Approach to the diagnosis of cytomegalovirus infection

INTRODUCTION — Cytomegalovirus (CMV) is a common infection, and, although serious disease is rare in immunocompetent individuals, CMV is a major pathogen for immunocompromised patients, including solid organ transplant recipients, hematopoietic cell transplant recipients, human immunodeficiency virus (HIV)-infected patients, and patients treated with immunomodulating drugs.

The range of clinical disease due to CMV in immunocompromised patients is broad and includes febrile syndromes, hepatitis, pneumonitis, retinitis, encephalitis, esophagitis, and colitis. Since the signs and symptoms of CMV disease often overlap with other infectious processes and rejection, the diagnosis is made by integrating the clinical history, clinical presentation, and laboratory data. Because CMV produces lifelong latent infection, distinguishing active disease from latent infection and asymptomatic reactivation presents an additional diagnostic challenge.

The diagnosis and monitoring of CMV infection and tissue-invasive disease will be discussed here, with an emphasis on the role of diagnostic tests and their strengths and limitations. An overview of the diagnostic tests for CMV is presented separately. The manifestations of CMV in immunocompetent and immunocompromised patients are also discussed elsewhere. (See "Overview of diagnostic tests for cytomegalovirus infection" and "Epidemiology, clinical manifestations, and treatment of cytomegalovirus infection in immunocompetent adults" and "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" and "Pathogenesis, clinical manifestations, and diagnosis of AIDS-related cytomegalovirus retinitis" and "AIDS-related cytomegalovirus gastrointestinal disease" and "AIDS-related cytomegalovirus neurologic disease" and "Overview of cytomegalovirus infections in children" and "Cytomegalovirus infection in pregnancy" and "Congenital cytomegalovirus infection: Clinical features and diagnosis" and "Congenital cytomegalovirus infection: Management and outcome".)

INFECTION VERSUS DISEASE — CMV infection and disease are not synonymous terms; not all patients with infection develop overt clinical disease.

●CMV infection refers to virus isolation or detection of viral proteins (antigens) or nucleic acid in any body fluid or tissue specimen regardless of symptoms or signs [1,2].

●CMV disease refers to evidence of CMV infection with attributable symptoms or signs; CMV disease may manifest as either a viral syndrome (eg, fever, malaise, leukopenia, neutropenia, atypical lymphocytosis, thrombocytopenia) or as tissue-invasive disease [1,2].

IMMUNOCOMPROMISED HOSTS — The diagnosis of CMV disease in immunocompromised patients relies on clinical history and presentation along with laboratory testing. It is essential to interpret laboratory tests in the clinical context, since the virus, CMV deoxyribonucleic acid (DNA), and CMV antigen can all be detected in some patients who do not have active disease.

The following discussion applies to patients who are immunocompromised due to solid organ transplantation, hematopoietic cell transplantation, HIV infection, or the use of immunomodulating drugs. The types of CMV disease seen in the different patient populations vary, but there are similarities to the diagnostic approach.

Approach to diagnosis — Appropriate diagnostic tests are essential for the management of CMV infection and disease in immunocompromised patients. Diagnostic modalities include serology, quantitative polymerase chain reaction (PCR), pp65 antigenemia, culture, and histopathology (table 1).

CMV DNA and antigen tests — There have been numerous studies supporting the clinical utility of CMV replication assays, particularly plasma or whole-blood quantitative PCR assays, in managing CMV disease in immunocompromised patients [3]. The combination of viral load in the initial phase of infection and the rate of increase in viral load may help to identify patients at risk for CMV disease. These assays are now used routinely in many transplant centers to diagnose active CMV disease, screen patients for the use of pre-emptive antiviral therapy, and monitor responses to antiviral therapy.

