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Journal of Clinical Microbiology, November 2009, p. 3682-3691, Vol. 47, No. 11
0095-1137/09/$08.00+0     doi:10.1128/JCM.00781-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination {triangledown}

Britta Moens,1* Giovanni López,2 Vanessa Adaui,2 Elsa González,2,3 Lien Kerremans,1 Daniel Clark,2,4 Kristien Verdonck,2,5 Eduardo Gotuzzo,2,3 Guido Vanham,5,6 Olivier Cassar,7 Antoine Gessain,7 Anne-Mieke Vandamme,1 and Sonia Van Dooren1,8

Rega Institute for Medical Research, KU Leuven, Leuven, Belgium,1 Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima 31, Peru,2 Departamento de Medicina, Facultad de Medicina, Universidad Peruana Cayetano Heredia, Lima, Peru,3 Laboratorios de Investigación y Desarrollo (LID), Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru,4 Virology Unit, Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium,5 Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium,6 Unite d'Epidemiologie et Physiopathologie des Virus Oncogenes, Institut Pasteur, Paris, France,7 Centre for Medical Genetics, UZ Brussels, Brussels, Belgium8

Received 17 April 2009/ Returned for modification 17 July 2009/ Accepted 28 August 2009

The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3. Our assay uses tax type-specific primers and dually labeled probes and has a dynamic range of 105 to 10 HTLV copies. One hundred sixty-three samples were analyzed, among which all of the different subtypes within each HTLV genotype could be detected. The performance of proviral load determination of our multiplex assay was compared with that of a previously published HTLV-1 singleplex quantitative PCR based on SYBR green detection, developed at a different institute. Linear regression analysis showed a statistically significant (P < 0.0001) and strong (r2 = 0.87) correlation between proviral load values measured with the two distinct real-time PCR assays. In conclusion, our novel assay offers an accurate molecular diagnosis and genotyping, together with the determination of the proviral load of HTLV-infected individuals, in a single amplification reaction. Moreover, our molecular assay could offer an alternative when current available serological assays are insufficient.

* Corresponding author. Mailing address: Rega Institute for Medical Research, KU Leuven, Minderbroederstraat 10, 3000 Leuven, Belgium. Phone: 016/33.21.51. Fax: 016/33.21.31. E-mail: britta.moens{at}rega.kuleuven.be

{triangledown} Published ahead of print on 9 September 2009.

Journal of Clinical Microbiology, November 2009, p. 3682-3691, Vol. 47, No. 11
0095-1137/09/$08.00+0     doi:10.1128/JCM.00781-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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