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.

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.

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.

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.

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.

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.

Proteomic map of Trypanosoma cruzi CL Brener: the reference strain of the genome project.

In this work two-dimensional gel electrophoresis combined with mass spectrometry was carried out in order to start the construction of a map of soluble proteins from epimastigote form of Trypanosoma cruzi CL Brener. This strain is a hybrid organism derived from two genotypes, T. cruzi I and T. cruzi II and was chosen for genome sequencing. The two-dimensional gel electrophoresis showed that most of proteins focused at 4-7 pH range. The identification demonstrated that several proteins were in multiple isoforms, such as tubulin and heat shock proteins. Potential targets for development of chemotherapeutic agents like arginine kinase, an enzyme absent from mammalian tissues that is involved in the energy supply of the parasite, were also detected.

Ancestral genomes, sex, and the population structure of Trypanosoma cruzi.

Acquisition of detailed knowledge of the structure and evolution of Trypanosoma cruzi populations is essential for control of Chagas disease. We profiled 75 strains of the parasite with five nuclear microsatellite loci, 24Salpha RNA genes, and sequence polymorphisms in the mitochondrial cytochrome oxidase subunit II gene. We also used sequences available in GenBank for the mitochondrial genes cytochrome B and NADH dehydrogenase subunit 1. A multidimensional scaling plot (MDS) based in microsatellite data divided the parasites into four clusters corresponding to T. cruzi I (MDS-cluster A), T. cruzi II (MDS-cluster C), a third group of T. cruzi strains (MDS-cluster B), and hybrid strains (MDS-cluster BH). The first two clusters matched respectively mitochondrial clades A and C, while the other two belonged to mitochondrial clade B. The 24Salpha rDNA and microsatellite profiling data were combined into multilocus genotypes that were analyzed by the haplotype reconstruction program PHASE. We identified 141 haplotypes that were clearly distributed into three haplogroups (X, Y, and Z). All strains belonging to T. cruzi I (MDS-cluster A) were Z/Z, the T. cruzi II strains (MDS-cluster C) were Y/Y, and those belonging to MDS-cluster B (unclassified T. cruzi) had X/X haplogroup genotypes. The strains grouped in the MDS-cluster BH were X/Y, confirming their hybrid character. Based on these results we propose the following minimal scenario for T. cruzi evolution. In a distant past there were at a minimum three ancestral lineages that we may call, respectively, T. cruzi I, T. cruzi II, and T. cruzi III. At least two hybridization events involving T. cruzi II and T. cruzi III produced evolutionarily viable progeny. In both events, the mitochondrial recipient (as identified by the mitochondrial clade of the hybrid strains) was T. cruzi II and the mitochondrial donor was T. cruzi III.

Cellular localization and expression of gp63 homologous metalloproteases in Leishmania (Viannia) braziliensis strains.

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that encompasses a broad spectrum of clinical manifestations. In a previous study, we showed that Brazilian and Colombian L. braziliensis strains, isolated from patients with distinct clinical manifestations, display different pattern of metalloprotease activities. Following these results, we investigated the cellular localization of these molecules and their relation to the major surface protease (gp63) of Leishmania. Comparative analyses of metalloprotease expression among different clinical isolates as well as an evaluation of the effect of long-term in vitro passage on the expression pattern of these metalloproteases were also performed. Western blot analysis, using an anti-gp63 antibody, revealed polypeptide patterns with a similar profile to that observed in zymographic analysis. Flow cytometry and fluorescence microscopy analyses corroborated the presence of metalloproteases with homologous domains to gp63 in the parasites and revealed differences in the expression level of such molecules among the isolates. The cellular distribution of metalloproteases, assessed by confocal analysis, showed the existence of intracellular metalloproteases with homologous domains to gp63, predominantly located near the flagellar pocket. Finally, it was observed that differential zymographic profiles of metalloproteases exhibited by L. (V.) braziliensis isolates remain unaltered during prolonged in vitro culture, suggesting that the proteolytic activity pattern is a stable phenotypic characteristic of these parasites.

