Impact of genomic sequence variability on quantitative PCR assays for diagnosis of polyomavirus BK infection

Knowledge of polyomavirus BK (BKV) genomic diversity has greatly expanded. The implications of BKV DNA sequence variation for the performance of molecular diagnostic assays is not well studied. We analyzed 184 publically available VP-1 sequences encompassing the BKV genomic region targeted by an in-house quantitative hydrolysis probe-based PCR assay. A perfect match with the PCR primers and probe was seen in 81 sequences. One Dun and 13 variant prototype oligonucleotides were synthesized as artificial targets to determine how they affected the performance of PCR. The sensitivity of detection of BKV in the PCR assay was a function of the viral genotype. Prototype 1 (BKV Dun) could be reliably detected at concentrations as low as 10 copies/μl. However, consistent detection of all BKV variants was possible only at concentrations of 10,000 copies/μl or higher. For BKV prototypes with 2 or more mismatches (representing genotype IV, genotype II, and genotype 1c strains), the calculated viral loads were 0.57 to 3.26% of the expected values. In conclusion, variant BKV strains lower the sensitivity of detection and may have a substantial effect on quantitation of the viral load. Physicians need to be cognizant of these effects when interpreting the results of quantitative PCR testing in transplant recipients, particularly if there is a discrepancy between the clinical impression and the measured viral load.     

Comparison of three real-time PCR for the quantification of polyomavirus BK

BackgroundReactivation of latent polyomavirus BK is associated with nephropathy (PVAN) after renal transplantation. BK viral load determinations are a highly sensitive and specific method for predicting risk for PVAN.Objectives and study designThe performance of three real-time PCR for BKV DNA quantification (MultiCode^®-RTx BK virus ASR [MC-RTx], MGB-Alert BKV ASR [MGB] and a laboratory developed assay [LDA]) were evaluated against a conventional PCR (test of record, TOR) in terms of linearity, dynamic range, and accuracy.ResultsThe LOD (log₁₀ copies/ml) were 2.0, 2.0 and 3.0 for MC-RTx, MGB and LDA, respectively with a commercial plasma panel and 2.0, 2.6 and 3.5 with a urine panel. These assays demonstrated excellent linearity (r² = 1.0) and reproducibility (CV range = 0.7–20.4%, 0.9–13.2%, and 0.5–13%, respectively). In an analysis of 100 clinical specimens, all 76 samples defined as true positive for BKV DNA (positive by two or more methods or a recent history of positivity) were detected with MC-RTx, while only 64 were detected with MGB and 55 were detected with LDA. BKV DNA was not detected by any method in the true negative specimens. Based on these results, the sensitivities were 100% for MC-RTx, 84% for MGB and 72% for LDA. The greatest linear correlation with the mean concentration was observed with MC-RTx (r² = 0.96) with two samples (3%) with greater than 0.5 log₁₀ variance in quantification versus seven (11%) with MGB and ten (18%) with LDA.ConclusionsThese real-time assays for BKV load demonstrated excellent performance characteristics, with the MC-RTx demonstrating the greatest sensitivity.     

Diagnostic assays for polyomavirus JC and progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a devastating and often fatal demyelinating disease of the central nervous system for which effective therapies are lacking. It is caused by the replication of polyomavirus JC (JCV) in the oligodendrocytes and astrocytes leading to their cytolytic death and loss of myelin from the subcortical white matter. While the virus is very common in human populations worldwide, the incidence of the disease is very low and confined almost exclusively to individuals with some form of immunological dysfunction. However, the number of people who constitute the at‐risk population is growing larger and includes individuals with HIV‐1/AIDS and patients receiving immunomodulatory therapies such as multiple sclerosis patients treated with natalizumab. Further adding to the public health significance of this disease are the difficulties encountered in the diagnosis of PML and the lack of useful biomarkers for PML progression. In this review, we examine the diagnostic assays that are available for different aspects of the JCV life cycle, their usefulness and drawbacks, and the prospects for improvements. Copyright © 2015 John Wiley & Sons, Ltd.     

