BACKGROUND Conventional microscopic counting is a widely utilised method for evaluating the trypanocidal effects of drugs on intracellular amastigotes. This is a low-cost approach, but it is time-consuming and reliant on the expertise of the microscopist. So, there is a pressing need for developing technologies to enhance the efficiency of low-cost anti-Trypanosoma cruzi drug screening. OBJECTIVES In our laboratory, we aimed to expedite the screening of anti-T. cruzi drugs by implementing a fluorescent method that correlates emitted fluorescence from green fluorescent protein (GFP)-expressing T. cruzi (Tc-GFP) with cellular viability. METHODS Epimastigotes (Y strain) were transfected with the pROCKGFPNeo plasmid, resulting in robust and sustained GFP expression across epimastigotes, trypomastigotes, and intracellular amastigotes. Tc-GFP epimastigotes and intracellular amastigotes were exposed to a serial dilution of benznidazole (Bz). Cell viability was assessed through a combination of microscopic counting, MTT, and fluorimetry. FINDINGS The fluorescence data indicated an underestimation of the activity of Bz against epimastigotes (IC50 75 µM x 14 µM). Conversely, for intracellular GFP-amastigotes, both fluorimetry and microscopy yielded identical IC50 values. Factors influencing the fluorimetry approach are discussed. MAIN CONCLUSIONS Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time-consuming microscopic counting of intracellular amastigotes. lowcost low cost timeconsuming time consuming microscopist So antiTrypanosoma anti Trypanosoma laboratory antiT. antiT T anti-T GFPexpressing expressing TcGFP Tc (Tc-GFP Y strain plasmid trypomastigotes Bz. . (Bz) MTT IC (IC5 7 1 µM. µM) Conversely GFPamastigotes, GFPamastigotes amastigotes, GFP-amastigotes IC5 values discussed (Bz (IC

BACKGROUND The availability of genes and protein sequences for parasites has provided valuable information for drug target identification and vaccine development. One such parasite is Bartonella quintana, a Gram-negative, intracellular pathogen that causes bartonellosis in mammalian hosts. OBJECTIVE Despite progress in understanding its pathogenesis, limited knowledge exists about the virulence factors and regulatory mechanisms specific to B. quintana. METHODS AND FINDINGS To explore these aspects, we have adopted a subtractive proteomics approach to analyse the proteome of B. quintana. By subtractive proteins between the host and parasite proteome, a set of proteins that are likely unique to the parasite but absent in the host were identified. This analysis revealed that out of the 1197 protein sequences of the parasite, 660 proteins are non-homologous to the human host. Further analysis using the Database of Essential Genes predicted 159 essential proteins, with 28 of these being unique to the pathogen and predicted as potential putative targets. Subcellular localisation of the predicted targets revealed 13 cytoplasmic, eight membranes, one periplasmic, and multiple location proteins. The three-dimensional structure and B cell epitopes of the six membrane antigenic protein were predicted. Four B cell epitopes in KdtA and mraY proteins, three in lpxB and BQ09550, whereas the ftsl and yidC proteins were located with eleven and six B cell epitopes, respectively. MAINS CONCLUSIONS This insight prioritises such proteins as novel putative targets for further investigations on their potential as drug and vaccine candidates. development quintana Gramnegative, Gramnegative Gram negative, negative Gram-negative hosts pathogenesis aspects identified 119 66 nonhomologous non homologous 15 2 1 cytoplasmic membranes periplasmic threedimensional dimensional BQ09550 BQ respectively candidates 11 6 BQ0955 BQ095 BQ09 BQ0

