Genetic diversity of Trypanosoma cruzi strains isolated from chronic chagasic patients and non-human hosts in the state of São Paulo, Brazil.

BACKGROUND: Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES: This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS: T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS: It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS: The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.

Isolation, typing, and drug susceptibility of Leishmania (Leishmania) infantum isolates from dogs of the municipality of Embu das Artes, an endemic region for canine leishmaniasis in Brazil.

The parasitic protozoa Leishmania (Leishmania) infantum is the etiological agent of human visceral leishmaniasis and canine leishmaniasis in South America, where Brazil is the most affected country. This zoonotic disease is transmitted by the bite of an infected phlebotomine sand fly and dogs constitute the main domestic reservoir of the parasite. In this study, we screened 2348 dogs of the municipality of Embu das Artes, Brazil, for antibodies against the parasite. Prevalence for canine leishmaniasis seropositivity was 2.81%, as assessed using a Dual-Path Platform rapid test for canine leishmaniasis. Twenty-five seropositive dogs were euthanized for parasite isolation and 14 isolates were successful obtained. Nucleotide sequencing of the internal transcribed spacer confirmed the isolates to be L. (L.) infantum, and very low sequence variability was observed among them. The in vitro susceptibility to miltefosine and paromomycin was assessed and moderate variation in paromomycin susceptibility was found among the isolates in the promastigote and intracellular amastigote stages. On the other hand, in vitro susceptibility to miltefosine of these isolates was homogenous, particularly in the amastigote stage (EC50 values from 0.69 to 2.07 µM). In addition, the miltefosine sensitivity locus was deleted in all the isolates, which does not corroborate the hypothesis that the absence of this locus is correlated with a low in vitro susceptibility. Our findings confirm that the municipality of Embu das Artes is endemic for canine leishmaniasis and that isolates from this region are susceptible to paromomycin and miltefosine, indicating the potential of these drugs to be clinically evaluated in the treatment of human visceral leishmaniasis in Brazil.

ELISA with recombinant antigen Lb6H validated for the diagnosis of American tegumentary leishmaniasis.

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL.

Toxoplasma gondii SAG2, SAG3 and GRA6 alleles and single nucleotide polymorphism in congenital infections with known parasite load and clinical outcome.

Amniotic fluid DNA samples were genotyped by multilocus-nested-PCR-RFLP, but only three of 11 markers amplified 113 of 122 (92.6%) samples, resulting in 12 untyped and 101 partial non-archetypal genotypes. The 101 typed samples were subdivided into four groups: G1 with 73 samples (5'and 3' SAG2 allele I + SAG3 allele III + GRA6 allele III), 53 had parasite load ≤ 102 parasites/mL (43 asymptomatic, 10 mild infections), 17 had load > 102 and ≤ 103 (one mild, 13 moderate and three severe), and three had load > 103 parasites/mL (three severe); G2 with 22 samples (5'and 3' SAG2 allele I + SAG3 allele III), all parasite load levels ≤ 102 parasites/mL (18 asymptomatic and four mild); G3 with five samples (5' and 3' SAG2 allele I + SAG3 allele II), parasite load ≤ 102 parasites/mL (three asymptomatic and two mild); G4 with one sample (5' and 3' SAG2 allele II + SAG3 allele II + GRA6 allele I), a parasite load < 102 parasites/mL in an asymptomatic infant. After DNA sequencing, restriction sites confirmed SAG2, SAG3 and GRA6 alleles in 98.7%, 100% and 100% of the cases, respectively, while single nucleotide polymorphisms confirmed 90% of 5'-SAG2 allele I; 98.7% of 3'-SAG2 allele I; 98% of SAG-3 allele III, but only 40% of GRA6 allele III results. For the moment, partial non-archetypal genotypes of parasites did not show any relationship with either parasite load in amniotic fluid samples or clinical outcome of infants at the age of 12 months.

New Allele-Specific Oligonucleotide (ASO) amplifications for Toxoplasma gondii rop18 allele typing: Analysis of 86 human congenital infections in Brazil.

