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Journal of Clinical Microbiology, September 1998, p. 2434-2438, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of Cytomegalovirus in Plasma and Cerebrospinal Fluid
Specimens from Human Immunodeficiency Virus-Infected Patients by
the AMPLICOR CMV Test
Christopher M.
Long,1
Lawrence
Drew,2
Richard
Miner,2
Dragana
Jekic-McMullen,2
Chaka
Impraim,1 and
Shaw-Yi
Kao1,*
Roche Molecular Systems, Inc., Alameda,
California,1 and
Mt. Zion Medical
Center, University of California, San Francisco,
California2
Received 24 March 1998/Returned for modification 27 April
1998/Accepted 8 June 1998
 |
ABSTRACT |
We have developed the AMPLICOR CMV Test, which is rapid and
sensitive for the detection of cytomegalovirus (CMV) in plasma and
cerebrospinal fluid (CSF) specimens. The test incorporated an internal
control in the reaction mixture to monitor the amplification efficiency
and the presence of inhibitors. The AMPLICOR CMV Test was very specific
in detecting 12 clinical CMV isolates and four laboratory CMV strains
tested. Cross-reactivity with 26 non-CMV pathogens was not observed.
The AMPLICOR CMV Test requires only 50 µl of specimen (plasma or CSF)
for processing. The performance of the AMPLICOR CMV Test was compared
to those of the CMV antigenemia assay and the conventional tube culture
method. Among 112 plasma specimens from 43 human immunodeficiency
virus-infected patients, CMV was detected in 20 (18%) of the specimens
by the AMPLICOR CMV Test, 21 (19%) of the specimens by the CMV
antigenemia assay, and 10 (9%) of the specimens by culture. In CSF
specimens from AIDS patients, CMV was detected in 10 of 58 (17%)
specimens tested by the AMPLICOR CMV Test, 5 of 28 (18%) specimens
tested by the antigen assay, and none of the 25 specimens tested by
culture. While the performance of the AMPLICOR CMV Test in this study
was comparable to that of the CMV antigen assay, processing of
specimens by the AMPLICOR CMV Test was much simpler than that by the
antigen assay; in addition, the antigen assay requires greater than
105 leukocytes from blood or 1 ml of CSF to perform the
assay. Our study suggested that the AMPLICOR CMV Test could provide a
rapid and sensitive assay for the detection of CMV in plasma and CSF specimens.
| |
INTRODUCTION |
Cytomegalovirus (CMV) is an
opportunistic pathogen in immunocompromised individuals, such as AIDS
patients and transplant recipients, as well as in neonates. Diseases
that are associated with CMV infections in immunocompromised hosts
include retinitis, gastrointestinal disease, central nervous system
diseases, interstitial pneumonia, and other visceral diseases
(2).
Diagnosis of disseminated CMV infection and active visceral disease
usually involves detection of the virus in peripheral blood leukocytes
(PBLs) by the conventional tube culture method or the shell vial
procedure. Both techniques require maintenance of tissue culture cell
lines, timely processing of specimens, and labor-intensive preparation
of PBLs by dextran sedimentation. CMV disease in AIDS patients and
transplant recipients can be difficult to diagnose because cultures are
often negative, despite the development of visceral syndromes.
Direct detection of CMV antigens in blood provides a more rapid
diagnosis of CMV infection than tissue culture (7, 14). However, it requires timely specimen processing, PBL preparation, and
scanning of slides with a fluorescence microscope, making analysis of
large numbers of specimens difficult. Direct detection of viral nucleic
acid in PBL cells by various methods can be sensitive, but it requires
leukocyte preparations and may detect latent CMV rather than active
infection (5). Direct detection of CMV DNA in plasma or sera
by PCR correlates well with PBL culture positivity and is a sensitive
method for the detection of CMV viremia and systemic CMV disease
(3, 17, 22).
