Live, Attenuated, Tetravalent Butantan-Dengue Vaccine in Children and Adults.

BACKGROUND: Butantan-Dengue Vaccine (Butantan-DV) is an investigational, single-dose, live, attenuated, tetravalent vaccine against dengue disease, but data on its overall efficacy are needed. METHODS: In an ongoing phase 3, double-blind trial in Brazil, we randomly assigned participants to receive Butantan-DV or placebo, with stratification according to age (2 to 6 years, 7 to 17 years, and 18 to 59 years); 5 years of follow-up is planned. The objectives of the trial were to evaluate overall vaccine efficacy against symptomatic, virologically confirmed dengue of any serotype occurring more than 28 days after vaccination (the primary efficacy end point), regardless of serostatus at baseline, and to describe safety up to day 21 (the primary safety end point). Here, vaccine efficacy was assessed on the basis of 2 years of follow-up for each participant, and safety as solicited vaccine-related adverse events reported up to day 21 after injection. Key secondary objectives were to assess vaccine efficacy among participants according to dengue serostatus at baseline and according to the dengue viral serotype; efficacy according to age was also assessed. RESULTS: Over a 3-year enrollment period, 16,235 participants received either Butantan-DV (10,259 participants) or placebo (5976 participants). The overall 2-year vaccine efficacy was 79.6% (95% confidence interval [CI], 70.0 to 86.3) - 73.6% (95% CI, 57.6 to 83.7) among participants with no evidence of previous dengue exposure and 89.2% (95% CI, 77.6 to 95.6) among those with a history of exposure. Vaccine efficacy was 80.1% (95% CI, 66.0 to 88.4) among participants 2 to 6 years of age, 77.8% (95% CI, 55.6 to 89.6) among those 7 to 17 years of age, and 90.0% (95% CI, 68.2 to 97.5) among those 18 to 59 years of age. Efficacy against DENV-1 was 89.5% (95% CI, 78.7 to 95.0) and against DENV-2 was 69.6% (95% CI, 50.8 to 81.5). DENV-3 and DENV-4 were not detected during the follow-up period. Solicited systemic vaccine- or placebo-related adverse events within 21 days after injection were more common with Butantan-DV than with placebo (58.3% of participants, vs. 45.6%). CONCLUSIONS: A single dose of Butantan-DV prevented symptomatic DENV-1 and DENV-2, regardless of dengue serostatus at baseline, through 2 years of follow-up. (Funded by Instituto Butantan and others; DEN-03-IB ClinicalTrials.gov number, NCT02406729, and WHO ICTRP number, U1111-1168-8679.).

A Double-blind, Randomized Trial to Evaluate Miltefosine and Topical Granulocyte Macrophage Colony-stimulating Factor in the Treatment of Cutaneous Leishmaniasis Caused by Leishmania braziliensis in Brazil.

BACKGROUND: The treatment of cutaneous leishmaniasis (CL) in Brazil using pentavalent antimony (Sbv) is associated with a high rate of failure. Miltefosine has proven efficacy for CL caused by L. braziliensis, with a cure rate (CR) of 75%. A combined treatment with granulocyte macrophage colony-stimulating factor (GM-CSF) and miltefosine could increase CR and decrease healing time. METHODS: A randomized, double-blind clinical trial to evaluate the efficacy of miltefosine combined with topical GM-CSF (M + GM) vs miltefosine and placebo (M + P) vs Sbv in 133 patients with CL caused by L. braziliensis in Bahia, Brazil. RESULTS: The final CR at 180 days after the initiation of treatment was 44.4% in the Sbv group, 76.6% in the M + P group (P = .003 vs Sbv), and 75.6% in the M + GM group (P = .004 vs Sbv). The median healing time for cure was 102 days for the Sbv group and 60 days for both miltefosine groups (P = .0009). During the 6-month follow-up period, 4 relapses were documented: 1 in the Sbv group, 1 in the M + P group, and 2 in the M + GM group. Mild adverse events occurred in 65% of patients from the Sbv group, 76% and 79% from the M + P and M + GM groups respectively. CONCLUSIONS: Miltefosine is more effective than Sbv for the treatment of CL caused by L. braziliensis in Brazil and accelerates the healing time. Association with GM-CSF does not improve therapeutic outcome. CLINICAL TRIALS REGISTRATION: NCT03023111.

