CMV Infection & Resistance
Cytomegalovirus (CMV) is a linear, double-stranded DNA virus with an icosahedral capsid. CMV, also known as HHV-5, is a member of the Herpesviridae family, along with herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpes virus 6 (HHV-6), human herpes virus 7 (HHV-7), and human herpes virus 8 (HHV-8), all of which infect humans. CMV, HHV-6, and HHV-7 are all members of the Betaherpesvirinae subfamily. The replication cycle of CMV is slow and induces the formation of large, multinucleated cells (cytomegalia). Once the virus has infected an individual, CMV establishes latency in lymphoreticular tissue, secretory glands, kidneys, and other tissues.
CMV CLINICAL MANIFESTATIONS
CMV is ubiquitous throughout the world. When the virus is acquired at a young age, it rarely causes noticeable illness. However, in developed Western countries, infection is often acquired later in life when it is more likely to cause significant illness. The prevalence of antibodies among adults in the U.S. is between 40 and 100%, depending largely upon socioeconomic conditions1. The infection rate gradually increases throughout childhood. Once infected, the individual carries the virus for life due to the ability of CMV to establish a latent state of infection. It is estimated that at any given time, up to 10% of the population is secreting CMV from various sources, such as urine, saliva, semen, or breast milk2. The virus is transmitted readily through any of these sources. Children, as well as daycare workers, are at high risk for contracting CMV, since it is shed frequently in urine. In adults, primary CMV infection is typically acquired through blood transfusions, contact with an infected cervix or semen, or transplanted organ tissues. In young adults and CMV seronegative recipients of CMV-positive blood transfusions, a syndrome resembling mild EBV mononucleosis is not uncommon. The patient often will present with prolonged fever, splenomegaly, abnormal liver function, and atypical lymphocytes. However, a positive heterophile antibody test does not occur in CMV mononucleosis as in EBV mononucleosis.
Currently, transplacental infection with CMV is the most common viral cause of prenatal damage to fetuses. Approximately 1% of fetuses are infected with CMV in utero; however, the majority of maternal infections are reactivations and rarely cause congenital CMV syndrome2. Primary infection of the mother during the first trimester of pregnancy puts the fetus at higher risk for congenital CMV syndrome. Primary infection carries a 30 to 40% risk of fetal infection with a 10 to 15% risk of clinical abnormalities2. A smaller percentage of those infants will suffer severe CMV syndrome, which can include microcephaly, thrombocytopenia, hepatosplenomegaly, petechial hemorrhages, jaundice, encephalitis, mental retardation, and hearing impairment. Neonates can also acquire the virus during passage through the birth canal or from contact with infected saliva and breast milk.
Immunocompromised Patient Population
The immunocompromised population, including transplant patients, HIV patients, and to a lesser extent cancer patients, are at highest risk for developing significant disease syndromes caused by CMV, including interstitial pneumonia, gastrointestinal infection, central nervous system disease, hepatitis, retinitis, and encephalitis. CMV reactivations have also been reported to occur frequently in critically ill immunocompetent patients and are associated with prolonged hospitalization or death3.
CMV infection is one of the most frequently occurring opportunistic infections in AIDS patients, with CMV retinitis accounting for approximately 80% of CMV disease cases4. The advent of highly active antiretroviral therapy (HAART) has resulted in an 80% decrease in the incidence of CMV retinitis, which previously affected an estimated 30% of AIDS patients5,6. However, some patients do not have access or respond well to HAART; thus, CMV still remains a concern in patients when CD4+ T-cell counts decline to 50 to 100 cells/μl7.
