Viral infections Monitoring cytomegalovirus infection by ... - Clonit

Bone Marrow Transplantation (2002) 30, 315–319  2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00 www.nature.com/bmt

Viral infections Monitoring cytomegalovirus infection by antigenemia assay and two distinct plasma real-time PCR methods after hematopoietic stem cell transplantation Y Tanaka1, Y Kanda2, M Kami3, S Mori1, T Hamaki4, E Kusumi4, S Miyakoshi4, Y Nannya2, S Chiba2, Y Arai5, K Mitani5, H Hirai2, Y Mutou4 for the Japan Hematology and Oncology Clinical Study Group (J-HOCS) 1

Department of Hematology, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan; 2Department of Cell Therapy and Transplantation Medicine, University of Tokyo, Tokyo, Japan; 3Stem Cell Transplant Unit, National Cancer Center Hospital, Tokyo, Japan; 4Department of Hematology, Toranomon Hospital, Tokyo, Japan; and 5Department of Hematology, Dokkyo University School of Medicine, Tochigi, Japan

Summary: We compared a CMV virus load determined by realtime PCR with an antigenemia value to analyze the correlation between these two methods. We also compared the values for virus load determined by the two distinct real-time PCR methods, which amplify the US17 region and immediate–early (IE) gene of CMV, respectively, to evaluate the reliability of these methods. Two hundred and sixty-five samples were obtained weekly from 29 patients, who had engraftment after unrelated bone marrow transplantation or HLA-mismatched related blood stem cell transplantation. CMV infection was detected in 115 samples from 22 patients by US17-PCR and 69 samples from 20 patients by the antigenemia assay. Fifty-eight samples were positive for both assays, but 57 and 11 samples were positive only for US17-PCR and antigenemia, respectively. A good correlation of the results of US17-PCR and antigenemia was demonstrated (r = 0.61). All antigenemia-positive samples and randomly selected antigenemia-negative samples were subjected to IE-PCR. The results of IE-PCR showed a good correlation with those of antigenemia (r = 0.64). Furthermore, the best correlation was observed between US17-PCR and IE-PCR (r = 0.83). In conclusion, both real-time PCR methods showed a good correlation with the antigenemia assay, and could be used to monitor CMV infection after hematopoietic stem cell transplantation. Bone Marrow Transplantation (2002) 30, 315–319. doi:10.1038/sj.bmt.1703661 Keywords: cytomegalovirus; real-time PCR; antigenemia; hematopoietic stem cell transplantation; pre-emptive therapy

Correspondence: Dr Y Kanda, Department of Cell Therapy and Transplantation Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan Received 28 January 2002; accepted 21 May 2002

Cytomegalovirus (CMV) disease is a major problem after allogeneic hematopoietic stem cell transplantation.1–3 Ganciclovir is a potent antiviral agent against CMV and is the mainstay of CMV prophylaxis.1–3 However, the administration of ganciclovir to all bone marrow transplant recipients (universal prophylaxis) did not improve survival because of neutropenia caused by ganciclovir.4,5 It also raised a problem of late CMV diseases.4,5 Therefore, a pre-emptive approach, that involves monitoring of CMV infection and using ganciclovir only in high-risk patients, has been investigated.1,2 The CMV antigenemia assay is a rapid and quantitative method to monitor CMV infection,6 and is widely used as a guide for starting a pre-emptive therapy with ganciclovir.7,8 However, this method is very laborious, because an examiner must count several tens of thousands of cells per sample. In addition, the results may be influenced by several factors including storage and fixation methods.9 Polymerase chain reaction (PCR) to detect CMV-DNA has also been used to detect CMV infection.10 Although it might be too sensitive for discrimination of high-risk patients for CMV disease,11 the development of real-time PCR has made it possible to quantitatively evaluate the virus load.12–14 However, the threshold of the virus load to discriminate high-risk patients has not been determined, although the threshold of the antigenemia value has been reported.15,16 Serially monitoring the virus load without administering ganciclovir is the only method to address this issue, but it is unethical. Therefore, in this study, we compared a CMV virus load determined by real-time PCR with an antigenemia value to estimate the threshold virus load. We also compared the viral load between two distinct realtime PCR methods to evaluate the reliability of these methods.

