College of American Pathologists
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  Viral virtuoso—RT-PCR as first-line test


CAP Today




April 2008
Cover Story

William Check, PhD

Nucleic acid amplification-based techniques have given rise to many clinically useful tests since the polymerase chain reaction was adapted for clinical laboratory use in the early 1990s. An important new application of PCR technology has been developed and expanded over the past several years, as quantitative assays for several pathogenic viruses—hepatitis B and C viruses, cytomegalovirus, Epstein-Barr virus, and the polyoma virus BK—have been introduced into the molecular pathology laboratory.

“There is definitely a trend toward real-time PCR replacing culture-based assays, and in some cases serology as well, for viruses,” says Margaret L. Gulley, MD, director of molecular pathology and professor of pathology and laboratory medicine at the University of North Carolina-Chapel Hill School of Medicine. “For many viruses real-time PCR is the first-line test, and we can use the same instruments for all viruses, only with different probes and primers.”

Karen Weck, MD, associate professor of pathology and laboratory medicine and director of molecular genetics at the University of North Carolina-Chapel Hill, groups these viruses into two categories. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and polyoma virus BK (BKV) are ubiquitous in the population and establish latent, persistent infection, she says. “Many people can be infected and have low-level viral DNA without it being clinically relevant. That is why you need quantitative assays to look at the viral load.” Latent infection with these viruses is usually without clinical consequences until a person becomes immunosuppressed, such as after transplantation. Then in a small fraction of patients the virus is reactivated. For latent viruses that are common in the general population, the purpose of quantitative monitoring is to determine when viral load rises to a dangerous level. “You will never achieve viral negativity for these viruses,” Dr. Weck says, “and it is not necessary for patient health.” For the other viruses—HIV, hepatitis C, and hepatitis B, where any amount of virus puts patients at risk for disease, qualitative PCR tests can be used for diagnosis and quantitative monitoring is used to follow the response to therapy.

Clinical viral load was widely measured first for human immunodeficiency virus, and it is from this precedent that more-recent quantitative clinical virus assays derive. “Insights from HIV are setting a trend in other viruses,” says Alexandra Valsamakis, MD, PhD, director of clinical virology and molecular microbiology, Division of Medical Microbiology, and associate professor of pathology, Johns Hopkins Medical Institutions. “HIV set the paradigm that has been tested, applied, and modified for other viruses. It is definitely true that if you build it, they will come,” Dr. Valsamakis has found. “Every time we expand our quantitative panel, the volumes grow. Quantitative EBV and BK virus testing are good examples.” In her laboratory requests for quantitative EBV measurement have grown from 100 in 2003 to more than 900 in 2007, while quantitative BKV testing volume has doubled from less than 300 in 2005 to more than 600 in 2007.

David R. Hillyard, MD, medical director of the Department of Molecular Pathology at ARUP Laboratories, says molecular virus quantification as a field was “jump-started by the need to do targets like HIV and HCV.

“With that experience people looked at other situations, such as CMV for transplantation patients, then expanded to BK virus and others. Where we are now,” Dr. Hillyard says, “is that there is a set of clinical conditions where quantification of certain viruses is absolutely required, as opposed to just qualitative detection. And that spectrum of viruses continues to increase.”

For quantitative virus measurement, Dr. Hillyard says, almost all labs today use some type of real-time PCR, which is intrinsically quantitative. The difference between qualitative and quantitative often depends on whether a standard curve has been generated. “As we run qualitative tests,” he says, “we are running uncalibrated quantitative tests. It is certainly a greater burden to support a quantitative test, but quantitative information is sitting there in every real-time assay.” Dr. Hillyard raises the possibility that people may start capturing quantitative information for viruses that are now being measured qualitatively to see if it is clinically important. This would accelerate the use of quantitative testing.

Dr. Gulley’s favorite technology in the clinical lab is real-time PCR. “It allows us not only to detect a pathogen,” she says, “but also to measure the amount. In the case of EBV, because the pathogen is within a tumor, it allows us to measure tumor burden.” Dr. Gulley also emphasizes the extra work entailed in bringing a real-time PCR assay into the laboratory. Increasingly, automated kits are becoming available, though there is no FDA-approved assay for EBV, she says. “In-house developed or ASR [analyte-specific reagent] assays being used in clinical labs work well—they are rapid, sensitive, specific, and precise—but one problem with not having kits is a lack of standardization among labs in terms of units of reporting or even the sample type on which testing is done.”

