Antiviral Therapy 2015; 20:49 55 (doi: /IMP2798)

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1 Antiviral Therapy 2015; 20:49 55 (doi: /IMP2798) Original article Comparison between virus shedding and fever duration after treating children with pandemic A H1N1/09 and children with A H3N2 with a neuraminidase inhibitor Norio Sugaya 1 *, Yuko Sakai-Tagawa 2, Masahiro Bamba 1, Rieko Yasuhara 1, Masahiko Yamazaki 3, Chiharu Kawakami 4, Yoshio Yamaguchi 5, Yoshiaki Ide 6, Masataka Ichikawa 7, Keiko Mitamura 8, Yoshihiro Kawaoka 9,10 1 Department of Pediatrics, Keiyu Hospital, Yokohama, Japan 2 Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan 3 Zama Children s Clinic, Zama, Japan 4 Yokohama City Institute of Health, Yokohama, Japan 5 Institute of Clinical Research, National Hospital Organization Tochigi Hospital, Utsunomiya, Japan 6 Department of Pediatrics, Isehara Kyodo Hospital, Isehara, Japan 7 Ichikawa Children s Clinic, Isehara, Japan 8 Department of Pediatrics, Eiju General Hospital, Tokyo, Japan 9 Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan 10 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin Madison, Madison, WI, USA *Corresponding author sugaya47@gmail.com Background: Shedding of the pandemic virus during an influenza pandemic is thought to persist longer than shedding of influenza viruses during annual influenza seasons, because people have much less immunity against a pandemic influenza. A correlation is thought to exist between the length of virus shedding and the clinical severity of influenza illness. Methods: We compared the virus isolation rates of children with pandemic A H1N1/09 influenza infection and children with A H3N2 influenza infection after the patients had been treated with one of three neuraminidase inhibitors (NAI) such as peramivir, laninamivir and oseltamivir. The clinical effectiveness of each NAI was assessed on the basis of the duration of the febrile period after the start of treatment. Results: Influenza viruses were isolated from 15 of the 34 patients in the A H3N2 group (mean age 6.2 years) and from 4 of the 25 patients in the A H1N1/09 (mean age 5.6 years) virus group (44.1% versus 16.0%; P<0.05). However, the differences between the duration of fever in the patients in the A H3N2 group and A H1N1/09 group after treatment with the NAIs were not significant. Conclusions: The virus isolation rates after treatment with each of the NAIs were significantly lower in the A H1N1/09 group, suggesting that the pandemic A H1N1/09 virus was more sensitive to the NAIs than the seasonal A H3N2 virus was. Clinically, there were no significant differences in the effectiveness of the NAIs between the H1N1/09 infected group and H3N2 infected group. Introduction During the season 20.7 million cases of pandemic influenza A H1N1/09 infection were reported in Japan but only 198 deaths were reported nationwide [1] and there were no reports of deaths of pregnant women [2]. Japan may have had the lowest incidence of severe cases in the H1N1/09 pandemic and the lowest case fatality rate for symptomatic illness (<0.001%; 198/20.7 million) among the countries where a widespread epidemic was reported. The very low mortality rate in Japan in the H1N1/09 epidemic was probably attributable to the universal implementation of early treatment with neuraminidase inhibitors (NAIs) [1]. Since the season, in addition to oseltamivir and zanamivir, the recently approved intravenous 2015 International Medical Press (print) (online) 49

2 N Sugaya et al. drug peramivir [3,4] and inhaled drug laninamivir [5,6] have been used in Japan, bringing to four the total number of NAIs currently being used in hospitals and clinics nationwide [7]. However, the widespread use of NAIs in Japan has given rise to concern with regard to persistent virus shedding as well as with regard to resistance to NAIs. There has been controversy with regard to the timing of the return to school of children with influenza who were treated with NAIs in Japan, because the duration of fever is shorter in children with influenza treated with NAIs, and if they return to school sooner, they may transmit the virus and cause a large outbreak in the school. With this possibility in mind the Japanese government recently changed the criterion for returning to school after an influenza infection. Based on the assumption that schoolchildren should stay home for at least 5 days after the onset of fever, they are now only permitted to return to school two days after their temperature has returned to normal. This change in criterion was made because in the absence of antiviral treatment adult influenza patients are known to shed viruses for 5 days after the onset of fever [8]. Virus shedding after treatment with NAIs is a more serious problem in a pandemic. Persistent virus shedding was frequently reported during the H1N1/09 pandemic [9 13]. The results of one study suggested that most patients (91%) shed H1N1/09 virus from