A multisite trial comparing two cytomegalovirus (CMV) pp65 antigenemia test kits, biotest CMV brite and Bartels/Argene CMV antigenemia

A total of 513 blood specimens, predominantly from organ transplant recipients, human immunodeficiency virus-positive patients, and bone marrow transplant recipients, were tested for cytomegalovirus (CMV) by culture and pp65 antigenemia across four test sites. Peripheral blood leukocytes were examined by using both the Biotest CMV Brite and the Bartels/Argene CMV Antigenemia kits. A total of 109 specimens were positive for CMV, 106 (97%) were positive by antigenemia, and 34 (31%) were positive by culture. According to the manufacturers' instructions, 150,000 cells were applied per slide for the Biotest kit and 200,000 cells per slide for the Bartels kit. A total of 93 specimens (88%) were positive by the Biotest kit, and 86 (81%) were positive by the Bartels kit. In specimens found to be positive by only one kit, the positive cell counts were low (median, 1; range, 1 to 7). When the data from all four sites were combined and analyzed, there was no statistical difference between the performance of the two kits; the Biotest and Bartels kits were found to be equivalent in sensitivity, specificity, and positive and negative predictive values for the detection of CMV pp65 antigenemia.     

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 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.     

Comparison of monoclonal antibodies for rapid detection of cytomegalovirus in spin-amplified plate cultures.

Cytomegalovirus (CMV) was detected in 56 of 275 specimens (20%); 50 of 56 (89%) were detected by conventional culture, and 37 (66%) were detected by rapid assay at 72 h with a commercial monoclonal antibody and a pooled monoclonal antibody. Although the two antibodies were equally sensitive at 72 h, the pooled antibody gave a brighter, more easily detected signal. Other viruses were isolated from 9 specimens (3.3%) by conventional culture. Use of rapid assays alone fails to detect slow-growing CMV and non-CMV viral pathogens.     

Rapid diagnosis of invasive cytomegalovirus infection by examination of tissue specimens in centrifugation culture

Two hundred eight tissue specimens from 81 patients were examined by centrifugation culture for the rapid diagnosis of invasive cytomegalovirus (CMV) infection. Results were compared with those obtained by both conventional viral cultures and histologic examination. CMV was identified by centrifugation culture in 52 (25%) specimens at 16 hours after inoculation, including 39 lung specimens and 12 gastrointestinal specimens. One additional esophageal specimen was positive (53 of 208 specimens; 25.5%) at 36 hours. In contrast, CMV was recovered from only 50 of these 53 specimens by conventional cell culture at a mean of 11 days after inoculation. Forty-three of the 208 specimens (20.5%) were positive for CMV when examined by histologic methods, including light microscopy and tissue immunofluorescence. All 43 were positive by centrifugation culture. Specimens that were positive by centrifugation culture but negative by histologic methods included three lung specimens and seven gastrointestinal specimens. The technic of centrifugation culture is more sensitive for diagnosis of invasive CMV infection by the examination of affected tissue than either conventional viral culture or histologic technics, including tissue immunofluorescence, and more rapid than conventional culture or routine histologic examination.     

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.     

Enhanced recovery of cytomegalovirus in conventional tube cultures with a spin-amplified adsorption.

Low-speed centrifugation-mediated adsorption was evaluated as an enhancement of infectivity of clinical and laboratory strains of cytomegalovirus (CMV) occurring with cells grown in conventional culture tubes. The time required for reporting of primary isolates of CMV from urine specimens adsorbed onto monolayers of WI-38 cells in culture tubes was calculated. Of 668 specimens adsorbed by the stationary phase (SP) method, 98 were positive by cytopathic effect (CPE) that required an average of 16.8 days for recovery in culture. However, the appearance of CPE required a shorter average time of 11.9 days for 70 CMV strains isolated from 283 specimens adsorbed in tube cultures by the spin-amplified (SA) method. In another phase of clinical CMV recovery, urine specimens were adsorbed by the SA method onto cell cultures grown in both shell vials and test tubes. Of 594 specimens inoculated, a total of 74 were positive by either CPE in test tubes or immunostaining-localized early antigen in shell vials. Approximately one-third of these CMV isolates were recovered only by CPE from specimens adsorbed by the SA method in test-tube cultures. In a related study to further evaluate differences between adsorption methods, the AD-169 laboratory strain of CMV was adsorbed by SP and SA methods onto MRC-5 cells grown in both culture vessels. Early antigen detection by immunomicroscopy was found in the infected cells at least 2 to 4 days prior to the appearance of CPE, regardless of adsorption procedure. In both vessels, the replication of AD-169 virus in cultures adsorbed by the SA method consistently exceeded that of virus adsorbed by the SP procedure. CPE occurred 24 to 48 h earlier and progressed two to four times more extensively; early antigen was expressed two- to fourfold greater within 24 to 48 h postinfection; and foci of infected cells containing late antigen were two to four times greater in number at 1, 2, and 5 days postinfection. Overall, the replication and enhancement of infectivity of laboratory and clinical strains of CMV as determined by CPE and early and late antigen expression occurred most efficiently with specimens adsorbed by the SA method onto cultures grown in conventional tubes or shell vials.     

