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Journal of Clinical Microbiology, May 1998, p. 1333-1337, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Evaluation of Murex CMV DNA Hybrid Capture Assay
for Detection and Quantitation of Cytomegalovirus Infection in Patients
following Allogeneic Stem Cell Transplantation
Holger
Hebart,1,*
Daphne
Gamer,1
Juergen
Loeffler,1
Claudia
Mueller,1
Christian
Sinzger,2
Gerhard
Jahn,2
Peter
Bader,3
Thomas
Klingebiel,3
Lothar
Kanz,1 and
Hermann
Einsele1
Medizinische Klinik und Poliklinik, Abteilung
II und Sektion für Transplantationsimmunologie und
Immunhämatologie,1
Institut
für Epidemiologie und Klinik der
Viruskrankheiten,2 and
Universitätskinderklinik, Abteilung Kinderheilkunde
1,3 72076 Tübingen, Germany
Received 2 October 1997/Returned for modification 2 December
1997/Accepted 26 January 1998
 |
ABSTRACT |
Murex hybrid capture DNA assay (HCS) is a solution hybridization
antibody capture assay for detection and quantitation of cytomegalovirus (CMV) DNA in leukocytes. To determine whether CMV HCS
is sensitive enough to initiate and monitor antiviral therapy after
allogeneic stem cell transplantation (SCT), 51 consecutive SCT
recipients were prospectively screened for the appearance of CMV
infection by HCS, PCR, and culture assays from blood samples. Preemptive antiviral therapy was initiated after the second positive PCR result in all patients, as previously reported, and HCS was not
considered for clinical decision making. A total of 417 samples were
analyzed. Of these, 21 samples were found to be positive by PCR and
HCS, 88 samples were PCR positive but HCS negative, and 308 were
negative by both assays. Concordance of results between PCR and HCS and
between HCS and blood culture was observed in 78.9 and 95.9% of the
samples assayed, respectively. PCR was found to be more sensitive than
HCS, and HCS was more sensitive than the blood culture assay
(P < 0.0001). Four patients with symptomatic CMV
infection were PCR positive prior to the onset of CMV-related symptoms,
whereas HCS detected CMV DNA in three patients prior to and one at
onset of CMV disease. The numbers of genomes per milliliter of blood
were higher in patients with symptomatic CMV infection than in those
with asymptomatic CMV infection (P = 0.06). None of
the HCS-negative patients developed CMV disease. Thus, all patients
with CMV disease were correctly identified by HCS; however, the lower
sensitivity limit of the HCS assay may still be insufficient to allow
diagnosis of CMV infection early enough to prevent CMV disease in
patients following allogeneic SCT.
| |
INTRODUCTION |
In spite of recent developments in
diagnosis and treatment, cytomegalovirus (CMV) infection remains a
major infectious complication in recipients of an allogeneic stem cell
transplantation (SCT) (15). Symptomatic CMV infection,
especially when leading to interstitial pneumonia, is still associated
with a high mortality rate in these patients (6, 16). Early
diagnosis of CMV infection and preemptive antiviral therapy, based on
the PCR technique, resulted in a significant reduction of the incidence
of CMV disease and of CMV-associated mortality following allogeneic SCT
(3-5). Thus, pp65 antigen detection in leukocytes
(1), and PCR analysis of leukocytes and plasma (3-5,
19) are widely used for the early diagnosis of CMV infection.
However, the various protocols applied are poorly standardized
(9). To make test results in international drug trials
comparable, the standardization of the diagnostic assays applied is
mandatory. Murex hybrid capture CMV DNA assay (HCS) is a commercially
available solution hybridization antibody capture assay for the
quantitative detection of CMV DNA in leukocytes. HCS allows early
diagnosis and monitoring of antiviral therapy in AIDS patients and in
solid-organ-transplant recipients (11, 13, 14, 18). To
determine whether HCS is sensitive enough to initiate and to monitor
antiviral therapy after allogeneic SCT, HCS was compared to PCR with
whole blood and to rapid and conventional virus culture techniques for
the detection and quantitation of CMV in blood samples.
| |
MATERIALS AND METHODS |
Patients.
