Multisite evaluation of a monoclonal antibody reagent (Syva) for rapid diagnosis of cytomegalovirus in the shell vial assay.

A pre-cytopathic effect (CPE) monoclonal antibody reagent (Syva Co., Palo Alto, Calif.) was evaluated in four laboratories for the rapid detection of cytomegalovirus (CMV) in shell vial cell cultures at 16 to 24 h and 40 to 48 h postinoculation. Results were compared with those obtained by inoculation of the specimen into conventional tube cell cultures that were examined for the presence of typical CMV CPE and subsequently tested by reaction with the monoclonal antibody reagent in an indirect immunofluorescence test. Of 937 specimens, CMV was positive in 184 (20%). CMV was detected twice as frequently in shell vials only (n = 29) as in conventional tube cell cultures (n = 14). Pre-CPE shell vial assay was 91% sensitive (range, 84 to 98%) and 96% specific (range, 93 to 98%) compared with the detection of CPE in conventional tube cell cultures. Overall, 137 of 166 (83%) and 143 of 166 (86%) of the CMV strains were detected at 16 to 24 h and 40 to 48 h postinoculation, respectively. The Syva reagent produced sensitive and specific results for the rapid detection of CMV infection in shell vial cell cultures and reliably confirmed the presence of the virus as detected by CPE in conventional tube cell cultures.     

Comparison of standard tube and shell vial cell culture techniques for the detection of cytomegalovirus in clinical specimens.

A monoclonal antibody was used to detect an early antigen of cytomegalovirus (CMV) by fluorescence 16 h after inoculation of MRC-5 monolayers in 1-dram (ca. 3.7-ml) shell vials and low-speed centrifugation. Of 770 specimens (urine, blood, lung tissue, sputum) processed in shell vials, 124 (16%) were positive for the virus at 16 h postinfection. CMV was isolated in standard tube cell cultures (average time, 9 days) from only 88 specimens, but there were no instances (with the exception of 2 blood specimens) in which CMV was recovered from tube cultures but not from shell vials. Additional specimens from 18 patients were positive in the shell vial assay but negative in the conventional tube cell culture assay. Other specimens from 14 of the 18 patients yielded CMV in conventional tube cell cultures. Of the 4 patients from whom CMV was not recovered from other specimens by conventional tube cell culturing, all had evidence of recent CMV infections, as indicated by a fourfold or greater rise in antibody titer. The specificity of the shell vial assay for the detection of CMV is supported by assays of other specimens from the same patients yielding the virus or serological evidence indicating recent infections, the known enhancement of CMV detection after centrifugation of the shell vials, and the distinct and easily recognizable fluorescence confined to the nuclei of CMV-infected cells. Our data indicate that the shell vial cell culture assay for the detection of CMV is as specific as and more sensitive than conventional tube cell culturing for the diagnosis of CMV infections.     

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)     

Detection of cytomegalovirus infections in specimens other than urine by the shell vial assay and conventional tube cell cultures.

Blood, bronchoscopy-lavage, biopsy (lung, liver, kidney), sputum, and other (cecum, bone) specimens were inoculated into shell vials and conventional cell tube cultures seeded with MRC-5 cells over a 23-month period. Of 1,472 specimens, 182 (12.4%) yielded cytomegalovirus (CMV)-positive results from 81 patients. Significantly more CMV-positive specimens were detected in shell vials (n = 154; 84.6%) than in conventional tube cell cultures (n = 126; 69.2%) (P less than 0.01). We found that 98 (53.8%) of the total 182 and 41 (42.7%) of the 96 blood specimens positive for CMV were detected by both the shell vial assay and conventional tube cell cultures. However, 56 (30.7%) of the total 182 and 31 (32.3%) of the 96 blood specimens positive for CMV were obtained exclusively in shell vials after detection with monoclonal antibody. Alternatively, 28 (15.4%) of the total 182 and 24 (25%) of the 96 blood specimens positive for the virus were isolated only in conventional tube cell cultures. Thus, although the shell vial assay was more sensitive and rapid than the conventional tube cell culture method, both systems must be used, especially for blood specimens, for the laboratory diagnosis of CMV infections.     

Detection of cytomegalovirus in bronchoalveolar lavage: a comparison of techniques.

