Significance of nested PCR and quantitative real time PCR for cytomegalovirus detection in renal transplant recipients

International Journal of Antimicrobial Agents 24 (2004) 455–462

Significance of nested PCR and quantitative real time PCR for cytomegalovirus detection in renal transplant recipients Atef Kanaana , Isabel Coura , Roberto Alvarez-Lafuentea , Mar Benedictoa , Esther Culebrasa , Dolores Pratsb , Cristina Fern´andezc , Juan J Picazoa,∗ a

Department of Microbiology, Hospital Cl´ınico San Carlos, C/ Profesor Mart´ın Lagos s/n, 28040 Madrid, Spain Department of Nephrology, Hospital Cl´ınico San Carlos, C/ Profesor Mart´ın Lagos s/n, 28040 Madrid, Spain Department of Preventive Medicine, Hospital Cl´ınico San Carlos, C/ Profesor Mart´ın Lagos s/n, 28040 Madrid, Spain b

c

Received 5 May 2004; accepted 18 June 2004

Abstract Immunocompromised renal transplant recipients are susceptible to severe cytomegalovirus (CMV) infection that makes its detection important in clinical practice. A total of 536 blood and 536 serum samples from 67 renal transplant recipients who had previously been diagnosed with terminal renal insufficiency were studied for CMV infection. In all samples, serology, shell vial culture, antigenaemia and nested polymerase chain reaction (PCR) in blood and serum were tested, and a real-time quantitative PCR was run on 90 specimens. Sixtyseven blood donors were used as controls. The results show that the quantitative real-time PCR assay could be of great interest for predicting CMV disease, and to monitor the onset of pre-emptive therapy. © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: CMV; Nested PCR; Quantitative PCR; Renal transplant

1. Introduction Cytomegalovirus (CMV) reinfections and reactivations occur frequently in immunocompromised patients and are still an important cause of morbidity and mortality in renal transplant recipients. Hence, a highly sensitive diagnostic technique is essential in order to indicate specific antiviral therapy. This high sensitivity, as many other studies have shown, can be achieved by molecular amplification assays. Nevertheless, CMV remains latent in leukocytes after primary infection, which makes its detection in peripheral blood of limited value in certain clinical situations. On the other hand, CMV DNA cell-free detection in plasma or serum has become feasible and appears to be a good marker of active infection [1–3]. Determination of viral load proved to be effective for monitoring active infection and thus could be ∗

Corresponding author. Tel.: +34 91 3303478; fax: +34 91 3303478. E-mail address: [email protected] (J.J. Picazo).

helpful in predicting the patient’s risk of developing CMV disease [4,5]. The recently developed real-time PCR assay to quantify CMV genome load is fast, automated and accurate for clinical applications [6]. Using blood samples for this assay may be more appropriate when CMV DNA load is low such as during antiviral therapy [7]. In this study, a nested PCR in blood and serum and a quantitative real-time PCR in blood were performed and compared with the different diagnostic assays used in our laboratory for management of CMV infection (serology, shell vial culture and antigenaemia).

2. Materials and methods 2.1. Patients and clinical samples A total of 536 total blood and a further 536 serum samples from 67 renal transplant recipients who had previously been diagnosed with terminal renal insufficiency were studied for

0924-8579/$ – see front matter © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2004.06.012

