Intervention reducing malaria parasite load in vector mosquitoes: No impact on Plasmodium falciparum extrinsic incubation period and the survival of Anopheles gambiae.

In the fight against malaria, transmission blocking interventions (TBIs) such as transmission blocking vaccines or drugs, are promising approaches to complement conventional tools. They aim to prevent the infection of vectors and thereby reduce the subsequent exposure of a human population to infectious mosquitoes. The effectiveness of these approaches has been shown to depend on the initial intensity of infection in mosquitoes, often measured as the mean number of oocysts resulting from an infectious blood meal in absence of intervention. In mosquitoes exposed to a high intensity of infection, current TBI candidates are expected to be ineffective at completely blocking infection but will decrease parasite load and therefore, potentially also affect key parameters of vector transmission. The present study investigated the consequences of changes in oocyst intensity on subsequent parasite development and mosquito survival. To address this, we experimentally produced different intensities of infection for Anopheles gambiae females from Burkina Faso by diluting gametocytes from three natural Plasmodium falciparum local isolates and used a newly developed non-destructive method based on the exploitation of mosquito sugar feeding to track parasite and mosquito life history traits throughout sporogonic development. Our results indicate the extrinsic incubation period (EIP) of P. falciparum and mosquito survival did not vary with parasite density but differed significantly between parasite isolates with estimated EIP50 of 16 (95% CI: 15-18), 14 (95% CI: 12-16) and 12 (95% CI: 12-13) days and median longevity of 25 (95% CI: 22-29), 15 (95% CI: 13-15) and 18 (95% CI: 17-19) days for the three isolates respectively. Our results here do not identify unintended consequences of the decrease of parasite loads in mosquitoes on the parasite incubation period or on mosquito survival, two key parameters of vectorial capacity, and hence support the use of transmission blocking strategies to control malaria.

A detailed kinetic model of glycolysis in Plasmodium falciparum-infected red blood cells for antimalarial drug target identification.

Upon infection by the malaria parasite Plasmodium falciparum, the glycolytic rate of a red blood cell increases up to 100-fold, possibly contributing to lactic acidosis and hypoglycemia in patients with severe malaria. This dramatic increase in glucose uptake and metabolism was correctly predicted by a newly constructed detailed enzyme kinetic model of glucose metabolism in the trophozoite-infected red blood cell. Subsequently, we expanded the model to simulate an infected red blood cell culture, including the different asexual blood-stage forms of the malaria parasite. The model simulations were in good agreement with experimental data, for which the measured parasitic volume was an important parameter. Upon further analysis of the model, we identified glucose transport as a drug target that would specifically affect infected red blood cells, which was confirmed experimentally with inhibitor titrations. This model can be a first step in constructing a whole-body model for glucose metabolism in malaria patients to evaluate the contribution of the parasite's metabolism to the disease state.

Development of a new assay for quantification of parasite load of intracellular Leishmania sp. in macrophages using flow cytometry.

Finding novel methodologies that enhance the precision, agility, and standardization of drug discovery is crucial for studying leishmaniasis. The slide count is the technique most used to assess the leishmanicidal effect of a given drug in vitro. Despite being consolidated in the scientific environment, it presents several difficulties in its execution, assessment, and results. In addition to being laborious, this technique takes time, both for the preparation of the material for analysis and for the counting itself. Our research group suggests a fresh approach to address this requirement, which involves utilizing nuclear labeling with propidium iodide and flow cytometry to determine the quantity of Leishmania sp. parasites present in macrophages in vitro. Our results show that the fluorescence of infected samples increases as the infection rate increases. Using Pearson's Correlation analysis, it was possible to establish a correlation coefficient (Pearson r = 0.9473) that was strongly positive, linear, and directly proportional to the fluorescence and infection rate variables. Thus, it is possible to infer a mathematical equation through linear regression to estimate the number of parasites in each sample using the Relative Fluorescence Units (RFU) values. This new methodology opens space for the possibility of using this methodological resource in the in vitro quantification of Leishmania in macrophages.

Potential i-Nos/Arg-1 Switch with NLRP3 and Parasitic Load Down Regulation in Experimental Schistosoma mansoni Infection via Chloroquine Repurposing.

