Studies of Cytotoxicity Effects, SARS-CoV-2 Main Protease Inhibition, and in Silico Interactions of Synthetic Chalcones.

SARS-CoV-2 main protease (Mpro ) plays an essential role in proteolysis cleavage that promotes coronavirus replication. Thus, attenuating the activity of this enzyme represents a strategy to develop antiviral agents. We report inhibitory effects against Mpro of 40 synthetic chalcones, and cytotoxicity activities, hemolysis, and in silico interactions of active compounds. Seven of them bearing a (E)-3-(furan-2-yl)-1-arylprop-2-en-1-one skeleton (10, 28, and 35-39) showed enzyme inhibition with IC50 ranging from 13.76 and 36.13 µM. Except for 35 and 36, other active compounds were not cytotoxic up to 150 µM against THP-1 and Vero cell lines. Compounds 10, and 35-39 showed no hemolysis while 28 was weakly hemotoxic at 150 µM. Moreover, molecular docking showed interactions between compound 10 and Mpro (PDBID 5RG2 and 5RG3) with proximity to cys145 and His41, suggesting a covalent binding. Products of the reaction between chalcones and cyclohexanethiol indicated that this binding could be a Michael addition type.

The presence of Giardia lamblia assemblage A in dogs suggests an anthropozoonotic cycle of the parasite in Rio de Janeiro, Brazil.

BACKGROUND: Giardia lamblia is a zoonotic protozoan that is classified into 8 genotypes and is distributed worldwide. Assemblages A and B were found to infect dogs and humans, whereas assemblages C and D are dog host-specific. Our objective was to investigate the G. lamblia genotypes circulating in a canine population in Rio de Janeiro, RJ. RESULTS: Sixty stool samples positive for G. lamblia from street dogs were characterized. Fragments of the conserved genes encoding beta-giardin (ß-gia) and glutamate dehydrogenase (gdh) were used as targets. The sequences from beta-giardin and glutamate dehydrogenase genes obtained from all 60 dog samples were 100% similar to G. lamblia genotype A. CONCLUSION: The detection of genotype A suggests that G. lamblia transmission in Rio de Janeiro has a predominantly anthropozoonotic cycle.

Identification of Giardia lamblia Assemblage E in Humans Points to a New Anthropozoonotic Cycle.

Giardia lamblia is a pathogen transmitted by water and food that causes infection worldwide. Giardia genotypes are classified into 8 assemblages (A-H). Assemblages A and B are detected in humans, but they are potentially zoonotic because they infect other mammalian hosts. Giardia in samples from 44 children was genotyped. Conserved fragments of the genes encoding ß-giardin and glutamate dehydrogenase were sequenced and their alignment were carried out with sequences deposited in GenBank. As expected for Rio de Janeiro, the majority of samples were related to assemblage A. Surprisingly, assemblage E was detected in 15 samples. Detection of assemblage E in humans suggests a new zoonotic route of Giardia transmission.

Hospital-associated methicillin-resistant Staphylococcus aureus carrying the PVL gene outbreak in a Public Hospital in Rio de Janeiro, Brazil

Hospital associated methicillin-resist Staphylococcus aureus has long been associated to outbreaks in the hospital environment. In this work, we investigated an outbreak of Hospital associated methicillin-resist Staphylococcus aureus carrying the Panton-Valentine leukocidin gene, which occurred in a large community hospital in Rio de Janeiro, Brazil.

The compositional landscape of minicircle sequences isolated from active lesions and scars of American cutaneous leishmaniasis.

BACKGROUND: American cutaneous leishmaniasis (ACL) is characterized by cutaneous lesions that heal spontaneously or after specific treatment. This paper reports on the analysis of kDNA minicircle sequences from clinical samples (typical lesions and scars) that were PCR-amplified with specific primers for Leishmania species of the subgenus Viannia. METHODS: From 56 clinical isolates we obtained a single amplified fragment (ca. 790 bp), which after cloning and sequencing resulted in 290 minicircle sequences from both active lesions and scars. We aimed to get a compositional profile of these sequences in clinical samples and evaluate the corresponding compositional changes. Sequences were analyzed with the compseq and wordcount (Emboss package) to get the composition of di-, tri-, tetra-, penta- and hexanucleotides. Additionally, we built a nucleotide dictionary with words of 7, 8, 9 and 10 nucleotides. RESULTS: This compositional analysis showed that minicircles amplified from active cutaneous lesions and scars have a distinct compositional profile as viewed by nucleotide composition of words up to 10mer. With regard to the most frequent nucleotide words above length 6, there is also a distinct pattern for 7, 8, 9 and 10mer. CONCLUSION: These results indicate that minicircle sequences can be monitored upon direct exposure to a selection/stressing environment (e.g. chemical action) by evaluating their nucleotide compositional profile. It might be useful as a molecular tool in research concerning the evolution of infecting Leishmania in both vector and vertebrate hosts.