The availability of anti-CMV therapy delays the development of disease
and increases the rate of patient survival (18, 19). In AIDS
patients, a longer survival time is also associated with increasing
incidences of CMV disease in the central nervous system that are
extremely difficult to diagnose by conventional culture of
cerebrospinal fluid (CSF). In autopsy studies, CMV-related neurological
disorders occur in >20% of AIDS patients (16). The
prognosis for patients with CMV-related neurological disorders is poor.
Thus, the availability of diagnostic methods that are more sensitive
than tissue culture for the detection of diseases of the central
nervous system would benefit patient management. The branched DNA
(bDNA) assay, the CMV antigen assay, and PCR-based assays have all been
shown to be more sensitive and rapid than culture in detecting CMV in
CSF (6, 15, 21). However, both CMV antigen and bDNA assays
require a large volume of CSF (1 ml) and a centrifugation step. Such a
large volume of CSF is not always available.
In this report, we describe the development of a simple and rapid
AMPLICOR CMV Test for the detection of CMV DNA in 50-µl plasma and
CSF specimens from human immunodeficiency virus (HIV)-infected patients
and report a comparison of the performance of the AMPLICOR CMV Test
with those of the antigen assay and tissue culture.
| |
MATERIALS AND METHODS |
Patients and specimens.
One hundred twelve plasma specimens
were obtained from 43 HIV-infected patients who were participating in
trials of anti-CMV drugs. Fifty-eight CSF samples were obtained from
HIV-infected patients who were suspected of having CMV or other
neurological diseases.
Viral and bacterial strains.
All viral stocks were obtained
from the American Type Culture Collection unless indicated otherwise.
Additional isolates were provided by R. Whitely (University of
Alabama). Toledo virus was provided by E. S. Mocarski (Stanford
University). Hepatitis C virus was from a clinical specimen that
was provided by S. Tsang at Roche Molecular Systems, Inc. (RMS). HIV
type 1 and type 2 (cloned provirus) were provided by C. Christopherson
at RMS. The Candida albicans DNA, Mycobacterium
sp. DNA, and DNAs from other bacterial strains in this study were
supplied by K. Greisen, K. Young, and D. Leong (all at RMS),
respectively (8). Viral DNA preparation was based on a
detergent and proteinase K digestion method (1). Viral RNA
preparation was based on a guanidine thiocyanate precipitation method
(13). The DNA or RNA concentration at an optical density at
260 nm (OD260) and molecular weight were used to determine
the copy number. If the concentration of nucleic acid was too low, an
endpoint dilution was used to estimate the copy number.
Specimen collection and processing.
Blood specimens were
drawn into tubes containing EDTA (0.1 ml EDTA per 10 ml of blood). CSF
specimens from patients were obtained from various hospitals and were
stored frozen at 
40°C.
Dextran sedimentation procedure for collection of
leukocytes.
A dextran sedimentation procedure was used to collect
PBLs for the direct quantification of CMV DNA. Specimens of whole
blood, which had been held at room temperature for up to 8 h, were
transferred to 15-ml centrifuge tubes. After the addition of 2 ml of
6% dextran (high-molecular-weight dextran [molecular weight,
76,000]; Sigma Chemical Co., St. Louis, Mo.) in phosphate-buffered
saline, the tubes were mixed gently by inversion and were then
incubated without additional mixing at 37°C for 15 to 30 min to allow
the blood to settle. The leukocyte-rich supernatant (top layer) was
collected, with care being taken not to disturb the erythrocyte layer,
and was transferred to a new 15-ml centrifuge tube. Puck's saline without Ca2+ (Gibco BRL, Grand Island, N.Y.) was then added
to bring the volume to 15 ml. The leukocyte suspension was centrifuged
at 400 to 450 × g for 15 min, and the supernatant was
decanted. The pellet was resuspended in 5 ml of sterile distilled
H2O with light vortexing to lyse the remaining
erythrocytes, and Puck's saline was added to bring the volume to 15 ml. After centrifugation at 400 to 450 × g for 15 min,
the supernatant was decanted and the pellet was resuspended to 2 ml
with culture medium.
Conventional tube culture.