In previous studies, the inhibitory effect of chloroquine on NLRP3 inflammasome and heme production was documented. This may be employed as a double-bladed sword in schistosomiasis (anti-inflammatory and parasiticidal). In this study, chloroquine's impact on schistosomiasis mansoni was investigated. The parasitic load (worm/egg counts and reproductive capacity index [RCI]), i-Nos/Arg-1 expression, splenomegaly, hepatic insult and NLRP3-immunohistochemical expression were assessed in infected mice after receiving early and late repeated doses of chloroquine alone or dually with praziquantel. By early treatment, the least RCI was reported in dually treated mice (41.48 ± 28.58) with a significant reduction in worm/egg counts (3.50 ± 1.29/2550 ± 479.58), compared with either drug alone. A marked reduction in the splenic index was achieved by prolonged chloroquine administration (alone: 43.15 ± 5.67, dually: 36.03 ± 5.27), with significantly less fibrosis (15 ± 3.37, 14.25 ± 2.22) than after praziquantel alone (20.5 ± 2.65). Regarding inflammation, despite the praziquantel-induced significant decrease in NLRP3 expression, the inhibitory effect was marked after dual and chloroquine administration (liver: 3.13 ± 1.21/3.45 ± 1.23, spleen: 5.7 ± 1.6/4.63 ± 2.41). i-Nos RNA peaked with early/late chloroquine administration (liver: 68.53 ± 1.8/57.78 ± 7.14, spleen: 63.22 ± 2.06/62.5 ± 3.05). High i-Nos echoed with a parasiticidal and hepatoprotective effect and may indicate macrophage-1 polarisation. On the flip side, the chloroquine-induced low Arg-1 seemed to abate immune tolerance and probably macrophage-2 polarisation. Collectively, chloroquine synergised the praziquantel-schistosomicidal effect and minimised tissue inflammation, splenomegaly and hepatic fibrosis.

Immunotherapy Combining Mimotopes Selected by Phage Display Plus Amphotericin B Is Effective for Treatment Against Visceral Leishmaniasis.

The treatment for visceral leishmaniasis (VL) causes toxicity in patients, entails high cost and/or leads to the emergence of resistant strains. No human vaccine exists, and diagnosis presents problems related to the sensitivity or specificity of the tests. Here, we tested two phage clones, B1 and D11, which were shown to be protective against Leishmania infantum infection in a murine model as immunotherapeutics to treat mice infected with this parasite species. The phages were used alone or with amphotericin B (AmpB), while other mice received saline, AmpB, a wild-type phage (WTP) or WTP/AmpB. Results showed that the B1/AmpB and D11/AmpB combinations induced polarised Th1-type cellular and humoral responses, which were primed by high levels of parasite-specific IFN-γ, IL-12, TNF-α, nitrite and IgG2a antibodies, which reflected in significant reductions in the parasite load in distinct organs of the animals when analyses were performed 1 and 30 days after the treatments. Reduced organic toxicity was also found in these animals, as compared with the controls. In conclusion, preliminary data suggest the potential of the B1/AmpB and D11/AmpB combinations as immunotherapeutics against L. infantum infection.

Screening of malaria infections in human blood samples with varying parasite densities and anaemic conditions using AI-Powered mid-infrared spectroscopy.

