Transglutaminase 2 Regulates Innate Immunity by Modulating the STING/TBK1/IRF3 Axis.

We have recently shown that type 2 transglutaminase (TG2) plays a key role in the host's inflammatory response during bacterial infections. In this study, we investigated whether the enzyme is involved in the regulation of the STING pathway, which is the main signaling activated in the presence of both self- and pathogen DNA in the cytoplasm, leading to type I IFN (IFN I) production. In this study, we demonstrated that TG2 negatively regulates STING signaling by impairing IRF3 phosphorylation in bone marrow-derived macrophages, isolated from wild-type and TG2 knockout mice. In the absence of TG2, we found an increase in the IFN-ß production and in the downstream JAK/STAT pathway activation. Interestingly, proteomic analysis revealed that TG2 interacts with TBK1, affecting its interactome composition. Indeed, TG2 ablation facilitates the TBK1-IRF3 interaction, thus indicating that the enzyme plays a negative regulatory effect on IRF3 recruitment in the STING/TBK1 complex. In keeping with these findings, we observed an increase in the IFNß production in bronchoalveolar lavage fluids from COVID-19-positive dead patients paralleled by a dramatic decrease of the TG2 expression in the lung pneumocytes. Taken together, these results suggest that TG2 plays a negative regulation on the IFN-ß production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.

Commercially available CD4 + and CD8 + IFN-γ release assays combined with an HBHA-induced IGRA improve the characterization of the tuberculosis spectrum and monitoring of treatment in children.

Commercially available Interferon-γ release assays (IGRAs), including the last-generation QuantiFERON TB-Plus (QFT-Plus), are effective in aiding the diagnosis of tuberculosis (TB) infection but cannot distinguish latent TB subjects from active TB patients. The aim of this study was to prospectively evaluate the performance of an HBHA-based IGRA, combined with commercially available IGRAs, to assess their usefulness as a prognostic biomarkers and aid in the monitoring of TB treatment in children. Following clinical, microbiological, and radiological assessment, children younger than 18 years of age classified as either LTBI or active TB were tested at baseline and during treatment by the QuantiFERON TB-Plus (QFT) assay and an aliquot of whole-blood was stimulated with HBHA. Among the 655 children evaluated, 559 (85.3%) were classified as "Non TB", 44 patients (6.7%) with active TB, and 52 (7.9%) with LTBI. The median HBHA-IGRA IFN-gamma responses were able to discriminate active TB from LTBI (0.13 IU/ml vs 1.995, (p < 0,0001), those with asymptomatic TB from those with symptomatic TB (1.01 IU/ml vs 0.115 IU/ml, p 0.017), or more severe TB (p 0.022), and significantly raised during successful TB treatment (p < 0.0001). Conversely, CD4 + and CD8 + responses were similar in all groups of patients, although active TB patients had higher CD4 + responses and LTBI higher CD8 + responses.  Conclusion: HBHA-based IGRA, combined with CD4 + and CD8 + responses assessed by commercially available IGRAs, is a useful support in the characterization of the TB spectrum in children and monitoring of TB-therapy. What is Known: • Current immune diagnostics are not able to discriminate active and latent Ttuberculosis, including the recently approved QFT-PLUS.. • New immunological assays with prognostic value are highly needed. What is New: • HBHA-based IGRA, combined with CD4+ and CD8+ responses assessed by commercially available IGRAs, is a useful support for the differentiation of active and latent TB in children.. • HBHA-based IGRA, combined with CD4+ and CD8+ responses assessed by commercially available IGRAs, is a useful support in the monitoring of TBtherapy in children..

Cysteamine/Cystamine Exert Anti-Mycobacterium abscessus Activity Alone or in Combination with Amikacin.

