Study Design and Study Subjects

The outbreak of pandemic (H1N1) 2009 in Taiwan began in July 2009. Our hospital, a 2,600-bed medical center that had experienced an outbreak of severe acute respiratory syndrome in 2003 (9–12), reorganized the severe acute respiratory syndrome research team to study pandemic (H1N1) 2009 virus in Taiwan. We established fever clinics (also called flu-like illness clinics) after a seriously ill child died of a pandemic (H1N1) 2009 virus infection at the beginning of this outbreak. Throat (tonsillopharyngeal) swab specimens from patients with influenza-like symptoms were obtained for the diagnosis of pandemic (H1N1) 2009 virus infection by differential and quantitative reverse RT-PCR or virus culture and RT-PCR. Patients confirmed as infected with pandemic (H1N1) 2009 virus received oseltamivir treatment for 5 days in a dosage based on body weight, according to the manufacturer’s recommendations.

Respiratory specimens were serially collected during the period of hospitalization or upon outpatient follow-up after informed consent was obtained. We collected the following clinical data: demographic characteristics, disease severity, coexisting conditions, fever-onset time, time to initiate antiviral treatment, and duration of fever after antiviral treatment. For the classification of disease severity, pneumonia was defined by the presence of patchy alveolar opacities on chest radiographs, and meningoencephalitis was diagnosed on the basis of presence of brain swelling with leptomeningeal enhancement seen on magnetic resonance imaging. This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital.

Virus Culture

Virus culture was performed by using a Madin-Darby canine kidney cell line obtained from American Type Culture Collection (Manassas, VA, USA). Supernatants from infected cultures were mixed with equal volumes of 1% guinea pig erythrocyte suspension and incubated for 1 h at 4°C. Culture cells with supernatants found positive by hemagglutination test were subjected to a direct fluorescent antibody assay with fluorescein-conjugated monoclonal antibodies against influenza viruses A and B and Evans blue dye (Oxoid, Ely, UK) to differentiate between influenza A and B viruses. Influenza A viruses were further differentiated from novel or seasonal influenza A viruses by real-time RT-PCR, as described below.

Differentiation and Quantification of Pandemic (H1N1) 2009 Virus

We extracted total nucleic acids from throat swab specimens or the supernatant of positive virus cultures with the Roche MagNA Pure Compact System (Roche Molecular Diagnostics, Mannheim, Germany) by using the manufacturer’s external lysis protocol and extraction reagents (Total Nucleic Acid Isolation Kit; Roche Molecular Diagnostics) to yield 100 μL of elutes. All PCRs were performed by using the standard real-time RT-PCR procedure of the Centers for Disease Control and Prevention for swine (H1N1) influenza (13). Briefly, 20 μL of RT-PCR mixture containing 1× Universal PCR Master Mix, 1× MultiScribe and RNase Inhibitor, 100 nmol/L TaqMan probe, and 5 μL extracted RNA, or water for no template controls, was subjected to RT-PCR in the presence of 250 nmol/L swine influenza nucleoprotein gene-specific primers or internal control (RNase P) primers. The reactions were performed and analyzed in a ABI PRISM 7000 sequence Detection System (Applied Biosystems, Branchburg, NJ, USA) under the following conditions: 30 min at 48ºC and 10 min at 95ºC, followed by 40 cycles of 30 s at 95ºC and 1 min at 60ºC.

For the quantitative assay, a reference standard was prepared by using the pUC57 vector (GeneDireX, Las Vegas, NV, USA) containing the corresponding specific viral sequence. Tenfold dilutions equivalent to 1 to 1.0 × 10⁷ copies per reaction were prepared to generate calibration curves and to be run in parallel with the test samples. The limit of detection of this real-time RT-PCR was 1 copy/mL. The viral load in each sample was calculated and corrected by using the threshold cycle value of the internal control (RNase P) (14).

Statistical Analysis

The correlation between the viral load and the number of days after the onset of symptoms was analyzed by using the Spearman rho correlation. Comparisons of the viral load among groups with different disease severity were analyzed by using Tukey post hoc multiple comparison after analysis of variance. The duration of viral shedding was calculated by using the Kaplan-Meier method and tested by the log-rank test. For multivariate analysis of the duration of viral shedding, we used the Cox proportional hazards model. All variables such as age, gender, disease severity, coexisting conditions, viral load from the first throat swab specimen, and time to initiate antiviral treatment were treated as categorized data in the multivariate analysis. The median value was taken as the cutoff value between the different groups in continuous variables such as age and viral load in the days after the onset of symptoms.

Demographic Data for the Patients Infected with Pandemic (H1N1) 2009

From August 1 through September 30, 2009, a total of 1,052 patients were analyzed for novel influenza (H1N1) infection by RT-PCR, and pandemic (H1N1) 2009 virus infections were confirmed in 602 patients. In the 425 samples analyzed by virus culture and RT-PCR, 222 were RT-PCR positive and 204 were culture positive. Of the 204 culture-positive samples, 192 (94%) were RT-PCR positive; of the 222 RT-PCR–positive samples, 194 (87%) were culture positive. The RT-PCR method is a quantitative assay and was quicker and more sensitive than virus culture.