Association of the presence of residual anti-Toxoplasma gondii IgM in pregnant women and their respective family groups in Miracema, Northwest Rio de Janeiro, Brazil.

The seroprevalence of toxoplasmosis in 832 pregnant women in Miracema, Rio de Janeiro, was determined and 75.1% (625) and 2.0% (17) were anti-Toxoplasma gondii IgG and IgM positive, respectively. Out of the 17 IgM positive pregnant women, only one had low avidity IgG corresponding to the acute phase of the infection. All the other women presented with high avidity IgG and also presented with residual IgM anti-T. gondii. Of this sample, 106 received home visits (this includes 11 family nuclei of pregnant women with residual IgM anti-T. gondii, 68 nuclei of only IgG positive pregnant women and 27 nuclei of pregnant women with no antibodies to anti-T. gondii), resulting in 267 individuals visited. Out of these 267 individuals, 21 were positive for IgG and IgM anti-T. gondii and were candidates for the IgG avidity test. All of them presented with high avidity IgG and residual IgM. Five of these IgM+ individuals were (5/238; 2.1%) relatives of IgM negative pregnant women. The other 16 (16/29; 55.2%) were relatives of IgM+ pregnant women who were positive for residual IgM anti-T. gondii. This association was statistically significant (p = 0.0000). The analysis presented herein raises questions regarding the presence of residual IgM anti-T. gondii such as genetic determinants or even constant antigenic stimuli for the same family cluster.

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.

Proteome analysis of Leishmania (Viannia) braziliensis by two-dimensional gel electrophoresis and mass spectrometry.

Leishmania (Viannia) braziliensis, a protozoan parasite widespread in the New World, is responsible for the infection of different mammal orders, including humans. This species is considered to be a major etiological agent of American cutaneous leishmaniasis. A proteomic study was carried out to identify proteins expressed by L. (V.) braziliensis. One hundred and one spots representing 75 protein entries were identified by MALDI-TOF-TOF. Isoelectric point values estimated by gel electrophoresis matched closely with predicted values, although some discrepancies existed suggesting that post-translational protein modifications may be common in L. braziliensis. Moreover, 20 hypothetical proteins were experimentally identified. Identified proteins were classified into 15 groups according to biological process. Among the proteins identified, approximately 40% have not been previously reported in a proteomic map of Leishmania. In addition, a number of potential virulence factors and drug targets were identified in this protein map, including some proteins associated with the metastatic phenotype. This study describes the first compilation of a proteomic reference map for L. braziliensis (pI 4-7, M(r) 10-130 kDa) and provides a very useful tool for comparative studies of strains isolated from patients presenting different clinical manifestations of leishmaniasis as well as a potential tool to identify markers for clinical diagnosis, therapeutics, and prognosis.

Cross sectional study reveals a high percentage of asymptomatic Plasmodium vivax infection in the Amazon Rio Negro area, Brazil.

A parasitological, clinical, serological and molecular cross-sectional study carried out in a highly endemic malaria area of Rio Negro in the Amazon State, Brazil, revealed a high prevalence of asymptomatic Plasmodium vivax infection. A total of 109 persons from 25 families were studied in five villages. Ninety-nine inhabitants (90.8%) had at least one previous episode of malaria. Serology showed 85.7% and 46.9% of positivity when P. falciparum antigens and P. vivax MSP-1, respectively, were used. Twenty blood samples were PCR positive for P. vivax (20.4%) and no P. falciparum infection was evidenced by this technique. No individual presenting positive PCR reaction had clinical malaria during the survey neither in the six months before nor after, confirming that they were cases of asymptomatic infection. Only one 12 year old girl presented a positive thick blood smear for P. vivax. This is the first description of asymptomatic Plasmodium infection in this area studied.

