Introduction
Cytomegalovirus (CMV) is considered the most important pathogen in transplant recipient patients as it can cause significant morbidity and mortality. Anti-CMV treatments have proven to be effective but are not without adverse side effects. Thus, there is a strong need for sensitive and reliable tests to diagnose and monitor active CMV infection. Several testing methodologies are available in today’s clinical laboratories to evaluate a patient’s CMV status: viral culture, serology, histopathology, pp65 antigenemia, and quantitative PCR. In this post, we will review the advantages and limitations of these tests.
Viral culture
Viral culture is performed most commonly by the shell vial assay (also known as rapid culture), in which a cell line (usually human fibroblast cells) is inoculated with patient sample by centrifugation. The virus is then detected by either direct or indirect fluorescent monoclonal antibody, providing results within 1-3 days. The centrifugation step greatly improves turnaround time when compared to traditional tube cell culture technique, which may take 2-3 weeks before a result can be reported as negative.
Culturing CMV has been largely replaced by newer methodologies like quantitative PCR and CMV antigenemia. This is due to relatively weaker sensitivity for diagnosing CMV infection compared to newer tests, as well as slower turnaround time. Viral cultures of urine, oral secretions, and stool are not recommended due to poor specificity; however, for diagnosis of congenital CMV, viral culture of urine or saliva samples is an acceptable alternative if PCR is not available.
Serology
CMV serostatus is an important metric to evaluate prior to patients receiving a hematopoietic or solid organ transplant. Serologic testing is done primarily via enzyme immunoassays and indirect immunofluorescence assays. These tests check for presence of anti-CMV immunoglobulin (Ig)M and IgG to provide evidence of recent or past infection. Outside of establishing serostatus (primarily in organ donors and recipients), serologic testing for CMV is not recommended in diagnosing or monitoring active CMV infection.
CMV IgM antibodies can be detected within the first two weeks of symptom development and can be present for another 4-6 months. IgG antibodies can be detected 2-3 weeks after symptoms develop, and remain present lifelong. These antibody measurements are particularly useful in determining risk of CMV acquisition in seronegative patients (negative for IgM and IgG) at time of transplantation. IgG titers can also be measured every 2-4 weeks to assess for CMV reactivation in seropositive patients. Since CMV IgG persistently remains in circulation, testing for it has a higher specificity compared to IgM, and thus is the preferred immunoglobulin to test for in establishing serostatus. Serologic tests can be falsely positive if patients have recently received IVIG or blood products, so testing on pretransfusion samples are preferred if possible.
Histopathology
Under the microscope, cells infected with CMV can express certain viral cytopathic effects. These infected cells classically show cytoplasmic and nuclear inclusions (owl eye nuclei) with cytoplasmic and nuclear enlargement. Additionally, immunohistochemistry (IHC) can stain antibodies specifically for CMV proteins to highlight infected cells, making histologic examination quicker and improving diagnostic sensitivity.
Histopathology can be useful in identifying tissue-invasive disease, such as CMV colitis or pneumonitis. Cases in which PCR testing is negative does not necessarily exclude tissue-invasive disease; thus, the diagnosis of tissue-invasive disease relies on histologic examination (with or without IHC) or possibly viral culture. On the other hand, a negative histologic result does not exclude tissue-invasive disease, possibly due to inadequate sampling, and shows the potential for weak diagnostic sensitivity.
pp65 antigenemia
CMV antigenemia testing uses indirect immunofluorescence to identify pp65 antigen, a CMV-specific matrix protein, in peripheral blood polymorphonuclear leukocytes. Whole blood specimens are lysed and then the leukocytes are cytocentrifuged onto a glass slide. Monoclonal antibodies to pp65 are applied, followed by a secondary FITC-labeled antibody. The slide is then read using a fluorescence microscope for homogenous yellow-green polylobate nuclear staining, indicating presence of CMV antigen-positive leukocytes. Studies have suggested that a higher number of positive cells correlates with an increased risk of developing active disease. The sensitivity of antigenemia testing is higher than that of viral culture and offers a turnaround time within several hours.
