XXI ExpoPPGIm Annual Meeting of the Graduate Program in Immunology, Online, 1-3 September 2021

These proceedings contains 30 articles that covered various topics related to immunology and related fields. The conference papers presented focused on investigating the role of genetics, microbiome, and immunological pathways in disease pathogenesis and treatment. Studies presented at the conference explored the genetic factors associated with obesity in Brazilian children, the role of flavonoids in reprogramming microglia towards a neuroprotective inflammatory profile, the gut microbiome in asthmatic individuals, and the involvement of the MTOR gene and its variants in the severity of COVID-19. Other studies evaluated the immunodiagnostic potential of a protein exclusive to Corynebacterium pseudotuberculosis, genetic markers associated with alcohol dependence and asthma, and the effects of nicotine on glial cells in Parkinson's disease. The conference also presented research on the molecular mechanisms associated with the anti-glioma and immunomodulatory effects of flavonoids, the influence of Trypanosoma cruzi co-infection on the immune response and clinical outcome of patients with cutaneous leishmaniasis, and the association of metalloproteinase gene variants with periodontitis. Furthermore, the papers presented discussed the production of Zika virus singular peptide for the development of serological immunassays, and the role of genetic polymorphisms in the IL1B and IL6 genes in periodontitis. Lastly, the conference included research on the immunological response of broiler chickens fed with diet supplemented with zinc, and the modulatory effects of Agatis flavone on the glial response in an ex vivo model of brain trauma.

Pfizer-BioNTech vaccine induces the production of cross-reactive antibodies against Trypanosoma cruzi proteins: A preliminary study.

OBJECTIVE: To evaluate the presence of cross-reactivity by anti-severe acute respiratory syndrome coronavirus 2 antibodies induced by the Pfizer-BioNTech vaccine against Trypanosoma cruzi proteins in a screening test. METHODS: Forty-three serum samples were obtained from personnel at the Hospital General Naval de Alta Especialidad in Mexico City who received one or two doses of the vaccine and were tested for T. cruzi infection using four tests: two 'in house' enzyme-linked immunosorbent assays (ELISAs), a commercial ELISA diagnostic kit and an immunoblot test. RESULTS: IgG antibodies against the T. cruzi proteins were present in the serum of unvaccinated subjects and subjects who had received one or two doses of the vaccine. The positivity of the samples against T. cruzi was ruled out by means of a Western Blot assay, where all samples were negative for T. cruzi. CONCLUSION: The data suggest that people convalescing from coronavirus disease 2019 and those who received the Pfizer-BioNTech vaccine exhibit cross-reactive antibodies against T. cruzi antigens in ELISA assays.

African animal trypanocide resistance: A systematic review and meta-analysis.

Background: African animal trypanocide resistance (AATr) continues to undermine global efforts to eliminate the transmission of African trypanosomiasis in endemic communities. The continued lack of new trypanocides has precipitated drug misuse and overuse, thus contributing to the development of the AATr phenotype. In this study, we investigated the threat associated with AATr by using the major globally available chemotherapeutical agents. Methods: A total of seven electronic databases were screened for an article on trypanocide resistance in AATr by using keywords on preclinical and clinical trials with the number of animals with treatment relapse, days taken to relapse, and resistant gene markers using the PRISMA checklist. Data were cleaned using the SR deduplicator and covidence and analyzed using Cochrane RevMan®. Dichotomous outputs were presented using risk ratio (RR), while continuous data were presented using the standardized mean difference (SMD) at a 95% confidence interval. Results: A total of eight publications in which diminazene aceturate (DA), isometamidium chloride (ISM), and homidium chloride/bromide (HB) were identified as the major trypanocides were used. In all preclinical studies, the development of resistance was in the order of HB > ISM > DA. DA vs. ISM (SMD = 0.15, 95% CI: -0.54, 0.83; I 2 = 46%, P = 0.05), DA vs. HB (SMD = 0.96, 95% CI: 0.47, 1.45; I 2 = 0%, P = 0.86), and HB vs. ISM (SMD = -0.41, 95% CI: -0.96, 0.14; I 2 = 5%, P = 0.38) showed multiple cross-resistance. Clinical studies also showed evidence of multi-drug resistance on DA and ISM (RR = 1.01, 95% CI: 0.71-1.43; I 2 = 46%, P = 0.16). To address resistance, most preclinical studies increased the dosage and the treatment time, and this failed to improve the patient's prognosis. Major markers of resistance explored include TbAT1, P1/P2 transporters, folate transporters, such as F-I, F-II, F-III, and polyamine biosynthesis inhibitors. In addition, immunosuppressed hosts favor the development of AATr. Conclusion: AATr is a threat that requires a shift in the current disease control strategies in most developing nations due to inter-species transmission. Multi-drug cross-resistance against the only accessible trypanocides is a major public health risk, justifying the need to revise the policy in developing countries to promote control of African trypanosomiasis.

