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Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which can lead to a disabling neurodegenerative condition. M. leprae preferentially infects skin macrophages and Schwann cells-glial cells of the peripheral nervous system. The infection modifies the host cell lipid metabolism, subverting it in favor of the formation of cholesterol-rich lipid droplets (LD) that are essential for bacterial survival. Although researchers have made progress in understanding leprosy pathogenesis, many aspects of the molecular and cellular mechanisms of host-pathogen interaction still require clarification. The purinergic system utilizes extracellular ATP and adenosine as critical signaling molecules and plays several roles in pathophysiological processes. Furthermore, nucleoside surface receptors such as the adenosine receptor A2AR involved in neuroimmune response, lipid metabolism, and neuron-glia interaction are targets for the treatment of different diseases. Despite the importance of this system, nothing has been described about its role in leprosy, particularly adenosinergic signaling (AdoS) during M. leprae-Schwann cell interaction. Methods: M. leprae was purified from the hind footpad of athymic nu/nu mice. ST88-14 human cells were infected with M. leprae in the presence or absence of specific agonists or antagonists of AdoS. Enzymatic activity assays, fluorescence microscopy, Western blotting, and RT-qPCR analysis were performed. M. leprae viability was investigated by RT-qPCR, and cytokines were evaluated by enzyme-linked immunosorbent assay. Results: We demonstrated that M. leprae-infected Schwann cells upregulated CD73 and ADA and downregulated A2AR expression and the phosphorylation of the transcription factor CREB (p-CREB). On the other hand, activation of A2AR with its selective agonist, CGS21680, resulted in: 1) reduced lipid droplets accumulation and pro-lipogenic gene expression; 2) reduced production of IL-6 and IL-8; 3) reduced intracellular M. leprae viability; 4) increased levels of p-CREB. Conclusion: These findings suggest the involvement of the AdoS in leprosy neuropathogenesis and support the idea that M. leprae, by downmodulating the expression and activity of A2AR in Schwann cells, decreases A2AR downstream signaling, contributing to the maintenance of LD accumulation and intracellular viability of the bacillus.

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Introduction: New diagnostic tools are needed to rapidly assess the efficacy of pulmonary tuberculosis (PTB) treatment. The aim of this study was to evaluate several immune biomarkers in an observational and cross-sectional cohort study conducted in Paraguay. Methods: Thirty-two patients with clinically and microbiologically confirmed PTB were evaluated before starting treatment (T0), after 2 months of treatment (T1) and at the end of treatment (T2). At each timepoint plasma levels of IFN-y, 17 pro- and anti-inflammatory cytokines/chemokines and complement factors C1q, C3 and C4 were assessed in unstimulated and Mtb-specific stimulated whole blood samples using QuantiFERON-TB gold plus and recombinant Mycobacterium smegmatis heparin binding hemagglutinin (rmsHBHA) as stimulation antigen. Complete blood counts and liver enzyme assays were also evaluated and correlated with biomarker levels in plasma. Results: In unstimulated plasma, C1q (P<0.001), C4 (P<0.001), hemoglobin (P<0.001), lymphocyte proportion (P<0.001) and absolute white blood cell count (P=0.01) were significantly higher in PTB patients at baseline than in cured patients. C1q and C4 levels were found to be related to Mycobacterium tuberculosis load in sputum. Finally, a combinatorial analysis identified a plasma host signature comprising the detection of C1q and IL-13 levels in response to rmsHBHA as a tool differentiating PTB patients from cured TB profiles, with an AUC of 0.92 (sensitivity 94% and specificity 79%). Conclusion: This observational study provides new insights on host immune responses throughout anti-TB treatment and emphasizes the role of host C1q and HBHA-specific IL-13 response as surrogate plasma biomarkers for monitoring TB treatment efficacy.

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Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which can lead to a disabling neurodegenerative condition. M. leprae preferentially infects skin macrophages and Schwann cells­glial cells of the peripheral nervous system. The infection modifies the host cell lipid metabolism, subverting it in favor of the formation of cholesterol-rich lipid droplets (LD) that are essential for bacterial survival. Although researchers have made progress in understanding leprosy pathogenesis, many aspects of the molecular and cellular mechanisms of host­pathogen interaction still require clarification. The purinergic system utilizes extracellular ATP and adenosine as critical signaling molecules and plays several roles in pathophysiological processes. Furthermore, nucleoside surface receptors such as the adenosine receptor A2AR involved in neuroimmune response, lipid metabolism, and neuron­glia interaction are targets for the treatment of different diseases. Despite the importance of this system, nothing has been described about its role in leprosy, particularly adenosinergic signaling (AdoS) during M. leprae­Schwann cell interaction. Methods: M. leprae was purified from the hind footpad of athymic nu/nu mice. ST88-14 human cells were infected with M. leprae in the presence or absence of specific agonists or antagonists of AdoS. nzymatic activity assays, fluorescence microscopy, Western blotting, and RT-qPCR nalysis were performed. M. leprae viability was investigated by RT-qPCR, and cytokines were evaluated by enzymelinked immunosorbent assay. Results: We demonstrated that M. leprae-infected Schwann cells upregulated CD73 and ADA and downregulated A2AR expression and the phosphorylation of the transcription factor CREB (p-CREB). On the other hand, activation of A2AR with its selective agonist, CGS21680, resulted in: 1) reduced lipid droplets accumulation and pro-lipogenic gene expression; 2) reduced production of IL-6 and IL-8; 3) reduced intracellular M. leprae viability; 4) increased levels of p-CREB. Conclusion: These findings suggest the involvement of the AdoS in leprosy neuropathogenesis and support the idea that M. leprae, by downmodulating the expression and activity of A2AR in Schwann cells, decreases A2AR downstream signaling, contributing to the maintenance of LD accumulation and intracellular viability of the bacillus.

