Comparison of enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and virus isolation for detection of respiratory viruses in nasopharyngeal secretions.

Nasopharyngeal secretions obtained from 94 children with acute respiratory illness were examined for the presence of respiratory syncytial virus (RSV), adenovirus, and influenza virus type A by virus culturing (virus isolation technique [VIT]), immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA). Similar results were obtained in at least two tests for RSV, influenza virus type A, and adenovirus in 92 (97.9%), 88 (93.6%), and 88 (93.6%) cases, respectively. Both rapid virus detection methods showed good specificity for the diagnosis of these virus infections (greater than or equal to 90.7%) and were more sensitive than was VIT for RSV detection. In a more accurate statistical analysis, the indexes of agreement between VIT and ELISA were substantial for RSV (kappa = 0.69; zeta = 5.5; P less than 0.0001), influenza virus type A (kappa = 0.67; zeta = 5.3; P less than 0.0001), and adenovirus (kappa = 0.71; zeta = 6.0; P less than 0.0001), while it was almost perfect for RSV when ELISA was compared with IFA (kappa = 0.88; zeta = 5.7; P less than 0.0001). Although the observed agreement was good in the comparison of these two tests for these three viruses (89%0, the indexes of agreement were moderate in the comparison of IFA and VIT for RSV (K = 0.55; Z = 2.0; P < 0.05), influenza virus type A (K = 0.42; Z = 9.7; P < 0.0001), and adenovirus (K = 0.41; Z = 6.5; P < 0.0001) and of ELISA and IFA for influenza virus type A (K = 0.55; Z = 7.0; P < 0.0001) and adenovirus (K = 0.59; Z = 6.8; P < 0.0001). All of the statistical evaluations demonstrated better agreement between ELISA and VIT for influenza virus type A and adenovirus.     

Detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay, indirect immunofluorescence, and virus isolation: a comparative study

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of respiratory syncytial virus (RSV) antigens in nasopharyngeal secretions (NPS) from children with acute respiratory disease. Antisera against RSV nucleocapsids were used as immunoreagents for this test system. The results obtained by RSV antigen ELISA were compared to those of indirect immunofluorescence (IF) and tissue culture virus isolation (TC). Of the 404 NPS obtained, 278 were tested in parallel by ELISA and IF and 205 by ELISA and TC, and 89 were screened in parallel by all three methods. The sensitivity of ELISA in relation to IF was 86.7%, the specificity 95.7%. Sensitivity and specificity obtained by ELISA were 89.9% and 94.4%, respectively, compared to TC. False-negative results were obtained with all three test systems used.     

Comparison of ortho respiratory syncytial virus enzyme-linked immunosorbent assay and HEp-2 cell culture.

Two hundred seventy nasopharyngeal aspirates were tested in duplicate with the Ortho Diagnostics, Inc. (Raritan, N.J.), respiratory syncytial virus antigen enzyme-linked immunosorbent assay. The test was sensitive (80 to 82%) and specific (96%) when compared with cell culture. The enzyme-linked immunosorbent assay detected seven antigen-positive specimens not among the 71 specimens that were positive for respiratory syncytial virus in cell culture. A blocking test confirmed those specimens as true positives (specificity, 100%).     

Comparison of two new tests for rapid diagnosis of respiratory syncytial virus infections by enzyme-linked immunosorbent assay and immunofluorescence techniques.

The sensitivity and the specificity of two new commercial reagent tests, an indirect fluorescent antibody test (FAT) with a mouse monoclonal antibody (MAb) against respiratory syncytial virus (RSV) and an enzyme-linked immunosorbent assay (ELISA) RSV antigen detection kit, were determined by a comparison of results from these tests with those of tissue culture isolation and an indirect FAT with bovine polyclonal antibody (BPA). Of 251 nasal aspirates from infants with suspected RSV infection, positive results were found for 99 (39%) by the FAT-MAb, 93 (37%) by the FAT-BPA, and 87 (35%) by the ELISA; 69 of 240 (29%) were positive by cultures. The FAT-MAb was a more sensitive technique than cultures, with 87% sensitivity for the FAT-MAb and 84% for the ELISA. It was also more sensitive than the FAT-BPA, with 97% sensitivity for the FAT-MAb and 85% for the ELISA. This could be caused only by the distinctive volume of suspended specimens used in these tests. Of 171 negative culture specimens, positive (but not false-positive) results were found for 18% by the FAT-MAb and for 12% by the ELISA. Inversely, 13% of 69 culture positive specimens were FAT-MAb negative and 16% were ELISA negative, emphasizing the importance of tissue cultures for the maximum recovery of RSV, as well as for detection of other respiratory viruses. The FAT-MAb and ELISA were easy to perform and interpret, thus facilitating wider use.     

