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It is well-known that Leishmania parasites can alter the behavior of the sand fly vector in order to increase their transmission potential. However, little is known about the contribution of the infecting host's blood composition on subsequent sand fly infection and survival. This study focused on the host's glucose metabolism and the insulin/insulin-like growth factor 1 (IGF-1) pathway as both metabolic processes are known to impact vector-parasite interactions of other protozoa and insect species. The focus of this study was inspired by the observation that the glycemic levels in the blood of infected Syrian golden hamsters inversely correlated to splenic and hepatic parasite burdens. To evaluate the biological impact of these findings on further transmission, Lutzomyia longipalpis sand flies were infected with blood that was artificially supplemented with different physiological concentrations of several monosaccharides, insulin or IGF-1. Normoglycemic levels resulted in transiently higher parasite loads and faster appearance of metacyclics, whereas higher carbohydrate and insulin/IGF-1 levels favored sand fly survival. Although the recorded effects were modest or transient of nature, these observations support the concept that the host blood biochemistry may affect Leishmania transmission and sand fly longevity.

BackgroundMiltefosine (MIL) is currently the only oral drug available to treat visceral leishmaniasis but its use as first-line monotherapy has been compromised by an increasing treatment failure. Despite the scarce number of resistant clinical isolates, MIL-resistance by mutations in a single aminophospholipid transporter gene can easily be selected in a laboratory environment. These mutations result in a reduced survival in the mammalian host, which can partially be restored by exposure to MIL, suggesting a kind of drug-dependency.Methodology/principal findingsTo enable a combined study of the infection dynamics and underlying immunological events for differential in vivo survival, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of MIL-resistant (MIL-R) and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results in C57Bl/6 and BALB/c mice show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-γ levels, finally resulting in a less efficient establishment in liver macrophages. The elevated IFN-γ levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites. In addition, we demonstrated that MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-γ production.Conclusions/significanceDifferential induction of innate immune responses in the liver was found to underlie the attenuated phenotype of a MIL-R parasite and its peculiar feature of drug-dependency. The impact of MIL on hepatic NK and NKT activation and IFN-γ production following recognition of a MIL-R strain indicates that this mechanism may sustain infections with resistant parasites and contribute to treatment failure.

Leishmaniasis is a major infectious disease with hundreds of thousands of new cases and over 20,000 deaths each year. The current drugs to treat this life-threatening infection have several drawbacks such as toxicity and long treatment regimens. A library of 1.8 million compounds, from which the hits reported here are publicly available, was screened against Leishmania infantum as part of an optimization program; a compound was found with a 2-aminobenzimidazole functionality presenting moderate potency, low metabolic stability and high lipophilicity. Several rounds of synthesis were performed to incorporate chemical groups capable of reducing lipophilicity and clearance, leading to the identification of compounds that are active against different parasite strains and have improved in vitro properties. As a result of this optimization program, a group of compounds was further tested in anticipation of in vivo evaluation. In vivo tests were carried out with compounds 29 (L. infantum IC50: 4.1 μM) and 39 (L. infantum IC50: 0.5 μM) in an acute L. infantum VL mouse model, which showed problems of poor exposure and lack of efficacy, despite the good in vitro potency.

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type N-glycans. In overlay assays, these sugars were differentially recognized by the mannose receptor and DC-SIGN C-type lectins. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.

In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the 'Mary kDNA PCR' and a newly designed 'LC kDNA PCR' for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available.

BACKGROUND:Cutaneous leishmaniasis (CL) is a major public health concern in Ethiopia. However, knowledge about the complex zoonotic transmission cycle is limited, hampering implementation of control strategies. We explored the feeding behavior and activity of the vector (Phlebotomus pedifer) and studied the role of livestock in CL transmission in southwestern Ethiopia. METHODS:Blood meal origins of engorged sand flies were determined by sequencing host DNA. A host choice experiment was performed to assess the feeding preference of P. pedifer when humans and hyraxes are equally accessible. Ear and nose biopsies from livestock were screened for the presence of Leishmania parasites. Sand flies were captured indoor and outdoor with human landing catches and CDC light traps to determine at which time and where P. pedifer is mostly active. PRINCIPAL FINDINGS:A total of 180 P. pedifer sand flies were found to bite hosts of 12 genera. Humans were the predominant blood meal source indoors (65.9%, p < 0.001), while no significant differences were determined outdoors and in caves. In caves, hyraxes were represented in blood meals equally as humans (45.5% and 42.4%, respectively), but the host choice experiment revealed that sand flies have a significant preference for feeding on hyraxes (p = 0.009). Only a single goat nose biopsy from 412 animal samples was found with Leishmania RNA. We found that P. pedifer is predominantly endophagic (p = 0.003), but occurs both indoors and outdoors. A substantial number of sand flies was active in the early evening, which increased over time reaching its maximum around midnight. CONCLUSION:In contrast to earlier suggestions of exclusive zoonotic Leishmania transmission, we propose that there is also human-to-human transmission of CL in southwestern Ethiopia. Livestock does not play a role in CL transmission and combined indoor and outdoor vector control measures at night are required for efficient vector control.

