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Background: Infection by nematodes is a problem for human health, livestock, and agriculture, as it causes deficits in host health, increases production costs, and incurs less food supply. The control of these parasites is usually done using anthelmintics, which, in most cases, have not been fully effective. Therefore, the search for new molecules with anthelmintic potential is necessary. Methods: In the present study, we isolated and characterized molecules from the nematophagous fungus Pochonia chlamydosporia and tested these compounds on three nematodes: Caenorhabditis elegans, Ancylostoma ceylanicum, and Ascaris suum. Results: The ethyl acetate extract showed nematicidal activity on the nematode model C. elegans. We identified the major substance present in two subfractions of this extract as ketamine. Then, we tested this compound on C. elegans and the parasites A. ceylanicum and A. suum using hamsters and mice as hosts, respectively. We did not find a difference between the animal groups when considering the number of worms recovered from the intestines of animals treated with ketamine and albendazole (P ˂ 0.05). The parasite burden of larvae recovered from the lungs of mice treated with ketamine was similar to those treated with ivermectin. Conclusion: These results presented here demonstrate the nematicidal activity of ketamine in vitro and in vivo. Thus, confirming the nematicidal potential of the molecule present in the fungus P. chlamydosporia may consist a new method of controlling parasites.

Hookworm infection is considered the most prevalent human soil-transmitted helminth infection affecting approximately 500 million people and accounting for 3.2 million disability-adjusted life years lost annually. As with many other neglected tropical diseases, no international surveillance mechanisms that show accurate data on the prevalence of hookworm infection are in place, thus hindering strategies to control parasite transmission. In this review, we unravel the current knowledge in immunopathology and immunoregulation of hookworm infection, and present discoveries in drug therapies based on the capability of hookworms to regulate inflammation to treat allergic, inflammatory, and metabolic diseases. Additionally, we highlight potential vaccine development and treatments, and propose avenues for further inquiry.

This study determined the time-course of recovery after resistance training (RT) sessions and the association between changes in performance with changes in biomechanical, physiological and perceptual parameters. After a 4-week familiarization period, 14 resistance-trained males performed 3 experimental conditions, each one including 2 sessions with a recovery interval of 24, 48 h or 72 h, in a randomized order. RT sessions consisted of 5 sets of 8-10RM on squat and leg press exercises. The resistance was equal for the 2 sessions of each condition and repetitions were performed until concentric failure. Volume load (VL) and first set volume load (FSVL) were compared between sessions. Tests before each session included countermovement jump (CMJ), maximal voluntary isometric contraction (MVIC), creatine kinase (CK) and delayed onset muscle soreness (DOMS). (2 × 3) ANOVA with effect sizes (ES) assessed the time-course of recovery and Kendall test the correlation between variables (

Trematodes have complex life cycles with multiple hosts. Biomphalaria snails commonly act as the first intermediate hosts of several species that can affect human and animal health. The specific identification of larval trematodes found in snails is difficult and limited, since the taxonomy of these flukes is based on morphological traits of the adults found in vertebrates. Despite recent advances worldwide, studies aiming at the use of molecular tools for the identification of cercariae found in snails are scarce in the South America. In fact, most studies are focused on Schistosoma mansoni, with few efforts directed towards the identification of larvae of other parasites found in planorbids. When reported, these other parasites are identified as cercarial types, an artificial morphological system of classification. Therefore, alternative strategies for a correct, rapid and inexpensive identification of larval trematodes found in Biomphalaria are needed. This work aimed at developing a methodology capable of distinguishing four important families of trematodes (Clinostomidae, Echinostomatidae, Schistosomatidae and Strigeidae) commonly found infecting species of Biomphalaria. Using the rDNA sequences of 34 species as input for the online tool TipMT, we designed trematode family-specific primers targeting the ITS region optimized to be used in multiplex PCR. The panel of primers identified in this study was effective at the same PCR condition. The specificity of the primers was confirmed, and the PCR sensitivity ranged from 0.1 ng to 1 ag of the DNA of the parasite. This methodology was also effective for the detection of coinfection. Through a simple, fast, accurate, and inexpensive methodology, it is possible to properly identify the trematode families included in this study in a single PCR reaction. A family level identification provides important information about probable hosts, pattern of life cycle and possible impacts that the infection generates in a specific region, thus allowing the design of better control strategies, especially for those infections that have medical and veterinary importance.

Leishmaniasis is a neglected tropical disease affecting more than 1.5 million people annually, with an annual mortality of over 20.000. The drugs used for its treatment are toxic, expensive, require extended treatment times and present variable efficacy. The disease severity and therapy limitations suggest the need for new antileishmanial agents. In this context, in order to identify new options for treatment, a number of studies based on nanotechnological strategies have been carried out. Poloxamers are triblock copolymers very often utilized for nanotherapeutic solutions, resulting in products with better solubility, higher stability, superior therapeutic efficacy and less toxicity. This review will discuss the physicochemical properties of the copolymers, as well as describe the use of poloxamers for the development of therapeutic formulations to treat leishmaniasis.

Trichuriasis and ascariasis are neglected tropical diseases caused by the gastrointestinal dwelling nematodes Trichuris trichiura (a whipworm) and Ascaris lumbricoides (a roundworm), respectively. Both parasites are staggeringly prevalent, particularly in tropical and subtropical areas, and are associated with substantial morbidity. Infection is initiated by ingestion of infective eggs, which hatch in the intestine. Thereafter, T. trichiura larvae moult within intestinal epithelial cells, with adult worms embedded in a partially intracellular niche in the large intestine, whereas A. lumbricoides larvae penetrate the gut mucosa and migrate through the liver and lungs before returning to the lumen of the small intestine, where adult worms dwell. Both species elicit type 2 anti-parasite immunity. Diagnosis is typically based on clinical presentation (gastrointestinal symptoms and inflammation) and the detection of eggs or parasite DNA in the faeces. Prevention and treatment strategies rely on periodic mass drug administration (generally with albendazole or mebendazole) to at-risk populations and improvements in water, sanitation and hygiene. The effectiveness of drug treatment is very high for A. lumbricoides infections, whereas cure rates for T. trichiura infections are low. Novel anthelminthic drugs are needed, together with vaccine development and tools for diagnosis and assessment of parasite control in the field.

The human hookworm Necator americanus infects more than 400 million people worldwide, contributing substantially to the poverty in these regions. Adult stage N. americanus live in the small intestine of the human host where they inject excretory/secretory (ES) products into the mucosa. ES products have been characterized at the proteome level for a number of animal hookworm species, but until now, the difficulty in obtaining sufficient live N. americanus has been an obstacle in characterizing the secretome of this important human pathogen. Herein we describe the ES proteome of N. americanus and utilize this information along with RNA Seq data to conduct the first proteogenomic analysis of a parasitic helminth, significantly improving the available genome and thereby generating a robust description of the parasite secretome. The genome annotation resulted in a revised prediction of 3,425 fewer genes than initially reported, accompanied by a significant increase in the number of exons and introns, total gene length and the percentage of the genome covered by genes. Almost 200 ES proteins were identified by LC-MS/MS with SCP/TAPS proteins, ‘hypothetical’ proteins and proteases among the most abundant families. These proteins were compared to commonly used model species of human parasitic infections, including Ancylostoma caninum, Nippostrongylus brasiliensis and Heligmosomoides polygyrus. SCP/TAPS proteins are immunogenic in nematode infections, so we expressed four of those identified in this study in recombinant form and showed that they are all recognized to varying degrees by serum antibodies from hookworm-infected subjects from a disease-endemic area of Brazil. Our findings provide valuable information on important families of proteins with both known and unknown functions that could be instrumental in host-parasite interactions, including protein families that might be key for parasite survival in the onslaught of robust immune responses, as well as vaccine and diagnostic targets., Author summary Hookworms infect hundreds of millions of people in tropical regions of the world. Adult worms reside in the small bowel where they feed on blood, causing iron-deficiency anemia when present in large numbers and contributing substantially to the poverty in these regions. Hookworms inject excretory/secretory (ES) products into the gut tissue when they feed, and while the protein constituents of ES products have been characterized for a number of animal hookworm species, difficulty in obtaining sufficient live human hookworms has thus far precluded characterization of the secreted proteome. Herein we describe the ES proteins of the major human hookworm, Necator americanus, and utilize this information to significantly improve the available genome sequence. Almost 200 ES proteins were identified and compared to the secreted proteomes of other parasitic roundworms to provide a molecular snapshot of the host-parasite interface. We produced recombinant forms of some of the identified proteins and showed that they are all recognized to varying degrees by antibodies from hookworm-infected subjects. Our work sheds light on important families of proteins that might be key for parasite survival in the human host, and presents a dataset that can now be mined in the search for vaccine, drug and diagnostic targets.