Evaluation of the PrimeCapture CMV DNA Detection Plate System for Detection of Cytomegalovirus in Clinical Specimens

With the availability of anticytomegalovirus (CMV) therapeutic agents, rapid detection of CMV is important in the care and management of the immunosuppressed patient. The PrimeCapture CMV DNA Detection Plate System (PC-PCR) was evaluated for the detection of CMV in blood and cerebrospinal fluid (CSF). The resolution of discordant results was performed by consensus testing utilizing a combination of conventional cell culture (TC-CPE), the CMV-antigenemia (CMV-Ag) assay, one or more in-house CMV nested PCR assays, and/or patient evaluation and follow-up. Of 51 blood specimens from 34 patients, 23 (45%) were identified as true positives. PC-PCR was significantly more sensitive than the CMV-Ag assay, TC-CPE, or a combination of both tests. The sensitivities, specificities, positive predictive values (PPV), and negative predictive values (NPV) for PC-PCR, the CMV-Ag assay, TC-CPE, and a combination of CMV-Ag and TC-CPE were 78, 75, 72, 81%; 46, 100, 100, 70%; 39, 100, 100, 67%; and 58, 100, 100, 73%, respectively. CMV was not detected or isolated in CSF, resulting in a combined PC-PCR sensitivity, specificity, PPV, and NPV of 77, 90, 68, and 93%, respectively. Among those laboratorians considering the incorporation of molecular CMV diagnostics into their clinical microbiology or virology laboratories, the CMV PC-PCR offers a relatively simple-to-perform and sensitive assay system.     

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.     

Multicenter comparison of the digene hybrid capture CMV DNA assay (version 2.0), the pp65 antigenemia assay, and cell culture for detection of cytomegalovirus viremia

We compared the Digene Hybrid Capture CMV DNA Assay version 2.0, the pp65 antigenemia assay, traditional tube culture, and shell vial culture for the detection of cytomegalovirus (CMV) viremia in several patient populations at three centers. Of 561 blood specimens collected from 402 patients, complete clinical and laboratory data were available for 489. Using consensus definitions for true positives and true negatives, the sensitivities of the Hybrid Capture assay, antigenemia, shell vial, and tube culture were 95, 94, 43, and 46%, respectively. The specificities of the Hybrid Capture assay and antigenemia were 95 and 94%, respectively. At all three study sites, the detected level of CMV viremia was significantly higher with the Hybrid Capture assay or antigenemia than with shell vial and tube culture. In a group of 131 healthy nonimmunosuppressed volunteers, the Hybrid Capture assay demonstrated a specificity of over 99%. The Hybrid Capture assay is a standardized assay that is simple to perform and can utilize whole blood specimens that have been stored for up to 48 h. The high sensitivity and specificity of the Hybrid Capture assay along with its simplicity and flexibility make it a clinically useful assay for the detection of CMV viremia in immunocompromised or immunosuppressed patients. Further evaluation to determine its role in predicting CMV disease and for monitoring the therapeutic response to anti-CMV therapy is needed.     

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.     

Increased sensitivity for rapid detection of cytomegalovirus by shell vial centrifugation assay using mink lung cell cultures

A comparative study was made of various human and non-human cell cultures to determine their sensitivity for cytomegalovirus (CMV) as detected by the production of CMV early antigen using the shell vial centrifugation assay. Mink lung cell cultures, frequently used for detection of herpes simplex virus in clinical specimens, were found to be significantly more sensitive to infection by CMV than other cell cultures tested. Using the shell vial centrifugation assay, the mink lung cell cultures were more sensitive than human diploid fibroblasts for the detection of the Davis strain of human CMV and CMV from clinical specimens.     

Cytomegalovirus (CMV) antigenemia assay is more sensitive than shell vial cultures for rapid detection of CMV in polymorphonuclear blood leukocytes.

