Not applicable.

Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important role in the life cycle of Trypanosoma brucei, the causative parasite of human African trypanosomiasis (HAT), also known as African sleeping sickness. We used homology modeling and docking studies to guide fragment growing into the parasite-specific P-pocket in the enzyme binding site. The resulting catechol pyrazolinones act as potent TbrPDEB1 inhibitors with IC₅₀ values down to 49 nM. The compounds also block parasite proliferation (e.g., VUF13525 (20b): T. brucei rhodesiense IC₅₀ = 60 nM, T. brucei brucei IC₅₀ = 520 nM, T. cruzi = 7.6 μM), inducing a typical multiple nuclei and kinetoplast phenotype without being generally cytotoxic. The mode of action of 20b was investigated with recombinantly engineered trypanosomes expressing a cAMP-sensitive FRET sensor, confirming a dose-response related increase of intracellular cAMP levels in trypanosomes. Our findings further validate the TbrPDEB family as antitrypanosomal target.

Background and methodologyDue to geographic overlap of malaria and visceral leishmaniasis (VL), co-infections may exist but have been poorly investigated. To describe prevalence, features and risk factors for VL-malaria co-infections, a case-control analysis was conducted on data collected at Amudat Hospital, Uganda (2000–2006) by Médecins sans Frontières. Cases were identified as patients with laboratory-confirmed VL and malaria at hospital admission or during hospitalization; controls were VL patients with negative malaria smears. A logistic regression analysis was performed to study the association between patients' characteristics and the occurrence of the co-infection.ResultsOf 2414 patients with confirmed VL, 450 (19%) were positively diagnosed with concomitant malaria. Most co-infected patients were males, residing in Kenya (69%). While young age was identified by multivariate analysis as a risk factor for concurrent VL and malaria, particularly the age groups 0–4 (odds ratio (OR): 2.44; 95% confidence interval (CI): 1.52–3.92) and 5–9 years (OR: 2.23, 95% CI: 1.45-3-45), mild (OR: 0.53; 95% CI: 0.32–0.88) and moderate (OR: 0.45; 95% CI: 0.27–0.77) anemia negatively correlated with the co-morbidity. VL patients harboring skin infections were nearly three times less likely to have the co-infection (OR: 0.35; 95% CI: 0.17–0.72), as highlighted by the multivariate model. Anorexia was slightly more frequent among co-infected patients (OR: 1.71; 95% CI: 0.96–3.03). The in-hospital case-fatality rate did not significantly differ between cases and controls, being 2.7% and 3.1% respectively (OR: 0.87; 95% CI: 0.46–1.63).ConclusionsConcurrent malaria represents a common condition among young VL patients living in the Pokot region of Kenya and Uganda. Although these co-morbidities did not result in a poorer prognosis, possibly due to early detection of malaria, a positive trend towards more severe symptoms was identified, indicating that routine screening of VL patients living in malaria endemic-areas and close monitoring of co-infected patients should be implemented.

BackgroundIn areas where visceral leishmaniasis (VL) and malaria are co-endemic, co-infections are common. Clinical implications range from potential diagnostic delay to increased disease-related morbidity, as compared to VL patients. Nevertheless, public awareness of the disease remains limited. In VL-endemic areas with unstable and seasonal malaria, vulnerability to the disease persists through all age-groups, suggesting that in these populations, malaria may easily co-occur with VL, with potentially severe clinical effects.MethodsA retrospective case-control study was performed using medical records of VL patients admitted to Tabarakallah and Gedarif Teaching Hospitals (Gedarif State) and Al`Azaza kala-azar Clinic (Sennar State), Sudan (2005-2010). Patients positively diagnosed with VL and malaria were identified as cases, and VL patients without microscopy-detectable malaria as controls. Associations between patient characteristics and the occurrence of the co-infection were investigated using logistic regression analysis. Confirmation of epidemiological outcomes was obtained with an independently collected dataset, composed by Médecins Sans Frontières (MSF) at Um-el-Kher and Kassab Hospitals, Gedarif State (1998).ResultsThe prevalence of malaria co-infection among VL surveyed patients ranged from 3.8 to 60.8%, with a median of 26.2%. Co-infected patients presented at hospital with deteriorated clinical pictures. Emaciation (Odds Ratio (OR): 2.46; 95% Confidence Interval (95% CI): 1.72-3.50), jaundice (OR: 2.52; 95% CI: 1.04-6.09) and moderate anemia (OR: 1.58; 95% CI: 1.10-2.28) were found to be positively associated with the co-infection, while severity of splenomegaly (OR: 0.53; 95% CI: 0.35-0.81) and, to a less extent, hepatomegaly (OR: 0.52; 95% CI: 0.27-1.01) appeared to be reduced by concomitant VL and malaria. The in-hospital case-fatality rates did not significantly differ between co- and mono-infected patients (OR: 1.13; 95% CI: 0.59-2.17). Conversely, a significantly increased mortality rate (OR: 4.38; 95% CI: 1.83-10.48) was observed by MSF amongst co-infected patients enrolled at Um-el-Kher and Kassab Hospitals, who also suffered an enhanced risk of severe anemia (OR: 3.44; 95% CI: 1.68-7.02) compared to VL mono-infections.ConclusionsIn endemic VL areas with unstable seasonal malaria, like eastern Sudan, VL patients are highly exposed to the risk of developing concomitant malaria. Prompt diagnosis and effective treatment of malaria are essential to ensure that its co-infection does not result into poor prognoses.