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CMV infection. Each recipient gave eight specimens, and the first sample was taken on the first day after transplant, then every 2 weeks for the first month and monthly thereafter during the following 6 months. Of these 67 kidney recipients, 40 (59.7%) were men (mean age 46.95 years, range 19–66) and 27 (40.3%) women (mean age 45.88 years, range 23–67). Forty-one (61.2%) patients received prophylaxis with ganciclovir. Single blood and serum samples from 67 blood donors with the same characteristics of age and sex, were used as controls in order to make a comparative study. Ninety blood samples were selected randomly and DNA–CMV load was determined by quantitative real-time PCR. For PCR assays, samples were collected from patients and controls after informed consent was obtained and were stored at −70 ◦ C until used. In all samples, serology, shell vial culture, antigenaemia, nested PCR in blood and serum were tested, and a real-time quantitative PCR was run on 90 specimens. Active infection was defined as detection of CMV IgM, pp65 antigen in peripheral blood leukocytes (PBLs) and/or viraemia. CMV disease was defined when any of the previous assays was positive and by the presence of unexplained fever, malaise, leukopenia, thrombocytopenia, transaminitis, unexplained elevation of the creatinine level or CMV tissue invasive disease with isolation from biopsy specimens as outlined by previous international CMV workshops [8]. 2.2. Serology The enzyme-linked immunosorbent assay (ELISA) was used to detect the specific CMV IgG and CMV IgM antibodies. The assay was performed using ELISA-processor III (Dade Behring Holding GmbH, Liederbach, Germany), as described by Huang and Kowalik [9]. The controls used were positive and negative serum (Behring). Results were read at 450 nm as indicated by the manufacturer. 2.3. Shell vial culture PBLs from 5 ml EDTA-treated peripheral blood samples were separated by sedimentation in an NH4 Cl solution (pH 7.4) for 15 min at 20–25 ◦ C and then centrifuged (3000 rpm, 15 min). Leukocytes were suspended in 3 ml of MEM (Minimal Essential Medium). For each sample, a fraction of 200 ␮l leucocytes was seeded in a vial covered by a monolayer of human fibroblasts (MRC-5 cells, Vircell, S.L. Granada, Spain, From ATCC, American Type Culture Collection). Vials were then centrifuged for 45 min at 2000 rpm. After the sample was incubated at 37 ◦ C for 1 h, the seeded leucocytes were removed and 2 ml of MEM with 2% of PBS was added. Each vial was incubated at 37 ◦ C for 48 h. CMV was identified by antihuman CMV antibody according to the manufacturers protocol (Chemicon International Inc., CA, USA). Cells infected by CMV could be observed, as the nuclei of fibroblasts were stained green.

2.4. Detection of pp65 antigen A fraction of 2.5 × 105 of PBLs (200 ␮l) prepared from EDTA-treated blood samples was spotted on a Cytospin slide (Shandon, Runcorn, United Kingdom), fixed with formalin plus 10% sucrose at room temperature and stained by immunofluorescence with monoclonal antibodies directed against human CMV lower matrix phosphoprotein pp65 according to the manufacturer’s protocol (Light Diagnostics, Chemicon International Inc., CA, USA). The test was considered positive when at least three fluorescent cells were observed. 2.5. DNA extraction from PBLs DNA was extracted from 6 cm3 of peripheral blood, which was diluted with STMT (sucrose 0.32 mol/l, Tris–HCl pH 7.51 mmol/l, MgCl2 5 mmol/l, Triton X-100 at 1% and water). This was immediately resuspended and left for 5 min at 4 ◦ C, then centrifuged at 3500 rpm for 20 min and the supernatant was carefully removed. Another wash with STMT was performed and the supernatant was removed. A volume of 4.5 ml of solution B (NaCl 0.075, EDTA 0.024 mol/l pH 8 and water) and 0.5 ml of solution C (proteinase K 20 mg/ml and SDS at 10%) were added and agitated by vortex so that the sediment was resuspended, and the sample was left overnight shaking at 37 ◦ C. The following day, 1.7 ml of NaCl 6 mol/l was added, mixed by vortex for 15 min and centrifuged at 3500 rpm for 20 min. The supernatant was then transferred to a sterile tube where DNA was precipitated by adding a volume of isopropanol. It was then extracted, dissolved in 400 ␮l of TE (10 mM of Tris pH 7.5, 1 mM of EDTA), shaken at 37 ◦ C for 1 h and cooled at 4 ◦ C for 2 days. Aliquots were stored at −40 ◦ C. 2.6. DNA extraction from serum The QIAamp blood kit (Qiagen S.A., Courtaboeuf, France) was used for DNA extraction from sera (200 ␮l) as indicated by the manufacturer. 2.7. Nested PCR The PCR primers used were previously described (2): the sequences of the outer primers were (5 AGAGTCTGCTCTCCTAGTGT-3 ) and (5 -CTATCTCAGACACTGGCTCA-3 ); the inner primers consisted of the upstream primer (5 -CCACCCGTGGTGCCAGCTCC3 ) and downstream primer (5 -CCCGCTCCTCCTGAGCACCC-3 ). Two hundred nanograms of DNA from each sample was amplified in a 50 ␮l reaction mixture, which contained (a) for the first round: 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, dATP, dGTP, dCTP and dTTP (400 ␮M each), 1.25 U of Taq polymerase, 1 ␮M each primer and distilled water to complete the final volume, and (b) for the nested PCR, a reaction volume of 50 ␮l

