HIV PIs on L. amazonensis Figure 2. Ultrastructural changes observed in L. amazonensis after HIV PIs treatment. Parasites (16108 cells) from 48-hours cultures were inoculated in fresh medium in the absence (A) or in the presence of nelfinavir at 15.12 mM (IC50) (B–H) or lopinavir at 16.47 mM (IC50) (I–J) and incubated for 4 (B–E), 6 (F–G), 8 (H) and 24 (I–J) hours. Subsequently, cells were processed for transmission electron microscopy. An intense flagellar and plasma membrane shedding (black arrowheads) was seen after 4 hours of treatment with both inhibitors (B–D). Some effects were exclusive of nelfinavir, such as cytoplasm shrink (B and E, ), increase in the number of intracellular vesicles, resembling acidocalcisomes (G, w) and lipid inclusions (E and F, v). Both drugs induced nuclear wrapping by the endoplasmic reticulum (G and H, black arrows), mithocondrial swelling (F, white arrowheads) and myelin-like structures (H, larger arrow). In lopinavir treated cells, blocks of condensed chromatin were observed close to the nuclear envelope (I, white arrow), as well as enlarged vesicles (J, ). n - nucleus; k - kinetoplast; f - flagellum and m - mithocondrion. Some of the ultrastructural alterations described for nelfinavir (B–H) were also visualized with lopinavir, for simplicity, these phenomena were represented only by nelfinavir treated cells. doi:10.1371/journal.pone.0004918.g002 dramatically, especially due to increased overlap of both diseases [2]. Additionally, the real impact of Leishmania/HIV co-infection is probably underestimated on the global scale due to deficiencies in the surveillance systems. The fact that leishmaniasis is not included among the AIDS-defining diseases contributes to this scarcity of information [24]. HIV infection can increase the risk of developing leishmaniasis by 100–2000 times [2]. In addition, visceralization of Leishmania strains, such as L. amazonensis, that are classically PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4918