After 21 patients who had received oseltamivir therapy before RT-PCR diagnosis were excluded, 581 patients were monitored for involvement of other organs beyond the upper respiratory tract. Of the 581 patients, the median age was 10.1 years (range 0.38–78.8 years) (Table 1). Twenty (3.4%) patients had a severe illness, including pneumonia and meningoencephalitis. Most patients (502, 86.4%) visited the hospital for diagnosis of pandemic (H1N1) 2009 virus infection within 2 days of the onset of fever. The viral load from the first throat swab specimen was >4 log₁₀ copies/mL in 278 (47.8%) patients. No deaths were found in this cohort study, but 1 patient died of severe pneumonia and cerebral hemorrhage before the start of the study.

Viral Load and Days after the Onset of Fever

Figure 1

Figure 1. A) Viral loads in afebrile and febrile patients infected with pandemic (H1N1) 2009 virus, Taiwan, before they received oseltamivir treatment. B) Correlation of the virus load with number of days after...

Most pandemic (H1N1) 2009–infected patients visited the hospital to receive treatment for fever; the mean time to visit the hospital after the onset of fever was 1.57 days. Eight patients infected with pandemic (H1N1) 2009 virus sought treatment without fever. The mean viral load was significantly lower in afebrile patients than in febrile patients (2.61 vs. 3.82 log₁₀ copies/mL, p = 0.004; Figure 1, panel A). The viral load was maintained at a high level during the first 3 days and was inversely correlated to the days after the onset of fever (Figure 1, panel B; Spearman correlation, R = –0.21, p<0.001).

Higher Viral Loads in Patients with Pneumonia

Figure 2

Figure 2. Viral load before treatment in relation to age (A) and disease severity (B) in patients infected with pandemic (H1N1) 2009 virus, Taiwan. Circles indicate individual values. URI, upper respiratory tract infection....

We analyzed whether the viral load was correlated with age or disease severity. The viral load in patients of all ages was not significantly different (Figure 2, panel A). We found that patients with pneumonia had significantly higher mean viral loads (4.92 log₁₀ copies/mL; 95% confidence interval [CI] 4.30–5.54; n = 17) than in those with upper respiratory tract infection (3.77 log₁₀ copies/mL, 95% CI 3.67–3.87; n = 518, p<0.001) or with bronchitis (3.74 log₁₀ copies/mL, 95% CI 3.43–4.05; n = 43, p = 0.002; Figure 2, panel B).

Prolonged Viral Shedding Time after Oseltamivir Treatment

Figure 3

Figure 3. A) Percentage of patients with positive reverse transcription–PCR (RT-PCR) results and B) viral load in throat swabs from 60 pandemic (H1N1) 2009 virus–infected patients treated with oseltamivir, according to the number...

Sixty patients agreed to have serial measurements taken of viral shedding after oseltamivir treatment by quantitative RT-PCR analysis of the number of viral RNA copies. Specimen collection was started from the day a participant agreed to join the study and continued in 2– 3-day intervals until viral RNA was undetectable. The results showed that the median viral shedding time was 9 days (95% CI 7.37–10.63) after the onset of symptoms, and 65% of patients had detectable influenza viral RNA for >7 days after the onset of symptoms (Figure 3, panel A). One patient with severe pneumonia had prolonged viral shedding for >27 days. The mean viral load in the throat quickly decreased from 4.13 log₁₀ copies/mL to 1.15 log₁₀ copies/mL from days 0–2 to days 6–8, respectively, after the onset of symptoms (Figure 3, panel B).

Prolonged Period of Viral Shedding in Patients <13 Years of Age

Figure 4

Figure 4. Kaplan-Meier estimates of patients with positive reverse transcription–PCR (RT-PCR) results for pandemic (H1N1) 2009 infection, by age group, Taiwan.

We evaluated the factors that could affect the rate of viral shedding, including age, gender, coexisting conditions, disease severity, viral load from the first throat swab specimen, and time from onset of symptoms to start of treatment. By using multivariate analysis, we found that only age was associated with a prolonged period of viral shedding (Table 2). To compare age differences, 13 years was chosen as the median cutoff age between the younger and older groups. We found that patients <13 years of age (n = 31) had a median viral shedding time of 11 days (95% CI 10.19–11.81) after the onset of symptoms; patients ages ≥13 years (n = 29) had a shorter median viral shedding time of 7 days (95% CI 5.85–8.15) after the onset of symptoms (p<0.001; Figure 4). Among patients <13 years of age, 89% had detectable influenza viral RNA for >7 days after the onset of symptoms, and 36% had detectable influenza viral RNA for >14 days (Figure 4).

The clinical characteristics and associated coexisting conditions for patients <13 and ≥13 years of age are shown in Table 3. No significant differences were found between the 2 groups in gender, disease severity, coexisting conditions, viral load from the first throat swab, time to initiate antiviral treatment, and duration of fever after antiviral treatment. Most of the patients in each group were afebrile within 24 h after the initiation of antiviral therapy (74.2% vs. 89.7%). Of the 60 patients, 14 (23.3%) had a coexisting condition, including 8 (25.8%) <13 years of age and 6 (20.7%) ≥13 years. Among patients ≥13 years, 1 patient had 3 underlying medical conditions (diabetes, hypertension, and congestive heart failure), and another patient had 2 conditions (hypertension and cerebrovascular disease). Asthma was the most common condition in patients <13 years of age (22.6%).