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Molecular Testing for Polyomaviruses
C H A P T E R
10 Molecular Testing for Polyomaviruses G.W. Procop and B. Yen-Lieberman Section of Clinical Microbiology, Department of Laboratory Medicine, Cleveland Clinic, Cleveland, OH, United States
INTRODUCTION Polyomaviruses are small (45 nm), nonenveloped viruses with icosahedral nucleocapsids. The genetic material is double-stranded DNA, with genomes that range 4.5 5.5 kb. The Polyomaviridae contains two groups: the Orthopolyomaviruses and the Wukipolyomaviruses [1,2]. The Orthopolyomavirus genus contains the three most important human pathogens: the BK virus, the JC virus, and the Merkel cell polyomavirus (MCPV) [2]. It also contains the trichodysplasia spinulosa-associated polyomavirus. The BK and JC viruses received their curious nomenclature from the initials of the patients from which they were originally isolated [3,4]. The Wukipolyomavirus genus contains the WU and KI viruses for which it was named, as well as the Human polyomaviruses 6, 7, 8, 9, and 10. Although the WU and KI polyomaviruses have been recovered from the respiratory, plasma, and/or urine specimens of transplant recipients, these have not been associated with disease [5]. The breadth of the Polyomaviridae is undetermined and the discovery of new polyomaviruses is expected. The most important etiologic agents of disease in this group, the BK and JC viruses, will command the attention of the majority of this chapter. Human polyomaviruses viruses cause minimal to no disease in the immunocompetent host. Serologic studies suggest that the majority of humans are infected early in life. Infections are either asymptomatic or produce subclinical disease, possibly involving the respiratory tract. The precise mode of transmission is a matter of debate. Some have advocated a respiratory route of infection for the BK virus, since BK viral DNA has been detected in tonsillar tissues [6]. However, others have studied body fluids, such as saliva, and the respiratory secretions of children with
Diagnostic Molecular Pathology DOI: http://dx.doi.org/10.1016/B978-0-12-800886-7.00010-8
upper respiratory tract infections for the presence of the BK and JC viruses, and these fluids did not harbor the viruses [7]. Uro oral, fecal oral, and transplacental transmission have also been postulated as modes of transmission, with the former seemingly very feasible given the permissive nature of urothelial cells for the replication of these viruses [8]. Regardless of the mode of transmission, these viruses are commonly found in the general population. The majority of children (ie, between 65% and 90%) are seropositive for the BK virus by the age of 10 [9]. Seropositivity rates for BK rise until around 40 years of age and then decrease slightly. The JC virus is also highly prevalent with 50 80% of the human population demonstrating serologic evidence of prior infection [10]. The seroprevalance of the other polyomaviruses has not been extensively studied. However, it has been demonstrated that up to 77% of the general population have been exposed to the MCPV [11]. After initial infection, a transient viremia likely ensues whereby the virus reaches the destination tissues for latency. It has been hypothesized that this dissemination could occur through the infection of mononuclear leukocytes in which BK virus DNA has been detected [12]. It is not known for certain whether these viruses become truly latent in the host cells or maintain subclinical (ie, minimal maintenance) replication once the viruses reach their destination tissues. It is known, however, that asymptomatic replication of both the BK and JC viruses occurs in the urothelium (ie, the lining epithelium of the bladder, ureters, and renal pelvis), with subsequent shedding of the virus into the urine [13]. Asymptomatic shedding of the BK virus has been documented in up to 10% of healthy adults [8]. Similarly, 25% of pregnant women have been shown to asymptomatically shed the BK virus in the urine [14]. Age-related shedding of the BK virus
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has been reported, with shedding occurring less frequently in individuals less than 30 years old and gradually increasing in individuals greater than or equal to 30 years old [15]. This asymptomatic shedding of virus may contribute to the transmission of the virus to those who were previously unexposed and is supportive of a uro oral route of infection.
MOLECULAR TARGETS The molecular targets used for the detection of the BK and JC viruses include the VP1 gene, the VP2 gene, and the T antigen gene. Variations in the VP1 sequence are associated with different subtypes [16]. Primer sets for the quantitative assessment of BK virus are commercially available (Table 10.1), as are numerous laboratory-developed tests. Similarly, laboratorydeveloped tests have been used for the detection of the JC virus and MCPV. The RealStar JC virus (Altona, Hamburg, Germany), which targets the large T antigen gene, is available. The linear range claimed is 1000 1012 copies/mL. Similarly, the GeneProof JC Virus assay (GeneProof, Brno, Czech Republic) that targets the junction of the VP1 and VP2 genes is also available. They claim a linear range from 528 to 1010 copies/mL. GeneProof also offers a combined BK/JC assay.