This study aimed to detect and differentiate Toxoplasma gondii by the allele typing of its polymorphic rop18 gene. For this purpose, a novel genotyping system using allele-specific oligonucleotides (ASOs) was designed, consisting of three ASO pairs. The first and third pairs specifically amplify rop18 allele I and allele III, while the second pair amplify both allele I and II. Genomic DNA from 86 congenital infections was analyzed by ASO-PCRs, successfully typing 82 (95.35%) samples. The remaining 4 samples (4.65%) required sequencing and single nucleotide polymorphism (SNP) analysis of the amplification products. The distribution of samples according to rop18 alleles was: 39.5% of allele III, 38.4% of allele II, 19.8% of mixed rop18 alleles (I/III or II/III), and 2.3% of allele I. The six severely compromised infants exhibited the highest parasite load levels and were infected during the first and early second trimesters of pregnancy. Among these cases, two were associated with rop18 allele I parasites, two with mixed rop18 alleles (I/III), one with allele II, and one with allele III parasites. In conclusion, all severe cases of congenital toxoplasmosis were infected during early pregnancy, but they were not exclusively associated with rop18 allele I parasites, as observed in murine toxoplasmosis. Furthermore, nearly one-fifth of parasites were non-archetypal, exhibiting more than one rop18 allele, indicating a higher genetic diversity of Toxoplasma gondii in this South American sample. Overall, a robust T. gondii rop18 allele typing was developed and suggested that congenital toxoplasmosis in humans involves complex mechanisms beyond the parasite genotype.

Assessment of biomarkers and clinical parameters as predictors of survival in patients with chagasic heart failure.

BACKGROUND: Chagas disease, endemic in Latin America and spreading globally due to emigration, has a significant health burden, particularly in relation to chagasic heart failure (HF). Chagasic cardiomyopathy (CCM) is characterized by chronic inflammatory myocardial disease. This study aimed to identify inflammatory parameters and biomarkers that could aid in the management of patients with chagasic HF. METHODS AND FINDINGS: A cohort study was conducted at a tertiary cardiology single-center over a mean follow-up period of 2.4 years. The study included patients with HF secondary to CCM enrolled between October 2013 and July 2017. Various clinical parameters, echocardiography findings, parasitemia status, brain natriuretic peptide (BNP) and troponin T (TnT) levels, and inflammatory biomarkers (IL-6, IL-10, IL-12p70, IL-17A, adiponectin, and IFN-γ) were assessed. The study encompassed a cohort of 103 patients, with a median age of 53 years and 70% being male. The left ventricular ejection fraction (LVEF) was 28%, with 40% of patients classified as NYHA II functional class. The median BNP level was 291 pg/ml. The observed mortality rate during the study period was 38.8%. Predictors of lower survival were identified as elevated levels of BNP, TnT, reduced LVEF, and increased adiponectin (thresholds: BNP > 309 pg/ml, TnT > 27.5 ng/ml, LVEF < 25.5%, adiponectin > 38 µg/mL). Notably, there was no evidence indicating a relationship between parasitemia and the inflammatory parameters with lower survival in these patients, including INF-γ, IL-6, IL-10, IL12-(p70), and IL17a. CONCLUSION: Despite the presence of a chronic inflammatory process, the evaluated inflammatory biomarkers in this cohort were not predictive of survival in patients with chagasic HF with reduced ejection fraction (HFrEF). However, reduced LVEF, elevated BNP, adiponectin levels, and troponin T were identified as predictors of lower survival in these patients.

Silent circulation of Chikungunya virus among pregnant women and newborns in the Western Brazilian Amazon before the first outbreak of chikungunya fever.

The prevalence of immunity to Chikungunya virus (CHIKV) in pregnant women and newborns in the Western Brazilian Amazon was assessed at a time when previous studies did not report chikungunya fever in the area. In 435 asymptomatic pregnant women and 642 healthy unrelated newborns, the presence of IgM and IgG antibodies to CHIKV were determined by a commercial ELISA. All participants were negative to IgM anti-CHIKV. Anti-CHIKV IgG was identified in 41 (9.4%) pregnant women and 66 (10.3%) newborns. The presence of anti-CHIKV IgG was positively associated with the lowest socioeconomic status in pregnant women (OR 2.54, 95% CI 1.15-5.62, p=0.021) and in the newborns' mothers (OR 5.10, 95% CI 2.15-12.09, p< 0.001). Anti-CHIKV IgG was also associated with maternal age in both, the pregnant women (OR 1.06, 95% CI 1.00-1.11, p=0.037) and the newborns'mothers (OR 1.08, 95% CI 1.03-1.12, p=0.001). Pregnancy outcomes in which the mother or the newborn was anti-CHIKV IgG positive proceeded normally. Negative CHIKV serology was associated with being positive for DENV antibodies and having had malaria during pregnancy. These findings showed that there was already a silent circulation of CHIKV in this Amazon region before the first outbreak of chikungunya fever. Furthermore, seropositivity for CHIKV was surprisingly frequent (10%) in both, pregnant women and newborns, affecting mainly low-income women.

