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Journal of Clinical Microbiology, September 1998, p. 2509-2513, Vol. 36, No. 9
Research Center in Infections Diseases
(CHUL)1 and
Division of Microbiology,
Received 12 March 1998/Returned for modification 20 May
1998/Accepted 26 June 1998
The AMPLICOR cytomegalovirus (CMV) test, a new qualitative assay
for the detection of CMV DNA in plasma, was compared to conventional methods and quantitative PCR (Q-PCR) assays by using leukocytes and
plasma from 179 blood samples from subjects with AIDS. For the
diagnosis of CMV disease, cell-based assays such as a Q-PCR with
polymorphonuclear leukocytes (Q-PCR-PMNL) and a pp65 antigenemia assay
had the highest sensitivities but suffered from a lack of specificity.
The best agreement between the results of the Q-PCR-PMNL assay and
those of the AMPLICOR test was found when a threshold diagnostic value
of 690 copies per 105 cells was selected for the Q-PCR-PMNL
assay. In that context, the AMPLICOR CMV test had a sensitivity of
96.4% and a specificity of 95.3% when results were compared to
results of the cell-based PCR assay. This threshold was close to the
one described as associated with the best sensitivity and specificity
for the diagnosis of CMV disease in a recently published study
(4). Blood samples that tested positive by the Q-PCR-PMNL
assay but negative by the AMPLICOR CMV test were associated with viral
loads (mean, 785 copies, median, 96 copies per 105
leukocytes) lower than the viral loads of blood samples that tested
positive by both assays (mean, 21,452 copies; median, 9,784 copies per
105 leukocytes) (P = 0.003). The AMPLICOR
CMV test gave positive results at least 48 days before the development
of symptomatic CMV disease in a longitudinal analysis of a limited
subset of patients (n = 6) from whom sequential
specimens were available for testing. In conclusion, the AMPLICOR CMV
test is a very convenient assay combining rapidity, simplicity, and the
possibility of batch testing. A positive result by this test seems
particularly important since this implies, in most instances, the
presence or the imminence of CMV disease, although a negative test
result does not rule out disease.
Cytomegalovirus (CMV) is an
important cause of morbidity and mortality in human immunodeficiency
virus (HIV)-infected subjects with low CD4 T-cell counts (8,
11). In addition to the level of cellular immunosuppression, the
presence of CMV viremia appears to be an important risk factor for the
development of CMV disease in that population (12, 20).
Recent studies have shown a good correlation between high viral loads
in the leukocytes of AIDS patients (as determined by sensitive methods
such as an antigenemia assay [15] and quantitative PCR
[Q-PCR] assays [4, 19]) and the development of
symptomatic CMV infections. Alternatively, the detection of CMV DNA in
plasma has been shown to be a good diagnostic and possibly predictive marker for the occurrence of CMV disease in some studies (16, 22,
23). The assay to detect CMV DNA in plasma offers many technical
advantages over cell-based assays, including a convenient specimen type
and simpler qualitative PCR testing.
However, most PCR protocols in use to detect CMV are still
time-consuming and require a fair amount of expertise. In that context,
we evaluated a new commercially available assay, the AMPLICOR CMV test
(Roche Diagnostic Systems, Inc., Branchburg, N.J.), which allows simple
batch testing of a large number of plasma samples. Results obtained by
this test were compared to results of other conventional CMV assays as
well as to those of in-house Q-PCR-based methods by using plasma and
leukocytes from HIV-infected subjects with and without CMV disease.
Subjects and samples.
HIV- and CMV-seropositive subjects
with CD4 T-cell counts below 250 per mm3 were enrolled in
this study. The vast majority of these individuals were not receiving
HIV protease inhibitors at that time. Subjects were divided into two
groups based on the presence or absence of CMV disease as previously
defined (21). Briefly, the diagnosis of CMV retinitis
required compatible ophthalmologic lesions whereas the diagnosis of
visceral CMV disease required the presence of typical intranuclear
inclusions in tissues or bronchoalveolar lavage cells. EDTA-treated
blood samples were collected before antiviral therapy was initiated,
and they were processed within 6 h. The blood was first
centrifuged for the recovery of plasma and cells. Plasma was filtered
through a 0.45-µm-pore-size filter (Corning Costar, Cambridge, Mass.)
and kept at Nonmolecular CMV assays.