Monitoring the drug susceptibility of Leishmania isolates still largely relies on standard in vitro cell-based susceptibility assays using (patient-isolated) promastigotes for infection. Although this assay is widely used, no fully standardized/harmonized protocol is yet available hence resulting in the application of a wide variety of host cells (primary cells and cell lines), different drug exposure times, detection methods and endpoint criteria. Advocacy for standardization to decrease inter-laboratory variation and improve interpretation of results has already repeatedly been made, unfortunately still with unsatisfactory progress. As a logical next step, it would be useful to reach at least some agreement on the type of host cell and basic experimental design for routine amastigote susceptibility determination. The present laboratory study using different L. infantum strains as a model for visceral leishmaniasis species compared primary cells (mouse peritoneal exudate (PEC), mouse bone marrow derived macrophages and human peripheral blood monocyte derived macrophages) and commercially available cell lines (THP-1, J774, RAW) for either their susceptibility to infection, their role in supporting intracellular amastigote multiplication and overall feasibility/accessibility of experimental assay protocol. The major findings were that primary cells are better than cell lines in supporting infection and intracellular parasite multiplication, with PECs to be preferred for technical reasons. Cell lines require drug exposure of >96h with THP-1 to be preferred but subject to a variable response to PMA stimulation. The fast dividing J774 and RAW cells out-compete parasite-infected cells precluding proper assay read-out. Some findings could possibly also be applicable to cutaneous Leishmania strains, but this still needs cross-checking. Besides inherent limitations in a clinical setting, susceptibility testing of clinical isolates may remain problematic because of the reliance on patient-derived promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity.

BACKGROUND:Since miltefosine monotherapy against visceral leishmaniasis (VL) caused by Leishmania donovani has been discontinued in the Indian subcontinent due to an increase in the number of treatment failures, single dose liposomal amphotericin B is now advocated as a treatment option of choice. Paromomycin-miltefosine combination therapy can be used as substitute first-line treatment in regions without cold-chain potential. Previous laboratory studies in the closely related species Leishmania infantum have demonstrated that paromomycin monotherapy fairly rapidly selects for resistance producing a phenotype with increased fitness. Given the possible clinical implications of these findings for the current field situation, the present study aimed to identify the potential hazards of paromomycin-miltefosine combination therapy. PRINCIPAL FINDINGS:Drug interaction studies using the fixed-ratio isobologram method revealed an indifferent interaction between paromomycin and miltefosine. In hamsters infected with L. infantum, the combination resulted in cumulative efficacy in reducing parasite burdens in the liver, spleen and bone marrow. Selected resistant lines against the single drugs did not display cross-resistance. When the intracellular amastigote stage was repeatedly exposed to the paromomycin-miltefosine combination, either in vitro or in vivo, no significant susceptibility decrease towards either drug was noted. CONCLUSIONS:These results suggest that implementation of paromomycin-miltefosine combination therapy indeed could represent a safe and affordable treatment option for L. donovani VL as miltefosine appears to overrule the anticipated rapid development of PMM resistance.

Host-directed therapies (HDTs) constitute promising alternatives to traditional therapy that directly targets the pathogen but is often hampered by pathogen resistance. HDT could represent a new treatment strategy for leishmaniasis, a neglected tropical disease caused by the obligate intracellular parasite Leishmania. This protozoan develops exclusively within phagocytic cells, where infection relies on a complex molecular interplay potentially exploitable for drug targets. We previously identified naloxonazine, a compound specifically active against intracellular but not axenic Leishmania donovani. We evaluated here whether this compound could present a host cell-dependent mechanism of action. Microarray profiling of THP-1 macrophages treated with naloxonazine showed upregulation of vATPases, which was further linked to an increased volume of intracellular acidic vacuoles. Treatment of Leishmania-infected macrophages with the vATPase inhibitor concanamycin A abolished naloxonazine effects, functionally demonstrating that naloxonazine affects Leishmania amastigotes indirectly, through host cell vacuolar remodeling. These results validate amastigote-specific screening approaches as a powerful way to identify alternative host-encoded targets. Although the therapeutic value of naloxonazine itself is unproven, our results further demonstrate the importance of intracellular acidic compartments for host defense against Leishmania, highlighting the possibility of targeting this host cell compartment for anti-leishmanial therapy.