BACKGROUND: Effective testing for malaria, including the detection of infections at very low densities, is vital for the successful elimination of the disease. Unfortunately, existing methods are either inexpensive but poorly sensitive or sensitive but costly. Recent studies have shown that mid-infrared spectroscopy coupled with machine learning (MIRs-ML) has potential for rapidly detecting malaria infections but requires further evaluation on diverse samples representative of natural infections in endemic areas. The aim of this study was, therefore, to demonstrate a simple AI-powered, reagent-free, and user-friendly approach that uses mid-infrared spectra from dried blood spots to accurately detect malaria infections across varying parasite densities and anaemic conditions. METHODS: Plasmodium falciparum strains NF54 and FCR3 were cultured and mixed with blood from 70 malaria-free individuals to create various malaria parasitaemia and anaemic conditions. Blood dilutions produced three haematocrit ratios (50%, 25%, 12.5%) and five parasitaemia levels (6%, 0.1%, 0.002%, 0.00003%, 0%). Dried blood spots were prepared on Whatman™ filter papers and scanned using attenuated total reflection-Fourier Transform Infrared (ATR-FTIR) for machine-learning analysis. Three classifiers were trained on an 80%/20% split of 4655 spectra: (I) high contrast (6% parasitaemia vs. negative), (II) low contrast (0.00003% vs. negative) and (III) all concentrations (all positive levels vs. negative). The classifiers were validated with unseen datasets to detect malaria at various parasitaemia levels and anaemic conditions. Additionally, these classifiers were tested on samples from a population survey in malaria-endemic villages of southeastern Tanzania. RESULTS: The AI classifiers attained over 90% accuracy in detecting malaria infections as low as one parasite per microlitre of blood, a sensitivity unattainable by conventional RDTs and microscopy. These laboratory-developed classifiers seamlessly transitioned to field applicability, achieving over 80% accuracy in predicting natural P. falciparum infections in blood samples collected during the field survey. Crucially, the performance remained unaffected by various levels of anaemia, a common complication in malaria patients. CONCLUSION: These findings suggest that the AI-driven mid-infrared spectroscopy approach holds promise as a simplified, sensitive and cost-effective method for malaria screening, consistently performing well despite variations in parasite densities and anaemic conditions. The technique simply involves scanning dried blood spots with a desktop mid-infrared scanner and analysing the spectra using pre-trained AI classifiers, making it readily adaptable to field conditions in low-resource settings. In this study, the approach was successfully adapted to field use, effectively predicting natural malaria infections in blood samples from a population-level survey in Tanzania. With additional field trials and validation, this technique could significantly enhance malaria surveillance and contribute to accelerating malaria elimination efforts.

Evaluation of blood based quantitative PCR as a molecular diagnostic tool for post kala-azar dermal leishmaniasis (PKDL).

BACKGROUND: Post kala-azar dermal leishmaniasis (PKDL) is a consequential dermal manifestation of visceral leishmaniasis (VL), serving as a parasite reservoir. The traditional diagnostic approach, which requires an invasive skin biopsy is associated with inherent risks and necessitates skilled healthcare practitioners in sterile settings. There is a critical need for a rapid, less invasive method for Leishmania detection. The main objective of this study was to evaluate and compare the diagnostic efficacy of PCR and qPCR in detecting PKDL, utilizing both skin and blood samples and to assess the utility of blood samples for molecular diagnosis. METHODS AND RESULTS: 73 individuals exhibiting clinical symptoms of PKDL and who had tested positive for rK39 rapid diagnostic test (RDT) were enrolled in this study. For the diagnosis of PKDL, both PCR and real-time quantitative PCR (qPCR), employing SYBR Green and TaqMan assays, were performed on blood and skin matched samples. qPCR results using both TaqMan and SYBR Green assay, indicated higher parasite loads in the skin compared to blood, as evident by the Ct values. Importantly, when blood samples were used for PKDL diagnosis by qPCR, an encouraging sensitivity of 69.35% (TaqMan assay) and 79.36% (SYBR Green) were obtained, compared to 8.2% with conventional PCR. CONCLUSION: The findings of the study suggest the potential utility of blood for molecular diagnosis by qPCR, offering a less invasive alternative to skin biopsies in field setting for the early detection of parasitaemia in PKDL patients and effective management and control of the disease.

The absence of eosinophils is associated with early metastatic lesions in Leishmania amazonensis-infected mice.

BACKGROUND: Eosinophils are granulocytes that rapidly increase frequency in the bloodstream during helminthic infections and allergic responses. They are found in tissue infected by Leishmania during early disease, but their role during infection is not entirely understood. OBJECTIVES: We aim to compare the disease due to Leishmania amazonensis in BALB/c and Δdbl-GATA1 mice, which lack eosinophils. METHODS: BALB/c and Δdbl-GATA1 mice infected with L. amazonensis were observed for several weeks. The parasite load and dissemination pattern were assessed. FINDINGS: The Δdbl-GATA1 mice developed an anticipated dissemination of L. amazonensis and a worsening disease. No differences were found in the lesion development or the parasite load in the footpad among Δdbl-GATA1 mice and BALB/c eight weeks after infection. However, nine weeks after infection, massive growth of metastatic lesions appeared in several parts of the skin in Δdbl-GATA1 mice, weeks earlier than BALB/c. We observed increased parasites in the bloodstream, probably an essential dissemination route. Thirteen weeks after infection, metastatic lesions were found in all Δdbl-GATA1 mice. MAIN CONCLUSION: These results suggest a protective role of eosinophils in delaying the disease caused by L. amazonensis, although several limitations of this mice strain must be considered.