Host-directed therapies are emerging as a promising tool in the curing of difficult-to-treat infections, such as those caused by drug-resistant bacteria. In this study, we aim to test the potential activity of the FDA- and EMA-approved drugs cysteamine and cystamine against Mycobacterium abscessus. In human macrophages (differentiated THP-1 cells), these drugs restricted M. abscessus growth similar to that achieved by amikacin. Here, we use the human ex vivo granuloma-like structures (GLS) model of infection with the M. abscessus rough (MAB-R) and smooth (MAB-S) variants to study the activity of new therapies against M. abscessus. We demonstrate that cysteamine and cystamine show a decrease in the number of total GLSs per well in the MAB-S and MAB-R infected human peripheral blood mononuclear cells (PBMCs). Furthermore, combined administration of cysteamine or cystamine with amikacin resulted in enhanced activity against the two M. abscessus morpho variants compared to treatment with amikacin only. Treatment with cysteamine and cystamine was more effective in reducing GLS size and bacterial load during MAB-S infection compared with MAB-R infection. Moreover, treatment with these two drugs drastically quenched the exuberant proinflammatory response triggered by the MAB-R variant. These findings showing the activity of cysteamine and cystamine against the R and S M. abscessus morphotypes support the use of these drugs as novel host-directed therapies against M. abscessus infections.

First description of the katG gene deletion in a Mycobacterium tuberculosis clinical isolate and its impact on the mycobacterial fitness.

Isoniazid (INH) is the cornerstone of the anti-tuberculosis regimens and emergence of Mycobacterium tuberculosis (Mtb) resistant strains is a major threat to our ability to control tuberculosis (TB) at global level. Mutations in the gene coding the catalase KatG confer resistance to high level of INH. In this paper, we describe for the first time a complete deletion of the genomic region containing the katG gene in an Mtb clinical strain isolated in Italy in a patient with HIV infection that previously completed INH preventive therapy. We genotypically characterized the Mtb strain and showed that katG deletion confers high-level resistance to INH (MIC > 25.6 µg/mL). The katG deletion did not impact significantly on Mtb fitness as we did not detect enhanced susceptibility to H2O2 compared to the wild type Mtb strains nor impaired growth in in vitro infection models. These findings highlight the ability of Mtb to acquire resistance to INH while maintaining fitness and pathogenic potential.

Lumipulse G SARS-CoV-2 Ag assay evaluation using clinical samples from different testing groups.

OBJECTIVES: Compared to RT-PCR, lower performance of antigen detection assays, including the Lumipulse G SARS-CoV-2 Ag assay, may depend on specific testing scenarios. METHODS: We tested 594 nasopharyngeal swab samples from individuals with COVID-19 (RT-PCR cycle threshold [Ct] values ≤ 40) or non-COVID-19 (Ct values >40) diagnoses. RT-PCR positive samples were assigned to diagnostic, screening, or monitoring groups of testing. RESULTS: With a limit of detection of 1.2 × 104 SARS-CoV-2 RNA copies/mL, Lumipulse showed positive percent agreement (PPA) of 79.9% (155/194) and negative percent agreement of 99.3% (397/400), whereas PPAs were 100% for samples with Ct values of <18 or 18-<25 and 92.5% for samples with Ct values of 25-<30. By three groups, Lumipulse showed PPA of 87.0% (60/69), 81.1% (43/53), or 72.2% (52/72), respectively, whereas PPA was 100% for samples with Ct values of <18 or 18-<25, and was 94.4, 80.0, or 100% for samples with Ct values of 25-<30, respectively. Additional testing of RT-PCR positive samples for SARS-CoV-2 subgenomic RNA showed that, by three groups, PPA was 63.8% (44/69), 62.3% (33/53), or 33.3% (24/72), respectively. PPAs dropped to 55.6, 20.0, or 41.7% for samples with Ct values of 25-<30, respectively. All 101 samples with a subgenomic RNA positive result had a Lumipulse assay's antigen positive result, whereas only 54 (58.1%) of remaining 93 samples had a Lumipulse assay's antigen positive result. CONCLUSIONS: Lumipulse assay was highly sensitive in samples with low RT-PCR Ct values, implying repeated testing to reduce consequences of false-negative results.

