Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.

Coronavirus NL63 was found in hospitalized children with upper and lower respiratory infections.

The 2 groups of human coronaviruses (HCoVs) represented by the prototype strains HCoV 229E and HCoV OC43 are mostly known as viruses responsible for common cold syndrome. HCoVs are difficult to detect, and epidemiological data are rare. From October 2000 through April 2001, we tested 1803 respiratory samples for HCoV by reverse-transcriptase polymerase chain reaction. From 8 February through 27 March 2001, HCoV OC43 was detected in samples obtained from 30 (6%) of 501 patients. The other viruses detected were respiratory syncytial virus (6.1%), parainfluenza virus 3 (1%), influenza virus A (7.8%), influenza virus B (7.2%), rhinovirus (6.4%), enterovirus (1%), and adenovirus (2%). Infection with HCoV OC43 was detected in patients of all age groups. The following clinical symptoms were noted: fever (in 59.8% of patients), general symptoms (in 30%), digestive problems (in 56.8%), rhinitis (in 36.6%), pharyngitis (in 30%), laryngitis (in 3.3%), otitis (in 13.3%), bronchitis (in 16.6%), bronchiolitis (in 10%), and pneumonia (in 6.6%). This study shows that an outbreak of HCoV OC43 respiratory infection was responsible for the lower respiratory tract symptoms observed in nearly one-third of patients identified by active surveillance for coronavirus infection.

The performances of four multiplex PCR (m-PCR) were compared to direct immunofluorescence assay (DFA) and HuH7 cell culture for the detection of viruses in 263 children admitted to hospital with an acute respiratory illness. One hundred fifty (57.6%) nasal aspirates were found DFA-positive; 188 (72.3%) were found positive by both DFA and HuH7 cell culture, and 242 (92%) were PCR-positive. The m-PCR detected 124 viruses which were not found by conventional methods: 68 rhinovirus, 17 human metapneumovirus, 15 respiratory syncytial virus (RSV), 8 parainfluenza virus (PIV), 5 coronavirus 229E, 3 OC43 and 3 NL63, 4 enterovirus, 2 influenza virus B and C virus. The m-PCR were more sensitive, had the advantages of a shorter delay in specific diagnosis, and a lower cost than DFA and culture. Using these m-PCR, the prevalence of each virus was compared between in-patient and out-patient groups of children attending the emergency unit of the hospital. Nasal aspirates from 411 (91.5%) children were found positive by the PCRs. RSV, rhinovirus, and influenza virus were the most frequent viruses detected in this population, representing 43.6%, 31.8%, and 8.8% of the virus found, respectively, followed by human metapneumovirus (4.4%), coronavirus (3.4%), parainfluenza virus (3.2%), adenovirus (2.3%), and enterovirus (2.1%). RSVs were detected more significantly in the in-patient group than in the out-patient group, and influenza viruses were detected more frequently in the out-patient group than in the in-patient group. Moreover, the use of m-PCR pointed out the frequency of rhinovirus and mixed viral detections in these patients. In conclusion, according to the requirements of speed and low cost of the methods, and to achieve the highest rate of detection of respiratory viruses, the combined use of DFA and m-PCR is today likely to be the best way to improve diagnosis of respiratory illnesses in children.

HCoV-HKU1 can be detected in respiratory and stool samples from children and adults in a part of the world other than Hong Kong. Our results suggest that HCoV-HKU1 could be associated with respiratory and enteric diseases, and its detection can be related to a persistent asymptomatic infection in patients with poor underlying conditions.