Previous Article | Next Article 
Journal of Clinical Microbiology, May 1999, p. 1431-1435, Vol. 37, No. 5
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Comparison of Cytomegalovirus Loads in Plasma and Leukocytes
of Patients with Cytomegalovirus Retinitis
Douglas A.
Jabs,1,2,*
Michael
Forman,3
Cheryl
Enger,4 and
J. Brooks
Jackson3,
for The
Cytomegalovirus Retinitis and Viral Resistance Study Group
Departments of
Ophthalmology,1
Medicine,2
Pathology,3 and
Oncology,4 The Johns Hopkins
University School of Medicine, Baltimore, Maryland
Received 3 November 1998/Returned for modification 21 December
1998/Accepted 28 January 1999
 |
ABSTRACT |
Cytomegalovirus (CMV) DNA loads in paired leukocyte and plasma
samples from 199 patient visits by 66 patients with CMV retinitis were
determined. Leukocyte CMV load determinations had a greater range of
values (mean, 24,587 copies/106 leukocytes; maximum,
539,000) than did plasma CMV load determinations (mean, 10,302 copies/ml; maximum, 386,000), and leukocyte viral loads were detectable
in a greater proportion of patients at the time of diagnosis of CMV
retinitis prior to initiation of anti-CMV therapy (82%) than were
plasma viral loads (64%) (P = 0.0078). Agreement with CMV blood cultures was slightly better for plasma (
= 0.68) than for leukocytes ( = 0.53), due to a greater
proportion of patients with detectable viral loads in leukocytes having
negative blood cultures.
| |
INTRODUCTION |
Cytomegalovirus (CMV) is an
important pathogen in patients with AIDS (6, 10, 11, 16).
Although CMV may affect multiple organs, CMV retinitis is the most
frequently encountered presentation in patients with AIDS, and it
accounts for 75 to 85% of CMV disease in these patients (6,
10).
Quantification of the amount of human immunodeficiency virus (HIV) RNA
in the plasma (HIV load) has become established as a useful clinical
tool for monitoring patients with HIV infection. It predicts the
progression of HIV disease and morbidity (14), and
suppression of the HIV load by using highly active antiretroviral therapy results in improvement in immune function, reduction in opportunistic infections, and increased survival (7, 8, 15).
In an analogous manner, the amount of CMV DNA in the blood (CMV load)
can be quantitated. It has been proposed that the CMV load may be
useful for identifying patients at high risk for the development of CMV
disease and/or for monitoring patients with CMV disease (2, 3, 17,
19). The detection of circulating CMV DNA in plasma
(17) or whole blood (2) predicts the subsequent development of CMV disease in patients with low CD4+-T-cell
counts, and the qualitative detection of CMV DNA in the whole blood
correlates with new episodes of CMV disease in patients with CMV
retinitis (3). Because latent CMV is thought to reside in
leukocytes, it is unclear whether quantification of the CMV load in
leukocytes or plasma is a better method for identifying and monitoring
these patients. Here we report our experience in simultaneously
quantifying the amount of CMV DNA from leukocytes and plasma in samples
obtained prospectively from a cohort of patients with CMV retinitis.
| |
MATERIALS AND METHODS |
The Cytomegalovirus Retinitis and Viral Resistance Study is a
prospective epidemiologic study of patients with CMV retinitis and AIDS
(5). Eligible patients had newly diagnosed CMV retinitis and
were untreated prior to enrollment. The diagnosis of CMV retinitis was
made on clinical grounds by indirect ophthalmoscopy through a dilated
pupil (5). At the time of enrollment, patients gave a
medical history, had an eye examination, and underwent phlebotomy for
culture of blood for CMV and assessment of the CMV loads in blood
leukocytes and plasma. All baseline determinations were performed on
specimens obtained prior to initiation of therapy. Patients were
treated according to best medical judgement, and therapy was selected
without knowledge of the baseline results. Patients were monitored
monthly for clinical outcomes. Blood cultures for CMV and assessment of
CMV load were performed at 1 and 3 months after enrollment and every 3 months thereafter.
Culture of blood for CMV.
Blood samples for viral culture
were transported to the Virology Laboratory within 60 min of
collection. Cultures for CMV were processed in tubes of MRC-5, WI-38,
and MRHF fibroblasts. Tubes were read daily for CMV-specific cytopathic
effect, and CMV was confirmed by fluorescence microscopy. All
uncontaminated negative cultures were held for 6 weeks before being
recorded as negative (5, 12, 13).
Quantification of CMV DNA in plasma and leukocytes.