Accuracy of QuantiFERON-TB Gold Plus Test for Diagnosis of Mycobacterium tuberculosis Infection in Children.

Compared to its predecessor QuantiFERON-TB Gold In Tube (QFT-IT), QuantiFERON-TB Gold Plus (QFT-Plus) contains an additional antigen tube (TB2), stimulating both CD4+ and CD8+ T cells. The ability to discriminate CD4+ and CD8+ responses is suggested to be useful in differentiating stages of Mycobacterium tuberculosis infection. While QFT-Plus has already been evaluated in adults, there are not enough data in children evaluated for suspected active tuberculosis (TB) or latent TB infection (LTBI). A prospective cross-sectional study was conducted among children aged 0 to 17 years who were evaluated for suspected active TB or screened for LTBI. All children underwent QFT-Plus and further clinical, radiological, and/or microbiological analyses according to clinical scenario. Of the 198 children enrolled, 43 (21.7%) were tested because of suspicion of active TB. A total of 12/43 (27.9%) were diagnosed with active TB, and among these, 10/12 (83.3%) had a positive QFT-Plus assay. Of the 155 children screened for LTBI, 18 (11.6%) had a positive QFT-Plus, and 5 (2.5%) had an indeterminate result. TB1 and TB2 quantitative responses were not able to discriminate active disease from latent infection. The percent agreement between TB1 and TB2 was 100%. QFT-Plus assay showed good sensitivity for active TB and was particularly useful for the evaluation of children with suspected LTBI, giving a low rate of indeterminate results in this group. More studies are needed to properly evaluate QFT-Plus ability in discriminating active disease from latent infection.

IP-10 contributes to the inhibition of mycobacterial growth in an ex vivo whole blood assay.

Interferon-γ inducible protein 10 (IP-10), is a potent chemoattractant that promotes migration of monocytes and activated T-cells to inflammation foci. IP-10 is elevated in serum of patients with chronic hepatitis C virus (HCV) and tuberculosis (TB) infections, although it remains to be determined the contribution of IP-10 in restricting Mycobacterium tuberculosis (Mtb) replication. Here, we investigated the impact of IP-10 on mycobacteria replication using the ex vivo model of human whole-blood (WB) assay. In particular, we compared the levels of IP-10 upon infection with different Mtb clinical strains and species of non-tuberculous mycobacteria (NTM) and evaluated how IP-10 may contain bacterial replication. Interestingly, we observed that the inhibition of the host enzyme dipeptidyl peptidase IV (DPP-IV), which inactivates IP-10 through cleavage of two amino acids at the chemokine N-terminus, restricted mycobacterial persistence in WB, supporting the critical role of full length IP-10 in mediating an anti-Mtb response. Addition of recombinant IP-10 expressed in eukaryotic cells enhanced the anti-mycobacterial activity in WB, although no differences were observed when IP-10 containing different proportions of cleaved and non-cleaved forms of the chemokine were added. Moreover, recombinant IP-10 did not exert a direct anti-mycobacterial effect. Our results underscore the clinical relevance of IP-10 in mycobacteria pathogenesis and support the potential outcomes that may derive by targeting the IP-10/CXCR3 pathway as host directed therapies for the treatment of Mtb or NTM infections.

PE_PGRS3 of Mycobacterium tuberculosis is specifically expressed at low phosphate concentration, and its arginine-rich C-terminal domain mediates adhesion and persistence in host tissues when expressed in Mycobacterium smegmatis.