A 1-ml aliquot of prepared
leukocytes and 0.5 to 1 ml of CSF samples were inoculated into
human diploid fibroblast cultures and were examined daily for
cytopathic effect. Suspected isolates were confirmed by
immunofluorescence by using a monoclonal antibody to CMV antigen.
CMV antigen assay.
The CMV antigen assay was performed by
using the CMV-vue (FITC) immunofluorescence kit (INCSTAR, Stillwater,
Minn.). This assay detects the lower matrix protein (pp65) of CMV in
peripheral blood leukocytes by use of immunocytochemical methods. The
dextran sedimentation procedure was used to prepare leukocytes.
Twenty-five microliters of cell suspension was spotted onto slides, air
dried, and fixed in an FITC-vue fixative. The fixed cells were then
incubated with a CMV FITC-vue monoclonal antibody directed against
pp65, followed by immunofluorescence staining with fluorescein
isothiocyanate (FITC)-labeled anti-mouse immunoglobulin G (IgG).
Positively stained cells were then visualized by light microscopy.
One milliliter of a CSF specimen was centrifuged in a microcentrifuge
at 14,000 × g for 5 min to pellet the cells. The cells were suspended in 40 µl of medium. Next, 15 µl of the cell
suspension was spotted onto slides, air dried, and stained by following
the protocol used for PBLs.
The results were reported qualitatively as either positive or negative;
quantitation of the number of positive cells was not
attempted.
Construction of CMV controls.
The CMV internal control
(pSYC31) was constructed to include the same primer-binding sequence
used in the AMPLICOR CMV Test but had a different probe sequence that
allowed the differentiation of the internal control from the CMV
target. The amplified product (amplicon) from the internal control was
the same size as the amplicon from the CMV target and had a G+C content
similar to that of the amplicon from the CMV target, ensuring the same
efficiency of the amplification between the internal control and the
target. The CMV-positive control (pCMA1) was constructed to include the entire amplicon region. It contains the same primer- and probe-binding sites as the CMV target DNA. The copy number of the control was determined by Poisson analysis.
AMPLICOR CMV Test.
The primers and probe were selected from
the 5' end of the CMV DNA polymerase gene based on the sequence of
strain AD169 (10). Both primers were biotinylated at their
5' ends. The size of the amplicon was 365 bp.
CMV DNA was isolated from 50 µl of a plasma or a CSF specimen by
lysing the viral particles in 500 µl of CMV extraction reagent
at
100°C for 30 min, and then 50 µl of the processed specimen
was
added to a PCR tube that contained 50 µl of the CMV master
mix and a
CMV internal control, yielding ~5 µl of plasma or CSF
equivalent
per PCR mixture. Amplification was conducted in a GeneAmp
PCR system
9600 (Perkin-Elmer, Norwalk, Conn.). To eliminate any
pre-PCR carryover
contamination, the amplification reaction was
run at 50°C for 10 min
to activate the AmpErase (uracil-
N-glycosylase)
included in
the master mix. This was followed by 40 cycles at
94 and 65°C, each
for 30 s. The final extension step was 72°C
for 10 min.
Detection procedures and reagents are the same as discussed previously
(
11) for the AMPLICOR PCR Diagnostics Kit for
Chlamydia trachomatis except that a CMV-specific capture
plate and a control
plate were used. The colorimetric results on the
microwell plate
were read on a spectrophotometric reader (Molecular
Devices Corporation,
Menlo Park, Calif.) at 450 nm. An OD of 0.35 was
the cutoff for
the assay, with values of <0.35 interpreted as negative
and those
of
0.3 interpreted as positive. The cutoff was determined
by
amplification of 220 plasma specimens from healthy individuals:
119 were CMV IgG seronegative specimens (mean OD = 0.091, minimum
OD = 0.060, maximum OD = 0.154, standard deviation = 0.017) and
101 were CMV IgG seropositive specimens (mean OD = 0.088, minimum
OD = 0.064, maximum OD = 0.146, standard
deviation = 0.017) (
20).
| |
RESULTS |
Inclusivity and exclusivity testing.
