Cytomegalovirus (CMV) Workup

Updated: Jul 07, 2021
  • Author: Ricardo Cedeno-Mendoza, MD; Chief Editor: Michael Stuart Bronze, MD  more...
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Workup

Laboratory Studies

In congenital CMV disease, laboratory abnormalities may include abnormal transaminases, bilirubin and platelet levels. To diagnose congenital CMV infection, testing should be performed within the first 3 weeks of life, because testing past this period does not differentiate intrauterine from perinatal acquisition of CMV infection. Saliva CMV PCR is the preferred diagnostic test for newborn congenital CMV screening, because high viral load are shed in both urine and saliva in infants with congenital CMV. Due to some false-positive results in breastfeeding patients, obtaining a sample of saliva at least 1 hour after could avoid potential contamination with CMV from breastmilk. Other recommended test include Polymerase chain reaction (PCR) in bood and urine as well. [15, 20]

The prognostic value of CMV viral load in neonatal samples, especially with asymptomatic infection, is unclear. The role of virus burden in the peripheral blood, urine and saliva in disease and outcome needs further study. [15]

Antigen testing

Antigenemia is defined as the detection of the CMV pp65 antigen in leukocytes. [8]

The pp65 assay is used to detect messenger matrix proteins on the CMV virus, with either immunofluorescence assay or messenger RNA amplification. These proteins are typically expressed only during viral replication.

Antigen tests are often the basis for institution of antiviral therapy in transplant recipients and may allow for the detection of subclinical disease in high-risk patients. The assay is sensitive and specific yields results quickly.

Antigen assays cannot be used in patients with leukopenia, as these tests detect antigen within neutrophils.

In immunocompromised patients, low or moderate CMV antigenemia may indicate reactivation or infection. [9]

It has been reported that the pp65 antigen assay and quantitative CMV PCR (COBAS Amplicor Monitor Test; see Quantitative polymerase chain reaction) yield similar effectiveness in diagnosing and monitoring patients with active CMV infection. [49]

Qualitative polymerase chain reaction

Qualitative PCR is used to detect CMV in blood and tissue samples.

PCR depends on the multiplication of primers specific for a portion of a CMV gene. The primers usually bind to the area of virus that codes for early antigen.

Qualitative PCR is extremely sensitive, but, because CMV DNA can be detected in patients with or without active disease, the clinical utility of qualitative PCR is limited. [50, 51, 52] Serial PCR may be more helpful clinically.

It yields a positive result before the antigenemia test in transplant recipients with viremia.

Results are typically negative in patients without CMV viremia.

In transplant recipients, a negative CMV PCR result goes against reactivation, but not infection. [53]

Commercially available qualitative PCR testing can be performed using the NucliSens CMV Test, a nucleic acid sequence-based amplification assay (NASBA, Organon Teknika Corporation, Durham, North Carolina).

Quantitative polymerase chain reaction

Quantitative PCR has been used to detect plasma CMV. The advantage of quantitative PCR over regular PCR is unknown. Ideally, quantitative PCR is as sensitive as qualitative PCR and provides an estimate of the number of CMV genomes present in plasma.

A study of newborns compared real-time PCR assays of liquid-saliva and dried-saliva specimens with rapid culture of saliva specimens obtained at birth. Both PCR assays showed high sensitivity and specificity for detecting CMV infection. [54]

A study of more than 3400 blood specimens from organ transplant recipients tested with CMV PCR and pp65 antigenemia found that quantitative real-time PCR for CMV DNA could be used in lieu of antigenemia for monitoring CMV infection and determining when to initiate preemptive treatment. [55]

In theory, the CMV viral load would indicate whether therapy is necessary because patients whose viral load is below a certain cutoff would not develop CMV disease. However, the level of viremia necessary for CMV disease to occur may vary depending on host factors and the type of organ transplant, and this may need to be determined empirically. For example, in CMV retinitis, the viral load has a poor positive predictive value, meaning its clinical utility is limited. A detectable CMV viral load at the time of CMV retinitis diagnosis was shown in one study to correlate with increased mortality (P = 0.007). [56] CMV involvement of the GI tract also has a poor correlation with CMV viremia.