Systemic cytokines/chemokines associated to radiographic abnormalities in pneumonia in children.

Community-acquired pneumonia (CAP) diagnosis remains a challenge in paediatrics. Chest radiography is considered gold standard for definition of pneumonia, however no previous study assessed the relationship between immune response and radiographic-confirmed-pneumonia. We assessed association between cytokines/chemokines levels and radiographic abnormalities in children with CAP. Children < 5-years-old hospitalized with CAP were investigated in a prospective study at the Federal University of Bahia Hospital, Brazil. On admission, clinical data and biological samples were collected to investigate 20 aetiological agents and determine serum cytokines/chemokines levels; chest radiographs were performed. Among 158 patients, radiographic diagnosis of pneumonia was confirmed in 126(79.7%) and 17(10.8%) had pleural effusion. Viral, bacterial and pneumococcal infection were detected in 80(50.6%), 78(49.4%) and 37(23.4%) cases. By comparing the median concentrations of serum cytokines/chemokines between children with or without pleural effusion, interleukin(IL)-6 was higher (26.6[18.6-103.7] vs 3.0[0.0-19.8]; p < 0.001) among those with pleural effusion; and between children with or without radiographic-confirmed-pneumonia, IL-6 was higher in the first subgroup (4.5[0.0-23.4] vs 0.0[0.0-3.6]; p = 0.02) after having excluded cases with pleural effusion. Stratified analyses according to aetiology showed IL-6 increase in the radiographic-confirmed-pneumonia subgroup inside the pneumococcal infection (28.2[5.9-64.1] vs 0.0[0.0-0.0]; p = 0.03) subgroup. By multivariable analysis, with IL-6 as dependent variable, pneumococcal infection and pleural effusion showed independent association with IL-6 elevation [respective OR: 5.071 (95%CI = 2.226-11.548; p < 0.001) and 13.604 (95%CI = 3.463-53.449; p = 0.0001)]. Considering the cases without pleural effusion, the area under the curve of IL-6 to predict pneumococcal infection was 0.76 (95%CI = 0.66-0.86; p < 0.001). IL-6 increase is a potential biomarker of pneumococcal infection among children with CAP without pleural effusion upon admission.

Investigating associations between intestinal alterations and parasite load according to Bifidobacterium spp. and Lactobacillus spp. abundance in the gut microbiota of hamsters infected by Leishmania infantum.

BACKGROUND: Visceral leishmaniasis (VL) is a tropical neglected disease with high associated rates of mortality. Several studies have highlighted the importance of the intestinal tract (IT) and gut microbiota (GM) in the host immunological defense. Data in the literature on parasite life cycle and host immune defense against VL are scarce regarding the effects of infection on the IT and GM. OBJECTIVES: This study aimed to investigate changes observed in the colon of Leishmania infantum-infected hamsters, including alterations in the enteric nervous system (ENS) and GM (specifically, levels of bifidobacteria and lactobacilli). METHODS: Male hamsters were inoculated with L. infantum and euthanised at four or eight months post-infection. Intestines were processed for histological analysis and GM analysis. Quantitative polymerase chain reaction (qPCR) was performed to quantify each group of bacteria: Bifidobacterium spp. (Bf) and Lactobacillus spp (LacB). FINDINGS: Infected hamsters showed histoarchitectural loss in the colon wall, with increased thickness in the submucosa and the mucosa layer, as well as greater numbers of intraepithelial lymphocytes. Forms suggestive of amastigotes were seen inside mononuclear cells. L. infantum infection induced changes in ENS, as evidenced by increases in the area of colonic enteric ganglia. Despite the absence of changes in the levels of Bf and LacB during the course of infection, the relative abundance of these bacteria was associated with parasite load and histological alterations. MAIN CONCLUSIONS: Our results indicate that L. infantum infection leads to important changes in the colon and suggest that bacteria in the GM play a protective role.

Determination and Profiling of Human Skin Odors Using Hair Samples.

