SIGNIFICANCE OF HUMAN CYTOMEGALOVIRUS DNA DETECTION IN IMMUNOCOMPROMISED HEART TRANSPLANT PATIENTS

Abstract

Peripheral blood samples (n=1240), obtained at variable intervals from 483 heart transplantation (HTx) patients under immunosuppressive therapy, and blood samples (n=1013) obtained upon blood donation from 1013 healthy anti-human cytomegalovirus (HCMV) positive blood donors, were tested for HCMV DNA by nested polymerase chain reaction (PCR). The detection limit of the nested PCR was determined to be less than 10 copies of the plasmid pRR 47, containing the HCMV immediate early gene.

HCMV DNA was detected in 79 of 483 HTx patients (17%). To the contrary, HCMV DNA was only detected in 1 of 1013 anti-HCMV positive, healthy blood donors (0.1%). This PCR positive donor had recently contracted a primary HCMV infection. The rate of HCMV PCR positive immunosuppressed HTx patients in our study was lower than the rate of HCMV PCR positive healthy blood donors in previous reports in the literature.

Blood samples (269 from 117 HTx patients) were as-sayed for HCMV DNA in peripheral blood leukocytes, HCMV DNA in plasma, and HCMV tegument protein 65 kDa (pp 65 antigen). Three laboratory diagnostic patterns were observed and related to clinical findings: (1) HCMV DNA only in leukocytes was observed in 26 patients, 7 of whom had HCMV disease, 5 of whom had graft rejection, and 14 of whom had no specific symptoms; (2) HCMV DNA both in leukocytes and in plasma (viremia) was observed in 3 patients, who were all symptomatic with HCMV disease; (3) HCMV DNA in leukocytes and in plasma (viremia) and pp 65 antigen were observed in 13 patients, all of whom were symptomatic (10 patients had HCMV disease, and 3 patients had graft rejection).

A similar sequence of diagnostic patterns was observed in all symptomatic HCMV infections and reactivations in this study: HCMV DNA appeared first in peripheral blood leukocytes, then also in plasma, followed by pp 65 antigen detectable in peripheral blood leukocytes. Upon clinical recovery, these findings disappeared in reverse order. However, HCMV DNA remained detectable in peripheral blood leukocytes for several weeks.

The detection of HCMV DNA in the peripheral blood is an exception, not the rule, even in severely immunosuppressed HTx patients. It indicates a pathological condition, albeit without clinical symptoms in some patients, and it is the earliest signal of HCMV replication. Of 42 patients in whom HCMV DNA was initially detected only in peripheral blood leukocytes, 16 patients progressed into viremia. Thus, HCMV-specific PCR performed on nucleic acid extracts from lysed peripheral blood is an appropriate method for the monitoring of HCMV infections in immunosuppressed HTx patients.

Human cytomegalovirus (HCMV^*) infection causes serious disease in immunosuppressed transplant patients ^{(1, 2)}. Laboratory diagnosis of HCMV infections and reactivations in their early stage is therefore required, ideally before the onset of major viremia. Anti-HCMV antibody detection is not suitable for this purpose; virus detection by cell culture is tedious and slow. Immunocytochemical detection of HCMV antigens (e.g., HCMV tegument protein 65 kDa [pp 65 antigen]) in peripheral blood has frequently been employed for the diagnosis of HCMV infections and reactivations ^(3-13). However, cytochemically detectable antigenemia is usually already the consequence of considerable virus replication ⁽¹⁴⁾, and many patients are already symptomatic at this stage. An earlier detection of virus replication would enable close monitoring of those patients at risk to develop symptomatic HCMV disease, and a decision about antiviral therapy may be reached earlier. The recent development of the polymerase chain reaction (PCR) for the in vitro amplification of nucleic acids ⁽¹⁵⁾ enables the detection of HCMV DNA at the level of single genome copies. Results may be obtained within 1 working day ⁽¹⁶⁾. Therefore, PCR seems to be a promising method for the monitoring of immunosuppressed and immunocompromised patients for HCMV infections and reactivations ^{(5, 8, 14, 17-36)}. Under the assumption that HCMV does not permanently persist in the peripheral blood at levels detectable by PCR, HCMV PCR should be the ideal method to detect HCMV infections or reactivations in their very early stage.

