Detection of cytomegalovirus by 24-well plate centrifugation assay using a monoclonal antibody to an early nuclear antigen and by conventional cell culture

During a 12-month period, two methods for detection of cytomegalovirus (CMV) in 1624 clinical specimens were compared: (1) centrifugal inoculation of MRC-5 cells on coverslips in 24-well plates and staining with a monoclonal antibody to CMV early nuclear antigen after incubation for 40 h (EA assay), and (2) conventional tube cell culture. CMV was identified in 183 (11.3%) specimens from 113 different patients. The EA assay was positive for CMV in 144/183 specimens (79%), and CMV was detected by recognition of specific cytopathic effect (CPE) in conventional cell culture in 143/183 (78%). Both methods yielded CMV in 56% of the specimens (104/183). CMV was detected by EA assay alone in 22% (40/183) and only by CPE in 21% (39/183) of the positive specimens. When all specimen types were considered, there was no significant difference in the detection of CMV between the two methods. However, bronchoalveolar lavage (BAL) fluids yielded CMV more frequently by EA assay than by CPE (58 compared to 48 of 574, p = 0.0178), and CMV was detected in blood specimens more often by CPE than by EA assay (20 compared to one of 149, p less than 0.0001). In addition to CMV, other viruses were recovered by conventional tube cell culture, including herpes simplex virus (HSV) type 1 from 17 BAL fluids (two of which were positive for CMV by EA assay) and one liver biopsy and adenovirus serotype 4 from four separate urine specimens and three gastrointestinal tract biopsies from one patient.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen.

A commercially available monoclonal antibody directed against an early nuclear protein of cytomegalovirus was used with low-speed centrifugation for the rapid detection of this virus from urine specimens inoculated onto MRC-5 cells. A total of 19 of 162 (11.7%) urine specimens inoculated were positive by both immunofluorescence and peroxidase-antiperoxidase procedures (sensitivity, 100%), whereas only 18 of the samples produced cytopathic effects in conventional cell culture (specificity, 94.7%). All specimens were positive by immunofluorescence and peroxidase-antiperoxidase procedures at 36 h postinfection, whereas an average of 9 days was required for cytopathic effects to develop in cell cultures.     

Rapid (24h) neutralization assay for the detection of antibodies to human cytomegalovirus using a monoclonal antibody to an HCMV early nuclear protein

Neutralizing antibodies to human cytomegalovirus (HCMV) may play an important role during the course of an HCMV infection but yet detection with conventional methods remains difficult and time consuming. A new neutralization test has been developed basically corresponding to the known microtitre technique. A monoclonal antibody to an HCMV early nuclear protein is utilized to detect HCMV (AD169) infected fibroblasts by biotin/streptavidin-enhanced immunoperoxidase staining. The test procedure was thereby confined to only 24 h. Evaluation of the new neutralization test (24h-NT) was done in comparison to the conventional method (NT). Preselected serum samples (n = 217), of which n = 169 were HCMV-seropositive in ELISA (reciprocal titre greater than or equal to 40), were tested for neutralizing antibodies (NAbs) to HCMV. ELISA titres were measured by a commercially available kit (Behring, F.R.G.). NAbs could be detected in 107 sera by 24h-NT (reciprocal titre greater than or equal to 5) and in 110 sera determined by NT (reciprocal titre greater than or equal to 5). Those sera were exclusively positive in ELISA. ELISA negative sera (n = 48; reciprocal titre less than 40) yielded no detectable neutralizing titres. This specific and rapid test should facilitate HCMV-NAbs detection for a wide variety of applications including routine virology diagnostics, evaluation of HCMV hyperimmunoglobulins and medical research.     

Rapid 24-well plate centrifugation assay for detection of influenza A virus in clinical specimens

Two methods for detection of influenza virus in 234 clinical respiratory specimens were compared: (i) a 24-well plate-centrifugation assay using Madin Darby canine kidney (MDCK) cells and staining with monoclonal antibody pools to influenza A and B (Centers for Disease Control, Atlanta, GA) after incubation for 16 h and 40 h, and (ii) conventional tube cell culture using MDCK cells and primary rhesus monkey kidney cells. Influenza A was identified in 23 specimens (10%). No influenza B was recovered. The rapid centrifugation and tissue culture methods were positive for influenza A in 21 (91%) and 16 (70%) of the 23 specimens, respectively. Fourteen specimens were positive by both methods, 2 were positive by tissue culture alone, and 7 were positive by rapid centrifugation only. Of the 21 specimens positive by rapid centrifugation, 16 (76%) were detected after overnight incubation, and 5 (24%) were positive only after incubation for 40 h. Cytopathic effect was observed in 13 (81%) of the 16 isolates identified by tissue culture after an average of 6 days, and 3 (19%) were identified only by hemadsorption and staining with monoclonal antibodies at day 10. Compared with conventional tissue culture, the 24-well plate centrifugation assay is a more rapid and more sensitive method for detecting influenza virus in clinical specimens.     