Background. There is no gold standard method for human skin odor determination; several techniques can be applied to collect, extract, transfer, and detect human skin odors. However, none of these methods are suitable for field sampling of a large number of individuals. Objective. The present study aimed to develop a simple, fast, non-invasive, and low-cost method for such a purpose. Methods. Considering that hair from legs can act as a retention mesh of volatile organic compounds (VOCs), samples of leg hairs provided by healthy adult males were collected and solid-phase microextraction (SPME), in headspace (HS) mode, coupled to gas chromatography (GC) and mass spectrometry (MS) analysis of the samples was carried out. A pilot test was applied to detect five quality markers that are frequently reported in human skin odors. Then, several steps were performed for method standardization. The method was applied to 36 different individuals (3 sampled under laboratory conditions and 33 under field conditions), aiming to evaluate its applicability in both environments. Findings. A total of 49 VOCs were identified, and 73.5% of these have been reported in previous studies. Main Conclusions. Hair from legs can be considered an efficient tool for human skin odor sampling and a suitable and practical matrix for human skin odor profile determination by using HS-SPME/GC-MS.

Systemic cytokines and chemokines on admission of children hospitalized with community-acquired pneumonia.

Community-acquired pneumonia (CAP) is the main cause of death in children under-5 years worldwide and Streptococcus pneumoniae is the most common bacterial agent. However, it is difficult to identify pneumococcal infection among children with CAP. We aimed to assess association between any cytokine/chemokine and pneumococcal infection in childhood CAP. Furthermore, we evaluated the diagnostic value of cytokine/chemokine for pneumococcal infection. This prospective study was conducted at an Emergency Room, in Salvador, Brazil. Children <5-years-old hospitalized with CAP in a 21-month period were evaluated. On admission, clinical and radiological data were collected along with biological samples to investigate 20 etiological agents and determine serum cytokines (interleukin (IL)-8, IL-6, IL-10, IL-1ß, IL-12, TNF-α, IL-2, IL-4, IL-5, γ-interferon), and chemokines (CCL2, CCL5, CXCL9, CXCL10) concentration. From 166 patients with etiology detected, pneumococcal infection was detected in 38 (22.9%) cases among which the median IL-6(pg/ml) was 31.2 (IQR: 12.4-54.1). The other 128 cases had other causative agents detected (Haemophilus influenzae, Moraxella catarrhalis, atypical bacteria and viruses) with the median IL-6 concentration being 9.0 (IQR: 4.1-22.0; p < 0.001). The area under the ROC curve for IL-6 to predict pneumococcal CAP was 0.74 (95%CI: 0.65-0.83; p < 0.001). By multivariate analysis, with pneumococcal CAP as dependent variable, IL-6 was an independent predictor for pneumococcal infection (OR = 5.56; 95%CI: 2.42-12.75, cut-off point = 12.5 pg/ml; p = 0.0001). The negative predictive value of IL-6 under 12.5 pg/ml for pneumococcal infection was 90% (95%CI: 82-95%). Independently significant difference was not found for any other cytokines/chemokines. Serum IL-6 concentration on admission is independently associated with pneumococcal infection among children under-5 years hospitalized with CAP.

Evolution of acute infection with atypical bacteria in a prospective cohort of children with community-acquired pneumonia receiving amoxicillin.