Haematogenous dissemination of Leishmania (Viannia) braziliensis in human American tegumentary leishmaniasis.

One of the potential dangers of American tegumentary leishmaniasis (ATL) caused by Leishmania (Viannia) braziliensis is the development of mucosal lesions. Haematogenous dissemination of the parasite is the most likely mechanism to explain this occurrence, but most attempts to isolate the parasite from blood have so far been unsuccessful. The presence of Leishmania in peripheral blood was therefore evaluated by PCR using DNA samples isolated from patients presenting active cutaneous or mucosal disease, and from individuals cured by antimonial treatment as well as individuals without a past history of leishmaniasis but with a positive Montenegro skin test, all living in L. (V.) braziliensis-endemic areas. Leishmania DNA was found not only in those patients presenting active cutaneous (24.8%) or mucosal (35%) lesions, but also in samples isolated from healed individuals (27.3%) as well as in asymptomatic skin-test-positive residents of endemic areas (37.5%). Overall, PCR showed the presence of parasite DNA in the blood of 26.2% of the 225 examined samples. These data suggest that persistence of parasites within the host may last for many years and, rather than being a risk factor, might be important in maintaining the protective response in those living in endemic areas.

Emerging Chagas disease in Amazonian Brazil.

In the Amazon Basin, Trypanosoma cruzi infection is enzootic, involving a variety of wild mammals and at least 10 of the 16 reported silvatic triatomine bug species. Human cases of Chagas disease are increasing, indicating that the disease may be emerging as a wider public health problem in the region: 38 cases from 1969 to 1992, and 167 in the past eight years. This article reviews the status of Chagas disease in Amazonian Brazil, including known reservoirs and vectors, and the genetic diversity of T. cruzi. At least three subspecific groups of T. cruzi-T. cruzilZ1, T. cruziZ3 and T. cruziZ3/Z1 ASAT--are present. It appears that T. cruzil has an extant capacity for genetic exchange. Attention is also drawn to the risk of domestic endemicity, in addition to the tasks facing the disease control authorities.

Chronic Chagas' disease in rhesus monkeys (Macaca mulatta): evaluation of parasitemia, serology, electrocardiography, echocardiography, and radiology.

Severe chronic damage to the heart and gastrointestinal tract in patients with Chagas' disease are often observed 10-20 years after the acute phase. The course of long-lasting infection with the Colombian strain of Trypanosoma cruzi was studied in seven rhesus monkeys infected for 15-19 years. Subpatent parasitemia was detected in all studied animals, using hemoculture (two of seven), artificial xenodiagnosis (three of seven), and a polymerase chain reaction PCR (six of six). High titers of specific IgG antibody to T. cruzi persisted throughout the chronic phase of infection. Abnormal electrocardiographic (three of six) and echocardiographic (one of six) patterns detected in the T. cruzi-infected monkeys were possibly related to parasite-triggered myocardial damage. The results suggest that rhesus monkeys experimentally infected with T. cruzi, besides reproducing the acute phase of Chagas' disease, also develop chronic chagasic cardiomyopathy.

Molecular markers for species identification in the Leishmania subgenus Viannia.

We have previously identified a novel genomic sequence of 500 bp, the beta 500-DNA sequence, in the subgenus Leishmania (Viannia). This sequence was localized upstream of the beta-tubulin gene. Restriction fragment length polymorphism and hybridization analysis has shown that the beta 500-DNA sequence is specific to this subgenus. A polymerase chain reaction (PCR) assay confirmed this specificity. The beta 500-DNA sequence was apparently absent from the genomic deoxyribonucleic acid of L. colombiensis and L. equatoriensis. These results indicate that a PCR assay based on the beta 500-DNA sequence is likely to be of use to detect and identify Leishmania parasites of this subgenus in clinical samples with high sensitivity, specificity and reliability. The beta 500-DNA sequence can be considered a molecular marker for the subgenus Viannia.

