Comparison of immunosorbent electron microscopy, enzyme immunoassay and counterimmunoelectrophoresis for detection of human rotavirus in stools

The detection of human rotaviruses by routine electron microscopy examination of stool specimens has been compared with the sensitivity of detection obtainable by three different immunoassays. These assays are: 1) immunosorbent electron microscopy (ISEM), which consists of the serological trapping of viruses on electron microscopy grids coated with protein A and specific viral antiserum; 2) an enzyme-linked immunosorbent assay (ELISA), in which the primary antibody is rabbit anti-rotavirus immunoglobulin, the secondary antibody is chicken anti-rotavirus immunoglobulin extracted from egg yolk of immunized hens, and the indicator antibody is alkaline phosphatase-conjugated rabbit anti-chicken immunoglobulin; 3) counterimmunoelectrophoresis (CIE). A total of 63 stool specimens from infants with gastroenteritis were examined. Of these, 23 and 24 specimens were found to contain rotavirus by electron microscopy and CIE, respectively. When scored by ELISA and ISEM, 37 and 39 were found to be positive, respectively. Confirmatory inhibition assays were necessary to eliminate some false positive reactions in ELISA. Detection of human rotaviruses in stools by ISEM is as sensitive as by ELISA, but in weakly positive specimens, ISEM offers the additional advantage of a direct visual demonstration of the presence of the aetiological agent.     

Comparison of a new, rapid enzyme immunoassay with a latex agglutination test for qualitative detection of rubella antibodies.

A total of 450 sera were tested for rubella virus antibodies by using a new, rapid enzyme immunoassay, SUDS Rubella. The results were compared with those obtained by using the Rubascan test, a well-established latex agglutination method. The sensitivity of the SUDS Rubella was 99.5%, and the specificity was 100%, when compared with Rubascan. The SUDS Rubella test can be performed in 10 min and provides an accurate screening test for the detection of rubella antibodies.     

Evaluation of new commercial enzyme immunoassay for rotavirus detection.

We evaluated a new commercial enzyme immunoassay (EIA) for rotavirus (Rotavirus EIA; International Diagnostic Laboratories, Chesterfield, Mo.). A total of 161 consecutive stool samples (including 18 from infants less than 30 days old) submitted to the diagnostic laboratory at Children's Hospital, Washington University Medical Center, St. Louis, Mo., for rotavirus detection were tested by Rotavirus EIA and by Rotazyme II (Abbott Laboratories, North Chicago, III.) according to the instructions of the manufacturer. In addition, 16 samples from infants less than 30 days old without diarrhea were tested by both assays. Samples showing discrepant results after repeat testing were examined by electron microscopy. Nine samples yielding discrepant results were also tested by using a reference EIA directly on the specimen and on culture supernatants from two passages in MA 104 cells. Rotavirus EIA and Rotazyme II yielded concordant results for 85% of the samples. All of the 26 discrepant samples tested negative by Rotavirus EIA and positive (15 samples) or equivocal (11 samples) by Rotazyme II. These samples included 11 from symptomatic infants more than 30 days old, 2 from symptomatic infants less than 30 days old (neonates), and 2 from neonates without diarrhea. Rotavirus was not detected in any of the 24 that were examined by electron microscopy or in any of the 9 that were tested by the reference EIA. The sensitivity, specificity, positive predictive value, and negative predictive value were 100% for Rotazyme EIA and 100, 90, 70, and 100%, respectively, for Rotazyme II. Rotavirus EIA was comparable to Rotazyme II in ease of performance. We conclude that Rotavirus EIA is equally sensitive and more specific than Rotazyme II for detecting rotavirus. Rotavirus EIA is a practical and accurate rotavirus assay for use in clinical laboratories.     

Comparison of an enzyme immunoassay with electron microscopic procedures for detecting rotavirus.

The sensitivity and specificity of an enzyme immunoassay (Rotazyme), an ongrid immunoelectron microscopy procedure, and conventional negative stain electron microscopic techniques were compared. By using partially purified human rotavirus and simian rotavirus (SA-11) of known particle concentration, the enzyme immunoassay was essentially equivalent to the immunoelectron microscopic procedure and significantly more sensitive than conventional electron microscopic techniques. The level of sensitivity was approximately 10(6) particles per ml for simian rotavirus SA-11 and 10(7) particles per ml for human rotavirus. In an evaluation of 455 clinical samples by these techniques, a sensitivity of 98% and specificity of 92% were demonstrated. Samples negative by the immunoelectron microscopic procedure and positive by enzyme immunoassay could be confirmed by a blocking assay.     