Background: Atypical bacteria are treatable causative agents of community-acquired pneumonia (CAP). However, there is no conclusive evidence that a child with CAP should receive empirical treatment against such agents. Objectives: We assessed the possibility of association between clinical failure and acute infection by these bacteria among children with CAP treated with amoxicillin. Patients and methods: Patients aged 2-59 months with non-severe CAP received amoxicillin during prospective follow-up. Acute and convalescent blood samples were collected. Probable acute infection by Mycoplasma pneumoniae (specific IgM antibodies), by Chlamydia pneumoniae or Chlamydia trachomatis (specific IgM antibodies and/or IgG/IgA titre change) was investigated. Outcomes were assessed during follow-up at 2, 5 and 14-28 days. Treatment failure included development of danger signs, persistent fever, tachypnoea or death. ClinicalTrials.gov: NCT01200706. Results: Of 787 children, 86 (10.9%; 95% CI = 8.9%-13.3%) had acute M. pneumoniae infection. C. pneumoniae acute infection was found in 79 of 733 (10.8%; 95% CI = 8.7%-13.2%) and C. trachomatis was found in 3 of 28 (10.7%; 95% CI = 2.8%-26.5%) <6 months old. Among patients with or without treatment failure at 2 days, acute M. pneumoniae infection (11.7% versus 10.7%; P = 0.7), acute C. pneumoniae infection (8.5% versus 11.3%; P = 0.3) and acute C. trachomatis infection (16.7% versus 9.1%; P = 0.5) were found. No significant differences were found with regard to treatment failure at the 5 day evaluation. Overall, amoxicillin was substituted in 3.5% versus 2.7% among patients with or without acute infection by one of these bacteria ( P = 0.6). Conclusions: The overall substitution rate of amoxicillin was very low. It is not necessary to give an empirical non-ß-lactam antibiotic as a first-line option to treat every child between 2 and 59 months old with non-severe CAP.

Phenotypic diversity and selection maintain Leishmania amazonensis infectivity in BALB/c mouse model.

Leishmania are protozoan parasites that show remarkable diversity, as revealed by the various clinical forms of leishmaniasis, which can range from mild skin lesions to severe metastatic cutaneous/mucosal lesions. The exact nature and extent of Leishmania phenotypic diversity in establishing infection is not fully understood. In order to try to understand some aspects of this diversity, we subcutaneously infected BALB/c mice with first and second generation subclones of a L. amazonensis strain isolated from a patient (BA125) and examined in vivo lesion growth rate and antimony susceptibility. In vivo fast-, medium- and slow-growing subclones were obtained; moreover, fast-growing subclones could generate slow-growing subclones and inversely, revealing the continuous generation of diversity after passage into mice. No antimony-resistant subclone appeared, probably a rare occurrence. By tagging subclone cells with a L. amazonensis genomic cosmid library, we found that only a very small number of founding cells could produce lesions. Leishmania clones transfected with in vivo selected individual cosmids were also diverse in terms of lesion growth rate, revealing the cosmid-independent intrinsic characteristics of each clone. Our results suggest that only a few of the infecting parasites are able to grow and produce lesions; later, within the cell mixture of each lesion, there coexist several parasite populations with different potentialities to grow lesions during the next infection round. This may reflect a sort of programmed heterogeneity of individual parasites, favoring the survival of some individuals in various environmental conditions.

Scoring clinical signs can help diagnose canine visceral leishmaniasis in a highly endemic area in Brazil.

Canine visceral leishmaniasis (CVL) diagnosis is still a challenge in endemic areas with limited diagnostic resources. This study proposes a score with the potential to distinguish positive CVL cases from negative ones. We studied 265 dogs that tested positive for CVL on ELISA and parasitological tests. A score ranging between 0 and 19 was recorded on the basis of clinical signs. Dogs with CVL had an overall higher positivity of the majority of clinical signs than did dogs without CVL or with ehrlichiosis. Clinical signs such as enlarged lymph nodes (83.93%), muzzle/ear lesions (55.36%), nutritional status (51.79%), bristle condition (57.14%), pale mucosal colour (48.21%), onychogryphosis (58.93%), skin lesion (39.28%), bleeding (12.50%), muzzle depigmentation (41.07%), alopecia (39.29%), blepharitis (21.43%), and keratoconjunctivitis (42.86%) were more frequent in dogs with CVL than in dogs with ehrlichiosis or without CVL. Moreover, the clinical score increased according to the positivity of all diagnostic tests (ELISA, p < 0.001; parasite culture, p = 0.0021; and smear, p = 0.0003). Onychogryphosis (long nails) [odds ratio (OR): 3.529; 95% confidence interval (CI): 1.832-6.796; p < 0.001], muzzle depigmentation (OR: 4.651; 95% CI: 2.218-9.750; p < 0.001), and keratoconjunctivitis (OR: 5.400; 95% CI: 2.549-11.441; p < 0.001) were highly associated with CVL. Interestingly, a score cut-off value ≥ 6 had an area under the curve of 0.717 (p < 0.0001), sensitivity of 60.71%, and specificity of 73.64% for CVL diagnosis. The clinical sign-based score for CVL diagnosis suggested herein can help veterinarians reliably identify dogs with CVL in endemic areas with limited diagnostic resources.