The development of an accurate protein-based antigen detection assay for diagnosis of active visceral leishmaniasis (VL) would represent a major clinical advance. VL is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani. The gold standard confirmatory diagnostic test for VL is the demonstration of parasites or their DNA from aspirates from spleen, lymph node, and bone marrow or from blood buffy coats. Here we describe the production and use of monoclonal antibodies (mAbs) for the development of a sensitive and specific antigen detection capture ELISA for VL diagnosis. This test simultaneously detects six leishmania protein biomarkers that we have previously described (Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1). The initial clinical validation of this new mAb-based multiplexed capture ELISA showed a sensitivity of ≥93%. The test was negative with 35 urine samples from healthy control subjects as well as with 30 patients with confirmed non-VL tropical diseases (cutaneous leishmaniasis, n = 6; Chagas disease, n = 6; schistosomiasis, n = 6; and tuberculosis, n = 12). These results strongly support the possible utility of this mAb-based multiplexed capture ELISA as a promising diagnostic test for active VL as well as for monitoring the treatment efficacy of this disease. The test is ready for upscaling and validation for clinical use., Author summary Visceral leishmaniasis (VL) or kala-azar is a systemic parasitic disease that is endemic in 75 countries with more than 500 million people at risk of infection. VL is caused primarily by the parasites known as Leishmania infantum and Leishmania donovani. It is estimated that VL affects 50,000–90,000 people each year, with 90% of the cases occurring in India, Kenya, Somalia, Sudan, South Sudan and Brazil. VL is a fatal disease if not treated. Laboratory diagnosis of this disease can be performed using several different approaches. However, there is still a need for a simple, non-expensive, sensitive and specific test that can be used not only for the accurate diagnosis of this serious disease but also to monitor the efficacy of its treatment. Here we describe a new test that can circumvent most of the drawbacks of existing approaches. This new test detects six leishmanial proteins or biomarkers that are eliminated in the urine of patients with VL. The test is a conventional ELISA assembled with monoclonal antibodies (mAbs) specific for each of the six different biomarkers and it is performed in urine samples of suspected patients. An initial clinical validation demonstrated that the test has a sensitivity of ≥93% and specificity of 100%. This new and accurate mAb-based multiplexed assay could be a useful resource to diagnose most clinical forms and/or the severity of the disease e.g., active VL, asymptomatic VL, post kala-azar dermal leishmaniasis, VL/HIV co-infection, etc.

Hygiene hypothesis dictates that the lack of microbial interaction during the first childhood increases the chance of developing autoimmune diseases due to not proper immune system maturation. Helminthes are known by their Th2 and modulatory immune response induction. Here, it was evaluated the influence of Necator americanus antigens during type 1 experimental mouse model (non-obese diabetic – NOD). Intraperitoneal injections for 18 weeks did not impair an inflammatory response, but induced a mixed Th1/Th2 response with presence of IL4 and IL10 from different sources. However, the induced immune response was not sufficient to decrease glucose levels but showed a change in the inflammatory infiltrate in the pancreas. It is necessary more refined studies to clarify the mechanisms of how Necator americanus could impair diabetes progression in mice.

Leishmaniasis is a neglected disease, caused by a parasite of Leishmania genus and widespread in the tropical and subtropical areas of the world. Currents drugs are limited due to their toxicity and parasite resistance. Therefore, the discovery of new treatment, more effective and less toxic, is urgent. In this study, we report the synthesis of six gem-dihydroperoxides (2a-2f), with yields ranging from 10 % to 90 %, utilizing a new methodology. The dihydroperoxides were converted into ten tetroxanes (3a-3j), among which six (3b, 3c, 3d, 3g, 3h and 3j) showed activity against intracellular amastigotes of Leishmania amazonensis. The cytotoxicity of all compounds was also evaluated against canine macrophages (DH82), human hepatoma (HepG2) and monkey renal cells (BGM). Most compounds were more active and less toxic than potassium antimonyl tartrate trihydrate, used as positive control. Amongst all tetroxanes, 3b (IC50 =0.64 μm) was the most active, being more selective than positive control in relation to DH82, HepG2 and BGM cells. In summary, the results revealed a hit compound for the development of new drugs to treat leishmaniasis.

Hookworms are intestinal nematode parasites that infect nearly half a billion people and are globally one of the most important contributors to iron-deficiency anemia. These parasites have significant impacts in developing children, pregnant women and working adults. Of all the soil-transmitted helminths or nematodes (STNs), hookworms are by far the most important, with disease burdens conservatively estimated at four million DALYs (Disability-Adjusted Life Years) and with productivity losses of up to US$139 billion annually. To date, mainly one drug, albendazole is used for hookworm therapy in mass drug administration, which has on average ∼80% cure rate that is lower (, Graphical abstract Image 1, Highlights • Bacillus thuringiensis (Bt) Cry5B is a new pan-hookworm cure. • Bt Cry5B is highly active against Ancylostoma caninum hookworm infections in dogs. • Bt Cry5B is highly active against Necator americanus. • Cry5B acts independent of the host immune system and directly on hookworms. • Cry5B retains full bioactivity after repeated dosing.

Ascariasis is considered the most neglected tropical disease, and is a major problem for the public health system. However, idiopathic pulmonary fibrosis (IPF) is a result of chronic extracellular deposition of matrix in the pulmonary parenchyma, and thickening of the alveolar septa, which reduces alveolar gas exchange. Considering the high rates of ascariasis and pulmonary fibrosis, we believe that these two diseases may co-exist and possibly lead to comorbidities. We therefore investigated the mechanisms involved in comorbidity of Ascaris suum (A. suum) infection, which could interfere with the progression of pulmonary fibrosis. In addition, we evaluated whether a previous lung fibrosis could interfere with the pulmonary cycle of A. suum in mice. The most important findings related to comorbidity in which A. suum infection exacerbated pulmonary and liver injury, inflammation and dysfunction, but did not promote excessive fibrosis in mice during the investigated comorbidity period. Interestingly, we found that pulmonary fibrosis did not alter the parasite cycle that transmigrated preferentially through preserved but not fibrotic areas of the lungs. Collectively, our results demonstrate that A. suum infection leads to comorbidity, and contributes to the aggravation of pulmonary dysfunction during pulmonary fibrosis, which also leads to significant liver injury and inflammation, without changing the A. suum cycle in the lungs., Author summary Ascariasis is considered a major problem for the public health system, which has an estimated 800 million infected people worldwide. It occurs in the United States, Africa, Asia, and Latin America, and is generally associated with poverty and precarious health conditions. Pulmonary fibrosis affects 14–63 people per 100,000 habitants/year, and is characterized by collagen deposition and alveolar wall thickening. The comorbidities caused by infections are commonly associated with pulmonary fibrosis exacerbations, poor prognosis, and high mortality. Despite the comorbidities caused by helminth infections, which display a pulmonary parasitic cycle such as that of Ascaris, there is no evidence relating to pulmonary fibrosis progression, possibly because Ascariasis is considered a neglected disease. We evaluated the role of Ascaris during pulmonary fibrosis. We considered two simple questions: (1) Whether Ascaris infection could protect or aggravate fibrosis (comorbidities) and (2) whether pulmonary fibrosis could change the cycle of Ascaris as a result of increased alveolar thickening, larvae retention, and the limitation of influx into airways. We answered both questions as follows: (1) Ascaris infection exacerbates pulmonary and liver injury and inflammation, but not fibrosis; and (2) Pulmonary fibrosis did not alter the course of Ascaris cycle in lungs during transmigration into airways, because Ascaris preferentially seeks and penetrates into the lung areas, which are thought to be preserved, but not into fibrotic areas.

The aim of this systematic review was to investigate the studies that evaluated the sensitivity and specificity of serologic tests using recombinant protein antigens from Mycobacterium leprae for leprosy diagnosis. We included 13 studies that were available in PubMed, Brazilian Virtual Library of Health, Web of Science, ScienceDirect and Scopus. From these studies, we found that the recombinant serine-rich 45-kDa protein of M. leprae (ML0411) demonstrated high performance for multibacillary (MB) also to paucibacillary (PB) patients, although this study was tested only for Indian population. Despite that, studies using the ND-O-LID antigen have been able to more accurately identify new cases of leprosy among people living in endemic or non-endemic areas and household contacts in Brazil, Colombia, and the Philippines, especially when combined with other biomarkers. Finally, low sensitivity values for PB patients’ antibodies response remain challenging for tests intended to diagnose clinical forms that comprise this classification in leprosy. O objetivo desta revisão sistemática foi investigar os estudos que avaliaram a sensibilidade e especificidade de testes sorológicos utilizando antígenos proteicos recombinantes do Mycobacterium leprae para o diagnóstico da hanseníase. Foram incluídos 13 estudos que estavam disponíveis no PubMed, Biblioteca Virtual Brasileira de Saúde, Web of Science, ScienceDirect e Scopus. A partir desses estudos, descobrimos que a proteína recombinante de 45 kDa rica em serina do M. leprae (ML0411) demonstrou alto desempenho para pacientes multibacilares (MB) e paucibacilares (PB), embora este estudo tenha sido testado apenas para a população indiana. Apesar disso, estudos com o antígeno ND-O-LID têm conseguido identificar com maior precisão novos casos de hanseníase entre pessoas residentes em áreas endêmicas ou não endêmicas e contatos domiciliares no Brasil, Colômbia e Filipinas, principalmente quando combinados com outros biomarcadores. Por fim, os valores de baixa sensibilidade para a resposta de anticorpos dos pacientes PB permanecem desafiadores para testes destinados a diagnosticar as formas clínicas que compõem essa classificação na hanseníase.

