Expansion of testing, isolation, quarantine, e-health and telemonitoring strategies in socioeconomically vulnerable neighbourhoods at primary healthcare in the fight against COVID-19 in Brazil: a study protocol of a multisite testing intervention using a mixed method approach.

INTRODUCTION: The key tools for mitigating the impact of COVID-19 and reducing its transmission include testing, quarantine and isolation, as well as telemonitoring. Primary healthcare (PHC) can be essential in increasing access to these tools. Therefore, the primary objective of this study is to implement and expand an intervention consisting of COVID-19 testing, isolation, quarantine and telemonitoring (TQT) strategies and other prevention measures at PHC services in highly socioeconomically vulnerable neighbourhoods of Brazil. METHODS AND ANALYSIS: This study will implement and expand COVID-19 testing in PHC services in two large Brazilian capital cities: Salvador and Rio de Janeiro. Qualitative formative research was conducted to understand the testing context in the communities and at PCH services. The TQT strategy was structured in three subcomponents: (1) training and technical support for tailoring the work processes of health professional teams, (2) recruitment and demand creation strategies and (3) TQT. To evaluate this intervention, we will conduct an epidemiological study with two stages: (1) a cross-sectional sociobehavioural survey among individuals from these two communities covered by PHC services, presenting symptoms associated with COVID-19 or being a close contact of a patient with COVID-19, and (2) a cohort of those who tested positive, collecting clinical data. ETHICS AND DISSEMINATION: The WHO Ethics Research Committee (ERC) (#CERC.0128A and #CERC.0128B) and each city's local ERC approved the study protocol (Salvador, ISC/UFBA: #53844121.4.1001.5030; and Rio de Janeiro, INI/Fiocruz: #53844121.4.3001.5240, ENSP/Fiocruz: #53844121.4.3001.5240 and SMS/RJ #53844121.4.3002.5279). Findings will be published in scientific journals and presented at meetings. In addition, informative flyers and online campaigns will be developed to communicate study findings to participants, members of communities and key stakeholders.

Repurposing tamoxifen as potential host-directed therapeutic for tuberculosis

The global burden of tuberculosis (TB) is aggravated by the continuously increasing emergence of drug resistance, highlighting the need for innovative therapeutic options. The concept of host-directed therapy (HDT) as adjunctive to classical antibacterial therapy with antibiotics represents a novel and promising approach for treating TB. Here, we have focused on repurposing the clinically used anticancer drug tamoxifen, which was identified as a molecule with strong host-directed activity against intracellular Mycobacterium tuberculosis (Mtb). Using a primary human macrophage Mtb infection model, we demonstrate the potential of tamoxifen against drug-sensitive as well as drug-resistant Mtb bacteria. The therapeutic effect of tamoxifen was confirmed in an in vivo TB model based on Mycobacterium marinum infection of zebrafish larvae. Tamoxifen had no direct antimicrobial effects at the concentrations used, confirming that tamoxifen acted as an HDT drug. Furthermore, we demonstrate that the antimycobacterial effect of tamoxifen is independent of its well-known target the estrogen receptor (ER) pathway, but instead acts by modulating autophagy, in particular the lysosomal pathway. Through RNA sequencing and microscopic colocalization studies, we show that tamoxifen stimulates lysosomal activation and increases the localization of mycobacteria in lysosomes both in vitro and in vivo, while inhibition of lysosomal activity during tamoxifen treatment partly restores mycobacterial survival. Thus, our work highlights the HDT potential of tamoxifen and proposes it as a repurposed molecule for the treatment of TB. Tuberculosis (TB) is the world's most lethal infectious disease caused by a bacterial pathogen, Mycobacterium tuberculosis. This pathogen evades the immune defenses of its host and grows intracellularly in immune cells, particularly inside macrophages. There is an urgent need for novel therapeutic strategies because treatment of TB patients is increasingly complicated by rising antibiotic resistance. In this study, we explored a breast cancer drug, tamoxifen, as a potential anti-TB drug. We show that tamoxifen acts as a so-called host-directed therapeutic, which means that it does not act directly on the bacteria but helps the host macrophages combat the infection more effectively. We confirmed the antimycobacterial effect of tamoxifen in a zebrafish model for TB and showed that it functions by promoting the delivery of mycobacteria to digestive organelles, the lysosomes. These results support the high potential of tamoxifen to be repurposed to fight antibiotic-resistant TB infections by host-directed therapy.

