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 10 5 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 (r 2 ؍ 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.Since the discovery of human T-lymphotropic virus type 1 (HTLV-1) in 1980 (16, 40), three other genotypes and 10 subtypes have been recognized. The precise geographical distribution and the clinical consequences of these infections are still a matter of debate. This can be attributed at least in part to the fact that there are insufficient accurate tools for HTLV diagnosis, genotyping, and measurement of viral burden.HTLV-1 is endemic in several geographical areas, including sub-Saharan Africa, South America, the Caribbean Islands, Japan, and Melanesia. It has been estimated that worldwide 10 to 25 million people are infected with this retrovirus (41, 53). Most HTLV-1-infected individuals remain asymptomatic throughout their lifetimes. However, 5 to 10% of infected people develop clinical complications, among which adult Tcell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are the most severe. Other manifestations of HTLV-1 infection include infective dermatitis (25), uveitis (34), arthritis (38), and Strongyloides stercoralis infection (53). Some of these manifestations could accelerate disease development and/or progression (12, 16). For HTLV-1, a distinction is made between seven subtypes: the worldwide, cosmopolitan subtype HTLV-1a; the Central African subtypes HTLV-1b, -d, -e, -f, and -g; and the Australo-Melanesic subtype 23,41,52).HTLV-2 was discovered in 1982. This retrovirus is endemic in Amerindian and pygmy populations and epidemic in intravenous drug users (16,49). In contrast to the case for HTLV-1, convincing epidemiological demonstra...