Plasmodium vivaxmalaria incidence has increased in Latin America and Asia and is responsible for nearly 74.1% of malaria cases in Latin America. Immune responses toP. vivaxare less well characterized than those toP. falciparum, partly becauseP. vivaxis more difficult to cultivate in the laboratory. While antibodies are known to play an important role inP. vivaxdisease control, few studies have evaluated responses toP. vivaxsexual stage antigens. We collected sera or plasma samples fromP. vivax-infected subjects from Brazil (n= 70) and Cambodia (n= 79) to assess antibody responses to domain 1 of the gametocyte/gamete stage protein Pvs230 (Pvs230D1M). We found that 27.1% (19/70) and 26.6% (21/79) of subjects from Brazil and Cambodia, respectively, presented with detectable antibody responses to Pvs230D1M antigen. The most frequent subclasses elicited in response to Pvs230D1M were IgG1 and IgG3. Although age did not correlate significantly with Pvs230D1M antibody levels overall, we observed significant differences between age strata. Hemoglobin concentration inversely correlated with Pvs230D1M antibody levels in Brazil, but not in Cambodia. Additionally, we analyzed the antibody response against Pfs230D1M, theP. falciparumortholog of Pvs230D1M. We detected antibodies to Pfs230D1M in 7.2 and 16.5% of Brazilian and CambodianP. vivax-infected subjects. Depletion of Pvs230D1M IgG did not impair the response to Pfs230D1M, suggesting pre-exposure toP. falciparum, or co-infection. We also analyzed IgG responses to sporozoite protein PvCSP (11.4 and 41.8% in Brazil and Cambodia, respectively) and to merozoite protein PvDBP-RII (67.1 and 48.1% in Brazil and Cambodia, respectively), whose titers also inversely correlated with hemoglobin concentration only in Brazil. These data establish patterns of seroreactivity to sexual stage Pvs230D1M and show similar antibody responses amongP. vivax-infected subjects from regions of differing transmission intensity in Brazil and Cambodia.

Made available in DSpace on 2018-12-11T16:53:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-02-01 Objective To investigate the effects of electroacupuncture (EA) at ST36 and CV12 on gastrointestinal transit and parasitological measurements during Strongyloides venezuelensis infection in rats. Design Rats were infected with S. venezuelensis and allocated to one of three groups that were infected and remained untreated (SV group, n=8), infected and treated with EA at CV12 (SV+CV12 group, n=8) or infected and treated with EA at ST36 (SV+ST36 group, n=8). EA was performed every 3 days over a 21-day period, at 4 mA intensity and 15 Hz frequency for 20 min. At 2 and 20 days post-infection (dpi), body weight, food and water intake, and faecal characteristics were monitored over a 24-hour period. Gastric emptying, caecal arrival time, small intestinal transit and eggs per gram (EPG) of faeces were calculated at 3, 9, 15 and 21 dpi. At 21 dpi, intestinal worm recovery was counted. Results EA at ST36 and CV12 slowed gastric emptying over the course of infection time. An accelerated intestinal transit was observed in the ST36 group, and after CV12 treatment the same effect was observed at 9 and 15 dpi. At 9 dpi, EPG was increased in the CV12 group. ST36 treatment decreased EPG at 9 and 15 dpi. At 21 dpi, both the ST36 and CV12 groups had increased EPG and worm numbers. No changes were observed in the other parameters analysed. Conclusions EA at ST36 and CV12 provoked changes in gastrointestinal transit that may be beneficial to the host during S. venezuelensis infection; however, based on the number of worms and EPG at 21 dpi, the indication for EA in the treatment of strongyloidiasis needs to be carefully assessed. Instituto de Ciências Biológicas e da Saúde UFMT-Universidade Federal de Mato Grosso, Avenida Valdon Varjão, no. 6.390. Instituto de Biociências UNESP-Universidade Estadual Paulista Instituto de Ciências Biológicas UFMG-Universidade Federal de Minas Gerais Centro de Ciências da Saúde UNCISAL-Universidade Estadual de Ciências da Saúde de Alagoas Instituto de Biociências UNESP-Universidade Estadual Paulista

Peptide-based vaccines have demonstrated to be an important way to induce long-lived immune responses and, therefore, a promising strategy in the rational of vaccine development. As to malaria, among the classic vaccine targets, the Apical membrane antigen (AMA-1) was proven to have important B cell epitopes that can induce specific immune response and, hence, became key players for a vaccine approach. The peptides selection was carried out using a bioinformatic approach based on Hidden Markov Models profiles of known antigens and propensity scale methods based on hydrophilicity and secondary structure prediction. The antigenicity of the selected B-cell peptides was assessed by multiple serological assays using sera from acute P.vivax infected subjects. The synthetic peptides were recognized by 45.5%, 48.7% and 32.2% of infected subjects for peptides I, II and III respectively. Moreover, when synthetized together (tripeptide), the reactivity increases up to 62%, which is comparable to the reactivity found against the whole protein PvAMA-1 (57%). Furthermore, IgG reactivity against the tripeptide after depletion was reduced by 42%, indicating that these epitopes may be responsible for a considerable part of the protein immunogenicity. These results represent an excellent perspective regarding future chimeric vaccine constructions that may come to contemplate several targets with the potential to generate the robust and protective immune response that a vivax malaria vaccine needs to succeed.

The development of T follicular helper cells (Tfh) is a multifactorial process that occurs in multiple stages. After their activation the Tfh cells interact with the B cells to complete their differentiation. During this process, the Tfh cells begin to express canonical molecules such as the transcription factor B-cell lymphoma 6 protein, the CXC chemokine receptors type 5, and the inducible T-cell costimulator, as well as secreting other molecules such as IL-21. This whole process is regulated positively and negatively by several factors so that the best response is offered in the face of diseases of various origins, among them helminthiasis. In this context, the role of circulating Tfh, IL-4 and IgG subtypes is essential for an effective response against these pathogens. In this review, the migration process and the differentiation of Tfh, the regulation, their cell subtypes and the role of Tfh in the context of helminth infections will be addressed.

Visceral leishmaniasis (VL) is caused by protozoa belonging to the Leishmania donovani complex and is considered the most serious and fatal form among the different types of leishmaniasis, if not early diagnosed and treated. Among the measures of disease control stand out the management of infected dogs and the early diagnosis and appropriate treatment of human cases. Several antigens have been characterized for use in the VL diagnosis, among them are the recombinant kinesin-derived antigens from L. infantum, as rK39 and rKDDR. The main difference between these antigens is the size of the non-repetitive kinesin region and the number of repetitions of the 39 amino acid degenerate motif (6.5 and 8.5 repeats in rK39 and rKDDR, respectively). This repetitive region has a high antigenicity score. To evaluate the effect of increasing the number of repeats on diagnostic performance, we designed the rKDDR-plus antigen, containing 15.3 repeats of the 39 amino acid degenerate motif, besides the absence of the non-repetitive portion from L. infantum kinesin. Its performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and rapid immunochromatographic test (ICT), and compared with the kinesin-derived antigens (rKDDR and rK39). In ELISA with human sera, all recombinant antigens had a sensitivity of 98%, whereas the specificity for rKDDR-plus, rKDDR and rK39 was 100%, 96% and 71%, respectively. When evaluated canine sera, the ELISA sensitivity was 97% for all antigens, and the specificity for rKDDR-plus, rKDDR and rK39 was 98%, 91% and 83%, respectively. Evaluation of the ICT/rKDDR-plus, using human sera, showed greater diagnostic sensitivity (90%) and specificity (100%), when compared to the IT LEISH (79% and 98%, respectively), which is based on the rK39 antigen. These results suggest that the increased presence of repetitive motifs in the rKDDR-plus protein improves the diagnostic performance of serological tests by increasing the specificity and accuracy of the diagnosis., Author summary Visceral Leishmaniasis (VL) is a neglected tropical disease caused by protozoa Leishmania infantum and L. donovani. This illness affects millions of people in various regions of the world and can be fatal if not early treated. In the Americas and Southern Europe, the VL is a zoonotic disease, being the dog its main reservoir. The early and correct diagnosis of dogs and humans infected with Leishmania is essential for the efficient disease control. However, current diagnostic methods have limitations, as they are not sufficiently sensitive and/or specific, and may not be able to identify all cases of VL or exhibit cross-reactions with other infections. We produced a recombinant protein, rKDDR-plus, constituted only by highly repetitive sequences kinesin-derived from L. infantum, and compare its performance with other kinesin-derived antigens (rK39 and rKDDR), but that have less repetitive sequences. The rKDDR-plus protein exhibited the best performance among the studied antigens, presenting a greater specificity in the VL diagnosis, both in the ELISA and rapid test platforms. Therefore, this antigen can contribute to the control of leishmaniasis in humans and dogs, through a diagnostic test with high specificity, achieved due to the use of the new rKDDR-plus antigen.