However, inconsistent data are presented in the literature about the detection of HCMV DNA in the peripheral blood of healthy individuals ^{(16, 37-43)}, about the significance of HCMV DNA detection in the peripheral blood of immunosuppressed or immunocompromised patients ^{(5, 7, 8, 14, 17-30, 32, 33, 36, 39, 44)}, and about the mode of virus persistence ^{(38, 42, 45-52)}.

In 2 studies with healthy blood donors, no HCMV DNA was detected in peripheral blood samples ^{(41, 43)}. No HCMV was detected in the peripheral blood of other healthy volunteers ⁽³⁹⁾ and even in HIV-infected individuals ⁽³²⁾. In other studies, HCMV DNA was frequently detected in peripheral blood, even in anti-HCMV negative blood donors ^{(16, 37, 38, 40, 42)}(Table 1).

Incoherent results were also reported from the monitoring of different groups of immunosuppressed transplant patients for HCMV in the peripheral blood ^{(5, 7, 8, 14, 19, 23, 26-28, 30, 33, 35, 44, 53)}. Although HCMV PCR is more sensitive and detects HCMV infections and reactivations earlier than pp 65 antigen detection ^{(5, 8, 14, 27, 28, 30, 35, 36, 53)}, the clinical significance of a positive HCMV PCR as compared to cyomegalovirus antigenemia (e.g., pp 65 antigen detection) in the peripheral blood is still unclear. Good correlation between clinical findings and pp 65 antigen detection in the peripheral blood was observed in renal transplant patients ⁽⁵⁴⁾ who usually receive less immunosuppresive drugs than HTx patients.

Many authors agree that the presence of HCMV DNA in the peripheral blood is a pathological condition ^{(14, 18-21, 30, 33, 55-57)}, and some authors recommend HCMV PCR for patient monitoring, because HCMV DNA is detectable several days before the onset of antigenemia ^{(7, 14, 19, 23, 26, 27, 33, 36, 53)}. Although 1 single positive HCMV PCR assay does not necessarily predict HCMV disease, negative HCMV PCR assays have a very high negative predictive value ^{(7, 19, 33)}. However, after an HCMV infection or reactivation has resolved, HCMV DNA may persist for a long time in the peripheral blood and may obscure PCR diagnosis for a while ⁽⁴⁴⁾.

We compared the versatility of HCMV PCR and pp 65 antigen detection for the diagnosis of HCMV infections and reactivations in heart transplant recipients. With a view to the divergent results obtained with different HCMV PCR systems (Table 1), we determined the lower detection limit and performed HCMV PCR on both the cellular and the plasma fraction of the blood samples (269 samples from 117 HTx patients). To test the hypothesis that HCMV permanently persists in peripheral blood cells, we also tested 1013 EDTA blood samples from anti-HCMV positive healthy blood donors.

PATIENTS, MATERIALS AND METHODS

Patients. Patients included 483 HTx patients under immunosuppressive therapy (cyclosporine/azathioprine/cortisone) (73 female and 410 male patients) and 1013 unselected healthy, anti-HCMV positive blood donors.

HTx inpatients (n=117), i.e., the patients immediately after transplantation, and patients who had been referred to the hospital because of complications were monitored at short but variable intervals (daily-every 4 days) on request. Outpatients (n=366) were monitored at rather regular intervals during their first year after transplantation: every 2 weeks during the first 3 months, every 3-4 weeks during months 4-9, and every 6 weeks after that. After the first year, HTx patients were monitored twice per year, including 1 stay at the hospital for 3 days. During the first post-HTx examinations, biopsies were performed regularly. On suspect biopsy, pp 65 antigen determination, HCMV PCR, and biopsy were performed every 7-10 days until the histological signs of graft rejection or inflammation resolved. All HTx patients received anti-HCMV immunoglobulin immediately after transplantation; therefore, post-HTx determinations of anti-HCMV IgG in these patients did not reflect their own immunostatus.

