Comparison of SYBR green I and lactate dehydrogenase antimalarial in vitro assay in Plasmodium falciparum field isolates.

Several assay methods are in use for monitoring the drug sensitivity of malaria parasites and screening new antimalarial drugs. Plasmodium lactate dehydrogenase (pLDH) and SYBR Green I in vitro assays were used to evaluate the drug efficacy of Chloroquine, Artemisinin and Azadirachta indica silver nano particles against Plasmodium falciparum 3D7 strain. The half-maximal inhibitory concentration (IC50) of each compound was estimated with non-linear regression model - dose-response analysis. The consistency between two methods was analysed with Cohen's kappa coefficient, interclass correlation and Bland-Altman plots. No statistical difference was found between IC50 values determined by both assays (p = 0.714). The proportion of resistant isolates to chloroquine according to SYBR green I (43.48%) and pLDH (34.78%) assays were similar (z = 0.302; p = 0.762) with significant concordant between methods (k = 0.819, p < 0.001). The results of pLDH Qualisa assay was comparable with classic SYBR green I assay and can be potentially useful in antimalarial drug efficacy surveillance.

Probing the distinct chemosensitivity of Plasmodium vivax liver stage parasites and demonstration of 8-aminoquinoline radical cure activity in vitro.

Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade.

In vitro screening as an anthelmintic discovery pipeline for Calicophoron daubneyi: nutritive media and rumen environment-based approaches.

Paramphistomosis can lead to morbidity and mortality of ruminant livestock within tropical and sub-tropical climates. In recent decades, rumen fluke has become an emerging infection in temperate climates across Western Europe, with Calicophoron daubneyi, the primary species present. Clinical outbreaks with C. daubneyi larvae are reported and adults might be responsible for production losses. There is not currently a widely licensed anthelmintic product available to control C. daubneyi. In this study, three existing flukicide anthelmintics were tested for efficacy against mature C. daubneyi, comparing a standard in vitro culturing assay and a new more relevant rumen fluid based in vitro compound screening protocol. The new rumen based screen confirmed that oxyclozanide was active against adult C. daubneyi and identified activity with praziquantel. The study highlighted the downstream value of incorporating relevant in vitro screening for anthelmintic discovery pipelines.

Establishment of an in vitro culture system to study the developmental biology of Onchocerca volvulus with implications for anti-Onchocerca drug discovery and screening.

BACKGROUND: Infections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after three decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology especially the mating behaviour and nodule formation as well as the development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite reproduction and behaviour. METHODOLOGY/PRINCIPAL FINDINGS: Prior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium damnosum flies were purified by centrifugation using a 30% Percoll solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both cell-free and cell-based co-culture systems and monitored daily by microscopic visual inspection. Exhausted culture medium was replenished every 2-3 days. The cell-free culture system (DMEM supplemented with 10% NCS) supported the viability and motility of O. volvulus larvae for up to 84 days, while the co-culture system (DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells) extended worm survival for up to 315 days. Co-culture systems alone promoted two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates (69.2±30%) observed in DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, while no moult was observed in DMEM supplemented with 10% NCS and seeded on LEC feeder cells. In DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, O. volvulus adult male worms attached to the vulva region of adult female worms and may have mated in vitro. Apparent early initiation of nodulogenesis was observed in both DMEM supplemented with 10% FBS and seeded on LLC-MK2 and DMEM supplemented with 10% NCS and seeded on LLC-MK2 systems. CONCLUSIONS/SIGNIFICANCE: The present study describes an in vitro system in which O. volvulus L3 larvae can be maintained in culture leading to the development of adult stages. Thus, this in vitro system may provide a platform to investigate mating behaviour and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for macrofilaricidal drug screening.

Progress and challenges in the use of fluorescence-based flow cytometric assays for anti-malarial drug susceptibility tests.

