Clinical-therapeutic follow-up of patients with American cutaneous leishmaniasis caused by different Leishmania spp. in Brazil.

American cutaneous leishmaniasis (ACL) may present different clinical manifestations, immune and therapeutic responses, depending on the Leishmania species, as well as inoculum size and factors inherent to the affected individual. Thus, the aim of this study was to carry out clinical-therapeutic follow-up of Brazilian patients with ACL caused by different Leishmania species. Between 2015 and 2018, patients with ACL from Amazonas and Pernambuco states (Brazil) were submitted to blood collection before and after treatment. The qPCR technique was used to quantify the parasite load. To identify the Leishmania species, one of the following techniques was employed: a conventional PCR performed from biopsy or blood DNA, followed by sequencing; or Multilocus Enzyme Electrophoresis from Leishmania isolated from biopsy/aspirated lesion. A total of 10.8% (23/213) of the patients included in positive cases were followed-up. All 23 patients were clinically and epidemiologically compatible with ACL and were also positive in parasitological tests (86.96%), molecular tests (73.91%) or both (60.87%). Seventeen samples collected before treatment and 11 collected after treatment were positive in the qPCR assay, with a mean parasite load (MPL) of 38.33 fg/µL and 11.81 fg/µL, respectively. Eight samples were positive in both collections. Thirteen patients (56.52%) were clinically cured (wound healing). Ten patients (43.47%) were not clinically cured at the time of return with the attending physician. Identification of Leishmania species was carried out in samples from nine patients, and six were identified as L. (Viannia) braziliensis, 2 as L (Viannia) guyanensis and 1 as L (Leishmania) amazonensis. One patient infected with L. guyanensis and other with L. braziliensis were not clinically cured and increased the mean parasite load after treatment. The data obtained from the followed-up patients and the relationship between clinical evolution and the infecting species demonstrate the need to understand its etiology to define the effective therapeutic protocol.

The effects of endoplasmic reticulum stressors, tunicamycin and dithiothreitol on Trypanosoma cruzi.

In higher eukaryotic cells, pertubations in ER environment, called ER stress, usually activate unfolded protein response (UPR) pathway in an attempt to re-stablish the ER homeostasis and prevent cell death. Because trypanosomatids appear to lack the classical UPR, it is not clear how these parasites respond to ER stress. Thus, the aim of this work was to evaluate the effects of ER stressors tunicamycin (TM) or dithiothreitol (DTT) on Trypanosoma cruzi. The TM treatment showed strong trypanostatic effect. At 2.5 µg/mL of TM, the mRNA levels of both binding protein (BiP) and calreticulin (CRT) increased significantly, whereas the protein levels of BiP remained stable. TM treatment induced ultrastructural changes compatible with an autophagic process. The DTT treatment inhibited the cell growth, induced drastic morphological changes, mitochondrial membrane depolarization and increased ROS production. The expression of BiP apparently was not affected by DTT, whereas the mRNA levels of BiP and CRT were significantly reduced. Our results suggest that TM induces autophagy/ER-phagy without causing substantial injury to the parasite. Conversely, the DTT treatment seems to rupture the mitochondrion homeostasis leading to parasite death. The comprehension of the mechanisms behind the susceptibility of T. cruzi to ER stress open perspectives for the development of chemotherapeutic agents addressed to these pathways.

Polymorphisms in GSTE2 is associated with temephos resistance in Aedes aegypti.

