The PE family of Mycobacterium tuberculosis includes 98 proteins which share a highly homologous N-terminus sequence of about 110 amino acids (PE domain). Depending on the C-terminal domain, the PE family can be divided in three subfamilies, the largest of which is the PE_PGRS with 61 members. In this study, we determined the cellular localization of three PE proteins by cell fractionation and immunoelectron microscopy by expressing chimeric epitope-tagged recombinant proteins in Mycobacterium smegmatis. We demonstrate that the PE domain of PE_PGRS33 and PE11 (a protein constituted by the only PE domain) contains the information necessary for cell wall localization, and that they can be used as N-terminal fusion partners to deliver a sufficiently long C-terminus-linked protein domain on the mycobacterial cell surface. Indeed, we demonstrate that PE_PGRS33 and Rv3097c (a lipase belonging to the PE family) are surface exposed and localize in the mycobacterial cell wall. Moreover, we found that PE_PGRS33 is easily extractable by detergents suggesting its localization in the mycobacterial outer membrane. Beyond defining the cellular localization of these proteins, and a function for their PE domains, these data open the interesting possibility to construct recombinant mycobacteria expressing heterologous antigens on their surface for vaccine purposes.

Tuberculosis (TB) still poses a major threat to mankind and during the last thirty years we have seen a recrudescence of the disease even in countries where TB was thought to be conquered. It is common opinion that more effective control tools such as new diagnostics, a new vaccine and new drugs are urgently needed to control the global pandemic, though the so far insufficient understanding of the Mycobacterium tuberculosis (Mtb) mechanism of pathogenesis is a major obstacle for the development of these control tools. In this review, we will summarize the recent advancement in the understanding of Mtb biology and on the pathogenesis of Mtb infection with emphasis on latent infection, with the change in paradigm of the last few years where the dichotomy between latent and active disease has been reconsidered in favor of a dynamic equilibrium between the host and the bacilli, encompassing a continuous spectrum of conditions that has been named TB spectrum. Implications for the diagnosis and control of disease in certain population will also be discussed.

Background:Human papillomavirus 16 infection has been proven to be associated with oropharyngeal squamous cell carcinomas (SCCs) and is probably the main reason of the reported increase in the incidence. The role of high-risk (HR) HPV for carcinogenesis of other sites in the head and neck awaits confirmation. With the aim to evaluate the prevalence of HPV infection and the reliability of different diagnostic tools in SCCs of different sites, 109 consecutive untreated head and neck SCCs were enroled, and fresh tumour samples collected.Methods:Human papillomavirus DNA was detected by Digene Hybrid Capture 2 (HC2). Human papillomavirus E6 and E7 mRNA were detected by NucliSENS EasyQ HPVv1. P16 expression was evaluated by immunohistochemistry.Results:In all, 12.84% of cases were infected by HR genotypes and 1.84% by low-risk genotypes. Human papillomavirus 16 accounted for 87% of HR infections. The overall agreement between DNA and RNA detection is 99.1%. Although p16 expression clearly correlates with HPV infection (P=0.0051), the inter-rater agreement is poor (k=0.27). The oropharynx showed the highest HR HPV infection rate (47.6%) and was also the only site in which p16 immunohistochemistry revealed to be a fair, but not excellent, diagnostic assay (κ=0.61).Conclusion:The prognostic role of HR HPV infection in oropharyngeal oncology, with its potential clinical applications, underscores the need for a consensus on the most appropriate detection methods. The present results suggest that viral mRNA detection could be the standard for fresh samples, whereas DNA detection could be routinely used in formalin-fixed, paraffin-embedded samples.

SummaryThe 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 Mtb DPE_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 Mtb DPE_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 nonpathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.

Background: To date, there are no comprehensive data on pediatric COVID-19 from Latin America. This study aims to assess COVID-19 and Multisystem Inflammatory Syndrome (MIS-C) in Latin American children, to appropriately plan and allocate resources to face the pandemic on a local and international level. Methods: Ambispective multicenter cohort study from 5 Latin American countries. Children 18 years of age or younger with microbiologically confirmed SARS-CoV-2 infection or fulfilling MIS-C definition were included. Findings: Four hundred nine children were included, with a median age of 3.0 years (interquartile range 0.6–9.0). Of these, 95 (23.2%) were diagnosed with MIS-C. One hundred ninety-one (46.7%) children were admitted to hospital and 52 (12.7%) required admission to a pediatric intensive care unit. Ninety-two (22.5%) patients required oxygen support: 8 (2%) were started on continuous positive airway pressure and 29 (7%) on mechanical ventilation. Thirty-five (8.5%) patients required inotropic support. The following factors were associated with pediatric intensive care unit admission: preexisting medical condition (P < 0.0001), immunodeficiency (P = 0.01), lower respiratory tract infection (P < 0.0001), gastrointestinal symptoms (P = 0.006), radiologic changes suggestive of pneumonia and acute respiratory distress syndrome (P < 0.0001) and low socioeconomic conditions (P = 0.009). Conclusions: This study shows a generally more severe form of COVID-19 and a high number of MIS-C in Latin American children, compared with studies from China, Europe and North America, and support current evidence of a more severe disease in Latin/Hispanic children or in people of lower socioeconomic level. The findings highlight an urgent need for more data on COVID-19 in Latin America.