Optimizing JC and BK polyomavirus IgG testing for seroepidemiology and patient counseling

BackgroundPolyomavirus JC (JCPyV) and BK (BKPyV) can cause significant diseases in immunocompromised patients including nephropathy, hemorrhagic cystitis, and leukoencephalopathy. Recently, JCPyV and BKPyV IgG have been explored as risk predictors in multiple sclerosis and transplant patients, but sensitivity, specificity and quantification issues limit current performance.ObjectiveTo improve JCPyV and BKPyV-specific antibody testing.Study designHealthy blood donor sera (N = 400) were tested at dilutions 1:100, 1:200, and 1:400 for JCPyV- and BKPyV-specific IgG using VP1 virus-like particle (VLP)-based ELISAs normalized to a laboratory reference serum. Normalized optical density 492 nm greater or equal 0.1 in all 3 dilutions was regarded as reactive. Sera with discordant reactivity in at least one dilution were retested after VLP preadsorption.ResultsAt dilutions 1:100, 1:200, and 1:400, IgG reactivity was 74%, 60% and 53% for JCPyV, and 93%, 86% and 74% for BKPyV, respectively. At these dilutions, JCPyV-VLP preadsorption identified 56, 4 and 0 false-positives and 0, 4 and 27 false-negatives, respectively. Dilution-dependent sensitivity was 100%, 98%, and 89%, and specificity 65, 98%, and 100%, respectively. For sera diluted 100-, 200-, and 400-fold, BKPyV-VLP preadsorption identified 28, 1 and 0 false-positives, and 0, 0 and 46 false-negatives, and sensitivity was 100%, 100%, 86%, and specificity 50%, 98%, 100%, respectively.ConclusionFor seroepidemiology studies, normalized JCPyV and BKPyV IgG ELISA at 1:200 serum dilution provides optimal sensitivity and specificity with the lowest false-positive and false-negative rate. For individual risk assessment, dilutions of 100, 200, and 400 combined with preadsorption for low-reactive sera may be most appropriate.     

Effect of sequence polymorphisms on performance of two real-time PCR assays for detection of herpes simplex virus

Herpes simplex virus (HSV) is the most common cause of acquired, sporadic encephalitis in the United States. PCR identification of HSV in spinal fluid has become the diagnostic gold standard due to its sensitivity and potential for speed, replacing other methods such as culture. We developed a real-time PCR assay to detect HSV, using a new type of hybridization probe, the Eclipse probe. In this study, we ran 323 samples (171 positives and 152 negatives) with the Eclipse real-time PCR assay and compared these results with another PCR assay using gel detection. The real-time assay agreed with our reference method for 319 out of the 323 samples tested (99%). Using two different real-time PCR platforms, we discovered that SNPs within the amplicon's probe binding region that are used to distinguish HSV-1 from HSV-2 can decrease assay sensitivity. This problem is potentially a general one for assays using fluorescent probes to detect target amplification in a real-time format. While real-time PCR can be a powerful tool in the field of infectious disease, careful sequence evaluation and clinical validation are essential in creating an effective assay.     

Age-specific seroprevalence of Merkel cell polyomavirus, BK virus, and JC virus

We produced capsids of Merkel cell polyomavirus (MCPyV) in a baculovirus expression system and developed a virus-like particle (VLP) enzyme-linked immunosorbent assay (ELISA). To determine age-specific seroprevalence, serum samples were collected from 947 individuals attending hospital outpatient clinics and ranging in age from 1 to 93 years. To evaluate the association between exposure to MCPyV and Merkel cell cancer (MCC), plasma samples were obtained from 33 MCC patients and 37 controls. MCPyV seroprevalence was 45% in children under 10 years of age, increased to 60% in the next decade of life, and peaked at 81% among those 60 to 69 years of age. Levels of MCPyV capsid antibodies were positively correlated with age (P = 0.007). Virus specificity of MCPyV seroreactivity was supported by competitive inhibition of reactivity by MCPyV VLPs and not by BK polyomavirus (BKPyV) VLPs. MCPyV seroprevalence was greater among MCC patients (91%) than controls (68%; age-adjusted P value, 0.32); the mean level of MCPyV antibodies was also greater (P = 0.04). The age-specific seroprevalence of MCPyV shares with previously known polyomaviruses, BKPyV and JC polyomavirus (JCPyV), evidence of widespread exposure in human populations beginning early in life. MCPyV age-specific seroprevalence also has unique features. Seroprevalence among children is higher than that of JCPyV but lower than that of BKPyV. Among older adults, MCPyV seroprevalence remains high, while that of BKPyV declines and that of JCPyV continues to rise. In agreement with results from other studies, we found an association between MCPyV seropositivity and MCC, and higher levels of serum MCPyV capsid antibodies in MCC patients than in controls.     