The glutathione S-transferases (GSTs) are enzymes involved in several distinct biological processes. In insects, the GSTs, especially delta and epsilon classes, play a key role in the metabolism of xenobiotics used to control insect populations. Here, we investigated its potential role in temephos resistance, examining the GSTE2 gene from susceptible (RecL) and resistant (RecR) strains of the mosquito Aedes aegypti, vector for several pathogenic arboviruses. Total GST enzymatic activity and the GSTE2 gene expression profile were evaluated, with the GSTE2 cDNA and genomic loci sequenced from both strains. Recombinant GSTE2 and mutants were produced in a heterologous expression system and assayed for enzyme kinetic parameters. These proteins also had their 3D structure predicted through molecular modeling. Our results showed that RecR has a profile of total GST enzymatic activity higher than RecL, with the expression of the GSTE2 gene in resistant larvae increasing six folds. Four exclusive RecR mutations were observed (L111S, I150V, E178A and A198E), which were absent in the laboratory susceptible strains. The enzymatic activity of the recombinant GSTE2 showed different kinetic parameters, with the GSTE2 RecR showing an enhanced ability to metabolize its substrate. The I150V mutation was shown to induce significant changes in catalytic parameters and a 3D modeling of GSTE2 mapped two of the RecR changes (L111S and I150V) near the enzyme's catalytic pocket, also implying an impact on its catalytic activity. Our results reinforce a potential role for GSTE2 in the metabolic resistance phenotype while contributing to the understanding of the molecular basis for the resistance mechanism.

Study of association of the rs2275913 IL-17A single nucleotide polymorphism and susceptibility to cutaneous leishmaniasis caused by Leishmania braziliensis.

Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is the most spread clinical form of leishmaniasis in Brazil. However, only a few part of the people infected develop clinically perceptive disease, suggesting the influence of human genetic components in the CL pathogeny. The rs2275913 SNP is the nucleotide variant of the IL17A gene. The A allele is associated with a vast number of infectious and non-infectious diseases. Here, we investigated the association of the rs2275913 SNP (G/A) from IL-17A and two forms of susceptibility to CL in Brazil by case-control study. Furthermore, we evaluated the functional relevance of this SNP during the immune response of the host and analyzed its impact in the parasite elimination. Weak associations of A allele with susceptibility to L. braziliensis infection or to symptomatic CL were observed, and a tendency of A allele carriers to be more susceptible to infection and cutaneous disease. Functional analysis of the Th17 cell phenotypes revealed lower frequencies of CD4+ IL-17+ cells in samples of infected people with AA/AG genotypes. Furthermore, people carrying the A allele maintain higher parasite loads, reinforcing the genetic susceptibility findings. This study adds knowledge about the influence of a significant genetic variation on IL-17 promoter on CL pathogenesis, and may contribute to enhance the knowledge about the role of IL-17 in the L. braziliensis infections.

The diagnosis of canine visceral leishmaniasis in Brazil: Confronting old problems.

In Brazil, the main strategy adopted to contain Visceral Leishmaniasis (VL) is the controversial culling of dogs with reagent serology for Canine VL (CVL). Despite there are studies showing that significant reduction of human cases has not been observed, as well as there are works demonstrating the occurrence of false-positive results in the confirmatory test, the protocol has been maintained. Researches that can reinforce the existence and persistence of this problem, as well as bring concrete alternatives are pivotal. In this context, the aim of this work was to evaluate and compare the serological, molecular and parasitological methods employed for CVL detection in Brazil, in dogs with diverse clinical profiles, from two endemic areas of Pernambuco state. Comparisons among TR-DPP®, EIE-LVC and qPCR (animals from Goiana-PE: 91) demonstrated that agreements varied from 'poor' to 'moderate' (kappa = 0.162-0.442), and a triple agreement occurred in 61.36% (54/88) of the samples. The highest percentage of agreement was obtained between TR-DPP® and EIE-LVC within the polysymptomatic group (93.33%; 14/15). Of the 34 dogs with reagent serology from Caruaru-PE, 17 (50%) and 29 dogs (85.29%) were positive for qPCR and parasitological exam, respectively. By comparing serology, qPCR and parasitological exam, the lowest percentage of agreements were obtained within the asymptomatic group (40%-72.72%). It was possible to observe that the percentage of agreement tended to decrease according to the absence of clinical manifestations in the dogs. Thus, from the fact that all diagnostic tools evaluated have their limitations, it is very important to be careful before to propose an alternative set of diagnostic criteria. Besides, the answer for better results in the control of CVL may not be in the choose of the best set of diagnostic tools, but it may be in the strategy of culling itself. In this context, it is very important to invest in alternative control measures, such as mass vaccination and treatment of dogs, thus reducing the transmission to the vector and helping to avoid new canine and human cases.