Quantification of CMV DNA (viral load) was performed for plasma and
leukocytes by using the COBAS AMPLICOR CMV MONITOR system according to
the manufacturer's instructions (4). Briefly, plasma (200 µl) was added to lysis reagent (600 µl) which contained a
quantitation standard. DNA was precipitated with isopropanol (800 µl)
and pelleted by centrifugation at 16,000 × g for 15 min. The pellet was washed with 70% ethanol and resuspended in
specimen diluent (400 µl).
Leukocytes were separated from EDTA-treated blood by sedimentation with
6% (wt/vol) dextran in phosphate-buffered saline. The leukocyte-rich
supernatant was centrifuged at 450 × g for 15 min, and
the pellet was resuspended in sterile distilled water (5 ml) to lyse
erythrocytes. Culture medium (10 ml of minimal essential medium with
10% fetal calf serum) was added, and the sample was again centrifuged
at 450 × g for 15 min. Erythrocyte lysis with
distilled water was repeated if necessary. Leukocytes were counted,
aliquoted into microcentrifuge tubes (2 × 106
cells/tube), and centrifuged at 12,000 × g for 10 min.
Ice-cold phosphate-buffered saline was added to the cell pellet, and
200 µl containing 8 × 105 cells was transferred to
600 µl of lysis buffer with a quantitation standard. This
concentration of leukocytes had been determined previously to give the
optimal results (13a). Subsequent specimen preparation steps
as described above for plasma were followed.
The extracted sample (50 µl) was added to PCR Master Mix (50 µl)
and placed in the COBAS AMPLICOR instrument for automated
amplification, detection of CMV DNA, and quantitation. This system
amplifies a 365-bp region of the CMV DNA polymerase gene (UL 54).
The
instrument captures the biotinylated amplification products
with
magnetic particles coated with specific oligonucleotide probes
and
detects the bound products calorimetrically. The quantitation
standard
contains DNA with primer binding sites identical to those
of the CMV
target and a unique probe binding region that allows
it to be
distinguished from the CMV amplicon. CMV DNA levels in
the test
specimen are determined by comparing the absorbance of
the specimen to
the absorbance obtained for the quantitation standard.
The dynamic
range for quantitation is approximately 3 log
10 units,
with
400 copies as the lower limit of
detection.
Statistical methods.
Because of the skewed nature of the
distribution of the viral load data, these values were transformed to
log10 for statistical analyses. The relationship between
results for CMV DNA from leukocytes and from plasma was assessed by
linear regression, both on the actual scale and on the log scale, and
the correlation coefficient was computed. McNemar's test for paired
data was used to determine whether there was any difference in the
frequency of positive CMV load measurements between leukocytes and
plasma (18). Kappa statistics were computed to estimate the
agreement beyond that of chance between positive cultures and
detectable CMV load.
| |
RESULTS |
Specimens for comparison of the quantity of CMV DNA in the blood
(CMV load) were available from 199 patient visits by 66 patients with
CMV retinitis. Of these, 44 patient visits were at the time of
diagnosis of CMV retinitis and prior to the initiation of anti-CMV therapy, and 155 visits were follow-up visits, at which time the patients were on treatment for CMV retinitis. Twenty-two patients had
only follow-up visits. The comparison of plasma CMV load and leukocyte
CMV load is outlined in Table 1. In
general, measurements of the CMV load in leukocytes had a greater range
of values than did measurements of the CMV load in plasma. A scatter
plot comparing leukocyte CMV load and plasma CMV load is presented in
Fig. 1. There was a good correlation
between leukocyte CMV load and plasma CMV load (r = 0.81;
P < 0.0001). Because of the large number of specimens with
undetectable CMV loads (<400 copies) from both sources, we also
analyzed the correlation between leukocyte CMV load and plasma CMV load
for the 84 specimens with a detectable viral load by either technique.
In this analysis, there still was a good correlation between leukocyte
and plasma CMV loads (r = 0.79; P < 0.0001).
Because most of the specimens came from follow-up visits, when patients
were on treatment, a large number of specimens had an undetectable CMV
load by either technique. However, the leukocyte CMV load was elevated
(detectable) in a greater proportion of patients than was the plasma
CMV load; 65% of the CMV load measurements from leukocytes were
undetectable, whereas 74% of the measurements from plasma were
undetectable (McNemar's test, P = 0.0009). There was a
barely significant difference between the two sources at follow-up
visits; 79% of follow-up visit measurements from leukocytes were
undetectable, whereas 85% from plasma were undetectable (P = 0.049). Therefore, we analyzed the subgroup of specimens
obtained from patients at the time of diagnosis of CMV retinitis, prior to the initiation of anti-CMV therapy, a time when most patients might
be expected to have a detectable CMV load. At the time of diagnosis of
CMV retinitis, a significantly greater proportion of patients had a
detectable CMV load in leukocytes than in plasma; 82% of patients had
a detectable CMV load in leukocytes, and 64% had a detectable load in
plasma (P = 0.0078). The measurements of CMV loads in
leukocytes at the time of diagnosis of CMV retinitis also had a greater
range of values than did those of loads in plasma (Table 1).
