Bonnie C. K. Wong

Viral Loads and Duration of Viral Shedding in Adult Patients Hospitalized with Influenza

Abstract BackgroundThe goal of this study was to characterize viral loads and factors affecting viral clearance in persons with severe influenza MethodsThis was a 1-year prospective, observational study involving consecutive adults hospitalized with influenza. Nasal and throat swabs were collected at presentation, then daily until 1 week after symptom onset. Real-time reverse-transcriptase polymerase chain reaction to determine viral RNA concentration and virus isolation were performed. Viral RNA concentration was analyzed using multiple linear or logistic regressions or mixed-effect models ResultsOne hundred forty-seven inpatients with influenza A (H3N2) infection were studied (mean age ± standard deviation, 72±16 years). Viral RNA concentration at presentation positively correlated with symptom scores and was significantly higher than that among time-matched outpatients (control subjects). Patients with major comorbidities had high viral RNA concentration even when presenting >2 days after symptom onset (mean ± standard deviation, 5.06±1.85 vs 3.62±2.13 log10 copies/mL; P=.005; β, +0.86 [95% confidence interval, +0.03 to +1.68]). Viral RNA concentration demonstrated a nonlinear decrease with time; 26% of oseltamivir-treated and 57% of untreated patients had RNA detected at 1 week after symptom onset. Oseltamivir started on or before symptom day 4 was independently associated with an accelerated decrease in viral RNA concentration (mean β [standard error], −1.19 [0.43] and −0.68 [0.33] log10 copies/mL for patients treated on day 1 and days 2–3, respectively; P<.05) and viral RNA clearance at 1 week (odds ratio, 0.10 [95% confidence interval, 0.03–0.35] and 0.30 [0.10–0.90] for patients treated on day 1–2 and day 3–4, respectively). Conversely, major comorbidities and systemic corticosteroid use for asthma or chronic obstructive pulmonary disease exacerbations were associated with slower viral clearance. Viral RNA clearance was associated with a shorter hospital stay (7.0 vs 13.5 days; P=.001) ConclusionPatients hospitalized with severe influenza have more active and prolonged viral replication. Weakened host defenses slow viral clearance, whereas antivirals started within the first 4 days of illness enhance viral clearance

Outcomes of adults hospitalised with severe influenza

Background The aim of this study was to investigate factors affecting clinical outcomes of adults hospitalised with severe seasonal influenza. Methods A prospective, observational cohort study was conducted over 24 months (2007–2008) in two acute, general hospitals. Consecutive, hospitalised adult patients were recruited and followed once their laboratory diagnosis of influenza A/B was established (based on viral antigen detection and virus isolation from nasopharyngeal aspirates collected per protocol). Outcomes studied included in-hospital death, length of stay and duration of oxygen therapy. Factors affecting outcomes were analysed using multivariate Cox proportional hazards models. Sequencing analysis on the neuraminidase gene was performed for available H1N1 isolates. Results 754 patients were studied (influenza A, n=539; >75% H3N2). Their mean age was 70±18 years; co-morbidities and serious complications were common (61–77%). Supplemental oxygen and ventilatory support was required in 401 (53.2%) and 41 (5.4%) patients, respectively. 39 (5.2%) patients died; pneumonia, respiratory failure and sepsis were the causes. 395 (52%) patients received antiviral (oseltamivir) treatment. Omission of antiviral treatment was associated with delayed presentation or negative antigen detection results. The mortality rate was 4.56 and 7.42 per 1000 patient-days in the treated and untreated patients, respectively; among those with co-morbidities, it was 5.62 and 11.64 per 1000 patient-days, respectively. In multivariate analysis, antiviral use was associated with reduced risk of death (adjusted HR (aHR) 0.27 (95% CI 0.13 to 0.55); p<0.001). Improved survival was observed with treatment started within 4 days from onset. Earlier hospital discharge (aHR 1.28 (95% CI 1.04 to 1.57); p=0.019) and faster discontinuation of oxygen therapy (aHR 1.30 (95% CI 1.01 to 1.69); p=0.043) was associated with early treatment within 2 days. Few (n=15) H1N1 isolates in this cohort had the H275Y mutation. Conclusions Antiviral treatment for severe influenza is associated with reduced mortality and improved clinical outcomes.

Cytokine response patterns in severe pandemic 2009 H1N1 and seasonal influenza among hospitalized adults.