Drug-resistant Plasmodium is a frequent global threat in malaria eradication programmes, highlighting the need for new anti-malarial drugs and efficient detection of treatment failure. Plasmodium falciparum culture is essential in drug discovery and resistance surveillance. Microscopy of Giemsa-stained erythrocytes is common for determining anti-malarial effects on the intraerythrocytic development of cultured Plasmodium parasites. Giemsa-based microscopy use is conventional but laborious, and its accuracy depends largely on examiner skill. Given the availability of nucleic acid-binding fluorescent dyes and advances in flow cytometry, the use of various fluorochromes has been frequently attempted for the enumeration of parasitaemia and discrimination of P. falciparum growth in drug susceptibility assays. However, fluorochromes do not meet the requirements of being fast, simple, reliable and sensitive. Thus, this review revisits the utility of fluorochromes, notes previously reported hindrances, and highlights the challenges and opportunities for using fluorochromes in flow cytometer-based drug susceptibility tests. It aims to improve drug discovery and support a resistance surveillance system, an essential feature in combatting malaria.

Aspectos relacionados com a positividade para a esquistossomose: estudo transversal em área de baixa prevalência em Alagoas, 2020 / Aspectos relacionados con la positividad para la esquistosomiasis: un estudio transversal en un área de baja prevalencia en Alagoas, Brasil, 2020 / Aspects related to positivity for schistosomiasis: a cross-sectional study in a low prevalence area in Alagoas, Brazil, 2020

Objetivo: Analisar aspectos relacionados com a positividade para esquistossomose em área de baixa prevalência, no Brasil. Métodos: Estudo transversal, realizado no primeiro semestre de 2020, quando foram analisadas a proporção de positividade, em função do número de lâminas de Kato-Katz, o desempenho diagnóstico do teste e a estimação da positividade a partir dos dados do Sistema de Informação do Programa de Vigilância e Controle da Esquistossomose (SISPCE). Resultados: Foram analisadas 2.088 lâminas de 348 indivíduos, sendo a proporção de positividade de 11,8%, 26,7% e 31,0% para 1, 4 e 6 lâminas analisadas, respectivamente. Houve concordância excelente (índice Kappa = 0,91) na comparação entre as leituras de 4 e 6 lâminas. Foi estimada subnotificação de 2,1 vezes nos dados do SISPCE. Conclusão: Ampliar o número de lâminas aumentou a positividade do Kato-Katz, o que pode contribuir para maximizar o controle da doença enquanto problema de Saúde Pública.

Objetivo: Analizar aspectos relacionados con la positividad para esquistosomiasis en área de baja prevalencia en Brasil. Métodos: Estudio transversal, realizado en el primer semestre de 2020, donde se analizó la proporción de positividad según el número de portaobjetos de Kato-Katz, el rendimiento diagnóstico de la prueba y la creación de un factor de estimación de positividad a partir de los datos del Sistema de Información del Programa de Vigilancia y Control de la Esquistosomiasis (SISPCE). Resultados: Se analizaron 2.088 láminas de 348 individuos, con proporción de positividad del 11,8%, 26,7% y 31,0% para 1, 4 y 6 láminas analizadas, respectivamente. Hubo una excelente concordancia (Kappa = 0,91) en la comparación entre la lectura de 4 y 6 láminas. Se estimó un subregistro de 2,1 veces en los datos del SISPCE. Conclusión: Aumentar el número de muestras aumentó la positividad de Kato-Katz, lo que puede contribuir a maximizar el control de la enfermedad como problema de Salud Pública.

Objective: To analyze aspects related to schistosomiasis positivity in an area of low prevalence in Brazil. Methods: This was a cross-sectional study, carried out in the first half of 2020, where we analyzed the proportion of positivity, according to the number of Kato-Katz slides, the diagnostic performance of the test and positivity estimates based on data from the Schistosomiasis Surveillance and Control Program Information System (SISPCE). Results: 2,088 slides from 348 individuals were analyzed, with proportion of positivity of 11.8%, 26.7% and 31.0% for 1, 4 and 6 slides analyzed, respectively. There was excellent agreement (Kappa = 0.91) between the readings of 4 and 6 slides. The SISPCE data was estimated to be underreported by up to 2.1 times. Conclusion: Increasing the number of slides increased Kato-Katz positivity, which can contribute to maximizing the control of the disease as a Public Health problem.

Cellular thermal shift assay for the identification of drug-target interactions in the Plasmodium falciparum proteome.