Samples. EDTA blood was used for HCMV PCR. Heparinized blood was used for immunocytochemical detection of HCMV pp 65 antigen, and serum was used for antibody detection. A total of 1240 EDTA blood samples were obtained from the 483 HTx patients in our study (1-28 samples per patient, mean 2.5). Also obtained were 269 sets of 10 ml heparinized blood (for pp 65 antigen detection), 5 ml EDTA blood (for PCR), and whole blood (for antibody determination) from 117 HTx patients; the other 971 EDTA blood samples were obtained from 366 HTx outpatients. These 971 samples had previously been frozen and were hemolytic, so that PCR could not differentiate between HCMV DNA in the cellular and in the plasma fraction of these samples. From 1013 anti-HCMV positive blood donors, EDTA blood samples were obtained on blood donation (1 sample per donor).

Preparation of plasma-free cell nuclei. Plasma-free cell nuclei were prepared by mixing 200 μl blood with 1.3 ml PBS containing 0.9% nonionic detergent (Tween 20, NP 40) and centrifugation at 10,000×g for 2 minutes. The pelleted nuclei were washed and centrifuged twice with 1.3 ml PBS without detergent.

Preparation of cell-free plasma. Cell-free EDTA plasma was prepared by centrifugation in 1.5 ml tubes at 10,000×g for 5 minutes. Absence of cellular DNA was proven by negative human leukocyte antigen (HLA) DQa specific PCR (see below).

Reagents. Thermostable DNA polymerase (AmpliTaq) was obtained from Applied Biosystems (Pfungstadt, Germany). Nucleoside triphosphates were from Pharmacia, Freiburg. Oligonucleotide primers were synthesized on an Applied Biosystems ABI 381 synthesizer, using phosphoamidite reagents from Milligen, Hamburg. Agarose (NuSieve 3:1) was from Biozym, Hameln. All other reagents were of analytical grade quality from Merck, Darmstadt. QIAamp nucleic acid extraction columns, proteinase K, digestion and washing buffers were obtained from Qiagen (Hilden, Germany). Anti-HCMV pp 65 protein mouse monoclonal antibodies HCMV-C10 and HCMV-C11 were obtained from Biotest (Dreieich, Germany). Rabbit antimouse IgG and IgM serum and peroxidase conjugated goat antirabbit serum were obtained from Dianova (Hamburg, Germany). Anti-HCMV enzyme immunoassays (IgG, IgM) were obtained from Abbott (Wiesbaden, Germany).

Nucleic acid extractions. To eliminate PCR inhibiting substances, nucleic acids were extracted by adsorption to silica ⁽⁵⁸⁾. QIAamp spin columns were used for this purpose according to the manufacturer's instructions unless stated otherwise in this section. For DNA extraction from plasma-free cell nuclei, the pellet of washed nuclei obtained from 200 μl EDTA blood was taken up in 200 μl buffer and 25 μl proteinase K solution as supplied by the manufacturer and digested at 70°C for 10 minutes. After addition of 200 μl isopropanol, the mixture was loaded on the column, centrifuged, and then washed twice with 500 μl washing buffer as supplied by the manufacturer, and the nucleic acid extract was eluted with 100 μl water at 70°C. For DNA extraction from cell-free plasma, 200 μl precentrifuged EDTA plasma were digested with proteinase K, and nucleic acids were extracted as described for nuclei.