More broadly, Dr. Gulley emphasizes her concern about the lack of support for analytic and clinical validation studies for in-house–developed and ASR assays. “Government needs to recognize that both analytic and clinical validation of lab tests requires substantial investment to do it well,” she says. Ana­lytic validation studies would optimize assay design, ensure sensitivity and specificity of assays, and address preanalytic factors such as sample collection, handling, and storage—all things that a manufacturer would do for an FDA-cleared kit.

“CLIA requires that all those analytic and preanalytic issues be addressed,” Dr. Gulley says, “and the CAP accreditation process and CLIA inspection help assure the public that clinical labs are performing well. Especially helpful are proficiency surveys.”

Clinical validation addresses indications for testing and how physicians would manage patients differently with assay results. For EBV, many studies have been published on the utility of viral load assays, but most are small, Dr. Gulley says. “We would like to find a source of funds for large prospective trials to show that optimized EBV viral load assays improve health.” One possibility is to work within existing cancer cooperative trials groups. “These groups should be encouraged to include lab correlation studies with every clinical trial,” Dr. Gulley suggests, “so that specimens will be made available for this type of analytic and clinical work.”

Viral load testing for CMV, EBV, and BKV suffers from a lack of standardization for quantitative testing. Most labs use commercial assay methods for HIV and HCV. Dr. Weck and others presented a workshop at an Association for Molecular Pathology meeting two years ago with interlaboratory comparisons for BKV testing. “Among the five or six participating labs we found very poor absolute correlation, though there was very good agreement between labs for rising and lowering viral titers,” Dr. Weck says. Standard curves were parallel but displaced, an observation that applies to EBV and CMV as well. “I think it is okay where we are right now,” she says. “Clinically, serial monitoring over time is more important than looking at a single isolated value.” She would like to see CAP Surveys, which now cover EBV and CMV, expanded to include BKV now that more laboratories are monitoring this virus. “I have had requests from maybe 10 labs in the last five years for my BKV method,” Dr. Weck says, “so it is not just being done by big labs.”

Indications for HIV and CMV viral load testing are well known. Applying quantitative testing to HBV is more recent, and the uses of quantitative monitoring for HCV have been expanded. Viral load monitoring for BKV and EBV, too, is a more recent innovation. Says Dr. Valsamakis, “Quantitative testing has rocked the world of therapeutic monitoring for hepatitis B virus.”

She points to a study from Taiwan published in 2006, “Risk of Hepatocellular Carcinoma Across a Biological Gradient of Serum Hepatitis B Virus DNA Level” (Chen CJ, et al. JAMA. 2006;295:65–73). The authors concluded: “Elevated serum HBV DNA level (=10,000 copies/mL) is a strong risk predictor of hepatocellular carcinoma independent of HBeAg, serum alanine aminotransferase level, and liver cirrhosis.” This finding was “revolutionary and, by inference,” Dr. Valsamakis says, “the best marker that a patient receiving therapy with antiviral agents such as nucleoside inhibitors will experience slowed progression of liver disease may well be a drop in HBV viral load. The lower the load and the longer it remains as low as possible, the slower these patients may progress.” Until recently, loss of hepatitis B e antigen was looked for—the antigen was thought to be a marker of active viral replication, and loss of e antigen suggested the virus had become inactive. “That may be true,” Dr. Valsamakis says, “but there is a population of e antigen-negative patients who have replicating virus and who progress to having significant inflammation and fibrosis. So having a single marker that can be followed over time in all patients has greatly simplified therapeutic monitoring in chronic HBV.”

To measure HBV viral load, most laboratories are using commercial ASR-based reagents, Dr. Valsamakis says. “There are currently no FDA-approved assays for HBV quantification,” she says, “though some manufacturers have data in front of FDA.”