Oral administration of buparvaquone nanostructured lipid carrier enables in vivo activity against Leishmania infantum.

Leishmaniasis, a neglected tropical disease, is prevalent in 98 countries with the occurrence of 1.3 million new cases annually. The conventional therapy for visceral leishmaniasis requires hospitalization due to the severe adverse effects of the drugs, which are administered parenterally. Buparvaquone (BPQ) showed in vitro activity against leishmania parasites; nevertheless, it has failed in vivo tests due to its low aqueous solubility. Though, lipid nanoparticles can overcome this holdback. In this study we tested the hypothesis whether BPQ-NLC shows in vivo activity against L. infantum. Two optimized formulations were prepared (V1: 173.9 ± 1.6 nm, 0.5 mg of BPQ/mL; V2: 232.4 ± 1.6 nm, 1.3 mg of BPQ/mL), both showed increased solubility up to 73.00-fold, and dissolution up to 83.29%, while for the free drug it was only 2.89%. Cytotoxicity test showed their biocompatibility (CC50 >554.4 µM). Besides, the V1 dose of 0.3 mg/kg/day for 10 days reduced the parasite burden in 83.4% ±18.2% (p <0.05) in the liver. BPQ-NLC showed similar leishmanicidal activity compared to miltefosine. Therefore, BPQ-NLC is a promising addition to the limited therapeutic arsenal suitable for leishmaniasis oral administration treatment.

Staphylococcus aureus-Cure-Associated Antigens Elicit Type 3 Immune Memory T Cells.

BACKGROUND: Staphylococcus aureus is one of the most frequently major mastitis pathogens that cause clinical and subclinical mastitis worldwide. Current antimicrobial treatments are usually ineffective, and the commercially available vaccines lack proven effectiveness. The immunological response elicited by the recombinant S. aureus-cure-associated proteins phosphoglycerate kinase (PGK), enolase (ENO), and elongation factor-G (EF-G) in combination with the granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA vaccination was studied in this work. METHODS: Here, twenty-three C57BL/6 mice were divided into four groups and vaccinated with: G1: none (control); G2: GM-CSF DNA plasmid DNA vaccine; G3: the combination of EF-G+ENO+PGK; and G4: the combinations of EF-G+ENO+PGK proteins plus GM-CSF plasmid DNA vaccine. After 44 days, spleen cells were collected for immunophenotyping and lymphocyte proliferation evaluation by flow cytometry upon S. aureus stimulus. RESULTS: Immunization with the three S. aureus recombinant proteins alone resulted in a higher percentage of IL-17A+ cells among CD8+ T central memory cells, as well as the highest intensity of IL-17A production by overall lymphocytes indicating that the contribution of the combined lymphocyte populations is crucial to sustaining a type 3 cell immunity environment. CONCLUSION: The immunization with three S. aureus-cure-associated recombinant proteins triggered type 3 immunity, which is a highly interesting path to pursue an effective bovine S. aureus mastitis vaccine.

SARS-CoV-2 and rhinovirus infections: are there differences in clinical presentation, laboratory abnormalities, and outcomes in the pediatric population?

This study aims to assess COVID-19 and other respiratory viruses in pediatric patients. Between April 17 and September 30, 2020, we collected 1,566 respiratory samples from 1,044 symptomatic patients who were younger than 18 years old to assess SARS-CoV-2 infection. Of these, 919 were analyzed for other respiratory pathogens (ORP). Patients with laboratory-confirmed COVID-19 or ORP were included. We evaluated 76 pediatric COVID-19 infections and 157 other respiratory virus infections. Rhinovirus occurred in 132/157 (84%). COVID-19 patients who were significantly older, had more fevers, headaches and pneumonia than those with ORP. The median white blood cell count was lower in patients with SARS-CoV-2 than in those with ORP (6,470 versus 8,170; p=0.02). COVID-19 patients had significantly worse symptoms than those with ORP.

Technical performance of a lateral flow immunoassay for detection of anti-SARS-CoV-2 IgG in the outpatient follow-up of non-severe cases and at different times after vaccination: comparison with enzyme and chemiluminescent immunoassays.

