International Association of Physicians in AIDS Care, February 2000 Journal
Vincent C. Emery, BSc, PhD
| Introduction Genetic Basis of CMV Resistance Methods to Identify CMV Resistance Incidence of CMV Drug Resistance Future Developments Acknowledgments References |
After ganciclovir (GCV) and foscarnet were introduced into clinical use in the late 1980s for the treatment of human cytomegalovirus (CMV) disease, several groups reported the appearance of clinical resistance to these compounds.1-4 Since DNA sequence analysis of the CMV genome had failed to identify a thymidine kinase homologue, it was originally assumed that cellular enzymes activated GCV to its active triphosphate form.5 This proved not to be true. In 1992 Littler and colleagues6 and Sullivan and coworkers7 showed that the UL97 gene of human CMV encodes a protein kinase that can phosphorylate GCV and acyclovir to their monophosphate moieties. Since these reports, many studies have detailed the molecular basis of resistance to GCV involving both UL97 and the CMV DNA polymerase (UL54), and more recent work defined the mutations leading to resistance to foscarnet and to cidofovir (reviewed in reference 8). This article will concentrate on the genetic basis of resistance, the methods available to identify CMV resistance in clinical samples, and the incidence of CMV resistance.
Thanks to the pioneering work of Chou and colleagues,9-12 the molecular basis of UL97 resistance to GCV has been mapped in clinically relevant settings. Overall, clinical strains of CMV resistant to GCV in vitro predominately possess mutations at amino acids 460, 520, 594, and 595. In addition, deletions have also been noted within the UL97 sequence between amino acids 591 and 594, 595, and 600, and between 590 and 603. The 591-594 deletion was the deletion present in the prototypic GCV resistance strain that led to the discovery of UL97 as a GCV kinase. In vivo, UL97 mutant strains appear to be less fit than their wild-type counterparts.13
Mutations in the CMV DNA polymerase (UL54) leading to GCV resistance are more complex but are predominately located in the exo-II and C regions of UL54. These mutations may also involve regions II, III, V, and VI (reviewed in reference 8). Resistance ranges from 3.5-fold for the L802M mutation to more than 20-fold for the D301N mutation in the exo-1 domain, for the L501F mutation, or for the T403I mutation in C domain. As expected, many UL54 mutations associated with GCV resistance also show cross-resistance to cidofovir, and some UL54 mutations (for example, D301N) also show cross-resistance to foscarnet.
Mutations leading to cidofovir resistance are predominately located in the exo II, C, and III domains of UL54. Resistance ranges from more than 10-fold (for mutations 408D + S897P, L501F, G678S, V781I) to between 20- to 90-fold (for K513N and D413E) (reviewed in 8). Most mutations associated with cidofovir resistance also confer cross-resistance to GCV, and some UL54 mutations associated with cidofovir resistance (such as D301N) confer cross-resistance to foscarnet.
The classic approach to identifying strains of CMV resistant to antivirals has been propagation of strains in vitro followed by plaque reduction assays in the presence of drug. Such assays allow an IC50 to be assigned to the drug in question and compared with the IC50 of fully sensitive strains. Although there is a spectrum of IC50 values for clinical strains of human CMV, the average value is substantially lower than those of genotypically resistant strains.
The major problems with in vitro propagation of clinical strains of CMV are (1) the low frequency with which strains can be grown from biologically relevant fluids such as blood, (2) the possibility that the complex mixture of resistant and sensitive strains may be disturbed upon passage, especially if the resistant strain is substantially less fit than its wild-type counterpart, and (3) the long time it takes for such assays to provide clinically relevant information. Consequently, molecular approaches to identifying resistance have been developed. At present, three molecular approaches can be used.
The first method uses restriction fragment length polymorphisms generated by the mutations to identify the acquisition or loss of restriction sites within the UL97 gene. Using such an approach, Chou and colleagues extensively detailed the frequency of mutations in UL97, and these studies led to our current understanding of the molecular basis of resistance at this genetic locus.9,10 Bowen and coworkers described an alternative approach in which a modified form of an assay used to detect genetic resistance to anti-HIV compounds--the point mutation assay--is used to target key codons in the UL97 protein.15 Both of these assays provide data quickly and efficiently, but, as with all molecular assays, they are confounded by the number of positions being analyzed and the possibility that compensatory mutations may affect the overall ability of the virus to escape from the antiviral in question.
The third approach, which has been used predominately to assess UL54 mutations, is direct sequencing. Although direct sequencing can also detect mixed populations, it is relatively time consuming and expensive. Until more rapid gene-based assays are available, for example, with gene chip technology, it is unlikely that clinical diagnostic laboratories will use such an approach routinely.
There are numerous case reports of clinically relevant resistance against GCV and foscarnet (reviewed in reference 16). Generally, the frequency of resistance increases with longer exposure to the antiviral in question. Hence, AIDS patients who require chronic maintenance therapy for CMV retinitis exhibit a higher incidence of resistance than do transplant recipients who often require only short-term therapy.