MOLECULAR TECHNOLOGIES Quantitative PCR is the most commonly used method in the assessment of clinical specimens for the detection and quantitation of BK and JC polyomaviruses. This has been done in conjunction with a variety of probe types. Less commonly, qualitative PCR is used to determine the presence of these viruses. In situ hybridization and immunohistochemistry are used in the assessment of biopsy specimens for these viruses [17,18]. Electron microscopy was used in the distant past, but it has largely been replaced by these TABLE 10.1
technologies [19]. However, electron microscopy remains an important tool for discovery and in instances wherein these other technologies fail or inconsistent results are produced. Quantitative PCR is used predominantly for the detection and quantitative monitoring of the BK virus in plasma. A wide variety of PCR assays have been described for the detection and/or quantitation of the BK virus. Many of these are laboratory-developed tests, but some are commercially available [20 25]. These assays usually employ some type of nucleic acid extraction method prior to PCR amplification. Although all aspects of tests need to undergo a thorough assessment during assay validation, most commercially-available extraction methods are robust in the removal of inhibitors and are largely comparable with respect to DNA yield [26]. The quantity of virus present in the plasma is important, since higher values are more predictive of BK nephropathy. Qualitative PCR for the BK virus in urine may be used to identify (or screen out) patients for BK nephropathy, but this method is not commonly used in this manner. However, the qualitative detection of the JC virus in the cerebrospinal fluid (CSF) of patients with progressive multifocal leukoencephalopathy (PML) is sufficient to support this diagnosis in the appropriate clinical setting, which includes supportive radiological findings. Given the relative simplicity of converting a qualitative rapid cycle PCR assay into a quantitative assay, quantitative values are often reported when the JC virus is detected in CSF or other body fluids. Beyond quantitative PCR assays, loop-mediated isothermal amplification (LAMP) techniques have been developed for the detection of the BK virus [27]. Although commercially-available LAMP methods for BK viruses are not available, this type of technology is attractive because it uses standard laboratory equipment, has an acceptable level of sensitivity, and may be used without DNA extraction and a thermocycler. Such a technology could conceivably be used to inexpensively screen for the presence of the BK virus in
Commercially-Available BK Virus Assays
Vendor/Assay
Assay specifics
Target region
Dynamic range (copies/mL)
ELiTechMGB Alert BK Virus Primers
MGB probes
Large T antigen
12.5 3 101 to 12.5 3 106
Focus DiagnosticsSimplexa BKV Kit
Scorpion
VP2
5.1 3 102 to 1.0 3 108
Luminex (Eragen)MultiCode BK Virus Primers
MultiCode
Large T antigen
5.0 3 102 to 5.0 3 106
QiagenArtus BK Virus RG Kit
Real-time PCR
Large T antigen
5.0 3 102 to 5.0 3 106
GeneProof BK Virus Kit
Real-time PCR
VP1/VP2 junction
5.96 3 102 to 1.0 3 1010
RealStar BK virus (Altona)
Real-time PCR
Unknown
1.0 3 103 to 1.0 3 1012
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at-risk populations. In addition to monoplex assays, multiplex assays have been developed. Some target only the most common polyomaviruses (ie, BK and JC), whereas others target additional viruses that may cause disease in the immunocompromised host, such as adenovirus [21,25]. Fluorescence resonance energy transfer probe sets that utilize broad-range polyomavirus primers have also been used to detect and differentiate the BK and JC viruses in a single reaction (Fig. 10.1) [22]. Such an assay may be useful in institutions that screen urine specimens, since either virus may be present. In addition to these, qualitative seminested PCR assays have also been described [28]. In situ hybridization and immunohistochemistry are technologies largely used by anatomic pathologists for the detection of these and other viruses in histologic preparations. The availability of these stains is important to confirm the nature of intranuclear inclusions thought to likely represent polyomaviral inclusions. Other viruses, such as adenovirus and cytomegalovirus, can cause intranuclear inclusions in the same at-risk patient population and are represented in the differential diagnosis. These tools are particularly useful when the intranuclear inclusions are not typical. Although biopsy suffers from sampling error, the demonstration of BK viral inclusions associated with interstitial nephritis is the gold standard for the diagnosis of BK nephropathy. Fortunately, tissue is regularly available for assessment, since renal biopsy is necessary to monitor and/or assess for transplant rejection. However, the same is not always true regarding brain biopsy and the diagnosis of PML. Tissue is not readily available in these instances and most would like to avoid a brain biopsy if at all possible. Although the
FIGURE 10.1 This post-amplification melt curve was produced following real-time PCR amplification using broad-range polyomavirus primers. Fluorescence resonance energy transfer probes that hybridized with complete complementarity with the BK virus amplicon had two mismatches with the JC virus amplicon and several mismatches with the SV40 virus amplicon. This demonstrates the qualitative detection and differentiation of the medically-important BK and JC polyomaviruses in a single assay.
demonstration of the JC virus in infected oligodendroglial cells in immunocompromised patients with a demyelinating disease may be the gold standard for the diagnosis of PML, most are satisfied with the demonstration of the JC virus in the CSF to support the diagnosis of PML in a patient with the appropriate clinical and radiological findings.
CLINICAL UTILITY Although most individuals become infected with a polyomavirus sometime during life, serious symptomatic diseases caused by polyomaviruses occur in immunocompromised patients. The type of immunologic compromise and the degree to which the immune system is suppressed are directly related to the type of polyomavirus infection that is most likely to occur, and in some instances with the severity of infection. The main types of diseases caused by polyomaviruses, as well as associated risk factors and the clinical utility of testing, will be discussed here.
The BK Virus The BK virus is responsible for two main types of disease. It causes nephropathy that can result in graft failure in renal transplant recipients and hemorrhagic cystitis in patients undergoing stem cell transplantation. Early studies described the severe tubulointerstitial nephritis caused by the BK virus in renal allografts [29]. Serologic studies are supportive of the hypothesis that symptomatic disease is most commonly the result of reactivation of latent, endogenous virus rather than primary infection [30,31]. BK nephropathy most commonly occurs in the transplanted kidney, but infection of the native kidney has also been described [10,32]. Disease usually presents 10 13 months after transplantation [10]. Risk factors include a seropositive donor status and/or a seronegative recipient status, older patient age, male gender, ischemic or immunologic injury, and the degree of HLA mismatch. This latter factor is likely more an indicator of the degree of immunosuppression that will be necessary to avoid transplant rejection. Otherwise stated, the greater the HLA mismatch, the higher the number of rejection episodes that are likely to occur, which will necessitate antilymphocyte therapy to control rejection, which facilitates the emergence of the BK virus [33]. However, there is debate regarding the contribution of either cold ischemia or rejection episodes to BK virus reactivation [34]. The diagnosis and monitoring of patients at risk for BK nephropathy is multifactorial and has evolved with
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the introduction of molecular methods. The BK virus can be grown in cell culture or shell vial assay, or directly detected in the urine by immunofluroescent methods. These methods, which were used in the past, have been replaced by quantitative PCR, which is faster, more specific, and less labor intense [35,36]. Quantitative PCR for the BK polyomavirus is predominantly performed on extracts from plasma specimens from renal transplant recipients. This is usually performed periodically throughout the lives of these patients and the presence and quantity of BK virus are measured and trends are established. A baseline should be established relatively soon after transplantation. Testing should be done with the greatest frequency through the 10- to 13-month window after transplantation when the incidence of disease is greatest, then at a regular interval. A recommended protocol for the screening of renal transplant recipients is to screen the urine every 3 months and the plasma every 1 3 months for the first 2 years after transplantation or when graft dysfunction occurs [37]. An increasing viral load in the presence of decreasing renal function correlates with BK nephropathy [38]. The same correlation is not as clear for high BK viral loads in the urine, since the BK virus replicates in the urothelium and relatively high viral loads may be present in the absence of renal involvement. However, Pang et al. undertook a 1-year prospective study evaluating urine and plasma specimens from renal transplant recipients [39]. They found that as the viral load in the urine increased from 7.0 to 10.0 log(10) copies/mL the percentage of patients with viremia increased from 22% to 100%, respectively. These authors suggested that plasma viral load testing could be reserved for those patients who have greater than or equal to 7.0 log(10) copies/mL of virus in the urine. The absence of BK virus in the urine has a high negative predictive value for BK nephropathy. The routine use of BK viral load testing represents an important advancement in the diagnosis and monitoring of patients for BK nephropathy. It is particularly important since it has direct implications on the immunosuppressive regimen given to the patient. Decreased kidney function may occur for a variety of reasons in the renal transplant recipient. In addition to BK nephropathy, decreased renal function may occur secondary to transplant allograft rejection. The differentiation of these conditions is critical, since transplant rejection is addressed by increasing the immunosuppression, whereas BK nephropathy is addressed by decreasing the immunosuppression. The BK virus is the most common cause of hemorrhagic cystitis in hematopoietic stem cell transplant recipients [40,41]. Hemorrhagic cystitis usually occurs
2 weeks after transplant and may affect up to 25% of these patients [42]. Hemorrhagic cystitis ranges in severity from mild (only microscopic hematuria) to severe (clots of blood become obstructive to the flow of urine). There is a great deal of morbidity associated with moderate to severe disease. When the BK virus is the cause of hemorrhagic cystitis the BK viral loads in the urine are extremely high and may be as great as 104 copies/mL. The absence of significant quantities of the BK virus in the urine of a patient with hemorrhagic cystitis is suggestive of another etiology. Other etiologies include infections by adenovirus or cytomegalovirus, as well as the chemotherapeutic treatment, albeit this latter cause has become less common as an etiology due to preventive measures taken to prepare the patient for therapy [1]. The BK virus has been associated with ureteral stenosis, which is not surprising given the locus of viral replication. Additionally, it has only rarely been associated with other diseases, such as native kidney infection in recipients of other types of transplants (eg, heart and stem cell), pneumonia, and encephalitis [43 45]. Individuals have sought associations of these viruses with other diseases, but these searches have largely been unfruitful. For example, an examination of lung tissue extracts from 33 patients with documented idiopathic pulmonary fibrosis found no evidence for the presence of either the BK or JC virus by PCR [46].
