Oral infectivity through carnivorism in murine model of Trypanosoma cruzi infection.

Introduction: Oral transmission of T. cruzi is probably the most frequent transmission mechanism in wild animals. This observation led to the hypothesis that consuming raw or undercooked meat from animals infected with T. cruzi may be responsible for transmitting the infection. Therefore, the general objective of this study was to investigate host-pathogen interactions between the parasite and gastric mucosa and the role of meat consumption from infected animals in the oral transmission of T. cruzi. Methods: Cell infectivity assays were performed on AGS cells in the presence or absence of mucin, and the roles of pepsin and acidic pH were determined. Moreover, groups of five female Balb/c mice were fed with muscle tissue obtained from mice in the acute phase of infection by the clone H510 C8C3hvir of T. cruzi, and the infection of the fed mice was monitored by a parasitemia curve. Similarly, we assessed the infective capacity of T. cruzi trypomastigotes and amastigotes by infecting groups of five mice Balb/c females, which were infected orally using a nasogastric probe, and the infection was monitored by a parasitemia curve. Finally, different trypomastigote and amastigote inoculums were used to determine their infective capacities. Adhesion assays of T. cruzi proteins to AGS stomach cells were performed, and the adhered proteins were detected by western blotting using monoclonal or polyclonal antibodies and by LC-MS/MS and bioinformatics analysis. Results: Trypomastigote migration in the presence of mucin was reduced by approximately 30%, whereas in the presence of mucin and pepsin at pH 3.5, only a small proportion of parasites were able to migrate (∼6%). Similarly, the ability of TCTs to infect AGS cells in the presence of mucin is reduced by approximately 20%. In all cases, 60-100% of the animals were fed meat from mice infected in the acute phase or infected with trypomastigotes or amastigotes developed high parasitemia, and 80% died around day 40 post-infection. The adhesion assay showed that cruzipain is a molecule of trypomastigotes and amastigotes that binds to AGS cells. LC-MS/MS and bioinformatics analysis, also confirmed that transialidase, cysteine proteinases, and gp63 may be involved in TCTs attachment or invasion of human stomach cells because they can potentially interact with different proteins in the human stomach mucosa. In addition, several human gastric mucins have cysteine protease cleavage sites. Discussion: Then, under our experimental conditions, consuming meat from infected animals in the acute phase allows the T. cruzi infection. Similarly, trypomastigotes and amastigotes could infect mice when administered orally, whereas cysteinyl proteinases and trans-sialidase appear to be relevant molecules in this infective process.

WITHDRAWN: Bacteroides thetaiotaomicron necrotizing fasciitis as presentation of colorectal cancer, with subsequent cutaneous exteriorization.

Ahead of Print article withdrawn by publisher. An 80-year-old woman presented necrotizing fasciitis on the right flank, requiring debridement. Tomography reported ascending colon neoplasm fistulized to the skin. Colonoscopy confirms adenocarcinoma. Intervention postponed due to rejection of surgery during the pandemic and SARS-COV-2 infection, producing progression with exteriorization of the neoplasm. A bloc laparotomic right hemicolectomy was performed (pT4bN0).

Three-dimensional modelling as a novel interactive tool for preoperative planning for complex perianal fistulas in Crohn's disease.

AIM: The aim of this study is to demonstrate the added value of three-dimensional (3D) reconstruction models and artificial intelligence for preoperative planning in complex perianal Crohn's disease. MRI is the gold standard for diagnosis of complex perianal fistulas and abscess due to its high sensitivity, but it lacks high specificity values. This creates the need for better diagnostic models such as 3D image processing and reconstruction (3D-IPR) with artificial intelligence (AI) algorithms. METHOD: This is a prospective study evaluating the utility of 3D reconstruction models from MRI in four patients with perineal Crohn's disease (pCD). RESULTS: Four pCD patients had 3D reconstruction models made from pelvic MRI. This provided a more visual representation of perianal disease and made possible location of the internal fistula orifice, seton placement in fistula tracts and abscess drainage. CONCLUSION: Three-dimensional reconstruction in CD-associated complex perianal fistulas can facilitate disease interpretation, anatomy and surgical strategy, potentially improving preoperative planning as well as intraoperative assistance. This could probably result in better surgical outcomes to control perianal sepsis and reduce the number of surgical procedures required in these patients.