Rapid detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay.

A new enzyme-linked immunosorbent assay (ELISA) respiratory syncytial virus antigen detection kit (Ortho Diagnostics, Inc., Raritan, N.J.) was compared with virus culture and with the indirect fluorescent antibody test (FAT) by using fresh nasal washings from children with suspected respiratory syncytial virus infection. Both uncentrifuged nasal washings and pellets from centrifuged split specimens were examined by ELISA. The ELISA was considered positive when the optical density was greater than the mean background optical density plus 0.15. Specimens positive by ELISA but negative by culture and FAT were reexamined in an ELISA blocking assay. Of 337 uncentrifuged specimens, 124 (37%) were positive by culture, 107 (32%) were positive by FAT, and 166 (49%) were positive by ELISA. Blocking assays showed that 21 of 30 (70%) of the seemingly false-positive ELISA tests were, in fact, true-positives and that the cultures and FATs were falsely negative. A patient specimen was considered positive when it was positive by virus culture, FAT, or blocking assay. The sensitivity, specificity, and positive predictive value of the ELISA test were 88, 94, and 95%, respectively. Centrifugation of nasal washings raised the sensitivity from 88 to 92% but reduced the specificity from 94 to 81%. We conclude that the Ortho ELISA test of uncentrifuged nasal washings is more sensitive than virus culture or indirect FAT and is highly specific.     

Detection of respiratory syncytial virus in nasopharyngeal secretions by inhibition of enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay developed for the demonstration of respiratory syncytial (RS) virus immunoglobulin G antibodies was used for the detection of RS virus in specimens of nasopharyngeal secretions (NPS) obtained from children with acute respiratory disease. Samples of NPS were incubated with a fixed amount of standard serum (human serum antibodies to RS virus) before being added to the enzyme-linked immunosorbent assay test plate. A decrease in the optical density value determined for this standard serum was seen with majority of NPS specimens from which RS virus had been isolated in tissue culture. The reliability and the specificity of this inhibiton test were supported by experiments with purified RS virus and by tests with NPS specimens containing other respiratory viruses.     

Comparison of washed nasopharyngeal cells and whole nasal secretions for detection of respiratory syncytial virus antigens by enzyme-linked immunosorbent assay.

We compared washed nasal epithelial cells with unfractionated nasal secretions as sources of respiratory syncytial virus (RSV) antigens in an indirect enzyme-linked immunosorbent assay (ELISA). Of 28 infants positive for RSV by virus isolation or direct immunofluorescence or both, 27 (96%) were positive by ELISA with whole nasal secretions, whereas only 19 (68%) were positive by ELISA with the matching washed-cell fractions. Furthermore, the ELISA absorbances obtained with nasal secretions were significantly greater than those seen with washed-cell fractions, indicating that whole nasal secretions contain relatively greater amounts of RSV antigens as measured by ELISA.     

Simplified enzyme-linked immunosorbent assay for specific antibodies to respiratory syncytial virus.

A simplified and reliable enzyme-linked immunosorbent assay (ELISA) was applied to the detection of serum antibodies against respiratory syncytial virus (RSV). RSV-infected cells were fixed and dried on 96-well microtiter plates and kept at 4 degrees C. The titers of reference sera were determined by endpoint dilution. A linear relation was found between the titers and the logarithm of absorbance values of sera diluted to 1:1,000 (r = 0.93, P less than 0.001). Measurement of RSV antibodies was done by using a single serum dilution (1:1,000) in conjunction with a standard curve. A strong correlation was found between complement fixation and ELISA results (r = 0.89, P less than 0.001). In addition, the ELISA method exhibited higher titers and a greater sensitivity than did complement fixation, although the applicability of the assay is limited with positive serum samples of low titer.     