The JC Virus The JC virus causes PML [47]. This disease was associated with a variety of malignancies or sarcoidosis prior to the HIV epidemic [48,49]. Patients with progressive HIV infection (ie, AIDS) are at high risk of PML. The CD4 1 T-cell reduction caused by HIV infection produces the perfect milieu for the emergence of the JC virus in the central nervous system. The lytic infection of myelin-producing oligodendroglial cells is the cause of this demyelinating disease. As with BK nephropathy, serologic studies are supportive of reactivation of endogenous virus rather than a primary infection [50,51]. This disease has become significantly less common in resource-rich countries wherein patients have access to highly active antiretroviral therapy. A new group of patients are now recognized as atrisk for PML. This group consists of patients who undergo immunotherapy with biologics for a variety of autoimmune conditions. PML has been reported in patients receiving natalizumab, for multiple sclerosis, efalizumab for psoriasis, and infliximab for Crohn’s disease and other autoimmune diseases [52 57]. In
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rare instances, PML may present in patients with minimal to occult immunosuppression, the possibility of this disease should remain in the differential diagnosis of patients with the appropriate clinical and radiological findings [58]. Quantitative or qualitative PCR for the JC virus on extracts from CSF provides the necessary supportive data to confirm the diagnosis of PML in the appropriate clinical setting [59]. PCR for the JC virus in this setting has largely replaced brain biopsy for the diagnosis of PML, just as temporal lobe biopsy for the diagnosis of herpes simplex virus (HSV) meningoencephalitis has been replaced by HSV PCR performed on CSF specimens. The JC virus, like the BK virus, has also been noted to be a cause of urethral stenosis. There have been rare instances of JC nephropathy in renal transplant recipients [60]. Therefore, some have suggested that this condition should be termed polyomavirus nephropathy. JC nephropathy should be suspected in patients with decreasing renal function, but with undetectable to very low BK viral loads in the plasma. An infection with a BK variant that is not detected or not quantitated well by the BK virus PCR should also be suspected in this scenario. The histopathologic assessment of the kidney biopsy for evidence is critically important in this type of situation. If JC nephropathy is suspected, then quantitative PCR for the JC virus should be performed on plasma specimens. Similarly, the detection and/or quantitation of the JC virus in the urine may be used to disclose the presence and quantity of this virus in patients with ureteral stenosis.
Other Polyomaviruses The Merkel cell carcinoma (MCC) polyomavirus has received a great deal of study recently, given the etiologic association of this virus with MCC. MCC is a neuroendocrine carcinoma that arises from the cell that bears the same name. These carcinomas occur most commonly in transplant recipients. However, even in this patient population the incidence of disease is low. The lesion is characterized by neoplastic cells with neuroendocrine features that in many ways resemble small cell carcinoma. The presence of immunoreactivity to cytokeratin 20 is useful in differentiating MCC from small cell carcinoma. Although the MCPV has been strongly associated with MCC, there are MCCs that lack the presence of the MCPV and likely have arisen through other mechanisms [61]. Therefore, the absence of the MCPV does not exclude the diagnosis of MCC. Currently there is no role for testing for this virus in routine medical practice laboratory.
There is currently no documented clinical utility in testing the numerous other polyomaviruses that have been described.
LIMITATIONS OF TESTING The presence of PCR inhibitors may occur in any clinical specimen, but these are minimal in specimens that are extracted using modern techniques for the recovery of DNA. The possibility of specimento-specimen contamination or extract-to-extract contamination predominantly concerns predominantly the processing and testing of urine specimen extracts where the viral load may be very high. These should not be processed in the same run as plasma specimens. Hayden et al. used results from a national proficiency testing provider to study factors that contributed to variability in quantitative PCR results for viral assays [62]. They reiterate the importance of standardized quantitative control material, but explore other potential factors. They found that the selection of the quantitative calibrator, the use of commerciallyprepared primers and probes, and the target gene selected for amplification all were associated with variability. The differences in these variables between different laboratories are in part responsible for interlaboratory variability of BK testing, which has been described [62]. Genomic sequence variability occurs in the BK virus and has an impact on the detection and reliable quantitation of this virus. Randhawa et al. studied the variability of the VP1 gene sequence, which was their target for a hydrolysis probe PCR. Of 184 publically-available sequences for review, only 44% (n 5 81) demonstrated a perfect match for either primers or probes [63]. Not surprisingly, they determined that BK genotypes with mismatches at the primers and probe sites would not be detected unless they were present in high concentrations (ie, the sensitivity of the assay was decreased for these subtypes). Furthermore, they reported that the calculated viral loads would be between 0.57% and 3.26% of the expected values for BK strains with greater than or equal to two mismatches [63]. Similarly, Hoffman et al. compared seven different BK virus quantitative PCR assays and reported substantial disagreement [64]. Like others, this was due to primer and probe mismatches. However, they noted that this primarily occurred with subtypes III and IV. The seven assays were described as typically uniform for the more common subtypes (ie, Ia, V, and VI). Others, similarly, have reported the failure to detect BK virus strains secondary to mismatches, which has necessitated assay redesign [23,24]. It has been a goal of many to design
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an assay that will detect all BK subtypes equally well [24,64]. Therefore, it is recommended that target sequences are annually reviewed against newly available sequences [24]. New subtypes of the BK virus are still being described [65]. Therefore, it remains important to continually assess the ability of the assays in routine use to detect and adequately quantitate these subtypes. It is important to consider the genomic variability that is known to occur among BK subtypes for particular genes and to consider this factor, if the BK virus is not detected or the viral load is inconsistent with the clinical findings [63]. The quantities of polyomavirus shed into the urine are often logarithmically greater than the quantities detected in the plasma. Therefore, an important practical consideration is to not mix specimen types in quantitative PCR runs. A contamination event from a specimen or a urine extract with a very high BK viral load could produce misleading, false-positive BK viral load results in nearby extracts from plasma. It is our recommendation that if both plasma and urine specimens are to be assessed for the presence and quantity of the BK virus, then these should be processed separately and the PCR assays run at different times. The cross-reactivity of in situ hybridization probes and immunohistochemistry reagents has been described. This is expected for the immunohistochemistry product, since the commercially-available antibody is directed against antigens on the SV40 polyomavirus. Therefore, these reagents should be considered sensitive for the visual detection of polyomavirus in tissue preparations that have been appropriately processed, but nonspecific with respect to which type of polyomavirus is present. This is usually not an issue since the polyomavirus associated with a demyelinating disease in the brain would be the JC virus and the polyomavirus associated with renal histopathology is most likely, but not exclusively, the BK virus. Therefore, we recommend correlating the results of in situ hybridization or immunohistochemistry studies with the results of PCR assay that target the respective polyomaviruses.
