College of American Pathologists
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  Putting molecular methods to work


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Advancing molecular testing

January 2000
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William Check, PhD

Nucleic acid-based assays are “very powerful but very expensive tools,” said Mary Jane Ferraro, PhD, MPH, director of microbiology laboratories, Massachusetts General Hospital, convening a symposium on molecular methods in infectious diseases this past fall at the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy.

Symposium speakers discussed the application and interpretation of these tests for human papilloma virus, hepatitis C virus, human immunode-ficiency virus, and cytomegalovirus, but they noted that the tests are useful for other pathogens as well.

Human papilloma virus. Discussing molecular methods for detecting human papilloma virus, Nancy Kiviat, MD, stated that many women are infected with HPV, but only a few get cervical cancer. Dr. Kiviat is chief of pathology, Harborview Hospital, and director of cytopathology, University of Washington Associated Hospitals, Seattle.

"Some women’s immune systems can’t handle the infection, and we think those individuals are at highest risk of HPV-related neoplasia," Dr. Kiviat said. Viral risk factors include the proteins E6 and E7, expressed during infection with high-risk HPV types. These proteins reduce the activity of the tumor-suppressor proteins p53 and RB105. "These viral genes set the stage for accumulation of chromosomal abnormalities that, in some cases, lead to progression to malignancy," Dr. Kiviat told CAP TODAY.

HPV testing, Dr. Kiviat added, plays a definite role in assisting triage for women with ASCUS, or atypical squamous cells of undetermined significance. Widespread Pap screening has reduced the rate of cervical cancer from 40 to seven cases per 100,000 women. But in addition to revealing squamous intraepithelial lesions, Pap smears also identify ASCUS in five to 10 percent of women screened, and all of these women are asked to return for a repeat Pap smear. Many eventually have colposcopy and biopsy.

Most of these examinations are unnecessary because the risk of cervical cancer with ASCUS alone is extremely low. Yet more than one-third of high-grade SILs identified during screening come from ASCUS smears. Testing for HPV can help identify the minority of women with ASCUS who have underlying disease, without requiring all women with ASCUS to undergo invasive examinations.

Evidence for this contention comes from studies, such as the one performed at Northern California Kaiser Permanente Medical Group (JAMA, 1999;281:1605-1610). The Kaiser study compared use of HPV testing versus repeat cytology for triage of women with ASCUS. Of 46,000 women screened with standard cytology, 995 who had ASCUS returned for colposcopy and repeat cytology. HPV testing was performed for these 995 women using a hybrid capture assay (Digene Corp.) on specimens collected in liquid medium during the initial screening.

Two major findings emerged. First, approximately the same fraction of women—39 percent—would have been sent for colposcopy and biopsy based on detecting HPV DNA in the initial cervical specimen as established by findings at repeat cytology. Second, based on colposcopy results, "The sensitivity and specificity of HPV testing for identification of women with underlying high-grade SIL was essentially the same as that of repeat Pap smear," reported Dr. Kiviat.

The investigators proposed that women with ASCUS who are HPV-positive undergo immediate colposcopy and all other women who have ASCUS undergo repeat Pap testing, which would provide 97 percent sensitivity for high-grade SIL. "Most important," the investigators concluded, "our proposed algorithm allows HPV testing and a subsequent triage decision based on the initial [ASCUS] Pap specimen." With this scheme, many women could avoid the cost and inconvenience of repeat physician office visits.

This approach requires using the same sample for Pap smear and HPV testing. Part of the sample is used for cytology, and part is held in liquid medium and probed for HPV if ASCUS is found.

To prepare for this tack, Dr. Kiviat told CAP TODAY, the University of Washington Hospitals switched to liquid-based cytology a few years ago. Now any woman with ASCUS automatically will have an HPV test: Those who are positive will be referred for colposcopy, and those who are HPV-negative will follow a routine Pap smear schedule.

To reduce the cost of the HPV assay, Dr. Kiviat uses only high-risk probes. "Virtually all samples with CIN II or III have a high-risk HPV type," she said.