Polymerase chain reaction. Five microliters of nucleic acid extract, representing the nucleic acid from 10 μl peripheral blood (about 5×10⁴ leukocytes), were used for the first PCR in a total volume of 20 μl. The reaction mix was 50 mmol/L KCl, 20 mmol/L Tris-Cl (pH 8.5), 1.5 mmol/L MgCl₂, 0.2 mmol/L deoxynucleoside triphosphate, 0.2 μmol/L outer primers MIE 4 (CCA AGC GGC CTC TGA TAA CCA AGC C, positions 1142-1166 of major immediate early gene, EMBL accession HEIE 1) and MIE 5 (CAG CAC CAT CCT CCT CTT CCT CTG G, positions 1576-1552), producing a 435 base pair (bp) amplificate. Cycle parameters: 35×(95°C, 60°C, 72°C), 60 seconds at each temperature. Then 2 μl of first round PCR product were transferred into an 18 μl reaction mix for the nested PCR, of similar composition, but with primers MIE 6 (AGT GTG GAT GAC CTA CGG GCC ATC G, positions 1267-1291) and MIE 7 (GGT GAC ACC AGA GAA TCA GAG GAG C, positions 1376-1352), yielding a 110 bp amplificate. Cycle parameters: 30×(95°C, 55°C, 72°C), 60 seconds at each temperature.

To ensure absence of cells, the HLA DQa specific primers GH 26 and GH 27 ⁽¹⁵⁾ were included at 0.2 μmol/L concentrations when DNA extracts from plasma were amplified (GH 26: GT GCT GCA GGT GTA AAC TTG TAC CAG; GH 27: C ACG GAT CCG GTA GCA GCG GTA GAG TTG, amplificate length 230 bp).

Analysis of PCR products. PCR products were detected by ethidium bromide stain after electrophoresis in 2% agarose gels. Nucleotide sequences of the 110 bp nested PCR products were confirmed by restriction enzyme digest with AluI (70 bp and 40 bp fragments) and BalI (86 bp and 24 bp fragments) (Fig. 1C).

Determination of PCR lower detection limit. Plasmid pRR 47 ⁽⁵⁹⁾ was sequentially diluted into samples with nominally 1000, 100, 10, 1, and 0.1 copies. Of each concentration, 15 samples were amplified; 100, 10, and 1 plasmid copies were amplified in parallel with every series of nucleic acid extracts to ensure consistent sensitivity.

pp 65 antigen detection. Peripheral blood granulocytes were obtained from heparinized blood by sedimentation in Ficoll, adjusted to 1.000 cells/μl, spun onto glass slides, and fixed in methanol/acetone. Incubation of alcohol-fixed cells was performed in 3 steps, using (1) commercial mouse monoclonal antibody against HCMV pp 65 antigen, (2) rabbit anti-mouse antiserum, and (3) peroxidase-labelled polyclonal goat anti-rabbit serum, followed by diaminobenzidine stain as described elsewhere ⁽³⁾.

Anti-HCMV antibody detection. Anti-HCMV IgG and IgM enzyme immunoassays were obtained from Abbott (Wiesbaden, Germany) and were used according to the manufacturer's instructions.

RESULTS

Lower detection limit of nested PCR. Nested PCR detected single copies of plasmid pRR 47 (containing a 6.7 kb fragment of HCMV IE DNA), when the plasmid dilution was directly introduced into the PCR tube (12 of 15 reactions positive) (Fig. 1A). Spiking experiments showed that more copies of HCMV DNA were required when the DNA was extracted from whole blood: 200 plasmid copies in 200 μl EDTA blood were detected in 11 of 15 samples; 2000 plasmid copies in 200 μl EDTA blood were detected in 15 of 15 samples (Fig. 1B). A portion of nucleic acid extract derived from 10 μl blood was routinely used for PCR (5 μl extract, approximately 5×10⁴ leukocytes). Thus, HCMV DNA was detected at the detection limit of about 1 genome copy per 5000 leukocytes.

Stability of HCMV DNA. HCMV DNA remained detectable in EDTA blood samples after storage of the original blood sample at room temperature for at least 2 days, after storage at 4°C for at least 1 week, and after storage at [minus]20°C for at least 6 months.

Stability of pp 65 antigen. pp 65 remained detectable for no longer than 36 hours both in heparinized blood and in EDTA blood stored at room temperature and no longer than 48 hours in heparinized blood and in EDTA blood stored at 4°C.