Choice of test — Quantitative PCR tests and the CMV pp65 antigenemia test are available for detecting viral DNA and antigen, respectively. Quantitative PCR assays offer several advantages over the antigenemia assay, including better assay standardization, increased stability of the specimen, smaller specimen volume, and the ability to test patients with leukopenia. For these reasons, quantitative PCR assays are more widely used than the antigenemia test, and we prefer them for the diagnosis and monitoring of immunocompromised patients with CMV infection and disease [4,5]. Quantitative PCR tests should be calibrated to the World Health Organization (WHO) International standard. Because viral replication can occur in the setting of asymptomatic reactivation, qualitative CMV DNA testing has limited clinical utility and should not be used routinely.

CMV replication assays are discussed in greater detail separately. (See "Overview of diagnostic tests for cytomegalovirus infection", section on 'Molecular assays' and "Overview of diagnostic tests for cytomegalovirus infection", section on 'CMV antigenemia assays'.)

Predictive role — Studies of solid organ transplant recipients using quantitative PCR assays developed in individual laboratories have shown a higher viral load in patients with active CMV disease compared with those with asymptomatic infection [6-10]. However, CMV DNA values were not comparable among the assays because of differences in assay design and quantification standards. As a result, the cutoff viral load associated with active CMV disease has not been established. Though agreement in viral-load values between assays has improved with the availability of the WHO international and US Food and Drug Administration (FDA)-approved tests calibrated to the WHO standard, differences still exist. This lack of standardization has also limited attempts to establish a viral-load cutoff that predicts the development of CMV disease. (See "Overview of diagnostic tests for cytomegalovirus infection", section on 'International standard' and "Overview of diagnostic tests for cytomegalovirus infection", section on 'Molecular assays'.)

Initial viral load — Several studies have reported on the clinical utility of PCR testing in the diagnosis and monitoring of CMV disease following solid organ transplantation [11-14]. These studies have shown that higher CMV loads were detected in patients with symptomatic disease compared with those with asymptomatic infection. A natural history study of CMV disease in liver transplant recipients showed that the optimal cutoff for predicting CMV disease was in the range of 2000 to 5000 copies/mL of plasma; at a cutoff of >5000 copies/mL, the sensitivity was 86 percent and the specificity was 87 percent [12]. All patients with a viral load >20,000 copies/mL developed CMV disease. An important limitation of these data is that these CMV DNA levels apply to the Amplicor Monitor test, which is not available clinically. In a more recent study of solid organ transplant recipients at lower risk for CMV infection, a cutoff of approximately 4000 international units/mL was established for initiating pre-emptive therapy [15]. However, these results may not be generalizable, as testing was done on plasma samples using an assay that is not FDA approved and the study population was solid organ transplant recipients at low risk of developing CMV disease. (See "Overview of diagnostic tests for cytomegalovirus infection", section on 'Molecular assays'.)

Rate of increase — Not only is the level of CMV load predictive of symptomatic disease, but the rate of increase can also be used to predict which patients are at risk for CMV disease [16,17]. In one study, patients with CMV disease had a significantly faster rate of increase in viral load between the last PCR-negative and first PCR-positive sample than those without CMV disease (0.33 log₁₀ versus 0.19 log₁₀ genomes/mL daily) [16].

Serology — Serologic tests have no role in diagnosing CMV disease in immunocompromised patients. They are used pretransplant to establish serostatus, which predicts risk of developing disease and guides the use of prophylactic antiviral therapy. (See "Overview of diagnostic tests for cytomegalovirus infection", section on 'Serology'.)

Testing based on type of disease

Viral syndrome — Both quantitative CMV PCR and antigenemia assays can be used to diagnose CMV-associated viral syndromes, but, as stated above, there are no established cutoff values to definitively diagnose active CMV infection. We prefer quantitative PCR for the diagnosis and monitoring of patients with CMV infection and disease. (See 'Choice of test' above and 'Predictive role' above.)

For transplant recipients, a positive viral load and symptoms consistent with CMV infection establish the diagnosis of CMV disease. However, transplant recipients and HIV-infected individuals may have low-level viremia that does not require therapy. The decision of whether a patient requires therapy must be made on a case-by-case basis. Both conventional CMV culture and the CMV shell vial assay lack the sensitivity needed to diagnose CMV-associated viral syndromes [5].