Based on preliminary results of studies she and her colleagues have conducted in Seattle and Africa, Dr. Kiviat believes HPV testing also will play a role in primary screening for cervical cancer. Detection of HPV DNA appears to have sensitivity and specificity as good as cytology, if not better, for high-grade SIL (CIN II or III). So, in the future, it may be more cost-effective and better for the purpose of detection to use HPV as a primary screen and then do a Pap smear on HPV-positive women.

Some laboratories are in an earlier stage of implementing HPV testing, however. Says Stephen Dumler, MD, director of the Division of Medical Microbiology, Johns Hopkins University Hospitals, "We are evaluating the Digene assay, predominantly for high-risk strains, in collaboration with cytologists and gynecology groups around the university." Physicians have adopted a liquid-based collection method and, for samples with ASCUS, the laboratory will reflexively test for high-risk HPV types. "We are assessing the feasibility of that approach," Dr. Dumler told CAP TODAY.

At Massachusetts General, "Clinicians are not knocking down the doors yet" for HPV testing, said James Versalovic, MD, PhD, the hospital’s assistant director of microbiology. "We are definitely talking about it," he commented to CAP TODAY. "Cytologists are still content with routine Pap smears. But the question of whether or when to do HPV testing for ASCUS smears is on the table."

Hepatitis C virus. Turning to molecular tests for detecting and quantitating hepatitis C virus, symposium speaker David Gretch, MD, PhD, associate professor of laboratory medicine, University of Washington, stated that during the past few years, the burgeoning epidemic of HCV in the United States increasingly has been recognized. About 4 million persons are infected. Approximately 85 percent of infections enter a chronic phase, and perhaps 20 percent of those progress to cirrhosis over about 20 years. Infection with HCV is the leading cause of the need for liver transplant, Dr. Gretch noted.

Qualitative detection of HCV viremia predicts liver disease in persons who are seropositive for HCV, said Dr. Gretch, referring to an earlier study. All 16 seropositive patients in the study who had qualitative evidence of HCV viremia had liver disease on biopsy, regardless of whether liver enzymes were elevated, and not one of seven seropositive patients with normal enzymes and no detectable plasma HCV RNA had biopsy evidence of liver disease. "In the presence of normal enzymes, viral RNA testing is very important to guide decisions about biopsy," Dr. Gretch concluded.

Reviewing available tests, Dr. Gretch noted that the qualitative RT-PCR assay for HCV confirms viremia and active infection and is the most sensitive. An isothermal qualitative assay based on transcription-mediated amplification technology is used in blood banking. Quantitative RT-PCR and bDNA assays help monitor and guide therapy. Treatment of HCV infection with interferon, now typically combined with ribavirin, is producing sustained five-year responses in 40 percent of patients, Dr. Gretch said. Virological response—reduction of viral load to below 100 copies/mL—is the most accurate indicator of response.

If patients clear viremia after one week of therapy, 76 percent have a sustained response; if viremia clears by two weeks, sustained response is seen in 36 percent; by three weeks, sustained response is seen in 13 percent.

HCV RNA testing is also used to determine length of treatment: If viremia does not clear by three months, therapy is stopped. (Six months is the cutoff for combination therapy.) Terminating therapy in the absence of a treatment response is especially valuable for patients who are not tolerating the drugs.

An additional application of molecular testing in HCV infection is for sequencing isolates to determine their genotype. About two-thirds of U.S. patients are infected with type 1 virus, which has only about a 10 percent response rate, while virus types 2 and 3 have a 35 to 40 percent response rate. "But typing should not be used to deny therapy," Dr. Gretch- cautioned. "Response rate for type 1 virus can be doubled by extending duration of interferon therapy to 48 weeks." Therapy can be stopped for patients with types 2 or 3 virus whose viremia clears by six months.

Massachusetts General’s Dr. Versalovic sends out samples for HCV, "But we are moving towards implementation of molecular HCV testing," he added. He initially plans to use quantitative viral load measurement to follow up patients in therapy. For budgetary reasons, quantitative testing will come first—right now that assay is in highest demand from gastroenterologists.