Viral load testing for HCV is having a large impact clinically, Dr. Hillyard says. “Quantitative testing for HCV is used for one of the most important clinical algorithms in medicine—determining the likelihood that an individual is responding to therapy with pegylated interferon and ribavirin,” he says. “Patients who do not show a decrease in viral load in response to those drugs have a very low chance of responding to therapy. On the other hand, patients whose viral load drops, especially if it drops early in treatment, have a much better likelihood of responding. Quantitative HCV testing may also guide decisions to shorten or extend the duration of therapy.”

At the 2007 AMP meeting, Mitchell L. Shiffman, MD, gave a plenary talk on viral load monitoring in the management of patients with chronic HCV. In an interview with CAP TODAY, Dr. Shiffman, who is chief of the hepatology section and medical director of the liver transplant program at Virginia Commonwealth University Medical Center, described the utility of monitoring HCV viral load during therapy. “The sooner viral load becomes negative, the higher the cure rate,” says Dr. Shiffman, who has participated in much of the work on HCV treatment and monitoring. (In practice, “cure” means sustained viral response, defined as an undetectable serum HCV RNA level 24 weeks after the end of treatment.) If viremia disappears by week four, the patient has a 90 percent chance of cure. For loss of viremia at week 12, this figure drops to 66 percent, and for week 24 it is 45 percent, a trend that is independent of genotype.

Standard therapy with pegylated interferon (pegIFN) and ribavirin is 48 weeks for genotype one and 24 weeks for genotype two/three. For genotype two/three, shortening treatment to 16 weeks decreases the overall incidence of sustained viral response from 70 percent to 62 percent (Shiffman ML, et al. N Engl J Med. 2007;357:124– 134). “For patients who respond with an undetectable viral load by week four, treatment can be stopped at 24 weeks and the sustained viral response is still excellent,” Dr. Shiffman says. “For patients who are not negative by week four, and particularly those who still have measurable virus RNA at week 12, extending therapy decreases the relapse rate.” For genotype one patients, treatment is extended to 72 weeks; for those with genotype two/three, it is extended to 48 weeks. “So the beauty of viral load testing is that we can find out when the patient goes negative and adjust treatment length accordingly.”

In his talk, Dr. Shiffman noted the importance of HCV infection: In the U.S., about 4 million people are infected with the virus, which is the leading cause of chronic liver disease, cirrhosis, liver cancer, and liver transplantation. The number of new cases of HCV infection rose substantially in the late 1960s and early 1970s. As a result, even though the incidence and prevalence of HCV infection are decreasing, the prevalence of chronic infection is increasing and may continue to do so until it peaks in 2015.

Infection with HCV is diagnosed by a rise in the liver enzyme ALT. Infection becomes chronic in about 85 percent of cases; it can be confirmed by measuring HCV RNA. Early measurement of viral RNA is meaningless because all patients have intermittent viremia in the first one to two months. “Wait until ELISA becomes positive at one to two months,” Dr. Shiffman says, “then confirm with a diagnostic molecular RNA assay. In chronic disease, RNA increases after two months; if RNA is positive, begin treatment.” In cases that resolve, viremia is not seen at that time point.

Nor is viral load monitoring useful for assessing severity of disease or risk of progression. “Serum HCV RNA level remains stable for up to four years,” Dr. Shiffman noted. “So it does not signal severity of disease. You do not need to follow the viral load in patients not on treatment.”

The main utility of viral load testing is during treatment, Dr. Shiffman emphasized. Randomized trials established the efficacy of pegIFN alfa-2b and -2a plus ribavirin (Fried MW, et al. N Engl J Med. 2002;347:975–982; Manns MP, et al. Lancet. 2001;358:958–965). “Molecular diagnosis becomes incredibly useful in this situation,” he said. It can detect how soon viral load goes below the limit of detection and thus guide duration of therapy. If viral RNA is still positive at four weeks, treatment is extended for 48 or 72 weeks. Extended treatment increased the sustained viral response rate from 32 percent to 45 percent and decreased the relapse rate from 47 percent to 13 percent (Sanchez-Tapias JM, et al. Gastroenterology. 2006;131:451–460). If viral RNA persists at 24 weeks, however, extended therapy is futile, Dr. Shiffman said. “If HCV RNA is not negative by week 24, the chance of it becoming negative later is virtually zero. So you should monitor viral load early and stop treatment if it does not become negative.”