Background Studying cytokine/chemokine responses in severe influenza infections caused by different virus subtypes may improve understanding on pathogenesis. Methods Adults hospitalized for laboratory-confirmed seasonal and pandemic 2009 A/H1N1 (pH1N1) influenza were studied. Plasma concentrations of 13 cytokines/chemokines were measured at presentation and then serially, using cytometric-bead-array with flow-cytometry and ELISA. PBMCs from influenza patients were studied for cytokine/chemokine expression using ex-vivo culture (Whole Blood Assay,±PHA/LPS stimulation). Clinical variables were prospectively recorded and analyzed. Results 63 pH1N1 and 53 seasonal influenza patients were studied. pH1N1 patients were younger (mean±S.D. 42.8±19.2 vs 70.5±16.7 years), and fewer had comorbidities. Respiratory/cardiovascular complications were common in both groups (71.4% vs 81.1%), although severe pneumonia with hypoxemia (54.0% vs 28.3%) and ICU admissions (25.4% vs 1.9%) were more frequent with pH1N1. Hyperactivation of the proinflammatory cytokines IL-6, CXCL8/IL-8, CCL2/MCP-1 and sTNFR-1 was found in pH1N1 pneumonia (2–15 times normal) and in complicated seasonal influenza, but not in milder pH1N1 infections. The adaptive-immunity (Th1/Th17)-related CXCL10/IP-10, CXCL9/MIG and IL-17A however, were markedly suppressed in severe pH1N1 pneumonia (2–27 times lower than seasonal influenza; P−values<0.01). This pattern was further confirmed with serial measurements. Hypercytokinemia tended to be sustained in pH1N1 pneumonia, associated with a slower viral clearance [PCR-negativity: day 3–4, 55% vs 85%; day 6–7, 67% vs 100%]. Elevated proinflammatory cytokines, particularly IL-6, predicted ICU admission (adjusted OR 12.6, 95%CI 2.6–61.5, per log10unit increase; P = 0.002), and correlated with fever, tachypnoea, deoxygenation, and length-of-stay (Spearmans rho, P-values<0.01) in influenza infections. PBMCs in seasonal influenza patients were activated and expressed cytokines ex vivo (e.g. IL-6, CXCL8/IL-8, CCL2/MCP-1, CXCL10/IP-10, CXCL9/MIG); their ‘responsiveness’ to stimuli was shown to change dynamically during the illness course. Conclusions A hyperactivated proinflammatory, but suppressed adaptive-immunity (Th1/Th17)-related cytokine response pattern was found in severe pH1N1 pneumonia, different from seasonal influenza. Cytokine/immune-dysregulation may be important in its pathogenesis.

Complications and Outcomes of Pandemic 2009 Influenza A (H1N1) Virus Infection in Hospitalized Adults: How Do They Differ From Those in Seasonal Influenza?

BACKGROUND It is unclear whether pandemic 2009 influenza A (pH1N1) infection caused more significant disease among hospitalized adults than seasonal influenza. METHODS A prospective, observational study was conducted in adults hospitalized with polymerase chain reaction-confirmed pH1N1 infection in 2 acute-care general hospitals from June 2009 to May 2010 (n = 382). Complications and outcomes were described and compared with those in a seasonal influenza cohort (2007-2008, same hospitals; n = 754). RESULTS Hospitalized patients with pH1N1 influenza were younger than those with seasonal influenza (mean age ± standard deviation, 47 ± 20 vs 70 ± 19 years) and fewer had comorbid conditions (48% vs 64%). The rate of positive immunofluorescence assay results was low (54% vs 84%), and antiviral use was frequent (96% vs 52%). Most patients in both cohorts developed complicated illnesses (67.8% vs 77.1%), but patients with pH1N1 influenza had higher rates of extrapulmonary complications (23% vs 16%; P = .004) and intensive care unit admission and/or death (patient age <35 years, 2.3% vs 0%; 35-65 years, 12.4% vs 3.2%; >65 years, 13.5% vs 8.5%; adjusted odds ratio [OR] 2.13; 95% confidence interval [CI], 1.25-3.62; P = .005). Patients who received antiviral treatment within 96 h after onset had better survival (log-rank test, P < .001). However, without timely treatment, the mortality risk was higher with pH1N1 infection (9.0% vs 5.8% for seasonal influenza; adjusted OR, 6.85; 95% CI, 1.64-28.65; P = .008]. Bacterial superinfection worsened outcomes. CONCLUSIONS Adults hospitalized for pH1N1 influenza had significant complications and mortality despite being younger than patients with seasonal influenza. Antiviral treatment within 96 h may improve survival.

Viral clearance and inflammatory response patterns in adults hospitalized for pandemic 2009 influenza A(H1N1) virus pneumonia.

