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Journal of Clinical Microbiology, September 1998, p. 2714-2717, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Comparative Evaluation of Colorimetric Microtiter Plate Systems
for Detection of Herpes Simplex Virus in Cerebrospinal Fluid
Yi-Wei
Tang,
Paul N.
Rys,
Barbara J.
Rutledge,
P.
Shawn
Mitchell,
Thomas F.
Smith, and
David H.
Persing*
Division of Clinical Microbiology, Department
of Laboratory Medicine and Pathology, Mayo Clinic, Rochester,
Minnesota 55905
Received 10 March 1998/Returned for modification 13 April
1998/Accepted 3 June 1998
 |
ABSTRACT |
In the past few years, application of the PCR to the detection of
herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) from
patients with encephalitis and meningitis has become standard
laboratory practice. However, from an operational perspective, the true
diagnostic value of PCR in this setting is yet to be realized because
most laboratories subject the amplification products to lengthy probe
hybridization procedures by Southern blotting. As alternatives to
Southern blotting, we evaluated colorimetric microtiter plate (MTP)
systems from ViroMed Laboratories, Inc. (PrimeCapture), CPG, Inc.
(Quanti-PATH), and Incstar Corp. (GEN-ETI-K), in addition to a
system developed at the Mayo Clinic with the PCR ELISA system
(Boehringer Mannheim Corp.). We tested PCR products from 86 clinical CSF specimens submitted to our Molecular Microbiology Laboratory. The CSF specimens used had to have sufficient volume for
comparative analysis. By conventional Southern blotting methods, 54 were positive and 32 were negative for HSV DNA. Compared with Southern
blotting, the sensitivity and specificity were 63.0 and 100.0%,
respectively, for the PrimeCapture system, 98.2 and 96.9%, respectively, for the Quanti-PATH system, 98.2 and 100.0%,
respectively, for the GEN-ETI-K system, and 100.0 and 96.9%,
respectively, for the Mayo system. All four MTP systems had turnaround
times 12 to 24 h less than that for Southern blotting. There were
no significant differences in costs or technologist time
between the Mayo system and Southern blotting. Other features of the
Mayo system include type-specific genotypic identification of
HSV and the potential for determination of drug resistance by DNA
sequencing. Overall, we found that colorimetric MTP systems were likely
to improve test turnaround times and patient care at no additional
cost.
| |
INTRODUCTION |
Herpes simplex virus (HSV) is a
ubiquitous agent responsible for a wide variety of human infections. In
addition to epithelial infections such as gingivostomatitis,
pharyngitis, genital herpes, whitlow, conjunctivitis, and keratitis,
HSV is an important cause of central nervous system (CNS) infections
and accounts for 2 to 19% of human encephalitis cases (9, 33,
38). The clinical spectrum of CNS diseases has recently been
expanded; for example, most cases of benign recurrent aseptic
meningitis (Mollaret's meningitis) are caused by HSV (39),
especially HSV type 2 (HSV-2) (36). Because specific
antiviral therapy is available, the rapid, definitive laboratory
diagnosis of HSV is important to support clinical findings. Moreover,
in the setting of possible HSV encephalitis, patients are often managed
as inpatients while awaiting test results.
Although cell culture is considered the standard method for laboratory
diagnosis of ulcerative HSV infections, HSV is rarely recovered in
cell cultures inoculated with cerebrospinal fluid (CSF). Brain biopsy
specimens may yield culturable virus, but this invasive surgical
procedure is controversial when performed solely to collect specimens
for the laboratory diagnosis of infectious disease. The sensitivities
of HSV antigen and antibody assays for CNS infections are very low
(13). In addition, antibodies may appear in the CSF as a
consequence of the breakdown in the blood-brain barrier, leading to
false-positive results (23). Since 1990, several studies
have shown that PCR detection of HSV DNA in CSF should be considered
the new standard for the laboratory diagnosis of CNS disease caused by
this virus (8, 11, 12, 15, 22, 24, 28-30).
Although the technology underlying PCR is relatively rapid, the PCR
product (amplicon) must be identified definitively as the sequence of
interest to provide adequate diagnostic specificity. The conventional
technique for this is hybridization of a specific probe to a Southern
blot, which increases both the sensitivity and the specificity of
the test. This step, however, takes an additional 12 to 24 h to
complete, delaying the use of test results for clinical
intervention. The ideal postamplification detection system would
combine the increased sensitivity and specificity of Southern
blotting with a rapid turnaround time (2, 14, 19, 20, 32).
For this purpose, enzyme-linked adsorbent microtiter plate (MTP)
systems have been adapted for amplicon detection (40).
