Background
Cytomegalovirus (CMV) infection still causes significant morbidity and mortality following allogeneic stem cell transplantation (SCT)
PCR-based methods have been recently evaluated for diagnosing and monitoring CMV infection after allogeneic SCT
Previous studies showed that the presence of CMV in plasma or serum is indicative of active viral replication being associated with a high predictive value for CMV disease in SCT recipients
There is much interest in the quantification of CMV load in blood for monitoring and prediction of CMV disease development and progression. Many studies have shown that the amount of CMV DNA is significantly associated with disease development
In this study, 38 patients were prospectively monitored for CMV infection during the first 100 days post-SCT. Two different commercially available assays were simultaneously used to compare clinical utility of a cell-based pp-65 Ag test with plasma PCR (COBAS AMPLICOR CMV MONITOR). Pre-emptive therapy, based on a positive pp65Ag assay result, was administered in all SCT recipients.
Methods
Patients and study design
Between January 2003 and February 2005, 42 consecutive SCT recipients at risk for CMV infection (donor and/or recipient CMV seropositive) were included in the study. Four out of 42 patients were excluded because survived less than 50 days after transplantation without any evidence of pp65 Ag positive cells and/or CMV-DNA. Therefore, 38 patients were considered valuable for this study. Blood samples were tested for CMV by both quantitative CMV-PCR in plasma and pp65 Ag at weekly intervals and, on suspicion of CMV infection, from day -7 to day 100 post-transplant.
Patients underwent SCT at the Transplant Unit of S. Eugenio Hospital, Tor Vergata University, Rome, Italy. From the time of hospital admission onwards, patients were given standard prophylaxis for bacterial (levofloxacin 500 mg daily p.o.), and fungal (fluconazole at 200 mg twice daily p.o.) infections. Acyclovir at standard (250 mg/m2 i.v. every 8 hours, or 200 mg four times a day) or high (500 mg/m2 i.v. every 8 hours, or 800 mg p.o. four times a day) dose was given for prophylaxis of herpes simplex virus infections in related or unrelated transplants, respectively. No patients received prophylactic intravenous immunoglobulins, ganciclovir, foscarnet, or cidofovir. The ethic Committee of S. Eugenio Hospital, Tor Vergata University, Rome approved the study and an informed written consent was received from each patient. Patient characteristics are summarized in Table
Clinical characteristics
Characteristics
Value
Patients
38
Age, y
Median
41
Range
(11–68)
Sex
Male
22
Female
16
Diagnosis
Acute leukemia
16
Lymphoma
6
Multiple myeloma
4
Myelodyspastic syndrome
4
Solid tumor
4
Non neoplastic disorders
3
Chronic lynphocytid leukemia
1
Acute GVHD
0–I
27
II–IV
11
Total body irradiation
Yes
15
No
23
Conditioning regimen
Standard
25
Reduced intensity conditioning
13
Type of transplant
Related
30
Unrelated
8
Hemopoietic reconstitution
Median days of PMN >0.5 × 109/L (range)
16 (11 – 41)
Median days of PLTS >20 × 109/L (range)
15 (9 – 65)
Median days of PLTS >50 × 109/L (range)
18 (11 – 88)
Source of stem cells
Peripheral blood
31
Bone marrow
4
Cord blood
3
Prophylaxis of aGVHD
CyA+MTX
25
CyA+MMF
6
CyA+PDN
4
Other
3
CMV status (donor/recipient)
Positive/positive
26
Negative/positive
10
Positive/negative
2
Deaths
4
GVHD: Graft versus host disease; CyA: Cyclosporin-A; MTX: Methotrexate; PDN: Prednisone; CMV: Cytomeglovirus. MMF: Micofenolate Mofetil; PMN: Peripheral Mononuclear cells; PLTS: Platelets
Criteria for the diagnosis of CMV infection and disease
Cytomegalovirus infection and disease were defined according to published recommendations
CMV serology
Serum samples were tested by a commercially available enzyme linked immunosorbent assay (Delta Biological, Italy), according to the manufacturer's instructions.