Chagas disease, caused by the parasite Trypanosoma cruzi, is considered endemic in more than 20 countries but lacks both an approved vaccine and limited treatment for its chronic stage. Chronic infection is most harmful to human health because of long-term parasitic infection of the heart. Here we show that immunization with a virus-like particle vaccine displaying a high density of the immunogenic α-Gal trisaccharide (Qβ-αGal) induced several beneficial effects concerning acute and chronic T. cruzi infection in α1,3-galactosyltransferase knockout mice. Approximately 60% of these animals were protected from initial infection with high parasite loads. Vaccinated animals also produced high anti-αGal IgG antibody titers, improved IFN-γ and IL-12 cytokine production, and controlled parasitemia in the acute phase at 8 days post-infection (dpi) for the Y strain and 22 dpi for the Colombian strain. In the chronic stage of infection (36 and 190 dpi, respectively), all of the vaccinated group survived, showing significantly decreased heart inflammation and clearance of amastigote nests from the heart tissue., Author summary Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a significant endemic infectious disease in Latin America and is spreading in the U.S. and Europe with the presence of its insect transmission vector. No approved vaccine against Chagas disease exists. We describe a vaccine candidate based on a carbohydrate found on the T. cruzi cell surface, linked in the vaccine to a virus-like particle that provides a strong and focused immune response. Mice were immunized and challenged with the Trypanosoma cruzi parasites from two strains (Y and Colombian). Vaccination conferred substantial protection of mice against infection, compared with the unvaccinated group. Vaccinated animals presented low parasitemia, increased production of pro-inflammatory cytokines IL-12 and IFN-γ, decreased cardiomyocyte damage, and rapid clearance of parasite nests from heart tissue. These effects were especially significant at time points modeling chronic disease, an important consideration for this pathogen. We, therefore, believe this is a valuable path to pursue in the development of vaccines against Chagas disease.

Secreted and surface-displayed carbohydrates are essential for virulence and viability of many parasites, including for immune system evasion. We have identified the α-Gal trisaccharide epitope on the surface of the protozoan parasites Leishmania infantum and Leishmania amazonensis, the etiological agents of visceral and cutaneous leishmaniasis, respectively, with the latter bearing larger amounts of α-Gal than the former. A polyvalent α-Gal conjugate on the immunogenic Qβ virus-like particle was tested as a vaccine against Leishmania infection in a C57BL/6 α-galactosyltransferase knockout mouse model, which mimics human hosts in producing high titers of anti-α-Gal antibodies. As expected, α-Gal-T knockout mice infected with promastigotes of both Leishmania species showed significantly lower parasite load in the liver and slightly decreased levels in the spleen, compared with wild-type mice. Vaccination with Qβ–α-Gal nanoparticles protected the knockout mice against Leishmania challenge, eliminating the i...

Leprosy is a chronic infectious disease whose disequilibrium in the host's genetic, immunological and clinical mechanisms leads to distinct manifestations defining the type of immunological response. This review focuses its attention on the influence of the Vitamin D Receptor and hepcidin expressions that can suggest the protection or severity of leprosy.

The aim of this work was to elucidate the immunopathological mechanisms of how helminths may influence the course of a viral infection, using a murine model. Severe virulence, a relevant increase in the virus titres in the lung and a higher mortality rate were observed in Ascaris and Vaccinia virus (VACV) co-infected mice, compared with VACV mono-infected mice. Immunopathological analysis suggested that the ablation of CD8+ T cells, the marked reduction of circulating CD4+ T cells producing IFN-γ, and the robust pulmonary inflammation were associated with the increase of morbidity/mortality in co-infection and subsequently with the negative impact of concomitant pulmonary ascariasis and respiratory VACV infection for the host. On the other hand, when evaluating the impact of the co-infection on the parasitic burden, co-infected mice presented a marked decrease in the total number of migrating Ascaris lung-stage larvae in comparison with Ascaris mono-infection. Taken together, our major findings suggest that Ascaris and VACV co-infection may potentiate the virus-associated pathology by the downmodulation of the VACV-specific immune response. Moreover, this study provides new evidence of how helminth parasites may influence the course of a coincident viral infection.

Leishmaniasis is a neglected tropical disease (NTDs), endemic in 88 countries that affect more than 12 million people. Current drugs are limited due to their toxicity, development of biological resistance, length of treatment and high cost. Thus, the search for new effective and less toxic treatments is an urgent need. In this study, we report the synthesis of 3 new amide derivatives of hederagenin (22–24) with yields between 70% and 90%, along with 57 other derivatives of hederagenin (1–21, 25–60) carrying different groups at C-28 previously reported by our group, and the results of their in vitro ability to inhibit the growth of Leishmania infantum. Some derivatives (3, 4, 44, 49 and 52), showed activity at micromolar level and low toxicity against BGM and HepG2 cells. Moreover, the ability of hederagenin derivatives 3 (IC50 = 9.7 μM), 4 (12 μM), 44 (11 μM) and 49 (2 μM), to prevent proliferation of intracellular amastigote forms of L. infantum and their higher selectivity index and low toxicity compared to commercial positive drug control of choice (potassium antimonyl tartrate trihydrate) (IC50 = 80 μM, SI = 0.1), make these compounds promising candidates for the treatment of leishmaniasis.

Among the various hypotheses put forward to explain the modulatory influence of helminth infection on allergic effector responses in humans, the IL-10–induced suppression of Th2-associated responses has been the leading candidate. To explore this helminth/allergy interaction more fully, parasite- and allergen-specific CD4+ T cell responses in 12 subjects with filarial infections, and coincident allergic sensitization (filarial [Fil]+allergy [A]+) were compared with the responses to three appropriate control groups (Fil−A− [n = 13], Fil−A+ [n = 12], Fil+A− [n = 11]). The most important findings revealed that Fil+A+ had marked (p < 0.0001 for all cytokines) increases in parasite Ag-driven Th2 (IL-4, IL-5, IL-13), Th9 (IL-9), and the regulatory (IL-10) cytokines when compared with Fil+A−. Moreover, using multiparameter flow cytometry, filarial parasite Ag induced a marked increase in not only the frequency of CD4+ T cells producing IL-4, IL-5, IL-2, and TNF-α in Fil+A+ when compared with Fil+A− patients, but also in the frequencies of polyfunctional Th2-like (CD4+IL-4+IL-5+ and CD4+IL-2+IL-4+IL-5+TNF-α+) cells. The Th2-associated responses seen in the Fil+A+ group were correlated with serum IgE levels (p < 0.01, r = 0.5165 for IL-4; p < 0.001, r = 0.5544 for IL-5; and p < 0.001, r = 0.4901 for IL-13) and levels of circulating eosinophils (p < 0.0116, r = 0.5656) and their degranulation/activation products (major basic protein [p < 0.001, r = 0.7353] and eosinophil-derived neurotoxin [p < 0.01, r = 0.7059]). CD4+ responses to allergen were not different (to a large extent) among the groups. Taken together, our data suggest that allergic sensitization coincident with filarial infection drives parasite Ag-specific T cell hyperresponsiveness, which is characterized largely by an augmented Th2-dominated immune response.

A 2-year-old captive gorilla (Gorilla gorilla gorilla) was diagnosed with visceral leishmaniasis in Brazil, and treated with a single dose of liposomal amphotericin B, which resulted in clinical cure. This is the first report of visceral leishmaniasis in gorillas, and the first reported liposomal amphotericin B treatment in great apes.