An aliquot of 106 PMNL
was inoculated onto human foreskin fibroblasts in a 24-well microplate
for conventional blood culture. The cells were observed for 28 days for
the appearance of typical CMV-induced cytopathic effect. A second
aliquot of 106 PMNL was centrifuged in a shell vial and
stained after 48 h with a monoclonal antibody (CMV early nuclear
protein; Dupont, NEN, Boston, Mass.) directed against the CMV p72
antigen. A third aliquot of PMNL was dedicated to the CMV pp65
antigenemia assay (1C3 clone; Argene, Parc Technologique Delta Sud,
France). The exact number of cells spotted on each well was counted
with a grid, and results were reported per 105 PMNL. The
remainder of the PMNL aliquot was frozen at Q-PCR-PMNL assay.
PMNL pellets were thawed and resuspended
in a lysing solution (1× PCR buffer; Promega Corporation, Madison,
Wis.) containing proteinase K (final concentration, 120 µg/ml; Sigma,
Mississauga, Ontario, Canada) to obtain a concentration of
104 cells per µl. The mixture was incubated for 1 h
at 56°C, and proteinase K was inactivated by heating the solution for
10 min at 95°C. Ten microliters (105 PMNL) of the mixture
was added to the PCR master mix. The CMV DNA load in PMNL was evaluated
by a recently described Q-PCR protocol that uses PMNL (Q-PCR-PMNL) and
nonisotopic hybridization (4). Hybrids were detected with a
commercial kit (SHARP Signal System; Digene Corporation, Silver Spring,
Md.). The lower limit of detection of this assay is 25 copies per
105 PMNL. All samples negative for CMV DNA by PCR were
screened for the presence of PCR inhibitors by testing for the
QC-PCR-plasma assay.
A 100-µl aliquot of filtered plasma
was added to a 100-µl solution containing (final concentrations) 100 mM KCl, 20 mM Tris-HCl (pH 8.3), 5 mM MgCl2, and 0.9%
Tween 20 solution. After proteinase K digestion (final concentration,
120 µg/ml) at 55°C for 1 h, the reaction was inactivated at
95°C for 10 min and the reaction mixture was microcentrifuged
(12,000 × g for 5 min), and then 10 µl of
supernatant (corresponding to 5 µl of initial plasma) was used for
PCR (1). A quantitative-competitive PCR assay used with
plasma (QC-PCR-plasma) has recently been described and is similar to
that used with PMNL except that 50 copies of an internal control (IC)
are added to each sample in order to rule out the presence of PCR
inhibitors in cell-free specimens (3). This competitor
consists of a plasmid containing the human papillomavirus type 31 genome with CMV major immediate-early primer sequences at its ends so
that both the IC and patient DNAs containing CMV are amplified by the
same CMV primers. After PCR amplification, half of the reaction mixture
is hybridized to a specific human papillomavirus type 31 probe, the
other half is hybridized to a specific CMV major immediate-early probe,
and a colorimetric signal is obtained by the SHARP Signal System
described above. The lower limit of detection of this assay is about 12 copies per 5 µl of plasma.
AMPLICOR CMV test.
The AMPLICOR CMV test is a qualitative
PCR test for the detection of CMV DNA in plasma and is based on four
major processes: specimen preparation, PCR amplification of target DNA,
hybridization of amplified products to specific probes, and detection
of probe-bound amplified products by colorimetric determination. In
addition, the test permits the simultaneous amplification of an IC
plasmid that has primer binding regions identical to those of the CMV target sequence but that has a different internal probe binding region.
The AMPLICOR CMV test was performed according to the manufacturer's recommendations except that plasma samples were filtered
(0.45-µm-pore-size filter) before use. Briefly, CMV DNA is first
isolated from 50 µl of plasma by lysis of virus particles at 100°C
for 30 min with a detergent solution containing proteinase K. Processed
specimens (equivalent to 5 µl of initial plasma) are then added to
the PCR master mix containing the IC. Specific biotinylated CMV primers amplify a sequence of approximately 365 bp within the human CMV DNA
polymerase gene (UL 54). Selective amplification of target nucleic
acids is achieved by the use of AmpErase (Roche Diagnostic Systems,
Inc.) and dUTP. Amplification is performed in a GeneAmp PCR System 9600 (Perkin-Elmer, Norwalk, Conn.) under the following conditions: 10 min
at 50°C and then 40 cycles of 30 s at 94°C followed by 30 s at 65°C and finally 10 min at 72°C.
Statistical analyses.
