Exploring nasopharyngeal microbiota profile in children affected by SARS-CoV-2 infection.

The relationship between COVID-19 and nasopharyngeal (NP) microbiota has been investigated mainly in the adult population. We explored the NP profile of children affected by COVID-19, compared to healthy controls (CTRLs). NP swabs of children with COVID-19, collected between March and September 2020, were investigated at the admission (T0), 72 h to 7 days (T1), and at the discharge (T2) of the patients. NP microbiota was analyzed by 16S rRNA targeted-metagenomics. Data from sequencing were investigated by QIIME 2.0 and PICRUSt 2. Multiple machine learning (ML) models were exploited to classify patients compared to CTRLs. The NP microbiota of COVID-19 patients (N = 71) was characterized by reduction of α-diversity compared to CTRLs (N = 59). The NP microbiota of COVID-19 cohort appeared significantly enriched in Streptococcus, Haemophilus, Staphylococcus, Veillonella, Enterococcus, Neisseria, Moraxella, Enterobacteriaceae, Gemella, Bacillus, and reduced in Faecalibacterium, Akkermansia, Blautia, Bifidobacterium, Ruminococcus, and Bacteroides, compared to CTRLs (FDR < 0.001). Exploiting ML models, Enterococcus, Pseudomonas, Streptococcus, Capnocytopagha, Tepidiphilus, Porphyromonas, Staphylococcus, and Veillonella resulted as NP microbiota biomarkers, in COVID-19 patients. No statistically significant differences were found comparing the NP microbiota profile of COVID-19 patients during the time-points or grouping patients on the basis of high, medium, and low viral load (VL). This evidence provides specific pathobiont signatures of the NP microbiota in pediatric COVID-19 patients, and the reduction of anaerobic protective commensals. Our data suggest that the NP microbiota may have a specific disease-related signature since infection onset without changes during disease progression, regardless of the SARS-CoV-2 VL. IMPORTANCE: Since the beginning of pandemic, we know that children are less susceptible to severe COVID-19 disease. A potential role of the nasopharyngeal (NP) microbiota has been hypothesized but to date, most of the studies have been focused on adults. We studied the NP microbiota modifications in children affected by SARS-CoV-2 infection showing a specific NP microbiome profile, mainly composed by pathobionts and almost missing protective anaerobic commensals. Moreover, in our study, specific microbial signatures appear since the first days of infection independently from SARS-CoV-2 viral load.

The 2020 International Alliance for the Control of Scabies Consensus Criteria for the Diagnosis of Scabies.

BACKGROUND: Scabies is a common parasitic skin condition that causes considerable morbidity globally. Clinical and epidemiological research for scabies has been limited by a lack of standardization of diagnostic methods. OBJECTIVES: To develop consensus criteria for the diagnosis of common scabies that could be implemented in a variety of settings. METHODS: Consensus diagnostic criteria were developed through a Delphi study with international experts. Detailed recommendations were collected from the expert panel to define the criteria features and guide their implementation. These comments were then combined with a comprehensive review of the available literature and the opinion of an expanded group of international experts to develop detailed, evidence-based definitions and diagnostic methods. RESULTS: The 2020 International Alliance for the Control of Scabies (IACS) Consensus Criteria for the Diagnosis of Scabies include three levels of diagnostic certainty and eight subcategories. Confirmed scabies (level A) requires direct visualization of the mite or its products. Clinical scabies (level B) and suspected scabies (level C) rely on clinical assessment of signs and symptoms. Evidence-based, consensus methods for microscopy, visualization and clinical symptoms and signs were developed, along with a media library. CONCLUSIONS: The 2020 IACS Criteria represent a pragmatic yet robust set of diagnostic features and methods. The criteria may be implemented in a range of research, public health and clinical settings by selecting the appropriate diagnostic levels and subcategories. These criteria may provide greater consistency and standardization for scabies diagnosis. Validation studies, development of training materials and development of survey methods are now required. What is already known about this topic? The diagnosis of scabies is limited by the lack of accurate, objective tests. Microscopy of skin scrapings can confirm the diagnosis, but it is insensitive, invasive and often impractical. Diagnosis usually relies on clinical assessment, although visualization using dermoscopy is becoming increasingly common. These diagnostic methods have not been standardized, hampering the interpretation of findings from clinical research and epidemiological surveys, and the development of scabies control strategies. What does this study add? International consensus diagnostic criteria for common scabies were developed through a Delphi study with global experts. The 2020 International Alliance for the Control of Scabies (IACS) Criteria categorize diagnosis at three levels of diagnostic certainty (confirmed, clinical and suspected scabies) and eight subcategories, and can be adapted to a range of research and public health settings. Detailed definitions and figures are included to aid training and implementation. The 2020 IACS Criteria may facilitate the standardization of scabies diagnosis.