A. Kanaan et al. / International Journal of Antimicrobial Agents 24 (2004) 455–462

contained 5 ␮l of the previously amplified product used as a template, deoxynucleoside triphosphates at 200 ␮M each, 1 ␮M each of the inner primers and 1 U of Taq DNA polymerase and the reaction buffer. The preparations were covered with 150 ␮l of mineral oil. A negative control was included to verify the absence of contamination in each reaction using bi-distilled water as a template. For internal positive control, primers were added to the reaction mixture to amplify human ␤-globin gene to ensure the absence of inhibiting factors as well as the presence of sufficient DNA, throughout the entire amplification procedures. Purified CMV genome (Sigma) was used as an external positive control. Amplification was carried out in a thermal cycler (Perkin-Elmer/Cetus). For the first round, denaturalization was by one 10 min cycle at 94 ◦ C followed by 352-min amplification cycles at 94 ◦ C, 1 min 30 s at 65 ◦ C and 1 min at 72 ◦ C. This was followed by one cycle of 7 min 30 s at 72 ◦ C. For the second round PCR, the 35 cycles were performed for 1 min at 94 ◦ C, 30 s at 62 ◦ C and 1 min at 72 ◦ C. 2.8. Sensitivity and specificity of nested PCR detection Using the conditions previously described, a series of reactions were performed in which the template used was known quantities of CMV genome. For this purpose, we used decreasing concentrations of the CMV genome. The copy number, which corresponded to the last band and was clearly visualized by electrophoresis, was 900 copies for the first amplification round and 10 copies for the second amplification or nested PCR. Primers were specific, since performing the assay with DNA from the rest of the herpesviruses gave negative results. 2.9. Detection of the amplified product Amplified DNA extracts were visualized using 3% agarose gel (BioRad) stained by ethidium bromide and observed under UV light. As well as the samples, a molecular weight marker (DNA Molecular Weight Marker VIII, Roche Diagnostics) was run in each gel. The product of the first PCR round was 289 bp in size and that of the nested PCR round was 162 bp. 2.10. Quantitative real-time PCR Quantitative real-time PCR was used to detect CMV DNA in a Real-Time Cycler (model Rotor-Gene 2000; Corbett Research. Sydney). Primers and TaqMan probe for the detection of CMV were defined in the UL83 gene sequence [10] as follows: forward primer, 5 GTCAGCGTTCGTGTTTCCCA-3 ; reverse primer, 5 GGGACACAACACCGTAAAGC-3 and TaqMan probe, 5 -FAM-CCCGCAACCCGCAACCCTTCATG-3 TAMRA. No cross-reactivity was observed when the specificity of the primers and probe was tested for other human herpes viruses