Cytomegalovirus (CMV) is a common cause of lower respiratory tract infections in immunocompromised individuals. Bronchoalveolar lavage (BAL) is a noninvasive means to procure large numbers of bronchial and alveolar cells from the lung. To assess various methods of detecting CMV in the lavage specimen, 26 BAL specimens from 16 patients at high risk for CMV infection were evaluated. The methods and time required for analysis were the following: cytologic examination of Papanicolaou-stained membrane filters (1 h); viral cytopathic effects in tissue culture (days to weeks); spin amplification followed by staining with a monoclonal antibody for detection of CMV early nuclear antigen (18 h); and in situ hybridization (IH) with a biotinylated complementary DNA (cDNA) CMV probe (5 h). CMV was detected in 11 of 26 (42%) specimens by the early antigen assay, ten of 26 (38%) by in situ hybridization, five of 26 (19%) by tissue culture, and three of 26 (12%) by routine cytology. The absence of diagnostic CMV nuclear and/or cytoplasmic inclusions in many specimens positive by in situ hybridization and/or early antigen detection assay may be in part due to low levels of viral replication, insufficient for the development of diagnostic inclusions. These data show that techniques using in situ hybridization or fluorescent anti-CMV antibodies are rapid and are more sensitive for CMV identification than both cytomorphological examination and traditional tissue culture methods. Additional studies are required to determine the clinical significance of early CMV detection by in situ hybridization and early nuclear antigen detection assays.     

Direct in situ hybridization for rapid detection of cytomegalovirus in bronchoalveolar lavage

Rapid detection of cytomegalovirus (CMV) from pulmonary specimens in immunosuppressed persons may provide an origin for pneumonia. In situ DNA hybridization has been effective for detection of CMV in otherwise nondiagnostic histologic material. Studies comparing bronchoalveolar lavage (BAL) with open-lung biopsy have shown the former to be superior in detecting most pulmonary pathogens affecting immunocompromised patients. Fifty consecutive BAL specimens were studied to compare direct in situ DNA hybridization, routine tissue culture, and conventional cytologic examination to assess the efficacy of the hybridization technic to rapidly detect CMV. Using tissue culture as the standard, a sensitivity of 90% (28 of 31) and specificity of 63% (12 of 19) were observed with the CMV probe. Discrepant results between the probe and tissue culture were present in ten cases. There were seven probe-positive, culture-negative cases, three of which had systemic CMV infection, including two patients with inclusions noted by conventional cytologic examination. Three probe-negative, culture-positive cases were found. In the authors' laboratory, the predictive value of a positive CMV probe is 80% (28 of 35). In contrast to the probe, conventional cytologic examination revealed CMV inclusions in only 23% (7 of 31) of the culture-positive cases. An average of 21 days was required for CMV cultures to become positive; probe results were available within 24 hours. The authors conclude direct in situ DNA hybridization is a useful rapid method for the detection of CMV in BAL specimens submitted for cytologic examination.     

Evaluation of number of shell vial cell cultures per clinical specimen for rapid diagnosis of cytomegalovirus infection.

Specimens submitted for the diagnosis of cytomegalovirus (CMV) infection were inoculated into three (blood) or two (urine, tissue, bronchoalveolar lavage [BAL]) shell vials seeded with MRC-5 cells for the diagnosis of CMV infection. We evaluated the detection of 993 specimens that were positive for CMV according to the number of shell vial cell cultures inoculated per specimen. For blood cultures, and considering one CMV-positive shell vial as 100%, inoculation of three shell vials versus one increased the detection rate of the virus by 51%. Inoculation of three shell vials compared with two yielded a 20% increase in the detection rate of CMV. For urine, tissue, and BAL specimens, inoculation of two shell vials compared with one resulted in increases of 7, 10, and 5%, respectively. For maximum detection of CMV in shell vial cell cultures, at least three vials should be inoculated with blood specimens, and two vials should be used for urine, tissue, and BAL samples.     

Rapid detection of infectious cytomegalovirus in blood with the aid of monoclonal antibodies

The recently developed early antigen immunofluorescence (IF) method for the detection of infectious cytomegalovirus (CMV) in clinical specimens has hardly been applied on blood samples. We compared the CMV early antigen detection technique with the conventional cell culture method in 415 different buffy coat samples from 85 different immunocompromised patients. Duplicate coverslips were stained with two different monoclonal antibodies 4-6 days after inoculation. The conventional cultures were examined for typical cytopathic effects (CPE) during 10 weeks. Forty samples from 19 patients were positive by the IF technique, most of them with both monoclonal antibodies. Only 22 of these samples were positive in the conventional cell culture assay, on average after 15.8 days. CMV viraemia was detected exclusively by the IF method in 18 samples, 7 of which were from five patients without any further evidence of an active CMV infection. CMV viraemia was detected exclusively by the CPE method in eight samples, on average after no less than 36.6 days. CMV viraemia was not found in blood samples from 10 patients with laboratory proven active CMV infections and 53 patients without any evidence of an active CMV infection. In our hands the early antigen method for the detection of infectious CMV in blood is nearly as specific (at least 98.1%) and clearly much faster and more sensitive than the conventional cell culture method. The early CMV antigen detection method is therefore a very useful tool for the rapid detection of infectious CMV in blood.     