A systems genomics approach identifies SIGLEC15 as a susceptibility factor in recurrent vulvovaginal candidiasis.

Candida vaginitis is a frequent clinical diagnosis with up to 8% of women experiencing recurrent vulvovaginal candidiasis (RVVC) globally. RVVC is characterized by at least three episodes per year. Most patients with RVVC lack known risk factors, suggesting a role for genetic risk factors in this condition. Through integration of genomic approaches and immunological studies in two independent cohorts of patients with RVVC and healthy individuals, we identified genes and cellular processes that contribute to the pathogenesis of RVVC, including cellular morphogenesis and metabolism, and cellular adhesion. We further identified SIGLEC15, a lectin expressed by various immune cells that binds sialic acid-containing structures, as a candidate gene involved in RVVC susceptibility. Candida stimulation induced SIGLEC15 expression in human peripheral blood mononuclear cells (PBMCs) and a polymorphism in the SIGLEC15 gene that was associated with RVVC in the patient cohorts led to an altered cytokine profile after PBMC stimulation. The same polymorphism led to an increase in IL1B and NLRP3 expression after Candida stimulation in HeLa cells in vitro. Last, Siglec15 expression was induced by Candida at the vaginal surface of mice, where in vivo silencing of Siglec15 led to an increase in the fungal burden. Siglec15 silencing was additionally accompanied by an increase in polymorphonuclear leukocytes during the course of infection. Identification of these pathways and cellular processes contributes to a better understanding of RVVC and may open new therapeutic avenues.

A biological model of scabies infection dynamics and treatment informs mass drug administration strategies to increase the likelihood of elimination.

Infections with Sarcoptes scabiei, or scabies, remain common in many disadvantaged populations. Mass drug administration (MDA) has been used in such settings to achieve a rapid reduction in infection and transmission, with the goal of eliminating the public health burden of scabies. While prevalence has been observed to fall substantially following such an intervention, in some instances resurgence of infection to baseline levels has occurred over several years. To explore the biology underpinning this phenomenon, we have developed a theoretical model of scabies life-cycle and transmission dynamics in a homogeneously mixing population, and simulate the impact of mass drug treatment strategies acting on egg and mite life cycle stages (ovicidal) or mites alone (non-ovicidal). In order to investigate the dynamics of the system, we first define and calculate the optimal interval between treatment doses. We calculate the probability of eradication as a function of the number of optimally-timed successive treatment doses and the number of years over which a program is run. For the non-ovicidal intervention, we first show that at least two optimally-timed doses are required to achieve eradication. We then demonstrate that while more doses over a small number of years provides the highest chance of eradication, a similar outcome can be achieved with fewer doses delivered annually over a longer period of time. For the ovicidal intervention, we find that doses should be delivered as close together as possible. This work provides a platform for further research into optimal treatment strategies which may incorporate heterogeneity of transmission, and the interplay between MDA and enhancement of continuing scabies surveillance and treatment strategies.

Association of a variable number tandem repeat in the NLRP3 gene in women with susceptibility to RVVC.

Vaginal infections with Candida spp. frequently occur in women of childbearing age. A small proportion of these women experience recurrent vulvovaginal candidosis (RVVC), which is characterized by at least three episodes of infection in one year. In addition to known risk factors such as antibiotics, diabetes, or pregnancy, host genetic variation and inflammatory pathways such as the IL-1/Th17 axis have been reported to play a substantial role in the pathogenesis of RVVC. In this study, we assessed a variable number tandem repeat (VNTR) polymorphism in the NLRP3 gene that encodes a component of the inflammasome, processing the proinflammatory cytokines IL-1ß and IL-18. A total of 270 RVVC patients and 583 healthy controls were analyzed, and increased diseases susceptibility was associated with the presence of the 12/9 genotype. Furthermore, functional studies demonstrate that IL-1ß production at the vaginal surface is higher in RVVC patients bearing the 12/9 genotype compared to controls, whereas IL-1Ra levels were decreased and IL-18 levels remained unchanged. These findings suggest that IL-1ß-mediated hyperinflammation conveyed by the NLRP3 gene plays a causal role in the pathogenesis of RVVC and may identify this pathway as a potential therapeutic target in the disease.

Electroporation Enhances Bleomycin Efficacy in Cats with Periocular Carcinoma and Advanced Squamous Cell Carcinoma of the Head.

