Chlorella vulgaris extract and Imiquimod as new therapeutic targets for leishmaniasis: An immunological approach.

The therapeutic regimen for the treatment of American Tegumentary Leishmaniasis (ATL) is targeted at the death of the parasite; therefore, it is essential to develop a treatment that can act on the parasite, combined with the modulation of the inflammatory profile. Thus, the aim of this study was to make an in vitro evaluation of the therapeutic potential of Chlorella vulgaris extract (CV) and Imiquimod for ATL. Selectivity indices (SI) were determined by inhibitory concentration assays (IC50) in L. braziliensis cells and cytotoxic concentrations (CC50) were measured in human cells using the MTT method, based on the CV microalgae extract (IC50 concentrations of 15.63 to 500 µg/mL; CC50 concentrations of 62.5-1000 µg/mL) in comparison with the reference drugs and Imiquimod. The immune response was evaluated in healthy human cells by gene expression (RT-qPCR) and cytokine production (Flow Cytometry). The CV extract (SI = 6.89) indicated promising results by showing higher SI than meglumine antimoniate (SI = 3.44) (reference drug). In all analyses, CV presented a protective profile by stimulating the production of Th1 profile cytokines to a larger extent than the reference drugs. Imiquimod showed a high expression for Tbx21, GATA3, RORc and Foxp3 genes, with increased production only of the TNF cytokine. Therefore, the data highlight the natural extract and Imiquimod as strong therapeutic or adjuvant candidates against ATL, owing to modulation of immune response profiles, low toxicity in human cells and toxic action on the parasite.

Benznidazole treatment decreases IL-6 levels in Trypanosoma cruzi-infected human adipocytes differentiated from adipose tissue-derived stem cells.

BACKGROUND: Trypanosoma cruzi, which causes Chagas disease (CD), is a versatile haemoparasite that uses several strategies to evade the host's immune response, including adipose tissue (AT), used as a reservoir of infection. As it is an effective barrier to parasite evasion, the effectiveness of the drug recommended for treating CD, Benznidazole (BZ), may be questionable. OBJECTIVE: To this end, we evaluated the parasite load and immunomodulation caused by BZ treatment in the culture of adipocytes differentiated from human adipose tissue-derived stem cells (ADSC) infected with T. cruzi. METHODS: The ADSC were subjected to adipogenic differentiation. We then carried out four cultures in which we infected the differentiated AT with trypomastigote forms of the Y strain of T. cruzi and treated them with BZ. After the incubation, the infected AT was subjected to quantitative polymerase chain reaction (qPCR) to quantify the parasite load and transmission electron microscopy (TEM) to verify the infection. The supernatant was collected to measure cytokines, chemokines, and adipokines. FINDINGS: We found elevated secretion of IL-6, CXCL-10/IP-10, CCL2/MCP-1, CCL5/RANTES, and leptin in infected fat cells. However, treatment with BZ promoted a decrease in IL-6. MAIN CONCLUSION: Therefore, we believe that BZ has a beneficial role as it reduces inflammation in infected fat cells.

Outbreak of Chagas disease in Brazil: Validation of a molecular diagnostic method.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi (T. cruzi), affects millions of people worldwide. Polymerase Chain Reaction (PCR) and real-time quantitative PCR (qPCR) have been used as tools to monitor parasitic levels in the bloodstream of individuals exposed to infection, thus enabling the monitoring of relapses and the effectiveness of therapy, for example. The aim of this study was to evaluate the TcSAT-IAM system, developed by our research group, on samples from patients with suspected Chagas disease infection. Initially, primer systems were developed for the detection of the nuclear DNA (SAT-DNA) from T. cruzi (TcSAT-IAM). The Cruzi system, predicted in the literature, and TcSAT-IAM were then evaluated in relation to their analytical sensitivity, specificity and efficiency. Afterwards, the applicability of the qPCR technique using both systems (separately) for the diagnosis of acute CD was evaluated in samples from 77 individuals exposed to the outbreak that occurred in Pernambuco-Brazil, relating the results obtained to those of the classical diagnostic methods recommended for this stage of the infection. TcSAT-IAM and Cruzi had a detection limit of 1 fg of target DNA (0,003 parasites). Thirty-eight cases were recorded, 28 by laboratory criteria and 10 by clinical and epidemiological criteria. Blood samples from 77 subjects were submitted to qPCR by both systems, reaching an agreement of 89.61% between them. After analyzes between systems and diagnostic criteria, the TcSAT-IAM showed sensitivity and specificity of 52.36% (CI 37.26-67.52) and 92.31% (CI 79.68-97.35), respectively, accuracy of 72.73% and moderate agreement. The TcSAT-IAM showed an accuracy of 72.58% and 75% in relation to parasitological and serological tests (IgM anti-T. cruzi), respectively. Therefore, quantitative PCR should be incorporated into the diagnosis of suspected acute cases of Chagas disease.

