Human cytomegalovirus double resistance in a donor-positive/recipient-negative lung transplant patient with an impaired CD4-mediated specific immune response

Fausto Baldanti1,2, Daniele Lilleri1, Giulia Campanini1, Giuditta Comolli1,2, Anna Lisa Ridolfo3, Stefano Rusconi3 and Giuseppe Gerna1,*

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background: Emergence of human cytomegalovirus (HCMV) resistance to ganciclovir in solid-organ transplant recipients has been found to be mostly associated with primary HCMV infection.

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


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human cytomegalovirus (HCMV) resistance to antiviral drugs is becoming an increasing problem in transplantation settings. Several groups have reported the emergence of drug-resistant HCMV strains in both solid-organ and haematopoietic stem cell transplant recipients.13 A common feature observed in this setting is that drug-resistant HCMV strains almost exclusively emerge during treatment of primary HCMV infection, in particular in lung transplant recipients.13 A possible explanation for the increased risk of emergence of drug-resistant HCMV strains in donor-positive/recipient-negative (D+/R) transplant recipients is the impairment of these patients in building up an efficient cell-mediated immune response. If this assumption is correct, then monitoring of the T cell response could be a valuable tool for the identification of patients at risk of developing HCMV drug resistance.

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Virological parameters

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-{alpha}-phycoerythrin and anti-CD69-PerCP. CD4 cells expressing TNF-{alpha} 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.


    Results
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In November 2001, a 57-year-old HCMV-seronegative male with idiopathic lung fibrosis received a single lung transplant from an HCMV-seropositive donor. Ciclosporin (150 mg twice a day), azathioprine (100 mg once daily) and prednisolone (25 mg four times a day) were administered to the patient as anti-rejection therapy. Following 1 month of prophylaxis with intravenous ganciclovir (5 mg/kg once daily), the patient was enrolled in a pre-emptive therapy protocol, in which treatment was intended to start upon first confirmed positive antigenaemia. Sixty days after transplant, the patient developed primary HCMV infection (Figure 1), and 2 weeks later ganciclovir (5 mg/kg twice daily) was administered on the basis of high antigenaemia (360 pp65-positive PBL/2 x 105 PBL), associated with fever, heartburn and hypertransaminasaemia. Treatment was interrupted after 4 weeks upon negative antigenaemia and viraemia, and lack of clinical symptoms. However, shortly thereafter, the patient showed a recurrence of HCMV infection which was treated with a 2-week course of ganciclovir. Subsequently, in the absence of antiviral treatment, antigenaemia and DNAaemia were consistently positive. Therefore, following an additional course of treatment with ganciclovir at full dosage, a ganciclovir maintenance regimen (5 mg/kg once daily) was started. The emergence of a ganciclovir-resistant strain with a mutation in UL97 (A594V) was documented 270 days after transplant by direct sequencing of HCMV DNA from blood. A subsequent foscarnet treatment (90 mg/kg twice daily) was effective in clearing HCMV from blood, but had to be discontinued after 2 weeks because of renal toxicity (creatinine levels >= 3 mg/dL), further exacerbated by ciclosporin administration. A new recurrence of HCMV infection was again treated with foscarnet at half the dosage (90 mg/kg once daily). At 360 days after transplant, as a result of positivity of cultures of lung secretions collected at bronchoscopy for Mycobacterium tuberculosis, rifampicin, isoniazid and ethambutol were administered.



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Figure 1. (a) Monitoring of HCMV infection by quantification of antigenaemia, viraemia and DNAaemia in a D+/R lung transplant recipient, is shown. Ganciclovir (GCV) and foscarnet (PFA) treatments are indicated by shaded areas. Arrows indicate the emergence of a GCV-resistant (GCVR) strain with a mutation in UL97 (A594V) and a strain cross-resistant to both GCV and cidofovir (GCVR CDVR) with mutations in UL97 (H520Q) and UL54 (P522S). (b) Level of CD4 cell response to HCMV during follow-up (starting 360 days after transplantation) is indicated by the continuous line. Mean levels of CD4 cell response to HCMV observed in five immunocompetent individuals 1 year after onset of primary HCMV infection, and five heart or lung transplant recipients 1 year after transplantation, are shown for comparison (bars).

