In Vitro Metacyclogenesis of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Clinical Field Isolates, as Evaluated by Morphology, Complement Resistance, and Infectivity to Human Macrophages

This study was designed to assess in vitro metacyclogenesis of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis clinical field isolates obtained from patient lesions (L. braziliensis IMG3 and PPS6m; L. amazonensis MAB6). Metacyclogenesis was evaluated by different criteria, namely, promastigote size (morphometric analysis and flow cytometry), surface modifications (loss of lectin or monoclonal antibody (mAb) binding, complement resistance), and infectivity to human macrophages. Growth curves were similar for all parasites evaluated. The various features analyzed were expressed in a high percentage of promastigotes at 6th and 10th days of culture and a low percentage at the 2nd day. However, in most isolates, these features, considered as markers of metacyclogenesis, seemed to develop with different time courses, since the percentages of metacyclic forms detected with each technique were usually different. Parasites from 6th or 10th day and those negatively selected with lectin or mAb similarly infected human macrophages. From all isolates analyzed, L. amazonensis PH8 and MAB6 showed the highest and the lowest levels of susceptibility, respectively, to leishmanicidal activity of IFN-gamma/LPS-activated macrophages. Our results showed that by using different techniques to evaluate different aspects of metacyclogenesis (morphological and biochemical modifications) different percentages of metacyclic promastigotes can be detected in each isolate culture.