Low Parasite Burden Research Articles

Characterization of memory T cells in individuals resistant to Schistosoma mansoni infection

To evaluate the frequency of memory CD4+ and CD8+ T cells from individuals resistant and susceptible to Schistosoma mansoni infection. We selected individuals with low (resistant) and high (susceptible) parasite burden using databases generated during previous studies carried out in the same endemic area. The cell surface markers were performed using flow cytometry. In this study, the resistant individuals showed an increase in the CD4+ memory T-cell pool associated with an increase in the central memory cell (TCM) and a decrease in the effector memory cell (TEM ). Individuals susceptible to infection had higher frequencies of effector memory cells compared to resistant individuals. These data suggest that resistance to Smansoni infection may be associated with an increase in the number of CD4+ memory T cells and susceptibility to infection is associated with a decrease in the central memory cell as well as high proportions of effector memory cells.

Detection of Spiked Fasciola hepatica Eggs in Stool Specimens Using LAMP Technique

Fascioliasis is one of the most important food-borne worm disease caused by Fasciola sp. Parasitological diagnosis is more difficult due to the low parasite burden and a few eggs shedding of helminths. Therefore, it will be valuable to development of simple, fast and reliable diagnostic tests for detection of human and animal fascioliasis. Infected liver collected from abattoir in Tehran, Iran in 2017. F. hepatica eggs were detached from the uterus of worms under a stereo microscope. Various numbers of eggs were spiked to 200 mgr. of negative feces samples. DNA was extracted and then target regions (nuclear IGS) were amplified by LAMP assay using six primers. Fecal specimens without egg and DNA of other helminths were used as negative controls. F. hepatica sample which confirmed by morphologic criteria and PCR-RFLP was used as positive control. LAMP products by using SYBR Green I could detect even a single egg in fecal samples which was visible by change of color from orange to green. There was no cross amplification by other helminths including; Taenia saginata, Dicrosolium dendriticum and F. gigantica. LAMP seems a rapid, sensitive, cost-effective technique for detection of human fascioliasis.

Disentangling the effect of host genetics and gut microbiota on resistance to an intestinal parasite

Resistance to infection is a multifactorial trait, and recent work has suggested that the gut microbiota can also contribute to resistance. Here, we performed a fecal microbiota transplant to disentangle the contribution of the gut microbiota and host genetics as drivers of resistance to the intestinal nematode Heligmosomoides polygyrus. We transplanted the microbiota of a strain of mice (SJL), resistant to H. polygyrus, into a susceptible strain (CBA) and vice-versa. We predicted that if the microbiota shapes resistance to H. polygyrus, the FMT should reverse the pattern of resistance between the two host strains. The two host strains had different microbiota diversities and compositions before the start of the experiment, and the FMT altered the microbiota of recipient mice. One mouse strain (SJL) was more resistant to colonization by the heterologous microbiota, and it maintained its resistance profile to H. polygyrus (lower parasite burden) independently of the FMT. On the contrary, CBA mice harbored parasites with lower fecundity during the early stage of the infection, and had an up-regulated expression of the cytokine IL-4 (a marker of H. polygyrus resistance) after receiving the heterologous microbiota. Therefore, while host genetics remains the main factor shaping the pattern of resistance to H. polygyrus, the composition of the gut microbiota also seems to play a strain-specific role.

Leishmania major p27 gene knockout as a novel live attenuated vaccine candidate: Protective immunity and efficacy evaluation against cutaneous and visceral leishmaniasis in BALB/c mice

