1 Servizio di Virologia and 2 Laboratori Sperimentali di Ricerca, IRCCS Policlinico San Matteo, 27100 Pavia; 3 Istituto di Malattie Infettive e Tropicali, Università degli Studi di Milano, Ospedale Luigi Sacco, Milan, Italy
Received 25 July 2003; returned 20 September 2003; revised 28 October 2003; accepted 10 November 2003
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Abstract |
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Materials and methods: The case of a donor-positive/recipient-negative (D+/R) lung transplant patient developing ganciclovir and cidofovir resistance is described. HCMV infection was monitored by weekly determination of antigenaemia, viraemia and DNAaemia. HCMV-specific CD4 cell immunity was determined by cytokine flow cytometry. The emergence of drug-resistant HCMV strains was documented by sequencing of UL97 and UL54 genes of HCMV directly in blood samples.
Results: Following primary HCMV infection, the patient showed repeated reactivations for over a year, eventually resulting in the selection of a ganciclovir-resistant HCMV strain with a mutation in the UL97 gene product (A594V). Determination of HCMV-specific CD4 cell immunity showed a persistently impaired immune response. Subsequent foscarnet treatment allowed only transitory virus clearance from blood owing to renal toxicity. Further ganciclovir treatment induced a new mutation in both UL97 (H520Q) and UL54 (P522S) with final emergence of double resistance to both ganciclovir and cidofovir. The patient eventually died of lung failure.
Discussion: Determination of HCMV-specific CD4 cell immunity could be of help in predicting the emergence of drug-resistant strains in D+/R transplant recipients.
Keywords: HCMV, drug resistance, D+R, CD4 cell response
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Introduction |
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Here, we report the case of a lung transplant recipient with HCMV infection developing virus resistance to both ganciclovir and cidofovir, 1 year after transplantation, in the presence of an impaired CD4 cell response to HCMV.
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Materials and methods |
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Virological monitoring of HCMV infection consisted of weekly determination of antigenaemia, viraemia and DNAaemia4 for a 16-month period. Antigenaemia was expressed as the number of pp65-positive peripheral blood leucocytes (PBL)/2 x 105 PBL examined, viraemia as the number of p72-positive human fibroblast nuclei after inoculation of 2 x 105 PBL into a shell vial culture, and DNAaemia as HCMV DNA copy number in 10 µL of whole blood.4 The emergence of HCMV drug-resistant strains was evaluated by sequencing of genes UL97 (encoding the viral phosphotransferase) and UL54 (encoding the viral DNA polymerase) directly in blood samples collected concomitantly with viral load rebounds during antiviral treatment.4
Intracellular cytokine staining
Evaluation of the CD4 cell response to HCMV infection was carried out by cytokine flow cytometry as previously described.5 In detail, the frequency of HCMV-specific CD4 cells was determined by culturing peripheral blood mononuclear cells in the presence of HCMV antigen (HCMV-infected fibroblast lysate) or its negative control antigen and anti-CD28 and anti-CD49d monoclonal antibodies. After 1 h of culture, brefeldin A was added to prevent cytokine release and cells were cultured for an additional 14 h before staining with anti-CD4-fluorescein isothiocyanate, anti-TNF--phycoerythrin and anti-CD69-PerCP. CD4 cells expressing TNF-
and CD69 were considered activated cells. For comparison, the HCMV-specific CD4 cell response was evaluated in five seropositive immunocompetent subjects 12 months after primary infection, and five solid-organ (heart or lung) transplant recipients (D+/R+) 12 months after transplantation.
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Results |
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Discussion |
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In contrast, the selection of drug-resistant HCMV variants in D+/R transplant recipients occurs following repeated recurrences of HCMV infection associated with a high viral load.1,7 Similar kinetics of HCMV infection were observed in patients with AIDS carrying drug-resistant HCMV strains,7 and a deficit in HCMV-specific CD4 cell response in the presence of high CD4 cell counts has been associated with development of HCMV disease both in transplant recipients (even in the presence of a positive CD8 cell response)8,9 and HIV-infected individuals.10
This report documents that, following primary HCMV infection, an impaired CD4 cell response to HCMV may indeed be associated with repeated recurrences of HCMV infection, eventually leading to the emergence of multidrug-resistant HCMV strains. Our data confirm and extend the observation recently reported by Benz et al.9 on the emergence of a ganciclovir-resistant HCMV strain in a pancreatic/renal transplant recipient undergoing primary HCMV infection in the presence of an incomplete CD4 cell response.
