Giardia lamblia-infected preschoolers present growth delays independent of the assemblage A, B or E.

BACKGROUND: Intestinal parasite Giardia can affect children's physical development mainly stunting even in asymptomatic cases. The protozoa G. lamblia is divided into assemblages A-H. However, it is still unclear whether clinical manifestations and pathogenesis may vary according to the infecting assemblage. OBJECTIVES: To investigate whether G. lamblia assemblages influence differently the physical development of preschoolers from a community of Rio de Janeiro, Brazil. METHODS: Anthropometric parameters were analysed from children attending a daycare centre and stool samples were obtained for the G. lamblia diagnosis. G. lamblia isolates from positive samples were genotyped. Data were analysed in order to verify whether there is a relationship between G. lamblia infection and the physical development of children according to the assemblage. FINDINGS: Herein we demonstrated that although eutrophic, G. lamblia-infected daycare preschoolers from a low-income community presented growth delay compared to non-infected ones. This effect was observed for the three assemblages (A, B or E) found infecting humans. MAIN CONCLUSION: G. lamblia causes growth delays on children independent of infecting assemblage (A, B or E).

DC-SIGN receptor is expressed by cells from cutaneous leishmaniasis lesions and differentially binds to Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis promastigotes.

BACKGROUND: Dendritic cells (DCs) specific intercellular adhesion molecule (ICAM)-3-grabbing non integrin receptor (DC-SIGN) binds to subgenera Leishmania promastigotes mediating its interaction with DC and neutrophils, potentially influencing the infection outcome. OBJECTIVES: In this work, we investigated whether DC-SIGN receptor is expressed in cells from cutaneous leishmaniasis (CL) lesions as well as the in vitro binding pattern of Leishmania (Viannia) braziliensis (Lb) and L. (L.) amazonensis (La) promastigotes. METHODS: DC-SIGN receptor was labeled by immunohistochemistry in cryopreserved CL tissue fragments. In vitro binding assay with CFSE-labeled Lb or La promastigotes and RAJI-transfecting cells expressing DC-SIGN (DC-SIGNPOS) or mock-transfected (DC-SIGNNEG) were monitored by flow cytometry at 2 h, 24 h and 48 h in co-culture. RESULTS: In CL lesion infiltrate, DC-SIGNPOS cells were present in the dermis and near the epidermis. Both Lb and La bind to DC-SIGNPOS cells, while binding to DC-SIGNNEG was low. La showed precocious and higher affinity to DC-SIGNhi population than to DC-SIGNlow, while Lb binding was similar in these populations. CONCLUSION: Our results demonstrate that DC-SIGN receptor is present in L. braziliensis CL lesions and interact with Lb promastigotes. Moreover, the differences in the binding pattern to Lb and La suggest DC-SIGN can influence in a difference way the intake of the parasites at the first hours after Leishmania infection. These results raise the hypothesis that DC-SIGN receptor could participate in the immunopathogenesis of American tegumentary leishmaniasis accounting for the differences in the outcome of the Leishmania spp. infection.

Biomarkers of disease severity in patients with visceral leishmaniasis co-infected with HIV.

Visceral leishmaniasis (VL) is caused by the protozoan Leishmania spp, transmitted by sand fly bites. VL is one of the deadliest tropical infection diseases, yet the coinfection with HIV virus drastically increases relapses, treatment failure and mortality. The concomitant action of these two pathogens leads to high cellular activation independently of the progression to AIDS. In addition, microbial translocation and bacterial infections are thought to contribute worsening the clinical picture. Identifying biomarkers associated with disease severity is of interest for clinical management of patients with VL-HIV/AIDS. Thus, we analyzed in the sera several markers including interleukins (IL-1ß, IL-6, IL-8, and IL-17), interferon-γ (IFN- γ), tumor necrosis factor (TNF), lipopolysaccharide (LPS), soluble CD14 (sCD14), macrophage migration inhibitory factor (MIF) and intestinal fatty acid-binding protein (IFABP). These markers were compared with disease severity in 24 patients with VL/HIV presenting different clinical outcomes. Disease severity was defined by the probability of death calculated using a score set system derived by the Kala-Cal® software. Probability of death ranged from 3.7% to 97.9%, with median of 28.8%. Five patients died (20%). At the univariate analysis, disease severity was correlated with TNF, IFN-γ and sCD14. LPS was positively correlated with sCD14 specifically in patients with low CD4+ count (CD4+ T-cell <200 cells/mL). Most importantly, the multivariate analysis including LPS, CD4+count and sCD14 showed that sCD14 was the only independent predictor for disease severity and death. Altogether, our results indicated that sCD14 is a powerful marker of pathogenicity and death for patients with VL-HIV/AIDS.