Despite decades of research, little is known about the cellular targets and the mode of action of the vast majority of antimalarial drugs. We recently demonstrated that the cellular thermal shift assay (CETSA) protocol in its two variants: the melt curve and the isothermal dose-response, represents a comprehensive strategy for the identification of antimalarial drug targets. CETSA enables proteome-wide target screening for unmodified antimalarial compounds with undetermined mechanisms of action, providing quantitative evidence about direct drug-protein interactions. The experimental workflow involves treatment of P. falciparum-infected erythrocytes with a compound of interest, heat exposure to denature proteins, soluble protein isolation, enzymatic digestion, peptide labeling with tandem mass tags, offline fractionation, and liquid chromatography-tandem mass spectrometry analysis. Methodological optimizations necessary for the analysis of this intracellular parasite are discussed, including enrichment of parasitized cells and hemoglobin depletion strategies to overcome high hemoglobin abundance in the host red blood cells. We outline an effective data processing workflow using the mineCETSA R package, which enables prioritization of drug-target candidates for follow-up studies. The entire protocol can be completed within 2 weeks.

Investigation of pyrimidine nucleoside analogues as chemical probes to assess compound effects on the proliferation of Trypanosoma cruzi intracellular parasites.

Trypanosoma cruzi parasites utilise de novo pyrimidine biosynthesis to produce DNA and survive within mammalian host cells. This pathway can be hijacked to assess the replication of intracellular parasites with the exogenous addition of a DNA specific probe. To identify suitable probe compounds for this application, a collection of pyrimidine nucleoside analogues was assessed for incorporation into T. cruzi intracellular amastigote DNA using image-based technology and script-based analysis. Associated mammalian cell toxicity of these compounds was also determined against both the parasite host cells (3T3 cells) and HEK293 cells. Incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into parasite DNA was the most effective of the probes tested, with minimal growth inhibition observed following either two or four hours EdU exposure. EdU was subsequently utilised as a DNA probe, followed by visualisation with click chemistry to a fluorescent azide, to assess the impact of drugs and compounds with previously demonstrated activity against T. cruzi parasites, on parasite replication. The inhibitory profiles of these molecules highlight the benefit of this approach for identifying surviving parasites post-treatment in vitro and classifying compounds as either fast or slow-acting. F-ara-EdU resulted in <50% activity observed against T. cruzi amastigotes following 48 hours incubation, at 73 µM. Collectively, this supports the further development of pyrimidine nucleosides as chemical probes to investigate replication of the parasite T. cruzi.

Cellular Markers for the Identification of Chemoresistant Isolates in Leishmania.

Markers to diagnose chemoresistance in infecting Leishmania parasites are urgently required. This is fundamental for patients who do not heal during or after treatment, as they are unresponsive, or patients who relapse at the end of the therapy, suffering from therapeutic failure. Glucose utilization is an indicator of cell viability that closely associates with metabolic activity. In Leishmania, glucose is a source of carbon atoms and is imported into the cell through specific transporters. In experimentally developed chemoresistant Leishmania parasites a significant decrease of the expression of glucose transporters as well as in the cellular accumulation glucose has been described. Alternatively, the electrical membrane potential is an essential parameter for the formation of the electromotive force needed for the acquisition of important nutrients and solutes (e.g., glucose) by cells, and changes in glucose concentration are suggested to constitute a physiological adaptation associated with a chemoresistant phenotype of Leishmania parasites. Here we describe easy methods to measure glucose uptake and the membrane potential in isolates from patient suffering leishmaniasis. Correlation between both parameters might be helpful to identify chemoresistant parasites. Results suggest that the measured kinetics of glucose utilization rate can be correlated with the plasma membrane potential and together used to differentiate between the performance of wild-type and reference parasites on the one hand and parasites isolated from patients with therapeutic failure on the other.

In Vitro Image-Based Assay for Trypanosoma cruzi Intracellular Forms.

The advances in development and popularization of automated fluorescence microscopes and pipetting robots allowed scientists to establish high-throughput compound screening using image-based assays for Trypanosoma cruzi intracellular forms, which are associated to chronic Chagas disease. An intracellular T. cruzi image-based assay is a valuable tool to early stage drug discovery for Chagas disease, because it allows scientists to assess a compound's efficacy and safety in the same experiment. During the last 10 years, several improvements have been incorporated into intracellular T. cruzi assay protocols to make them more predictable in what happens with parasites within an infected organism. In the present chapter, a protocol will be presented for an intracellular T. cruzi assay, but at a low-throughput scale, more compatible with facilities in many academic laboratories.