8p.-3 fig.-1 tab., Clinically available drugs for mucocutaneous and cutaneous leishmaniases (CL) include mainly pentavalent antimony (Sb(V)) complexes, liposomal amphotericin B, and miltefosine (HePC). However, they present at least one of the following limitations: long-term parenteral administration through repeated doses, severe side effects, drug resistance, and high cost. HePC is the only oral drug available, but the appearance of resistance has resulted in changes of its use from monotherapy to combination therapy. Amphiphilic Sb(V) complexes, such as SbL8 obtained from reaction of Sb(V) with N-octanoyl-N-methylglucamide, were recently found to be orally active against experimental CL. The property of SbL8 to self-assemble in aqueous solution, forming nanostructures, led us to investigate the incorporation of HePC into SbL8 nanoassemblies and the therapeutic efficacy of SbL8/HePC nanoformulation by oral route in a murine model of CL. HePC incorporation into the SbL8 nanosystem was evidenced by using a fluorescent analog of HePC. The antileishmanial activity of SbL8/HePC nanoassemblies was evaluated after daily oral administration for 30 days in Leishmania amazonensis-infected BALB/c mice, in comparison with monotherapies (SbL8 or HePC) and saline control. All the treatments resulted in significant reduction in the lesion size growth, when compared with control. Strikingly, only SbL8/HePC nanoassemblies promoted a significant decrease of the parasite burden in the lesion. This work establishes the therapeutic benefit of SbL8/HePC association by oral route in a CL model and constitutes an important step towards the development of new orally active drug combination., This work was supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number: 425332/2018–7), Coordenação de Aperfeicoamento de Pessoal de Nível Superior (CAPES, grant number: PNPD20131163), and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG, grant number: APQ-03129-16) for financial support. F.F. was recipient of fellowship from CNPq (grant number: 305659/2017-0) and Chaire Jean d’Alembert, Université Paris-Saclay, France (ANR-11-IDEX-0003-02). L.R was supported by Program of Redes Temáticas de Investigación Cooperativa RETICS-FEDER (grant number: RD16/0027/0010).

The serodiagnosis of visceral leishmaniasis (VL) presents problems related to the sensitivity and/or specificity of the tests. In this context, more refined antigens should be identified and applied for the improvement of disease diagnosis. In the present study, DNA with an encoding of a Leishmania infantum hypothetical protein, LiHyC, was cloned, and the recombinant protein was expressed, purified, and evaluated for the serodiagnosis of canine and human VL. In addition, a specific B-cell epitope present in the LiHyC sequence was predicted; the peptide was both synthetized and evaluated in the ELISA experiments. For comparison, commercial diagnostic kits were used against positive (VL hosts) and negative (healthy hosts) samples. Results showed that the recombinant protein (rLiHyC) and synthetic peptide (PeptC) were highly sensitive and specific to diagnose canine and human VL, with 100% sensitivity and specificity, while no false-positive or false-negative result was detected. When the DPP® CVL kit was used to identify canine samples, 44 and 52 of the 60 L. infantum-infected animals, without or with clinical signals of disease, respectively, were identified, while eight and four samples were considered as false-negatives, respectively. For human VL, an IT LEISH® kit was used, and 33 of the 40 VL patients were identified, while seven samples were considered to be false-negatives. Post-therapeutic serological follow-up testing sera samples from treated and untreated VL patients showed a significant drop in the anti-PeptC and anti-rLiHyC antibody levels, thus suggesting the feasibility to use the recombinant protein and/or synthetic peptide in future studies as diagnostic and/or prognostic markers for VL.

Visceral leishmaniasis (VL) is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani The gold standard diagnostic test for VL is the demonstration of parasites or their DNA in spleen, lymph node, or bone marrow aspirates. Serological tests exist but cannot distinguish active VL from either prior exposure to the parasites or previously treated VL disease. Using mass spectroscopy, we have previously identified three L. infantum protein biomarkers (Li-isd1, Li-txn1, and Li-ntf2) in the urine of VL patients and developed a sensitive and specific urine-based antigen detection assay for the diagnosis of VL that occurs in Brazil (where VL is caused by L. infantum). However, unpublished observations from our laboratory at DetectoGen showed that these biomarkers were detected in only 55% to 60% of VL patients from India and Kenya, where the disease is caused by L. donovani Here, we report the discovery and characterization of two new biomarkers of L. donovani (Ld-mao1 and Ld-ppi1) present in the urine of VL patients from these two countries. Capture enzyme-linked immunosorbent assays using specific rabbit IgG and chicken IgY were developed, and the assays had sensitivities of 44.4% and 28.8% for the detection of Ld-mao1 and Ld-ppi1, respectively. In contrast, a multiplexed assay designed to simultaneously detect all five leishmanial biomarkers markedly increased the assay sensitivity to 82.2%. These results validate the utility of leishmanial protein biomarkers found in the urine of VL patients as powerful tools for the development of an accurate diagnostic test for this disease.

American visceral leishmaniasis (VL) is a vector-borne disease transmitted by some species of phlebotomine sandflies from the genus Lutzomyia. This neglected tropical zoonosis shows increasing urbanization process, since the end of the 1980s. After the emergence of foci of the disease in urban areas, VL has assumed an important role in public health. Although VL is widely prevalent in several parts of the world, diagnosing the illness is still difficult. We present a case of a 12-year-old girl with a history of recurrent fever, anorexia, cachexia, chronic fatigue, weight loss, left palpebral unilateral edema, persistent cough and pancytopenia. A diagnosis of VL was performed using a reference immunochromatographic rapid test. Identification of the infecting protozoan was directly obtained by PCR of bone marrow. The patient responded favorably to treatment using liposomal amphotericin B. This is the first report of human visceral leishmaniasis in the city of Lavras in the South of Minas Gerais State. This first report of VL highlighted the need of maintenance of permanent surveillance and control programs in the city of Lavras, including the active search of sandflies, human and canine cases. The current situation of Lavras should also be taken as an alert to other near cities where favorable eco-epidemiological conditions may exist.

Visceral leishmaniasis (VL) is a zoonosis and the dog is considered the most important urban reservoir. Cases in cats have been reported, but little is known about Leishmania infection and disease in wild felids and canids kept in captivity in endemic areas. Thus, the serological pattern of wild felids and canids kept in captivity at the Belo Horizonte Zoological Garden was investigated using two primary antigens for conventional ELISA: k39 and rKDDR, as well as three serological rapid kits: Dual Path Platform (DPP®) immunochromatographic test, rKDDR Immunochromatographic assay and ELISA SNAP Leishmania IDEXX®. A total of 21 serum samples, 13 of wild felids and 8 wild canids of varying age and sex were evaluated. The results obtained in the tests were analyzed by agreement using Kappa coefficient, and between ELISA antigens all the analysis performed had showed significant agreement among both of them, as well between the three immunochromatographic tests. The results demonstrated that there is serological evidence of wild animals seropositive for Leishmania antibodies at the Belo Horizonte Zoological Garden, and that all the antigens and rapid tests used can be employed in serological screening for VL in wild felids and canids.

The Kato-Katz (KK) technique is the mainstay mapping tool for the diagnosis of Schistosoma mansoni infection, despite showing poor sensitivity in cases of low-intensity infections. As an alternative, a rapid point-of-care circulating cathodic antigen diagnostic test (POC-CCA) has been commercially developed that involves a simple urine assay to detect S. mansoni, rather than a stool-based parasitological examination. Although POC-CCA has proven to be a more sensitive test than KK, it is not yet clear how to interpret discordant results between the two tests, particularly for situations in which the KK result is positive and the POC-CCA result is negative. Thus, the objective of this study was to evaluate the degree of diagnostic variability between different POC-CCA batches with respect to results obtained with KK. For this purpose, we collected urine and stool samples of school-aged children from areas of low and moderate endemicity in Brazil, and compared different POC-CCA batches results with those of KK-positive individuals. We found a statistically significant difference between the results obtained from various POC-CCA batches using the same urine samples, regardless of the degree of endemicity and the intensity of infection in positive KK samples. In addition, there was poor agreement between the KK and POC-CCA results in some batches of the rapid test, resulting in false negatives. These findings raise concerns around quality control checks of POC-CCA, especially in light of the high cost and increasing reliance on this new diagnostic method as control programs move towards a goal of elimination.

Background Trypanosoma cruzi, the etiologic agent of Chagas disease, is currently divided into six discrete typing units (DTUs), named TcI-TcVI. TcII is among the major DTUs enrolled in human infections in South America southern cone, where it is associated with severe cardiac and digestive symptoms. Despite the importance of TcII in Chagas disease epidemiology and pathology, so far, no genome-wide comparisons of the mitochondrial and nuclear genomes of TcII field isolates have been performed to track the variability and evolution of this DTU in endemic regions. Results In the present work, we have sequenced and compared the whole nuclear and mitochondrial genomes of seven TcII strains isolated from chagasic patients from the central and northeastern regions of Minas Gerais, Brazil, revealing an extensive genetic variability within this DTU. A comparison of the phylogeny based on the nuclear or mitochondrial genomes revealed that the majority of branches were shared by both sequences. The subtle divergences in the branches are probably consequence of mitochondrial introgression events between TcII strains. Two T. cruzi strains isolated from patients living in the central region of Minas Gerais, S15 and S162a, were clustered in the nuclear and mitochondrial phylogeny analysis. These two strains were isolated from the other five by the Espinhaço Mountains, a geographic barrier that could have restricted the traffic of insect vectors during T. cruzi evolution in the Minas Gerais state. Finally, the presence of aneuploidies was evaluated, revealing that all seven TcII strains have a different pattern of chromosomal duplication/loss. Conclusions Analysis of genomic variability and aneuploidies suggests that there is significant genomic variability within Minas Gerais TcII strains, which could be exploited by the parasite to allow rapid selection of favorable phenotypes. Also, the aneuploidy patterns vary among T. cruzi strains and does not correlate with the nuclear phylogeny, suggesting that chromosomal duplication/loss are recent and frequent events in the parasite evolution. Electronic supplementary material The online version of this article (10.1186/s12864-018-5198-4) contains supplementary material, which is available to authorized users.