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SARS-CoV-2 variant detection relies on resource-intensive whole-genome sequencing methods. We sought to develop a scalable protocol for variant detection and surveillance in Paraguay, pairing rRT-PCR for spike mutations with Nanopore sequencing. A total of 201 acute-phase nasopharyngeal samples were included. Samples were positive for the SARS-CoV-2 N2 target and tested with the Spike SNP assay to detect mutations associated with the following variants: alpha (501Y), beta/gamma (417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). Spike SNP calls were confirmed using amplicon (Sanger) sequencing and whole-genome (Nanopore) sequencing on a subset of samples with confirmed variant lineages. Samples had a mean N2 Ct of 20.8 (SD 5.6); 198/201 samples (98.5%) tested positive in the Spike SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198 samples (51.5%), which was consistent with the P.1 lineage (gamma variant) in Paraguay. No mutations (K417 only) were found in 64/198 (32.3%), and K417/484K was identified in 22/198 (11.1%), consistent with P.2 (zeta). Seven samples (3.5%) tested positive for 452R without 478K, and one sample with genotype K417/501Y was confirmed as B.1.1.7 (alpha). The results were confirmed using Sanger sequencing in 181/181 samples, and variant calls were consistent with Nanopore sequencing in 29/29 samples. The Spike SNP assay could improve population-level surveillance for mutations associated with SARS-CoV-2 variants and inform the judicious use of sequencing resources.

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OBJECTIVES: Tuberculosis (TB) is the leading infectious cause of death in the world. Cheaper and more accessible TB treatment monitoring methods are needed. Here, we evaluated white blood cell (WBC) absolute counts, lymphocyte, and monocyte proportions during TB treatment, and characterized their association with treatment failure. METHODS: This multicentered prospective cohort study was based in Bangladesh, Georgia, Lebanon, Madagascar, and Paraguay. Adult, non-immunocompromised patients with culture-confirmed pulmonary TB were included and followed up after two months of treatment and at the end of therapy. Blood counts were compared to treatment outcome using descriptive statistics, logistic regression, and Receiver Operating Characteristic (ROC) analyses. RESULTS: Between December 2017 and August 2020, 198 participants were enrolled, and 152 completed treatment, including 28 (18.5%) drug-resistant patients. The rate of cure at the end of treatment was 90.8% (138/152). WBC absolute counts decreased, and lymphocyte proportions increased throughout treatment. In multivariate analyses, baseline high WBC counts and low lymphocyte proportions were associated with positive sputum culture results at the end of treatment (WBC > 11,450 cells/mm3: p = 0.048; lymphocytes <16.0%: p = 0.039; WBC > 11,450 cells/mm3 and lymphocytes <16.0%: p = 0.024). CONCLUSION: High WBC counts and low lymphocyte proportions at baseline are significantly associated with the risk of TB treatment failure.

