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Given the distinct epidemiological profiles and clinical manifestations associated with HTLV-1 and HTLV-2 infections, establishing an accurate differential diagnosis is of critical importance. The serological\/molecular approaches currently available for differential diagnosis are expensive, time-consuming, and require multiple complementary assays, and still lead to inconclusive results. In this context, the development of a single-platform assay referred to as FC-Duplex IgG1 (HTLV-1\/2) assay represented a valuable advance for the differential diagnosis of HTLV infections in clinical settings, while counseling HTLV-seropositive patients and\/or conducting seroepidemiological surveys. The present study aimed at qualifying the FC-Duplex IgG1 (HTLV-1\/2) kit prototype for differential serodiagnosis of HTLV-1 and HTLV-2 infections. A biorepository library, composed of serum samples from four HTLV reference centers (HTLV-1(+)\/n\u2009=\u2009225; HTLV-2(+)\/n\u2009=\u200980 and HTLV1\/2(-)\/n\u2009=\u200955), was tested for anti-HTLV-1 and anti-HTLV-2 IgG1 reactivity using three classification criteria. Discriminant performance analysis supported the high accuracy of FC-Duplex IgG1 (HTLV-1\/2) with elevated proportion of correct classification according to PCR &amp; Western Blot reference standards (Criterion 3\u2009=\u200989%; Criterion 2\u2009=\u200990% and Criterion 1\u2009=\u200999%). Decision tree accuracy and Leave One Out Cross-Validation re-enforced the robustness, confirming the minimal overfitting of our findings. Kappa agreement indices further confirmed the substantial to near perfect agreement of FC-Duplex IgG1 (HTLV-1\/2) with reference standard methods (Criterion 3\u2009=\u20090.72; Criterion 2\u2009=\u20090.75 and Criterion 1\u2009=\u20090.97). Altogether, these findings qualified the FC-Duplex IgG1 (HTLV-1\/2) assay for differential diagnosis of HTLV-1\/2 infections.<\/jats:p>","DOI":"10.1371\/journal.pntd.0013770","type":"journal-article","created":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T18:37:11Z","timestamp":1765219031000},"page":"e0013770","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["FC-duplex IgG1 (HTLV-1\/2): Qualifying the flow cytometry IgG1 duplex assay for differential diagnosis of HTLV-1 and HTLV-2 infections"],"prefix":"10.1371","volume":"19","author":[{"given":"J\u00falia Pereira","family":"Martins","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Felipe Araujo","family":"Santos","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kelly 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