HCV, CMV viral load—treatment, testing, timing

On the other hand, whether to treat is a complicated decision, and the use of IL28B genotyping is evolving. “For a patient who has failed treatment or who may be a borderline candidate for treatment due to factors like state of liver decompensation, IL28B genotyping might help in the decision to treat, especially given the many emerging therapeutic options that are around the corner,” Dr. Hillyard says. IL28B testing is being included in clinical trials of new drugs, he adds, and it may well be integral to future testing algorithms.

CMV infection can present with a spectrum of disease in those with a compromised immune system, Dr. Mehta said in the AMP session. In HIV-positive patients, it may manifest as retinitis, polyradiculopathy, dementia, colitis, or cholangitis. In immunosuppressed transplant recipients, it can cause “CMV syndrome”—unexplained fever lasting more than 48 hours, malaise, and a decreased neutrophil count. It can also cause CMV tissue disease: pneumonitis, hepatitis, colitis, or encephalitis. In either situation, the risk of allograft rejection rises, as does the risk of bacterial or fungal infections or EBV post-transplant lymphoproliferative disorder (PTLD) (Fishman J. N Engl J Med. 2007;357:2601–2614).

Transplant programs employ a variety of strategies to prevent CMV infection. Universal prophylaxis requires giving antivirus therapy to all at-risk patients in the early post-transplant period. In preemptive therapy, patients are monitored at regular intervals—for instance weekly—and a rising viral load is treated to prevent symptomatic disease. “Resistance has been observed with both strategies,” Dr. Mehta said, later adding in an interview: “There are no large studies on this question so far, although some are ongoing. Choosing a strategy is part of the art of medicine and local culture.”

In the Emory transplant programs, clinicians employ what Dr. Mehta calls “a mixed bag of strategies,” which differ by organ and physician culture. Recipients of a donor-positive liver are monitored weekly for three months, then biweekly for three months. Universal prophylaxis is used for three or six months in heart, lung, and kidney transplants.

For diagnosis of CMV, PCR is the most widely used method, Dr. Mehta says. He uses an in-house PCR based on commercial primers and probes. Quantitative PCR has become widespread at transplant centers, he notes, though correlation of absolute value to disease state is difficult. Only one group has attempted to establish an ROC curve relating viral load to development of disease (Humar A, et al. Transplantation. 1999;68:1305–1311). In this study, viral load greater than 2,000 copies/mL had 91 percent sensitivity and 75 percent specificity for predicting clinical illness, with a positive predictive value of 50 percent and a negative predictive value of 99.6 percent. “In reality, there is considerably more heterogeneity in transplant patients than in that clinical trial,” Dr. Mehta says. Cutoffs should be different for different organs, in his view: The lower the immunosuppressive regimen, the higher the treatment threshold. “Different organ transplants receive different degrees of immunosuppression,” Dr. Mehta said. His laboratory has converted to the WHO standard for expressing viral load. In this system, the 2,000 copies/mL in the Humar study equals about 500 IU/mL, which is what they use as a rough cutoff.

Kinetics of increase of CMV DNA is also important. Initial viral load detected and rate of viral load increase are independent risk factors for disease, Dr. Mehta said. “The combination of initial viral load and rate of change can identify patients at imminent risk of CMV disease.” During therapy, viral load should be monitored every one to two weeks; an undetectable viral load is the goal of treatment. In one study, all 16 non-relapsing patients had an undetectable viral load after therapy (Sia IG, et al. JID. 2000;181:717–720).

“CMV resistance remains a major problem in all forms of transplantation,” Dr. Mehta said. “Fortunately, it is not that frequent,” he added later, even though anti-CMV drugs are used as monotherapy. A typical patient in whom to suspect resistance is one who had an initial good response to therapy but then starts to exhibit an increasing viral load. To detect resistance, phenotypic (plaque reduction) assays are available, but genotypic (sequencing) assays are more commonly used. Dr. Mehta sends out samples for genotypic testing. To interpret sequence results, an understanding of the genetics of resistance is necessary (Lurain NS, Chou S. Clin Microbiol Rev. 2010;23:689–712).