Commercially available quantitative PCR assays include the COBAS Amplicor CMV Monitor test (Roche Diagnostics, Indianapolis, IN) and various laboratory-developed PCR assays. The COBAS Amplicor CMV Monitor test measures viremia in the range of 600-100,000 copies/mL. [57]

In the quest to standardize viral load testing for CMV, the FDA approved marketing in July 2012 of a fully automated assay, the COBAS AmpliPrep/COBAS TaqMan CMV test (CAP/CTM CMV test, Roche Molecular Diagnostics, Pleasanton, CA), which uses an international standard to quantitate plasma CMV load. This assay is available in Europe, but not yet in the United States. It measures viremia in a range of 150-10,000,000 copies/mL. [58]

Because viral loads are not comparable among different assays, it is important to use the same test and same sample type (whole blood or plasma) when monitoring patients over time. [59]

Shell vial assay

The shell vial assay is performed by adding the clinical specimen to a vial that contains a permissive cell line for CMV. The shell vials are centrifuged at a low speed and placed in an incubator. After 24 and 48 hours, the tissue culture medium is removed and the cells are stained using a fluorescein-labeled anti-CMV antibody. The cells are read using a fluorescent microscope. Alternatively, the cells are stained with an antibody against CMV, followed by a fluorescein-labeled anti–immune globulin.

This test has been found to be as sensitive as traditional tissue culture.

Cytopathology

Intracellular inclusions surrounded by a clear halo may be demonstrated with various stains (Giemsa, Wright, hematoxylin-eosin, Papanicolaou). This gives the appearance of an "owl's eye" (see Pathophysiology).

Hematoxylin-eosin–stained lung section showing typ Hematoxylin-eosin–stained lung section showing typical owl-eye inclusions (480X). Courtesy of Danny L Wiedbrauk, PhD, Scientific Director, Virology & Molecular Biology, Warde Medical Laboratory, Ann Arbor, Michigan.
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Imaging Studies

The diagnosis of CMV pneumonia can be suggested by chest radiography findings, but these findings cannot be used to differentiate between other common causes of pneumonia in immunocompromised hosts. A chest radiograph finding consistent with pneumonia and a BAL result that is CMV positive is a common method for diagnosis.

CT scan is more sensitive for the identification of infiltrate. It has been valuable in patients who present with hypoxia and no infiltrate visible on chest roentgenography.

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Other Tests

Cytomegalovirus resistance testing

If resistant CMV infection is suspected (refractory to IV ganciclovir or valganciclovir therapy), an alternative therapy should be used based on genetic resistance. UL54 mutation is associated with ganciclovir resistance and a cross-resistance with Cidofovir. UL97 mutation is also associated with ganciclovir resistance. If this is noted, Foscarnet is the recommended therapy. Letermovir could be an alternate option due to a different mechanism of action (however this is currently recommended only for prophylaxis). If low levels of resistance is suspected, higher doses of Ganciclovir could be used. [60]

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Histologic Findings

The hallmark of CMV infection is the finding of intranuclear inclusions consistent with herpesvirus infection. CMV infection may be confirmed using in situ hybridization or direct or indirect staining of intranuclear inclusions using CMV-specific antibodies linked to an indicator system (eg, horseradish peroxidase, fluorescein).

Hematoxylin-eosin–stained lung section showing typ Hematoxylin-eosin–stained lung section showing typical owl-eye inclusions (480X). Courtesy of Danny L Wiedbrauk, PhD, Scientific Director, Virology & Molecular Biology, Warde Medical Laboratory, Ann Arbor, Michigan.
Here, using immunofluorescent technique, a specime Here, using immunofluorescent technique, a specimen of human embryonic lung (25X) reveals the presence of cytomegalovirus. Courtesy of the CDC and Dr Craig Lyerla.
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