
i n f o
Article history: Received 21 April 2015 Received in revised form 9 June 2015 Accepted 23 June 2015 Keywords: Influenza Influenza vaccination Viral shedding
a b s t r a c t We report a case of a virologist – who is in age-appropriate medical condition with no relevant chronic diseases – who shed influenza A H3N2 virus RNA for 70 days while infectious virus could be detected by cell culture only up to 5 days after onset of symptoms despite a 5-day course of oseltamivir. The case might have implications for infection control in hospital settings and the weighting of the predictive value of PCR results. © 2015 Elsevier B.V. All rights reserved.
Even though this isn’t a fairy tale. . . “Once upon the time there was a virologist. Every year he got his flu vaccine in late autumn, likewise he has been vaccinated against influenza in November 2013. However, in April 2014 he suddenly felt sick. . .Remember – he has been a virologist for years. What did he do? Of course – he went to the lab and got tested. . ..” 1. Why this case is important To our knowledge this is the first time that long-term (70 days) influenza A H3N2 virus RNA shedding occurs in an otherwise healthy individual while infectious virus could be detected by cell culture only up to 5 days after onset of symptoms. 2. Case description A 55-year old virologist has been vaccinated with a trivalentinactivated influenza vaccine (TIV) by the Occupational Health Service on November 8, 2013. He has been vaccinated against influenza each year since 1988. The virologist has been in an age appropriate medical condition with no severe chronic disease. He does not take any immunomodulatory drugs. On April 2, 2014 he developed a cough and on April 3, a more severe cough, chills, and fatigue. On that day, a throat swab was
∗ Corresponding author. Fax: +49 69 630183061. E-mail addresses: [email protected] (S. Wicker), [email protected] (H.F. Rabenau). http://dx.doi.org/10.1016/j.jcv.2015.06.099 1386-6532/© 2015 Elsevier B.V. All rights reserved.
positive for influenza A, RNA detected by real-time reverse transcriptase polymerase chain reaction (rRT-PCR), and inoculation of MDCK cultures, revealed the growth of infectious influenza A virus (see Fig. 1). On April 4 and 5 the symptoms got worse with additional headache and body aches and he felt feverish. Starting from April 6, significant signs of improvement could have been seen. While the severe cough and chills vanished, the physical exhaustion and fatigue continued until April 23. The influenza A virus was subtyped as H3N2. Throat secretions were positive on culture for 5 days (day 2–6 following the onset of symptoms) and viral RNA could be detected until day 70 (June 11, 2014) by rRT-PCR (see Fig. 1), with temporarily negative results. A final PCR test on day 175 (September 22, 2014) was negative. He was treated with a standard 5-day course of oral oseltamivir (75 mg two times a day) beginning 20 h after symptom onset. The cough persisted until the end of the oseltamivir therapy on day 5, and through day 25 he felt uncomfortable and was not able to perform his duties in a normal manner. 2.1. Virological methods For culture of the throat secretions flocked swabs (UTM, cat. no. 355CW, Copan, Brescia, Italy) with 1 ml universal transport medium were used. Throat cultures were performed every day (from day 1 to 47, and additional on day 70, 76, 77, 175 after onset of symptoms) at morning time (7–8 am) in a standardized manner, i.e. 10 to 15 sec slowly rotating the swab in the posterior oropharynx without preparatory drinking or teeth brushing. For automated RNA/DNA samples extraction the QiaSymphony DSP Virus/Pathogen Midi kit and QIAsymphony instrument (Qiagen, Hilden, Germany) was used. All nucleic acid
S. Wicker, H.F. Rabenau / Journal of Clinical Virology 69 (2015) 200–202
201
Fig. 1. Influenza protocol: detection of influenza H3N2 in standardized deep throat swabs: Day 0 = start of symptoms (dry cough, sub-febrile). PCR Ct-values indicate the semiquantitative amount of Influenza A-RNA. Last positive PCR result on day 70, final test on day 176 was PCR negative. 䊉 = PCR result (Ct 40 means PCR negative) Cell culture: − = negative, no infectious virus detectable, (+) = only few, single cells positive, +++ = indication a highly positive cell culture.