Trypanosoma cruzi pathogenicity involves virulence factor expression and upregulation of bioenergetic and biosynthetic pathways.

The molecular repertoire of Trypanosoma cruzi effects its virulence and impacts the clinical course of the resulting Chagas disease. This study aimed to determine the mechanism underlying the pathogenicity of T. cruzi. Two T. cruzi cell lines (C8C3hvir and C8C3lvir), obtained from the clone H510 C8C3 and exhibiting different virulence phenotypes, were used to evaluate the parasite's infectivity in mice. The organ parasite load was analysed by qPCR. The proteomes of both T. cruzi cell lines were compared using nLC-MS/MS. Cruzipain (Czp), complement regulatory protein (CRP), trans-sialidase (TS), Tc-85, and sialylated epitope expression levels were evaluated by immunoblotting. High-virulence C8C3hvir was highly infectious in mice and demonstrated three to five times higher infectivity in mouse myocardial cells than low-virulence C8C3lvir. qPCR revealed higher parasite loads in organs of acute as well as chronically C8C3hvir-infected mice than in those of C8C3lvir-infected mice. Comparative quantitative proteomics revealed that 390 of 1547 identified proteins were differentially regulated in C8C3hvir with respect to C8C3lvir. Amongst these, 174 proteins were upregulated in C8C3hvir and 216 were downregulated in C8C3lvir. The upregulated proteins in C8C3hvir were associated with the tricarboxylic acid cycle, ribosomal proteins, and redoxins. Higher levels of Czp, CRP, TS, Tc-85, and sialylated epitopes were expressed in C8C3hvir than in C8C3lvir. Thus, T. cruzi virulence may be related to virulence factor expression as well as upregulation of bioenergetic and biosynthetic pathways proteins.

Demodex folliculorum (Trombidiformes: Demodicidae) and Demodex brevis Prevalence in an Extreme Environment of Chile.

The prevalence of mites of the genus Demodex and their associations with host age, gender, workplace, and comorbid skin and ocular conditions were studied in participants in Antofagasta, Chile, which is in a region with an extreme environment. We examined 680 participants aged 18-88 yr using standardized surface skin biopsies. Among them, Demodex had a prevalence of 13.5 % (95% confidence interval, 10.88-16.17). A slightly higher prevalence was observed in males (51.1%; 95% confidence interval, 40.9-61.3; nonsignificant) and participants in the 69-88 yr age group (50.0%; 95% confidence interval, 23.8-76.2; P < 0.05). Regarding the species involved, Demodex folliculorum was found in 89.1% (CI 82.7-95.5) of cases, while D. brevis was found in the remaining 10.9% of cases. A higher prevalence (25.0% CI 16.1-33.91) was observed in participants who worked indoors in generally enclosed and dust-rich environments (such as theaters, libraries, and administrative offices). There was also a strong association between Demodex prevalence and conjunctival hyperemia, with 35.9% (95% confidence interval, 9.1-35; OR 17.9) of the Demodex positive participants having this pathology compared to 10.3% of the noninfested participants. In summary, the prevalence of Demodex in Antofagasta, Chile (13.5%) was toward the lower end of the range reported among other regions around the world. Environmental factors such as exposure to the sun (including ultraviolet rays) or environmental pollution may affect the mites. In addition, Demodex genetics (related to virulence) and the ocular or skin microbiota may positively or negatively influence infestation and pathology.

Toxicological Characterization and Phospholipase D Activity of the Venom of the Spider Sicarius thomisoides.