Dissection of Barrier Dysfunction in Organoid-Derived Human Intestinal Epithelia Induced by Giardia duodenalis.

BACKGROUND & AIMS: The protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed at overcoming these limitations and recapitulating the authentic series of pathogenic events in the primary human duodenal tissue by using the human organoid system. METHODS: We established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological, and tight junction components. RESULTS: Organoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of the epithelial barrier upon G. duodenalis infection. Gene set enrichment analysis suggested major expression changes, including gene sets contributing to ion transport and tight junction structure. Solute carrier family 12 member 2 and cystic fibrosis transmembrane conductance regulator-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization, and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. Functionally, barrier loss was linked to the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase A-cAMP response element-binding protein signaling pathway. CONCLUSIONS: Data suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection during which alterations of cellular ion transport were followed by breakdown of the tight junctional complex and loss of epithelial integrity, events involving a cAMP/protein kinase A-cAMP response element-binding protein mechanism. These findings and the newly established organoid-derived model to study G. duodenalis infection may help to explore new options for intervening with disease and infection, in particular relevant for chronic cases of giardiasis.

The influence of parasite load on transcriptional activity and morphology of a cestode and its ant intermediate host.

Parasites with complex life cycles are known to induce phenotypic changes in their intermediate hosts to increase transmission to the final host. The magnitude of these changes could increase with the number of parasites, which would be beneficial to co-infecting parasites. Yet, adverse effects of high parasite load (i.e. many parasites in a single host) might stress both hosts and parasites (e.g. through an increased immune response). We investigated the consequences of parasite load on the transcriptional activity and morphology of the cestode Anomotaenia brevis and its intermediate host, the ant Temnothorax nylanderi. We demonstrated that many differentially expressed host genes shifted with parasite load, and their functions indicate a stronger immune response and fight against oxidative stress in heavily infected hosts. The expression of other host genes responded to infection in an all-or-nothing manner, as did the morphology of the host workers. However, the cestodes became smaller when they competed with other parasites for resources from a single host. Their expression profile further indicated shifts in host immune avoidance, starvation resistance and vesicle-mediated transport. In summary, our study reveals clear consequences of parasite load and highlights specific processes and traits affected by this.

Enhanced apoptotic index in hepatocytes, Kupffer cells, and inflammatory infiltrate showed positive correlation with hepatic lesion intensity, parasite load, and clinical status in naturally Leishmania-infected dogs.

It is unknown if Leishmania amastigote infections affect hepatocytes and Kupffer cell apoptosis, and the role played by apoptosis in liver lesions in leishmaniasis is still unclear. Clinically affected and subclinically infected dogs with leishmaniosis and uninfected controls were assessed. Parasite load, biochemical markers for evaluation of liver damage, morphometry (area, perimeter, number of inflammatory focus, major and minor diameters), apoptosis in hepatic tissue (hepatocytes, Kupffer cells, and inflammatory infiltrates) and cellularity in inflammatory foci were quantified. The parasite load in clinically affected dogs proved to be higher than in the other groups. All morphometric parameters (area, perimeter, number of inflammatory focus, major and minor diameters) from clinically affected were higher than the values found in the subclinically infected and uninfected control dogs. Only clinically affected dogs presented high levels of ALT, FA, GGT and cholesterol in serum. Strong positive correlation was observed between biochemical markers for evaluation of liver damage (ALT, FA, GGT and cholesterol) and hepatic apoptosis (hepatocytes, Kupffer cells, and inflammation). Clinically affected dogs showed a more intense hepatic lesion. Hepatocytes showed a higher rate of apoptosis in Leishmania-infected dogs than in uninfected control dogs. The Kupffer cell apoptotic index and apoptosis within the inflammatory infiltrates were higher in clinically affected dogs. The apoptotic index evaluated in hepatocytes, Kupffer cells, and inflammatory infiltrates showed a positive correlation with the intensity of the hepatic lesion, parasite load, and clinical status. Apoptotic cells also showed positive immunostaining for TUNEL, Bcl2, and Bax. Our data showed that hepatic apoptosis was related to the severity of liver damage, the progression of infection, and the parasite load in leishmaniasis. Apoptotic regulated cell recruitment modulated the inflammatory response and favored the survival and dissemination of parasites, depending on the clinical status of the Leishmania-infected dogs.