Evidence of human polyomavirus BK and JC infection in normal brain tissue.

Infection with the polyomaviruses JC and BK is ubiquitous in the human population and JCV is the only virus associated with the central nervous system disease progressive multifocal leukoencephalopathy. In the attempt to analyze the pathogenesis of polyomavirus infections we asked whether human polyomaviruses invade the brain during persistence. Brain autopsy material from 67 individuals with disorders other than PML was examined for the presence of polyomavirus DNA. Southern blot analysis demonstrated JCV-specific full-length virus genomes in healthy brain tissue in about 20% of the patients. Type-specific analysis with polymerase chain reaction and sequencing confirmed these data. Additionally, the presence of BKV DNA sequences covering an early gene fragment and the control region with flanking early and late protein coding sequences was detected. Cloning of the complete BKV genome from two cases supported the assumption that not only full-length JCV DNA was present in those tissue specimens but also BKV genomes. The data obtained demonstrate that dual infection of the brain with the polyomaviruses JCV and BKV is a common event and give strong evidence that both viruses frequently establish a latent CNS infection.     

Marked variability of BK virus load measurement using quantitative real-time PCR among commonly used assays

BK virus (BKV) is the infectious cause of polyomavirus-associated nephropathy. Screening guidelines for renal-transplant recipients define levels of viremia and viruria that are actionable for additional testing or intervention. However, standardized real-time PCR primers, probes, and standards are unavailable, and the extent of agreement among published assays is unknown. We compared seven TaqMan real-time PCR primer/probe sets (three designed at this institution, three described in the literature, and one purchased) in conjunction with two different standards to prospectively measure BKV titers in 251 urine specimens submitted to our clinical laboratory. We observed substantial disagreement among assays attributable both to features of primer and probe design and to choice of reference material. The most significant source of error among individual specimens was primer or probe mismatch due to subtype-associated polymorphisms, primarily among subtype III and IV isolates. In contrast, measurement of the most abundant subtypes (Ia, V, and VI) were typically uniform among all seven assays. Finally, we describe and validate a new clinical assay designed to reliably measure all subtypes encountered in our study population (Ia, Ic, III, IV, and VI). Consideration of available BKV sequence information in conjunction with details of subtype distribution allowed us to develop a redesigned assay with markedly improved performance. These results suggest that both accurate BKV measurement and the uniform application of BKV screening guidelines could be significantly improved by the use of standardized reference materials and PCR primers and probes.     

Detection and Differentiation of Human Polyomaviruses JC and BK by LightCycler PCR

Human polyomaviruses JC and BK may cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy and hemorrhagic cystitis. Molecular detection by PCR is recognized as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, a real-time PCR assay using the LightCycler platform was evaluated and compared to an "in-house" PCR assay using a conventional detection method. A total of 122 urine specimens were tested, and human polyomavirus was detected in 49 specimens (40%) by both conventional PCR and LightCycler PCR. The remaining 73 specimens (60%) were found negative by both assays. For 46 of the 49 positive specimens, LightCycler PCR and conventional PCR identified the same polyomavirus type. These samples included 30 samples with JC virus (JCV), 14 samples with BK virus (BKV), and 2 samples in which both viruses were detected. In the remaining three samples, both JCV and BKV were detected by the conventional assay, but only JCV was detected by the LightCycler assay. The results of this study show that the LightCycler PCR assay displays sensitivity and specificity similar to those of a conventional PCR assay. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid detection and differentiation of JCV and BKV in the clinical laboratory.     