Hospital-associated methicillin-resistant Staphylococcus aureus carrying the PVL gene outbreak in a Public Hospital in Rio de Janeiro, Brazil.

Hospital associated methicillin-resist Staphylococcus aureus has long been associated to outbreaks in the hospital environment. In this work, we investigated an outbreak of Hospital associated methicillin-resist Staphylococcus aureus carrying the Panton-Valentine leukocidin gene, which occurred in a large community hospital in Rio de Janeiro, Brazil.

Genetic polymorphism for IFNγ +874T/A in patients with acute toxoplasmosis / Polimorfismo genético para IFNγ +874T/A em pacientes com toxoplasmose aguda

INTRODUCTION: A single nucleotide polymorphism (SNP) in the gene encoding gamma interferon influences its production and is associated with severity of infectious diseases. This study aimed to evaluate the association of IFNγ+874T/A SNP with duration of disease, morbidity, and development of retinochoroiditis in acute toxoplasmosis. METHODS: A case-control study was conducted among 30 patients and 90 controls. RESULTS: Although statistical associations were not confirmed, A-allele was more common among retinochoroiditis cases and prolonged illness, while T-allele was more frequent in severe disease. CONCLUSIONS: Despite few cases, the results could indicate a relation between IFNγ+874T/A single nucleotide polymorphism and clinical manifestations of toxoplasmosis.

INTRODUÇÃO: Um polimorfismo de nucleotideo único (SNP) no gene codificante para interferon gama influencia a sua produção e pode estar associado à gravidade de diversas doenças infecciosas. O objetivo deste estudo foi avaliar a associação entre SNP para IFNγ+874T/A com a duração da doença, a morbidade e o desenvolvimento de retinocoroidite na toxoplasmose aguda. MÉTODOS: Estudo de caso-controle incluindo 30 pacientes e 90 controles. RESULTADOS: Apesar da ausência de associação estatística, o alelo A foi mais comum entre os casos com retinocoroidite e doença prolongada e o alelo T nas formas mais severas. CONCLUSÕES: Os dados encontrados sugerem uma relação entre o polimorfismo de base única em IFNγ+874T/A com a morbidade e com o desenvolvimento de retinocoroidite por toxoplasmose.

Investigation of host candidate malaria-associated risk/protective SNPs in a Brazilian Amazonian population.

The Brazilian Amazon is a hypo-endemic malaria region with nearly 300,000 cases each year. A variety of genetic polymorphisms, particularly in erythrocyte receptors and immune response related genes, have been described to be associated with susceptibility and resistance to malaria. In order to identify polymorphisms that might be associated with malaria clinical outcomes in a Brazilian Amazonian population, sixty-four human single nucleotide polymorphisms in 37 genes were analyzed using a Sequenom massARRAY iPLEX platform. A total of 648 individuals from two malaria endemic areas were studied, including 535 malaria cases (113 individuals with clinical mild malaria, 122 individuals with asymptomatic infection and 300 individuals with history of previous mild malaria) and 113 health controls with no history of malaria. The data revealed significant associations (p<0.003) between one SNP in the IL10 gene (rs1800896) and one SNP in the TLR4 gene (rs4986790) with reduced risk for clinical malaria, one SNP in the IRF1 gene (rs2706384) with increased risk for clinical malaria, one SNP in the LTA gene (rs909253) with protection from clinical malaria and one SNP in the TNF gene (RS1800750) associated with susceptibility to clinical malaria. Also, a new association was found between a SNP in the CTL4 gene (rs2242665), located at the major histocompatibility complex III region, and reduced risk for clinical malaria. This study represents the first association study from an Amazonian population involving a large number of host genetic polymorphisms with susceptibility or resistance to Plasmodium infection and malaria outcomes. Further studies should include a larger number of individuals, refined parameters and a fine-scale map obtained through DNA sequencing to increase the knowledge of the Amazonian population genetic diversity.

Echinococcus granulosus genotypes circulating in alpacas (Lama pacos) and pigs (Sus scrofa) from an endemic region in Peru.