Since knowing a detailed viral load pattern greatly assists disease management, the following schedule of monitoring is recommended:

  • Genotype one: baseline, weeks 4, (8, 12), (12–36), 36, 48, 60, 72.
  • Genotype two/three: baseline, weeks 4 (8, 12, 16, 20), 24, 36.

In his summary, Dr. Shiffman reiterated three main points:

  1. The level of HCV RNA in untreated patients does not change over time or affect the severity of liver disease.
  2. The time at which a patient becomes HCV RNA undetectable during treatment is the most important predictor of sustained viral response.
  3. Monitoring HCV RNA frequently during treatment will enable you to determine which patients are capable of responding and how long these patients should be treated.

CMV is the HIV of latent viruses—it has seniority and its indications are familiar to most people. Most at risk are AIDS patients and immunosuppressed transplant patients, both solid organ and bone marrow transplant recipients.

More recently, about five percent of renal transplant patients have been found to develop BKV-associated nephropathy. “It is now standard of care to do PCR-based testing in renal transplantation patients to look for those who may be at risk for BKV-associated nephropathy,” says Dr. Weck, who took part in some of the seminal work on BKV monitoring (Randhawa P, et al. J Clin Micro. 2004;42:1176– 1180). “Many labs test BKV viral load either in blood or in urine in all patients receiving a renal transplant,” she continues. “We found a very good correlation between blood and urine viral load, although viral load in urine is two to three logs higher than in blood.”

An older cytopathologic test for acute nephropathy is microscopic examination of urine for decoy cells, which are sloughed off during kidney damage. “Decoy cells are still used in some institutions,” Dr. Weck says, “but most centers have replaced them with PCR, which is more specific.” However, the gold standard is still histology from a biopsy. “You can also do in situ hybridization to look for BKV transcripts,” Dr. Weck says. This technique requires biopsy material. “So the great utility of BKV PCR,” she concludes, “is to monitor patients noninvasively and identify those who need a biopsy. The disadvantage of biopsy is that there can be sampling error, so PCR can also be more sensitive.” In a situation in which viral load is positive and biopsy is negative, Dr. Weck says one option is to follow viral load and think about another biopsy. “In some centers,” she says, “PCR results would be indicative of BKV-associated nephropathy and clinicians would treat the disease.” As with EBV, the best treatment for it is to back off on immunosuppression. Some antiviral therapies have been used, especially cidofovir, but this drug has nephrotoxicity. “It is a hard choice to balance graft rejection against viral infection against a nephrotoxic antiviral therapy,” Dr. Weck says.

BKV has also been associated with hemorrhagic cystitis of the bladder in bone marrow transplant recipients. “We don’t monitor for hemorrhagic cystitis as much as for BKV-associated nephropathy,” Dr. Weck says. “The association between BKV viral load and hemorrhagic cystitis is not as clear and needs more work.”

Virtually everyone has been exposed to Epstein-Barr virus. Between 20 percent and 80 percent of people expel virus in saliva and have low levels (one per million) of EBV-infected white blood cells circulating in their blood. However, less than one percent of latently infected people develop an EBV-related tumor. “That is why it is important to have quantitative rather than qualitative assays,” Dr. Gulley says. A clinical assay needs to measure high levels of EBV-infected WBCs or high cell-free DNA levels in plasma that are indicative of neoplasia or impending neoplasia. Dr. Gulley has done important work establishing the validity of viral load monitoring in patients with EBV-related lymphoma (Ryan JL, et al. J Mol Diagn. 2004;6:378–385). She and her colleagues concluded, “The findings suggest that Q-PCR is an effective method of distinguishing disease-associated virus from incidental virus in paraffin-embedded tissue and in plasma samples.”