BACKGROUND: Advanced carcinoma of the head represents a substantial health problem in cats for local control and overall survival. OBJECTIVES: Evaluate the capability of electrochemotherapy (ECT) to improve bleomycin efficacy in cats with periocular carcinoma and advanced carcinoma of the head. ANIMALS: Twenty-one cats with periocular carcinoma (17 squamous cell carcinoma [SCC] and 4 anaplastic carcinoma) and 26 cats with advanced SCC of the head. METHODS: Nonrandomized prospective controlled study. Periocular carcinoma cohorts: 12 cats were treated with bleomycin (15 mg/m(2) i.v.) coupled with ECT under anesthesia; 9 cats were treated with bleomycin alone. Advanced head SCC cohorts: 14 cats were treated with bleomycin (15 mg/m(2) i.v.) coupled with ECT administered under sedation; 12 control cats were treated with bleomycin alone. ECT treatments (2-8) were performed every other week until complete remission (CR) or tumor progression occurred. RESULTS: Toxicities were minimal and mostly treated symptomatically. Overall response rate in the ECT treated animals was 89% (21 Complete Response [CR] and 2 Partial Response [PR]) whereas controls had response rate of 33% (4 CR and 3 PR). Median time to progression in ECT group was 30.5 months, whereas in controls it was 3.9 months (P < .0001). Median time to progression for ECT cohorts was 24.2 months for periocular cohort and 20.6 in advanced head SCC cohort, respectively. CONCLUSIONS: Electrochemotherapy is well tolerated for advanced SCC of the head in cats; its use may be considered among loco-regional strategies for cancer therapy in sensitive body regions such as periocular region.

Fine-tuning of Th17 Cytokines in Periodontal Disease by IL-10.

Periodontitis (PD) is a chronic disease caused by the host inflammatory response to bacteria colonizing the oral cavity. In addition to tolerance to oral microbiome, a fine-tuned balance of IL-10 levels is critical to efficiently mount antimicrobial resistance without causing immunopathology. Clinical and animal studies support that adaptive T-helper (Th) cytokines are involved in the pathogenesis of alveolar bone destruction in PD. However, it remains unclear what type of Th response is related to human PD progression and what role IL-10 has on this process. We addressed the contribution of IL-10 in limiting Th1 and Th17 inflammatory response in murine and human PD. Through a combination of basic and translational approaches involving selected cytokine-deficient mice as well as human genetic epidemiology, our results demonstrate the requirement for IL-10 in fine-tuning the levels of Th17 (IL-17A and IL-17F) cytokines in experimental and human PD. Of novelty, we found that IL-17F correlated with protection in murine and human PD and was positively regulated by IL-10. To our knowledge, this is the first demonstration of the protective role for IL-17F in PD, its positive regulation by IL-10, and the potential differential role for IL-17A and IL-17F in periodontal disease.

Five years of experience in nocturnal enuresis and urinary incontinence in children: where we are and where we are going.

BACKGROUND: Nocturnal enuresis (NE) is a very common pediatric disorder. The aim of this study was to evaluate the characteristics of patients with NE or urinary incontinence (UI) during a period of 5 years to increase the knowledge on these conditions and optimize their diagnosis and treatment. METHODS: We enrolled 278 children with NE or UI referred to the pediatric nephrology ambulatory, 'A. Gemelli' University Hospital of Rome, from December 2006 to December 2011. RESULTS: We observed that heredity, parasomnias, left-handedness, polythelia and constipation are correlated to NE and UI. CONCLUSIONS: We wanted to clarify the definition of NE and UI and describe our experience on the main characteristics of these conditions by referring to the latest knowledge reported in the literature.

Immune resistance and tolerance to fungi.

Fungal diseases represent an important paradigm in immunology, since they can result from either the lack of recognition or over-activation of the inflammatory response. Current understanding of the pathophysiology underlying fungal infections and diseases highlights the multiple cell populations and cell-signaling pathways involved in these complex conditions beyond the dysregulated chaos in which fungal infection and disease are perceived. A systems biology approach that integrates investigations of immunity at the systems-level is required to generate novel insights into this complexity and to decipher the dynamics of the host-fungus interaction. Recent advances in the immune response to fungi have highlighted the cellular and molecular mechanisms of immune adaptations that maintain homeostasis with the fungal biota and its possible rupture in fungal infections and diseases. Functionally distinct modules of immunity, i.e., resistance and tolerance, evolved for the achievement of the best-fitted host-fungus interaction in mammals, are now essential components of the host-fungus interaction in the vertebrate host.