one day before to 8 days after onset, and H1N1/09 is often shed for a longer period of time than seasonal influenza viruses are [9]. The purpose of this study was to compare virus shedding by children infected with pandemic A H1N1/09 and children infected with A H3N2 who had been treated with NAIs, and to investigate whether there was an association between the duration of virus shedding and the clinical effectiveness of the NAIs against A H1N1/09 and A H3N2. Methods Virus shedding To compare the virus isolation rates of the influenza patients, nasal or throat swab specimens were obtained before the start of NAI treatment (day 1) and again 3 to 6 days after the start of treatment (day 4 to day 7). Virus isolation rate in this study means the percentage of subjects from whom the virus was isolated at a given interval (3 to 6 days) after the start of NAI treatment. The virus isolation rate after NAI treatment was compared in the influenza A H3N2 group and the pandemic A H1N1/09 group. There were three subgroups in each virus infection group, that is, subgroups treated with peramivir, laninamivir and oseltamivir, respectively. Virus infection titres before and after the start of treatment were also compared. All of the patients included in this study arrived at one of our hospitals with a fever >38 C within 48 h after the onset of the fever. We assumed that if each of the influenza viruses was highly sensitive to the NAIs and as a result that the virus replication was suppressed, the virus isolation rate and mean virus infection titre after NAI treatment would be lower. Virus shedding and the clinical effectiveness of NAI treatment were investigated in children with influenza who came to our hospitals for the treatment of influenza-like illness in the season and in the season. In the season the main epidemic strains were A H1N1/09 and A H3N2, and in the season the main epidemic strains were A H3N2 and B viruses. Because NAI treatment is now standard practice in Japan, there were no cases that had not been treated with an NAI to serve as controls. The patients who were tested for viral shedding and the patients who were tested for clinical effectiveness (duration of fever) were different groups, although some patients overlapped, especially among the children treated with peramivir. Clinical effectiveness of the NAIs Because a prolonged high fever is one of the most important symptoms of influenza virus infection in children, we assessed the clinical effectiveness of the NAIs on the basis of the duration of the febrile period after the start of treatment [14]. The ages of the patients in the subgroups treated with each of the NAIs were matched as closely as possible within each of the virus infection groups. We mainly selected patients between 5 and 10 years old to compare the effectiveness of the NAIs, because, in Japan, oseltamivir can only be used to treat children from 1 year to 10 years of age because of the neuropsychiatric disorders suspected of being adverse reactions to oseltamivir that have been reported in older children [9]. The inhaled NAIs, that is, zanamivir and laninamivir, on the other hand, can be used starting at 5 years of age, and intravenous peramivir has been approved in Japan for the treatment of children over 2 months of age. We excluded patients treated with zanamivir from the evaluation of effectiveness against A H1N1/09, because the patients mean age was higher than 10 years old. The choice of NAI was based on patients or parents wishes, except in the group treated with peramivir. The patients treated with oseltamivir or laninamivir were outpatients with mild influenza, a fever >38 C and upper respiratory symptoms such as cough or rhinorrhoea. No other NAIs had been used to treat the patients treated with peramivir before the intravenous infusion of peramivir. A total of 9 of the 38 patients treated with International Medical Press

3 Virus shedding and fever duration peramivir were hospitalized patients, although their condition was not severe. On admission, five of the nine hospitalized patients treated with peramivir had febrile convulsions and four had bronchitis. In 17 of the 29 outpatients treated with peramivir, peramivir was selected because of nausea and vomiting. All patients in this study arrived at one of our hospitals with a fever of over 38.0 C within 48 h after the onset of fever and they were tested with rapid diagnostic tests and diagnosed with influenza A or B before the start of NAI treatment. The diagnoses were later confirmed by virus isolation and/or PCR. Oseltamivir was prescribed in weight-based unit doses to be administered in divided doses twice daily for 5 days. Peramivir is administered to children as a single 10 mg/kg intravenous dose infused over 15 min (upper limit, 600 mg) on the first day of treatment [3,4]. Laninamivir is administered in the form of a single inhalation (20 mg) on the first day of treatment [5,6]. Parents were instructed to take