Different inoculum of Leishmania braziliensis concentrations influence immunopathogenesis and clinical evolution in the ear dermis hamster model of cutaneous leishmaniasis.

The golden hamster (Mesocricetus auratus) is commonly used as a promising model for Leishmania braziliensis infection developing skin-ulcerated lesions. However, different protocols using high concentration of parasites inoculated in the footpad result in severe clinical disease. Here, we further investigate the outcome of the site of infection and concentration of L. braziliensis parasites inoculated on the immunopathogenesis and clinical evolution. Initially, hamsters were infected in the ear dermis or hind footpad with a concentration of 1 × 105 parasites. Animals infected in the ear dermis developed a disease, with an increased parasite load that more closely resembled human cutaneous leishmaniasis lesions comparing to the group infected in the footpad. Next, we evaluated if different parasite concentrations (104 , 105 and 106 ) inoculated in the ear dermis would impact the course and clinical aspects of infection. Hamsters infected with 104 and 105 parasites developed mild lesions compared to the group infected with 106 that presented severe and persistent lesions. The parasite load varied between the different parasite concentrations. The inflammatory response was more intense when infection was initiated with 106 parasites accompanied by an increased initial expression of IL-4, IL-10 and arginase in the lymph node followed by expression of both pro-and anti-inflammatory cytokines comparing to groups infected with 104 and 105 parasites. In conclusion, the number of parasites inoculated, and the initial site of infection could influence the inflammatory response, and clinical presentation. Our results suggest that the ear dermis infection model induces a chronic disease that relates to immunopathological aspects of CL natural infection.

High levels of anti-Leishmania IgG3 and low CD4+ T cells count were associated with relapses in visceral leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) is severe and potentially fatal. Brazil is one of the countries with the greatest endemicity for the disease in the world. The reduction of CD4+ T lymphocytes, B cells activation and high levels of inflammatory cytokines (IL-6/IL-8/TNF/IL-1ß), plasma LPS, soluble CD14, anti-Leishmania IgG3 and low leptin levels are involved in the immunopathogenesis of VL, most associated with severe VL. Despite relapses occurring in about 4-5% of patients with VL not associated with HIV infection, the factors underlying relapses are little known. Our aim was to identify clinical, laboratory and immunological parameters that may be associated with recurrences in VL. METHODS: Fifteen VL patients recruited from Hospital Eduardo de Menezes (BH-MG) were grouped into relapsing (R-VL, n = 5) and non-relapsing (NR-VL, n = 10) and evaluated during active disease, immediately after treatment (post-treatment) and 6 months post-treatment (6mpt). Clinical and laboratory data obtained from medical records were correlated with CD4+ and CD8+ T cell counts and anti-Leishmania Igs and IL-6 plasma levels and compared to those parameters of ten healthy controls. RESULTS: During the active phase of VL, despite similarity in the clinical symptoms, the rates of thrombocytopenia, elevated transaminases (AST and ALT) and hyperbilirubinemia were higher in the NR-VL group compared to R-VL (p < 0.05), a profile reversed during the post-treatment phase. All patients had low CD4+ T counts in active phase, however, NR-VL patients had a higher gain of this cell type than R-VL in the post-treatment (p < 0.05). There was a significant reduction in IgG3 levels during the follow-up in the NR-VL group compared to the R-VL, especially at 6mpt (p < 0.05). In addition, IgG3 levels were negatively correlated with CD4+ T counts in the R-VL group (r = - 0.52). Elevated levels of IL-6 were observed in active VL and correlated with clinical markers of severity. CONCLUSIONS: During active phase of VL, the NR-VL patients presented more severe laboratorial abnormalities compared to R-VL, probably because the latter had already received previous treatment. On the other hand, R-VL exhibited greater impairment of immune reconstitution and a high degree of B lymphocyte activation, which must be a factor that favored relapses.