Relation between neonatal malnutrition and gene expression: inflammasome function in infections caused by Candida Albicans.

PURPOSE: To investigate the effects of neonatal malnutrition followed by nutritional replacement on the signaling mechanisms developed by the inflammasome complex by analyzing the expression of the targeted TLR2, TLR4, NLRP3, caspase-1 and release of IL-1ß and IL-18 by alveolar macrophages infected in vitro with Candida albicans. METHODS: Male Wistar rats (n = 24), 90-120 days, were suckled by mothers whose diet during lactation contained 17 % protein in the nourish group and 8 % protein in the malnourished group. After weaning, both groups were fed a normal protein diet. Macrophages were obtained after tracheostomy, through the collection of bronchoalveolar lavage fluid. The quantification of the expression levels of targets (TLR2, TLR4, NLRP3 and caspase-1) was performed by real-time RT-PCR. Production of cytokines was performed by ELISA. RESULTS: The malnourished animals during lactation showed reduced body weight from the fifth day of life, remaining until adulthood. Further, the model applied malnutrition induced a lower expression of TLR4 and caspase-1. The quantification of the TLR2 and NLRP3, as well as the release of IL-1ß and IL-18, was not different between groups of animals nourished and malnourished. The system challenged with Candida albicans showed high expression levels of all targets in the study. CONCLUSIONS: The tests demonstrate nutritional restriction during critical periods of development, although nutritional supplementation may compromise defense patterns in adulthood in a timely manner, preserving distinct signaling mechanism, so that the individual does not become widely vulnerable to infections by opportunistic pathogens.

Effect of neonatal malnutrition on expression of nitric oxide synthase enzyme, production of free radicals and in vitro viability of alveolar macrophages infected with methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

OBJECTIVE: Evaluate the effects of neonatal malnutrition on the microbicidal response and viability of in vitro macrophages infected with Staphylococcus aureus sensitive/resistant to methicillin. METHODS: Male Wistar rats (n = 24) were divided into two distinct groups: nourished (rats breast-fed by mothers undergoing diet with 17% casein) and malnourished (rats breast-fed by mothers undergoing diet with 8% casein). Macrophages were recovered after surgical tracheostomy procedure by collecting bronchoalveolar lavage. Four systems were established: negative control, composed only by phagocytes; positive control, macrophages plus lipopolysaccharide; and two test systems, macrophages plus Staphylococcus aureus sensitive and resistant to methicillin. Plates were incubated at 37 °C for 24 h. After this period, tests for the analysis of cell viability and microbicidal response were performed. In the statistical analysis, the Student's t and ANOVA tests were used, accepting p < 0.05. RESULTS: The neonatal malnutrition impaired the animals' body weight. There was a lower expression of the inducible nitric oxide enzyme (iNOS), nitric oxide production, and viability of macrophages infected with methicillin-resistant Staphylococcus aureus. However, increased production of superoxide anion in the malnourished group was detected. CONCLUSION: Neonatal malnutrition focusing on critical periods of development promoted lower expression of iNOS, nitric oxide production, cell viability, and exacerbated reactive oxygen species production. The high levels of reactive oxygen species may favor the onset of serious and systemic infections with fatal outcome if associated with methicillin-resistant Staphylococcus aureus.

Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques.