their child s temperature twice daily, in the morning and in the evening, on 5 consecutive days, and record it on a fever record sheet we prepared. The record sheet was returned to us on the day of the final visit. We considered a patient s fever to have resolved when the patient s temperature had fallen to 37.5 C and remained there for two more measurements. Virus isolation and titration Clinical specimens found to be virus-positive by testing with rapid diagnostic kits were stored at -80 C until virus isolation. Madin Darby canine kidney (MDCK) cells and MDCK cells overexpressing the b-galactoside a2,6-sialyltransferase I gene (MDCK-ST6GalI) [15] were used for viral isolation and for plaque assays. Monolayer cell cultures in 12-well plates were infected with 10-fold serial dilutions of each nose or throat swab specimen. After adsorption at 33 C under 5% CO 2 for 1 h, the infected cells were overlaid with 0.75% SeaKem agarose (Lonza, Rockland, ME, USA) in the presence of tosyl-phenylalanine-chloromethyl-ketonetreated trypsin (0.5 mg/ml; Worthington Biochemical, Lakewood, NJ, USA) in MEM containing 7.5% bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA) and were then incubated at 33 C under 5% CO 2 for 3 days. After fixation with 20% formalin, the plaques were stained with amido black. Virus infection titres are expressed as log 10 plaque forming units/ml. Viral subtypes were determined by Multiplex PCR with four primers designed to differentiate between the pandemic H1N1 virus and seasonal H1N1 and H3N2 viruses [16]. Statistical methods We used Kaplan Meier curves for the duration of fever after the start of treatment in each treatment group as the basis for making the comparisons, and tested them for differences by the log-rank test, by using the R version 2.15 statistical analysis software program [17]. We used Student s t-test or Welch s t-test to analyse mean values for statistically significant differences and the χ 2 test to evaluate differences in virus isolation rate and virus infection titre between the A H1N1/09 group and the A H3N2 group. All data are expressed as means ±sd. Written informed consent to inclusion in this study was obtained from each child s parent or guardian. This study was conducted with the approval of the ethics committee of each of our hospitals. Results Comparison between the viral shedding patterns in the influenza A H3N2 group and the influenza A H1N1/09 group after treatment with NAIs There were 14 patients (mean age 6.1 ±3.5 years) with influenza A H3N2 who had been treated with peramivir. Their mean virus titres before and after peramivir infusion were 4.73 and 0.82, respectively. A H3N2 viruses were isolated from 6 (42.9%) of these 14 patients after treatment. There were 14 patients (mean age 4.0 ±4.0 years) with influenza A H1N1/09 who had been treated with peramivir. Their mean virus titres before and after treatment were 4.36 and 0.34, respectively. Viruses were isolated from 2 (14.3%) of these 14 patients after treatment. After peramivir treatment the virus isolation rate was higher in the influenza A H3N2 group than in the A H1N1/09 group. There were 10 patients (mean age 8.5 ±2.3 years) with influenza A H3N2 who had been treated with laninamivir. Their mean virus titres before and after treatment were 3.47 and 0.97, respectively. A H3N2 viruses were isolated from 5 (50.0%) of these 10 patients after treatment. There were six patients (mean age 10.8 ±3.4 years) in the influenza A H1N1/09 group who had been treated with laninamivir. Their mean virus titres before and after treatment were 3.43 and 0.30, respectively. Viruses were isolated from one (16.7%) of these six patients after treatment. The same as after treatment with peramivir, the rate of virus isolation after laninamivir treatment was higher in the influenza A H3N2 group than in the A H1N1/09 group. There were 10 patients (mean age 4.1 ±3.2 years) with influenza A H3N2 who had been treated with oseltamivir. Their mean virus titres before and after treatment were 4.55 and 1.00, respectively. A H3N2 viruses were isolated from 4 (40.0%) of these 10 patients after treatment. There were five patients (mean age 3.6 ±2.0 years) in the influenza A H1N1/09 subgroup who had been treated with oseltamivir. Their mean virus titres before and after treatment were 5.02 and 0.26, respectively. Viruses were isolated from one (20.0%) of these five Antiviral Therapy

