Simultaneous seeding and infecting of shell vials for rapid detection of cytomegalovirus infection.

Fifty cytomegalovirus isolates were used to infect shell vials containing confluent MRC-5 cell monolayers and shell vials which were seeded with MRC-5 cells at the time of inoculation. All 50 of the isolates were detected by immunofluorescence in shell vials containing confluent monolayers, whereas 39 (78%) of the isolates were detected in shell vials that had been seeded and inoculated simultaneously (P less than 0.001). Preformed monolayers of cells in shell vials provide the most sensitive system for the detection of cytomegalovirus infection.     

Evaluation of R-Mix FreshCells in shell vials for detection of respiratory viruses

The performance of a mixture of mink lung and A549 cell lines in shell vials (MSVs) for the detection of respiratory viruses in 159 specimens was evaluated. MSVs, conventional culture, and direct immunofluorescence assay identified 96, 85, and 67% of the influenza A virus-positive specimens, respectively. MSVs provided both a high degree of sensitivity and rapid turnaround times for the detection of influenza A virus.     

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 of shell vials and conventional tubes seeded with rhabdomyosarcoma and MRC-5 cells for the rapid detection of herpes simplex virus.

Shell vials (SV) and conventional tubes (CT) were seeded with rhabdomyosarcoma (RD) and MRC-5 cells and inoculated with clinical specimens, and the systems were evaluated for the rapid diagnosis of herpes simplex virus (HSV) infections by detection of cytopathic effects (CPE) (for CT, for 7 days) and by using fluoresceinated monoclonal antibodies (for SV, 16 h postinoculation). Of 245 genital specimens (16 from males and 229 from females) 56 (23%) seeded with MRC-5 cells (14 type 1 and 42 type 2) and 55 (22%) seeded with RD cells were detected in CT; however, CPE were recognized in only 26 (46%) of the total HSV-positive cultures 1 day postinoculation. Forty-eight (86% sensitivity, MRC-5) and 46 (84% sensitivity, RD) HSV strains were detected immunologically in SV 16 h postinoculation. Early CPE in CT or fluorescent foci in SV were easier to detect in MRC-5 than in RD cell cultures. MRC-5 and RD cells were equally sensitive to infection with HSV. CT cell cultures were more sensitive than SV but less rapid for the detection of HSV infection (P less than 0.01). We recommend using SV for the rapid diagnosis of HSV infections, but in addition, CT must be inoculated with MRC-5 or RD to ensure maximum detection of this virus.     

Rapid detection of varicella-zoster virus in clinical specimens using monoclonal antibodies on shell vials and smears

Monoclonal antibodies were used for detecting varicella-zoster virus (VZV) by immunofluorescence (IF) in clinical specimens inoculated on shell vial cultures. Vesicles of 74 patients with varicella or herpes zoster-like eruptions were tested by this method and by conventional cell culture. Further diagnostic tests were direct IF on smears (42 patients), the cytological Tzanck test (28 patients), and serology (30 patients). Both IF assays were performed with the commercial VZV-specific monoclonal antibody 3B3 (Ortho). Using the shell vial technique, we found VZV in 37 (50%) of the patients, on average after 3 days. The conventional culture method yielded 26 positives (35%), on average after 7.5 days. Twenty-nine of the shell vial IF-positive patients were also positive by direct IF on smears (30 tested), while 15 gave positive Tzanck smears (18 tested). The sensitivities of the shell vial IF test, the direct IF test, the conventional culture method, and the Tzanck test were about 95%, 97%, 70%, and 79%, respectively. For the specificities, we found at least 97% for the shell vial IF test, at least 91% for the direct IF test, and about 78% for the Tzanck test. We conclude that both VZV IF tests are much more reliable than the conventional cell culture method and the Tzanck test for the laboratory diagnosis of VZV infections.     

Effect of centrifuging shell vials at 3,500 x g on detection of viruses in clinical specimens.

