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A Real-Time Reverse Transcriptase-Polymer Chain Reaction To Evaluate Natural History of Viral Shedding in Outpatient Children and Adolescents With Pandemic 2009 Influenza A(H1N1) FREE TO VIEW

Luisa Galli, MD; Alberta Azzi, BSc; Elena Chiappini, MD, PhD; Fabiana Corcioli, PhD; Tommaso Bianconi, MD; Francesco Mannelli, MD; Maurizio de Martino, MD
Author and Funding Information

From the Department of Sciences for Woman and Child’s Health (Drs Galli, Chiappini, Bianconi, and de Martino), University of Florence, Anna Meyer Children’s University Hospital; the Department of Public Health (Ms Azzi and Dr Corcioli), University of Florence; and the Emergency Department (Dr Mannelli), Anna Meyer Children’s University Hospital.

Correspondence to: Luisa Galli, MD, Department of Sciences for Woman and Child’s Health, University of Florence, Italy Division of Pediatric Infectious Diseases at the Anna Meyer Children’s University Hospital, Viale Pieraccini, 24 I-50131 Florence, Italy; e-mail: luisa.galli@unifi.it


Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestpubs.org/site/misc/reprints.xhtml).


© 2010 American College of Chest Physicians


Chest. 2010;138(2):456-457. doi:10.1378/chest.10-0323
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To the Editor:

Li et al1 report in a recent issue of CHEST (April 2010) a retrospective cohort study in which the viral load (VL) profile was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in 27 untreated patients with pandemic 2009 influenza A(H1N1) [A(H1N1)] and 118 patients treated with oseltamivir. The mean age for the cohort was 17.6 years, but none of the patients was aged < 1 year. This study provided interesting data on the effect of oseltamivir on viral shedding in children and adults, but the data are less reliable when describing natural history of shedding in untreated patients. As a matter of fact, VL on nasopharyngeal aspirates (NPAs) were reported in seven patients at 1 day, 10 at 2 to 3 days, six at 4 to 5 days, and eight at 6 to 7 days, thus suggesting that different patients were evaluated at each interval postsymptom onset or that a complete follow-up was performed on six patients at most.

By contrast, we prospectively evaluated VL in NPAs from 23 untreated infants, children, and adolescents (median age, 12.7 years; range, 0.7 to 17 years) without underlying comorbidities and with uncomplicated influenza attending to the Anna Meyer Children’s University Hospital (Florence, Italy) during July 2009. With parental consent, clinical assessment and NPAs were repeated at 5 and 10 days from symptom onset in children with positive results. RNA was extracted by the QIAamp Viral RNA minikit (Qiagen; Milan, Italy). Two real-time RT-PCRs,2 using the SuperScript III Platinum One-Step qRT_PCR (Invitrogen; Carlsbad, CA), and primers and probes targeting the influenza A virus M gene and the hemagglutinin gene of the A(H1N1), respectively, were performed. The positive control was the strain A/Italy/05/2009(H1N1)v isolated in our laboratory (accession numbers GQ251032 to GQ251039). For the quantitative assay targeting the hemagglutinin gene, serial dilutions (-2 to -7 containing 1 × 105.5 to 1 × 100.5 tissue culture infectious doses 50 equivalents [TCID50]/mL) of the positive control strain were used to construct the calibration curve. Approximately one TCID50 contained 1,000 viral genome copies. The estimated limit of the quantitative RT-PCR was 3.1 TCID50 equivalents/mL (approximately 3,100 copies/mL). Virus concentrations (expressed in TCID50 equivalents) were log transformed and reported (Table 1) as mean ± SD. One girl still had a positive result 12 days after onset of fever. Duration of fever in all 23 children was 3 days (interquartile range, 3 to 5 days).

Table Graphic Jump Location
Table 1 —Viral RNA Concentrations in All Positive Nasopharyngeal Aspirates

First vs second NPA, P = .184; second vs third NPA, P = .242; first vs third NPA, P = .102 (comparisons by Student t test). IQR = interquartile range; NPA = nasopharyngeal aspirate; TCID50 = tissue culture infectious doses 50 equivalents.

a 

N = 23.

Our findings indicate that about three-fourths of children and adolescents longitudinally evaluated and with a mild A(H1N1) illness shed the virus at 5 days and that a small but discrete (17%) proportion shed the virus up to 9 days after symptom onset. VL was evaluated by molecular methods only, which also may amplify viral RNA from nonviable virus particles. However, quantitative VLs detected 9 days after onset of symptoms were compatible with the presence of viable virus, as found in our in vitro study (data not shown). So far, even if the relationship between viral shedding measured by a quantitative RT-PCR and viral transmission has not been clearly demonstrated, observations of prolonged A(H1N1) detection in respiratory specimens from infants, children, and adolescents several days after fever disappearance may have important implications for infection control in the community and in hospital settings.

Li IW, Hung IF, To KK, et al. The natural viral load profile of patients with pandemic 2009 influenza A(H1N1) and the effect of oseltamivir treatment. Chest. 2010;1374:759-768. [CrossRef] [PubMed]
 
World Health Organization (WHO)World Health Organization (WHO) CDC protocol of realtime RTPCR for influenza A(H1N1).Accessed April 30, 2009 http://www.who.int/csr/resources/publications/swineflu/realtimeptpcr/en/index.xhtml.
 

Figures

Tables

Table Graphic Jump Location
Table 1 —Viral RNA Concentrations in All Positive Nasopharyngeal Aspirates

First vs second NPA, P = .184; second vs third NPA, P = .242; first vs third NPA, P = .102 (comparisons by Student t test). IQR = interquartile range; NPA = nasopharyngeal aspirate; TCID50 = tissue culture infectious doses 50 equivalents.

a 

N = 23.

References

Li IW, Hung IF, To KK, et al. The natural viral load profile of patients with pandemic 2009 influenza A(H1N1) and the effect of oseltamivir treatment. Chest. 2010;1374:759-768. [CrossRef] [PubMed]
 
World Health Organization (WHO)World Health Organization (WHO) CDC protocol of realtime RTPCR for influenza A(H1N1).Accessed April 30, 2009 http://www.who.int/csr/resources/publications/swineflu/realtimeptpcr/en/index.xhtml.
 
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