CMV is among the most common and important infectious agents among transplant recipients, both solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) patients. In transplant recipients, the factor that most strongly influences the degree of morbidity and mortality caused by CMV is the type and extent of immunosuppressive therapy. Reactivation can occur in any individual who is latently infected. However, no transplant patient is safe from CMV since this pathogen can also be acquired from the transplanted organ. This is referred to as a primary infection resulting from a CMV seronegative recipient and a CMV seropositive donor (D+/R-). CMV can also be community acquired following transplantation and is of particular concern in pediatric transplant patients. In SOT patients, particularly those who develop a primary infection during the first 3 months post-transplant, a specific CMV syndrome consisting of fever, malaise, arthralgia, and neutropenia may be observed7.
CMV infections have been associated with indirect effects, such as dysfunction or rejection of the transplanted organ; increased risk for bacterial or fungal opportunistic infections; development of EBV associated post-transplant lymphoproliferative disease; accelerated atherosclerosis in heart transplant patients; and decreased patient and graft survival7,8. Symptomatic CMV infections occur most frequently in D+/R- patients7. In the absence of antiviral intervention, symptomatic CMV infections occur in approximately:
39 to 41% of heart-lung transplant recipients;
9 to 35% of heart transplant recipients;
22 to 29% of liver and pancreas transplant recipients;
8 to 32% of kidney transplant recipients;
50% of kidney-pancreas transplant recipients; and
22% of small-bowel transplant recipients9.
In HSCT recipients, pneumonia and enteritis are the most common clinical manifestations of CMV disease7. CMV seropositive patients are at highest risk. Approximately 70% of these patients demonstrate reactivated latent CMV, with 35 to 40% developing disease without preemptive antiviral therapy with ganciclovir10. In seronegative recipients with a seropositive donor, 20% develop primary infection and 10% develop disease; in seropositive autograft recipients, 25 to 40% demonstrate reactivated endogenous infection and 5 to 7% develop disease10. Seronegative autograft recipients and seronegative allograft recipients with a seronegative donor both demonstrate a 1 to 3% infection rate and a 1 to 2% disease rate10. Preemptive antiviral therapy has reduced the incidence of CMV disease to less than 5% in most high-risk CMV-seropositive HSCT patients during the first 100 days after transplant11. As a result, CMV disease now occurs most commonly after day 100 following transplantation. Patients with CMV reactivation before day 100 and those receiving steroids for graft-versus-host disease (GVHD) are at highest risk(approximately 30%) for late onset CMV disease12.
CMV LABORATORY DIAGNOSIS
A diagnosis of CMV disease cannot be made solely on clinical grounds; laboratory confirmation is required. Culture has been the traditional method to diagnose CMV infection; however, culture has several significant limitations:
CMV can take up to 6 weeks to grow.
The virus is temperature labile and may be inactivated before it reaches the laboratory, leading to false negative results.
Culture is not quantitative so viral load cannot be assessed.
Most significantly, the amount of virus needed to cause disease in a transplant patient is far less than the amount of virus needed to grow in culture.
Another widely accepted diagnostic method is the CMV antigenemia assay. A major step forward from culture, antigenemia is more sensitive, semi-quantitative, and the assay can be performed in one day. In this assay, the patient’s white cells are attached to a glass slide. The cells are then stained with CMV-specific monoclonal antibodies that are conjugated to a fluorescent molecule. The laboratory scientist then visualizes the patient’s white cells under a fluorescent microscope and looks for cells containing CMV inclusions, which indicate that CMV is replicating in that cell. While this method is generally acceptable, there are notable limitations:
The blood specimen must be less than 6 hours old to be tested
The assay is quite labor intensive and technically demanding
If patient has a very low cell count, the specimen may not be suitable for testing
The need for a rapid, sensitive, specific, and quantitative CMV detection system that overcomes the limitations of previous methods has been well established. The advent of quantitative real-time polymerase chain reaction (PCR) has dramatically improved CMV detection, thereby positively impacting patient survival. Quantitative real-time PCR can be used to monitor the patient’s response to antiviral drug treatment. Of further advantage, it can be performed on a wide variety of specimen sources including blood, cerebral spinal fluid (CSF), urine, bronchial alveolar lavage, ocular specimens, tissue biopsies, and bone marrow biopsies, among others. Due to the highly sensitive nature of molecular testing, utilizing whole-blood can yield positive results due to latent CMV in white blood cells. To avoid detection of latent CMV, plasma can be used instead of whole blood or buffy coat for testing. In addition, CMV nucleotide changes may interfere with efficient binding of the primers or probe resulting in significant underquantification of viral load or a false negative result. Utilizing a multiple-target assay can avoid this occurrence by providing an alternate binding site in the event of a sequence variation, thereby providing an accurate view of the patient’s viral burden13.