We compared the cytomegalovirus (CMV) antigenemia assay with shell vial cultures of polymorphonuclear leukocyte (PMNL)-enriched blood fractions for rapid diagnosis of CMV viremia. PMNL fractions of 280 blood specimens from 171 patients (170 solid-organ transplant recipients and 1 patient undergoing pretransplant evaluation) were inoculated in shell vial and conventional CMV cultures. A commercially available kit (CMV-vue kit; INCSTAR Corp.) was used for the CMV antigenemia assay, in which PMNL preparations were stained with monoclonal antibodies directed against the CMV protein pp65. Mixed-leukocyte blood fractions from the same blood specimens were inoculated in parallel shell vial and conventional cultures. CMV viremia (defined by the isolation of CMV in conventional cultures) was detected in 32 (13%) of 245 PMNL fractions included in the final analysis. Twenty-eight (87.5%) were also positive in the CMV antigenemia assay, whereas 22 (69%) were positive in shell vial cultures. Ten (4%) additional PMNL fractions positive only in the CMV antigenemia assay were from eight patients with active CMV infections (six patients), who had previous or subsequent episodes of CMV viremia (seven patients), or in whom CMV was isolated in cultures of simultaneously obtained mixed-leukocyte fractions (three patients). Overall, the CMV antigenemia assay was significantly more sensitive than shell vial cultures for detection of CMV in the PMNL fraction of blood leukocytes (P < 0.01, McNemar's test), and we recommend it as the method of choice for rapid diagnosis of CMV viremia.     

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.     

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.     

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.     

Practical protocol for cytomegalovirus isolation: use of MRC-5 cell monolayers incubated for 2 weeks.

Isolation of cytomegalovirus (CMV) in tissue culture is presently the most reliable means of proving active CMV infection. To improve both the cost-effectiveness and clinical usefulness of procedures for the isolation of CMV from fresh clinical specimens, we analyzed results obtained with standard isolation procedures and compared them with results obtained under different conditions. Cell monolayers from commercial sources were inoculated with fresh specimens and then were observed for a cytopathic effect typical of CMV. Of 1,375 specimens submitted over a 12-month period, 6.4% were CMV positive in WI-38 monolayers within 28 days after inoculation. The mean day of CMV detection for 45 urine, 13 cervical-vaginal, and 5 saliva specimens was 6.7 +/- 3.1 (mean +/- standard deviation), 9.9 +/- 3.3, and 7.7 +/- 3.3 days, respectively, and 92% were positive within 14 days. When 1,058 subsequent specimens were inoculated in parallel onto WI-38 and MRC-5 cell monolayers, 8.7% were positive for CMV. MRC-5 cells were significantly more sensitive than WI-38 cells: 98% of all positive specimens appropriate for comparison were detected in MRC-5 cultures, but only 85% were detected in WI-38 cells. Although 1 specimen was positive in WI-38 cells only, 38% of all isolates were positive earlier (16 specimens) or only (10 specimens) in MRC-5 cultures. Based on these data, we have developed a practical 2-week protocol for CMV isolation from fresh clinical specimens that includes the use of MRC-5 cell monolayers incubated at 36 degrees C.     

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)     

Direct detection of cytomegalovirus from bronchoalveolar lavage samples by using a rapid in situ DNA hybridization assay.

An in situ DNA hybridization assay was compared with centrifugation culture for rapid detection of cytomegalovirus (CMV) from bronchoalveolar lavage (BAL) samples. Eighty BAL samples were inoculated into both centrifugation culture and standard culture. Cytospin preparations of the BAL samples were studied in a 75-min in situ DNA hybridization assay using the PathoGene CMV kit (Enzo Biochem, Inc., New York, N.Y.). Of the 80 samples, 39 (49%) were positive for CMV; 37 of 39 (95%) were positive by centrifugation culture, 34 of 39 (87%) were positive in standard culture, 24 of 39 (62%) were positive by in situ hybridization, and 20 of 39 (56%) were positive by histologic and/or immunofluorescence techniques. The in situ hybridization assay detected 23 of the 37 samples positive in centrifugation culture, for a sensitivity of 62% and a specificity of 98%. We conclude that the in situ hybridization assay is a specific and more rapid test than centrifugation culture and standard culture for diagnosis of CMV pulmonary infection. For the clinical laboratory, however, current hybridization methods are not sufficiently sensitive to replace centrifugation culture for detection of CMV in BAL specimens.