Rapid detection of influenza virus by shell vial assay with monoclonal antibodies.

Of 45 influenza virus strains (43 type A and 2 type B) detected in conventional tube cell cultures (average time, 4 days), 25 (56%) were detected by immunofluorescence in the shell vial assay 24 h postinoculation. The specific fluorescence produced should allow this procedure to be readily adapted by laboratories with various degrees of experience with immunofluorescence methodology.     

Identification of polyoma BK virus in kidney transplant recipients by shell vial cell culture assay and urine cytology

Urine cytology often is used to identify BK virus in kidney transplant recipients in the cytology laboratory. To assess the usefulness of the shell vial cell culture assay to identify BK virus, urine samples from 42 kidney transplant recipients were tested by the urine cytology and shell vial cell culture assays. The shell vial cell culture assay is just as sensitive and specific as urine cytology for the identification of BK virus in kidney transplant recipients.     

Diagnosis of cytomegalovirus infections by shell vial assay and conventional cell culture during antiviral prophylaxis.

A total of 3,552 specimens for conventional cytomegalovirus (CMV) culture and shell vial assay for CMV immediate-early antigen were obtained during a prospective randomized trial for prophylaxis of CMV disease after liver transplantation. Prophylaxis with ganciclovir for 2 weeks and then high-dose acyclovir for 2.5 months was compared with high-dose acyclovir alone for 3 months. During the first 12 weeks after transplantation, when the patients were on prophylaxis, there were significantly more clinical samples positive by the shell vial assay and negative by standard culture in comparison with the number of samples obtained from weeks 13 to 24, after prophylaxis was discontinued, that were positive by the shell vial assay and negative by standard culture. In contrast, significantly fewer samples were positive by both the shell vial assay and standard culture during the first 12 weeks compared with the number obtained 13 to 24 weeks after transplantation that were positive by both methods. Samples positive by the shell vial assay only were obtained significantly more frequently from patients with asymptomatic than symptomatic CMV infections, while samples positive by both methods were obtained significantly more often from patients with symptomatic CMV infection. It was concluded that antiviral prophylaxis with high-dose acyclovir or ganciclovir and then high-dose acyclovir and asymptomatic CMV infection are associated with a decrease in the level of CMV isolation by standard cell culture in comparison with that by the shell vial assay.     

Relevance of influenza a virus detection by PCR, shell vial assay, and tube cell culture to rapid reporting procedures

Influenza A virus was detected at higher rates and for more extended time periods with real-time PCR than with cell cultures. We show here that, using the theranostic approach, rapid viral detection and reporting can provide for early implementation and assessment of available antiviral therapy.     

Isolation of Rickettsia prowazekii from blood by shell vial cell culture.

A blood sample from a patient who returned from Algeria with a fever inoculated on human embryonic lung fibroblasts by the shell vial cell culture technique led to the recovery of Rickettsia prowazekii. The last clinical strain was isolated 30 years ago. Shell vial cell culture is a versatile method that could replace the classic animal and/or embryonated egg inoculation.     

Optimization of detection of cytomegalovirus viremia in transplantation recipients by shell vial assay.

Cytomegalovirus (CMV) viremia is a widely used laboratory marker of CMV disease following transplantation and is additionally used to trigger preemptive antiviral therapy. Despite this, the optimal method for diagnosing CMV viremia in transplantation recipients remains unknown. To determine the sampling frequency and blood volume required for the optimal diagnosis of viremia by shell vial assay, a prospective study of 46 viremic transplantation recipients was conducted. Blood specimens (2.5 and 5 ml) were collected twice, 3 h apart, at a median of 1.4 days (range, 1 to 3 days) after the triggering shell vial-positive blood had been collected. Considering a single 2.5-ml specimen, an average of only 40% of previously viremic patients had documented CMV in their blood: this increased to 50% when a second 2.5-ml sample of blood was collected 3 h later. The yields of two 2.5-ml versus two 5-ml samples were 50 versus 61%, respectively. Viremia as detected by shell vial assay is intermittent, and increasing the frequency and volume of blood sampling increases its diagnosis. These results have implications in diagnosis of CMV infection and its preemptive therapy.     

Rapid detection of respiratory viruses by shell vial culture and direct staining by using pooled and individual monoclonal antibodies.

