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Overview of diagnostic tests for cytomegalovirus infection

Overview of diagnostic tests for cytomegalovirus infection
Literature review current through: May 2024.
This topic last updated: Sep 18, 2022.

INTRODUCTION — Cytomegalovirus (CMV) generally produces an asymptomatic or minimally symptomatic acute illness in immunocompetent patients. In immunocompromised hosts, however, CMV infection can result in a broad array of clinical presentations, including a nonspecific syndrome (which may include fever, malaise, weakness, myalgias, arthralgias, leukopenia, and/or thrombocytopenia in the setting of viremia) or end-organ disease such as retinitis, pneumonitis, encephalitis, hepatitis, or gastrointestinal tract ulceration. These manifestations are associated with significant morbidity and mortality.

An overview of the diagnostic tests for the diagnosis of CMV infections will be presented here. The approach to the diagnosis of CMV is discussed separately. The manifestations of CMV in immunocompetent and immunocompromised patients are also presented elsewhere.  

(See "Approach to the diagnosis of cytomegalovirus infection".)

(See "Epidemiology, clinical manifestations, and treatment of cytomegalovirus infection in immunocompetent adults".)

(See "Cytomegalovirus infection in pregnancy".)

(See "Overview of cytomegalovirus infections in children", section on 'Laboratory diagnosis'.)

(See "Pathogenesis, clinical manifestations, and diagnosis of AIDS-related cytomegalovirus retinitis", section on 'Diagnosis'.)

(See "AIDS-related cytomegalovirus gastrointestinal disease".)

(See "Clinical manifestations, diagnosis, and management of cytomegalovirus disease in kidney transplant patients".)

(See "Prevention of cytomegalovirus infection in lung transplant recipients".)

(See "Clinical manifestations, diagnosis, and treatment of cytomegalovirus infection in lung transplant recipients".)

(See "Prevention of cytomegalovirus disease in kidney transplant recipients".)

(See "Pathogenesis, clinical manifestations, and diagnosis of AIDS-related cytomegalovirus retinitis".)

(See "AIDS-related cytomegalovirus neurologic disease".)

The clinical features, diagnosis, and management of congenital CMV infection are also presented separately. (See "Congenital cytomegalovirus infection: Clinical features and diagnosis" and "Congenital cytomegalovirus infection: Management and outcome".)

BACKGROUND — The testing methods described in this topic have advantages and disadvantages and must be interpreted in the context of the clinical presentation and other diagnostic assessments. Molecular amplification methods are now the standard of care for diagnosing CMV infection; in the past, serologic testing and viral cultures from multiple sites were the cornerstones of diagnosis.

Appropriate diagnostic tests are essential for the management of CMV infection and disease in immunocompromised patients. Several diagnostic modalities are available, including serology, quantitative polymerase chain reaction, pp65 antigenemia, culture, and histopathology (table 1).

The approach to diagnosis of CMV infection is discussed separately. (See "Approach to the diagnosis of cytomegalovirus infection".)

SEROLOGY — Serology provides indirect evidence of recent or prior CMV infection based upon changes in antibody titers at different time points during a clinical illness. Many different antibody detection techniques are available. Enzyme immunoassays and indirect and anticomplement immunofluorescence assays are the most commonly used serologic tests in clinical laboratories; complement-fixation techniques, immune adherence hemagglutination, indirect hemagglutination, and neutralization tests are performed in a limited number of diagnostic laboratories [1]. Serologic tests measure the presence of anti-CMV immunoglobulin (Ig)M and IgG.

Recent or acute infection — A diagnosis of recent or acute CMV is considered probable (though not definite) in the following circumstances:

The detection of CMV-specific IgM antibodies (suggesting recent seroconversion)

A fourfold or greater increase in CMV-specific IgG titers in paired specimens obtained at least two to four weeks apart

Although the sensitivity and specificity of serologic tests are adequate, the requirement for paired serum samples limits the utility of these tests in establishing a timely diagnosis. CMV-specific IgM antibodies are typically detectable within the first two weeks after the development of symptoms and may persist for several months [2]. Among patients with acute CMV infection who had serial serum samples available for analysis, CMV-specific IgM antibodies were detectable for a period of four to six months after the onset of symptoms [2]. Therefore, a positive CMV-specific IgM antibody alone may provide misleading information if a prior baseline test is not available (in cases in which it represents prior infection rather than current infection).