BACKGROUND: Cutaneous leishmaniasis (CL) is a parasitic disease caused by various Leishmania species. Several studies have shown that real time quantitative PCR (qPCR) can be used for Leishmania spp. identification by analyzing the melting temperature (Tm). Thus, the aim of this study was to evaluate the viability of qPCR for differentiating eight closely related Leishmania species that cause the same clinical form of the disease and to compare the results with classical techniques. METHODS: qPCR assays for standardizing the Tm using reference strains were performed. After the CL diagnosis on blood samples of domestic animals, positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by Tm. A Restriction Fragment Length Polymorphism (RFLP) assay, a reference test, was also standardized, by using the reference strains. RESULTS: Through standardization of Tm for Leishmania spp., two Tm ranges were created for analysis: 1 (Tm = 78-79.99 °C) included Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis and Leishmania (V.) shawi; and 2 (Tm = 80-82.2 °C) included Leishmania (V.) naiffi, Leishmania (L.) amazonensis and Leishmania (L.) mexicana. A total of 223 positive blood samples were analyzed, with 58 included in range 1 and 165 in range 2. L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis were identified by sequencing, while L. (V.) braziliensis, L. (L.) mexicana and L. (V.) panamensis were identified by RFLP analysis. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by qPCR Tm analysis; five were classified in range 1 and five in range 2. A concordance of 80% was calculated between qPCR and the gold-standard (MLEE) with no significant difference between the methods (p = 0.6499); a similar result was observed for sequencing and qPCR (p = 0.2566). In contrast, a highly significant difference was observed for qPCR and RFLP (p < 0.001). CONCLUSIONS: In this study, we demonstrated the potential use of qPCR as a tool for Leishmania species identification using two Tm ranges.

Quantitative real time PCR assays for the detection of Leishmania (Viannia) braziliensis in animals and humans.

American cutaneous leishmaniasis (ACL) caused by Leishmania (Viannia) braziliensis is a neglected disease of humans in the New World that may also cause irreversible skin and eventually mucocutaneous lesions. This parasite can also infect dogs and represents a diagnostic challenge for veterinarians. Methods currently available for the diagnosis of ACL have a low sensitivity and may be time-consuming, representing a limit for treatment expedition of ACL. Quantitative real time PCR assays (qPCR) for the detection of L. (V.) braziliensis in canine blood samples were developed herein, and the detection limit and specificity of different molecular targets (kDNA and rDNA) evaluated. Of the protocols assessed, two qPCR assays, one targeting the kDNA and other the SSU rDNA of L. (V.) braziliensis, performed better, with detection limits of 100 fg and 10 pg, respectively. These assays were also used to test skin samples from humans with suspected ACL. The results indicate that the qPCR protocols developed represent an advance for the diagnosis of ACL in dogs and humans from this region, and provide a rapid and non-invasive diagnosis of the infection by L. (V.) braziliensis. Considering the quantitative nature of the assays, they will also be useful for monitoring treatment efficacy and preventing relapses in human patients in Brazil, although further studies are needed to critically evaluate the specificity of the qPCRs for their capacity to distinguish different Leishmania species and subspecies (represented by zymodemes) in other countries. Finally, molecular assays established may represent new tools for future basic and applied research focused on species identification, host-parasite associations, and infection dynamics in host and vector populations.

Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR.

Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.

High levels of infectiousness of asymptomatic Leishmania (Viannia) braziliensis infections in wild rodents highlights their importance in the epidemiology of American Tegumentary Leishmaniasis in Brazil.

