Porównanie testów real-time PCR do ilościowego oznaczania DNA wirusa cytomegalii z użyciem systemu LightCycler 2.0 u pacjentów hematologicznych
[Results comparison of cytomegalovirus viral load in hematological patients using real-time PCR assays designed for LightCycler 2.0 system]

STRESZCZENIE

Przeprowadzono porównanie laboratoryjnej użyteczności pięciu diagnostycznych testów do wykrywania DNA CMV opartych na technice real-time PCR. Wykazano celowość wprowadzenia międzynarodowego układu kalibratorów, jako narzędzia dla międzylaboratoryjnej standaryzacji wyników wiremii CMV uzyskiwanych metodami molekularnymi.

ABSTRACT

Introduction: Human cytomegalovirus infections are the major viral complications associated with the post-transplant period in haematopoietic stem cell and solid organ transplant recipients. Clinical research suggests a role for viral load measurement in predicting and monitoring CMV-associated diseases. Early detection of cytomegalovirus infection with molecular biology methods is favorable for the efficacy of antiviral chemotherapy. Our point of interest was comparing specify and level of detection of cytomegalovirus DNA in plasma and whole blood specimens with different commercial CMV kits, as well as “in-house“ method, designated for the LightCycler 2.0 system.

Materials and Methods: 59 plasma and 37 whole blood samples (total number of 96), taken from patients after allogeneic stem cells transplantation, were tested for the presence of CMV DNA using the LightCycler 2.0 instrument with qPCR tests coming from Roche®, Artus/Qiagen®, GeneProof® and ALPCO®. The fifth method used was “in-house” realtime PCR assay, based on TaqMan chemistry.

Results: Results for samples containing a low cytomegalovirus load were more accurate with the Artus/Qiagen® and Roche® tests, than were results obtained with different kits, which underestimated the viral load of samples containing low DNA copy numbers.

Conclusions: Comparison of all methods used in present work indicates that employment of real-time PCR is advisable in common post-transplantation diagnostics. The high level of sensitivity and specificity provided by most of the tests using LightCycler instrument are favorable for the use of this system in the detection of CMV DNA. In addition, convenience of usage and short time of test performance makes this method suitable for both routine cytomegalovirus screening in different clinical specimens and monitoring of the proper antiviral therapy.

Key words: cytomegalovirus, post-transplant infections, molecular diagnostics, real-time PCR