Deaths Related to Chagas Disease and COVID-19 Co-Infection, Brazil, March-December 2020.

We analyzed epidemiologic characteristics and distribution of 492 deaths related to Chagas disease and coronavirus disease (COVID-19) co-infection in Brazil during March‒December 2020. Cumulative co-infected death rates were highest among advanced age groups, persons of Afro-Brazilian ethnicity and with low education levels, and geographically distributed mainly in major Chagas disease‒endemic areas.

SYSTEMATIC LITERATURE REVIEW on the SCREENING and PROPHYLAXIS of CHRONIC and OPPORTUNISTIC INFECTIONS

Background: Opportunistic and chronic infections can arise in the context of treatment used for Autoimmune Rheumatic Diseases (ARDs). Although it is recognized that screening procedures and prophylactic measures must be followed, clinical practice is largely heterogeneous, with relevant recommendations not currently developed or disparately located across the literature. Objectives: To conduct a systematic literature review (SLR) focusing on the screening and prophylaxis of opportunistic and chronic infections in ARDs. This is preparatory work done by members of the respective EULAR task force (TF). Methods: Following the EULAR standardised operating procedures, we conducted an SLR with the following 5 search domains;1) Infection: infectious agents identifed by a scoping review and expert opinion (TF members), 2) Rheumatic Diseases: all ARDs, 3) Immunosuppression: all immunosuppressives/immunomodulators used in rheumatology, 4) Screening: general and specifc (e.g mantoux test) terms, 5) Prophylaxis: general and specifc (e.g trimethop-rim) terms. Articles were retrieved having the terms from domains 1 AND 2 AND 3, plus terms from domains 4 OR 5. Databases searched: Pubmed, Embase, Cochrane. Exclusion criteria: post-operative infections, pediatric ARDs, not ARDs (e.g septic arthritis), not concerning screening or prophylaxis, Covid-19 studies, articles concerning vaccinations and non-Εnglish literature. Quality of studies included was assessed as follows: Newcastle Ottawa scale for non-randomized controlled trials (RCTs), RoB-Cochrane tool for RCTs, AMSTAR2 for SLRs. Results: 5641 studies were initially retrieved (Figure 1). After title and screening and removal of duplicates, 568 full-text articles were assessed for eligibility. Finally, 293 articles were included in the SLR. Most studies were of medium quality. Reasons for exclusion are shown in Figure 1. Results categorized as per type of microbe, are as follows: For Tuberculosis;evidence suggests that tuberculin skin test (TST) is affected by treatment with glucocorticoids and conventional synthetic DMARDs (csDMARDs) and its performance is inferior to interferon gamma release assay (IGRA). Agreement between TST and IGRA is moderate to low. Conversion of TST/IGRA occurs in about 10-15% of patients treated with biologic DMARDs (bDMARDs). Various prophylactic schemes have been used for latent TB, including isoniazide for 9 months, rifampicin for 4 months, isoniazide/rifampicin for 3-4 months. For hepatitis B (HBV): there is evidence that risk of reactivation is increased in patients positive for hepatitis B surface antigen. These patients should be referred for HBV treatment. Patients who are positive for anti-HBcore antibodies, are at low risk for reactivation when treated with glucocorticoids, cDMARDs and bDMARDs but should be monitored periodically with liver function tests and HBV-viral load. Patients treated with rituximab display higher risk for HBV reactivation especially when anti-HBs titers are low. Risk for reactivation in hepatitis C RNA positive patients, treated with bDMARDs is low. However, all patients should be referred for antiviral treatment and monitored periodically. For pneumocystis jirovecii: prophylaxis with trimeth-oprim/sulfamethoxazole (alternatively with atovaquone or pentamidine) should be considered in patients treated with prednisolone: 15-30mg/day for more than 4 weeks. Few data exist for screening and prophylaxis from viruses like E B V, CMV and Varicella Zoster Virus. Expert opinion supports the screening of rare bugs like histoplasma and trypanosoma in patients considered to be at high risk (e.g living in endemic areas). Conclusion: The risk of chronic and opportunistic infections should be considered in all patients prior to treatment with immunosuppressives/immunomod-ulators. Different screening and prophylaxis approaches are described in the literature, partly determined by individual patient and disease characteristics. Collaboration between different disciplines is important.