Specimen processing
A 15 ml volume of EDTA-treated blood was collected from each patient. Ten ml were used for CMV Ag assay and processed within 4 hours; plasma obtained from the remaining blood was used for CMV PCR assay.
Virological assays
Pp65 antigen test (Antigenemia assay)
The CMV pp65 antigenemia test was carried out with commercially available monoclonal antibodies, according to the standard protocol [CINA Kit, Argene]. Briefly, EDTA-treated whole blood samples were fractioned by dextran-sedimentation and lysis of erythrocytes. Slides were incubated with monoclonal anti-pp65 [pool of monoclonal antibodies (1C3+AYM-1), Biosoft, Paris, France]. The pp65 Ag results were reported as the total number of positive cells/200.000 PBL examined. All clinical decision regarding pre-emptive antiviral therapy were based on CMV Ag.
Quantification of CMV-DNA
CMV-DNA was quantified on plasma samples by using the Cobas Amplicor CMV Monitor, according to the manufacturer's instructions (Roche Molecular Systems). The dynamic range for quantification is approximately 3 log10 units, with 400 copies as the lower limit of detection.
Preemptive therapy for prevention of CMV disease
Pre-emptive therapy was started at the first detection of pp65-Ag positive cells (≥ 1 positive cell/200,000 cells). Plasma CMV DNA results were not considered for clinical decision making. Antiviral pre-emptive therapy was based on intravenous infusion of either ganciclovir at 10 mg/Kg/day or foscarnet at 180 mg/Kg/day for 2 to 3 weeks followed by either ganciclovir at 5 mg/Kg/day or foscarnet at 90 mg/Kg/day for 2 to 3 weeks.
Statistical analysis
The agreement between CMV detection assay was evaluated using the Kappa coefficient. Values of kappa above 0.75 indicate strong agreement; values between 0.40 and 0.75 represent fair to good agreement and value less than 0.40 reflect poor agreement
The mean period of time between transplant and pp65 Ag or PCR positivity (whichever first occurred) were compared by the Student's t-test. Differences between median time of disappearance of pp65 Ag or DNA were tested using Mann-Withney test. After normalization of the pp65 Ag values by logarithmic transformation (log10), the correlation between the two test was evaluated with Pearson's correlation coefficient. Differences in percentages were tested using the Fisher's exact test (2-tailed). P-values less than 0.05 were considered statistically significant.
Results
Clinical outcome
Of the 38 valuable patients, 25 (70%) had active CMV infection (Table
Patients with CMV infection
UPN
ASSAY
DAYS FROM TRANSPLANT
-7
0
+7
+14
+21
+28
+35
+42
+49
+56
+63
+70
+77
+84
+91
+100
0119.1
pp65 Ag
ND
ND
ND
- •
- •
- •
1•
-
-
-
- •
- •
- •
- •
- •
- •
Plasma PCR
ND
ND
ND
- •
- •
- •
- •
-
-
-
- •
- •
- •
- •
- •
- •
0120.1
pp65 Ag
ND
- •
- •
- •
- •
7•
-
-
†
Plasma PCR
ND
- •
- •
- •
- •
970•
-
-
†
0122.1
pp65 Ag
- •
- •
NE
- •
- •
- •
- •
4•
-
-
†
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
- •