We report a comparison of four colorimetric MTP systems: the
PrimeCapture system from ViroMed Laboratories, Inc., the Quanti-PATH system from CPG, Inc., the GEN-ETI-K system from Incstar
Corp., and a system developed at the Mayo Clinic by PCR ELISA
(Boehringer Mannheim Corp.). We tested all four systems
against standard Southern blotting for the detection of HSV PCR
products resulting from clinical CSF specimens.
(This study was presented in part at the 14th Annual Meeting of the Pan
American Society of Clinical Virology, 26 to 29 April 1998, Clearwater
Beach, Fla.)
| |
MATERIALS AND METHODS |
Specimens.
Eighty-six CSF specimens submitted to the
Molecular Microbiology Laboratory at the Mayo Clinic for the diagnosis
of HSV CNS disease by PCR were selected retrospectively for the study.
Colorimetric MTP systems.
We evaluated four colorimetric MTP
systems. Three were commercial kits specifically designed for the
detection of HSV DNA. In one type of format (PrimeCapture [lot
9705309116; ViroMed Laboratories, Inc., Minneapolis, Minn.];
Quanti-PATH [lot 116; CPG, Inc., Lincoln Park, N.J.]),
amplification of the HSV target was performed with the biotinylated
primers included in the kit. The amplified product was denatured and
hybridized to the MTP wells, which were precoated with a
sequence-specific capture probe. Unbound amplicons were removed by
washing, and a streptavidin-enzyme conjugate was added followed by
colorimetric detection (17, 20, 37). A second format
(GEN-ETI-K [lot 98B04; Incstar Corp., Stillwater, Minn.]) also had
HSV-specific probes bound to the MTP wells. In this system, however, the target (also amplified with primers included in the kit)
was not biotinylated. Instead, an enzyme-linked antibody recognized
target DNA which has hybridized to the specific probes on the MTP well
surface (21). In another format, developed by Boehringer
Mannheim Corp. (the PCR ELISA kit), digoxigenin-dUTP was incorporated
with the PCR product. A sequence-specific, biotinylated capture probe
was hybridized to the denatured amplicon, and the complexes were
captured in avidin-coated MTP wells. Detection was completed with
enzyme-linked anti-digoxigenin antibodies (10, 16, 27). We
developed the Mayo system with the PCR ELISA kit. Type-specific
5'-biotinylated capture probes for HSV-1 (TK-G, 5'-ACAAACATCGTGTTGGGGGC-3') and HSV-2 (TK-H,
5'-ACGAACCTGGTCCTGGGTGT-3') were designed to differentiate
HSV genotypes (7, 34).
Extraction of nucleic acid.
Nucleic acid from 200 µl of
CSF was extracted with IsoQuick (Orca Research, Inc., Bothell, Wash.)
according to the manufacturer's instructions. Extracted DNAs were
resuspended in 50 µl of water. Five microliters of each specimen
extract was used for PCR amplification.
PCR amplification.
When MTP systems from ViroMed
Laboratories, Inc., CPG, Inc., Incstar Corp., or Boehringer Mannheim
Corp. were used, the manufacturers' recommendations were followed. For
the Mayo-developed system, the reaction mixtures were placed in a
Perkin-Elmer 9600 thermal cycler programmed for a three-step PCR
procedure by a previously described protocol (5, 22,
35). The primer set used at Mayo (TK-A,
[5'-GACMAGCGCCCAGATAACAA-3'] and TK-B
[5'-MCAGCATRGCCAGGTCAAGC-3']) amplified a 335-bp portion of the
thymidine kinase (TK) gene (22).
Amplicon identification.
All specimens were amplified by PCR
followed by gel electrophoresis, conventional Southern blotting, and
probe hybridization as described previously (5, 22, 35).
Amplicon identification with MTP systems from ViroMed Laboratories,
Inc., CPG, Inc., and Incstar Corp. was performed according to the
manufacturers' instructions.
The Mayo system was developed with Boehringer Mannheim's PCR ELISA kit
for the detection of digoxigenin-labeled PCR products. Aliquots of the
denatured amplicon were mixed with each of two hybridization solutions,
one of which contained a 5' biotin-labeled DNA capture probe (TK-G)
specific for HSV-1 and the other of which contained a probe (TK-H)
specific for HSV-2. If the corresponding target DNA sequence was
present, the probes hybridized and the resulting biotinylated DNA
complexes were captured on streptavidin-coated microtiter plate wells.