 
A deficit in the cell-mediated immune response to HCMV was suspected, and therefore CD4 and CD8 cell counts as well as the HCMV-specific CD4 cell response were determined. Results showed that the frequency of HCMV-specific CD4 cells (0.48/mm3 blood; Figure 1) was 3–5-fold lower than the mean value of either five seropositive immunocompetent individuals at 12 months after onset of primary HCMV infection (mean, 2.80; S.E.M. ± 1.19, range 0.61–6.21) or five heart or lung transplant recipients at 1 year after transplantation (mean, 1.87; S.E.M. ± 0.79, range 0.52–4.76). Total CD4 cell count (483 cells/mm3 blood) was comparable with that of the other transplanted patients (mean, 655; S.E.M. ± 168, range 323–1297 cells/mm3 blood) and lower than that of immunocompetent subjects (mean, 1137; S.E.M. ± 244, range 439–1913 cells/mm3 blood). At 380 days after transplant, in the presence of negative HCMV antigenaemia and viraemia, foscarnet treatment was interrupted, even in the presence of a low level of HCMV DNA (10 copies/10 µL) because of renal impairment (creatinine levels >= 3 mg/dL). One week after treatment discontinuation, the patient showed high levels of HCMV antigenaemia, viraemia and DNAaemia. Despite the compassionate administration of ganciclovir at full dosage in an attempt to at least partially block virus replication, HCMV load remained stable at a high level. In addition, a ganciclovir-resistant HCMV strain with mutations in both UL97 (H520Q) and UL54 (P522S) emerged, the latter conferring cross-resistance to cidofovir. The CD4 cell response to HCMV remained consistently negative (mean value of the five assays carried out, 0.40; S.E.M. ± 0.18, range 0.21–0.56). The patient eventually died shortly thereafter of lung failure.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
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The emergence of HCMV drug resistance in the transplantation setting is still a relatively rare event, which appears to be primarily restricted to the D+/R subset of transplanted patients.13 However, this event is more and more frequently reported, in parallel with the increasing number of transplant patients. The reason why D+/R transplant recipients appear to be at such a high risk of developing drug-resistant HCMV strains is still unclear. In fact, these patients are treated either prophylactically or pre-emptively upon first appearance of HCMV in blood, in contrast to D+/R+ and D/R+ patients who, according to pre-emptive treatment protocols, are treated when in the presence of a higher HCMV load.4 In this situation, antiviral treatment should be most effective, HCMV should be rapidly cleared and selection of mutant virus strains should be unlikely to occur. Indeed, in the great majority of D+/R transplant patients with HCMV infection,4 early antiviral treatment determines sustained virus clearance from blood because, despite immunosuppression, their HCMV-specific CD4 cell response reaches levels comparable to those observed in immunocompetent individuals.6

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.


    Acknowledgements
 
We thank Luca Dossena, Lucia Chezzi and Cinzia Zanello for skilful technical assistance. We also thank Linda D’Arrigo for revision of the English. This work was partially supported by Ministero della Salute, IRCCS Policlinico San Matteo, Ricerca Finalizzata (grants 0AG/F16, convenzione 126 and convenzione 127) and Ricerca Corrente (grant no. 80425).


    Footnotes
 
* Corresponding author. Tel: +39-0382-502644; Fax: +39-0382-502599; E-mail: g.gerna{at}smatteo.pv.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Baldanti, F., Simoncini, L., Sarasini, A. et al. (1998). Ganciclovir resistance as a result of oral ganciclovir in a heart transplant recipient with multiple human cytomegalovirus strains in blood. Transplantation 66, 324–9.[ISI][Medline]

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, 645–9.[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, 760–8.[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, 155–75.[ISI]

5 . 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, 1225–30.[Abstract/Free Full Text]

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, 541–8.[Abstract/Free Full Text]

7 . Baldanti, F. & Gerna, G. (2003). Human cytomegalovirus resistance to antiviral drugs: diagnosis, monitoring and clinical impact. Journal of Antimicrobial Chemotherapy 52, 324–30.

8 . 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, 2686–92.[Abstract/Free Full Text]

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, 724–7.[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, 1285–9.[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, 35–43.[Abstract/Free Full Text]