Some species of the fungus Sporothrix cause a chronic granulomatous infection in humans and animals called sporotrichosis. In the last decades, some research into serological tests has been carried out by different groups for the rapid detection of this infection. We performed a systematic review of the literature with meta-analysis to evaluate studies using Sporothrix spp. antigens and to evaluate their accuracy for sporotrichosis diagnostic. We searched Scopus, MEDLINE, Web of Science, GALE, Technology Research Database, DOA, Elsevier, SciELO, and Google Scholar Databases. The united results of sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio with their corresponding 95% confidence intervals (CI) were assessed. A total of 15 assays from 8 studies using 7 different serological methods and 8 different antigens were analyzed. The studies were performed in the USA, Brazil, and Venezuela from 1973 until 2015 and presented good quality. A high heterogeneity for sensitivity [I2 = 90.7%; 87% CI = (84–89), P

Mycobacterium leprae infection depends on the competence of the host immune defense to induce effective protection against this intracellular pathogen. The present study investigated the serum levels of vitamin D and the antimicrobial peptide cathelicidin, to determine the statistical correlation between them in leprosy patients before and post-six months of multidrug therapy (MDT), household contacts, and healthy individuals. Previous studies associated these molecules with high risks to develop mycobacterial diseases, such as tuberculosis and leprosy. A total of 34 leprosy patients [paucibacillary (n = 14), multibacillary (n = 20)], and 25 household contacts were recruited. Eighteen healthy adults were selected as a control group. Serum concentrations of vitamin D (25(OH)VD3) and cathelicidin were measured using high-performance liquid chromatography (HPLC), and an enzyme-linked immunosorbent assay (ELISA) kit, respectively. There were no significant differences in serum levels of 25(OH)VD3 between all groups, and the overall prevalence rate of vitamin D deficiency was 67.1%. Cathelicidin levels were significantly lower in both untreated and treated patients when compared to controls and household contacts (p

Objectives To identify immunodominant antigens of Toxocara canis recognised by Toxocara-infected sera as recombinant reagents for immunodiagnosis of toxocariasis. Methods Pooled sera from human cases of toxocariasis were used to identify immunodominant antigens by immunoscreening a T. canis larval expression cDNA library. The positive clones were sequenced to reveal the identity of the antigens. The recombinant proteins were expressed in E. coli and then used to confirm their immunoreaction with sera of humans with toxocariasis. Two chosen antigens were also used to differentiate Toxocara infection from other helminth infections in mice. Results Eleven antigens with immunodiagnostic potential were identified, including two C-type lectins (CTLs) that reacted strongly with the Toxocara-positive serum pool. The first CTL (Tc-CTL-1) is the same as TES-32, previously identified as a major immunodominant component of TES; the second CTL (Tc-CTL-2) is a novel C-type lectin sharing 83% amino acid sequence identity within the functional domain of Tc-CTL-1. The E. coli-expressed recombinant Tc-CTL-1 was strongly recognised by the Toxocara-positive serum pool or sera from animals experimentally infected with T. canis. Reactivity with recombinant Tc-CTL-1 was higher when the unreduced protein was used in an enzyme-linked immunosorbent assay (ELISA), dot-blot assay or Western blot test compared to the protein under reduced condition. Both recombinant Tc-CTL-1- and Tc-CTL-2-based ELISAs were able to differentiate T. canis infection from other helminth infections in experimentally infected mice. Conclusions Both Tc-CTL-1 and Tc-CTL-2 were able to differentiate Toxocara infection from other helminth infections and could potentially be used as sensitive and specific immunodiagnostic antigens. Objectifs Identifier des antigenes immunodominants de Toxocara canis reconnus par des serums infectes par Toxocara, comme recombinants reactifs pour le diagnostic immunologique de la toxocarose. Methodes Des serums pooles provenant de cas humains de toxocarose ont ete utilises pour identifier des antigenes immunodominants par immunocriblage d'une banque d'expression d’ADNc de larves de T. canis. Les clones positifs ont ete sequences pour reveler l'identite des antigenes. Les proteines recombinantes ont ete exprimees dans E. coli et ensuite utilisees pour confirmer leur reaction immunologique avec des serums humains de toxocarose. Deux antigenes ont egalement ete utilises pour differencier la toxocarose d'autres helminthiases chez la souris. Resultats 11 antigenes a potentiel d'immunodiagnostic ont ete identifies, dont deux lectines de type C (CTL) qui ont reagi fortement avec le pool de serums positifs pour Toxocara. La premiere CTL (Tc-CTL-1) est la meme que TES-32, precedemment identifiee comme une composante immunodominante majeure de TES; la deuxieme CTL (Tc-CTL-2) est une nouvelle lectine de type C partageant une identite de 83% avec la sequence d'acides amines dans la region fonctionnelle de Tc-CTL-1. Le recombinant Tc-CTL-1 exprime dans E. coli a ete fortement reconnu par le pool de serums positifs pour Toxocara ou des serums provenant d'animaux infectes experimentalement avec T. canis. La reactivite avec le recombinant Tc-CTL-1 etait plus forte lorsque la proteine non reduite a ete utilisee dans des essais ELISA, Dot-blot ou Western-blot par rapport a la proteine sous sa forme reduite. Les ELISA bases sur les deux recombinants Tc-CTL-1 et Tc-CTL-2 ont ete en mesure de differencier l'infection a T. canis d'autres helminthiases chez la souris infectee experimentalement. Conclusions Tc-CTL-1 et Tc-CTL-2 ont ete en mesure de differencier la toxocarose d'autres helminthiases et pourraient potentiellement etre utilises comme antigenes d'immunodiagnostic sensibles et specifiques. Objetivos Identificar antigenos inmunodominantes de Toxocara canis reconocidos por sueros infectados con Toxocara como reactivos recombinantes para el inmunodiagnostico de la toxocariasis. Metodos Se utilizo un pool de sueros de casos de toxocariasis humana para identificar los antigenos inmunodominantes mediante inmunoscreening en una biblioteca de ADNc de larvas de T. canis. Los clones que dieron positivo fueron secuenciados para identificar los antigenos. Las proteinas recombinantes se expresaron en E. coli y se utilizaron para confirmar su inmunoreactividad con sueros de humanos con toxocariasis. Dos antigenos especificos se utilizaron tambien para diferenciar la infeccion por Toxocara de otras infecciones por helmintos en raton. Resultados Se identificaron 11 antigenos con potencial inmunodiagnostico, incluyendo dos lectinas del tipo C (CLT) que reaccionaban fuertemente con el pool de sueros Toxocara-positivos. La primera LTC (Tc- CLT -1) es la misma que TES-32, previamente identificada como el principal componente inmunodominante de TES; la segunda LTC (Tc- CLT -2) es una nueva lectina tipo C que comparte un 83% de la secuencia de aminoacidos dentro del dominio funcional de Tc- CLT -1. La Tc- CLT -1 recombinante expresada en E. coli era altamente reconocida por el pool de sueros Toxocara-positivos o sueros de animales infectados experimentalmente con T. canis. La reactividad con el Tc- CLT -1 recombinante era mayor cuando se utilizaba la proteina bajo condiciones no reductoras en un ELISA, Dot blot o Western blot comparado con la proteina en condiciones reductoras. Las ELISAs en las que se utilizaban la Tc- CLT -1 recombinante y la Tc- CLT -2 recombinante eran capaces de diferenciar la infeccion por T. canis de otras infecciones helminticas en ratones infectados experimentalmente. Conclusiones Tanto Tc- CLT-1 como Tc- CLT-2 eran capaces de diferenciar la infeccion por Toxocara de otras infecciones helminticas y potencialmente podrian utilizarse como antigenos sensibles y especificos para inmunodiagnostico.