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[23] Lamontagne B, Girard N, Boucher A, Labbe AC. Improved detection and quantitation of human BK polyomavirus by PCR assay. J Clin Microbiol 2011;49:2778. [24] Dumoulin A, Hirsch HH. Reevaluating and optimizing polyomavirus BK and JC real-time PCR assays to detect rare sequence polymorphisms. J Clin Microbiol 2011;49:1382 8. [25] Funahashi Y, Iwata S, Ito Y, Kojima S, Yoshikawa T, Hattori R, et al. Multiplex real-time PCR assay for simultaneous quantification of BK polyomavirus, JC polyomavirus, and adenovirus DNA. J Clin Microbiol 2010;48:825 30. [26] Tang YW, Sefers SE, Li H, Kohn DJ, Procop GW. Comparative evaluation of three commercial systems for nucleic acid extraction from urine specimens. J Clin Microbiol 2005;43:4830 3. [27] Bista BR, Ishwad C, Wadowsky RM, Manna P, Randhawa PS, Gupta G, et al. Development of a loop-mediated isothermal amplification assay for rapid detection of BK virus. J Clin Microbiol 2007;45:1581 7. [28] Held TK, Biel SS, Nitsche A, Kurth A, Chen S, Gelderblom HR, et al. Treatment of BK virus-associated hemorrhagic cystitis and simultaneous CMV reactivation with cidofovir. Bone Marrow Transplant 2000;26:347 50. [29] Smith RD, Galla JH, Skahan K, Anderson P, Linnemann Jr. CC, Ault GS, et al. Tubulointerstitial nephritis due to a mutant polyomavirus BK virus strain, BKV(Cin), causing end-stage renal disease. J Clin Microbiol 1998;36:1660 5. [30] Hogan TF, Borden EC, McBain JA, Padgett BL, Walker DL. Human polyomavirus infections with JC virus and BK virus in renal transplant patients. Ann Intern Med 1980;92:373 8. [31] Nickeleit V, Singh HK, Mihatsch MJ. Latent and productive polyomavirus infections of renal allografts: morphological, clinical, and pathophysiological aspects. Adv Exp Med Biol 2006;577:190 200. [32] Nickeleit V, Mihatsch MJ. Polyomavirus nephropathy in native kidneys and renal allografts: an update on an escalating threat. Transplant Int 2006;19:960 73. [33] Awadalla Y, Randhawa P, Ruppert K, Zeevi A, Duquesnoy RJ. HLA mismatching increases the risk of BK virus nephropathy in renal transplant recipients. Am J Transplant 2004;4(10):1691 6. [34] Priftakis P, Bogdanovic G, Tyden G, Dalianis T. Polyomaviruria in renal transplant patients is not correlated to the cold ischemia period or to rejection episodes. J Clin Microbiol 2000;38:406 7. [35] Marshall WF, Telenti A, Proper J, Aksamit AJ, Smith TF. Rapid detection of polyomavirus BK by a shell vial cell culture assay. J Clin Microbiol 1990;28:1613 15. [36] Hogan TF, Padgett BL, Walker DL, Borden EC, McBain JA. Rapid detection and identification of JC virus and BK virus in human urine by using immunofluorescence microscopy. J Clin Microbiol 1980;11:178 83. [37] Hirsch HH, Brennan DC, Drachenberg CB, Ginevri F, Gordon J, Limaye AP, et al. Polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations. Transplantation 2005;79:1277 86. [38] Randhawa P, Ho A, Shapiro R, Vats A, Swalsky P, Finkelstein S, et al. Correlates of quantitative measurement of BK polyomavirus (BKV) DNA with clinical course of BKV infection in renal transplant patients. J Clin Microbiol 2004;42:1176 80. [39] Pang XL, Doucette K, LeBlanc B, Cockfield SM, Preiksaitis JK. Monitoring of polyomavirus BK virus viruria and viremia in renal allograft recipients by use of a quantitative real-time PCR assay: one-year prospective study. J Clin Microbiol 2007;45:3568 73. [40] Schechter T, Liebman M, Gassas A, Ngan BY, Navarro OM. BK virus-associated hemorrhagic cystitis presenting as mural nodules in the urinary bladder after hematopoietic stem cell transplantation. Pediatr Radiol 2010;40 1430-1423.