Detection of cytomegalovirus in clinical specimens by virus isolation and by a monoclonal antibody against the early nuclear antigen

A commercially available monoclonal antibody against the 72000 Dalton early nuclear protein (EA) of cytomegalovirus (CMV) strain AD169 was used in an indirect immunofluorescence staining procedure (IF) for rapid detection of CMV-infected cells in tissue cultures inoculated with clinical specimens (200 urines, 22 throat washings, 5 stools, 4 bronchoalveolar lavage fluids). The results obtained by this method were compared with those obtained by virus isolation with and without centrifugal enhancement of viral infectivity. In 66 (28.6%) of the 231 samples, CMV was detected by at least one of the methods used. Of 59 specimens producing CMV-specific cytopathic effect (CPE) in tissue culture, 46 (78%) were also positive in the EA test 16 hours after inoculation. Seven CPE-negative samples were, however, positive in the EA test. Five (38%) of the false negative EA test results were due to CMV strains that did not react with the monoclonal antibody used.     

Production and characterisation of a human monoclonal antibody to cytomegalovirus and its use in an early nuclear fluorescence assay

A human monoclonal antibody to cytomegalovirus (CMV) was produced by transforming peripheral blood mononuclear cells of a patient with recent CMV infection. It is directed against a late antigen located in the nucleus of CMV infected fibroblasts at 24-72 hours postinfection and immuneprecipitates 65K and 48K proteins from 35S-labelled CMV infected cells. Results of its use in an early nuclear fluorescence assay for rapid diagnosis are presented.     

Detection of adenovirus by rapid 24-well plate centrifugation and conventional cell culture with dexamethasone

Two methods for rapid detection of adenovirus were tested: (i) 24-well plate centrifugation followed by staining with a monoclonal antibody after incubation for 24 h and 48 h, and (ii) pretreatment of A549 cells used in conventional cell culture and 24-well plate centrifugation with 10(-5)M dexamethasone. Twenty-seven clinical isolates of adenovirus and 12 specimens from which adenovirus had been recovered were included in the analysis. Both isolates and specimens had been frozen at -70 degrees C for up to 6 months. By 24-well plate centrifugation both with and without dexamethasone, 21 (78%) and 27 (100%) isolates were positive for adenovirus at 24 h and 48 h, respectively. Of the specimens, 6 (50%) and 8 (67%) were positive by 24-well plate centrifugation without dexamethasone at 24 h and 48 h, respectively, whereas with dexamethasone 3 (25%) were positive at 24 h and 7 (58%) were positive at 48 h. Overall, combining isolates and specimens, the sensitivity of 24-well plate centrifugation for detection of adenovirus at 24 h was 69% without dexamethasone and 62% with dexamethasone, and at 48 h the sensitivity was 90% without dexamethasone and 87% with dexamethasone. The specificity under all conditions tested was 100%. In conventional tissue culture dexamethasone inhibited recovery of adenovirus. Without dexamethasone, adenovirus was recovered from all 39 samples within 7 days after inoculation; however with dexamethasone pretreatment, the virus was detected in only 31 (79%) of the samples tested in the same period of time.     

Detection of cytomegalovirus in shell vial cultures by using a DNA probe and early nuclear antigen monoclonal antibody.

An in situ biotinylated DNA probe assay was evaluated as an adjunct to anti-cytomegalovirus early nuclear antigen indirect immunofluorescence and cytopathic effect on cytomegalovirus-infected monolayers in shell vial cultures. Viral infection was detected by early nuclear antigen indirect immunofluorescence at 24 h and by DNA probe assay and shell vial cytopathic effect at 5 days.     

Rapid detection of cytomegalovirus in bronchoalveolar lavage specimens by a monoclonal antibody method.

Cytomegalovirus (CMV), a common cause of pneumonia in immunocompromised subjects, is conventionally diagnosed in the laboratory by tube cell culture assays or by detection of characteristic inclusions in histologic sections. Of 160 immunocompromised patients, CMV infection was diagnosed in 19 subjects by bronchoalveolar lavage (BAL), using a monoclonal antibody directed against an early nuclear antigen of the virus. Cytospin preparations from BAL and MRC-5 cell cultures inoculated with the BAL specimens yielded positive results for 6 (31.6%) and 18 (94%) of the 19 subjects, respectively, within hours of the bronchoscopic procedure, whereas conventional tube cell cultures were positive for 11 of the 19 subjects (57.9%) only after an average of 9.3 days. The monoclonal antibody method permitted easy and rapid detection of CMV in BAL specimens.     