Point of care (PoC) diagnostics are at the focus of government initiatives, NGOs and fundamental research alike. In high-income countries, the hope is to streamline the diagnostic procedure, minimize costs and make healthcare processes more efficient and faster, which, in some cases, can be more a matter of convenience than necessity. However, in resource-limited settings such as low-income countries, PoC-diagnostics might be the only viable route, when the next laboratory is hours away. Therefore, it is especially important to focus research into novel diagnostics for these countries in order to alleviate suffering due to infectious disease. In this review, the current research describing the use of PoC diagnostics in resource-limited settings and the potential bottlenecks along the value chain that prevent their widespread application is summarized. To this end, we will look at literature that investigates different parts of the value chain, such as fundamental research and market economics, as well as actual use at healthcare providers. We aim to create an integrated picture of potential PoC barriers, from the first start of research at universities to patient treatment in the field. Results from the literature will be discussed with the aim to bring all important steps and aspects together in order to illustrate how effectively PoC is being used in low-income countries. In addition, we discuss what is needed to improve the situation further, in order to use this technology to its fullest advantage and avoid “leaks in the pipeline”, when a promising device fails to take the next step of the valorization pathway and is abandoned.

Background Ascaris lumbricoides is one of the three major soil-transmitted gastrointestinal helminths (STHs) that infect more than 440 million people in the world, ranking this neglected tropical disease among the most common afflictions of people living in poverty. Children infected with this roundworm suffer from malnutrition, growth stunting as well as cognitive and intellectual deficits. An effective vaccine is urgently needed to complement anthelmintic deworming as a better approach to control helminth infections. As37 is an immunodominant antigen of Ascaris suum, a pig roundworm closely related to the human A. lumbricoides parasite, recognized by protective immune sera from A. suum infected mice. In this study, the immunogenicity and vaccine efficacy of recombinant As37 were evaluated in a mouse model. Methodology/Principal findings As37 was cloned and expressed as a soluble recombinant protein (rAs37) in Escherichia coli. The expressed rAs37 was highly recognized by protective immune sera from A. suum egg-infected mice. Balb/c mice immunized with 25 μg rAs37 formulated with AddaVax™ adjuvant showed significant larval worm reduction after challenge with A. suum infective eggs when compared with a PBS (49.7%) or adjuvant control (48.7%). Protection was associated with mixed Th1/2-type immune responses characterized by high titers of serological IgG1 and IgG2a and stimulation of the production of cytokines IL-4, IL-5, IL-10 and IL-13. In this experiment, the AddaVax™ adjuvant induced better protection than the Th1-type adjuvant MPLA (38.9%) and the Th2-type adjuvant Alhydrogel (40.7%). Sequence analysis revealed that As37 is a member of the immunoglobulin superfamily (IgSF) and highly conserved in other human STHs. Anti-As37 antibodies strongly recognized homologs in hookworms (Necator americanus, Ancylostoma ceylanicum, A. caninum) and in the whipworm Trichuris muris, but there was no cross-reaction with human spleen tissue extracts. These results suggest that the nematode-conserved As37 could serve as a pan-helminth vaccine antigen to prevent all STH infections without cross-reaction with human IgSF molecules. Conclusions/Significance As37 is an A. suum expressed immunodominant antigen that elicited significant protective immunity in mice when formulated with AddaVax™. As37 is highly conserved in other STHs, but not in humans, suggesting it could be further developed as a pan-helminth vaccine against STH co-infections., Author summary Ascaris infection is the most common infection of humans living in poverty worldwide and can result in malnutrition and stunted physical and mental development in children. A preventive vaccine is urgently needed as a complementary approach to anthelmintic deworming to increase the efficiency of STH infection control. To develop a vaccine against Ascaris infection, an immunodominant antigen, As37 of A. suum, was cloned and expressed as a soluble recombinant protein in E. coli. The recombinant As37 protein (rAs37) was highly recognized by protective immune sera from A. suum infected mice. Balb/c mice immunized with 25 μg rAs37 formulated with the adjuvant AddaVax™ showed significant larval worm reduction against challenge with A. suum infective eggs when compared to a PBS (49.7%) or adjuvant control (48.7%). Protection was associated with a mixed Th1/2-type immune response characterized by high titers of serological IgG1 and IgG2a and stimulation of the production of cytokines IL-4, IL-5, IL-10 and IL-13. The AddaVax™ adjuvant induced better protection than the Th1-type adjuvant MPLA (38.9%) and the Th2-type adjuvant Alhydrogel (40.7%). Sequence analysis revealed that As37 was a member of the immunoglobulin superfamily (IgSF) and highly conserved in other human STHs. Anti-As37 antibodies strongly recognized homologs in hookworms (Necator americanus, A. ceylanicum, A. caninum) and in the whipworm T. muris, but there was no cross-reaction with human spleen tissue extracts. These results indicate that the nematode-conserved As37 protein could be developed as a pan-helminth vaccine antigen to prevent all STH infections without reacting with human IgSF molecules.

Intestinal parasites cause a significant public health problem worldwide due to the associated morbidities, mainly in infected school-aged children (SAC). The strategy of large-scale deworming in SAC to control the transmission of soil-transmitted helminths (STH) has been advocated by the World Health Organization and was recently adopted in Brazil; however, the long-term effects of mass deworming on the larger parasitological profile have been less studied. After a five-year period of school-based large-scale treatment for STH using an annual single dose of albendazole in a community of Sergipe state, Brazil, a marked reduction in prevalence was observed (15.4%% vs.7.4% for Ascaris sp., 6.0%% vs. 0.4% for hookworm, and 12.8%% vs. 4.5%% for Trichuris trichiura), with the exception of Strongyloides stercoralis, which had no statistically significant change in prevalence. There was, however, an increase in the prevalence of intestinal protozoans, specifically Entamoeba histolytica/E. dispar (0.0%% vs. 36.0%), Blastocystis hominis (0.0%% vs. 40.1%), and Giardia duodenalis (5.6%% vs. 14.5%). Although the findings showed a dramatic reduction in the prevalence of STH after four rounds of preventive chemotherapy, there was an increase in intestinal protozoan infections, indicating a change in the epidemiological profile.

SummaryThe human hookworm Necator americanus infects more than 400 million people worldwide, contributing substantially to the poverty in these regions. Adult stage N. americanus live in the small intestine of the human host where they inject excretory/secretory (ES) products into the mucosa. ES products have been characterized at the proteome level for a number of animal hookworm species, but until now, the difficulty in obtaining sufficient live N. americanus has been an obstacle in characterizing the secretome of this important human pathogen. Herein we describe the ES proteome of N. americanus and utilize this information to conduct the first proteogenomic analysis of a parasitic helminth, significantly improving the available genome and thereby generating a robust description of the parasite secretome. The genome annotation resulted in a a revised prediction of 3,425 fewer genes than initially reported, accompanied by a significant increase in the number of exons and introns, total gene length and the percentage of the genome covered by genes. Almost 200 ES proteins were identified by LC-MS/MS with SCP/TAPS proteins, ‘hypothetical’ proteins and proteases among the most abundant families. These proteins were compared to commonly used model species of human parasitic infections, including Ancylostoma caninum, Nippostrongylus brasiliensis and Heligmosomoides polygyrus. Our findings provide valuable information on important families of proteins with both known and unknown functions that could be instrumental in host-parasite interactions, including protein families that might be key for parasite survival in the onslaught of robust immune responses, as well as vaccine and drug targets.

The laboratorial diagnosis of leishmaniasis is based on parasitological methods, which are invasive, present high cost, require laboratorial infrastructure and/or trained professionals; as well as by immunological methods, which usually present variable sensitivity and/or specificity, such as when they are applied to identify asymptomatic cases and/or mammalian hosts presenting low levels of antileishmanial antibodies. As consequence, new studies aiming to identify more refined antigens to diagnose visceral (VL) and tegumentary (TL) leishmaniasis are urgently necessary. In the present work, the Leishmania eukaryotic elongation factor-1 beta (EF1b) protein, which was identified in L. infantum protein extracts by antibodies in VL patients' sera, was cloned and its recombinant version (rEF1b) was expressed, purified and tested as a diagnostic marker for VL and TL. The post-therapeutic serological follow-up was also evaluated in treated and untreated VL and TL patients, when anti-rEF1b antibody levels were measured before and after treatment. Results showed that rEF1b was highly sensitive and specific to diagnose symptomatic and asymptomatic canine VL, as well as human TL and VL. In addition, low cross-reactivity was observed when sera from healthy subjects or leishmaniasis-related diseases patients were tested. The serological follow-up showed also that rEF1b-specific antibodies declined significantly after treatment, suggesting that this protein could be also evaluated as a prognostic marker for human leishmaniasis.