Human rotavirus detection by counterimmunoelectrophoresis versus enzyme immunoassay and electron microscopy after direct ultracentrifugation.

The sensitivity of the counterimmunoelectrophoresis test with NCDV, Wa, and SA-11 rotavirus antisera was 60, 60, and 67%, respectively. The counterimmunoelectrophoresis specificity was greater than 99%, but the low sensitivity is a limiting feature of this test as a first-line immunodiagnostic test for rotavirus detection.     

Choice of reference assay for the detection of rotavirus in fecal specimens: electron microscopy versus enzyme immunoassay.

Two previously demonstrated sensitive and specific enzyme immunoassays (EIAs) for rotavirus, one using polyclonal and monoclonal antisera (TestPack Rotavirus [TPK]; Abbott Laboratories) and the other using only monoclonal anti-rotavirus antibodies (Rotaclone [RTC]; Cambridge BioScience Corporation), were evaluated as potential reference assays for rotavirus testing in comparison with direct negative-staining electron microscopy (EM), the current laboratory standard. Two hundred and seven stool samples collected consecutively during the winter of 1989 from children with acute diarrhea admitted to a ward for infants from 0 to 2 years of age were tested by the EIAs and by EM. TPK specimens were read visually; RTC results were read spectrophotometrically. Specimens with discordant EIA and EM results were further evaluated by a fluorescent focus assay. Specimens positive by EM and those negative by EM but positive by fluorescent focus assay were considered to be positive for rotavirus. Of the 207 stools tested, 35 (17%) were positive for rotavirus by these criteria. EM had a sensitivity of only 80%. Specificities were 100% for RTC and EM and 89% for TPK. These findings indicate that EM, although very specific, is relatively insensitive compared with a highly sensitive monoclonal antibody-based EIA. An EIA with high sensitivity and specificity, such as RTC, is a more appropriate reference standard for rotavirus testing.     

Latex immunoassay for rapid detection of rotavirus.

A latex agglutination (LA) test was evaluated for the detection of human rotaviruses in stool specimens. Both antiserum and immunoglobulin G (IgG)-sensitized latex particles were used, with IgG-coated beads being more sensitive for human rotavirus antigen detection. Latex beads sensitized with anti-simian-SA-11 IgG were stable for at least 8 months when stored at 4 degrees C. The sensitivity of the test was compared with that of the Rotazyme (Abbott Laboratories, Diagnostics Div., North Chicago, Ill.) test. The least number of particles detected was 9.0 X 10(5) particles by the LA test versus 4.5 X 10(5) particles by the Rotazyme test. When 10 stool specimens were serially diluted for antigen endpoint determinations, the geometric mean titer by the LA test was 592 versus 1,280 by the Rotazyme test. Forty-three stool samples positive by the Rotazyme test were all positive by the LA test, and no false negative results were detected. Unconfirmed false positive reactions ranged between 8 and 24%. The LA test for rotavirus antigen detection is direct, easy to perform, sensitive, quick, and may have application for use in diagnostic laboratories, emergency rooms, and physician's offices.     

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 CAPTIA syphilis G enzyme immunoassay with rapid plasma reagin test for detection of syphilis.

The CAPTIA Syphilis G enzyme-linked immunosorbent assay compared favorably with the rapid plasma reagin test when used to screen for syphilis in a low-risk population. The sensitivity and specificity of the CAPTIA Syphilis G test were 100 and 97.8%, respectively, for 646 routine specimens and 100 and 99.2%, respectively, for 265 specimens from obstetrics patients. Overall, for 911 specimens, the CAPTIA Syphilis G test showed a sensitivity of 100%, a specificity of 98.2%, and positive and negative predictive values of 78.9 and 100%, respectively. For the same population, the rapid plasma reagin test showed a sensitivity and a specificity of 96.4 and 97.5%, respectively, and positive and negative predictive values of 72 and 99.8%, respectively.     

Detection of rotavirus with a new polyclonal antibody enzyme immunoassay (Rotazyme II) and a commercial latex agglutination test (Rotalex): comparison with a monoclonal antibody enzyme immunoassay.