An increase in shell vial centrifugation force to 3,500 x g and a concomitant reduction in spin time to 15 min did not decrease the sensitivity of detecting viruses in clinical specimens compared with the accepted practice of using 700 x g for 40 min. No damage to the cell monolayer (ML) at the higher g force was observed. Toxicity to the ML is decreased with the shorter spin, probably because of reduced time of contact between the specimen and the ML.     

Evaluation of four methods for rapid detection of adenovirus

Four methods for rapid detection of adenovirus were evaluated by testing retrospectively 28 frozen clinical specimens from which an adenovirus strain had been isolated. After thawing all specimens were retested for the presence of adenovirus by conventional culture on KB cells and found to be positive. The four tests used for rapid detection of adenovirus were a 48-hour culture technique, and an immunoassay, a latex agglutination test and an immunofluorescence assay for direct detection of viral antigen using commercially available reagents. Of the 28 specimens all were positive in the 48-hour culture, 25 (89 %) positive in the immunoassay and 10 (36 %) positive in the latex agglutination test. Six of eight nasopharyngeal aspirate specimens were positive in the immunofluorescence assay. Twenty-five clinical specimens negative for adenovirus on conventional culture were also negative in the 48-hour culture technique. Overall, the rapid (48-hour) culture technique was 100 % sensitive and 100 % specific compared to conventional culture. The direct detection of viral antigen by immunoassay was less sensitive, however results were available within a few hours. Prospective comparative studies are warranted to determine whether these rapid techniques could replace conventional culture in the routine diagnosis of adenovirus infection.     

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.     

Modification of shell vial centrifugation method for detection of herpes simplex virus.

The shell vial centrifugation method for the detection of herpes simplex virus was modified with the addition of maintenance medium before specimen inoculation rather than after. Sensitivity and specificity values were comparable to those obtained with postcentrifugation medium addition.     

Polymerase chain reaction for rapid detection of ocular adenovirus infection

Adenoviruses are associated with endemic and epidemic acute conjunctivitis, large nosocomial outbreaks reflecting virus transmission on unwashed hands or inadequately sterilised ophthalmic instruments. The polymerase chain reaction (PCR) proved more sensitive than antigen detection by immune dot-blot test for the rapid diagnosis of ocular adenovirus infection (sensitivities in a retrospective study 112/123 (91%) versus 72/123 (59%), P< 0.001). Indeed, in a prospective comparison, DNA amplification and virus isolation generated similar numbers of positive results (34 versus 32), though five PCR positive results were possibly false positives. The sensitivity of the PCR was largely independent of adenovirus subgenus or serotype, though reduced sensitivity with subgenus B strains could not be excluded. Specimen preparation for DNA amplification using a simple lysis buffer proved more effective than phenol-chloroform extraction. The immune dot-blot test gave unavoidable false positive results, but with the PCR this problem could be minimized by technical modifications. The PCR could replace antigen detection and virus isolation as the initial test for adenoviruses in conjunctival swabs, with cell culture only being retained for adenovirus serotyping in PCR positive specimens and for other viruses such as herpes simplex. © 1995 Wiley-Liss, Inc.     

一种快速检测人腺病毒的免疫层析分析方法的建立

目的:建立一种基于免疫层析法(Immunochromatographic assay, ICA)的快速简便的人腺病毒(Human adenoviruses,HAdVs)检测方法。方法:利用抗原结合和病毒斑点实验检测发现单克隆抗体3C11和7E6能特异性结合所有测试的人腺病毒,以这两个抗体建立ICA方法。使用培养病毒及1...     

