2.2.2. BNZ activity against intracellular amastigote forms 
BNZ activity against amastigote forms was performed by seeding 1 × 104 Vero cells·well–1 in 24-well plates with rounded coverslips at 37º C in humidified 95 % air, 5 % CO2 atmosphere in Gibco RPMI 1640 medium supplemented with 10% (v /v ) hiFBS. After 24h of incubation, Vero cells were infected with culture-derived trypomastigotes at a multiplicity of infection (MOI) ratio of 10:1. After 24 h of infection, nonphagocyted trypomastigotes were washed, and infected Vero cells were treated by adding BNZ at a concentration range from 100 to 0.4 μM (500 μL·well–1 volumes) at 37º C in humidified 95 % air, 5 % CO2 atmosphere for 72 h. Untreated growth controls were also included. Counting of amastigotes and infected cells was performed in methanol-fixed and Giemsa-stained preparations by random analysis of 500 host cells (Martín-Escolano et al. , 2020b). The infectivity index, defined as the average number of amastigote forms in infected cells multiplied by the percentage of infected cells, was also determined. The trypanocidal activity was expressed as the IC50 and IC90 (inhibitory concentrations 50 and 90) using GraphPad Prism 6 software. Each BNZ concentration was tested in triplicate in four separate determinations.