Epidemiology of Giardia duodenalis assemblages in Brazil: there is still a long way to go.

Giardia duodenalis infection is distributed worldwide and can achieve prevalence around 60%, especially in developing countries. This protozoan is divided into eight assemblages, in which A and B have high zoonotic potential, whereas C to H are host-specific. This scenario is changing as molecular studies progress, highlighting that knowledge on host-specificity still has a long way to go. Understanding the players involved in transmission routes enables rational designs of control strategies. Considering the high prevalence of giardiasis, this review aims to gather together the data on available studies on the distribution of G. duodenalis assemblages in Brazil until September 2020.

CD49d Is Upregulated in Circulating T Lymphocytes from HTLV-1-Infected Patients.

INTRODUCTION: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic progressive myelopathy associated with an inflammation of the central nervous system (CNS), being characterized by perivascular infiltration of inflammatory cells. HTLV-1-infected cells have the capacity to migrate through endothelial layers by enhancing adhesion receptor expression and corresponding ligands. T cells interact with the extracellular matrix via integrin receptors and these interactions affect both cell migration and proliferation. The importance of these interactions in retrovirus-induced diseases, however, remains less clear. METHODS: Herein we studied the expression of 3 integrin alpha chains (CD49d, CD49e, and CD49f) on the membrane of T-cell subsets in patients infected by HTLV-1, both HAM/TSP patients and oligo/asymptomatic subjects who were asymptomatic or presented slight manifestations related to the virus infection. RESULTS: We observed higher peripheral blood frequency of CD49dhiCD4+ and CD49dhiCD8+ T cells in HTLV-1-infected patients. CONCLUSION: Our findings suggest that the increased expression of adhesion molecules, such as CD49d on T lymphocytes from HTLV-1-infected patients may contribute to the pathogenesis of the disease, in both oligo/asymptomatic and HAM/TSP-infected subjects. Accordingly, it is conceivable that there is a potential use of CD49d as target for a therapeutic approach aiming at blocking migration of activated T cells from HTLV-1-infected patients into the CNS, thus avoiding the progression to HAM/TSP.

In vitro-induction of metronidazole-resistant Giardia duodenalis is not associated with nucleotide alterations in the genes involved in pro-drug activation.

Giardiasis is an infectious disease caused by Giardia duodenalis. The pro-drug metronidazole (MTZ) is the first-line treatment for giardiasis. Parasite's proteins as pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxin (Fd), nitroreductase-1 (NR-1) and thioredoxin reductase (TrxR) participate in MTZ activation. Here, we showed Giardia trophozoites long-term exposed to MTZ presented higher IC50 than controls, showing the drug influenced the parasite survival. That reduction in MTZ's susceptibility does not seem to be related to mutations in the genes pfor, fd, nr-1 or trxr. It points that different mechanism as alterations in other metabolic pathways can account for Giardia resistance to MTZ therapy.

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents.

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

Apoptosis and frequency of total and effector CD8+ T lymphocytes from cutaneous leishmaniasis patients during antimonial therapy.

