Detection of M. tuberculosis in the environment as a tool for identifying high-risk locations for tuberculosis transmission Renu Verma, Flora Martinez Figueira Moreira, Agne Oliveira do Prado Morais, Katharine S. Walter, Paulo César Pereira dos Santos, Eugene Kim, Thiego Ramon Soares, Rafaele Carla Pivetta de Araujo, Bruna Oliveira da Silva, Andrea da Silva Santos, Julio Croda, Jason R. Andrews Science of the Total Environment, 2022 Tuberculosis (TB) remains a leading cause of infectious mortality globally, yet most cases cannot be epidemiologically linked even with extensive contact investigations and whole genome sequencing. Consequently, there remain major gaps in our understanding of where and when M. tuberculosis (Mtb) exposures occur. We aimed to investigate whether Mtb can be detected in environments where TB patients were recently present, which could serve as a tool for characterizing exposure risk. We collected 389 environment surface (ES) swabs from two high TB burden prisons in Brazil, sampling 41 (n = 340) cells occupied by individuals with active TB and 7 (n = 49) cells from individuals without TB. In a subset of pooled swabs (n = 6) and a swab from a cigarette lighter from the cell with active TB patients, we enriched Mtb DNA using RNA-bait hybrid capture assays and performed whole genome sequencing. In prison cells, Mtb DNA was detected in 55/340 (16 %) of ES swabs from cells occupied by active TB patients and none (0/49) from cells in which no active TB patients were present. Mtb was detected in 13/16 (81 %) prison cells occupied by the individuals with high/medium sputum Xpert Mtb load and 8/25 (32 %) with low/very low sputum Mtb load (p = 0.003). Seven hybrid capture samples had a median genomic coverage of 140×. rpoB mutations conferring high-level rifampin resistance were detected in 3/7 ES swabs. Mtb was frequently detectable in environments recently occupied by individuals with active TB. This approach could be applied in congregate environments to identify and characterize high-risk settings for Mtb exposure.
The role of prisons in disseminating tuberculosis in Brazil: A genomic epidemiology study Katharine S. Walter, Paulo César Pereira dos Santos, Thais Oliveira Gonçalves, Bruna Oliveira da Silva, Andrea da Silva Santos, Alessandra de Cássia Leite, Alessandra Moura da Silva, Flora Martinez Figueira Moreira, Roberto Dias de Oliveira, Everton Ferreira Lemos, Eunice Cunha, Yiran E. Liu, Albert I. Ko, Caroline Colijn, Ted Cohen, Barun Mathema, Julio Croda, Jason R. Andrews Lancet Regional Health Americas, 2022 Globally, prisons are high-incidence settings for tuberculosis. Yet the role of prisons as reservoirs of M. tuberculosis, propagating epidemics through spillover to surrounding communities, has been difficult to measure directly. To quantify the role of prisons in driving wider community M. tuberculosis transmission, we conducted prospective genomic surveillance in Central West Brazil from 2014 to 2019. We whole genome sequenced 1152 M. tuberculosis isolates collected during active and passive surveillance inside and outside prisons and linked genomes to detailed incarceration histories. We applied multiple phylogenetic and genomic clustering approaches and inferred timed transmission trees. M. tuberculosis sequences from incarcerated and non-incarcerated people were closely related in a maximum likelihood phylogeny. The majority (70.8%; 46/65) of genomic clusters including people with no incarceration history also included individuals with a recent history of incarceration. Among cases in individuals with no incarceration history, 50.6% (162/320) were in clusters that included individuals with recent incarceration history, suggesting that transmission chains often span prisons and communities. We identified a minimum of 18 highly probable spillover events, M. tuberculosis transmission from people with a recent incarceration history to people with no prior history of incarceration, occurring in the state's four largest cities and across sampling years. We additionally found that frequent transfers of people between the state's prisons creates a highly connected prison network that likely disseminates M. tuberculosis across the state. We developed a framework for measuring spillover from high-incidence environments to surrounding communities by integrating genomic and spatial information. Our findings indicate that, in this setting, prisons serve not only as disease reservoirs, but also disseminate M. tuberculosis across highly connected prison networks, both amplifying and propagating M. tuberculosis risk in surrounding communities. Brazil's National Council for Scientific and Technological Development and US National Institutes of Health.
