Curt A. Gleaves

Evaluation of an enzyme immunoassay for the detection of herpes simplex virus (HSV) antigen from clinical specimens in viral transport media

A total of 301 clinical specimens (229 culture positive and 72 culture negative) were assayed retrospectively by an enzyme immunoassay. Specimens were transported to the virus lab in viral transport media (VTM), and were inoculated into HF and A549 cell culture tubes for viral isolation with the remainder of the sample being saved at -70 degrees C. Specimens were thawed, vortexed and resuspended in 10 x Herptran concentrate and each sample was then added in duplicate to designated wells of a microtiter plate for the EIA assay. The EIA detected 147/150 (98%) culture positive specimens from symptomatic patients, 63/79 (79.7%) culture positive specimens from patients considered asymptomatic and 210/229 (91.7%) culture positive specimens overall. The EIA was negative for 70/72 (97.2%) culture negative specimens. These data suggest that the EIA test can be used with clinical specimens submitted in conventional VTM. However, VTM samples which are EIA negative, particularly with EIA values close to the EIA positive cutoff value, need to be cultured.

An enzyme immunoassay for the direct detection of adenovirus in clinical specimens

A total of 414 clinical samples were tested by an enzyme immunoassay (EIA) for direct detection of adenovirus as well as cultured in Graham 293, A549, and human diploid foreskin fibroblast cells. Adenovirus was detected in 69 (16.7%) of 414 clinical specimens. The EIA detected adenovirus in 51 (12.3%) of 414 specimens; two were EIA positive only. Adenovirus was isolated from 67 (16.2%) of 414 specimens; 18 were culture positive only. Compared with viral isolation, EIA had a sensitivity of 73.4% and a specificity of 99.4%, with a positive predictive value of 96% and a negative predictive value of 95%.

Use of serum substitutes for the growth of four cell lines commonly used for virus isolation

Four serum extenders (Opti-MEM I, BioRich, Excyte I and Excyte III) and two serum substitutes (Omni Serum and Serum Plus) were evaluated for the reduction or replacement of FBS for the growth and maintenance of four representative cell lines used for virus isolation. MRC-5, human fetal foreskin fibroblast (HF), A549 and Hep-2 cells were seeded into culture flasks containing MEM with 10% FBS or with the serum extenders or substitutes and subcultured into 16 x 125 mm glass tubes and 1-dram shell vials. Only cells cultured with Opti-MEM I and Omni Serum grew consistently in tubes and vials and these reagents were compared to FBS for viral isolation and detection. Laboratory stocks of CMV, HSV, VZV, adenovirus (Ad) and RSV were tested. In HF and MRC-5 cells, CMV was isolated in cells cultured in either Opti-MEM I or Omni Serum before CPE appeared in cultures containing FBS. Ad and VZV CPE were observed first in HF and MRC-5 cells containing Omni Serum. HSV CPE was seen at the same time in HF and MRC-5 cells with all 3 media. HSV and Ad CPE in A549 cells were also seen at the same time in all 3 media. RSV and Ad CPE in Hep-2 cells were observed first in FBS containing media. Both Opti-MEM I and Omni Serum performed well as substitutes for FBS for growth of stock viruses without loss of cell sensitivity.

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.

Serologic confirmation and typing of clinical herpes simplex virus isolates: comparison of two different monoclonal antibody immunofluorescence kits

Abstract Two direct innunofluorescence assays using murine monoclonal antibody reagents (Microtrak ™ , Syva Co.; Pathfinder ™ , Kallestad Laboratories, Inc.), were evaluated for culture confirmation and typing of clinical herpes simplex virus (HSV) isolates. Of 101 HSV isolates initially tested with both sets of reagents, 82 of 101 isolates were confirmed and typed as HSV-I and 19 were typed as HSV-II with the Microtrak ™ reagents. The Pathfinder ™ reagents confirmed and typed 70 of 101 HSV isolates as HSV-I and the same 19 as HSV-II. Twelve isolates could not be confirmed or typed with the Pathfinder ™ reagents. Restriction endonuclease analysis showed these 12 HSV isolates to be HSV-I. Subsequently, a new HSV-I monoclonal antibody was added to the original HSV-I Pathfinder ™ reagent for re-evaluation. The 12 discrepant isolates and 118 new HSV isolates were tested both with the original Microtrak ™ reagents and with the original HSV-II and the new HSV-I Pathfinder ™ reagents. One hundred isolates were confirmed and typed as HSV-I and 30 as HSV-II by both sets of reagents. These evaluations emphasize the continual need for quality control within the clinical laboratory to insure that commercially available reagents indeed perform as expected.

CULTURE CONFIRMATION AND TYPING OF HERPES SIMPLEX VIRUS ISOLATES FROM CLINICAL SPECIMENS WITH NEW MONOCLONAL ANTIBODY TYPING REAGENTS

Abstract FITC-labelled monoclonal antibody typing reagents for herpes simplex virus (HSV) (PathoDx, Diagnostic Products Corp., Los Angeles, California) were compared to another commercially available FITC-labelled monoclonal antibody typing reagent for culture confirmation and typing of HSV isolates from clinical specimens. A total of 344 clinical specimens were evaluated for culture confirmation and typing by the two systems. There were 296 HSV-positive cultures identified by cytopathic effect (CPE). The PathoDx typing reagents identified 143 isolates as HSV-1, and 151 isolates as HSV-2, with two isolates staining with both the type 1 and type 2 reagent. The PathoDx typing reagents had a 100% concordance with the other set of typing reagents.

Detection of herpes simplex virus from clinical specimens by centrifugation enhanced cell culture in MRC-5, primary rabbit kidney and mink lung cells☆

Abstract The relative sensitivity of monoclonal antibody staining in centrifugation culture for detecting herpes simplex virus from clinical specimens was compared in three separate cell lines: MRC-5, primary rabbit kidney (RK) and mink lung (ML). Of 169 specimens tested, 42 (24.8%) specimens were positive for HSV. Of these, 40 specimens were positive in centrifugation culture ( 36 42 in MRC-5; 37 42 in RK; 37 42 in ML) and 41 specimens in standard cell culture ( 37 42 in MRC-5; 38 42 in RK; 38 42 in ML). This study indicates that all three cell lines are equivalent for the detection of HSV in centrifugation culture.