Natural resistance to meglumine antimoniate is associated with treatment failure in cutaneous leishmaniasis caused by Leishmania (Viannia) panamensis.

The multifactorial basis of therapeutic response can obscure the relation between antimicrobial drug susceptibility and clinical outcome. To discern the relationship between parasite susceptibility to meglumine antimoniate (SbV) and therapeutic outcome of cutaneous leishmaniasis, risk factors for treatment failure were considered in evaluating this relationship in ninety-one cutaneous leishmaniasis patients and corresponding clinical strains of Leishmania (Viannia) panamensis. Parasite susceptibility to 32 µg SbV/mL (plasma Cmax) was evaluated in primary human macrophages, PBMCs, and U937 macrophages. Early parasitological response to treatment was determined in lesions of a subgroup of patients, and pathogenicity of Sb-resistant and sensitive clinical strains was compared in BALB/c mice. Parasite survival in cell models and patient lesions was determined by qRT-PCR of Leishmania 7SLRNA transcript. Parasite loads in BALB/c mice were quantified by limiting dilution analysis. The disparate Sb-susceptibility of parasite subpopulations distinguished by isoenzyme profiles (zymodemes) was manifest in all cell models. Notably, Sb-resistance defined by parasite survival, was most effectively discerned in U937 macrophages compared with primary human host cells, significantly higher among strains from patients who failed treatment than cured and, significantly associated with treatment failure. Each unit increase in transformed survival rate corresponded to a 10.6-fold rise in the odds of treatment failure. Furthermore, treatment failure was significantly associated with naturally Sb-resistant zymodeme 2.3 strains, which also produced larger lesions and parasite burdens in BALB/c mice than Sb-sensitive zymodeme 2.2 strains. The confounding effect of host risk factors for treatment failure in discerning this association was evidenced in comparing strains from patients with and without the defined risk factors for treatment failure. These results establish the association of natural resistance to meglumine antimoniate with treatment failure, the importance of host risk factors in evaluating drug susceptibility and treatment outcome, and the clinical and epidemiological relevance of natural Sb-resistance in L. (V.) panamensis subpopulations.

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.

Leishmania blood parasite dynamics during and after treatment of visceral leishmaniasis in Eastern Africa: A pharmacokinetic-pharmacodynamic model.

BACKGROUND: With the current treatment options for visceral leishmaniasis (VL), recrudescence of the parasite is seen in a proportion of patients. Understanding parasite dynamics is crucial to improving treatment efficacy and predicting patient relapse in cases of VL. This study aimed to characterize the kinetics of circulating Leishmania parasites in the blood, during and after different antileishmanial therapies, and to find predictors for clinical relapse of disease. METHODS: Data from three clinical trials, in which Eastern African VL patients received various antileishmanial regimens, were combined in this study. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative PCR (qPCR) before, during, and up to six months after treatment. An integrated population pharmacokinetic-pharmacodynamic model was developed using non-linear mixed effects modelling. RESULTS: Parasite proliferation was best described by an exponential growth model, with an in vivo parasite doubling time of 7.8 days (RSE 12%). Parasite killing by fexinidazole, liposomal amphotericin B, sodium stibogluconate, and miltefosine was best described by linear models directly relating drug concentrations to the parasite elimination rate. After treatment, parasite growth was assumed to be suppressed by the host immune system, described by an Emax model driven by the time after treatment. No predictors for the high variability in onset and magnitude of the immune response could be identified. Model-based individual predictions of blood parasite load on Day 28 and Day 56 after start of treatment were predictive for clinical relapse of disease. CONCLUSION: This semi-mechanistic pharmacokinetic-pharmacodynamic model adequately captured the blood parasite dynamics during and after treatment, and revealed that high blood parasite loads on Day 28 and Day 56 after start of treatment are an early indication for VL relapse, which could be a useful biomarker to assess treatment efficacy of a treatment regimen in a clinical trial setting.

Development of a topical treatment for tegumentary leishmaniasis using 8-hydroxyquinoline.