The Bartels respiratory virus panel detection kit is an indirect fluorescent-antibody (IFA) method that uses pooled and individual antisera for tissue culture confirmation of seven respiratory viruses. We evaluated these reagents for detecting viral antigen in shell vial cultures and by direct staining of cells from respiratory specimens. The isolation from 254 specimens of respiratory viruses in shell vial cultures compared with standard tube cultures was highly sensitive (94%) and specific (97.3%). The numbers of viral isolates detected in three consecutive years of testing with shell vial cultures were 68 of 254 (26.8%), 101 of 381 (26.5%), and 122 of 430 (28.4%). IFA direct staining of all 1,065 specimens resulted in 183 (17.2) being uninterpretable because of inadequate numbers of cells or interfering fluorescence. The sensitivity and specificity of the interpretable IFA direct stains in comparison with shell vial cultures were 85.9 and 87.1%, respectively. For detection of 881 adequate specimens, Bartels respiratory syncytial virus IFA direct staining compared with an Ortho Diagnostics Systems direct fluorescent-antibody test for respiratory syncytial virus RSV was highly sensitive (95.5%) and specific (97%). Shell vial cultures combined with Bartels IFA reagents are a rapid alternative to standard tube cultures. Bartels IFA direct staining with individual antisera provides useful same-day screening of respiratory specimens, but the antiserum pool was not effective in screening for positive specimens because of excessive amounts of nonspecific fluorescence.     

Rapid diagnosis of respiratory viral infections by using a shell vial assay and monoclonal antibody pool.

We compared the detection of seven respiratory viruses by using a commercially available monoclonal antibody pool in a 2-day shell vial assay with that by using standard cell culture with respiratory syncytial virus (RSV) enzyme-linked immunosorbent assay (ELISA)-negative nasal secretions from hospitalized children. We found 179 respiratory virus isolates by either method in 675 specimens. Overall, the shell vial assay detected 147 of 179 (79%) of the positives after 2 days; cell culture detected 148 of 179 (80%) after a mean incubation period of 7.6 days (range, 1 to 14 days). The sensitivity of the shell vial assay was 78% for RSV, 94% for influenza B virus, 83% for adenovirus, and 80% for parainfluenza viruses. The sensitivity of the cell culture was 70% for RSV, 79% for influenza B virus, 90% for adenovirus, and 89% for parainfluenza viruses. The 2-day shell vial assay allowed the detection of respiratory viruses in a clinically relevant time frame and rapidly detected RSV in specimens lacking RSV antigen by ELISA.     

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.     

Comparison between indirect immunofluorescence assay and shell vial culture for detection of mumps virus from clinical samples

We report a prospective comparison of the efficacies of an indirect immunofluorescence assay (IFA) and shell vial culture (SVC) of throat swab and urine samples from patients with mumps. Throat swab samples were used for the IFA; the urine samples and throat swabs were inoculated into vials of Vero cells. We studied 62 patients by using 62 throat swabs and 50 urine samples (50 patients with both samples). Sixty (96.7%) throat samples were positive in the SVC, and 61 (98.3%) were positive in the IFA. For the 50 patients from whom both samples were available, the IFA was positive in 50 (100%) cases, the urine sample was positive in 49 (98%) cases, and the throat swab was positive in 48 (96%) cases (P > 0.05). This comparison of throat swabs and urine samples has shown that the two clinical samples are similar in efficacy.     

Detection of enteroviruses from clinical specimens by spin amplification shell vial culture and monoclonal antibody assay.

Conventional tube cell culture was compared with a 72-h, spin-amplified shell vial indirect immunofluorescence assay for the detection of enterovirus from clinical specimens. The sensitivity for the shell vial assay after resolution of discrepant results were 93 and 100%, respectively. The shell vial assay detected 93% of the positive cultures within 72 h of incubation while conventional tube culture detected only 51% of the positive cultures within the same time interval. The data suggest that a spin-amplified shell vial indirect immunofluorescence assay may be useful for the detection of enterovirus from clinical specimens.     

Comparison of standard culture methods, a shell vial assay, and a DNA probe for the detection of herpes simplex virus.

A nonradioactive, biotinylated herpes simplex virus (HSV) DNA probe, a shell vial (rabbit kidney cell) culture assay enhanced by a direct fluorescent (HSV monoclonal)-antibody stain at 16 to 20 h postinoculation, and conventional tube cultures with confirmation via HSV-specific (polyclonal antibody) immunoperoxidase assay were compared for 199 specimens. The predictive values of the positive results were 54.5% for the probe, 95.9% for the shell vial assay, and 100% for the conventional culture methods, while the predictive values of the negative tests were 68.1, 84.0, and 98.4%, respectively. We conclude that the DNA probe (sensitivity, 24.5%; specificity, 88.3%) and the shell vial assay (sensitivity, 66.2%; specificity, 98.4%) cannot be substituted for conventional tube culture techniques (sensitivity, 97.1%; specificity, 100%) in the routine identification of HSV in our laboratory.     