HCMV DNA, pp 65 antigen, anti-CMV IgM, and clinical findings in HTx patients. A total of 1240 whole blood samples from 483 HTx patients were assayed. Of the 1240 samples, 275 (21.8%) were HCMV PCR positive. These 275 samples were from 79 HTx patients: 79 of 483 HTx patients (17%) were HCMV PCR positive at least on 1 occasion during our study. The difference in the proportion of HCMV DNA positive samples and HCMV DNA positive patients is explained by the fact that more samples per patient were obtained from symptomatic inpatients (Table 2).

In 269 samples (from 117 inpatients), these 4 HCMV diagnostic parameters were determined: (1) HCMV DNA in cells, (2) HCMV DNA in plasma, (3) pp 65 antigen, and (4) anti-HCMV IgM. Of the 117 inpatients, 75 (64%) were HCMV DNA and pp 65 antigen negative (Table 2); 42 of 117 inpatients had HCMV DNA in their peripheral blood leukocytes during our study, and 26 of 117 inpatients (22%) were HCMV DNA positive only in the cellular fraction of their peripheral blood. Three of 117 inpatients (3%) were positive for HCMV DNA in cells and in plasma, and 13 of 117 inpatients (9%) were positive for pp 65 antigen, for HCMV DNA in cells, and for HCMV DNA in plasma. Anti-HCMV IgM developed in 11 of 117 inpatients (9%); in 3 of these patients, anti-HCMV IgM appeared without prior detection of pp 65 antigen (Table 3). The time interval between first detection of HCMV DNA in the peripheral blood and the appearance of HCMV pp 65 antigen in our patients ranged from 1 to 35 days (mean, 8.5 days).

In patients with symptomatic primary HCMV infections (i.e., pre-HTx anti-HCMV IgG negative) and in patients with symptomatic HCMV reactivations (pre-HTx anti-HCMV IgG positive), we observed a hierarchy of laboratory findings. The first and basic finding was HCMV DNA in peripheral blood leukocytes. When the patients progressed from this condition into viremia, HCMV DNA was also detected in plasma. No patient was observed with HCMV only in the plasma but not in the cellular fraction. pp 65 antigen was detected only after HCMV DNA had appeared in the plasma; there were no patients with pp 65 antigen who did not have HCMV DNA in both the plasma fraction and the leukocytes of the peripheral blood. Anti-HCMV IgM appeared, or rose in titer, several days later, but some patients did not respond with IgM titer. Upon clinical improvement, pp 65 antigen, HCMV DNA in plasma, and HCMV DNA in leukocytes disappeared in this order. After resolution of clinical symptoms (i.e., pathological electrocardiogram, pneumonia, fever, renal insufficiency, water retention), HCMV DNA remained detectable in the cellular fraction of the peripheral blood for several weeks.

An example of such a diagnostic pattern during an HCMV infection is given in Figure 2a). Not all patients, however, developed anti-HCMV IgM (Fig. 2b). In some patients, because of the rather random sampling, we missed the viremic phase, and appearance of anti-HCMV IgM gave indirect evidence of infection later (Fig. 2c). Patients with HCMV DNA in plasma and patients with pp 65 antigenemia in this study were all symptomatic, whereas some patients with HCMV DNA only in the cellular fraction of the peripheral blood were asymptomatic (observed during the resolving phase).

HCMV detection in blood donors. No HCMV DNA could be detected by nested PCR in the plasma or in the peripheral leukocytes of 1012/1013 unselected healthy anti-HCMV positive blood donors. The only blood donor with HCMV DNA in the peripheral blood in our study (female, 30 years of age) had contracted a primary HCMV infection a few weeks before donation. She had previously been anti-HCMV negative and was found to be anti-HCMV IgM positive upon donation.