With the availability of potent CMV-specific antiviral drugs and treatment strategies, the incidence of CMV disease has decreased dramatically14. However, treatment of chronic viral infections such as CMV in immunocompromised patients presents challenges, including drug toxicity, delayed onset of disease after discontinuing therapy, and emergence of CMV genomic mutations that confer drug resistance.
In SOT and HSCT recipients, either preemptive or prophylactic therapies are used with the goal of avoiding the initiation of treatment after clinical signs and symptoms8. In institutions that follow prophylactic treatment protocols, therapy is administered to patients during the period of highest risk of infection to prevent development of CMV viremia and disease8. However, studies have shown that up to 30% of CMV D+/R- SOT recipients develop delayed-onset primary CMV disease after cessation of prophylaxis15. In institutions that follow preemptive treatment protocols, CMV viremia is monitored and treatment is initiated when CMV viremia reaches a specified threshold prior to the patient becoming symptomatic8. Preemptive treatment has proven to be effective in preventing development of CMV-related symptoms with the advantage of avoiding unnecessary treatment and related drug toxicity16. Baldanti and colleagues demonstrated that the preemptive treatment approach in transplant patients shows advantages in treating a lower number of patients for shorter periods of time as well as avoiding the emergence of CMV disease after interruption of prophylactic treatment regimens for CMV17. Although preemptive therapy is widely utilized, no consensus on standard treatment protocols or clinically validated thresholds has been reached17. A major factor in the lack of consensus is the use of different viral parameters among laboratories to monitor CMV infection16. In addition, varying threshold values are used to determine treatment for different patient populations16. For example, it is common to treat HSCT recipients and D+/R- SOT recipients upon the first appearance of CMV in blood; whereas, in SOT recipients, higher viral load levels have been set as a threshold for treatment initiation16.
A meta-analysis, evaluating prophylactic versus preemptive therapy for CMV disease prevention, found both strategies beneficial in preventing CMV disease and reducing allograft rejection18. Only universal prophylaxis reduced bacterial and fungal infections and mortality and only prophylaxis seemed to reduce the incidence of CMV end-organ disease in subgroups of patients at highest risk of infection and in those who received induction therapy with antilymphocyte antibodies18. Another meta-analysis reported results of randomized, controlled clinical trials comparing ganciclovir prophylaxis with preemptive ganciclovir therapy; there was no statistically significant difference in preventing CMV disease between strategies19. CMV antiviral drugs include ganciclovir, valganciclovir, foscarnet, and cidofovir. Intravenous (IV) ganciclovir remains the first-line treatment for CMV disease8. Valganciclovir, an oral prodrug that is metabolized to active ganciclovir, has replaced oral ganciclovir due to its increased bioavailability in universal prophylaxis and preemptive therapy8. Foscarnet is only used as a second-line therapy due to its association with renal and neural toxicity. Cidofovir has been used for treatment of CMV retinitis in AIDS patients. While data are limited for the use of cidofovir in the transplant setting, findings suggest that it can be used for patients who experienced failure of ganciclovir or foscarnet therapy8.
Interestingly, Campath® (alemtuzumab) has been associated with an incidence of symptomatic CMV reactivation ranging from 4 to 29%20. Campath is indicated for the treatment of B-cell chronic lymphocytic leukemia (BCLL) in patients who have been treated with alkylating agents and who have failed fludarabine therapy. This incidence may be clinically significant and, as a result, some oncologists prescribe prophylactic therapy with CMV antiviral drug(s). An alternative strategy that would avoid unnecessary and costly drug use would be to treat preemptively when the virus reaches a certain threshold, similar to treatment strategies for transplant patients.
