Chiharu Kawakami

Resistant influenza A viruses in children treated with oseltamivir: descriptive study

BACKGROUND Oseltamivir is an effective inhibitor of influenza virus neuraminidase. Although viruses resistant to oseltamivir emerge less frequently than those resistant to amantadine or rimantadine, information on oseltamivir-resistant viruses arising during clinical use of the drug in children is limited. Our aim was to investigate oseltamivir resistance in a group of children treated for influenza. METHODS We analysed influenza A viruses (H3N2) collected from 50 children before and during treatment with oseltamivir. We sequenced the genes for neuraminidase and haemagglutinin and studied the mutant neuraminidases for their sensitivity to oseltamivir carboxylate. FINDINGS We found neuraminidase mutations in viruses from nine patients (18%), six of whom had mutations at position 292 (Arg292Lys) and two at position 119 (Glu119Val), which are known to confer resistance to neuraminidase inhibitors. We also identified another mutation (Asn294Ser) in one patient. Sensitivity testing to oseltamivir carboxylate revealed that the neuraminidases of viruses that have an Arg292Lys, Glu119Val, or Asn294Ser mutation were about 10(4)-10(5)-fold, 500-fold, or 300-fold more resistant than their pretreatment neuraminidases, respectively. Oseltamivir-resistant viruses were first detected at day 4 of treatment and on each successive day of the study. More than 10(3) infectious units per mL of virus were detected in some of the patients who did not shed drug-resistant viruses, even after 5 days of treatment. INTERPRETATION Oseltamivir-resistant mutants in children being treated for influenza with oseltamivir arise more frequently than previously reported. Furthermore, children can be a source of viral transmission, even after 5 days of treatment with oseltamivir.

Lower Clinical Effectiveness of Oseltamivir against Influenza B Contrasted with Influenza A Infection in Children

BACKGROUND Recently, many Japanese physicians have claimed that oseltamivir is less effective in children with influenza B virus infection. This study assesses the effectiveness of oseltamivir against influenza A (H3N2) and influenza B in children on the basis of the duration of febrile illness. METHODS We used oseltamivir to treat 127 children with influenza A (H3N2; mean age, 6.97 years [range, 1-15 years]) and 362 children with influenza B (mean age, 5.16 years [range, 1-15 years]) in outpatient clinics. The duration of fever after the start of oseltamivir therapy was compared in the influenza A group and the influenza B group. RESULTS The mean duration of fever after the start of oseltamivir therapy was significantly greater in the influenza B group than in the influenza A (H3N2) group (2.18 days vs. 1.31 days, respectively; P<.001). The difference was marked in young children (1-5 years old; 2.37 days for the influenza B group vs. 1.42 days for the influenza A group) but was not significant among older children (11-15 years old). The 50% inhibitory concentration of oseltamivir against influenza B virus was 75.4+/-41.7 nmol/L and was substantially higher than that for type A (H3N2) virus (0.3+/-0.1 nmol/L). Only 3 (1.6%) of 192 influenza B viruses were resistant to oseltamivir. CONCLUSIONS Oseltamivir is much less effective against influenza B virus infection in young children, probably because of the low sensitivity of influenza B viruses to oseltamivir. The effectiveness of oseltamivir against influenza B is influenced by age and host immunity. A few oseltamivir-resistant influenza B strains were isolated before the start of oseltamivir therapy.