Summary We have recently developed a sensitive and specific urine‐based antigen detection ELISA for the diagnosis of visceral leishmaniasis (VL). This assay used rabbit IgG and chicken IgY polyclonal antibodies specific for the Leishmania infantum proteins iron superoxide dismutase 1 (Li‐isd1), tryparedoxin1 (Li‐txn1) and nuclear transport factor 2 (Li‐ntf2). However, polyclonal antibodies have limitations for upscaling and continuous supply. To circumvent these hurdles, we began to develop immortalized monoclonal antibodies. We opted for recombinant camelid VHHs because the technology for their production is well established and they do not have Fc, hence providing less ELISA background noise. We report here an assay development using VHHs specific for Li‐isd1 and Li‐ntf2. This new assay was specific and had analytical sensitivity of 15‐45 pg/mL of urine. The clinical sensitivity was comparable to that obtained with the ELISA assembled with conventional rabbit and chicken antibodies to detect these two antigens. Therefore, similar to our former studies with conventional antibodies, the future inclusion of VHH specific for Li‐txn1 and/or other antigens should further increase the sensitivity of the assay. These results confirm that immortalized VHHs can replace conventional antibodies for the development of an accurate and reproducible antigen detection diagnostic test for VL.

In the present study, a conserved Leishmania hypothetical protein, LiHyE, was evaluated for the serodiagnosis of leishmaniasis. Results showed that it presented high sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) to serologically identify visceral leishmaniasis (VL) dogs when 40 positive sera and 95 cross-reactive samples were used. rLiHyE also showed the best results of sensitivity, specificity, PPV, and NPV to identify tegumentary leishmaniasis (TL) and VL patients when 45 leishmaniasis patients' sera and 90 cross-reactive samples were used. Results were better in comparison to those obtained when rA2 or Leishmania antigenic extract was employed as controls. The posttreatment follow-up showed that rLiHyE-specific antibodies declined significantly after the end of treatments, and a predominance of the IgG2 subclass was found in comparison to IgG1 levels in both TL and VL patients. In conclusion, rLiHyE can be considered a candidate for the serodiagnosis of canine and human leishmaniasis.

Toxic effects of Peruvian Hadruroides lunatus scorpion venom on different biochemical and enzymatic parameters in blood serum of Wistar rats and Swiss mice were determined after experimental envenomation. An increase in enzymatic activities of Aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH) and levels of serum protein and albumin were observed while a decrease in creatinine level in serum was perceived after 30 min of envenomation. No alterations in urea levels and in kidney histology were detected in the envenomed rats. The global leukocytes count was diminished, with decrease in lymphocytes, eosinophils and neutrophils levels in the bloodstream, while no alterations were found in hematological parameters of red series in rats injected with H. lunatus venom. IL-2, IL-4, IL-6, INF-γ, TNF, IL-17A and IL-10 levels were evaluated 0.5, 3 and 6 h after experimental envenomation of mice with H. lunatus venom. From all the analyzed cytokines, only IL-6 showed an increase in serum levels. Taken together, these results point out that envenomation by H. lunatus can impair hematological and immunological parameters and therefore might be monitored in accidents involving this species.

New methods for evaluating the canine immune system are necessary, not only to monitor immunological disorders, but also to provide insights for vaccine evaluations and therapeutic interventions, reducing the costs of assays using dog models, and provide a more rational way for analyzing the canine immune response. The present study intended to establish an in vitro toll to assess the parasitological/immunological status of dogs, applicable in pre-clinical trials of vaccinology, prognosis follow-up and therapeutics analysis of canine visceral leishmaniasis. We have evaluated the performance of co-culture systems of canine Leishmania chagasi-infected macrophages with different cell ratios of total lymphocytes or purified CD4(+) and CD8(+) T-cells. Peripheral blood mononuclear cells from uninfected dogs were used for the system set up. Employing the co-culture systems of L. chagasi-infected macrophages and purified CD4(+) or CD8(+) T-cell subsets we observed a microenvironment compatible with the expected status of the analyzed dogs. In this context, it was clearly demonstrated that, at this selected T-cell:target ratio, the adaptive immune response of uninfected dogs, composed by L. chagasi-unprimed T-cells was not able to perform the in vitro killing of L. chagasi-infected macrophages. Our data demonstrated that the co-culture system with T-cells from uninfected dogs at 1:5 and 1:2 ratio did not control the infection, yielding to patent in vitro parasitism (≥ 80%), low NO production (≤ 5 μM) and IL-10 modulated (IFN-γ/IL-10 ≤ 2) immunological profile in vitro. CD4(+) or CD8(+) T-cells at 1:5 or 1:2 ratio to L. chagasi-infected macrophages seems to be ideal for in vitro assays. This co-culture system may have great potential as a canine immunological analysis method, as well as in vaccine evaluations, prognosis follow-up and therapeutic interventions.

Live attenuated Leishmania donovani parasites such as LdCen(-/-) have been shown elicit protective immunity against leishmanial infection in mice and hamster models. Previously, we have reported on the induction of strong immunogenicity in dogs upon vaccination with LdCen(-/-) including an increase in immunoglobulin isotypes, higher lymphoproliferative response, higher frequencies of activated CD4(+) and CD8(+) T cells, IFN-γ production by CD8(+) T cells, increased secretion of TNF-α and IL-12/IL-23p40 and, finally, decreased secretion of IL-4. To further explore the potential of LdCen(-/-) parasites as vaccine candidates, we performed a 24-month follow up of LdCen(-/-) immunized dogs after challenge with virulent Leishmania infantum, aiming determination of parasite burden by qPCR, antibody production (ELISA) and cellular responses (T cell activation and cytokine production) by flow cytometry and sandwich ELISA. Our data demonstrated that vaccination with a single dose of LdCen(-/-) (without any adjuvant) resulted in the reduction of up to 87.3% of parasite burden after 18 months of virulent challenge. These results are comparable to those obtained with commercially available vaccine in Brazil (Leishmune(®)). The protection was associated with antibody production and CD4(+) and CD8(+) proliferative responses, as well as T cell activation and significantly higher production of IFN-γ, IL-12/IL-23p40 and TNF-α, which was comparable to responses induced by immunization with Leishmune(®), with significant differences when compared to control animals (Placebo). Moreover, only animals immunized with LdCen(-/-) expressed lower levels of IL-4 when compared to animals vaccinated either with Leishmune(®) or PBS. Our results support further studies aiming to demonstrate the potential of genetically modified live attenuated L. donovani vaccine to control L. infantum transmission in endemic areas for CVL.

In this study we assessed the basal transcription of genes associated with innate immunity (i.e. Nramp1, NOD1, NOD2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR9) in canine monocyte-derived macrophages from Leishmania-free dogs. Additionally, secretion of cytokines (IL-10, IL-12, TNF-α and IFN-γ) and nitric oxide in culture supernatants of macrophages with higher or lower resistance to intracellular survival of Leishmania infantum was also measured. Constitutive transcription of TLR9 and NOD2 were negligible; NOD1, TLR1, and TLR7 had low levels of transcription, whereas Nramp1 and TLR2, 3, 4, 5, and 6 had higher levels of constitutive transcription in canine monocyte-derived macrophages. There were no significant differences in transcription between macrophages with higher or lower resistance to intracellular survival of L. infantum. Secretion of TNF-α was higher in more resistant macrophages (designated as resistant) at 24h after infection when compared to less resistant macrophages (designated as susceptible), as well as the secretion of IFN-γ at 72 h post infection. Secretion of IL-10 was lower in resistant macrophages at 24h after infection. No detectable production of nitric oxide was observed. Interestingly, there was a negative correlation between NOD2 transcript levels and intracellular survival of L. infantum in resistant macrophages. This study demonstrated that decreased intracellular survival of L. infantum in canine macrophages was associated with increased production of TNF-α and IFN-γ and decreased production of IL-10; and that constitutive transcription of Nramp1, TLR and NLR does not interfere in intracellular survival of L. infantum.

In this paper we report the synthesis and characterization of four new nitroaromatic compounds, 2-{6-nitrobenzo[1,3]dioxol-5-(methyleneamino)}benzoic acid (1), 2-{[5-(2-nitrophenyl)furan-2-yl]methylene-amino}benzoic acid (2), 2-{(6-nitrobenzo[1,3]dioxol-5-yl)methylene}hydrazinecarboxamide (3) and 2-{[5-(2-nitrophenyl)furan-2-yl]methylene}hydrazinecarboxamide (4). Compounds (1)–(4) have been authenticated by infrared and NMR spectroscopy, and the structure of (1), (2) and (4) have been determined by X-ray diffraction. In addition, the in vitro ability of compounds (1)–(4) to inhibit the growth of Leishmania infantum has been evaluated. Comparisons of the redox potential of the compounds and leishmanicidal activity indicate that the presence of the electroactive nitro group is important for the biological activity. The inhibition activity of compound (3) is comparable to that of the reference drug, SbCl3. Considering the important side effects and the low efficiency of SbCl3 in the case of resistance, compound (3) deserves further attention as a promising anti-leishmanicidal drug for veterinary use.