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.

Benznidazole treatment decreases IL-6 levels in Trypanosoma cruzi-infected human adipocytes differentiated from adipose tissue-derived stem cells

BACKGROUND Trypanosoma cruzi, which causes Chagas disease (CD), is a versatile haemoparasite that uses several strategies to evade the host's immune response, including adipose tissue (AT), used as a reservoir of infection. As it is an effective barrier to parasite evasion, the effectiveness of the drug recommended for treating CD, Benznidazole (BZ), may be questionable. OBJECTIVE To this end, we evaluated the parasite load and immunomodulation caused by BZ treatment in the culture of adipocytes differentiated from human adipose tissue-derived stem cells (ADSC) infected with T. cruzi. METHODS The ADSC were subjected to adipogenic differentiation. We then carried out four cultures in which we infected the differentiated AT with trypomastigote forms of the Y strain of T. cruzi and treated them with BZ. After the incubation, the infected AT was subjected to quantitative polymerase chain reaction (qPCR) to quantify the parasite load and transmission electron microscopy (TEM) to verify the infection. The supernatant was collected to measure cytokines, chemokines, and adipokines. FINDINGS We found elevated secretion of IL-6, CXCL-10/IP-10, CCL2/MCP-1, CCL5/RANTES, and leptin in infected fat cells. However, treatment with BZ promoted a decrease in IL-6. MAIN CONCLUSION Therefore, we believe that BZ has a beneficial role as it reduces inflammation in infected fat cells.

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.

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.

Aedes aegypti Strain Subjected to Long-Term Exposure to Bacillus thuringiensis svar. israelensis Larvicides Displays an Altered Transcriptional Response to Zika Virus Infection.

Bacillus thuringiensis svar. israelensis (Bti) larvicides are effective in controlling Aedes aegypti; however, the effects of long-term exposure need to be properly evaluated. We established an Ae. aegypti strain that has been treated with Bti for 30 generations (RecBti) and is still susceptible to Bti, but females exhibited increased susceptibility to Zika virus (ZIKV). This study compared the RecBti strain to a reference strain regarding: first, the relative transcription of selected immune genes in ZIKV-challenged females (F30) with increased susceptibility detected in a previous study; then, the whole transcriptomic profile using unchallenged females (F35). Among the genes compared by RT-qPCR in the ZIKV-infected and uninfected females from RecBti (F30) and the reference strain, hop, domeless, relish 1, defensin A, cecropin D, and gambicin showed a trend of repression in RecBti infected females. The transcriptome of RecBti (F35) unchallenged females, compared with a reference strain by RNA-seq, showed a similar profile and only 59 differentially expressed genes were found among 9202 genes analyzed. Our dataset showed that the long-term Bti exposure of the RecBti strain was associated with an alteration of the expression of genes potentially involved in the response to ZIKV infection in challenged females, which is an important feature found under this condition.

Molecular and biological features of Culex quinquefasciatus homozygous larvae for two cqm1 alleles that confer resistance to Lysinibacillus sphaericus larvicides.