Detection of respiratory syncytial virus in clinical specimens by viral culture, direct and indirect immunofluorescence, and enzyme immunoassay.

We evaluated prospectively the detection of respiratory syncytial virus (RSV) by culture and by direct antigen detection using an indirect immunofluorescence assay (IFA), a direct monoclonal immunofluorescence assay (DFA), and a monoclonal enzyme immunoassay (EIA). Of 221 specimens, 95 (43%) were culture positive for RSV, 4 (1.8%) contained more than one virus, and 17 (7.6%) contained a virus other than RSV. Overall, HEp-2 and Flow 6000 cells grew significantly more RSV isolates (82 and 72%, respectively) than A549 cells, which grew only 29% of the isolates. The mean time for RSV detection with HEp-2 cells was 2.9 days. This was significantly less than the mean time for RSV detection with either Flow 6000 cells (6.1 days) or A549 cells (6.4 days). Of 221 specimens, 129 were tested simultaneously by culture, IFA, and DFA. Of these 129 specimens, 62 (48%) were positive by culture, 69 (53%) were positive by IFA, and 70 (54%) were positive by DFA. For 92 specimens screened simultaneously by culture, IFA, and EIA, positive results were obtained for 33 (36%) of the specimens by both culture and IFA and for 29 (32%) of the specimens by EIA. Of 126 culture-negative specimens, 21 (17%) were positive for RSV when determined by IFA. Conversely, 14 (15%) of 95 RSV culture-positive specimens were negative by IFA, whereas DFA missed 19% of the culture-positive specimens. Compared with culture, the Kallestad EIA kit had a sensitivity and specificity of 73 and 92% respectively, but missed 9 (27%) of 33 culture-positive specimens. These data demonstrate that isolation by culture continues to be important for viral diagnosis of REV infections and that for valid comparative studies between viral isolations and rapid detection methods, both sensitive host cells and appropriate test conditions are required.     

Detection of respiratory syncytial virus and rotavirus by enhanced chemiluminescence enzyme-linked immunosorbent assay.

The horseradish peroxidase-catalyzed oxidation of luminol was used in a chemiluminescence avidin-biotin enzyme-linked immunosorbent assay (CL-ABE) to detect respiratory syncytial virus (RSV) and rotavirus (ROV). This CL-ABE was carried out in a new apparatus constructed for the photographic registration of the light emission produced. When measured in a spectrophotometer, the light emission produced by the oxidation of luminol showed a peak emission at 425 nm. The chemiluminescence output reached maximum within 1 min after the initiation of luminol oxidation and diminished to one-half of maximum emission within 8 min. When titrations of purified ROV by avidin-biotin enzyme-linked immunosorbent assay (ABE) were monitored by CL-ABE and by conventional orthophenylenediamine (OPD) staining (OPD-ABE), the detection limits were 0.01 and 0.04 ng of ROV protein, respectively. Similar titrations of purified RSV gave detection limits of 0.2 and 0.8 ng of RSV protein by CL-ABE and OPD-ABE, respectively. When 26 RSV-positive samples of nasopharyngeal secretions (NPS) were titrated by CL-ABE and by OPD-ABE, the mean titers obtained were 737 and 254, respectively. When 19 ROV-positive fecal samples were titrated by CL-ABE and by OPD-ABE, the mean titers obtained were 82,000 and 29,000, respectively. When samples of NPS from 123 infants and children with acute lower respiratory disease were tested for the presence of RSV, 31 NPS samples were RSV positive by CL-ABE, and 29 of these 31 NPS samples were RSV positive by OPD-ABE.     

Comparison of direct and indirect immunofluorescence staining of clinical specimens for detection of respiratory syncytial virus antigen.

Immunofluorescence staining methods for respiratory syncytial virus antigen detection were compared. Of 50 specimens originally positive for respiratory syncytial virus by direct immunofluorescence and culture, 49 were positive by repeat direct immunofluorescence and 32 were positive by indirect immunofluorescence. Additional results obtained on specimens originally negative for respiratory syncytial virus by direct immunofluorescence, culture, or both indicate that direct immunofluorescence staining for respiratory syncytial virus antigen was more sensitive than was indirect immunofluorescence.     