PE_PGRSs of Mycobacterium tuberculosis (Mtb) represent a family of complex and peculiar proteins whose role and function remain elusive. In this study, we investigated PE_PGRS3 and PE_PGRS4, two highly homologous PE_PGRSs encoded by two contiguous genes in the Mtb genome. Using a gene-reporter system in Mycobacterium smegmatis (Ms) and transcriptional analysis in Mtb, we show that PE_PGRS3, but not PE_PGRS4, is specifically expressed under low phosphate concentrations. Interestingly, PE_PGRS3, but not PE_PGRS4, has a unique, arginine-rich C-terminal domain of unknown function. Heterologous expression of PE_PGRS3 in Ms was used to demonstrate cellular localisation of the protein on the mycobacterial surface, where it significantly affects net surface charge. Moreover, expression of full-length PE_PGRS3 enhanced adhesion of Ms to murine macrophages and human epithelial cells and improved bacterial persistence in spleen tissue following infection in mice. Expression of the PE_PGRS3 functional deletion mutant lacking the C-terminal domain in Ms did not enhance adhesion to host cells, showing a phenotype similar to the Ms parental strain. Interestingly, enhanced persistence of Ms expressing PE_PGRS3 did not correlate with increased concentrations of inflammatory cytokines. These results point to a critical role for the ≈ 80 amino acids long, arginine-rich C-terminal domain of PE_PGRS3 in tuberculosis pathogenesis.

Incidental intraoperative diagnosis of Mycobacterium abscessus meningeal infection: a case report and review of the literature.

PURPOSE: Mycobacterium abscessus, and rapidly growing mycobacteria in general, are rare but increasing causes of central nervous system (CNS) infections. The aim of this study is to highlight the importance of considering these microorganism in the differential diagnosis of CNS infections, obtaining a prompt diagnosis, and improving clinical outcomes. METHODS: Case report and literature review. RESULTS: We report a case of meningeal infection in a patient who underwent decompressive craniectomy after a craniofacial trauma. The diagnosis was made analyzing a sample obtained during a second operation of cranioplasty. A regimen of amikacin, clarithromycin, and imipenem/cilastatin was started. In the following days, the patient experienced a variety of side effects. So, first clarithromycin was replaced with linezolid, then amikacin was stopped and cefoxitin added to the therapy and at the end all the antibiotics were withdrawn. The patient was discharged in good conditions and a clinical interdisciplinary follow-up was started. After 12 months, the patient is still doing well. After a literature analysis, 15 cases of M. abscessus CNS infections were identified. Various modes of acquisition, underlying disease and therapeutic schemes were evident. CONCLUSIONS: Considering the results of the literature analysis and the increasing incidence of M. abscessus, all specialists involved in the management of CNS infection should be aware of the importance of atypical microorganisms in differential diagnosis.

Host Directed Therapies for Tuberculosis: Futures Strategies for an Ancient Disease.

The emergence and spread of drug-resistant strains of Mycobacterium tuberculosis is worsening the global threat of tuberculosis (TB). There is a need and urgency for the development of new treatments for TB, for the management of drug resistant TB (MDR-TB) and for improved regimens against drug-susceptible TB, with the goal of reducing toxicity and length of therapy that will boost patience compliance. The paucity of new drugs is a major obstacle to design new regimens while host-directed therapies (HDTs) are emerging as a promising area of research and are opening new avenues to fight TB. In this review, we discuss examples of potentially promising strategies aimed at improving the host response to M. tuberculosis, and argue how a better understanding of TB pathogenesis, with the fine characterization of the immunological mediators involved, may pave the way for the design of new therapies, the identification of new drugs or the repurposing of some already in use for other diseases. We emphasize that any HDTs shall be included as adjunct therapy to the drug-combination regimens already in use for TB, with the goal to reduce tissue damage and immunopathology and enhance bacterial clearance. We anticipate that the benefits of HDTs against TB will be highest against MDR-TB, where the activity of current regimens is poor and the cost high.

Unraveling the potential of graphene quantum dots against Mycobacterium tuberculosis infection.