In Vitro Drug Efficacy Testing Against Trypanosoma brucei.

The recent endorsement of fexinidazole by the European Medicines Agency for the treatment of human African trypanosomiasis has demonstrated the high predictive value of cell-based assays for parasite chemotherapy. Here we describe three in vitro drug susceptibility tests with Trypanosoma brucei that have served as the basis for the identification of fexinidazole as a promising lead: (1) a standard assay with end-point measurement to determine drug efficacy; (2) a wash-out assay to test for reversibility and speed of drug action; (3) isothermal microcalorimetry for real-time measurement of onset of drug action and time to kill. Together, these assays allow to estimate pharmacodynamic parameters in vitro and to devise appropriate treatment regimens for subsequent in vivo experiments.

In Vitro Growth Inhibition Assays of Leishmania spp.

In vitro growth (inhibition) assays have a dual application, either supporting the discovery of novel drugs or as a monitoring tool of drug resistance in patient isolates. From an experimental design point of view, both are quite different with regard to the infecting Leishmania species and strain, the wide variety of permissive host cells (primary cells versus cell lines), drug exposure times, detection methods and endpoint criteria. Recognizing the need for enhanced assay standardization to decrease interlaboratory variation and improve proper interpretation of results, a detailed description is given of the basic fundamental procedures and requirements for routine in vitro growth of Leishmania spp. with specific focus on the intracellular amastigote susceptibility assay. Although the described experimental procedures focus on visceral Leishmania species, the same assay principles may apply for the cutaneous species as well.

In Vivo Bioluminescence Imaging to Assess Compound Efficacy Against Trypanosoma brucei.

Traditional animal models for human African trypanosomiasis rely on detecting Trypanosoma brucei brucei parasitemia in the blood. Testing the efficacy of new compounds in these models is cumbersome because it may take several months after treatment before surviving parasites become detectable in the blood. To expedite compound screening, we have used a Trypanosoma brucei brucei GVR35 strain expressing red-shifted firefly luciferase to monitor parasite distribution in infected mice through noninvasive whole-body bioluminescence imaging. This protocol describes the infection and in vivo bioluminescence imaging of mice to assess compound efficacy against T. brucei during the two characteristic stages of disease, the hemolymphatic phase (stage 1) and the encephalitic or central nervous system phase (stage 2).

In-depth comparison of cell-based methodological approaches to determine drug susceptibility of visceral Leishmania isolates.

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.

Comparison of in vitro assays to study the effectiveness of antiparasitics against Acanthamoeba castellani trophozoites and cysts.

We aimed to compare LDH release assay, trypan blue and fluorescent stainings, and non-nutrient Escherichia coli plate assay in determining treatment efficacy of antiamoebic agents against Acanthamoeba castellanii trophozoites/cysts, in vitro. 1BU trophozoites/cysts were challenged with 0.02% polyhexamethylene biguanid (PHMB), 0.1% propamidine isethionate (PD), and 0.0065% miltefosine (MF). Efficacies of the drugs were determined by LDH release and trypan blue assays, by Hoechst 33343, calcein-AM, and ethidium homodimer-1 fluorescent dyes, and by a non-nutrient agar E. coli plate assay. All three antiamoebic agents induced a significant LDH release from trophozoites, compared to controls (p < 0.0001). Fluorescent-dye staining in untreated 1BU trophozoites/cysts was negligible, but using antiamoebic agents, there was 59.3%-100% trypan blue, 100% Hoechst 33342, 0%-75.3% calcein-AM, and 100% ethidium homodimer-1 positivity. On E. coli plates, in controls and MF-treated 1BU trophozoites/cysts, new trophozoites appeared within 24 h, encystment occurred after 5 weeks. In PHMB- and PD-treated 1BU throphozoites/cysts, irregularly shaped, smaller trophozoites appeared after 72 h, which failed to form new cysts within 5 weeks. None of the enzymatic- and dye-based viability assays tested here generated survival rates for trophozoites/cysts that were comparable with those yielded with the non-nutrient agar E. coli plate assay, suggesting that the culture-based assay is the best method to study the treatment efficacy of drugs against Acanthamoeba.

