Significance of HTLV-1 proviral load quantification by real-time PCR as a surrogate marker for HTLV-1-infected cell count.
We developed a real-time (RT) PCR quantitative assay to measure the level of the integrated viral genome of HTLV-1 in host peripheral blood-mononuclear cells (PB-MNC) from healthy carriers and patients with adult T-cell leukemia (ATL). All of the clinical specimens were serologically and molecularly characterized by enzyme-linked immunosorbent assay (ELISA) and Southern blot hybridization (SBH) analyses. The assay system for quantifying the proviral copy level was sensitive, accurate, and reproducible over a wide range of density from 100 to 0.1% with a coefficient of variation (%) of 4.5 to 9.6. The proviral load of the healthy carriers and patients with ATL was 301 +/- 339 copies per 10(4) MNC (3 +/- 3.4%) on average and varied depending on the ATL cell number and the SBH band-status of single or multiple bands. In ATL cases with multiple bands detected by SBH analysis, their ATL cells were shown to harbor multiple copies within one ATL cell, so that the corrected copy number interpolated by the band number in SBH was closely equivalent to the expected ATL cell number in PB, corresponding to the virus-infected cell burden. The proviral load in healthy carriers ranged from 0.1 to 15% of PB-MNC, and, in combination with the fraction (%) of ATL-like flower cells defined by PB smear morphology, enabled carriers to be subgrouped into three categories. This result indicates that the detection of proviral load by(RT) PCR is sufficient and relevant to monitor the infected cell number in the PB and to evaluate the HTLV-1 pathologic status.
Kamihira
S
S
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki City, Japan. kamihira@net.nagasaki-u.ac.jp
Dateki
N
N
Sugahara
K
K
Hayashi
T
T
Harasawa
H
H
Minami
S
S
Hirakata
Y
Y
Yamada
Y
Y
D015415:Biomarkers / Q000032:analysis; D015139:Blotting, Southern; D002353:Carrier State / Q000097:blood / Q000821:virology; D004279:DNA, Viral / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay; D018628:Gene Dosage; D015490:HTLV-I Infections / Q000097:blood / Q000821:virology; D015368:Human T-lymphotropic virus 1 / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D015458:Leukemia, T-Cell / Q000097:blood / Q000821:virology; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000097:blood / Q000821:virology; D007958:Leukocyte Count; D007963:Leukocytes, Mononuclear / Q000821:virology; D011533:Proviruses / Q000235:genetics* / Q000254:growth & development; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D019562:Viral Load
D015415:Biomarkers; D004279:DNA, Viral