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[10]. A set of primers and an exonuclease probe located in the human ␤-globin gene [11] were used as amplification control (forward primer, 5 -CAGAGCCATCTATTGCTTAC-3 ; reverse primer 5 -CATGGTGTCTGTTTGAGGTT-3 ; and TaqMan probe, 5 -JOE-ACACAACTGTGTTCACTAGC-3 TAMRA). The reaction mixture was: Tris–HCl 10 mM (pH 8.3), KCl 50 mM, MgCl2 2.5 mM, dATP, dGTP, dCTP and dTTP (200 mM from each), 0.25 mM of primers, 0.125 mM of TaqMan probes, 1 U Taq DNA polymerase (AmpliTaq Gold. PE Applied Biosystems), 200 ng of whole blood purified DNA and water to achieve a final reaction volume of 25 ml. Each sample was analysed in quadruplicate, that is, two consecutive PCRs with duplicated samples. The samples were placed into a Rotor-Gene 2000 cycler and preincubated at 95 ◦ C for 15 min to activate AmpliTaq Gold DNA polymerase. Two-step thermocycling was then performed for 45 cycles: denaturation at 95 ◦ C for 15 s and annealing/extension for 50 s at 60 ◦ C. With these primers and conditions, the sensitivity of the real-time PCR assay was only one copy. For the interpolation of viral load of patient samples, a standard curve of known amounts of purified and quantified DNA (Advanced Bio-Technologies) was designed (10,000, 1000, 100 and 10 copies). Calculations of CT and Rn, standard curve preparation and quantification of DNA in the samples were performed using the software provided with the Real-Time Cycler Rotor-Gene 2000. 2.11. Statistical analyses The software packages used for data analysis were SPSS version 10 and Epidat version 2.0. Independent qualitative variables such as CMV DNA detection were compared using the χ2 test or Fisher’s exact test. Paired qualitative variables between nested PCR in serum and the other assays were compared using Mac Nemar’s test. Quantitative association between these variables was analysed using Student’s t-test. The levels of concordance among the assays were estimated by the Kappa coefficient of concordance (κ). The non-parametric Mann–Whitney test was used to compare viral load of symptomatic and asymptomatic groups. A Receiver Operating Characteristic (ROC) curve was constructed to evaluate the capacity of each assay to discriminate the presence of CMV, which is represented by the area under the curve and the optimal cut-off value with the highest combination of sensitivity and specificity to compare the different assays. Sensitivity, specificity, positive and negative predictive values (PPV, NPV) and likelihood ratios were determined with 95% confidence intervals (C.I.s).

3. Results 3.1. Prevalence of CMV detection by different assays When the total number of patients is considered, CMV DNA was detected by nested PCR in at least one sample

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Table 1 Prevalence of CMV detection by each assay in the patient group and the corresponding total positive samples Total

Patients 67 Samples 536

PCR–sera

Antigenemia

Culture

CMV–IgM

No.

%

No.

%

No.

%

No.

50 120

74.6 33 22.4 54

49.3 10.1

20 25

29.9 24 4.7 69

% 35.5 12.8

throughout the follow up in the blood of 65 (97%) transplanted renal recipients and in that of 38 (56%) controls. In serum, CMV DNA was detected in 50 (74.6%) patients and in three (4.5%) controls. There was a significant difference between both groups in blood and serum (P < 0.001). When the total number of positive samples was considered, the frequency of CMV DNA detection in blood of 32 patients (65.7%) and 38 controls (56.7%) was similar (P > 0.05). Nevertheless, there was a significant difference between the total positive serum samples: 120 (22.4%) for patients and 3 (4.5%) for controls (P < 0.001). Prevalence of CMV infection by antigenaemia, culture and detection of CMV-IgG and IgM are shown in Table 1.