Tissue-invasive disease — The gold standard for diagnosing CMV tissue-invasive disease is the identification of CMV inclusions or positive CMV-specific immunohistochemistry staining on histopathology. A positive culture from a biopsy specimen is also considered consistent with CMV disease, but, since tissue may be contaminated with blood or body secretions and viral shedding can occur from some sites (urine, bronchoalveolar lavage [BAL] fluid, stool) in the absence of clinical disease, it is optimal to perform both culture and histopathology on the tissue specimen.

Individuals with suspected CMV tissue-invasive disease should also have plasma or whole-blood PCR testing, since PCR results are often available prior to the biopsy results and may influence the decision to initiate antiviral therapy. In addition, it is important to establish the baseline viral load because serial testing is used to monitor response to therapy. (See 'Monitoring response to treatment' below.)

A negative plasma or whole-blood PCR does not exclude tissue-invasive CMV disease, especially in patients with CMV gastrointestinal disease, pneumonia, or retinitis. The absence of CMV inclusions on histopathology also does not exclude tissue-invasive disease since such inclusions can be missed as a result of sampling error.

In some patients, it may not be possible to obtain a tissue biopsy. When this is the case, viral-load testing or culture of body fluids may be performed. Culture of urine specimens should be avoided since a positive urine culture does not correlate with CMV disease (with the exception of congenital CMV) [5].

Pneumonitis

●For pneumonitis in transplant recipients, the identification of CMV inclusions in cells obtained from a BAL or lung biopsy supports the diagnosis of CMV disease. A positive culture is often difficult to interpret, since CMV is commonly shed in respiratory secretions in the absence of disease.

●In lung transplant recipients, quantitative CMV PCR can be performed on BAL specimens. Viral-load testing of BAL fluid has higher sensitivity than culture, but, as with culture, positive viral-load results can represent either viral shedding or pulmonary disease [5]. Some studies have shown the level of CMV DNA in BAL correlates with pneumonitis [18-20], but this correlation is not consistently observed [21]. One study evaluating lung transplant recipients (using the COBAS AmpliPrep/COB TaqMan test) found that patients with CMV pneumonia have higher BAL viral loads compared with those without pneumonia; the optimal cutoff was 4545 international units/mL with a sensitivity of 91 percent and specificity of 77 percent [20]. (See "Clinical manifestations, diagnosis, and treatment of cytomegalovirus infection in lung transplant recipients", section on 'Pneumonitis'.)

●In HIV-infected individuals, culture of BAL fluid has both low sensitivity and low specificity for the diagnosis of CMV pneumonitis. There have been several studies documenting the low sensitivity of CMV culture of BAL fluid; one reported a sensitivity of 50 percent [22] and another reported a sensitivity of 60 percent [23] for the diagnosis of CMV pneumonitis compared with autopsy findings. Regarding specificity, a positive CMV culture of BAL does not correlate with gas exchange, chest radiographic abnormalities, or acute morbidity due to pulmonary disease [24,25]. In one study, 9 of 19 asymptomatic HIV-infected individuals (47 percent) had a positive CMV BAL culture [26].

Gastrointestinal disease

●For gastrointestinal disease, plasma or whole-blood CMV DNA load tests are sometimes negative, so the diagnosis relies upon culture and histopathology of a tissue biopsy. Gastrointestinal disease may be focal and patchy, so multiple biopsies may be needed to confirm the diagnosis of colitis or esophagitis.

