Persistence of SARS-CoV-2 infection and viral intra- and inter-host evolution in COVID-19 hospitalized patients.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) persistence in COVID-19 patients could play a key role in the emergence of variants of concern. The rapid intra-host evolution of SARS-CoV-2 may result in an increased transmissibility, immune and therapeutic escape which could be a direct consequence of COVID-19 epidemic currents. In this context, a longitudinal retrospective study on eight consecutive COVID-19 patients with persistent SARS-CoV-2 infection, from January 2022 to March 2023, was conducted. To characterize the intra- and inter-host viral evolution, whole genome sequencing and phylogenetic analysis were performed on nasopharyngeal samples collected at different time points. Phylogenetic reconstruction revealed an accelerated SARS-CoV-2 intra-host evolution and emergence of antigenically divergent variants. The Bayesian inference and principal coordinate analysis analysis showed a host-based genomic structuring among antigenically divergent variants, that might reflect the positive effect of containment practices, within the critical hospital area. All longitudinal antigenically divergent isolates shared a wide range of amino acidic (aa) changes, particularly in the Spike (S) glycoprotein, that increased viral transmissibility (K417N, S477N, N501Y and Q498R), enhanced infectivity (R346T, S373P, R408S, T478K, Q498R, Y505H, D614G, H655Y, N679K and P681H), caused host immune escape (S371L, S375F, T376A, K417N, and K444T/R) and displayed partial or complete resistance to treatments (G339D, R346K/T, S371F/L, S375F, T376A, D405N, N440K, G446S, N460K, E484A, F486V, Q493R, G496S and Q498R). These results suggest that multiple novel variants which emerge in the patient during persistent infection, might spread to another individual and continue to evolve. A pro-active genomic surveillance of persistent SARS-CoV-2 infected patients is recommended to identify genetically divergent lineages before their diffusion.

Changing and evolution of influenza virus: is it a trivial flu?

BACKGROUND: Influenza viruses are etiological agents which cause contagious respiratory, seasonal epidemics and, for Influenza A subtypes, pandemics. The clinical picture of Influenza has undergone continuous change over the years, due to intrinsic viral evolution as well as "reassortment" of its genomic segments. The history of Influenza highlights its ability to adapt and to rapidly evolve, without specific circumstances. This reflects the complexity of this pathology and poses the fundamental question about its assumption as a "common illness" and its impact on public health. SUMMARY: The global influenza epidemics and pandemics claimed millions of deaths, leaving an indelible mark on public health, and showing the need for a better comprehension of the influenza virus. The clear understanding of genetic variations during the Influenza seasonal epidemics is a crucial point for developing effective strategies for prevention, treatment, and vaccine design. The recent advance in Next Generation Sequencing approaches, model systems to virus culture and bioinformatics pipeline played a key role in the rapid characterization of circulating Influenza strains. In particular, the increase of computational power allowed to perform complex tasks in healthcare setting through Machine Learning (ML) algorithms, which analyze different variables, such as medical and laboratory outputs, to optimize medical research and to improve public health systems. The early detection of emerging and re-emerging pathogens is of matter importance to prevent next pandemics. KEY MESSAGES: The perception of influenza as a "trivial flu" or a more serious public health concern is a subject of ongoing debate, reflecting the multifaceted nature of this infectious disease. The variability in the severity of influenza shed the light on the unpredictability of the viral characteristics, coupled with the challenges in accurately predicting circulating strains. This adds complexity to the public health burden of Influenza and highlights the need of targeted interventions.

Role of the T2Dx magnetic resonance assay in patients with suspected bloodstream infection: a single-centre real-world experience.

