Hopital Sainte-Justine, Montreal, Quebec H3T 1C5, Canada. Received 27 .... comparison in detecting RSV, we chose to define a specimen as truly positive if ...
Vol. 32, No. 11
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1994, p. 2861-2864
0095-1137/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Validation of Respiratory Syncytial Virus Enzyme Immunoassay and Shell Vial Assay Results LOUISE PEDNEAULT,l.2* LOUISE ROBILLARD,2 AND JEAN P. TURGEON3 Department of Microbiology and Immunology, Universite de Montreal, Montreal, Quebec H3C 3J7,1 and Department of Microbiology and Infectious Diseases2 and Department of Pediatrics,3 Hopital Sainte-Justine, Montreal, Quebec H3T 1C5, Canada Received 27 April 1994/Returned for modification 16 June 1994/Accepted 23 August 1994
The Pathfinder respiratory syncytial virus (RSV) enzyme immunoassay (EIA) (Kallestad), the shell vial (SV) technique, and conventional cell culture (CC) were compared for detection of RSV in nasopharyngeal aspirates. We found sensitivities, specificities, and positive and negative predictive values of 58.4, 100, 100, and 68.2%; 80.7, 97.2, 97.0, and 81.9%; and 77.6, 97.2, 96.9, and 79.5% for the CC, EIA, and SV methods, respectively. The SV and ETA techniques were both more sensitive than CC (P < 0.001). Finally, 29 respiratory viruses other than RSV were identified by CC. Respiratory syncytial virus (RSV) is well recognized as the single most important pathogen accounting for acute viral infections of the lower respiratory tract in infants and young children. Outbreaks of RSV infections occur each year in the winter and early spring (for a complete review, see reference 5). Rapid detection of RSV is mandatory for early diagnosis, isolation measures, and, if judged necessary, antiviral therapy. Several rapid diagnostic methods, including the enzyme immunoassay (ETA) and immunofluorescence, which rely on detection of RSV antigen in respiratory secretions, have been increasingly used for that purpose. RSV ETA allows detection of nonviable as well as viable virus particles; the former are not always found when a method involving a cell culture (CC) step is used (10, 12). There is, however, discrepancy among different groups concerning the sensitivity and specificity reported for RSV antigen detection (1, 3, 6-9, 11, 12, 15, 17, 18). The shell vial (SV) procedure and conventional CC allow detection of small amounts of infectious virus that can be missed by the rapid antigen detection assays. However, because the SV technique involves a spin amplification prior to short-term culture of the inoculated specimens, it can provide a result much faster than standard CC, often before the appearance of a cytopathic effect (CPE) (9, 13, 14). Finally, RSV being a labile virus, appropriate guidelines for collection, transport, and inoculation of specimens must be followed (19). Because of these concerns, the present study was undertaken in order to validate the significance of results obtained by EIA or the SV assay for the detection of RSV. A total of 320 nasopharyngeal aspirates obtained from pediatric patients seen at Sainte-Justine Hospital during the 1991-to-1992 and 1992-to-1993 winter epidemic seasons and submitted to the virology laboratory for RSV detection were evaluated. Specimens were collected by using a sterile, infant suction trap (Luki; American Cyanamid, Danbury, Conn.) and immediately transported to the laboratory, where they were stored at 40C until further processing. One portion of the sample was tested by EIA for RSV, a second portion was inoculated into CC tubes and one SV, and a third portion was ultimately frozen at
-70°C for future studies. Nasopharyngeal aspirates were processed once daily within 24 h of collection. The Pathfinder RSV direct antigen detection system (Kallestad Diagnostics, Austin, Tex.) was used according to the manufacturer's instructions. According to its absorbance value, a clinical sample could be classified as positive, negative, or equivocal. Specimens found equivocal by spectrophotometric evaluation were excluded from the test performance analysis (see Table 2). The centrifugation culture was a modification of that previously described by Gleaves et al. (4). All specimens were diluted in 3 ml of viral transport medium (minimum essential medium with Earle's balanced salt solution, 0.5% gelatin, and antibiotics), vortexed, and inoculated at a volume of 0.4 ml into one SV each in which HEp-2 cells (ViroMed Laboratories, Minnetonka, Minn.) had been grown for 2 to 4 days. The vials were then centrifuged at 700 x g for 1 h. Excess inoculum was removed, fresh medium was added, and the vials were incubated at 37°C for approximately 40 h. A positive control consisting of a known strain of RSV and a negative control were included with each specimen run. For immunofluorescent staining, the medium was removed from the vials, and the coverslips with infected monolayers were rinsed twice with phosphate-buffered saline (PBS), fixed in cold acetone for 10 min, and air dried. Coverslips were then removed from the SV, dipped in PBS, and placed with the monolayer facing up on a glass microscope slide. One drop of the Bartels affinity-purified mouse monoclonal antibody against RSV (Baxter Diagnostics, Inc., Deerfield, Ill.) was added to each of them. Slides were incubated for 45 min at 37°C in a humidity chamber. After two more rinses of the coverslips with PBS, they were placed, cells facing down, in one drop of goat anti-mouse fluorescein isothiocyanate conjugate (Bartels, Baxter Diagnostics, Inc.) applied on a clean microscope slide. The latter were incubated at 37°C for 30 min. After two last washes in PBS, coverslips were mounted on glass microscope slides and examined at X400 magnification on a Zeiss epifluorescence microscope. A specimen was considered positive for RSV if typical punctate cytoplasmic fluorescence with small inclusions was observed in at least two cells. Clinical samples, processed as described above, were inoculated at a volume of 0.2 ml per tube into two tubes of primary rhesus monkey kidney (RhMK) cells (ViroMed), one tube of human foreskin fibroblasts (gift of B. Brodeur, National Lab-