BACKGROUND Leprosy is a highly neglected disease that is considered a serious public health problem in many countries. This illness is characterised by a variety of clinical and histopathological manifestations that are related to the patient immune response. OBJECTIVES This work aimed evaluate the profile of circulating immune mediators in the plasma from patients classified clinically as paucibacillary (PB), multibacillary (MB), households contacts (HHC), type1 leprosy reaction (T1R), type2 leprosy reaction (T2R) and control individuals without medical history of leprosy (CTL). METHODS To assessment of the plasma immune mediators was used multiplex microbeads immunoassay “Luminex”. FINDINGS The results showed that patients (PB) had a regulatory-biased profile, while MB revealed a pro-inflammatory trend of highly expressed biomarkers. HHC display conspicuously increased levels in the plasma of the chemokines (CCL2, CCL3, CCL4, CCL5 and CXCL8), pro-inflammatory cytokines (IFN-γ,TNF and IL-1β), modulating cytokines (IL-9 and IL-1Ra) and growth factors (PDGF, G-CSF and IL-2). Interestingly, HHC displayed superior production of IFN-γ as compared to other leprosy groups, indicating a putative protective role for this cytokine during chronic Mycobacterium leprae exposure. MAIN CONCLUSION Further investigations are currently underway to elucidate the potential of these mediators as biomarkers applicable to the diagnosis/prognosis of leprosy and also T1R and T2R leprosy reactions. countries response PB, PB , MB, (MB) HHC, (HHC) type T1R, TR T R (T1R) (T2R CTL. CTL . (CTL) Luminex. Luminex “Luminex” (PB regulatorybiased regulatory biased proinflammatory pro inflammatory CCL2, CCL2 CCL (CCL2 CCL3 CCL4 CXCL8, CXCL8 CXCL CXCL8) IFNγ,TNF IFNγTNF IFN γ,TNF γ TNF IL1β, IL1β ILβ IL 1β β IL-1β) IL9 9 (IL- IL1Ra ILRa 1Ra Ra IL-1Ra PDGF, PDGF (PDGF GCSF G CSF IL2. IL2 2 IL-2) Interestingly IFNγ groups exposure diagnosisprognosis diagnosis prognosis reactions (MB (HHC (T1R (CTL “Luminex (CCL γTNF IL-1β (IL IL-2 IL-

BACKGROUND Leishmaniases encompass a spectrum of neglected diseases caused by parasites of the genus Leishmania, grouped in two forms: tegumentary and visceral leishmaniasis. OBJECTIVES In this study, we propose Friend Virus B NIH Jackson (FVB/NJ) mouse strain as a new experimental model of infection with Leishmania (Leishmania) amazonensis, the second most prevalent agent of tegumentary leishmaniasis in Brazil. METHODS AND FINDINGS We performed in vitro infections of FVB/NJ macrophages and compared them with BALB/c macrophages, showing that BALB/c cells have higher infection percentages and a higher number of amastigotes/cell. Phagocytosis assays indicated that BALB/c and FVB/NJ macrophages have similar capacity to uptake parasites after 5 min incubations. We also investigated promastigotes’ resistance to sera from FVB/NJ and BALB/c and observed no difference between the two sera, even though FVB/NJ has a deficiency in complement components. Finally, we subcutaneously infected FVB/NJ and BALB/c mice with 2 × 106 parasites expressing luciferase. Analysis of lesion development for 12 weeks showed that FVB/NJ and BALB/c mice have similar lesion profiles and parasite burdens. MAIN CONCLUSIONS This work characterises for the first time the FVB/NJ mouse as a new model for tegumentary leishmaniasis caused by Leishmania (L.) amazonensis. forms study FVBNJ FVB NJ (FVB/NJ (Leishmania amazonensis Brazil BALBc BALB c amastigotescell amastigotes cell amastigotes/cell incubations promastigotes components Finally 10 luciferase 1 burdens L. L (L. (L

BACKGROUND In Brazil, transmission of visceral and cutaneous leishmaniasis has expanded geographically over the last decades, with both clinical forms occurring simultaneously in the same area. OBJECTIVES This study characterised the clinical, spatial, and temporal distribution, and performed entomological surveillance and natural infection analysis of a leishmaniasis-endemic area. METHODS In order to characterise the risk of leishmaniasis transmission in Altos, Piauí, we described the clinical and socio-demographic variables and the spatial and temporal distribution of cases of American visceral leishmaniasis (AVL) and American cutaneous leishmaniasis (ACL) cases and identified potential phlebotomine vectors. FINDINGS The urban area concentrated almost 54% of ACL and 86.8% of AVL cases. The temporal and spatial distribution of AVL and ACL cases in Altos show a reduction in the number of risk areas, but the presence of permanent disease transmission foci is observed especially in the urban area. 3,808 phlebotomine specimens were captured, with Lutzomyia longipalpis as the most frequent species (98.45%). Of the 35 females assessed for natural infection, one specimen of Lu. longipalpis tested positive for the presence of Leishmania infantum and Leishmania braziliensis DNA. MAIN CONCLUSION Our results indicate the presence of risk areas for ACL and AVL in the municipality of Altos and highlight the importance of entomological surveillance to further understand a possible role of Lu. longipalpis in ACL transmission. Brazil decades leishmaniasisendemic endemic Piauí sociodemographic socio demographic (AVL (ACL vectors 54 868 86 8 86.8 3808 3 808 3,80 captured 98.45%. 9845 98.45% . 98 45 (98.45%) Lu DNA 5 86. 380 80 3,8 984 98.45 9 4 (98.45% 38 3, 98.4 (98.45 98. (98.4 (98. (98 (9 (