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 diagnosis of cytomegalovirus infection in renal transplant and dialysis patients by DNA-DNA hybridisation assay

One hundred forty-eight urine specimens were collected from 47 renal transplant and dialysis patients and screened for the detection of cytomegalovirus (CMV). Diagnosis of CMV infection was suggested in 17 out of 47 patients (36.2%) by more than one of the five methods used. DNA hybridisation assay (DNA HA) using 32P-labelled probe detected CMV DNA in 15 (31.9%) of 47 patients, whereas virus isolation on conventional tube cell cultures (CTC), immunofluorescence incorporating monoclonal antibodies on centrifugation vial cultures (IF), complement fixation test (CFT), and electron microscopy (EM) yielded positive results in only nine (19.2%), 12 (25.2%), 11 (23.4%), and one (2.1%) of 47 patients, respectively. The significance of these results obtained by DNA HA lies not only in the apparent increase in number of patients diagnosed, but also in both early and rapid detection of CMV DNA. More importantly, the DNA HA is highly specific in that it correlates accurately with clinical and laboratory data characteristic of CMV disease. In respect of clinically manifest CMV disease, the specificity of DNA HA, CTC, IF, CFT, and EM was 87.5, 43.7, 56.3, 43.7, and 6.3%, respectively. These advantages of DNA HA make it the test of choice for early diagnosis of CMV infections in immunosuppressed patients.     

Rapid detection of cytomegalovirus in bronchoalveolar lavage specimens from marrow transplant patients: evaluation of a direct fluorescein-conjugated monoclonal antibody reagent

An FITC-conjugated monoclonal antibody reagent containing three CMV-specific monoclonal antibodies was evaluated for the rapid detection of CMV in bronchoalveolar lavage (BAL) cytospin preparations by direct IF (DFA). Eighty-six BAL samples from 72 marrow transplant patients were inoculated into both centrifugation and standard cell culture. CMV was detected in 49/86 (57%) BAL samples. DFA detected 37/46 (80%) samples which were positive in centrifugation culture. While DFA staining lacked the sensitivity (overall sensitivity 38/49, 78%) to replace either standard or centrifugation culture, the total laboratory time needed to complete the DFA was only 1.5 h and its concurrent use with centrifugation culture can provide rapid specific diagnosis of CMV pneumonia.     

Comparative sensitivity of three methods for the diagnosis of cytomegalovirus lung infection

Conventional cell culture (CCC), an immunofluorescent assay for the rapid detection of CMV-induced early antigens (DEAFF) in infected fibroblasts and an immunofluorescent assay for the detection of CMV-infected cells obtained directly from clinical material were compared prospectively for their ability to detect CMV in bronchoalveolar lavage (BAL) material. CMV was detected by at least one method in 47 of 139 BALs (33.8%). The mean sensitivities of each of the three assays were 93% for DEAFF, 46% for CCC and 22% for the direct method. The mean time to diagnosis was 24 h, 15 days and 4 h for each of the methods, respectively. The use of monoclonal antibodies in the DEAFF test on BAL specimens provided a simple and rapid method for the diagnosis of CMV lung infection and was shown to be more reliable than conventional culture methods in achieving a diagnosis from BAL specimens.     

Detection of viral and chlamydial antigens in open-lung biopsy specimens

The recovery of viruses and Chlamydia trachomatis from cell cultures and the detection of their antigens in impression smears prepared from open-lung biopsy (OLB) specimens from immunocompromised adults were compared. Touch impression smears were prepared on three slides, each containing eight wells. OLB tissue was homogenized (Stomacher) and inoculated into MRC-5, primary monkey kidney, and McCoy cell cultures. The direct and indirect immunofluorescence (IF) tests were used to detect antigens to the following organisms: herpes simplex virus, adenovirus, parainfluenza types 1 and 3, respiratory syncytial virus, cytomegalovirus (CMV), Chlamydia trachomatis, influenza types A and B, and varicella-zoster virus. Of 105 OLB specimens, 21 viral isolates (20%) were recovered in cell culture; 20 were CMV and one was an influenza virus type A (H3N2). Both culture and IF results were positive with 12 specimens, but in nine instances a virus was isolated and IF was negative or eight times culture results did not yield the organism but IF test results were positive. Chlamydia trachomatis was never isolated or detected by IF. The authors recommend that for optimal detection of CMV from OLB specimens a new rapid centrifugation-enhanced cell culture system be used in preference, or in conjunction with a preliminary IF screen of impression smears for CMV detection.     

Detection of herpes simplex virus in conventional tube cell cultures and in shell vials with a DNA probe kit and monoclonal antibodies.