Leishmaniasis is a growing health problem in many parts of the world and efforts to find vaccine against the disease are a public health priority. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania spp. Defined genetic alteration of the Leishmania genome can be achieved using a gene-targeted disruption strategy that allows for the selection of parasites lacking genes essential for long-term survival and virulence. Previously, we demonstrated that genetically modified live attenuated Leishmania major, lacking the p27gene (Lmp27−/−) is safe and induces cellular immunity in BALB/c mice. p27 is a component of the COX complex that is responsible for ATP synthesis. In the current study, the Lmp27−/− strain was assessed as a live attenuated vaccine. Overall protective immunity and efficacy were evaluated at various time periods following Leishmania major (L. major) and Leishmania infantum (L. infantum) challenges separately in BALB/c mice. Cytokine and anti-Leishmania antibody levels, splenocyte proliferation, delayed type hypersensitivity (DTH), skin lesion development, and parasite burden in the liver and spleen were the measured variables. The results demonstrated that immunized mice had a significant T-helper type 1 (Th1) response, smaller skin lesions and lower parasite burdens in their liver and spleens following a L. major challenge. Furthermore, the Lmp27−/− mutant also granted cross-protection against L. infantum infection.These results suggest that immunization with Lmp27−/− parasites provide significant protective immunity and efficacy against infection with homologous as well as heterologous species of Leishmania parasites.

Evaluation of Immune Response against Leishmaniasis in BALB/c Mice Immunized with Cationic DOTAP/DOPE/CHOL Liposomes Containing Soluble Leishmania major Antigens

Whole killed Leishmania vaccine reached phase III clinical trials but failed to display significant efficacy in human mainly due to limited Th1 inducer adjuvant. Liposomes consisting of 1, 2-dioleoyl-3trimethylammonium-propane (DOTAP) bearing an inherent adjuvanticity and 1, 2-dioleoyl-L-α-glycero-3-phosphatidylethanolamine (DOPE) is well known to intensify the efficacy of positively charged liposomes. Soluble Leishmania major antigens (SLA) encapsulated in cationic liposomes using lipid film method in 2016). BALB/c mice were immunized subcutaneously (SC), three times in a 2-wk interval, with Lip (DOTAP)-SLA+, Lip (DOTAP/DOPE)-SLA+, Lip (DOTAP/DOPE/CHO)-SLA+, Lip (DOTAP/DOPE/CHO), Lip (DOPE/CHO), SLA or HEPES buffer. At week 2 after the last booster injection, immunized mice have challenged SC in the footpad with L. major parasites. To investigate the rate of protection and the type of immune response generated in mice, lesions development was assessed, IL-4 and IFN-γ levels with the ratio of IgG2a/IgG1 isotype were studied to describe the type of generated immune response. Mice immunized with all liposomal form of SLA showed smaller footpad swelling and lower parasite burden in the spleen and footpad compared to the group of mice received buffer. However, these formulations did not show protection against leishmaniosis because of a generated mixed Th1/Th2 response in mice characterized by high production of IFN-γ and IL4 and a high titer of IgG1 and IgG2a antibody. Immunization with Lip (DOTAP/DOPE/CHO)-SLA+ was not an appropriate strategy to protect mice against leishmaniosis.

WILLIS METHOD MODIFICATION WITH A VIEW TO IMPROVING ROUTINE COPROPARASITOLOGICAL DIAGNOSIS

Intestinal parasites still cause high morbidity and mortality, due to poor hygiene and sanitaryconditions, and therefore indiscriminate treatment has been routine practice advocated by thePublic Health staff. Although there is a consensus regarding the need to diagnose such diseases,this is not performed with the necessary care, due to great demand and the lack of a wide rangedand highly sensitive technique. In this sense, most clinical laboratories use routine methods forfecal examination such as the Lutz sedimentation or modified Ritchie methods, which are completeand easy to execute, but do not have adequate sensitivity to detect low density eggs and protozoancysts, especially when there is a predominance of low parasite burdens. In contrast, there aremethods that are based on the flotation of low density developmental forms, namely, the Willismethod (NaCl flotation d=1.120), which is rapid, easy to perform and allows high density eggflotation but with low sensitivity for protozoan cysts; and the Faust method, which is based oncentrifugal flotation of developmental forms in a 33% ZnSO4 (d=1.200) solution, but with thedisadvantage of being lengthy and requiring a centrifuge. In this study, we verified the applicabilityof introducing an alteration in the Willis method, which consisted in the substitution of NaCl byZnSO4 in order to combine the advantages of this method with the Faust method. 208 samples wereassessed by the Willis and Ritchie methods and by the proposed method (modified Willis). Thelatter proved superior to the other two (ρ <0,0001 – X2) regarding the detection of protozoan cysts,but similar to the Ritchie method in regard to other diagnosed parasites, therefore demonstratingthe high potential for the introduction of this modified method in the routine of fecal diagnosis.