Enhanced Expression of an α2,6-Linked Sialic Acid on MDCK Cells Improves Isolation of Human Influenza Viruses and Evaluation of Their Sensitivity to a Neuraminidase Inhibitor

ABSTRACT The extensive use of neuraminidase (NA) inhibitors to treat influenza virus infections mandates close monitoring for resistant variants. Cultured cells do not provide a reliable means of evaluating the susceptibility of human influenza virus isolates to NA inhibitors. That is, the growth of such viruses in cell lines (e.g., Madin-Darby canine kidney [MDCK] cells) is not inhibited by these drugs, even though their sialidase activity is drug-sensitive. Matrosovich et al. (J. Virol. 77:8418-8425, 2003) showed that an MDCK cell line overexpressing the human β-galactoside α2,6-sialyltransferase I (ST6Gal I) gene has the potential to assess the sensitivity of human influenza virus isolates to NA inhibitors, based on studies with a limited number of viruses. Here, we asked whether clinical isolates of influenza virus are universally sensitive to an NA inhibitor (oseltamivir) in an MDCK cell line expressing the ST6Gal I gene. The sensitivity of viruses to oseltamivir correlated with the sensitivity of viral sialidase to the compound, demonstrating the potential utility of this modified cell line for detecting NA inhibitor-resistant viruses. Moreover, in ST6Gal I-overexpressing cells, the growth of human influenza viruses was up to 2 logs higher than in MDCK cells. We conclude that the human ST6Gal I-expressing MDCK cell line is useful not only for evaluating their sensitivity to NA inhibitors, but also for isolation of influenza viruses from clinical samples.

Comparison of the Clinical Effectiveness of Oseltamivir and Zanamivir against Influenza Virus Infection in Children

BACKGROUND We compared the clinical effectiveness of oseltamivir and zanamivir in children with influenza A (H1N1) virus, influenza A (H3N2) virus, and influenza B virus infections. METHODS Total febrile period and the duration of fever after the start of treatment were compared between an oseltamivir-treated group (mean age, 8.9 years; range, 4.0-15.9 years) and a zanamivir-treated group (mean age, 10.0 years; range, 4.0-15.7 years) in the pediatric outpatient clinics of our hospitals. Oseltamivir was used to treat 91 children with influenza A (H3N2) infection and 24 children with influenza A (H1N1) infection. Zanamivir was used to treat 35 children with influenza A (H3N2) infection and 12 children with influenza A (H1N1) infection. Oseltamivir was also used to treat 128 children with influenza B virus infection, and zanamivir was used to treat 59 with influenza B virus infection. RESULTS There was no statistically significant difference in total febrile period or duration of fever after the start of treatment between the oseltamivir-treated group and the zanamivir-treated group of children with influenza A (H3N2) infection (mean duration of febrile period, 2.40 days vs. 2.39 days; mean duration of fever after the start of treatment, 1.35 days vs. 1.40 days), influenza A (H1N1) (mean duration of febrile period, 2.60 days vs. 2.46 days; mean duration of fever after the start of treatment, 1.79 days vs, 1.54 days), or influenza B (mean duration of febrile period, 2.95 days vs. 2.84 days; mean duration of fever after the start of treatment, 1.86 days vs. 1.67 days). Oseltamivir was more effective against influenza A (H3N2) than against influenza A (H1N1) or influenza B. CONCLUSIONS Oseltamivir and zanamivir were equally effective in reducing the febrile period of children with influenza A (H1N1), influenza A (H3N2), and influenza B virus infection.

Oseltamivir-Resistant Influenza A Viruses Circulating in Japan

ABSTRACT Surveillance studies of the influenza viruses circulating in Europe and other countries in 2007 and 2008 have revealed rates of resistance to oseltamivir of up to 67% among H1N1 viruses. In the present study, we examined 202 clinical samples obtained from patients infected with H1N1 virus in Japan in 2007 and 2008 for oseltamivir resistance and found that three were oseltamivir resistant (1.5%). The 50% inhibitory concentrations (IC50s), as measured by a sialidase inhibition assay with these drug-resistant viruses, were >100-fold higher than those of the nonresistant viruses (median IC50, 12.6 nmol/liter). The His274Tyr (strain N2 numbering) mutation of the neuraminidase protein, which is known to confer oseltamivir resistance, was detected in these three isolates. Phylogenetic analysis showed that one virus belonged to a lineage that is composed of drug-resistant viruses isolated in Europe and North America and that the other two viruses independently emerged in Japan. Continued surveillance studies are necessary to observe whether these viruses will persist.

