FC-duplex IgG1 (HTLV-1/2): Qualifying the flow cytometry IgG1 duplex assay for differential diagnosis of HTLV-1 and HTLV-2 infections

Human T-lymphotropic virus (HTLV) infection has been implicated in a broad spectrum of clinical conditions, encompassing both neoplastic and inflammatory-degenerative disorders, associated with relevant immunological dysfunctions. 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 = 225; HTLV-2(+)/n = 80 and HTLV1/2(-)/n = 55), 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 & Western Blot reference standards (Criterion 3 = 89%; Criterion 2 = 90% and Criterion 1 = 99%). 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 = 0.72; Criterion 2 = 0.75 and Criterion 1 = 0.97). Altogether, these findings qualified the FC-Duplex IgG1 (HTLV-1/2) assay for differential diagnosis of HTLV-1/2 infections.