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[41] Bogdanovic G, Priftakis P, Giraud G, Kuzniar M, Ferraldeschi R, Kokhaei P, et al. Association between a high BK virus load in urine samples of patients with graft-versus-host disease and development of hemorrhagic cystitis after hematopoietic stem cell transplantation. J Clin Microbiol 2004;42:5394 6. [42] Dropulic LK, Jones RJ. Polyomavirus BK infection in blood and marrow transplant recipients. Bone Marrow Transplant 2008;41:11 18. [43] Galan A, Rauch CA, Otis CN. Fatal BK polyoma viral pneumonia associated with immunosuppression. Hum Pathol 2005;36:1031 4. [44] Sandler ES, Aquino VM, Goss-Shohet E, Hinrichs S, Krisher K. BK papova virus pneumonia following hematopoietic stem cell transplantation. Bone Marrow Transplant 1997;20:163 5. [45] Cubukcu-Dimopulo O, Greco A, Kumar A, Karluk D, Mittal K, Jagirdar J. BK virus infection in AIDS. Am J Surg Pathol 2000;24:145 9. [46] Procop GW, Kohn DJ, Johnson JE, Li HJ, Loyd JE, YenLieberman B, et al. BK and JC polyomaviruses are not associated with idiopathic pulmonary fibrosis. J Clin Microbiol 2005;43:1385 6. [47] Major EO, Amemiya K, Tornatore CS, Houff SA, Berger JR. Pathogenesis and molecular biology of progressive multifocal leukoencephalopathy, the JC virus-induced demyelinating disease of the human brain. Clin Microbiol Rev 1992;5 49 73. [48] Astrom KE, Mancall EL, Richardson Jr. EP. Progressive multifocal leukoencephalopathy. Brain 1958;81:93 127. [49] Richardson Jr. EP. Progressive multifocal leukoencephalopathy. N Engl J Med 1961;265:815 23. [50] Padgett BL, Walker DL. Virologic and serologic studies of progressive multifocal leukoencephalopathy. Prog Clin Biol Res 1983;105:107 17. [51] Brooks BR, Walker DL. Progressive multifocal leukoencephalopathy. Neurol Clin 1984;2:299 313. [52] Di Lernia V. Progressive multifocal leukoencephalopathy and antipsoriatic drugs: assessing the risk of immunosuppressive treatments. Int J Dermatol 2010;49:631 5. [53] Warnke C, Menge T, Hartung HP, Racke MK, Cravens PD, Bennett JL, et al. Natalizumab and progressive multifocal leukoencephalopathy: what are the causal factors and can it be avoided? Arch Neurol 2010;67:923 30. [54] Lysandropoulos AP, Du Pasquier RA. Demyelination as a complication of new immunomodulatory treatments. Curr Opin Neurol 2010;23:226 33. [55] Bellizzi A, Barucca V, Fioriti D, Colosimo MT, Mischitelli M, Anzivino E, et al. Early years of biological agents therapy in Crohn’s disease and risk of the human polyomavirus JC reactivation. J Cell Physiol 2010;224:316 26. [56] Clifford DB, De Luca A, Simpson DM, Arendt G, Giovannoni G, Nath A. Natalizumab-associated progressive multifocal leukoencephalopathy in patients with multiple sclerosis: lessons from 28 cases. Lancet Neurol 2010;9:438 46. [57] Tan CS, Koralnik IJ. Progressive multifocal leukoencephalopathy and other disorders caused by JC virus: clinical features and pathogenesis. Lancet Neurol 2010;9:425 37. [58] Gheuens S, Pierone G, Peeters P, Koralnik IJ. Progressive multifocal leukoencephalopathy in individuals with minimal or occult immunosuppression. J Neurol Neurosurg Psychiatry 2010;81:247 54. [59] Hammarin AL, Bogdanovic G, Svedhem V, Pirskanen R, Morfeldt L, Grandien M. Analysis of PCR as a tool for detection of JC virus DNA in cerebrospinal fluid for diagnosis of progressive multifocal leukoencephalopathy. J Clin Microbiol 1996;34:2929 32.
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[60] Drachenberg CB, Hirsch HH, Papadimitriou JC, Gosert R, Wali RK, Munivenkatappa R, et al. Polyomavirus BK versus JC replication and nephropathy in renal transplant recipients: a prospective evaluation. Transplantation 2007;84:323 30. [61] Miner AG, Patel RM, Wilson DA, Procop GW, Minca EC, Fullen DR, et al. Cytokeratin 20-negative Merkel cell carcinoma is infrequently associated with the Merkel cell polyomavirus. Mod Pathol 2015;28:498 504. [62] Hayden RT, Yan X, Wick MT, Rodriguez AB, Xiong X, Ginocchio CC, et al. Factors contributing to variability of quantitative viral PCR results in proficiency testing samples: a multivariate analysis. J Clin Microbiol 2012;50:337 45.
[63] Randhawa P, Kant J, Shapiro R, Tan H, Basu A, Luo C. Impact of genomic sequence variability on quantitative PCR assays for diagnosis of polyomavirus BK infection. J Clin Microbiol 2011;49:4072 6. [64] Hoffman NG, Cook L, Atienza EE, Limaye AP, Jerome KR. Marked variability of BK virus load measurement using quantitative real-time PCR among commonly used assays. J Clin Microbiol 2008;46:2671 80. [65] Kapusinszky B, Chen SF, Sahoo MK, Lefterova MI, Kjelson L, Grimm PC, et al. BK polyomavirus subtype III in a pediatric renal transplant patient with nephropathy. J Clin Microbiol 2013;51:4255 8.