The specificity of
the AMPLICOR CMV Test was examined with a panel of nucleic
acids from CMV isolates and non-CMV pathogens. Twelve
clinical CMV isolates and four laboratory CMV strains
(strains Davis, Towne, Toledo, and AD169) were included in
the CMV panel used for examination of specificity. All clinical
isolates and laboratory strains of CMV were detectable by the
AMPLICOR CMV Test with a target input of less than 100 copies of
DNA.
The panel used for examination of exclusivity consisted of nucleic
acids isolated from 5 non-CMV herpesviruses and 21 nonherpesvirus
pathogens. At 5 × 10
3 copies of DNA or RNA input
molecules or more, these pathogens
showed no cross-reactivity with the
CMV-specific primers and probe,
and the internal control in each
test was amplified and detected.
A 10-copy sensitivity was
demonstrated with the CMV-positive control
and with purified DNA of CMV
AD169. The five non-CMV herpesviruses
were herpes simplex virus
type 1, herpes simplex virus type 2,
Epstein-Barr virus,
varicella-zoster virus, and human herpesvirus
6. The 21 non-herpesvirus
pathogens included two fungi (
C. albicans and
Aspergillus niger), seven viruses (HIV types 1 and
2, human
hepatitis C virus, human hepatitis B virus, and human
papillomavirus
types 6, 16, and 18), and 12 bacteria
(
Mycobacterium tuberculosis,
Mycobacterium
avium,
Mycobacterium intracellulare,
Klebsiella pneumoniae,
Staphylococcus
aureus,
Staphylococcus epidermidis,
Streptococcus
pneumoniae,
Propionibacterium acnes,
Neisseria
meningitidis,
Haemophilus influenzae,
Listeria
monocytogenes, and
Escherichia coli).
Analysis of plasma specimens by AMPLICOR CMV Test.
One hundred
twelve plasma specimens from 43 HIV-infected patients who were enrolled
in anti-CMV drug studies were tested by the AMPLICOR CMV Test.
Matched leukocyte specimens from these patients were also
tested by a CMV antigenemia assay and the conventional tube culture
method. Among the 112 plasma specimens, 20 (18%) were positive by the
PCR assay. The CMV antigen assay detected 21 (19%) CMV-positive
specimens, and the conventional tube culture method detected 10 (9%)
CMV-positive specimens. The 20 AMPLICOR CMV Test-positive specimens
were from eight patients. A scattergram of AMPLICOR CMV Test results
(Fig. 1A) demonstrated a clear-cut separation between CMV-positive and CMV-negative plasma
specimens.
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FIG. 1.
Detection of CMV from plasma and CSF specimens by
AMPLICOR CMV Test. An OD of 0.35 was the cutoff for the assay, with
values of <0.35 being interpreted as negative and those of 0.35
being interpreted as positive. Among 112 plasma specimens tested by the
AMPLICOR CMV Test, 20 were positive and 92 were negative. Among 58 CSF
specimens tested, 10 were positive and 48 were negative.
|
|
In order to compare the three tests, a consensus analysis was performed
as follows. If two of the three assays detected CMV,
the specimens were
designated consensus positive, and if two of
the three assays were
negative, the specimens were designated
consensus negative. The
consensus analysis showed that 93 specimens
were consensus negative and
that 19 specimens were consensus positive.
The results of the three
assays relative to the consensus results
are shown in Table
1. The AMPLICOR CMV Test had a
sensitivity
of 95% and a specificity of 98%. The sensitivity and
specificity
of the antigen assay were 100 and 98%, respectively, and
those
of the conventional tube culture method were 42 and 98%,
respectively.
Compared to the consensus results, three specimens had
discordant
results by the AMPLICOR CMV Test. One specimen was PCR
negative
but consensus positive, and two specimens were consensus
negative
but PCR positive.
View this table:
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TABLE 1.
Consensus comparison of AMPLICOR CMV Test, antigen assay,
and conventional culture assay for plasma specimens from
HIV-infected patients
|
|
The only specimen that was negative by PCR but positive by the antigen
assay and culture was from patient A, from whom a total
of six
sequential specimens were tested in the study (Table
2).
