Development of a real-time polymerase chain reaction assay for the diagnosis of human herpesvirus-6 infection and application to bone marrow transplant patients.
A quantitative real-time PCR assay was developed for human herpesvirus-6 (HHV-6) genome based on TaqMan technology. After choosing a region of interest into the U65-U66 genes of HHV-6 genome, its nucleotide sequence was determined among four HHV-6 strains (one variant A and three variants B) to exclude a variability of sensitivity due to interstrain sequence differences. A plasmid containing HHV-6 target sequences identical to those of reference type viruses was constructed with the aim of standardisation. This HHV-6 genomic quantitation assay has a threshold sensitivity of ten copy equivalents (EqCop) per reaction. In order to test the feasibility of this assay directly on human samples, the technique was applied to the quantitation of HHV-6 genome in 30 blood samples from healthy subjects as well as 31 blood samples and three samples of cerebrospinal fluid (CSF) from 21 bone marrow transplant (BMT) recipients and four patients with a haematological disease but not treated by bone marrow transplantation. HHV-6 load ranged between 0.00015 and 0.0008 equivalent DNA copy number (EqCop) per 100 peripheral blood mononuclear cells (PBMCs) in healthy subjects whereas it ranged from <10 to 7500 EqCop per 100 PBMCs, and from <10 to 415,820 EqCop per 100 microl of whole CSF in patients. The efficacy of treatment with antiherpetic drug was associated with a decrease of the viral load in the CSF of one patient. This method leads to relevant results in term of range of quantitation, sensitivity, and safety against contamination by amplicons, and might constitute a useful tool for the follow-up of BMT recipients particularly in the presence of antiherpetic therapy.
Gautheret-Dejean
Agnès
A
Laboratoire de Virologie, UPRES EA 2387, Groupe Hospitalier Pitié-Salpêtrière, 83 boulevard de l'Hôpital, 75013 Paris, France. agnes.gautheret@psl.ap-hop-paris.fr
Manichanh
Chaysavanh
C
Thien-Ah-Koon
Florence
F
Fillet
Anne-Marie
AM
Mangeney
Nicole
N
Vidaud
Michel
M
Dhedin
Nathalie
N
Vernant
Jean-Paul
JP
Agut
Henri
H
D000328:Adult; D001483:Base Sequence; D016025:Bone Transplantation; D004279:DNA, Viral / Q000032:analysis*; D015654:Herpesvirus 6, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D009711:Nucleotides; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011933:Reagent Kits, Diagnostic; D019349:Roseolovirus Infections / Q000175:diagnosis* / Q000821:virology; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors
D004279:DNA, Viral; D009711:Nucleotides; D011933:Reagent Kits, Diagnostic