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Journal of Clinical Microbiology, August 2001, p. 2984-2986, Vol. 39, No. 8
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.2984-2986.2001
Simplified Microneutralization Test for Serotyping
Adenovirus Isolates
Marietta D.
Malasig,1,*
Pulak R.
Goswami,1,
Leta K.
Crawford-Miksza,2
David P.
Schnurr,2 and
Gregory
C.
Gray1
Department of Defense Center for Deployment
Health Research, Naval Health Research Center, San
Diego,1 and Viral and Rickettsial
Disease Laboratory, California Department of Health Services,
Richmond,2 California
Received 18 December 2000/Returned for modification 7 March
2001/Accepted 7 May 2001
 |
ABSTRACT |
A simplified microneutralization procedure is described that
uses an empirically determined virus challenge dose, a single dilution
of antiserum, and observation of cytopathic effect to determine the
adenovirus serotype. The simplified test has faster turnaround time and
was 96% concordant with a confirmatory test using serial dilutions of
type-specific sera. This method will find utility in high-volume
serotyping work.
| |
TEXT |
Adenovirus (Ad) type 4 and 7 vaccines effectively controlled Ad infections in a military population,
but the virus reemerged as a major cause of acute respiratory
disease when vaccine production was stopped (1, 4, 5).
This prompted the Department of Defense to establish surveillance for
Ad infections among trainees to determine the serotype distribution and
to assess the effect of vaccine loss. Because of large numbers of Ads
isolated, an efficient and rapid method for serotyping isolates was devised.
Ad serotype classification by neutralization with type-specific
antisera in a microplate format has been previously described (2,
3, 6). This technique requires prior titration of viral isolates
to determine the virus challenge dose and takes 3 to 7 days to
complete. In this report, we describe a simplified microneutralization
test using an empirically determined virus dilution and a single
antiserum concentration. This research has been conducted in compliance
with all applicable federal regulations governing the protection of
human subjects in research.
A549 cells (American Type Culture Collection, Manassas, Va.) were
maintained by monthly subculturing at a 1:4 split ratio, using Eagle's
minimum essential medium in Earle's balanced salt solution, fetal
bovine serum (10%), antibiotics, and amino acids (Biowhittaker,
Walkersville, Md.). Prototype Ads were purchased from the American Type
Culture Collection and subpassaged in A-549 cells. Type-specific rabbit
immune sera to Ad types 1 to 5, 7, and 21 (Viral and Rickettsial
Disease Laboratory [VRDL], California Department of Health Services,
Berkeley, Calif.) were standardized to contain 20 antibody units per 10 µl. Each antibody unit neutralizes 100 50% tissue culture infective
doses (TCID50) of the prototype virus. The
serotypes used in the test were those most frequently associated with
respiratory disease (7).
Ad surveillance was conducted from October 1996 to June 1998 (5) among symptomatic military recruits from four training camps in the United States. Throat specimens were inoculated into A-549
cells. When 75% cytopathic effect (CPE) was observed, the cells were
scraped and resuspended in culture medium, spotted, and stained with
Ad-specific fluorescence-labeled monoclonal antibody (Chemicon
International, Temecula, Calif.).
Simplified microneutralization.
An assay medium consisting of
Eagle's minimum essential medium in Earle's balanced salt solution,
fetal bovine serum (10%), NaHCO3 (0.09 g),
L-glutamine (2 mM), penicillin (200 U/ml), streptomycin (200 µg/ml), and Fungizone (1.0 µg/ml) was used in the test. Single dilutions (10 µl) of type-specific hyperimmune rabbit sera were loaded in each well and diluted with 40 µl of assay medium. Isolates were vortexed, diluted 1:10, and loaded into designated wells at 50 µl per well. The infectivities of the isolates were assayed in a
logarithmic serial dilution using 50 µl of the isolate at 1:10
dilution. The plates were incubated for 1 h at 37°C in 5% CO2, and then 5,000 A549 cells were added to each
well. The plates were further incubated for 7 days and observed after
72 h and on days 5 and 7. The serotypes were determined by
inhibition of CPE in wells containing type-specific antiserum. Isolates
not neutralized by any antiserum were retested at the dilution where 75% CPE was first observed in the infectivity assay. Isolates that did
not type after repeat tests were considered nontypeable.
Validation.