BACKGROUND Little is known about the virological and inflammatory responses of severe pandemic 2009 influenza A(H1N1) virus pneumonia during antiviral treatment. METHODS In a prospective observational study, we recruited consecutive adults hospitalized with confirmed pandemic 2009 H1N1 infection during a 16-week period. Nasopharyngeal aspirate and non-respiratory samples (blood, stool and urine) were collected at presentation, and serial nasopharyngeal flocked swabs (NPFS) and tracheal aspirates (TA) were collected after initiating oseltamivir treatment for quantitative viral RNA assay, using real-time reverse transcriptase-PCR. Serial plasma samples were collected for cytokine/chemokine assay using cytometric bead array. Patients with severe pneumonia (lung infiltrates and hypoxaemia) were compared to those with milder illnesses. RESULTS A total of 66 patients were studied (mean age 43 ±20 years); 28 (42%) developed severe pneumonia, of whom 10 (15%) required intubation. Severe pneumonia was associated with older age, dyspnoea, delayed presentation >2 days from onset, extrapulmonary virus detection (13-28%) and higher viral concentration despite late-presentation (multiple linear regression, β=0.94, 95% confidence interval 0.15-1.74; P=0.02). Patients with severe pneumonia exhibited slow viral clearance with oseltamivir treatment, particularly in the lower respiratory tract (median [interquartile range] durations of RNA positivity after antiviral initiation were NPFS 6.0 days [3.0-8.0], TA 11.0 days [7.8-14.3] versus milder illness group NPFS of 2.0 days [1.0-3.0] days; P<0.01). High viral load in lower respiratory tract despite upper-tract RNA negativity and viral rebound after stopping treatment were noted in some patients. H275Y mutation was absent. High plasma levels of interleukin (IL)-6, CXCL-8 (IL-8), CCL2 (monocyte chemoattractant protein-1) and soluble tumour necrosis factor receptor-1 were observed, which correlated with the extent and progression of pneumonia in hospital. CONCLUSIONS In severe 2009 H1N1 pneumonia, viral clearance is slow with treatment, particularly in the lower respiratory tract. A more sustained antiviral regime appears warranted.

Hypercytokinemia and Hyperactivation of Phospho-p38 Mitogen-Activated Protein Kinase in Severe Human Influenza A Virus Infection

BACKGROUND We postulate that hypercytokinemia plays a role in immunopathogenesis of severe human influenza. METHODS We prospectively studied 39 consecutive patients who were hospitalized with severe influenza A virus infection. On laboratory confirmation of the diagnosis, paired acute-phase (obtained at hospital admission) and convalescent-phase (obtained >10 days after hospital admission) plasma samples were collected for assay of 11 cytokines and chemokines (interleukin [IL] 1 beta; IL-6; IL-10; IL-12p70; tumor necrosis factor alpha; IL-8; monokine induced by interferon [IFN]-gamma; IFN-inducible protein 10; monocyte chemoattractant protein 1; regulated upon activation, normal T cell-expressed and secreted; and IFN-gamma) using cytometric bead-array analysis and enzyme-linked immunosorbent assay. Simultaneously, virus concentration in the acute-phase nasopharyngeal aspirate was determined using real-time quantitative reverse-transcriptase polymerase chain reaction. Intracellular signaling molecules regulating lymphocyte activation, phospho-p38 mitogen-activated protein kinase and phospho-extracellular signal-regulated protein kinase in CD4+ and CD8+ T lymphocytes were studied in the acute-phase samples using flow cytometric analysis and were compared with results for samples from healthy control subjects. RESULTS Statistically significant increases in plasma IL-6 (3.7-fold increase), IL-8 (2.6-fold increase), IFN-induced protein 10 (4.9-fold increase), and monokine induced by IFN-gamma (2.3-fold increase) concentrations were detected during acute illness (P < .01 for all, by Wilcoxon signed-rank test); the highest concentrations were observed on symptom days 3 and 4. Corresponding plasma cytokine and chemokine concentrations and nasopharyngeal viral loads showed statistically significant correlations (rho = 0.41, 0.49, 0.54, and 0.46, respectively; P < or = .01). Phospho-p38 mitogen-activated protein kinase expression in CD4+ lymphocytes was increased, correlating with cytokine concentrations (e.g., for IFN-induced protein 10, rho = 0.78; P < .01); phospho-extracellular signal-regulated protein kinase was suppressed. Advanced age and comorbidity were associated with aberrant IL-6, IL-8, and monokine induced by IFN-gamma responses (P < .05, by Mann-Whitney U test). An elevated IL-6 concentration was independently associated with prolonged hospitalization (hospitalization for >5 days; P = .02), adjusted for age, comorbidity, and virus load. CONCLUSIONS Hypercytokinemia (of proinflammatory and T helper 1 cytokines) is detected in severe influenza, correlating with clinical illness and virus concentration. Hyperactivation of phospho-p38 mitogen-activated protein kinase (in T helper cells) is possibly involved. Early viral suppression may attenuate these potentially deleterious cytokine responses.

