title: Multiplex real-time PCR for the detection and quantitation of HTLV-1 and HTLV-2 proviral load: addressing the issue of indeterminate HTLV results.
abstract: BACKGROUND
  Routine diagnosis of Human T Lymphotropic virus (HTLV) infection is primarily serologically based; however the proportion of unresolved and indeterminate Western blot results range from 0.02% to 50% in endemic areas.
  
  
  OBJECTIVE
  To validate a sensitive in-house quantitative multiplex real-time assay (mqRT-PCR), capable of detecting and quantifying HTLV-1 and HTLV-2, and use it to differentiate unresolved serological profiles, and monitor infection in HTLV-1 infected patients.
  
  
  METHODS
  The mqRT-PCR was designed as a single-tube assay. Quantitative results were reported as copy number of HTLV provirus per 10(6) cells and the numbers of cells were calculated based on the quantitative result for albumin, of which there are 2 copies/cell. Assay standards were amplified from HTLV-1 infected MT-2 cells and HTLV-2 transfected CEM cells. Blood samples were obtained from HTLV seropositive former blood donors.
  
  
  RESULTS
  The mqRT-PCR assay was efficient (98.8-101.2%), reproducible (coefficient of variance<5%) and sensitive to 1 copy for HTLV-1, HTLV-2 and Albumin. The assay resolved the infection profile in 16/17 patients, with undetermined subtype, all of which were reassigned as HTLV-1 infections. In addition, the average PVL detected in patients suffering from HTLV-1 associated HAM/TSP (n=23, 13,450 copies/10(6) cells) was significantly higher than those detected in asymptomatic carriers (n=21, 6665 copies/10(6) cells).
  
  
  CONCLUSIONS
  We propose a new testing algorithm for the laboratory diagnosis of HTLV infection, which includes HTLV specific mqRT-PCR for resolving HTLV serological results. Furthermore, quantitation of PVL load by real-time PCR may be useful in assessing the link between infection and disease, and in monitoring patients undergoing therapy.
authors.affiliation: National Virus Reference Laboratory (NVRL), University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Allison
authors.initials: A
authors.lastname: Waters
authors.forename: André L A
authors.initials: AL
authors.lastname: Oliveira
authors.forename: Suzie
authors.initials: S
authors.lastname: Coughlan
authors.forename: Carla
authors.initials: C
authors.lastname: de Venecia
authors.forename: Doris
authors.initials: D
authors.lastname: Schor
authors.forename: Ana-Claudia
authors.initials: AC
authors.lastname: Leite
authors.forename: Abelardo Q C
authors.initials: AQ
authors.lastname: Araújo
authors.forename: William W
authors.initials: WW
authors.lastname: Hall
mesh_terms: D015153:Blotting, Western; D004279:DNA, Viral / Q000032:analysis; D003955:Diagnostic Tests, Routine / Q000379:methods; D015490:HTLV-I Infections / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D015491:HTLV-II Infections / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D015367:Human T-lymphotropic virus 2 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019562:Viral Load
chemical_list: D004279:DNA, Viral