The rs5743836 polymorphism in TLR9 confers a population-based increased risk of non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) has been associated with immunological defects, chronic inflammatory and autoimmune conditions. Given the link between immune dysfunction and NHL, genetic variants in toll-like receptors (TLRs) have been regarded as potential predictive factors of susceptibility to NHL. Adequate anti-tumoral responses are known to depend on TLR9 function, such that the use of its synthetic ligand is being targeted as a therapeutic strategy. We investigated the association between the functional rs5743836 polymorphism in the TLR9 promoter and risk for B-cell NHL and its major subtypes in three independent case-control association studies from Portugal (1160 controls, 797 patients), Italy (468 controls, 494 patients) and the US (972 controls, 868 patients). We found that the rs5743836 polymorphism was significantly overtransmitted in both Portuguese (odds ratio (OR), 1.85; P=7.3E-9) and Italian (OR, 1.84; P=6.0E-5) and not in the US cohort of NHL patients. Moreover, the increased transcriptional activity of TLR9 in mononuclear cells from patients harboring rs5743836 further supports a functional effect of this polymorphism on NHL susceptibility in a population-dependent manner.

Immunotherapy of aspergillosis.

Management of invasive aspergillosis in high-risk patients remains challenging. There is an increasing demand for novel therapeutic strategies aimed at enhancing or restoring antifungal immunity in immunocompromised patients. In this regard, modulation of specific innate immune functions and vaccination are promising immunotherapeutic strategies. Recent findings have also provided a compelling rationale for assessment of the contribution of the individual genetic profile to the immunotherapy outcome. Altogether, integration of immunological and genetic data may contribute to the optimization of therapeutic strategies exerting control over immune pathways, ultimately improving the management of fungal infections in high-risk settings.

A simple methodology for analyzing association effects on response functions via Monte Carlo simulations.

A simple methodology was developed to analyze association effects on the thermodynamic response functions for a pure self-associated fluid via Monte Carlo simulations. The procedure essentially involves expressing the residual energy and volume of the fluid in terms of these properties for two hypothetical fluids consisting of monomers and associated molecules, respectively. This allows the thermodynamic response functions to be expressed in a perturbative form as a combination of the values for the property in the monomeric fluid and the contribution of association (the perturbative term). The proposed methodology was used to determine both contributions to the isobaric heat capacity and to the temperature and pressure derivatives of the volume for OPLS methanol along the 50 MPa isobar from 220 to 1500 K. Based on the results, both terms exert a substantial influence on the isobaric heat capacity; by contrast, the association term for the volumetric properties is negligible. These results are consistent with those of a previous work involving simulations with the same model under identical thermodynamic conditions but a different approach. They are also compared with others previously reported in context. Moreover, a comprehensive study of the different types of clusters present in the fluid was performed and the results were related to thermodynamic properties. A strong correlation between the heat capacity of the monomeric fluid and this structural analysis was found.

IL-22 defines a novel immune pathway of antifungal resistance.

The role of IL-17 and Th17 cells in immunity vs. pathology associated with the human commensal Candida albicans remains controversial. Both positive and negative effects on immune resistance have been attributed to IL-17/Th17 in experimental candidiasis. In this study, we provide evidence that IL-22, which is also produced by Th17 cells, has a critical, first-line defense in candidiasis by controlling the growth of infecting yeasts as well as by contributing to the host's epithelial integrity in the absence of acquired Th1-type immunity. The two pathways are reciprocally regulated, and IL-22 is upregulated under Th1 deficiency conditions and vice versa. Whereas both IL-17A and F are dispensable for antifungal resistance, IL-22 mediates protection in IL-17RA-deficient mice, in which IL-17A contributes to disease susceptibility. Thus, our findings suggest that protective immunity to candidiasis is made up of a staged response involving an early, IL-22-dominated response followed by Th1/Treg reactivity that will prevent fungal dissemination and supply memory.

Electrochemotherapy treatment for bilateral pleomorphic rhabdomyosarcoma in a cat.

A 13-year-old male neutered cat was presented for the sudden growth of two nodular lesions close to the upper eyelid of both eyes. Fine-needle aspiration cytology was suggestive of mesenchymal neoplasia. The cat had conservative surgical excision in order to preserve the eyelids' functionality; however, the histopathological report came with a diagnosis of incompletely excised bilateral pleomorphic rhabdomyosarcoma. Due to the local aggressiveness of this neoplasm, the cat was treated with two sessions of cisplatin-based electrochemotherapy, delivered 14 days apart. Systemic or local toxicities were not detected during the whole course of therapy. The cat is still in complete remission after 12 months. Electrochemotherapy is a safe and efficacious adjuvant therapy for aggressive sarcomas and warrants further investigations in order to standardise its protocols.