In the context of neglected diseases, tegumentary leishmaniasis (TL) presents an emerging and re-emerging character in the national territory and in the world. The treatment of TL has limitations, such as intravenous administration route, high toxicity, and high treatment costs. Thus, several researchers work on new therapeutic strategies to improve the effectiveness of the treatment of leishmaniasis. In this light, the present study used a topical formulation, containing 8-hydroquinoline (8-HQN), for the treatment of Balb/c mice infected with L. amazonensis. After the treatment, the mean diameter of the lesion was measured, as well as the parasite load in organs and immunological parameters associated with the treatment. The results showed that the animals treated with 8-HQN 5%, when compared to controls, showed a reduction in the mean diameter of the lesion and in the parasite load. The animals treated with the ointment showed a type 1 cellular immune response profile associated with the production of cytokines such as INF-γ and TNF-α. In addition, the treatment did not demonstrate toxicity to mice. Therefore, the topical formulation containing 8-HQN 5% is a promising candidate in the topical treatment and could be considered, in the future, as an alternative for the treatment of TL.

Dynamics of the Trypanosoma cruzi infection in adipose tissue: Assessing gene expression of PNPLA2, FASN, and ACAT1 under Benzonidazole treatment and indirect mononuclear immune cells interaction.

Trypanosoma cruzi is a parasite with a high capacity to adapt to the host. Animal models have already demonstrated that the tropism of this parasite occurs not only in cardiac/digestive tissues but also in adipose tissue (AT). That said, the consequences ofT. cruziinfection for AT and the implications of treatment with Benzonidazole in this tissue are under discussion. Here, we tested the hypothesis that T. cruzi infection in adipose tissue upon treatment with Benzonidazole (Bz) and the interaction of mononuclear immune cells (PBMC) influences the relative expression of ACAT1, FASN, and PNPLA2 genes. Thus, stem cells derived from adipose tissue (ADSC) after adipogenic differentiation were indirectly cultivated with PBMC after infection with the T. cruzi Y strain and treatment with Bz. We use the TcSAT-IAM system and RT-qPCR to evaluate the parasite load and the relative quantification (ΔCt) of the ACAT1, FASN, and PNPLA2 genes. Our results demonstrate that treatment with Bz did not reduce adipocyte infection in the presence (p-value: 0.5796) or absence (p-value: 0.1854) of cultivation with PBMC. In addition, even though there is no statistical difference when compared to the control group (AT), T. cruzi induces the FASN expression (Rq: 14.00). However, treatment with Bz in AT suggests the increases of PNPLA2 expression levels (Rq: 12.58), even in the absence of T. cruzi infection. During indirect cultivation with PBMC, T. cruzi smooths the expression of PNPLA2 (Rq: 0.824) and instigates the expression of ACAT1 (Rq: 1.632) and FASN (Rq: 1.394). Furthermore, the treatment with Bz during infection induces PNPLA2 expression (Rq: 1.871), maintaining FASN expression levels (Rq: 1.334). Given this, our results indicate that treatment with Benzonidazole did not decrease T. cruzi infection in adipose tissue. However, treating the adipocyte cells with Bz during the interaction with PBMC cells influences the lipid pathways scenario, inducing lipolytic metabolism through the expression of PNPLA2.

Blood parameters and parasite burden in cattle with chronic fascioliasis.

Fascioliasis is a trematodiasis that affects domestic and wild animals as well as humans worldwide. It is a well-recognized disease in livestock, were it produces serious economic losses. Yet in cattle, there is limited information about the burden of liver flukes and its relation to the eggs per gram shed to the environment. There is also lack of knowledge on the effect of parasite load in blood parameters of infected animals, which is important to evaluate the severity and progression of the disease. The objective of this work was to gain insight in these aspects. Cattle from Mendoza province, Argentina, were inspected at a farm and at the abattoir determining the presence or absence of Fasciola hepatica. Each animal was sampled for blood and feces and in the slaughterhouse the livers were inspected. Hematology and blood chemistry parameters were determined, feces were examined for F. hepatica eggs by a quantitative sedimentation technique and livers were thoroughly inspected to determine the number of flukes. Infected cattle presented a mild burden of liver flukes per animal, strongly correlated (r = 0.72) to the number of eggs per gram of feces. The total number of eggs (X̄=35,100) shed per animal to the environment and the type of livestock management techniques in the region exacerbate the role of cattle as efficient reservoirs of this disease. Statistically significant lower red blood cell, lymphocyte and neutrophil counts were observed in infected compared to uninfected animals. All hepatic parameters tested showed highly statistically significant differences (p < 0.001) as well as proteins by cause of rise of globulins in infected cattle. The correlation between the amount of flukes in the liver and the number of eggs per gram of faces indicates coprology as a reliable and cost-effective method to infer parasite burden. The impact of fascioliasis on blood parameters can be of aid for the veterinary practitioner on the assessment of this disease on cattle.

