Analysis of human cytomegalovirus DNA in urines of newborns and infants by means of a new ultrarapid real-time PCR-system.
BACKGROUND
Amplification techniques such as PCR are becoming increasingly popular in the field of diagnosis of human cytomegalovirus (HCMV) also, thus substituting conventional techniques like the time consuming HCMV antigen or cell culture assays. Current PCR protocols however, are labor intensive, and moreover, the need for extensive postamplification manipulations increases the risk of false positive results due to contamination with amplified products.


OBJECTIVE
to overcome these shortcomings, the new ultrarapid and semi-automated real-time LightCycler PCR-system (LC-PCR), which combines amplification and detection in a closed capillary system, was tested for its suitability in diagnosis of HCMV in urines.


METHODS
73 urine samples from 64 newborns and infants suspected of having congenitally or postnatally acquired HCMV were tested with the LC-PCR and results were compared with those obtained in parallel with a conventional PCR-ELISA and the rapid shell vial assay for detection of HCMV early antigen (EA-assay).


RESULTS
with these methods, 31 newborns/infants were found to be infected with HCMV. HCMV DNA was detected in 39 urines while the EA-assay was positive in 33 urines. All the EA positive samples were also positive for HCMV DNA. In the urines of the remaining 33 newborns (34 urine samples) neither HCMV DNA nor EA were detectable. The overall agreement of the two PCR tests was 100% while a 92% agreement was obtained between the PCR and the EA-assays. As the sensitivity of the three tests turned out to be quite similiar, the discrepancy observed in the positive rate between PCR and EA-assay is due to other factors which will be discussed in detail. However, while LC-PCR takes only about 2 h from sample preparation to result generation, the EA-assay, such as the conventional PCR-ELISA, needs 24-48 h. Furthermore, due to its capability to perform cycle-by-cycle monitoring, the LC instrument enables semi-quantitative analysis of HCMV viral-load.


CONCLUSIONS
LC-PCR is a suitable new tool for routine analysis of HCMV in the urines of newborns and infants. Compared to the conventional PCR-ELISA a considerable increase in test rapidity and reliability is achieved without the need to sacrifice sensitivity.
Schalasta
G
G
Institute for Virology, Infectiology and Epidemiology and Medical Diagnostic Laboratory, Rosenbergstrasse 85, D-70193, Stuttgart, Germany. schalasta@labor-enders.de
Eggers
M
M
Schmid
M
M
Enders
G
G
D000956:Antigens, Viral / Q000032:analysis; D002675:Child, Preschool; D015331:Cohort Studies; D003587:Cytomegalovirus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000652:urine / Q000821:virology*; D004279:DNA, Viral / Q000652:urine*; D004797:Enzyme-Linked Immunosorbent Assay; D005453:Fluorescence; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity
D000956:Antigens, Viral; D004279:DNA, Viral; D011933:Reagent Kits, Diagnostic