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Background: Tuberculosis (TB) is a leading infectious cause of death. To improve treatment efficacy, quicker monitoring methods are needed. The objective of this study was to monitor the response to a heparin-binding hemagglutinin (HBHA) interferon-γ (IFN-γ) release assay (IGRA) and QuantiFERON-TB Gold Plus (QFT-P) and to analyze plasma IFN-γ levels according to sputum culture conversion and immune cell counts during treatment. Methods: This multicentered cohort study was based in Bangladesh, Georgia, Lebanon, Madagascar, and Paraguay. Adult, non-immunocompromised patients with culture-confirmed pulmonary TB were included. Patients were followed up at baseline (T0), after two months of treatment (T1), and at the end of therapy (T2). Clinical data and blood samples were collected at each timepoint. Whole blood samples were stimulated with QFT-P antigens or recombinant methylated Mycobacterium tuberculosis HBHA (produced in Mycobacterium smegmatis; rmsHBHA). Plasma IFN-γ levels were then assessed by ELISA. Findings: Between December 2017 and September 2020, 132 participants completed treatment, including 28 (21.2%) drug-resistant patients. rmsHBHA IFN-γ increased significantly throughout treatment (0.086 IU/ml at T0 vs. 1.03 IU/ml at T2, p < 0.001) while QFT-P IFN-γ remained constant (TB1: 0.53 IU/ml at T0 vs. 0.63 IU/ml at T2, p = 0.13). Patients with low lymphocyte percentages (<14%) or high neutrophil percentages (>79%) at baseline had significantly lower IFN-γ responses to QFT-P and rmsHBHA at T0 and T1. In a small group of slow converters (patients with positive cultures at T1; n = 16), we observed a consistent clinical pattern at baseline (high neutrophil percentages, low lymphocyte percentages and BMI, low TB1, TB2, and MIT IFN-γ responses) and low rmsHBHA IFN-γ at T1 and T2. However, the accuracy of the QFT-P and rmsHBHA IGRAs compared to culture throughout treatment was low (40 and 65% respectively). Combining both tests improved their sensitivity and accuracy (70-80%) but not their specificity (<30%). Conclusion: We showed that QFT-P and rmsHBHA IFN-γ responses were associated with rates of sputum culture conversion. Our results support a growing body of evidence suggesting that rmsHBHA IFN-γ discriminates between the different stages of TB, from active disease to controlled infection. However, further work is needed to confirm the specificity of QFT-P and rmsHBHA IGRAs for treatment monitoring.

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BACKGROUND: The Latin American & Mediterranean (LAM) spoligotype family is one of the most successful genotype of Mycobacterium tuberculosis worldwide and particularly prevalent in South-America. Within this family, a sublineage named Region of Difference Rio (RDRio) was reported initially in Brazil and is characterized by a genomic deletion of about 26.3 kb. This lineage seems to show a specific adaptation to the Euro-Latin American population. In this context, we sought to evaluate the LAM family and the presence of the RDRio genotype in samples from three Latin American countries including Paraguay, Venezuela and Argentina. To detect LAM strains reliably we applied a typing scheme using spoligotyping, 12 loci MIRU-VNTR, the Ag85C103 SNP and the regions of difference RDRio and RD174. IS6110-RFLP results were also used when available. RESULTS: Genotyping of 413 M. tuberculosis isolates from three Latin-American countries detected LAM (46%) and the ill-defined T clade (16%) as the most frequent families. The highest clustering rate was detected in the sample population from the city of Caracas in Venezuela. We observed considerable differences in the presence of the RDRio lineage, with high frequency in Caracas-Venezuela (55%) and low frequency in Buenos Aires-Argentina (11%) and Paraguay (10%). The molecular markers (RD174, Ag85C103, MIRU02-MIRU40 signature) of the RDRio lineage were essentially confirmed. For the LAM family, the most polymorphic loci were MIRU40, MIRU31, MIRU10, MIRU26, MIRU16 and the least polymorphic MIRU24, MIRU20, MIRU04, MIRU23. CONCLUSIONS: Our results suggest a differential adaptation of LAM-sublineages in neighboring populations and that RDRio strains spread regionally with different rates of distribution. The Ag85C SNP and RDs (RD174, RDRio) tested in this study can in fact facilitate molecular epidemiological studies of LAM strains in endemic settings and low-income countries.

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Mycobacterium leprae, an obligate intracellular bacillus, infects Schwann cells (SCs), leading to peripheral nerve damage, the most severe leprosy symptom. In the present study, we revisited the involvement of phenolic glycolipid I (PGL I), an abundant, private, surface M. leprae molecule, in M. leprae-SC interaction by using a recombinant strain of M. bovis BCG engineered to express this glycolipid. We demonstrate that PGL I is essential for bacterial adhesion and SC internalization. We also show that live mycobacterium-producing PGL I induces the expression of the endocytic mannose receptor (MR/CD206) in infected cells in a peroxisome proliferator-activated receptor gamma (PPARγ)-dependent manner. Of note, blocking mannose recognition decreased bacterial entry and survival, pointing to a role for this alternative recognition pathway in bacterial pathogenesis in the nerve. Moreover, an active crosstalk between CD206 and the nuclear receptor PPARγ was detected that led to the induction of lipid droplets (LDs) formation and prostaglandin E2 (PGE2), previously described as fundamental players in bacterial pathogenesis. Finally, this pathway was shown to induce IL-8 secretion. Altogether, our study provides evidence that the entry of live M. leprae through PGL I recognition modulates the SC phenotype, favoring intracellular bacterial persistence with the concomitant secretion of inflammatory mediators that may ultimately be involved in neuroinflammation.