* Corresponding author. Mailing address: H6pital Sainte-Justine, Departement de Microbiologie et Immunologie, 3175 Chemin Cote Sainte-Catherine, Montreal, Quebec, Canada H3T 1C5. Phone: (514) 345-4643. Fax: (514) 345-4818.
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NOTES
oratory for Immunology, Laboratory Center for Disease Control, Ottawa, Canada) and, during the 1991-to-1992 season, into one HEp-2 CC tube as well. CCs were incubated at 36°C. The fibroblast tubes were kept for 4 weeks, and the others were kept for 2 weeks; all tubes were examined on alternate days for the appearance of CPE. Because the vast majority of viruses produced a CPE shortly after inoculation, hemadsorption with 0.4% guinea pig erythrocytes performed on an RhMK tube was used as a screening procedure to detect infected CPE-negative cultures before discarding them after 2 weeks of incubation. When a CPE or a positive hemadsorption was observed, definitive virus identification was achieved by performing an indirect fluorescence assay with monoclonal antibodies specific for the suspected isolate. Influenza viruses A and B; parainfluenza viruses types 1, 2, and 3; adenovirus; and RSV were confirmed with the appropriate monoclonal antibody from the Bartels viral respiratory panel (Bartels, Baxter Diagnostics Inc.), and herpes simplex virus types 1 and 2 were confirmed with monoclonal antibodies from Patho Dx (Diagnostic Products Corporation, Los Angeles, Calif.). Because the conventional CC is probably not sufficiently sensitive to be used as a completely reliable gold standard for comparison in detecting RSV, we chose to define a specimen as truly positive if standard CC was positive or if a positive result by one or both rapid techniques was accompanied by a clinical diagnosis compatible with an RSV infection (expanded "gold standard" in Table 2). When clinical samples were found positive solely by one or both rapid diagnostic methods, patients' medical records were reviewed to verify the clinical significance of such results. Since RSV-related lower respiratory tract disease cannot always be distinguished from a non-RSV-related illness strictly on the basis of clinical signs and symptoms (16), we considered solely diagnoses of uncomplicated bronchiolitis, pneumonia, apnea, croup, and upper respiratory tract infection, in the absence of a positive bacterial isolate, to be compatible with an RSV infection. However, if a patient had other predisposing conditions (such as a bacterial respiratory illness) that could explain or interfere with the clinical presentation of an RSV infection, the diagnosis was not considered compatible and the result was thus not considered to be a true positive. Sensitivity, specificity, and positive and negative predictive values were calculated with standard formulas. The statistical significance of differences in proportions was assessed with the chi-square test. A bilateral P value of less than 0.05 was considered significant. Table 1 summarizes the results obtained with each assay. All three methods identified 71 positive and 136 negative specimens, resulting in a global agreement of 67.6%. Overall, 174 of 320 (54.4%) specimens were positive for RSV by one or more of the methods; RSV was isolated by standard CC in 96 of 320 (30.0%) nasopharyngeal aspirates examined. Of the 75 cases in which RSV was identified only by one or both rapid diagnostic techniques but not by CC, 74 patients' charts were available for review. Applying stringent criteria, we found that 67 of the 74 patients (90.5%) had a clinical diagnosis fully compatible with an RSV infection (Table 1). All 27 patients with clinical specimens found positive by RSV EIA and by the SV method but negative by CC for the presence of RSV had a clinical diagnosis compatible with an RSV infection. Sensitivity, specificity, and positive and negative predictive values are presented in Table 2. Fifteen specimens giving equivocal results by EIA were excluded from the performance analysis for each test (Table 2 [n = 305]). According to the definition of our expanded gold standard, both rapid diagnostic methods appeared to be more sensitive than conventional CC
TABLE 1. Comparison of EIA, the SV technique, and conventional CC for detection of RSV on 320 nasopharyngeal aspirate specimens Result by': RSV EIA
CC
RSV SV
+ + -
+
-
+ and ov
-
+
+
-
-
+
+
+
-
+
+
ov
+ +
-
Equivocal
+
-
Equivocal Equivocal
+ +
ov ov +
+
+ + +
+ and ov +
No. with given result
69 2 7 1 11 4 26 1 26 2 20 115 21 10 3 2
No. of cases with diagnosis compatible with RSV infectionb
NDC
ND ND ND ND ND 26/26 1/1 22/26
2/2 16/19 ND ND ND ND ND
' +, positive for the presence of RSV; -, negative for the presence of RSV; ov, other virus (see text for more details on these isolates). b Number of cases with RSV diagnosis/number of charts reviewed. cND, not done.