Twenty-five years have passed since the initial observation of endemic zoonotic sporotrichosis in Rio de Janeiro, Brazil. Since then, this disease has spread throughout South America. Accompanying the emergence of this mycosis, some progress has been made, including the expansion of a research network in this field and higher visibility of sporotrichosis within government authorities and funding agencies. However, there are still some challenges to curbing the expansion of this disease in the coming years. These include the development of rapid and accurate diagnostic tests, new antifungal drugs, particularly for the treatment of extracutaneous manifestations of sporotrichosis, and more comprehensive care for cats with sporotrichosis. Including these actions in the sporotrichosis research agenda is required so as to change the development of this disease in the years to come. Twentyfive Twenty five Janeiro Brazil then America mycosis made agencies However tests drugs come

BACKGROUND The incidence of visceral leishmaniasis (VL) has increased in the Southern region of Brazil in recent years, especially in the State of Paraná. New species have been suggested with potential to act as vector in VL endemic areas. OBJECTIVES Identify the Leishmania species in sand fly specimens collected from 2016 to 2018 in the municipality of Itaperuçu, Vale do Ribeira, Paraná, Brazil. METHODS Light traps were used for collections and for the analysis of sand fly were used the multiplex polymerase chain reaction (PCR) methodology and subsequent sequencing. FINDINGS Among the collected specimens, 88.62% were attributed to the species Nyssomyia neivai, which were grouped into 176 pools. Three positive pools were detected: two with Leishmania (Viannia) braziliensis and one with L. (Leishmania) infantum. The positivity rate for the parasite was 0.25% based on the presence of at least one infected insect in the pool. MAIN CONCLUSIONS The detection of L. infantum in Ny. neivai draws attention due to its abundance and anthropophily in the State of Paraná. Moreover, this finding is considered as an alert and suggests that the vector competence of Ny. neivai and the criteria for its incrimination should be carried out, given its wide distribution in southern of Brazil. (VL years Paraná areas 201 Itaperuçu Ribeira PCR (PCR sequencing 8862 88 62 88.62 17 detected Viannia (Viannia L (Leishmania 025 0 25 0.25 pool Ny Moreover out 20 886 8 6 88.6 1 02 2 0.2 88. 0.

BACKGROUND Malaria is an infectious disease caused by protozoan parasites belonging to the genus Plasmodium. Human-to-human transmission depends on a mosquito vector; thus, the interruption of parasite transmission from humans to mosquitoes is an important approach in the fight against malaria. The parasite stages infectious to mosquitoes are the gametocytes, sexual stages that are ingested by the vector during a blood meal and transform into male and female gametes in the midgut. Immunity against sexual stage antigens expressed by gametocytes, gametes, and the zygote formed after fertilisation can interrupt the parasite sexual cycle in the mosquito. This transmission blocking immunity is mediated by specific antibodies ingested during the mosquito blood feed, inhibiting the parasite development in the midgut. Merozoite thrombospondin related anonymous protein (MTRAP) is a merozoite and gametocyte surface protein essential for gamete egress from erythrocytes and for parasite transmission to mosquitoes. OBJECTIVES Here, we evaluated the potential of the P. berghei MTRAP to elicit antibodies with the ability to inhibit gamete fertilisation in vitro. METHODS We expressed a soluble recombinant PbMTRAP and used it to immunise BALB/c mice. The transmission blocking activity of the anti-rPbMTRAP antibodies was tested through in vivo challenge experiments followed by in vitro conversion assays. FINDINGS Immunisations with the rPbMTRAP induced a strong antibody response and the antibodies recognised the native protein by Western Blot and IFA. Anti-rPbMTRAP present in the blood stream of immunised mice partially inhibited gamete conversion into ookinetes. CONCLUSION Our results indicate that antibodies to PbMTRAP may reduce but are not sufficient to completely block transmission. Plasmodium Humantohuman Human human thus malaria gametocytes midgut feed (MTRAP Here P BALBc BALB c antirPbMTRAP anti assays IFA AntirPbMTRAP Anti ookinetes