Specimens submitted for diagnosis of herpes simplex virus (HSV) infection were inoculated into shell vials and reacted with a commercial DNA probe kit (Pathogene; Enzo Biochem, Inc., New York, N.Y.) and an immunofluorescence assay at 16 h postinoculation. The results were compared with isolation of the virus in conventional tube cell cultures. Of 504 specimens, 105 (20.8%) were positive for HSV. Of the 105, 93 HSV-positive specimens (89%) were detected by all three assay systems. Maximum detection of HSV (100 of 105 [95%]) was obtained by probe or monoclonal antibody assay in shell vials, which had sensitivities of 98 and 97%, respectively, compared with viral recovery in conventional tube cell cultures (mean time for recognition of cytopathic effects, 2 days). Both shell vial assays were 99% specific. The DNA probe kit may be used as an alternative to a monoclonal antibody and fluorescence assay in shell vials as a diagnostic method for rapid laboratory detection of HSV infection.     

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.     

Detection of cytomegalovirus from blood leukocytes separated by sepracell-MN and Ficoll-Paque/Macrodex methods.

Two density gradient separation techniques for separation of blood leukocytes were compared for the laboratory diagnosis of cytomegalovirus (CMV) viremia. Of 510 blood specimens processed by both methods, 76 (14.9%) yielded CMV. Of the 76 positive specimens, 66 (87%) and 65 (86%) were processed by the Ficoll-Paque/Macrodex (F-P/M; Macrodex is dextran 70 in normal saline; Pharmacia, Pisataway, N.J.) and Sepracell-MN methods, respectively. Of the 76 CMV-positive blood specimens, 72 (95%) were detected in shell vial cell cultures, whereas only 42 (55%) were detected in conventional tube cell cultures. The time for recognition of specific cytopathic effects due to CMV in tube cell cultures (8.0 versus 7.1 days), the number of fluorescent foci in each positive shell vial culture (19.3 versus 20.1), and the costs of the reagents ($3.50 versus $2.80) were similar and independent of the leukocyte separation method (F-P/M versus Sepracell-MN). Recovery of CMV from heparinized blood (F-P/M method) was similar to that from EDTA-anticoagulated blood (Sepracell-MN method). The Sepracell-MN method is a rapid and sensitive method for detection of CMV from blood specimens and is recommended as a replacement for the more tedious and time-consuming F-P/M procedure.     

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 detection of cytomegalovirus in clinical specimens by immunofluorescent staining of shell vial cultures

A recently described rapid technique for detection of cytomegalovirus (CMV) was evaluated in clinical specimens utilizing indirect immunofluorescent staining (IFA) of shell vial cultures. A total of 266 clinical specimens received for viral isolation were inoculated to commercially available shell vials seeded with human lung fibroblasts (MRC-5), centrifuged at 700 X g for one hour, and stained after 18 hours incubation with monoclonal antibody to CMV early nuclear protein (Biotech Research Laboratories) and fluorescein conjugated goat antimouse IgG (Cappel Laboratories). All specimens were also inoculated to tubes of human lung fibroblasts and observed for cytopathic effect (CPE) for 28 days. Of 54 specimens positive for CMV, 36 were positive by both IFA and CPE, 3 were positive by CPE only, and 15 were positive by IFA only (P less than 0.01 by the chi-square test). Failure to detect CMV associated CPE in 10 of these 15 samples was probably due to concomitant infection with herpes simplex virus or heavy bacterial or fungal contamination. Nine of the 13 patients with IFA-positive CPE-negative specimens had CMV infection documented by other positive cultures. It was concluded that the shell vial IFA rapid technique for detection of CMV is highly specific, more sensitive than conventional isolation, and well suited for application in a clinical virology laboratory.     

Comparison of culture and the antigenemia assay for detection of cytomegalovirus in blood specimens submitted to a reference laboratory.

We compared the antigenemia assay (AA) with tandem shell vial cultures (SVCs) and tube cultures (TCs) for detection of cytomegalovirus (CMV) in 343 blood specimens. For 249 specimens, the AA was performed in duplicate with two different commercially available monoclonal antibody reagents (Biotest Diagnostic Corporation and Argene Biosoft). Specimens considered true positives were positive in either culture system or both AAs. Only specimens which were negative in both cultures and positive in a single AA were tested retrospectively with a CMV PCR assay. CMV recovery rates were also calculated to determine if increased specimen age resulted in decreased positivity. CMV recovery rates for the AA and the combination of both cultures were 20.0 and 5.0% at 3 to 18 h, 20.2 and 14.0% at 18 to 35 h, 12.5 and 7.8% at 36 to 52 h, and 18.8 and 6.3% at 64 to 75 h, respectively. The sensitivities and specificities of the Biotest AA, the Argene AA, SVC, and TC were 84.4 and 100.0, 100.0 and 99.6, 44.4 and 100.0, and 46.0 and 100.0%, respectively. The AA was significantly more sensitive than either culture method alone and was also more sensitive than the two culture methods used in tandem (the tandem culture sensitivity was 63.5%); the Argene AA identified more positives than the Biotest AA.     

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.