Envenomation by Loxosceles spiders (Sicariidae family) has been thoroughly documented. However, little is known about the potential toxicity of members from the Sicarius genus. Only the venom of the Brazilian Sicarius ornatus spider has been toxicologically characterized. In Chile, the Sicarius thomisoides species is widely distributed in desert and semidesert environments, and it is not considered a dangerous spider for humans. This study aimed to characterize the potential toxicity of the Chilean S. thomisoides spider. To do so, specimens of S. thomisoides were captured in the Atacama Desert, the venom was extracted, and the protein concentration was determined. Additionally, the venoms were analyzed by electrophoresis and Western blotting using anti-recombinant L. laeta PLD1 serum. Phospholipase D enzymatic activity was assessed, and the hemolytic and cytotoxic effects were evaluated and compared with those of the L. laeta venom. The S. thomisoides venom was able to hydrolyze sphingomyelin as well as induce complement-dependent hemolysis and the loss of viability of skin fibroblasts with a dermonecrotic effect of the venom in rabbits. The venom of S. thomisoides showed intraspecific variations, with a similar protein pattern as that of L. laeta venom at 32-35 kDa, recognized by serum anti-LlPLD1. In this context, we can conclude that the venom of Sicarius thomisoides is similar to Loxosceles laeta in many aspects, and the dermonecrotic toxin present in their venom could cause severe harm to humans; thus, precautions are necessary to avoid exposure to their bite.

Observational cross-sectional study of Trichomonas tenax in patients with periodontal disease attending a Chilean university dental clinic.

BACKGROUND: The oral flagellated protozoan Trichomonas tenax has been associated with patients with periodontal disease. However, no recent studies have been conducted on the prevalence of T. tenax in Chile. The aim of this study was to determine the presence of T. tenax in patients with periodontal disease, admitted to the Dental Clinic of the University of Antofagasta, Chile, through Polymerase Chain Reaction (PCR) amplification of the beta-tubulin gene. METHODS: An observational, cross-sectional study was conducted on 50 patients diagnosed with periodontal disease, 20 of them with gingivitis and 30 with periodontitis. T. tenax was identified by PCR amplification of the beta-tubulin gene. Associations between the protozoan and periodontal disease or the presence of risk factors to establish T. tenax infection were determined using the chi-square test and binary logistic regression analysis. RESULTS: T. tenax was present in 28 out of 50 (56%) of patients with periodontal disease, and was more prevalent when associated with periodontitis (21 out of 30; 70%) than dental plaque-induced gingivitis (7 out of 20; 35%). Non-statistically-significant associations were observed between the presence of T. tenax and age, gender, smoking habit or diabetes. Statistically significant associations were observed between the presence of T. tenax and periodontal disease, and between T. tenax and the Periodontal Screening and Recording (PSR) index. CONCLUSION: T. tenax showed a high presence in patients with progressive states of periodontal diseases. Consequently, T. tenax detection is strongly recommended in patients with periodontal disease diagnosis and with a PSR index greater than 3.

Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders.

BACKGROUND: Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. METHODS: The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. RESULTS: We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. CONCLUSIONS: People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.

Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.(AU)

Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Background Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.

Phospholipase D from Loxosceles laeta Spider Venom Induces IL-6, IL-8, CXCL1/GRO-α, and CCL2/MCP-1 Production in Human Skin Fibroblasts and Stimulates Monocytes Migration.