●CMV gastrointestinal disease cannot be excluded based on a negative plasma or whole-blood PCR result. In a retrospective study of 81 solid organ transplant recipients (mostly kidney and liver transplant recipients) with gastrointestinal symptoms who underwent gastrointestinal biopsies and quantitative plasma PCR testing, 20 cases of biopsy-proven gastrointestinal disease were identified [27]. The sensitivity of PCR for diagnosing CMV gastrointestinal disease was 85 percent and the specificity was 95 percent. The mean plasma CMV load in patients with gastrointestinal disease was 38,334 copies/mL, but plasma CMV DNA was undetectable in three patients (15 percent) with biopsy-proven CMV gastrointestinal disease. The diagnosis was established by immunohistochemistry in two of the three patients and by viral cytopathic effect in one patient. Plasma viral-load testing has high sensitivity for the diagnosis of gastrointestinal disease in high-risk recipients (ie, CMV-seronegative recipients who receive organs from CMV-seropositive donors). However, the sensitivity of this assay decreases substantially in CMV-seropositive organ transplant recipients. [27,28]

●CMV culture of stool specimens has no role for the diagnosis of CMV colitis, since a positive culture may represent either asymptomatic viral shedding or disease.

The diagnosis of CMV gastrointestinal disease in HIV-infected patients is discussed in detail separately. (See "AIDS-related cytomegalovirus gastrointestinal disease", section on 'Diagnosis and differential diagnosis'.)

Central nervous system disease

●For the diagnosis of CMV encephalitis, myelitis, or polyradiculopathy, viral-load testing of cerebrospinal fluid (CSF) has become the standard of care. CMV culture of CSF specimens has poor sensitivity. Most of the studies evaluating viral load or antigenemia for the diagnosis of CMV neurologic disease were performed in HIV-infected individuals from the pre-antiretroviral therapy (ART) era, when the disease was more common. These studies showed that the detection of CMV DNA or antigen is very sensitive and specific [29-31]. Based on clinical experience, CMV DNA can be detected in the CSF of HIV-1-infected individuals in the absence of CMV disease; quantitative viral-load testing may be helpful in assessing the level of CMV DNA, which may influence treatment decisions.

●Central nervous system disease in solid organ transplant recipients is very rare, and the diagnostic utility of viral-load testing has not been well studied. The recommendation is to treat solid organ transplant recipients who have detectable viral load in the CSF [5].

Retinitis — The diagnosis of CMV retinitis in HIV-infected patients is discussed separately. (See "Pathogenesis, clinical manifestations, and diagnosis of AIDS-related cytomegalovirus retinitis", section on 'Diagnosis'.)

Monitoring response to treatment — Viral-load assays are useful in monitoring responses to antiviral therapy in transplant recipients. The following principles should be considered when monitoring the response to treatment:

●Viral-load values among different CMV tests are not comparable [5]. Patients should therefore be monitored using the same assay. (See "Overview of diagnostic tests for cytomegalovirus infection", section on 'Molecular assays'.)

●Viral-load values in most patients are one log₁₀ higher in whole-blood specimens compared with plasma, so the same specimen type should be used when monitoring response to therapy [32]. (See "Overview of diagnostic tests for cytomegalovirus infection", section on 'Whole blood versus plasma'.)

●When monitoring patients, a baseline viral load should be obtained the day that antiviral therapy is initiated and should be repeated weekly. Based on the half-life of CMV in plasma, testing should be performed no more frequently than every five to seven days [5,33].

●The reproducibility of viral-load tests is such that changes in viral load need to exceed three- to fivefold to represent meaningful changes in viral replication [5,33].

●We typically give treatment doses of an antiviral agent until the CMV load is negative (and for a minimum of two weeks) [5]. When using a highly sensitive viral-load test (lower limit of quantification <200 international units/mL), treatment can be discontinued after one negative result. Confirmatory viral-load testing should be done one week after discontinuing therapy [5]. Viral-load values usually become undetectable several weeks after initiating therapy, although clearance of DNA will take longer for higher initial viral-load values and when testing is done on whole-blood samples compared with plasma [12,34,35]. An overly sensitive viral-load test could lead to overtreatment if low (clinically insignificant) levels of CMV DNA are detected [32]. The approach to antiviral therapy is discussed in greater detail separately. (See "Clinical manifestations, diagnosis, and management of cytomegalovirus disease in kidney transplant patients", section on 'Treatment' and "Clinical manifestations, diagnosis, and treatment of cytomegalovirus infection in lung transplant recipients", section on 'Monitoring on therapy'.)

