Comparison of Cytomegalovirus Antigenemia and Shell Vial Culture in Allogeneic Marrow Transplantation Recipients Receiving Ganciclovir Prophylaxis
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Bone Marrow Transplantation, (1997) 19, 37–41 1997 Stockton Press All rights reserved 0268–3369/97 $12.00 Comparison of cytomegalovirus antigenemia and shell vial culture in allogeneic marrow transplantation recipients receiving ganciclovir prophylaxis VA Nicholson1, E Whimbey1, R Champlin2, D Abi-Said1, D Przepiorka2, J Tarrand3, K Chan4, GP Bodey1 and JM Goodrich5 1Section of Infectious Disease, 2Department of Hematology, and 3Division of Laboratory Medicine, 4Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX; and 5Division of Infectious Diseases, Southern Illinois University School of Medicine, Springfield, IL, USA Summary: be diagnosed with CMV disease coincident with or prior to having a positive CMV surveillance culture.1,7–9 Prophy- We prospectively monitored 61 allogeneic BMT patients lactic strategies result in administration of antiviral drugs for evidence of CMV infection and disease starting 7 to 30–40% of patients who would not have CMV infection days prior to transplant until day 110 after transplant. or disease.1,5 Patients receiving pre- and post-transplantation gan- To employ effectively an early treatment strategy for ciclovir prophylaxis were followed for the incidence of CMV infection requires a diagnostic test which is rapid and infection by the CMV antigenemia assay and shell vial risk stratifies patients for subsequent disease. Previously, cultures. The median age of all patients was 32 years centrifugation cultures have been used as a marker of CMV (range 5–54 years). Fourteen (25%) of 57 evaluable replication to initiate antiviral therapy in MT patients but patients became CMV antigenemia or culture positive. have lacked sensitivity.1,8 The CMV antigen assay appears The incidence of culture or antigenemia positivity in to be more sensitive than viral culture10–13 and provides CMV seropositive or seronegative patients with a sero- diagnostic results in a few hours as opposed to culture tech- positive donor was 29% (14 of 49 patients). The anti- niques which may take a few days to weeks.10,14,15 The genemia assay became positive a median of 29 days assay is based on the detection from the peripheral blood (range 12–89 days) after BMT as compared to 46 days of patients to a CMV late matrix tegument protein pp65.16,17 (range 26–98 days) by shell vial assay (P , 0.001). There The assay depends on the use of a pool of monoclonal anti- were no cases of CMV disease in the first 110 days after bodies directed against epitopes on this protein. In this transplant. This study demonstrates that despite the use study we investigated the use of the CMV antigen assay to of prophylactic ganciclovir, BMT patients developed monitor the development of CMV infection in allogeneic CMV infection but did not progress to disease in this BMT patients receiving prophylactic ganciclovir for the study, the CMV antigenemia assay may be used to prevention of CMV disease. The assay was compared with monitor for CMV infection during prophylaxis, and the viral culture and the centrifugation assay for sensitivity current regimens for CMV prophylaxis with ganciclovir and specificity. may require further evaluation to determine an optimal regimen to prevent CMV infection. Keywords: CMV; bone marrow transplant; antigenemia; Patients and methods diagnosis; viremia Patients The study group was comprised of 61 consecutive patients Cytomegalovirus (CMV) remains an important opportu- undergoing allogeneic MT between December 1992 and nistic infection following allogeneic BMT.1 Recently, September 1993 (Table 1). Patients were followed from day effective strategies for the prevention of CMV disease have −7 to day 100 after transplant. Blood was obtained weekly been developed in which ganciclovir has been given to for virus culture and the antigenemia assay. Urine cultures BMT patients either prophylactically (to all CMV seroposi- were collected weekly. Virus cultures were obtained from tive patients)1–6 or as early treatment (evidence of active other clinically relevant sites as needed (throat, tracheal CMV replication).1,7,8 Each of these strategies has limi- aspirate and bronchoalveolar lavage). tations. Twelve to 32% of patients given an antiviral based on an early treatment strategy (pre-emptive therapy based on a positive surveillance culture from blood or BAL) will CMV prophylaxis and treatment From March 1991 to April 1992, ganciclovir was given Correspondence: Dr J Goodrich, SIU School of Medicine, PO Box 19230, three times per week beginning 30 days post-transplant. In Springfield, Illinois 62794-1311, USA May 1992, patients who received T cell-depleted marrow Received 1 February 1996; accepted 18 July 1996 (TCD) were given ganciclovir prophylaxis five times per CMV antigenemia and allogeneic BMT VA Nicholson et al 38 Table 1 Characteristics of study population CMV antigenemia assay Characteristic