Differential Expression of the Eicosanoid Pathway in Patients With Localized or Mucosal Cutaneous Leishmaniasis.

Unfettered inflammation is thought to play critical role in the development of different clinical forms of tegumentary leishmaniasis. Eicosanoids are potent mediators of inflammation and tightly associated with modulation of immune responses. In this cross-sectional exploratory study, we addressed whether targets from the eicosanoid biosynthetic pathway, assessed by multiplexed expression assays in lesion biopsy and plasma specimens, could highlight a distinct biosignature in patients with mucocutaneous leishmaniasis (MCL) or localized cutaneous leishmaniasis (LCL). Differences in immunopathogenesis between MCL and LCL may result from an imbalance between prostaglandins and leukotrienes, which may serve as targets for future host-directed therapies.

The microbiological signature of human cutaneous leishmaniasis lesions exhibits restricted bacterial diversity compared to healthy skin.

Localised cutaneous leishmaniasis (LCL) is the most common form of cutaneous leishmaniasis characterised by single or multiple painless chronic ulcers, which commonly presents with secondary bacterial infection. Previous culture-based studies have found staphylococci, streptococci, and opportunistic pathogenic bacteria in LCL lesions, but there have been no comparisons to normal skin. In addition, this approach has strong bias for determining bacterial composition. The present study tested the hypothesis that bacterial communities in LCL lesions differ from those found on healthy skin (HS). Using a high throughput amplicon sequencing approach, which allows for better populational evaluation due to greater depth coverage and the Quantitative Insights Into Microbial Ecology pipeline, we compared the microbiological signature of LCL lesions with that of contralateral HS from the same individuals.Streptococcus, Staphylococcus,Fusobacterium and other strict or facultative anaerobic bacteria composed the LCL microbiome. Aerobic and facultative anaerobic bacteria found in HS, including environmental bacteria, were significantly decreased in LCL lesions (p < 0.01). This paper presents the first comprehensive microbiome identification from LCL lesions with next generation sequence methodology and shows a marked reduction of bacterial diversity in the lesions.

Interleukin 10-Dominant Immune Response and Increased Risk of Cutaneous Leishmaniasis After Natural Exposure to Lutzomyia intermedia Sand Flies.

BACKGROUND: Leishmaniasis is caused by parasites transmitted to the vertebrate host by infected sand flies. During transmission, the vertebrate host is also inoculated with sand fly saliva, which exerts powerful immunomodulatory effects on the host's immune response. METHODS: We conducted a prospective cohort analysis to characterize the human immune response to Lutzomyia intermedia saliva in 264 individuals, from an area for cutaneous leishmaniasis (CL) caused by Leishmania braziliensis. RESULTS: Antibodies were found in 150 individuals (56.8%); immunoglobulin G1 and G4 were the predominant subclasses. Recall responses to salivary gland sonicate showed elevated production of interleukin 10 (IL-10), interleukin 13, interferon γ, CXCL9, and CCL2 compared with controls. CD4(+)CD25(+) T cells, including Foxp3(+) cells, were the main source of IL-10. L. braziliensis replication was increased (P < .05) in macrophages cocultured with saliva-stimulated lymphocytes from exposed individuals and addition of anti-IL-10 reverted this effect. Positive correlation between antibody response to saliva and cellular response to Leishmania was not found. Importantly, individuals seropositive to saliva are 2.1 times more likely to develop CL (relative risk, 2.1; 95% confidence interval, 1.07-4.2; P < .05). CONCLUSIONS: Exposure to L. intermedia sand flies skews the human immune response, facilitating L. braziliensis survival in vitro, and increases the risk of developing CL.

Arginase I, polyamine, and prostaglandin E2 pathways suppress the inflammatory response and contribute to diffuse cutaneous leishmaniasis.