BACKGROUND: T2Dx was approved by the US Food and Drug Administration for the rapid detection of a modified panel of ESKAPE bacterial species or Candida spp. causing bloodstream infection (BSI). PATIENTS AND METHODS: We performed a retrospective, observational study from January 1, 2018 to December 31, 2019 of all hospitalised patients with suspected BSI who underwent assessment using T2Dx in addition to standard blood culture (BC). T2-positive patients (cases) were compared to a matched group of patients with BSI documented only by BC (1:2 ratio) to investigate the possible impact of T2Dx on the appropriateness of empirical antimicrobial therapy and 21-day mortality. RESULTS: In total, 78 T2Dx-analysed samples (49 patients) were analysed. The T2Dx assay result was positive for18 patients and negative for 31 patients. The concordance rates of the T2Bacteria Panel and T2Candida Panel results with those of standard BC were 74.4% and 91.4%, respectively. In the matched analysis, inappropriate empiric antimicrobial therapy administration was significantly less frequent in cases than in comparators (5.5% vs. 38.8%). The 21-day mortality rate was twofold lower in cases than in comparators (22.2% vs. 44.4%), although the difference was not significant. No other analysed variables were significantly different between the two groups. CONCLUSIONS: This study illustrated that T2Dx might be associated with an increase in the appropriateness of empiric antimicrobial therapy in patients with BSI. Further studies are needed to evaluate whether the T2Dx assay can improve patient outcomes.

Identification of pmrB mutations as putative mechanism for colistin resistance in A. baumannii strains isolated after in vivo colistin exposure.

Colistin resistance among extensively-resistant Acinetobacter baumannii isolates is a serious health-care problem. Alterations in PmrA-PmrB two-component system have been associated with resistance to colistin. We investigated three pairs of colistin-susceptible and colistin-resistant A. baumannii, sequentially isolated from three patients before and after colistin treatment, respectively. The pmrA and pmrB genes were sequenced by Sanger method. Amino acidic positions and their effect on protein were predicted by InterPro and PROVEAN tools. Expression of pmrA, pmrB and pmrC genes was assessed by semi-quantitative reverse transcription-PCR (qRT-PCR). We found three different nonsynonymous substitutions P233T, E301G and L168K in pmrB coding region, each one in a different colistin resistance strain. The E301G and L168K substitutions represent novel mutations in pmrB, not previously described. Relative expression of pmrA, pmrB and pmrC mRNA increased in all colistin resistant strains. In our study, pmrB substitutions were associated with pmrC over-expression and colistin resistance. Further studies are necessary to understand their impact on modification of lipid A components.

Prevalence, molecular epidemiology, and antimicrobial resistance of methicillin-resistant Staphylococcus aureus from swine in southern Italy.

BACKGROUND: Colonization by livestock-associated MRSA (LA-MRSA) has increasingly been reported in the swine population worldwide. The aim of this study was to assess the prevalence of MRSA nasal carriage in healthy pigs, including the black (Calabrese) breed, from farms in the Calabria Region (Southern Italy). Between January and March 2018, a total of 475 healthy pigs reared in 32 farms were sampled by nasal swabbing. MRSA isolates were characterized by spa, MLST and SCCmec typing, and susceptibility testing to 17 antimicrobials. RESULTS: 22 of 32 (66.8%) pig farms resulted positive for MRSA. The prevalence of MRSA was 46.1% (219 MRSA culture-positive out of 475 samples). MRSA colonization was significantly higher in intensive farms and in pigs with a recent or ongoing antimicrobial treatment. All 219 MRSA isolates were assigned to ST398. The most common spa types were t011 (37.0%), t034 (22.4%) and t899 (15.1%). A novel spa type (t18290) was detected in one isolate. An insertion of IS256 in the ST398-specific A07 fragment of the SAPIG2195 gene was detected in 10 out of 81 t011 isolates. Nearly all isolates carried the SCCmec type V element, except 11 isolates that carried the SCCmec type IVc. None of the isolates was positive for the Panton-Valentine leukocidin. All isolates were resistant to tetracycline. High resistance rates were also found for clindamycin (93.1%), trimethoprim/sulfamethoxazole (68.4%), fluoroquinolones (47.9-65.3%) and erythromycin (46.1%). None of the isolates was resistant to vancomycin and fusidic acid. Overall, a multidrug resistant phenotype was observed in 88.6% of isolates. CONCLUSIONS: We report a high prevalence of MRSA among healthy swine in Southern Italy farms, with higher isolation frequency associated with intensive farming. The epidemiological types identified in our study reflect those reported in other European countries. Our findings underscore the importance of monitoring the evolution of LA-MRSA in pig farms in order to implement control measures and reduce the risk of spread in the animal population.