The identification of the genotypes of Echinococcus granulosus present in livestock and wild animals within regions endemic for cystic echinococcosis (CE) is epidemiologically important. Individual strains display different biological characteristics that contribute to outbreaks of CE and that must be taken into account in the design of intervention programs. In this study, samples of hydatid cysts due to E. granulosus were collected from alpacas (4) in Puno and pigs (8) in Ayacucho in Peru, an endemic region for CE. Polymerase chain reaction amplification and DNA sequencing of specific regions of the mitochondrial cytochrome C oxidase subunit 1 and NADH dehydrogenase subunit 1 genes confirmed the presence of a strain common to sheep, the G1 genotype, in alpacas. Two different strains of E. granulosus were identified in pigs: the G1 and the G7 genotypes. This is the first report of the G1 genotype of E. granulosus in alpacas in endemic regions of CE in Peru.

Echinococcus granulosus genotypes circulating in alpacas (Lama pacos) and pigs (Sus scrofa) from an endemic region in Peru

The identification of the genotypes of Echinococcus granulosus present in livestock and wild animals within regions endemic for cystic echinococcosis (CE) is epidemiologically important. Individual strains display different biological characteristics that contribute to outbreaks of CE and that must be taken into account in the design of intervention programs. In this study, samples of hydatid cysts due to E. granulosus were collected from alpacas (4) in Puno and pigs (8) in Ayacucho in Peru, an endemic region for CE. Polymerase chain reaction amplification and DNA sequencing of specific regions of the mitochondrial cytochrome C oxidase subunit 1 and NADH dehydrogenase subunit 1 genes confirmed the presence of a strain common to sheep, the G1 genotype, in alpacas. Two different strains of E. granulosus were identified in pigs: the G1 and the G7 genotypes. This is the first report of the G1 genotype of E. granulosus in alpacas in endemic regions of CE in Peru.

Genetic polymorphism for IFNγ +874T/A in patients with acute toxoplasmosis.

INTRODUCTION: A single nucleotide polymorphism (SNP) in the gene encoding gamma interferon influences its production and is associated with severity of infectious diseases. This study aimed to evaluate the association of IFNγ+874T/A SNP with duration of disease, morbidity, and development of retinochoroiditis in acute toxoplasmosis. METHODS: A case-control study was conducted among 30 patients and 90 controls. RESULTS: Although statistical associations were not confirmed, A-allele was more common among retinochoroiditis cases and prolonged illness, while T-allele was more frequent in severe disease. CONCLUSIONS: Despite few cases, the results could indicate a relation between IFNγ+874T/A single nucleotide polymorphism and clinical manifestations of toxoplasmosis.

Giardiasis as zoonosis: between proof of principle and paradigm in the Northwestern region of São Paulo State, Brazil.

OBJECTIVE: In order to evaluate the potential zoonotic transmission of Giardia duodenalis, isolates from humans and dogs in the Northwestern region of the São Paulo State, Brazil were characterized based on the ß-giardin gene. METHODS: The samples were analyzed by sequencing of the Nested-PCR products. RESULTS: The A1 and A2 subgenotypes were detected in human and dogs. Cysts of assemblage B, C and D have not been found in any isolates studied. CONCLUSIONS: These results are consistent with the view that giardiasis in the largest endemic region of the Brazil should not be seen as a single entity.

Giardiasis as zoonosis: between proof of principle and paradigm in the Northwestern region of São Paulo State, Brazil

OBJECTIVE: In order to evaluate the potential zoonotic transmission of Giardia duodenalis, isolates from humans and dogs in the Northwestern region of the São Paulo State, Brazil were characterized based on the β-giardin gene. METHODS: The samples were analyzed by sequencing of the Nested-PCR products. RESULTS: The A1 and A2 subgenotypes were detected in human and dogs. Cysts of assemblage B, C and D have not been found in any isolates studied. CONCLUSIONS: These results are consistent with the view that giardiasis in the largest endemic region of the Brazil should not be seen as a single entity.

Molecular characterization of Echinococcus granulosus from Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene.

Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosus worldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

Effects of doxycycline on the endosymbiont Wolbachia in Dirofilaria immitis (Leidy, 1856)--naturally infected dogs.

Dirofilaria immitis carries intracellular endosymbiotic bacteria of the genus Wolbachia, known to be vital for the worms and sensitive to tetracycline antibiotics. With the purpose of studying the interaction between D. immitis and the endosymbiont Wolbachia sp., heartworm naturally infected microfilaremic or antigenemic dogs were treated with doxycycline (10mg/kg/day of the drug in three cycles of 21 days each, with 6-month intervals). Blood samples were collected on days 0, 7 and 21 of each treatment as well as on day 111 after the beginning of each cycle. A final sample was collected on day 723 from the beginning of the first treatment. The samples were examined for the presence and number of microfilariae and the presence of antigen as well as the presences of D. immitis and Wolbachia sp. DNA using PCR (polymerase chain reaction). With this approach, an evaluation of the effect of doxycycline on antigenemia and on the presence of Wolbachia sp. DNA in dogs with heartworm infection was possible. Doxycycline treatment did not alter the detection of adult parasite antigens with the exception of two animals, though the number of animals carrying Wolbachia sp. DNA decreased, despite the presence of the microfilariae. The effect of the antibiotic therapy on the worms may have interfered with the transmission of heartworm disease because the population of microfilariae and the number of microfilaremic dogs were reduced and the microfilariae positive samples that were found did not test positive for Wolbachia sp. in many cases. These findings suggest that in areas were doxycycline is extensively used D. immitis transmission may be impaired by the reduction on the number of microfilariae and on the endosymbiotic bacteria in the larvae turning them incapable of completing development once they infected a new host.

