Validation of respiratory syncytial virus enzyme immunoassay and shell vial assay results.

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 EIA techniques were both more sensitive than CC (P < 0.001). Finally, 29 respiratory viruses other than RSV were identified by CC.     

Diagnostic efficacy of two rapid tests for detection of respiratory syncytial virus antigen.

With the availability of ribavirin therapy for serious respiratory syncytial virus (RSV) infections, rapid diagnostic tests for the detection of RSV antigen are increasingly important. Efficacies of a commercially available enzyme immunoassay (EIA) (Abbott Laboratories, North Chicago, Ill.) and a fluorescent-antibody assay (FA) were evaluated in a study involving 135 specimens from children with respiratory symptoms. A nasal wash specimen was cultured immediately on RSV-sensitive A549 cells; the nasal wash was also used for EIA. FA was performed on a nasopharyngeal swab specimen with bovine anti-RSV and anti-bovine immunoglobulin G antisera (Burroughs Wellcome Co., Research Triangle Park, N.C.). A total of 39 specimens (28%) were tissue culture positive, including 35 EIA-positive and 37 FA-positive samples (sensitivities, 90 and 95%, respectively). All 96 tissue culture-negative specimens were EIA negative (specificity, 100%); 94 of these 96 specimens were FA negative (specificity, 98%). Positive and negative predictive values for the tests were as follows: 100 and 96% for EIA, respectively, and 95 and 98% for FA, respectively. Other viruses, including influenza A virus, adenovirus, enterovirus, and herpes simplex virus, were isolated in nine cases. One adenovirus-positive specimen had a false-positive RSV FA result; all nine specimens were RSV EIA negative. Both tests performed well in our study and provide cost-effective alternatives to tissue culture. The RSV EIA, in particular, uses standard serologic techniques and equipment and does not require expertise in virology. More widespread availability of rapid diagnostic tests for RSV will hopefully result in early and appropriate use of antiviral therapy in patients at risk for serious RSV infections.     

Prospective comparison of R-mix shell vial system with direct antigen tests and conventional cell culture for respiratory virus detection.

BACKGROUND AND OBJECTIVES: Conventional cell culture (CC) has limited clinical utility as a result of the extended incubation period often required for virus isolation. Alternative methodologies have been introduced in an effort to improve turnaround times. One such system, the R-mix shell vial is discussed herein. The study objectives were: (a) to establish R-mix testing parameters as compared to direct antigen testing (DAT) and CC, and (b) to assess technical aspects and cost of R-mix in a high volume clinical virology laboratory. STUDY DESIGN: A prospective analysis of respiratory samples submitted to the clinical virology laboratory between November 2004 and April 2005 was performed. All specimens were inoculated onto R-mix shell vials (SV) and CC tubes; and a subset also underwent DAT for influenza A and B and/or RSV. A retrospective estimated cost analysis was made. RESULTS: A total of 563 samples were included in the study, which collectively revealed a total of 207 viruses. Sensitivity of R-mix for seven major respiratory viruses ranged from 45% to 83% compared to CC and DAT, while mean time to detection (TTD) varied from 1.1 to 1.4 days. In addition to these viruses, 23 picornaviruses, 11 CMV isolates and 5 HSV isolates were detected by CC alone. CONCLUSIONS: The R-mix system has similar sensitivity as CC for the detection of parainfluenza 1-3 and influenza A/B while dramatically reducing the TTD. Furthermore, it is significantly more sensitive and produces more timely results for RSV than CC; yet, neither method offers a diagnostic benefit over rapid DAT for RSV detection. The sensitivity of R-mix for adenovirus appears to be significantly lower than that of CC. Lastly, methodologies other than R-mix must remain in place under circumstances where identification of other potential viral respiratory pathogens, including herpesviruses and picornaviruses, is desired.     

Comparison of two rapid streptococcal antigen detection assays with culture for diagnosis of streptococcal pharyngitis.