The Relationship Between Epidemiological Factors and the Parasite Load of Sarcocystis in Yezo-Deer (Cervus nippon yesoensis) in Hokkaido, Japan.

Several cases of gastrointestinal symptoms including diarrhea and vomiting due to the consumption of Sarcocystis-infected venison have been reported in Japan. However, the control of case incidence is difficult, as epidemiological information concerning Sarcocystis in venison in Japan is insufficient. We examined the prevalence and parasite load of Sarcocystis in 89 samples of Yezo-deer (Cervus nippon yesoensis) venison in Hokkaido by quantifying the copy numbers of the 18S rRNA gene of Sarcocystis, followed by a statistical analysis that considered the sampling area, age, and sex to clarify the parameters related to the parasite load. The copy numbers per gram of venison in samples ranged from 4.8 to 8.8 log. Wilcoxon rank-sum test, the one-way factorial analysis of variance (ANOVA), Steel-Dwass test, and a two-way factorial ANOVA showed significant differences in the copy numbers among sampling areas, not by age or sex, suggesting that the load of Sarcocystis in wild deer depended on the sampling area in Hokkaido. Notably, more than 80% of Hokkaido venison has a higher gene copy number than the meat that caused Sarcocystis fayeri-food poisoning. This information is expected to contribute to the establishment of hygiene standards for safe venison consumption and the control of gastrointestinal symptom cases due to consumption of Sarcocystis-infected venison.

Increasing a microscope's effective field of view via overlapped imaging and machine learning.

This work demonstrates a multi-lens microscopic imaging system that overlaps multiple independent fields of view on a single sensor for high-efficiency automated specimen analysis. Automatic detection, classification and counting of various morphological features of interest is now a crucial component of both biomedical research and disease diagnosis. While convolutional neural networks (CNNs) have dramatically improved the accuracy of counting cells and sub-cellular features from acquired digital image data, the overall throughput is still typically hindered by the limited space-bandwidth product (SBP) of conventional microscopes. Here, we show both in simulation and experiment that overlapped imaging and co-designed analysis software can achieve accurate detection of diagnostically-relevant features for several applications, including counting of white blood cells and the malaria parasite, leading to multi-fold increase in detection and processing throughput with minimal reduction in accuracy.

Increased parasite load is associated with reduced metabolic rates and escape responsiveness in pumpkinseed sunfish.

Wild animals have parasites that can compromise their physiological and/or behavioural performance. Yet, the extent to which parasite load is related to intraspecific variation in performance traits within wild populations remains relatively unexplored. We used pumpkinseed sunfish (Lepomis gibbosus) and their endoparasites as a model system to explore the effects of infection load on host aerobic metabolism and escape performance. Metabolic traits (standard and maximum metabolic rates, aerobic scope) and fast-start escape responses following a simulated aerial attack by a predator (responsiveness, response latency and escape distance) were measured in fish from across a gradient of visible (i.e. trematodes causing black spot disease counted on fish surfaces) and non-visible (i.e. cestodes in fish abdominal cavity counted post-mortem) endoparasite infection. We found that a higher infection load of non-visible endoparasites was related to lower standard and maximum metabolic rates, but not aerobic scope in fish. Non-visible endoparasite infection load was also related to decreased responsiveness of the host to a simulated aerial attack. Visible endoparasites were not related to changes in metabolic traits or fast-start escape responses. Our results suggest that infection with parasites that are inconspicuous to researchers can result in intraspecific variation in physiological and behavioural performance in wild populations, highlighting the need to more explicitly acknowledge and account for the role played by natural infections in studies of wild animal performance.

Fasting blood glucose in a Ghanaian adult is causally affected by malaria parasite load: a mechanistic case study using convergent cross mapping.