"I’m not entirely in agreement with our volume distributions in HCV testing," Dr. Versalovic told CAP TODAY. But once he sets it up and gets it under control, he plans to introduce qualitative testing and genotyping for diagnosis and followup. "I agree that qualitative testing and genotyping are probably more important than quantitative testing," he stated. The qualitative test is more sensitive than the quantitative assay by one order of magnitude, 100 copies versus 1,000 copies.

"Qualitative testing may become the preferred confirmatory diagnostic test for HCV," Dr. Versalovic said, although he noted it is not yet approved for that purpose.

Human immunodeficiency virus. Assays for HCV are the highest-volume molecular tests in many laboratories. In others, HIV testing takes the top spot.

Quantitative HIV assays are used to monitor viral load during therapy and soon may be adopted in some centers for diagnosing acute HIV infection, explained Angela Caliendo, MD, PhD, medical director of microbiology/molecular diagnostics, Department of Pathology and Laboratory Medicine, Emory University, Atlanta.

The only FDA-approved quantitative assay for HIV is the Roche RT-PCR, which is used for an estimated 70 percent of tests. The other primary tests for HIV viral load are Quantiplex bDNA from Bayer and NucliSense (formerly NASBA) from Organon Teknika.

In their standard versions, all three methods are sensitive down to 400 to 500 copies/mL. In their ultra sensitive versions, the lower limit of detection is 50 to 100 copies/mL. Even though there is good correlation between bDNA 3.0 and Roche’s ultrasensitive method, Dr. Caliendo said, "I still do not recommend that you switch technologies for monitoring an individual patient."

A qualitative assay (that detects proviral DNA) is available that is used primarily for neonatal diagnosis. Even for this application, Dr. Caliendo prefers a quantitative RNA assay.

One difference among assays is dynamic range: the ultrasensitive RT-PCR has an upper limit of 75,000 copies/mL, while bDNA version 3.0 goes up to 500,000 copies/mL, and NucliSense goes up to 10 million copies/mL. Therefore it would be necessary to use both the standard and ultrasensitive versions of RT-PCR, while one could use the ultrasensitive versions of the other two assays alone.

"The need for a standard and ultrasensitive assay may be a problem for laboratory workflow and turnaround time," said Dr. Caliendo, particularly for laboratories that have a small volume of HIV testing. Even though her laboratory does not do a high volume of HIV testing, Dr. Caliendo uses RT-PCR, arranging workflow to accommodate it.

Dr. Caliendo chose RT-PCR for HIV testing because she had considerable research experience with the assay and was satisfied with its performance. She uses the NucliSense assay for various research studies. She likes having an assay with an internal control. "It is very important to have a handle on efficiency of extraction," she said.

Inherent in the RT-PCR and NucliSense assays is the ability to evaluate efficiency of specimen extraction. "When you get a negative result," Dr. Caliendo commented, "you know this is not due to inhibitors or poor specimen extraction."

The methods also differ in specificity. The specificity is nearly 100 percent for RT-PC-R and NucliSense and is 95 to 98 percent for bDNA.

RT-PCR and bDNA are less labor-intensive to perform than NucliSense. But an automated extractor, priced at about $60,000, has simplified NucliSense considerably.

A precaution that should be taken with all assays is not to overinterpret small changes—for example, from undetectable to 200 copies/mL. For all assays, intra-assay variation and biologic variation of viral load together are about 0.5 log. So a change in viral load doesn’t represent clinical change until it exceeds 0.5 log, about a threefold change. "This is especially important at lower values, below 500 copies/mL," Dr. Caliendo stressed. "You might want to ask for a four- to fivefold change at that level." Patients have "blips" where they go below 50 copies/mL, then go back above that level, and then come down again. "Make sure clinicians repeat a test before changing therapy," Dr. Caliendo advised.