BACKGROUND: The epidemiological significance of wildlife infections with aetiological agents causing human infectious diseases is largely determined by their infection status, contact potential with humans (via vectors for vector-borne diseases), and their infectiousness to maintain onward transmission. This study quantified these parameters in wild and synanthropic naturally infected rodent populations in an endemic region of tegumentary leishmaniasis in northeast Brazil. METHODS: Capture-mark-recapture (CMR) of rodents was conducted over 27 months in domestic/peri domestic environs, household plantations and nearby Atlantic Forest (9,920 single trap nights). Rodent clinical samples (blood and ear tissue) were tested for infection by conventional PCR and quantitative PCR (qPCR) for Leishmania (Viannia) braziliensis, and xenodiagnosis to measure infectiousness to the local sand fly vector. RESULTS: A total 603 individuals of 8 rodent species were (re)captured on 1,051 occasions. The most abundant species were Nectomys squamipes (245 individuals, 41% of the total catch), Rattus rattus (148, 25%), and Necromys lasiurus (83, 14%). All species were captured in greater relative frequencies in plantations; R. rattus was the only species captured in all three habitats including in and around houses. Four species, comprising 22.6% of individuals captured at least twice, were geolocated in more than one habitat type; 78.6% were infected with L. (V.) braziliensis, facilitating inter-species and inter-habitat transmission. Species specific period prevalence ranged between 0%-62% being significantly higher in N. squamipes (54-62%) and Hollochillus sciureus (43-47%). Xenodiagnosis was performed on 41 occasions exposing 1,879 Nyssomyia whitmani sand flies to five rodent species (37 individuals). Similar mean levels of infectiousness amongst the more common rodent species were observed. Longitudinal xenodiagnosis of the N. squamipes population revealed a persistent level of infectiousness over 13 months follow-up, infecting a median 48% (IQR: 30.1%-64.2%) of exposed blood-fed vectors. The proportion of exposed flies infected was greater in the low compared to in the high seasonal period of vector abundance. L. (V.) braziliensis parasite loads in rodent blood quantified by qPCR were similar across rodent species but did not represent a reliable quantitative marker of infectiousness to sand flies. The standardised risk of rodent infection in plantations was 70.3% relative to 11.3% and 18.4% in peri domestic and forest habitats respectively. R. rattus was the only exception to this trend indicating greatest risk in the peri domestic environment. CONCLUSIONS: The results support the view that a collective assemblage of wild and synanthropic rodent species is an important wild reservoir of L. (V.) braziliensis in this region, with N. squamipes and R. rattus probably playing a key role in transmission within and between habitat types and rodent species. Rodents, and by implication humans, are at risk of infection in all sampled habitats, but more so in homestead plantations. These conclusions are based on one of the longest CMR study of small rodents in an American Tegumentary Leishmaniasis (ATL) foci.

Therapeutic Potential of Photosynthetic Microorganisms for Visceral Leishmaniasis: An Immunological Analysis.

New therapeutic strategies for visceral leishmaniasis (VL) have been studied, and the development of an immunotherapeutic agent that modulates the host's immune response is necessary. The aim of this study was to evaluate in vitro the bioactive extracts of photosynthetic microorganisms (PMs) for their leishmanicidal/leishmanistatic and immunomodulatory potentials. Bioactive extracts from PMs (Arthrospira platensis and Dunaliella tertiolecta) were obtained by sonication. Reference drugs, miltefosine (MTF) and N-methylglucamine antimoniate (SbV), were also evaluated. The selectivity index (SI) of treatments was determined by assays of inhibitory concentration (IC50) in Leishmania infantum cells and cytotoxic concentrations (CC50) in human peripheral blood mononuclear cells by the MTT method. The immune response was evaluated in healthy human cells by the production of cytokines and nitric oxide (NO) and the gene expression of Tbx21, GATA3, RORc, and FOXP3, using four concentrations (CC50, ½ CC50, » CC50, and IC50) for in-vitro stimulation. Based on the data obtained, we observed that the extracts of D. tertiolecta (SI = 4.7) and A. platensis (SI = 3.8) presented better results when compared to SbV (SI = 2.1). When analyzing the immune response results, we identified that the extracts of PMs stimulated the production of cytokines of the Th1 profile more than the reference drugs. The extracts also demonstrated the ability to stimulate NO synthesis. Regarding gene expression, in all concentrations of A. platensis extracts, we found a balance between the Th1/Th2 profile, with the average expression of the Tbx21 gene more than the GATA3 in the highest concentration (CC50). Regarding the extract of D. tertiolecta, we can observe that, in the lowest concentrations, a balance between all the genes was present, with the average expression of the GATA3 gene being lower than the others. The best result was found in the ½ CC50 concentration, stimulating a balanced positive expression between the Th1×Th17×Treg profiles, with a negative expression of GATA3. Thus, PM extracts showed promising results, presenting low toxicity, leishmanicidal/leishmanistatic activity, and induction of the immune response, which could be potential therapeutic candidates for VL.

Transovarial passage of Leishmania infantum kDNA in artificially infected Rhipicephalus sanguineus.