Comparison of fluorescent-antibody-to-membrane-antigen test, indirect immunofluorescence assay, and a commercial enzyme-linked immunosorbent assay for determination of antibody to varicella-zoster virus.

The demand for sensitive and specific assays to determine immune status to varicella can be expected to increase with the anticipated availability of a varicella-zoster virus vaccine for use in nonimmune adults, especially health care personnel, and in immunosuppressed children. Although the fluorescent-antibody-to-membrane-antigen (FAMA) test remains the reference standard to which other tests are compared, simpler alternative assays are needed. In this study, the FAMA was compared with a simple indirect immunofluorescence assay (IFA) and a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to varicella-zoster virus. One hundred and twelve serum samples were screened by the FAMA test and IFA at a 1:5 dilution, and 100% agreement was found. Of these samples, 101 were available for testing by ELISA, and identical results were obtained with 97 samples (96% agreement). When the samples were screened at a 1:2 dilution, 99 of 101 results agreed. In addition, 31 spinal fluid samples were tested by all three methods. When screening was at a 1:2 dilution, there was 96.8% agreement between the FAMA test and IFA. When the cutoff value established for sera was used for the spinal fluid samples, there was 90.3% agreement between the ELISA and the FAMA test. Thus, both IFA and ELISA can be considered sensitive and specific alternatives to the FAMA test, and in addition, both use commercially available reagents.     

An enzyme-linked immunosorbent assay using monoclonal antibodies for the detection of respiratory syncytial virus in clinical specimens

Summary An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of respiratory syncytial virus in nasopharyngeal secretions. This assay employed as immunoreagents two monoclonal antibodies directed against two distinct epitopes of the viral nucleocapsid. One of them (RSV4) was used for antigen capture and the other (NC4) was labelled with N-hydroxy-succinimide-epsilon-caproil biotin and used for antigen detection. Streptavidin biotin-peroxydase complexes were employed as amplification mode. The immunoassay was performed in 6 hours and was able to detect as little as 1 ng/ml of purified nucleocapsid. When 87 nasopharyngeal secretions were analyzed by an indirect immunofluorescence assay using commercial reagents and by the newly developed ELISA, the sensitivity and the specificity of the two assays were found to be very similar.     

Comparative study of nasopharyngeal aspirate and nasal swab specimens for diagnosis of acute viral respiratory infection

Paired nasopharyngeal aspirate (NPA) and nasal swab (NS) samples from 475 children hospitalized for acute respiratory infection were studied for the detection of influenza virus, parainfluenza virus, respiratory syncytial virus, and adenovirus by immunofluorescence test, viral culture, and multiplex PCR assay. The overall sensitivity of viral detection with NPA specimens was higher than that obtained with NS specimens.     

Comparison of enzyme-linked immunosorbent assay, Western blotting, microagglutination, indirect immunofluorescence assay, and flow cytometry for serological diagnosis of tularemia

The serodiagnostic efficiencies of five different approaches to detecting antibodies (immunoglobulins G, A, and M) developed in clinically proven infections with Francisella tularensis have been assessed. Fifty serum samples from patients suffering from tularemia during an outbreak in Sweden were compared with samples from 50 healthy blood donors (controls) by using an enzyme-linked immunosorbent assay (ELISA), microagglutination (MA), Western blotting (WB), an indirect immunofluorescence assay (IIFA), and flow cytometry (FC). ELISA, WB, and FC were based on the use of preparations of lipopolysaccharides (LPS) of the live vaccine strain of Francisella tularensis subsp. holarctica (ATCC 29684) as a capture antigen. Whole methanol-fixed bacteria were used for IIFA and MA. Optimized protocols yielded a diagnostic sensitivity and specificity of 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. A total of 6,632 serum samples from individuals between the ages of 18 and 79 years, representatively recruited from all regions of Germany, were screened to estimate and confirm the positive predictive value (PVpos) of the ELISA. Serum samples from 15 (0.226%) individuals tested positive for F. tularensis-specific antibodies by ELISA and confirmatory WB. The resulting prevalence-dependent PVpos of 10.2% and specificity of 98.1% were consistent with our findings for tularemia patients and controls. We conclude that the combined usage of a screening ELISA and a confirmatory WB based on LPS as a common antigen, as well as the MA, is a suitable serodiagnostic tool, while the quality of the IIFA is hampered by subjective variations of the results. FC is a promising new approach that might be improved further in terms of multiplex analyses or high-throughput applications.     