Between December 1994 and February 1996, 51 consecutive patients after allogeneic bone marrow transplantation
(n = 46) or allogeneic peripheral blood SCT
(n = 5) (median age, 33 years; range, 2 to 57 years)
were included in the study. Conditioning therapy consisted either of
busulfan (four doses of 1 mg/kg of body weight on days 
7 to 4) and
cyclophosphamide (two doses of 60 mg/kg of body weight on days 3 and
2) or of total body irradiation (two doses of 2 Gy on days 6 to
4) and cyclophosphamide (two doses of 60 mg/kg of body weight, days
3 and 2). Patient characteristics at the time of transplant are
shown in Table 1. Oral acyclovir was
administered in all cases at a dosage of 400 mg four times a day for
prophylaxis of herpes simplex virus infection until day 100 after SCT.
Nineteen CMV-seropositive patients received a transplant from a
seropositive donor, and four patients received a transplant from a
seronegative donor; another ten CMV-seronegative patients received a
transplant from a seropositive donor. Thus, 33 of the 51 patients
analyzed were at risk to develop CMV infection and disease. All
CMV-seropositive patients and/or patients receiving a transplant from a
CMV-seropositive donor received blood products which had not been
screened for CMV, whereas all CMV-seronegative patients receiving a
transplant from a CMV-seronegative donor received CMV-seronegative
blood products. Informed consent was obtained from patients, according
to the declaration of Helsinki, and the study was approved by an
institutional review board.
Study design.
All 51 patients were monitored weekly for
signs of CMV reactivation or infection by Murex HCS, PCR with whole
blood, and conventional as well as rapid virus culture assays beginning
at the time of neutrophil recovery (>500/µl) until day 100 posttransplantation. CMV-seronegative patients receiving a transplant
from a CMV-seronegative donor were included as negative controls.
Therapy for CMV infection and CMV disease.
All patients were
included in a clinical trial used to evaluate PCR-based preemptive
therapy with ganciclovir. Murex HCS was not considered for clinical
decision making. According to the study protocol, PCR-based preemptive
therapy with ganciclovir (two doses of 5 mg/kg of body weight/day) was
initiated within 24 to 48 h from the time of the second
consecutive positive PCR result as previously described (3).
Patients with documented CMV disease received only combination therapy
with ganciclovir and CMV-hyperimmunoglobulin. To reduce the duration of
antiviral therapy, ganciclovir was stopped after 14 days if clearance
of the virus could be documented by PCR. Thus, maintenance therapy with
ganciclovir at 5 mg/kg/day five times weekly was administered only when
PCR remained positive after a 14-day course of antiviral therapy.
CMV disease.
CMV disease was diagnosed according to the
criteria defined by the Fourth International Cytomegalovirus Workshop
in Paris 1993 (12), as follows. CMV pneumonia was indicated
by dyspnea, interstitial infiltrates on the chest radiograph, and
positive CMV culture of bronchial washings. CMV enteritis was indicated by gastrointestinal symptoms and the demonstration of CMV by histology or immunohistology. CMV hepatitis was indicated by abnormal liver function, typical histological changes, and the detection of CMV in the
liver biopsy by culture or DNA hybridization.
PCR.
DNA extraction of 5 ml of EDTA-anticoagulated blood was
performed as previously reported (5). A 147-bp DNA fragment
located between positions 1767 and 1913 of the fourth exon of the
immediate-early gene of the human CMV strain AD169 was amplified by PCR
with specific primers. In all assays 0.1, 1, 10, and 100 fg of cloned
HCMV-DNA fragment pCM5018 were amplified. The intensity of the signals of the titration curve was compared with the signal from each clinical
sample and a semiquantitative assessment was performed as recently
described by our group (10). The median time for a complete
PCR test was 12 h.
Virus culture.
Human foreskin fibroblasts grown in glass
tubes were inoculated for 45 min at 37°C with 0.2 ml of a leukocyte
suspension (1 × 106 to 2 × 106
cells) prepared by dextran sedimentation. After removal of the clinical
material, human foreskin fibroblasts were maintained in culture in
minimal essential medium at 37°C. Growth of CMV was identified by
production of its characteristic cytopathic effects on human foreskin
fibroblasts. In a separate identically inoculated culture, CMV-specific
antigens were detected 48 h after inoculation by an indirect
peroxidase technique. Cultures were fixed with cold methanol for 10 min. Monoclonal antibody E13 (dilution, 1:750; Biosoft, Paris, France),
which is directed against CMV immediate-early antigen, was added for 60 min at 37°C. Subsequent incubation with peroxidase-conjugated rabbit
anti-mouse immunoglobulin antibody (dilution, 1:750; Dako, Hamburg,
Germany) and with the chromogen aminoethylcarbazol (Sigma Chemical Co.,
St. Louis, Mo.) yielded brown nuclear staining of infected cells.
Cultures were read with a Leitz Fluovert microscope at a 63-fold
magnification.
