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.

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

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.

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

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.

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.

Genotipagem de isolados de Mycobacterium tuberculosis do Paraguai, da Argentina e da Venezuela

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 15% 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 freqüente (30%) no Rio de Janeiro e dados preliminares sugerem que cepas pertencentes a este genótipo apresentam 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 genótipos 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 freqüência da linhagem 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 freqüência 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 contem 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 ser abordados no futuro.