This study assessed the technical performance of a rapid lateral flow immunochromatographic assay (LFIA) for the detection of anti-SARS-CoV-2 IgG and compared LFIA results with chemiluminescent immunoassay (CLIA) results and an in-house enzyme immunoassay (EIA). To this end, a total of 216 whole blood or serum samples from three groups were analyzed: the first group was composed of 68 true negative cases corresponding to blood bank donors, healthy young volunteers, and eight pediatric patients diagnosed with other coronavirus infections. The serum samples from these participants were obtained and stored in a pre-COVID-19 period, thus they were not expected to have COVID-19. In the second group of true positive cases, we chose to replace natural cases of COVID-19 by 96 participants who were expected to have produced anti-SARS-CoV-2 IgG antibodies 30-60 days after the vaccine booster dose. The serum samples were collected on the same day that LFIA were tested either by EIA or CLIA. The third study group was composed of 52 participants (12 adults and 40 children) who did or did not have anti-SARS-CoV-2 IgG antibodies due to specific clinical scenarios. The 12 adults had been vaccinated more than seven months before LFIA testing, and the 40 children had non-severe COVID-19 diagnosed using RT-PCR during the acute phase of infection. They were referred for outpatient follow-up and during this period the serum samples were collected and tested by CLIA and LFIA. All tests were performed by the same healthcare operator and there was no variation of LFIA results when tests were performed on finger prick whole blood or serum samples, so that results were grouped for analysis. LFIA's sensitivity in detecting anti-SARS-CoV-2 IgG antibodies was 90%, specificity 97.6%, efficiency 93%, PPV 98.3%, NPV 86.6%, and likelihood ratio for a positive or a negative result were 37.5 and 0.01 respectively. There was a good agreement (Kappa index of 0.677) between LFIA results and serological (EIA or CLIA) results. In conclusion, LFIA analyzed in this study showed a good technical performance and agreement with reference serological assays (EIA or CLIA), therefore it can be recommended for use in the outpatient follow-up of non-severe cases of COVID-19 and to assess anti-SARS-CoV-2 IgG antibody production induced by vaccination and the antibodies decrease over time. However, LFIAs should be confirmed by using reference serological assays whenever possible.

Nucleoprotein-based ELISA for detection of SARS-COV-2 IgG antibodies: Could an old assay be suitable for serodiagnosis of the new coronavirus?

OBJECTIVES: We evaluated the performance of a nucleoprotein-based enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies to SARS-CoV-2. METHODS: The ELISA was based on serum IgG reactivity to a 46-kDa protein derived from the recombinant SARS-CoV2 nucleoprotein. Assay sensitivity was assessed using serum samples from 134 COVID-19 confirmed cases obtained > 15 days after symptom onset. Specificity was determined by testing sera from 94 healthy controls. Cross-reactivity was evaluated with sera from 96 individuals with previous dengue or zika virus-confirmed infections, with 44 sera from individuals with confirmed infections to other respiratory viruses or with bacterial and fungal infections that cause pneumonia and with 40 sera negative for SARS-CoV-2 nucleoprotein by commercial ELISA kits. RESULTS: The majority of subjects were male and ≥ 60 years old. Assay sensitivity was 90.3 % (95 % confidence interval 84.1 %-94.2 %) and specificity was 97.9 % (92.6 %-99.4 %). There was no cross-reactivity with sera from individuals diagnosed with dengue, zika virus, influenza virus, rhinovirus, adenovirus, respiratory syncytial virus, seasonal coronavirus, Mycobacterium tuberculosis, Staphylococcus (S. aureus and coagulase-negative), Streptococcus pneumoniae, Klebsiella pneumoniae and the fungus Aspergillus fumigatus. The level of concordance of our test with results from commercial ELISA kits was 100 %. CONCLUSION: The nucleoprotein-based ELISA was specific for detection of IgG anti-nucleoprotein antibodies to SARS-CoV-2. It utilizes a frequently employed low expense assay protocol and is easier to perform than other currently available commercial SARS-CoV2 antibody detection tests.

SARS-CoV-2 infections with emphasis on pediatric patients: a narrative review.

