Evaluation of number of shell vial cell cultures per clinical specimen ...

Vol. 26, No. 2

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1988, p. 198-200 0095-1137/88/020198-03$02.00/0

Copyright © 1988, American Society for Microbiology

Evaluation of Number of Sheli Vial Cell Cultures Per Clinical Specimen for Rapid Diagnosis of Cytomegalovirus Infection CARLOS V. PAYA,' ARLO D. WOLD,2 DUANE M. ILSTRUP,3 AND THOMAS F. SMITH2* Department of Internal Medicine and Infectious Diseases,' Section of Clinical Microbiology,2 and Section of Medical Statistics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905 Received 19 June 1987/Accepted 9 November 1987

Specimens sùbmitted for the diagnosis of cytomegalovirus (CMV) infection were inoculated into three (blood) 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 shel! 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 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 sheli 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. or

a

Cytomegalovirus (CMV) infection is a common cause of morbidity and mortality in the immunocompromised host (3). With the use of specific therapeutic agents, the use of rapid diagnostic methods for these infections has become essential for effective patient management (1; C. V. Paya, P. E. Hermans, and R. A. F. Krom, 3rd Eur. Congr. Clin. Microbiol., The Hague, The Netherlands, May 1987, abstr. no. 160). The shell vial assay using monoclonal antibodies against an early antigen of CMV provides diagnostic results within 16 h after inoculation of the specimen and a greater sensitivity compared with conventional tube cell cultures for detecting the virus in clinical specimens (2, 6, 7). We have retrospectively evaluated the sensitivity of the assay according to the specimen source and the number of shell vials inoculated for the maximum detection of CMV. Three shell vials were required for maximal detection of CMV from blood cultures; however, inoculation of two shell vials rather than one improved the positivity rate of CMV to a minor degree (0

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Number shell vials FIG. 1. Increase in positivity in relation to the number of shell vials. (A) Percent cumulative positivity if one, two, or three shell vials are considered for viremia; (B) percent cumulative positivity if one or two shell vials are considered for other tissue or body fluids.

for culture of C. trachomatis. However, of 50 specimens positive in our laboratory for this organism, inclusions were detected in cells on the first two of four cover slips examined in 49 of 50 (98%) specimens. Based on these results, we arbitrarily inoculated two shell vials for the diagnosis of CMV infection with all specimens except from blood. Separated leukocytes from blood specimens were suspended in MEM. One-half of this preparation (1 ml) was inoculated into a conventional tube cell culture; the remaining 1 ml was distributed to three shell vials (0.3 ml per vial). The shell vial inoculum was based on studies that indicated that increasing the specimen volume higher than 0.3 ml led to specimen toxicity without a concomitant increase in the sensitivity of the test (7). Because of the clinical importance of the diagnosis of CMV infections, together with the practical considerations of the cost and timeliness of specimen collection, the laboratory has the responsibility for using diagnostic methodology that yields the maximum results from an individual sample. In this regard, 20 (2%) of the 1,032 total specimens positive could not be examined in one of the shell vials because of deficient cell monolayèrs. Interestingly, only 7 of 83 (8.4%) blood or 1,032 (0.6%) total specimens were toxic to monolayers. Generally, toxicity and microbial contamination of cell monolayers have been more problematic with conventional tube cell culture specimens than with shell

vials. For example, in a previous study, microbial contamination of conventional tube cell cultures occurred in 8 (15%) of 55 specimens that yielded positive CMV results by the shell vial assay but were negative for this virus by examination for specific cytopathic effect (6). Presumably, the higher rate of toxic reaction in tube cell cultures was due to the longer contact times of the specimen material with cells required for incubation relative to the rapid shell vial assay. Quantitative results were not available in our retrospective analysis; however, the number of fluorescent foci present in monolayers of shell vial cultures was generally low (