Comparative Analysis of Parasite Load on Recently Established Invasive Pumpkinseed Lepomis gibbosus (Actinopterygii: Centrarchidae) in Europe.

PURPOSE: The aim of this study was the comparative analysis of the parasite communities of new populations of invasive pumpkinseed (Lepomis gibbosus) in western Ukraine with pumpkinseed from Czechia, where populations have rapidly expanded over the last two decades. METHODS: Sampling took place at three localities in the western part of Ukraine (i.e. Dobrotvir Reservoir (Vistula basin), Burshtyn Reservoir (Dniester basin), Mynai Pond (Danube basin)) and four in Czechia (i.e. Oxbow D2, Herspický Pond (Danube basin), and Kolín oxbow and Rímov Reservoir (Elbe basin). RESULTS: In total, 11 parasite taxa were recorded in Ukraine and 17 in Czechia. Four species were co-introduced from North America with their host, i.e. the myxosporean Myxobolus dechtiari, the monogeneans Onchocleidus dispar and Onchocleidus similis, and metacercariae of a trematode Posthodiplostomum centrarchi. High dominance indices were related to a high abundance of co-introduced parasites, i.e. O. similis in Mynai pond and P. centrarchi in Dobrotvir Reservoir. Overall abundance of acquired parasites was generally low. CONCLUSION: This study shows that parasite communities in recently established pumpkinseed populations in the western part of Ukraine and Czechia are less diverse than those established in Europe for decades. The generally low parasite load in these new populations may play an important role in their ability to successfully establish and create strong populations by providing a competitive advantage over local species.

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.

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.

The interplay between temperature, Trypanosoma cruzi parasite load, and nutrition: Their effects on the development and life-cycle of the Chagas disease vector Rhodnius prolixus.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi transmitted by blood-sucking insects of the subfamily Triatominae, is a major neglected tropical disease affecting 6 to 7 million of people worldwide. Rhodnius prolixus, one of the most important vectors of Chagas disease in Latin America, is known to be highly sensitive to environmental factors, including temperature. This study aimed to investigate the effects of different temperatures on R. prolixus development and life-cycle, its relationship with T. cruzi, and to gather information about the nutritional habits and energy consumption of R. prolixus. We exposed uninfected and infected R. prolixus to four different temperatures ranging from 24°C to 30°C, and monitored their survival, developmental rate, body and blood meal masses, urine production, and the temporal dynamics of parasite concentration in the excreted urine of the triatomines over the course of their development. Our results demonstrate that temperature significantly impacts R. prolixus development, life-cycle and their relationship with T. cruzi, as R. prolixus exposed to higher temperatures had a shorter developmental time and a higher mortality rate compared to those exposed to lower temperatures, as well as a lower ability to retain weight between blood meals. Infection also decreased the capacity of the triatomines to retain weight gained by blood-feeding to the next developmental stage, and this effect was proportional to parasite concentration in excreted urine. We also showed that T. cruzi multiplication varied depending on temperature, with the lowest temperature having the lowest parasite load. Our findings provide important insights into the potential impact of climate change on the epidemiology of Chagas disease, and can contribute to efforts to model the future distribution of this disease. Our study also raises new questions, highlighting the need for further research in order to understand the complex interactions between temperature, vector biology, and parasite transmission.

Molecular monitoring of treatment efficacy in human visceral leishmaniasis.

