Quantitative analysis of cytomegalovirus load using a real-time PCR assay

Abstract

A novel real-time PCR assay system was developed to quantify the cytomegalovirus (CMV) genome load. The real-time PCR assay could detect from 6 to over 10(6) copies of CMV-DNA with a wide linear range. The virus load of immunocompromised patients with symptomatic CMV infections was quantified and compared to that of asymptomatic ones. In symptomatic patients, all 17 peripheral blood leukocytes were positive for CMV DNA, and its mean value was 10(3.3) copies/10(6) cells. On the other hand, only 9 of 38 samples (24%) were positive in the asymptomatic patients, and its mean titer was lower (10(2.0) copies/10(6) cells) than that of the symptomatic group (P = 0.002). In plasma, the virus genome was detected in 13 out of 17 samples from symptomatic patients (76%), and its mean value was 10(4.0) copies/ml. In contrast, for the asymptomatic group, only one out of 36 samples were positive (3%). Finally, this system was used to monitor two patients with CMV infections serially. The CMV DNA copy number changed with their clinical symptoms and anti-CMV therapy, and virtually paralleled the result of the pp65 antigenemia assay in both cases. In one patient with the cord blood transplantation, however, the CMV DNA became positive faster than the antigenemia assay. These results indicate that this assay is sensitive and useful for estimating the CMV genome load not only in peripheral blood leukocytes but also in plasma. It can be very helpful for diagnosing CMV-related diseases and monitoring the virus load in patients with CMV infections.

Transmission of cytomegalovirus via breast milk in extremely premature infants

“…13 Real-time PCR was performed using a TaqMan PCR Kit and Model 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA), as described previously. 9,14,15 The detection limit of this assay was 100 copies ml -1 .…”

Signature profiles of CMV-specific T-cells in patients with CMV reactivation after hematopoietic SCT

“…The viral loads were normalized to 10 000 human genomic equivalents obtained by quantification of the albumin gene. 25 Normalized viral load above 100 was considered clinically significant and preemptive therapy with ganciclovir was initiated. Patients were treated with i.v.…”

Monitoring cytomegalovirus infection by antigenemia assay and two distinct plasma real-time PCR methods after hematopoietic stem cell transplantation

“…12,13 US17-PCR was performed in Otsuka Assay Laboratories (Otsuka Pharmaceutical, Tokyo, Japan) and IE-PCR was performed in SRL (Tokyo, Japan). Briefly, DNA extracted from 100 l of plasma was subjected to PCR using TaqMan Universal PCR Master Mix (PE Biosystems, Foster City, CA, USA) and the PCR product was detected as an increase in the fluorescent intensity using ABI Prism 7700 (PE Biosystems).…”

“…10 Although it might be too sensitive for discrimination of high-risk patients for CMV disease, 11 the development of real-time PCR has made it possible to quantitatively evaluate the virus load. [12][13][14] However, the threshold of the virus load to discriminate high-risk patients has not been determined, although the threshold of the antigenemia value has been reported. 15,16 Serially monitoring the virus load without administering ganciclovir is the only method to address this issue, but it is unethical.…”