Internally controlled triplex quantitative PCR assay for human polyomaviruses JC and BK

We have developed a triplex TaqMan-based quantitative PCR assay for the human polyomaviruses JC (JCPyV) and BK (BKPyV). The assay simultaneously detects and quantifies both JCPyV and BKPyV in human clinical samples, and it includes an internal amplification control consisting of murine polyomavirus (MuPyV) plasmid DNA. We developed the assay for the Roche LightCycler 480 platform with the reporter dyes VIC, 6-FAM, and Cy5 for MuPyV, BKPyV, and JCPyV, respectively. The assay had a high specificity for BKPyV and JCPyV when either viral genome was present alone or in mixed samples over a range of 10¹ to 10⁷ copy numbers per reaction. The analytical sensitivity was 50 copies for BKPyV and 10 copies for JCPyV. The use of the MuPyV internal control ensured monitoring of the quality of the extraction and of PCR inhibition, even in samples such as cerebrospinal fluid and plasma in which controls based on host genes cannot be effectively used. In addition, we developed a similar assay using a different dye configuration (6-FAM, VIC, and NED) that could be used on an ABI 7500 Fast platform. This assay had sensitivities similar to those of the LightCycler 480 configuration for BKPyV and JCPyV when either viral genome was present alone, but the sensitivity of detection of BKPyV was greatly decreased when an excess of JCPyV (>100-fold) was present in the sample. This internally controlled combined assay offers greater convenience and cost-effectiveness compared to separate assays for each virus and can also detect unexpected PyV activations by testing for both viruses in all samples.     

Low frequency of SV40, JC and BK polyomavirus sequences in human medulloblastomas, meningiomas and ependymomas

Several reports have suggested a role for polyomaviruses in the pathogenesis of human brain tumors. This potential involvement is not conclusively resolved. For the present study, a highly sensitive PCR-assay with fluorescence-labelled primers was developed to search for polyomavirus sequences in human brain tumor and control DNA samples. The assay was shown to detect approximately one viral large T-antigen (TAg) gene per 250 cells. We identified simian virus 40 (SV40)-like sequences in 2/116 medulloblastomas, in 1/131 meningiomas, in 1/25 ependymomas and in 1/2 subependymomas. A single case of ependymoma contained SV40 VP-1 late gene sequences. Moreover, one of the meningioma samples showed JC virus sequences. In contrast, 60 hepatoblastoma samples and 31 brain samples from schizophrenic patients were consistently negative. BK virus sequences were not detectable in any of our samples. Immunohistochemical analysis of two SV40 positive tumor biopsies failed to detect large TAg in the tumor cells. In the JC positive meningioma, immunoreactivity for the viral late gene product (VP-1) was not observed. Our data do not entirely rule out SV40 and JC virus as an initiative agent with a hit-and-run mechanism. However the low frequency of virus sequences and the absence of TAg protein expression argue against a major role of these viruses in the pathogenesis of human medulloblastomas, meningiomas and ependymomas.     

Comparison of PCR-southern hybridization and quantitative real-time PCR for the detection of JC and BK viral nucleotide sequences in urine and cerebrospinal fluid

The presence of the human polyomaviruses JCV and BKV in immunocompromised patients can lead to lethal diseases and conditions including progressive multifocal leukoencephalopathy (PML), interstitial nephritis, hemorrhagic cystitis, and kidney allograft rejection. Typically, detection of JCV and BKV in clinical samples has employed standard PCR amplification for viral nucleotide sequences, with subsequent confirmation for viral genome specificity of PCR products by southern blot hybridization. Here, we directly tested a validated PCR-southern protocol with a TaqMan real-time PCR protocol (Applied Biosystems) to assay clinical samples of urine and cerebrospinal fluid. We found equal specificity and sensitivity with both methods. However, real-time allowed for absolute viral-genome quantitation without the use of radionucleotides and was performed more rapidly, in as little as 24 h. Such advantages are important to consider in the effort to establish international standardization of controls for the detection of JCV and BKV, which would aid in screening confidence and the reliable assessment of anti-viral therapies.     