Introduction: The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains has underscored the urgent need for novel therapeutic approaches. Carbon-based nanomaterials, such as graphene oxide (GO), have shown potential in anti-TB activities but suffer from significant toxicity issues. Methods: This study explores the anti-TB potential of differently functionalized graphene quantum dots (GQDs) - non-functionalized, L-GQDs, aminated (NH2-GQDs), and carboxylated (COOH-GQDs) - alone and in combination with standard TB drugs (isoniazid, amikacin, and linezolid). Their effects were assessed in both axenic cultures and in vitro infection models. Results: GQDs alone did not demonstrate direct mycobactericidal effects nor trapping activity. However, the combination of NH2-GQDs with amikacin significantly reduced CFUs in in vitro models. NH2-GQDs and COOH-GQDs also enhanced the antimicrobial activity of amikacin in infected macrophages, although L-GQDs and COOH-GQDs alone showed no significant activity. Discussion: The results suggest that specific types of GQDs, particularly NH2-GQDs, can enhance the efficacy of existing anti-TB drugs. These nanoparticles might serve as effective adjuvants in anti-TB therapy by boosting drug performance and reducing bacterial counts in host cells, highlighting their potential as part of advanced drug delivery systems in tuberculosis treatment. Further investigations are needed to better understand their mechanisms and optimize their use in clinical settings.

Solute Transporter OCTN1/Slc22a4 Affects Disease Severity and Response to Infliximab in Experimental Colitis: Role of Gut Microbiota and Immune Modulation.

BACKGROUND: Inflammatory bowel diseases are chronic disabling conditions with a complex and multifactorial etiology, still incompletely understood. OCTN1, an organic cation transporter, could have a role in modulating the inflammatory response, and some genetic polymorphisms of this molecule have been associated with increased risk of inflammatory bowel diseases. Until now, limited information exists on its potential in predicting/modulating patient's response to therapies. The aim of this study was to evaluate the role of OCTN1 in modifying gut microbiota and mucosal immunity in response to infliximab therapy in murine colitis. METHODS: A dextran sodium sulphate model of colitis was used to assess the clinical efficacy of infliximab administered intravenously in ocnt1 gene knockout mice and their C57BL/6 controls. Stool, colon, and mesenteric lymph node samples were collected to evaluate differences in gut microbiota composition, histology, and T cell populations, respectively. RESULTS: Octn1 -/- influences the microbiota profile and is associated with a worse dysbiosis in mice with colitis. Infliximab treatment attenuates colitis-associated dysbiosis, with an increase of bacterial richness and evenness in both strains. In comparison with wild type, octn1-/- mice have milder disease and a higher baseline percentage of Treg, Tmemory, Th2 and Th17 cells. CONCLUSIONS: Our data support the murine model to study OCTN1 genetic contribution to inflammatory bowel diseases. This could be the first step towards the recognition of this membrane transporter as a biomarker in inflammatory conditions and a predictor of response to therapies.

In this article, we evaluated the role of OCTN1, an organic cation transporter, in modifying gut microbiota and immune T cell populations, as well as its effects on experimental colitis and the response to infliximab treatment.

PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis.

The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome-lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C-terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non-pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.

Alternative therapies against Mycobacterium abscessus infections.

BACKGROUND: Mycobacterium abscessus (Mab) is considered as the most pathogenic rapid-growing mycobacteria in humans, causing pulmonary and extra-pulmonary diseases, especially in patients with cystic fibrosis. Mab shows intrinsic and acquired resistance to many drugs, leaving limited treatment options that lead to a generally poor prognosis. The standard therapeutic regimen last for more than 6 months and consists of a drug cocktail that ideally includes a macrolide and amikacin. Yet, toxicity and efficacy are suboptimal due also to the high toxicity. There is a need to introduce innovative and out-of-the-box approaches to improve treatments. OBJECTIVES: In this narrative review, we summarize the recent research on the alternative strategies proposed and discuss the importance of using appropriate experimental assays to assess their activity. SOURCES: Included articles were identified by searching PubMed and MEDLINE until June 2023. The search terms were 'Mycobacterium abscessus', 'antimicrobial', and 'alternative therapies'. Additional relevant references were obtained from articles retrieved from the primary search. CONTENT: Therapies against Mab including host directed therapies, repurposed drugs, phage therapy, anti-virulence strategies, essential oils, and inhalation therapies. IMPLICATIONS: Alternative treatments may represent a valid tool to cope the burden of antimicrobial resistance in Mab-caused diseases.