Background To date, there are no comprehensive data on pediatric COVID19 from Latin America. This study aims to assess COVID-19 and Multisystem Inflammatory Syndrome (MIS C) in Latin American children, in order to appropriately plan and allocate resources to face the pandemic on a local and International lever Methods Ambispective multicentre cohort study from five Latin American countries. Children aged 18 years or younger with microbiologically confirmed SARS CoV 2 infection were included. Findings 409 children were included, with a median age of 53.0 years (IQR 0.6 9.0). Of these, 95 191 (23.2%) were diagnosed with MIS C. 191 (46.7%) children were admitted to hospital and 52 (12.7%) required admission to a Pediatric Intensive Care Unite (PICU). 92 (22.5%) patients required oxygen support: 8 (2%) were started on continuous positive airway pressure (CPAP) and 29 (7%) on mechanical ventilation. 35 (8.5%) patients required inotropic support. The following factors were associated with PICU admission: pre-existing medical condition (P < 0.0001), immunodeficiency (P = 0.01), lower respiratory tract infection (P< 0.0001), gastrointestinal symptoms (P = 0.006), radiological changes suggestive of pneumonia and acute respiratory distress syndrome (P< 0.0001), low socioeconomic conditions (P 0.009). Conclusions This study shows a generally more severe form of COVID 19 and a high number of MIS C in Latin American children, compared with studies from China, Europe and North America, and support current evidence of a more severe disease in Latin/Hyspanic children or in people of lower socioeconomic level. The findings highlight an urgent need of more data of COVID 19 in South America.

PE_PGRS represent a large family of proteins typical of pathogenic mycobacteria whose members are characterized by an N-terminal PE domain followed by a large Gly-Ala repeat-rich C-terminal domain. Despite the abundance of PE_PGRS-coding genes in the Mycobacterium tuberculosis (Mtb) genome their role and function in the biology and pathogenesis still remains elusive. In this study, we generated and characterized an Mtb H37Rv mutant (MtbΔ33) in which the structural gene of PE_PGRS33, a prototypical member of the protein family, was inactivated. We showed that this mutant entered macrophages with an efficiency up to ten times lower than parental or complemented strains, while its efficiency in infecting pneumocytes remained unaffected. Interestingly, the lack of PE_PGRS33 did not affect the intracellular growth of this mutant in macrophages. Using a series of functional deletion mutants of the PE_PGRS33 gene to complement the MtbΔ33 strain, we demonstrated that the PGRS domain is required to mediate cell entry into macrophages, with the key domain encompassing position 140–260 amino acids of PE_PGRS33. PE_PGRS33-mediated entry into macrophages was abolished in TLR2-deficient mice, as well as following treatment with wortmannin or an antibody against the complement receptor 3 (CR3), indicating that PE_PGRS33-mediated entry of Mtb in macrophages occurs through interaction with TLR2.

Extended-spectrum-␤-lactamase (ESBL)-producing strains of Escherichia coli are a significant cause of bloodstream infections (BSI) in hospitalized and nonhospitalized patients. We previously showed that delaying effective antimicrobial therapy in BSI caused by ESBL producers significantly increases mortality. The aim of this retrospective 7-year analysis was to identify risk factors for inadequate initial antimicrobial therapy (IIAT) (i.e., empirical treatment based on a drug to which the isolate had displayed in vitro resistance) for inpatients with BSI caused by ESBL-producing E. coli. CI, 1.11 to 6.29; P ‫؍‬ 0.02). IIAT was the strongest risk factor for 21-day mortality and significantly increased the length of hospitalization after BSI onset. Our results underscore the need for a systematic approach to the management of patients with serious infections by ESBL-producing E. coli. Such an approach should be based on sound, updated knowledge of local infectious-disease epidemiology, detailed analysis of the patient's history with emphasis on recent contact with the health care system, and aggressive attempts to identify the infectious focus that has given rise to the BSI.Extended-spectrum ␤-lactamases (ESBLs) are a heterogeneous group of plasmid-mediated bacterial enzymes that confer significant resistance to oxyimino cephalosporin and monobactam antimicrobials (8,28,30). Throughout the world, increasing attention is being focused on the growing involvement of ESBL-producing strains of Escherichia coli in serious infections of hospitalized and nonhospitalized patients (3, 26, 28-30, 34, 35, 39, 45). This trend is due largely to the emergence of CTX-M type ESBLs, a rapidly expanding group of enzymes that are being encountered with increasing frequency, especially in E. coli (24,25,35,36,48). They are encoded by transferable plasmid genes captured from the chromosomes of Kluyvera spp. (6, 36). Acquisition of any ESBL determinant reduces the number of antimicrobial agents to which the microorganism is susceptible (18,25,38). This problem is compounded by the fact that ESBL producers often carry other antimicrobial resistance genes, which are located near the bla gene on the mobile DNA elements that are involved in their dissemination. In these cases, there is a substantial risk that the infecting pathogen will be resistant to the empirically prescribed antimicrobial treatment.In a previous study we found that failure to provide adequate antimicrobial therapy in the initial stages of bloodstream infections (BSI) caused by ESBL-producing Enterobacteriaceae was associated with a strong increase in the risk of 21-day mortality (45). These findings are consistent with those of other investigators (1,18,19,35,39,40). However, much less is known about the specific factors that increase the likelihood of ineffective empirical treatment in these cases (18). Identification of risk factors that are predictive of resistance to empirically prescribed antimicrobials should facilitate attempts to define more-effective managemen...