Real-life 3D therapy failure: Analysis of NS5A 93H RAS plus 108 K polymorphism in complex with ombitasvir by molecular modeling.

We report a real-life 3D therapy failure in a patient treated with ombitasvir (OMV)/paritaprevir/ritonavir and dasabuvir without ribavirin (3D-R). He had therapy failure at week 12 after the end of treatment. We detected resistance-associated substitutions (RASs) plus polymorphisms on NS3, NS5A, and NS5B target regions by population sequencing (15% cut-off) at baseline, at relapse and during follow-up. About this, NS5A RASs generally persist longer than resistances in the other target genes and may impact treatment outcome. Therefore, to evaluate OMV drug-resistance mechanism, we studied the acquired RAS plus polymorphisms on NS5A phosphoprotein by computational studies. OMV showed a higher affinity towards baseline and 93H/108 K mutant structure (follow-up) with respect to 93H/R108 mutant structure (relapse) on phosphoprotein. By Molecular Dynamics simulations (MDs), structural information about the protein stability in presence of OMV were observed. According to our data, molecular modeling approach has proved to be a powerful method to evaluate the impact of these RASs plus specific amino acid (AA) changes on phosphoprotein.

Detection of Natural Resistance-Associated Substitutions by Ion Semiconductor Technology in HCV1b Positive, Direct-Acting Antiviral Agents-Naïve Patients.

Naturally occurring resistance-associated substitutions (RASs) can negatively impact the response to direct-acting antivirals (DAAs) agents-based therapies for hepatitis C virus (HCV) infection. Herein, we set out to characterize the RASs in the HCV1b genome from serum samples of DAA-naïve patients in the context of the SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology, 2014) project. We deep-sequenced the NS3/4A protease region of the viral population using the Ion Torrent Personal Genome Machine, and patient-specific majority rule consensus sequence summaries were constructed with a combination of freely available next generation sequencing data analysis software. We detected NS3/4A protease major and minor variants associated with resistance to boceprevir (V36L), telaprevir (V36L, I132V), simeprevir (V36L), and grazoprevir (V36L, V170I). Furthermore, we sequenced part of HCV NS5B polymerase using Sanger-sequencing and detected a natural RAS for dasabuvir (C316N). This mutation could be important for treatment strategies in cases of previous therapy failure.

Gut inflammation and immunity: what is the role of the human gut virome?

The human virome comprises viruses that infect host cells, virus-derived elements in our chromosomes, and viruses that infect other organisms, including bacteriophages and plant viruses. The development of high-throughput sequencing techniques has shown that the human gut microbiome is a complex community in which the virome plays a crucial role into regulation of intestinal immunity and homeostasis. Nevertheless, the size of the human virome is still poorly understood. Indeed the enteric virome is in a continuous and dynamic equilibrium with other components of the gut microbiome and the gut immune system, an interaction that may influence the health and disease of the host. We review recent evidence on the viruses found in the gastrointestinal tract, discussing their interactions with the resident bacterial microbiota and the host immune system, in order to explore the potential impact of the virome on human health.

Virological Mechanisms in the Coinfection between HIV and HCV.