Comparison between Nasopharyngeal and Saliva Samples for the Detection of Respiratory Viruses in Children with Acute Lower Respiratory Tract Infections: A Pilot Study.

PURPOSE: During the COVID-19 pandemic, the use of salivary swabs (SS) to detect the SARS-CoV-2 virus has been implemented and widely studied in adults and children. However, the role of SS in detecting other common respiratory viruses in children is poorly investigated. METHODS: Children younger than 18 years of age admitted with respiratory signs and symptoms underwent both nasopharyngeal and SS procedures. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of SS were calculated, considering the nasopharyngeal swab result as the gold standard. RESULTS: A total of 83 patients (44 females, 53%) underwent both nasopharyngeal and SS procedures. Overall, the sensitivity of SS was 49.4%. Sensitivity according to different respiratory viruses ranged from 0% to 71.43%, while the specificity ranged from 96% to 100%. Negative predictive value ranged from 68.06% to 98.8%, while positive predictive value ranged from 0 to 100%. SS sensitivity in patients younger than 12 months of age was 39.47%, while in patients older than or equal to 12 months of age it was 57.78%. Patients with negative SS had a significantly lower median age (8.5 months (15.25) vs. 23 months (34), p = 0.001) and a significantly lower quantity of median saliva collected for salivary analysis (0 µL (213) vs. 300 µL (100), p < 0.001). CONCLUSIONS: SS has a relatively low sensitivity in detecting common respiratory viruses in children with LRTI, with a lower probability in younger children (and in particular those younger than 6 months of age) or those from whom we have collected lesser amounts of saliva. New strategies to improve saliva collection are needed for testing on a larger study population.

The ubiquitin ligase TRIM32 promotes the autophagic response to Mycobacterium tuberculosis infection in macrophages.

Mycobacterium tuberculosis (Mtb) is known to evade host immune responses and persist in macrophages for long periods. A mechanism that the host uses to combat Mtb is xenophagy, a selective form of autophagy that targets intracellular pathogens for degradation. Ubiquitination of Mtb or Mtb-containing compartments is a key event to recruit the autophagy machinery and mediate the bacterial delivery to the lysosome. This event relies on the coordinated and complementary activity of different ubiquitin ligases, including PARKIN, SMURF1, and TRIM16. Because each of these factors is responsible for the ubiquitination of a subset of the Mtb population, it is likely that additional ubiquitin ligases are employed by macrophages to trigger a full xenophagic response during Mtb infection. In this study, we investigated the role TRIM proteins whose expression is modulated in response to Mtb or BCG infection of primary macrophages. These TRIMs were ectopically expressed in THP1 macrophage cell line to assess their impact on Mtb replication. This screening identified TRIM32 as a novel player involved in the intracellular response to Mtb infection, which promotes autophagy-mediated Mtb degradation. The role of TRIM32 in xenophagy was further confirmed by silencing TRIM32 expression in THP1 cells, which causes increased intracellular growth of Mtb associated to impaired Mtb ubiquitination, reduced recruitment of the autophagy proteins NDP52/CALCOCO2 and BECLIN 1/BECN1 to Mtb and autophagosome formation. Overall, these findings suggest that TRIM32 plays an important role in the host response to Mtb infection through the induction of autophagy, representing a promising target for host-directed tuberculosis therapies.

Evaluation of the Toxic Activity of the Graphene Oxide in the Ex Vivo Model of Human PBMC Infection with Mycobacterium tuberculosis.