Reasons for the deficit in the immune response that we observed were not identified. However, the development of M. tuberculosis infection in our patient would seem to indicate a broad impairment of the immune function. In the patient herein described, the sustained impairment of the HCMV-specific CD4 cell-mediated immune response caused, first, the appearance of a single mutation in UL97 conferring resistance to ganciclovir and, second, a double mutation in both UL97 (different from the previous one) and UL54, the latter conferring cross-resistance to cidofovir. At this time, therapeutic options with antiviral drugs were restricted to foscarnet. However, foscarnet could be administered only at a reduced dosage because of renal toxicity.
Most often, clinical conditions of transplanted patients carrying drug-resistant HCMV strains allow administration of alternative antiviral regimens able to control viral infection. However, impairment of the renal function may often limit the availability of valid therapeutic options. In fact, foscarnet is excreted in the urine and at the moment there are no recommended dose adjustments for advanced renal failure or patients undergoing dialysis. Moreover, following primary HCMV infection, the level of impairment of the immune system may dramatically decrease the chance of clearing HCMV from blood. In these cases, few therapeutic options are left. While waiting for new drugs to become available in clinical settings, one possibility for intervention relies on the administration of HCMV-specific autologous cytotoxic T lymphocytes.11 However, although suggested as an effective tool for treatment of HCMV drug-resistant infections, this therapeutic approach is available only in a few specialized centres. Moreover, the efficacy of this approach in large patient series remains to be established.
In conclusion, an emerging pattern from this as well as other case reports indicates that monitoring of HCMV-specific cell-mediated immunity in D+/R transplant patients (including HCMV-specific CD8 cell response) could be of great help in predicting the emergence of drug-resistant HCMV strains. In addition, introduction of new anti-HCMV molecules in the clinical settings is warranted.
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Acknowledgements |
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Footnotes |
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References |
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2 . Limaye, A. P., Corey, L., Koelle, D. M. et al. (2000). Emergence of ganciclovir-resistant cytomegalovirus disease among recipients of solid-organ transplants. Lancet 356, 6459.[CrossRef][ISI][Medline]
3 . Lurain, N. S., Bhorade, S. M., Pursell, K. J. et al. (2002). Analysis and characterization of antiviral drug-resistant cytomegalovirus isolates from solid organ transplant recipients. Journal of Infectious Diseases 186, 7608.[CrossRef][ISI][Medline]
4 . Gerna, G., Baldanti, F., Grossi, P. et al. (2001). Diagnosis and monitoring of human cytomegalovirus infection in transplant recipients. Reviews in Medical Microbiology 12, 15575.[ISI]
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.
Piccinini, G., Comolli, G., Genini, E. et al. (2001). Comparative analysis of human cytomegalovirus-specific CD4+ T cell frequency and lymphoproliferative response in HIV-positive patients. Clinical and Diagnostic Laboratory Immunology 8, 122530.
6
.
Rentenaar, R. J., Gamadia, L. E., van DerHoek, N. et al. (2000). Development of virus-specific CD4(+) T cells during primary cytomegalovirus infection. Journal of Clinical Investigation 105, 5418.
7 . Baldanti, F. & Gerna, G. (2003). Human cytomegalovirus resistance to antiviral drugs: diagnosis, monitoring and clinical impact. Journal of Antimicrobial Chemotherapy 52, 32430.
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.
Gamadia, L. E., Remmerswaal, E. B., Weel, J. F. et al. (2003). Primary immune responses to human CMV: a critical role for IFN-gamma-producing CD4+ T cells in protection against CMV disease. Blood 101, 268692.
9 . Benz, C., Holz, G., Michel, D. et al. (2003). Viral escape and T-cell immunity during ganciclovir treatment of cytomegalovirus infection: case report of a pancreatico-renal transplant recipient. Transplantation 75, 7247.[CrossRef][ISI][Medline]
10 . Komanduri, K. V., Feinberg, J., Hutchins, R. K. et al. (2001). Loss of cytomegalovirus-specific CD4+ T cell responses in human immunodeficiency virus type 1-infected patients with high CD4+ T cell counts and recurrent retinitis. Journal of Infectious Diseases 183, 12859.[CrossRef][ISI][Medline]
11
.
Riddell, S. R. & Greenberg, P. D. (2000). T-cell therapy of cytomegalovirus and human immunodeficiency virus infection. Journal of Antimicrobial Chemotherapy 45, Suppl. T3, 3543.