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Abstract
Background
Direct antigen test (DAT) and culture are primary tests to diagnose infections by respiratory viruses, but are mainly available for the traditional viral pathogens such as respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PIV), and adenovirus in clinical laboratories. The objective of this study was to evaluate a multiplex reverse transcriptase-PCR method using Seeplex™ RV Detection kit (Seegene, Korea) for the detection of rhinovirus, coronavirus, and human metapneumovirus (hMPV).
Methods
From January to May 2007, nasopharyngeal aspirates (NPAs) from pediatric patients negative for culture and DAT of traditional viral pathogens were tested with Seeplex™. All the amplicons were directly sequenced and homology of the sequences was searched in the National Center for Biotechnology Information (NCBI) database. Patients’ medical records were reviewed for clinical and demographic features.
Results
Forty-seven (26.4%) of 178 NPAs were positive: 18 rhinovirus, 15 hMPV, 4 RSV A, 3 coronavirus OC43, 3 influenza virus A, 2 adenovirus, 1 coronavirus NL63, and 1 RSV B. Based on maximum identity, each of the sequences indicating rhinovirus, hMPV, and coronavirus OC43 matched to the corresponding viruses with homology of 94-98%, 96-99%, and 98-100%, respectively. Seeplex™-positive patients were 0-11 yr old with a male:female ratio of 1.5:1. Clinical diagnoses included 9 pneumonia, 6 bronchiolitis, 2 cold, 1 asthma exacerbation for rhinovirus; 10 pneumonia, 4 bronchiolitis, and 1 clinical sepsis for hPMV; and 1 pneumonia, 2 croup, and 1 cold for coronavirus.
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Fig. 1.
Phylogenetic analysis using Neighbour-Joining method showed a close relationship between the isolates of this study (A. Human rhinovirus [HRV] R1, B. Human metapneumovirus [hMPV] M1, C. Coronavirus [CoV] OC43 C1, D. CoV NL63 C4) and archival strains of corresponding viruses. Distances are presented as a number of substitutions per site (a scale of ‘0.1’ means 0.1 nucleotide substitution per site).
Abbreviations: hRSV, human respiratory syncytial virus; bRSV, bovine respiratory syncytial virus; PVM, pneumonia virus of mice; APV, avian pneumovirus; SARS, severe acute respiratory syndrome.
Table 1.
Clinical diagnoses of 47 patients positive for rhinovirus, human metapneumovirus, coronavirus, and other viruses by multiplex reverse transcriptase PCR
| Respiratory viruses | Total | N of patients | |||||
|---|---|---|---|---|---|---|---|
| Pneumonia | Bronchiolitis | Croup | Asthma exacerbation | Clinical sepsis | Common cold | ||
| HRV | 18 | 9 | 6 | 0 | 1 | 0 | 2 |
| hMPV | 15 | 10 | 4 | 0 | 0 | 1 | 0 |
| CoV OC43 | 3 | 1 | 0 | 1 | 0 | 0 | 1 |
| CoV NL63 | 1 | 0 | 0 | 1 | 0 | 0 | 0 |
| Others∗ | 10 | 3 | 5 | 0 | 0 | 2 | 0 |
| Total | 47 | 23 | 15 | 2 | 1 | 3 | 3 |
Table 2.
Similarities between the sequences of the respiratory viral isolates in this study and the archival sequences from the GenBank
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