Murex HCS.
The assay was provided by Murex Diagnostica GmbH
(Burgwedel, Germany) and was performed within 8 h according to the
manufacturer's instructions. Briefly, 3.5 ml of EDTA-anticoagulated
blood was lysed with the manufacturer's lysis solution. The leukocytes
were transferred to hybridization tubes, pelleted, and either stored at
20°C for up to 2 months or immediately processed. The denaturing agents were added to the samples for 25 min at 70°C. The samples were
transferred to fresh hybridization tubes and incubated for an
additional 25 min at 70°C. Thereafter, the CMV-specific RNA probe was
added, and the tubes were incubated for 2 h at 70°C. The RNA-DNA
hybrids were transferred to specific-antibody-coated capture tubes that
were placed on a rotary shaker for 60 min at 1,100 rpm. An alkaline
phosphatase-conjugated antibody specific for RNA-DNA hybrids was added,
and the tubes were incubated for 30 min at room temperature. The tubes
were then washed and the substrate was added; after a further
incubation step, the tubes were read with a luminometer. CMV-DNA was
quantitated by comparing the relative light units (RLU) of the samples
to the RLU of a calibration standard curve, and the results were
expressed as genomes per milliliter. A ratio of sample RLU to the
positive cutoff value of >1.0 was considered positive for CMV DNA
according to the manufacturer's instructions.
Statistical evaluation.
Primary study endpoints were the
time from SCT to a positive PCR, HCS, and culture test result.
Differences in sensitivity were analyzed by log rank analysis for
censored data. Comparison of the viral load in patients with
symptomatic and asymptomatic CMV infection and with culture-positive
and culture-negative blood samples as well as with semiquantitatively
assessed PCR- and HCS-positive blood samples was performed by using the
Wilcoxon rank test (GraphPad Prism, version 2; GraphPad Software, Inc.,
San Diego, Calif.).
| |
RESULTS |
Diagnosis of CMV infection.
A total of 417 blood samples from
51 patients who underwent an allogeneic SCT (mean, 8 samples per
patient; range, 3 to 18) were analyzed. CMV viremia was diagnosed in 23 patients by PCR (109 of 417 samples), in 12 patients by HCS (21 of 417 samples), and in 6 patients by the culture method (6 of 417 samples).
When the times to the first positive test result were compared in
patients who received preemptive antiviral therapy as mandated in the
study, PCR proved to be more sensitive than HCS (P < 0.0001) and HCS was more sensitive than the culture method
(P < 0.0001). CMV infection was diagnosed by PCR a
median of 37.5 days (mean, 39.9 days; standard error [SE], 3.83 days;
range, 14 to 68 days), by HCS a median of 43 days (mean, 50 days; SE,
7.2 days; range, 14 to 88 days), and by blood culture a median of 50.5 days (mean, 55.8 days; SE, 9.4 days; range, 31 to 100 days)
posttransplantation (Table 2). None of
the 18 CMV-seronegative patients receiving grafts from a seronegative
donor had a positive HCS result, and only one had a single slightly
positive PCR assay result.
Results were 95.9% concordant between blood cultures and HCS, 75.3%
concordant between blood cultures and PCR, and 78.9% concordant
between PCR and HCS. All PCR-negative samples were HCS and culture
negative. Eighty-eight samples were PCR positive but HCS negative.
Of
these, 18 discordant samples were taken prior to and 64 after
initiation of antiviral therapy. Because five HCS-negative patients
were PCR positive only once, these patients did not receive antiviral
therapy. One HCS-negative blood sample proved to be culture positive
(Fig.
1). This sample was drawn 10 days
after initiation of antiviral
therapy and in the absence of clinical
signs of CMV disease. Moreover,
due to toxicity related to ganciclovir,
the leukocyte count had
dropped below 4,000/µl in this patient.
Additionally, sensitivity
was not found to be affected by the leukocyte
counts when the
viral loads in PCR-positive blood samples between those
above
and those below 4,000 leukocytes/µl were compared
(
P = 0.1).
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FIG. 1.