A total of 176 human fecal specimens were examined for the presence of rotavirus by four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, North Chicago, Ill. (Rotazyme I); a modification of this assay which is now commercially available (Rotazyme II); and a latex agglutination test (Rotalex) recently introduced by Medical Technology Corp., Somerset, N.J. In addition, selected specimens were examined for the presence of rotavirus by electron microscopy, immune electron microscopy, and RNA gel electrophoresis. A total of 40 specimens were positive in the monoclonal antibody enzyme immunoassay, and 136 were negative. Using the results obtained with this procedure as the reference standard, we found the sensitivities of the Rotazyme I, Rotazyme II, and Rotalex tests to be 97.4, 100, and 81.6%, respectively. The specificities of these three procedures were 88.8, 83.9, and 100%, respectively.     

Comparison of latex agglutination with enzyme immunoassay for detection of rotavirus in fecal specimens

Human rotaviruses are the most important etiologic agents of acquired diarrhea in infants and young children worldwide. Early diagnosis is essentialfor effective patient treatment. The latex agglutination (LA) assays for rotavirus diagnosis are rapid, inexpensive, and the most widely used to screen specimens. The performance of the LA Rotagen (Biokit S.A., Barcelona, Spain) was evaluated for rotavirus detection infecal samples of outpatients with acute gastroenteritis. This assay was compared with the enzyme immunoassay (EIA) EIARA (Bio-Manguinhos, Rio de Janeiro, Brazil). From January to October 2000, 285 fecal specimens were analyzed. Forty-four samples (15.4%) were reactive, 214 (75.4%) were nonreactive, and 27 (9.5%) were indeterminate by LA. All LA-positive samples were positive by EIA, and 2 LA-negative samples were positive by EIA. Of specimens indeterminate by LA, 67% were positive by EIA. The sensitivity, specificity, and accuracy of LA were 69%, 100%, and 93%, respectively. These results indicate that assay is as sensitive and specific as the EIA, and it could be applied on a large scale for screening stool specimens in suspected rotavirus diarrhea. However, the indeterminate results must be confirmed by other methods, such as EIA.     

Occurrence of nonspecific reactions among stool specimens tested by the Abbott TestPack rotavirus enzyme immunoassay.

Sixty-five stool specimens obtained from children suffering from gastroenteritis were tested for the presence of antigen to rotavirus by the Abbott TestPack Rotavirus (TestPack) enzyme immunoassay kit. The Kallestad Pathfinder enzyme immunoassay, polyacrylamide gel electrophoresis, immune electron microscopy, and virus isolation were utilized as reference assays. Fifty-four specimens were in accord by TestPack and Kallestad Pathfinder. Among 11 discordant specimens positive with TestPack but negative by Kallestad Pathfinder, rotavirus was not identified by polyacrylamide gel electrophoresis, immune electron microscopy, or isolation in primary African green monkey kidney cell cultures. TestPack displayed a performance specificity of 83%. The inordinately high number of stool specimens reported as false-positive by TestPack precludes the incorporation of this antigen detection kit into our routine regimen of diagnostic virologic testing.     

Comparison of direct electron microscopy, immune electron microscopy, and rotavirus enzyme-linked immunosorbent assay for detection of gastroenteritis viruses in children.

An approximate 10% suspension in water of the first available stool sample from 411 infants and young children with acute gastroenteritis was examined by electron microscopy (EM) after 2 min of negative staining. This procedure enabled the detection of 88% of the 199 rotavirus infections, all of the 22 adenovirus infections, and 47% of the 15 approximately 27-nm virus infections ultimately detected by a combination of techniques, including immune electron microscopy (IEM) and rotavirus enzyme-linked immunosorbent assay (ELISA). Of the 204 infections detected by direct EM of stools, 76% were detected within 2 min of viewing, and 94% were detected within 6 min of viewing. Type 1 and type 2 rotavirus particles were visualized with approximately equal efficiency, although type 2 rotavirus infections were more common. Rectal swab preparations were clearly inferior to stool preparations for the detection of virus infection by direct EM. IEM examination was required for efficient visualization of viruses in rectal swab specimens. ELISA was the most sensitive method for the detection of rotaviruses; with this method, all infections in which rotavirus particles were visualized by EM or IEM were detected. However, 73% of the 1,834 specimens which were presumptively positive for rotavirus by conventional indirect ELISA proved to be falsely positive on the basis of EM, IEM, blocking ELISA, confirmatory ELISA, or a combination of these methods. False-positive rotavirus ELISA reactions apparently were eliminated when fecal specimens were tested in a modified confirmatory ELISA with a lower dilution of rotavirus-negative (pre-immunization) than rotavirus-positive (post-immunization) capture antibody from the same animal.     