Number CMV culture Number CMV culture Peripheral blood leukocytes (PBL) were isolated from or antigenemia or antigenemia 10 ml heparinized blood by the dextran sedimentation positive (%) negative (%) method. PBL were suspended in PBS at a concentration of 1.5 × 106 cells/ml and cytospin preparations were made. Male/Female 9/5 31/12 Slides were fixed in acetone, air-dried, and stored at 4°C Age median (range) 32 (9–48) 34 (5–54) if not stained within 24 h. Slides were stained with 35 mlof Underlying disease CML 4 (29) 11 (26) C10 and C11 monoclonal antibody directed against CMV AML 7 (50) 15 (35) phosphoprotein pp65 (Clonab, Biotest). Slides were coun- Myelodysplastic 0 (0) 3 (7) terstained with FITC-conjugated goat anti-mouse IG F(ab) syndrome (Biotest, Denville, NJ, USA) diluted in PBS 1% human CLL 1 (7) 4 (9) albumin and 0.0005% Evans blue. Positive cells showed a Lymphoma 1 (7) 3 (7) Aplastic anemia 0 (0) 1 (2) yellow–green nuclear staining. Positive controls consisted ALL 1 (7) 6 (14) of infected fibroblast or leukocytes from patients known to Donor be CMV antigenemia positive. Matched related 9 (64) 26 (60) Matched unrelated 2 (14) 12 (28) Mismatched 3 (2) 5 (12) CMV serology (D/R) Results Pos/Pos 11 (78) 18 (42) Pos/Neg 1 (7) 8 (19) Neg/Pos 2 (14) 9 (21) Sixty-one patients were enrolled in the study but four Neg/Neg 0 (0) 8 (19) patients were excluded from the analysis. Two patients Graft-versus-host excreted CMV prior to the start of monitoring, one patient disease died during conditioning therapy, and one patient was lost 0–1 6 (43) 23 (53) 2–4 8 (57) 20 (46) to follow-up prior to obtaining blood for culture and anti- genemia assays. Fifty-seven patients were evaluated for the CML = chronic myelogenous leukemia; AML = acute myelogenous leuke- final analysis (Table 1). Eight patients who were CMV mia; CLL = chronic lymphocytic leukemia; ALL = acute lymphocytic leu- seronegative with seronegative donors were included. kemia. Forty-three (75%) of the patients remained culture and anti- gen negative and represented the control group. There were no significant differences between the culture and antigen negative and culture and antigen positive groups, except for positive donor recipients pairs (P = 0.02). week. Beginning in October 1992, most patients received Fourteen (25%) of 57 patients (29% of 49 CMV sero- the five times per week regimen unless the attending phys- positive donor/patient combinations) became culture or ician directed differently. At day −1, patients were switched antigenemia positive (Table 2). Eleven of the 14 patients to acyclovir 500 mg/m2 every 8 h until engraftment (ANC had a positive viral culture at some site (blood = 9, >1 × 109/l for 2 consecutive days). Three patients received urine = 1, sputum = 1). Thirteen of the fourteen patients ganciclovir 5 mg/kg three times per week (Table 2). All were positive for CMV antigenemia. Ten of the 13 patients patients received intravenous immunoglobulin 500 mg/kg who were antigenemia positive had corresponding positive weekly from admission to day 110. Patients who became virus cultures. All patients who were culture positive were CMV culture positive received ganciclovir treatment at a antigenemia positive. The exception was a patient who had dose of 5 mg/kg twice per day or foscarnet 60 mg/kg three a positive sputum culture on day 45 after transplant who times per day. The decision to use foscarnet was made by remained antigenemia negative. The median time to CMV the attending physician and was based on ganciclovir toxi- antigenemia or culture positivity was 29 days (range 12– city (neutropenia or thrombocytopenia). The treatment dur- 89 days) and 46 days (range 26–98 days), respectively (P ation was determined by the clinical judgement of the , 0.0001, paired log rank test) (Figure 1). All nine patients attending physician. Patients who were CMV seronegative who were blood culture positive were antigenemia positive. and received a marrow from a CMV seronegative donor A positive antigenemia test preceded all positive blood received CMV seronegative blood products. shell vial cultures by a median of 14 days. One patient who was antigenemia positive on day 89 after transplant had a positive urine culture on day 98 after transplant but Statistical evaluation remained blood culture negative. Three patients were antigenemia positive on days 12, 26 The statistical analysis was performed using the x2 test and and 29 after transplant without having a positive CMV cul- a two-tailed Fisher exact test as appropriate for examination ture from any site. Two of the three patients were on inad- of differences in proportions, and the Mann–Whitney test equate anti-CMV prophylaxis (acyclovir and ganciclovir for evaluation of differences between medians. Time-to- three times per week) when they became antigenemia event variables were estimated according to the method of positive. Both patients became antigenemia negative upon Kaplan and Meier18 and compared using paired and the institution of ganciclovir five times per week.