●Patients with documented ganciclovir resistance may have persistently elevated viral-load values while on therapy [35]. (See "Overview of diagnostic tests for cytomegalovirus infection", section on 'Resistance testing' and "Clinical manifestations, diagnosis, and treatment of cytomegalovirus infection in lung transplant recipients", section on 'Ganciclovir resistance'.)

●A persistently detectable CMV load following two weeks of therapy has been associated with relapsing CMV infection [14], leading many experts to recommend treatment until CMV DNA is no longer detected in plasma or whole blood [5]. (See "Clinical manifestations, diagnosis, and treatment of cytomegalovirus infection in lung transplant recipients", section on 'Monitoring on therapy'.)

●The baseline viral load and the presence of viral-load suppression during the first several weeks of antiviral therapy are predictive of the response to therapy. One study evaluated CMV load in plasma samples using the COBAS AmpliPrep/COBAS TaqMan CMV test calibrated to the international CMV standard; the samples were from patients who were enrolled in the VICTOR study, an international randomized trial that compared the efficacy of intravenous ganciclovir with oral valganciclovir in solid organ transplant recipients with CMV disease [36]. Of 267 patients, 251 had CMV disease resolution by day 49 of antiviral therapy. Patients with pretreatment CMV DNA concentrations <18,200 (4.3 log₁₀) international units/mL had significantly faster time to disease resolution (adjusted hazard ratio [aHR] 1.56). Patients with CMV load suppression (<137 international units/mL [<2.1 log₁₀]) at days 7, 14, and 21 had significantly faster times to clinical disease resolution (aHRs 1.61, 1.73, and 1.64). Relative viral-load reductions from baseline were not associated with faster resolution of CMV disease.

●CMV load kinetics can also be useful for identifying patients who are more likely to develop recurrent CMV disease. In one study, the mean time to viral clearance was significantly longer in patients with recurrent CMV disease compared with those without recurrence (mean 33.8 versus 17.2 days, respectively) [37]. CMV load half-life was 8.8 days in patients with recurrence compared with 3.17 days in patients without recurrence. Monitoring viral-load kinetics allows for the identification of patients at risk for recurrent CMV disease at a very early stage in treatment, thus allowing for optimization of antiviral therapy.

Resistance testing — Although uncommon, antiviral resistance is a serious complication of CMV disease [38]. Development of resistance usually occurs after prolonged drug exposure (>6 weeks) [5]. Viral-load testing and antigenemia tests are useful tools to identify patients at risk for developing drug resistance. Patients who have a persistently elevated viral load, a rising viral load while on appropriate therapy, or a rebound in viral load after an initial response should be evaluated for drug resistance. An increase or lack of reduction of viral load during the first two weeks of therapy is not a reliable indicator of drug resistance [5].

●Resistance testing is done by automated sequencing methods directly from whole-blood or plasma specimens. More reliable results are obtained when viral-load values exceed 1000 copies/mL [5]. (See "Overview of diagnostic tests for cytomegalovirus infection", section on 'Resistance testing'.)

●Three drug classes are approved for the treatment of CMV, all of which target the DNA polymerase: ganciclovir (and the prodrug valganciclovir), foscarnet, and cidofovir. The initial phosphorylation of ganciclovir requires the viral UL97 kinase. Resistance to ganciclovir can occur due to mutations in the UL97 phosphotransferase gene or the UL54 polymerase gene, whereas resistance to foscarnet and cidofovir is due to mutations in the UL54 polymerase gene.