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10 Molecular Testing for Polyomaviruses G.W. Procop and B. Yen-Lieberman Section of Clinical Microbiology, Department of Laboratory Medicine, Cleveland Clinic, Cleveland, OH, United States
INTRODUCTION Polyomaviruses are small (45 nm), nonenveloped viruses with icosahedral nucleocapsids. The genetic material is double-stranded DNA, with genomes that range 4.5 5.5 kb. The Polyomaviridae contains two groups: the Orthopolyomaviruses and the Wukipolyomaviruses [1,2]. The Orthopolyomavirus genus contains the three most important human pathogens: the BK virus, the JC virus, and the Merkel cell polyomavirus (MCPV) [2]. It also contains the trichodysplasia spinulosa-associated polyomavirus. The BK and JC viruses received their curious nomenclature from the initials of the patients from which they were originally isolated [3,4]. The Wukipolyomavirus genus contains the WU and KI viruses for which it was named, as well as the Human polyomaviruses 6, 7, 8, 9, and 10. Although the WU and KI polyomaviruses have been recovered from the respiratory, plasma, and/or urine specimens of transplant recipients, these have not been associated with disease [5]. The breadth of the Polyomaviridae is undetermined and the discovery of new polyomaviruses is expected. The most important etiologic agents of disease in this group, the BK and JC viruses, will command the attention of the majority of this chapter. Human polyomaviruses viruses cause minimal to no disease in the immunocompetent host. Serologic studies suggest that the majority of humans are infected early in life. Infections are either asymptomatic or produce subclinical disease, possibly involving the respiratory tract. The precise mode of transmission is a matter of debate. Some have advocated a respiratory route of infection for the BK virus, since BK viral DNA has been detected in tonsillar tissues [6]. However, others have studied body fluids, such as saliva, and the respiratory secretions of children with
Diagnostic Molecular Pathology DOI: http://dx.doi.org/10.1016/B978-0-12-800886-7.00010-8
upper respiratory tract infections for the presence of the BK and JC viruses, and these fluids did not harbor the viruses [7]. Uro oral, fecal oral, and transplacental transmission have also been postulated as modes of transmission, with the former seemingly very feasible given the permissive nature of urothelial cells for the replication of these viruses [8]. Regardless of the mode of transmission, these viruses are commonly found in the general population. The majority of children (ie, between 65% and 90%) are seropositive for the BK virus by the age of 10 [9]. Seropositivity rates for BK rise until around 40 years of age and then decrease slightly. The JC virus is also highly prevalent with 50 80% of the human population demonstrating serologic evidence of prior infection [10]. The seroprevalance of the other polyomaviruses has not been extensively studied. However, it has been demonstrated that up to 77% of the general population have been exposed to the MCPV [11]. After initial infection, a transient viremia likely ensues whereby the virus reaches the destination tissues for latency. It has been hypothesized that this dissemination could occur through the infection of mononuclear leukocytes in which BK virus DNA has been detected [12]. It is not known for certain whether these viruses become truly latent in the host cells or maintain subclinical (ie, minimal maintenance) replication once the viruses reach their destination tissues. It is known, however, that asymptomatic replication of both the BK and JC viruses occurs in the urothelium (ie, the lining epithelium of the bladder, ureters, and renal pelvis), with subsequent shedding of the virus into the urine [13]. Asymptomatic shedding of the BK virus has been documented in up to 10% of healthy adults [8]. Similarly, 25% of pregnant women have been shown to asymptomatically shed the BK virus in the urine [14]. Age-related shedding of the BK virus
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has been reported, with shedding occurring less frequently in individuals less than 30 years old and gradually increasing in individuals greater than or equal to 30 years old [15]. This asymptomatic shedding of virus may contribute to the transmission of the virus to those who were previously unexposed and is supportive of a uro oral route of infection.
MOLECULAR TARGETS The molecular targets used for the detection of the BK and JC viruses include the VP1 gene, the VP2 gene, and the T antigen gene. Variations in the VP1 sequence are associated with different subtypes [16]. Primer sets for the quantitative assessment of BK virus are commercially available (Table 10.1), as are numerous laboratory-developed tests. Similarly, laboratorydeveloped tests have been used for the detection of the JC virus and MCPV. The RealStar JC virus (Altona, Hamburg, Germany), which targets the large T antigen gene, is available. The linear range claimed is 1000 1012 copies/mL. Similarly, the GeneProof JC Virus assay (GeneProof, Brno, Czech Republic) that targets the junction of the VP1 and VP2 genes is also available. They claim a linear range from 528 to 1010 copies/mL. GeneProof also offers a combined BK/JC assay.
MOLECULAR TECHNOLOGIES Quantitative PCR is the most commonly used method in the assessment of clinical specimens for the detection and quantitation of BK and JC polyomaviruses. This has been done in conjunction with a variety of probe types. Less commonly, qualitative PCR is used to determine the presence of these viruses. In situ hybridization and immunohistochemistry are used in the assessment of biopsy specimens for these viruses [17,18]. Electron microscopy was used in the distant past, but it has largely been replaced by these TABLE 10.1
technologies [19]. However, electron microscopy remains an important tool for discovery and in instances wherein these other technologies fail or inconsistent results are produced. Quantitative PCR is used predominantly for the detection and quantitative monitoring of the BK virus in plasma. A wide variety of PCR assays have been described for the detection and/or quantitation of the BK virus. Many of these are laboratory-developed tests, but some are commercially available [20 25]. These assays usually employ some type of nucleic acid extraction method prior to PCR amplification. Although all aspects of tests need to undergo a thorough assessment during assay validation, most commercially-available extraction methods are robust in the removal of inhibitors and are largely comparable with respect to DNA yield [26]. The quantity of virus present in the plasma is important, since higher values are more predictive of BK nephropathy. Qualitative PCR for the BK virus in urine may be used to identify (or screen out) patients for BK nephropathy, but this method is not commonly used in this manner. However, the qualitative detection of the JC virus in the cerebrospinal fluid (CSF) of patients with progressive multifocal leukoencephalopathy (PML) is sufficient to support this diagnosis in the appropriate clinical setting, which includes supportive radiological findings. Given the relative simplicity of converting a qualitative rapid cycle PCR assay into a quantitative assay, quantitative values are often reported when the JC virus is detected in CSF or other body fluids. Beyond quantitative PCR assays, loop-mediated isothermal amplification (LAMP) techniques have been developed for the detection of the BK virus [27]. Although commercially-available LAMP methods for BK viruses are not available, this type of technology is attractive because it uses standard laboratory equipment, has an acceptable level of sensitivity, and may be used without DNA extraction and a thermocycler. Such a technology could conceivably be used to inexpensively screen for the presence of the BK virus in
Commercially-Available BK Virus Assays
Vendor/Assay
Assay specifics
Target region
Dynamic range (copies/mL)
ELiTechMGB Alert BK Virus Primers
MGB probes
Large T antigen
12.5 3 101 to 12.5 3 106
Focus DiagnosticsSimplexa BKV Kit
Scorpion
VP2
5.1 3 102 to 1.0 3 108
Luminex (Eragen)MultiCode BK Virus Primers
MultiCode
Large T antigen
5.0 3 102 to 5.0 3 106
QiagenArtus BK Virus RG Kit
Real-time PCR
Large T antigen
5.0 3 102 to 5.0 3 106
GeneProof BK Virus Kit
Real-time PCR
VP1/VP2 junction
5.96 3 102 to 1.0 3 1010
RealStar BK virus (Altona)
Real-time PCR
Unknown
1.0 3 103 to 1.0 3 1012
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at-risk populations. In addition to monoplex assays, multiplex assays have been developed. Some target only the most common polyomaviruses (ie, BK and JC), whereas others target additional viruses that may cause disease in the immunocompromised host, such as adenovirus [21,25]. Fluorescence resonance energy transfer probe sets that utilize broad-range polyomavirus primers have also been used to detect and differentiate the BK and JC viruses in a single reaction (Fig. 10.1) [22]. Such an assay may be useful in institutions that screen urine specimens, since either virus may be present. In addition to these, qualitative seminested PCR assays have also been described [28]. In situ hybridization and immunohistochemistry are technologies largely used by anatomic pathologists for the detection of these and other viruses in histologic preparations. The availability of these stains is important to confirm the nature of intranuclear inclusions thought to likely represent polyomaviral inclusions. Other viruses, such as adenovirus and cytomegalovirus, can cause intranuclear inclusions in the same at-risk patient population and are represented in the differential diagnosis. These tools are particularly useful when the intranuclear inclusions are not typical. Although biopsy suffers from sampling error, the demonstration of BK viral inclusions associated with interstitial nephritis is the gold standard for the diagnosis of BK nephropathy. Fortunately, tissue is regularly available for assessment, since renal biopsy is necessary to monitor and/or assess for transplant rejection. However, the same is not always true regarding brain biopsy and the diagnosis of PML. Tissue is not readily available in these instances and most would like to avoid a brain biopsy if at all possible. Although the
FIGURE 10.1 This post-amplification melt curve was produced following real-time PCR amplification using broad-range polyomavirus primers. Fluorescence resonance energy transfer probes that hybridized with complete complementarity with the BK virus amplicon had two mismatches with the JC virus amplicon and several mismatches with the SV40 virus amplicon. This demonstrates the qualitative detection and differentiation of the medically-important BK and JC polyomaviruses in a single assay.