The internal control for this
specimen, which was AMPLICOR CMV
Test negative but consensus positive,
was positive, indicating
that the specimen was noninhibitory to the PCR
and that the detection
procedure worked well. Repeated assay of this
specimen showed
sporadic positive results, and the specimen was
consistently positive
if the plasma input was increased to ~25 µl
of plasma equivalent
per PCR by a different specimen processing method,
suggesting
that the plasma had a low viral load (data not shown). The
fifth
specimen received from patient A was positive by the PCR assay
but consensus negative. Even though two specimens from patient
A had
discordant results, the detection of CMV in the plasma of
patient A by
the AMPLICOR CMV Test occurred 43 days earlier than
the diagnosis of
retinitis.
The other consensus-negative but PCR-positive specimen was from patient
B, from whom a total of three sequential plasma samples
were obtained.
By a prototype quantitative CMV assay (
12), the viral loads
of the two specimens from patient A with discordant results
were <400
copies per ml of plasma and the viral load of the specimen
from patient
B with discordant results was 2,610 copies per ml
of plasma. By the CMV
antigen assay two specimens had discordant
results: antigen assay
positive but consensus negative. Both specimens
were weakly positive,
with low numbers of positive cells by the
antigen assay (1 positive
cell among 2 × 10
5 PBLs).
Analysis of CSF specimens by AMPLICOR CMV Test.
The AMPLICOR
CMV Test was used to evaluate 58 CSF specimens from AIDS patients
suspected of having an infection of the central nervous system. The
antigen assay and tissue culture results were also available for some
but not all of the CSF specimens. The AMPLICOR CMV Test detected 17%
(10 of 58) CMV-positive CSF specimens (Fig. 1B). The antigen assay
detected 18% (5 of 28) positive specimens, and tissue culture detected
no CMV-positive specimens among the 25 CSF specimens tested. Results of
all three tests were available for only 23 of the 58 specimens. Among
the 23 specimens, PCR detected CMV in 1 specimen, and the antigen assay
also detected CMV in this specimen. Of the remaining 35 of the 58 specimens, 9 of them were positive by PCR and 4 of them were confirmed
to be positive by the CMV antigen assay. Of the other five PCR-positive
specimens, four were from patients with polyradiculopathy, and the
fifth specimen was from a patient who showed varicella-zoster virus infection and neurological weakness (Table
3). The CMV culture results for the fifth
patient were negative. For the 48 negative specimens, the internal
controls, which were coamplified in the PCR, were positive, suggesting
that there were no detectable inhibitors in these specimens. The
clinical symptoms of the patients with PCR-negative results revealed
that, except for the specimen from one patient, these specimens were
unlikely to be from patients who had CMV infection. The one exception
was a specimen from a patient who was diagnosed with polyradiculopathy
but who was undergoing treatment.
| |
DISCUSSION |
The AMPLICOR CMV Test amplifies a portion of the coding region of
the CMV DNA polymerase gene. The primers and probe selected for the
test are specific for CMV. Twelve clinical CMV isolates and four
laboratory CMV strains could be detected by the AMPLICOR CMV Test with
a target input of <100 copies. The test had a 10-copy sensitivity, as
demonstrated with CMV-positive control DNA and viral DNA. With DNA or
RNA inputs of 5 × 103 molecules, the CMV-specific
primers and probe did not cross-react with the 26 non-CMV pathogens
tested. These pathogens include the herpesviruses and blood pathogens,
such as HIV type 1, HIV type 2, hepatitis C virus, hepatitis B virus,
and C. albicans.
The AMPLICOR CMV Test results for 112 plasma specimens showed that, by
comparison with the consensus results, the sensitivity and specificity
of the test are 95 and 98%, respectively, and the sensitivity and
specificity of the CMV antigen assay are 100 and 98%, respectively.
