Comparison of an Immunochromatography Test with Multiplex Reverse Transcription-PCR for Rapid Diagnosis of Respiratory Syncytial Virus Infections

Abstract

A new commercial rapid 10-min one-step immunochromatography (IC) test, SAS RSV test, was compared to another IC test, Directigen EZ RSV, employing RT-PCR as the “gold standard” for detecting respiratory syncytial virus. Of 102 clinical samples, 79 were positive by RT-PCR, 66 (82.5%) were positive with the SAS RSV test, and 55 (69.6%) were positive with Directigen EZ RSV. The specificity of the new test was 91.3% (21 of 23), similar to that of Directigen EZ RSV (100% [23 of 23]). This test performs well enough to be used for patient care.

Human respiratory syncytial virus (RSV) is the most important viral pathogen causing lower respiratory tract infections in infants, immunocompromised hosts, and the elderly (1, 2, 10). Epidemics of RSV infection occur every winter in temperate climates, during rainy seasons, or year-round in tropical regions (11). RSV can infect the same individual repeatedly as well as infants under 6 months of age who still possess maternal antibodies against the virus (7). Many of these infections are difficult to distinguish clinically from other respiratory viral infections and some bacterial infections. Laboratory diagnosis by cell culture and viral serology is usually necessary to identify the etiologic agent, although the final results of RSV isolation by tissue culture usually require several days. A more rapid diagnosis can be made by direct detection methods, such as enzyme immunoassay and immunochromatography (IC) testing with nasopharyngeal swabs or aspirates (4, 6, 9). Recently these test have been widely used; however, they are only moderately sensitive in detecting RSV in respiratory tract specimens. On the other hand, immunofluorescent-antibody tests are highly sensitive for detecting RSV; however, they need special equipment and take at least 1 h to complete (4).

We evaluated a new IC test, the SAS RSV test (SA Scientific, San Antonio, Tex.), for the first time to assess its clinical usefulness in detecting RSV antigens in nasopharyngeal swabs from subjects with RSV respiratory tract infections. This test takes 10 min to perform and relies on two specific monoclonal antibodies against RSV antigen. The sensitivity, specificity, and convenience of the test were assessed and compared to those of an existing IC test, Directigen EZ RSV (BD Biosciences, San Jose, Calif.). As a “gold standard ” for detecting RSV in clinical samples, we used a multiplex reverse transcription-PCR (multiplex RT-PCR) method that was developed by Stockton et al. (12) for detecting and subtyping RSV (groups A and B). Recent studies suggest that RT-PCR is more sensitive than viral culture for detecting respiratory viruses in clinical specimens (13).

One hundred two patients from 8 days to 9 years old (median = 11 months) were included; 9, 1, 52, 23, and 17 subjects were diagnosed as having upper respiratory tract infection, laryngitis, tracheobronchitis, bronchiolitis, and pneumonia, respectively. The patients were seen during 2003 to 2004 at three institutions. Nasopharyngeal swabs obtained from each subject were suspended in 2 ml of 2% fetal calf serum-minimal essential medium and were used for two IC tests. The other was stored at −20°C until further analysis.

For RNA extraction, we used 280-μl samples according to the spin protocol of the QIAamp viral RNA minikit (Qiagen, Valencia, Calif.). For cDNA synthesis, 22.2 μl of RNA solution was added to a reaction mixture (17.8 μl) containing random hexamer (Takara, Otsu, Japan) and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, Calif.). The reaction mixture was incubated at room temperature for 10 min, 37°C for 45 min, and 95°C for 5 min and quenched on ice (12).

For multiplex RT-PCR, the oligonucleotide primers were designed to amplify the nucleoprotein (N) and phosphoprotein (P) genes of RSV, because they are highly conserved and are regions of the RSV genome which allow subgrouping of RSV strains into A and B types (12). For the primary PCR, primers were set to cover the region from the N to the P gene. For the second PCR, primers were arranged to amplify the N gene. Each primer pair was used at 5 pmol in the primary amplification and 25 pmol in the second amplification. For the primary PCR, 20 μl of cDNA solution was added to 80 μl of reaction mixture, and 2 μl of primary product was then transferred to 48 μl of the secondary amplification mixture. Amplicons were visualized by ethidium bromide staining following electrophoresis on 2% agarose gels.