BACKGROUND American tegumentary leishmaniasis (ATL) is an endemic neglected tropical disease (NTD), its conventional treatment is toxic, slow, and invasive. Rapid diagnosis is crucial for the clinical management of suspected patients, so the development and use of low-cost, miniaturised and portable devices could be the key. OBJECTIVES This work aimed to develop a simple paper-based electrochemical platform for the serological detection of ATL. METHODS Platform was fabricated in Whatman N°1 paper, contains a hydrophobic zone generated by wax printing, two pencil graphite electrodes, and uses specific crude extracts (CA) antigens for ATL immuno-determination. The platform performance was analysed by measuring the relative impedance change for different antigen-antibody combinations. Then, 10 serum human samples previously diagnosed by the gold standard (five positive ATL cases and five non-ATL cases) were evaluated. FINDINGS The platform presented a linear response for the charge transfer resistance (ΔRct) and the interface reactance (ΔXc). Also, optimal working conditions were established (1/60 serum dilution and 180 µg/mL CA concentration). Then, the platform permits to distinguish between ATL and non-ATL (p < 0.05) human serum samples. MAIN CONCLUSIONS Our platform could allow the diagnosis, management, and monitoring of leishmaniasis while being an extremely simple and environmentally friendly technology. (ATL NTD, NTD , (NTD) toxic slow invasive patients lowcost, lowcost low cost, cost low-cost key paperbased paper based N1 N 1 N° printing electrodes (CA immunodetermination. immunodetermination immuno determination. determination immuno-determination antigenantibody antigen antibody combinations Then nonATL non evaluated ΔRct (ΔRct ΔXc. ΔXc . (ΔXc) Also 1/60 160 60 (1/6 18 µgmL µg mL concentration. concentration concentration) p 0.05 005 0 05 technology (NTD (ΔXc 1/6 16 6 (1/ 0.0 00 1/ (1 0. (

The incorporation of different molecules by eukaryotic cells occurs through endocytosis, which is critical to the cell’s survival and ability to reproduce. Although this process has been studied in greater detail in mammalian and yeast cells, several groups working with pathogenic protists have made relevant contributions. This review analysed the most relevant data on the endocytic process in anaerobic protists (Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, and Tritrichomonas foetus). Many protozoa can exert endocytic activity across their entire surface and do so with great intensity, as with E. histolytica. The available data on the endocytic pathway and the participation of PI-3 kinase, Rab, and Rho molecular complexes is reviewed from a historical perspective. endocytosis cell s reproduce contributions Entamoeba histolytica intestinalis vaginalis foetus. foetus . foetus) intensity E PI3 PI 3 PI- kinase Rab perspective

BACKGROUND Giant viruses have brought new insights into different aspects of virus-cell interactions. The resulting cytopathic effects from these interactions are one of the main aspects of infection assessment in a laboratory routine, mainly reflecting on the morphological features of an infected cell. OBJECTIVES In this work, we follow the entire kinetics of the cytopathic effect in cells infected by viruses of the Mimiviridae family, spatiotemporally quantifying typical features such as cell roundness, loss of motility, decrease in cell area and cell lysis. METHODS Infections by Acanthamoeba polyphaga mimivirus (APMV), Tupanvirus (TPV) and M4 were carried out at multiplicity of infection (MOI) 1 and MOI 10 in Acanthamoeba castellanii. Monitoring of infections was carried out using time lapse microscopy for up to 72 hours. The images were analyzed using ImageJ software. FINDINGS The data obtained indicate that APMV is the slowest virus in inducing the cytopathic effects of rounding, decrease in cell area, mobility and cell lysis. However, it is the only virus whose MOI increase accelerates the lysis process of infected cells. In turn, TPV and M4 rapidly induce morphological and behavioral changes. MAIN CONCLUSIONS Our results indicate that mimiviruses induce different temporal responses within the host cell and that it is possible to use these kinetic data to facilitate the understanding of infection by these viruses. viruscell routine work family roundness motility APMV, , (APMV) (TPV M (MOI castellanii 7 hours software rounding However turn changes (APMV

Chagas disease is a tropical neglected disease that affects millions of people worldwide, still demanding a more effective and safer therapy, especially in its chronic phase which lacks a treatment that promotes substantial parasitological cure. The technical note of Romanha and collaborators published in 2010 aimed establish a guideline with the set of minimum criteria and decision gates for the development of new agents against Trypanosoma cruzi with the focus on developing new antichagasic drugs. In this sense, the present review aims to update this technical note, bringing the state of the art and new advances on this topic in recent years. worldwide therapy cure 201 drugs sense years 20 2