BACKGROUND: Leishmaniasis is an important parasitic disease affecting millions worldwide. Human cutaneous leishmaniasis (CL) is endemic in Rio de Janeiro, Brazil, where is caused by Leishmania braziliensis. The adaptive immune response is accountable for the healing of CL and despite of key role of CD8+ T cells in this immune response little is known about the CD8+ T lymphocytes frequencies, apoptosis and antigen-responsive CD8+ T lymphocytes of CL patients during antimonial therapy. METHODS: Using flow cytometry, we examined total and effector CD8+ T cells from CL patients before (PBT), during (PDT) and after (PAT) treatment for apoptosis and frequencies upon isolation and after in vitro L. braziliensis antigens (LbAg)-stimulation culture. Besides, a correlation study between immunological findings and lesion size was done. RESULTS: PDT showed lower frequencies of total CD8+ T lymphocytes and higher levels of apoptosis of these cells, which were also observed following LbAg-stimulation culture. Regarding effector CD8+ T cells, high frequencies were observed in PDT, while lower frequencies were observed in PAT. Interestingly, PDT showed higher frequencies of apoptotic-effector CD8+ T lymphocytes. Similar results were seen after in vitro antigenic-stimulation assays. Correlation analysis showed that the greater the size of lesion, the smaller the frequency of effector CD8+ T lymphocytes in PDT and PAT, as well as a positive correlation between apoptotic-effector CD8+ T cells frequency and lesion size of PDT. CONCLUSIONS: Changes in effector CD8+ T-lymphocyte frequencies, during and after treatment, seem to represent a critical stage to generate an efficient immune response and suggest that these cells would be evolved in the triggering or in the resolution of lesion, under the influence of therapy. This hypothesis opens new perspectives to clarify controversial statements about the protective or deleterious role of CD8+ T cells in the cure or aggravation of CL and the new approach of evaluating patients during treatment proved to be of utmost importance for understanding the immune response in the healing process of human CL.

Leishmania (Viannia) naiffi: rare enough to be neglected?

In the Brazilian Amazon, American tegumentary leishmaniasis (ATL) is endemic and presents a wide spectrum of clinical manifestations due, in part, to the circulation of at least seven Leishmania species. Few reports of Leishmania (Viannia) naiffi infection suggest that its occurrence is uncommon and the reported cases present a benign clinical course and a good response to treatment. This study aimed to strengthen the clinical and epidemiological importance of L. (V.) naiffi in the Amazon Region (Manaus, state of Amazonas) and to report therapeutic failure in patients infected with this species. Thirty Leishmania spp samples isolated from cutaneous lesions were characterised by multilocus enzyme electrophoresis. As expected, the most common species was Leishmania (V.) guyanensis (20 cases). However, a relevant number of L. (V.) naiffi patients (8 cases) was observed, thus demonstrating that this species is not uncommon in the region. No patient infected with L. (V.) naiffi evolved to spontaneous cure until the start of treatment, which indicated that this species may not have a self-limiting nature. In addition, two of the patients experienced a poor response to antimonial or pentamidine therapy. Thus, either ATL cases due to L. (V.) naiffi cannot be as uncommon as previously thought or this species is currently expanding in this region.

T-cell receptor Vß repertoire of CD8+ T-lymphocyte subpopulations in cutaneous leishmaniasis patients from the state of Rio de Janeiro, Brazil.

In human cutaneous leishmaniasis (CL), the immune response is mainly mediated by T-cells. The role of CD8+ T-lymphocytes, which are related to healing or deleterious functions, in affecting clinical outcome is controversial. The aim of this study was to evaluate T-cell receptor diversity in late-differentiated effector (LDE) and memory CD8+ T-cell subsets in order to create a profile of specific clones engaged in deleterious or protective CL immune responses. Healthy subjects, patients with active disease (PAD) and clinically cured patients were enrolled in the study. Total CD8+ T-lymphocytes showed a disturbance in the expression of the Vß2, Vß9, Vß13.2, Vß18 and Vß23 families. The analyses of CD8+T-lymphocyte subsets showed high frequencies of LDE CD8+T-lymphocytes expressing Vß12 and Vß22 in PAD, as well as effector-memory CD8+ T-cells expressing Vß22. We also observed low frequencies of effector and central-memory CD8+ T-cells expressing Vß2 in PAD, which correlated with a greater lesion size. Particular Vß expansions point to CD8+ T-cell clones that are selected during CL immune responses, suggesting that CD8+ T-lymphocytes expressing Vß12 or Vß22 are involved in a LDE response and that Vß2 contractions in memory CD8+T-cells are associated with larger lesions.