In 2005, the Member States of WHO adopted a resolution encouraging countries to develop health financing systems capable of achieving and/or maintaining universal coverage of health services - where all people have access to needed health services without the risk of severe financial consequences. In doing this, a major challenge for many countries will be to move away from out-of-pocket payments, which are often used as an important source of fund collection. Prepayment methods will need to be developed or expanded but, in addition to questions of revenue collection, specific attention will also have to be paid to pooling funds to spread risks and to enable their efficient and equitable use. Developing prepayment mechanisms may take time, depending on countries' economic, social and political contexts. Specific rules for health financing policy will need to be developed and implementing organizations will need to be tailored to the level that countries can support and sustain. In this paper we propose a comprehensive framework focusing on health financing rules and organizations that can be used to support countries in developing their health financing systems in the search for universal coverage.

BACKGROUND:Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts. METHODOLOGY/PRINCIPAL FINDINGS:A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%). CONCLUSION/SIGNIFICANCE:We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.

BACKGROUND: Tsetse flies are obligate blood-feeding insects that transmit African trypanosomes responsible for human sleeping sickness and nagana in livestock. The tsetse salivary proteome contains a highly immunogenic family of the endonuclease-like Tsal proteins. In this study, a recombinant version of Tsal1 (rTsal1) was evaluated in an indirect ELISA to quantify the contact with total Glossina morsitans morsitans saliva, and thus the tsetse fly bite exposure. METHODOLOGY/PRINCIPAL FINDINGS: Mice and pigs were experimentally exposed to different G. m. morsitans exposure regimens, followed by a long-term follow-up of the specific antibody responses against total tsetse fly saliva and rTsal1. In mice, a single tsetse fly bite was sufficient to induce detectable IgG antibody responses with an estimated half-life of 36-40 days. Specific antibody responses could be detected for more than a year after initial exposure, and a single bite was sufficient to boost anti-saliva immunity. Also, plasmas collected from tsetse-exposed pigs displayed increased anti-rTsal1 and anti-saliva IgG levels that correlated with the exposure intensity. A strong correlation between the detection of anti-rTsal1 and anti-saliva responses was recorded. The ELISA test performance and intra-laboratory repeatability was adequate in the two tested animal models. Cross-reactivity of the mouse IgGs induced by exposure to different Glossina species (G. m. morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and other hematophagous insects (Stomoxys calcitrans and Tabanus yao) was evaluated. CONCLUSION: This study illustrates the potential use of rTsal1 from G. m. morsitans as a sensitive biomarker of exposure to a broad range of Glossina species. We propose that the detection of anti-rTsal1 IgGs could be a promising serological indicator of tsetse fly presence that will be a valuable tool to monitor the impact of tsetse control efforts on the African continent.

BACKGROUND: The discovery of Nanobodies (Nbs) with a direct toxic activity against African trypanosomes is a recent advancement towards a new strategy against these extracellular parasites. The anti-trypanosomal activity relies on perturbing the highly active recycling of the Variant-specific Surface Glycoprotein (VSG) that occurs in the parasite's flagellar pocket. METHODOLOGY/PRINCIPAL FINDINGS: Here we expand the existing panel of Nbs with anti-Trypanosoma brucei potential and identify four categories based on their epitope specificity. We modified the binding properties of previously identified Nanobodies Nb_An05 and Nb_An33 by site-directed mutagenesis in the paratope and found this to strongly affect trypanotoxicity despite retention of antigen-targeting properties. Affinity measurements for all identified anti-trypanosomal Nbs reveal a strong correlation between trypanotoxicity and affinity (K(D)), suggesting that it is a crucial determinant for this activity. Half maximal effective (50%) affinity of 57 nM was calculated from the non-linear dose-response curves. In line with these observations, Nb humanizing mutations only preserved the trypanotoxic activity if the K(D) remained unaffected. CONCLUSIONS/SIGNIFICANCE: This study reveals that the binding properties of Nanobodies need to be compatible with achieving an occupancy of >95% saturation of the parasite surface VSG in order to exert an anti-trypanosomal activity. As such, Nb-based approaches directed against the VSG target would require binding to an accessible, conserved epitope with high affinity.

Many low-income countries need to substantially increase expenditure to meet universal coverage goals for essential health services but, because they have very low-incomes, most will be unable to raise adequate funds exclusively from domestic sources in the short to medium term. Increased aid for health will be required. However, there has long been a concern that the rapid arrival of large amounts of foreign exchange in a country could lead to an increase in inflation and loss of international competitiveness, with an adverse impact on exports and economic growth, an economic phenomenon termed 'Dutch disease'. We review cross-country and country-level empirical studies and propose a simple framework to gauge the extent of macroeconomic risks. Of the 15 low-income countries that are increasing aid-financed health spending, 7 have high macroeconomic risks that may constrain the sustained expansion of spending. These conditions also apply in one-quarter of the 42 countries not presently increasing spending. Health authorities should be aware of the multiple risk factors at play, including factors that are health-sector specific and others that generally are not. They should also realize that there are effective means for mitigating the risk of Dutch disease associated with increasing development assistance for health. International partners also have an important role to play since more sustainable and predictable flows of donor funding will allow more productivity enhancing investment in physical and human capital, which will also contribute to ensuring there are few harmful macroeconomic effects of increases in aid.