Recombinant BCG-LTAK63 vaccine candidate for tuberculosis induces an inflammatory profile in human macrophages

Tuberculosis (TB) is one of the top 10 leading causes of death worldwide. The recombinant BCG strain expressing the genetically detoxified A subunit of the thermolabile toxin from Escherichia coli (LTAK63) adjuvant (rBCG-LTAK63) has previously been shown to confer superior protection and immunogenicity compared to BCG in a murine TB infection model. To further investigate the immunological mechanisms induced by rBCG-LTAK63, we evaluated the immune responses induced by rBCG-LTAK63, BCG, and Mycobacterium tuberculosis (Mtb) H37Rv strains in experimental infections of primary human M1 and M2 macrophages at the transcriptomic and cytokine secretion levels. The rBCG-LTAK63-infected M1 macrophages more profoundly upregulated interferon-inducible genes such as IFIT3, OAS3, and antimicrobial gene CXCL9 compared to BCG, and induced higher levels of inflammatory cytokines such as IL-12(p70), TNF-β, and IL-15. The rBCG-LTAK63-infected M2 macrophages more extensively upregulated transcripts of inflammation-related genes, TAP1, GBP1, SLAMF7, TNIP1, and IL6, and induced higher levels of cytokines related to inflammation and tissue repair, MCP-3 and EGF, as compared to BCG. Thus, our data revealed an important signature of immune responses induced in human macrophages by rBCG-LTAK63 associated with increased inflammation, activation, and tissue repair, which may be correlated with a protective immune response against TB.

Repurposing diphenylbutylpiperidine-class antipsychotic drugs for host-directed therapy of Mycobacterium tuberculosis and Salmonella enterica infections

The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide’s and Fluspirilene’s efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms.

Challenges and proposals for scaling up COVID-19 testing and diagnosis in Brazil. / Desafios e propostas para ampliação da testagem e diagnóstico para COVID-19 no Brasil.

The Brazilian context of social inequalities and barriers in accessing health services may deteriorate the situation of the COVID-19 pandemic, which already affects all Brazilian federative states, with the growing curve of increasing confirmed cases and deaths. National governments and scientific field agents have been looking for evidence for the best practices of prevention and control of transmission, and care of infection and disease, including diagnosis, treatment, and health care measures. The large-scale testing strategy, aimed at early diagnosis, quarantine of the mild cases identified, as well as those of the contacts, and adequate care of severe cases, has been revised and indicated as one of the efficient pandemic control measures in several countries in the world. This paper aims to discuss the challenges of COVID-19 testing and diagnosis in Brazil.

Desafios e propostas para ampliação da testagem e diagnóstico para COVID-19 no Brasil / Challenges and proposals for scaling up COVID-19 testing and diagnosis in Brazil

Resumo O contexto brasileiro de desigualdades sociais e barreiras no acesso aos serviços de saúde pode agravar a situação da pandemia de COVID-19, que já afeta todos os estados da federação, com a curva crescente de aumento de casos confirmados e mortes. O governo dos países e os agentes do campo científico têm buscado evidências para as melhores práticas de prevenção e controle da transmissão, e cuidado da infecção e doença, incluindo medidas de diagnóstico, tratamento e de atenção à saúde. A estratégia de testagem em larga escala, visando o diagnóstico precoce, quarentena dos casos leves identificados, bem como dos contactantes, e cuidado adequado dos casos graves, tem sido revisada e indicada como uma das medidas eficientes para o controle da pandemia em vários países do mundo. O artigo tem como objetivo discutir os desafios da testagem e do diagnóstico de COVID-19 no Brasil.