specimens were tested using real-time PCR according to a standard protocol (adapted from Ward et al. [1]), using IAV matrix gene-specific primers and Taqman probes (influenza matrix F: 5‘-AAg-ACC-AAT-CCT-gTC-ACC-TCT-gA-3‘ and Influenza A25A: 5‘-CAA-AgC-gTC-TAC-gCT-gCA-gTC-C-3‘ and as Influenza A-probe: 5‘-FAM-TTT-gTg-TTC-ACG-CTC-ACC-GT-TAMRA-3‘). The laboratory-developed influenza A one-step real-time RT-PCR runs on an ABI 7900HT real-time PCR instrument. The semiquantitative PCR results are expressed as threshold cycle (Ct) values. The Ct-values correlate with the relative amount of viral nucleic acid detected. The cut off value of Ct 40 means a negative result. The higher the Ct-value the lower the viral amount (Ct-values ≥ 37 indicate a (very) low virus amount). As internal control bovine viral diarrhea virus was used. The possibility of false-positive swab results were excluded by the routine inclusion of negative controls using the same swabs submerged in cell culture medium. For the detection of infectious influenza virus a shell vial assay in 96-well microtitre plates (Greiner Bio-One, Frickenhausen, Germany) was performed. Specimens were inoculated in eight wells (100 L/well) on seeded MDCK monolayer cells (Mardin Darby kidney cells; ATCC: CCL-34, Manassas, VA, USA). After centrifugation of the plate (700 × g, 40 min) the inoculum was aspirated, fresh minimal essential medium (MEM, Biochrom AG, Berlin, Germany, supplemented with 10% foetal bovine serum, 100 IU/ml penicillin, 100 g/ml streptomycin) was added and the plates incubated at 33 ◦ C for 72 h. Afterwards the medium was discarded and the cells fixed with acetone/methanol (40:60). Staining of the cells (four wells per influenza type) were performed using influenza A-specific monoclonal antibodies (Chemicon, Limburg, Germany) and biotinylated secondary antibodies. Virus specific staining was determined by microscopic analysis. 3. Other similar and contrasting cases in the literature A randomised placebo-controlled trial showed that oseltamivir treatment started within 2 days following illness onset significantly reduced virus shedding on day 2, 4, and 7 in patients with uncomplicated influenza infections [2]. However, in a study from 45 public and private outpatient clinics in Hong Kong oseltamivir treatment
was not associated with statistically significant reduction in the duration of viral shedding. Patients who had taken oseltamivir within 48 h experienced a statistically non-significant 24% reduction in time to cessation of viral shedding [3].
4. Discussion and references Influenza infections occurring among vaccinated individuals (vaccine failures) have been described in the literature [4,5]. So far this case is not uncommon however, this case is striking because, influenza virus RNA was detected until day 70 by PCR (with repeatedly negative results in between on day 10, 14, 16, 23, 25, 33, 34, 36, 38, 40–47), but infectious virus only for 5 days by cell culture (with negative results from day 6 day after symptom onset and day 5 after initiation of oseltamivir therapy; no further cell culture testing were performed after 8 consecutive days of negative results) (see Fig. 1). In line with previous reports we found that the H3N2 virus load in throat swabs peaked 2 days after the onset of acute respiratory symptoms [6] (see Fig. 1). It is known that comorbidities and immunosuppression can be associated with longer influenza virus shedding and slower viral clearance [7]. Previous reports have described shedding of influenza A in immunocompromised patients for up to 18 months [8–15]. However, our virologist has been otherwise healthy.
4.1. What does this mean for virology and infection control? In our case, semi-quantitative PCR testing indicated the presence of influenza A virus RNA for up to 70 days, which might have implications for disease transmission and infection control in hospital settings. The question arises on the clinical significance of positive PCR results: in particular, regarding patient management it needs to be decided, whether isolation procedures at a ward can be stopped or whether a patient is able to work again (e.g. as a healthcare worker) or whether he may have contact to immunosuppressed individuals. It is questionable whether a patient despite negative results in cell culture but positive PCR results might be still infectious.
