INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The presence of cytomegalovirus (CMV) DNA in blood as detected by PCR is used to monitor transplant patients at risk of active CMV infection (for reviews, see references 1 and 16). Both peripheral blood leukocyte (PBL) and plasma fractions are used for CMV PCR, but the clinical significance of results varies and is dependent on the PCR approach. By quantitative PCR techniques, demonstration of high-level CMV leukocyte DNAemia and plasma DNAemia usually precedes the development of clinical features. Although levels of leukocyte DNAemia and plasma DNAemia are usually well correlated, CMV DNA load is considerably higher in PBLs, which can reasonably be explained by the strong cell association of the virus (2, 6, 18, 22). As the performance of quantitative PCR is technically demanding and cost-intensive, which limits its high-throughput use, qualitative CMV PCR assays with reliable significance remain desirable. Leukocyte DNAemia, however, is often detected in CMV immunoglobulin G (IgG)-positive patients with no further evidence of active CMV infection, whereas a positive CMV PCR with plasma has been reported to be more specifically associated with clinical manifestations (1, 16).

Despite the important diagnostic role of CMV plasma DNAemia, its biological properties are not fully understood. One notion is that it reflects active virus replication (20-22). On the other hand, the high abundance of CMV leukocyte DNAemia in actively CMV-infected patients has led to the hypothesis that plasma DNAemia may be, at least partly, a result of PBL lysis (6, 22). If cell turnover was a major mechanism that elicits CMV plasma DNAemia, this would suggest that in patients with CMV leukocyte DNAemia long-term storage of whole blood samples before separation may cause release of viral DNA into the plasma fraction, thus distorting PCR results. This question is of great importance not only in the clinical setting but also for test reproducibility and standardization, especially since commercial kits for CMV PCR of plasma samples are available (3, 10). Some investigators argue that contamination of freshly prepared plasma fractions with cellular DNA can be overcome by ultrafiltration, whereas others have reported that PCR for cellular target sequences is regularly positive even with ultrafiltered samples (6, 20, 21). None of these investigations, however, tested the effect of delays in sample processing under controlled conditions. This problem has been addressed in the present study.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Patients. The blood samples from kidney allograft recipients investigated in this study were obtained between days 18 and 66 posttransplantation. Sixty CMV IgG-positive recipients with CMV leukocyte DNAemia were enrolled in the study. Of these, 30 patients were latently infected with CMV and 30 experienced active CMV infection (see below for definitions). Of the latently infected patients, 19 received a graft from a CMV-seronegative donor; 21 of the donors among the recipients who became actively infected with CMV were seronegative. Ten CMV IgG-positive patients without leukocyte DNAemia (seven donors were seronegative) and five CMV IgG-negative patients (all five donors were seronegative) served as controls. To rule out factors apart from the storage conditions which might influence CMV PCR results, patients receiving antiviral therapy at the time of the investigation were excluded.

Blood sample processing. Twenty milliliters of whole blood was freshly collected from each participant and placed into tubes that contained EDTA. One-milliliter aliquots were prepared immediately postdrawing. One aliquot was processed right away for baseline PCR of PBLs and plasma, and one was used for the antigenemia assay (see below). The remaining unprocessed sample aliquots were stored at room temperature (RT) and at 4°C. From these, PBLs and plasma were prepared for PCR analysis after storage for 2, 4, 6, 12, and 24 h.

PCR assays. CMV DNA was quantitated in all leukocyte and plasma extracts. To assess the role of cell lysis, samples were additionally tested for the single-copy human -globin gene. Target sequences were quantitated by competitive PCR with cloned standard (ST) sequences as described previously (11, 14), with slight modifications. The CMV ST sequence was generated in a PCR by site-directed mutagenesis and was subcloned into the vector pSPT19 (Boehringer Mannheim, Mannheim, Germany) (14). It contained three successive point mutations within the amplified coding region of CMV glycoprotein B compared to the sequence derived from laboratory strain AD169 (4). The cloning of the -globin ST sequence was described previously (9).

Antigenemia assay. Aliquots of 10⁵ PBLs obtained from freshly prepared sample aliquots were centrifuged in duplicate onto glass slides, fixed, and permeabilized with 5% paraformaldehyde-0.5% Nonidet P-40, and pp65 antigens were detected in polymorphonuclear cells by indirect immunofluorescence as described previously (5) by using Clonab CMV (Biotest, Dreieich, Germany) according to the manufacturer's instructions.