BACKGROUND Chagas disease is a systemic illness with widespread microvascular involvement. Experimental and clinical studies suggest that functional and structural microcirculatory abnormalities might be relevant to the disease progression. OBJECTIVES To show the presence of sublingual microcirculatory alterations in patients with chronic Chagas disease. METHODS This was a cross-sectional study including adult patients with serologic diagnosis of Chagas disease (n = 41) and control volunteers with negative serology (n = 38), from an endemic rural population. Study participants underwent clinical, electrocardiographic, echocardiographic, and sublingual videomicroscopic assessment. Videos were acquired by a sidestream-dark-field (SDF) imaging device and evaluated by a software-assisted analysis (AVA 3.2 software). FINDINGS Most of Chagas disease patients were in the indeterminate phase (n = 34) and had lower heart rate and more echocardiographic abnormalities than control group (50 vs. 26%, p = 0.03). They also exhibited higher small microvessels total and perfused vascular density (20.12 ± 2.33 vs. 19.05 ± 2.25 and 20.03 ± 2.28 vs. 19.01 ± 2.25 mm/mm2, p < 0.05 for both). Other microvascular variables did not differ between groups. MAIN CONCLUSIONS Patients with chronic Chagas disease exhibited increases in sublingual total and perfused microvascular density. Angiogenesis might be the underlying mechanism. The videomicroscopic assessment of mucosal sublingual microcirculation might be an additional tool in the monitoring of Chagas disease. involvement progression crosssectional cross sectional n 41 38, 38 , 38) population electrocardiographic sidestreamdarkfield sidestream dark field SDF (SDF softwareassisted software assisted AVA 32 3 2 3. software. . software) 34 50 (5 vs 26 26% 0.03. 003 0.03 0 03 0.03) 20.12 2012 20 12 (20.1 233 33 2.3 1905 19 05 19.0 225 25 2.2 2003 20.0 228 28 1901 01 mmmm2 mmmm mm mm2 mm/mm2 005 0.0 both. both both) groups mechanism 4 5 ( 00 20.1 201 1 (20. 23 2. 190 19. 22 200 20. mm/mm 0. (20 (2

BACKGROUND The immunological response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and immunisation is variable. OBJECTIVES To describe the humoral immune response by correlating IgA and IgG antibodies with NAbs titration following CoronaVac® immunisation and an mRNA (Comirnaty®) booster among healthcare workers (HCWs) and to compare the cytokine and interleukin profiles between HCWs vaccinated with CoronaVac and coronavirus disease 2019 (COVID-19) infected patients. METHODS Samples from 133 HCWs collected at 20 (T1) and 90 (T2) days after CoronaVac immunisation and 15 (T3) days after a booster dose with the Comirnaty vaccine were analysed for IgA and IgG EIA and neutralisation assay. Cytokine levels from vaccinated individuals at T1 day and COVID-19 patients were compared. FINDINGS Neutralising antibodies (NAbs) were observed in 81.7% of participants at T1, but only 49.2% maintained detectable NAbs after 90 days. The booster dose increased NAbs response in all participants. The cytokines with the highest levels post-vaccination were IL-6 and MCP-1. The MCP-1, IL-18, and IFN- γ levels were higher in COVID-19 patients than in vaccinated HCWs, while IL-22 levels increased in the vaccinated HCWs group. MAIN CONCLUSIONS The neutralisation titres in the T2 samples decreased, and antibody levels detected at T2 showed a more significant reduction than the neutralisation. The higher IL-22 expression in immunised individuals compared to those with COVID-19 suggests that IL-22 may be beneficial in protecting against severe disease. SARSCoV2 SARSCoV SARS CoV (SARS-CoV-2 variable Comirnaty® (Comirnaty® (HCWs 201 COVID19 COVID 19 (COVID-19 13 T (T1 9 (T2 1 T3 (T3 assay COVID-1 (NAbs 817 81 7 81.7 492 49 49.2 postvaccination post vaccination IL6 IL 6 IL- MCP1. MCP1 MCP 1. MCP-1 MCP1, 1, IL18, IL18 18, 18 IL-18 IFN IL22 22 IL-2 group decreased (SARS-CoV- (Comirnaty COVID1 (COVID-1 (T COVID- 8 81. 4 49. MCP- IL1 IL-1 IL2 (SARS-CoV (COVID- (COVID