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Chikungunya was introduced into the Americas in 2015 causing a pandemic across the continent. Testing during the acute phase of infection relies on qRT-PCR, but available assays have a number of limitations. A qRT-PCR assay specific to the chikungunya E1 gene was designed using sequence data from contemporary strains. A probit analysis established the 95% limit of detection as 19.6 copies per reaction. We compared the assay with a US Centers for Disease Control (CDC) chikungunya qRT-PCR as the reference standard. The assay had a sensitivity and specificity of 98.4% and 100% in 90 samples retrospectively collected in Guatemala. In a further 74 febrile samples prospectively collected in Ecuador and Guatemala the test had a sensitivity and specificity of 100% and 98.4%, respectively. Sequencing the nsp4 gene of the discordant positive sample indicated the presence of chikungunya RNA, and mismatches to the primer binding sites of the CDC assay., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

During its intra-erythrocytic development, the malaria parasite Plasmodium falciparum synthesizes insoluble hemozoin (HZ) crystals that are released into the circulation upon rupture of parasitized red blood cells, and rapidly phagocytized by host mononuclear cells. Here, HZ persists undigested, causing functional impairment and possibly leading to increased host susceptibility to secondary infections. In patients with malaria and visceral leishmaniasis (VL) co-infections, HZ-loaded macrophages are likely to co-harbor Leishmania donovani parasites, but whether this might influence the course of the Leishmania infection is unknown. In this study, L. donovani amastigote growth was monitored in mouse RAW 264.7 macrophages and PMA-differentiated THP-1 cells previously exposed to increasing amounts of HZ or its synthetic analogue β-hematin (BH). Latex beads were used as a phagocytic control. Data demonstrate that phagocytosis of HZ and BH by RAW 264.7 cells promoted infection therein by L. donovani parasites in a dose-dependent fashion. Similar results were not observed when using THP-1 cells, despite a clear persistence of undigested heme up to 48h after phagocytosis. Conditioning with lipopolysaccharide (LPS)/interferon (IFN)-γ prior to Leishmania infection triggered the release in RAW 264.7 cells of nitric oxide (NO), a highly leishmanicidal metabolite. However, neither HZ nor BH pre-ingestion were able to inhibit NO production following stimulation with LPS/IFN-γ, suggesting that the HZ- and BH-promoting effect on L. donovani infection occurred with an NO-independent mechanism. In conclusion, these preliminary findings highlight a possible detrimental effect of HZ on the course of VL, warranting further investigation into the clinical relevance of the current models., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The neglected tropical disease (NTD) visceral leishmaniasis (VL) has been targeted by the WHO for elimination as a public health problem on the Indian subcontinent by 2017 or earlier. To date there is a surprising scarcity of mathematical models capable of capturing VL disease dynamics, which are widely considered central to planning and assessing the efficacy of interventions. The few models that have been developed are examined, highlighting the necessity for better data to parameterise and fit these and future models. In particular, the characterisation and infectiousness of the different disease stages will be crucial to elimination. Modelling can then assist in establishing whether, when, and how the WHO VL elimination targets can be met., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Currently available drugs for treatment of Leishmania infections are highly toxic and drug resistance to first line therapies has been observed. New, safer and more effective drugs are urgently needed to improve clinical resolution of the disease and reduce the risks associated with it. High-throughput screening of new compounds against cultured promastigotes is easy to perform, but the results are poorly predictive of in vivo efficacy. Intra-macrophage amastigote models provide a better proxy of the clinically relevant stage of disease and should be routinely implemented in the search for new anti-leishmanial agents, despite being labor intensive. This study describes the use of a duplex quantitative Reverse-Transcriptase PCR (qRT-PCR) for assessment of drug activity against Leishmania intracellular amastigotes and their host cells. The assay simultaneously quantifies Leishmania 18S ribosomal RNA and the human β2-microglobulin (β-2M) mRNA, used for monitoring drug cytotoxicity and test performance. Accurate determination of parasite viability by the newly developed qRT-PCR was confirmed by parallel assessment of compound performance against standard microscopy. Highly reproducible anti-leishmanial activities were obtained with a set of structurally- and pharmacologically-diverse compounds, whose toxicity against host cells correlated with a low β-2M amplification. Sensitive and versatile, this duplex qRT-PCR offers a valuable tool for assessment of drug activities against Leishmania amastigotes and their host cells.