The two RSV antigen IC tests were performed according to the manufacturer's directions. The SAS RSV test is a sandwich immunoassay that uses a paper membrane with a monoclonal antibody in the liquid phase and two polyclonal antibodies in the solid phase. The liquid-phase antibody is a gold colloid-conjugated mouse monoclonal antibody to RSV glycoprotein (signal antibody), while the two solid-phase antibodies are monoclonal antibodies to RSV and polyclonal antibody to mouse immunoglobulin. The signal antibody segment is adjacent to the round well of the sample aliquot. Briefly, the 10-min, one-step procedure is as follows. A 150-μl portion of a specimen is transferred to the round well of the testing device. The specimen migrates via capillary action along the membrane, and RSV reacts with the signal antibody. The RSV-signal antibody complex also reacts with the monoclonal antibody to RSV and forms a colored line that develops within 10 min. The excess signal antibody which does not bind to RSV migrates further until it reacts with the polyclonal antibody to mouse immunoglobulin, producing a separate, second color line. In the absence of RSV, only one colored line develops, as a result of the reaction between the signal antibody and the antibody to mouse immunoglobulin. Directigen EZ RSV consists of similar antibody components and is handled in the same way as the SAS RSV test.

Of 102 samples, 79 (77.5%) were positive for RSV by multiplex RT-PCR; 63 (79.7%) and 16 (20.3%) of the isolates were identified as groups A and B, respectively. Of the 79 RSV strains, 6 (7.6%), 37 (46.8%), 22 (27.9%), and 14 (17.7%) were isolated from patients with upper respiratory tract infection, tracheobronchitis, bronchiolitis, and pneumonia, respectively. The results of the multiplex RT-PCR and two IC tests are summarized in Table 1. The SAS RSV test was more sensitive (66 of 79; 83.5%) than Directigen EZ RSV (55 of 79: 69.6%). However, the SAS RSV test showed a little less specificity (21 of 23; 91.3%) than Directigen EZ RSV (23 of 23; 100%). The SAS RSV test detected group B RSV (15 of 16; 93.4%) with greater sensitivity than group A RSV (51 of 63; 81.0%) (Table 2), although this was not statistically significant (χ² test, P > 0.3). A similar tendency was observed with Directigen EZ RSV (group A, 43 of 63 [68.3%]; group B, 12 of 16 [75.0%]).

TABLE 1.

Comparison of two IC tests with Multiplex RT-PCR in RSV detection

Multiplex RT-PCR result	No. of specimens
	SAS RSV test		Directigen EZ RSV		Total
	Positive	Negative	Positive	Negative
Positive	66	13	55	24	79
Negative	2	21	0	23	23
Total	68	34	55	47	102

TABLE 2.

Sensitivity of two IC tests according to RSV subgroup (A or B)

RSV subgroup	No. of specimens positive by:
	Multiplex RT-PCR	SAS RSV test	Directigen EZ RSV
A	63	51	43
B	16	15	12
Total	79	66	55

Differentiation of bacterial and other viral infections from RSV infection is a common clinical problem. With RSV, lower respiratory tract infections in infants and young children, especially those with congenital heart disease or chronic lung disease, are often severe (3, 7). Consequently, a simple, sensitive, and rapid diagnostic test for RSV infections would be invaluable to those caring for children. Rapid confirmation or elimination of RSV would allow a pediatrician to counsel a child's parents about the prognosis and would facilitate prompt and adequate measures to restrict transmission of the virus in a children's ward containing high-risk infants.

In this study, we demonstrate the higher sensitivity and specificity of a rapid, one-step IC test, the SAS RSV test, for the diagnosis of RSV respiratory tract disease. We had two false-positive results, although the reactions were very weak. One specimen was bloody, and the other had a high viscosity. Therefore, the sample condition might be important in achieving absolute specificity.

Until now, a biotin-enhanced enzyme immunoassay for RSV antigen detection kit (Testpack RSV; Abbott Laboratories, Abbott Park, Ill.) has been widely used in Japan. This kit was reported to have moderate sensitivity compared with cell culture (82.7%) (8) or multiplex RT-PCR (77.1%) (5). However, the method involves several steps and requires more than 25 min to complete.

In conclusion, the SAS RSV test can be completed within 10 min without special instruments. It rapidly provides helpful information for the diagnosis and for developing a treatment plan for patients with suspected RSV respiratory diseases. These results can be available during the patient's first examination, at the bedside or in the outpatient clinic.