Cutaneous loxoscelism envenomation by Loxosceles spiders is characterized by the development of a dermonecrotic lesion, strong inflammatory response, the production of pro-inflammatory mediators, and leukocyte migration to the bite site. The role of phospholipase D (PLD) from Loxosceles in the recruitment and migration of monocytes to the envenomation site has not yet been described. This study reports on the expression and production profiles of cytokines and chemokines in human skin fibroblasts treated with catalytically active and inactive recombinant PLDs from Loxosceles laeta (rLlPLD) and lipid inflammatory mediators ceramide 1-phosphate (C1P) and lysophosphatidic acid (LPA), and the evaluation of their roles in monocyte migration. Recombinant rLlPLD1 (active) and rLlPLD2 (inactive) isoforms induce interleukin (IL)-6, IL-8, CXCL1/GRO-α, and CCL2/monocyte chemoattractant protein-1 (MCP-1) expression and secretion in fibroblasts. Meanwhile, C1P and LPA only exhibited a minor effect on the expression and secretion of these cytokines and chemokines. Moreover, neutralization of both enzymes with anti-rLlPLD1 antibodies completely inhibited the secretion of these cytokines and chemokines. Importantly, conditioned media from fibroblasts, treated with rLlPLDs, stimulated the transmigration of THP-1 monocytes. Our data demonstrate the direct role of PLDs in chemotactic mediator synthesis for monocytes in human skin fibroblasts and indicate that inflammatory processes play an important role during loxoscelism.

Decreased cruzipain and gp85/trans-sialidase family protein expression contributes to loss of Trypanosoma cruzi trypomastigote virulence.

Two cell lines derived from a single Trypanosoma cruzi clone by long-term passaging generated a highly virulent (C8C3hvir) and a low virulent (C8C3lvir) cell line. The C8C3hvir cell line was highly infective and lethal to Balb/c mice, and the C8C3lvir cell line was three- to five-fold less infective to mouse cardiomyocytes than C8C3hvir. The highly virulent T. cruzi cell line abundantly expressed the major cysteine proteinase cruzipain (Czp), complement regulatory protein (CRP) and trans-sialidase (TS), all of which are known to act as virulence factors in this parasite. The in vitro invasion capacity and in vivo Balb/c mouse infectiveness of the highly virulent strain was strongly reduced by pre-treatment with antisense oligonucleotides targeting TS or CRP or with E64d. Based on these results, we conclude that decreased levels of TS, CRP and Czp expression could contribute to loss of T. cruzi trypomastigote virulence.

Tryptophan and aspartic acid residues present in the glycerophosphoryl diester phosphodiesterase (GDPD) domain of the Loxosceles laeta phospholipase D are essential for substrate recognition.

It is known that the family of phospholipases D (PLD) from spiders of the genus Loxosceles, hydrolyze the substrates sphingomyelin and lisophosphatidylcholine, by their catalytic acid-base action which involves two histidines. However, little is known about the amino acids that participate on substrate recognition. In this study we identified highly conserved amino acids of the glycerophosphoryl diester phosphodiesterase (GDPD) domain of recombinant LlPLD1, which interact with the substrate sphingomyelin. The mutation of W256 to serine and D259 to glycine decreased significantly the sphingomyelinase and hemolytic activity when compared to wild type LlPLD1. The interaction of LlPLD1 with sphingomyelin was also strongly reduced in both mutants LlPLD1-W256S and LlPLD1-D259G. The results show the importance of these residues in the interaction of the protein with its substrate sphingomyelin in cell membranes.

Loxoscelismo: revisión de la epidemiología, clínica, diagnóstico y tratamiento actuales / Loxoscelism: a review of the epidemiology, diagnosis and current clinical treatments

Spiders of the genus Loxosceles have a wide world distribution. In Chile, the brown recluse spider Loxosceles laeta, commonly called the "corner spider", is one of the species which causes the clinical syndrome known as loxoscelism, poisoning by the accidental bite of this arachnid. Loxoscelism is characterized by dermonecrotic lesions (cutaneous loxoscelism), which in more severe cases (systemic loxoscelism) may be accompanied by intravascular hemolysis of erythrocytes and renal insufficiency with a high associated mortality. Treatment requires the precocious application of anti-venom, along with complementary measures based on the use of antihistamines, corticoids and antibiotics. The diagnosis, which currently is eminently clinical, should be accompanied by the identification of the spider when possible, due to the lack of diagnostic methods of rapid response applicable at the level of emergency clinics. The introduction of new tools based on recombinant proteins of highly immunogenic components present in the venom is a recently explored alternative. Preventive methods based on avoidance of contact with the spider continue to be the principal mechanism to avoid an endemic pathology with high risk of death in our region. The objective of this revision is to bring up to date fundamental aspects of loxoscelism, especially its epidemiology, diagnosis and clinical treatment.