Comparative evaluation of lesion development, tissue damage, and cytokine expression in golden hamsters (Mesocricetus auratus) infected by inocula with different Leishmania (Viannia) braziliensis concentrations.

The golden hamster (Mesocricetus auratus) is a susceptible model to Leishmania (Viannia) spp.; however, available studies employ different infection protocols, which account for clinical and pathological presentation differences. Herein, L. (V.) braziliensis preparations were standardized to contain 10(4), 10(5), or 10(6) parasites to determine an optimal inoculum that ensured cutaneous lesions without causing a disseminated infection in hamsters. Lesion development was followed for 105 days by size measurements, and skin, draining lymph node, spleen, and sera were investigated to check parasite load, spleen visceralization, cytokine expression, histopathological changes, and anti-Leishmania IgG levels. The lesion emergence time was inversely proportional to the parasite concentration in the inocula. Animals infected by 10(4) parasites presented nodular lesions, while those infected with 10(6) parasites often exhibited ulcerated lesions. The differences in the final lesion sizes were observed between 10(4) and 10(5) inocula or 10(4) and 10(6) inocula. High IFNG expression, anti-Leishmania IgG levels, and parasite load occurred independently of the inoculum used. A mild inflammatory skin involvement was observed in animals infected with 10(4) parasites, while extensive tissue damage and parasite spleen visceralization occurred with 10(5) and 10(6) parasites. These results indicate that inocula with different concentrations of parasites generate differences in the time of lesion emergence, clinical presentation, and systemic commitment, despite high and similar IFNG expression and parasite load. This suggests that a modulation in the immune response to different parasite numbers occurs in an early phase of the infection, which could dictate the establishment and magnitude of the chronic phase of the disease.

A link between circulating immune complexes and acute kidney injury in human visceral leishmaniasis.

Visceral leishmaniasis (VL) is an infectious disease caused by Leishmania infantum. Clinically, VL evolves with systemic impairment, immunosuppression and hyperactivation with hypergammaglobulinemia. Although renal involvement has been recognized, a dearth of understanding about the underlying mechanisms driving acute kidney injury (AKI) in VL remains. We aimed to evaluate the involvement of immunoglobulins (Igs) and immune complexes (CIC) in the occurrence of AKI in VL patients. Fourteen VL patients were evaluated between early treatment and 12 months post-treatment (mpt). Anti-Leishmania Igs, CIC, cystatin C, C3a and C5a were assessed and correlated with AKI markers. Interestingly, high levels of CIC were observed in VL patients up to 6 mpt. Concomitantly, twelve patients met the criteria for AKI, while high levels of cystatin C were observed up to 6 mpt. Plasmatic cystatin C was positively correlated with CIC and Igs. Moreover, C5a was correlated with cystatin C, CIC and Igs. We did not identify any correlation between amphotericin B use and kidney function markers in VL patients, although this association needs to be further explored in subsequent studies. Our data reinforce the presence of an important renal function impairment during VL, suggesting the involvement of Igs, CIC, and C5a in this clinical condition.

Candidate gene case-control and functional study shows macrophage inhibitory factor (MIF) polymorphism is associated with cutaneous leishmaniasis.

American tegumentary leishmaniasis (ATL) is an infectious disease caused mostly by Leishmania(Viannia)braziliensis in Southeast Brazil. The clinical manifestations are vast, ranging from asymptomatic to severe mucosal leishmaniasis (ML). It has been suggested that variation of the pathogen does not fully explain the response spectrum and the variability of clinical manifestations. Previous data have shown that host genetics also play a role in disease outcome. Herein, we have tested the association of TNF, IL10, IL12 and MIF single nucleotide polymorphisms (SNPs) using a case-control study design including 110 cutaneous leishmaniasis (CL) patients and 682 healthy subjects. The genotype-phenotype correlation was also assessed using leishmania antigens to stimulate peripheral blood mononuclear cells obtained from cured CL patients. Results demonstrated that the MIF -173C allele is associated with leishmaniasis outcome and also with lower levels of MIF in culture supernatants. Also, the TNF -308AA genotype was statistically increased among leishmaniasis patients. The results showed here suggest that the lower levels of MIF produced by MIF -173C carriers could influence the host-Leishmania interaction, favoring infection and disease progression. On the other hand, high TNF levels can contribute to tissue damage, consequently leading to skin lesions.