We evaluated the agreement between CMV load measurements and blood
cultures for CMV (Table 2). In general,
if a patient had a positive blood culture for CMV, then the CMV load
was detectable with either leukocytes (94%) or plasma (91%) as the
source, and there were no significant differences between the two
sources (P = 1.00). The results were similar both at
diagnosis of CMV retinitis (95% for plasma and 100% for leukocytes;
P = 1.00) and at follow-up (87% for plasma and 87%
for leukocytes; P = 1.00). However, a CMV load was
detectable for a greater proportion of patients than was a positive
blood culture, whether from plasma (51 specimens) or from leukocytes
(68 specimens). A higher proportion of specimens with a detectable
viral load in plasma showed a positive blood culture for CMV (63%)
than did those with a detectable viral load in leukocytes (49%).
Similar results were seen at the time of diagnosis of CMV retinitis and
at follow-up visits (Table 2). Overall, the agreement between blood
cultures and CMV loads was slightly greater for plasma ( = 0.68)
than for leukocytes ( = 0.53). The agreement was better for plasma
both at the time of diagnosis of CMV retinitis ( = 0.56 for plasma
versus = 0.31 for leukocytes) and at follow-up visits ( = 0.64 for plasma and = 0.48 for leukocytes).
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Comparison of CMV loads in plasma and leukocytes with
blood culture results for patients with CMV retinitis
|
|
| |
DISCUSSION |
Assessment of the amount of HIV RNA present in plasma (HIV load)
has become an important tool in the clinical management of patients
with AIDS, particularly in the era of highly active antiretroviral therapy (7, 8). Similarly, it appears that the assessment of
the amount of CMV DNA in the blood (CMV load) may become a useful tool
for identifying patients at high risk for the development of CMV
disease and for monitoring patients with CMV disease on treatment.
Zipeto et al. (19) reported that the CMV load was elevated
in patients with CMV disease compared to patients without CMV disease,
whether the source of CMV DNA was leukocytes or plasma. Both Shinkai et
al. (17) and Bowen et al. (2) have reported that
qualitative assessment of plasma CMV load correlated with the
subsequent development of CMV disease in patients at high risk for CMV
disease because of their low CD4+-T-cell counts. Bowen et
al. (3) reported that CMV DNA was detectable in blood in
patients with CMV retinitis on treatment and that a high proportion of
patients with a positive plasma CMV load developed new episodes of CMV
disease. These studies all suggested that the assessment of CMV load
could be a useful tool in the management of patients.
In our study we used the Roche COBAS AMPLICOR assay for CMV load to
compare the results with plasma and leukocytes as the sources of CMV
DNA. Measurement of the CMV load in leukocytes detected CMV DNA in the
blood in a greater proportion of patients both at the time of diagnosis
of CMV retinitis and during follow-up and had a greater range of values
than did measurement of the CMV load in plasma. The two sources use
different denominators (milliliters of plasma versus 106
leukocytes) and therefore are not directly comparable. However, given
the typical number of leukocytes per milliliter of blood (106 to 107), a calculated comparison likely
would show an even greater disparity. However, measurements for the two
sources were highly correlated, as indicated by the high value of the
correlation coefficient.
Viral load measurements can be performed more rapidly than cultures for
CMV. Both sources of CMV DNA for measurement of the CMV load were
successful in identifying patients who had positive blood cultures for
CMV, and there were no significant differences between assays with
leukocytes and plasma in the ability to identify patients with a
positive blood culture either at diagnosis of CMV retinitis or during
follow-up. Conversely, a CMV load from either source was detectable for
a greater proportion of patients than was a positive blood culture for
CMV, and leukocyte CMV loads were detectable in a greater proportion of
blood culture-negative patients than were plasma CMV loads, resulting
in better agreement (higher 's) between blood cultures and plasma
CMV loads than between blood cultures and leukocyte CMV loads. Boivin
et al. (1) compared plasma CMV load and leukocyte CMV load
in a group of patients with and without CMV disease. They concluded
that quantitative leukocyte CMV load was superior to qualitative plasma CMV load for detecting patients with CMV disease, based upon a superior
specificity and positive predictive value. However, their quantitative
assay used a threshold of detection of 16,000 copies, which is
substantially different from ours, and our study evaluated only
patients with CMV disease at diagnosis and during follow-up. Whether
the greater number of patients detected by leukocyte CMV load will
prove to be more clinically useful remains to be determined. Long-term
follow-up studies of a greater number of patients might determine
whether assessment of the CMV load in plasma or in leukocytes will be
better correlated with clinical outcomes, such as progression of
retinitis and occurrence of resistant CMV.