(P < 0.001), and a statistically significant lower negative predictive value was observed for standard CC compared with the more rapid procedures (P < 0.001 for CC versus EIA, and P < 0.01 for CC versus SV). Conventional CC documented infection with viruses other than RSV in 27 specimens, including 8 cases of mixed infection. Parainfluenza virus type 3 was recovered from 10 specimens, influenza A virus was recovered from 10 specimens, influenza B virus was recovered from 5 specimens, adenovirus was recovered from 3 specimens, and herpes simplex virus type 1 was recovered from one specimen. Eight mixed infections were found: two with RSV and parainfluenza virus 3, two with RSV and influenza A virus, one with RSV and influenza B virus, one with RSV and adenovirus, one with parainfluenza virus 3 and influenza A virus, and one with influenza A virus and adenovirus. Conventional CC could detect RSV in three of the six mixed infections in which the virus was recovered, whereas one or both rapid diagnostic procedures could document its presence in all six cases. The complementarity between conventional CC and rapid methods for detection of RSV has already been well documented (10). In our study, however, the standard CC was found to be less sensitive than the two rapid methods for the TABLE 2. Analysis of test performance for the RSV ETA, the RSV SV assay, and the conventional CC for the detection of RSV (n = 305)' Predictive value (%) Test
% Sensitivity
% Specificity
CC RSV ETA RSV SV
58.4
100 97.2 97.2
80.7b 77.6b
Positive
Negative
100 97.0 96.9
68.2
81.9b 79.5C
' A specimen was considered truly positive if standard CC was positive or if a positive result by one or both rapid techniques was accompanied by a clinical diagnosis compatible with an RSV infection (expanded gold standard). b p < 0.001 for sensitivities of CC versus EIA and CC versus SV and for the negative predictive value of CC versus EIA. c P < 0.01 for the negative predictive value of CC versus SV.
VOL. 32, 1994
detection of RSV. We used aspirated secretions that had not previously- been frozen, and a combination of fibroblasts, RhMK, and HEp-2 CCs, all conditions that were shown to yield maximal recovery of RSV (2, 19). However, factors such as specimen collection in the later stages of RSV infection, when the virus may no longer be recoverable in culture (10), were not controlled for. Furthermore, infectious virus present at very low titers in a clinical specimen may absolutely need the additional step of centrifugation amplification in the SV assay for detection. Unequal distribution of the original inoculum from specimens containing low titers of virus could also account for a single positive result obtained by one of the three techniques compared in the present study. Finally, we decided not to inoculate one HEp-2 cell culture tube during the 1992-to-1993 season, because we and others have found that RhMK cells and human fibroblasts were very sensitive cell lines for isolation of RSV (unpublished data and reference 2) as well as other respiratory viruses, and because we were using SVs seeded with HEp-2 cells. However, this may have slightly lowered the positivity rate obtained with the CC in our study. The definition of a completely reliable gold standard to be used in comparative studies for the detection of RSV has been a matter of concern. In general, the sensitivities and specificities reported for RSV antigen detection by ETA varied from 61 to 96% and 75 to 100%, respectively (1, 3, 6-9, 11, 12, 15, 17, 18). It seems very likely that the ETA-positive but conventional CC-negative samples represent false-negative culture results rather than false-positive ETA results. This is reinforced in our study by the fact that RSV was detected by both rapid diagnostic methods, but not by CC, in 27 of 27 patients with a clinical diagnosis compatible with an RSV infection. Since it is well known that some positive ETA results may be falsely positive (10), several investigators confirmed the specificity of EIA-positive but CC-negative results by a blocking assay (1, 3, 11, 18). Because there was insufficient quantity of such specimens left in our study, we could not document whether they were indeed true positives by using a blocking assay. In the few studies in which the Pathfinder RSV EIA has been evaluated, various sensitivities and specificities were reported (1, 8, 9, 15). However, several differences in study designs could explain such variability; Johnston and Siegel reported a sensitivity of 87% for the ETA, but they only