A rapid pharmacogenomic assay to detect nat2 polymorphisms and guide isoniazid dosing for tuberculosis treatment Renu Verma, Sunita Patil, Nan Zhang, Flora M. F. Moreira, Marize T. Vitorio, Andrea da S. Santos, Ellen Wallace, Devasena Gnanashanmugam, David H. Persing, Rada M. Savic, Julio Croda, Jason R. Andrews American Journal of Respiratory and Critical Care Medicine, 2021 RATIONALE Standardized dosing of anti-tubercular drugs contributes to a substantial incidence of toxicities, inadequate treatment response, and relapse, in part due to variable drug levels achieved. Single nucleotide polymorphisms (SNPs) in the N-acetyltransferase-2 (NAT2) gene explain the majority of interindividual pharmacokinetic variability of isoniazid (INH). However, an obstacle to implementing pharmacogenomic-guided dosing is the lack of a point-of-care assay. OBJECTIVES To develop and test a NAT2 classification algorithm, validate its performance in predicting isoniazid clearance, and develop a prototype pharmacogenomic assay. METHODS We trained random forest models to predict NAT2 acetylation genotype from unphased SNP data using a global collection of 8,561 phased genomes. We enrolled 48 pulmonary TB patients, performed sparse pharmacokinetic sampling, and tested the acetylator prediction algorithm accuracy against estimated INH clearance. We then developed a cartridge-based multiplex qPCR assay on the GeneXpert platform and assessed its analytical sensitivity on whole blood samples from healthy individuals. MEASUREMENTS AND MAIN RESULTS With a 5-SNP model trained on two-thirds of the data (n=5,738), out-of-sample acetylation genotype prediction accuracy on the remaining third (n=2,823) was 100%. Among the 48 TB patients, predicted acetylator types were: 27 (56.2%) slow, 16 (33.3%) intermediate and 5 (10.4%) rapid. INH clearance rates were lowest in predicted slow acetylators (median 14.5 L/hr), moderate in intermediate acetylators (median 40.3 L/hr) and highest in fast acetylators (median 53.0 L/hr). The cartridge-based assay accurately detected all allele patterns directly from 25 ul of whole blood. CONCLUSIONS An automated pharmacogenomic assay on a platform widely used globally for tuberculosis diagnosis could enable personalized dosing of isoniazid.
Toxicological analysis and efficacy of 2-phenylchromone on mycobacteria viability and inflammatory response induced by Mycobacterium bovis Flora Martinez Figueira Moreira, Joyce Alencar Santos Radai, Vanessa Vilamaior de Souza, Claudia Rodrigues Berno, Flavio Henrique Souza de Araújo, Magaiver Andrade-Silva, Rodrigo Juliano Oliveira, Arielle Cristina Arena, Maria das Graças Müller de Oliveira Henriques, Candida Aparecida Leite Kassuya, Julio Croda Phytomedicine Plus, 2021 Background: Studies have reported that compounds similar to flavone present antimicrobial and anti-inflammatory properties; however these effects are poorly described in the context of mycobacterium infection. Purpose: This study investigated the efficacy of 2-phenylchromone (Flavone) on mycobacteria viability and inflammatory response induced by Mycobacterium bovis, as well as its toxicological potential in in vitro and in vivo models. Methods: In vitro antimycobacterial activity was performed using the resazurin microtiter assay method. In order to investigate the anti-inflammatory action, C57Bl/6 mice were pretreated orally with 2-phenylchromone (1–100 mg/kg) 1 h before the pleurisy induction. The leukocyte migration and cytokine production were evaluated. Acute oral toxicity test, genotoxicity evaluations and splenic phagocytosis assay were also evaluated. Results: The minimum inhibitory concentration of 2-phenylchromone in the presence of the Mycobacterium tuberculosis strain was 28.90 µg/mL. In the BCG-induced pleurisy model, the oral treatment with 2-phenylchromone (1, 10, or 100 mg/kg) caused a significant reduction in the tumor necrosis factor (TNF) levels in the pleural exudate, as well as in the total and differential leukocyte counts. The oral treatment with 2-phenylchromone also did not present acute toxicity or genotoxicity in comet assay and micronucleus tests. However, 2-phenylchromone induced an increase of 1.48-times in splenic phagocytosis in the animals. Conclusion: Our results showed that 2-phenylchromone did not induced toxicity signs and is effective against Mycobacterium viability and reduced the inflammatory response elicited by mycobacteria.