BACKGROUND: Focused efforts of the visceral leishmaniasis elimination program have led to a drastic decline in cases, and the present challenge is disease monitoring, which this study aimed to assess. METHODS: A Leishmania kinetoplastid-targeted qPCR quantified parasite load at disease presentation, and following treatment completion (n=49); an additional 80 cases were monitored after completion of treatment. RESULTS: The parasite load at disease presentation was 13 461.00 (2560.00-37764.00)/µg gDNA, which upon completion of treatment reduced in 47 of 49 cases to 1(1-1)/µg gDNA, p<0.0001. In 80 cases that presented >2 months post-treatment, their parasite burden similarly decreased to 1(1-1)/µg gDNA except in 6 of 80 cases, which were qPCR positive. CONCLUSION: In 129 cases of visceral leishmaniasis, qPCR by quantification of parasite burden proved effective for monitoring treatment.

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.

MicroRNA-194 regulates parasitic load and IL-1ß-dependent nitric oxide production in the peripheral blood mononuclear cells of dogs with leishmaniasis.

Domestic dogs are the primary urban reservoirs of Leishmania infantum, the causative agent of visceral leishmaniasis. In Canine Leishmaniasis (CanL), modulation of the host's immune response may be associated with the expression of small non-coding RNAs called microRNA (miR). miR-194 expression increases in peripheral blood mononuclear cells (PBMCs) of dogs with leishmaniasis with a positive correlation with the parasite load and in silico analysis demonstrated that the TRAF6 gene is the target of miR-194 in PBMCs from diseased dogs. Here, we isolated PBMCs from 5 healthy dogs and 28 dogs with leishmaniasis, naturally infected with L. infantum. To confirm changes in miR-194 and TRAF6 expression, basal expression of miR-194 and gene expression of TRAF6 was measured using qPCR. PBMCs from healthy dogs and dogs with leishmaniasis were transfected with miR-194 scramble, mimic, and inhibitor and cultured at 37° C, 5% CO2 for 48 hours. The expression of possible targets was measured: iNOS, NO, T-bet, GATA3, and FoxP3 were measured using flow cytometry; the production of cytokines IL-1ß, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and TGF-ß in cell culture supernatants was measured using capture enzyme-linked immunosorbent assays (ELISA). Parasite load was measured using cytometry and qPCR. Functional assays followed by miR-194 inhibitor and IL-1ß blockade and assessment of NO production were also performed. Basal miR-194 expression was increased in PBMC from dogs with Leishmaniasis and was negatively correlated with TRAF6 expression. The mimic of miR-194 promoted an increase in parasite load. There were no significant changes in T-bet, GATA3, or FoxP3 expression with miR-194 enhancement or inhibition. Inhibition of miR-194 increased IL-1ß and NO in PBMCs from diseased dogs, and blockade of IL-1ß following miR-194 inhibition decreased NO levels. These findings suggest that miR-194 is upregulated in PBMCs from dogs with leishmaniasis and increases parasite load, possibly decreasing NO production via IL-1ß. These results increase our understanding of the mechanisms of evasion of the immune response by the parasite and the identification of possible therapeutic targets.

Ivermectin decreases parasite load, testosterone, and potentially antler length in a group of captive red deer males (Cervus elaphus).

Secondary sex traits (SSTs) can favour males in intra-sexual competition, allowing females to reliably assess their quality. They can also be connected to other aspects of fitness, such as resistance to parasites and pathogens, as parasites have negative effects on the development of SSTs. Antlers are one of the most recognizable examples of SSTs whose development is regulated by testosterone and reflects the actual condition of the bearer. Elevated testosterone can exaggerate the size of SSTs while impairing the function of the immune system ("The Immunocompetence Handicap Hypothesis") posing a trade-off between antler development and immune function. In this study, we experimentally manipulated the parasite load in captive red deer (Cervus elaphus) males with Ivermectin during antler development for two consecutive years. Expecting an inverse proportionality between parasite load and antler size, we hypothesized the treated deer to have larger antlers than the untreated ones. Our results showed that, following the immunocompetence handicap hypothesis, parasite load was positively associated with testosterone levels. However, the application of Ivermectin suppressed the parasite load of the treated animals but did not lead to the development of larger antlers. Instead, it significantly suppressed the concentration of testosterone in the treated animals, whilst the animals that had higher testosterone also had the highest parasite load. Our findings show that Ivermectin can potentially decrease the levels of testosterone and, consequently, antler size. These findings have important implications for the management of captive populations, especially in contexts where the development of large trophies is desired.

