Respiratory Viruses Detected by Sensitive Molecular Technique

The diagnosis of respiratory viruses is presently based on virus isolation, detection of antigens, or serology—all of which are too time-consuming and, in some cases, have low sensitivity.

A real-time reverse transcriptase polymerase chain reaction (RT-PCR) detection method has been used to determine the frequency of a wide range of respiratory viruses in specimens from outpatients of all ages with acute respiratory infection (ARI).


Virologists at the Universidade Federal do Espírito Santo (Vitória, Brazil) collected nasopharyngeal aspirates (NPAs) from 162 patients of all ages who were attended at either emergency rooms or primary care units between August 2007 and August 2009. All samples were tested by indirect immunofluorescence assay (IIF) and Fast-Track Diagnostics Respiratory Pathogens 21 PLUS (FTDRP) multiplex real-time RT-PCR assay. Twenty-three respiratory pathogens, including 18 human respiratory viruses and 5 bacterial species, were tested using the multiplex protocol.

Seven respiratory viruses, respiratory syncytial virus (RSV), parainfluenza viruses 1-3 (PIV 1-3), influenza viruses A and B (Inf A, Inf B) and human adenovirus (HAdV) were screened in all specimens by IIF using the Respiratory Panel 1 Viral Screening & Identification Kit (Chemicon International, Millipore; Billerica, MA, USA). Multiplex real-time RT-PCR was performed using the FTDRP 21 plus (Fast-track Diagnostics; Sliema, Malta). Amplification was performed in the ABI 7500 real-time PCR system thermocycler (Applied Biosystems; Foster City, CA, USA).

The IIF method detected 33 positive specimens (20.4%) with the influenza virus the most common etiologic agent detected (23/33). Inf A and Inf B corresponded to 8% (13/162) and 6.2% (10/162) of the samples, respectively, and the IIF technique detected only single infections. The investigators reported that 88 (54.3%) specimens were positive for one or more respiratory viruses by multiplex real-time RT-PCR. The influenza virus (15.4%), human rhinovirus (HRV) (8%) and RSV (7.4%) were the viruses most frequently detected and accounted for one-third of the positive samples. Six cases of viral co-detection were observed, mainly involving RSV. The following bacteria were detected: Streptococcus pneumoniae in 43 samples, Staphylococcus aureus in 20, and 2 with Haemophilus influenza.

The authors concluded that the use of multiplex real-time RT-PCR increased the viral detection by 33.9% and revealed a larger number of respiratory viruses implicated in ARI cases, including the most recently described respiratory viruses, human bocavirus, Human metapneumovirus, influenza A (H1N1) pdm09 virus, Human coronavirus NL63 (HCoV) and HCoV HKU1. The study was published in the September 2014 issue of the Memorias do Instituto Oswaldo Cruz.

Related Links:
Universidade Federal do Espírito Santo
Chemicon International 
Fast-track Diagnostics