Graphene Oxide has been proposed as a potential adjuvant to develop improved anti-TB treatment, thanks to its activity in entrapping mycobacteria in the extracellular compartment limiting their entry in macrophages. Indeed, when administered together with linezolid, Graphene Oxide significantly enhanced bacterial killing due to the increased production of Reactive Oxygen Species. In this work, we evaluated Graphene Oxide toxicity and its anti-mycobacterial activity on human peripheral blood mononuclear cells. Our data show that Graphene Oxide, different to what is observed in macrophages, does not support the clearance of Mycobacterium tuberculosis in human immune primary cells, probably due to the toxic effects of the nano-material on monocytes and CD4+ lymphocytes, which we measured by cytometry. These findings highlight the need to test GO and other carbon-based nanomaterials in relevant in vitro models to assess the cytotoxic activity while measuring antimicrobial potential.

Evaluation of Everolimus Activity against Mycobacterium tuberculosis Using In Vitro Models of Infection.

Even though Everolimus has been investigated in a phase II randomized trial as a host-directed therapy (HDT) to treat tuberculosis (TB), an oncological patient treated with Everolimus for a neuroendocrine pancreatic neoplasia developed active TB twice and a non-tuberculous mycobacterial (NTM) infection in a year and a half time span. To investigate this interesting case, we isolated and genotypically characterized the Mycobacterium tuberculosis (Mtb) clinical strain from the patient and tested the effect of Everolimus on its viability in an axenic culture and in a peripheral blood mononuclear cell (PBMCs) infection model. To exclude strain-specific resistance, we tested the activity of Everolimus against Mtb strains of ancient and modern lineages. Furthermore, we investigated the Everolimus effect on ROS production and autophagy modulation during Mtb infection. Everolimus did not have a direct effect on mycobacteria viability and a negligible effect during Mtb infection in host cells, although it stimulated autophagy and ROS production. Despite being a biologically plausible HDT against TB, Everolimus does not exert a direct or indirect activity on Mtb. This case underlines the need for a careful approach to drug repurposing and implementation and the importance of pre-clinical experimental studies.

Evaluation of the New NucliSENS EasyQ KPC test for rapid detection of Klebsiella pneumoniae carbapenemase genes (blaKPC).

KPC-type carbapenemases are emerging in Klebsiella pneumoniae and other Gram-negative pathogens worldwide. Rapid and sensitive detection of these resistance determinants has become relevant to clinical management and infection control. We evaluated the bioMérieux EasyQ real-time PCR assay for bla(KPC) detection with 300 members of the Enterobacteriaceae, including 29 control strains producing known carbapenemases and 271 nonreplicate clinical isolates. The EasyQ assay correctly detected all of the 111 isolates harboring bla(KPC) genes, with no false positives, and results were available within 2 h.

Immunopathology of SARS-CoV-2 Infection: A Focus on T Regulatory and B Cell Responses in Children Compared with Adults.

While the clinical impact of COVID-19 on adults has been massive, the majority of children develop pauci-symptomatic or even asymptomatic infection and only a minority of the latter develop a fatal outcome. The reasons of such differences are not yet established. We examined cytokines in sera and Th and B cell subpopulations in peripheral blood mononuclear cells (PBMC) from 40 children (<18 years old), evaluating the impact of COVID-19 infection during the pandemic's first waves. We correlated our results with clinical symptoms and compared them to samples obtained from 16 infected adults and 7 healthy controls. While IL6 levels were lower in SARS-CoV-2+ children as compared to adult patients, the expression of other pro-inflammatory cytokines such as IFNγ and TNFα directly correlated with early age infection and symptoms. Th and B cell subsets were modified during pediatric infection differently with respect to adult patients and controls and within the pediatric group based on age. Low levels of IgD− CD27+ memory B cells correlated with absent/mild symptoms. On the contrary, high levels of FoxP3+/CD25high T-Regs associated with a moderate−severe clinical course in the childhood. These T and B cells subsets did not associate with severity in infected adults, with children showing a predominant expansion of immature B lymphocytes and natural regulatory T cells. This study shows differences in immunopathology of SARS-CoV-2 infection in children compared with adults. Moreover, these data could provide information that can drive vaccination endpoints for children.