CMV assays were compared by both
observation of concordance and analysis with the kappa coefficient,
which is a chance-corrected measure of agreement. For most purposes,
kappa values greater than 0.75 may be taken to represent excellent
agreement beyond chance, values below 0.40 may be taken to represent
poor agreement, and values between 0.40 and 0.75 may be taken to
represent fair-to-good agreement (9). Student's
t test was used to compare the viral loads from concordant
(Q-PCR- and AMPLICOR CMV test-positive) and discordant (Q-PCR-positive
and AMPLICOR CMV test-negative) assay results.
Concordance of results and kappa coefficient agreement among CMV
assays.
Blood samples were obtained from 168 HIV- and
CMV-seropositive subjects with CD4 T-cell counts of
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Evaluation of the AMPLICOR Cytomegalovirus Test
with Specimens from Human Immunodeficiency Virus-Infected
Subjects
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C until extraction. The cells were separated on a
6% dextran gradient, and polymorphonuclear leukocytes (PMNL) were
counted and used in nonmolecular and molecular assays.
70°C until cell lysis.
-actin gene (7).
0.35 are considered positive for CMV regardless of
the IC result. Specimens with a CMV OD of <0.35 and an IC OD of 0.35
are considered negative for CMV, whereas those with both CMV and IC ODs
of <0.35 are considered invalid negative results.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
250 per
mm3, including 26 patients with newly diagnosed CMV disease
(20 with retinitis, 4 with gastrointestinal disease, and 2 with
pneumonitis). Additionally, blood samples from 11 HIV-infected subjects
with high CD4 T-cell counts and/or CMV-seronegative status were
included as controls. The observed concordance (number of concordant
results divided by the number of concordant plus discordant
results) and the agreement (kappa coefficients) among the results of
the AMPLICOR CMV test and other CMV assays are reported in Table
1. Excellent agreement (Kappa value of
>0.75) was found among the results of the AMPLICOR CMV test, the shell
vial assay, and the QC-PCR-plasma assay. The agreement between the
results of the AMPLICOR CMV test and those of the Q-PCR-PMNL assay was
low (kappa value = 0.35) but it increased considerably (kappa
value = 0.81) when a diagnostic cutoff value of 690 copies per
105 PMNL was selected for the cell-based assay (see below).
TABLE 1.
Concordance and kappa coefficient agreement among results
of CMV assays and the AMPLICOR CMV test
Comparison between results of the AMPLICOR CMV test and those of
the QC-PCR-plasma assay.
The AMPLICOR CMV test was compared to the
in-house QC-PCR-plasma assay by using 179 plasma samples. Results of
both tests were concordant for 169 specimens (147 negative and 22 positive). The two assays showed discordant results for 10 samples
(seven AMPLICOR-positive and three QC-PCR-plasma-positive tests). Based on results obtained with the other CMV assays and on clinical information, all seven AMPLICOR CMV test results were considered true
positives. Four of the seven false-negative test results obtained with
the QC-PCR-plasma assay were due to the presence of PCR inhibitors.
Such inhibitors were encountered in none of the samples tested by the
AMPLICOR CMV test but were encountered in 3.4% (6 of 179) of those
tested by the QC-PCR-plasma assay. The three samples that tested
positive only by the QC-PCR-plasma assay were all considered true
positives. The viral DNA loads in these three samples were very low
(mean, 18 copies per 5 µl; median, 24 copies per 5 µl [all
contained 25 copies]) compared to the viral loads in the 22 samples
that were positive by both assays (mean, 1,548 copies per 5 µl;
median, 216 copies per 5 µl; range, <25 to 10,091 copies).
Comparison between results of the AMPLICOR CMV test and those of the Q-PCR-PMNL assay. The AMPLICOR CMV test was also compared to the Q-PCR-PMNL assay by using 177 blood samples. Results were concordant for 122 samples (94 negative and 28 positive samples). On the other hand, discordant results were found for 55 specimens (all positive by the Q-PCR-PMNL assay only). Specimens that tested positive by the Q-PCR-PMNL assay only were from two different groups of subjects. Forty-six of the 55 (83.6%) samples were from asymptomatic HIV-infected subjects (mean, 242 CMV DNA copies per 105 PMNL; range, <25 to 4,376 copies), whereas 9 of the 55 (16.4%) samples were from subjects with established CMV disease (mean, 3,563 CMV DNA copies per 105 PMNL; range, <25 to 16,259 copies). Overall, the mean viral load for the 55 samples positive by the Q-PCR-PMNL assay but negative by the AMPLICOR CMV test was 785 copies per 105 PMNL (median, 96 copies; range, <25 to 16,259 copies). In comparison, the mean CMV DNA load for the 28 samples that tested positive by the two assays was 21,452 copies per 105 PMNL (median, 9,784 copies; range, 292 to 140,430 copies) (P = 0.003). The AMPLICOR CMV test had sensitivity and specificity values of 33.7 and 100.0%, respectively, when results were compared to results of the Q-PCR-PMNL assay with no preselected cutoff value. The best agreement between the two tests was found when a threshold of 690 copies per 105 cells was selected for the Q-PCR-PMNL assay. In that context, the AMPLICOR CMV test had a sensitivity of 96.4% and a specificity of 95.3% when results were compared to results of the cell-based PCR assay.