CMV-specific IgG antibodies are often not detectable until two to three weeks following the onset of symptoms and persist lifelong [2].

Past infection — Serologic tests are also helpful in determining past exposure to CMV infection. When assessing past CMV exposure or infection, any IgG result above the cutoff of the test is considered positive. The cutoff may vary depending on the method used, and it is generally provided with test results. CMV IgG results are particularly useful for the management of immunocompromised hosts at risk for CMV reactivation. As an example, human immunodeficiency virus (HIV)-infected patients with serologic evidence of past exposure to CMV warrant monitoring for CMV retinitis in the setting of progressive immunocompromise.

Risk of acquisition — Serologic tests are also helpful in determining risk of acquisition of CMV. For example, a seronegative patient is not at risk for CMV reactivation; on the other hand, the same patient is at high risk for new acquisition of infection if transplanted with an organ from a CMV-seropositive donor. (See "Clinical manifestations, diagnosis, and treatment of cytomegalovirus infection in lung transplant recipients" and "Clinical manifestations, diagnosis, and management of cytomegalovirus disease in kidney transplant patients".)

MOLECULAR ASSAYS — Most laboratories use quantitative real-time polymerase chain reaction (PCR; viral load) tests for monitoring patients at risk for CMV disease, diagnosing active CMV disease, and monitoring response to therapy [3]. Qualitative CMV PCR assays are very sensitive but cannot distinguish between latent deoxyribonucleic acid (DNA) and actively replicating virus. They are also unable to differentiate low-level from high-level viral replication, which is important for predicting the risk of CMV disease and for monitoring the response to therapy. Given these limitations, qualitative PCR has a limited role for the diagnosis or management of patients with CMV infection.

Quantitative CMV DNA assays include:

COBAS AmpliPrep/COBAS TaqMan CMV test – The COBAS AmpliPrep/COBAS TaqMan CMV test is a real-time PCR test that targets the polymerase gene and is calibrated to the World Health Organization (WHO) international standard to quantify the CMV load in plasma with a reported range from 137 to 9,100,000 international units/mL [4] (see 'International standard' below). This test has been approved by the US Food and Drug Administration (FDA) for serial testing to evaluate changes in CMV load in solid organ transplant recipients [5].

COBAS CMV test – The COBAS CMV test is a real-time PCR test that targets the polymerase gene and is calibrated to the WHO international standard to quantify the CMV load in plasma with a reported range from 34.5 to 10,000,000 international units/mL. The test has been approved by the FDA as an aid in the management of CMV in solid organ transplant recipients and hematopoietic stem cell recipients.

Artus CMV RGQ MDx test – The Artus CMV RGQ MDx test is a real-time PCR test that targets the CMV major immediate early gene and is calibrated to the WHO international standard to quantify CMV DNA from plasma with a reported range of 159 to 7,940,000 international units/mL [6] (see 'International standard' below). This test has been approved by the FDA to use as an aid in the management of solid organ transplant patients undergoing treatment for CMV.

RealTime CMV test – The RealTime CMV test is a real-time PCR test that targets the UL34 and UL80.5 genes and is calibrated to the WHO international standard to quantify the CMV load in plasma with a reported range from 50 to 156,000,000 international units/mL. The test has been approved by the FDA to use as an aid in the management of hematopoietic stem cell transplant recipients who are undergoing anti-CMV therapy.

Alinity m CMV assay – The Alinity m assay is a real-time PCR test for use with the automated Alinity m System, which targets the UL34 and UL80.5 genes and is calibrated to the WHO international standard to quantify the CMV load in plasma with a reported range from 30 to 100,000,000 international units/mL. The test is FDA approved and is intended for use as an aid in the management of hematopoietic stem cell transplant and solid organ transplant patients who are undergoing anticytomegalovirus therapy. The test can be used to assess virologic response to anticytomegalovirus therapy.

