1 Servizio di Virologia, 2 Laboratori Sperimentali di Ricerca, IRCCS Policlinico San Matteo, 27100 Pavia, Italy
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Abstract |
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Keywords: HCMV, antivirals, drug resistance
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Introduction |
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This report will describe the mechanism of action of anti-HCMV drugs and the molecular basis of HCMV drug resistance. In addition, techniques for monitoring HCMV infection and response to antiviral treatment and for detecting the emergence of drug-resistant HCMV strains will be reviewed. Special attention will be paid to discuss the advantages and drawbacks of the techniques for phenotypic and genotypic drug resistance determination and to highlight the peculiar clinical features relevant to the emergence of drug-resistant HCMV strains in transplantation settings.
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Assays for diagnosis and monitoring of HCMV infection |
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Viraemia consists of a rapid virus isolation technique based on 1624 h co-cultivation of 2 x 105 peripheral blood leucocytes (PBL) with human fibroblast monolayers grown in shell vials, followed by counting of infected fibroblasts after nuclear staining with a monoclonal antibody (MAb) directed to the major HCMV immediate-early (IE) antigen p72.40
Antigenaemia consists of quantitative determination of HCMV pp65-positive cells in 2 x 105 PBL examined in a cytospin preparation.41
The number of HCMV-infected CCEC is determined in the cytospin preparations used for antigenaemia quantification after staining with pp65-specific MAbs.42
DNAaemia can be determined by quantitative PCR either in PBLs,43 plasma43,44 or whole blood45 and is expressed as HCMV DNA genome equivalents (or copy number) per 2 x 105 PBL, or a determined volume of whole blood or plasma, respectively. Other methods, such as the branched DNA (bDNA) technique, have been used for HCMV DNA quantification in immunocompromised patients.46
RNAaemia consists of qualitative or quantitative determination of IE- or late-mRNAs in blood by RTPCR47 or NASBA (nucleic acid sequence based amplification).48,49
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Anti-HCMV drugs available clinical practice |
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Ganciclovir is usually administered at the dosage of 5 mg/kg body weight twice a day for induction treatment, whereas primary or secondary prophylaxis treatments (see below) are usually carried out with half this dosage. Ganciclovir is currently administered intravenously (iv), although an oral formulation of the drug is available. Valganciclovir is a recently developed oral prodrug of ganciclovir with improved pharmacokinetic characteristics.51 In a recent study, it was shown that using valganciclovir at a dosage of 900 mg twice a day for induction treatment and 900 mg once a day for maintenance treatment, systemic exposure to ganciclovir was similar to that obtained following iv administration of ganciclovir, eventually leading to a comparable efficacy of the two drugs.51
Foscarnet is a DNA polymerase inhibitor acting as a competitor of pyrophosphate. Foscarnet does not require metabolic activation and is administered at a dosage of 90 mg/kg body weight twice a day for induction treatment and at half that dosage for primary or secondary prophylaxis. The administration is iv.
Cidofovir is a nucleotide analogue inhibiting the viral DNA polymerase. Being already phosphorylated, it does not require activation by UL97. A peculiar characteristic of cidofovir is its extended half-life in vivo allowing weekly or bi-weekly administration. The drug is administered iv at a dosage of 5 mg/kg once a week during induction and 5 mg/kg once every other week during maintenance treatment.
A schematic of the chemical structures of ganciclovir, foscarnet and cidofovir is shown in Figure 1.