-
-
†
0123.1
pp65 Ag
- •
- •
- •
- •
- •
- •
- •
1•
-
-
-
- •
- •
- •
- •
- •
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
- •
-
-
-
- •
- •
- •
- •
- •
0124.1
pp65 Ag
- •
- •
- •
- •
- •
4•
-
-
-
- •
1•
-
-
-
- •
- •
Plasma PCR
- •
- •
- •
- •
- •
- •
-
-
-
- •
- •
-
-
-
- •
- •
0125.1
pp65 Ag
ND
- •
- •
- •
- •
- •
- •
- •
7•
-
-
-
- •
- •
- •
- •
Plasma PCR
ND
- •
770•
- •
- •
- •
- •
- •
- •
-
-
-
- •
- •
- •
- •
5244.2
pp65 Ag
- •
- •
NE
- •
- •
- •
40•
2
80
7
-
-
-
ND
2•
-
Plasma PCR
- •
- •
- •
- •
- •
- •
1980•
1500
10000
1010
-
-
-
ND
- •
-
0127.1
pp65 Ag
NE
NE
NE
NE
NE
- •
- •
20•
-
-
-
- •
- •
- •
- •
- •
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
- •
948
-
-
- •
- •
- •
- •
- •
0128.1
pp65 Ag
- •
- •
NE
- •
- •
- •
5•
-
-
-
- •
- •
- •
- •
- •
- •
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
-
-
ND
- •
- •
- •
- •
- •
- •
0130.1
pp65 Ag
- •
5•
-
-
-
- •
- •
- •
ND
- •
1•
-
-
-
- •
- •
Plasma PCR
- •
-•
-
-
-
- •
- •
- •
ND
- •
- •
-
-
-
- •
- •
0134.1
pp65 Ag
- •
- •
NE
NE
- •
- •
40•
6
2
-
-
-
- •
- •
8•
-
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
-
-
-
-
-
- •
- •
- •
-
0135.1
pp65 Ag
- •
- •
NE
- •
- •
- •
- •
1•
-
-
-
- •
- •
- •
- •
- •
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
- •
-
-
-
- •
- •
- •
- •
- •
0137.1
pp65 Ag
- •
- •
NE
- •
- •
- •
- •
- •
5•
-
-
-
- •
- •
- •
- •
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
- •
- •
-
-
-
- •
- •
- •
- •
0139.1
pp65 Ag
- •
ND
- •
- •
- •
- •
- •
74•
30
6
-
-
-
ND
- •
- •
Plasma PCR
- •
ND
- •
- •
- •
- •
- •
6490•
22400
9690
-
-
-
ND
- •
- •
5224.2
pp65 Ag
- •
- •
- •
- •
- •
- •
- •
30•
-
5
-
-
2
-
-
-
Plasma PCR
- •
- •
- •
- •
- •
- •
550•
- •
-
825
-
-
-
-
-
-
5298.1
pp65 Ag
- •
- •
- •
- •
- •
- •
- •
- •
2•
-
-
-
- •
- •
- •
ND
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
- •
- •
-
-
-
- •
- •
- •
ND
5176.1
pp65 Ag
- •
- •
- •
- •
- •
- •
- •
- •
- •
60•
-
-
ND
- •
- •
- •
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
470•
3830•
NE
1400
-
ND
- •
- •
- •
0149.1
pp65 Ag
- •
- •
NE
- •
- •
- •
- •
- •
- •
- •
- •
- •
2•
-
NE
-
Plasma PCR
- •
- •
-
- •
- •
- •
- •
- •
- •
- •
- •
- •
-
-
-
-
0150.1
pp65 Ag
- •
- •
- •
- •
- •
- •
- •
- •
10•
4
-
-
-
- •
- •
- •
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
- •
- •
832
1530
680
-
- •
- •
- •
0151.1
pp65 Ag
- •
- •
- •
- •
- •
5•
3
-
1
-
-
-
- •
- •
- •
- •
Plasma PCR
- •
- •
- •
- •
- •
438•
-
695
-
-
-
-
- •
- •
- •
- •
0152.1
pp65 Ag
- •
- •
NE
ND
32•
-
144
-
13
30
3
-
-
-
- •
422•
Plasma PCR
- •
- •
- •
ND
- •
568
8960
955
2670
1720
11110
3070
1250
659
- •
21200•
0155.1
pp65 Ag
- •
- •
- •
NE
- •
- •
ND
- •
- •
- •
- •
351•
9
-
-
-
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
463•
7860•
6510•
12600•
6790•
4770
-
-
-
0164.1
pp65 Ag
- •
- •
NE
NE
- •
- •
- •
- •
400•
4
7
-
-
-
- •
- •
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
891•
8280•
5830
7360
-
-
-
- •
- •
5278.2
pp65 Ag
- •
- •
NE
NE
- •
1•
1
-
-
-
- •
- •
- •