202
S. Wicker, H.F. Rabenau / Journal of Clinical Virology 69 (2015) 200–202
CDC guidance recommends the isolation of hospitalized symptomatic patients for 7 days after illness onset, because shedding of influenza viruses generally diminishes over the course of 7 days and virus transmission correlates with fever [16]. Thus, the observations in this case are sufficiently unique to warrant further study of influenza shedding more extensively using PCR. The clinical relevance of the laboratory results for influenza virus in this case are uncertain, because it is not clear whether an individual with detection of viral nucleic acid is still infectious and if, there might be a difference between immunocompetent and immunocompromised patients [9]. Despite using a standardised throat swab procedure, quite variable Ct-values (ranging from 40 to 27.9 [on day 18]) were noted after the acute infection period. One reason for this phenomenon might be that the patient may have had one or multiple re-infections with other circulating H3N2 viruses. While this is not totally excludable we would have expected lower Ct-values if a re-infection would have happened. Like Leekha et al. [12], we were able to detect viral shedding significantly longer by PCR, compared with cell culture. Even though the virus load was undulant, it remains unclear why influenza virus RNA was so long detectable. Due to the PCR technology, not only viral RNA from infectious virus but also from non-infectious virus in principle can be detected. What rings particularly incredible is that an undulant amount of residual virus remains present in a throat. It cannot be ruled out that cell associated virus aggregates, which might have been created through the neuraminidase inhibitor activity, might be released over the time. Because of this point of principle the predictive value of (lower positive) PCR results in the context of infection control procedures must be critically evaluated. Conflict of interest The views in this article are the personal views of the authors and do not necessarily represent the views of the professional organizations or institutions of which we are members. Funding None. Competing interests Sabine Wicker is a member of the German Standing Committee on Vaccination (STIKO). She has received honoraria for non-product-related talks on influenza vaccination from GlaxoSmithKline, Sanofi Pasteur, AstraZeneca and Novartis and has participated in workshops about healthcare workers and vaccination sponsored by Abbot. Holger Rabenau has no conflict of interest. Ethical approval Not needed.
Contribution SW and HR drafted the manuscript; HR performed the laboratory analysis and the interpretation of the data. Both authors read and approved the final manuscript. References [1] C.L. Ward, M.H. Dempsey, C.J. Ring, et al., Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement, J. Clin. Virol. 29 (3) (2004) 179–188. [2] A.M. Fry, D. Gaswami, K. Nahar, et al., Efficacy of oseltamivir treatment started within 5 days of symptom onset to reduce influenza illness duration and virus shedding in an urban setting in Bangladesh: a randomised placebo-controlled trial, Lancet Infect. Dis. 14 (2014) 109–118. [3] S. Ng, B.J. Cowling, V.J. Fang, et al., Effects of oseltamivir treatment on duration of clinical illness and viral shedding and household transmission of influenza virus, Clin. Infect. Dis. 50 (2010) 707–714. [4] J.G. Petrie, S.E. Ohmit, E. Johnson, et al., Efficacy studies of influenza vaccines: effect of end points used and characteristics of vaccine failures, J. Infect. Dis. 203 (2011) 1309–1315. [5] S. Ng, M.Y. Ni, V.J. Fang, et al., Characteristics of vaccine failure in a randomized placebo-controlled trial of inactivated influenza vaccine in children, Pediatr. Infect. Dis. J. 33 (2014) e63–6. [6] M. Loeb, P.K. Singh, J. Fox, et al., Longitudinal study of influenza molecular viral shedding in Hutterite communities, J. Infect. Dis. 206 (2012) 1078–1084. [7] N. Lee, P.K.S. Chan, D.S.C. Hui, et al., Viral load and duration of viral shedding in adult patients hospitalized with influenza, J. Infect. Dis. 200 (2009) 492–500. [8] G. Boivin, N. Goyette, H. Bernatchez, Prolonged excretion of amantadine-resistant influenza A virus quasi species after cessation of antiviral therapy in an immunocompromised patient, Clin. Infect. Dis. 34 (2002) e23–e25. [9] J. Bruminhent, P.J. Deziel, J.T. Wotton, J. Binnicker, R.R. Razonable, Prolonged shedding of pandemic influenza A(H1N1) 2009 virus in a pancreas-after-kidney transplant recipient, J. Clin. Virol. 