Management of Cardiovascular Disease in Patients With COVID-19 and Chronic Chagas Disease: Implications to Prevent a Scourge Still Larger.

Cardiovascular diseases (CVD) are the most important cause of morbidity and mortality in the general population. Because the high prevalence of COVID-19 and chronic Chagas disease (CCD) where the latter is endemic, all such diseases will likely be observed in the same patient. While COVID-19 can provoke generalized endotheliitis, which can lead to a cytokine storm and a hyper-coagulable state culminating into in-site and at a distance thrombosis. Therefore, small-vessel coronary artery disease (CAD), cerebrovascular disease, thromboembolism, and arrhythmias are prominent findings in COVID-19. In CCD, small-vessel CAD, cardioembolic stroke, pulmonary embolism, heart failure and arrhythmias are frequently observed as a result of a similar but less intense mechanism. Consequently, the association of CCD and COVID-19 will likely increase the incidence of CVD. Thus, doctors on the frontline should be on the alert for this diagnostic possibility so that the proper treatment can be given without any delay.

Impact of COVID-19 In-hospital Mortality in Chagas Disease Patients.

The COVID-19 virus infection caused by the new SARS-CoV-2 was first identified in Rio de Janeiro (RJ), Brazil, in March 2020. Until the end of 2021, 504,399 COVID-19 cases were confirmed in RJ, and the total death toll reached 68,347. The Evandro Chagas National Institute of Infectious Diseases from Oswaldo Cruz Foundation (INI-Fiocruz) is a referral center for treatment and research of several infectious diseases, including COVID-19 and Chagas disease (CD). The present study aimed to evaluate the impact of COVID-19 on in-hospital mortality of patients with CD during the COVID-19 pandemic period. This observational, retrospective, longitudinal study evaluated all patients with CD hospitalized at INI-Fiocruz from May 1, 2020, to November 30, 2021. One hundred ten hospitalizations from 81 patients with CD (58% women; 68 ± 11 years) were evaluated. Death was the study's main outcome, which occurred in 20 cases. The mixed-effects logistic regression was performed with the following variables to test whether patients admitted to the hospital with a COVID-19 diagnosis would be more likely to die than those admitted with other diagnoses: admission diagnosis, sex, age, COVID-19 vaccination status, CD clinical classification, and the number of comorbidities. Results from multiple logistic regression analysis showed a higher risk of in-hospital mortality in patients diagnosed with COVID-19 (OR 6.37; 95% CI 1.78-22.86) compared to other causes of admissions. In conclusion, COVID-19 infection had a significant impact on the mortality risk of INI-Fiocruz CD patients, accounting for one-third of deaths overall. COVID-19 presented the highest percentage of death significantly higher than those admitted due to other causes during the COVID-19 pandemic.