Local and travel-associated transmission of tuberculosis at Central Western border of Brazil, 2014–2017 Katharine S. Walter, Mariana Bento Tatara, Kesia Esther da Silva, Flora Martinez Figueira Moreira, Paulo Cesar Pereira dos Santos, Dândrea Driely de Melo Ferrari, Eunice Atsuko Cunha, Jason R. Andrews, Julio Croda Emerging Infectious Diseases, 2021 International migrants are at heightened risk for tuberculosis (TB) disease. Intensified incarceration at international borders may compound population-wide TB risk. However, few studies have investigated the contributions of migration, local transmission, or prisons in driving incident TB at international borders. We conducted prospective population-based genomic surveillance in 3 cities along Brazil’s central western border from 2014–2017. Although most isolates (89/132; 67%) fell within genomic transmission clusters, genetically unique isolates disproportionately occurred among participants with recent international travel (17/42; 40.5%), suggesting that both local transmission and migration contribute to incident TB. Isolates from 40 participants with and 76 without an incarceration history clustered together throughout a maximum-likelihood phylogeny, indicating the close interrelatedness of prison and community epidemics. Our findings highlight the need for ongoing surveillance to control continued introductions of TB and reduce the disproportionate burden of TB in prisons at Brazil’s international borders.
Blood-based host biomarker diagnostics in active case finding for pulmonary tuberculosis: A diagnostic case-control study Flora Martinez Figueira Moreira, Renu Verma, Paulo Cesar Pereira dos Santos, Alessandra Leite, Andrea da Silva Santos, Rafaele Carla Pivetta de Araujo, Bruna Oliveira da Silva, Júlio Henrique Ferreira de Sá Queiroz, David H. Persing, Erik Södersten, Devasena Gnanashanmugam, Purvesh Khatri, Julio Croda, Jason R. Andrews Eclinicalmedicine, 2021 BackgroundThere is a need to identify scalable tuberculosis screening strategies among high burden populations. The WHO has identified a non-sputum-based triage test as a development priority.MethodsWe performed a diagnostic case-control study of point-of-care C-reactive protein (CRP) and Prototype-Xpert-MTB-Host-Response (Xpert-MTB-HR) assays in the context of a mass screening program for tuberculosis in two prisons in Brazil. All incarcerated individuals irrespective of symptoms were screened by sputum Xpert MTB/RIF and sputum culture. Among consecutive, Xpert MTB/RIF or culture-confirmed cases and Xpert MTB/RIF and culture-negative controls, CRP was quantified in serum by a point-of-care assay (iChroma-II) and a 3-gene expression score was quantified from whole blood using the Xpert-MTB-HR cartridge. We evaluated receiver operating characteristic area under the curve (AUC) and assessed specificity at 90% sensitivity and sensitivity at 70% specificity, consistent with WHO target product profile (TPP) benchmarks.FindingsTwo hundred controls (no TB) and 100 culture- or Xpert MTB/RIF-positive tuberculosis cases were included. Half of tuberculosis cases and 11% of controls reported any tuberculosis symptoms. AUC for CRP was 0·79 (95% CI: 0·73–0·84) and for Xpert-MTB-HR was 0·84 (95% CI: 0·79–0·89). At 90% sensitivity, Xpert-MTB-HR had significantly higher specificity (53·0%, 95% CI: 45·0–69·0%) than CRP (28·1%, 95% CI: 20·2–41·8%) (p = 0·003), both well below the TPP benchmark of 70%. Among individuals with medium or high sputum Xpert MTB/RIF semi-quantitative load, sensitivity (at 70% specificity) of CRP (90·3%, 95% CI: 74·2–98·0) and Xpert-MTB-HR (96·8%, 95% CI: 83·3–99·9%) was higher.InterpretationFor active case finding in this high tuberculosis-burden setting, CRP and Xpert-MTB-HR did not meet TPP benchmarks for a triage test. However, Xpert-MTB-HR was highly sensitive in detecting individuals with medium or high sputum bacillary burden.FundingNational Institutes of Health (R01 AI130058 and R01 AI149620) and Brazilian National Council for Scientific and Technological Development (CNPq-404182/2019-4).