61 (2014) 302–304. [10] I. Ganhoke, D.S. Rawat, A. Rai, S. Khare, R.L. Ichhpujani, Pandemic influenza A (H1N1) 2009 in India: duration of virus shedding in patients under antiviral treatment, Indian J. Med. Microbiol. 29 (2011) 37–41. [11] S. Kanchana, S. Kanchana, C. Chuntrakul, K. Malathum, S. Prachayangprecha, Y. Poovorawan, Pandemic (H1N1) 2009 virus infection: persistent viral shedding after oseltamivir treatment, J. Infect. 63 (2011), 2. [12] S. Leekha, N.L. Zitterkopf, M.J. Espy, T.F. Smith, R.L. Thompson, P. Sampathkumar, Duration of influenza A virus shedding in hospitalized patients and implications for infection control, Infect. Control Hosp. Epidemiol. 28 (2007) 1071–1076. [13] A.C. Nilsson, M. Brytting, F. Serifler, P. Björkman, K. Persson, A. Widell, Longitudinal clearance of seasonal influenza A viral RNA measured by real-time polymerase chain reaction in patients identified at a hospital emergency department, Scand. J. Infect. Dis. 42 (2010) 679–686. [14] A. Pinsky, S. Mix, J. Rowe, et al., Long-term shedding of influenza A virus in stool of immunocompromised child, Emerg. Infect. Dis. 16 (7) (2010) 1165–1167. [15] D.M. Weinstock, L.V. Gubareva, G. Zuccotti, Prolonged shedding of multidrug-resistant influenza A virus in an immunocompromised patient, N. Engl. J. Med. 348 (9) (2003) 867–888. [16] Centers for Disease Control and Prevention, Interim Guidance on Infection Control Measures for 2009 H1N1 Influenza in Healthcare Settings, Including Protection of Healthcare Personnel. CDC 2010; http://www.cdc.gov/h1n1flu/ guidelines infection control.htm
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Case report
The virologist and the flu Sabine Wicker a , Holger F. Rabenau b,∗ a b
Occupational Health Service, University Hospital Frankfurt, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany Institute of Medical Virology, University Hospital Frankfurt, Goethe University, Paul-Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany
a r t i c l e
i n f o
Article history: Received 21 April 2015 Received in revised form 9 June 2015 Accepted 23 June 2015 Keywords: Influenza Influenza vaccination Viral shedding
a b s t r a c t We report a case of a virologist – who is in age-appropriate medical condition with no relevant chronic diseases – who shed influenza A H3N2 virus RNA for 70 days while infectious virus could be detected by cell culture only up to 5 days after onset of symptoms despite a 5-day course of oseltamivir. The case might have implications for infection control in hospital settings and the weighting of the predictive value of PCR results. © 2015 Elsevier B.V. All rights reserved.
Even though this isn’t a fairy tale. . . “Once upon the time there was a virologist. Every year he got his flu vaccine in late autumn, likewise he has been vaccinated against influenza in November 2013. However, in April 2014 he suddenly felt sick. . .Remember – he has been a virologist for years. What did he do? Of course – he went to the lab and got tested. . ..” 1. Why this case is important To our knowledge this is the first time that long-term (70 days) influenza A H3N2 virus RNA shedding occurs in an otherwise healthy individual while infectious virus could be detected by cell culture only up to 5 days after onset of symptoms. 2. Case description A 55-year old virologist has been vaccinated with a trivalentinactivated influenza vaccine (TIV) by the Occupational Health Service on November 8, 2013. He has been vaccinated against influenza each year since 1988. The virologist has been in an age appropriate medical condition with no severe chronic disease. He does not take any immunomodulatory drugs. On April 2, 2014 he developed a cough and on April 3, a more severe cough, chills, and fatigue. On that day, a throat swab was
∗ Corresponding author. Fax: +49 69 630183061. E-mail addresses: [email protected] (S. Wicker), [email protected] (H.F. Rabenau). http://dx.doi.org/10.1016/j.jcv.2015.06.099 1386-6532/© 2015 Elsevier B.V. All rights reserved.
