Fibrosis

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DISPATCHES

Rhinovirus C and Respiratory Exacerbations in Children with Cystic Fibrosis
Marina B. de Almeida, Rodrigo M. Zerbinati, Adriana F. Tateno, Cristina M. Oliveira, Renata M. Romão, Joaquim C. Rodrigues, Cláudio S. Pannuti, and Luiz Vicente F. da Silva Filho
To investigate a possible role for human rhinovirus C in respiratory exacerbations of children with cystic fibrosis, we conductedmicrobiologic testing on respiratory specimens from 103 such patients in São Paulo, Brazil, during 2006– 2007. A significant association was found between the presence of human rhinovirus C and respiratory exacerbations.

Instituto da Crinaça, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. The Study A total of 103 CF patients (49 girls, 54 boys; median age 8.9years; age range 3.8 months–17.8 years) were enrolled in the study from September 6, 2006 through September 4, 2007. Nasopharyngeal aspirates or nasal mucus specimens for viral investigation, as well as sputum or oropharyngeal samples for microbiology culture, were collected during scheduled visits or unscheduled visits on 408 occasions, with a median ± SD of 4 ± 1.74 visits per patient (range 1–9visits). Clinical and lung function data were obtained at all visits. Exacerbation of respiratory disease was defined as the presence of >2 of the following signs or symptoms: fever, increase in the amount of secretion or cough intensity, change in sputum’s color, worsening of dyspnea, loss of appetite, a decrease of forced expiratory volume in 1 s >10%, and weight loss. Total nucleic acids wereextracted from nasopharyngeal samples by using a QIAmp Viral RNA Mini Kit (QIAGEN, Hamburg, Germany), according to manufacturer’s instructions. Reverse transcription was conducted with High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) by using 20 μL of the previously extracted RNA. Respiratory viruses were identified by individual reverse transcription–PCRs or PCRs selective forRSV; influenza viruses A and B; human parainfluenza viruses 1, 2, and 3; human coronavirus; human metapneumovirus; adenovirus; human bocavirus; picornavirus; and the β-actin gene (9). For picornavirus, we used the primer pair OL26–OL27, which include a portion of the 5′ noncoding region (NCR) common to all picornaviruses, in the same conditions previously described (10). After sequence amplification,products were examined by capillary electrophoresis in an automated DNA sequencer (MegaBace, General Electric Healthcare–Amersham Biosciences, Little Chalfont, UK), and results were visualized through the MegaBace FragmentProfiler software, which discriminates fragment sizes and fluorescent intensities. Samples in which picornavirus cDNA had been identified were submitted to a TaqMan-based real-timePCR protocol (11) to identify HRV and enterovirus. Only samples in which rhinovirus had been identified by real-time PCR were submitted to 5′ ΝCR sequencing with an ABI 377 automated sequencer (Applied Biosystems), and results were submitted to the GenBank database, accession nos. GU933027–GU933118. Sequencing of the 5′ NCR region has been shown to accurately discriminate among subtypes ofrhinovirus, including genotypes C and A2 (12). All sequencing chromatograms obtained were edited manually to obtain contiguous fragments (contigs), by using Sequence

C

ystic fibrosis (CF) is an autosomal inherited disease characterized by recurrent and chronic respiratory infections that ultimately lead to the need for a lung transplant early in life or to death (1). The role of bacterial infectionsin CF is well established, and most treatments focus on eradication or suppression of bacterial infections (mainly those caused by Pseudomonas aeruginosa) (1). Respiratory viruses such as respiratory syncytial virus (RSV) and influenza also seem to cause early damage or increase the risk for respiratory exacerbations (2,3) in these patients. However, the role of newly described respiratory...
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