Evaluation of a direct fluorescein-conjugated monoclonal antibody for detection of cytomegalovirus in centrifugation culture.

A fluorescein-conjugated murine monoclonal antibody (MAb) reactive with cytomegalovirus (CMV) was evaluated for the detection of CMV in centrifugation culture. Of 188 specimens, 90 were positive for CMV in centrifugation culture. The fluorescein-conjugated MAb detected CMV in 86 of 90 (95%) specimens at 16 h postinoculation, and 88 of 90 (98%) were positive at 36 h. The fluorescein-conjugated MAb can be used in a direct immunofluorescence assay that can be completed in 15 min following cover slip fixation. Use of this antibody in centrifugation culture provides a convenient and rapid assay for the identification of CMV.     

Combined use of sonication and monoclonal antibodies for the detection of early and late cytomegalovirus antigens in centrifugation cultures

A total of 157 clinical specimens was inoculated into shell vials and conventional tube cell cultures containing confluent monolayers of human embryonic lung fibroblasts (HELF). Of 31 clinical cytomegalovirus (CMV) isolates, 30 specimens (96.8%) were positive by the immunofluorescence method on centrifugation vial cultures (CVC-IF), whereas the cytopathic effects (CPE) of CMV were detected in only 14 specimens (45.2%) in conventional tube cell cultures (CCC), P less than 0.001 and in 22 specimens (70.9%) in centrifugation vial cultures (CVC-P), P less than 0.1. Significantly more fluorescent foci were detected in centrifugation cultures inoculated with sonicated urine samples (P less than 0.001). CVC-P is more sensitive than CCC for the diagnosis of CMV (P less than 0.05), and a highly significant difference was observed when we compared the mean day to initial detection of CPE (P less than 0.001). For optimal detection of CMV, both CVC-IF and CVC-P should be used for the laboratory diagnosis of this virus infection.     

Early detection of cytomegalovirus in cell culture by a monoclonal antibody

A commercially available monoclonal antibody directed against early cytomegalovirus (CMV) antigen was used for the demonstration of CMV by immunofluorescence (IF) in cell culture within 2 days. The results were compared with the appearance of CMV-specific cytopathogenic effect (CPE). Urine specimens from 31 healthy children in day-care centers were inoculated on human embryonic fibroblasts. In addition, 45 CMV strains that had been stored at -70 degrees C were reinoculated. CMV was detected in 8/31 urine specimens by IF and 7 of these gave a specific CPE at an average of 16 days post-inoculation. One specimen was negative by IF but specific CPE was found at day 13. After reinoculation, CMV was detected in 76% by IF while 44 specimens developed CPE within a 6-week period. Demonstration of early CMV antigen in cell culture was found to be a rapid method for early diagnosis of CMV. Since the conventional cell culture with detection of CPE was more sensitive it may be useful to combine the two methods.     

Rapid detection of group B streptococcal antigen by monoclonal antibody sandwich enzyme assay.

Group B Streptococcus (GBS) is the most common cause of neonatal sepsis and meningitis. Infants at greatest risk to develop invasive disease are delivered to women colonized with GBS in their birth canals and lacking immunity to the colonizing serotype. We have investigated the sensitivity and specificity of a recently developed monoclonal antibody sandwich enzyme immunoassay for detection of GBS antigen. The sandwich enzyme immunoassay detected types II and III GBS at a concentration of 5 X 10(4) CFU/ml and types Ia and Ib GBS at 5 X 10(5) CFU/ml. No cross-reactions were noted when each of the GBS serotypes was reacted with antibodies of differing serotypes specificities. Type III GBS native antigen was detected at a concentration of 1 ng/ml. The sandwich enzyme assay is more sensitive than other methods currently in use for rapid detection of GBS and is serotype specific. This assay system should prove useful for the detection of GBS colonization during labor and for identification of neonates with invasive disease.     

Effect of dexamethasone on detection of herpes simplex virus in clinical specimens by conventional cell culture and rapid 24-well plate centrifugation.