HSV-specific DNA complexes were detected with an
anti-digoxigenin-peroxidase conjugate, which recognized digoxigenin-11-dUTP substitutions incorporated into the amplicon during
PCR. The peroxidase substrate was then added for color development. A
positive result was defined as an optical density (OD) at 405 nm
(OD405) 
OD490 value greater than or equal to 0.1. The HSV genotype was determined by which probe gave positive results. A uracil-N-glycosylase-based inactivation system
was adapted to control for possible amplicon carryover contamination (5, 18).
Turnaround time and cost assessment.
The turnaround time for
amplicon identification was calculated for each protocol. Direct cost
was determined on an annualized basis and included the costs for test
kits, materials, reagents, and equipment and laboratory personnel
salaries. We added 8% to each direct cost to cover utilities. Variable
allied health effort (hands-on time) was calculated per specimen. Fixed
effort (specimen processing, buffer preparation, maintenance, bench
cleaning, and data entry) was calculated on a per-day basis. Each
procedure was outlined on a flowchart and timed by laboratory personnel in a manner consistent with other cost analyses performed routinely in
the Department of Laboratory Medicine and Pathology at the Mayo Clinic.
Statistics.
Comparisons of sensitivities (based on rate
ratios) were performed with Epiinfo software (version 6; Centers for
Disease Control and Prevention, Atlanta, Ga.).
| |
RESULTS |
Performance evaluation.
Of 86 CSF specimens tested, 54 were
HSV DNA positive and 32 were HSV DNA negative, as determined by
detection of an amplification product in ethidium bromide-stained
agarose gels or by film exposure after Southern blotting with probe
hybridization. This technique is referred to as the "conventional
method" in the present study. All four MTP systems had high
specificities (
96.9%) compared to the results of conventional
methods (Table 1). The MTP systems from
CPG, Inc., Incstar Corp., and Mayo also had excellent sensitivities (98.2%) compared with the sensitivity of the system from ViroMed Laboratories, Inc., which was significantly lower (63.0%; 95% confidence interval, 51 to 77%).
With MTP assays, the difference in OD between positive and negative
controls is the objective basis for interpreting colorimetric
data. All
four MTP systems had mean ratios of the OD for a specimen
versus the
average OD for a negative control (S/N ratios) of >10
for specimens
positive by conventional methods and mean S/N ratios
of
1 for
specimens negative by conventional methods (Fig.
1).
Thus, the mean cutoff values which
distinguished positive results
from negative results were well
separated for all four systems
evaluated.
View larger version (12K):
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|
FIG. 1.
S/N ratios determined with four MTP systems. HSV DNA
positivity or negativity was determined by Southern blotting followed
by probe hybridization. The large dots represent 10 specimens, and the
small dots represent 1 specimen. A short bar stands for the cutoff
value for each test.
|
|
The Mayo system was able to differentiate HSV genotypes by using
type-specific capture probes. Among 55 CSF specimens which
yielded
positive tests with the Mayo system, 20 (36.4%) were infected
with
HSV-1, 32 (58.2%) were infected with HSV-2, and 3 (5.5%)
were
infected with both genotypes of the virus. Of the 54 CSF
specimens
identified as positive both with the Mayo system and
by the
conventional method, the MTP system from ViroMed Laboratories,
Inc.,
was positive for 13 (65.0%) of 20 HSV-1-positive specimens,
20 (64.5%) of 31 HSV-2-positive specimens, and 1 (33.3%) of 3
HSV-1- and
HSV-2-positive specimens. Hence, the prevalence of
false-negative
results for the system from ViroMed Laboratories,
Inc., was not
associated with a specific genotype.
Turnaround time estimation and cost analysis.
Southern
blotting followed by probe hybridization required a minimum of 15 h for amplicon identification in our laboratory, including sample
loading and running of the gel (2 h), denaturation and neutralization
(1 h), blotting (6 h), prehybridization (30 min), hybridization (4 h),
two washings (40 and 10 min), and film exposure and development (30 min). All four MTP systems needed a test time of <4 h for completion
of the identification of the HSV amplicon (Table
2). The cost and technologist time
including specimen processing and amplification steps for the
Mayo-developed MTP system were compared with those for conventional
methods on the basis of an estimated laboratory volume of 3,040 procedures in 1996. Costs and technologist times were similar for the
Mayo assay and the conventional methods (Table
3).
| |
DISCUSSION |
The generation of rapid laboratory test results has had a major
impact on the clinical management of patients with CNS infections. This
is especially true for HSV infections since early treatment with
acyclovir is very effective, reducing the rate of mortality from HSV
encephalitis from 70% to 19 to 30% (33, 38). In the present study, we used Southern blotting followed by probe
hybridization as our evaluation standard, which has a demonstrated
sensitivity of 97.7 to 98.1% compared to the detection of HSV antigens
or recovery of the virus from brain biopsy specimens (1,
15). PCR performed with CSF has a reduced turnaround time
and a reduced level of patient risk for the diagnosis of HSV infection
of the CNS compared to those for cell culture technology; however, 2 days is usually required to complete conventional Southern blotting and
probe hybridization in our laboratory.