Due to shared transmission routes, coinfection with Hepatitis C Virus (HCV) is common in patients infected by Human Immunodeficiency Virus (HIV). The immune-pathogenesis of liver disease in HIV/HCV coinfected patients is a multifactorial process. Several studies demonstrated that HIV worsens the course of HCV infection, increasing the risk of cirrhosis and hepatocellular carcinoma. Also, HCV might increase immunological defects due to HIV and risk of comorbidities. A specific cross-talk among HIV and HCV proteins in coinfected patients modulates the natural history, the immune responses, and the life cycle of both viruses. These effects are mediated by immune mechanisms and by a cross-talk between the two viruses which could interfere with host defense mechanisms. In this review, we focus on some virological/immunological mechanisms of the pathogenetic interactions between HIV and HCV in the human host.

Does the Human Gut Virome Contribute to Host Health or Disease?

The human gastrointestinal (GI) tract harbors eukaryotic and prokaryotic viruses and their genomes, metabolites, and proteins, collectively known as the "gut virome". This complex community of viruses colonizing the enteric mucosa is pivotal in regulating host immunity. The mechanisms involved in cross communication between mucosal immunity and the gut virome, as well as their relationship in health and disease, remain largely unknown. Herein, we review the literature on the human gut virome's composition and evolution and the interplay between the gut virome and enteric mucosal immunity and their molecular mechanisms. Our review suggests that future research efforts should focus on unraveling the mechanisms of gut viruses in human homeostasis and pathophysiology and on developing virus-prompted precision therapies.

Role of Treg, Breg and other cytokine sets in host protection and immunopathology during human leishmaniasis: Are they potential valuable markers in clinical settings and vaccine evaluation?

Leishmaniasis is a vector-borne disease caused by obligate intracellular protozoan parasites that can infect humans and other mammals. Pro- and anti-inflammatory cytokines are important regulators of innate and specific responses in Leishmania infection. Resistance to leishmaniasis is related to T helper 1 (Th1) response with the production of pro-inflammatory cytokines: IL-12, IL-1ß, IFN-γ, TNF-α, IL-2 leading to activation of macrophages and parasite killing. Instead, a more intense Th2 (IL-4, IL-5, IL-13), Treg (IL-10 and TGF-ß) and Breg response (IL-10 and IL-35) are related to parasite persistence through the inhibition of macrophage activation, which promotes the escape from host immune system. Interestingly, a cytokine involved in the parasite killing in one form of leishmaniasis may be "pathogen friendly" in another form of the disease. To date, few studies are focusing on the role of Treg and Breg cytokines in human models of leishmaniasis; therefore, further investigations are needed to clarify their potential role in the diagnosis and prognosis of such protozoan infections, as well as in the development of vaccines against leishmaniasis. This review summarizes the current knowledge about the role of cytokines produced by Th1, Th2, Treg, and Breg cells involved in Leishmania disease progression and host protection. Some cytokines might play a role as diagnostic and prognostic clinical markers, or they could represent a novel approach leading to new anti-leishmaniasis therapies. Overall, advances in knowledge of the complex network of cytokines secreted by immune cells could help to better understand signaling pathways and host immune responses during Leishmania infection. This approach would allow these mediators to be used as therapeutic strategies against leishmaniasis.

COVID-19 Vaccine Booster Shot Preserves T Cells Immune Response Based on Interferon-Gamma Release Assay in Inflammatory Bowel Disease (IBD) Patients on Anti-TNFα Treatment.