Golden hamster (Mesocricetus auratus) as an experimental model for Leishmania (Viannia) braziliensis infection.

The lack of an adequate model for Leishmania (Viannia) braziliensis infection is a limiting factor for studying American tegumentary leishmaniasis (ATL). The golden hamster (Mesocricetus auratus) is a promising model because besides being highly susceptible to dermotropic Leishmania infection, the lesions are very similar to cutaneous leishmaniasis (CL) in humans. However, different Leishmania isolates or species and/or protocols have resulted in different outcomes, whereas no study has evaluated the reproducibility of L. braziliensis infection in this model. The natural history of L. braziliensis infection in 34 hamsters was evaluated by using a single parasite isolate in 8 independent experiments under similar experimental conditions. Clinical, histological and immunological analyses were performed. The hamsters presented skin ulcers similar to those observed in ATL. The intra-experiment lesion increment tended to show an intermediary variance. Histological analysis of infected skins showed granulomatous reaction, scarce amastigotes, and Schaumann's bodies. Blood lymphocytes proliferated in response to leishmanial antigens. The severity of the infection was positively correlated to spleen weight, and the titres of anti-Leishmania IgG antibodies. Our findings indicate that the hamster is an appropriate model for immunopathogenesis studies of CL caused by L. braziliensis, supporting its use in clinical, vaccine and chemotherapy experimental protocols.

Enterobius vermicularis in Brazil: An integrative review.

Enterobius vermicularis, an intestinal helminth, is transmitted through the ingestion of eggs found in food, water, dust, or other fomites, including infected individuals. This review aimed to examine the frequency and distribution of E. vermicularis infections in Brazil between 1991 and 2022. The conducted bibliographic survey revealed that the frequency of E. vermicularis infections in Brazil ranged from 0.1 to 26.1%, depending on factors such as population ethnicity, individual age group, geographic area, time frame, and diagnostic method. However, these findings were based on a limited number of publications, suggesting that the actual prevalence rates of E. vermicularis infection may still be unknown and potentially underestimated.

Identification of Immunodominant Proteins of the Leishmania (Viannia) naiffi SubProteome as Pan-Specific Vaccine Targets against Leishmaniasis.

Leishmaniasis is a wide-spectrum disease caused by parasites from Leishmania genus. A well-modulated immune response that is established after the long-lasting clinical cure of leishmaniasis can represent a standard requirement for a vaccine. Previous studies demonstrated that Leishmania (Viannia) naiffi causes benign disease and its antigens induce well-modulated immune responses in vitro. In this work we aimed to identify the immunodominant proteins present in the soluble extract of L. naiffi (sLnAg) as candidates for composing a pan-specific anti-leishmaniasis vaccine. After immunoblotting using cured patients of cutaneous leishmaniasis sera and proteomics approaches, we identified a group of antigenic proteins from the sLnAg. In silico analyses allowed us to select mildly similar proteins to the host; in addition, we evaluated the binding potential and degree of promiscuity of the protein epitopes to HLA molecules and to B-cell receptors. We selected 24 immunodominant proteins from a sub-proteome with 328 proteins. Homology analysis allowed the identification of 13 proteins with the most orthologues among seven Leishmania species. This work demonstrated the potential of these proteins as promising vaccine targets capable of inducing humoral and cellular pan-specific immune responses in humans, which may in the future contribute to the control of leishmaniasis.

Low doses of 3-phenyl-lawsone or meglumine antimoniate delivery by tattooing route are successful in reducing parasite load in cutaneous lesions of Leishmania (Viannia) braziliensis-infected hamsters.