3.2. Concordance of nested PCR in serum with the different assays

3.3. Test validation criteria Culture and nested PCR in serum were used as reference assays, culture, since it is considered to be a standard assay, and nested PCR in serum because it presented the highest positivity rate. Sensitivity, specificity, PPV and NPV values are shown in Tables 3 and 4. In comparison with culture, the positive likelihood ratio having a positive result in a patient with CMV infection was 13, C.I. 95%: 8.9–18.8 for antigenaemia, 5.4, C.I. 95%: 4.5–6.4 for nested PCR in serum, and 1.3, C.I. 95%: 0.5–3.1 for the detection of CMV-IgM: 3.4. Seronegative recipients The follow-up results of the different assays in this group of patients are shown in Table 5. Seven of the transplant recipients were seronegative before renal transplantation (R−). Three of them received their transplant from seronegative donors (D−). Only one of these had a positive result, which was detected by nested PCR in blood. Four of the seronegative patients received their transplant from seropositive donors (D+). Three of these patients had detectable CMV DNA by nested PCR in serum and were positive by at least another assay. Only one developed symptoms, which were compatible with CMV disease. All four were receiving prophylaxis with ganciclovir. 3.5. CMV load by quantitative real-time PCR

Concordance of nested PCR in serum with culture, antigenaemia and the detection of CMV-IgM is shown in Table 2. CMV was detected by nested PCR in serum in 17.7, 16.4 and 13.4% more than by culture, CMV IgM and antigenaemia, respectively.

As mentioned above, 90 blood samples were selected randomly and a quantitative real-time PCR was applied. Seventyseven samples corresponded to 36 patients receiving ganciclovir, 15 of these samples were from nine patients who devel-

Table 2 Concordance of nested PCR in serum with the other assays used for diagnosis of CMV infection Assay A

Assay B

Concordance (%)

Discrepancy (%) A+/B−

B+/A−

PCR–serum PCR–serum PCR–serum

Culture IgM Antigenaemia

82.3 76.7 85.5

17.7 16.4 13.4

0 6.9 1.1

a b

Pa

κb

<0.001 <0.001 <0.001

0.29 0.21 0.47

Mac Nemar’s Test. Kappa coefficient.

Table 3 Sensitivity, specificity, positive and negative predictive values of each assay when shell vial culture was considered as the reference technique Assay

Sensitivity

Specificity

Positive predictive value

Negative predictive value

CMV IgM C.I. 95% Antigenaemia C.I. 95% Nested PCR–blood C.I. 95% Nested PCR–serum C.I. 95%

16.0 5.3–36.9 84.0 83.0–94.7 100 83.4–99.6 100 83.4–99.6

87.3 84.0–90.0 93.5 90.9–95.4 36.0 31.8–40.3 81.4 77.7–84.6

5.8 1.9–14.9 38.9 26.2–53.1 7.1 4.7–10.4 20.8 14.1–29.4

95.5 93.0–97.1 99.1 97.7–99.7 100 97.5–99.9 100 98.9–99.9

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Table 4 Sensitivity, specificity, positive and negative predictive values of each assay when nested PCR in serum was considered as the reference technique Assay

Sensitivity

Specificity

Positive predictive value

Negative predictive value

Shell vial C.I. 95% CMV IgM C.I. 95% Antigenaemia C.I. 95% Nested PCR–blood C.I. 95%

20.8 14.2–29.4 26.7 19.2–35.7 39.2 30.5–48.5 89.2 81.9–93.9

100 98.9–100 91.1 87.8–93.6 98.3 96.4–99.2 41.1 36.4–46.0

100 83.4–99.6 46.4 34.4–58.7 87.0 74.5–94.2 30.4 25.7–35.5

81.4 77.7–84.6 81.2 77.2–84.5 8.9 81.3–87.9 92.9 88.0–96.0

Table 5 Seronegative recipients group Serological status (IgG) Pre-trasplant Donor