Immune-modifying treatment in inflammatory bowel disease (IBD) impairs the humoral response. The role of T lymphocytes in this setting is still unclear. This study aims to assess if a booster shot (third dose) of BNT162b2 mRNA COVID-19 vaccine enhanced the humoral response and elicited cellular immunity in IBD patients on different immuno-therapy regimens compared to healthy controls (HCs). Five months after a booster dose, serological and T-cell responses were assessed. The measurements were described using geometric means with 95% confidence intervals. The differences between study groups were assessed by Mann-Whitney tests. Seventy-seven subjects (n = 53 IBD patients and n = 24 HCs), who were fully vaccinated and not previously SARS-CoV-2 infected, were recruited. Regarding the IBD patients, 19 were affected by Crohn's disease and 34 by ulcerative colitis. During the vaccination cycle, half of the patients (53%) were on stable treatment with aminosalicylates, and 32% were on biological therapy. No differences in antibody concentrations between IBD patients and HCs, nor T-cell responses, were found. Stratifying IBD patients based on the type of treatment (anti-TNFα agents vs. other treatment regimens), a decrease only in antibody titer (p = 0.008), but not in cellular response, was observed. Even after the COVID-19 vaccine booster dose, the TNFα inhibitors selectively decreased the humoral immune response compared to patients on other treatment regimens. The T-cell response was preserved in all study groups. These findings highlight the importance of evaluating T-cell immune responses following COVID-19 vaccination in a routine diagnostic setting, particularly for immunocompromised cohorts.

Evaluation of IL-35, as a Possible Biomarker for Follow-Up after Therapy, in Chronic Human Schistosoma Infection.

The host response to helminth infections is characterized by systemic and tissue-related immune responses that play a crucial role in pathological diseases. Recently, experimental studies have highlighted the role of regulatory T (Tregs) and B (Bregs) cells with secreted cytokines as important markers in anti-schistosomiasis immunity. We investigated the serical levels of five cytokines (TNFα, IFN-γ, IL-4, IL-10 and IL-35) in pre- and post-treatment samples from chronic Schistosoma infected patients to identify potential serological markers during follow-up therapy. Interestingly, we highlighted an increased serum level of IL-35 in the pre-therapy samples (median 439 pg/mL for Schistosoma haematobium and 100.5 pg/mL for Schistsoma mansoni infected patients) compared to a control group (median 62 pg/mL and 58 pg/mL, respectively, p ≤ 0.05), and a significantly lower concentration in post-therapy samples (181 pg/mL for S. haematobium and 49.5 pg/mL for S. mansoni infected patients, p ≤ 0.05). The present study suggests the possible role of IL-35 as a novel serological biomarker in the evaluation of Schistosoma therapy follow-up.

Orthopedic Device-Related Infections Due to Emerging Pathogens Diagnosed by a Combination of Microbiological Approaches: Case Series and Literature Review.

Orthopedic and trauma device-related infections (ODRI) due to high virulence microorganisms are a devastating complication after orthopedic surgery. Coagulase-negative Staphylococci (CoNS) are mainly involved but commensal bacteria, located in human mucous membranes, are emerging pathogens in ODRI. Currently, bacterial culture is the gold standard for ODRI but the diagnostic process remains time consuming and laborious. We evaluated a combination of microbiological approaches in the diagnosis of emerging pathogens involved in ODRI. We analyzed two synovial fluids, five tissue samples and five surgical wound swabs from two different patients with ODRI, attending the Department of Orthopedic and Trauma Surgery of Mater Domini Teaching Hospital, Catanzaro, Italy. Identification was carried out with a combination of microbiological approaches (culture, mass spectrometry and 16s rRNA gene sequencing). We demonstrated the importance of a combination of microbiological approaches for the diagnosis of emerging pathogens in ODRI, because the low number of cases in the literature makes it very difficult to formulate guidelines for the management of patients.

Seroprevalence and phylogenetic characterization of hepatitis E virus in pig farms in Southern Italy.

Hepatitis E virus (HEV) infection is an emerging public health problem in industrialized countries. The infection is associated with waterborne epidemics and transmitted via faecal-oral route. Zoonotic cases of HEV in humans have increased in Europe, and HEV genotype 3 (HEV-3) is the most frequent among humans and animals. Nevertheless, HEV surveillance in the Italian pig farming industry is patchy. Here, HEV prevalence in pig farms located in the Calabria region in Southern Italy was investigated. A total of 692 serum samples were collected from 26 farms and tested for anti-HEV IgG antibody detection. The percentage of HEV-seropositive pigs was 56.8 %. Small farm size, farrow-to-finishing production, and infrequent cleaning procedures were associated with higher HEV seroprevalence. In 12 of the HEV-seropositive farms, 67 faecal samples were collected and 10 of these (10.6 %) tested positive for HEV RNA. Seven of 10 viral RNA sequences were genotyped for phylogenetic analysis, five of which belonged to subtype HEV-3f and two to subtype HEV-3e. The high HEV seroprevalence and the circulation of HEV-3 strains among domestic pigs in the Calabria region pose a risk for the zoonotic transmission of HEV from pigs to occupational exposed workers.

