Real-Time PCR Diagnostics Failure Caused by Nucleotide Variability within Exon 4 of the Human Cytomegalovirus Major Immediate-Early Gene

Lengerová, Martina; Ráčil, Zdeněk; Volfová, Pavlína; Lochmanová, Jana; Berkovcová, Jitka; Dvořáková, Dana; Vorlíček, Jiří; Mayer, Jiřı́

doi:10.1128/jcm.01109-06

Cited by 18 publications

(18 citation statements)

References 22 publications

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“…The gB sequence variability of the CMV present in the saliva specimens most likely resulted in a mismatch between our primers and probes, leading to significant lowering of the sensitivity of the real-time PCR assay. Similar findings were also reported by Lengerova et al, who experienced about a 5% false-negative rate with an in-house assay due to mismatches in the target region of primers/probes located within the major immediateearly exon 4 region (12).…”

supporting

confidence: 88%

Diagnostic Consequences of Cytomegalovirus Glycoprotein B Polymorphisms

et al. 2011

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supporting

confidence: 88%

Diagnostic Consequences of Cytomegalovirus Glycoprotein B Polymorphisms

et al. 2011

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“…Only in the last few years have a substantial number of full-genome BKV DNA sequences been published (3,10,17,27,29); thus, the extent of BKV genetic diversity is not necessarily reflected in the design of commonly used PCR reagents. The importance of locating real-time PCR primers and probes within well-conserved genomic regions is well known (15,19). The experiments in this study illustrate some potential manifestations of primer/probe mismatches.…”

Section: Discussionmentioning

confidence: 80%

Marked Variability of BK Virus Load Measurement Using Quantitative Real-Time PCR among Commonly Used Assays

Hoffman¹,

Cook²,

Atienza³

et al. 2008

J Clin Microbiol

142

105

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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.BK virus (BKV) is a small, nearly ubiquitous DNA virus in the family Polyomaviridae that causes chronic, usually asymptomatic infection in immunocompetent individuals. Since the advent of potent antirejection therapy, BKV has emerged as the primary infectious cause of polyomavirus-associated nephropathy (PVAN), affecting 1 to 10% of renal-transplant recipients, and with frequencies of graft loss between 10% and 80% (7).Quantitative assays for BKV in urine and serum are used as both screening and diagnostic tests for PVAN in renal-transplant recipients. Prospective studies of renal-allograft patients have demonstrated that high levels of BKV viruria precede sustained viremia, which in turn precedes evidence of allograft dysfunction (2). Furthermore, screening blood or urine for high levels of BKV replication in conjunction with interventions, such as reduction of immunosuppression (2) or treatment with cidofovir (12), triggered at defined levels of viremia or viruria has been associated with improved clinical outcomes.Renal-transplant programs have instituted BKV screening protocols, and quantitative BKV testing is increasingly performed in molecular virology laboratories. In 2005, an expert panel recommended the use of either urine cytology or nucleic-acid-based testing to screen renal-transplant recipients during the first 2 years after transplantation who present with evidenc...

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“…One potential explanation for the discrepant quantitation is that the CMV isolate from this patient contains a mutation in the proprietary major immediateearly gene sequences targeted by the artus CMV PCR. Such mutations have been reported to cause decreased amplification in other assays that target this gene, but they have not been reported previously for the artus CMV PCR (6,16).…”

Section: Discussionmentioning

confidence: 81%

Clinical Significance of Low Cytomegalovirus DNA Levels in Human Plasma

Waggoner

Libiran

et al. 2012

J Clin Microbiol

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The clinical significance of the detection of low copy numbers of cytomegalovirus (CMV) DNA in immune-suppressed patients remains unclear. In this study, we compared the artus CMV Rotor-Gene PCR, utilizing an automated nucleic acid extraction and assay setup (the artus CMV protocol), with the COBAS Amplicor CMV Monitor test (our reference protocol). We then analyzed the results of all CMV PCR tests ordered following the implementation of the artus CMV protocol at our institution and followed 91 adult patients with positive test results. The artus CMV protocol had a linear range extending from 2.0 to 7.0 log 10 copies/ml and had a lower limit of 95% detection of 57 copies/ml. With archived plasma samples, this protocol demonstrated 100% sensitivity and 94% specificity for the detection of CMV DNA. Following implementation of the artus CMV protocol, 320 of 1,403 (22.8%) plasma samples tested positive (compared with 323/3,579 [9.0%] samples in the preceding 6 months), and 227 (16.2%) samples had copy numbers of <400/ml. Ninety-one adult patients had at least one positive test. The data were analyzed using a threshold of 200 copies/ml, and in 22 episodes, the viral load increased from <200 copies/ml to >200 copies/ml on sequential tests. In 21 of these 22 episodes, either the viral load continued to increase or antiviral treatment was initiated in response to the repeat value. In summary, we evaluate the performance characteristics of a protocol utilizing the artus CMV PCR and identify clinically meaningful changes in CMV DNA copy numbers even when they are initially detected at a low level.

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Real-Time PCR Diagnostics Failure Caused by Nucleotide Variability within Exon 4 of the Human Cytomegalovirus Major Immediate-Early Gene

Cited by 18 publications

References 22 publications

Diagnostic Consequences of Cytomegalovirus Glycoprotein B Polymorphisms

Diagnostic Consequences of Cytomegalovirus Glycoprotein B Polymorphisms

Marked Variability of BK Virus Load Measurement Using Quantitative Real-Time PCR among Commonly Used Assays

Clinical Significance of Low Cytomegalovirus DNA Levels in Human Plasma

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