In this study, 801 pharyngeal specimens were cultured for group A streptococci and tested with the Biostar Strep A Optical Immunoassay (Strep A OIA). The respective sensitivities and specificities were as follows: culture, 97.1 and 100%; Strep A OIA, 91.5 and 94.8%. Of the 801 specimens, 597 were also tested with the Abbott TestPack Strep A Assay (TP-ST). For those specimens tested by all three methods, the respective sensitivities and specificities were as follows: culture, 98.1 and 100%; Strep A OIA, 92.3 and 95.4%; and TP-ST, 79.4 and 100%. The Strep A OIA is significantly more sensitive than TP-ST and compares favorably with culture.     

Comparison between indirect immunofluorescence assay and shell vial culture for detection of mumps virus from clinical samples

We report a prospective comparison of the efficacies of an indirect immunofluorescence assay (IFA) and shell vial culture (SVC) of throat swab and urine samples from patients with mumps. Throat swab samples were used for the IFA; the urine samples and throat swabs were inoculated into vials of Vero cells. We studied 62 patients by using 62 throat swabs and 50 urine samples (50 patients with both samples). Sixty (96.7%) throat samples were positive in the SVC, and 61 (98.3%) were positive in the IFA. For the 50 patients from whom both samples were available, the IFA was positive in 50 (100%) cases, the urine sample was positive in 49 (98%) cases, and the throat swab was positive in 48 (96%) cases (P > 0.05). This comparison of throat swabs and urine samples has shown that the two clinical samples are similar in efficacy.     

Enhanced detection of respiratory syncytial virus by shell vial in children hospitalised with respiratory illnesses in northern Jordan

During the period between November 1997 and May 1998, a total of 350 nasopharyngeal aspirates were obtained from children admitted to the Respiratory Disease Unit at Princess Rahma Hospital, northern Jordan, and diagnosed clinically as suffering from respiratory tract infections. Nasopharyngeal aspirates were investigated for the presence of respiratory syncycial virus (RSV) by using shell vial (SV) culture assay, conventional culture assay, and direct immunofluorescence assay. Out of 350 nasopharyngeal aspirates, 101(28.9%) were found positive by any of SV, conventional culture, and immunofluorescence; 91 (90.1%) by SV, 87(86.1%) by culture, and 83(82.2%) by immunofluorescence. The maximum number of virus isolations was noted in children below the age of 1 year and was associated with bronchiolitis. SV assay showed the highest sensitivity (94.3%) and specificity (96.9%) for detecting RSV from nasopharyngeal aspirates. These results emphasise the importance of SV culture assay for diagnosis of RSV, although immunofluorescence is a valuable, rapid diagnostic assay.     

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.     

Comparison of a new commercial enzyme immunoassay for rapid detection of respiratory syncytial virus

Two rapid methods for detection of respiratory syncytial virus in respiratory specimens were compared: direct immunofluorescence assay (DFA) with monoclonal antibody and an enzyme immunoassay (EIA) (Test-Pack RSV). Ninety-five nasopharyngeal washings and aspirates from 51 children were examined; the patients were hospitalized during a winter outbreak of RSV infection in the first trimester of 1990. A total of 41.0 % and 56.8 % of these samples were positive by EIA and DFA respectively. Considering only the 51 specimens collected at the onset of illness, EIA detected 72.5 % positive samples and DFA detected 78.4 %. In comparison with DFA, EIA was 92.5 % sensitive and 100 % specific for the acute phase of illness. When all the samples were taken into account, specificity was maintained but sensitivity fell to 72.2 %. The results show that both methods are useful during the acute phase of the illness, when the viral load is important. However, later on in the course of the infection DFA appears to be more sensitive than EIA.     

Comparison of three enzyme-linked immunosorbent assays and a direct fluorescent-antibody test for detection of respiratory syncytial virus antigen.

We prospectively evaluated three enzyme immunoassays (EIAs) and a direct fluorescent-antibody (DFA) test for respiratory syncytial virus detection. Of 90 specimens, 79% gave the same results in all four tests (30 positive and 41 negative) and 97% were in agreement in three of the four assays. The agreement between the direct fluorescent-antibody test and each enzyme immunoassay was greater than or equal to 86%.     