This narrative review summarizes the main aspects underlying the new coronavirus SARS-CoV-2, its epidemiology, pathophysiology, pointing to differences of SARS-CoV-2 main receptors ACE2, in terms of expression and the amount of soluble ACE2 in the circulation of children, men and women, and also in those with risk factors such as the smokers and pregnant women or presenting with comorbidities (diabetes, obesity, hypertension and other cardiovascular diseases, renal and CNS pre-existing diseases). Clinical manifestations in adults and children were also described, emphasizing the particularities already seen in children, regarding signs, symptoms, viral excretion time and the involvement of all organs and systems. The COVID-19 in the pediatric population was divided into two sections: one dedicated to previously healthy children and adolescents with COVID-19, and the other to those who live with comorbidities and acquired COVID-19. A few paragraphs were reserved to the recently described severe multisystemic inflammatory syndrome associated with COVID-19 (MIS-C) that shares certain characteristics with Kawasaki disease. Some studies on the infection in pregnant and postpartum women, as well as neonates were shown. This review has also covered the laboratory diagnosis of COVID-19, passing through the imaging diagnosis made by the chest tomography revealing ground glass patching opacities, and results of non-specific exams such as the total blood with lymphopenia, the coagulation tests with increased prothrombin times, as well as marked increments of the D-dimer, troponin and proinflammatory cytokines. In the section devoted to the specific laboratory diagnosis of COVID-19, the most used RT-PCR protocols were described and some studies on the serological diagnosis with IgA, IgM and IgG detection were detailed, including the use of rapid immunochromatographic assays and discussing the ideal period after the onset of symptoms to perform each type of test. In the end, the management of pediatric patients with COVID-19 based mainly on supportive measures has been briefly commented.

Severe clinical spectrum with high mortality in pediatric patients with COVID-19 and multisystem inflammatory syndrome.

OBJECTIVES: To assess the outcomes of pediatric patients with laboratory-confirmed coronavirus disease (COVID-19) with or without multisystem inflammatory syndrome in children (MIS-C). METHODS: This cross-sectional study included 471 samples collected from 371 patients (age<18 years) suspected of having severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The study group comprised 66/371 (18%) laboratory-confirmed pediatric COVID-19 patients: 61 (92.5%) patients tested positive on real-time reverse transcription-polymerase chain reaction tests for SARS-CoV-2, and 5 (7.5%) patients tested positive on serological tests. MIS-C was diagnosed according to the criteria of the Center for Disease Control. RESULTS: MIS-C was diagnosed in 6/66 (9%) patients. The frequencies of diarrhea, vomiting, and/or abdominal pain (67% vs. 22%, p=0.034); pediatric SARS (67% vs. 13%, p=0.008); hypoxemia (83% vs. 23%, p=0.006); and arterial hypotension (50% vs. 3%, p=0.004) were significantly higher in patients with MIS-C than in those without MIS-C. The frequencies of C-reactive protein levels >50 mg/L (83% vs. 25%, p=0.008) and D-dimer levels >1000 ng/mL (100% vs. 40%, p=0.007) and the median D-dimer, troponin T, and ferritin levels (p<0.05) were significantly higher in patients with MIS-C. The frequencies of pediatric intensive care unit admission (100% vs. 60%, p=0.003), mechanical ventilation (83% vs. 7%, p<0.001), vasoactive agent use (83% vs. 3%, p<0.001), shock (83% vs. 5%, p<0.001), cardiac abnormalities (100% vs. 2%, p<0.001), and death (67% vs. 3%, p<0.001) were also significantly higher in patients with MIS-C. Similarly, the frequencies of oxygen therapy (100% vs. 33%, p=0.003), intravenous immunoglobulin therapy (67% vs. 2%, p<0.001), aspirin therapy (50% vs. 0%, p<0.001), and current acute renal replacement therapy (50% vs. 2%, p=0.002) were also significantly higher in patients with MIS-C. Logistic regression analysis showed that the presence of MIS-C was significantly associated with gastrointestinal manifestations [odds ratio (OR)=10.98; 95%CI (95% confidence interval)=1.20-100.86; p=0.034] and hypoxemia [OR=16.85; 95%CI=1.34-211.80; p=0.029]. Further univariate analysis showed a positive association between MIS-C and death [OR=58.00; 95%CI=6.39-526.79; p<0.0001]. CONCLUSIONS: Pediatric patients with laboratory-confirmed COVID-19 with MIS-C had a severe clinical spectrum with a high mortality rate. Our study emphasizes the importance of investigating MIS-C in pediatric patients with COVID-19 presenting with gastrointestinal involvement and hypoxemia.

