[Problems of cytomegalovirus diagnostics by real-time polymerase chain reaction.].
OBJECTIVE
The main goal was to explain the discrepancies between two PCR methods used for detection of cytomegalovirus (CMV) in peripheral blood samples of patients of Department of Internal Medicine-Hematooncology, University Hospital, Brno.


METHODS
In past we used exon 4 of major immediate-early (MIE) gene as the target for quantitative detection of the CMV in clinical samples, but sometimes this method failed to detect the viral load in samples that were positively tested using less sensitive qualitative method targeting another region (exon 2-4) of the same gene. From January 2004 to January 2005 we totally tested samples from 363 patients. 64 patients were at least once CMV positive using quantitative method, but 20 patients were repeatedly false negative.To find the cause of this discrepancy we performed partial sequence analysis of this region (nt positions 2719-2919, GenBank M21295) and glycoprotein B (gB) genotyping. We sequenced samples from 35 patients-15 giving true positive (both in qualitative and quantitative method) and 20 giving false negative (negative in quantitative but positive in qualitative method) results in several consecutive blood samples.


RESULTS
The 15 true positive samples were 100% homological, whereas all 20 false negative samples showed high degree of variation from the laboratory strain AD169. These changes are not random and indicate that the two groups of patients were infected by different CMV genotypes. Moreover, sequence alignment showed similarity to laboratory strains Toledo and Towne. No preferential concordance was observed between clinical context, MIE exon 4 sequence and gB groups.


CONCLUSIONS
Because of high sequence variability exon 4 of MIE gene can not be used for routine diagnostics. Genetic varibility among the pathogenic strains may seriously affect its proper diagnostics.
Lengerová
Martina
M
Center of Molecular Biology and Gene Therapy, Department of Internal Medicine Hematooncology, University Hospital Brno, Czech Republic.
Volfová
Pavlína
P
Lochmanová
Jana
J
Rácil
Zdenek
Z
Minaríková
Dagmar
D
Dvoráková
Dana
D
Vorlícek
Jirí
J
Mayer
Jirí
J
D003587:Cytomegalovirus; D003586:Cytomegalovirus Infections / Q000821:virology; D004279:DNA, Viral; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019562:Viral Load
D004279:DNA, Viral