BACKGROUND: Adults with diabetes mellitus (DM) in malaria-endemic areas might be more susceptible to Plasmodium infection than healthy individuals. Herein, the study was aimed at verifying the hypothesis that increased fasting blood glucose (FBG) promotes parasite growth as reflected by increased parasite density. METHODS: Seven adults without DM were recruited in rural Ghana to determine the relationships between FBG and malaria parasite load. Socio-economic data were recorded in questionnaire-based interviews. Over a period of 6 weeks, FBG and Plasmodium sp. Infection were measured in peripheral blood samples photometrically and by polymerase chain reaction (PCR)-assays, respectively. Daily physical activity and weather data were documented via smartphone recording. For the complex natural systems of homeostatic glucose control and Plasmodium sp. life cycle, empirical dynamic modelling was applied. RESULTS: At baseline, four men and three women (median age, 33 years; interquartile range, 30-48) showed a median FBG of 5.5 (5.1-6.0 mmol/L); one participant had an asymptomatic Plasmodium sp. infection (parasite density: 240/µL). In this participant, convergent cross mapping (CCM) for 34 consecutive days, showed that FBG was causally affected by parasite density (p < 0.02), while the reciprocal relationship was not discernible (p > 0.05). Additionally, daily ambient temperature affected parasite density (p < 0.01). CONCLUSION: In this study population living in a malaria-endemic area, time series analyses were successfully piloted for the relationships between FBG and Plasmodium sp. density. Longer observation periods and larger samples are required to confirm these findings and determine the direction of causality.

Phenological variation in parasite load and inflammatory response in a lizard with an asynchronous reproductive cycle.

We present the first study that compares phenological variation in parasite load and inflammatory response in a lizard with asynchronous male and female gonadal cycles. Other studies have used many species with seasonal and synchronous reproductive cycles, in which it is difficult to decouple the effects of internal and external factors that can affect parasite abundance in each sex. Species with asynchronous reproductive cycles provide the opportunity to study the effects of seasonality and reproductive condition separately, but few studies have documented variation in parasite abundance in these species. We made an extensive comparison of parasite load and inflammatory response of the lizard Sceloporus torquatus, a species with asynchronous reproductive cycles, throughout its active period. We hypothesized that the parasite load would be higher in the period of maximum gonadal activity for each sex, negatively related to body condition and inflammatory response. Our results partially support the hypothesis; males had more parasites in summer than in spring and autumn, while females had more parasites in spring and summer than in autumn; however, we do not find a relationship between parasite load, body condition and inflammatory response. Our results indicated that host-parasite interactions are complex and depend upon both environmental and internal factors. Therefore, longer-term studies may provide a more comprehensive picture of host-parasite dynamics in populations of wild lizards.

MHC Class II (DRB) Promoter Polymorphism and Its Role in Parasite Control among Malaria Patients.

MHC class II (MHCII) molecules are cell surface glycoproteins that play an important role to develop adaptive immune responses. MHCII-disease association is not restricted to structural variation alone but also may extend to genetic variations, which may modulate gene expression. The observed variations in class II gene expression make it possible that the association of MHCII polymorphism with diseases may relate to the level of gene expression in addition to the restriction of response to Ag. Understanding the extent of, and the mechanisms underlying, transcription factor DNA binding variation is therefore key to elucidate the molecular determinants of complex phenotypes. In this study, we investigated whether single nucleotide polymorphisms in MHCII-DRB regulatory gene may be associated with clinical outcomes of malaria in Plasmodium-infected individuals. To this end, we conducted a case-control study to compare patients who had mild malaria with those patients who had asymptomatic Plasmodium infection. It demonstrates that GTAT haplotype exerts an increased DRB transcriptional activity, resulting in higher DRB expression and subsequently perturbed Ag presentation and T cell activation, higher TLR-mediated innate immune gene expression, and Ag clearance, so low parasitemia in comparison with haplotypes other than GTAT (GTAC, GGGT). Hence, we hypothesized that DRB gene promoter polymorphism might lead to altered DRB gene expression, which could possibly affect the TLR-triggered innate immune responses in malaria patients. These genetic findings may contribute to the understanding of the pathogenesis of malaria and will facilitate the rational vaccine design for malaria.

Quantitative monitoring of experimental and human leishmaniasis employing amastigote-specific genes.