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.     

Rapid detection of cytomegalovirus by fluorescent monoclonal antibody staining and in situ DNA hybridization in a dram vial cell culture system.

By using dram vial cell culture methods, three commercially available tests for cytomegalovirus (CMV) detection were compared: direct fluorescent monoclonal antibody staining for CMV-specific early and late antigens (direct FA), indirect fluorescent monoclonal antibody staining for a CMV-specific early antigen (indirect FA), and in situ DNA hybridization with a biotinylated CMV-specific DNA probe kit (DNA probe). Of those tests, only the indirect FA provided consistent, reliable virus detection within the initial 24 h postinfection for serial 10-fold dilutions of CMV AD169 (laboratory strain) and for three selected urine samples. However, when used prospectively, the indirect FA failed to detect virus within the initial 10 days postinfection in 15 of 78 consecutive specimens that were eventually positive by cell culture. Although the indirect FA was more sensitive than the direct FA or DNA probe, its utility appeared limited to specimens with high CMV concentrations. On the basis of these data, we recommend that indirect FA be reserved as an adjunct to standard cell culture for selected samples in diagnostic hospital laboratories.     

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.     

Rapid detection of respiratory viruses using mixtures of monoclonal antibodies on shell vial cultures.

Eleven hundred and thirty-three clinical specimens submitted to the laboratory for diagnosis of respiratory virus infections were tested by direct immunofluorescence (DIF) for respiratory syncytial virus (RSV), by shell vial culture, and by conventional cell culture. The shell vial cultures were stained with 8 different monoclonal antibodies both 1 day and 3-7 days after inoculation. In order to limit the cost and the workload, mixtures of monoclonal antibodies were used. Coverslips with HEp-2 cells were incubated with a mixture of FITC-labeled monoclonal antibody to RSV and nonlabeled monoclonal antibody to adenovirus. When no RSV positive IF staining was observed after the first incubation step, the same coverslip was incubated once more with FITC-labeled anti-mouse antibody. A positive reaction at this stage indicated the presence of adenovirus. Similarly, cultures of tertiary monkey kidney cells were investigated with a mixture of two FITC-labeled monoclonals to the influenza viruses A and B and three nonlabeled monoclonals to the parainfluenza viruses 1, 2 and 3. If influenza virus or parainfluenza virus was detected, the exact type was determined by staining different parts of a duplicate coverslip. Shell vial cultures for cytomegalovirus (CMV) were always performed separately on human embryonic lung fibroblasts. Using this approach, we detected RSV (n = 248), CMV (n = 42), parainfluenza virus (n = 31), influenza virus (n = 28), and adenovirus (n = 6), in most cases after only one day of culture. For RSV, the sensitivity of the shell vial method was too low (74%) to allow omission of DIF (sensitivity 95%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Prospective comparison of R-mix shell vial system with direct antigen tests and conventional cell culture for respiratory virus detection.

BACKGROUND AND OBJECTIVES: Conventional cell culture (CC) has limited clinical utility as a result of the extended incubation period often required for virus isolation. Alternative methodologies have been introduced in an effort to improve turnaround times. One such system, the R-mix shell vial is discussed herein. The study objectives were: (a) to establish R-mix testing parameters as compared to direct antigen testing (DAT) and CC, and (b) to assess technical aspects and cost of R-mix in a high volume clinical virology laboratory. STUDY DESIGN: A prospective analysis of respiratory samples submitted to the clinical virology laboratory between November 2004 and April 2005 was performed. All specimens were inoculated onto R-mix shell vials (SV) and CC tubes; and a subset also underwent DAT for influenza A and B and/or RSV. A retrospective estimated cost analysis was made. RESULTS: A total of 563 samples were included in the study, which collectively revealed a total of 207 viruses. Sensitivity of R-mix for seven major respiratory viruses ranged from 45% to 83% compared to CC and DAT, while mean time to detection (TTD) varied from 1.1 to 1.4 days. In addition to these viruses, 23 picornaviruses, 11 CMV isolates and 5 HSV isolates were detected by CC alone. CONCLUSIONS: The R-mix system has similar sensitivity as CC for the detection of parainfluenza 1-3 and influenza A/B while dramatically reducing the TTD. Furthermore, it is significantly more sensitive and produces more timely results for RSV than CC; yet, neither method offers a diagnostic benefit over rapid DAT for RSV detection. The sensitivity of R-mix for adenovirus appears to be significantly lower than that of CC. Lastly, methodologies other than R-mix must remain in place under circumstances where identification of other potential viral respiratory pathogens, including herpesviruses and picornaviruses, is desired.