obtained a 17% positivity rate for the conventional CC and defined their true positive as a specimen positive by one or more tests (9). On the other hand, two other groups used the standard CC as their gold standard and obtained sensitivities and specificities for the RSV ETA of 90 and 80% (15) and 73 and 92% (8), respectively. Such differences could probably be explained in part by the fact that Subbarao et al. obtained a high positivity rate for conventional CC (59.4%) (15), whereas 35.7% of the CCs were found positive in the study of Hughes et al., who also analyzed a smaller sample size (n = 92) (8). Therefore, factors such as conventional CC positivity rate, definitions of true positives, sample sizes, and timing of specimen collection could all have played a role to explain such variability in sensitivity and specificity rates. Finally, Ahluwalia and Hammond obtained a sensitivity of 79% and a specificity of 98% for the Pathfinder EIA and a CC positivity rate of 28.9% in a study performed on 211 samples, in which a true positive was defined as a specimen confirmed positive by conventional CC or by an EIA blocking assay (1). Our sensitivity and specificity results for the Pathfinder EIA as well as our conventional CC positivity rate (30.0%) were very comparable to those published by Ahluwalia and Hammond. Furthermore, we both stringently defined an expanded gold standard.
NOTES
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In our study, we found a sensitivity of 78% for the detection of RSV by the SV assay. Two investigators reported sensitivities of 73 and 78% for the SV technique (9, 13), while a third team found a sensitivity of 92% for the same assay (14). In this last case, however, calculations were based on the evaluation of the SV assay and standard CC performed on 50 specimens that had originally tested positive for RSV by direct fluorescence assay (14). Conventional CC documented infection with viruses other than RSV in 27 specimens. These findings emphasize the importance of standard CC for identifying viruses missed or not looked for by the more rapid techniques. On the other hand, in six cases of mixed infections in which RSV was identified, the presence of another virus did not interfere with the results obtained by EIA or the SV assay (six of six specimens were positive for RSV by one or both rapid techniques), while RSV was recovered by standard CC in only three of the six specimens. The growth of other viruses or the occasional lack of quality of CC for its ability to develop syncytia may have rendered the RSV CPE undetectable, resulting in a seemingly false-negative CC for RSV. In summary, the EIA and SV procedures represent valid alternatives to the conventional CC for detecting RSV during the winter epidemic season. The Pathfinder ETA was easy to perform and provided a rapid turnaround time for results. Like other ETAs, the Pathfinder ETA does not require intact respiratory tract cells and allows detection of extracellular antigen (in contrast to immunofluorescence assays), it is applicable for large-scale testing, and the results can be objectively obtained by spectrophotometry. On the other hand, optimal results with the SV technique require the availability of a fluorescence microscope and a well-trained, experienced microscopist; as is true of all assays involving immunofluorescence, interpretation is subjective. In addition, the assay is not suitable for largescale testing or automation. Therefore, we do not recommend that the SV assay replace the ETA as an initial screening procedure for the detection of RSV. On the basis of our results, we would rather advocate that all appropriate respiratory specimens from pediatric patients with a clinical diagnosis compatible with an RSV infection be screened for RSV by ETA as an initial procedure and that only the EIA-negative specimens be tested by the SV assay during the winter epidemic season. We also advocate culturing all respiratory specimens from patients who are immunocompromised, who are afflicted with cardiac malformations or pulmonary abnormalities, or who have severe lower respiratory tract disease to ensure comprehensive recovery of viruses. Louise Pedneault is a clinical research scholar supported in part by the Fonds de la Recherche en Sante du Quebec. We thank the staff of the H6pital Sainte-Justine's Diagnostic Virology Laboratory for excellent technical assistance. REFERENCES 1. Ahluwalia, G. S., and G. W. Hammond. 1988. Comparison of cell
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