Establishing the presence of EBV-encoded RNA (EBER) in paraffin-embedded tissues by in situ hybridization is the best test to prove that a tumor is EBV-related, Dr. Gulley says. “You can’t use immunostains because no single viral protein is consistently expressed in every kind of EBV-related tumor, whereas EBER is expressed in every EBV-related malignancy,” she says. By correlating viral load to the presence or absence of EBV-encoded RNA in situ hybridization in 61 AIDS patients with lymphoma, Dr. Gulley and her colleagues established that “EBV viral load shows promise as a tool to assist in diagnosis and management of EBV-related lymphoma patients” (Fan H, et al. J Med Virol. 2005;75: 59–69).

Quantitative EBV measurement is most useful in transplant patients who are immunosuppressed and at risk for developing EBV-related neoplasia, Dr. Gulley says. It’s also useful in AIDS patients, who are immunosuppressed and predisposed to develop lymphoma, and it’s useful in nasopharyngeal carcinoma, another EBV-related malignancy. “In each of these instances,” she says, “we are measuring EBV viral load in the blood or plasma, so this is a noninvasive way to identify the presence of tumor and to monitor the efficacy of therapy.” She points out that EBV-related diseases are common in a worldwide context. “It is estimated that about one percent of all humans will develop some kind of EBV-related malignancy.”

In the U.S., the major clinical indication for quantitative EBV testing is in pediatric patients who have received allogeneic transplants of marrow, stem cells, or solid organs. “These patients are iatrogenically immunosuppressed, and EBV viral load assays can be used to help diagnose post-transplant lymphoproliferative disorder [PTLD],” Dr. Gulley says, “or even in a preemptive way to predict a future PTLD so that early intervention can be started before the patient becomes symptomatic.” Once intervention is underway, viral load testing can monitor its efficacy. In a Dutch study, preemptive treatment—withdrawal of immunosuppression along with administration of the monoclonal antibody rituximab—in transplant recipients whose EBV viral titer exceeded 1,000 genome equivalents/mL reduced the incidence of PTLD and completely abrogated PTLD-related mortality (van Esser JW, et al. Blood. 2002; 99:4364– 4369).

EBV-related lymphomas in AIDS patients can be Hodgkin-like or non-Hodgkin-like. They can also present within the central nervous system. “Those are always EBV-related, and EBV levels are detectable within the cerebrospinal fluid in those cases,” Dr. Gulley says. “In the setting of an AIDS patient who has a solitary brain mass and positive EBV PCR in cerebrospinal fluid, that is very consistent with a diagnosis of primary lymphoma of the brain. In some cases, clinicians have chosen not to do brain biopsy but to treat for lymphoma based on the combination of clinical, radiographic, and CSF findings.” Once a patient is diagnosed with brain lymphoma, the laboratory can continue to test CSF for EBV DNA as a way to follow the efficacy of treatment. “That requires a spinal tap but is still better than brain biopsy,” Dr. Gulley notes.

EBV-related nasopharyngeal carcinoma (NPC) is much more common in Southeast Asia, especially southern China, than in the United States. “Immigrants from those areas to the U.S. retain a much higher rate of nasopharyngeal carcinoma, although as they adopt the Western diet it tends to diminish, suggesting tumorigenesis is at least partly a dietary effect,” Dr. Gulley says. “In cities like San Francisco and New York, where there is a large Asian population, there is a high number of NPC patients, largely of Asian descent.”

Studies from Hong Kong showed that, in NPC patients, higher pretherapy EBV DNA viral load correlated with advanced stage, affected relapse rate and survival, and was an independent prognostic factor to conventional factors in staging (Leung SF, et al. Cancer. 2003;98:288–291; Leung SF, et al. J Clin Oncol. 2006;24:5414–5418). “Testing using viral load is even more valuable than serological testing for monitoring these patients for residual disease and for early relapse,” Dr. Gulley says. “There is exciting data suggesting that you could collect nucleic acid from the surface of the nasopharynx with a swab and use EBV RNA as a very specific marker for local recurrence.”

For all of these assays, optimal performance requires standards and calibrators. “That is absolutely the critical issue,” Dr. Hillyard says. “Clinical literature and technical lab studies demonstrate that when you go beyond HIV, HCV, and HBV, standardization really begins to fall off. If you read a study for EBV or BKV and try to compare the results from your lab, you don’t know whether your results apply to the numbers described in that manuscript. So it is difficult to be informed by the literature about what these quantitative values mean.” The heart of the problem, Dr. Hillyard says, is a lack of international standards and controls.