EMERGENCE OF CMV ANTIVIRAL RESISTANCE
CMV antiviral resistance first emerged in the HIV-infected patient population in the pre-HAART era. At that time, specific treatment was only initiated in the presence of symptomatic CMV disease due to the lack of available rapid and sensitive laboratory assays to monitor asymptomatic individuals16. Secondary prophylaxis was administered on a lifelong basis at a reduced dosage due to the high risk of CMV disease relapse16. Prophylaxis did not prevent CMV infections due to the inability to effectively control HIV replication and the subsequent decay of the immune system16. Thus, prophylaxis led to the emergence of multidrug-resistant CMV strains. With the introduction of HAART, the incidence of CMV infection in HIV patients declined sharply16. However, CMV remains an issue for patients who fail to respond to HAART for reasons that include drug-related toxicity, intolerance, poor adherence to treatment, and emergence of HIV drug-resistant strains16.
Antiviral-resistant CMV is an emerging entity in transplant patients and presents many challenges that directly or indirectly threaten the success of transplantation15. The overall outcome of patients with drug-resistant CMV disease is generally poor, as the treatment of these cases is prolonged, highly complex, and associated with other morbidity complications15. The frequency of CMV drug resistance in the transplant population varies considerably according to tissue type and risk factors21. Factors associated with an increased risk of developing antiviral resistance include CMV D+/R- serostatus, high levels of CMV replication, the use of very potent immunosuppressive therapy, and prolonged exposure to subtherapeutic ganciclovir levels15. Ganciclovir resistance has been reported to most commonly affect lung transplant22,23 and kidney transplant recipients, with or without pancreas allograft22-25.
Valganciclovir, on the basis of increased bioavailability, is the most commonly utilized drug for prevention of CMV disease after SOT in an effort to avoid ganciclovir resistance15. Studies that assess the burden of ganciclovir resistance in the context of valganciclovir prophylaxis are limited, and the available studies are conflicting. While one study did not observe the occurrence of antiviral resistance, Eid and colleagues report that delayed-onset primary CMV disease caused by clinically suspected or genotypically confirmed antiviral-resistant CMV is not uncommon among D+/R- patients receiving valganciclovir prophylaxis, especially among kidney transplant recipients15,23,26. Eid and colleagues observed that the clinical presentation of drug-resistant CMV is predominantly tissue-invasive gastrointestinal disease and that the clinical course is protracted15. In addition, Eid and colleagues suggested that CMV load in the blood of patients with gastrointestinal CMV disease may not necessarily reflect the extent of tissue involvement and may not be an appropriate marker of assessing therapeutic response in these cases15. An important conclusion of this study is that a proof-of-cure colonoscopy should be performed to ensure disease resolution prior to cessation of therapy15.