CMV monitoring by antigenemia assay and two distinct real-time PCR methods Y Tanaka et al

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Patients and methods Patients CMV sero-positive patients or sero-negative patients with a sero-positive donor, who underwent unrelated bone marrow transplantation or HLA-mismatched related blood stem cell transplantation in centers that belong to the Japan Hematology and Oncology Clinical Study Group (J-HOCS), were enrolled in the study. Written informed consent was obtained from all patients. Preparative regimens varied among the cases. Graft-versus-host disease (GVHD) prophylaxis was given with cyclosporine A or tacrolimus combined with short-course methotrexate. CMV infection was monitored by real-time PCR and the antigenemia assay weekly after neutrophil engraftment (⬎0.5 ⫻ 109/l) until day 100 after transplantation. Pre-emptive therapy with ganciclovir at 5 mg/kg twice daily was started at the detection of positive antigenemia and continued until it became negative. CMV antigenaemia assay CMV antigenemia assay was performed by the modified method described previously.15 Briefly, 1.5 ⫻ 105 peripheral blood leukocytes were attached to a slide using a cytocentrifuge and fixed with cold acetone. In fact, from onethird to half of the centrifuged cells were fixed on the slides. The cells were incubated with monoclonal antibody HRPC7 (Teijin, Tokyo, Japan) raised against CMV immediate– early gene antigen, and stained by the direct immunoperoxidase method. These cells were analyzed under a light microscope and the results are presented as the number of positive cells per 50 000 cells. Diagnosis of CMV diseases Patients with signs and symptoms compatible with CMV disease, such as interstitial pneumonitis, gastroenteritis, hepatitis, or retinitis, underwent a biopsy for extensive pathological and microbiological examinations if possible, by which a definite diagnosis of CMV disease was established. However, biopsy of the liver was not performed in patients with thrombocytopenia or ascites. Early and late CMV diseases were defined as those occurring before and after day 100, respectively. CMV real-time PCR assay Real-time PCR was performed with two distinct sets of primers and a TaqMan probe for US17 region and the immediate–early (IE) gene.12,13 US17-PCR was performed in Otsuka Assay Laboratories (Otsuka Pharmaceutical, Tokyo, Japan) and IE-PCR was performed in SRL (Tokyo, Japan). Briefly, DNA extracted from 100 ␮l of plasma was subjected to PCR using TaqMan Universal PCR Master Mix (PE Biosystems, Foster City, CA, USA) and the PCR pro-

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duct was detected as an increase in the fluorescent intensity using ABI Prism 7700 (PE Biosystems). Real-time fluorescent measurements were taken and a threshold cycle (CT) value for each sample was calculated by determining the point at which the fluorescence exceeded 10 times the baseline fluorescence. A standard curve was constructed using the CT values obtained from serially diluted DNA extracted from a plasmid that contains the respective region of CMV. The CT values from the clinical samples were plotted on the standard curve and the copy number was calculated automatically using Sequence Detection System version 1.6 (PE Biosystems). All samples were prospectively tested by the antigenemia assay and US17-PCR. After the completion of these tests, all available antigenemia-positive samples and several randomly selected antigenemia-negative samples were subjected to IE-PCR. Results of each assay were masked to the examiner of other assays. Statistical analyses The results of the real-time PCR and the antigenemia assay were analyzed after logarithmic transformation. Pearson’s correlation coefficient was calculated to compare the results of the two monitoring methods. Samples negative for either of the two tests were excluded from the analysis of correlation.