Las arañas del género Laxosceles, tienen una amplia distribución mundial. En Chile, Laxosceles laeta (comúnmente conocida como “araña de los rincones”) es una de las especies causante del cuadro clínico conocido como loxoscelismo, envenenamiento por la mordedura de este arácnido por accidente. El loxoscelismo se caracteriza por lesiones dermonecróticas (loxoscelismo cutáneo), que en los casos más graves (loxoscelismo sistemáticos), se puede acompañar con hemólisis intravascular de los eritrocitos e insuficiencia renal con elevada mortalidad asociada. El tratamiento requiere la aplicación precoz del antisuero, junto a mediadas complementarias basada con el uso del antihistamínicos, corticoides y antibióticos. El diagnóstico, en la actualidad eminentemente clínico, debe ser acompañado por la identificación de los ejemplares, cuando es posible, ante la falta de métodos diagnóstico de respuesta precoz aplicable a nivel asistencial de urgencia. La introducción de nuevas herramientas basadas en proteínas recombinantes de componentes altamente inmunogénicos presentes en el veneno, es una alternativa explorada recientemente. Las medidas de prevención basadas en evitar el contacto con la araña, sigue siendo los principales mecanismos para evitar una patología endémica y con elevado riesgo de muerte en nuestra región. El objetivo de la presente revisión es actualizar aspectos fundamentales del loxoscelismo, especialmente sobre su epidemiología, clínica, diagnóstico y tratamiento.

[Effectiveness of a repellent paint against the spider Loxosceles laeta]. / Evaluación experimental de la eficiencia de una pintura repelente para arañas del género Loxosceles.

BACKGROUND: Loxoscelism is a severe reaction to the bite of the spider Loxosceles laeta. In recent years, a paint with repellent properties has been promoted in the commerce. However, there are no reports of experiments evaluating its effectiveness. AIM: To evaluate experimentally the repellent properties of a paint against Loxosceles laeta. MATERIAL AND METHODS: Males, females and nymphs of L laeta were deposited in cockpits that allow the free displacement of the spider. Half of the cockpit was covered with repellent paint. Daily observations during one week, determined how frequently the spiders occupied the space covered with repellent paint. The experiments were run in triplicate. RESULTS: No statistical differences in the occupancy of spaces covered with repellent paint or not covered with it were observed for nymphs (87% and 67%, respectively), males (72% and 77%, respectively) or females (91% and 84%, respectively). CONCLUSIONS: The tested paint does not have a repellent action against the spider Loxosceles laeta.

Evaluación experimental de la eficiencia de una pintura repelente para arañas del género Loxosceles / Effectiveness of a repellent paint against the spider Loxosceles laeta

Background: Loxoscelism is a severe reaction to the bite of the spider Loxosceles laeta. In recent years, a paint with repellent properties has been promoted in the commerce. However, there are no reports of experiments evaluating its effectiveness. Aim: To evaluate experimentally the repellent properties of a paint against Loxosceles laeta. Material and methods: Males, females and nymphs of L laeta were deposited in cockpits that allow the free displacement of the spider. Half of the cockpit was covered with repellent paint. Daily observations during one week, determined how frequently the spiders occupied the space covered with repellent paint. The experiments were run in triplicate. Results: No statisticaldifferences in the occupancy of spaces covered with repellent paint or not covered with it were observed for nymphs (87% and 67%, respectively), males (72% and 77%, respectively) orfemales (91% and 84%, respectively). Conclusions: The tested paint does not have a repellent action against the spider Loxosceles laeta.