Testing cell-mediated immunity may help to select patients who no longer need monotherapy (Kumar D, et al. Am J Transplant. 2009;9:1214–1222). “The data don’t give us a clear indication yet” for how useful such tests will be, Dr. Mehta says, but there is high clinical interest and ongoing research.

Dr. Arcenas, in an interview, said one of the caveats in laboratory testing for CMV is to distinguish true active infection from latent virus. “On the anatomic pathology side, we can suspect CMV lung infection from an immuno­stain on tissue, and try to correlate it with a bronchoalveolar lavage sample, then do PCR.” Sometimes there is a question of whether CMV is the primary infection. “In that case we can do studies to rule out other viral causes,” he says. “We may tell our doctors that the test is detecting CMV DNA but we can’t tell whether it is an active or latent infection.”

Sequential monitoring for viral load is important to detect true increases. “Sometimes we get low level ‘blips,’” Dr. Arcenas says. “The next week virus may be undetectable. So we need to watch it over time.”

Since many laboratories, including Dr. Arcenas’, have developed their own in-house PCR assay, the WHO international standard is valuable. “Not all LDTs [lab-developed tests] are equal,” he says. Having a standard allows patients to get testing at another laboratory and still get the benefit of serial test results. He is now making the conversion from copies/mL to reporting out IU/mL.

Quantitative PCR is preferable for monitoring viral load in plasma, in Dr. Arcenas’ view, since undetectable viremia is the crucial factor. “With other sample types,” he says, “qualitative PCR is acceptable as long as the results support the clinical diagnosis.” These would include urine, as well as white blood cells. “It is kind of up in the air as to what a viral load means in lymphocytes,” he says. “And just the presence of the CMV in retinitis is important.”

Laboratorians should be ready to address the question of possible resistance. “We had a couple of cases where the doctor called us because the viral load wasn’t going down or wasn’t negative,” Dr. Arcenas says. “They asked, ‘Is my patient developing resistance against ganciclovir?’ We don’t do resistance testing in-house. We send out.” When a physician feels that a patient isn’t responding as they should, Dr. Arcenas, in consultation with the clinician, may recommend changing therapy or sending out for resistance testing.

“There is no FDA-approved test for detecting resistance in CMV,” Dr. Hillyard said in an interview. “Most often resistance testing is performed by Sanger sequencing following amplification of the two genes where resistance can occur.” These are UL97, a phosphotransferase gene with associated ganciclovir resistance mutations, and UL54, the viral DNA polymerase gene, with mutations conferring resistance to ganciclovir, cidofovir, and foscarnet.

Dr. Hillyard enumerated several challenges in resistance testing for CMV. “From the analytical perspective, patients failing therapy sometimes have relatively low levels of CMV,” he says. “So we need to have reasonably sensitive resistance assays. Also, minor populations of resistant virus sometimes need to be characterized against a background of wild-type virus.” Sanger sequencing struggles when resistant virus is less than 20 percent of the major population, he says. “We can use pyrosequencing to get down to the five percent level, but other issues make its use problematic.”

One of the most critical problems, Dr. Hillyard says, is distinguishing between polymorphisms and resistance variants. A growing number of mutations in UL54 and UL97 genes are being associated with resistance. However, new polymorphisms not associated with significant resistance are also showing up. “An exciting way forward in this field is the use of genetic recombineering technology, in which specific DNA base changes can be quickly introduced into well-characterized drug-sensitive strains. This provides a much clearer assignment of which mutations cause resistance and which are irrelevant polymorphisms.” Dr. Hillyard sees a big need for well-curated databases of CMV resistance mutations that have been validated in this fashion.

William Check is a writer in Ft. Lauderdale, Fla.