Expires: May 13, 2025
Matthew Binnicker, Ph.D., D(ABMM), is the Director of Clinical Virology and Vice Chair of Practice in the Department of Laboratory Medicine and Pathology at Mayo Clinic. He holds the academic rank of Associate Professor of Laboratory Medicine and Pathology.
Hi, I’m Bobbi Pritt, Director of the Clinical Parasitology Lab and Vice Chair of Education in the Department of Laboratory Medicine and Pathology at Mayo Clinic. Cytomegalovirus, or CMV, is a common cause of disease in the transplant population. In some patients who are diagnosed with CMV and are on antiviral treatment for infections, the virus may develop resistance to the drugs. In this “Hot Topic,” my colleague, Dr. Matthew Binnicker, will provide an overview of a new test developed by Mayo Clinic Laboratories, which uses next-generation sequencing (NGS) technology to identify mutations in the genome of CMV that may be associated with antiviral resistance. I hope you enjoy this month’s Hot Topic, and I want to personally thank you for allowing Mayo Clinic the opportunity to be a partner in your patients' health care.
Thanks for the introduction, and thanks for joining me for this Hot Topic on antiviral resistance testing for cytomegalovirus in the transplant population.
Before we begin, I should mention that I don’t have any financial disclosures related to the information that will be presented.
So with that, let’s get started.
CMV is a herpesvirus that is a common cause of disease in the transplant population. Many people in the population have been exposed to CMV, so there is a high seroprevalence. Because it is a member of the Herpesviridae family, cytomegalovirus can cause acute disease, but then also undergo latency and potentially cause reactivated infection if an individual becomes immune-suppressed.
Because of this ability to cause latent infection, CMV is one of the most common complications affecting transplant recipients.
Whether CMV will cause disease or not in a transplant recipient depends on a variety of factors, including the type of transplant—for example, whether a patient underwent a stem cell transplant or a solid organ transplant. Another important factor includes the serostatus of the recipient and donor, with the greatest risk of disease typically being in situations where the donor is seropositive for CMV and the recipient is seronegative, otherwise known as a D+/R- serostatus. And finally, the degree of immunosuppression can influence whether a patient is more or less likely to develop a disease related to CMV infection.
Although many patients who are infected with CMV will remain asymptomatic or have a subclinical infection, there are a number of potential clinical manifestations in the transplant population. One of the more common manifestations is known as CMV syndrome, which is categorized by patients experiencing a fever of unknown origin for greater than two days, accompanied by malaise and a decreased neutrophil count. In addition to this nonspecific syndrome, CMV can also cause a range of other illnesses in transplant recipients, including pneumonitis, hepatitis, gastrointestinal disease, and central nervous system disease.
When CMV disease is suspected based on a patient’s clinical presentation, the diagnosis can be supported by a number of laboratory tests. In recent years, molecular detection of CMV nucleic acid in clinical specimens (e.g., using real-time PCR) has become a common approach. In addition to qualitative detection of the virus in various clinical samples, like tissue or respiratory specimens, quantitative viral-load testing is also recommended in the transplant population, with an increasing viral load in serially collected plasma samples being an indicator of active infection. Another indication of tissue-invasive disease is the presence of “owl’s-eye” viral inclusions in histopathology.
The diagnosis of CMV disease generally requires the documented detection or identification of CMV in clinical specimens accompanied by clinical manifestations that are consistent with this virus.
If a transplant recipient is diagnosed with CMV disease, a typical first-line treatment is to reduce the patient’s immunosuppressive medications, in the hopes that the immune system will manage the infection. Also, there are a number of antiviral therapies available, with ganciclovir often being used initially to treat CMV disease. Unfortunately, as more and more patients receive antiviral therapy, resistance may occur, with an estimated 1‒5% of transplant recipients being infected with a viral strain that is resistant, or develops resistance, to commonly used antivirals. The vast majority of resistance results from specific mutations in two genes in CMV. The most common mutations occur in the gene UL97 and can confer resistance to ganciclovir. In rare situations, mutations may arise in the gene UL54, and when this happens, there is a greater risk for cross-resistance to multiple antiviral drugs, including ganciclovir, cidofovir, and foscarnet.
When should you suspect CMV antiviral resistance? It should typically be considered when there is evidence of rising or persistently elevated CMV viral loads, despite a patient being on appropriate therapy for at least two weeks. To help illustrate this, let’s look at an example case. In this situation, we have a patient who presents eight weeks after his transplant with fever, myalgias, and a decreased neutrophil count. A CMV viral load is ordered and reported as elevated at approximately 20,000 international units per mL (IU/mL). This is highly suspicious for CMV disease, but to confirm, a repeat viral load is performed one week later and is now over 100,000 IU/mL. Ganciclovir therapy is initiated, and the CMV viral load shows a nice response to therapy over the next four weeks. The clinical team then decides to back off to every other week for viral load monitoring, and for the next month, things seem pretty stable. But because of a slight uptick in the viral load at week 10, the team decides to watch this more closely, and sure enough, there is an appreciable increase in the viral load over the next two weeks. This would definitely raise concern for the emergence of antiviral resistance in this patient.
In patients for whom there is concern for antiviral resistance, there have historically been a few different laboratory options. Years ago, phenotypic testing could be performed, in which patient specimens were inoculated into viral culture in the presence and absence of an antiviral drug. This method has a prolonged turnaround time, and because of this, is no longer used in routine clinical practice. More recently, Sanger sequencing has been the go-to approach for prediction of antiviral resistance. This approach utilizes conventional sequencing of short regions, typically 300‒900 base pairs, of the UL97 and UL54 genes of CMV. A limitation of Sanger sequencing is that the sequence quality typically drops after about 700 base pairs, so it may not be able to accurately sequence long stretches of DNA. Also, it has limited ability to accurately differentiate between mixed populations of sequence (e.g., if there are both wild type and mutant strains of a virus in the same specimen).
To address these limitations, Mayo Clinic Laboratories has developed a new approach, based on NGS technology, for detection of mutations in UL97 and UL54 that are associated with antiviral resistance. In this test, plasma is extracted to obtain purified nucleic acid, which is then subjected to PCR using specific primers for the CMV genes UL97 and UL54. After the PCR has amplified the entire UL97 and UL54 genes, the amplicons are mixed and sequenced on an NGS platform, the sequence data are subsequently analyzed, and a report with resistance and susceptibility predictions to ganciclovir, cidofovir, and foscarnet is generated.
This novel assay was implemented at Mayo Clinic Laboratories on May 2, 2019, and requires a plasma sample with a CMV viral load of at least 500 IU/mL. During validation of this assay, it was demonstrated that the NGS-based assay showed 97.7% overall agreement with traditional Sanger sequencing and could identify resistance-associated mutations at a prevalence threshold of at least 15%.
In summary, CMV is an important cause of disease in transplant patients, and as laboratorians and providers we should be aware that resistance to antivirals can occur, typically resulting from specific mutations in the UL97 and/or UL54 genes of CMV. Mayo Clinic Laboratories has fully validated and is now offering a novel NGS assay for the detection of mutations associated with antiviral resistance.
I’d like to finish up this presentation by referring you to several published articles related to this topic that you may find beneficial. Thanks again for joining me for this Hot Topic on antiviral resistance testing for CMV.
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