Minimal effect of delayed sample processing on results of quantitative PCR for cytomegalovirus DNA in leukocytes compared to results of an antigenemia assay

Schäfer, Peter; Tenschert, W; Gutensohn, K.; Laufs, Rainer

doi:10.1128/jcm.35.3.741-744.1997

Cited by 60 publications

(21 citation statements)

References 22 publications

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“…Thus, a single plasma CMV PCR level of Ն10,000 copies/ml or a Ն5fold increase between two sequential measurements seemed to indicate high risk of CMV disease and could be used to initiate anti-CMV preemptive therapy. Although the number of specimens available at the time CMV disease declared itself was limited, our findings are strengthened by agreement with data from previous studies of other solid-organ and bone marrow transplant recipients (13,24). In this study, the number of CMV DNA copies/ml underwent a 10-fold reduction in the first month of therapy in successfully treated patients.…”

Section: Discussionsupporting

confidence: 91%

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Comparison of PCR, Antigenemia Assay, and Rapid Blood Culture for Detection and Prevention of Cytomegalovirus Disease after Lung Transplantation

Weinberg

Hodges

et al. 2000

J Clin Microbiol

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The goal of this study was to evaluate serial cytomegalovirus (CMV) blood culture, antigenemia testing, and qualitative and quantitative plasma CMV PCR for their ability to predict CMV disease and thus to direct preemptive therapy after lung transplantation. Forty-one patients provided 414 samples for blood culture, 290 samples for antigenemia testing, and 432 samples for PCR. Seven patients developed 11 episodes of CMV disease. CMV PCR had sensitivity, specificity, and positive predictive and negative predictive values of 79, 99, 84, and 99%, respectively, compared with 48, 99, 85, and 98%, respectively, for antigenemia testing, and 8, 100, 100, and 97%, respectively, for culture. Only quantitative CMV PCR correlated with disease stage: asymptomatic patients had a mean of 1,500 CMV DNA copies/ml, whereas patients who developed CMV disease had 5,087 copies/ml 12 to 4 weeks before symptoms and 32,000 copies/ml at diagnosis. Furthermore, CMV PCR-measured DNA increased 5- to 10-fold immediately preceding symptoms. PCR and antigenemia test values decreased with anti-CMV therapy. CMV DNA (as detected by PCR), but not antigenemia, persisted in patients who later developed recurrent CMV disease. The data indicate that lung transplant recipients will benefit from monitoring of CMV disease by plasma CMV PCR.

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Section: Discussionsupporting

confidence: 91%

“…Finally, the utility of antigenemia testing is limited by the rapid loss of signal during storage because specimens have to be processed within 6 h of collection (3,19). In contrast, the results of quantitative CMV PCR are stable for at least 1 week at refrigerator temperature (21,24).…”

Section: Discussionmentioning

confidence: 99%

Comparison of PCR, Antigenemia Assay, and Rapid Blood Culture for Detection and Prevention of Cytomegalovirus Disease after Lung Transplantation

Weinberg

Hodges

et al. 2000

J Clin Microbiol

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“…First, qualitative CMV PCR of polymorphonuclear leukocyte fractions depleted of the mononuclear cells by Ficoll density centrifugation is promising for the monitoring of renal allograft recipients (18). Its specificity is comparable to that plasma PCR and it is even more sensitive than plasma PCR (15), and storage of unprocessed samples for at least 72 h has no effect on the results (17). Second, serum CMV PCR is supposed to be equivalent to plasma PCR for prediction of active infections (8,13) and therefore should also be investigated for the adverse effects of delayed sample preparation.…”

Section: Discussionmentioning

confidence: 99%

False-Positive Results of Plasma PCR for Cytomegalovirus DNA due to Delayed Sample Preparation