Notwithstanding these observations, relatively few published studies report the incidence of resistance in large cohorts. Using cell culture analysis of urine cultures from 72 AIDS patients receiving GCV therapy for CMV retinitis, Drew and colleagues scored the overall incidence of resistance as 7.6 percent.2
Using more sensitive molecular methods, Bowen and coworkers prospectively followed 45 AIDS retinitis patients receiving 21 days of intravenous GCV induction therapy followed by oral GCV maintenance therapy.17 During follow-up 14 of these patients became DNAemic, all of whom had progression of their retinitis. Of these 14 patients, 10 (71 percent) had GCV-resistant strains of CMV detected by a rapid molecular assay (see above). As expected, none of the patients who exhibited genotypic resistance became PCR negative after reinduction therapy with GCV. The population-based incidence of resistance in this study (22 percent) was higher than the figure determined by Drew and associates.2
The advent of prophylactic strategies to prevent CMV disease after organ transplantation has allowed assessment of the incidence of resistance in these immunocompromised cohorts. In a series of 240 solid organ transplant recipients treated with GCV prophylaxis, GCV-resistant CMV disease developed in 2.1 percent at a median of 10 months after transplantation.18 In the 20 patients with CMV disease by one year, 25 percent had resistant virus. It is likely that the incidence of GCV resistance determined in this study represents a minimum value, and further studies using genotypic approaches are awaited.
The advent of highly active antiretroviral therapy (HAART) for HIV-infected individuals has led to a dramatic reduction in the incidence of CMV infection and disease. Consequently, the proportion of patients requiring induction and maintenance therapy for CMV retinitis has also decreased substantially. Since these individuals were most at risk for developing CMV drug resistance, at the time of writing the incidence of GCV resistance has declined. How long this situation will last is unclear. However, it is likely that failure of HAART due to long-term toxicity and poor adherence will lead to concomitant increases in the risk of CMV-related disease. If that happens, chronic therapy for CMV infection which will again be required.
For immunocompromised groups other than HIV-positive individuals, the availability of newer therapies or new formulations (such as valganciclovir, the oral prodrug of GCV) will almost certainly result in increased use in preemptive and prophylactic management strategies. Such conditions create an ideal environment for generation of CMV drug resistance, and it would be naive to suggest that drug resistance to newer anti-CMV agents undergoing phase I/II trials will neither develop nor have important clinical implications.
I would like to acknowledge my colleagues Dr. E.F. Bowen, Ms. C. Shannon-Lowe, Dr. Margaret A Johnson, and Professor P.D. Griffiths for their contributions to the work reported here. Work in my laboratory is supported by the Medical Research Council (UK), Wellcome Trust, the European Community, and the National Institutes of Health, USA.
2. Drew WL, Miner RC, Busch DF, et al. Prevalence of resistance in patients receiving ganciclovir for serious cytomegalovirus infection. J Infect Dis 1991;163:716-719.
3. Sullivan V, Coen DM. Isolation of foscarnet-resistant human cytomegalovirus patterns of resistance and sensitivity to other antiviral drugs. J Infect Dis 1991;164:781-784.
4. Knox KK, Dorbyski WR, Carrigan DR. Cytomegalovirus isolate resistant to ganciclovir and foscarnet from a marrow transplant patient. Lancet 1991;337:1292-1293.
5. Chee MS, Bankier AT, Beck S, et al. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 1990;154:125-169.
6. Littler E, Stuart AD, Chee MS. Human cytomegalovirus UL97 open reading frame encodes a protein that phosphorylates the antiviral nucleoside analogue ganciclovir. Nature 1992;358:160-162.
7. Sullivan V, Talarico CL, Stanat SC, et al. A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells. Nature 1992;358:162-164. Published errata appear in Nature 1992 Sep 3;359(6390):85 and 1993 Dec 23-30;366(6457):756.
8. Tatti KM, Smith IL, Schinazi RF. Mutations in human cytomegalovirus (HCMV) DNA polymerase associated with antiviral resistance. Int Antiviral News 1998;6:6-9.
9. Chou S, Guentzel S, Michels KR, et al. Frequency of UL97 phosphotransferase mutations related to ganciclovir resistance in clinical cytomegalovirus isolates. J Infect Dis 1995;172:239-242.
10. Chou S, Erice A, Jordan MC, et al. Analysis of the UL97 phosphotransferase coding sequence in clinical cytomegalovirus isolates and identification of mutations conferring ganciclovir resistance. J Infect Dis 1995;171:576-583.
11. Chou S, Marousek G, Guentzel S, et al. Evolution of mutations conferring multidrug resistance during prophylaxis and therapy for cytomegalovirus disease. J Infect Dis 1997;176:86-89.
12. Boivin G, Chou S, Quirk MR, et al. Detection of ganciclovir resistance mutations by quantitation of cytomegalovirus (CMV) DNA in leukocytes of patients with fatal disseminated CMV disease. J Infect Dis 1996;173:523-528.
13. Emery VC, Cope AV, Bowen EF, et al. The dynamics of human cytomegalovirus replication in vivo. J Exp Med 1999;190:177-182.
14. De Clerq E. Therapeutic potential of HPMPC as an antiviral drug. Rev Med Virol 1993;3:85-96.
15. Bowen EF, Johnson MA, Griffiths PD, Emery VC. Development of a point mutation assay for the detection of human cytomegalovirus UL97 mutations associated with ganciclovir resistance. J Virol Methods 1997;68:225-234.
16. Chou S. Antiviral drug resistance in human cytomegalovirus. Transplant Infect Dis 1999;1:105-114.
17. Bowen EF, Emery VC, Wilson P, et al. Cytomegalovirus polymerase chain reaction viraemia in patients receiving ganciclovir maintenance therapy for retinitis. AIDS 1998;12:605-611.
18. Limaye AP, Corey L, Koelle DM, et al. Emergence of ganciclovir resistant cytomegalovirus disease among solid organ transplant recipients. Presented at: 39th Interscience Conference on Antimicrobial Agents and Chemotherapy; September 26-29, 1999; San Francisco. Abstract LB-1.
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This information is designed to support, not replace, the relationship that exists between you and your doctor.