Multi-center screening of the Pathogen Box collection for schistosomiasis drug discovery.

BACKGROUND: Over the past five years, as a public service to encourage and accelerate drug discovery for diseases of poverty, the Medicines for Malaria Venture (MMV) has released box sets of 400 compounds named the Malaria, Pathogen and Stasis Boxes. Here, we screened the Pathogen Box against the post-infective larvae (schistosomula) of Schistosoma mansoni using assays particular to the three contributing institutions, namely, the University of California San Diego (UCSD) in the USA, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Fundação Oswaldo Cruz (FIOCRUZ) in Brazil. With the same set of compounds, the goal was to determine the degree of inter-assay variability and identify a core set of active compounds common to all three assays. New drugs for schistosomiasis would be welcome given that current treatment and control strategies rely on chemotherapy with just one drug, praziquantel. METHODS: Both the UCSD and Swiss TPH assays utilize daily observational scoring methodologies over 72 h, whereas the FIOCRUZ assay employs XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) at 72 h to measure viability as a function of NAD+/NADH redox state. Raw and transformed data arising from each assay were assembled for comparative analysis. RESULTS: For the UCSD and Swiss TPH assays, there was strong concordance of at least 87% in identifying active and inactive compounds on one or more of the three days. When all three assays were compared at 72 h, concordance remained a robust 74%. Further, robust Pearson's correlations (0.48-0.68) were measured between the assays. Of those actives at 72 h, the UCSD, Swiss TPH and FIOCRUZ assays identified 86, 103 and 66 compounds, respectively, of which 35 were common. Assay idiosyncrasies included the identification of unique compounds, the differential ability to identify known antischistosomal compounds and the concept that compounds of interest might include those that increase metabolic activity above baseline. CONCLUSIONS: The inter-assay data generated were in good agreement, including with previously reported data. A common set of antischistosomal molecules for further exploration has been identified .

20S Proteasome as a Drug Target in Trichomonas vaginalis.

Trichomoniasis is a sexually transmitted disease with hundreds of millions of annual cases worldwide. Approved treatment options are limited to two related nitro-heterocyclic compounds, yet resistance to these drugs is an increasing concern. New antimicrobials against the causative agent, Trichomonas vaginalis, are urgently needed. We show here that clinically approved anticancer drugs that inhibit the proteasome, a large protease complex with a critical role in degrading intracellular proteins in eukaryotes, have submicromolar activity against the parasite in vitro and on-target activity against the enriched T. vaginalis proteasome in cell-free assays. Proteomic analysis confirmed that the parasite has all seven α and seven ß subunits of the eukaryotic proteasome although they have only modest sequence identities, ranging from 28 to 52%, relative to the respective human proteasome subunits. A screen of proteasome inhibitors derived from a marine natural product, carmaphycin, revealed one derivative, carmaphycin-17, with greater activity against T. vaginalis than the reference drug metronidazole, the ability to overcome metronidazole resistance, and reduced human cytotoxicity compared to that of the anticancer proteasome inhibitors. The increased selectivity of carmaphycin-17 for T. vaginalis was related to its >5-fold greater potency against the ß1 and ß5 catalytic subunits of the T. vaginalis proteasome than against the human proteasome subunits. In a murine model of vaginal trichomonad infection, proteasome inhibitors eliminated or significantly reduced parasite burden upon topical treatment without any apparent adverse effects. Together, these findings validate the proteasome of T. vaginalis as a therapeutic target for development of a novel class of trichomonacidal agents.

The egg hatch test: A useful tool for albendazole resistance diagnosis in Fasciola hepatica.

In the current study, the egg hatch test (EHT) has been evaluated as an in vitro technique to detect albendazole (ABZ) resistance in Fasciola hepatica. The intra- and inter-assay variations of the EHT were measured by means of the coefficient of variation in different fluke isolates and over time; then, the results of the EHT were compared with the "gold standard" controlled efficacy test, which assesses the in vivo anthelmintic efficacy. The EHT was used later to evaluate the intra-herd variability regarding the level of ABZ resistance in calves infected by the same fluke isolate. Finally, several factors of the initial protocol were modified to improve the simplicity of the assay, including the incubation time of eggs with the drug and the use of eggs collected from faeces. The greatest uniformity between results within the assay and over time until 8 weeks after gallbladder collection (the deadline proposed for egg analysis) was obtained with an ABZ concentration of 0.5 µM. The length of exposure to ABZ was shown to be critical, as prolonged incubation (15 days) led to a change of ovicidal activity. The ABZ concentration of 0.5 µM is suggested as a possible discriminating dose to predict ABZ resistance, due to the close agreement between the results of the EHT at an ABZ concentration of 0.5 µM and those of the in vivo assays.

