Mammalian cell invasion by the protozoan parasite, Trypanosoma cruzi, is facilitated by the activation of host cell phosphatidylinositol 3 (PI 3)-kinases. We demonstrate that the well-characterized Ca2+-regulated lysosome-mediated parasite entry pathway is abolished by wortmannin pretreatment. In addition, we have characterized a novel route of T. cruzi invasion unexpectedly revealed in the course of this study. For over a decade, targeted exocytosis of lysosomes at the host cell plasma membrane was considered as the primary mechanism for T. cruzi entry into non-professional phagocytic cells. We now provide evidence that a significant fraction (50% or greater) of invading T. cruzi trypomastigotes exploit an alternate actin-independent entry pathway that involves formation of a tightly associated host cell plasma membrane-derived vacuole enriched in the lipid products of class I PI 3-kinases, PtdInsP3/PtdIns(3,4)P2. Initially devoid of lysosomal markers, the resultant parasite-containing vacuoles gradually acquire lysosome associated membrane protein 1 (lamp-1) and fluid phase endocytic tracer from the lysosomal compartment. In striking contrast to latex bead phagosomes, few T. cruzi vacuoles associate with the early endosomal marker, EEA1 and the 'maturation' process becomes refractory to PI 3-kinase inhibition immediately following parasite internalization. Jointly, these data provide a new paradigm for T. cruzi invasion of non-professional phagocytic cells and reveal a novel vacuole maturation process that appears to bypass the requirement for EEA1.

SummaryMammalian cell invasion by the protozoan pathogen Trypanosoma cruzi is critical to its survival in the host. To promote its entry into a wide variety of nonprofessional phagocytic cells, infective trypomastigotes exploit an arsenal of heterogenous surface glycoproteins, secreted proteases and signalling agonists to actively manipulate multiple host cell signalling pathways. Signals initiated in the parasite upon contact with mammalian cells also function as critical regulators of the invasion process. Whereas the full spectrum of cellular responses modulated by T. cruzi is not yet known, mounting evidence suggests that these pathways impinge on a number of cellular processes, in particular the ubiquitous wound-repair mechanism exploited for lysosomemediated parasite entry. Furthermore, differential engagement of host cell signalling pathways in a cell type-specific manner and modulation of host cell gene expression by T. cruzi are becoming recognized as essential determinants of infectivity and intracellular survival by this pathogen.

SummaryOne of the hallmarks of Trypanosoma cruzi invasion of non-professional phagocytes is facilitation of the process by host cell actin depolymerization. Host cell entry by invasive T. cruzi trypomastigotes is accomplished by exploiting a cellular wound repair process involving Ca 2+ -regulated lysosome exocytosis (i.e. lysosome-dependent) or by engaging a recently recognized lysosome-independent pathway. It was originally postulated that cortical actin microfilaments present a barrier to lysosome-plasma membrane fusion and that transient actin depolymerization enhances T. cruzi entry by increasing access to the plasma membrane for lysosome fusion. Here we demonstrate that cytochalasin D treatment of host cells inhibits early lysosome association with invading T. cruzi trypomastigotes by uncoupling the cell penetration step from lysosome recruitment and/or fusion. These findings provide the first indication that lysosome-dependent T. cruzi entry is initiated by plasma membrane invagination similar to that observed for lysosome-independent entry. Furthermore, prolonged disruption of host cell actin microfilaments results in significant loss of internalized parasites from infected host cells. Thus, the ability of internalized trypomastigotes to remain cell-associated and to fuse with host cell lysosomes is critically dependent upon host cell actin reassembly, revealing an unanticipated role for cellular actin remodelling in the T. cruzi invasion process.

Better targeting of antimalarials to people who need them will maximize the impact of interventions in the private sector.