BACKGROUND: Culex quinquefasciatus resistance to the binary toxin from Lysinibacillus sphaericus larvicides can occur because of mutations in the cqm1 gene that prevents the expression of the toxin receptor, Cqm1 α-glucosidase. In a resistant laboratory-selected colony maintained for more than 250 generations, cqm1REC and cqm1REC-2 resistance alleles were identified. The major allele initially found, cqm1REC , became minor and was replaced by cqm1REC-2 . This study aimed to investigate the features associated with homozygous larvae for each allele to understand the reasons for the allele replacement and to generate knowledge on resistance to microbial larvicides. RESULTS: Homozygous larvae for each allele were compared. Both larvae displayed the same level of resistance to the binary toxin (3500-fold); therefore, a change in phenotype was not the reason for the replacement observed. The lack of Cqm1 expression did not reduce the total specific α-glucosidase activity for homozygous cqm1REC and cqm1REC-2 larvae, which were statistically similar to the susceptible strain, using artificial or natural substrates. The expression of eight Cqm1 paralog α-glucosidases was demonstrated in resistant and susceptible larvae. Bioassays in which cqm1REC or cqm1REC-2 homozygous larvae were reared under stressful conditions showed that most adults produced were cqm1REC-2 homozygous (69%). Comparatively, in the offspring of a heterozygous sub-colony reared under optimal conditions for 20 generations, the cqm1REC allele assumed a higher frequency (0.72). CONCLUSION: Homozygous larvae for each allele exhibited a similar resistant phenotype. However, they presented specific advantages that might favor their selection and can be used in designing resistance management practices. © 2021 Society of Chemical Industry.

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.

Duplex qPCR for Leishmania species identification using lesion imprint on filter paper.

BACKGROUND: American cutaneous leishmaniasis (ACL) is caused by different Leishmania parasites, which stimulate and direct the immune response against the infection. OBJECTIVE: To evaluate the TaqMan probe technology applicability to diagnose and identifying of Leishmania spp. related to the ACL etiology. METHODOLOGY: Through the MEGA 6.0 software, performed an in silico analysis using multiple alignments of Leishmania spp. which were available on GenBank for different genomic targets. The efficiency (e), specificity and detection limit (DL) were calculated for each system, these were associated to compose a duplex-qPCR (DqPCR). The samples of blood, lesion biopsy and lesion imprint on filter paper from patients residing in states of Amazonas (AM) and Pernambuco (PE)-Brazil, (cases and controls) were used to perform the DqPCR technique. The capacity to identify the Leishmania species was determined by comparison with isoenzymes method and sequencing analysis. RESULTS: Internal Transcribed Spacer 1 (rDNA) was the target selected. Two sets of primers and probes were designed and combined: SVS for subgenus Viannia and LaS for L. (L.) amazonensis. The results were: SVSe = 93.24%, SVS DL = 50 fg/µL; LaSe = 89.3%, LaSLD = 5 fg/µL presented 100% of specificity. In total, 236 individuals participated of the present study, wherein were 101 blood samples, 33 biopsies and 147 lesion imprints. The imprint was the most sensitive sample, showing 83.06% of sensitivity, 86.96% of specificity and substantial agreement between the techniques analysis (k = 0.531; p < 0,001). Regarding the species identification, DqPCR and sequencing/isoenzymes have agreed at 100%, since the infection is caused by a single Leishmania species. CONCLUSION: The DqPCR technique was applicable in diagnosis and identification of Leishmania spp. (subgenus Viannia and L. amazonensis). Furthermore, the lesion imprint is less invasive, allowing a fewer discomfort and greater acceptance by the patients, in addition of being low cost and easy handling.

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.

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.

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.

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.

Impact of neonatal malnutrition on expression TLR-9, NF-kB and cytokines of macrophages infected in vitro with methicillin resistant Staphylococcus aureus.