In that regard, Dr. Valsamakis says, there are reasons to be optimistic. “As soon as a CMV standard preparation is available, which from my understanding should be coming fairly soon from NIST [National Institute of Standards and Technology], the aim is to go back and replicate that work for EBV.”

In the meantime, it is up to laboratories to validate their in-house–developed or ASR assays. Melissa B. Miller, PhD, D(ABMM), assistant professor of pathology and laboratory medicine and director of the molecular microbiology laboratory at the University of North Carolina-Chapel Hill, provides an example of how time-consuming and laborious this can be. “In 2004 we offered an endpoint research-use-only PCR assay for HCV,” she says. “In reality it was not to be used for clinical management of patients.” A major problem with this assay for viral load testing was that it had very limited dynamic range, so two assays were needed—a very sensitive qualitative assay to measure sustained viral response and a quantitative assay for patients who were fluctuating over time. “We offered the quantitative assay here and sent out for the qualitative assay,” Dr. Miller says. “At that time there was not a huge market, so there were not many options.” There wasn’t much demand for HBV viral load measurement either. “As more data became available and more drugs were approved,” Dr. Miller says, “monitoring HBV viral load became an integral part of managing patients, so we brought that in-house.”

With the need to do clinical HCV viral load testing in-house, Dr. Miller’s laboratory evaluated two real-time Q-PCR ASR products, one from Abbott and another from Roche. “We saw very good correlation between the two assays for all genotypes,” Dr. Miller says. “Our decision came down to workflow and hands-on time.” They chose the Roche system for both HCV and HBV for convenience and efficiency. Both have a broad dynamic range and replaced their existing qualitative and quantitative tests.

Dr. Miller says validating these two assays during the evaluation period was “very painful” for the fellow in the lab at that time, Jennifer Goodrich, PhD. “She spent a lot of time doing side-by-side studies. With ASRs you have to determine the performance characteristics yourself,” Dr. Miller notes. “The manufacturer is not allowed to give input.” Validation was done by the laboratory’s standard algorithm, in which the first step is to define the lower limit of detection and the lower limit of quantification. (The lower limit of detection is the viral load at which you can detect that quantity at least 95 percent of the time; the lower limit of quantification is the level at which you can quantitate accurately within the linear range.) “Doing just this first step required many reproducibility studies,” Dr. Miller says.

For an ASR-based assay the laboratory also needs to evaluate specificity with different specimen types, possible cross-reacting material, and other viruses, such as HIV and HSV, which requires large panels. “You want to test actual patient samples and sometimes cultured material,” Dr. Miller says.

Next the performance of the assay in patients diagnosed with disease is evaluated. As a gold standard reference test for HCV, they used the endpoint research-use-only assay, “realizing that it could be a flawed gold standard, which happens all the time with molecular products,” Dr. Miller says. For HBV they used the reference laboratory assays because that was the result they had been reporting to physicians. With HCV, the situation was “rather nice,” Dr. Miller says, because they were comparing the endpoint assay to two real-time PCR assays. “We could see that the so-called gold standard was flawed,” she says. “If there was a discrepancy of more than one log between the gold standard assay and the two real-time assays, the real-time assays always agreed.”

In all, this entire process took about six months, which leads Dr. Miller to stress how important it is for diagnostic companies to continue to take their products for full FDA approval. “Many labs don’t have the time and expertise and resources to do this,” she says. Real-time Q-PCR products for HCV and HBV from Roche and Abbott are now under review. “Both systems are completely automated and have very wide dynamic ranges,” Dr. Miller says. She’s hoping her lab can switch to an FDA-approved platform soon.

But the FDA-approved products are for high-volume tests only, Dr. Miller points out. Assays for CMV, EBV, and BKV are used commonly with the transplant population, as well as adenovirus and human herpes virus 6, yet she knows of no company looking to bring assays for these viruses through the FDA process.

She implores diagnostic companies to realize they can’t pursue just the high-volume testing. “If they want to help us in patient care,” she says, “they have to develop lower-volume tests as well.”

William Check is a medical writer in Wilmette, Ill.