Design, Synthesis and Antitubercular Activity of Novel Isoniazid‑Cyclic‑Amine‑Azachalcones Hybrids Jefferson Oliveira, Cristiane Shiguemoto, Amarith das Neves, Flora Moreira, Giovana Gomes, Renata Perdomo, Sandro Barbosa, Palimécio Guerrero Jr., Júlio Croda, Adriano Baroni Journal of the Brazilian Chemical Society, 2020 In this work, it is described the design of twenty-four heterocyclic amine-azachalcones compounds through molecular hybridization of chalcone scaffold and fragments of isoniazid, fluoroquinolones, and linezolid with antituberculosis potential. The new compounds were synthesized via Claisen-Schmidt condensation, providing yields of 36-95%. Fifteen compounds showed antituberculosis activity against Mycobacterium tuberculosis H37Rv strain. Two amine-azachalcones 15 and 17 showed relevant biological activity with minimum inhibitory concentration (MIC) values of 6.62 and 4.85 μM, respectively. Compound 12 showed the best profile of antitubercular activity with MIC = 9.54 μM and selectivity index (SI) = 9.33. It was found that morpholine group is important to increase potency of antimycobacterial activity but also to add some toxicity to the chalcone molecular framework. The results described herein would be a guide in the designing of novel and optimized antitubercular derivatives based on the chalcone scaffold.
Assessment of Validity of a Blood-Based 3-Gene Signature Score for Progression and Diagnosis of Tuberculosis, Disease Severity, and Treatment Response Hayley C. Warsinske, Aditya M. Rao, Flora M. F. Moreira, Paulo Cesar P. Santos, Andrew B. Liu, Madeleine Scott, Stephaus T. Malherbe, Katharina Ronacher, Gerhard Walzl, Jill Winter, Timothy E. Sweeney, Julio Croda, Jason R. Andrews, Purvesh Khatri JAMA Network Open, 2018 Key Points Question How does a previously described blood-based 3-gene tuberculosis score perform as a screening test and a disease monitoring tool for all stages of tuberculosis? Findings In this nested case-contral study, the 3-gene tuberculosis score was associated with progression from latent Mycobacterium tuberculosis infection to active tuberculosis 6 months prior to sputum conversion with 86% sensitivity and 84% specificity, diagnosed patients with active tuberculosis with 90% sensitivity and 70% specificity, and correlated with treatment response and the severity of lung pathology. Meaning The 3-gene tuberculosis score can be implemented as a rapid, blood-based screening and triage test with the required World Health Organization target product profiles for the accurate detection and tracking of progressive, active, and treated tuberculosis disease.
Antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities of the essential oil of Psidium guineense Sw. and spathulenol Kamilla Felipe do Nascimento, Flora Martinez Figueira Moreira, Joyce Alencar Santos, Candida Aparecida Leite Kassuya, Julio Henrique Rosa Croda, Claudia Andrea Lima Cardoso, Maria do Carmo Vieira, Ana Lúcia Tasca Góis Ruiz, Mary Ann Foglio, João Ernesto de Carvalho, Anelise Samara Nazari Formagio Journal of Ethnopharmacology, 2018 ETHNOPHARMACOLOGICAL RELEVANCE Leaves from Psidium guineense Sw. are used in popular medicine for the treatment of inflammatory disease. However, there is no scientific evidence demonstrating this activity. AIM OF THE STUDY To evaluate the antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities of the essential oil of P. guineense and spathulenol (a major constituent). The study was conducted in part to provide evidence supporting the ethnobotanical use of the leaves of this species. MATERIAL AND METHODS The essential oil (EOPG) was extracted from the leaves of P. guineense by hydrodistillation and analysed by gas chromatography-mass spectrometry (GC-MS). The major compound, spathulenol (PG-1), was isolated in a chromatographic column and characterized by nuclear magnetic resonance (NMR). EOPG and PG-1 were evaluated in vitro for antioxidant activity by DPPH, ABTS and MDA methods; anti-inflammatory potential was assessed using two models, including pleurisy and oedema, in mice. The impact of EOPG and PG-1 on cell proliferation was determined via spectrophotometric quantification of the cellular protein content using a sulforhodamine B assay, and anti-Mycobacterium tuberculosis activity was determined using the REMA method. RESULTS A total of 38 components were identified from the EOPG, with the sesquiterpenic alcohol spathulenol (PG-1) (80.7%) being the major constituent. EOPG and PG-1 exhibited the highest antioxidant activities in the DPPH and MDA system compared with reference standard, with IC50 values ranging from 26.13 to 85.60μg/mL. Oral administration of EOPG and PG-1 showed significant inhibition in the Cg-induced mice paw oedema and pleurisy model. The EOPG (GI50 = 0.89μg/mL) and PG-1 (GI50 = 49.30μg/mL) were particularly effective against the ovarian cancer cell line. Both showed moderate antimycobacterial activity. CONCLUSION For the first time, this study demonstrated the antioxidant, anti-inflammatory, antiproliferative and antimycobacterial properties of the essential oil of P. guineense (leaves were collected in Dourados-MS) and spathulenol, collaborating the etnhopharmacologycal use of this plant due to its an anti-inflammatory effect.
Anti-inflammatory, antimycobacterial and genotoxic evaluation of Doliocarpus dentatus Raissa Borges Ishikawa, Maicon Matos Leitão, Roberto Mikio Kassuya, Luis Fernando Macorini, Flora Martinez Figueira Moreira, Claudia Andrea Lima Cardoso, Roberta Gomes Coelho, Arnildo Pott, Guilherme Martins Gelfuso, Julio Croda, Rodrigo Juliano Oliveira, Candida Aparecida Leite Kassuya Journal of Ethnopharmacology, 2017 ETHNOPHARMACOLOGICAL RELEVANCE Doliocarpus dentatus is a medicinal plant widely used in Mato Grosso do Sul State for removing the swelling pain caused by the inflammation process and for treating urine retention. AIM OF THE STUDY The genotoxic aspects and the anti-inflammatory and antimycobacterial activity of the ethanolic extract obtained from the leaves of D. dentatus (EEDd) were investigated. MATERIALS AND METHODS The EEDd was evaluated against Mycobacterium tuberculosis, and the compound composition was evaluated and identified by nuclear magnetic resonance (NMR). The mice received oral administration of EEDd (30-300mg/kg) in carrageenan models of inflammation, and EEDd (10-1000mg/kg) was assayed by the comet, micronucleus, and phagocytosis tests and by the peripheral leukocyte count. RESULTS Phenols (204.04mg/g), flavonoids (89.17mg/g), and tannins (12.05mg/g) as well as sitosterol-3-O-β-D-glucopyranoside, kaempferol 3-O-α-L-rhamnopyranoside, betulinic acid and betulin were present in the EEDd. The value of minimal inhibitory concentration (MIC) of EEDd was 62.5µg/mL. The EEDd induced a significant decrease in the edema, mechanical hypersensitivity and leukocyte migration induced by carrageenan. The comet and micronucleus tests indicated that the EEDd was not genotoxic. The EEDd also did not change the phagocytic activity or the leukocyte perLipheral count. CONCLUSIONS The EEDd does not display genotoxicity, phagocytosis and could act as an antimycobacterial and anti-inflammatory agent. This study should contribute to ensuring the safe use of EEDd.