Schäfer

Tenschert

Schröter

et al. 2000

Journal of Clinical Microbiology

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Positive results by cytomegalovirus (CMV) PCR of plasma are considered predictive of active CMV infection in kidney allograft recipients. To assess whether contamination with leukocyte-derived CMV DNA can distort the results, aliquots of whole-blood samples from 60 CMV immunoglobulin G-positive patients with leukocyte CMV DNAemia were stored for up to 24 h at room temperature (RT) and at 4°C before plasma preparation. Native and ultrafiltered plasma samples were tested by CMV and ␤-globin PCRs. Among 30 latently infected patients (negative for CMV pp65 antigens), low baseline rates (10%) and levels (median number of copies, 10 [per 10 l]) of CMV plasma DNAemia in native plasma samples increased significantly over time (after 4 h at RT, 37% [P < 0.001]; median number of copies, 45 [P < 0.001]). Similar effects were found during storage at 4°C. Ultrafiltration reduced the levels of CMV plasma DNAemia, but by 6 h of storage the levels were significantly elevated as well. CMV and ␤-globin DNA kinetics in plasma were parallel. In contrast, 30 actively infected patients (pp65 positive) had high baseline rates (87% in native samples) and levels (median number of copies, 75) of CMV plasma DNAemia. No significant effects of storage or ultrafiltration and no concordance with ␤-globin DNA kinetics were seen. In conclusion, delayed preparation of plasma samples bears a significant risk of false-positive CMV PCR results, probably due to leukocyte lysis. This has important implications in the clinical setting and for PCR standardization.

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“…The major drawback of this test is the need for immediate (within 6 h) processing of blood samples to achieve optimal sensitivity (406). Delays in sample processing for longer than 24 h result in significant decreases in the number of detectable pp65-positive cells in blood specimens (41,406); with some modifications and the use of stabilization reagents, this obstacle may be overcome (55). False-negative results may occur in neutropenic patients, since the antigen-emia test depends on the presence of a sufficient number of polymorphonuclear leukocytes (261).…”

Section: Antigenemia Assaymentioning

confidence: 99%

“…These assays should also be standardized for the number of cells from which DNA is extracted (379). An advantage of amplification methods over culture and antigen detection assays is that samples can be stored at room temperature for up to 72 h with no significant alteration in the level of detectable DNA (50,380,406). As well, PCR can detect CMV DNA from the blood of patients with localized disease.…”

Section: Pcr Amplificationmentioning

confidence: 99%

New Strategies for Prevention and Therapy of Cytomegalovirus Infection and Disease in Solid-Organ Transplant Recipients

2000

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SUMMARY In the past three decades since the inception of human organ transplantation, cytomegalovirus (CMV) has gained increasing clinical import because it is a common pathogen in the immunocompromised transplant recipient. Patients may suffer from severe manifestations of this infection along with the threat of potential fatality. Additionally, the dynamic evolution of immunosuppressive and antiviral agents has brought forth changes in the natural history of CMV infection and disease. Transplant physicians now face the daunting task of recognizing and managing the changing spectrum of CMV infection and its consequences in the organ recipient. For the microbiology laboratory, the emphasis has been geared toward the development of more sophisticated detection assays, including methods to detect emerging antiviral resistance. The discovery of novel antiviral chemotherapy is an important theme of clinical research. Investigations have also focused on preventative measures for CMV disease in the solid-organ transplant population. In all, while much has been achieved in the overall management of CMV infection, the current understanding of CMV pathogenesis and therapy still leaves much to be learned before success can be claimed.

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Minimal effect of delayed sample processing on results of quantitative PCR for cytomegalovirus DNA in leukocytes compared to results of an antigenemia assay

Cited by 60 publications

References 22 publications

Comparison of PCR, Antigenemia Assay, and Rapid Blood Culture for Detection and Prevention of Cytomegalovirus Disease after Lung Transplantation

Comparison of PCR, Antigenemia Assay, and Rapid Blood Culture for Detection and Prevention of Cytomegalovirus Disease after Lung Transplantation

False-Positive Results of Plasma PCR for Cytomegalovirus DNA due to Delayed Sample Preparation

New Strategies for Prevention and Therapy of Cytomegalovirus Infection and Disease in Solid-Organ Transplant Recipients

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