DOADORES DE SANGUE DO HEMOCENTRO REGIONAL DE SANTA MARIA COM SOROLOGIA REAGENTE OU INCONCLUSIVA PARA DOENÇA DE CHAGAS DURANTE O PERÍODO DA PANDEMIA DE COVID-19

Objetivos: Analisar o número de doadores de sangue do Hemocentro Regional de Santa Maria (HEMOSM), os quais apresentaram sorologia reagente ou inconclusiva para Doença de Chagas (DC), durante o período de fevereiro/2020 a julho/2021. Material e métodos: Trata-se de um estudo observacional retrospectivo, de abordagem quantitativa, realizado por meio da coleta de dados do Sistema HEMOVIDA (Sistema Nacional de Gerenciamento em Serviços de Hemoterapia) e dos arquivos do Laboratório de Sorologia do HEMOSM durante o período da Pandemia de COVID-19. As amostras de sangue dos doadores foram coletadas em tubos para a obtenção do soro empregado nos ensaios. A técnica para a detecção de anticorpos anti Trypanosoma cruzi foi a eletroquimioluminescência. Resultados: Dos 295 doadores que apresentaram algum impedimento durante a triagem sorológica, 23 deles tiveram as bolsas de sangue descartadas por Doença de Chagas, representando 8% das bolsas desprezadas pela triagem sorológica no mesmo período. Ainda, 4 apresentaram coinfecção por outros agentes etiológicos, sendo 3 por Treponema pallidum (sífilis) e 1 por HIV. Daqueles 23 doadores, 16 apresentaram sorologia reagente com detecção de anticorpos contra o T. cruzi, enquanto 7 apresentaram sorologia inconclusiva. Em relação ao sexo dos doadores, com resultados reagentes ou inconclusivos para a DC, 40% são mulheres e 60% homens. 70% dos doadores que apresentaram marcador sorológico para a DC são residentes da cidade de Santa Maria e os 30% restantes são residentes de cidades próximas pertencentes ao estado do RS. Discussão: A Doença de Chagas é uma condição crônica que leva aproximadamente 40% dos indivíduos infectados a desenvolverem sinais clínicos com envolvimento cardíaco ou digestivo. Trata-se de uma patologia parasitária decorrente da infecção pelo protozoário hemoflagelado T. cruzi, tendo como vetores os insetos triatomíneos. A transmissão pode ocorrer de forma vetorial (maior relevância epidemiológica), congênita, por transplante de órgãos, transmissão oral, através de acidentes de laboratórios e por transfusões sanguíneas, sendo este último modo de transmissão o que faz com que a DC seja pesquisada para doadores de sangue. A pesquisa da DC é importante pois 60% dos portadores do parasita não apresentam manifestações clínicas da doença. Os doadores com sorologia reagente ou indeterminada são convocados ao hemocentro para realização de nova coleta de sangue para confirmação dos resultados e para orientação a respeito do acompanhamento desta infecção. Nesse sentido, conforme os dados encontrados na presente amostra, percebe-se que foi identificado um número significativo de soropositivos para DC durante o período analisado, percentual aproximado ao do HIV, o que fundamenta a importância desse rastreio e da notificação em serviços de saúde. Conclusão: Em comparação com outras causas de descarte de hemocomponentes, como a triagem sorológica para sífilis e hepatites virais, a DC não tem grande destaque, mas de qualquer forma, mesmo que o resultado seja inconclusivo, todos os hemocomponentes produzidos a partir das doações desses voluntários devem ser descartados para a garantia da segurança transfusional.

Plant Terpenoids as Hit Compounds against Trypanosomiasis.

Human African trypanosomiasis (sleeping sickness) and American trypanosomiasis (Chagas disease) are vector-borne neglected tropical diseases, caused by the protozoan parasites Trypanosoma brucei and Trypanosoma cruzi, respectively. These diseases were circumscribed to South American and African countries in the past. However, human migration, military interventions, and climate changes have had an important effect on their worldwide propagation, particularly Chagas disease. Currently, the treatment of trypanosomiasis is not ideal, becoming a challenge in poor populations with limited resources. Exploring natural products from higher plants remains a valuable approach to find new hits and enlarge the pipeline of new drugs against protozoal human infections. This review covers the recent studies (2016-2021) on plant terpenoids, and their semi-synthetic derivatives, which have shown promising in vitro and in vivo activities against Trypanosoma parasites.

Epidemiological aspects of Chagas disease in the state of Piauí (Northeast Brazil) in the period 2010-2019.