In contrast, the turnaround time for colorimetric MTP systems was about
10 h, including <4 h for amplicon identification. Providing
that CSF specimens were processed in the morning, test results could be
available on the same day as specimen submission. If test results
were positive, unnecessary antibacterial treatment could be eliminated
and appropriate antiviral therapy could be provided. In addition,
negative PCR results for HSV DNA in some cases provide sufficient
evidence to mitigate against unnecessary intravenous acyclovir
treatment ($180.00/day) and would expand the diagnostic consideration
for other etiologic causes of CNS infection.
Colorimetric MTP systems and Southern blotting use a sequence-specific
probe that provides two technical enhancements to PCR (10, 20,
21). First, the sensitivity for the detection of PCR products
with MTP systems is 10- to 100-fold greater than that with ethidium
bromide-stained agarose gels. Second, the use of a specific probe
confirms the specificity of the PCR product. Unlike Southern blotting
and probe hybridization, use of the MTP allows the simultaneous rapid
analysis of multiple PCR mixtures. Importantly, the assay is
potentially automatable when performed in a 96-well format. Finally,
the physical containment of the amplification products in the MTP is
superior to that by Southern blotting-based methods and thus may be
less prone to contamination problems.
Additional advantages of the Mayo-developed system include
identification of virus genotypes and the recognition of polymorphisms that may be responsible for drug resistance. The TK gene of HSV has
been targeted by the Mayo MTP system. Because significant heterogeneity
between HSV-1 and HSV-2 exists in the 335-bp amplicon, allele-specific
probes can be used for genotype determination. Interestingly, our
results have indicated that some HSV-amplified DNAs react
with both type-specific probes, suggesting mixed infections with
the two genotypes. Point mutations in the TK gene may also be
responsible for acyclovir resistance (3, 4, 25), and polymorphisms in this locus may be associated with neurotropism (31). Determination of acyclovir resistance or neurotropism by detection of these point mutations may be important for patients undergoing long-term therapy and immunocompromised hosts (6, 26). A single test that includes amplification of the TK gene followed by determination of sequence polymorphisms may be able to
predict the HSV genotype and possibly drug resistance and neurotropism without the need for a second amplification reaction. Therefore, by
using separate MTP wells for HSV-1- and HSV-2-specific hybridizations, the viral type can be identified immediately. Acyclovir resistance could be determined by direct sequencing of the amplicon based on
clinical management.
Our data indicate that one need not sacrifice sensitivity to obtain
rapid results. Three of the four MTP systems tested had sensitivities
of >98% compared with the results of Southern blotting followed by
probe hybridization. The Mayo system had the best sensitivity (100%)
of the four systems tested. We noted that among 36 CSF specimens
determined to be HSV DNA negative by conventional methods, one was
positive by MTP analysis with the systems from both CPG, Inc., and
Mayo. This result was obtained for a patient who was clinically
diagnosed with aseptic meningitis on the basis of a headache, increased
leukocyte counts, and elevated total protein levels in her CSF,
suggesting a likely false-negative result by the conventional method.
Colorimetric MTP analysis for HSV PCR amplicon identification can be
performed in less than 4 h. These assays do not require toxic
chemical agents or an electrophoretic apparatus. Substitution of MTP
for Southern blot analysis is cost neutral, with no compromise in test
sensitivity. MTP systems may be adapted for automation, which is a
compelling requirement for PCR testing, which, in many laboratories, is
expanding at a rapid rate. Collectively, these factors make the
implementation of this technology in routine diagnostic testing a
fundamental goal in our laboratory.
| |
ACKNOWLEDGMENTS |
We thank Jill Thorvilson, T. J. Berg, Carl Greiner, Heather
Skarhus, Paul Heimgartner, Ann Wimmer, and David Majewski for technical
assistance and Jonathan Hibbs for critically reviewing the manuscript.
| |
ADDENDUM IN PROOF |
The data generated with the PrimeCapture HSV DNA detection system
were derived using a system lot number recalled by the manufacturer. The data may not accurately reflect the current performance of the
assay system.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Laboratory Medicine and Pathology, Hilton 470, Mayo Clinic, 200 First St., S.W., Rochester MN 55905. Phone: (507) 284-2876. Fax: (507) 284-4272. E-mail: persing.david{at}mayo.edu.
| |
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Journal of Clinical Microbiology, September 1998, p. 2714-2717, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