positive for influenza A, RNA detected by real-time reverse transcriptase polymerase chain reaction (rRT-PCR), and inoculation of MDCK cultures, revealed the growth of infectious influenza A virus (see Fig. 1). On April 4 and 5 the symptoms got worse with additional headache and body aches and he felt feverish. Starting from April 6, significant signs of improvement could have been seen. While the severe cough and chills vanished, the physical exhaustion and fatigue continued until April 23. The influenza A virus was subtyped as H3N2. Throat secretions were positive on culture for 5 days (day 2–6 following the onset of symptoms) and viral RNA could be detected until day 70 (June 11, 2014) by rRT-PCR (see Fig. 1), with temporarily negative results. A final PCR test on day 175 (September 22, 2014) was negative. He was treated with a standard 5-day course of oral oseltamivir (75 mg two times a day) beginning 20 h after symptom onset. The cough persisted until the end of the oseltamivir therapy on day 5, and through day 25 he felt uncomfortable and was not able to perform his duties in a normal manner. 2.1. Virological methods For culture of the throat secretions flocked swabs (UTM, cat. no. 355CW, Copan, Brescia, Italy) with 1 ml universal transport medium were used. Throat cultures were performed every day (from day 1 to 47, and additional on day 70, 76, 77, 175 after onset of symptoms) at morning time (7–8 am) in a standardized manner, i.e. 10 to 15 sec slowly rotating the swab in the posterior oropharynx without preparatory drinking or teeth brushing. For automated RNA/DNA samples extraction the QiaSymphony DSP Virus/Pathogen Midi kit and QIAsymphony instrument (Qiagen, Hilden, Germany) was used. All nucleic acid
S. Wicker, H.F. Rabenau / Journal of Clinical Virology 69 (2015) 200–202
201
Fig. 1. Influenza protocol: detection of influenza H3N2 in standardized deep throat swabs: Day 0 = start of symptoms (dry cough, sub-febrile). PCR Ct-values indicate the semiquantitative amount of Influenza A-RNA. Last positive PCR result on day 70, final test on day 176 was PCR negative. 䊉 = PCR result (Ct 40 means PCR negative) Cell culture: − = negative, no infectious virus detectable, (+) = only few, single cells positive, +++ = indication a highly positive cell culture.
specimens were tested using real-time PCR according to a standard protocol (adapted from Ward et al. [1]), using IAV matrix gene-specific primers and Taqman probes (influenza matrix F: 5‘-AAg-ACC-AAT-CCT-gTC-ACC-TCT-gA-3‘ and Influenza A25A: 5‘-CAA-AgC-gTC-TAC-gCT-gCA-gTC-C-3‘ and as Influenza A-probe: 5‘-FAM-TTT-gTg-TTC-ACG-CTC-ACC-GT-TAMRA-3‘). The laboratory-developed influenza A one-step real-time RT-PCR runs on an ABI 7900HT real-time PCR instrument. The semiquantitative PCR results are expressed as threshold cycle (Ct) values. The Ct-values correlate with the relative amount of viral nucleic acid detected. The cut off value of Ct 40 means a negative result. The higher the Ct-value the lower the viral amount (Ct-values ≥ 37 indicate a (very) low virus amount). As internal control bovine viral diarrhea virus was used. The possibility of false-positive swab results were excluded by the routine inclusion of negative controls using the same swabs submerged in cell culture medium. For the detection of infectious influenza virus a shell vial assay in 96-well microtitre plates (Greiner Bio-One, Frickenhausen, Germany) was performed. Specimens were inoculated in eight wells (100 L/well) on seeded MDCK monolayer cells (Mardin Darby kidney cells; ATCC: CCL-34, Manassas, VA, USA). After centrifugation of the plate (700 × g, 40 min) the inoculum was aspirated, fresh minimal essential medium (MEM, Biochrom AG, Berlin, Germany, supplemented with 10% foetal bovine serum, 100 IU/ml penicillin, 100 g/ml streptomycin) was added and the plates incubated at 33 ◦ C for 72 h. Afterwards the medium was discarded and the cells fixed with acetone/methanol (40:60). Staining of the cells (four wells per influenza type) were performed using influenza A-specific monoclonal antibodies (Chemicon, Limburg, Germany) and biotinylated secondary antibodies. Virus specific staining was determined by microscopic analysis. 3. Other similar and contrasting cases in the literature A randomised placebo-controlled trial showed that oseltamivir treatment started within 2 days following illness onset significantly reduced virus shedding on day 2, 4, and 7 in patients with uncomplicated influenza infections [2]. However, in a study from 45 public and private outpatient clinics in Hong Kong oseltamivir treatment
was not associated with statistically significant reduction in the duration of viral shedding. Patients who had taken oseltamivir within 48 h experienced a statistically non-significant 24% reduction in time to cessation of viral shedding [3].