Diagnostic values of the assays for CMV disease.
The
sensitivities, specificities, and positive and negative predictive
values of the conventional and molecular assays obtained with data from
the 168 HIV- and CMV-seropositive subjects with 250 CD4 T-cells per
mm3 are reported in Table 2.
The incidence of CMV disease for these individuals during the study
period was 15.5%. The highest sensitivity and negative predictive
values were obtained by using the leukocyte-based assays (Q-PCR-PMNL
and pp65 antigenemia assays), whereas the highest specificity and
positive predictive values were obtained by using the plasma-based
assays (in-house QC-PCR-plasma and AMPLICOR CMV test). Twenty-four of
the 29 (82.8%) samples that were positive by the AMPLICOR CMV test
were from subjects with documented CMV disease (n = 17)
or from patients who developed disease within 4.3 months of testing
(n = 7).
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Longitudinal analysis of the CMV viral load. Sequential blood samples were analyzed from six subjects before they developed CMV disease (Table 3). Plasma samples from five of six patients were positive by the AMPLICOR CMV test at the time CMV disease was diagnosed, although all six patients had positive samples prior to development of disease. The AMPLICOR test was positive for at least 48 days before the onset of CMV disease (up to 350 days for one subject). There was generally a good concordance between a positive AMPLICOR CMV test and a high viral load in PMNL as determined by the Q-PCR-PMNL and the antigenemia assays.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Until recently, PCR testing of blood samples has been used mainly to confirm the diagnosis of CMV disease especially when retinal lesions are atypical or to complement histopathological examination in the case of visceral disease (4, 19, 23). Recently, CMV PCR assays have also been evaluated for their potential to identify high-risk individuals in the context of preemptive therapeutic strategies (16, 22). Finally, molecular CMV assays can also be used for monitoring antiviral therapy and assessing the emergence of drug-resistant mutants (2, 5, 6, 13). However, many basic problems, such as the need for a Q-PCR instead of a qualitative PCR assay in some of the clinical situations described above, remain unsolved. Similarly, there exists a controversy over the type of specimen to use for PCR testing, i.e., PMNL or plasma. More importantly, there is a lack of standardization for CMV PCR testing, in part because simple and reproducible commercial kits have not been developed. The purpose of our study was twofold. First, we compared various nonmolecular and molecular CMV assays (using both PMNL and plasma specimens) for their potential to confirm CMV disease and also, to a lesser extent, to predict the development of disease in subjects with AIDS. At the same time, we performed an evaluation of a new commercially available PCR kit, the AMPLICOR CMV test.
The results of this study demonstrate an excellent agreement (kappa value > 0.75) between the results of the AMPLICOR CMV test and those of another plasma-based PCR assay in use at our institution (3). The agreement between the results of the AMPLICOR CMV test and those of other cell-based assays was not as good in general. In particular, a very low correlation (kappa value = 0.35) was found with the results of the Q-PCR-PMNL assay that we recently described (4). However, a better correlation was found between the results of the two assays when a cutoff of 690 copies per 105 cells was selected for the PCR-PMNL test (kappa value = 0.81). This cutoff value is close to the one (1,000 copies per 105 PMNL) associated with the best sensitivity (87.5%) and specificity (96.2%) for the diagnosis of CMV disease in a recently published study (4). A positive result by the AMPLICOR CMV test was also clearly associated with a high viral DNA load, and conversely, a negative test was associated with a low viral DNA load. This finding can be illustrated by comparing the mean CMV DNA load in samples positive by both PCR assays (AMPLICOR test and Q-PCR assays) with the one found in samples positive by the Q-PCR assays only. Indeed, we found an 84.1- and 27.3-fold difference in the mean viral loads of concordant and discordant samples using, respectively, QC-PCR-plasma and Q-PCR-PMNL results as a reference. Similarly, Freymuth et al. showed that the presence of CMV DNA in plasma as detected by nested PCR was in correlation with high levels of antigenemia in AIDS patients and organ transplant recipients (10).