Early nutritional aggressions promote epigenetic adjustments that culminate in the loss of phenotype plasticity (with permanent long-term modifications). Maternal diet and inadequate neonatal nutrition can result in fetal programming that presents susceptibility to infections in adult life. Thus, it becomes essential to verify the impacts of neonatal malnutrition (even following nutritional replacement) on the immunological response to methicillin resistant Staphylococcus aureus (MRSA) infections. Male rats were divided into two distinct groups: Nourished and Malnourished. After isolation of mononuclear cells, four systems were established: negative control, positive control and two testing systems, (MSSA and MRSA). Tests were performed to analyze expression of TLR-9, NF-kB, IL-1ß, IL-18 and IL-33. For statistical analysis, we used the Student t and ANOVA tests p < 0.05. Even after nutritional replacement, malnutrition in the neonatal period compromised the animals' weight gains p < 0.05. There was a reduction in the expression of the immunological response in the positive control, however deregulation was observed in the gene expression of MRSA-infected macrophages, with a reduction in TLR-9 expression, and overexpression in NF-kB and cytokines p < 0.05. Puppies inflicted with protein-calorie malnutrition were compromised; (long-term) body growth and immune response. In the infectious scenario, immune collapse is reflected in inflammatory response exacerbation with a likely histolytic character. Immune disabling (resulting from gene expression deregulation) causes susceptibility to infections due to ineffective recognition, intense pro-inflammatory mediation, and cell death. It is suggested that neonatal malnutrition can program susceptibility to multiresistant bacterial infections, and generally favors a triggering of more intense confrontations with fatal outcomes.

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.

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.

Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach.

INTRODUCTION:: Molecular techniques have been shown to be alternative methods for the accurate detection of infectious and parasitic diseases, such as the leishmaniases. The present study describes the optimization and evaluation of a duplex real-time quantitative PCR (qPCR) protocol developed for the simultaneous detection of Leishmania infantum DNA and sample quality control. METHODS:: After preliminary tests with the newly designed TaqMan® probes for the two targets ( L. infantum and glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene), the duplex qPCR protocol was optimized. For the evaluation of the standardized protocol, human blood samples were tested (n=68) and the results were compared to those obtained by reference diagnostic techniques. Statistical analyses included percentage agreement and the Kappa ( k ) coefficient. RESULTS:: The detection limit of L. infantum DNA reached 2x10 2 fg (corresponding to ~1 parasite) per µL of blood (ε: 93.9%). The percentage agreement obtained between the duplex VL qPCR and the reference techniques was individually obtained as follows: molecular: 88.3% ( k =0.666; 95% CI 0.437-0.894, good), and serological: 81.7% ( k =0.411; 95% CI 0.125-0.697, moderate). Between the reference techniques, the percentage agreement was 86.7% ( k =0.586; 95% CI 0.332-0.840, moderate). CONCLUSIONS:: The new duplex VL qPCR protocol indicated good potential for the accurate, fast, and reliable detection of L. infantum DNA, when applied as a complement to the classical diagnostic tools already available, especially in health or research reference centers.

Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach

Abstract INTRODUCTION: Molecular techniques have been shown to be alternative methods for the accurate detection of infectious and parasitic diseases, such as the leishmaniases. The present study describes the optimization and evaluation of a duplex real-time quantitative PCR (qPCR) protocol developed for the simultaneous detection of Leishmania infantum DNA and sample quality control. METHODS: After preliminary tests with the newly designed TaqMan® probes for the two targets ( L. infantum and glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene), the duplex qPCR protocol was optimized. For the evaluation of the standardized protocol, human blood samples were tested (n=68) and the results were compared to those obtained by reference diagnostic techniques. Statistical analyses included percentage agreement and the Kappa ( k ) coefficient. RESULTS: The detection limit of L. infantum DNA reached 2x10 2 fg (corresponding to ~1 parasite) per µL of blood (ε: 93.9%). The percentage agreement obtained between the duplex VL qPCR and the reference techniques was individually obtained as follows: molecular: 88.3% ( k =0.666; 95% CI 0.437-0.894, good), and serological: 81.7% ( k =0.411; 95% CI 0.125-0.697, moderate). Between the reference techniques, the percentage agreement was 86.7% ( k =0.586; 95% CI 0.332-0.840, moderate). CONCLUSIONS: The new duplex VL qPCR protocol indicated good potential for the accurate, fast, and reliable detection of L. infantum DNA, when applied as a complement to the classical diagnostic tools already available, especially in health or research reference centers.