Most human metapneumovirus and human respiratory syncytial virus in infant nasal secretions is cell free

Abstract

Background

Nasopharyngeal secretions aspirated from infants with bronchiolitis (NPA) are a valuable resource for the study of virus dynamics and local inflammatory responses, however samples are small and difficult to manipulate.

Objectives

To improve yield of NPA from infants. To establish if removal of the cellular component of NPA affects quantification of human metapneumovirus (hMPV) or human respiratory syncytial virus (hRSV) genome.

Study design

Weight of NPA collected into traps from 30 infants was compared with that collected in trap plus catheter and washed through with saline from another 30 infants. hMPV (n = 33) and hRSV (n = 30) genome was measured by reverse-transcribed real-time polymerase chain reaction (RT-RT-PCR) in paired whole and cell-free NPA collected by the improved method.

Results

The improved method of NPA collection gave near two-fold greater weight (p = 0.002) of NPA (mean = 0.52 g (S.D. = 0.30 g)) than the traditional method (0.32 g (S.D. 0.19)). There was strong agreement and no significant difference between viral load measured in whole and cell-free fractions of NPA for both viruses (samples (n), correlation coefficient (cc) and significance (p)); hMPV (n = 33, cc = 0.938, p < 0.001) and hRSV (n = 30, cc = 0.977 and p < 0.001).

Conclusions

The majority of hRSV and hMPV in nasal secretions is not associated with cells. Removal of the cellular component of NPA does not interfere with quantification of hRSV and hMPV.

Introduction

Bronchiolitis is the clinical diagnosis and histopathological description of a seasonal respiratory tract infection in human infants. A spectrum of disease severity is observed ranging from coryza with cough through to failure of gas exchange requiring mechanical ventilation. The commonest causal pathogen is human respiratory syncytial virus (hRSV) followed by human metapneumovirus (hMPV) (Garcia et al., 2006). Nasopharyngeal respiratory secretions collected from infants with bronchiolitis by direct aspiration (nasopharyngeal aspirate, NPA) are a valuable resource for the study of virus dynamics and local inflammatory responses. The cellular and cell-free fractions of respiratory secretions can be used for different purposes such as immunocytochemistry and cytokine quantitation. NPA samples are difficult to handle, as they are viscous, tenacious and small (Bont et al., 2001).

We describe a method for improved collection and preparation of NPA from infants and quantification of hMPV and hRSV virus genome load by reverse-transcribed real-time polymerase chain reaction (RT-RT-PCR). We show that these methods provide ample quantities of respiratory secretions providing nucleic acids for use in quantitative assays. We demonstrate that removal of the cellular component of NPA for use in other studies does not alter quantification of hMPV or hRSV. We deduce that the majority of both viruses in nasal secretions is not associated with cells.