Laboratory-developed PCR assays – There are a variety of analyte-specific reagents available to assist laboratories in the development of assays. Most laboratory-developed assays use real-time PCR technologies, which have several advantages compared with standard PCR methods, including decreased risk of carryover contamination, broader linear range, and more rapid turnaround time. The performance, sensitivity, and linear range of laboratory-developed assays vary because of different specimen types, nucleic acid extraction methods, gene targets, primers, probes, detection methods, and quantitation standards. As a result, there may not be close agreement in viral load values among different laboratories.

International standard — In 2010, an international standard developed by the WHO and composed of a standardized amount of CMV became available from the National Institute of Biological Standards and Controls in the United Kingdom [7]. The availability of this reference reagent is an important advance in CMV testing, since it should allow for the standardization of viral load values among different laboratory-developed tests when results are expressed as international units/mL [8]. It was hoped that this would facilitate clinical studies to determine the viral load values that correlate with the development of clinical disease. However, one study has shown that using the international standard, variability in CMV DNA results reported on individual specimens has been reduced but clinically relevant variability persists, limiting meaningful inter-assay result comparison [9]. A study that used digital PCR showed that quantitative results for patient specimens showed good correlation in most comparisons, with some showing poorer correlations when testing samples with low viral loads [10]. Commercial assays using digital PCR methods are not available.

As more laboratories convert to using commercial assays calibrated to the international standard and move away from individual laboratory-based tests, the agreement between laboratories should continue to improve. However, DNA extraction techniques, PCR targets, and specimen types used (eg, plasma or whole blood) still differ among testing centers. Thus, variability among assays will continue to exist. As an example, the size of the DNA target that is amplified impacts the viral load. Because most CMV DNA in plasma consists of small fragments [11], viral load assays that amplify small targets (eg, approximately 100 base pairs or less) yield higher viral loads than assays that amplify larger targets [9]. Moreover, the extraction efficiency for small DNA fragments varies considerably among different commercial extraction methods [12]. These factors contribute to the variability in viral load values between commercial assays, despite the availability of the WHO international standard. For these reasons, the same viral load test should be used when monitoring patients over time.

The variability in viral load values among tests has hindered studies aimed at establishing the appropriate viral load cutoff for the diagnosis of clinically significant CMV infection. These cut-off values will likely depend upon a variety of factors, including:

Assay used

Specimen type (eg, plasma, whole blood)

Organ transplanted

Serostatus of both the recipient and donor

Type of immunosuppression

Whole blood versus plasma — Plasma or whole-blood specimens are typically used for CMV load testing. When comparing viral load values between the two compartments, the DNA levels follow similar kinetics [13]. However, viral DNA is detected more frequently and viral load values are often higher in whole blood compared with plasma because both cell-free and intracellular viruses are detected. One study showed that viral load values in most patients are about 1 log10 (10-fold) higher in whole blood compared with plasma [14]. Although testing of whole-blood specimens is more sensitive than plasma, low levels of CMV DNA in whole blood or plasma do not always correlate with active CMV disease [15].

CMV DNA is stable in whole blood and plasma for up to five days when stored at 4°C [16,17] and for three to four days when stored at room temperature [17,18]. Ethylenediaminetetraacetic acid (EDTA) is the preferred anticoagulant for quantitative CMV PCR assays. One study showed that, once plasma is separated from whole blood, the plasma DNA is stable for 14 days at 4°C [19].

CMV ANTIGENEMIA ASSAYS — CMV antigenemia assays permit the rapid and direct detection of CMV proteins (pp65) in peripheral blood leukocytes. This technique employs fluorescently labeled monoclonal antibodies specific to the pp65 lower matrix protein of CMV in peripheral blood polymorphonuclear leukocytes. Positive results are reported as the number of cells with staining per total number of cells counted. Results are generally available within 24 hours, and the test performs well in HIV-infected patients and recipients of solid organ transplants [20-22]. In these patients, antigenemia appears to correlate with viremia [4].

The antigenemia test is more sensitive than culture for the detection of CMV in blood [20-23], and studies have shown that a higher number of positive cells correlates with risk of developing active disease (10 to 20 positive cells/200,000 leukocytes) [24,25].