As a diagnostic test for CMV disease in HIV-infected subjects, the AMPLICOR test was highly specific (92%) but not as sensitive (65%) as other cell-based assays. The lower sensitivity of the AMPLICOR test than those of the Q-PCR-PMNL and antigenemia assays is in agreement with our own data obtained by another PCR-plasma assay (sensitivity, 65%) as well as results from other groups who reported a higher viral load in PMNL than in plasma for an equivalent blood volume (14, 24). However, some investigators have found much higher sensitivity values (ranging from 74 to 83%) using other plasma- or serum-based PCR assays (16, 23). The reasons for such discrepancies need to be further examined, but it might be related to sample preparation, the volume of sample tested, or the PCR conditions. Also, the use of filtered plasma in our study may have resulted in a lower than expected sensitivity value for the AMPLICOR CMV test, although preliminary data indicate that filtration does not seem to alter test results significantly (data not shown). Recently, the AMPLICOR CMV test was found to be more sensitive than the antigenemia assay in a small study realized with samples from bone marrow and solid organ transplant recipients (17). Although those results differ somewhat from ours, the population studied and the definition of CMV disease were not the same. In particular, both cases of active CMV infection and CMV disease were considered in the analysis of the latter study whereas only proven cases of CMV disease were included in our analysis. The discrepant results emphasize the fact that CMV diagnostic tests should be evaluated in each of the different clinical settings.
Interestingly, the AMPLICOR CMV test readily identified the risk of progression to CMV disease in a small subset of patients who had longitudinal PCR evaluations (Table 3). For these subjects, the AMPLICOR test was positive at least 48 days before the development of disease. A recent larger study showed that the median time between a first positive result by an in-house PCR-plasma assay and diagnosis of CMV disease was approximately 6.0 months (22). In that context, PCR-plasma assays may be a better laboratory marker than PCR-PMNL assays for enacting preemptive therapy for AIDS patients, since cell-based assays may be positive for an unacceptably long period of time before development of disease (3, 14). Clearly, a larger prospective longitudinal study is needed to verify this aspect.
Overall, the AMPLICOR CMV test is a very convenient assay that combines rapidity (<6 h), simplicity, and the possibility of batch testing. Unlike with the antigenemia assay, specimens do not need to be processed immediately and require minimal preparation before testing (18). Also, compared to most PCR protocols in use, the AMPLICOR CMV test minimizes contamination risks by taking advantage of the AmpErase- dUTP system and facilitates detection of PCR products with the use of microwell plates and a colorimetric detection system. On the other hand, this test does not provide quantitative results and still may need to be used in conjunction with additional methodologies for virus recovery and antiviral susceptibility testing.
In conclusion, the place of the AMPLICOR CMV test is not completely defined at present and is likely to depend on the type of clinical situations encountered and the workload of the virology laboratory. For large reference laboratories, the AMPLICOR CMV test appears particularly suitable, since blood samples may be kept for up to 24 h at room temperature before being processed and batch testing is possible. Clinicians need to keep in mind that a negative test does not rule out CMV disease but that a positive test almost always identifies an individual with an established CMV disease or one at high risk for subsequent disease. Indeed, our results show that 82.8% (24 of 29) of the positive AMPLICOR CMV tests were found with samples from patients with active CMV disease or subjects who developed disease within 4.3 months. For smaller laboratories, the CMV antigenemia assay is still a very good alternative, but it has the technical limitations described previously. Although, we did not address this aspect of testing in our study, it is probable that Q-PCR assays will be necessary for monitoring anti-CMV therapy. In that context, a Q-PCR assay, which can be used with either plasma or PMNL specimens, from Roche Diagnostic Systems (COBAS AMPLICOR CMV MONITOR test) is under evaluation.
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ACKNOWLEDGMENTS |
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This work was supported by a grant (MA-13924) from the Medical Research Council of Canada and by Roche Molecular Systems, Inc.
Guy Boivin is a scholar of the Medical Research Council of Canada.
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FOOTNOTES |
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* Corresponding author. Mailing address: CHUL, room RC-709, 2705 Blvd. Laurier, Sainte-Foy, Québec, Canada G1V 4G2. Phone: (418) 654-2705. Fax: (418) 654-2715. E-mail: Guy.Boivin{at}crchul.ulaval.ca.
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Abstract
Introduction
Materials & Methods
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Discussion
References
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Journal of Clinical Microbiology, September 1998, p. 2509-2513, Vol. 36, No. 9
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