4. Discussion and references Influenza infections occurring among vaccinated individuals (vaccine failures) have been described in the literature [4,5]. So far this case is not uncommon however, this case is striking because, influenza virus RNA was detected until day 70 by PCR (with repeatedly negative results in between on day 10, 14, 16, 23, 25, 33, 34, 36, 38, 40–47), but infectious virus only for 5 days by cell culture (with negative results from day 6 day after symptom onset and day 5 after initiation of oseltamivir therapy; no further cell culture testing were performed after 8 consecutive days of negative results) (see Fig. 1). In line with previous reports we found that the H3N2 virus load in throat swabs peaked 2 days after the onset of acute respiratory symptoms [6] (see Fig. 1). It is known that comorbidities and immunosuppression can be associated with longer influenza virus shedding and slower viral clearance [7]. Previous reports have described shedding of influenza A in immunocompromised patients for up to 18 months [8–15]. However, our virologist has been otherwise healthy.
4.1. What does this mean for virology and infection control? In our case, semi-quantitative PCR testing indicated the presence of influenza A virus RNA for up to 70 days, which might have implications for disease transmission and infection control in hospital settings. The question arises on the clinical significance of positive PCR results: in particular, regarding patient management it needs to be decided, whether isolation procedures at a ward can be stopped or whether a patient is able to work again (e.g. as a healthcare worker) or whether he may have contact to immunosuppressed individuals. It is questionable whether a patient despite negative results in cell culture but positive PCR results might be still infectious.
202
S. Wicker, H.F. Rabenau / Journal of Clinical Virology 69 (2015) 200–202
CDC guidance recommends the isolation of hospitalized symptomatic patients for 7 days after illness onset, because shedding of influenza viruses generally diminishes over the course of 7 days and virus transmission correlates with fever [16]. Thus, the observations in this case are sufficiently unique to warrant further study of influenza shedding more extensively using PCR. The clinical relevance of the laboratory results for influenza virus in this case are uncertain, because it is not clear whether an individual with detection of viral nucleic acid is still infectious and if, there might be a difference between immunocompetent and immunocompromised patients [9]. Despite using a standardised throat swab procedure, quite variable Ct-values (ranging from 40 to 27.9 [on day 18]) were noted after the acute infection period. One reason for this phenomenon might be that the patient may have had one or multiple re-infections with other circulating H3N2 viruses. While this is not totally excludable we would have expected lower Ct-values if a re-infection would have happened. Like Leekha et al. [12], we were able to detect viral shedding significantly longer by PCR, compared with cell culture. Even though the virus load was undulant, it remains unclear why influenza virus RNA was so long detectable. Due to the PCR technology, not only viral RNA from infectious virus but also from non-infectious virus in principle can be detected. What rings particularly incredible is that an undulant amount of residual virus remains present in a throat. It cannot be ruled out that cell associated virus aggregates, which might have been created through the neuraminidase inhibitor activity, might be released over the time. Because of this point of principle the predictive value of (lower positive) PCR results in the context of infection control procedures must be critically evaluated. Conflict of interest The views in this article are the personal views of the authors and do not necessarily represent the views of the professional organizations or institutions of which we are members. Funding None. Competing interests Sabine Wicker is a member of the German Standing Committee on Vaccination (STIKO). She has received honoraria for non-product-related talks on influenza vaccination from GlaxoSmithKline, Sanofi Pasteur, AstraZeneca and Novartis and has participated in workshops about healthcare workers and vaccination sponsored by Abbot. Holger Rabenau has no conflict of interest. Ethical approval Not needed.