Diffuse cutaneous leishmaniasis (DCL) is a rare clinical manifestation of tegumentary leishmaniasis. The molecular mechanisms underlying DCL pathogenesis remain unclear, and there is no efficient treatment available. This study investigated the systemic and in situ expression of the inflammatory response that might contribute to suppression in DCL. The plasma levels of arginase I, ornithine decarboxylase (ODC), transforming growth factor ß (TGF-ß), and prostaglandin E2 (PGE2) were higher in patients with DCL, compared with patients with localized cutaneous leishmaniasis (LCL) or with controls from an area of endemicity. In situ transcriptomic analyses reinforced the association between arginase I expression and enzymes involved in prostaglandin and polyamine synthesis. Immunohistochemistry confirmed that arginase I, ODC, and cyclooxygenase2 expression was higher in lesion biopsy specimens from patients with DCL than in those from patients with LCL. Inhibition of arginase I or ODC abrogates L. amazonensis replication in infected human macrophages. Our data implicate arginase I, ODC, PGE2, and TGF-ß in the failure to mount an efficient immune response and suggest perspectives in the development of new strategies for therapeutic intervention for patients with DCL.

Impact of visceral leishmaniasis and curative chemotherapy on cytochrome P450 activity in Brazilian patients.

AIMS: The aim of the present study was to investigate the impact of human visceral leishmaniasis (VL) and curative chemotherapy on the activity of cytochrome P450 (CYP) 3A, CYP2C9 and CYP2C19 in patients from an endemic region in Brazil. METHODS: Adult patients with parasitologically confirmed VL were given a CYP phenotyping cocktail, comprising midazolam, omeprazole and losartan, immediately before (Study phase 1), 2-3 days (phase 2) and 3-6 months (phase 3) after curative VL chemotherapy. CYP activity was assessed by the apparent clearance of midazolam (CYP3A), omeprazole/5-hydroxyomeprazol ratio in plasma (CYP2C19) and losartan/E3174 ratio in urine (CYP2C9). RESULTS: Mean values (95% confidence interval) in phases 1, 2 and 3 were, respectively: log apparent midazolam clearance, 1.21 (1.10-1.31), 1.45 (1.32-1.57) and 1.35 (1.26-1.44) ml min(-1) kg(-1) ; omeprazole/5-hydroxyomeprazole ratio, 0.78 (0.61-0.94), 0.45 (0.27-0.63) and 0.37 (0.20-0.55); losartan/E3174 ratio, 0.66 (0.39-0.92), 0.35 (0.20-0.50) and 0.35 (0.16-0.53). Analysis of variance revealed significant differences in CYP3A (P = 0.018) and CYP2C19 (P = 0.008), but not CYP2C9 (P = 0.11) phenotypic activity, across the three study phases. CONCLUSION: The phenotypic activities of CYP3A4 and CYP2C19 were significantly reduced during acute VL compared with post-chemotherapy. We propose that increased plasma concentrations of proinflammatory cytokines during active disease account for the suppression of CYP activity. The failure to detect significant changes in CYP2C9 activity in the overall cohort may reflect differential effects of the inflammatory process on the expression of CYP isoforms, although the possibility of insufficient statistical power cannot be dismissed.

SOD1 plasma level as a biomarker for therapeutic failure in cutaneous leishmaniasis.

We show that increased plasma superoxide dismutase 1 (SOD1) levels are statistically significant predictors of the failure of pentavalent antimony treatment for cutaneous leishmaniasis caused by Leishmania braziliensis. In Leishmania amazonensis-infected patients, host SOD1 levels can be used to discriminate between localized and drug-resistant diffuse cutaneous leishmaniasis. Using in situ transcriptomics (nCounter), we demonstrate a significant positive correlation between host SOD1 and interferon α/ß messenger RNA (mRNA) levels, as well as interkingdom correlation between host SOD1 and parasite SOD2/4 mRNA levels. In human macrophages, in vitro treatment with SOD1 increases the parasite burden and induces a diffuse cutaneous leishmaniasis-like morphology. Thus, SOD1 is a clinically relevant biomarker and a therapeutic target in both localized and diffuse cutaneous leishmaniasis.

Comparison of oral amoxicillin given thrice or twice daily to children between 2 and 59 months old with non-severe pneumonia: a randomized controlled trial.