Phlebotomine sand flies are the only proven biological vectors of Leishmania parasites. However, Rhipicephalus sanguineus ticks have long been suspected to transmit Leishmania infantum in studies carried out in laboratory and natural conditions. In the present study, 5 microl of L. infantum promastigotes (1x10(6) cells per ml) was injected into the hemocel through the coxa I of four engorged females (F1, F2, F3 and F4). Control ticks (F5 and F6) were injected with sterile phosphate-buffered saline (PBS) using the same procedure. Then, these females, their eggs, and the originated larvae were tested by real time polymerase chain reaction (real-time PCR) for the presence of L. infantum kinetoplast DNA (kDNA). Females and eggs were tested after the end of the oviposition period (about 5 weeks post-inoculation) whereas larvae were tested about 4 months after the inoculation of females. All artificially infected females were positive for L. infantum kDNA. In addition, two pools of eggs (one from F2 and other from F4) and four pools of larvae (one from each F1 and F4 and two from F2) were positive for L. infantum kDNA. These results showed, for the first time, the transovarial passage of L. infantum kDNA in R. sanguineus ticks, thus suggesting that the transovarial transmission of L. infantum protozoa in ticks is worth to be investigated.

Detection of Leishmania infantum in Rhipicephalus sanguineus ticks from Brazil and Italy.

Canine leishmaniosis is a widespread disease caused by Leishmania parasites, which are transmitted by phlebotomine sand flies. However, in some areas where canine leishmaniosis is endemic, but the primary vectors have not been found, ticks have been suspected to play a role in transmitting the infection. Herewith, we report the detection of Leishmania infantum kinetoplast minicircle DNA (kDNA) in ticks collected from naturally infected dogs living in rural areas of Southern Italy (site A) and Northeastern Brazil (site B). Between March and October 2007, ticks were collected from 26 dogs positive to anti-Leishmania antibodies (one from site A and 25 from site B) and either placed directly into vials containing 70% ethanol or maintained alive for identification and subsequent dissection. All the 95 ticks collected were morphologically identified as Rhipicephalus sanguineus. After identification, their genomic DNA was extracted (either individually or in pools) and processed by polymerase chain reaction (PCR) for the detection of L. infantum kDNA. Two pools of salivary glands from ticks (one from five females and other from five males) found on a dog from site A and tested by a conventional PCR were positive. Amplicon sequencing confirmed the identity of the parasite. In addition, nine (12.3%) out of the 73 ticks found on dogs from site B and tested by a real-time PCR were positive, with a low parasite load (less than 1 parasite/ml). The retrieval of L. infantum kDNA in salivary glands of R. sanguineus ticks has been here reported for the first time. Therefore, further studies are needed to assess the competence of ticks as vectors of Leishmania parasites from dog to dog.

Analysis of the IGS rRNA Region and Applicability for Leishmania (V.) braziliensis Characterization.

The causative species is an important factor influencing the evolution of American cutaneous leishmaniasis (ACL). Due to its wide distribution in endemic areas, Leishmania (V.) braziliensis is considered one of the most important species in circulation in Brazil. Molecular targets derived from ribosomal RNA (rRNA) were used in studies to identify Leishmania spp.; however, the Intergenic Spacer (IGS) region has not yet been explored in parasite species differentiation. Besides, there is a shortage of sequences deposited in public repositories for this region. Thus, it was proposed to analyze and provide sequences of the IGS rRNA region from different Leishmania spp. and to evaluate their potential as biomarkers to characterize L. braziliensis. A set of primers was designed for complete amplification of the IGS rRNA region of Leishmania spp. PCR products were submitted to Sanger sequencing. The sequences obtained were aligned and analyzed for size and similarity, as well as deposited in GenBank. Characteristics of the repetitive elements (IGSRE) present in the IGS rRNA were also verified. In addition, a set of primers for L. braziliensis identification for qPCR was developed and optimized. Sensitivity (S), specificity (σ), and efficiency (ε) tests were applied. It was found that the mean size for the IGS rRNA region is 3 kb, and the similarity analysis of the sequences obtained demonstrated high conservation among the species. It was observed that the size for the IGSRE repetitive region varies between 61 and 71 bp, and there is a high identity between some species. Fifteen sequences generated for the IGS rRNA partial region of nine different species were deposited in GenBank so far. The specific primer system for L. braziliensis showed S = 10 fg, ε = 98.08%, and logσ = 103 for Leishmania naiffi; logσ = 104 for Leishmania guyanensis; and logσ = 105 for Leishmania shawi. This protocol system can be used for diagnosis, identification, and quantification of a patient's parasite load, aiding in the direction of a more appropriate therapeutic management to the cases of infection by this etiological agent. Besides that, the unpublished sequences deposited in databases can be used for multiple analyses in different contexts.