Blinded to the serotype results, VRDL validated
105 randomly selected isolates using the colorimetric
microneutralization test described elsewhere (2). Serial
2-fold dilutions of the type-specific hyperimmune sera described above
were used. The criterion for confirmation of a serotype was for the
isolate to be neutralized by a dilution of the type-specific serum
within 16-fold of the titer of the same serum required to neutralize the prototype virus. The type identification was considered
confirmed when the titer of the isolate was at least 32 TCID50 and the neutralization titer observed was
within 16-fold of the titer observed when the same antiserum was tested
against the homologous prototype virus.
One thousand eight hundred fourteen (53.1%) of the total 3,413 throat swabs collected were positive for Ad. Serotyping was attempted
on 1,808 isolates, as 66 isolates were excluded due to possible
contamination with the positive control used during culture.
The simplified typing method took approximately 3 to 7 days per test
and successfully typed 1,567 (90%) of 1,742 isolates studied on the
first attempt (Fig. 1). One hundred
seventy-five isolates required additional attention (Fig. 1): 141 required retesting due to all serotype wells showing CPE
(n = 118), two wells showing CPE (n = 13), or no wells showing CPE (n = 10); 18 had
discrepant results after they were inadvertently tested twice; and 16 were contaminated with other organisms. Of the 118 isolates that were
CPE positive with all antisera used, 31 typed after retesting using the
same virus dilution, 72 typed after retesting at a dilution where 75%
CPE was first observed in back titration, and 9 failed to type after
retesting (Fig. 1). The remaining six isolates were not retested.
All 13 isolates exhibiting two serotypes were successfully typed upon
retesting. The 10 isolates that were CPE negative with all antisera
used were not retested. Of the samples tested, 1,697 (97.4%) were
successfully typed. Overall, 4 (0.2%) of the isolates were Ad type 1, 32 (1.9%) were type 2, 142 (8.4%) were type 3, 974 (57.4%) were type
4, 407 (24.0%) were type 7, and 138 (8.1%) were type 21.
Validation results revealed agreement of the simplified
microneutralization test with the standard test for 99 of 105 selected isolates. Two type 2 isolates could not be validated because of coinfection with another virus. Upon retest by the simplified procedure, two of the four discrepant isolates were in agreement with
the standard (Table 1). The two remaining
discrepant isolates were retested by VRDL, and serotype agreement was
established. After these adjustments, a 98% concordance was observed
between the results of the simplified microneutralization test and
those of the standard test.
View this table:
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TABLE 1.
Samples with discrepant results by the simplified
microneutralization test compared to the confirmatory test
|
|
The highly concentrated antiserum used in the simplified procedure was
the key factor in the successful performance of the test. Each
antiserum was standardized to contain 20 antibody units that neutralize
100 TCID50 of the prototype at a 20-fold
dilution, making it possible to use an approximate test dilution,
thereby cutting the assay time from 14 to 7 days.
Our results indicate that the simplified microneutralization test works
well with rapidly growing, relatively high-Ad-titer viral isolates but
may not perform as well for serotypes which fail to produce high-titer
virus in vitro. However, the same performance may be achieved for
low-titer isolates if a higher viral challenge dose is used.
The performance characteristics of the simplified test make it useful
for serotyping large numbers of isolates in laboratory-based epidemiological surveillance, outbreak investigations, and infection control in clinical settings where nosocomial infection is suspected. Its rapid turnaround time leads to cost savings, making it a valuable tool in these settings.
| |
ACKNOWLEDGMENTS |
We gratefully thank the members of the Adenovirus Surveillance
Group, Colleen McDonough, Cassandra Morn, Pamela Poblete, Debbie Kamens, Jason Unruh, Anthony Hawksworth, Heather Taylor, Rosana Magpantay, and Tuan Pham, for their assistance in this project.
This is report no. 00-45 supported by the DoD Global Emerging
Infections Systems.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: DoD Center for
Deployment Health Research, Naval Health Research Center, P.O. Box
85122, San Diego, CA 92186. Phone: (619) 553-8771. Fax: (619) 553-8449. E-mail: malasig{at}nhrc.navy.mil.
Present address: Amsterdam Medical Center, University of Amsterdam,
The Netherlands.
| |
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Journal of Clinical Microbiology, August 2001, p. 2984-2986, Vol. 39, No. 8
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.2984-2986.2001
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