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 (Table1). 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 DNA extraction of urine samples did not improve the positivity rate in our hands (unpublished data), suggesting that direct detection of CMV DNA in urine will suffice, as previously reported (4, 7).
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
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 (1-1), 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
- 1-1.↵
- Copyright © 2001 American Society for Microbiology