Immunoprophylactic Potential of a New Recombinant Leishmania infantum Antigen for Canine Visceral Leishmaniasis: An In Vitro Finding.

The development and application of safe and effective immunoprophylactic/immunotherapeutic agents against canine visceral leishmaniasis (CanL) have been pointed out as the only means for the real control of the disease. Thus, this study aimed to evaluate the in vitro cellular immune response of dogs, elicited by the new recombinant proteins of Leishmania infantum, Lci10 and Lci13, in order to investigate their potential for vaccinology. Twenty-four dogs were submitted to clinical, parasitological, serological and molecular tests, and then separated into two study groups: 12 infected (InD) and 12 non-infected dogs (NInD), and six of each group were directed for Lci10 and Lci13 evaluation. Peripheral blood mononuclear cells (PBMC) were cultured and stimulated with Lci10 (10 µg/ml) or Lci13 (5 µg/ml), and with L. infantum soluble antigen (LSA) (25 µg/ml) or no stimulus (NS) as controls. Afterwards, the mRNA levels of different cytokines were quantified through qPCR, and Nitric Oxide (NO) production was assessed in the culture supernatants. Significant differences were considered when p ≤ 0.05. The comparative analysis revealed that, in the NInD group, Lci13 promoted a significant increase in the expression of IFN-γ in relation to LSA (p = 0.0362), and the expression of this cytokine in NInD was significantly higher than that presented in the InD (p = 0.0028). A negative expression for TGF-ß was obtained in both groups. Lci13 also induced a greater production of NO in relation to the NS sample in the NInD group. No significant differences were observed after stimulation with Lci10. In conclusion, the results suggest a protective role of Lci13 for uninfected animals, thus with a potential for immunoprophylaxis. The results will help to direct the antigen Lci13 for further studies (pre-clinical trials), in order to determine its immunogenicity and reactogenicity effects, as a way to consolidate its real applicability for vaccinology against CanL.

TLR and NLRP3 inflammasome expression deregulation in macrophages of adult rats subjected to neonatal malnutrition and infected with methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Nutritional aggression in critical periods may lead to epigenetic changes that affect gene expression. The aim of this study was to assess the effect of neonatal malnutrition on the expression of toll-like receptor (TLR)-2, TLR-4, and NLRP3 receptors, caspase-1 enzyme, and interleukin (IL)-1 ß production in macrophages infected with methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) Staphylococcus aureus. METHODS: Wistar rats (N = 24) were divided in two distinct groups: nourished (17% casein) and malnourished (8% casein). Four systems were established after the isolation of mononuclear cells: negative control, positive control, MRSA, and MSSA. The plates were incubated at 37°C for 24 h in humidified atmosphere and 5% carbon dioxide. Tests were performed after this period to analyze the expression of standard recognition receptors, caspase-1 enzyme, and the production of IL-1 ß. Student's t test and analysis of variance were used in the statistical analysis; P < 0.05 was statistically significant. RESULTS: Malnutrition reduced animal growth and the expression of TLR-2, TLR-4, and NLRP3 receptors, the caspase-1 enzyme, and the IL-1 ß levels in macrophages infected with lipopolysaccharides in the present study. However, the interaction between the S. aureus and the macrophages promoted greater gene expression of receptors and enzymes. CONCLUSION: The neonatal malnutrition model compromised the expression of standard recognition receptors, of the caspase-1 enzyme as well as the production of IL-1 ß. However, the S. aureus and neonatal malnutrition combination led to intense transcription of such innate immunity components. Therefore, the deregulation in the expression of TLR and NLRP3 receptors and of the caspase-1 enzyme may induce extensive tissue injury and favor the permanence and spread of these bacteria, especially those that are methicillin resistant.