This test has been utilized since the 1980s, but has seen less use recently due to the increasing popularity of quantitative PCR. Antigenemia testing is labor intensive, and requires experienced and trained personnel to interpret the results (which can be somewhat subjective). This test also must be performed on whole blood specimens within 6-8 hours of collection due to decreasing sensitivity over time, which limits transportability of specimens. Additionally, It is not recommended to be run on patients with absolute neutrophil counts below 1000/mm3, due to decreased sensitivity. Despite these limitations, CMV antigenemia testing is still considered a viable choice for diagnosing and monitoring CMV infection, especially when viral load testing is not available.
Quantitative PCR
Quantitative real-team polymerase chain reaction (PCR) is the most commonly used method to monitor patients at risk for CMV disease and response to therapy, as well as for diagnosing active CMV infection. The advantages of using a quantitative PCR assay include increased sensitivity over antigenemia testing, quick turnaround time, flexibility of using whole blood or plasma specimens for up to 3-4 days at room temperature, and the availability of an international reference standard published by the World Health Organization (WHO).
Several assays from Roche, Abbott, and Qiagen are available and FDA-approved. The targets of these assays vary, with some targeting polymerase and other targeting CMV major immediate early gene. These assays are all calibrated with the WHO international standard, which was developed in 2010 to help standardize viral load values among different labs when results are reported in international units/mL. The goal of this international standard is to decrease the interlaboratory variability of viral load, and determine the appropriate cut-offs for determining clinical CMV disease. There is still improvement to be made in this area, as variability still exists between labs.
Conclusion
There are several tests to determine the CMV status of patients. Some of these tests are suited for particular goals, such as serology for determining serostatus prior to organ transplantation, or histology and IHC to diagnose tissue-specific CMV disease. For diagnosis and monitoring of general CMV disease, the test of choice in most laboratories is quantitative PCR, which offers automated, quick and sensitive results. Antigenemia, while dated and labor intensive, is still an acceptable alternative when PCR is neither available nor cost-effective for smaller labs. Both of these testing methods are preferred over viral culture, which has poorer diagnostic sensitivity and relatively longer turnaround time.
Despite the numerous advantages quantitative PCR has, there is still variability in viral load counts among laboratories. This is due to varying DNA extraction techniques, gene targets used by PCR, and specimen types used. There is still a lot of work to be done in standardizing testing in regards to not just CMV, but also other viral pathogens like Epstein-Barr virus, BK virus, adenovirus and HHV6. Updated standards and increased use of standardized assays will hopefully decrease this variability between labs to improve testing results and in turn, improve patient care.
References
- https://www.uptodate.com/contents/overview-of-diagnostic-tests-for-cytomegalovirus-infection#H104411749
- https://www.uptodate.com/contents/congenital-cytomegalovirus-infection-clinical-features-and-diagnosis?topicRef=8305&source=related_link#H9542666
- Kotton CN, Kumar D, Caliendo AM, et al. Updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation. 2013;96(4):333-60.
- Hayden RT, Sun Y, Tang L, et al. Progress in Quantitative Viral Load Testing: Variability and Impact of the WHO Quantitative International Standards. J Clin Microbiol. 2017;55(2):423-430.
- Kotton CN, Kumar D, Caliendo AM, et al. The Third International Consensus Guidelines on the Management of Cytomegalovirus in Solid-organ Transplantation. Transplantation. 2018;102(6):900-931.
-David Joseph, MD is a 2nd year anatomic and clinical pathology resident at Houston Methodist Hospital in Houston, TX. He is planning on pursuing a fellowship in forensic pathology after completing residency. His interests outside of work include photography, playing bass guitar and video games, making (and eating) homemade ice cream, and biking.
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