Overview of Laboratory Tests for Cytomegalovirus

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

  1. https://www.uptodate.com/contents/overview-of-diagnostic-tests-for-cytomegalovirus-infection#H104411749
  2. https://www.uptodate.com/contents/congenital-cytomegalovirus-infection-clinical-features-and-diagnosis?topicRef=8305&source=related_link#H9542666
  3. 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.
  4. 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.
  5. 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.

2019 Call for Abstracts

The Abstract Submission site is open for ASCP 2019. Last year, ASCP had a record number of submissions and we aim to bring in even more this year. ASCP’s selection process is known as highly competitive, and as a result, presenters receive close attention from big-name faculty and industry contacts. Some have even gained immediate funding opportunities to expand their research.

Abstracts can be submitted until 11:59pm CST on March 20th. Submit your abstract HERE.

Call for Abstracts 2019

History of Generations: Baby Boomers

One of most well-known names of generations, besides perhaps Millennials, are the Baby Boomers. The Baby Boomers generation is currently the largest generation. Of all the generations, they cover the largest span of time (those born from 1946 to 1964). In large parts of the world, there was a big surge in births after the Second World War. That war had a significant influence on their values, perceptions, attitude, and approach to work.

One of the major aspects that make Baby Boomers stand out from previous generations is that this was really the first generation in which women started to work outside the home in large numbers, at least in the Western Hemisphere. This has a major influence on the home and work environment. In the United States, the children of Baby Boomers often had a latchkey around their neck so that they could go home after school without their parents being there.

Baby Boomers played a large role in shaping today’s society; they used music as a political tool, they increased focus environmental conservation, they were involved with the civil rights and women’s rights movements, and they are politically informed and outspoken. It is also the first generation in which both divorce and homosexuality became accepted. Overall, this generation is known for optimism, adaptability, having a strong work ethic, and being team-oriented.

Even though technology did not become part of daily life until Generation X, Baby Boomers witnessed enormous technological milestones, such as the first orbit around earth, landing a man on the moon, and the creation of the first nuclear power plant. All these events set the stage for later advances, and Baby Boomers are typically interested in learning how to use technology, although it does not come as natural to them as future generations. They also have tend to work longer and retire later in age, mainly because they link their self-worth to their job. In other words, their work ethic becomes their “worth ethic.” Knowing this when working with them is important, as they appreciate recognition in forms of awards, title changes, and public acknowledgement for their contributions.

Because this generation spans such a long time (and because some Boomers had children later in life due to second and third marriages), Baby Boomers are parents to both Generation X and Generation Y.  There is a lot to learn from this generation, so next time you work with one ask for some of their insights and understanding. This generation makes great mentors, especially because they are likely to have children of mentee age.

 

lotte-small

-Lotte Mulder earned her Master’s of Education from the Harvard Graduate School of Education in 2013, where she focused on Leadership and Group Development. She’s currently working toward a PhD in Organizational Leadership. At ASCP, Lotte designs and facilitates the ASCP Leadership Institute, an online leadership certificate program. She has also built ASCP’s first patient ambassador program, called Patient Champions, which leverages patient stories as they relate to the value of the lab.

 


 

Here is an interesting fact: there are two sub-sets of Baby Boomers. The first ones are the “Save-The-World Revolutionaries” of the ’60s and ’70s. The second set of Boomers are the career climbers, the yuppies, of the ’70s/’80s. The most profound characteristic of a Baby Boomer is their work ethic. They identify with their job, profession, or their career. So much so, that this generation has remained in the workforce beyond the age of 70.

In a lot of ways, I’m the typical Baby Boomer woman. I married the first time just before I was 20 years old. Divorced in my early 30’s and moved forward in my career because that’s what the “Boomer Women” did. They worked inside and outside of the home.

As a laboratory professional that left the bench many decades ago, and now working in the field of Organizational Leadership and Development, I am approaching the age of 70. I’m starting to realize my retirement day is closer than I’d like.    Like others of my generation, this concerns me because I am defined by my career! The thought of not working left me searching for my identity so much that I started seeing a therapist last year. I was, and am fortunate to work for an incredible organization that doesn’t judge one by their age. They look at the skills and competencies one brings to the table. I’m consciously working on succession planning so that my institutional knowledge remains with the organization and its people. It also helps to have two gifted professionals who wanted to learn from me and grow. Then it takes a manager like mine who supports me through this often painful process. I am blessed with that kind of support. Sometimes the work ethic equals “worth ethic” in the body, mind, and spirit of a Baby Boomer, which is something to keep in mind when working with this generation.

 

 

Stakenas-small

-Catherine Stakenas, MA, is the Senior Director of Organizational Leadership and Development and Performance Management at ASCP. She is certified in the use and interpretation of 28 self-assessment instruments and has designed and taught masters and doctoral level students.