Molecular characterization of Echinococcus granulosusfrom Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene

Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosusworldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

Proteomic analysis of two Trypanosoma cruzi zymodeme 3 strains.

Two Trypanosoma cruzi Z3 strains, designated as 3663 and 4167, were previously isolated from insect vectors captured in the Brazilian Amazon region. These strains exhibited different infection patterns in Vero, C6/36, RAW 264.7 and HEp-2 cell lineages, in which 3663 trypomastigote form was much less infective than 4167 ones. A proteomic approach was applied to investigate the differences in the global patterns of protein expression in these two Z3 strains. Two-dimensional (2D) protein maps were generated and certain spots were identified by mass spectrometry (MS). Our analyses revealed a significant difference in the expression profile of different proteins between strains 3663 and 4167. Among them, cruzipain, an important regulator of infectivity. This data was corroborated by flow cytometry analysis using anti-cruzipain antibody. This difference could contribute to the infectivity profiles observed for each strain by in vitro assay using different cell lines.

Detection of Wolbachia DNA in blood from dogs infected with Dirofilaria immitis.

Dirofilaria immitis is the causative agent of heartworm disease in canines and felines, and pulmonary dirofilariasis in man. It harbors a symbiotic intracellular bacterium from the genus Wolbachia that plays an important role in its biology and contributes to the inflammatory pathology of the heartworm. This endosymbiont is sensitive to the tetracycline family of antibiotics prompting its use in the treatment of filariasis. To track Wolbachia during treatment, primers were designed based on the FtsZ gene from Wolbachia. These primers amplify a single PCR product with the expected size from DNA samples derived from various species of worms that harbor Wolbachia (D. immitis, Brugia malayi and Brugia pahangy). The detection limit of Wolbachia DNA in the assay was 80 pg of D. immitis DNA. Furthermore, the primer set successfully amplified the expected PCR product using blood samples from dogs harboring the heartworm and circulating microfilariae.

Isotopic biomarker discovery and application in translational medicine.

Rational drug discovery relies on pathognomonic molecular reporters of disease or biomarkers. Therefore biomarkers contain relational or contextual information about disease pathophysiology. Two broad pathways can be taken to identify biomarkers: a 'top-down', holistic approach that makes no assumptions about biomarker type, or the 'bottom-up' approach, which is hypothesis driven and relies on a priori information. Both approaches involve parallel or sequential methods that include genomic and proteomic profiling. Biomarker discovery and translational medicine owe much to isotopic techniques because these provide near-real-time information about disease status as diagnostics, in drug delivery and for monitoring treatment. Here, we provide an overview of recent developments and some insight into the future role of isotopes in biomarker discovery and disease therapy.

Analysis of an acute Chagas disease outbreak in the Brazilian Amazon: human cases, triatomines, reservoir mammals and parasites.

An outbreak of Chagas disease occurred in Mazagão, Amapá, Brazilian Amazon in 1996. Seventeen of 26 inhabitants presented symptoms compatible with acute Chagas disease and were submitted to parasitological and serological tests. All 17 were positive in at least one parasitological test and 11 were also IgM or IgG anti-Trypanosoma cruzi positive. The nine asymptomatic patients were negative for parasites and one was positive for IgG anti-T. cruzi. Sixty-eight triatomines were captured (66 Rhodnius pictipes; two Panstrongylus geniculatus); 45 were infected with T. cruzi (43 R. pictipes; two P. geniculatus). Thirteen trypanosomatid strains were isolated: eight from humans and five from R. pictipes. Four were genotyped as T. cruzi I (two from humans; two from R. pictipes), seven as T. cruzi Z3 (six from humans; one from R. pictipes) and two as T. cruzi Z3 and T. rangeli (from R. pictipes). Treatment started for all patients leading to a decrease in parasitaemia in 16 during the follow-up period (6 months, 1, 5 and 7 years). All were serologically negative 7 years post-treatment. There was an overlap of genotypes in the same ecotope, raising the possibility of transmission through the oral route and the need for early therapeutic intervention for better patient management in the Brazilian Amazon.