- •
- •
- •
Plasma PCR
- •
- •
- •
- •
- •
- •
-
-
-
-
- •
- •
- •
- •
- •
- •
0173.1
pp65 Ag
NE
NE
NE
- •
- •
- •
ND
- •
- •
- •
- •
2•
-
-
-
- •
Plasma PCR
- •
- •
- •
ND
- •
- •
- •
- •
- •
- •
1710•
1520•
-
-
ND
- •
UPN: unique patient number; Ag: antigenemia; PCR: polymerase chain reaction; ND: not done; NE: not evaluate; -: negative result; Ag and PCR values are expressed as N° of positive leucocytes or viral copies, respectively; †: death. Dots represent samples of patients not on pre-emptive therapy
Patients with CMV disease
UPN
ASSAY
DAYS FROM TRANSPLANT
-7
0
+7
+14
+21
+28
+35
+42
+49
+56
+63
+70
+77
+84
+91
+100
0133.1
Pp65 Ag
- •
- •
- •
ND
- •
- •
- •
- •
- •
-** •
-
-
-
-
-
2
Plasma PCR
- •
- •
- •
ND
- •
- •
- •
- •
- •
-** •
-
-
-
-
-
-
0136.1
pp65 Ag
- •
- •
- •
- •
- •
- •
- •
- •
400** •
83
1
5
2
-
4
-
Plasma PCR
- •
- •
- •
- •
- •
- •
- •
- •
22600** •
12300
8810
6000
1290
-
-
-
0138.1
pp65 Ag
- •
- •
- •
7•
-**
-
-
- •
60- •
23
4
-
-
-
-
19
Plasma PCR
- •
- •
- •
- •
-**
-
-
- •-
1230- •
4070
16700
2240
-
-
758
2250
UPN: unique patient number; Ag: antigenemia; PCR: polymerase chain reaction; ND: not done; NE: not evaluate; -: negative result; **: CMV disease. Dots represent samples of patients not on pre-emptive therapy.
All patients developed intestinal CMV disease and UPN 0136.1 died on day + 111 for CMV disease associated with aGVHD.
Four of the engrafted patients died within 100 days after SCT: two for progression of acute myeloid leukemia and colon carcinoma, respectively; one for acute GVHD and one for CMV disease associated with acute GVHD.
Detection of CMV by pp65 antigenemia, and by the Cobas CMV PCR assays
Overall, 568 blood samples were analysed for pp65Ag and/or PCR; of the 35 (6.2%) samples obtained from patients in whom the pp65 Ag assay could not be performed because of the low number of cells (i.e. before engraftment or during severe neutropenia with absolute neutrophil counts of less than 200/μl), none was positive by plasma PCR assay. Of the 534 samples tested simultaneously for pp65 Ag and PCR, 31 (5.8%) were positive by PCR and pp65 Ag, 30 (5.6%) were pp65 Ag positive but PCR CMV negative, 22 (4.1%) were PCR positive/pp65Ag negative, and 451 (84.4%) were negative by both assays. CMV was detected in 83 samples (15%) by a single or both methods.
Relative sensitivities, specificities, and predictive values for detection of plasma DNA and pp65 Ag were calculated for both assays considering all specimens obtained from patients with and without anti-CMV therapy at the time of testing. Using as reference any positive test (either pp65 Ag or PCR assays), sensitivity/specificity/positive-predictive-value/negative-predictive-value for detection of CMV plasma DNA were 64%/100%/100%/94%, respectively. Using as reference any positive test (either pp65 Ag or PCR assays), sensitivity/specificity/positive-predictive-value/negative-predictive-value for detection of pp65 Ag were 73%/100%/100%/95%, respectively.