Many HSCT programs have adopted preemptive antiviral therapy with ganciclovir and valganciclovir in order to reduce CMV disease. In the HSCT setting, the development of antiviral resistance is reported to be uncommon and generally limited to case reports. Marfori and colleagues observed the development of known and new antiviral resistance sequence variations in two adult cases, in association with rising antigenemia and ensuing morbidity and mortality21. Marfori and colleagues suggested that CMV antiviral resistance in adult HSCT patients may not be infrequent21. In the pediatric population there have been reports of rapid emergence of resistance. Springer and colleagues described two pediatric HSCT recipients who died with persistent antiviral-resistant CMV infection27. Resistance in these patients developed rapidly, after only 6 weeks of foscarnet treatment in the first patient and after 11 weeks of ganciclovir treatment in the second patient27. Wolf and colleagues showed the emergence of resistant strains within 10 days to 3 weeks from initiation of therapy in 4 of 6 children with combined immunodeficiency (CID) after T-cell depleted bone marrow transplantation28. Eckle and colleagues reported the rapid emergence of ganciclovir-resistant mutations in 3 pediatric patients within 25, 53, and 30 days, respectively, from initiation of treatment following allogeneic peripheral blood stem cell transplantation29. The emergence of mutations in blood specimens was observed within 12, 29, and 9 days after the last negative genotypic test result29. Following genotypic detection of resistance, viral isolates were obtained 10, 124, and 129 days later and assayed retrospectively for phenotypic drug resistance29. Sequencing analysis of one patient’s CSF sample revealed a new mutation restricted to this anatomical location, which demonstrates the difficulty of establishing effective antiviral therapy in the presence of different resistance mutations in various body sites29. This patient went on to develop multidrug resistance after 7 months of antiviral therapy29. Of note, genotypic resistance screening restricted to blood specimens may miss existing mutant strains localized to a particular body site, as another patient described by Eckle and colleagues had a resistance mutation uniquely in a urine isolate29. Wolf and colleagues suggest the need for early and frequent genotypic monitoring and prompt therapeutic monitoring in the pediatric HSCT patient population28.
MOLECULAR BASIS OF CMV ANTIVIRAL RESISTANCE
In order to have anti-CMV activity, ganciclovir requires initial phosphorylation and activation by the CMV protein phosphotransferase, encoded by the CMV UL97 gene, followed by conversion to ganciclovir triphosphate by cellular enzymes30. Point mutations or small deletions in the UL97 phosphotransferase gene lead to changes that reduce or prevent the initial phosphorylation of ganciclovir7. These sequence variations are present in >90% of ganciclovir-resistant clinical isolates8. The majority of ganciclovir-resistant sequence variations cluster at UL97 codons 460, 520, and between 590 and 60731. Common sequence variations include M460V/I, C592G, H520Q, A594V, L595S, and C603W, most of which confer a 5-fold to 10-fold increase in ganciclovir IC50 (i.e., the concentration of drug that inhibits viral growth by 50%). Some sequence variations such as C592G and A594T confer a 2-fold to 3-fold increase in IC5031. Modest increases in IC50 may not prevent a response to continued full-dose ganciclovir, but they may facilitate the emergence of other sequence variations causing higher levels of resistance31.
Sequence variations in the CMV DNA polymerase, encoded by the CMV UL54 gene, are less common but may confer resistance to ganciclovir and cross-resistance to cidofovir or foscarnet. Variations in the UL54 gene typically occur after development of UL97 mutations and increase the overall level of ganciclovir resistance32. The UL54 variations resulting from ganciclovir therapy usually confer cidofovir cross-resistance; whereas, foscarnet resistance (sometimes with low-grade ganciclovir cross-resistance) typically involves a separate set of variations31. Triple-drug resistance is more infrequent but has been associated with sequence variations, including A834P and deletion of codons 981 through 982 of the UL54 CMV gene31. A more detailed review of characterization studies of UL97 and UL54 mutations in antiviral-resistant CMV strains has been published by Alejo Erice33.
DIAGNOSIS OF CMV ANTIVIRAL RESISTANCE
Monitoring the response to CMV antiviral treatment is one of the most powerful tools for detecting the emergence of drug resistance, as an increase in viral load has been associated with the emergence of drug-resistant strains16. However, recipients of high-dose corticosteroids are likely to demonstrate poor virologic responses to preemptive therapy34. Thus, in ganciclovir-naïve patients early after transplantation, increasing viral load usually indicates poor host control rather than ganciclovir resistance, which indicates the need for higher doses of antivirals (either continued or resumed induction dosing) rather than switching to an alternative antiviral drug34. Pescovitz and colleagues reported that patients with persistent viremia at 21 days should continue on valganciclovir treatment doses and are not likely to develop resistance mutations, whereas patients with viremia at 49 days should be evaluated for resistance35.