Influenza Encephalopathy Associated with Infection with Human Herpesvirus 6 and/or Human Herpesvirus 7

Influenza-associated encephalopathy is often reported in young Japanese children, but its pathogenesis is unknown. Although influenza virus can be demonstrated by throat culture for patients with encephalopathy, cultures of samples of cerebrospinal fluids (CSF) do not yield the virus. Eight patients with encephalopathy or complicated febrile convulsions had influenza virus infection diagnosed by means of culture, polymerase chain reaction (PCR), or rapid diagnosis using throat swabs. In all 8 cases, the results of PCR testing of CSF specimens for influenza virus were negative. On the other hand, human herpesvirus 6 (HHV-6) DNA was demonstrated in CSF specimens obtained from 2 of 8 patients. In 3 of 8 patients, the presence of human herpesvirus 7 (HHV-7) DNA was demonstrated in CSF specimens. Some cases of influenza-associated encephalopathy reported in Japan may be attributable to a dual infection with influenza virus and HHV-6, -7, or both. Another possibility is that latent HHV-6 or HHV-7 in the brain is reactivated by influenza, causing encephalopathy or febrile convulsions.

Frequency of Drug-resistant Viruses and Virus Shedding in Pediatric Influenza Patients Treated With Neuraminidase Inhibitors

BACKGROUND Although influenza virus resistance to the neuraminidase inhibitor zanamivir is reported less frequently than is resistance to the neuraminidase inhibitor oseltamivir in clinical settings, it is unknown whether this difference is due to the limited use of zanamivir or to an inherent property of the drug. We therefore compared the prevalence of drug-resistant viruses and virus shedding in seasonal influenza virus-infected children treated with either oseltamivir or zanamivir. METHODS Clinical specimens (throat or nasal swab) were collected from a total of 144 pediatric influenza patients during the 2005-2006, 2006-2007, 2007-2008, and 2008-2009 influenza seasons. Neuraminidase inhibitor-resistant mutants were detected among the isolated viruses by sequencing the viral hemagglutinin and neuraminidase genes. Sensitivity of the viruses to neuraminidase inhibitors was tested by neuraminidase inhibition assay. RESULTS In oseltamivir- or zanamivir-treated influenza patients who were statistically comparable in their age distribution, vaccination history, and type or subtype of virus isolates, the virus-shedding period in zanamivir-treated patients was significantly shorter than that in oseltamivir-treated patients. Furthermore, the frequency of zanamivir-resistant viruses was significantly lower than that of oseltamivir-resistant viruses. CONCLUSION In comparison with treatment with oseltamivir, treatment of pediatric patients with zanamivir resulted in the emergence of fewer drug-resistant influenza viruses and a shorter virus-shedding period. We conclude that zanamivir shows promise as a better therapy for pediatric influenza patients.

Evaluation of Influenza Virus A/H3N2 and B Vaccines on the Basis of Cross-Reactivity of Postvaccination Human Serum Antibodies against Influenza Viruses A/H3N2 and B Isolated in MDCK Cells and Embryonated Hen Eggs