Role of Aryl Hydrocarbon Receptor (AhR) in the Regulation of Immunity and Immunopathology During Trypanosoma cruzi Infection.

Resistance to Trypanosoma cruzi infection is dependent on a rapid induction of Th1-type and CD8+ T cell responses that should be promptly balanced to prevent immunopathology. T. cruzi-infected B6 mice are able to control parasite replication but show a limited expansion of Foxp3+regulatory T (Treg) cells that results in the accumulation of effector immune cells and the development of acute liver pathology. AhR is a ligand-activated transcription factor that promotes Treg cell development and suppression of pro-inflammatory cytokine production in dendritic cells, altering the course of adaptive immune response and the development of immunopathology. Here, we used different AhR-dependent activation strategies aiming to improve the Treg response, and B6 congenic mice carrying a mutant AhR variant with low affinity for its ligands (AhRd) to evaluate the role of AhR activation by natural ligands during experimental T. cruzi infection. The outcome of TCDD or 3-HK plus ITE treatments indicated that strong or weak AhR activation before or during T. cruzi infection was effective to regulate inflammation improving the Treg cell response and regularizing the ratio between CD4+ CD25- to Treg cells. However, AhR activation shifted the host-parasite balance to the parasite replication. Weak AhR activation resulted in Treg promotion while strong activation differentially modulated the susceptibility and resistance of cell death in activated T and Treg cells and the increase in TGF-β-producing Treg cells. Of note, T. cruzi-infected AhRd mice showed low levels of Treg cells associated with strong Th1-type response, low parasite burden and absence of liver pathology. These mice developed a Treg- and Tr1-independent mechanism of Th1 constriction showing increased levels of systemic IL-10 and IL-10-secreting CD4+ splenocytes. In addition, AhR activation induced by exogenous ligands had negative effects on the development of memory CD8+ T cell subsets while the lack/very weak activation in AhRd mice showed opposite results, suggesting that AhR ligation restricts the differentiation of memory CD8+T cell subsets. We propose a model in which a threshold of AhR activation exists and may explain how activation or inhibition of AhR-derived signals by infection/inflammation-induced ligands, therapeutic interventions or exposure to pollutants can modulate infections/diseases outcomes or vaccination efficacy.

Modulation of Allergic Reactivity in Humans Is Dependent on Schistosoma mansoni Parasite Burden, Low Levels of IL-33 or TNF-α and High Levels of IL-10 in Serum.

Helminth infections and allergies are characterized by a predominant type-2 immune response. In schistosomiasis, the Th-2 response is usually accompanied by induction of immunoregulatory mechanisms that contribute to worm survival and less severe schistosomiasis. Although helminth-induced immunomodulatory mechanisms seem to affect atopy, epidemiological studies on the relationship between helminths and allergy have been inconsistent, and data suggest that the modulatory effects may be influenced by helminth species, chronicity of infection, and parasite burden. Here we performed a cross-sectional study to investigate the effects of Schistosoma mansoni parasite burden and immune response on allergic reactivity of individuals living in a schistosomiasis endemic area in Brazil. Fecal samples from the participants were collected for extensive parasitological examinations by spontaneous sedimentation, Kato-Katz, Helmintex and Saline Gradient tests and molecular detection of S. mansoni by qPCR. Additionally, the concentrations of cytokines and chemokines, total IgE and IgE-reactivity to common house dust allergens were quantified from serum samples. IgE reactivity to dust allergens was detected in 47 individuals (23.8%), and 140 individuals (54.4%) were diagnosed with S. mansoni infection. Most of the infected population (108 individuals) presented very low parasite burden (≤12 eggs/g of feces). The frequency and intensity (p ≤ 0.03) of allergic reactivity were lower in S. mansoni-infected compared with non-infected individuals. Multivariable logistic regression models adjusted by age revealed that allergic reactivity was positively associated with low IL-10 response (OR, 4.55, 95% CI, 0.56–7.36) and high concentration of the inflammatory mediators IL-33 (OR, 2.70, 95% CI, 1.02–7.15) or TNF-α (OR, 6.88, 95% CI, 0.32–143.39) in serum, and inversely associated with S. mansoni infection (OR, 0.38, 95% CI, 0.16–0.87). Most importantly, the logistic regression demonstrated that the modulatory effects of Schistosoma infection depend on parasite burden, with individuals infected with ≤12 eggs/g of feces showing allergic IgE-reactivity similar to non-infected individuals Altogether, our data show that immunomodulation of allergic reactivity depends on S. mansoni burden, low type-2 inflammatory response, and high level of IL-10.