Detection of respiratory syncytial virus in nasopharyngeal secretions by shell vial technique.

A shell vial technique was used to recover respiratory syncytial virus (RSV) from frozen nasopharyngeal specimens previously tested by rapid diagnostic methods. With specimens determined to be positive by direct fluorescence assay (DFA), the shell vial technique was at least as sensitive as conventional tissue culture (92 versus 90%). The majority of RSV isolates were detected within 16 h postinoculation, versus an average of 4.5 days by conventional techniques. Also, the shell vial method recovered RSV from 16 of 17 specimens (94%) which had previously tested positive by enzyme immunoassay (EIA). In addition, the shell vial method detected RSV in 4 and 11% of specimens previously determined to be negative by DFA and EIA, respectively. Therefore, we recommend the use of the shell vial technique for specimens testing negative by the rapid methods of DFA or EIA.     

Rapid detection of influenza virus by shell vial assay with monoclonal antibodies.

Of 45 influenza virus strains (43 type A and 2 type B) detected in conventional tube cell cultures (average time, 4 days), 25 (56%) were detected by immunofluorescence in the shell vial assay 24 h postinoculation. The specific fluorescence produced should allow this procedure to be readily adapted by laboratories with various degrees of experience with immunofluorescence methodology.     

Comparison of standard culture methods, a shell vial assay, and a DNA probe for the detection of herpes simplex virus.

A nonradioactive, biotinylated herpes simplex virus (HSV) DNA probe, a shell vial (rabbit kidney cell) culture assay enhanced by a direct fluorescent (HSV monoclonal)-antibody stain at 16 to 20 h postinoculation, and conventional tube cultures with confirmation via HSV-specific (polyclonal antibody) immunoperoxidase assay were compared for 199 specimens. The predictive values of the positive results were 54.5% for the probe, 95.9% for the shell vial assay, and 100% for the conventional culture methods, while the predictive values of the negative tests were 68.1, 84.0, and 98.4%, respectively. We conclude that the DNA probe (sensitivity, 24.5%; specificity, 88.3%) and the shell vial assay (sensitivity, 66.2%; specificity, 98.4%) cannot be substituted for conventional tube culture techniques (sensitivity, 97.1%; specificity, 100%) in the routine identification of HSV in our laboratory.     

Relevance of influenza a virus detection by PCR, shell vial assay, and tube cell culture to rapid reporting procedures

Influenza A virus was detected at higher rates and for more extended time periods with real-time PCR than with cell cultures. We show here that, using the theranostic approach, rapid viral detection and reporting can provide for early implementation and assessment of available antiviral therapy.     

Rapid detection of polyomavirus BK by a shell vial cell culture assay.

Polyomavirus BK (BKV) causes asymptomatic latent infection in the human host that is reactivated during periods of immune suppression. Detection by conventional tube cell culture is difficult and time consuming because BKV exhibits slow growth with late (14 to 28 days) and subtle cytopathic effects. We developed a shell vial cell culture assay (SVA) using a cross-reactive monoclonal antibody to the T antigen of simian virus 40 to detect BKV rapidly by indirect immunofluorescence. Nuclear fluorescence was seen in BKV-infected cells as early as 16 h postinoculation; 6 to 28 times more foci were present at 36 h postinoculation. Human embryonic kidney cells infected with BKV produced 7 to 42 times more fluorescent foci than MRC-5 or rhabdomyosarcoma cells did. Centrifugation enhanced the infectivity of BKV in the SVA. To define the clinical utility of SVA, urine specimens from organ transplant patients were tested. Of 27 patients, 4 (15%) were found to be positive by SVA. SVA offers a simple and rapid method for detection of BKV that can be of use in clinical studies of this virus.     

