Genotipificación por MIRU-VNTR de aislados de mycobacterium tuberculosis remitidos al Hospital Km 81 en el año 2008

La tuberculosis (TB) es una enfermedad infecto-contagiosa causada por Mycobacterium tuberculosis (Mtb) que aún constituye un problema de salud pública en Paraguay. Este estudio piloto de tipo observacional descriptivo de corte transverso tuvo como objetivo caracterizar aislados de Mtb mediante la técnica molecular "Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats" (MIRU-VNTR). Se han utilizado 19 aislados colectados durante el 2008 en el Hospital Km 81, departamento de Cordillera. Las muestras fueron sometidas a amplificación de 12 loci MIRU-VNTR seguido de electroforesis en gel de poliacrilamida. Los patrones genéticos fueron clasificados haciendo uso de la herramienta bioinformática MIRU-VNTRplus. El Índice de Diversidad de Hunter Gaston (HGDI) determinó que los loci MIRU 10 y MIRU 40 presentaron el mayor poder discriminatorio. Se encontraron 4 cepas en 2 clados con 100% de similitud. Además, se hallaron 12/19 cepas en 4 clados con 80% de similitud. Estudios adicionales son necesarios para determinar asociaciones epidemiológicas. MIRU-VNTR agrupó las cepas estudiadas con miembros de las familias LAM, Haarlem y X con predominio de la familia LAM (79%)Fil: Duarte Fleitas, Mara Melissa. Universidad Nacional de Asunción (Paraguay).Fil: Díaz Acosta, Chyntia Carolina . Universidad Nacional de Asunción (Paraguay).Fil: Russomando Álvarez, Graciela Mabel. Universidad Nacional de Asunción (Paraguay)

C1q and HBHA-specific IL-13 levels as surrogate plasma biomarkers for monitoring tuberculosis treatment efficacy: a cross-sectional cohort study in Paraguay

IntroductionNew 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.MethodsThirty-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.ResultsIn 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%).ConclusionThis 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

Adenosine A2A receptor as a potential regulator of Mycobacterium leprae survival mechanisms: new insights into leprosy neural damage

BackgroundLeprosy 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.MethodsM. 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.ResultsWe 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.ConclusionThese 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

Exploring the "Latin American Mediterranean" family and the RDRio lineage in Mycobacterium tuberculosis isolates from Paraguay, Argentina and Venezuela

Submitted by Sandra Infurna ([email protected]) on 2020-03-28T17:15:58Z No. of bitstreams: 1 HarrrisonGomes_SidraVasconcelos_etal_IOC_2019.pd.pdf: 1746618 bytes, checksum: 5564ed65983e850073abda10c0053dea (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2020-03-28T17:43:14Z (GMT) No. of bitstreams: 1 HarrrisonGomes_SidraVasconcelos_etal_IOC_2019.pd.pdf: 1746618 bytes, checksum: 5564ed65983e850073abda10c0053dea (MD5)Made available in DSpace on 2020-03-28T17:43:14Z (GMT). No. of bitstreams: 1 HarrrisonGomes_SidraVasconcelos_etal_IOC_2019.pd.pdf: 1746618 bytes, checksum: 5564ed65983e850073abda10c0053dea (MD5) Previous issue date: 2019Universidad Nacional de Asunción. Instituto de Investigaciones en Ciencias de la Salud. Departamento de Biología Molecular y Biotecnología. Asunción, Paraguay / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular aplicada às Micobactérias. Rio de Janeiro, RJ, Brasil.Universidad Nacional de Asunción. Instituto de Investigaciones en Ciencias de la Salud. Departamento de Biología Molecular y Biotecnología. Asunción, Paraguay.Universidad Nacional de Asunción. Instituto de Investigaciones en Ciencias de la Salud. Departamento de Biología Molecular y Biotecnología. Asunción, Paraguay.Instituto Nacional de Enfermedades Infecciosas, ANLIS “Carlos G. Malbran”. Servicio de Micobacterias. Buenos Aires. Argentina.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular aplicada às Micobactérias. Rio de Janeiro, RJ, Brasil..Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular, Rio de Janeiro, RJ, Brasil.Laboratorio Central de Salud Pública, MSP y BS. Asunción, Paraguay.Instituto Nacional de Enfermedades Respiratorias Emilio Coni. Buenos Aires, Argentina.Instituto de Biomedicina. Laboratorio de Tuberculosis. Caracas, Venezuela / Universidad de Las Américas Facultad de Ciencias de la Salud. One Health Research Group. Quito, Ecuador.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular aplicada às Micobactérias. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular aplicada às Micobactérias. Rio de Janeiro, RJ, Brasil.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

SARS-CoV-2 Variants in Paraguay: Detection and Surveillance with an Economical and Scalable Molecular Protocol

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

Image_1_C1q and HBHA-specific IL-13 levels as surrogate plasma biomarkers for monitoring tuberculosis treatment efficacy: a cross-sectional cohort study in Paraguay.jpeg

IntroductionNew 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.MethodsThirty-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.ResultsIn unstimulated plasma, C1q (PConclusionThis 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.</p

Table_1_C1q and HBHA-specific IL-13 levels as surrogate plasma biomarkers for monitoring tuberculosis treatment efficacy: a cross-sectional cohort study in Paraguay.docx

IntroductionNew 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.MethodsThirty-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.ResultsIn unstimulated plasma, C1q (PConclusionThis 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.</p

Association of baseline white blood cell counts with tuberculosis treatment outcome: a prospective multicentered cohort study

International audienceObjectives: 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 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

PGL I expression in live bacteria allows activation of a CD206/PPARγ cross-talk that may contribute to successful Mycobacterium leprae colonization of peripheral nerves.

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

Multi-country evaluation of RISK6, a 6-gene blood transcriptomic signature, for tuberculosis diagnosis and treatment monitoring

There is a crucial need for non-sputum-based TB tests. Here, we evaluate the performance of RISK6, a human-blood transcriptomic signature, for TB screening, triage and treatment monitoring. RISK6 performance was also compared to that of two IGRAs: one based on RD1 antigens (QuantiFERON-TB Gold Plus, QFT-P, Qiagen) and one on recombinant M. tuberculosis HBHA expressed in Mycobacterium smegmatis (IGRA-rmsHBHA). In this multicenter prospective nested case–control study conducted in Bangladesh, Georgia, Lebanon and Madagascar, adult non-immunocompromised patients with bacteriologically confirmed active pulmonary TB (ATB),