Chagas disease (ChD), caused by the hemoflagellate protozoan Trypanosoma cruzi, is an important morbidity that affects approximately six million people in the American continent. T. cruzi parasites are mainly transmitted to human by the infected feces of blood-sucking triatomine insects. The persistent disease is endemic in many regions of South America, mostly affecting residents of rural areas. The aim of this study was to evaluate epidemiological aspects of ChD in the state of Pi-auí located in northeastern Brazil. This is an analytical cross-sectional study carried out from the collection of data of the Notifiable Diseases Information System (SINAN, in Portuguese, Sistema de Informações de Agravos de Notificação) of suspected and confirmed cases of acute ChD in the state of Piauí, in the period 2010-2019. Associations between T. cruzi positivity and the study variables were determined by the chi-square test or Fisher's exact test and were raised as prevalence ratios (PR) with 95% confidence interval. According to this survey, 517 suspected cases of acute ChD were reported in Piauí, with 70 cases (13.5%) confirmed. In 88.5% of confirmed cases, confirmation occurred by laboratory diagnosis. Most of the confirmed cases occurred in municipalities located in the semiarid region, with the municipality of São João do Piauí presenting the highest number of cases. Regarding sociodemographic data, females represent 55.7% of cases, people over 50 years of age (55.7%), being three cases in people up to 18 years of age, and less than 8 years of schooling (67.1%). 77.9% of confirmed cases had vector transmission as the probable form of infection. The data available in this study conclude that vectorial transmission of ChD in the state of Piauí remains active. This fact is corroborated by the number of notified and confirmed cases of acute ChD, requiring housing improvement programs and more effective epidemiological surveillance to control the transmission of the disease in the state.

Mechanism of Xuanbai Qingfei Jiedu decoction in the treatment of COVID-19 based on network pharmacology

Objective: To explore the pharmacological mechanism of Xuanbai Qingfei Jiedu Decoction in the treatment of coronavirus disease 2019 (COVID-19) on account of network pharmacology.

Catch me if you can: Under-detection of Trypanosoma cruzi (Kinetoplastea: Trypanosomatida) infections in Triatoma dimidiata s.l. (Hemiptera: Reduviidae) from Central America.

Assays for parasite detection in insect vectors provide important information for disease control. American Trypanosomiasis (Chagas disease) is the most devastating vector-borne illness and the fourth most common in Central America behind HIV/AIDS and acute respiratory and diarrheal infections (Peterson et al., 2019). Under-detection of parasites is a general problem which may be influenced by parasite genetic variation; however, little is known about the genetic variation of the Chagas parasite, especially in this region. In this study we compared six assays for detecting the Chagas parasite, Trypanosoma cruzi: genomic reduced representation sequencing (here referred to as genotype-by-sequencing or GBS), two with conventional PCR (i.e., agarose gel detection), two with qPCR, and microscopy. Our results show that, compared to GBS genomic analysis, microscopy and PCR under-detected T. cruzi in vectors from Central America. Of 94 samples, 44% (50/94) were positive based on genomic analysis. The lowest detection, 9% (3/32) was in a subset assayed with microscopy. Four PCR assays, two with conventional PCR and two with qPCR showed intermediate levels of detection. Both qPCR tests and one conventional PCR test targeted the 195 bp repeat of satellite DNA while the fourth test targeted the 18S gene. Statistical analyses of the genomic and PCR results indicate that the PCR assays significantly under detect infections of Central American T. cruzi genotypes.

An Ebola, Neisseria and Trypanosoma human protein interaction census reveals a conserved human protein cluster targeted by various human pathogens.

Filovirus ebolavirus (ZE; Zaire ebolavirus, Bundibugyo ebolavirus), Neisseria meningitidis (NM), and Trypanosoma brucei (Tb) are serious infectious pathogens, spanning viruses, bacteria and protists and all may target the blood and central nervous system during their life cycle. NM and Tb are extracellular pathogens while ZE is obligatory intracellular, targetting immune privileged sites. By using interactomics and comparative evolutionary analysis we studied whether conserved human proteins are targeted by these pathogens. We examined 2797 unique pathogen-targeted human proteins. The information derived from orthology searches of experimentally validated protein-protein interactions (PPIs) resulted both in unique and shared PPIs for each pathogen. Comparing and analyzing conserved and pathogen-specific infection pathways for NM, TB and ZE, we identified human proteins predicted to be targeted in at least two of the compared host-pathogen networks. However, four proteins were common to all three host-pathogen interactomes: the elongation factor 1-alpha 1 (EEF1A1), the SWI/SNF complex subunit SMARCC2 (matrix-associated actin-dependent regulator of chromatin subfamily C), the dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 (RPN1), and the tubulin beta-5 chain (TUBB). These four human proteins all are also involved in cytoskeleton and its regulation and are often addressed by various human pathogens. Specifically, we found (i) 56 human pathogenic bacteria and viruses that target these four proteins, (ii) the well researched new pandemic pathogen SARS-CoV-2 targets two of these four human proteins and (iii) nine human pathogenic fungi (yet another evolutionary distant organism group) target three of the conserved proteins by 130 high confidence interactions.