The Equivocal Role of Th17 Cells and Neutrophils on Immunopathogenesis of Leishmaniasis.

Advances in the understanding of leishmaniasis progression indicate that cellular interactions more complex than the Th1/Th2 paradigm define the course of infection. Th17 cells are a crucial modulator of adaptive immunity against Leishmania parasites acting mainly on neutrophil recruitment and playing a dual role at the site of infection. This review describes the roles of both these cell types in linking innate defense responses to the establishment of specific immunity. We focus on the Th17-neutrophil interaction as a crucial component of anti-Leishmania immunity, and the clinical evolution of cutaneous or visceral leishmaniasis. To date, information obtained through experimental models and patient evaluations suggests that the influence of the presence of interleukin (IL)-17 (the main cytokine produced by Th17 cells) and neutrophils during Leishmania infections is strictly dependent on the tissue (skin or liver/spleen) and parasite species. Also, the time at which neutrophils are recruited, and the persistence of IL-17 in the infection microenvironment, may also be significant. A clearer understanding of these interactions will enable better measurement of the influence of IL-17 and its regulators, and contribute to the identification of disease/resistance biomarkers.

Evaluation of urine for Leishmania infantum DNA detection by real-time quantitative PCR.

The availability of some sorts of biological samples which require noninvasive collection methods has led to an even greater interest in applying molecular biology on visceral leishmaniasis (VL) diagnosis, since these samples increase the safety and comfort of both patients and health professionals. In this context, this work aimed to evaluate the suitability of the urine as a specimen for Leishmania infantum kinetoplast DNA detection by real-time quantitative PCR (qPCR). Subsequent to the reproducibility analysis, the detection limit of the qPCR assay was set at 5fg (~0.025 parasites) per µL of urine. From the comparative analysis performed with a set of diagnostic criteria (serological and molecular reference tests), concordance value of 96.08% was obtained (VL-suspected and HIV/AIDS patients, n=51) (P>0.05). Kappa coefficient (95% CI) indicated a good agreement between the test and the set of diagnostic criteria (k=0.778±0.151). The detection of Leishmania DNA in urine by qPCR was possible in untreated individuals, and in those with or without suggestive renal impairment. Fast depletion of the parasite's DNA in urine after treatment (from one dose of meglumine antimoniate) was suggested by negative qPCR results, thus indicating it as a potential alternative specimen to follow up the efficacy of therapeutic approaches. Even when evaluated in a clinically heterogeneous set of patients, the urine showed good prospect as sample for VL diagnosis by qPCR, also indicating a good negative predictive value for untreated suspected patients.

Inclusion of quality controls on leishmaniases molecular tests to increase diagnostic accuracy in research and reference laboratories.

Early detection of leishmaniases and prompt institution of treatment are paramount for individuals and communities affected by these diseases. To overcome the remaining limitations inherent to molecular methods currently used and to ensure the accuracy of results in leishmaniases diagnosis, two triplex polymerase chain reaction (PCR) assays with quality controls for the reactions were developed. Validity indicators were assessed in 186 dog blood samples from endemic areas in Brazil. The level of agreement between the new tools and their singleplex protocols was assessed by kappa analysis. The triplex PCR for visceral leishmaniasis showed sensitivity (S) = 78.68 %, specificity (E) = 85.29 %, and efficiency (e) = 81.05 %. The cutaneous leishmaniasis protocol showed S = 97.29 %, E = 79.16 %, and e = 90.16 %. Both protocols showed good agreement with gold standards. These new tools enable, in a single reaction, the diagnosis of the diseases and the evaluation of the sample quality and DNA extraction process, thus reducing the cost of reagents and avoiding the eventual need for collecting a second sample.