OBJECTIVES: Oral amoxicillin (50 mg/kg/day) thrice daily is the first-line therapy for non-severe childhood pneumonia. Compliance could be enhanced if two daily doses are employed. We assessed the equivalence of oral amoxicillin (50 mg/kg/day) thrice or twice daily in those patients. PATIENTS AND METHODS: This randomized (1 : 1), controlled, triple-blinded investigation conducted at one centre in Brazil included children aged 2-59 months with non-severe pneumonia diagnosed by trained paediatricians based on respiratory complaints and radiographic pulmonary infiltrate/consolidation. Participants were randomly assigned to receive one bottle (Amoxicillin 1) at 6 am, 2 pm and 10 pm and the other bottle (Amoxicillin 2) at 8 am and 8 pm: one bottle contained amoxicillin and the other placebo and vice versa. Only the pharmacist knew patients' allocation. Follow-up assessments were done at 2, 5 and 14 days after enrolment. Chest radiographs were read by three independent radiologists. Primary outcome was treatment failure (development of danger signs, persistence of fever, tachypnoea, development of serious adverse reactions, death and withdrawal from the trial) at 48 h. ClinicalTrials.gov: identifier NCT01200706. RESULTS: Four hundred and twelve and 408 participants received amoxicillin thrice or twice daily, respectively. Treatment failure was detected in 94 (22.8%) and 94 (23.0%) patients in intention-to-treat analysis (risk difference 0.2%; 95% CI: -5.5%-6.0%) and in 80 (20.1%) and 85 (21.3%) patients in per-protocol analysis (risk difference 1.2%; 95% CI: -4.4%-6.8%). Pneumonia was radiologically confirmed by concordant reading in 277 (33.8%) cases, among whom treatment failure was registered in 25/133 (18.8%) and 27/144 (18.8%) participants from the thrice and twice daily doses subgroups, respectively (risk difference -0.05%; 95% CI: -9.3%-9.2%). CONCLUSIONS: Oral amoxicillin (50 mg/kg/day) twice daily is as efficacious as thrice daily.

Serological survey of Leishmania infection in blood donors in Salvador, Northeastern Brazil.

BACKGROUND: Visceral Leishmaniasis is endemic to Brazil, where it is caused by Leishmania infantum (syn. Leishmania chagasi). Following parasite inoculation, individuals may experience asymptomatic infection, raising the possibility of parasite transmission through the transfusion of contaminated blood products. In the present work, we evaluated the prevalence of asymptomatic Leishmania infection among blood donors in Salvador, northeastern Brazil. METHODS: Peripheral blood was collected from 700 blood donors attending the Blood Bank of Bahia (HEMOBA/SESAB), from January to September 2010. We evaluated anti-Leishmania serology by ELISA, employing Soluble Leishmania Antigen (sensitivity 90% and specificity 95%). The presence of parasite DNA was determined by qPCR, targeting a single copy gene (G6PD), and by end-point PCR, targeting multiple targets, namely a segment located in the Leishmania rRNA locus (ITS) and the conserved region of kinetoplastid DNA (kDNA) minicircles. RESULTS: The blood-donor population was comprised of 74.5% of males with a mean age of 34 years. Anti-Leishmania serology by ELISA was positive in 5.4% (38/700) individuals. One individual was also positive for Chagas' disease and another tested positive for Syphilis. Employing qPCR, parasite DNA was not found in any of 38 seropositive samples. However, by ITS PCR, 8/38 (21%) samples were positive and this positivity increased to 26/38 (68%) when we targeted kDNA amplification. Agreement between both techniques (ITS and kDNA PCR) was fair (kappa = 0.219). CONCLUSIONS: These results indicate that asymptomatic infection is present among the blood donor population of Salvador, a finding that warrants a broader discussion regarding the need to implement specific screening strategies.

Heme oxygenase-1 promotes the persistence of Leishmania chagasi infection.

