Semi-quantitative detection of cytomegalovirus DNA from native serum and plasma by nested PCR: influence of DNA extraction procedures.
The diagnostic implications of different procedures of DNA extraction were examined for the detection of HCMV DNA from sera and plasma of immunosuppressed patients. The detection limit of HCMV plasmid DNA from cell free seronegative plasma and serum by limiting dilution nested PCR decreased in the following sequence: phenol/chloroform > NaI-single tube method > proteinase K digestion equal to amplification of native sera and plasma. Nested PCR from native sera and plasma performed well and surpassed the proteinase K method in sensitivity for detection of serum DNAemia. Semi-quantitative determination of HCMV DNA titer present in native sera of immunosuppressed patients did not seem to be correlated to HCMV disease. When compared to the persistence of leukoDNAemia, the viral DNA titer in native plasma could only be observed in the acute phase of HCMV infection, an important phenomenon for diagnostic purposes. Correlation of serum DNAemia to virus culture revealed low positive and high negative predictive values. Predictive values of nested PCR from native sera for HCMV infection were not lower than those following organic DNA extraction. Despite its low correlation to viremia and virus isolation from any site, nested PCR from organic DNA extracts of serum or plasma is the most sensitive diagnostic tool of an ongoing HCMV infection. Additionally, semi-quantitative end point dilution nested PCR from native serum or plasma promises to be a rapid and easy tool for the monitoring of antiviral therapy.
Hamprecht
K
K
Abt. Medizinische Virologie und Epidemiologie der Viruskrankheiten, Hygiene-Institut der Universität Tübingen, Germany. klaus.hamprecht@uni-tuebingen.de
Mikeler
E
E
Jahn
G
G
D017088:AIDS-Related Opportunistic Infections / Q000175:diagnosis; D000998:Antiviral Agents / Q000627:therapeutic use; D016026:Bone Marrow Transplantation; D002725:Chloroform / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000188:drug therapy; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D019286:Endopeptidase K / Q000494:pharmacology; D015774:Ganciclovir / Q000627:therapeutic use; D006801:Humans; D016867:Immunocompromised Host; D019800:Phenol / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D012974:Sodium Iodide / Q000494:pharmacology; D014766:Viremia
D000998:Antiviral Agents; D004279:DNA, Viral; D019800:Phenol; D002725:Chloroform; D019286:Endopeptidase K; D012974:Sodium Iodide; D015774:Ganciclovir