Biodegradable nanocarriers coated with polymyxin B: Evaluation of leishmanicidal and antibacterial potential.

Most treatments of leishmaniasis require hospitalization and present side effects or parasite resistance; innovations in drug formulation/reposition can overcome these barriers and must be pursued to increase therapeutic alternatives. Therefore, we tested polymyxin B (polB) potential to kill Leishmania amazonensis, adsorbed or not in PBCA nanoparticles (PBCAnp), which could augment polB internalization in infected macrophages. PBCAnps were fabricated by anionic polymerization and analyzed by Dynamic Light Scattering (size, ζ potential), Nanoparticle Tracking Analysis (size/concentration), vertical diffusion cell (release rate), drug incorporation (indirect method, protein determination) and in vitro cell viability. Nanoparticles coated with polB (PBCAnp-polB) presented an adequate size of 261.5 ± 25.9 nm, low PDI and ζ of 1.79 ± 0.17 mV (stable for 45 days, at least). The 50% drug release from PBCAnp-polB was 6-7 times slower than the free polB, which favors a prolonged and desired release profile. Concerning in vitro evaluations, polB alone reduced in vitro amastigote infection of macrophages (10 µg/mL) without complete parasite elimination, even at higher concentrations. This behavior limits its future application to adjuvant leishmanicidal therapy or antimicrobial coating of carriers. The nanocarrier PBCAnp also presented leishmanicidal effect and surpassed polB activity; however, no antimicrobial activity was detected. PolB maintained its activity against E. coli, Pseudomonas and Klebsiella, adding antimicrobial properties to the nanoparticles. Thus, this coated drug delivery system, described for the first time, demonstrated antileishmanial and antimicrobial properties. The bactericidal feature helps with concomitant prevention/treatment of secondary infections that worst ulcers induced by cutaneous L. amazonensis, ultimately ending in disfiguring or disabling lesions.

Experimental nodel of C6 brain tumors in athymic rats.

Malignant brain tumor experimental models tend to employ cells that are immunologically compatible with the receptor animal. In this study, we have proposed an experimental model of encephalic tumor development by injecting C6 cells into athymic Rowett rats, aiming at reaching a model which more closely resembles to the human glioma tumor. In our model, we observed micro-infiltration of tumor cell clusters in the vicinity of the main tumor mass, and of more distal isolated tumor cells immersed in normal encephalic parenchyma. This degree of infiltration is superior to that usually observed in other C6 models.

Toxoplasma gondii SAG2, SAG3 and GRA6 alleles and single nucleotide polymorphism in congenital infections with known parasite load and clinical outcome

ABSTRACT Amniotic fluid DNA samples were genotyped by multilocus-nested-PCR-RFLP, but only three of 11 markers amplified 113 of 122 (92.6%) samples, resulting in 12 untyped and 101 partial non-archetypal genotypes. The 101 typed samples were subdivided into four groups: G1 with 73 samples (5'and 3' SAG2 allele I + SAG3 allele III + GRA6 allele III), 53 had parasite load ≤ 102 parasites/mL (43 asymptomatic, 10 mild infections), 17 had load > 102 and ≤ 103 (one mild, 13 moderate and three severe), and three had load > 103 parasites/mL (three severe); G2 with 22 samples (5'and 3' SAG2 allele I + SAG3 allele III), all parasite load levels ≤ 102 parasites/mL (18 asymptomatic and four mild); G3 with five samples (5' and 3' SAG2 allele I + SAG3 allele II), parasite load ≤ 102 parasites/mL (three asymptomatic and two mild); G4 with one sample (5' and 3' SAG2 allele II + SAG3 allele II + GRA6 allele I), a parasite load < 102 parasites/mL in an asymptomatic infant. After DNA sequencing, restriction sites confirmed SAG2, SAG3 and GRA6 alleles in 98.7%, 100% and 100% of the cases, respectively, while single nucleotide polymorphisms confirmed 90% of 5'-SAG2 allele I; 98.7% of 3'-SAG2 allele I; 98% of SAG-3 allele III, but only 40% of GRA6 allele III results. For the moment, partial non-archetypal genotypes of parasites did not show any relationship with either parasite load in amniotic fluid samples or clinical outcome of infants at the age of 12 months.

Targeting Leishmania amazonensis amastigotes through macrophage internalisation of a hydroxymethylnitrofurazone nanostructured polymeric system.