SARs for the Antiparasitic Plant Metabolite Pulchrol. 3. Combinations of New Substituents in A/B-Rings and A/C-Rings.

The natural products pulchrol and pulchral, isolated from the roots of the Mexican plant Bourreria pulchra, have previously been shown to possess antiparasitic activity towards Trypanosoma cruzi, Leishmania braziliensis and L. amazonensis, which are protozoa responsible for Chagas disease and leishmaniasis. These infections have been classified as neglected diseases, and still require the development of safer and more efficient alternatives to their current treatments. Recent SARs studies, based on the pulchrol scaffold, showed which effects exchanges of its substituents have on the antileishmanial and antitrypanosomal activity. Many of the analogues prepared were shown to be more potent than pulchrol and the current drugs used to treat leishmaniasis and Chagas disease (miltefosine and benznidazole, respectively), in vitro. Moreover, indications of some of the possible interactions that may take place in the binding sites were also identified. In this study, 12 analogues with modifications at two or three different positions in two of the three rings were prepared by synthetic and semi-synthetic procedures. The molecules were assayed in vitro towards T. cruzi epimastigotes, L. braziliensis promastigotes, and L. amazonensis promastigotes. Some compounds had higher antiparasitic activity than the parental compound pulchrol, and in some cases even benznidazole and miltefosine. The best combinations in this subset are with carbonyl functionalities in the A-ring and isopropyl groups in the C-ring, as well as with alkyl substituents in both the A- and C-rings combined with a hydroxyl group in position 1 (C-ring). The latter corresponds to cannabinol, which indeed was shown to be potent towards all the parasites.

Kinetoplastid cell biology and genetics, from the 2020 British Society for Parasitology Trypanosomiasis and Leishmaniasis symposium, Granada, Spain.

The British Society for Parasitology (BSP) holds a biannual symposium devoted to the kinetoplastids, and seeks to cover the full gamut of research into these important organisms, and alternates with the Woods Hole Kinetoplastid Molecular Cell Biology meeting that serves a similar community. While normally embedded within the main BSP Spring meeting, on several occasions the symposium has enjoyed the opportunity of being hosted on mainland Europe. In 2020, the BSP was fortunate to spend some time in Granada in Spain, where a superb meeting with excellent science in a spectacular setting was overshadowed by news of an emerging novel coronavirus. In this editorial, we hope to have captured some of that excellent science and to highlight aspects of the many great papers and reviews in this special issue, as well as provide a few images from the meeting, which we hope for this who attended will bring back some fond memories.

Quinoline-based Compounds as Key Candidates to Tackle Drug Discovery Programs of Microbicidal Agents.

Quinolines are heterocyclic nitrogen compounds, ubiquitous in nature and largely used as a structural component of dyes, solvent for resins, terpenes as well as during the production of several other chemical stuffs, including pesticides. Quinolines, such as quinine and chloroquine, exhibit various pharmacological properties, acting as antimalarial drugs, antiparasitic, antibacterial, antiviral, antifungal, and anticancer agents, besides being in clinical use for autoimmune diseases. A brief review has been presented regarding the biological effect and clinical use of quinolines and derivatives upon three trypanosomatids agents of important neglected tropical diseases; Trypanosoma cruzi, Trypanosoma brucei spp and Leishmania spp, which trigger Chagas disease, sleeping sickness and leishmaniasis, respectively, also extending to a glance update of their potential application towards other microbes relevant for emerging illness caused by fungi, bacteria and virus, including the pandemic Covid-19.