Receptor

− − − + + + +

− − − − − − −

Sero-conversion

IgM

Culture

Antigenaemia

PCR–blood

PCR–sera

Symptoms

− − − + + + +

− − − + − − +

− − − − − + −

− − − − − + +

− − + + + + +

− − − + − + +

− − − − − − +

oped symptoms compatible with CMV disease and the range of viral load was between 0 and 36,380 copies per 1 ␮g of total DNA. The other 62 samples were from 27 patients who did not present symptoms of CMV disease and the range of viral load was between 0 and 115,495 copies per 1 ␮g of total DNA. There was a significant difference between the median DNA copy number detected in samples from symptomatic patients (4845 copies per 1 ␮g of DNA) and that detected in asymptomatic patients (18 copies per 1 ␮g of DNA) (P < 0.001). 3.6. Evaluation of the diagnostic assays for detection of CMV infection and disease The ROC curve (Fig. 1) shows that the different assays could easily distinguish the presence of CMV infection since they displayed an area under the curve, which was 0.78 for the detection of IgM, 0.83 for nested PCR in serum, 0.89 for shell vial culture and 0.90 for antigenaemia with overlapping C.I.s of 95%, (P < 0.001). However, there was a difference between the optimal cut-off point, which was 590 copies per 1 ␮g of DNA for shell vial culture, 125 copies per 1 ␮g of DNA for nested PCR in serum, and 126 copies per 1 ␮g of DNA for antigenemia and 51 copies per 1 ␮g of DNA for the detection of CMV IgM. Quantitative PCR (Fig. 2) can also discriminate between patients with CMV disease and healthy individuals, since the area under the curve was 0.90 (C.I. of 95% between 0.79 and 1.00). The optimal cut-off point was 380 copies per 1 ␮g of total DNA, which had a sensitivity of 93.3% (C.I. 95%: 66.0–99.6%), specificity of 85.5% (C.I. 95%: 73.7–92.7%). PPV of 60.9% (C.I. 95%: 38.8–79.5%) and NPV of 98.1% (C.I. 95%: 88.8–99.9%).

4. Discussion 4.1. Prevalence of CMV DNA detected by nested PCR in total blood and serum If we consider patients and controls who had at least one positive sample throughout the study, there was a significant difference in detecting CMV DNA in blood and serum. This would suggest an active infection in at least some patients since more positivity was detected in patients than controls. But when the total number of positive samples was considered, the finding of a significant difference between the total positive serum samples of patients and controls and not between their total blood positive samples confirms that detection of DNA in serum could be used as a marker for active infection. This also confirms that applying PCR to blood samples is not suitable for diagnosis of active CMV infection because CMV remains latent in the leukocytes of healthy adults [12]. This is particularly important in clinical practice since detection of active infection could allow patients to begin therapy before symptoms appear. Gerna et al. [13] reported that the majority of reinfections and reactivations by CMV after transplant are often asymptomatic. 4.2. Concordance of nested PCR in serum with the different assays In patients with at least one positive sample during the entire follow-up, the frequency of CMV DNA detection by nested PCR in blood and that of CMV IgG were similar. On the other hand, there was a significant difference when total blood positive samples were considered (P <

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Fig. 1. Receiver operating characteristic curve for CMV detection with the cut-off values for (A) nested PCR in serum, (B) antigenaemia, (C) shell vial culture and (D) detection of CMV IgM.

0.001). Although both assays detect latent infection, there was a discrepancy due to the fact that when IgG turns positive, it remains positive forever. This was not the case with PCR.

The discrepancy between a positive nested PCR in serum and negative culture may be attributed to ganciclovir therapy. Some studies [14,15] confirm that most samples, which were negative by shell vial culture, were from patients treated with ganciclovir, and other authors [16] do not recommend using shell vial culture to monitor active infection in patients who are receiving ganciclovir. For the detection of CMV active infection, there was a significant difference between PCR in serum and specific IgM in patients as well as in samples (P < 0.001). Discrepancy between a positive PCR and a negative CMV IgM could occur, since in immunocompromised patients, CMV IgM only appears in 50% of reactivations or reinfections and could be detected later than CMV DNA by nested PCR [17–19]. As described by other authors [7,12,20–21], the two assays, which showed greater concordance (85.5%) for detection of active infection, were nested PCR in serum and antigenemia (κ = 0.47). 4.3. Test validation criteria

Fig. 2. Receiver operating characteristic curve and the optimal cut-off value for CMV disease diagnosis by quantitative real-time PCR.