Correlations between pp65 antigenemia and Cobas CMV PCR assays
To exclude any effect due to specific anti-CMV therapy, correlation analysis of pp65 AG and PCR results was only performed on 405 blood samples collected from patients not on ganciclovir, foscarnet or cidofovir therapy; the observed Kappa coefficient of agreement for both CMV assays was 0.304 (poor agreement). The correlation for both CMV assays were not significant among 43 out of 405 samples positive by both methods or by either methods (R2 = 0.000144, p = 0.94). On the contrary, when both tests were positive, a significant correlation was found (R2 = 0.68, p = 0.006) (FIG.
shows on a logarithmic graph the correlation of CMV quantitation by CMV pp65 antigenemia and plasma PCR among 8 samples obtained from patients not on CMV treatment determined positive by both methods (R2 = 0.68, P = 0.006)
shows on a logarithmic graph the correlation of CMV quantitation by CMV pp65 antigenemia and plasma PCR among 8 samples obtained from patients not on CMV treatment determined positive by both methods (R2 = 0.68, P = 0.006).
Time to detection and disappearance of pp65 antigenemia and PCR in transplanted patients
Of the 38 patients, 28 (73.6%) developed positive pp65 Ag after a median of 41 days (range 0–100 days), while 15 (39.5%) developed a positive PCR after a median of 43 days (range 5–67 days) (p = 0.64, Student's t-test) from SCT (Table
Interval from transplantation to detection of first positive CMV test of samples from 38 SCT transplant recipient
Test method
No. positive/total (% positive)
Time (days to positivity)
Mean
Median
Range
SD
Pp65 antigenemia
28/38 (73.7)
42.93
41
0–100
20.06
Plasma PCR
15/38 (39.5)
40.20
43
5–67
14.49
Incidence of positive pp65Ag and plasma PCR after SCT
For transplanted patients, the frequency to have a positive pp65 Ag assay (28/38 patients, 73.6%) was significantly higher than a positive PCR assay (15/38 patients, 39.4%) (p = 0.003). When the type of transplant was analyzed, the frequency to have a positive pp65 Ag assay (21/30 patients, 70%) was significantly higher than plasma PCR assay (10/30 patients, 33.3%) (p = 0.013) in HLA-identical sibling, but was similar in unrelated donor transplant (pp65 Ag positive in 7/8 patients, PCR positive in 5/8 patients) (p = 0.375). The frequency of CMV active infection was also similar between HLA-identical sibling transplant recipients and unrelated donor transplant, demonstrated by both the pp65 Ag assay (21/30 patients, 70% vs 7/8 patients, 87.5%), respectively, (p = 0.653) and PCR assay (10/30 patients, 33.3% vs 5/8 patients, 62.5%), respectively (p = 0.223).
When the incidence of CMV reactivation was analyzed according to the severity of acute GVHD, patients with grade II–IV acute GVHD developed CMV infection at a frequency similar to those with grade 0–1 acute GVHD. The frequency of positive pp65 Ag was 63.6% in 11 patients with grade II–IV acute GVHD and 77.7% in 27 patients with grade 0–I acute GVHD (p = 0.62). Similarly, the frequency of positive PCR was 36.4% in 11 patients with grade II–IV acute GHVD and 40.7% in 27 patients with grade 0–1 acute GVHD (p = 0.99).
No differences between positive pp65 Ag and plasma PCR assays were also found when patients were evaluated according to the dose of acyclovir. The frequency of positive pp65Ag was 72.4% in 29 patients who received acyclovir at standard dose and 77.8% in 9 patients who received acyclovir at high dose (p = 0.90). Similarly, the frequency of positive plasma PCR assay was 34.5% in 29 patients who received acyclovir at standard dose and 55.5% in 9 patients who received acyclovir at high dose (p = 0.43).
Patients with CMV diseases
Three patients developed intestinal CMV disease (7.9%): of these, one had negative pp65Ag and PCR assays before CMV disease, one had a concomitant positivity of both methods (400 Ag positive cells and 22600 copies/ml) and in the remaining patient only pp65 Ag was positive (7 positive cells) 1 week before the onset of CMV disease (Table
The UPN 0136.1 patient (Table
The UPN 0138.1 patient (Table
The UPN 0133.1 patient (Table
Discussion
Despite major advances in treatment and prevention, CMV infections remain an important cause of morbidity and mortality in SCT recipients. Prophylaxis with ganciclovir during the first 100 days after transplantation results effective in preventing CMV disease in high risk patients, but is also associated with significant myelotoxicity, increased incidence of invasive fungal infection, and late CMV disease due to delayed CMV-specific T-cell response recovery
In our study, we prospectively compared the results of plasma PCR and pp65 Ag assay; two commercially available detection assays to monitor CMV infections in SCT recipients receiving a pre-emptive antiviral therapy initiated because of a positive pp65 Ag assay result. A clinical finding of our study is the slight earlier positivity, although not statistically significant, of pp65 Ag assay over quantitative plasma PCR assay, finding in agreement to those already reported by Boeckh et al.