多重实时PCR检测三种呼吸道病毒

目的建立多重实时PCR检测体系可同时快速检测引起人呼吸道感染的甲型流感病毒（IAV）、呼吸道合胞病毒（RSV）和SARS冠状病毒（SARS-CoV）。方法通过对PCR反应条件的优化，建立了同时检测IAV、RSV和SARS-CoV的多重实时PCR方法。结果经熔解曲线分析，证实了该法可同时或分别检测3种重要呼吸道病毒。检测灵敏度分别为10^0.7 TCID50／ml、1 TCID50／ml和10^-0.5 TCID50／ml。采用相同的反应体系和条件，分别对其他7种呼吸道病毒（包括乙型流感病毒，副流感病毒2型，柯萨奇病毒B3和B5血清型及腺病毒2、3和7血清型）进行扩增，均未检测出扩增产物。此种方法可在4h内完成。结论建立的多重实时PCR检测方法适用于3种重要呼吸道病毒的快速检测。     

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

We have developed a real-time quantitative PCR (rt-QPCR) assay to detect and kinetically monitor BK virus viruria and viremia in renal transplant recipients (RTRs). A total of 607 urine and 223 plasma samples were collected from 203 individuals including those with BK virus-associated nephropathy (BKVAN) (n = 8), those undergoing routine posttransplant surveillance (SV) (n = 155), those with nontransplant chronic kidney disease (NT-CKD) (n = 20), and healthy living kidney donors (LD) (n = 20). The rt-QPCR assay was found to be highly sensitive and specific, with a wide dynamic range (2.4 to 11 log(10) copies/ml) and very good precision (coefficient of variation, approximately 5.9%). There was a significant difference in the prevalences of viruria and viremia between the BKVAN (100% and 100%) and SV (23% and 3.9%) groups (P < 0.001). No viruria or viremia was detected in LD or in NT-CKD patients. The median (range) peak levels of BK virus viruria and viremia, in log(10) copies/ml, were 10.26 (9.04 to 10.83) and 4.83 (3.65 to 5.86) for the BKVAN group versus 0 (0 to 10.83) and 0 (0 to 5.65) for the SV group, respectively (P < 0.001). When the BK virus load in the urine was <7.0 log(10) copies/ml, no BK virus viremia was detected. When the BK virus load in the urine reached 7.0, 8.0, 9.0, and > or =10.0 log(10) copies/ml, the corresponding detection of BK virus viremia increased to 20, 33, 50, and 100%, respectively. We propose monitoring of BK virus viruria in RTRs, with plasma BK virus load testing reserved for those with viruria levels of > or =7.0 log(10) copies/ml.     

Multiplex Real-Time PCR Assay for Simultaneous Quantification of BK Polyomavirus,JC Polyomavirus,and Adenovirus DNA