| |
ACKNOWLEDGMENTS |
This study was supported by grant EY-10268 from the
National Eye Institute, National Institutes of Health. Roche Molecular Systems, Inc., provided the equipment and reagents.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 550 North
Broadway, Suite 700, Baltimore, MD 21205. Phone: (410) 955-1966. Fax:
(410) 955-0629. E-mail:
dajabs{at}welchlink.welch.jhu.edu.
Other group members include John Bartlett, Stephen G. Bolton, James
P. Dunn, Susan LaSalvia, Laura G. Neisser, Earline Nanan, Richard D. Semba, John H. Kempen, Quan D. Nguyen, Shirley Quaskey, Judy Southall,
Joseph B. Margolick, Elvia Ramirez, Matthew D. Davis, Larry Hubbard,
Judy Brickbauer, Linda Kastorff, and Tamieca Hamlin.
| |
REFERENCES |
| 1.
|
Boivin, G.,
J. Handfield,
E. Toma,
G. Murray,
R. Lalonde, and M. G. Bergeron.
1998.
Comparative evaluation of the cytomegalovirus DNA load in polymorphonuclear leukocytes and plasma of human immunodeficiency virus-infected subjects.
J. Infect. Dis.
177:355-360[Medline].
|
| 2.
|
Bowen, E. F.,
C. A. Sabin,
P. Wilson,
P. D. Griffiths,
C. C. Davey,
M. A. Johnson, and V. C. Emery.
1997.
Cytomegalovirus (CMV) viraemia detected by polymerase chain reaction identifies a group of HIV-positive patients at high risk of CMV disease.
AIDS
11:889-893[Medline].
|
| 3.
|
Bowen, E. F.,
V. C. Emery,
P. Wilson,
M. A. Johnson,
C. C. Davey,
C. A. Sabin,
D. Farmer, and P. D. Griffiths.
1998.
Cytomegalovirus polymerase chain reaction viraemia in patients receiving ganciclovir maintenance therapy for retinitis.
AIDS
12:605-611[Medline].
|
| 4.
|
DiDomenico, N.,
H. Link,
R. Knobel,
T. Caratsch,
W. Weschler,
Z. G. Loewy, and M. Rosenstraus.
1996.
COBAS AMPLICORTM: fully automated RNA and DNA amplification and detection system for routine diagnostic PCR.
Clin. Chem.
42:1915-1993[Abstract/Free Full Text].
|
| 5.
|
Enger, C.,
D. A. Jabs,
J. P. Dunn,
M. S. Forman,
N. M. Bressler,
J. B. Margolick, and P. Charache for the CRVR Research Group.
1997.
Viral resistance and CMV retinitis: design and methods of a prospective study.
Ophthalmic Epidemiol.
4:41-48[Medline].
|
| 6.
|
Gallant, J. E.,
R. D. Moore,
D. D. Richman,
J. Keruly, and R. E. Chaisson.
1992.
Incidence and natural history of cytomegalovirus disease in patients with advanced human immunodeficiency virus disease treated with zidovudine. The Zidovudine Epidemiology Group.
J. Infect. Dis.
166:1223-1227[Medline].
|
| 7.
|
Gulick, R. M.,
J. W. Mellors,
D. Havlir,
J. J. Eron,
C. Gonzalez,
D. McMahon,
D. D. Richman,
F. T. Valentine,
L. Jonas,
A. Meibohm,
E. A. Emini, and J. A. Chodakewitz.
1997.
Treatment with indinavir, zidovudine, and lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy.
N. Engl. J. Med.
337:734-739[Abstract/Free Full Text].
|
| 8.
|
Hammer, S. M.,
K. E. Squires,
M. D. Hughes,
J. M. Grimes,
L. M. Demeter,
J. S. Currier,
J. J. Eron, Jr.,
J. E. Feinberg,
H. H. Balfour, Jr.,
L. R. Deyton,
J. A. Chodakewitz, and M. A. Fischl.
1997.
A controlled trial of two nucleoside analogues plus indinavir in persons with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic millimeter or less.