Statistical analyses. The frequencies of positive results of the CMV PCR and the -globin PCR for the plasma samples were compared between different groups by the ² test. The frequencies of positive -globin PCRs were compared between CMV PCR-positive and CMV PCR-negative samples of the same groups by the McNemar test. Quantities of CMV and -globin target sequences were compared (i) in PBLs and in plasma over the storage period by the Friedman test combined with the Wilcoxon rank test, (ii) in PBLs and in plasma between actively and latently CMV-infected patients by the Mann-Whitney U test, and (iii) between native and ultrafiltered plasma samples also by the Mann-Whitney U test. The correlation of the number of CMV DNA copies in PBLs with those in plasma fractions was determined by Spearman regression analysis.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

The effects of storage time and ultrafiltration on PCR results presented here were found to be independent of the storage temperature. Therefore, detailed data are shown only for sample aliquots stored at RT.

Qualitative CMV PCR of plasma. The influence of storage time and ultrafiltration on plasma CMV PCR results for the samples from 60 patients with leukocyte DNAemia (30 were latently CMV infected, and were 30 actively infected) is depicted in Fig. 1. At the baseline, among the native plasma samples only 10% from the latently CMV-infected group but 87% from the actively infected patients were CMV PCR positive (P < 0.001). Similar observations were made with the ultrafiltered plasma samples (3% positive for latently infected patients and 80% positive for actively infected individuals [P < 0.001]). In actively CMV-infected patients, the total rates of positive CMV PCRs did not increase significantly over the storage period, and no differences in the overall frequencies of positive CMV PCRs were evident between native and ultrafiltered samples. In contrast, among patients with latent CMV infection, the frequency of positive CMV PCR results was significantly higher than the baseline value by 4 h for native plasma samples (37% [P = 0.033]) and by 6 h for ultrafiltered samples (27% [P = 0.03]). The differences between actively and latently infected patients in the frequencies of positive CMV PCRs lost statistical significance by 12 and 24 h of storage regarding native and ultrafiltered plasma specimens, respectively.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Positivity rates for CMV PCR of plasma samples obtained from latently (A) and actively (B) CMV-infected patients, dependent on storage time (at RT) and ultrafiltration (N, native samples; F, ultrafiltered samples). Black bars, positivity both by CMV PCR and by -globin PCR; white bars, positivity by CMV PCR but negativity by -globin PCR. The storage times at which the frequencies of positivity of the CMV PCR became significantly different from the frequencies at the baseline are marked by asterisks.

Quantitative CMV PCR of plasma. To further analyze the data obtained by qualitative PCR, the kinetics of CMV DNA and -globin DNA were determined in all PBL and plasma sample aliquots over the storage period.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Effect of storage time (at RT) and of ultrafiltration on quantitative CMV PCR of plasma samples. The copy numbers are for 10 µl of plasma. Median values are shown for latently () and actively () CMV-infected patients. Error bars span the maximum and minimum values. The storage times at which differences from the baseline results became statistically significant are marked by asterisks.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In latently CMV-infected renal allograft recipients CMV PCR performed with plasma yields false-positive results with significant frequency when specimens are prepared with delay, regardless of the storage temperature. In contrast, storage factors do not seem to have any relevant effect on the results of CMV PCR for patients with active CMV infection. These findings have important implications for the diagnostic significance of plasma PCR.

The presence of CMV leukocyte DNAemia proved to be necessary for positive plasma PCR results. As isolated CMV leukocyte DNAemia in transplant recipients is rated as a pathological condition per se by some investigators (1), it must be noted that in this study the definition of latent CMV infection in leukocyte DNAemia-positive patients ruled out any episodes of pp65 antigenemia, virus shedding from body fluids, or CMV-related symptoms during the stay at hospital. In contrast, truly active CMV infection was assumed only when pp65 antigenemia was confirmed, because the prognostic significance of CMV DNAemia is dubious in pp65-negative patients with other signs of virus activity, e.g., virus isolation from urine or throat swabs (11, 19).