Results Transplantation outcome Thirty-one patients were enrolled between June 2000 and June 2001. All patients achieved sustained engraftment except for two who died before engraftment. Thus, CMV monitoring with the antigenemia assay and US17-PCR was performed in 29 patients. The underlying disease was acute myeloblastic leukemia in 12, acute lymphoblastic leukemia in four, chronic myelogenous leukemia in eight, myelodysplastic syndrome in three, non-Hodgkin’s lymphoma in one, and adult T cell leukemia in one. Seventeen patients (59%) developed grade II–IV acute GVHD. Two patients developed pathologically diagnosed CMV colitis and one developed CMV adrenalitis diagnosed at autopsy. CMV pneumonitis was suspected in another patient by an interstitial pneumonitis pattern on chest computed tomography and concurrent positive antigenemia. None of these were fatal. Finally, six of the engrafted patients died before 100 days after transplantation due to disease progression in two and acute GVHD, bacterial pneumonia, acute respiratory distress syndrome, and liver failure in one each, respectively. Incidence of CMV infection Two hundred and sixty-five samples were analyzed. CMV infection was detected in 115 samples from 22 patients by US17-PCR and 69 samples from 20 patients by the anti-


genemia assay. Fifty-eight samples were positive for both assays, but 57 and 11 samples were positive only for US17-PCR and antigenemia, respectively. The median duration from transplantation to the first detection of positive results of US17-PCR and antigenemia was 32 and 33 days, respectively (P = 0.11 by Wilcoxon signed rank test). Positive US17-PCR developed earlier than positive antigenemia in eight patients, whereas antigenemia became positive earlier in two. The relationship between US17-PCR and antigenemia is shown in Figure 1. A good correlation of the results of US17-PCR and antigenemia was demonstrated (r = 0.61, P ⬍ 0.0001). Eighteen patients received pre-emptive therapy with ganciclovir for a median of 22 days. Antigenemia became negative a median of 2 weeks after starting ganciclovir. No patient developed antiviral resistance.

randomly selected antigenemia-negative samples (n = 66) were subjected to IE-PCR. As shown in Figure 3, results of IE-PCR showed a good correlation with those of antigenemia assay (r = 0.64, P = 0.0002). Furthermore, the best correlation was observed between US17-PCR and IE-PCR (Figure 4, r = 0.83, P ⬍ 0.0001). However, in general, US17-PCR reported a higher (approximately three- to 10fold) virus load than IE-PCR. To rule out the possibility that this discrepancy was due to long preservation of the plasma samples, we subjected the control CMV samples of US17-PCR methods to IE-PCR. The control samples of US17-PCR at 300 copies/ml and 3000 copies/ml were determined as 150 copies/ml and 1000 copies/ml, respectively, by IE-PCR. Therefore, the discrepancy did not result from the preservation. Threshold of viral load to start ganciclovir

Time course of US17-PCR and antigenemia

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Figure 1 Correlation between US17-PCR virus load and the number of positive cells on antigenemia assay (r = 0.61, P ⬍ 0.0001).

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Assuming the threshold of the antigenemia value to start ganciclovir is 10 and three positive cells per 50 000 cells in HSCT from an HLA-identical sibling donor and HSCT

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Figure 2 shows the representative serial changes in US17PCR and antigenemia values in three patients. Both assays showed a similar pattern with a synchronized peak. The patient shown in the top panel developed suspected CMV pneumonitis at the peak of CMV virus load. Of the two patients who developed CMV colitis, one developed colitis at the peak of CMV infection (200 000 copies/ml by US17PCR and eight positive cells among 50 000 cells by the antigenemia assay), but the other developed colitis without prior detection of CMV infection. In the patient with CMV adrenalitis, CMV infection was detected only by US17PCR (500 copies/ml) a week before she died. CMV detection at a low virus load (⬍1000 copies/ml) by PCR assay without positive antigenemia was frequently (n = 41) observed, but was not associated with CMV disease, except for the patient with CMV adrenalitis.