N. Engl. J. Med.
337:725-733[Abstract/Free Full Text].
|
| 9.
|
Hiyoshi, M.,
S. Tagawa,
T. Takubo,
K. Tanaka,
T. Nakao,
Y. Higeno,
K. Tamura,
M. Shimaoka,
A. Fujii,
M. Higashihata,
Y. Yasui,
T. Kim,
A. Hiraoka, and N. Tatsumi.
1997.
Evaluation of the AMPLICOR CMV test for direct detection of cytomegalovirus in plasma specimens.
J. Clin. Microbiol.
35:2692-2694[Abstract].
|
| 10.
|
Hoover, D. R.,
Y. Peng,
A. Saah,
R. Semba,
R. R. Detels,
C. R. Rinaldo, Jr., and J. P. Phair.
1996.
Occurrence of cytomegalovirus retinitis after human immunodeficiency virus immunosuppression.
Arch. Ophthalmol.
114:821-827[Abstract/Free Full Text].
|
| 11.
|
Jabs, D. A.
1995.
Ocular manifestations of HIV infection.
Trans. Am. Ophthalmol. Soc.
93:623-683[Medline].
|
| 12.
|
Jabs, D. A.,
J. P. Dunn,
C. Enger,
M. S. Forman,
N. Bressler, and P. Charache for the CMV Retinitis and Viral Resistance Study Group.
1996.
Cytomegalovirus retinitis and viral resistance. 2. Prevalence of resistance at diagnosis.
Arch. Ophthalmol.
114:809-814[Abstract/Free Full Text].
|
| 13.
|
Jabs, D. A.,
C. L. Enger,
J. P. Dunn, and M. S. Forman for the CMV Retinitis and Viral Resistance Study Group.
1998.
Cytomegalovirus retinitis and viral resistance. 4. Ganciclovir resistance.
J. Infect. Dis.
177:770-773[Medline].
|
| 13a.
| Kao, S. Y. Personal communication.
|
| 14.
|
Mellors, J. W.,
G. P. Rinaldo, Jr.,
R. M. White,
J. A. Todd, and L. A. Kingsley.
1996.
Prognosis in HIV-1 infection predicted by the quantity of virus in plasma.
Science
272:1167-1170[Abstract].
|
| 15.
|
Palella, F. J., Jr.,
K. M. Delaney,
A. C. Moorman,
M. O. Loveless,
J. Fuhrer,
G. A. Satten,
D. J. Aschman,
S. D. Holmberg, and The HIV Outpatients Study Investigators.
1998.
Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection.
N. Engl. J. Med.
338:853-860[Abstract/Free Full Text].
|
| 16.
|
Peter, P.,
R. E. Hirschtick,
J. Phair,
J. S. Chmiel,
L. Poggensee, and R. Murphy.
1992.
Risk of developing cytomegalovirus in persons infected with the human immunodeficiency virus.
J. Acquired Immune Defic. Syndr.
5:1069-1074.
|
| 17.
|
Shinkai, M.,
S. A. Bozzette,
W. Powderly,
P. Frame, and S. A. Spector.
1997.
Utility of urine and leukocyte cultures and plasma DNA polymerase chain reaction for identification of AIDS patients at risk for developing human cytomegalovirus disease.
J. Infect. Dis.
175:302-308[Medline].
|
| 18.
|
Siegel, S.
1956.
The McNemar test for the significance of changes, p. 63-67.
In
H. Harlow (ed.), Nonparametric statistics for the behavioral sciences. McGraw-Hill Book Company, New York, N.Y.
|
| 19.
|
Zipeto, D.,
S. Morris,
C. Hong,
A. Dowling,
R. Wolitz,
T. C. Merigan, and L. Rasmussen.
1995.
Human cytomegalovirus (CMV) DNA in plasma reflects quantity of CMV DNA present in leukocytes.
J. Clin. Microbiol.
33:2607-2611[Abstract].
|
Journal of Clinical Microbiology, May 1999, p. 1431-1435, Vol. 37, No. 5
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Diaz-Mitoma, F., Leger, C., Miller, H., Giulivi, A., Frost, R., Shaw, L., Huebsch, L.
(2003). Comparison of DNA Amplification, mRNA Amplification, and DNA Hybridization Techniques for Detection of Cytomegalovirus in Bone Marrow Transplant Recipients. J. Clin. Microbiol.
41: 5159-5166
[Abstract]
[Full Text]
-
Tong, C. Y. W., Cuevas, L. E., Williams, H., Bakran, A.
(2000). Comparison of Two Commercial Methods for Measurement of Cytomegalovirus Load in Blood Samples after Renal Transplantation. J. Clin. Microbiol.
38: 1209-1213
[Abstract]
[Full Text]
Top
Abstract
Introduction