Comparative study of respiratory syncytial virus in nasopharyngeal aspirates using conventional cell culture, shell viral centrifugation culture, immunofluorescence and biotin-avidin enzyme linked immunosorbent assays.

133 nasopharyngeal aspirates (NPA) were simultaneously tested for the presence of respiratory syncytial virus (RSV) by conventional cell culture (CCC), shell vial centrifugation culture (SVC), immunofluorescence assay (IFA) and biotin-avidin enzyme linked immunosorbent assay (B-A ELISA). These yielded positive results in 32(24%), 45(33.8%), 36(27%) and 40(30%) of specimens, respectively. Specimens positive by IFA and B-A ELISA were all also positive by SVC. The sensitivity of CCC, IFA, and B-A ELISA comparing to SVC was 71%, 80%, and 88.9%, respectively. For rapid detection of RSV, we recommend the SVC method where a cell culture laboratory is available and the B-A ELISA method where a cell culture laboratory is not available.     

Respiratory syncytial virus serology by a simplified enzyme-linked immunosorbent assay.

A simplified enzyme-linked immunosorbent assay (ELISA) which utilized commercially available reagents was developed for respiratory syncytial virus (RSV)-specific immunoglobulin G. An analysis of the inherent variation of the assay allowed the setting of strict criteria for determining a significant change in RSV antibodies. The ELISA was more sensitive than the standard complement fixation or microneutralization tests in a carefully studied group of 32 RSV-infected adults. The ELISA correlated closely with complement fixation serological testing in 25 patients. The use of purified antigens might allow the development of a more sensitive ELISA.     

Comparison of throat and nasopharyngeal swab specimens for culture diagnosis of Bordetella pertussis infection.

During a 9-month period, we evaluated the relative sensitivity of throat and nasopharyngeal swab cultures for isolation of Bordetella pertussis. Of 38 pertussis cases, 36 (95%) had positive nasopharyngeal cultures, while only 16 of 36 (44%) had positive throat cultures. There were no cases of nasopharyngeal-negative, throat-positive cultures. The sensitivity of the direct fluorescent-antibody test was 70% when compared with culture.     

Comparison of cell culture with two direct Chlamydia tests using immunofluorescence or enzyme-linked immunosorbent assay

Two direct tests for diagnosis of infection due to Chlamydia trachomatis were evaluated on 417 specimens collected from a population with a low disease prevalence of 8.1 %. The intensity of positive results was graded according to the number of inclusions or elementary bodies and the optical density of the reaction. Thirty-four specimens were positive in cell culture, 39 positive with MicroTrak and 43 positive with Chlamydiazyme assay. The sensitivity of the two direct tests was 91.2 % (31 of 34); the specificity was 97.9 % (381 of 389) for MicroTrak and 96.9 % (377 of 389) for Chlamydiazyme assay. The positive predictive values were 79.5 % (31 of 39) for MicroTrak and 72.1 % (31 of 43) for Chlamydiazyme assay. None of the specimens negative by the culture method were positive by the two direct methods. Discrepancies were restricted to the slightly positive specimens. The direct tests seem to be an alternative for diagnosing Chlamydia trachomatis infections, but slightly positive results require cell culture confirmation.     

Early use of indirect immunofluorescence for the detection of respiratory syncytial virus in HEp-2 cell culture

Respiratory syncytial virus is detected in cell culture by the presence of cytopathic effect. To detect RSV before cytopathic effect is usually seen, slides were evaluated retrospectively from 482 HEp-2 cell cultures on days 2-4 after inoculation. Indirect immunofluorescent staining detected RSV in 57 of 94 cultures that eventually were found positive by cytopathic effect. In an additional 19 cases that ultimately showed no cytopathic effect, RSV also was detected. In 15 of the latter cases, the presence of RSV was confirmed in the original specimen. Use of indirect immunofluorescence can be used to augment the sensitivity of cell culture for the detection of RSV because cytopathic effect may not always be evident.