During a 4-month period, two methods for rapid detection of herpes simplex virus (HSV) were examined: (i) pretreatment of A549 cells with dexamethasone for conventional tissue culture (277 specimens) and (ii) 24-well plate centrifugation using A549 cells with and without dexamethasone pretreatment and staining with serotype-specific monoclonal antibodies (Syva Co., Palo Alto, Calif.) after incubation for 16 to 18 h (153 specimens). By conventional tube cell culture, both with and without dexamethasone, HSV was identified in 88 of 277 (32%) specimens. Significantly more specimens were positive for HSV at 24 h (46 versus 27 specimens) and at 48 h (a total of 72 versus 59 specimens) (P less than 0.0001) in dexamethasone-treated A549 cells. Of the 153 specimens tested by conventional culture and 24-well plate centrifugation, HSV was detected in 44 (29%) by conventional culture, and by 24-well plate centrifugation with and without dexamethasone, HSV was detected in 32 (21%) and 30 (20%) specimens, respectively. The sensitivity, specificity, and positive and negative predictive values of 24-well plate centrifugation with A549 cells for detection of HSV were 73 (71% without dexamethasone), 100, 100, and 90%, respectively. In conventional tube cell culture, pretreatment of A549 cells with dexamethasone results in more rapid detection of HSV. Centrifugal inoculation of dexamethasone-treated and untreated A549 cells in 24-well plates and staining with monoclonal antibodies after incubation for 16 to 18 h is an insensitive means to detect HSV in clinical specimens and should not replace conventional tube cell culture.     

Clinical comparison of two assays for rapid detection of cytomegalovirus early nuclear antigen

Three methods for detection of cytomegalovirus (CMV) in 218 clinical specimens were compared: (1) shell vial assay to detect the early nuclear antigen after incubation for 16 hours and 40 hours (Syva Company); (2) 24-well plate assay to detect the early nuclear antigen after incubation for 16 hours (DuPont); and (3) convention tissue cell culture. CMV was detected in 26 specimens (12%) by one or more of these methods. With the shell vial assay, 12 (46%) and 15 (58%) specimens were positive after incubation for 16 hours and 40 hours, respectively. CMV was detected in 17 specimens (65%) by the 24-well plate assay. There was no significant difference in the detection of CMV between these assays. CMV was identified by conventional tissue culture in 15 of 22 (68%) evaluable cultures after an average of 14.2 days. More specimens were positive by conventional culture than by the 16-hour shell vial assay (P = 0.035). For optimal detection of CMV in clinical specimens, both conventional tissue cell culture and an early antigen assay should be performed. The two early antigen assays evaluated in this study yielded comparable results. However, the 24-well plates are more easily manipulated, and the 24-well plate assay, as performed, was easier to interpret and more cost efficient.     

Rapid detection of respiratory syncytial virus with a monoclonal antibody.

We developed an indirect fluorescent-antibody test employing a mouse monoclonal antibody directed against the nucleoprotein of RSV for rapid detection of respiratory syncytial virus (RSV). This test demonstrated distinctive fluorescent inclusions in HEp-2 cells infected with 24 RSV isolates collected during 6 previous years but not in cells infected with 13 other respiratory viruses. Examination of nasal cells of 100 infants with acute respiratory illness showed that the indirect fluorescent-antibody test employing the monoclonal antibody was 79% sensitive and 100% specific, as compared with the combination of both culturing and a similar indirect fluorescent-antibody test with commercial anti-RSV serum. This monoclonal antibody is an easily produced, well-characterized, sensitive, and specific reagent for the rapid detection of RSV antigen.     

Rapid immunodiagnosis of active cytomegalovirus infection by monoclonal antibody staining of blood leucocytes

The appearance of cytomegalovirus (CMV) antigen positive blood leucocytes (CMV antigenaemia) was investigated in 52 renal transplant recipients during the first three months after transplantation. Using a mixture of three monoclonal antibodies, CMV (immediate early) antigens were detected in cytocentrifuged blood leucocytes within 3-5 h after sampling. The results were related to virus isolation from buffy coats (CMV viraemia), serology with a sensitive enzyme-linked immunosorbent assay (ELISA), and clinical symptoms of CMV disease. The antigen test was positive in all 14 patients with CMV viraemia, in 25 out of 27 of patients with serological evidence of primary or secondary CMV infection, and in 2 out of 25 patients without active infection. In patients with a clinical CMV syndrome the presence of CMV antigen (CMV-Ag) positive blood cells correspond with the period of signs and symptoms. CMV antigens were not detected in 23 out of 25 patients without active infection, nor in healthy controls and patients with other herpesvirus infections. CMV-Ag positive blood cells appeared, on average, nine days before serological signs of active infection. This method provides a rapid and sensitive approach to CMV detection, enabling early clinical diagnosis and subsequent tapering of immunosuppression or commencement of antiviral therapy.     

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.