In acute infection, less than six weeks after exposure, there is a mononucleosis-like syndrome with negative or indeterminate enzyme-linked immunosorbent assay and Western blot and very high viral load. In one study, viral load during acute infection ranged from 250,000 to 100 million copies/mL of HIV RNA. Bruce Walker, MD, Eric Rosenberg, MD, and colleagues at Massachusetts General Hospital focused on whether there is a pool of acutely infected persons who could be identified with appropriate testing. They tested 560 individuals who presented to physicians with an acute mononucleosis-like syndrome that turned out to be Monospot negative. Seven (approximately one percent) patients had definite or possible acute HIV infection. "There is more acute HIV infection out there than people appreciate," Dr. Caliendo concluded.

To find such cases, the clinician should take a history for HIV risk factors when someone presents with a viral syndrome and then determine whether to test for HIV. If the viral load is greater than 200,000 on two consecutive tests, the clinician should follow the patient to verify seroconversion and make a treatment decision.

"You need to appreciate that you are using this test in a way for which it was not licensed," Dr. Caliendo noted, "so the clinician needs to get patient consent and send a sample for concurrent ELISA." If the patient does not have HIV infection, the chart should reflect a negative ELISA along with the negative viral load result.

From the laboratory’s viewpoint, Dr. Caliendo said, "Clinicians are using viral load testing for the diagnosis of acute infection, and the laboratory doesn’t always know. Clinicians and laboratories need to work together on this."

At Massachusetts General, HIV is the third most ordered DNA amplification test performed directly on a primary clinical specimen, with more than 4,000 tests conducted annually, according to Dr. Versalovic. (First is chlamydia; second is gonococcus.) For diagnosis, Dr. Versalovic uses ELISA confirmed by immunoblot or Western blot. "Quantitative HIV viral load is not a primary diagnostic tool," he emphasized. "It is not FDA-approved for that."

Quantitative viral load is done to follow patients on therapy or to establish a pretherapy baseline. Some physicians also order quantitative RT-PCR for diagnosing acute infection. "It is useful in the acute HIV setting because you see sky-high viral loads," Dr. Versalovic said, often above the 750,000 copies/mL upper limit of detection for the RT-PCR assay. He agreed that one must also obtain informed consent and a diagnostic ELISA in this context.

Cytomegalovirus. Nucleic acid-based assays for detecting cyto-megalo-virus encompass qualitative and quantitative PCR, hybrid capture, NucliSense, and bDNA, said Vincent Emery, PhD, reader in virology, Royal Free and University College Medical School, London. They are used in transplant recipients and HIV-infected persons.

PCR and NucliSense are used most often, in Dr. Emery’s experience. Hybrid capture and bDNA will be used more in the future because the companies that market them have assays for other viruses, and it is easier to add one more virus by the same method.

"But working out specificity and sensitivity for disease for those assays is in [its infancy]," said Dr. Emery, who runs Bayer bDNA, Roche PCR, and an in-house assay for CMV. "That is historic—we were interested in CMV for several years, and commercial assays have only come online in the last few years," he told CAP TODAY.

With culture, CMV background is usually negative—sensitivity is only 25 percent. Even with a seropositive recipient of a seropositive organ, one would not expect to culture virus unless an infectious virus was on board. "So there is a high threshold of detection—if you do detect virus in culture, it has a high negative predictive value," Dr. Emery said.

For molecular assays, most samples—whole blood, plasma, leukocytes—will work, although viral load in plasma is about 20-fold lower than in whole blood.

Volume is another issue. With PCR, if you make it very sensitive and take a large amount of sample, then you will find positivity in most people who are antibody-positive. "That is clearly of no value," Dr. Emery commented. "What you want is high levels of DNA post-transplant indicative of active infection. You can do that two ways: take a small amount of blood and have a very sensitive assay or a rather large amount of sample and have a relatively insensitive assay." Dr. Emery has chosen the former route.

With this strategy, a viral load below 200 genomes/mL provides reasonable confidence that there is no replication or very low-grade replication. "With herpes viruses and their latent infection," Dr. Emery said, "there may be a low level of reactivation all the time that a healthy immune system controls." Only with immunocompromise does loss of control occur.

