Ganciclovir sensitivity of cytomegalovirus at diagnosis and during treatment of cytomegalovirus pneumonia in marrow transplant recipients

Abstract

Cytomegalovirus (CMV) often persists in the lungs of marrow transplant patients with CMV pneumonia, despite ganciclovir (GCV) treatment. To determine whether GCV resistance contributes to viral persistence, the susceptibilities of CMV isolates from diagnostic bronchoalveolar lavage samples and CMV isolates obtained during treatment or from autopsy lung tissue from 12 patients were compared by DNA hybridization. Resistance (50% effective dose, > 12 ,uM) was detected in an isolate from only one patient who had also received several courses of GCV. GCV resistance did not explain the persistence of CMV in the lung.Although the combination of ganciclovir (GCV) and intravenous immunoglobulin (i.v.-Ig) has decreased the rate of mortality from cytomegalovirus (CMV) pneumonia in marrow transplant recipients, mortality remains high at 30 to 50% (3, 20), and as many as 10% of treated patients have relapses (2). CMV was still detected in 80% of patients with CMV pneumonia at our institution in a routine follow-up bronchoalveolar lavage (BAL) specimens after 9 days of treatment (22), without a reduction in viral titer (23). These findings raised the question of whether resistance to GCV could explain the persistence of CMV in the lungs of some patients, despite treatment.All 12 patients examined in the present study were allogeneic bone marrow transplant recipients with CMV pneumonia. CMV pneumonia was defined as described previously (23) and was treated with GCV, 5 mg/kg of body weight twice daily for 2 weeks, and i.v.-Ig (Cutter Biological, Miles Laboratories Inc., Berkeley, Calif.), 500 mg/kg on alternate days for 2 weeks. GCV was continued at 5 mg/kg daily for up to 30 days. Relapse was defined as the recurrence of symptoms, worsening pulmonary infiltrates on chest radiograph (20), and detection of CMV in BAL specimens after the completion of a course of treatment for the initial episode. Only two patients had received GCV prior to the episode of CMV pneumonia being studied. With the exception of one patient, all patients had received acyclovir (ACV) intravenously as CMV prophylaxis; ACV was given at 500 mg/M2 every 8 h from the day of pretransplant conditioning to the day of discharge from the hospital (17).CMV isolates were obtained from the initial diagnostic BAL specimens, from follow-up BAL specimens 9 days after starting GCV and i.v.-Ig, when routine follow-up BAL was being performed to assess the subsequent duration of GCV therapy, or from autopsy lung tissue after 7 to 32 days of treatment. Samples from urine, throat, and blood were obtained weekly for culture of CMV. All For susceptibility testing, isolates from patients and isolates 8708 and 8704 were first thawed and were then reinoculated into tube cultures of human foreskin fibroblasts and passaged one to four times until the cytopathic effect had progressed to 75 to 100% of the monolayer. CMV-infected cells were then removed with 0.05% trypsin and were resuspended in Dulbecco's modification of Eagle's medium with 10% fetal bovine serum (DME-10). Fifty mil...

Prevention of Cytomegalovirus Disease in Recipients of Allogeneic Stem Cell Transplants

“…GCV resistance was determined for CMV isolates obtained from BAL fluid or from autopsy lung tissue by DNA hybridization. In only 1 of 12 patients was a GCV-resistant isolate detected (91). In a study of 50 allogeneic SCT recipients, 10 patients exhibited sustained or recurrent Ag despite GCV treatment.…”

“…Samples from these 10 patients were screened for the presence of the most frequent CMV UL97 mutations by restriction enzyme analysis, and none of these mutations were detected (30). Overall, antiviral drug resistance in adult SCT recipients has been reported only sporadically (24,30,84,91). There is some evidence that it might be more frequent in pediatric SCT recipients, especially in patients with primary immunodeficiencies (18,78,101).…”

Isolation and expansion of cytomegalovirus-specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA-tetramers

“…Each 25-L PCR reaction contained 1 ϫ PCR buffer II, 2 mM MgCl 2 , 0.8 mM dNTP, 0.5 L target complementary DNA (cDNA), 0.625 U AmpliTaq Gold DNA polymerase, one of 23 forward primers specific for the TCR V␤ 1, 2, 3, 4, 5.1, 5. 2,6,7,8,9,11,12,13,14,15,16,17,18,20,21,22,23, and 24 genes (each at a concentration of 1 M), and a common constant region-specific reverse primer labeled at its 5Ј end with a 6-FAM moiety (also at a concentration of 1 M). All primers were supplied by Life Technologies.…”

“…1,8,9 Continued treatment with antiviral drugs after bone marrow transplantation may induce drug resistance and delay endogenous reconstitution of CMVspecific immunity in patients, resulting in late CMV disease. 8,[10][11][12][13][14] These complications are especially encountered after transplantation from partially mismatched related donors and other mismatched transplants that require T-cell depletion.…”

High-dose acyclovir and pre-emptive ganciclovir to prevent cytomegalovirus disease in myeloablative and non-myeloablative allogeneic stem cell transplantation

“…5,6 However, these antiviral agents are not without disadvantages: ganciclovir causes neutropenia and an increased risk of bacterial and fungal infection; [7][8][9][10][11][12] resistance to antiviral drugs can develop and the treatment can delay recovery of CMV-specific immunity. 8,[12][13][14][15] In order to minimize the use of GCV or foscarnet, many investigators use a pre-emptive treatment strategy performing regular blood monitoring for CMV antigen or DNA (using techniques which have varying sensitivity) and treating at the first indication of viremia. [16][17][18][19] An alternative way to reduce the need for ganciclovir or foscarnet is to use highdose acyclovir (HDACV) (1.5 g/m 2 /day) as prophylaxis for CMV disease.…”