Fundamentally different effects of storage on plasma CMV PCR were observed between plasma from latently and actively CMV-infected patients. The data suggest that lysis of latently infected PBLs is the driving force for CMV plasma DNAemia in patients with latent infection but that this does not play a key role in active CMV infection.

In latently infected leukocytes, CMV DNA has been localized to the cell nucleus (7). Preanalytical turnover of these cells, with nucleus-associated DNA entering the plasma fraction, provides a reasonable explanation for the finding that the plasma CMV DNAemia that was low grade at early storage times continuously rose over time and strongly diminished with ultrafiltration, in parallel with -globin DNA levels in plasma. Accordingly, low absolute CMV copy numbers in PBLs and low ratios of CMV DNA in PBLs/CMV DNA in plasma gradually diminished over time.

Interpretation of the data for the actively infected patients is more complex. Plasma CMV DNA representing cell-free target sequences (6, 20, 21) is the most obvious explanation for the high-grade plasma CMV DNAemia at the baseline which remained stable over time; therefore, it did not correlate with -globin DNA kinetics and was largely uninfluenced by ultrafiltration. Moreover, CMV PCR-positive but -globin PCR-negative samples were present throughout storage. The notion of cell-free virus would fit reports on the recovery of infectious virus from the plasma fraction (6, 20). The release of virus DNA into the plasma fraction from other surrounding sites of infection (e.g., endothelial cells) is conceivable.

However, cell-free virus is not the only possible explanation. The significant correlation of CMV DNA copies between PBLs and plasma and the similar ratios of CMV DNA in PBLs/CMV DNA in plasma throughout storage suggests that plasma DNAemia in actively infected patients is also, to some extent, leukocyte associated (6, 18, 22). This is not necessarily contradictory to the statements made above because the CMV DNA circulating in PBLs during active infection has been localized to the cytoplasm (7) rather than the nucleus, most likely as a result of virus uptake from surrounding sites of replication. Accordingly, in actively infected patients CMV DNA and -globin DNA behaved consistently different in terms of ultrafiltrability. During CMV dissemination there may be a steady state in the bloodstream between the viral components present in the cytoplasms of cells and in the plasma fraction, leading to a comparatively high plasma CMV DNA load. Such a hypothesis could explain why, despite the high CMV DNA load in PBLs, the plasma CMV levels found at the baseline increased only marginally during storage, although PBL lysis must have resulted in the release of cytoplasmic CMV DNA.

Although samples of actively CMV-infected patients probably also contained CMV DNA derived from latently infected PBLs which entered the plasma over time, this was unlikely to have a great influence on the interpretation of the results because the levels of latent CMV DNA derived from PBLs (predominantly mononuclear cells) are very low in both actively and latently CMV-infected patients (18).

The -globin PCR data may reconcile some discrepant previous statements. Gerna et al. (6) regularly detected -globin DNA by qualitative PCR in plasma samples after ultrafiltration using a highly efficient DNA extraction method. Others have reported the loss of -globin signals by ultrafiltration, but the investigators used a proteinase K-based extraction protocol which refrained from DNA precipitation (20, 21). Therefore, the most likely reason for these discrepancies is the different sensitivities of the methods. In agreement with this interpretation, quantitation of -globin DNA in our study revealed that only a limited amount of cellular material was effectively ultrafiltered.

The preanalytical pitfalls of plasma CMV PCR may be overcome by alternative PCR approaches that are less error prone. First, qualitative CMV PCR of polymorphonuclear leukocyte fractions depleted of the mononuclear cells by Ficoll density centrifugation is promising for the monitoring of renal allograft recipients (18). Its specificity is comparable to that plasma PCR and it is even more sensitive than plasma PCR (15), and storage of unprocessed samples for at least 72 h has no effect on the results (17). Second, serum CMV PCR is supposed to be equivalent to plasma PCR for prediction of active infections (8, 13) and therefore should also be investigated for the adverse effects of delayed sample preparation.

In conclusion, it has been demonstrated that a mere positive CMV PCR result obtained with the plasma of a renal allograft patient with CMV leukocyte DNAemia should be interpreted with extreme caution. For quantitative CMV PCR delays in specimen processing are probably less crucial. It is urgently necessary to take notice of this matter in the clinical setting and when any measures for standardization of plasma CMV PCR procedures are devised.