Exploiting Genetically Modified Dual-Reporter Strains to Monitor Experimental Trypanosoma cruzi Infections and Host-Parasite Interactions.

Trypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. Despite a global research effort, there have been no significant treatment advances for at least 40 years. Gaps in our knowledge of T. cruzi biology and pathogenesis have been major factors in limiting progress. In addition, the extremely low parasite burden during chronic infections has complicated the monitoring of both disease progression and drug efficacy, even in predictive animal models. To address these problems, we genetically modified T. cruzi to express a red-shifted luciferase. Mice infected with these highly bioluminescent parasites can be monitored by in vivo imaging, with exquisite sensitivity. However, a major drawback of bioluminescence imaging is that it does not allow visualization of host-parasite interactions at a cellular level. To facilitate this, we generated T. cruzi strains that express a chimeric protein that is both bioluminescent and fluorescent. Bioluminescence allows the tissue location of infection foci to be identified, and fluorescence can then be exploited to detect parasites in histological sections derived from excised tissue. In this article, we describe in detail the in vivo imaging and confocal microscopy protocols that we have developed for visualizing T. cruzi parasites expressing these dual-reporter fusion proteins. The approaches make it feasible to locate individual parasites within chronically infected murine tissues, to assess their replicative status, to resolve the nature of host cells, and to characterize their immunological context.

In-vitro and In-vivo Antileishmanial Activity of a Compound Derived of Platinum, Oxaliplatin, against Leishmania Major.

This study aimed to evaluate the antileishmanial efficacy of oxaliplatin against Leishmania major (L. major) both in-vitro and in-vivo. The IC50, CC50, and SI of oxaliplatin against promastigotes, murine macrophages, Raw 264.7 cells, and intramacrophage amastigotes of L. major were investigated in-vitro. The effects of this drug on intracellular amastigotes were also assayed, and the percentage of infectivity and IIR were calculated. Flow cytometry was performed to assay apoptosis, using 50 and 100 µg/mL of oxaliplatin in the promastigotes and macrophages. In-vivo, the BALB/c mice were classified into three groups, receiving oxaliplatin, glucantime, and phosphate-buffered saline for one month, respectively. The lesion size, IFN-γ, and IL-4 levels, and parasite burden were also evaluated in the animals. After 72 h, the IC50 and CC50 of oxaliplatin against promastigotes and macrophages were respectively 0.5 and 66.78 µg/mL. The apoptosis of promastigotes and macrophages using 50 µg/mL of oxaliplatin was 7.25% and 2.14%, respectively, while apoptosis induced at 100 µg/mL was 15.48% and 2.80%, respectively. Similar to the glucantime group, the mice treated with oxaliplatin showed a lower parasite burden and smaller lesions, compared with the PBS group (p < 0.01). Furthermore, higher IFN-γ levels were reported in mice receiving oxaliplatin in comparison with those receiving PBS (p < 0.01). The current findings indicated the efficacy of oxaliplatin against promastigote and amastigote forms of Leishmania and L. major-induced leishmaniasis.

