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 of two antigen assays for rapid intrapartum detection of vaginal group B streptococcal colonization.

As part of a clinical investigation evaluating the efficacy of intrapartum antigen detection for screening for heavy vaginal colonization with group B streptococci (GBS), we compared the performance of modified Bactigen and Directigen GBS latex particle agglutination (LPA) kits. Paired vaginal swabs obtained from women in labor were rapidly transported to the laboratory and used for culturing (both swabs) and LPA testing (one swab by each method). GBS growth was estimated semiquantitatively and further designated as light or heavy growth. Performance specifications for each method were determined by comparing LPA and culture results from the same swab. A total of 4,251 paired swabs were evaluated during the study period. The performance specifications for detecting GBS growth of any degree for Bactigen and Directigen, respectively, were as follows: sensitivity, 20 and 24%; specificity, 99 and 99%. The performance specifications for heavy colonization for Bactigen and Directigen, respectively, were as follows: sensitivity, 57 and 62%; specificity, 99 and 99%. Neither LPA kit was a sensitive indicator of vaginal colonization with GBS or neonatal infection.     

Comparison of dot-blot DNA hybridisation and immediate early nuclear antigen production in cell culture for the rapid detection of human cytomegalovirus in urine

The sensitivity and specificity of four modes of two assays, immediate early nuclear antigen detection in cell culture (IENAD) at 24 and 48 h post-infection (p.i.) by immunofluorescence using a murine monoclonal antibody, and dot-blot DNA hybridisation with overnight or prolonged autoradiography using the 32P-labelled HindIII J fragment of human cytomegalovirus (HCMV) DNA as probe, were compared for the rapid detection of HCMV in urine. The sensitivity of IENAD was enhanced by low-speed centrifugation at the time of inoculation. DNA hybridisation with overnight autoradiography was significantly less sensitive than IENAD at 24 h p.i. (P less than 0.001), and even with prolonged autoradiography the hybridisation assay was slower and significantly less sensitive than IENAD at 48 h p.i. (P less than 0.02). The specificity of the two assays was virtually 100%. The sensitivity of DNA hybridisation was thus clearly inferior to that of IENAD.     

Diagnosis of cytomegalovirus infection by detection of the early antigen of cytomegalovirus in cell cultures

Conventional virus isolation and detection of cytomegalovirus (CMV) early antigen by immunofluorescence staining of cultured cells were compared in the diagnosis of CMV infection from urine specimens. By virus isolation, 33 specimens out of 333 studied were positive, and the mean length of culturing time for a positive result was 23 days (range from 6 to 45 days). By early antigen detection, 35 specimens were positive after 20 hours in culture, but the number of positive findings increased to as high as 49 after 7 days in culture. It is recommended that, in addition to the early antigen staining after one day in culture, cells should also be stained after one week in culture, because the sensitivity is essentially improved by extended culturing time.     

Comparison of two rapid culture methods for detection of cytomegalovirus in clinical specimens.

The conventional virus isolation technique was compared with a 24-h shell vial centrifugation culture technique and with a 48-h tube culture method for the detection of cytomegalovirus (CMV) in MRC-5 cells. Of 200 clinical specimens tested, 41 were positive for CMV by at least one procedure. Indirect immunoperoxidase staining was positive for 32 (78.0%) of 41 specimens in the tube culture method and for 30 (73.2%) of 41 specimens in the shell vial centrifugation method. CMV was detected in 23 (56.1%) of 41 specimens by the development of cytopathic effect within 14 days.     

Comparison of two commercial enzyme-linked immunosorbent assays for detection of herpes simplex virus antigen

Two commercial enzyme-linked immunosorbent assays (ELISA) for detection of herpes simplex virus (HSV) antigen in direct clinical specimens were compared with cell culture in primary rabbit kidney and MRC-5. With a total of 238 specimens, the sensitivity and specificity of the Dako ELISA method were determined to be 70.1% and 82.4%, respectively, and 60.4% and 88.2%, respectively, for the Ortho Antigen ELISA.     

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.     

Rapid detection of cytomegalovirus in cell culture by indirect immunoperoxidase staining with monoclonal antibody to an early nuclear antigen.

A method for the rapid detection of cytomegalovirus (CMV) in MRC-5 cells 48 h after inoculation with clinical specimens was developed. A commercially available monoclonal antibody to a CMV early nuclear antigen was used in an indirect immunoperoxidase (IPA) staining procedure performed directly on acetone-fixed cell monolayers in standard tubes (16 by 125 mm). Of 190 clinical specimens tested, 30 specimens produced CMV cytopathic effect in tissue culture (TC-CPE) within 14 days after inoculation and, of these 30, 28 were positive for CMV after 48 h by the IPA staining procedure (sensitivity, 93%). Of the remaining 160 clinical specimens negative by TC-CPE within 14 days, 7 were positive by the IPA stain (specificity, 96%). However, three of these seven specimens were positive by TC-CPE upon subculture after the initial 14-day incubation period, and one specimen was overgrown by herpes simplex virus type 2 before CMV cytopathic effect could develop. The mean time to appearance of cytopathic effect for the 30 specimens positive by TC-CPE within 14 days was 6.7 days. These findings indicate that this IPA staining is a useful method for the rapid detection of CMV in cell monolayers inoculated with clinical specimens.     

