Rapid identification viruses from nasal pharyngeal aspirates in acute viral respiratory infections by RT-PCR and electrospray ionization mass spectrometry

Abstract

Diagnosis of the etiologic agent of respiratory viral infection relies traditionally on culture or antigen detection. This pilot evaluation compared performance characteristics of the RT-PCR and electrospray ionization mass spectrometry (RT-PCR/ESI-MS) platform to conventional virologic methods for identifying multiple clinically relevant respiratory viruses in nasopharyngeal aspirates. The RT-PCR/ESI-MS respiratory virus surveillance kit was designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, adenoviridae types A-F, coronaviridae, human bocavirus, and human metapneumovirus. Patients (N=192) attending an emergency department during the 2007-2008 respiratory season consented, and "excess" frozen archived nasopharyngeal aspirates were analysed; 46 were positive by conventional virology and 69 by RT-PCR/ESI-MS, among which there were six samples with multiple viral pathogens detected. The sensitivity and specificity of the assay were 89.1% and 80.3%, respectively. Additional viruses that were not identified by conventional virology assays were detected (4 human bocaviruses and 7 coronaviruses). Samples in which the RT-PCR/ESI-MS results disagreed with conventional virology were sent for analysis by a third method using a commercial RT-PCR-based assay, which can identify viruses not detectable by conventional virologic procedures. Time to first result of RT-PCR/ESI-MS was 8h. RT-PCR/ESI-MS demonstrated capacity to detect respiratory viruses identifiable and unidentifiable by conventional methods rapidly.

Fig. 1

Algorithm of clinical reference diagnostic test in clinical virology laboratory for respiratory samples. Diagnostic algorithm used in clinical virology laboratory to detect respiratory viruses mainly divided into respiratory season or non-respiratory season. Respiratory season defined as October to the following January (October to November for RSV, December to January for influenza). Immunochromatographic assays were used in respiratory season as the sole screening test for RSV and influenza, which will stop the testing algorithm if results are positive. Respiratory panel cocktail DFA tests serve for adenovirus, influenza A and B, parainfluenza types 1–3 and RSV, which will stop the testing algorithm if results are positive in non-respiratory season or for those screened negative by immunochromatographic assays in respiratory season as well. RSV, respiratory syncytial virus; PIV, parainfluenza virus; HSV, herpes simplex virus; CMV, cytomegalovirus; hMPV, human metapneumovirus; DFA, direct fluorescent antibody test; CPE, cytopathic effect; RBC, red blood cells.

Fig. 2

Flow diagram of recruitment and performance analysis process. Bocavirus that were not detectable in both clinical virology laboratory and secondary RT-PCR based platform were excluded (2 subjects with bocavirus only detection and 2 bocavirus detection). After excluding bocavirus and coronavirus detections that clinical virology laboratory had no protocol to detect, 188 were included in the primary performance analysis as in Table 3. Six coronavirus and 31 samples for which clinical virology laboratory and RT-PCR/ESI-MS did not agree with sufficient volume left were sent to secondary RT-PCR based assay for secondary analysis.