Diagnosis of cytomegalovirus
Author
Timothy J Friel, MD
INTRODUCTION — Cytomegalovirus (CMV) generally produces an asymptomatic or minimally symptomatic acute illness in immunocompetent patients. In the immunocompromised host, however, CMV infection can result in a broad array of clinical presentations, including retinitis, pneumonia, encephalitis, hepatitis, and gastrointestinal tract ulceration -- complications associated with significant morbidity and mortality.
The diagnosis of CMV infections will be reviewed here. The manifestations of CMV in immunocompetent and immunocompromised patients are discussed separately. (See appropriate topic cards). The diagnosis of CMV during pregnancy is discussed separately (see "Cytomegalovirus infection in pregnancy").
BACKGROUND — All testing methods described below have their advantages and disadvantages and need to be interpreted in the context of the clinical presentation and other diagnostic assessments. In the past, serologic testing and viral cultures from multiple sites were the cornerstones of diagnosis of CMV infection. However, more recently, molecular amplification methods have gained prominence as diagnostic tools.
SEROLOGY — Serology provides indirect evidence of recent CMV infection based upon changes in antibody titers at different time points during a clinical illness. Many different antibody detection techniques are available, including complement-fixation techniques, enzyme-linked immunosorbent assays (ELISA), latex agglutination, radioimmunoassays, and indirect hemagglutination assays [1]. Investigators have also developed liquid-phase luciferase immunoprecipitation systems to provide qualitative assessments of anti-CMV antibodies [2].
In early studies, a diagnosis of recent or acute CMV was considered probable (though not definite) in the following circumstances:
The detection of CMV-specific IgM antibodies (suggesting recent seroconversion)
The observation of at least a fourfold 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 [3]. CMV-specific IgG antibodies are often not detectable until two to three weeks following the onset of symptoms [3]. Among a subset of 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 [3]. Therefore, a positive CMV-specific IgM antibody alone may provide misleading information if a prior baseline test is not available.
Serologies are also helpful in determining past exposure to CMV infection. This information is particularly relevant to the management of immunosuppressed hosts at risk for CMV reactivation syndromes. As an example, HIV-infected patients with serologic evidence of past exposure to CMV warrant monitoring for CMV retinitis with progressive immunosuppression. Likewise, serologies are also helpful in determining risk of acquisition of infection. 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 a CMV seropositive organ. (See "Pathogenesis, clinical manifestations, and diagnosis of AIDS-related cytomegalovirus retinitis" and "Infection in the solid organ transplant recipient".)
CULTURES — Using human fibroblast cultures, CMV can be isolated from multiple sites, including the blood, urine, throat washings, cerebrospinal fluid, bronchial washings, and biopsy specimens. However, there are several limitations of cell culture techniques:
CMV grows slowly in cell culture; depending on the level of virus present, one to six weeks must pass before characteristic cytopathic changes can be detected [1]. Thus, cultures cannot be relied upon as a rapid confirmatory test.
The detection of CMV in culture indicates the presence of the virus but does not confirm active CMV disease. CMV may be shed from the urine and throat intermittently for several months after acute CMV infection [4]. Immunosuppressed patients often shed virus for prolonged periods of time.
Though generally more sensitive than urine cultures, even isolation of CMV from the blood (peripheral blood leukocytes) is not diagnostic since viremia can persist in the absence of clinical disease, especially in the immunocompromised patient [5]. The overall sensitivity is limited, especially in comparison to newer techniques like CMV antigenemia assays and molecular amplification.
Therefore, cultures are not often performed, but can be useful when determining the presence of drug resistance [6].
EARLY ANTIGEN DETECTION — CMV early antigen detection (shell vial cultures) allows clinicians to detect CMV in culture before the development of characteristic cytopathic effects, thus accelerating the time to diagnosis. Following centrifugation of clinical samples (eg, urine, blood, bronchial washings) to increase the absorption of virus, cell monolayers are exposed to monoclonal antibodies. Binding of antibodies is indicative of "early" CMV replication within the cells [1]. Results are typically available within two to three days after specimens are obtained.
Monoclonal antibodies are also used to detect CMV proteins in tissue specimens submitted for pathologic evaluation by immunocytochemical techniques.
CMV ANTIGENEMIA ASSAYS — CMV antigenemia assays permit the rapid detection of CMV proteins in peripheral blood leukocytes. This technique employs tagged 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 patients with HIV infection and recipients of solid organ transplants [7-9]. In these patients, antigenemia appears to correlate with viremia [10].
Though extensively evaluated in immunosuppressed patients, the role of the CMV antigenemia assay in the diagnosis of CMV disease in immunocompetent patients has not been rigorously explored. One study evaluated this test in 40 patients with a suspected CMV mononucleosis-like syndrome over a two-year period [11]. CMV infection was ultimately confirmed in 10 patients; a positive CMV antigenemia assay was obtained in 9 of these 10 patients. Seven of the nine patients had low level antigenemia (less than 20 positive cells). Of the 30 remaining patients with other diagnoses, only one patient with systemic lupus erythematosus had detectable antigenemia. In this study the sensitivity and specificity of the test was 90 and 96 percent, respectively.