Comparison of two rapid diagnostic assays for detection of immunoglobulin M antibodies to dengue virus

Two easy-to-use commercial diagnostic assays, a dipstick enzyme-linked immunosorbent assay (ELISA) (Integrated Diagnostics, Baltimore, Md.) and an immunochromatographic card assay (PanBio, Brisbane, Australia) were evaluated for detection of immunoglobulin M (IgM) antibody to dengue virus with an in-house IgM antibody capture microplate ELISA as a reference assay. The dipstick ELISA was based on the indirect-ELISA format using dengue 2 virus as the only antigen and enzyme-labeled goat anti-human IgM antibody as the detector. The total assay time was 75 min. The immunochromatographic card assay was based on the antibody capture format and separately measured both anti-dengue virus IgM and IgG in the same test. Colloidal-gold-labeled anti-dengue virus monoclonal antibody bound with dengue virus 1 to 4 antigen cocktail was the detector, and anti-human IgM and IgG were the capture antibodies. The total assay time was <10 min. Sera from 164 individuals classified as either anti-dengue virus IgM positive (94) or anti-dengue virus IgM negative (70) in the reference microplate ELISA with a dengue virus 1 to 4 antigen cocktail were tested in the two commercial assays. The dipstick ELISA missed 7 of 94 positive samples, for a sensitivity of 92.6%, while the immunochromatographic card assay missed two positive samples, for a sensitivity of 97.9%. Of the 70 negative samples, four were false positive by the dipstick ELISA and two were false positive in the immunochromatographic card assay, resulting in specificities of 94.3 and 97.1%, respectively. Both commercial assays provide sensitive and specific detection of anti-dengue virus IgM antibody and could prove useful in settings where the microplate ELISA is impractical.     

Comparison of antibodies for rapid detection of cytomegalovirus.

Antibodies were compared for use in the spin-amplified shell vial method for rapid cytomegalovirus detection. Commercial antibodies by Du Pont Co., Whittaker M.A. Bioproducts, Bartels Immunodiagnostic, Virostat, and Serono Diagnostics were compared at incubation times of 16 to 48 h on 22 patient specimens. Only the Du Pont antibody showed 100% sensitivity and specificity and no nonspecific reactions.     

Evaluation and comparison of two assays for detection of immunity to rubella infection.

Two commercially available rapid screening tests, Rubacell (Abbott Laboratories; passive hemagglutination) and FIAX (International Diagnostic Technology; indirect immunofluorescence) were compared with a standard hemagglutination inhibition assay for detection of immunity to rubella infection. In tests of approximately 300 sera, both rapid assays were specific and sensitive and showed a high predictive value of a positive result. Within-run reproducibility studies were excellent for both tests; however, Rubacell was superior to FIAX with respect to time-cost analysis.     

The detection of human cytomegalovirus immediate early antigen in peripheral blood leucocytes.

Recently, Van der Bij et al. (1988) reported that active human cytomegalovirus (HCMV) infection could be diagnosed by the detection of HCMV immediate early antigen (IEA) directly in the peripheral blood leucocytes of renal transplant recipients. However, the indirect peroxidase technique used resulted in high background staining due to endogenous peroxidase activity and thus the detection of HCMV-IEA positive leucocytes, which are sometimes present in extremely low numbers, was not always reliable. In an attempt to solve this problem, we have evaluated the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique, immunogold-silver staining (IGSS), and several fixatives. Fixation with acetone: methanol 1:1 in conjunction with the APAAP technique proved to be the most successful method. In 155 blood samples obtained from 44 patients following renal transplantation and from three AIDS patients, the number of positive cells ranged between 1 and 700 out of 400,000 (median 2). In 23 samples from 11 patients (one AIDS patient) at least one positive cell was found. In this series there were no problems with the evaluation since strong positive signals were obtained without any background staining. We therefore recommend the use of this protocol for the rapid and reliable detection of HCMV-IEA in peripheral blood leucocytes.     

Rapid detection of cytomegalovirus by 24-well plate centrifugation with the use of a monoclonal antibody to an early nuclear antigen

