New indications for molecular virology testing

As our ability to detect viruses has grown, so has our understanding of the varied clinical manifestations these multifaceted agents can cause. This virtuous cycle continues to improve our ability to care for our sickest patients.

One of the greatest advances in clinical medicine of the last two decades has been the development of effective antiviral drugs. At the same time there has been increased recognition of the significance of viral infections in the immunocompromised host. Because of these changes, there is an urgent need to rapidly diagnose viral infections, monitor antiviral therapy, and test for antiviral resistance. Recent technological advances have led to the development of molecular methods to address these needs. These molecular methods provide rapid diagnosis and quantitative measurements of viral infections, and have markedly improved clinical outcomes.

Transplant patients.

Almost all patients undergoing transplantation have immune defects, ranging from mild in certain solid organ transplants to severe after hematopoietic stem cell transplants. As such, transplant patients are at elevated risk for a variety of viral infections, which traditionally have been a major cause of morbidity and mortality. Molecular virology testing has revolutionized the management of such infections, in many cases rendering them largely treatable or even preventable given optimal care.

Cytomegalovirus (CMV) presents the greatest viral threat to transplant recipients. CMV causes pneumonia and gastrointestinal disease in immunocompromised individuals. Historically, untreated CMV disease led to death in up to 25% of seropositive recipients, and CMV pneumonia had a mortality rate of greater than 70%. Among renal and liver transplant patients, CMV is a leading cause of graft rejection. Thanks to the development of effective antivirals such as ganciclovir, and their use in prophylactic regimens, early CMV disease has been greatly reduced. At the same time, however, we have seen an increase in the incidence of late CMV disease, occurring 100 or more days post-transplant.

Since prolonged exposure to ganciclovir carries very high risks of toxicity (neutropenia, often treated with very expensive products such as G-CSF) and the development of viral resistance, our institution has adopted a preemptive therapy approach. At-risk patients are monitored weekly for CMV by polymerase chain reaction (PCR), which provides greater accuracy and sensitivity than cell culture techniques. Samples for PCR can be shipped easily, allowing access to the same laboratory assay as patients move from inpatient to outpatient care. Therapy is initiated if testing shows evidence of CMV reactivation, regardless of the presence or absence of symptoms. While monitoring has a cost, these patients are spared prolonged treatment with prophylactic ganciclovir. Thus, much of the cost of testing can be recouped in drug cost savings alone. From the medical viewpoint, the value of frequent testing is unambiguous – patients avoid unnecessary drug exposure and reduce their risk of hospitalization and death.

As more patients survive transplants and viral infections, a new problem has emerged – viral resistance to ganciclovir – and thus we recently established a rapid assay for ganciclovir resistance. With an enlarging pallet of antivirals with activity against CMV from which to choose (maribavir, cidofovir, foscarnet), the rapid identification of ganciclovir-resistant strains is becoming critical to optimal patient care, providing clear indications for alternative drugs with distinct mechanisms of action.

Epstein Barr virus (EBV) is another threat to transplant recipients. EBV infects B cells, and in the context of immunosuppression can lead to the unchecked growth of these cells, causing post-transplant lymphoproliferative disorder (PTLD), a uniformly fatal disease unless treated early and aggressively. PTLD is recognized increasingly in children receiving renal or hepatic transplants, and in patients undergoing stem cell transplantation. PTLD can be treated with anti-B cell therapies such as rituximab, and early therapy offers the best chance of success. At our institution, at-risk patients are monitored weekly for circulating EBV by PCR, and those with elevated or rising EBV are evaluated carefully for signs of PTLD and for possible rituximab therapy. This approach has markedly reduced mortality from PTLD.

Kidney transplant recipients are not as severely immunosuppressed as recipients of stem cell transplants, but they are at risk of nephropathy caused by BK virus. The most insidious feature of BK nephropathy is that its clinical presentation is nearly identical with that of renal transplant rejection. While rejection requires increased immunosuppression, BK nephropathy calls for a marked decrease in immunosuppression – hence, the differentiation of these entities is critical. Serum PCR for BK virus is extremely useful in this regard, since BK viremia in this setting is nearly diagnostic of BK nephropathy. Thus, for renal transplant recipients with rising creatitine, PCR testing of serum for BK virus is now standard of care before considering increased immunosuppression.

CNS syndromes

Molecular testing for viruses has revolutionized the management of central nervous system (CNS) syndromes. An excellent example is PCR testing for enteroviruses, the most common cause of meningitis. Children with enterovirus meningitis present to the emergency department with fever, lethargy, headache, and nuchal rigidity. Enterovirus meningitis is self-limited, with no lasting consequences. Unfortunately, the clinical presentation of enterovirus meningitis is nearly identical to that of a true killer, bacterial meningitis. Thus, children with a benign infection are unnecessarily subjected to hospitalization and multiple diagnostic procedures. This is expensive for the health care system, and traumatic for our patients.

Enter molecular testing for enterovirus. PCR-based testing can be performed rapidly (within hours for our assay), to identify patients with benign viral meningitis. Once CSF is obtained for bacterial culture and a long-acting antibiotic administered (to deal with the remote possibility of co-infection with enterovirus and bacteria), these patients can be safely discharged from the hospital, to await final confirmation of negative bacterial cultures. The cost savings of this approach can be substantial, decreasing the mean hospital stay by 24-48 h, and reducing total hospital fees by $3000 or more.