The limitations of the antigenemia test include lack of stability of the whole-blood specimen. insensitivity when the patient has a low neutrophil count (<1000 cells/microL), and more difficulty standardizing test results [26]. For these reasons, most laboratories have moved away from antigenemia testing and prefer the use of quantitative molecular methods. (See "Approach to the diagnosis of cytomegalovirus infection", section on 'Approach to diagnosis'.)

HISTOPATHOLOGY — Histologic examination of tissue biopsies is useful for the diagnosis of CMV tissue-invasive disease. Diagnosis is based on the presence of inclusion bodies, typically basophilic intranuclear inclusions, although eosinophilic cytoplasmic inclusions may also be seen; the diagnosis of CMV in tissue sections can be confirmed with immunohistochemical staining [26]. Some experts recommend immunohistochemical staining of biopsy specimens in which inclusions bodies are not seen as a method to improve diagnostic sensitivity [26]. Detection of CMV inclusions in a lung biopsy or in a bronchoalveolar lavage specimen supports the diagnosis of tissue invasive disease and increases the predictive value of a positive culture. (See "Approach to the diagnosis of cytomegalovirus infection", section on 'Tissue-invasive disease'.)

CULTURE — Using human fibroblast cultures, CMV can be isolated from multiple specimen types, including the blood, cerebrospinal fluid (CSF), throat washings, bronchoalveolar lavage fluid, urine, and biopsy specimens. Shell vial culture has largely replaced conventional culture in clinical virology laboratories, since the former method is much faster than the latter method.

Conventional culture — There are several limitations of conventional cell culture techniques.

CMV grows slowly in conventional cell culture; depending on the level of virus present, it may take one to six weeks before characteristic cytopathic changes are observed [27]. Thus, culture cannot be relied upon as a rapid diagnostic test.

Conventional culture for CMV lacks sensitivity, particularly when culturing blood (peripheral blood leukocytes). As noted above, CMV can be cultured from of variety of body fluids and tissues, but it is most useful in the diagnosis of congenital CMV (urine) and tissue-invasive disease. The sensitivity of culture from blood samples is inadequate for the diagnosis of CMV disease; molecular methods or antigenemia should be used for this purpose. (See 'Molecular assays' above and 'CMV antigenemia assays' above.)

A positive blood culture is very specific for and predictive of CMV disease [28]. However, the detection of CMV in culture from other sites indicates the presence of the virus but does not confirm active CMV disease. CMV may be shed in the urine intermittently for several months after acute CMV infection, even in otherwise healthy individuals [29]. Immunosuppressed patients often shed virus in the urine and other specimen types (bronchoalveolar lavage fluid, stool) for prolonged periods of time in the absence of clinical disease [3,26].

Shell vial culture — Shell vial culture is a rapid culture method based on low-speed centrifugation and detection of CMV early antigen prior to the development of characteristic cytopathic effects in tissue culture, thus accelerating the time to diagnosis. Following centrifugation of clinical samples (eg, urine, bronchial washings, tissue) to increase the absorption of virus, cell monolayers are exposed to monoclonal antibodies against early antigen. Binding of antibodies is indicative of "early" CMV replication within the cells [27]. Results are typically available within two to three days after specimens are obtained. The sensitivity of shell vial culture varies with specimen type and is lower for blood samples compared with other body fluids. As with conventional culture, the sensitivity of shell vial culture for blood samples is inadequate for clinical use. Rapid shell vial cultures also have the same limitations regarding specificity as conventional culture. Most laboratories that culture CMV rely on shell vial culture (rather than conventional culture) due to the rapid turnaround time.

RESISTANCE TESTING — Monitoring CMV load is a useful approach for assessing the likelihood of drug resistance. Markers suggestive of CMV resistance include a rising viral load, rebounding viral load, and a persistently elevated viral load in the setting of antiviral therapy. In the past, resistance testing was performed using phenotypic methods, which required isolation of the virus in cell culture, determining the viral titer, and then culturing the virus in the presence of drug (usually ganciclovir). This was a tedious process that took weeks to months to complete and had little impact on patient care. Currently, resistance testing is done using genotypic assays to identify specific resistance mutations using automated sequencing methods. The assays are done directly from clinical specimens (plasma, cerebrospinal fluid, bronchoalveolar lavage) and usually require a viral load of >1000 copies/mL [30]. Genotypic testing is available from commercial laboratories with a turnaround time of approximately one week. The clinical applications of CMV resistance testing are discussed elsewhere. (See "Approach to the diagnosis of cytomegalovirus infection", section on 'Resistance testing' and "Clinical manifestations, diagnosis, and treatment of cytomegalovirus infection in lung transplant recipients", section on 'Ganciclovir resistance'.)