DISCUSSION

PCR has found widespread application in the monitoring of immunosuppressed patients because this method permits the detection of very small numbers of infectious agents in clinical samples (e.g., peripheral blood) within 1 day. However, the application of this method as an early and sensitive indicator of HCMV infection (or reactivation of a latent infection) is based on the assumption that the respective infectious agent is normally not present in the sample material. Whether HCMV is present in the peripheral blood during virus latency has long been obscured. The sites of latency are not precisely known ^{(1, 60)}. HCMV DNA has been found in different cell types in the peripheral blood during infections ^{(24, 38, 46, 47)} but also in asymptomatic individuals ^{(38, 40, 45)}. Surprisingly, the frequent detection of HCMV DNA in the peripheral blood of immunocompetent anti-HCMV negative individuals was also reported ^{(38, 16, 42)}(Table 1). Antibodies against HCMV are usually formed within weeks after infection and persist for many years ⁽⁶¹⁾. The prevalence of anti-HCMV antibodies is close to 100% in populations in developing countries and between 50% and 80% in most European populations. Annual anti-HCMV seroconversion rates of 1-2% are observed in European adults. In these populations, detection of HCMV DNA in the peripheral blood should be a very rare event rather than the rule. A rate of 34% anti-HCMV negative, HCMV DNA positive individuals, as reported in 1 study ⁽¹⁶⁾, is incompatible with these epidemiologic experiences and results most probably from carryover of amplified DNA. The problem of false positive PCR results and their impact on the literature is illustrated by reports about detection of HIV infections by PCR in blood samples from anti-HIV negative individuals who remained anti-HIV negative for many months ^(62-64).

Our investigation of 1013 anti-HCMV positive blood donors was performed with a very sensitive PCR, capable of detecting few genome copies. However, only 1 donor, who had recently recovered from a primary HCMV infection (with seroconversion), was HCMV DNA positive in the peripheral blood. This is strong evidence that HCMV is not permanently present in the peripheral blood of healthy anti-HCMV positive individuals. It is supported by data from 2 smaller cohorts ^{(41, 43)}.

Using a very sensitive nested PCR, we detected HCMV DNA in 79 of 483 (17%) of immunosuppressed HTx patients. Thus, even under immunosuppression, the presence of HCMV DNA in the peripheral blood of HTx patients is an exception, not the rule. This is also illustrated by the fact that the proportion of HCMV DNA positive HTx patients was higher in symptomatic inpatients (42 of 117 inpatients, 36%) than in asymptomatic outpatients who underwent routine testing (37 of 366 patients, 10%). It is remarkable that, despite the high sensitivity of HCMV PCR, we detected less HCMV PCR positive individuals among immunosuppressed HTx patients than others reported from studies with healthy blood donors.

HCMV DNA in the peripheral blood leukocytes of HTx patients remains detectable for long periods of time after an HCMV primary infection, or a reactivation, has resolved. This phenomenon was also reported for other transplant patients ⁽⁴⁴⁾. Therefore, a single detection of HMCV DNA in peripheral blood leukocytes without simultaneous detection of HCMV DNA in the plasma (i.e., viremia) and without information about the previous HCMV DNA status of this patient, may also indicate the remnants of resolved HCMV disease or the early stage of incipient virus replication, which has not yet progressed to general viremia. However, the appearance of HCMV DNA in the peripheral blood of a previously HMCV DNA negative patient is related to incipient virus replication and is the earliest indicator of imminent viremia, which is presently available. The potential of HCMV PCR as an early indicator of virus replication is underlined by our observations with 42 HTx inpatients who had HCMV DNA only in their peripheral blood leukocytes upon first testing: 16 of 42 patients (38%) progressed to viremia. These findings strongly suggest that all initially HCMV PCR positive HTx patients should be followed closely, until resolving and incipient viremia are clearly distinguished from each other.

HCMV DNA is stable in EDTA blood for many days, so that blood samples from outpatients may be mailed to and assayed in a central laboratory, ideally situated in the transplantation center, where PCR is performed under controlled, uniform conditions. The importance of quality control in PCR diagnosis is evident from the contradictory reports in the literature not only about the detection of HCMV by PCR ^{(16, 34, 38-43)} but also of other infectious agents, e.g., hepatitis C virus ⁽⁶⁵⁾. Presently, HCMV DNA detection in peripheral blood samples from HTx patients is an ideal tool for the early detection of HCMV primary infections and reactivations before the advent of symptomatic disease, but standardization of this method is urgently required.