Fecal Viral Concentration and Diarrhea in Norovirus Gastroenteritis

Fecal viral concentrations of 40 patients infected with norovirus genogroup GII.4 correlated with diarrhea duration and frequency of vomiting. Higher viral concentration and older age were independently associated with prolonged diarrhea (>4 days). These findings provide information on the pathogenesis and transmission of norovirus infections.

Possible Role of Aerosol Transmission in a Hospital Outbreak of Influenza

Abstract Background. We examined the role of aerosol transmission of influenza in an acute ward setting. Methods. We investigated a seasonal influenza A outbreak that occurred in our general medical ward (with open bay ward layout) in 2008. Clinical and epidemiological information was collected in real time during the outbreak. Spatiotemporal analysis was performed to estimate the infection risk among patients. Airflow measurements were conducted, and concentrations of hypothetical virus-laden aerosols at different ward locations were estimated using computational fluid dynamics modeling. Results. Nine inpatients were infected with an identical strain of influenza A/H3N2 virus. With reference to the index patients location, the attack rate was 20.0% and 22.2% in the “same” and “adjacent” bays, respectively, but 0% in the “distant” bay (P=.04). Temporally, the risk of being infected was highest on the day when noninvasive ventilation was used in the index patient; multivariate logistic regression revealed an odds ratio of 14.9 (95% confidence interval, 1.7–131.3; P=.015). A simultaneous, directional indoor airflow blown from the “same” bay toward the “adjacent” bay was found; it was inadvertently created by an unopposed air jet from a separate air purifier placed next to the index patients bed. Computational fluid dynamics modeling revealed that the dispersal pattern of aerosols originated from the index patient coincided with the bed locations of affected patients. Conclusions. Our findings suggest a possible role of aerosol transmission of influenza in an acute ward setting. Source and engineering controls, such as avoiding aerosol generation and improving ventilation design, may warrant consideration to prevent nosocomial outbreaks.

A Prospective Intervention Study on Higher-Dose Oseltamivir Treatment in Adults Hospitalized With Influenza A and B Infections

BACKGROUND It is unclear if higher-dose oseltamivir provides benefit beyond the standard dose in influenza patients who require hospitalization. METHODS A prospective intervention study was performed in 2 acute care general hospitals in Hong Kong over 4 seasonal peaks (2010-2012). Adults (≥18 years) with laboratory-confirmed influenza (85 A/H3N2, 34 A/H1N1pdm09, 36 B) infections who presented within 96 hours were recruited. Study regimen of either 150 mg or 75 mg oseltamivir twice daily for 5 days was allocated by site, which was switched after 2 seasons. Subjects with preexisting renal impairment (creatinine clearance, 40-60 mL/minute) received 75 mg oseltamivir twice daily. Viral clearance by day 5 and clinical responses were compared between groups. Plasma steady-state trough oseltamivir carboxylate (OC) concentration was measured by high-performance liquid chromatography-tandem mass spectrometry. RESULTS Altogether, 41 and 114 patients received 150 mg and 75 mg twice-daily oseltamivir, respectively; their enrollment characteristics (mean age, 61 ± 18 vs 66 ± 16 years) and illness severity were comparable. Trough OC levels were higher in the 150-mg group (501.0 ± 237.0 vs 342.6 ± 192.7 ng/mL). There were no significant differences in day 5 viral RNA (44.7% vs 40.2%) or culture negativity (100.0% vs 98.1%), RNA decline rate, and durations of fever, oxygen supplementation, and hospitalization. Results were similar when analyzed by study arm (all cases and among those without renal impairment). Subanalysis of influenza B patients showed faster RNA decline rate (analysis of variance, F = 4.14; P = .05) and clearance (day 5, 80.0% vs 57.1%) with higher-dose treatment. No oseltamivir resistance was found. Treatments were generally well tolerated. CONCLUSIONS We found no additional benefit of higher-dose oseltamivir treatment in adults hospitalized with influenza A, but an improved virologic response in influenza B. CLINICAL TRIALS REGISTRATION ClinicalTrials.gov, NCT01052961.

Role of human Toll‐like receptors in naturally occurring influenza A infections

We investigated the roles of Toll‐like receptors (TLRs) in naturally occurring influenza.