4 N Sugaya et al. patients after treatment. The same as after treatment with peramivir or laninamivir, the rate of virus isolation after treatment with oseltamivir was higher in the influenza A H3N2 group than in the A H1N1/09 group. A total of 15 A H3N2 strains were isolated after NAI treatment, and there were no significant differences between the virus isolation rates in the three A H3N2 treatment groups: peramivir 42.9%, laninamivir 50% and oseltamivir 40%. By contrast, only four A H1N1/09 strains were isolated after treatment, and there were no significant differences between the virus isolation rates in the three A H1N1/09 treatment groups: peramivir 14.3%, laninamivir 16.7% and oseltamivir 20%. The virus shedding patterns of the patients with A H3N2 infection (n=34, mean age 6.2 ±3.5 years) and patients with A H1N1/09 infection (n=25, mean age 5.6 ±4.6 years) are summarized in Figure 1. Throat or nasal specimens were collected from three patients (one in the A H3N2 infection group and two in the A H1N1/09 infection group) on day 2 or day 3, and their specimens were already virus-negative. Specimens were collected from the same three patients again during the period between day 4 to day 7, and the results were still negative. There were no significant differences between the A H3N2 infection group and A H1N1/09 infection group in mean age or mean virus infection titre before treatment. After the start of treatment, influenza viruses were Figure 1. Viral shedding by patients treated with the neuraminidase inhibitors, peramivir, laninamivir and oseltamivir 7.0 H3N2 (n=34) 6.0 Virus infectivity, pfu/ml Day 7.0 H1N1/09 (n=25) 6.0 Virus infectivity, pfu/ml Day International Medical Press

5 Virus shedding and fever duration isolated from 15 (44.1%) of the 34 patients infected with the A H3N2 virus but from only 4 (16.0%) of the 25 patients infected with the A H1N1/09 virus. The virus isolation rate was significantly higher in the A H3N2 infected group than in the A H1N1/09 infected group (P<0.05) and the mean virus infection titre after treatment was significantly higher in the A H3N2 group than in the A H1N1/09 group (0.98 ±1.28 versus 0.31 ±0.76; P<0.05). After NAI treatment the mean virus infection titres of the virus-positive patients were similar: A H3N2 group 2.20 ±1.28 (n=15) versus A H1N1/09 group 1.94 ±0.58 (n=4). The viral titres before treatment of the group of patients with persistent virus shedding and the group of patients without virus shedding were compared. The mean titres of the A H3N2 patients before NAI treatment were 4.02 ±1.03 (n=19) in the non-shedding group and 4.68 ±1.05 (n=15) in the persistent virus shedding group. The mean titres of the A H1N1/09 patients before NAI treatment were 4.21 ±1.22 (n=21) in the non-shedding group and 4.57 ±1.83 (n=4) in the persistent virus shedding group. There were no significant differences in viral titres between the non-shedding group and persistent virus shedding group before treatment of the H3N2 patients or the A H1N1/09 patients. The mean age of the group of patients without virus shedding (n=40) and the group of patients with persistent virus shedding (n=19) were compared. The mean age of the patients were 6.5 ±4.1 years in the non-shedding group and 4.8 ±3.5 years in the persistent virus shedding group. There were no significant differences in the mean age between the non-shedding group and persistent virus shedding group (P>0.1). Clinical effectiveness Comparison of the clinical effectiveness of the three NAIs against influenza A H3N2 infection The mean duration of fever after the start of treatment of the patients with influenza A H3N2 was shortest in the peramivir group (Table 1). The results of the log-rank test showed that the duration of fever after the start of treatment was significantly shorter in Table 1. Comparison between the effectiveness of three neuraminidase inhibitors Peramivir Oseltamivir Laninamivir A H3N2 Patients, n Mean age, years ±sd 7.1 ± ± ±1.9 Mean duration of fever, days ±sd 0.89 ± ± ±1.52 A H1N1/09 Patients, n Mean age, years ±sd 8.0 ± ± ±2.1 Mean duration of fever, days ±sd 0.75 ± ± ±1.19 the peramivir group than laninamivir group (P<0.01), but the difference in duration of fever between the peramivir group and the oseltamivir group was not significant (P>0.05). Comparison between the effectiveness of the three NAIs against influenza A H1N1/09 infection The mean duration of fever after the start of treatment of the patients with A H1N1/09 infection was shortest in the peramivir group (Table 1). According to the results of the log-rank test the duration of fever in the peramivir group was significantly shorter than in the oseltamivir group (P<0.01) or the laninamivir group (P<0.05). There were no significant differences in the effectiveness of each of the NAIs between the H1N1/09 infection group and H3N2 infection group. Discussion To our knowledge, this is the first study to compare virus shedding by pandemic A H1N1/09 patients and seasonal A H3N2 patients who were treated with NAIs. The results of our study showed a higher rate of influenza virus shedding by A H3N2 virus infected patients than by patients infected with A H1N1/09 virus (44.1% versus 16.0%; P<0.05). The mean virus infection titres of the virus-positive patients according to the results of cultures of specimens after NAI treatment of the A H3N2 patients and the A H1N1/09 patients were similar: 2.20 (n=15) versus 1.94 (n=4). Based on these findings, patients, especially the A H3N2 patients, will