Current therapeutic ways adopted for the treatment of leishmaniasis are toxic and expensive including parasite resistance is a growing problem. Given this scenario, it is urgent to explore treatment alternatives for leishmaniasis. The aim of this study was to evaluate the effect of 3-phenyl-lawsone (3-PL) naphthoquinone on Leishmania (Viannia) braziliensis infection, both in vitro and in vivo, using two local routes of administration: subcutaneous (higher dose) and tattoo (lower dose). In vitro 3-PL showed low toxicity for macrophages (CC50 >3200 µM/48h) and activity against intracellular amastigotes (IC50 = 193 ± 19 µM/48h) and promastigotes (IC50 = 116 ± 26 µM/72h), in which induced increased ROS generation. Additionally, 3-PL up-regulated the production of cytokines such as tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP-1), interleukin-6 (IL-6) and IL-10 in infected macrophages. However, the anti-amastigote action was independent of nitric oxide production. Treatment of hamsters infected with L. (V.) braziliensis from one week after infection with 3-PL by subcutaneous (25 µg/Kg) or tattooing (2.5 µg/Kg) route, during 3 weeks (3 times/week) or 2 weeks (2 times/week) significantly decreased the parasite load (p<0.001) in the lesion. The reduction of parasite load by 3-PL treatment was comparable to reference drug meglumine antimoniate administered by the same routes (subcutaneous 1mg/Kg and tattoo 0.1mg/Kg). In addition, treatment started from five weeks after infection with 3-PL per tattoo also decreased the parasite load. These results show the anti-leishmanial effect of 3-PL against L. (V.) braziliensis and its efficacy by subcutaneous (higher dose) and tattoo (lower dose) routes. In addition, this study shows that drug delivery by tattooing the lesion allows the use of lower doses than the conventional subcutaneous route, which may support the development of a new therapeutic strategy that can be adopted for leishmaniasis.

Recombinant protein KR95 as an alternative for serological diagnosis of human visceral leishmaniasis in the Americas.

In the Americas, visceral leishmaniasis (VL) is caused by the protozoan Leishmania infantum, leading to death if not promptly diagnosed and treated. In Brazil, the disease reaches all regions, and in 2020, 1,933 VL cases were reported with 9.5% lethality. Thus, an accurate diagnosis is essential to provide the appropriate treatment. Serological VL diagnosis is based mainly on immunochromatographic tests, but their performance may vary by location, and evaluation of diagnostic alternatives is necessary. In this study, we aimed to evaluate the performance of ELISA with the scantily studied recombinant antigens, K18 and KR95, comparing their performance with the already known rK28 and rK39. Sera from parasitologically confirmed symptomatic VL patients (n = 90) and healthy endemic controls (n = 90) were submitted to ELISA with rK18 and rKR95. Sensitivity (95% CI) was, respectively, 83.3% (74.2-89.7) and 95.6% (88.8-98.6), and specificity (95% CI) was 93.3% (85.9-97.2) and 97.8% (91.8-99.9). For validation of ELISA with the recombinant antigens, we included samples from 122 VL patients and 83 healthy controls collected in three regions in Brazil (Northeast, Southeast, and Midwest). When comparing the results obtained with the VL patients' samples, significantly lower sensitivity was obtained by rK18-ELISA (88.5%, 95% CI: 81.5-93.2) compared with rK28-ELISA (95.9%, 95% CI: 90.5-98.5), but the sensitivity was similar comparing rKR95-ELISA (95.1%, 95% CI: 89.5-98.0), rK28-ELISA (95.9%, 95% CI: 90.5-98.5), and rK39-ELISA (94.3%, 95% CI: 88.4-97.4). Analyzing the specificity, it was lowest with rK18-ELISA (62.7%, 95% CI: 51.9-72.3) with 83 healthy control samples. Conversely, higher and similar specificity was obtained by rKR95-ELISA (96.4%, 95% CI: 89.5-99.2), rK28-ELISA (95.2%, 95% CI: 87.9-98.5), and rK39-ELISA (95.2%, 95% CI: 87.9-98.5). There was no difference in sensitivity and specificity across localities. Cross-reactivity assessment, performed with sera of patients diagnosed with inflammatory disorders and other infectious diseases, was 34.2% with rK18-ELISA and 3.1% with rKR95-ELISA. Based on these data, we suggest using recombinant antigen KR95 in serological assays for VL diagnosis.