Dextran-coated poly (n-butyl cyanoacrylate) nanoparticles (PBCA-NPs) were prepared and were evaluated for enhanced delivery of a promising anti-Leishmania drug candidate, hydroxymethylnitrofurazone (NFOH), to phagocytic cells. Currently available chemotherapy for leishmaniasis, such as pentavalent antimonials, presents low safety and efficacy. Furthermore, widespread drug resistance in leishmaniasis is rapidly emerging. To overcome these drawbacks, the use of nanosized delivery systems can reduce systemic drug toxicity and increase the drug concentration in infected macrophages, therefore improving treatment of leishmaniasis. PBCA-NPs containing NFOH (PBCA-NFOH-NPs) were prepared by an anionic emulsion polymerisation method. The z-average and polydispersity index (PDI) were determined by photon correlation spectroscopy, the zeta potential by microelectrophoresis and the entrapment efficiency by HPLC. Cytotoxicity was determined using macrophages from BALB/c mice. Efficacy tests were performed using Leishmania amazonensis promastigotes and amastigotes. The z-average of PBCA-NFOH-NPs was 151.5 ± 61.97 nm, with a PDI of 0.104 ± 0.01, a zeta potential of -10.1 ± 6.49 mV and an entrapment efficiency of 64.47 ± 0.43%. Efficacy in amastigotes revealed IC50 values of 0.33 µM and 31.2 µM for the nanostructured and free NFOH, respectively (95-fold increase). The cytotoxicity study indicated low toxicity of the PBCA-NFOH-NPs to macrophages. The selectivity index was 370.6, which is 49-fold higher than free NFOH (7.6). Such findings indicated that improved efficacy could be due to NP internalisation following site-specific drug delivery and reactivation of immune protective reactions by the NP components. Thus, PBCA-NFOH-NPs have the potential to significantly improve the treatment of leishmaniasis, with reduced systemic side effects.

Technical performance of a lateral flow immunoassay for detection of anti-SARS-CoV-2 IgG in the outpatient follow-up of non-severe cases and at different times after vaccination: comparison with enzyme and chemiluminescent immunoassays

ABSTRACT This study assessed the technical performance of a rapid lateral flow immunochromatographic assay (LFIA) for the detection of anti-SARS-CoV-2 IgG and compared LFIA results with chemiluminescent immunoassay (CLIA) results and an in-house enzyme immunoassay (EIA). To this end, a total of 216 whole blood or serum samples from three groups were analyzed: the first group was composed of 68 true negative cases corresponding to blood bank donors, healthy young volunteers, and eight pediatric patients diagnosed with other coronavirus infections. The serum samples from these participants were obtained and stored in a pre-COVID-19 period, thus they were not expected to have COVID-19. In the second group of true positive cases, we chose to replace natural cases of COVID-19 by 96 participants who were expected to have produced anti-SARS-CoV-2 IgG antibodies 30-60 days after the vaccine booster dose. The serum samples were collected on the same day that LFIA were tested either by EIA or CLIA. The third study group was composed of 52 participants (12 adults and 40 children) who did or did not have anti-SARS-CoV-2 IgG antibodies due to specific clinical scenarios. The 12 adults had been vaccinated more than seven months before LFIA testing, and the 40 children had non-severe COVID-19 diagnosed using RT-PCR during the acute phase of infection. They were referred for outpatient follow-up and during this period the serum samples were collected and tested by CLIA and LFIA. All tests were performed by the same healthcare operator and there was no variation of LFIA results when tests were performed on finger prick whole blood or serum samples, so that results were grouped for analysis. LFIA's sensitivity in detecting anti-SARS-CoV-2 IgG antibodies was 90%, specificity 97.6%, efficiency 93%, PPV 98.3%, NPV 86.6%, and likelihood ratio for a positive or a negative result were 37.5 and 0.01 respectively. There was a good agreement (Kappa index of 0.677) between LFIA results and serological (EIA or CLIA) results. In conclusion, LFIA analyzed in this study showed a good technical performance and agreement with reference serological assays (EIA or CLIA), therefore it can be recommended for use in the outpatient follow-up of non-severe cases of COVID-19 and to assess anti-SARS-CoV-2 IgG antibody production induced by vaccination and the antibodies decrease over time. However, LFIAs should be confirmed by using reference serological assays whenever possible.

Genetic diversity of Trypanosoma cruzi strains isolated from chronic chagasic patients and non-human hosts in the state of São Paulo, Brazil

BACKGROUND Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.