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Figure 2 Representative serial changes in US17-PCR and antigenemia values in three patients. Both assays showed a similar pattern with a synchronized peak. An arrow in the top panel shows the detection of interstitial pneumonitis pattern on chest computed tomography. DHPG, ganciclovir; FCV, foscavir. Bone Marrow Transplantation


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Figure 4 Correlation between virus load determined by US17-PCR and IE-PCR (r = 0.83, P ⬍ 0.0001).

from a mismatched or unrelated donor,15,16 respectively, the threshold of US17-PCR and IE-PCR in the future study should be set at 10 000 (␬ = 0.24) and 1000 (␬ = 0.57) copies/ml, respectively, in HSCT from an HLA-identical sibling donor and 500 (␬ = 0.39) and 200 (␬ = 0.48) copies/ml, respectively, in HSCT from a mismatched or unrelated donor. Discussion Real-time PCR based on TaqMan technology enabled accurate quantification of viral DNA.17 In this study, we observed a good correlation between the antigenemia value and the virus load estimated by two distinct real-time PCR methods. US17-PCR appeared to be more sensitive than the antigenemia assay, but samples positive for US17-PCR and negative for the antigenemia assay were not associated with Bone Marrow Transplantation

CMV diseases except for a patient with CMV adrenalitis. Therefore, we considered it important to determine a threshold of the viral load estimated by real-time PCR. We provisionally determined the threshold of each PCR method as described above, but it should be validated in a prospective study. There have been several reports on the application of real-time PCR to the measurement of CMV virus load.12–14,18–21 However, they utilized different methods including regions for primers and TaqMan probe, and the source of DNA extraction (plasma, whole blood or leukocyte). Therefore, we must carefully interpret the results of real-time PCR. Actually, we observed a discrepancy in the virus load between two distinct PCR methods. A universal control sample may be required to solve this problem. Nevertheless, a better correlation was observed between the two independently performed real-time PCR assays employing distinct primers than that between the antigenemia assay and either of the two PCR methods. This implies that PCR methods which employ totally automated procedures, could produce more constant results than the antigenemia assay, which may be affected by human subjectivity. PCR assay using plasma can be used during leukocytopenia. In contrast, the antigenemia assay does not produce reliable data without sufficient leukocytes. This point is important, since some patients develop CMV disease before neutrophil engraftment and also, pre-emptive therapy with ganciclovir is frequently associated with subsequent neutropenia. Another reason why we used plasma as a source of DNA was the fact that PCR using leukocytes appeared to be too sensitive.11,12 It has been reported that CMV is detected in plasma only in an active CMV infection, whereas CMV can be detected in leukocytes in a latent infection.22 Therefore, we hypothesized that the correlation of the results between these two methods could be changed among phases of CMV infection, because the antigenemia assay reflects CMV in leukocytes. However, the correlation of the results of these two methods was equivalent between early and late phases of CMV infection, divided by before and after the peak antigenemia value (data not shown). A well-known pitfall of the antigenemia assay is the lack of sensitivity for CMV gastroenteritis.7,8 In this series, two patients developed CMV colitis; one after the detection of both positive antigenemia and positive PCR, and the other without prior detection of CMV infection by either method. Therefore, this issue might not be resolved by the use of real-time PCR. Our experience suggested that CMV adrenalitis is also difficult to detect by these assays. In conclusion, both real-time PCR methods showed a good correlation with the antigenemia assay, and the threshold to start ganciclovir was tentatively determined as described above. This threshold should be evaluated in a prospective study. Also, a randomized controlled trial is required to determine whether the real-time PCR method or the antigenemia assay is more suitable for CMV monitoring after hematopoietic stem cell transplantation.


Acknowledgements 11 The authors thank Ms Tomomi Nakae, Ms Eriko Oyama, and Ms Yuko Deguchi for the data management. 12

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