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The chronic course of lepromatous leprosy may be interrupted by acute inflammatory episodes known as erythema nodosum leprosum (ENL). Despite its being a major cause of peripheral nerve damage in leprosy patients, the immunopathogenesis of ENL remains ill-defined. Recognized by distinct families of germline-encoded pattern recognition receptors, endogenous and pathogen-derived nucleic acids are highly immunostimulatory molecules that play a major role in the host defense against infections, autoimmunity, and autoinflammation. The aim of this work was to investigate whether DNA sensing via TLR-9 constitutes a major inflammatory pathway during ENL. Flow cytometry and immunohistochemistry analysis showed significantly higher TLR-9 expression in ENL when compared with nonreactional lepromatous patients, both locally in the skin lesions and in circulating mononuclear cells. The levels of endogenous and pathogen-derived TLR-9 ligands in the circulation of ENL patients were also higher. Furthermore, PBMCs isolated from the ENL patients secreted higher levels of TNF, IL-6, and IL-1ß in response to a TLR-9 agonist than those of the nonreactional patients and healthy individuals. Finally, E6446, a TLR-9 synthetic antagonist, was able to significantly inhibit the secretion of proinflammatory cytokines by ENL PBMCs in response to Mycobacterium leprae lysate. Our data strongly indicate that DNA sensing via TLR-9 constitutes a major innate immunity pathway involved in the pathogenesis and evolution of ENL. Thus, the use of TLR-9 antagonists emerges as a potential alternative to more effectively treat ENL aiming to prevent the development of nerve injuries and deformities in leprosy.

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Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as "LAM-like", 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.

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A tuberculose (TB) é uma importante causa de mortalidade, sendo Mycobacterium tuberculosis (Mtb) o agente etiológico. Globalmente a família Latin American-Mediterranean (LAM) é responsável por aproximadamente 15por cento dos casos de TB. Dentro desta família, recentemente foi descrito um novo polimorfismo caracterizado por uma deleção de 26,3 kb e designado como LSP RDRio. Esta linhagem é frequente (30por cento) no Rio de Janeiro e dados preliminares sugerem que cepas pertencentes a este genótipo apresentem maior virulência. Neste contexto, é de grande importância avaliar a distribuição e transmissibilidade deste genótipo em nível regional e global. O presente estudo descritivo teve como objetivo, primeiramente, estudar a variabilidade genética entre isolados de Mtb do Paraguai, da Argentina e da Venezuela pelas técnicas de Spoligotyping e MIRU-VNTR 12loci, países onde existe pouca informação sobre a presença e natureza de genótipo de Mtb. Outro foco abordado foi o estudo da família LAM, que alberga genótipos de grande importância em nível regional. Para detectar esta família recentemente foi descrito um novo marcador genético que implica o estudo da presença do SNP Ag85C (G103A) por PCR-RFLP. Este marcador pode servir como complemento ao método de Spoligotyping, para ajudar a classificar certos spoligotypes mal definidos ou convergentes. O mesmo pode também constituir uma alternativa rápida e simples para detectar isolados LAM, o que pode ser de grande importância para países de menos recursos. Finalmente, estudamos a frequência da linguagem RDRio ao analisarmos além da presença do LSP RDRio, outros marcadores para esta linhagem como a deleção de RD174, a presença de 2 cópias alélicas no MIRU 2 e 1 cópia no MIRU 40 e genótipo LAM. Quanto ao primeiro objetivo, foi observada uma distribuição ampla diferença nas famílias de spoligotyping, de acordo com a região estudada. A maior taxa de agrupamentos de isolados observou-se na população de isolados venezuelanos, sugerindo um processo de transmissão contínua ou introdução de algumas linhagens muito tempo atrás. Quanto ao segundo objetivo, foi observado um comportamento variado do SNP Ag85C em referência ao seu papel como marcador da família LAM. Nos isolados paraguaios e venezuelanos houve um grau significativo de concordância com os resultados de spoligotyping enquanto nos pacientes argentinos houve baixa concordância. A relação entre a presença do SNP Ag85C (G103A) e o genótipo LAM, entretanto deve ser melhor estudada. Finalmente, foi observada a presença da linhagem RDRio nos três países, com maior frequência na Venezuela. A baixa frequência no Paraguai chama a atenção e deve ser mais bem estudada. As características genéticas da linhagem RDRio, salvo poucas exceções, estiveram presentes nos isolados dos três países estudados. Após construção de árvores tipo MST com base nos spoligotypes, observamos, como previsto, a família LAM9 como nodo central que contém a linhagem RDRio e do qual derivam os outros subtipos da família LAM. No entanto, os agrupamentos dos isolados RDRio nos MST de MIRU-VNTR devem ser melhor estudados. Concluindo, no presente estudo descrevemos os genótipos circulantes de Mtb nos três países estudados. Os resultados geraram perguntas interessantes que devem que devem ser abordados no futuro.

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