The gold standard for diagnosis of leishmaniasis is the microscopic detection of amastigotes/Leishman Donovan (LD) bodies, but its moderate sensitivity necessitates the development of molecular approaches. This study aimed to quantify in experimental animal models and human leishmaniasis the expression of amastigote-specific virulence genes, A2 and amastin by droplet digital polymerase chain reaction (ddPCR). Total RNA was isolated from L. donovani-infected hamsters or murine peritoneal macrophages and lesional biopsies from patients with post kala-azar dermal leishmaniasis (PKDL). Following cDNA conversion, EvaGreen-based ddPCR was performed using specific primers for A2 or amastin and parasite load expressed in copies per µL. Assay was optimized and the specificity of amastigote-specific A2 and amastin was confirmed. In hepatic and splenic tissues of L. donovani-infected hamsters and peritoneal macrophages, ddPCR demonstrated a greater abundance of A2 than amastin. Treatment of L. donovani-infected peritoneal macrophages with conventional anti-leishmanials, miltefosine and amphotericin B translated into a dose-dependent reduction in copies per µL of A2 and amastin, and the extrapolated IC50 was comparable with results obtained by counting LD bodies in Giemsa-stained macrophages. Similarly, in dermal biopsies of patients with PKDL, A2 and amastin were detected. Overall, monitoring of A2 by ddPCR can be an objective measure of parasite burden and potentially adaptable into a high throughput approach necessary for drug development and monitoring disease progression when the causative species is L. donovani.

Flau-A, a naphthoquinone derivative, is a promising therapeutic candidate against visceral leishmaniasis: A preliminary study.

Visceral leishmaniasis (VL) is a neglected tropical disease found in tropical and subtropical regions in the world. The therapeutics used for the treatment against disease presents problems, mainly related to drug toxicity, route of administration, high cost and/or by emergence of resistant strains. In this context, the search for alternative antileishmanial candidates is desirable. Recently, a naphthoquinone derivative namely 2-(2,3,4-tri-O-acetyl-6-deoxy-ß-L-galactopyranosyloxy)-1,4-naphthoquinone or Flau-A showed an effective in vitro biological action against Leishmania infantum. In the present study, the efficacy of this naphthoquinone derivative was evaluated in an in vivo infection model. BALB/c mice (n = 12 per group) were infected and later received saline or were treated with empty micelles (B/Mic), free Flau-A or it incorporated in Poloxamer 407-based micelles (Flau-A/Mic). The products were administered subcutaneously in the infected animals, which were then euthanized one (n = 6 per group) and 15 (n = 6 per group) days post-therapy, when immunological and parasitological evaluations were performed. Results showed that animals treated with Flau-A or Flau-A/Mic produced significantly higher levels of antileishmanial IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibody, when compared to data found in the control (saline and B/Mic) groups; which showed significantly higher levels of parasite-specific IL-4, IL-10 and IgG1 antibody. In addition, animals receiving free Flau-A or Flau-A/Mic presented also significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, when compared to the controls. A low hepatic and renal toxicity was also found. Overall, Flau-A/Mic showed better immunological and parasitological results, when compared to the use of free molecule. In conclusion, preliminary data suggest that this composition could be considered in future studies as promising therapeutic candidate against VL.

Combination of infra-red light with nanogold targeting macrophages in the treatment of Leishmania major infected BALB/C mice.

PURPOSE: In the treatment of cutaneous leishmaniasis (CL), developing drug resistance, existing toxic effects of drugs and failure respond to treatment cause the need to try different treatment methods. We investigated the effect of gold-conjugated macrophage-specific antibody on amastigotes under infra-red light for the treatment of CL. METHODS: Female BALB/c (4-8 weeks old, 20 ± 5 g weight) mice were used in the study. The L. major strain was inoculated on the soles of mice in amastigote form and subpassed. Nanogold (Au), Au + macrophage-specific antibody (MSA) modification and near infra-red (NIR) (5 seconds) were applied to mice groups that developed cutaneous leishmaniasis on their soles. On the 5th and 10th days of the treatment, the lesions were examined clinically and pathologically. RESULTS: When the erythema values were examined, the highest decrease was calculated in the Au + MSA + NIR group in the measurements made on the 10th day (p < 0.014). The best improvement in 10th day measurements is in the NIR and Au + MSA + NIR groups when area values were examined (p = 0.011, p = 0.001). There was a statistically significant difference between the groups in terms of parasite load (PL) (p < 0.005) in pathological evaluation. According to PL grouping, the best result is NIR (p = 0.002). When both main titles (clinical and pathological) are examined, the Au + MSA + NIR group is thought to have an optimal therapeutical feature. CONCLUSIONS: Au + MSA + NIR combination could be a new treatment approach for CL treatment.