Comparison of four-layer radioimmunoassay and electron microscopy for detection of human rotavirus.

A four-layer radioimmunoassay (RIA) using polystyrene beads as the solid phase, anti-rota guinea pig IgG as primary antibody, anti-rota rabbit IgG as secondary antibody, and 125I-labelled sheep anti-rabbit immunoglobulin as indicator antibody has been developed for the detection of human rotavirus in stool specimens. A comparison was made of the developed RIA, routine electron microscopy, and research electron microscopy of 147 unconcentrated stool specimens from patients with infantile gastroenteritis. In routine electron microscopy 17 (11.6%) false-positive or false-negative results were obtained when compared with research electron microscopy. Each specimen positive in research electron microscopy was positive in RIA, and six additional RIA positives were found from 58 electron microscopy negative specimens. A confirmatory test was necessary to find out marginally positive but nonspecific reactions in RIA. The developed radioimmunoassay is slightly more sensitive than research electron microscopy of unconcentrated stool specimens and considerably more sensitive and more specific than routine electron microscopy.     

TestPack Chlamydia, a new rapid assay for the direct detection of Chlamydia trachomatis.

TestPack Chlamydia (Abbott Laboratories) is a rapid enzyme immunoassay for the direct antigen detection of Chlamydia trachomatis in endocervical specimens. The assay is self-contained, requires no specialized equipment, and yields results in less than 30 min. The clinical performance of TestPack Chlamydia versus chlamydial cell culture was evaluated with a total of 1,694 paired endocervical specimens. Discordant samples were further investigated by immunofluorescent staining and by Chlamydiazyme immunoassay, with confirmatory procedures. The sensitivity of TestPack Chlamydia with less-than-48-h-old specimens was 76.5%, while culture sensitivity was 86.7%. TestPack Chlamydia specificity was determined to be 99.5%. These results indicate that TestPack Chlamydia is an accurate test for chlamydial infection, with a positive predictive value of 96.2%. This assay is suitable for low-volume chlamydial testing in physician offices, clinics, and smaller laboratories.     

Diagnosis of human Rotavirus infections: Comparison of an electrophoretic method,a modified complement fixation test and electron microscopy for Rotavirus detection

Summary A modified complement fixation test and a counter-immuno-electrophoresis on cellulose acetate were compared to electron microscopy, in detecting Rotavirus from stool specimens.Out of 75 samples from patients with acute gastroenteritis, 40 per cent yielded positive results.The main advantages of the above two methods in routine diagnosis are discussed.With 1 Figure     

Comparative evaluation of a commercial enzyme-linked immunoassay and solid-phase immune electron microscopy for rotavirus detection in stool specimens.

Using solid-phase immune electron microscopy (SPIEM) as a reference test, we examined 151 stool specimens from infants and young children with acute gastroenteritis for rotavirus detection by a one-step commercial enzyme-linked immunosorbent assay (ELISA) with labeled monoclonal antibody. Of the 83 samples determined to be positive for rotavirus by SPIEM, 82 were detected as positive by the monoclonal antibody ELISA (sensitivity, 98.7%), while 67 of the 68 specimens determined to be negative by SPIEM were correctly detected as negative by the ELISA (specificity, 98.5%). The diagnostic accuracy of the ELISA kit was 98.6%. Thus, the one-step monoclonal antibody ELISA, which can be completed in less than 90 min, appears to be highly suitable for the rapid and reliable detection of rotavirus in stools.     

Comparison of lateral-flow immunoassay and enzyme immunoassay with viral culture for rapid detection of influenza virus in nasal wash specimens from children

The performance of two commercially available rapid test kits for influenza virus detection was compared to that of viral culture by using 356 nasal wash specimens collected during the 2001 to 2002 influenza season. Overall, the two rapid tests were easy to perform and showed comparable sensitivities (70.4 and 72.2%) and specificities (97.7 and 98.3%); for both test kit groups, most of the specimens that yielded false-negative results were found to be growing influenza B virus.     

Comparison of five enzyme immunoassays, electron microscopy, and latex agglutination for detection of rotavirus in fecal specimens

Five different enzyme immunoassays, electron microscopy, and latex agglutination (Slidex; bioMerieux) were compared for the rapid detection of human rotavirus in fecal specimens. The enzyme immunoassay using rotavirus polyclonal antiserum (Dakopatts) with simple in-house modifications was shown by the use of confirmatory tests to be the most sensitive and specific procedure.