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Whole blood real-time PCR for cytomegalovirus DNA quantification: analysis of PCR data and pp65 antigen test in a cohort of solid-organ transplant recipients

Abstract number: 1733_132

Cerutti F., Pittaluga F., Varetto S., Gabella S., Franchello A., Rinaldi M., Ghisetti V., Allice T.

Cytomegalovirus (CMV) is a major opportunistic agent in solid organ transplantation (SOT). Pre-emptive therapy and a strict infection monitoring with highly sensitive methods have significantly decreased CMV morbidity and mortality. Recently introduced real-time PCR tests for routine CMV DNA quantification require correlation studies with pp65-antigen test as the gold standard. Moreover, there is no consensus as the most appropriate blood compartment (i.e. whole blood, leukocytes, plasma) for PCR test.

Aims: 1) to study the correlation between pp65-antigen test and CMV DNA as quantified by real-time PCR in whole blood (WB) in a cohort of SOT recipients and 2) the identify a CMV DNA cut-off level for pre-emptive anti-CMV therapy.

Methods: WB samples (n = 397) from 41 asymptomatically infected patients (18/41 undergoing pre-emptive therapy with ganciclovir) were monitored the first year after SOT by pp65 antigen test and real-time PCR for the UL123 gene, IE1 exon 4 (Nanogen, I). Extraction was carried out from 220 ul of WB with a fully automated system based on nucleic acid silica-gen affinity (BioRobot 9604, Qiagen, I).

Results: Pp65 and CMV DNA were highly correlated (r = 0.750, 78% concordant samples). CMV DNA level was much higher in pre-emptive treated patients than in not treated ones (mean+SD: 5.8+0.8 log10 copies/mL vs. 4.5+0.6, p < 0.0001). Pre-emptive therapy was started for pp65 values >90 positive cells, corresponding to a median CMV DNA level of 5.6 log10 copies/mL (mean+SD: 5.4+0.8, range 3.6–6.8). According to pp65 positive cells/200,000 examined cells of 0, 1–10, 11–50, 51–100 and >100, median levels of CMV DNA in WB were 3.8, 3.9, 4.6, 5.2 and 5.7 log10 copies/mL, respectively. A plasmid carring the UL123 viral amplified gene was used to probe the couppled extraction and real-time PCR platform sensitivity (100% at 3 copies/ul from the extraction step, 100% at 2 copies/ul from the amplification step) and reproducibility (between assay CV% <19).

Conclusion: CMV DNA evaluation in WB by real-time PCR significantly simplifies and accelerates the production of a reliable quantification for clinical purposes with a good correlation with pp65 antigen tests, excellent sensitivity and reproducibility. Levels of DNA >5.4 log10 copies/mL that is >250,000 copies/mL are highly suggestive of pre-emptive anti-CMV therapy.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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