Another sampling issue surfaces when monitoring response to therapy. Viral load goes below the limit of detection with plasma more quickly than with whole blood. To look at small changes in a short period, Dr. Emery’s laboratory uses whole blood and plots viral load decline quantitatively.

Earlier detection of CMV infection has become an important clinical issue with the advent of effective chemotherapy. Identifying persons at risk of disease and targeting them for pre-emptive therapy increases survival time and allows low-risk individuals to avoid treatment.

Median viral load is significantly higher in those who have symptoms or will develop symptoms, although, for unknown reasons, some persons sustain viral load up to 1 million copies/mL and don’t get disease.

Most clinicians will consider pre-emptive therapy with two consecutive positive qualitative PCR results. But because of earlier and more frequent treatment, the rate of CMV disease has declined. "So it is important to validate your approach to make sure you don’t call a positive too early or your clinicians will intervene too often," Dr. Emery cautioned.

In HIV-infected patients, quantitative viral load of CMV predicts progression to CMV disease and is related to survival even more strongly than HIV viral load. But therapy can’t be denied on the basis of a low CMV viral load. As a result, Dr. Emery said, "We don’t routinely use VL to trigger treatment. We use two consecutive [qualitative] results, even though we know that leads to overtreatment." In the future, Dr. Emery said, "Hopefully where we are heading is individualized patient management."

Although newer, more active therapeutic regimens for HIV, such as highly active antiretroviral therapy, or HAART, have reduced CMV disease, Dr. Emery’s laboratory still monitors patients with a history of CMV disease or a history of CD4 cells less than 100/mL. But CMV disease is very low in those who respond to HAART, so it is difficult to justify screening unless CD4 cell count deteriorates or viral load rises. "We have had patients with a CD4 level of 25 who now have 250 cells," he noted.

"Molecular assays for CMV will play an increasingly important role in management of CMV infections," Dr. Emery concluded. He recommended testing for CMV in all post-transplant patients and selected persons infected with HIV.

Dr. Emery also routinely monitors post-transplant patients for human herpes virus 6 and 7. "We have had a few cases where marrow engraftment was problematic, almost certainly due to HHV 6 infection," he told CAP TODAY. "But in order of importance, CMV is still the dominant infection post-transplant."

Johns Hopkins’ Dr. Dumler tests for CMV on cerebrospinal fluid or vitreous humor. His view of clinical context and optimal assays differs from that of Dr. Emery. "I don’t like the PCR qualitative assay for CMV," he stated. He believes CMV is ubiqitous, so it can be amplified under many circumstances. "And the qualitative test doesn’t correlate with disease so well," he added. "Antigenemia measures viral load quantitatively and correlates well with disease. To get a molecular assay for viral load that quantifies virus and much more closely correlates with clinical disease is a very important objective for the future."

Standardization of assays. Molecular testing may have become too successful too fast. Such methodology was discussed at the 1999 meeting of the Association for Molecular Pathology. "What was very striking was the great variation in how people prepare tests," said David Hillyard, MD, director of molecular pathology and associate medical director for infectious disease testing, ARUP Laboratories, Salt Lake City. "We need to begin to consider the issues that contribute to a good test. People need to exchange samples and see how tests compare. And for quantitative tests, we need to see whether we are all getting the same values."

"It is essential for us to get standardization of these assays," agreed Dr. Caliendo. "It is especially critical right now for hepatitis C." The two quantitative assays, RT-PCR and bDNA, provide very different results. "We anticipate that both companies will standardize their assays to the World Health Organization standard that is now available," she said.

Dr. Caliendo is involved with a working group of the AIDS Clinical Trials Group, which is evaluating CMV assays, and an NCCLS group writing a general guideline for quantitative molecular diagnostics. "With HCV and CMV, we are back to where we were with HIV assays three to five years ago," she said.

William Check is a freelance medical writer in Wilmette, Ill.