High Efficacy of Praziquantel in Schistosoma haematobium-Infected Children in Taraba State, Northeast Nigeria: A follow-up study

This study aimed to assess the efficacy of praziquantel in reducing urinary schistosomiasis prevalence, parasite burden and morbidity rates among a previously reported sample of Schistosoma haematobium-infected children. In addition, predisposing factors for reinfection one year post-treatment were also determined. This prospective follow-up study was conducted between March 2014 and February 2015 among 675 previously reported children with urinary schistosomiasis in the Murbai and Surbai communities of Ardo Kola, Taraba State, Nigeria. A single dose of 40 mg/kg of praziquantel was administered to each infected child, with a second dose administered one month later if necessary. The number of S. haematobium eggs in urine samples was calculated at baseline and post-treatment. At four weeks post-treatment, the overall cure rate was 98.1%. Among children with low and heavy parasite burdens at baseline, egg reduction rates (ERRs) were 100% and 96.5%, respectively. The vast majority of children with microhaematuria (98.7%) and proteinuria (98.6%) at baseline were cured at follow-up. Following a second dose, the ERR, overall and morbidity cure rates increased to 100%. At one year post-treatment, 272 infected children (40.3%) were re-assessed; of these, 51 children (18.8%) were reinfected. Close proximity to bodies of water (odds ratio [OR] = 1.23, 95% confidence interval [CI]: 0.998-1.530; P = 0.05) and fishing (OR = 2.23, 95% CI: 0.828-6.040; P = 0.01) were significant factors that predisposed children to reinfection. A moderate rate of reinfection was noted. Governmental and nongovernmental organisations in Nigeria should collaborate on mass treatment and health education campaigns to reduce the incidence of urinary schistosomiasis reinfections.

Systemic Cytokine and Chemokine Profiles in Individuals With Schistosoma mansoni Infection and Low Parasite Burden.

Intestinal schistosomiasis, caused by the parasitic trematode Schistosoma mansoni, is a chronic disease and the prolonged and continuous exposure to S. mansoni antigens results in a deviation of the host's immune response. For diagnosis, the Kato-Katz (KK) method is recommended, however, this method showed low accuracy in areas of low endemicity. This study aimed to characterize the cytokine and chemokine profile of individuals with an extremely low parasite load (<4 eggs per gram of feces), e.g., individuals who were detected by alternative parasitological methods, such as the saline gradient and/or Helmintex®. In order to search for immunological markers for infection, the immunological profile in serum samples of these individuals was then compared with patients detected with the KK method and with a higher parasite load and with individuals repetitively negative by extensive stool exams. The study was conducted in Northern Minas Gerais in a rural area of the Municipality of Januária. Serum samples of a total of 139 parasitologically well-characterized individuals were assessed for the following immunological markers by commercially available immunoassays: TNF-α, IL-1β, IL-6, IL-17A, IL-5, IL-10, IL-13, IL-33, IL-27, CCL3, CCL5, CXCL10, CCL11, and CCL17. As a result, there were no significant differences in concentrations or frequencies for immunological markers between egg-negative individuals or individuals with ultra-low (<4 epg) or low (4–99 epg) parasite loads. However, we found significant correlations between egg counts and eosinophil counts and between egg counts and IL-1β or TNF-α concentrations. The most striking alterations were found in individuals with the highest parasite load (≥100 epg). They had significantly higher TNF-α concentrations in serum when compared with individuals with a low parasite load (4–99 epg) and CCL17 concentrations were significantly elevated when compared with egg-negative individuals. Radar diagrams of frequencies for cytokine and chemokine responders in each infection group confirmed a distinct profile only in the infection group with highest parasite loads (≥100 epg).

Schistosomiasis mansoni in families of fishing workers of endemic area of Alagoas

Abstract Objective: To analyze the epidemiological and clinical aspects involved in the transmission and manifestation of schistosomiasis in a community of fishermen from the endemic area of Alagoas. Methods: Epidemiological, transversal, prospective, descriptive, quantitative study. The coproparasitological survey included 275 family units of fish workers and the epidemiological and clinical ones, those with a positive diagnosis for S. mansoni. Results: The prevalence of schistosomiasis was 13.9% (fishermen), 2.1% (shellfish) and 2.1% (family members). The occurrence of the infection varied according to gender, age, ethnicity and socioeconomic status. Exposure occurred near the home. Autochthonous and predominant low parasite burden, intestinal clinical presentation, abdominal pain and diarrhea were observed. There were no nutritional and pressure changes associated with parasitosis. Adherence to drug therapy and use of the schistosomicidal drug was feared. Conclusion: Fish workers are at high risk for contracting the disease with clinical hepatointestinal manifestation, which requires more intense health actions.