transmit influenza virus to others even on day 4 to day 7 after treatment with an NAI. However, our study was small and non-randomized. Because people have much less immunity against A H1N1/09 virus than against seasonal influenza viruses such as A H3N2 virus, it is generally thought that shedding of pandemic A H1N1/09 virus persists longer than shedding of seasonal influenza viruses [9 13]. Our results conflict with the general concept mentioned above. The cause of the different results is unknown, but is probably attributable to our method, virus isolation and the early timing of the start of treatment, that is, within 48 h after the onset of a fever that had reached >38 C. In most reports, virus shedding was detected by PCR. A detailed analysis of the 50% inhibitory concentration values and strains that are resistant to peramivir, laninamivir and oseltamivir will be reported elsewhere. Because the patients in this study were children, the patients immunity status against A H1N1/09 was not associated with a shorter virus shedding of A H1N1/09 patient group. Moreover, there were no significant differences between the virus shedding patterns of the peramivir, laninamivir and oseltamivir subgroups in either the A H1N1/09 patient group or A H3N2 patient group. Therefore, the difference in the duration Antiviral Therapy

6 N Sugaya et al. of virus shedding between A H1N1/09 patient group or A H3N2 patient group must have been attributable to higher sensitivity of the H1N1/09 virus to the NAIs. Virus shedding is an important factor in evaluating the effectiveness of NAIs. Longer virus shedding is associated with greater transmission of influenza viruses and the emergence of resistant strains [18]. The lower rate of virus shedding by the patients with A H1N1/09 virus infection that was demonstrated in this study has probably been responsible for the less frequent emergence of H1N1/09 resistant strains [19,20] than expected, and for the higher effectiveness of oseltamivir in protecting against severe illness in patients with H1N1/09 infection that has been confirmed in many countries [9,21,22]. Based on the duration of fever after the start of treatment, peramivir was the most effective of the three NAIs against influenza A H1N1/09 infection. However, the fact that this was a non-randomized, open-label study and the number of patients treated with peramivir was small was a limitation of this study with regard to the statistical significance of the clinical effectiveness data. One study conducted in Japan reported more rapid resolution of fever among A H3N2 patients treated with peramivir than among A H3N2 patients treated with other NAIs [23]. The virus isolation rates after treatment with peramivir, laninamivir and oseltamivir in this study were similar in the A H3N2 patient group (42.9%, 50% and 40%, respectively) and in the A H1N1/09 patient group (14.3%, 16.7% and 20%, respectively). However, duration of fever after NAI treatment was shortest in the peramivir group of influenza A H1N1/09 patients. Thus, the duration of virus shedding is not correlated with the duration of influenza patients fever in children. In other words, we could not determine the infectiousness of influenza patients based on their duration of fever or the NAI that was used to treat them, although high fever is one of the most important symptoms of influenza in children [14,24]. The duration of respiratory symptoms has been reported to be correlated with virus shedding by influenza patients in adults, whereas duration of fever was not found to be correlated with duration of virus shedding in the same study [12]. Our study showed a higher rate of virus shedding by A H3N2 infected patients than by patients infected with A H1N1/09 virus, although based on the duration of fever there were no significant differences in the effectiveness of the NAIs between the H1N1/09 infected group and H3N2 infected group. Acknowledgements This work was supported by a grant-in-aid for Specially Promoted Research and by a contract research fund for the Program for Funding Research Centers for Emerging and Reemerging Infectious Diseases from the Ministries of Education, Culture, Sports, Science, and Technology of Japan, and by grants-in-aid of Health, Labor, and Welfare of Japan, by ERATO (Japan Science and Technology Agency) and by National Institute of Allergy and Infectious Diseases Public Health Service Research grants. Disclosure statement NS has received speaker s honoraria from Daiichi Sankyo, Shionogi, Chugai and GlaxoSmithKline. YK has received speaker s honoraria from Toyama Chemical and Astellas Inc.; grant support from Chugai Pharmaceuticals, Daiichi Sankyo Pharmaceutical, Toyama Chemical, Otsuka Pharmaceutical Co., Ltd; is a consultant for Crucell; and is a founder of FluGen. The remaining authors declare no competing interests. References 1. Sugaya N, Shinjoh M, Mitamura K, Takahashi T. 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