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Journal of Clinical Microbiology, June 2001, p. 2364-2365, Vol. 39, No. 6
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2364-2365.2001
LETTERS TO THE EDITOR
LightCycler-Based Quantitative PCR for Detection of
Cytomegalovirus in Blood, Urine, and Respiratory Samples
 |
LETTER |
In response to a recently published article by Schaade et al.
(12) describing the value of LightCycler technology for
quantitative analysis of cytomegalovirus (CMV) in clinical material, we
wish to add our experience. Using a different LightCycler (LC; Idaho Technology Inc., Idaho Falls, Idaho), we too have developed a real-time
quantitative PCR (QPCR) assay for CMV (11). Our assay targets a shorter fragment of the glycoprotein B gene (150 bp) and
incorporates a cyanine 5-labeled reverse primer and a sequence-specific fluorescein-labeled hybridization probe, allowing the identification of
PCR product by fluorescence resonance energy transfer. We have designed
a quantitative standard (plasmid cloned) with a 2-bp mismatch in the
probe-binding region to facilitate its differentiation from CMV target
sequence by using melting curve analysis (2, 3). The assay
is sensitive (detection limit 
10 copies), with an overall dynamic
range of 2 × 103 to 5 × 108 copies/ml. When
applied to blood samples, the results compared well with an in-house
modification of a qualitative assay (5) and the
quantitative data correlated (r = 0.88; P < 0.001) with an independent TaqMan-based QPCR assay (9).
PCR has previously proved suitable for the detection and quantification
of CMV DNA in urine (4, 7, 10), whereas standard detection
of early antigen fluorescent foci (DEAFF) testing (8) is
slow (up to 72 h), lacks sensitivity, and may be hampered by cytotoxicity (1). We therefore sought to extend the
clinical utility of our assay to the examination of urine and
respiratory samples (including sputum, bronchoalveolar lavage fluid,
and nasopharyngeal secretion) to aid the identification of patients at
risk of developing CMV disease. We have tested such specimens from
pediatric and adult transplant patients, including 46 urine samples
(assayed directly, without DNA extraction) and 86 respiratory samples
(extracted using the QIAamp DNA minikit; Qiagen, West Sussex, United Kingdom).
QPCR proved to be more sensitive than DEAFF testing, giving three- and
five-fold increases in the positivity rate in urine and respiratory
samples, respectively, with viral loads ranging from <2 × 103 to 1 × 108 copies/ml (Table
1). All samples were readily evaluated by
PCR, whereas 21 of 132 samples (16%) were toxic in the DEAFF test, 7 (33.3%) of which were PCR positive. DNA extraction of urine samples
did not improve the positivity rate in our hands (unpublished data),
suggesting that direct detection of CMV DNA in urine will suffice, as
previously reported (4, 7).
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Comparison of LightCycler real-time PCR and DEAFF testing
for detection of CMV in urine and respiratory
samples
|
|
Our data confirm and extend those of Schaade et al. (12).
The LC QPCR assay is rapid (<2 h), provides timely results, and is
suitable for the detection of CMV DNA in a range of clinical specimens.
The application of LC-based QPCR to testing urine, respiratory, and
other specimens, in addition to the determination of CMV viral load in
blood, may assist in the early identification of patients with a high
risk of progression to CMV disease and in determining appropriate
parameters for therapeutic intervention. Prospective monitoring, using
QPCR technology to determine the rate of increase in viral load, may
refine attempts to identify patients at greatest risk of CMV disease
(6).
| |
FOOTNOTES |
*
Phone: 44 191 2261074 Fax: 44 191 2260365 E-mail:
newakear{at}north.phls.nhs.uk
| |
REFERENCES |
| 1.
|
Aitken, C.,
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1999.
Use of molecular assays in diagnosis and monitoring of cytomegalovirus disease following renal transplantation.
J. Clin. Microbiol.
37:2804-2807[Abstract/Free Full Text].
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M. J. Lay, and C. T. Wittwer.
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Anal. Biochem.
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Use of real-time PCR and fluorimetry to detect lamivudine resistance-associated mutations in hepatitis B virus.
Antimicrob. Agents Chemother.
43:1600-1608[Abstract/Free Full Text].
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Cope, A. V.,
P. Sweny,
C. Sabin,
L. Rees,
P. D. Griffiths, and V. C. Emery.
1997.
Quantity of cytomegalovirus viruria is a major risk factor for cytomegalovirus disease after renal transplantation.
J. Med. Virol.
52:200-205[CrossRef][Medline].
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| 5.
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Darlington, J.,
M. Super,
K. Patel,
J. E. Grundy,
P. D. Griffiths, and V. C. Emery.
1991.
Use of the polymerase chain reaction to analyse sequence variation within a major neutralising epitope of glycoprotein B (gp58) in clinical isolates of human cytomegalovirus.