Venn diagram showing the correlation of test results in
109 blood samples found to be positive in each of the three assays
analyzed: PCR (PCR from whole blood), HCS, and blood culture (BC).
|
|
When the viral DNA loads quantified by HCS were compared with those
found by semiquantitative assessment by PCR (scored as
+, ++, and +++)
in 21 blood samples positive in both assays, a
lower viral load as
assessed by HCS was found in samples with
a weakly positive PCR signal
(PCR
+: median, 11,770; mean, 13,980; SE, 2,438; range,
6,097 to 25,900
genomes/ml) than in samples with a moderately positive
signal
(PCR
++: median, 22,860; mean, 25,110; SE, 6,997;
range, 9,000 to 45,320
genomes/ml) and those with a strongly positive
PCR signal (PCR
+++: median, 23,680; mean, 254,100; SE,
89,850; range, 5,772 to 717,500
genomes/ml). However, due to the low
number of HCS-positive samples,
this result did not reach statistical
significance (PCR
+ versus PCR
++,
P = 0.09; PCR
+ versus PCR
+++,
P = 0.07) (Fig.
2).
Moreover, CMV DNA levels as assessed by
HCS were higher in
culture-positive than in culture-negative blood
samples (culture
positive: median, 35,510; mean, 166,400; SE,
137,900; range, 11,530 to
717,500 genomes/ml; culture negative:
median, 11,840; mean, 35,120; SE,
18,650; range, 5,772 to 311,700
genomes/ml) (
P = 0.06).
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FIG. 2.
Evaluation of CMV DNA quantified by HCS (genomes per
milliliter) versus semiquantitatively assessed PCR results for 21 HCS-positive blood samples. The median DNA values were 11,770 (PCR+), 22,860 (PCR++), and 23,680 (PCR+++) genomes/ml, respectively (for PCR+
versus PCR+++, P = 0.07). Horizontal lines
represent the median values.
|
|
Clinical course and monitoring of antiviral therapy.
Eighteen
patients received antiviral therapy until day 100 per study-mandated
rules. Four of these 18 patients developed proven CMV disease at the
time of the second consecutive positive PCR result (3 had CMV
interstitial pneumonia and 1 had CMV hepatitis). Two of these patients
had received a transplant from an unrelated donor, and two had received
a partially T-cell-depleted graft. All four patients recovered from CMV
disease under combined therapy with ganciclovir and CMV
hyperimmunoglobulin.
CMV infection was diagnosed by HCS in 12 of 18 patients receiving
antiviral therapy based on PCR-positive findings in two
consecutive
blood samples. All four patients who developed CMV
disease were
correctly identified by HCS a median of 5.5 days
prior to onset of
CMV-related symptoms (range, 0 to 11 days),
whereas only three were
identified by analyzing culture from whole
blood (Table
2). The median
peak titer of CMV DNA, as measured
by the HCS assay, was greater in the
four patients with CMV disease
(median, 28,190; mean, 197,800; SE
173,300; range, 17,270 to 717,500
genomes/ml) than in patients with
asymptomatic CMV infection (median,
11,570; mean, 14,670; SE, 3,595;
range, 4,576 to 45,320 genomes/ml)
(
P = 0.06) (Fig.
3).
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FIG. 3.
Relationship between CMV DNA and the severity of CMV
infection. Horizontal lines represent the median values.
|
|
When CMV disease was used as the standard of comparison, the positive
predictive value for a positive HCS assay result was
33.3% and for two
consecutive positive PCR results it was 22.2%.
The negative predictive
value was 100% for both assays.
PCR positivity persisted for a median of 4 weeks (range, 2 to 8 weeks)
after initiation of antiviral therapy, whereas HCS
was found to be
negative in one patient after 3 weeks, in three
patients after 2 weeks,
and in 8 patients after 1 week of antiviral
therapy. All patients with
asymptomatic CMV infection were HCS
negative 1 week after initiation of
antiviral therapy. Thus, the
efficacy of antiviral therapy could be
monitored by using HCS
and PCR.
| |
DISCUSSION |
To determine whether CMV HCS is sensitive enough to initiate and
monitor antiviral therapy after allogeneic SCT, 51 patients were
prospectively screened by HCS, a homemade PCR assay, and a blood
culture technique. As expected, PCR was more sensitive than HCS
(P < 0.0001) and HCS was more sensitive than viral
blood culture (P < 0.0001). PCR and HCS findings and
HCS and blood culture findings were found to be concordant in 78.9 and
95.9% of the samples, respectively. All patients with CMV disease were
correctly identified by HCS and PCR. The viral burden was found to be
higher in patients with CMV disease and those with a positive blood
culture assay (P = 0.06). HCS results became negative
after an average of 8 days of antiviral therapy and remained positive
for up to 3 weeks in patients with CMV-related symptoms, indicating
that HCS might be appropriate for monitoring the effectivity of
antiviral drugs.