BackgroundThe immune system plays a critical role in the development of co-infections, promoting or preventing establishment of multiple infections and shaping the outcome of pathogen-host interactions. Its ability to mediate the interplay between visceral leishmaniasis (VL) and malaria has been suggested, but poorly documented. The present study investigated whether concomitant infection with Leishmania donovani complex and Plasmodium falciparum in naturally co-infected patients altered the immunological response elicited by the two pathogens individually.ResultsCirculating levels of interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12p70, IL-13, IL-17A and tumor necrosis factor (TNF) were assessed in sera of patients infected with active VL and/or malaria and healthy individuals from Gedarif State, Sudan. Comparative analysis of cytokine profiles from co- and mono-infected patients highlighted significant differences in the immune response mounted upon co-infection, confirming the ability of L. donovani and P. falciparum to mutually interact at the immunological level. Progressive polarization towards type-1 and pro-inflammatory cytokine patterns characterized the co-infected patients, whose response partly reflected the effect elicited by VL (IFN-γ, TNF) and malaria (IL-2, IL-13), and partly resulted from a synergistic interaction of the two diseases upon each other (IL-17A). Significantly reduced levels of P. falciparum parasitaemia (P <0.01) were detected in the co-infected group as opposed to the malaria-only patients, suggesting either a protective or a non-detrimental effect of the co-infection against P. falciparum infection.ConclusionsThese findings suggest that a new immunological scenario may occur when L. donovani and P. falciparum co-infect the same patient, with potential implications on the course and resolution of these diseases.

The S. lycocarpum preparation may be useful against leishmaniasis and may have a good safety index, warranting further investigations into its active constituents and mechanism(s) of action.

Background Despite WHO guidelines for testing all suspected cases of malaria before initiating treatment, presumptive malaria treatment remains common practice among some clinicians and in certain low-resource settings the capacity for microscopic testing is limited. This can lead to misdiagnosis, resulting in increased morbidity due to lack of treatment for undetected conditions, increased healthcare costs, and potential for drug resistance. This is particularly an issue as multiple conditions share the similar etiologies to malaria, including brucellosis, a rare, under-detected zoonosis. Linking rapid diagnostic tests (RDTs) and digital test readers for the detection of febrile illnesses can mitigate this risk and improve case management of febrile illness. Methods This technical advance study examines Connected Diagnostics, an approach that combines the use of point-of-care RDTs for malaria and brucellosis, digitally interpreted by a rapid diagnostic test reader (Deki Reader) and connected to mobile payment mechanisms to facilitate the diagnosis and treatment of febrile illness in nomadic populations in Samburu County, Kenya. Consenting febrile patients were tested with RDTs and patient diagnosis and risk information were uploaded to a cloud database via the Deki Reader. Patients with positive diagnoses were provided digital vouchers for transportation to the clinic and treatment via their health wallet on their mobile phones. Results In total, 288 patients were tested during outreach visits, with 9% testing positive for brucellosis and 0.6% testing positive for malaria. All patients, regardless of diagnosis were provided with a mobile health wallet on their cellular phones to facilitate their transport to the clinic, and for patients testing positive for brucellosis or malaria, the wallet funded their treatment. The use of the Deki Reader in addition to quality diagnostics at point of care also facilitated geographic mapping of patient diagnoses in relation to key risk areas for brucellosis transmission. Conclusions This study demonstrates that the Connected Dx approach can be effective even when addressing a remote, nomadic population and a rare disease, indicating that this approach to diagnosing, treatment, and payment for healthcare costs is feasible and can be scaled to address more prevalent diseases and conditions in more populous contexts.

f Critical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity against Leishmania clinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of a Leishmania native enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth of Leishmania parasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assays.