Contribution SW and HR drafted the manuscript; HR performed the laboratory analysis and the interpretation of the data. Both authors read and approved the final manuscript. References [1] C.L. Ward, M.H. Dempsey, C.J. Ring, et al., Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement, J. Clin. Virol. 29 (3) (2004) 179–188. [2] A.M. Fry, D. Gaswami, K. Nahar, et al., Efficacy of oseltamivir treatment started within 5 days of symptom onset to reduce influenza illness duration and virus shedding in an urban setting in Bangladesh: a randomised placebo-controlled trial, Lancet Infect. Dis. 14 (2014) 109–118. [3] S. Ng, B.J. Cowling, V.J. Fang, et al., Effects of oseltamivir treatment on duration of clinical illness and viral shedding and household transmission of influenza virus, Clin. Infect. Dis. 50 (2010) 707–714. [4] J.G. Petrie, S.E. Ohmit, E. Johnson, et al., Efficacy studies of influenza vaccines: effect of end points used and characteristics of vaccine failures, J. Infect. Dis. 203 (2011) 1309–1315. [5] S. Ng, M.Y. Ni, V.J. Fang, et al., Characteristics of vaccine failure in a randomized placebo-controlled trial of inactivated influenza vaccine in children, Pediatr. Infect. Dis. J. 33 (2014) e63–6. [6] M. Loeb, P.K. Singh, J. Fox, et al., Longitudinal study of influenza molecular viral shedding in Hutterite communities, J. Infect. Dis. 206 (2012) 1078–1084. [7] N. Lee, P.K.S. Chan, D.S.C. Hui, et al., Viral load and duration of viral shedding in adult patients hospitalized with influenza, J. Infect. Dis. 200 (2009) 492–500. [8] G. Boivin, N. Goyette, H. Bernatchez, Prolonged excretion of amantadine-resistant influenza A virus quasi species after cessation of antiviral therapy in an immunocompromised patient, Clin. Infect. Dis. 34 (2002) e23–e25. [9] J. Bruminhent, P.J. Deziel, J.T. Wotton, J. Binnicker, R.R. Razonable, Prolonged shedding of pandemic influenza A(H1N1) 2009 virus in a pancreas-after-kidney transplant recipient, J. Clin. Virol. 61 (2014) 302–304. [10] I. Ganhoke, D.S. Rawat, A. Rai, S. Khare, R.L. Ichhpujani, Pandemic influenza A (H1N1) 2009 in India: duration of virus shedding in patients under antiviral treatment, Indian J. Med. Microbiol. 29 (2011) 37–41. [11] S. Kanchana, S. Kanchana, C. Chuntrakul, K. Malathum, S. Prachayangprecha, Y. Poovorawan, Pandemic (H1N1) 2009 virus infection: persistent viral shedding after oseltamivir treatment, J. Infect. 63 (2011), 2. [12] S. Leekha, N.L. Zitterkopf, M.J. Espy, T.F. Smith, R.L. Thompson, P. Sampathkumar, Duration of influenza A virus shedding in hospitalized patients and implications for infection control, Infect. Control Hosp. Epidemiol. 28 (2007) 1071–1076. [13] A.C. Nilsson, M. Brytting, F. Serifler, P. Björkman, K. Persson, A. Widell, Longitudinal clearance of seasonal influenza A viral RNA measured by real-time polymerase chain reaction in patients identified at a hospital emergency department, Scand. J. Infect. Dis. 42 (2010) 679–686. [14] A. Pinsky, S. Mix, J. Rowe, et al., Long-term shedding of influenza A virus in stool of immunocompromised child, Emerg. Infect. Dis. 16 (7) (2010) 1165–1167. [15] D.M. Weinstock, L.V. Gubareva, G. Zuccotti, Prolonged shedding of multidrug-resistant influenza A virus in an immunocompromised patient, N. Engl. J. Med. 348 (9) (2003) 867–888. [16] Centers for Disease Control and Prevention, Interim Guidance on Infection Control Measures for 2009 H1N1 Influenza in Healthcare Settings, Including Protection of Healthcare Personnel. CDC 2010; http://www.cdc.gov/h1n1flu/ guidelines infection control.htm