demonstration of the JC virus in infected oligodendroglial cells in immunocompromised patients with a demyelinating disease may be the gold standard for the diagnosis of PML, most are satisfied with the demonstration of the JC virus in the CSF to support the diagnosis of PML in a patient with the appropriate clinical and radiological findings.
CLINICAL UTILITY Although most individuals become infected with a polyomavirus sometime during life, serious symptomatic diseases caused by polyomaviruses occur in immunocompromised patients. The type of immunologic compromise and the degree to which the immune system is suppressed are directly related to the type of polyomavirus infection that is most likely to occur, and in some instances with the severity of infection. The main types of diseases caused by polyomaviruses, as well as associated risk factors and the clinical utility of testing, will be discussed here.
The BK Virus The BK virus is responsible for two main types of disease. It causes nephropathy that can result in graft failure in renal transplant recipients and hemorrhagic cystitis in patients undergoing stem cell transplantation. Early studies described the severe tubulointerstitial nephritis caused by the BK virus in renal allografts [29]. Serologic studies are supportive of the hypothesis that symptomatic disease is most commonly the result of reactivation of latent, endogenous virus rather than primary infection [30,31]. BK nephropathy most commonly occurs in the transplanted kidney, but infection of the native kidney has also been described [10,32]. Disease usually presents 10 13 months after transplantation [10]. Risk factors include a seropositive donor status and/or a seronegative recipient status, older patient age, male gender, ischemic or immunologic injury, and the degree of HLA mismatch. This latter factor is likely more an indicator of the degree of immunosuppression that will be necessary to avoid transplant rejection. Otherwise stated, the greater the HLA mismatch, the higher the number of rejection episodes that are likely to occur, which will necessitate antilymphocyte therapy to control rejection, which facilitates the emergence of the BK virus [33]. However, there is debate regarding the contribution of either cold ischemia or rejection episodes to BK virus reactivation [34]. The diagnosis and monitoring of patients at risk for BK nephropathy is multifactorial and has evolved with
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the introduction of molecular methods. The BK virus can be grown in cell culture or shell vial assay, or directly detected in the urine by immunofluroescent methods. These methods, which were used in the past, have been replaced by quantitative PCR, which is faster, more specific, and less labor intense [35,36]. Quantitative PCR for the BK polyomavirus is predominantly performed on extracts from plasma specimens from renal transplant recipients. This is usually performed periodically throughout the lives of these patients and the presence and quantity of BK virus are measured and trends are established. A baseline should be established relatively soon after transplantation. Testing should be done with the greatest frequency through the 10- to 13-month window after transplantation when the incidence of disease is greatest, then at a regular interval. A recommended protocol for the screening of renal transplant recipients is to screen the urine every 3 months and the plasma every 1 3 months for the first 2 years after transplantation or when graft dysfunction occurs [37]. An increasing viral load in the presence of decreasing renal function correlates with BK nephropathy [38]. The same correlation is not as clear for high BK viral loads in the urine, since the BK virus replicates in the urothelium and relatively high viral loads may be present in the absence of renal involvement. However, Pang et al. undertook a 1-year prospective study evaluating urine and plasma specimens from renal transplant recipients [39]. They found that as the viral load in the urine increased from 7.0 to 10.0 log(10) copies/mL the percentage of patients with viremia increased from 22% to 100%, respectively. These authors suggested that plasma viral load testing could be reserved for those patients who have greater than or equal to 7.0 log(10) copies/mL of virus in the urine. The absence of BK virus in the urine has a high negative predictive value for BK nephropathy. The routine use of BK viral load testing represents an important advancement in the diagnosis and monitoring of patients for BK nephropathy. It is particularly important since it has direct implications on the immunosuppressive regimen given to the patient. Decreased kidney function may occur for a variety of reasons in the renal transplant recipient. In addition to BK nephropathy, decreased renal function may occur secondary to transplant allograft rejection. The differentiation of these conditions is critical, since transplant rejection is addressed by increasing the immunosuppression, whereas BK nephropathy is addressed by decreasing the immunosuppression. The BK virus is the most common cause of hemorrhagic cystitis in hematopoietic stem cell transplant recipients [40,41]. Hemorrhagic cystitis usually occurs
2 weeks after transplant and may affect up to 25% of these patients [42]. Hemorrhagic cystitis ranges in severity from mild (only microscopic hematuria) to severe (clots of blood become obstructive to the flow of urine). There is a great deal of morbidity associated with moderate to severe disease. When the BK virus is the cause of hemorrhagic cystitis the BK viral loads in the urine are extremely high and may be as great as 104 copies/mL. The absence of significant quantities of the BK virus in the urine of a patient with hemorrhagic cystitis is suggestive of another etiology. Other etiologies include infections by adenovirus or cytomegalovirus, as well as the chemotherapeutic treatment, albeit this latter cause has become less common as an etiology due to preventive measures taken to prepare the patient for therapy [1]. The BK virus has been associated with ureteral stenosis, which is not surprising given the locus of viral replication. Additionally, it has only rarely been associated with other diseases, such as native kidney infection in recipients of other types of transplants (eg, heart and stem cell), pneumonia, and encephalitis [43 45]. Individuals have sought associations of these viruses with other diseases, but these searches have largely been unfruitful. For example, an examination of lung tissue extracts from 33 patients with documented idiopathic pulmonary fibrosis found no evidence for the presence of either the BK or JC virus by PCR [46].