●In patients initially treated with ganciclovir, the most common mutations are UL97 phosphotransferase mutations, which confer resistance to ganciclovir [5,39,40]. UL54 polymerase mutations may occur as second-step mutations in patients who already have a UL97 mutation; CMV isolates possessing both UL97 and UL54 mutations often have high-level ganciclovir resistance [41-44]. The ganciclovir resistance conferred by UL54 mutations cannot be overcome by increasing the dose of ganciclovir. UL54 DNA polymerase mutations confer various combinations of resistance to ganciclovir, foscarnet, and/or cidofovir [45].

Genotypic resistance testing is available from commercial laboratories. Testing can be ordered for the UL97 gene, the UL54 gene, or both. Results are usually available within one week. If the patient has been treated only with ganciclovir, sequencing of both the UL97 and UL54 genes should be done. UL97 resistance mutations alone do not alter foscarnet or cidofovir susceptibility [5].

The management of resistant CMV infection in lung transplant recipients is discussed in detail separately. (See "Clinical manifestations, diagnosis, and treatment of cytomegalovirus infection in lung transplant recipients", section on 'Ganciclovir resistance'.)

IMMUNOCOMPETENT HOSTS — Although CMV infection is common in immunocompetent hosts, most infections are asymptomatic; the most common clinical presentation is a mononucleosis-like syndrome. (See "Epidemiology, clinical manifestations, and treatment of cytomegalovirus infection in immunocompetent adults", section on 'CMV mononucleosis'.)

Diagnosis of primary CMV infection in immunocompetent hosts is usually made using serologic studies, either the detection of CMV-specific immunoglobulin (Ig)M or a fourfold rise in CMV-specific IgG; these test results provide a presumptive diagnosis in the appropriate clinical setting. Serologic tests have limitations. A fourfold rise in IgG may take several weeks to occur, and IgM may persist for months following primary infection, so detection may not always represent acute infection. When assessing past CMV exposure or infection, any IgG result above the cutoff of the test is considered positive. The cutoff value may vary depending on the method used and is generally provided with test results.

One study compared the performance of peripheral blood leukocyte culture, CMV polymerase chain reaction (PCR), and CMV antigenemia assays in a group of 52 immunocompetent patients with primary CMV infection diagnosed by serology [46]. 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 peripheral blood leukocyte cultures were much less sensitive, with positive results in only 50 and 21 percent of patients, respectively, during the first four weeks of symptoms. However, DNA remained detectable 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. Testing for CMV DNA in whole blood or plasma in conjunction with CMV-specific IgG could improve the specificity of the DNA test. The presence of CMV DNA combined with the absence of CMV-specific IgG support the diagnosis of acute CMV infection.

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 (21 percent) patients using three different commercially available assays [47]. The case patients had clinical findings suggestive of acute mononucleosis and laboratory evidence of CMV seroconversion or CMV-specific IgM; diagnostic testing for Epstein-Barr virus and human herpesvirus 6 were negative.

Critically ill patients — CMV infection occurs in immunocompetent adults with critical illness and usually appears to be due to reactivation of latent infection rather than primary infection. The rate of CMV infection in this patient population ranges from <1 to 35 percent [48].

●A study monitoring reactivation of CMV by measuring CMV load in plasma showed that CMV reactivation occurred in 33 percent of patients and was associated with prolonged hospitalization or death [49]. Another study showed that reactivation of CMV infection in critically ill patients is associated with an increase in all-cause mortality and increased hospital and/or intensive care unit (ICU) length of stay, rates of nosocomial infection, and duration of mechanical ventilation [48].

●Another study of 97 immunocompetent CMV-seropositive adults with new-onset sepsis showed that mortality rates were similar in those with and without CMV reactivation, but CMV reactivation was independently associated with increased ICU and hospital length of stay and prolonged mechanical ventilation [50].

●Not all studies support these findings. A retrospective study of 1504 immunocompetent critically ill patients with an ICU stay of greater than three days did not show an association between CMV serostatus and mortality or time to discharge [51].