Lack of technical specificity in the molecular diagnosis of toxoplasmosis.

The polymerase chain reaction amplification of a fragment of the B1 gene of Toxoplasma gondii coupled to hybridization was performed in 42 patients from Rio de Janeiro, Brazil. The results showed 50% of positivity in the IgM positive toxoplasmosis group, and 12.5% in the positive IgG and negative IgM individuals. The data presented here revealed a lack of specificity of the molecular approach, clearly indicating that the primers used may co-amplify human sequences.

High prevalence of Trypanosoma rangeli and Trypanosoma cruzi in opossums and triatomids in a formerly-endemic area of Chagas disease in Southeast Brazil.

In Brazil Trypanosoma rangeli has been detected in humans, sylvatic mammals and vectors in the Amazon Basin and in wild rodents in a Southern State. Here we report for the first time a high prevalence of T. rangeli in opossums and triatomids captured in peridomestic environments in a formerly-endemic area of Chagas disease in Southeast Brazil. Five molecular typing tools clearly indicate the presence of T. rangeli and Trypanosoma cruzi in mammalian reservoirs and triatomids. Twenty-one opossums (Didelphis albiventris) were captured and flagellates were detected in the blood of 57.1% (12/21) of the animals. Single infections with T. rangeli or T. cruzi were diagnosed, respectively, in 58.4 and 8.3% of the opossums. Mixed infections were observed in 33.3%. Forty-four triatomids (38 Rhodnius neglectus and 6 Panstrongylus megistus) were collected in palm trees within 50 m from human dwellings. Flagellates were observed in the digestive tract and feces of 50% of the insects. PCR assays performed in DNA samples obtained from 16 cultures of the intestinal tract revealed single infection with T. cruzi (68.7%) or T. rangeli (6.3%), as well as mixed infections (25%). T. rangeli was also detected in the hemolymph of two specimens. Genotyping revealed predominance of T. cruzi I. The data suggest that R. neglectus in conjunction with D. albiventris may be significant factors in the maintenance of the sylvatic and peridomestic cycles of T. rangeli in the region. The finding of T. cruzi and T. rangeli in triatomine species capable of domiciliation and therefore considered as alternative vectors for the parasite transmission opens up the possibility of re-establishment of Chagas disease following reinfestation of houses.

Trypanosoma cruzi transmission in a captive primate unit, Rio de Janeiro, Brazil.

A breeding in captivity program of neotropical primates for subsequent reintroduction in nature is in progress at the Primatology Center of Rio de Janeiro (CPRJ). Almost 200 animals of 20 species that include both wild captured animals and specimens born in captivity are maintained in CPRJ. Here, we examined 198 primates of CPRJ for infection with the protozoan parasite Trypanosoma cruzi. The animals included 18 species of eight genera. We also performed an "ad lib" search for triatomines that could be incriminated as putative transmitters of the protozoan in this scenario. Anti-T. cruzi antibodies were observed (by indirect immunofluorescence assay-IFA) in 40 monkeys (26.5%). Four Panstrongylus megistus were collected in the monkey's food storage room near the cages and in human dwellings in the proximity to CPRJ. T. cruzi were isolated from nine primates of two genera (Leontopithecus and Saguinus) and from two individuals of the vector P. megistus. The transmission inside the cages could be attested by the isolation of the T. cruzi from primates born in captivity. Multi-locus enzyme electrophoresis (MLEE) demonstrated that the two isolates from Saguinus bicolor bicolor displayed a zymodeme 1 profile in four out of five tested enzymes, while all isolates derived from Leontopithecus showed zymodeme 2 for four out of the five tested enzymes. Mini-exon gene analysis genotyped all isolates as T. cruzi II, which is associated with human disease in Brazil. A wild primate unit such as CPRJ, located inside the forest and near to human dwellings and with T. cruzi II infected animals, deserves a careful surveillance in order to prevent expansion of the infection.