Two methods for the detection of cytomegalovirus (CMV) in 457 clinical specimens were compared: (1) centrifugal inoculation of MRC-5 cells seeded on coverslips in 24-well plates and staining with a monoclonal antibody to CMV early nuclear antigen after incubation for both 16-18 hours (EA-1) and four days (EA-4); and (2) conventional tube cell culture. CMV was identified in 50 (11%) specimens from 34 different patients. EA-1 and EA-4 had positive results for CMV in 32 (64%) and 36 (73%) of the specimens, respectively. Positive inclusions were present on only one coverslip in 31% of the cases by EA-1 and in 10% by EA-4. The number of inclusions was not necessarily predictive of tissue culture results. CMV was recovered by conventional tissue culture from 27 specimens (54%) after an average of 17 days (range, 6-26 days). One specimen, positive for CMV by EA-4, yielded herpes simplex virus (HSV), and from 9 of the 407 CMV-negative specimens, another virus was recovered: HSV from 6 specimens and varicella zoster virus, adenovirus, and enterovirus from one specimen each. CMV was detected in significantly more specimens by EA-4 than by tissue culture (P = 0.037). However, there was no significant difference in the detection of CMV between EA-1 and EA-4 or between EA-1 and conventional culture. The authors' data suggest that for maximum recovery of CMV from clinical specimens, both an early antigen assay and conventional tissue culture should be performed. For urine specimens it appears that inoculation of two coverslips followed by staining after overnight incubation is adequate. To optimize the yield of the early antigen assay when testing specimens other than urine, the authors recommend inoculating three coverslips, two of which should be stained after overnight incubation, and, if necessary, the third coverslip could be stained after a more prolonged incubation period.     

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.     

Rapid detection of cytomegalovirus by tissue culture, centrifugation, and immunofluorescence with a monoclonal antibody to an early nuclear antigen

A rapid, sensitive and specific assay for the detection of cytomegalovirus (CMV) was developed utilizing MRC-5 cells in 24-well plates containing round coverslips. Centrifugation expedited the detection of CMV early antigen with monoclonal antibody. Immunofluorescent staining 16 h after inoculation with a stock CMV preparation (AD-169), demonstrated an 11-fold increase in the number of nuclear inclusions when the specimens were centrifuged (18 +/- 2.2) as compared to the non-centrifuged specimen (1.6 +/- 0.9). However, the number of nuclear inclusions depended on the age of the MRC-5 cells. They were more sensitive to CMV infection between 4 and 11 days after the cells were seeded into plates. Among 159 patient samples cultured for CMV, 23 (14%) were positive by the rapid method (mean of 32 h) and 18 (11%) by routine tissue culture (mean of 12 days). Cytomegalovirus in urine was detected within 1.3 days, whereas buffy coats (2.3 days) and bronchial washings (2.5 days) took longer. Staining for CMV inclusions at more than one time point was necessary for the optimal detection of CMV by the rapid method. We recommend using this assay system as it is rapid, specific, sensitive and versatile for the detection of CMV in many biological specimens.     

Clinical experience with cytomegalovirus isolation using conventional cell cultures and early antigen detection in centrifugation-enhanced shell vial cultures.

A total of 1,915 clinical samples was inoculated by low-speed centrifugation into shell vials (Bartels Immunodiagnostics, Bellvue, Wash.) containing cover slip monolayers of MRC-5 fibroblasts. At 1 and 2 days postinoculation, one cover slip was stained by an indirect immunofluorescence technique using a monoclonal antibody (Biotech Research Laboratories for Dupont, Billerica, Mass.) to cytomegalovirus (CMV) early antigen (EA). Clinical samples were also inoculated into three MRC-5 or MRHF cell cultures which were observed for 30 days for the appearance of a cytopathic effect (CPE). Of 157 CMV-positive samples, 92 (59%) were identified by centrifugation-enhanced EA (CE-EA) and 131 (83%) produced a CPE. CE-EA was less sensitive than CPE for all types of samples, although 17% of CMV-positive samples were detected by CE-EA alone. Evaluation of the CMV status of patients with CE-EA-positive-CPE-negative samples indicated that these samples likely represented true CMV-positive results. The average elapsed time between culture inoculation and identification of CMV decreased as follows when both CE-EA and CPE, rather than CPE alone, were used: urines, 15 to 7 days; buffy coats, 18 to 9 days; lung samples, 13 to 8 days; throat samples, 18 to 7 days. Although CE-EA was less sensitive than 30-day cell culture, both CE-EA and CPE were identified as valuable in CMV detection, and neither could be discontinued without a decrease in the CMV isolation rate or an increase in the turnaround time.     

Sensitive non-isotopic DNA hybridisation assay or immediate-early antigen detection for rapid identification of human cytomegalovirus in urine.

A sensitive non-radioactive DNA hybridisation assay employing digoxigenin-labelled probes was compared with immediate-early antigen detection and conventional virus isolation for the identification of human cytomegalovirus (HCMV) in 249 urine samples. Of 44 specimens yielding HCMV by virus isolation, more were positive by DNA hybridisation (32; 73%) than by immediate-early antigen detection (25; 52%) (P = 0.05). The specificity of the hybridisation assay in 45 apparently falsely positive specimens was supported by detection of HCMV DNA in 40 of these specimens using the polymerase chain reaction. Many urine specimens may thus contain large amounts of non-viable virus or free viral DNA. Evaluation of various protocols for the extraction and denaturation of virus DNA prior to hybridisation showed that proteinase K digestion with phenol/chloroform extraction was the most sensitive and reliable procedure. We conclude that the non-radioactive DNA hybridisation assay described is a potentially valuable routine diagnostic test.