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Approaches to treatment of HCMV infection in immunocompromised patients |
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In solid organ (SOTR) or haematopoietic stem cell (HSCTR) transplant recipients, pre-symptomatic treatment approaches are usually adopted. In particular, both primary prophylaxis and pre-emptive therapy of HCMV infections have been utilized. Primary prophylaxis is based on the administration of ganciclovir to SOTR for several weeks after transplantation in order to suppress viral replication during a period of time when the patient is considered to be at high risk for development of HCMV infection. In contrast, pre-emptive therapy is based on virological monitoring of patients in the post-transplant period in order to identify and treat only those actually at risk for HCMV disease since reaching threshold values of viral load. The latter approach was proven to be able to prevent development of HCMV-related symptoms with the advantage of avoiding unnecessary treatment and related drug toxicity. Although pre-emptive therapy is now widely utilized, no consensus on standardized treatment protocols has been reached. The major reason for this is the fact that different viral parameters have been used in different laboratories to monitor HCMV infection. The most utilized viral parameters have been antigenaemia and DNAaemia. However, these techniques still lack standardization and patients at risk for HCMV disease are thus far defined mostly on the basis of locally established threshold values. In addition, different patient populations are treated on the basis of different threshold values of viral load. In this respect, it is common to treat HSCTR at the first confirmed appearance of HCMV in blood,55 whereas in SOTR higher viral load levels have been set as a threshold for treatment initiation. An exception is represented by treatment of donor-positive, recipient-negative (D+/R) SOTR which is treated upon the first appearance of HCMV in blood.55
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Monitoring of HCMV infection and response to antiviral treatment |
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Molecular basis for drug resistance |
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Acquisition of mutations in UL97 phosphotransferase (Figure 2) appears to be a crucial step in the selection of ganciclovir-resistant HCMV strains. HCMV strains with mutation in key regions (domains VIII, VI and IX) of the viral enzyme have been proven to be highly resistant to ganciclovir.68,14,17,18,2528,31,56,6268 In addition to UL97, mutations in UL54 impact ganciclovir susceptibility.13,15,53,56,69,70 However, mutations in UL54 are less common and have been reported only in patients harbouring ganciclovir-resistant UL97-mutant strains maintained on ganciclovir treatment.53 The isolates with mutations in both UL97 and UL54 showed very high levels of resistance to ganciclovir and simultaneous cross-resistance to cidofovir,53 but retained foscarnet susceptibility. Finally, it is worth mentioning that, according to the limited data so far available, valganciclovir does not appear to select for an increased number of ganciclovir-resistant strains.71
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Mutations in domains II, III and VI of HCMV DNA polymerase (Figure 2) are responsible for foscarnet resistance.5,57,66,69,70 Interestingly, no cross resistance to either ganciclovir or cidofovir is associated with foscarnet-induced UL54 mutations.66,69,70 Thus, the reported emergence of double ganciclovir- and foscarnet-resistant HCMV strains was due to the accumulation of specific mutations in the two viral genes induced by sequential treatment with the two drugs.9,5,66 However, mutations in domain III have been proven to induce simultaneous resistance to foscarnet, ganciclovir and cidofovir.56
Cidofovir
Mutations in the HCMV DNA polymerase are responsible for simultaneous resistance to cidofovir and ganciclovir.66,70 Because of its high renal toxicity, cidofovir is not widely utilized for treatment of HCMV infection. Thus, selection of cidofovir-resistant strains is an extremely rare event and it appears mostly driven by sustained ganciclovir administration.53
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Methods for phenotypic determination of antiviral drug resistance |
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Plaque reduction assay (PRA)
This assay measures the reduction in number of cytopathic effect foci following infection of fibroblast monolayers with titrated virus isolates (CPE-PRA).72 Alternatively, the number of IE antigen or late antigen plaques can be determined (IEA- or LA-PRA).4,73 This assay is regarded as the reference method for HCMV drug susceptibility determination and, following titration of the virus isolate, requires 410 days to be completed. In detail, IEA-PRA provides results in 4 days, LA-PRA is completed in 67 days and CPE-PRA takes longer. Although PRA has been known for several years and widely utilized, it is not yet standardized and comparison of results from different laboratories appears sometimes difficult. The lack of standardization is primarily related to:(i) the use of different fibroblast strains, (ii) the use of cell-free virus as inoculum versus cell-associated virus, (iii) the virus plaque staining technique; (iv) the use of laboratory adapted HCMV strains (AD169, Towne) versus clinical isolates as controls, (v) the subjectivity in reading test results.
