Journal of Hematology - Supplements - Haematologica

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haematologica 2000; 85(supplement to n. 11):54-57 original paper Surveillance of cytomegalovirus infections in bone marrow transplant in Trieste: seven years’ experience P. D’AGARO, M. ANDOLINA, # P. BURGNICH, E. SAMAR, C. CAMPELLO Laboratory of Virology, Dept. Public Health; # Bone Marrow Transplantation Unit, IRCCS “Burlo Garofolo”, Trieste, Italy ABSTRACT Forty-five consecutive patients submitted to a bone marrow transplant (BMT) were followed up weekly in order to evaluate the incidence of cytomegalovirus (CMV) infections on the basis of CMV antigenemia and polymerase chain reaction. All but one transplanted patients engrafted; fourteen patients out of these were CMV antigenemia positive after 16-184 days (median 32.5, mean 43.4) with an 31.8% incidence. CMV infections were associated with graftversus-host disease and immunogenetic relationship between the donor and the recipient. No CMV infection was detectable in autologous transplants while antigenemia was demonstrated in 3/11 and 6/7 patients with BMT from respectively mismatched related and matched unrelated donors. ©2000, Ferrata Storti Foundation Key words: Cytomegalovirus, bone marrow transplant, surveillance, PCR. Viral infections are important complications after allogeneic bone marrow transplantation (BMT) and have a heavy impact on morbidity and mortality. The most frequent and clinically relevant are related to members of the Herpesvirus family. 1,2 In fact, a cytomegalovirus (CMV) infection is demonstrable in 50-70% of allogeneic BMT recipients and one third of these develops interstitial pneumonia (IP) with a case fatality ratio of 85%. 3-5 The main risk factors for viral complications in allogeneic BMT are latent viral infections of donors and of recipients, donor HLA compatibility, conditioning and graft-versus-host disease (GVHD). 6, 7 Post-transplant immune reconstitution after BMT is characterized by an early leukopenic granulocytopenic phase lasting one month, followed by a period between days 30 – 100 with a marked lack of T-cell function and characterized by acute GVHD; significative immune competence is recovered by the sixth month. 8-10 Curiously viral infections are not Correspondence: Dr. P. D’Agaro, E-mail: dagaro@burlo.trieste.it evenly distributed in the post-transplant period: the most frequently occurring infections in the first month are HSV oral mucositis and respiratory virus infections; CMV infections are the main complications in the period ranging from day 30 to 100 although they have recently begun to frequently demonstrated later with similar mortality. 11, 12 Epstein-Barr virus reactivations are relatively frequent between the 2 nd and 4 th months while Varicella Zoster virus reactivations between the 4 th and 12 th month. In recent years preventive measures and preemptive therapy have reduced CMV interstitial pneumonia associated mortality from 80% to 10-20%; 13,14 this underlines the relevance of virologic monitoring in order to detect systemic CMV infections early. An emerging problem is the selection of mutant strains resistant to the antivirals used in prophylaxis. 15 In this study we report the incidence of CMV infections in a two-year follow-up of forty-five consecutive BMT patients evaluated by pp65 antigenemia and polymerase chain reaction (PCR) investigations. Design and Methods Patients Forty-five consecutive patients who underwent BMT performed in the BMT Unit of our hospital between 10 January 1992 and 25 November 1999 were included in the study. Clinical data of the 44 engrafted BMT patients are reported in Table 1. In brief, 21 were female and 23 male, their median age was 10.6 years (11.6 F, 8.7 M); indications for BMT included acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myeloid leukemia, solid tumours, autoimmune diseases, myelodysplastic syndromes, and thalassemi. Fourteen patients needed a further BMT for relapse or lack of engraftment. All patients were screened for CMV antigenemia weekly until day +100; samples of polymorphonuclear cells were store at –20°C for further analysis by PCR amplification. haematologica vol. 85(supplement to n. 11):November 2000
55 Table 1. Summary of clinical data of the 44 engrafted patients. Table 2. Comparison of CMV detection in PMN by pp65 antigen assay and PCR. N° CMV infections p # Sex Male 23 9 0.223 Female 21 5 Age (years) 1-4 11 3 5-9 10 2 10-14 12 4 0.595 15-19 7 4 >19 4 1 CMV serol. Pre-transpl. Negative 12 2 0.186 Positive 32 12 CMV serol. Donor Negative 10 2 Positive 32 11 0.763 Unknown 2 1 Diagnosis AL 1 0 ALL 15 8 AML 4 2 CML 4 2 0.121 ST 7 0 AID 6 0 GEN 6 2 MDS 1 0 Donor Autologous 14 0 0.001 MRD 11 3 Haploidentical 12 5 MUD 7 6 GVHD No 25 2 0.001 Acute I 8 5 Acute II-IV 9 7 chronic 2 0 AL: acute leukemia; ALL: acute lymphoblastic leukemia; AML: acute myelogenous leukemia; CML: chronic myeloid leukemia; ST: solid tumor; AID: autoimmune disease; GEN: genetic disease; MDS: myelodysplastic syndrome. MRD: matched related donor; MUD: matched unrelated donor; # Pearson Chisquare test. CMV antigenemia The antigenemia assay was performed using a CINApool kit (Argene-Biosoft) according to the manufacturer’s instructions. Specimens were processed within 4 h of collection and a positive result was defined as ≥ 2 cells with fluorescent nuclei in duplicate spots containing 2×10 5 white blood cells. PCR A subset of 60 samples of polymorphonuclear DNA was examined by PCR. Amplification of a 435 bp fragment in the CMV major immediate early gene and of a 400 bp region in the late antigen gene was performed by single step PCR using primers MIE4–MIE5 and LA1–LA6 16 respectively in a Perkin Elmer thermal cycler. After a 5’ DNA denaturation step, 45 cycles of denaturation at 95°C for 30”, primer annealing at 60°C for 30” and DNA elongation at 72°C for 30” were followed by a final DNA extension at 72°C for 5’. Each run included one positive viral control (AD169) and two negative controls. PCR positive negative total Antigen assay positive 19 0 19 negative 1 40 41 total 20 39 60 The amplified products were demonstrated by gel electrophoresis, stained with ethidium bromide and examined on an ultraviolet transilluminator. Statistical analyses Differences in proportions between groups were examined using Pearson’s chi-squared test and Fisher’s exact test. Statistical calculations was performed using SPSS 10 software. Results The comparison between the results obtained with antigenemia and PCR techniques in a subset of 60 samples is reported in Table 2. PCR detected all 20 infected patients while antigenemia resulted negative in a PCR positive sample, thus giving a test sensitivity of 95%. The PCR positive-antigenemia negative result occurred in a patient with low levels of CMV in white blood cells and he become pp65 positive one week later. Of the 44 engrafted patients 14 were CMV positive by the antigenemia test. None of the 14 autologous BMT patients developed a CMV infection while the incidence was 46.7% in the group of patients receiving an allogeneic BMT. In detail 3 out 11 recipients of a transplant from an HLA matched related donor, 5 out 12 from a haploidentical donor and 6 out 7 from a matched unrelated donor were CMV-infected. All but one CMV infections developed within 100 days after BMT, the median time being 32.5 days (range: 16-184; first and third quartiles 23.8, 47.5). Sex, age, CMV antibody prevalence in donors and recipients and underlying diseases did not differ significantly between infected and noninfected patients. A strong association was demonstrated between antigenemia positivity and GVHD score (p
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