Visceral leishmaniasis (VL) remains a major public health problem worldwide. This disease is highly associated with chronic inflammation and a lack of the cellular immune responses against Leishmania. It is important to identify major factors driving the successful establishment of the Leishmania infection to develop better tools for the disease control. Heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, and its role in VL has not been investigated. In this study, we evaluated the role of HO-1 in the infection by Leishmania infantum chagasi, the causative agent of VL cases in Brazil. We found that L. chagasi infection or lipophosphoglycan isolated from promastigotes triggered HO-1 production by murine macrophages. Interestingly, cobalt protoporphyrin IX, an HO-1 inductor, increased the parasite burden in both mouse and human-derived macrophages. Upon L. chagasi infection, macrophages from Hmox1 knockout mice presented significantly lower parasite loads when compared with those from wild-type mice. Furthermore, upregulation of HO-1 by cobalt protoporphyrin IX diminished the production of TNF-α and reactive oxygen species by infected murine macrophages and increased Cu/Zn superoxide dismutase expression in human monocytes. Finally, patients with VL presented higher systemic concentrations of HO-1 than healthy individuals, and this increase of HO-1 was reduced after antileishmanial treatment, suggesting that HO-1 is associated with disease susceptibility. Our data argue that HO-1 has a critical role in the L. chagasi infection and is strongly associated with the inflammatory imbalance during VL. Manipulation of HO-1 pathways during VL could serve as an adjunctive therapeutic approach.

High-throughput prioritization of target proteins for development of new antileishmanial compounds.

Leishmaniasis, a vector-borne disease, is caused by the infection of Leishmania spp., obligate intracellular protozoan parasites. Presently, human vaccines are unavailable, and the primary treatment relies heavily on systemic drugs, often presenting with suboptimal formulations and substantial toxicity, making new drugs a high priority for LMIC countries burdened by the disease, but a low priority in the agenda of most pharmaceutical companies due to unattractive profit margins. New ways to accelerate the discovery of new, or the repositioning of existing drugs, are needed. To address this challenge, our study aimed to identify potential protein targets shared among clinically-relevant Leishmania species. We employed a subtractive proteomics and comparative genomics approach, integrating high-throughput multi-omics data to classify these targets based on different druggability metrics. This effort resulted in the ranking of 6502 ortholog groups of protein targets across 14 pathogenic Leishmania species. Among the top 20 highly ranked groups, metabolic processes known to be attractive drug targets, including the ubiquitination pathway, aminoacyl-tRNA synthetases, and purine synthesis, were rediscovered. Additionally, we unveiled novel promising targets such as the nicotinate phosphoribosyltransferase enzyme and dihydrolipoamide succinyltransferases. These groups exhibited appealing druggability features, including less than 40% sequence identity to the human host proteome, predicted essentiality, structural classification as highly druggable or druggable, and expression levels above the 50th percentile in the amastigote form. The resources presented in this work also represent a comprehensive collection of integrated data regarding trypanosomatid biology.

Role of Toll-like receptor 9 signaling in experimental Leishmania braziliensis infection.

Infection with Leishmania braziliensis causes cutaneous or mucocutaneous leishmaniasis in humans. Toll-like receptor 9 (TLR9) expression has been found in granulomas of lesions in L. braziliensis-infected individuals. L. braziliensis inoculation in mice induces very small lesions that are self-healing, whereas deficiency in the TLR adaptor molecule, MyD88, renders mice susceptible to infection. The TLR involved has not been identified, prompting us to investigate if TLR9 triggering by the parasite contributes to the strong resistance to infection observed in L. braziliensis-inoculated mice. The parasites activated wild-type (WT) dendritic cells (DCs) in vitro but not DCs derived from TLR9(-/-) mice. TLR9(-/-) mice inoculated with L. braziliensis exhibited a transient susceptibility characterized by increased lesion size and parasite burden compared to those of WT mice. Surprisingly, elevated levels of gamma interferon (IFN-γ) were measured at the site of infection and in draining lymph node T cells of TLR9(-/-) mice at the peak of susceptibility, suggesting that unlike observations in vitro, the parasite could induce DC activation leading to the development of Th1 cells in the absence of TLR9 expression. Taken together, these data show that TLR9 signaling is important for the early control of lesion development and parasite burden but is dispensable for the differentiation of Th1 cells secreting IFN-γ, and the high levels of this cytokine are not sufficient to control early parasite replication following L. braziliensis infection.