Abstract The Brazilian context of social inequalities and barriers in accessing health services may deteriorate the situation of the COVID-19 pandemic, which already affects all Brazilian federative states, with the growing curve of increasing confirmed cases and deaths. National governments and scientific field agents have been looking for evidence for the best practices of prevention and control of transmission, and care of infection and disease, including diagnosis, treatment, and health care measures. The large-scale testing strategy, aimed at early diagnosis, quarantine of the mild cases identified, as well as those of the contacts, and adequate care of severe cases, has been revised and indicated as one of the efficient pandemic control measures in several countries in the world. This paper aims to discuss the challenges of COVID-19 testing and diagnosis in Brazil.

Recombinant BCG expressing the LTAK63 adjuvant induces increased early and long-term immune responses against Mycobacteria

The development of more effective vaccines against Mycobacterium tuberculosis has become a world priority. Previously, we have shown that a recombinant BCG expressing the LTAK63 adjuvant (rBCG-LTAK63) displayed higher protection than BCG against tuberculosis challenge in mice. In order to elucidate the immune effector mechanisms induced by rBCG-LTAK63, we evaluated the immune response before and after challenge. The potential to induce an innate immune response was investigated by intraperitoneal immunization with BCG or rBCG-LTAK63: both displayed increased cellular infiltration in the peritoneum with high numbers of neutrophils at 24 h and macrophages at 7 d. The rBCG-LTAK63-immunized mice displayed increased production of Nitric Oxide at 24 h and Hydrogen Peroxide at 7 d. The number of lymphocytes was higher in the rBCG-LTAK63 group when compared to BCG. Immunophenotyping of lymphocytes showed that rBCG-LTAK63 immunization increased CD4+ and CD8+ T cells. An increased long-term Th1/Th17 cytokine profile was observed 90 d after subcutaneous immunization with rBCG-LTAK63. The evaluation of immune responses at 15 d after challenge showed that rBCG-LTAK63-immunized mice displayed increased TNF-a-secreting CD4+ T cells and multifunctional IL-2+ TNF-a+ CD4+ T cells as compared to BCG-immunized mice. Our results suggest that immunization with rBCG-LTAK63 induces enhanced innate and long-term immune responses as compared to BCG. These results can be correlated with the superior protection induced against TB

Recombinant BCG expressing the LTAK63 adjuvant induces increased early and long-term immune responses against Mycobacteria

The development of more effective vaccines against Mycobacterium tuberculosis has become a world priority. Previously, we have shown that a recombinant BCG expressing the LTAK63 adjuvant (rBCG-LTAK63) displayed higher protection than BCG against tuberculosis challenge in mice. In order to elucidate the immune effector mechanisms induced by rBCG-LTAK63, we evaluated the immune response before and after challenge. The potential to induce an innate immune response was investigated by intraperitoneal immunization with BCG or rBCG-LTAK63: both displayed increased cellular infiltration in the peritoneum with high numbers of neutrophils at 24 h and macrophages at 7 d. The rBCG-LTAK63-immunized mice displayed increased production of Nitric Oxide at 24 h and Hydrogen Peroxide at 7 d. The number of lymphocytes was higher in the rBCG-LTAK63 group when compared to BCG. Immunophenotyping of lymphocytes showed that rBCG-LTAK63 immunization increased CD4+ and CD8+ T cells. An increased long-term Th1/Th17 cytokine profile was observed 90 d after subcutaneous immunization with rBCG-LTAK63. The evaluation of immune responses at 15 d after challenge showed that rBCG-LTAK63-immunized mice displayed increased TNF-a-secreting CD4+ T cells and multifunctional IL-2+ TNF-a+ CD4+ T cells as compared to BCG-immunized mice. Our results suggest that immunization with rBCG-LTAK63 induces enhanced innate and long-term immune responses as compared to BCG. These results can be correlated with the superior protection induced against TB