Background A large proportion of artemisinin-combination therapy (ACT) anti-malarial medicines is consumed by individuals that do not have malaria. The over-consumption of ACTs is largely driven by retail sales in high malaria-endemic countries to clients who have not received a confirmatory diagnosis. This study aims to target ACT sales to clients receiving a confirmatory diagnosis using malaria rapid diagnostic tests (mRDTs) at retail outlets in Kenya and Nigeria. Methods This study comprises two linked four-arm 2 × 2 factorial cluster randomized controlled trials focused on malaria diagnostic testing and conditional ACT subsidies with the goal to evaluate provider-directed and client-directed interventions. The linked trials will be conducted at two contrasting study sites: a rural region around Webuye in western Kenya and the urban center of Lagos, Nigeria. Clusters are 41 and 48 participating retail outlets in Kenya and Nigeria, respectively. Clients seeking care at participating outlets across all arms will be given the option of paying for a mRDT—at a study-recommended price—to be conducted at the outlet. In the provider-directed intervention arm, the outlet owner receives a small monetary incentive to perform the mRDT. In the client-directed intervention arm, the client receives a free ACT if they purchase an mRDT and receive a positive test result. Finally, the fourth study arm combines both the provider- and client-directed interventions. The diagnosis and treatment choices made during each transaction will be captured using a mobile phone app. Study outcomes will be collected through exit interviews with clients, who sought care for febrile illness, at each of the enrolled retail outlets. Results The primary outcome measure is the proportion of all ACTs that are sold to malaria test-positive clients in each study arm. For all secondary outcomes, we will evaluate the degree to which the interventions affect purchasing behavior among people seeking care for a febrile illness at the retail outlet. Conclusions If our study demonstrates that malaria case management can be improved in the retail sector, it could reduce overconsumption of ACTs and enhance targeting of publicly funded treatment reimbursements, lowering the economic barrier to appropriate diagnosis and treatment for patients with malaria. Trial registration ClinicalTrials.govNCT04428307, registered June 9, 2020, and NCT04428385, registered June 9, 2020.

Background A large proportion of artemisinin-combination therapy (ACT) anti-malarial medicines is consumed by individuals that do not have malaria. The over-consumption of ACTs is largely driven by retail sales in high malaria-endemic countries to clients who have not received a confirmatory diagnosis. This study aims to target ACT sales to clients receiving a confirmatory diagnosis using malaria rapid diagnostic tests (mRDTs) at retail outlets in Kenya and Nigeria. Methods This study comprises two linked four-arm 2x2 factorial cluster randomized controlled trials focused on malaria diagnostic testing and conditional ACT subsidies with the goal to evaluate provider directed- and client-directed interventions. The study will be conducted at two contrasting study sites: a rural region around Webuye in western Kenya and the urban center of Lagos, Nigeria. Clusters are 41 and 48 participating retail outlets in Kenya and Nigeria, respectively. Clients seeking care at participating outlets across all arms will be given the option of paying for a mRDT – at a study-recommended price – to be conducted at the outlet. In the provider-directed intervention arm, the outlet owner receives a small monetary incentive to perform the mRDT. In the client-directed intervention arm, the client receives a free ACT if they purchase an mRDT and receive a positive test result. Finally, the fourth study arm combines both the provider- and client- directed interventions. The diagnosis and treatment choices made during each transaction will be captured using a mobile phone app. Study outcomes will be collected through exit interviews with clients, who sought care for febrile illness, at each of the enrolled retail outlets. Results The primary outcome measure is the proportion of all ACTs that are sold to malaria test-positive clients in each study arm. For all secondary outcomes we will evaluate the degree to which the interventions affect purchasing behavior among people seeking care for a febrile illness at the retail outlet. Conclusions If our study demonstrates that malaria case management can be improved in the retail sector, it could reduce overconsumption of ACTs and enhance targeting of publicly funded treatment reimbursements, lowering the economic barrier to appropriate diagnosis and treatment for patients with malaria.

No abstract

An amendment to this paper has been published and can be accessed via the original article.