J. Gen. Virol.
72:1985-1989[Abstract/Free Full Text].
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| 6.
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Emery, V. C.,
C. A. Sabin,
A. V. Cope,
D. Gor,
A. F. Hassan-Walker, and P. D. Griffiths.
2000.
Application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation.
Lancet
355:2032-2036[CrossRef][Medline].
|
| 7.
|
Fox, J.,
C. M. Kidd,
P. D. Griffiths,
P. Sweny, and V. C. Emery.
1995.
Longitudinal analysis of cytomegalovirus load in renal transplant recipients using a quantitative polymerase chain reaction: correlation with disease.
J. Gen. Virol.
76:309-319[Abstract/Free Full Text].
|
| 8.
|
Griffiths, P. D.,
D. D. Panjwani,
P. R. Stirk,
M. G. Ball,
M. Ganezakowski,
H. A. Blacklock, and H. G. Prentice.
1984.
Rapid diagnosis of cytomegalovirus infection in immunocompromised patients by detection of early antigen fluorescent foci.
Lancet
ii:1242-1245.
|
| 9.
| Guiver, M., A. J. Fox, K. Mutton, N. Mogulkoc, and J. Egan. Comparative evaluation of CMV viral load using TaqMan CMV
quantitative PCR with CMV antigenaemia in heart and lung transplant
recipients. Transplantation, in press.
|
| 10.
|
Jones, R. N.,
M. L. Neal,
B. Beattie,
D. Westmoreland, and J. D. Fox.
2000.
Development and application of a PCR-based method including an internal control for diagnosis of congenital cytomegalovirus infection.
J. Clin. Microbiol.
38:1-6[Abstract/Free Full Text].
|
| 11.
| Kearns, A. M., M. Guiver, V. James, and J. King.
Development and evaluation of a real-time quantitative PCR for the
detection of human cytomegalovirus. J. Virol. Methods, in press.
|
| 12.
|
Schaade, L.,
P. Kockelkorn,
K. Ritter, and M. Kleines.
2000.
Detection of cytomegalovirus DNA in human specimens by LightCycler PCR.
J. Clin. Mirobiol.
38:4006-4009[Abstract/Free Full Text].
|
| | | | |
Angela M. Kearns*
Brenda Draper
Wendy Wipat
Andrew J. L. Turner
Janice Wheeler
Roger Freeman
Newcastle Public Health
Laboratory
Newcastle General Hospital, Westgate
Rd.
Newcastle upon Tyne NE4 6BE, United
Kingdom
|
| | | | |
Jayne Harwood
F. Kate Gould
John H. Dark
Freeman
Hospital Freeman Road, High Heaton, Newcastle upon
Tyne NE7 7DN, United Kingdom
|
| |
AUTHORS' REPLY |
The findings of Kearns et al. focus on the detection of CMV DNA in
urine and respiratory samples and on the prospective monitoring of CMV
load for the prediction of CMV disease with the LightCycler instrument.
Indeed, we are convinced that these approaches will lead to the early
and specific diagnosis of CMV disease and the evaluation of appropriate
therapeutic options, too. Thus, the successful application of the
LightCycler technology for the detection and quantification of CMV DNA
from various materials is important. The quantitative measurement of
CMV DNA in saliva, respiratory swabs, urine, and blood will
enlighten the relation between local CMV replication and
systemic infection.
The speed of the LightCycler system will, on the one hand, support
insights in CMV pathogenesis and replication and, on the other, help to
establish the wide clinical use of the application of CMV load kinetics
for the prediction of CMV disease (1), an approach that
will be of major relevance in the future.
The anticipated short-term CMV DNA follow-up of risk patients will
require the automated extraction of CMV DNA from variant types of
samples. The clinical evaluation of principles that are suitable for
automated nucleic acid extraction from clinical samples and the
evaluation of extraction instruments themselves are needed now.
Otherwise, the prospective monitoring of CMV DNA load for the
identification of risk patients will not be established as a standard
procedure due to economically restricted man power and laboratory capacity.
| |
FOOTNOTES |
*
Phone: 49 241 8088460 Fax: 49 241 8888483 E-mail:
mkleines{at}post.klinikum.rwth-aachen.de
| |
REFERENCE |
| 1.
|
Emery, V. C.,
C. A. Sabin,
A. V. Cope,
D. Gor,
A. F. Hassan-Walker, and P. D. Griffiths.
2000.
Application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation.
Lancet
355:2032-2036.
|
| | | | |
Lars Schaade
Michael Kleines*
Division of Virology
Department of Medical
Microbiology
University Hospital
RWTH
Aachen
D-52057 Aachen, Germany
|
Journal of Clinical Microbiology, June 2001, p. 2364-2365, Vol. 39, No. 6
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2364-2365.2001
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