The increasing rate of immigration towards European countries and global tourism has determined a new approach by national health committees to control the spread of some infectious diseases previously confined to specific endemic areas. In particular, this new global situation elicits European countries to monitor the local epidemiology of these emerging diseases. In Italy, HTLV-I infection is still sporadic and is confined to immigrants arriving from endemic areas. In particular, sexual transmission, HIV-1 seropositivity and intravenous drug abuse are the preferential infection routes that may lead an increase in HTLV-I infection incidence in the next few years. Our study, focused on disclosing the presence of HTLV-1 in Italy, revealed two HTLV-1 infections in a selected group of individuals originating from African countries. The specific presence of infection in Africa-born patients demonstrated that the infection is noticeable in individuals coming from endemic areas whereas no positive cases were found in the in Italy-born patient group [35–39]. These data are in accordance with several studies on HTLV-I epidemiology in Italy [10, 18, 19, 40] even though a high prevalence of HTLV-I infection was more common in subjects co-infected with human immunodeficiency virus . Our results disclosed HTLV-1 infection in two HIV-1 negative women living in Italy from several years. Even though we found a low prevalence (0.058%: two out the 3408 individuals enrolled in our study) of HTLV-1 infection, up-to-date information is necessary in non endemic countries to gain more knowledge on the spread of this virus.
In our study, both serological techniques used (ELISA and WB) revealed clear-cut positive results in two samples but Western blot did not rule out inconclusive results in three serum samples from high risk individuals. However, the finding of inconclusive results both by ELISA and immunoblotting analysis suggests the need to confirm virus presence by molecular methods. The identification of specific viral sequences in infected cells is essential to confirm the serological diagnosis in subjects with positive or indeterminate results [16, 21, 30]. In addition to determining the distribution of virus in the organism, amplification techniques also document the pathogenesis of infection and the effectiveness of antiviral therapy [41, 42].
The clinical interest in molecular biology assays for HTLV diagnosis is increasing since proviral DNA levels represent a measure of integrated genome  and a surrogate marker of HTLV-I viral replication [44–46].
Our study also shows the application of a SYBR Green based real time PCR committed to HTLV-I provirus detection and quantitation. We optimised the conditions of SYBR Green real time PCR for HTLV-I DNA proviral detection with a high level of specificity (all 20 healthy blood donors' samples did not show any detectable fluorescent signal). Moreover, the assay has excellent dynamic range from 105 to 101 copies with a detection limit established at ten copies: a sensitivity comparable to other PCR formats for HTLV-I. SYBR Green was chosen instead of different real time approaches such as TaqMan or beacons to generate fluorescence signals, for several reasons. In particular, SYBR Green is less expensive than labelled probes that could also determine PCR artefacts beyond the 30th cycle during the amplification. In addition, probe selected sequences may be prone to specific mutations .
The lack of any signal both in seronegative blood donors, used as negative controls and in samples with inconclusive serological results suggests a high specificity level. Some literature data suggest that an indeterminate serological profile (seroreactivity to env proteins, in particular to GD21) could reflect true HTLV-1 infection . Interpretation of doubtful HTLV-1/2 Western blot patterns has therefore been enigmatic since the initiation of screening for these viruses in the late 1980s and several hypotheses have emerged in the last decade. Although HTLV-1 genomic sequences have been detected in the peripheral blood lymphocytes (PBL) of seropositive individuals, previous studies repeatedly demonstrated that PBL from the vast majority of HTLV-1/2 seroindeterminate individuals are PCR negative for HTLV-1 (48). However the possibility of a molecular biology technique characterized by an increased throughput over conventional PCR and quality performance must be taken into consideration for its wide application.
Since our SYBR green based real time PCR technique is a specific and simple assay for quantitative detection of HTLV-I proviral DNA, its application to monitor disease progression and verify the effectiveness of therapy offers an interesting option not only for first level diagnosis, but also for ongoing epidemiological surveillance.
HTLV-1 proviral DNA quantification opens interesting prospects. Cell-free viremia in plasma is not a prominent aspect of HTLV-associated diseases, unlike HIV infection, where the quantitative determination of RNA copies, the main prognostic parameter for disease evolution, directly mirrors viral replication . Moreover, the determination of proviral load, in combination with other biomarkers, could be an important step in the pathogenesis of HTLV-associated disease. In particular, a significant correlation between the proviral load and neopterin concentration (related to inflammatory process in the spinal cord lesion) has been found in the CSF of HAM/TSP patients .
Our method may also be useful to analyze PBMCs from blood donors with serological indeterminate results. Blood donor screening for HTLV was introduced in Japan in the mid 1980s, in the United States and Canada in 1988 and finally in France in 1991 , and even though the probability of collecting blood products from a viremic donor is extremely low, it is not negligible.