Figure 1: A, Determination of lower detection limit of human cytomegalovirus (HCMV) polymerase chain reaction (PCR) with plasmid pRR 47. Agarose gel electrophoresis of HCMV PCR products. From left to right (15 lanes): lane 1, deoxyribonucleic acid (DNA) chain length marker (top to bottom: 1114 bp, 900 bp, 692 bp, double band 501/489 bp, 404 bp, 320 bp, 242 bp, 190 bp, 147 bp, 124 bp, 110 bp faintly visible); lanes 2-4, nominally 0.1 plasmid copies per reaction; lanes 5-7, 1 plasmid copy per reaction; lanes 8-10, 10 plasmid copies per reaction; lanes 11-13, 100 plasmid copies per reaction; lane 14, first round PCR product (435 base pair); lane 15, DNA chain length marker. B, Determination of lower detection limit of HCMV PCR with nucleic acid extracts from plasmid pRR 47 spiked ethylenediaminetetraacetic acid (EDTA) blood. Agarose gel electrophoresis of HCMV PCR products; 200 μl EDTA blood were spiked with 20, 200, 2000, and 20,000 plasmid copies. Extracted DNA was taken up in 100 μl water, 5 μl was used per PCR assay. This corresponds to 1, 10, 100, and 1000 plasmid copies in the fraction of the extracted blood that was assayed in 1 PCR. From left to right (15 lanes): lane 1, DNA chain length marker (top to bottom: 1114 bp, 900 bp, 692 bp, double band 501/489 bp, 404 bp, 320 bp, 242 bp, 190 bp, 147 bp, 124 bp, 110 bp faintly visible); lanes 2-4, 1000 plasmid copies per reaction; lanes 5-7, 100 plasmid copies per reaction; lanes 8-10, 10 plasmid copies per reaction; lanes 11-13, 1 plasmid copy per reaction; lane 14, negative control (water instead of nucleic acid extract); lane 15, DNA chain length marker. C, Nucleotide sequence confirmation of HCMV PCR products by restriction endonuclease digest. Polyacrylamide gel electrophoresis of HCMV PCR products and restriction digests. From left to right (4 lanes): lane 1, DNA chain length marker (top to bottom: 1114 bp, 900 bp, 692 bp, double band 501/489 bp, 404 bp, 320 bp, 242 bp, 190 bp, 147 bp, 124 bp, 110 bp, 67 bp, 37 bp (faintly visible); lane 2, nested HCMV PCR product from major immediate early gene region (EMBL: HEIE 1, pos 1267-1376), 110 bp; lane 3, BalI digest (70 bp and 40 bp); lane 4, AluI digest (86 bp and 24 bp).

Figure 2: a, Scheme of a typical sequence of laboratory diagnostic findings in an anti-human cytomegalovirus (HCMV) positive heart transplantation (HTx) patient. The HCMV DNA appears first in leukocytes and then in the plasma fraction (viremia). After resolution of the reactivation, HCMV remains detectable in the leukocytes for a long time. This type of pattern was most frequently observed. R, rejection; F, fever. b, Scheme of a diagnostic pattern in a patient in whom HCMV disease developed rapidly with subsequent rejection. No blood samples were available from the first 27 days post HTx, and no samples from the days 29-35 post-HTx. Anti-HCMV IgM was not developed. One more episode of HCMV viremia was observed at day 125 post-HTx. R, rejection. c, Scheme of a diagnostic pattern in a clinically inconspicuous patient in whom an HCMV primary infection was retrospectively diagnosed from the detection of anti-HCMV IgM. This pattern illustrates that PCR monitoring should preferably be performed at rather short regular intervals to not miss short periods of HCMV viremia. R, rejection; F, fever.