Cytomegalovirus (CMV) is a linear, double-stranded DNA virus with an icosahedral capsid and is a member of the Herpesviridae family, which infects humans along with HSV-1, HSV-2, VZV, EBV, HHV-6, HHV-7, and HHV-8. CMV is also known as HHV-5. CMV, HHV-6, and HHV-7 are all members of the Betaherpesvirinae subfamily. The replication cycle of CMV is slow and produces large, multinucleated cells (cytomegalia). Once the virus has infected an individual, it establishes latency in lymphoreticular tissue, secretory glands, kidneys, and other tissues. Human cytomegalovirus (HCMV) infects only humans and will grow in the laboratory only in cell lines of human origin.
CYTOMEGALOVIRUS 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, at any given time, that 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 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.
The immunocompromised population, including transplant patients, HIV patients, and to a lesser extent cancer patients, are those at highest risk for developing the significant disease syndromes caused by CMV, including interstitial pneumonia, gastrointestinal infection, central nervous system (CNS) 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. 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. Reactivation can occur in any individual who is latently infected and no transplant patient is safe from CMV. This pathogen can also be acquired from the transplanted organ, resulting from a transplant between a CMV seropositive donor to a seronegative recipient (D+/R-), which is referred to as a primary infection. Additionally, CMV can also be community acquired following transplantation.
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 observed5. 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 Epstein-Barr virus-associated post-transplant lymphoproliferative disease; accelerated atherosclerosis in heart transplant patients; and decreased patient and graft survival5,6. Symptomatic CMV infections occur most frequently in D+/R- patients5. 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 recipients7.
In HSCT recipients, pneumonia and enteritis are the most common clinical manifestations of CMV disease5. 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 ganciclovir8. 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 disease8. 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 rate8. 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 transplant9. 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 disease10.
CYTOMEGALOVIRUS 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; and if the 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. 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 a cell-based assay can yield positive results due to latent CMV in white blood cells. To avoid detection of latent CMV, plasma should 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 burden11.
With the availability of potent CMV-specific antiviral drugs and treatment strategies, the incidence of CMV disease has decreased dramatically12. CMV antiviral drugs include ganciclovir, valganciclovir, foscarnet, and cidofovir. Intravenous (IV) ganciclovir remains the first-line treatment for CMV disease6. Valganciclovir, an oral prodrug that is metabolized to active ganciclovir, has replaced oral ganciclovir due to its increased bioavailability in universal prophylaxis and preemptive therapy6. Foscarnet is only used as a second-line therapy due to its association with renal toxicity13. 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 mixed results14. 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 initiating treatment prior to onset of clinical symptoms6. Many SOT institutions utilize prophylactic therapy with ganciclovir, which is administered to patients during the period of highest risk of infection to prevent development of CMV viremia and disease6. Conversely, due to the potential for negative side-effects in HSCT patients, including exacerbated neutropenia, most HSCT centers utilize preemptive treatment protocols, where CMV viremia is monitored and treatment is initiated when CMV viremia reaches a specified threshold prior6. Patients on extended courses of anti-CMV treatment are at increased for developing drug-resistance. Specifically, mutations in the CMV UL97 and UL54 are known to cause resistance to ganciclovir, foscarnet, or cidofovir. Recent guidelines indicate genotypic testing should be utilized to confirm the occurrence of CMV UL97 and UL54 antiviral resistance mutations, so that alternative treatment options may be considered13.
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