Shell vial culture showed very low sensitivity (20.8%, C.I. 95%:14.2–29.4%) compared with nested PCR in serum. This

A. Kanaan et al. / International Journal of Antimicrobial Agents 24 (2004) 455–462

makes it unsuitable for CMV detection since a large proportion of active infection will be missed. CMV IgM has low sensitivity and PPV in comparison with culture and nested PCR in serum. Hence, the assay is not adequate for the detection and monitoring of CMV active infection in renal transplant recipients, since it may underestimate the presence of viral infection. This agrees with other studies [13,22,23]. The sensitivity of antigenaemia was high compared with culture and was similar to that of PCR in serum, although it is still low when compared with nested PCR in serum (39.2% with C.I. of 95%: 30.5–48.5%). This assay has an acceptable PPV when compared with nested PCR in serum (87% with C.I. of 95%: 74.5–94.2%). It is, therefore, a useful procedure for monitoring active infection since its results can also be quantified. Furthermore, nested PCR in serum had the best sensitivity compared with culture (100% with C.I. of 95%: 83.4–99.6%) and a similar PPV to antigenaemia when both assays are compared with culture. It would thus be more appropriate for the detection and prediction of active infection [24,25]. CMV infection could be ruled out by any of these assays, since all of them had a high specificity and NPV. This agrees with other studies [22]. 4.4. Seronegative recipients In the pretransplant period, serology is essential since seronegative candidates for transplant are susceptible to developing primary infection. One of the three patients who received their transplant from seronegative donors (D−) had a positive result by nested PCR in blood. Larsson et al., [26] observed that CMV DNA could be detected by PCR in leukocytes of peripheral blood from all seropositive patients and in most seronegative blood donors over time. The fact that three of the four seronegative patients who received a seropositive transplant were found to have detectable CMV infection by more than one assay supports the idea that the transplanted organ is the principal source of CMV in the seronegative recipients, as previously suggested [27]. Moreover, only one of these patients developed symptoms compatible with CMV disease. Since all four were receiving prophylaxis with ganciclovir, in contrast to other studies [22], specific therapy could be indicated when any of these assays was positive in high-risk patients. 4.5. Quantitative real-time PCR There was a significant difference between the median CMV DNA copy number in patients presenting symptoms and those who did not, even though all of them were receiving prophylaxis. Taking into consideration the high number of asymptomatic patients treated, quantitative PCR could therefore have been useful to monitor these patients in order to avoid giving antiviral therapy when unnecessary.

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4.6. Evaluation of the diagnostic assays for detection of CMV infection and disease Although the optimal cut-off point for detection of CMV IgM was 51 copies per 1 ␮g of DNA, it could suggest the presence of CMV IgM at an early stage of CMV infection. Its lack of detection, however, could be explained by immunosuppression in these patients, as has already been mentioned [17–19].The similar optimal cut-off points of nested PCR in serum and antigenaemia, 125 and 126 copies per 1 ␮g of DNA, respectively, make these two assays more adequate for indicating when to start antiviral therapy to prevent the development of CMV disease, since the presence of symptoms, as shown in Fig. 2, had an optimal cut-off value of 380 copies per 1 ␮g of total DNA. The finding that this optimal cut-off value has a PPV of 60.9% of patients who had detectable CMV DNA developed CMV disease favours using quantitative PCR when available to monitor the onset of pre-emptive therapy. Furthermore, viral load would have been higher [31] if it had not been for the high number of patients who received prophylaxis with ganciclovir. This agrees with the findings of other authors [20,28–30]; however, the overlap of symptomatic and asymptomatic patients is too great and just the opposite conclusion should be drawn. Further studies should be conducted in order to validate the cut-off values obtained by quantitative PCR in this study.

Acknowledgements This work was supported in part by FIS 98/0614.

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