PCR has also revolutionized the management of disease caused by herpes simplex virus (HSV), especially in the neonate. Infected mothers (particularly those with primary HSV infection) can transmit HSV to the neonate during delivery. Untreated, neonatal herpes can cause CNS or disseminated disease, often resulting in devastating long-term sequelae or death. Acyclovir is an effective treatment, but early detection of disease and prompt initiation of therapy is critical. PCR for HSV is the test of choice, because it is far more sensitive than viral culture, and because of the speed with which results become available. PCR may also play a role in identifying women in late-term pregnancy who are actively shedding virus, and thus at elevated risk of transmitting HSV during delivery.

HSV can cause encephalitis in adults as well. Untreated disease frequently leads to severe long-term neurological sequelae or death. Again, the sensitivity of viral culture from CSF in this situation is very low, and PCR is the test of choice. As acyclovir is an effective therapy for HSV when started early, prompt diagnosis is critical. Relapses are common, and thus monitoring the CSF for resolution of infection is a necessary part of successful therapy.

One of the challenges of CNS viral infections is the fact that different viruses can have very similar presentations. Thus, a definitive diagnosis is not possible on clinical grounds alone. Since a given PCR test will only detect the virus for which it was designed, the clinician must have a high index of suspicion, and generate an inclusive list of the most likely viral culprits. Consultation with an infectious disease specialist is often helpful in this regard.

A good example is primary infection of children with human herpesvirus 6 (HHV-6). These children can present with fever and lethargy, and are often evaluated for possible bacterial infection, with blood and CSF culture. In such presentations, consideration should be given to both enterovirus and HHV-6. Importantly, HHV-6 appears to be shed for an extended period after primary infection, increasing the possibility of concomitant viral and bacterial infection, and therefore the presence of virus should not be misinterpreted as evidence that bacteria are absent. HHV-6 has also been increasingly recognized as a threat during transplantation, in large part due to its ability to induce systemic immunosuppression.

In children and adults, other viral causes of CNS infection include CMV, varicella zoster virus (VZV), and EBV. Although less common, the symptoms caused by these viruses can be similar to those caused by enterovirus or HSV. CNS infections with CMV, VZV, and EBV occur more frequently in the setting of immunosuppression, where the presentation of many syndromes is atypical. In elderly patients, VZV encephalitis is being recognized increasingly – even without the pathognomonic finding of shingles. Thus, a high index of suspicion is required, and the prudent clinician will cast a wide net.

Idiopathic pneumonia

Perhaps nowhere has the use of molecular testing for viruses undergone such profound evolution in the last three years as in what has been termed “idiopathic pneumonia”. The recognition of new pathogens such as human metapneumovirus (MPV), avian influenza, and SARS has filled the lay and medical press with new concerns, and new recognition of the role viruses play in idiopathic pneumonia. Respiratory infections account for a large and increasing proportion of hospital visits during the winter and spring, due in part to greater awareness of respiratory viruses, but also to the increasing availability of effective antiviral medications. Rapid and accurate diagnosis is important to guide therapy and provide surveillance, and often can decrease overall hospital costs. While much attention has been given to potential pandemics of avian influenza and SARS, most viral respiratory infections are caused by a familiar set of culprits: respiratory syncytial virus (RSV), influenza, parainfluenza (PIV) types 1, 2 and 3, and adenovirus, along with the more recently described human metapneumovirus (MPV). We have developed and evaluated a respiratory PCR panel for these viruses. Several findings suggest that molecular testing is extremely useful in the setting of acute respiratory illness. First, over half of patients referred for testing prove to have infection by one of these viruses. For many, infection is detectable by PCR but not by the traditional fluorescent antibody test. This is especially true for PIV1, PIV2, adenovirus, and MPV, for which the large majority of cases are detectable by PCR only. Also surprising is the frequency of mixed infections with two or more viruses - nearly 10% of cases. Molecular testing is rapidly altering our understanding of acute respiratory illness, and promises to greatly improve our care for these patients.

In every area where it has been evaluated, molecular virology testing is superior to traditional culture methods, often remarkably so. As such, few leaders advocate going back to the old days. Nevertheless, the cost of many molecular tests can cause sticker shock. In many cases it is helpful to view molecular testing in terms of its overall effect on the cost of managing patients, rather than a simple comparison of list prices.

Nevertheless, the reality remains that molecular testing for viruses is not cheap. How can institutions maximize the benefit of this new technology for their patients, while still being careful stewards of limited healthcare dollars? First, not all laboratories need to offer every test. Setting up a new molecular virology test is not trivial. In addition to capital expenses for specialized instrumentation, there are significant costs associated with assay design, validation, and on-going quality control. In fact, except for the highest-volume situations, it will often make financial sense to send such testing to outside laboratories. Fortunately, with the widespread reliability of overnight shipping, such sendouts no longer exact a significant penalty in terms of turnaround time, which is often an important determinant of quality of patient care. For example, our laboratory runs specialized molecular virology testing for outside clients located throughout the United States. Overnight shipping is available from anywhere in the US, and most testing is completed by late afternoon of the day the specimen is received. Results of testing can be available to clients immediately via electronic information system. Given the turnaround time of less than 36 hours, more and more clients are rethinking their mix of in-house and sendout testing. Regardless of the choice of in-house vs. sendout testing, however, the fact remains that molecular techniques have proven their worth in the management of viral infections, and are quickly becoming standard of care for our patients.