Laboratory testing should be used to confirm the occurrence of antiviral resistance and to provide guidance for alternative treatment options based on the type of resistance detected36. Two different approaches have been developed to assess antiviral resistance, namely phenotypic and genotypic assays.
Phenotypic assays are regarded as the “gold standard” for drug-resistance testing. These assays directly measure the drug susceptibilities of viral isolates by growing the virus in the presence of various concentrations of an antiviral drug in order to determine the concentration that will inhibit a percentage (most commonly 50%) of viral growth in cell culture7. Phenotypic assays do not depend on an understanding of the genetic mechanisms of drug resistance. However, these assays are not standardized; they may be too time-consuming to be used to guide clinical decisions; the results are subject to the selection bias introduced during growth of mixed viral populations in the artificial environment of cell culture; and they may lack sensitivity to detect low-level resistance or minor resistant subpopulations7. In addition, current methods for diagnosis of CMV infection usually do not yield a live viral isolate for phenotypic testing7.
Genotypic assays detect the presence of viral sequence variations known to be associated with antiviral resistance and are rapidly replacing phenotypic assays in clinical practice. The use of viral gene sequencing offers distinct advantages over other methods, including a rapid turnaround time, a broader range of antiviral resistance information, and the ability to provide information concerning new drugs as they become available7. However, special attention must be paid to the interpretation of new sequence variations or constellations of variations, as genotypic identification of mutant CMV strains is indirect evidence of drug resistance16. Confirmatory marker transfer experiments are performed prior to labeling a new mutant strain as resistant, as illustrated in the case of UL54 where some sequence variations appear to confer multiple drug resistance16. Clinical diagnostic sequencing assays typically include mutations that have been confirmed to confer antiviral resistance by marker transfer experiments, as well as mutations associated with resistance in multiple studies. Sporadically identified mutations in clinical isolates thought to confer resistance are not commonly included in assays for clinical use.
TREATMENT OF CMV ANTIVIRAL RESISTANCE
One of the most basic principles in avoiding development of CMV drug resistance is the consistent delivery of adequate levels of potent therapeutic agents without dose-limiting adverse effects, since resistant viral mutants are selected when the virus continues to replicate in the presence of subtherapeutic drug concentrations36. Additional strategies to avoid development of resistance include:
optimization of host immunity;
selection and adherence to dosing of bioavailable drug regimens;
avoidance of D+/R- mismatches in SOT recipients (although this is quite challenging due to the limited supply of available organs);
avoidance of excessive immunosuppression; or
enhancement of immunity with immunologic therapies36.
Unfortunately, development of antiviral-resistant CMV disease will likely continue to occur despite precautions36.
Ganciclovir resistance is the most frequently encountered type of CMV drug resistance with an incidence of 3.6 to 9% in nonspecific SOT cases and 15.8 to 27% in D+/R- lung transplant recipients, with the median onset of resistance estimated to be 5 to 6 months posttransplant37. In patients with ganciclovir-resistant CMV, foscarnet is the standard alternate treatment; however, this is often associated with acute renal failure31. Combination treatment with ganciclovir and foscarnet has been suggested, though data are conflicting31. Cidofovir may have moderate efficacy against ganciclovir-resistant CMV, though data are limited and some studies report treatment failure31. In some cases, clinical improvement may be observed with intensification of ganciclovir treatment even in the presence of genotypically or phenotypically resistant virus24,25.
While there have been many advances in detecting and managing CMV infections, the virus continues to be one of the most important infectious diseases among immunocompromised patients. Antiviral drugs in the form of prophylaxis or preemptive treatment strategies have reduced morbidity and mortality, though adverse effects including neutropenia and toxicity remain a challenge. The emergence of resistance to antiviral drugs presents many challenges to laboratory medicine and patient care. Genotypic testing aids in quickly identifying antiviral resistance, though effective alternative treatments are limited. Development of potent and less toxic therapeutic agents is needed to address antiviral-resistant CMV disease.
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