Consensus is lacking in the definition of drug resistance: some authors indicate 6 µM ganciclovir and 400 µM foscarnet as ID50 cut-off values for ganciclovir- and foscarnet-sensitive HCMV strains, respectively. In our laboratory we define as drug-resistant the HCMV isolates showing ID50 values 5-fold the mean ID50 values determined on a series of HCMV isolates from untreated patients.4,9,12,28,74 Other authors consider as resistant HCMV isolates with ID50 values increased by
2 S.D. of the mean ID50 values determined on a series of HCMV control field isolates.6
Recently, a rapid drug susceptibility screening assay has been developed based on the concept of PRA.58 This assay measures the reduction in IEA-plaques following inoculation onto fibroblast monolayers of a predetermined number of PBL and incubation of cell cultures for 46 days in the presence of a single drug concentration (ganciclovir, 20 µM; foscarnet, 400 µM). A decrease of 50% in the number of IEA-plaques with respect to the no-drug control is considered as associated with the presence of drug resistance.74 A similar approach has been described by Pepin et al.75
In situ ELISA
This technique measures the reduction in LA production in fibroblast cultures infected with cell-free HCMV, which reflects the number of cells undergoing a complete virus replication cycle. The assay is carried out in 96-well microtitre plates and viral titration can be carried out in the context of the assay by simultaneously challenging increasing virus dilutions with decreasing drug concentrations in the same plate. After 46 days of incubation, the amount of viral antigen in each well is determined by immunoenzymic staining followed by spectrophotometric determination of absorbance. The assay is completed in 46 days.76,77
Virus yield reduction assay
The effect of antiviral drugs on viral replication can also be determined by quantifying the reduction in infectious viral titres, measured either as cell-free or cell-associated virus.78 This technique is cumbersome and extremely time-consuming and is currently utilized for research in drug development.
Flow cytometry-based assays
The reduction in viral antigen production can also be determined using flow cytometry by counting the number of trypsinized fibroblasts expressing late viral proteins.79,80
DNA reduction assay
This assay measures the reduction in viral DNA synthesis in fibroblast cultures in the presence of increasing drug concentrations.81 Quantification of HCMV DNA is obtained by hybridization of the viral genome with a 125I-labelled probe followed by counting the -emission by the bound probe. Following preparation of a titrated virus stock, results are available in 1 week. However, this assay is not widespread, primarily because of the need for dedicated facilities and expensive equipment (
-counter).
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Methods for genotypic determination of antiviral drug resistance |
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Sequencing
Analysis of UL97 and UL54 genes is usually carried out following amplification of gene fragments from viral isolates or directly from clinical samples by PCR. When using an automated sequencer, results are available in a couple of days. This assay is the reference technique for the identification of mutations emerging during treatment failure. However, despite its powerful diagnostic potential, sequence analysis is currently restricted to specialized laboratories, requiring expensive equipment and personnel with specific expertise.
Restriction fragment length polymorphism (RFLP)
This assay allows detection of specific mutations associated with drug resistance by evaluating the restriction pattern of PCR products digested with endonucleases, in which restriction sites have been modified (suppressed or generated) by single mutations.28,63 In addition, deletions modifying the size of digested fragments can be readily detected.64 Thus far, this technique has been widely applied for identification of UL97 mutations conferring ganciclovir resistance.
Probe-specific hybridization or primer-specific amplification
Probes discriminating single nucleotide substitutions can be used for hybridization of PCR products. Alternatively, primers can be designed to anneal only with the wild-type or the mutant sequence, thus allowing specific amplification by PCR or ligase chain reaction (LCR).82,83
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Advantages and disadvantages of phenotypic testing for drug resistance |
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Advantages and disadvantages of genotypic testing for drug resistance |
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Conclusions |
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Acknowledgements |
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Footnotes |
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References |
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