The JC Virus The JC virus causes PML [47]. This disease was associated with a variety of malignancies or sarcoidosis prior to the HIV epidemic [48,49]. Patients with progressive HIV infection (ie, AIDS) are at high risk of PML. The CD4 1 T-cell reduction caused by HIV infection produces the perfect milieu for the emergence of the JC virus in the central nervous system. The lytic infection of myelin-producing oligodendroglial cells is the cause of this demyelinating disease. As with BK nephropathy, serologic studies are supportive of reactivation of endogenous virus rather than a primary infection [50,51]. This disease has become significantly less common in resource-rich countries wherein patients have access to highly active antiretroviral therapy. A new group of patients are now recognized as atrisk for PML. This group consists of patients who undergo immunotherapy with biologics for a variety of autoimmune conditions. PML has been reported in patients receiving natalizumab, for multiple sclerosis, efalizumab for psoriasis, and infliximab for Crohn’s disease and other autoimmune diseases [52 57]. In
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rare instances, PML may present in patients with minimal to occult immunosuppression, the possibility of this disease should remain in the differential diagnosis of patients with the appropriate clinical and radiological findings [58]. Quantitative or qualitative PCR for the JC virus on extracts from CSF provides the necessary supportive data to confirm the diagnosis of PML in the appropriate clinical setting [59]. PCR for the JC virus in this setting has largely replaced brain biopsy for the diagnosis of PML, just as temporal lobe biopsy for the diagnosis of herpes simplex virus (HSV) meningoencephalitis has been replaced by HSV PCR performed on CSF specimens. The JC virus, like the BK virus, has also been noted to be a cause of urethral stenosis. There have been rare instances of JC nephropathy in renal transplant recipients [60]. Therefore, some have suggested that this condition should be termed polyomavirus nephropathy. JC nephropathy should be suspected in patients with decreasing renal function, but with undetectable to very low BK viral loads in the plasma. An infection with a BK variant that is not detected or not quantitated well by the BK virus PCR should also be suspected in this scenario. The histopathologic assessment of the kidney biopsy for evidence is critically important in this type of situation. If JC nephropathy is suspected, then quantitative PCR for the JC virus should be performed on plasma specimens. Similarly, the detection and/or quantitation of the JC virus in the urine may be used to disclose the presence and quantity of this virus in patients with ureteral stenosis.
Other Polyomaviruses The Merkel cell carcinoma (MCC) polyomavirus has received a great deal of study recently, given the etiologic association of this virus with MCC. MCC is a neuroendocrine carcinoma that arises from the cell that bears the same name. These carcinomas occur most commonly in transplant recipients. However, even in this patient population the incidence of disease is low. The lesion is characterized by neoplastic cells with neuroendocrine features that in many ways resemble small cell carcinoma. The presence of immunoreactivity to cytokeratin 20 is useful in differentiating MCC from small cell carcinoma. Although the MCPV has been strongly associated with MCC, there are MCCs that lack the presence of the MCPV and likely have arisen through other mechanisms [61]. Therefore, the absence of the MCPV does not exclude the diagnosis of MCC. Currently there is no role for testing for this virus in routine medical practice laboratory.
There is currently no documented clinical utility in testing the numerous other polyomaviruses that have been described.
LIMITATIONS OF TESTING The presence of PCR inhibitors may occur in any clinical specimen, but these are minimal in specimens that are extracted using modern techniques for the recovery of DNA. The possibility of specimento-specimen contamination or extract-to-extract contamination predominantly concerns predominantly the processing and testing of urine specimen extracts where the viral load may be very high. These should not be processed in the same run as plasma specimens. Hayden et al. used results from a national proficiency testing provider to study factors that contributed to variability in quantitative PCR results for viral assays [62]. They reiterate the importance of standardized quantitative control material, but explore other potential factors. They found that the selection of the quantitative calibrator, the use of commerciallyprepared primers and probes, and the target gene selected for amplification all were associated with variability. The differences in these variables between different laboratories are in part responsible for interlaboratory variability of BK testing, which has been described [62]. Genomic sequence variability occurs in the BK virus and has an impact on the detection and reliable quantitation of this virus. Randhawa et al. studied the variability of the VP1 gene sequence, which was their target for a hydrolysis probe PCR. Of 184 publically-available sequences for review, only 44% (n 5 81) demonstrated a perfect match for either primers or probes [63]. Not surprisingly, they determined that BK genotypes with mismatches at the primers and probe sites would not be detected unless they were present in high concentrations (ie, the sensitivity of the assay was decreased for these subtypes). Furthermore, they reported that the calculated viral loads would be between 0.57% and 3.26% of the expected values for BK strains with greater than or equal to two mismatches [63]. Similarly, Hoffman et al. compared seven different BK virus quantitative PCR assays and reported substantial disagreement [64]. Like others, this was due to primer and probe mismatches. However, they noted that this primarily occurred with subtypes III and IV. The seven assays were described as typically uniform for the more common subtypes (ie, Ia, V, and VI). Others, similarly, have reported the failure to detect BK virus strains secondary to mismatches, which has necessitated assay redesign [23,24]. It has been a goal of many to design
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an assay that will detect all BK subtypes equally well [24,64]. Therefore, it is recommended that target sequences are annually reviewed against newly available sequences [24]. New subtypes of the BK virus are still being described [65]. Therefore, it remains important to continually assess the ability of the assays in routine use to detect and adequately quantitate these subtypes. It is important to consider the genomic variability that is known to occur among BK subtypes for particular genes and to consider this factor, if the BK virus is not detected or the viral load is inconsistent with the clinical findings [63]. The quantities of polyomavirus shed into the urine are often logarithmically greater than the quantities detected in the plasma. Therefore, an important practical consideration is to not mix specimen types in quantitative PCR runs. A contamination event from a specimen or a urine extract with a very high BK viral load could produce misleading, false-positive BK viral load results in nearby extracts from plasma. It is our recommendation that if both plasma and urine specimens are to be assessed for the presence and quantity of the BK virus, then these should be processed separately and the PCR assays run at different times. The cross-reactivity of in situ hybridization probes and immunohistochemistry reagents has been described. This is expected for the immunohistochemistry product, since the commercially-available antibody is directed against antigens on the SV40 polyomavirus. Therefore, these reagents should be considered sensitive for the visual detection of polyomavirus in tissue preparations that have been appropriately processed, but nonspecific with respect to which type of polyomavirus is present. This is usually not an issue since the polyomavirus associated with a demyelinating disease in the brain would be the JC virus and the polyomavirus associated with renal histopathology is most likely, but not exclusively, the BK virus. Therefore, we recommend correlating the results of in situ hybridization or immunohistochemistry studies with the results of PCR assay that target the respective polyomaviruses.
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