Discordances between the positivity rates of the two assays were found in our study. This discrepancy may be explained: 1) by the choice of pp65 Ag assay for guiding antiviral therapy, which may have modified CMV viral load to be detected by plasma PCR; 2) the two methods target different markers of virus replication, detecting free virions in plasma and virus protein in PBL, with different levels of sensitivity
Because a low CMV viral load is highly significant in the context of SCT, the primary objective should be on improving sensitivity
As the pp65Ag assay was found to be positive earlier than PCR, it became negative faster after initiation of antiviral therapy, although these differences did not reach statistical significance. The finding of faster clearance of pp65 Ag compared to plasma CMV DNA after treatment has been observed in the context of SCT
In particular, the kinetics of pp65AG and plasma DNA assays in a patient with CMV disease (UPN 0.138.1) (Table
In our study, the probability of patients to have a pp65 Ag positive assay was higher than to have a PCR positive assay. When we analyzed this finding, pp65 Ag positive result was statistically more frequent than PCR result in patients receiving an HLA-identical sibling transplant. However, 11 of these 21 patients had a positivity only for pp65 Ag with a low level of positive cells (median 2 positive cells, range 1–5). This suggests a CMV late phagocytosis by PBL, despite the absence of virus replication
Interestingly, a trend of more frequent CMV infection (either pp65 Ag assay or PCR assay) was shown in unrelated donor transplant recipients than HLA-identical sibling patients, while patients with acute GVHD II–IV grade did not develop more CMV infection than those with grade 0–1 acute GVHD. These findings could be explained by the lower number of unrelated patients compared to related who entered in our study. Further, in our study, statistical analyses may be influenced by the small number of patients, by the preexisting heterogeneity of conditioning regimens, GVHD prophylaxis, hematological diseases and donor type.
The choice of the ideal sample material for PCR (plasma, serum, leukocytes, or whole blood), remains to be elucidated. Furthermore, viral loads obtained in whole blood, where higher than those measured in plasma, serum, PBL and peripheral blood mononuclear cells
Conclusion
Both pp65Ag and quantitative plasma PCR assays (COBAS AMPLICOR CMV MONITOR) are useful in early detection of CMV infection and prevention of CMV disease after allogeneic SCT. Moreover, in the three patients who developed CMV disease, PCR anticipated the onset of CMV disease in one patient, while pp65 Ag in two, showing that both tests are useful in diagnosing CMV diseases. Lower thresholds should be probably adopted for quantitative assays in the context of SCT, because small amounts of CMV load require immediate therapy. Therefore, further studies are required to determine the diagnostic value of each method to guide pre-emptive therapy by the most clinically suitable method in SCT recipients.
Abbreviations
CMV: Cytomegalovirus,
pp65Ag: pp65 antigenemia,
SCT: stem cell transplantation,
PCR polymerase chain reaction,
PBL: peripheral blood leukocytes,
GVHD: Graft-versus-host disease,
CyA: Cyclosporin-A,
MTX: Methotrexate,
PDN: Prednisone,
PMN:polymorphonuclear cells
PLTS: platelets
MMF: micofenolato mofetil
ND: not done
NE: not evaluable
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All the authors contributed substantially to the study. GG and PDF designed the study, contributed to data analysis and wrote the manuscript. AP collected the data, contributed to data analysis and drafted the manuscript. AV contributed to design of the study and in writing the manuscript. AS performed the statistical analysis. LC, TD, AT, and LC collected the data. AC and MC conducted the laboratory studies. SA and PM provided critical comments for the manuscript. All authors read and approved the final manuscript.