Eryptosis of non-parasitized erythrocytes is related to anemia in Plasmodium berghei low parasitema malaria of Wistar rats.

It is known that premature elimination of non-parasitized RBCs (nRBCs) plays an important role in the pathogenesis of malarial anemia, in which suicidal death process (eryptosis) of nRBCs has been suggested to be involved. To check this possibility, we investigate eryptosis during infection of P. berghei ANKA in Wistar rats, a malaria experimental model that, similar to human malaria, the infection courses with low parasitemia and acute anemia. As expected, P. berghei ANKA infection was marked by low parasite burdens that reached a mean peak of 3% between days six and nine post-infection and solved spontaneously. A significant reduction of the hemoglobin levels (~ 30%) was also observed on days subsequent to the peak of parasitemia, persisting until day 16 post-infection. In eryptosis assays, it was observed a significant increase in the levels of PS-exposing nRBC, which coincided with the reduction of hemoglobin levels and was positively related to anemia. In addition to PS externalization, eryptosis of nRBC induced by P. berghei infection was characterized by cytoplasm calcium influx, but not caspases activity. These results confirm our previous studies evidencing a pro-eryptotic effect of malaria infection on nRBCs and show that a caspase-independent eryptotic process is implicated in anemia induced by P. berghei ANKA infection in Wistar rats.

Cross-protective efficacy of a conserved Leishmania braziliensis recombinant protein as a candidate vaccine against visceral leishmaniasis

Background: Leishmaniasis represents a disease complex in the world ranging from self-limiting cutaneous leishmaniasis (CL) to the fatal visceral leishmaniasis (VL). The development of new vaccines to prevent VL is a desirable measure to control the disease. An important challenge for the development of an effective antileishmanial vaccine is the low efficacy of the tested antigens to protect against distinct Leishmania spp., since candidates usually offer a species-specific protection, although there significant protein homology between species. Aiming to solve this issue, in the present study, the L. braziliensis enolase protein, was evaluated as a protective antigen, associated with saponin, to offers heterologous protection against L. infantum infection. Methods & Materials: The DNA sequence was cloned and the recombinant protein (rEnolase) was evaluated as a vaccine, associated with saponin, as an immune adjuvant. The protective efficacy of the vaccine was investigated in BALB/c mice (n = 16 per group) against experimental visceral leishmaniasis. Three doses were administered at two-week intervals in all animals. Thirty days after the last immunization, mice (n = 8, in each group) were euthanized and their sera samples and spleen were collected to analyze the immune response. At the same time, remaining animals were subcutaneously infected with 1 × 107 stationary-phase promastigotes of L. infantum. Ten weeks after challenge these animals were euthanized to analyze the immune response and parasite burden. Results: The results revealed that the vaccine induced higher levels of IFN-γ, IL-12, and GM-CSF when a capture ELISA and flow cytometry were performed, as well as an antileishmanial nitrite production after using in vitro stimulation with rEnolase and an antigenic Leishmania preparation. The vaccinated animals, when compared to the control groups, showed a lower parasite burden in the liver, spleen, bone marrow, and paws’ draining lymph nodes when both a limiting dilution technique and RT-PCR assay were performed. In addition, these mice showed low levels of antileishmanial IL-4, IL-10, and anti-Leishmania IgG1 isotype antibodies. Partial protection was associated with IFN-γ production, which was mainly mediated by CD4+ T cells. Conclusion: In conclusion, the present study's data showed that the L. braziliensis enolase protein could be considered a vaccine candidate that offers heterologous protection against VL.

Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis

Vaccination seems to be the best approach to control visceral leishmaniasis (VL). Resistance against infection is based on the development of a Th1 immune response characterized by the production of interferons-γ (IFN-γ), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α), among others. A number of antigens have been tested as potential targets against the disease; few of them are able to stimulate human immune cells. In the present study, 1 prediction of MHC class I and II molecules-specific epitopes in the amino acid sequences of 3 Leishmania proteins: 1 hypothetical, prohibitin, and small glutamine-rich tetratricopeptide repeat-containing proteins, was performed using bioinformatics tools, and a T-cell epitopes-based recombinant chimeric protein was constructed, synthetized and purified to be evaluated in invitro and in vivo experiments. The purified protein was tested regarding its immunogenicity in peripheral blood mononuclear cells (PBMCs) from healthy subjects and VL patients, as well as to its immunogenicity and protective efficacy in a murine model against Leishmania infantum infection. Results showed a Th1 response based on high IFN-γ and low IL-10 levels derived from in chimera-stimulated PBMCs in both healthy subjects and VL patients. In addition, chimera and/or saponin-immunized mice presented significantly lower parasite burden in distinct evaluated organs, when compared to the controls, besides higher levels of IFN-γ, IL-2, IL-12, and GM-CSF, and an IgG2a isotype-based humoral response. In addition, the CD4+ and CD8+ T-cell subtypes contributed to IFN-γ production in the protected animals. The results showed the immunogenicity in human cells and the protective efficacy against L. infantum in a murine model, and well indicate that this recombinant chimera can be considered as a promising strategy to be used against human disease.

Evaluating a point-of-care circulating cathodic antigen test (POC-CCA) to detect Schistosoma mansoni infections in a low endemic area in north-eastern Brazil

Schistosomiasis is still a public health problem in Brazil. The Kato-Katz test is the most frequently used diagnostic method for Schistosoma mansoni infection. However, it lacks sensitivity in areas of low prevalence. We have assessed the positivity rate of S. mansoni infection in Bananeiras, a village on Capistrano, Ceara, Brazil by performing a point-of-care test in urine to determine the circulating cathodic antigens (POC-CCA), and we compared the findings with those of the Kato-Katz technique for egg detection in stool and an enzyme-linked immunosorbent assay for specific antibodies against adult worms (SWAP-ELISA) in serum before treatment (baseline). Additionally, the POC-CCA and Kato-Katz test results were compared at one and two years post-treatment, and only POC-CCA strips were utilised for follow-up testing on urine samples at 3–6 weeks. Only one sample of stool and urine was collected per event. Overall, 258 individuals were investigated at the baseline. The POC-CCA test detected 10 (3.9%) positive cases; however, this amount increased to 30 (11.6%) when considering trace readings as positive (t + ), whereas the Kato-Katz method found only 4 (1.6%) positive cases and the SWAP-ELISA detected 105 (40.7%) positive cases. The consistency observed between a single POC-CCA (t + ) or (t-) and the Kato-Katz (three slides) was poor (Kappa indexes <0.20). The highest positivity rate as determined by CCA and Kato-Katz was found in adults. At the baseline, a praziquantel treatment was administered to all individuals regardless of their infection status. According to the POC-CCA test, 93% of the previous positive cases became negative by the third week after the treatment; this rate reached 100% at the sixth week assessment. The follow-up showed that of the 175 individuals evaluated at one year post-treatment, only one (0.6%) showed ‘trace’ results, and all the individuals were negative for eggs in the stool. At two years, all 185 examined individuals were negative by the Kato-Katz method, and 11 (5.9%) presented traces by POC-CCA. Our results indicate that a single POC-CCA test reveals a significantly higher number of positive cases than the Kato-Katz technique for diagnosing S. mansoni in a low endemic setting, when trace results are considered as positive cases. Nevertheless, the true significance of the trace is not clear. These findings reinforce the need to associate different tools for improved schistosomiasis diagnosis in individuals with low parasite burdens.

Plasma levels of innate immune mediators are associated with liver fibrosis in low parasite burden Schistosoma mansoni-infected individuals.