ABSTRACT The vaccine strains against influenza virus A/H3N2 for the 2010-2011 season and influenza virus B for the 2009-2010 and 2010-2011 seasons in Japan are a high-growth reassortant A/Victoria/210/2009 (X-187) strain and an egg-adapted B/Brisbane/60/2008 (Victoria lineage) strain, respectively. Hemagglutination inhibition (HI) tests with postinfection ferret antisera indicated that the antisera raised against the X-187 and egg-adapted B/Brisbane/60/2008 vaccine production strains poorly inhibited recent epidemic isolates of MDCK-grown A/H3N2 and B/Victoria lineage viruses, respectively. The low reactivity of the ferret antisera may be attributable to changes in the hemagglutinin (HA) protein of production strains during egg adaptation. To evaluate the efficacy of A/H3N2 and B vaccines, the cross-reactivities of postvaccination human serum antibodies against A/H3N2 and B/Victoria lineage epidemic isolates were assessed by a comparison of the geometric mean titers (GMTs) of HI and neutralization (NT) tests. Serum antibodies elicited by the X-187 vaccine had low cross-reactivity to both MDCK- and egg-grown A/H3N2 isolates by HI test and narrow cross-reactivity by NT test in all age groups. On the other hand, the GMTs to B viruses detected by HI test were below the marginal level, so the cross-reactivity was assessed by NT test. The serum neutralizing antibodies elicited by the B/Brisbane/60/2008 vaccine reacted well with egg-grown B viruses but exhibited remarkably low reactivity to MDCK-grown B viruses. The results of these human serological studies suggest that the influenza A/H3N2 vaccine for the 2010-2011 season and B vaccine for the 2009-2010 and 2010-2011 seasons may possess insufficient efficacy and low efficacy, respectively.

Effectiveness of Trivalent Inactivated Influenza Vaccine in Children Estimated by a Test-Negative Case-Control Design Study Based on Influenza Rapid Diagnostic Test Results

We assessed vaccine effectiveness (VE) against medically attended, laboratory-confirmed influenza in children 6 months to 15 years of age in 22 hospitals in Japan during the 2013–14 season. Our study was conducted according to a test-negative case-control design based on influenza rapid diagnostic test (IRDT) results. Outpatients who came to our clinics with a fever of 38°C or over and had undergone an IRDT were enrolled in this study. Patients with positive IRDT results were recorded as cases, and patients with negative results were recorded as controls. Between November 2013 and March 2014, a total of 4727 pediatric patients (6 months to 15 years of age) were enrolled: 876 were positive for influenza A, 66 for A(H1N1)pdm09 and in the other 810 the subtype was unknown; 1405 were positive for influenza B; and 2445 were negative for influenza. Overall VE was 46% (95% confidence interval [CI], 39–52). Adjusted VE against influenza A, influenza A(H1N1)pdm09, and influenza B was 63% (95% CI, 56–69), 77% (95% CI, 59–87), and 26% (95% CI, 14–36), respectively. Influenza vaccine was not effective against either influenza A or influenza B in infants 6 to 11 months of age. Two doses of influenza vaccine provided better protection against influenza A infection than a single dose did. VE against hospitalization influenza A infection was 76%. Influenza vaccine was effective against influenza A, especially against influenza A(H1N1)pdm09, but was much less effective against influenza B.

Trivalent inactivated influenza vaccine effective against influenza A(H3N2) variant viruses in children during the 2014/15 season, Japan.

The 2014/15 influenza season in Japan was characterised by predominant influenza A(H3N2) activity; 99% of influenza A viruses detected were A(H3N2). Subclade 3C.2a viruses were the major epidemic A(H3N2) viruses, and were genetically distinct from A/New York/39/2012(H3N2) of 2014/15 vaccine strain in Japan, which was classified as clade 3C.1. We assessed vaccine effectiveness (VE) of inactivated influenza vaccine (IIV) in children aged 6 months to 15 years by test-negative case–control design based on influenza rapid diagnostic test. Between November 2014 and March 2015, a total of 3,752 children were enrolled: 1,633 tested positive for influenza A and 42 for influenza B, and 2,077 tested negative. Adjusted VE was 38% (95% confidence intervals (CI): 28 to 46) against influenza virus infection overall, 37% (95% CI: 27 to 45) against influenza A, and 47% (95% CI: -2 to 73) against influenza B. However, IIV was not statistically significantly effective against influenza A in infants aged 6 to 11 months or adolescents aged 13 to 15 years. VE in preventing hospitalisation for influenza A infection was 55% (95% CI: 42 to 64). Trivalent IIV that included A/New York/39/2012(H3N2) was effective against drifted influenza A(H3N2) virus, although vaccine mismatch resulted in low VE.