Studies that have monitored patients for CMV reactivation have primarily used PCR tests to quantify CMV DNA in plasma, whole blood, or respiratory secretions. It is unclear what threshold viral load predicts adverse outcomes, and there is no evidence that treatment of such patients alters outcome. Further studies are needed to address these important clinical questions.

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 AND RECOMMENDATIONS

●Background − Cytomegalovirus (CMV) is a common infection, and, although serious disease is rare in immunocompetent individuals, CMV is a major pathogen for immunocompromised patients, including solid organ transplant recipients, hematopoietic cell transplant recipients, HIV-infected patients, and patients treated with immunomodulating drugs. The range of clinical disease due to CMV in immunocompromised patients is broad and includes febrile syndromes, hepatitis, pneumonitis, retinitis, encephalitis, esophagitis, and colitis. Since the signs and symptoms of CMV disease often overlap with other infectious processes and rejection, the diagnosis is made by integrating the clinical history, clinical presentation, and laboratory data. (See 'Introduction' above.)

●Types of diagnostic tests − Diagnostic modalities include serology, qualitative and quantitative polymerase chain reaction (PCR), pp65 antigenemia, culture, and histopathology (table 1).

●Tests for detecting viral replication − Quantitative PCR tests and the CMV pp65 antigenemia test are available for detecting viral replication.

•Quantitative PCR assays offer several advantages over the antigenemia assay, including better assay standardization, increased stability of the specimen, smaller specimen volume, and the ability to test patients with leukopenia. For these reasons, quantitative PCR assays are more widely used than the antigenemia test, and we prefer them for the diagnosis and monitoring of immunocompromised patients with CMV infection and disease. Quantitative PCR tests should be calibrated to the World Health Organization International standard. (See 'Approach to diagnosis' above and 'Choice of test' above.)

•Serologic tests have no role in diagnosing CMV disease in immunocompromised patients; they are used pretransplant to establish serostatus. (See 'Serology' above.)

●Magnitude of the viral load − Studies of solid organ transplant recipients using quantitative PCR assays have shown higher viral loads in patients with active CMV disease compared with those with asymptomatic infection. The rate of increase in viral load can be used to predict patients at risk for CMV disease. Patients with CMV disease have a significantly faster rate of increase in CMV load between the last PCR-negative and first PCR-positive sample than those without CMV disease. (See 'Approach to diagnosis' above.)

●Diagnosis of tissue-invasive disease − For the diagnosis of CMV tissue-invasive disease, we recommend histopathology and CMV-specific immunostaining of tissue biopsies. Individuals with suspected CMV tissue-invasive disease should also have plasma or whole-blood PCR testing, since PCR results are often available prior to the biopsy results and may influence the decision to initiate antiviral therapy. In addition, it is important to establish the baseline viral load because serial testing is used to monitor the response to therapy. Detailed recommendations for testing based upon the site of infection are presented above. (See 'Tissue-invasive disease' above.)

●Viral load monitoring − In patients with CMV infection or disease, viral load should be monitored weekly using the same assay and the same specimen type (whole blood or plasma). (See 'Monitoring response to treatment' above.)

●Resistance testing − Patients who have a persistently elevated viral load, a rising viral load while on appropriate therapy, or a rebound in viral load after an initial response should be evaluated for drug resistance. Resistance testing is performed using automated sequencing methods directly from clinical specimens. (See 'Resistance testing' above.)

●Role of serology − Diagnosis of primary CMV infection in immunocompetent individuals is usually based on serologic studies; the detection of CMV-specific immunoglobulin (Ig)M or a fourfold rise in CMV-specific IgG provide a presumptive diagnosis. (See 'Immunocompetent hosts' above.)

●Diagnosis of primary CMV infection in immunocompetent patients − CMV infection occurs in immunocompetent adults with critical illness and is typically due to reactivation of latent infection rather than primary infection. Some, but not all, studies have shown that CMV reactivation (as measured by CMV viral load) is associated with prolonged hospitalization and death. (See 'Critically ill patients' above.)