Clinical Significance of Low Cytomegalovirus DNA Levels in Human Plasma

Abstract

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.

Cytomegalovirus load at treatment initiation is predictive of time to resolution of viremia and duration of therapy in hematopoietic cell transplant recipients

“…Testing was performed using the artus CMV Rotor-Gene PCR (Qiagen, Germantown, MD) as previously described(16). Viral load values were expressed in international units (IU/mL) based on the test's calibration to the primary CMV WHO standard obtained from the National Institute for Biological Standards and Control (Hertfordshire, UK).…”

Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing

“…For the clinical plasma specimens, DNA was extracted from 1.0 ml plasma using the Qiagen virus/bacteria midi kit on the QIAsymphony SP device and eluted in 90 l of Tris-EDTA (TE) buffer (Qiagen, Germantown, MD). Viral loads were measured using artus CMV PCR reagents (Qiagen, Germantown, MD) on the Rotor-Gene Q, as previously described (31). For the reference control, cultured CMV strain AD169 (ATCC VR-538) (American Type Culture Collection, Manassas, VA) was diluted in defibrinated human plasma (SeraCare, Milford, MA) at 1:1,000 (high copy number) and 1:100,000 (low copy number), processed by the same protocol as the clinical specimens, and the viral load in copies/ml plasma was measured with the artus CMV PCR.…”

Early Kinetics of Plasma Cytomegalovirus DNA Load in Allogeneic Stem Cell Transplant Recipients in the Era of Highly Sensitive Real-Time PCR Assays: Does It Have Any Clinical Value?

“…The fact is that the limits of detection (LOD) and quantitation (LOQ) of most "in-house" and commercially available qRT-PCRs are well below the plasma CMV DNA load thresholds that are widely accepted for triggering the initiation of preemptive antiviral therapy (between 500 and 1,000 copies/ml at most centers, in the setting of no risk-adapted strategies) (1). In our experience (6), CMV DNA loads at the beginning of episodes of active CMV infection (first and even second positive qRT-PCR results) are frequently of low magnitude (below 500 copies/ml); this seems to be the experience of other groups as well (7,8). However, whether information on clinical utility could be derived from these early measurements remains to be determined.…”