Comparison of MRC-5 and U-373MG astrocytoma cells for detection of cytomegalovirus in shell vial centrifugation cultures

U-373MG astrocytoma cells are susceptible to human cytomegalovirus (CMV) infection and offer the advantage of a continuous cell line for clinical laboratory use. U-373MG to MRC-5 cells for detection of CMV by centrifugation culture were therefore compared. At 20 h, 10 (6.1 %) versus 12 (7.4 %) of 163 clinical specimens were positive for CMV, and at 40 h, 12 (7.4 %) versus 17 (10.4 %) were positive in U-373MG and MRC-5 cells, respectively. Substantial toxicity was found in U-373MG cells (84 %) when inoculated with blood specimens. For detection of CMV in centrifugation culture, MRC-5 cells are superior due both to higher sensitivity and lesser toxicity.     

Evaluation of R-Mix shell vials for the diagnosis of viral respiratory tract infections.

Respiratory viruses cause significant morbidity and mortality. The management of these infections can be improved by a rapid diagnosis and administration of available virus-specific therapy. The goal of this study was to compare R-Mix, an engineered tissue monolayer for rapid shell vial (SV) diagnosis of viral respiratory infections, with conventional tissue culture (TC) and conventional respiratory SV (primary rhesus monkey kidney (RhMK) and Hep2 monolayers). The primary outcome measure was sensitivity for detection of influenza A and B, respiratory syncytial virus, parainfluenza 1-3, and adenovirus. The study was performed in two phases: (1) the three methods were compared using 250 nasal washes from children with lower respiratory tract infections; (2) a modified R-Mix SV harvesting schedule (SV were harvested at 24 and 120 h) was compared with TC and conventional RhMK/Hep2 SV using 311 respiratory specimens. A total of 110 viruses were identified in the first and 55 in the second phase. Diagnostic accuracies of R-Mix harvested at 24, 48, and 120 h were 98%, whereas for TC varied between 99 and 100%, and for RhMK/Hep2 SV between 98 and 99%. Sensitivities of R-Mix harvested at 24, 48, and 120 h were 26, 75, and 47%, respectively, whereas for TC varied between 60 and 94%, and for RhMK/Hep2 SV between 62 and 85%. R-Mix harvested at 48 h represent a valuable substitute for RhMK/Hep2 SV because they have comparable sensitivities and diagnostic accuracies, but R-Mix offers several technical advantages. In contrast, R-Mix harvested at 24h did not seem a very useful diagnostic tool. The utility of R-Mix harvested at 120 h, which accelerated the diagnosis of 16% of positive specimens in study phase 2, needs further investigation.     

Successful use of shell vial centrifugation and 16 to 18-hour immunofluorescent staining for the detection of influenza A and B in clinical specimens

The rapid diagnosis of influenza A and B infections is beneficial for the proper management of patients with acute respiratory illness. The authors evaluated a shell vial centrifugation method to detect these viruses 16-18 hours postinoculation and compared it with conventional tube cell culture. Rhesus monkey kidney cells were used in both methods. Conventional culture of 334 respiratory specimens recovered 64 influenza isolates; the average time to positivity was 4.1 days. Low-speed shell vial centrifugation with polyclonal immunofluorescent staining 16-18 hours postinoculation was performed on 96 fresh specimens and on an additional 38 frozen specimens. These 134 specimens contained 49 of the 64 total influenza-positive specimens. The shell vial method yielded a sensitivity of 90.9% and 87.5% for fresh and frozen specimens, respectively, as compared with conventional tube cell culture. The authors conclude that the shell vial method is an important adjunct to conventional culture for the rapid detection of influenza A and B in clinical specimens.     

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.     

Isolation of seven respiratory viruses in shell vials: a practical and highly sensitive method.

The isolation of respiratory viruses in shell vials was compared with isolation in tube cultures in order to determine the sensitivity of the former, rapid method. Twenty of 21 influenza virus and 15 of 15 parainfluenza virus isolates were recovered in shell vials. One hundred twenty-seven of 138 respiratory syncytial virus isolates were detected in shell vials, but only 10 of 21 adenovirus isolates were positive by the rapid method. Shell vials are very effective for the diagnosis of respiratory viral infections, except for those caused by adenovirus.