Non-MHC immunity genes do not affect parasite load in European invasive populations of common raccoon.

Understanding the evolutionary mechanisms behind invasion success enables predicting which alien species and populations are the most predisposed to become invasive. Parasites may mediate the success of biological invasions through their effect on host fitness. The evolution of increased competitive ability (EICA) hypothesis assumes that escape from parasites during the invasion process allows introduced species to decrease investment in immunity and allocate resources to dispersal and reproduction. Consequently, the selective pressure of parasites on host species in the invasive range should be relaxed. We used the case of the raccoon Procyon lotor invasion in Europe to investigate the effect of gastrointestinal pathogen pressure on non-MHC immune genetic diversity of newly established invasive populations. Despite distinct differences in parasite prevalence between analysed populations, we detected only marginal associations between two analysed SNPs and infection intensity. We argue that the differences in parasite prevalence are better explained by detected earlier associations with specific MHC-DRB alleles. While the escape from native parasites seems to allow decreased investment in overall immunity, which relaxes selective pressure imposed on immune genes, a wide range of MHC variants maintained in the invasive range may protect from newly encountered parasites.

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.

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.

Vitamin D status, vitamin D receptor gene polymorphism, and haplotype in patients with cutaneous leishmaniasis: Correlation with susceptibility and parasite load index.

BACKGROUND: CL endemicity was reported worldwide including in Saudi Arabia, imposing a major challenge on the health authorities. Vitamin D and its receptor (VDR) are key modulators of the immune response where the VDR is expressed. A remarkable lack of data exists in humans about the contribution of vitamin D and polymorphisms of the VDR gene in protozoan infections, especially cutaneous leishmaniasis (CL). OBJECTIVE: This is the first work conducted to assess the relationship between vitamin D status, polymorphisms of the VDR gene (BsmI, ApaI, TaqI, and FokI), and VDR haplotype with parasite tissue load and susceptibility to CL. METHODS: Fifty-two patients with confirmed CL (21 patients receiving vitamin D medication and 31 patients not receiving it) and 46 control subjects participated in this cross-sectional investigation. VDR genotyping was determined by restriction fragment length polymorphism analysis. Serum levels of 25-OH vitamin D were assessed using the ELISA method in all participants. The skin biopsy quantified the parasite load based on the Ridley parasitic index. RESULTS: The mean serum level of 25-OH vitamin D in CL patients who were not receiving vitamin D therapy was significantly lower compared to CL patients on vitamin D therapy and controls (p <0.001 for both) and CL patients with no history of vitamin D therapy had a significantly higher frequency of vitamin D deficiency compared to CL patients on vitamin D therapy and controls (p < 0.05). Compared to CL patients with no history of vitamin D therapy, CL patients receiving vitamin D therapy had a significantly lower mean size of the lesion and RPI (p = 0.02, .03 respectively). The frequency of genotype "aa" and its "a" allele in ApaI SNP of VDR was significantly lower in CL patients compared to controls (p = 0.006 and 0.03 respectively). However, patients with CL had a considerably greater frequency of the "A" allele than the controls (p = 0.03), suggesting its role in CL susceptibility. There was no statistically significant difference between the two groups in the genotype and allele frequency distributions of BsmI, TaqI, and FokI (p > 0.05). When compared to controls, CL cases had a considerably greater frequency of the "B-A-T-F" haplotype (p = 0.04), and a significantly lower frequency of the "B-a-T-F" haplotype (p = 0.01) suggesting that these haplotypes may have the potential susceptibility or protection against CL respectively. The "Aa" genotype in ApaI SNP of VDR had considerably lower levels of vitamin D with higher parasite load compared to the "AA" and: aa" genotypes (p = 0.02,0.02 respectively). A significant negative correlation was found between the parasite load and 25-OH vitamin D levels (r2 = -0.53, p< 0.001). CONCLUSIONS: According to these findings, vitamin D levels and "ApaI" VDR gene polymorphisms could affect the parasite load and susceptibility to infection, whereas BsmI, FokI, and TaqI polymorphisms did not. Correction of vitamin D levels may aid in CL management.

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.