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Cytomegalovirus in solid organ transplant recipients".)

SUMMARY

Clinical manifestations − Cytomegalovirus (CMV) generally produces an asymptomatic or minimally symptomatic acute illness in immunocompetent patients. In immunocompromised hosts, however, CMV infection can result in a broad array of clinical presentations, including viral syndrome, retinitis, pneumonitis, encephalitis, hepatitis, esophagitis, and colitis. These complications are associated with significant morbidity and mortality. (See 'Introduction' above.)

Types of diagnostic tests − Appropriate diagnostic tests are essential for the management of CMV infection and disease in immunocompromised patients. Several diagnostic modalities are available, including serology, quantitative polymerase chain reaction (PCR), pp65 antigenemia, culture, and histopathology (table 1). (See 'Background' above.)

Serologic diagnosis of recent or acute CMV infection − A diagnosis of recent or acute CMV is considered probable in the following circumstances (see 'Serology' above):

The detection of CMV-specific immunoglobulin (Ig)M antibodies (suggesting recent seroconversion)

A fourfold or greater increase in CMV-specific IgG titers in paired specimens obtained at least two to four weeks apart

Duration of IgM positivity − IgM antibody can persist for several months and, therefore, may provide misleading information if a prior baseline test is not available (in cases in which it represents prior infection rather than current infection). (See 'Serology' above.)

Use of serology to determine past exposure − Serologic tests are also helpful in determining past exposure to CMV infection. This information is particularly useful for the management of immunosuppressed hosts at risk for CMV reactivation. (See 'Serology' above.)

Quantitative PCR − Real-time PCR tests for the quantification of CMV DNA are widely used for diagnosis and monitoring of CMV disease. Agreement in viral load values is improving with the availability of an international standard and the US Food and Drug Administration's approval of tests that are calibrated to the international standard. In spite of these advances, viral load values vary between tests. (See 'Molecular assays' above.)

Plasma versus whole blood testing − Viral load testing can be done using plasma or whole-blood specimens. Higher values are observed in whole-blood specimens compared with plasma. The same assay and specimen type should be used for monitoring patients over time. (See 'Whole blood versus plasma' above.)

CMV antigenemia − CMV antigenemia assays permit the rapid detection of CMV proteins in peripheral blood leukocytes; the test is more sensitive than culture for the detection of CMV in blood, and higher numbers of positive cells correlate with risk of developing active disease. The limitations of the antigenemia test include lack of stability of the whole-blood specimen, insensitivity when the patient has a low neutrophil count, and difficulties with assay standardization. (See 'CMV antigenemia assays' above.)

Pathology − Histopathology and immunohistochemical staining of tissue samples are useful for the diagnosis of tissue invasive disease. (See 'Histopathology' above.)

CMV culture − CMV culture can be performed on blood, tissue, and a variety of body fluids but is most useful for the diagnosis of congenital CMV and tissue-invasive disease. Shell viral culture has replaced conventional culture in many clinical laboratories because of the rapid time to results (one to three days) of the former culture type. Culture of blood (peripheral blood leukocytes) for CMV lacks sensitivity and should not be used for diagnosis of CMV diseases. The detection of CMV in culture of other specimen types does not confirm active CMV disease, since immunosuppressed patients often shed virus in the urine, bronchoalveolar lavage fluid, and stool for prolonged periods of time in the absence of clinical illness. (See 'Conventional culture' above.)

Resistance testing − CMV antiviral resistance testing is performed directly from clinical specimens (usually plasma or whole blood); specific drug-related mutations are identified using automated sequencing. Optimal results are obtained when the viral load value is >1000 copies/mL. (See 'Resistance testing' above.)

Approach to diagnosis − The approach to the diagnosis of CMV infection is discussed separately. (See "Approach to the diagnosis of cytomegalovirus infection".)

ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges Dr. Timothy Friel, who contributed to an earlier version of this topic review.

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