Three specimens had discordant results by the AMPLICOR CMV Test
compared to the consensus results. These three specimens were from two
patients: patients A and B. The two specimens from patient A had
discordant results due to a low viral load, since the viral loads in
these two specimens were <400 copies per ml of plasma when plasma
specimens were analyzed by a prototype quantitative test for CMV
(12). The viral load in the specimen from patient B with
discordant results was 2,610 copies per ml of plasma (12).
It was unclear why the antigen assay failed to detect CMV in PBLs while
there was a moderate viral load in plasma. Regardless of the
discordance, the detection of CMV by the AMPLICOR CMV Test in both
patients occurred much earlier than the diagnosis of CMV disease. For
patient A, PCR of plasma detected CMV 43 days before the diagnosis of
retinitis, and for patient B, PCR of plasma detected CMV 41 days before
the diagnosis of colitis. Overall, the performance of the AMPLICOR CMV
Test is comparable to the performance of the CMV antigen assay. However, the CMV antigen assay with blood specimens requires
>105 PBLs. Recently, it has been reported that the bDNA
assay can also detect CMV in PBLs, but it requires 2 × 106 to 6 × 106 PBLs for the assay
(4). Large numbers of PBLs may be hard to obtain from bone
marrow transplant recipients and AIDS patient with low CD4 cell counts.
The AMPLICOR CMV Test requires the processing of only 50 µl of
plasma. The requirement of such a small volume of plasma also makes the
detection of CMV in neonates possible.
The clinical utility of the AMPLICOR CMV Test with plasma
specimens has recently been demonstrated in a study with bone marrow transplant recipients (9). That study showed that plasma PCR is more sensitive than the antigen assay at detecting CMV disease.
Analysis of CSF specimens demonstrated that both the PCR assay and the
antigen assay detected the same CMV-positive specimens. Clinical
information suggested that these CMV-positive CSF specimens were
collected from patients who had developed CMV disease (Table 3).
Detection of CMV in CSF by the conventional tube culture method is
insensitive. In fact, the culture technique detected no CMV in 25 CSF
specimens tested in our study, and 5 of 25 specimens were positive by
the AMPLICOR CMV Test, and one AMPLICOR CMV Test-positive specimen was
also positive by the antigen assay (Table 3). PCR, the antigen assay,
and the bDNA assay have been demonstrated to be more sensitive than
culture for the detection of CMV in CSF specimens (6, 15,
21). However, both the CMV antigen assay and the bDNA assay
require at least 1 ml of CSF for the test. In addition, the CMV antigen
assay requires centrifugation of CSF in a microcentrifuge for 5 min to
pellet the cells, while the bDNA assay requires an ultracentrifugation
step of 1 h. Although the CSF specimens tested in this study were
limited in number and more work needs to be done in well-designed
studies, these results demonstrate the potential for the use of CSF in
the AMPLICOR CMV Test.
In this report, we show that the performance of the
AMPLICOR CMV Test is similar to the performance of the CMV
antigen assay. The advantage of the AMPLICOR CMV Test is that it
requires the processing of only 50 µl of plasma or CSF and does not
require the preparation of leukocytes. The specimen processing
procedure for the AMPLICOR CMV Test involves a single 30-min heating
step before the PCR. The test incorporates an internal control that is
coamplified with the CMV target in clinical specimens, providing a
useful tool for monitoring specimen amplification and specimen inhibition. In this study, the results for plasma and CSF by the AMPLICOR CMV Test indicated a clear separation between the positive and
the negative specimens (Fig. 1). The test is easy to perform and
requires less than 5 h for specimen processing, amplification, and
detection. It is therefore more rapid and technically easier for
large-scale screening than the CMV antigen assay.
| |
ACKNOWLEDGMENTS |
We thank Sheng-Yung Chang for the construction of the CMV
internal control and Beverly Dale for critical reading of the
manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Roche Molecular
Systems, Inc., 1145 Atlantic Ave., Alameda, CA 94501. Phone: (510) 814-2808. Fax: (510) 522-1285. E-mail:
Shaw-Yi.Kao{at}Roche.com.
| |
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0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
