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- MATERIALS AND METHODS
Objective: To develop a reverse transcription-polymerase chain reaction (RT-PCR)-based assay to identify the subgroup of the infecting respiratory syncytial virus (RSV) strain directly from nasopharyngeal aspirates (NPAs).
Method: A total of 154 NPAs which were positive for RSV antigen by direct immunofluorescence were subjected to RT-PCR. The primers used were designed to give products for subgroup A and B which were of different sizes and easily visualized on agarose electrophoresis. The PCR products were further analyzed by restriction analysis using enzymes which were unique or rare cutters within the PCR amplimer.
Results: It was possible to confirm RSV infection in 70% of the NPA samples studied. Of these, 92.6% belonged to the A group, and only 7.4% to the B group. Within the A group, six subgroups were identified using restriction analysis, while all B-group samples were identical to the prototype B strain, 18537.
Conclusion: RT-PCR performed on RNA isolated directly from NPAs provides a quick, easy-to-use, reasonably sensitive method to identify and group the infecting RSV strain.
Respiratory syncytial virus (RSV) is the major viral pathogen causing severe lower respiratory tract infections in infants, and it also causes significant morbidity amongst the elderly and in immunocompromised adults. Annual epidemics occur in late autumn and winter in temperate regions or during the rainy season in tropical countries . It has been known for some time that there are two antigenic variants of RSV, designated A and B . Group A and B often co-circulate during epidemics, A usually accounting for 70–80% of cases and causing more severe disease than B .
RSV infections are currently diagnosed by recovery of virus in cell culture, or by detection of viral antigens in nasopharyngeal secretions by immunofluorescence or enzyme immunoassays [4,5]. In this report, we describe a rapid method of detection of RSV which also differentiates between the two groups, and which can be used for subgrouping. The method is based on reverse transcription and polymerase chain reaction (RT-PCR) amplification of virion mRNA isolated from nasopharyngeal aspirates . In addition to providing rapid detection, this method provides group information which would be useful for treatment, epidemiologic and transmission studies, and the development of an RSV vaccine.
- Top of page
- MATERIALS AND METHODS
This study shows that it is technically possible to detect RSV mRNA directly in frozen NPAs by an RT-PCR technique which also yields information about the serogroup (A or B) of the infecting strain. Although the sensitivity of the technique was not ideal (70%), the NPA samples had been frozen at −20°C for varying periods of time, without RNase inhibitors, which would probably result in substantial RNA degradation.
Consistent with previous studies [8,9], the majority of infecting strains in the 1995 season belonged to group A. There is only one recorded instance of both groups occurring to the same extent . The different patterns obtained by restriction digestion show that there is genetic variability within the groups prevalent in one season. Antigenic variability within a group of RSV is well documented  and there is a recent report on genetic variation among the A-group RSV isolated at various times in different locations . Sullender et al. reported the presence of two genetically different isolates within an antigenically identical group of isolates from a single season. To our knowledge, this is the first time that multiple genetically distinct isolates have been demonstrated within an infecting group in one season.
Traditionally, diagnosis of RSV infection has been made by observation of typical cytopathic effects in mammalian cell culture, although electron microscopy might be considered in certain cases . Culture requires viable virus in the NPAs, and, as RSV is a slow-growing virus, positive identification can take up to 12 days, especially in the case of B-group virus. In recent years, detection of RSV antigens in NPAs by enzyme immunoassay and detection by immunofluorescence techniques have come into use as alternative methods of RSV diagnosis [4,5]. These have appreciably reduced the time required for diagnosis, although there is some loss in sensitivity as compared with culture. PCR technology has also been developed as means of rapid, accurate diagnosis of numerous infectious agents [6,8,13–16] with the same level of success. Paton et al.  have described a method for diagnosis of RSV infection based on RT-PCR which could be applied to NPAs directly. The PCR product so obtained was only 243 bp, and was occasionally difficult to visualize in agarose gels. In addition, no grouping information was obtained. Sullender et al.  have described an RT-PCR-based method which can reliably differentiate groups of RSV using RNA isolated from infected cell lysates. RT-PCR performed on nasal aspirates followed by hybridization by group-specific probes in a DNA enzyme immunoassay has been used recently  to increase the level of detection over previously described RT-PCR methods, although this is much less practical than simple RT-PCR for clinical use.
The method we describe here is relatively simple, reasonably sensitive, and, in contrast to other methods, provides full information about virus groups, which is useful for research studies. This method also has potential for use as a routine clinical test in a molecular diagnostic laboratory once it has been optimized and is as sensitive as the currently used techniques.