Prognostic significance of genetic variants in the IL-23/Th17 pathway for the outcome of T cell-depleted allogeneic stem cell transplantation.

T helper (Th) 17 cells have emerged as important mediators in infectious and inflammatory diseases and, recently, in transplant rejection. We analyzed the associations between five common genetic variants in the IL-23/Th17 signaling pathway, namely in IL17A, IL17F and IL23R genes, and clinical outcome in T cell-depleted allogeneic SCT (allo-SCT). In the multivariate analysis, variants in IL23R and IL17A genes were the most important prognostic factors. Thus, patient GA genotype at rs11209026 in IL23R was associated with improved overall survival (hazard ratio (HR)=0.48; P=0.028) and, in donor, with decreased risk of fungal infections (P=0.05). In contrast, patient TC and CC genotypes at rs8193036 in IL17A gene were associated with increased risk of CMV infection (HR=3.68; P=0.011) and patient acute GVHD (HR=7.08; P=0.008), respectively. These results suggest that genetic variants in the IL-23/Th17 inflammatory pathway are important prognostic factors for the clinical outcome of allo-SCT. Although validation studies are ultimately required, our results would suggest the potential usefulness of IL-23/Th17 genotyping in donor selection and patient evaluation.

Dynamics of extracellular release of Aspergillus fumigatus DNA and galactomannan during growth in blood and serum.

Aspergillus fumigatus is the major cause of invasive aspergillosis (IA), a disease associated with high rates of morbidity and mortality in patients undergoing treatment for haematological malignancies. This study investigated A. fumigatus growth in vitro and in a murine model of IA in order to provide insights into the dynamics of extracellular DNA and galactomannan (GM) release and their relevance to early diagnosis of IA. Following inoculation of whole blood with 20 A. fumigatus conidia ml(-1), DNA that corresponded to the inoculum could be detected by PCR but GM was not detected in plasma separated from the blood sample, indicating that the fungus did not grow in whole blood. The quantities of DNA detected by PCR, and GM, were proportional to the amount of fungal biomass present in vitro. Fungal DNA could be detected in the sera of mice experimentally infected with A. fumigatus with maximum detection in cyclophosphamide-treated mice.

Intranasally delivered siRNA targeting PI3K/Akt/mTOR inflammatory pathways protects from aspergillosis.

Innate responses combine with adaptive immunity to generate the most effective form of anti-Aspergillus immune resistance. Although some degree of inflammation is required for protection, progressive inflammation may worsen disease and ultimately prevents pathogen eradication. To define molecular pathways leading to or diverting from pathogenic inflammation in infection, we resorted to dendritic cells (DCs), known to activate distinct signaling pathways in response to pathogens. We found that distinct intracellular pathways mediated the sensing of conidia and hyphae by lung DCs in vitro, which translate in vivo in the activation of protective Th1/Treg responses by conidia or inflammatory Th2/Th17 responses by hyphae. In vivo targeting inflammatory (PI3K/Akt/mTOR) or anti-inflammatory (STAT3/IDO) DC pathways by intranasally delivered small interfering RNA (siRNA) accordingly modified inflammation and immunity to infection. Thus, the screening of signaling pathways in DCs through a systems biology approach may be exploited for the development of siRNA therapeutics to attenuate inflammation in respiratory fungal infections and diseases.

Balancing inflammation and tolerance in vivo through dendritic cells by the commensal Candida albicans.

We analyzed the contribution of intracellular signaling to the functional plasticity of dendritic cells (DCs) presenting Candida albicans, a human commensal associated with severe diseases. Distinct intracellular pathways were activated by recognition of different fungal morphotypes in distinct DC subsets and in Peyer's patches DCs. Inflammatory DCs initiated Th17/Th2 responses to yeasts through the adaptor myeloid differentiation factor-88 (MyD88), whereas tolerogenic DCs activate Th1/T regulatory cell (Treg) differentiation programs to hyphae involving Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) as an intermediary of signaling. In addition, signal transducer and activator of transcription 3 (STAT3), affecting the balance between canonical and non-canonical activation of nuclear factor-kappaB (NF-kappaB) and 2,3 indoleamine dioxygenase (IDO), pivotally contributed to DC plasticity and functional specialization. As Candida-induced tolerogenic DCs ameliorated experimental colitis, our data qualify Candida as a commensal with immunoregulatory activity, resulting from the orchestrated usage of multiple, yet functionally distinct, receptor-signaling pathways in DCs. Ultimately, affecting the local Th17/Treg balance might likely be exploited by the fungus for either commensalism or pathogenicity.