In the murine model, it was demonstrated that pro-inflammatory cytokines and chemokines are essential to the formation and modulation of Schistosoma-induced granulomatous inflammation. However, the relationship of these immune mediators and disease severity is hard to be established in naturally infected individuals. The current study evaluates the association between plasma concentrations of MIF, sTNF-R1, CCL3, CCL7 and CCL24 and schistosomiasis morbidity in Schistosoma mansoni-infected patients with a low parasite burden. For this propose, 97 S.mansoni-infected individuals were subjected to abdominal ultrasound analysis and clinical examination. Among them, 88 had plasma concentration of immune mediators estimated by ELISA assay. Multivariate linear regression models were used to evaluate the relationship between the plasma concentration of immune mediators and the variables investigated. Although most individuals presented low parasite burden, over 30% of them showed signs of fibrosis defined by ultrasound measurements and 2 patients had a severe form of schistosomiasis. No association between parasite burden and the plasma levels of chemokine/cytokines or disease severity was observed. There was a positive association between plasma concentration of CCL4, sTNF-R1, CCL3 and MIF with gall bladder thickness and/or with portal vein thickness that are liver fibrosis markers. In contrast, no association was found between CCL7 plasma concentrations with any of the schistosomiasis morbidity parameters evaluated. The data showed that CCL24, sTNFR1, MIF and CCL3 can be detected in plasma of S.mansoni-infected individuals and their concentration would be used as prognostic makers of Schistosoma-induced liver fibrosis, even in individuals with low parasite burden.

Identification and Preliminary Evaluation of a Novel Recombinant Protein for Serodiagnosis of Strongyloidiasis.

Strongyloides stercoralis is a human parasite that can cause a long-term infection. In immunosuppressed patients, strongyloidiasis may be fatal when there is overwhelming autoinfection resulting in the migration of large numbers of larvae through many organs. Definitive diagnosis is still a challenge, and a combination of symptoms, microscopic identification, and serology test results are often used to arrive at a clinical decision. However, intermittent larval excretion, low parasite burden, and occult infections are challenges with parasitological diagnosis of infection with S. stercoralis. Meanwhile, serologic tests using immunoglobulin G and parasite antigen extract have problems of cross-reactivity with other helminthic infections. Recombinant antigen-based serodiagnosis is a good alternative to overcome the laboratory diagnostic issues. Herein, we report on the isolation of cDNA clone encoding an antigen of potential diagnostic value identified from immunoscreening of a S. stercoralis cDNA library. The translated protein had highest similarity to Strongyloides ratti immunoglobulin-binding protein 1. The recombinant antigen produced, rSs1a, was assessed using western blot and enzyme-linked immunosorbent assay. The latter showed 96% diagnostic sensitivity and 93% specificity; thus, rSs1a has good potential for use in serodiagnosis of human strongyloidiasis.

Antileishmanial activity of extracts from Libidibia ferrea: development of in vitro and in vivo tests

ABSTRACT Treatment of cutaneous leishmaniasis (CL) is difficult due to the scarce number of drugs able to eliminate completely the intracellular form of the parasite. In the present study, the aim was to evaluate: i) phytochemical properties of extracts from Libidibia ferrea; ii) antileishmanial activity of extracts from L. ferrea against promastigotes and amastigotes of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) guyanensis; iii) the effects of topical treatment using hydrogel containing active extract of L. ferrea on golden hamsters infected with L. (L.) amazonensis. Extracts from leaves, branches and fruits of L. ferrea were obtained with hexane and methanol and were tested by in vitro assays in promastigotes and murine macrophages J774 experimentally infected with amastigotes of Leishmania. Groups of hamsters with CL received topical treatment with a formulation of extract (10%) hydrogels, 50 mg.day-1 for 40 days. In vitro activity of FrMeOH (methanolic extract from fruits without seeds) resulted in significant reduction of viable promastigotes of L. (L.) amazonensis (IC50 of 15.4 µg.mL-1) and demonstrated inhibition potential of amastigote forms of L. (L.) amazonensis and L. (V.) guyanensis and low cytotoxicity in macrophages. The overall data of topical treatment with extract hydrogels (GelFrMeOH) showed that lesion sizes were significantly reduced (42.78%), with low parasite burden by RT-qPCR and culture analysis by microscopy examination, and with histopathological findings such as lower inflammatory cell infiltration 40 days after treatment. Chemical analysis demonstrated FrMeOH contains high levels of phenolic compounds. The results indicate a possible alternative therapy for CL using phytotherapics.