Susceptibility testing of Leishmania spp. against amphotericin B and fluconazole using the Sensititre™ YeastOne™ YO9 platform.

BACKGROUND: Current drug regimens for cutaneous leishmaniasis (CL) include toxic systemic therapies such as amphotericin B (AB) and pentavalent antimonials. Fluconazole (FZ) is a well-tolerated potential oral alternative for the management CL. To date, few objective data exist to guide clinical decision-making when selecting a therapeutic agent a priori, and standardized, clinically-approved drug susceptibility testing platforms for Leishmania spp. have yet to be established. The Sensititre™ YeastOne™ YO9 plate is a commercialized drug susceptibility plate including AB and FZ used for routine testing of non-fastidious yeast. Our objective was to adapt the readily available Sensititre™ YeastOne™ YO9 plate, to determine drug susceptibility profiles of AB and FZ in cultured isolates of Old World and New World Leishmania spp. for the treatment of CL. METHODS: Promastigotes were cultured in Tobie's medium with Locke's overlay until log phase growth was achieved, inoculated into the Sensititre™ system, and incubated over 96 H. minimum inhibitory concentrations (MICs) were determined colorimetrically, and promastigote death was assessed by conventional microscopy out to 96- h. Colour change correlated to MIC values. RESULTS: All strains tested exhibited MIC values for FZ that were ≥ 256 µg/mL. New World strains demonstrated reduced susceptibility to AB (0.25 µg/mL - 0.50 µg/mL AB) compared to Old World strains at 0.12 µg/mL AB (p = 0.02). Seventeen (61%) of 28 Viannia isolates versus 82% (27/33) of non-Viannia isolates were resistant at 0.12 µg/mL AB (p = 0.09). For L. V. braziliensis isolates, mean MIC for AB was 0.375 ± 0.14 µg/mL (range 0.25-0.50 µg/mL), while for isolates of L. V. panamensis it was 0.314 ± 0.26 µg/mL (range 0.12-1.0 µg/mL). CONCLUSIONS: We adapted the Sensititre™ YeastOne™ YO9 plate for testing of Leishmania spp. susceptibility profiles for commonly used antifungals in the treatment of CL, including AB and FZ. Given its current utility in mycology, optimization of the system for potential clinical implementation in parasitology should be pursued. However evaluation of clinically relevant amastigote-stage stages, and higher concentrations of FZ beyond the upper limit concentration of the Sensititre™ YeastOne™ Y09 plate would be required.

Expanding the Biological Application of Fluorescent Benzothiadiazole Derivatives: A Phenotypic Screening Strategy for Anthelmintic Drug Discovery Using Caenorhabditis elegans.

The current methodologies used to identify promising new anthelmintic compounds rely on subjective microscopic examination of worm motility or involve genetic modified organisms. We describe a new methodology to detect worm viability that takes advantage of the differential incorporation of the fluorescent molecular marker propidium iodide and the 2,1,3-benzothiadiazole core, which has been widely applied in light technology. The new assay developed could be validated using the "Pathogen Box" library. By use of this bioassay, it was possible to identify three molecules with activity against Caenorhabditis elegans that were previously described as effective in in vitro assays against other pathogens, such as Schistosoma mansoni, Mycobacterium tuberculosis, and Plasmodium falciparum, accelerating the identification of molecules with anthelmintic potential. The current fluorescence-based bioassay may be used for assessing C. elegans viability. The described methodology replaces the subjectivity of previous assays and provides an enabling technology that is useful for rapid in vitro screens of both natural and synthetic compound libraries. It is expected that the results obtained from these robust in vitro screens would select the most effective compounds for follow-up in vivo experimentation with pathogenic helminths.