HCS has been shown to allow the rapid, quantitative, and objective
measurement of the CMV load in HIV-infected patients and in recipients
of an organ allograft (11, 13, 14, 18). Moreover, a
reasonable quantitative correlation of HCS and pp65 antigenemia has
been demonstrated in patients with a high viral burden, whereas
patients with low-level antigenemia (<20 positive/105
cells) were less likely to be HCS positive, indicating that HCS is less
sensitive than the pp65 antigen assay (14, 18). Our homemade
PCR assay performed with DNA extracted from whole blood showed a
sensitivity comparable to that of PCR performed on DNA extracted from
plasma or pp65 antigenemia following SCT (10). Thus, the
higher sensitivity of the PCR than of HCS was expected.
Preemptive antiviral therapy based on PCR has been shown to
significantly reduce CMV-related morbidity and mortality
(3). To further reduce the duration of potentially toxic
antiviral therapy, PCR-based preemptive ganciclovir is discontinued at
our institution once patients have cleared the virus from the blood after 2 to 4 weeks of therapy (3). A much lower number of
samples were found to be positive by HCS than by PCR prior to and also after initiation of antiviral therapy. Since all patients received preemptive antiviral therapy after the second consecutive positive PCR
assay irrespective of their HCS status, the prevalence of a positive
HCS result following allogeneic SCT may have been underestimated. Thus,
whether HCS is suitable to initiate and monitor antiviral therapy in
recipients of an allogeneic SCT remains debatable. However, because of
the very high mortality of CMV disease in recipients of an allogeneic
SCT (15), the lower sensitivity limit of HCS may be critical
in allowing a diagnosis of CMV infection early enough in this patient
cohort. Four patients developed CMV disease at the time of the second
consecutive positive PCR result. Three of these four patients were
found to be HCS positive prior to and one patient was positive at the
onset of clinical symptoms, whereas only three of four patients were
correctly identified by the blood culture assay after the onset of
clinical symptoms. Thus, HCS was more sensitive in identifying patients
with CMV disease than the blood culture assay.
As methods for CMV detection become more sensitive, quantitation of CMV
is becoming more important in assessing the predictive value of a
positive test result. In solid organ transplant recipients and in
patients with AIDS, CMV-related symptoms are associated with a high
viral burden in the blood, whereas in recipients of a bone marrow
transplant, even fatal disease does not seem to be associated with
higher levels of virus in blood leukocytes (11, 13, 17).
More-sensitive quantitative blood assays in recipients of an allogeneic
SCT may help to define a threshold and thus reduce the incidence of
overtreatment (8, 20). In this study, we have demonstrated
that quantitation of the CMV load in the blood can be easily achieved
by HCS. Moreover, a high viral burden correlated with CMV-related
symptoms. In contrast, because quantitative competitive PCR protocols
are still time-consuming and labor-intensive and are not a part of the
daily routine in most facilities, only limited data for these assays
are currently available.
HCS allows storage of samples for several days without a loss of
sensitivity (14), in contrast to the pp65 antigen assay, which requires immediate sample processing (2). Moreover,
quantitative PCR and pp65 antigenemia analyses are still poorly
standardized. Recently, a quantitative plasma PCR plate assay adopting
enzyme-linked immunosorbent assay technology to measure the amount of
CMV DNA was developed, and a good correlation with a quantitative
competitive PCR assay was demonstrated for this method (7).
Further prospective comparisons of different methods are needed to
achieve a better standardization of CMV diagnosis and treatment
monitoring.
In conclusion, HCS was more sensitive than the blood culture technique
but less sensitive than the PCR assay. HCS can be used to monitor the
efficacy of antiviral therapy by quantitation of CMV DNA in the blood.
The short interval between a positive test result and the onset of
CMV-related symptoms indicates that the sensitivity of the assay is
still insufficient to allow initiation of antiviral therapy in
recipients of an allogeneic SCT early enough to prevent CMV disease. A
second-generation HCS with improved sensitivity has been developed and
is being assessed for patients following allogeneic SCT in an ongoing
study.
| |
ACKNOWLEDGMENTS |
This work was supported by Deutsche Forschungsgemeinschaft (DFG)
grant SFB 510 B3.
CMV HCS DNA kits (version 1) were kindly provided by Murex.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Medizinische
Klinik und Poliklinik, Abteilung II, Otfried-Müller-Str.10,
D-72076 Tübingen, Germany. Phone: 0049 7071 2982712. Fax: 0049 7071 293179. E-mail: holger.hebart{at}uni-tuebingen.de.
| |
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Journal of Clinical Microbiology, May 1998, p. 1333-1337, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