MOLECULAR AMPLIFICATION — Molecular amplification assays are fast and reliable. Commercially available tests include the COBAS AMPLICOR CMV Monitor test (Roche Diagnostics, Indianapolis, IN), the Hybrid Capture System (Digene Corporation, Gaithersburg, MD) and the NucliSens CMV Test, a nucleic acid sequence-based amplification assay (NASBA, Organon Teknika Corporation, Durham, North Carolina).
The COBAS Amplicor test is a PCR assay that amplifies a 365 base pair region of the CMV polymerase gene. The manufacturer reports that the dynamic range of the assay is between 400 and 100,000 copies/mL. The assay has been designed for use with plasma, leukocyte, and whole blood specimens.
The Hybrid Capture System CMV DNA test is a signal amplification method using an RNA probe that targets 17 percent of the CMV genome. The target is detected employing antibodies that specifically bind RNA:DNA hybrids. The dynamic range is 1400 to 600,000 copies/mL. The assay has been designed for whole blood specimens.
Nucleic acid sequence-based amplification (NASBA) was developed to detect both immediate-early gene UL123 (IE1) and late gene expression (pp67) of CMV infection. This assay can also be performed on whole blood samples. Unlike quantitative PCR and signal amplification techniques, NASBA provides a qualitative result.
The sensitivity and specificity of NASBA was evaluated in a study of 21 solid organ transplant recipients; 90 percent of clinical samples that were positive by culture or immunofluorescence were positive by NASBA [12]. In contrast, NASBA was negative in 50 CMV seronegative and 50 seropositive asymptomatic blood donors. In another study of 30 renal transplant recipients, the sensitivity of the assay was 100 percent, but the specificity was only 76 percent [13].
Like the antigenemia assay, molecular amplification techniques have been studied most extensively in immunocompromised patients. Multiple reports cite a high sensitivity and specificity in patients with the acquired immunodeficiency syndrome (AIDS) [14] or following transplantation [15]. The use of molecular amplification assays in transplant patients is discussed elsewhere. (See "Viral load testing for cytomegalovirus in solid organ transplant recipients".)
Little data exist on the use of these assays in immunocompetent hosts. One investigation compared the performance of peripheral blood leukocyte (PBL) cultures, CMV PCR, and antigenemia assays in a group of 52 immunocompetent patients with primary CMV infection diagnosed by serology [16]. CMV DNA was detected in 100 percent of the 25 patients who had the PCR assay performed within one month of symptoms, but in none of the controls. In comparison, antigenemia assays and PBL cultures were much less sensitive, with positive results in only 50 percent and 21 percent of patients, respectively, during the first four weeks of symptoms. However, assays remained positive by PCR in 81 percent of patients two months after presentation. The prolonged detection of CMV DNA may decrease the specificity of the test when used to detect active disease.
Not all studies have supported the utility of CMV PCR assays in the diagnosis of acute CMV infections in immunocompetent individuals. Using serum obtained from 34 patients with confirmed CMV mononucleosis, investigators detected CMV DNA in the sera of only seven (20.5 percent) patients using three different commercially available assays. The case patients had clinical findings suggestive of acute mononucleosis and laboratory evidence of CMV seroconversion or CMV-specific IgM; diagnostic testing for EBV and HHV-6 were negative [17].
At present, the comparison of quantitative results obtained using different techniques is not possible. Therefore, it is important to use the same test method when monitoring changes in viral load in patients over time.
SUMMARY
Cytomegalovirus (CMV) generally produces an asymptomatic or minimally symptomatic acute illness in immunocompetent patients. In the immunocompromised host, however, CMV infection can result in a broad array of clinical presentations, including retinitis, pneumonia, encephalitis, hepatitis, and gastrointestinal tract ulceration -- complications associated with significant morbidity and mortality.
In the past, serologic testing and viral cultures from multiple sites were the cornerstones of diagnosis of CMV infection. However, more recently, molecular amplification methods have gained prominence as diagnostic tools.
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)
The observation of at least a fourfold increase in CMV-specific IgG titers in paired specimens obtained at least two to four weeks apart.
It should be noted that IgM antibody can persist for several months and, therefore, may provide misleading information if a prior baseline test is not available.
Serologies are also helpful in determining past exposure to CMV infection. This information is particularly relevant to the management of immunosuppressed hosts at risk for CMV reactivation syndromes.
CMV cultures are not often performed, but can be useful when determining the presence of drug resistance.
CMV early antigen detection (shell vial cultures) allows clinicians to detect CMV in culture before the development of characteristic cytopathic effects, thus accelerating the time to diagnosis. Following centrifugation of clinical samples (eg, urine, blood, bronchial washings) to increase the absorption of virus, cell monolayers are exposed to monoclonal antibodies. Binding of antibodies is indicative of "early" CMV replication within the cells.
Monoclonal antibodies are also used to detect CMV proteins in tissue specimens submitted for pathologic evaluation by immunocytochemical techniques.
CMV antigenemia assays permit the rapid detection of CMV proteins in peripheral blood leukocytes. This technique employs tagged 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 patients with HIV infection and recipients of solid organ transplants.
Molecular amplification assays are fast and reliable. Several testing methods are available, including the polymerase chain reaction, a hybrid capture assay, and nucleic acid sequence-based amplification (NASBA).
But the fact that there is an antecedent with CMV and the same symptoms precisely can not be a coincidence. Maybe this reaction comes only with CMG and Herpes like viruses?