In recent years, virus-induced nephropathy caused mainly by BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), and adenovirus has emerged as a problem in renal transplant patients. In the present study, we developed a multiplex real-time PCR assay to quantify the viral load of BKPyV, JCPyV, and adenovirus simultaneously. The dynamic range covered at least 6 orders of magnitude. This system was specific and reproducible, even in the presence of large amounts of DNA of other viruses. To validate this assay, urine samples from 124 renal transplant patients and serum samples from 18 hemorrhagic cystitis patients after hematopoietic stem cell transplantation were examined. In the urine samples from renal transplant patients, BKPyV was detected in 28 patients (22.6%), JCPyV was detected in 51 patients (41.1%), and adenovirus was detected in 2 patients (1.6%). The maximum amounts of each virus detected were 2.7 × 10⁹, 8.7 × 10⁸, and 1.2 × 10² copies/ml, respectively. Decoy cells were observed in 31 patients. The quantities of both BKPyV and JCPyV DNA were greater in samples with decoy cells. Two patients whose BKPyV viral loads exceeded 10⁸ copies/ml had elevated serum creatinine levels and were diagnosed with BKPyV nephropathy based on graft biopsies. In serum samples from hemorrhagic cystitis patients, BKPyV, JCPyV, and adenovirus was detected in six, two, and three patients, respectively. Strong correlations were observed between the viral DNA copy numbers determined in the multiplex assays and those determined in single assays. Since this new assay is rapid, sensitive, and specific, it can be used for quantitative analyses of viruses in urine and serum samples after transplantation.Although immunological rejection after renal transplantation has decreased with advances in immunosuppressive therapy, virus-induced nephropathy, which was rare with conventional immunosuppressants, has become a clinical problem. BK polyomavirus (BKPyV) is a prevalent pathogen causing virus-induced nephropathy, and other viruses, including JC polyomavirus (JCPyV) and adenovirus, can cause nephropathy (1, 4, 13). These viruses commonly infect a large number of people in childhood and remain latent in the urinary tract after primary infection. Reactivation with asymptomatic viruria may occur in both immunocompetent subjects and immunocompromised patients.For the early diagnosis and treatment of BKPyV nephropathy, urine cytology is useful for screening high-risk patients (1). The virus-infected urothelial cells called “decoy cells,” identified by their typical ground glass intranuclear inclusions on cellular smears stained by the Papanicolaou method, are observed in most patients with BKPyV nephropathy. However, decoy cells are not specific for the presence of BKPyV in urine and can be found in JCPyV and adenovirus infection (1, 4, 22). Therefore, the detection of viral DNA is essential for a precise diagnosis and the identification of patients at high risk for nephropathy. In addition, hemorrhagic cystitis can be observed after hematopoietic stem cell transplantation and sometimes after renal transplantation. This involves sustained hematuria and symptoms of lower urinary tract irritability, such as dysuria with frequency and urgency. It is caused most often by adenovirus with serotypes 11, 34, and 35 in subgroup B, although BKPyV and JCPyV can also cause hemorrhagic cystitis (6, 22).Recently, real-time PCR methods have been introduced into clinical virology for the quantitative detection of viral copy numbers, and the exact determination of virus genome copy numbers provides useful information about the magnitude of the infection, which is beneficial for evaluating whether the detected virus is pathogenic, especially with latent virus infections. Previously, we developed multiplex real-time PCR systems that can quantify three viruses simultaneously (20, 21) and put the technique into clinical practice after stem cell transplantation. The use of these systems reduces significantly the time and labor required for the examination of each virus.The present study developed a multiplex real-time PCR assay for the simultaneous quantification of BKPyV, JCPyV, and adenovirus and validated it for the quantitative determination of viral loads. This method was then further validated on samples of urine and serum from posttransplant patients.     

Development of a real-time fluorescence resonance energy transfer PCR to detect arcobacter species

A real-time PCR targeting the gyrase A subunit gene outside the quinolone resistance-determining region has been developed to detect Arcobacter species. The species identification was done by probe hybridization and melting curve analysis, using fluorescence resonance energy transfer technology. Discrimination between Arcobacter species was straightforward, as the corresponding melting points showed significant differences with the characteristic melting temperatures of 63.5 degrees C, 58.4 degrees C, 60.6 degrees C, and 51.8 degrees C for the Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter cibarius, and Arcobacter nitrofigilis type strains, respectively. The specificity of this assay was confirmed with pure cultures of 106 Arcobacter isolates from human clinical and veterinary specimens identified by phenotypic methods and 16S rRNA gene sequencing. The assay was then used to screen 345 clinical stool samples obtained from patients with diarrhea. The assay detected A. butzleri in four of these clinical samples (1.2%). These results were confirmed by a conventional PCR method targeting the 16S rRNA gene with subsequent sequencing of the PCR product. In conclusion, this real-time assay detects and differentiates Arcobacter species in pure culture as well as in the competing microbiota of the stool matrix. The assay is economical since only one biprobe is used and multiple Arcobacter species are identified in a single test.