The in vitro susceptibilities of the reference strain Leishmania donovani MHOM/ET/67/L82 to sodium stibogluconate, amphotericin B, miltefosine, and the experimental compound PX-6518 were determined for extracellular log-phase promastigotes, established axenic amastigotes, fresh spleen-derived amastigotes, and intracellular amastigotes in primary mouse peritoneal macrophages. Susceptibility to amphotericin B did not differ across the various axenic models (50% inhibitory concentrations [IC 50 ], 0.6 to 0.7 M), and amphotericin B showed slightly higher potency against intracellular amastigotes (IC 50 , 0.1 to 0.4 M). A similar trend was observed for miltefosine, with comparable efficacies against the extracellular (IC 50 , 0.4 to 3.8 M) and intracellular (IC 50 , 0.9 to 4.3 M) stages. Sodium stibogluconate, used either as Pentostam or as a crystalline substance, was inactive against all axenic stages (IC 50 , >64 g Sb V /ml) but showed good efficacy against intracellular amastigotes (IC 50 , 22 to 28 g Sb V /ml); the crystalline substance was about two to three times more potent (IC 50 , 9 to 11 g Sb V /ml). The activity profile of PX-6518 was comparable to that of sodium stibogluconate, but at a much higher potency (IC 50 , 0.1 g/ml). In conclusion, the differential susceptibility determines which in vitro models are appropriate for either drug screening or resistance monitoring of clinical field isolates. Despite the more complex and labor-intensive protocol, the current results support the intracellular amastigote model as the gold standard for in vitro Leishmania drug discovery research and for evaluation of the resistance of field strains, since it also includes host cell-mediated effects. Axenic systems can be recommended only for compounds for which no cellular mechanisms are involved, for example, amphotericin B and miltefosine.Current first-line chemotherapy of leishmaniasis relies on a rather limited arsenal of drugs including sodium stibogluconate, meglumine antimoniate, amphotericin B, and miltefosine, but these entail either problems of emerging resistance, severe side effects, or high costs (5). Since vaccines are not yet on the horizon (23), maintenance and improvement of existing treatment regimens, combined with new drug discovery initiatives, appear to be the only ways to guarantee continued control of this important tropical disease (3,11,27).Both for drug screening and for determination of the susceptibility of field strains, different laboratory methods are being used that focus on the promastigote, the axenic amastigote, or the intracellular amastigote stage. However, it remains unclear how these models cross-validate each other. The differences in environmental conditions between promastigotes and amastigotes in vivo are reflected in their needs for in vitro cultivation. While promastigotes are easily cultured in suspension (8), amastigotes are more difficult to maintain in vitro, since they require macrophages as host cells to meet the highly acidic intracellular environment (15). Cultivat...

BackgroundPathogens causing acute fever, with the exception of malaria, remain largely unidentified in sub-Saharan Africa, given the local unavailability of diagnostic tests and the broad differential diagnosis.MethodologyWe conducted a cross-sectional study including outpatient acute undifferentiated fever in both children and adults, between November 2015 and June 2016 in Kinshasa, Democratic Republic of Congo. Serological and molecular diagnostic tests for selected arboviral infections were performed on blood, including PCR, NS1-RDT, ELISA and IFA for acute, and ELISA and IFA for past infections.ResultsInvestigation among 342 patients, aged 2 to 68 years (mean age of 21 years), with acute undifferentiated fever (having no clear focus of infection) revealed 19 (8.1%) acute dengue–caused by DENV-1 and/or DENV-2 –and 2 (0.9%) acute chikungunya infections. Furthermore, 30.2% and 26.4% of participants had been infected in the past with dengue and chikungunya, respectively. We found no evidence of acute Zika nor yellow fever virus infections. 45.3% of patients tested positive on malaria Rapid Diagnostic Test, 87.7% received antimalarial treatment and 64.3% received antibacterial treatment.DiscussionChikungunya outbreaks have been reported in the study area in the past, so the high seroprevalence is not surprising. However, scarce evidence exists on dengue transmission in Kinshasa and based on our data, circulation is more important than previously reported. Furthermore, our study shows that the prescription of antibiotics, both antibacterial and antimalarial drugs, is rampant. Studies like this one, elucidating the causes of acute fever, may lead to a more considerate and rigorous use of antibiotics. This will not only stem the ever-increasing problem of antimicrobial resistance, but will–ultimately and hopefully–improve the clinical care of outpatients in low-resource settings.Trial registrationClinicalTrials.gov NCT02656862.

Fonds Wetenschappelijk Onderzoek, Vlaamse Interuniversitaire Raad-Universitaire Ontwikkelings Samenwerking, European and Developing Countries Clinical Trials Partnership, and the Belgian Technical Cooperation-Programme d'Etudes et d'Expertises-in the Democratic Republic of Congo.

In medical research, the ethical principle of respect for persons is operationalized into the process of informed consent. The consent tools should be contextualized and adapted to the different socio-cultural environment, especially when research crosses the traditional boundaries and reaches poor communities. We look at the challenges experienced in the malaria Quinact trial, conducted in the Democratic Republic of Congo, and describe some lessons learned, related to the definition of acceptable representative, the role of independent witness and the impact of socio-economic vulnerability. To ensure children's protection, consent is required by the parents or, in their absence, by a legally mandated representative. In our setting, children's responsibility is often entrusted permanently or temporarily to relatives or friends without a tribunal mandate. Hence, a notion of 'culturally acceptable representative' under supervision of the local Ethics Committee may be more suitable. To ensure protection of illiterate subjects, an independent witness is required to confirm that the consent was freely given. However, in low-literacy contexts, potential witnesses often don't have any previous relationship with patient and there may be power-unbalance in their relationship, rather than genuine dialogue. In poor communities, trial participation may be seen as an opportunity to secure access to healthcare. Poverty may also lead to 'competition' to access the research-related benefits, with a risk of disturbance at societal or household level. Adjusting consent procedures to sociocultural and socioeconomic realities is essential for fulfilling the underlying ethical principles. This requires a collaborative dialogue between researchers, regulators and ethics committees.

Maesa saponins with the 13,28-epoxy-oleanane triterpene core skeleton were described recently to possess strong and selective in vitro and in vivo antileishmania activity. In the absence of direct chemical derivatization possibilities, a structure-based literature search was carried out to explore a structure-activity relationship. Crude alcohol extracts from several plant species of Myrsinaceae, Primulaceae, Aceraceae and Icacinaceae were evaluated for in vitro activity against Leishmania infantum intracellular amastigotes and cytotoxicity on MRC-5(SV2) cells, while the saponin content was evaluated qualitatively by TLC. A clear correlation was found between the presence of close analogue 13,28-epoxy-oleanane triterpene saponins and potent and selective antileishmania activity. This was most striking in Maesa species, except for M. macrosepala. Interesting activities were also found in extracts that did not exactly match the TLC characteristics of the Maesa saponin references, as was the case for Ardisia angusta, A. amherstiana, A. caudata, A. gigantifolia, A. roseiflora, Myrsine affinis, Acer brevipes and A. laurinum var. petelotii. This study indicates that the 13,28-epoxy-oleanane triterpene moiety is essential for selective antileishmania potential and that several other plant species could still be explored for antileishmania drug discovery.