Evaluation of three rapid enzyme immunoassays and cell culture for detection of respiratory syncytial virus

Three rapid enzyme immunoassay techniques for the detection of respiratory syncytial virus antigen (Becton Dickinson Directigen RSV, Abbott RSV Testpack and Abbott RSV EIA) and cell culture were evaluated in a total of 250 nasal washings. The sensitivity and specificity were 62 % and 76 % respectively for Directigen, 64 % and 86 % for RSV Testpack, and 76 % and 81 % for RSV EIA, taking cell culture as the reference method. Agreement between cell culture and EIA techniques was 79 % (70 positive and 128 negative results). All three EIA techniques gave positive results in 69 samples (52 positive and 17 negative in the cell culture). In 121 samples all three EIA techniques gave negative results (103 negative and 18 positive in the cell culture). Using the cell culture technique 46 strains other than respiratory syncytial virus were isolated.     

Reliability of two new test kits for rapid diagnosis of respiratory syncytial virus infection.

Two new rapid enzyme immunoassays (EIAs) for detecting respiratory syncytial virus (RSV), Directigen (Becton Dickinson Microbiology Systems) and TestPack (Abbott Diagnostics) were compared with virus isolation and direct immunofluorescence by using fresh specimens. The sensitivities of both EIAs were low (72 to 73%), but when initial specimens were used, TestPack had a high sensitivity (92%) in contrast to that of Directigen (76%). Because of its high sensitivity and specificity, TestPack can be used for diagnosis of RSV in acute disease.     

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 enzyme-linked immunosorbent assays for detection of herpes simplex virus antigen.

Two enzyme-linked immunosorbent assays (ELISAs) for herpes simplex virus (HSV) detection were compared with culture in a prospective, blinded study with 153 patients with suspected recurrent oral or genital HSV. A subset of 15 of these subjects were studied daily until symptom resolution during a single episode of recurrent HSV. Direct-site specimens were collected and either placed in viral transport media (for Ortho ELISA and fresh inoculation into primary rabbit kidney cells) or frozen in ELISA collection media (DuPont). One hundred eighty-six culture-ELISA comparisons were analyzed. On the basis of culture positivity, the DuPont and Ortho ELISAs differed substantially with regard to sensitivity (93 versus 35%) but had similar specificities (95 versus 100%) and positive (85 versus 100%) and negative (98 versus 85%) predictive values. There were seven DuPont ELISA-positive, culture-negative samples which were confirmed positive for HSV by blocking antibody test (revised specificity, 100%; positive predictive value, 100%). Six of these discrepant samples were from previously culture-positive subjects. These results demonstrate that currently available ELISA kits vary substantially as to their sensitivities in detecting HSV antigen from direct-site specimens. In addition, antigen detection, by ELISA technology, is not always synonymous with state of viral infectivity as judged by tissue culture cytopathic effect.     

Peroxidase-antiperoxidase assay for rapid detection of respiratory syncytial virus in nasal epithelial specimens from infants and children.

A peroxidase-antiperoxidase (PAP) assay for the rapid detection of respiratory syncytial virus was compared with the indirect immunofluorescence method and with viral culture. Nasal epithelial specimens from 147 infants and children with acute respiratory infections were obtained and evaluated for the presence of respiratory syncytial virus antigens. Sensitivity, specificity, and accuracy by PAP were 91.7, 84.8, and 87.1%, respectively, and 87.0, 88.5, and 88.0%, respectively, by immunofluorescence compared with viral culture. The PAP assay was found to be as accurate as the indirect immunofluorescence method and more convenient to perform, since the color reaction and cell morphology were more easily observable by light microscopy. A new specimen collection method is reported; gentle scraping of the superficial nasal mucosa by the Rhino-probe method provided sufficient numbers of epithelial cells to perform multiple assays.     

Performance characteristics of VIDAS and directigen respiratory syncytial virus (RSV) antigen detection assays and culture for the identification of RSV in respiratory specimens

In a comparison of the Directigen and VIDAS respiratory syncytial virus antigen detection assays with viral culture, the sensitivity, specificity, positive and negative predictive values, and testing efficiency were 86, 93.1, 82.7, 94.6, and 91.2% for Directigen; 96.1, 90.8, 80.3, 98.3, and 92.3% for VIDAS; and 88.2, 100, 100, 95.7, and 96.8% for viral culture, respectively.