Dispersal and molecular characterisation of the Echinococcus granulosus (Batsch, 1786) complex isolated from various intermediate hosts in the Calabria region, southern Italy.

Cystic echinococcosis (CE) is a zoonotic disease caused by the tapeworms of the Echinococcus granulosus sensu lato complex, which have worldwide distribution. No data on the circulation of genotypes of the E. granulosus complex in intermediate hosts in endemic areas in Calabria are available. The aims of our study were to evaluate the dispersal of genotypes of the E. granulosus complex in Calabria and to characterise parasite isolates by Sanger sequencing and phylogenetic analysis. We collected 71 animal samples from pigs, wild boars, sheep, cattle and goats. The first PCR screening analysis targeted three partial genomic regions: the cytochrome c oxidase subunit 1 (cox1), calreticulin protein (cal) and NADH dehydrogenase subunit 1 (nad1); this identified 28 parasitic cysts. Bidirectional sequencing of cox1 amplicons and phylogenetic analysis allowed us to characterise all isolates. Molecular analyses of 28 newly generated cox1 sequences revealed that most wild boars (n = 16) and three pigs were parasitised by the larval stage of Taenia hydatidena Pallas, 1766, called cysticercus tenuicollis. Two isolates from wild boars were identified as Echinococcus canadensis Webster and Cameron, 1961 (G7), while five sheep and two goats were infected with E. granulosus G1 (sheep strain) and G1 microvariant (previously reported as G2 genotype or Tasmanian sheep strain), respectively. These molecular findings should prompt further and more extensive studies, to elucidate regional transmission patterns and to guide control programs.

Unidirectional animal-to-human transmission of methicillin-resistant Staphylococcus aureus ST398 in pig farming; evidence from a surveillance study in southern Italy.

Background: Livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) belonging to clonal complex 398 is recognized as an occupational hazard for workers employed in intensive animal husbandry, especially in the swine-breeding chain. In this study, we compared the prevalence and epidemiological type of MRSA isolates from swine and farm workers in a large area of southern Italy. Methods: Between January and March 2018, 88 workers from 32 farms where we had previously performed a survey for MRSA colonization of farmed pigs, were sampled by nasal swabbing. A follow-up investigation was conducted on seven workers 1 year after primary screening. MRSA isolates were characterized by MLST, spa and SCCmec typing, and tested for susceptibility to 15 antimicrobials. Epidemiological correlations between human and swine MRSA isolates were supported by Rep-MP3 and RAPD PCR fingerprinting, and whole-genome sequencing. Results: The overall colonization rate of MRSA in swine farm workers was 21.6%, being significantly higher in intensive farms and in workers with direct animal contact. All human MRSA isolates were multi-drug resistant, belonged to the ST398 livestock clade, and did not carry Panton-Valentine leukocidin and enterotoxin genes. Notably, 94.1% of human MRSA isolates belonged to the same epidemiological type as swine MRSA isolates from the corresponding farm. Persistent MRSA carriage was documented in some workers 1 year after primary sampling. Conclusions: We report a high prevalence of MRSA among swine farm workers, with higher colonization rates associated with intensive breeding and animal exposure. Our findings suggest unidirectional animal-to-human transmission of LA-MRSA and denote the high zoonotic transmissibility of the ST398 livestock clade.

Clinical, Virological Characteristics, and Outcomes of Treatment with Sofosbuvir/Ledipasvir in Two Pediatric Patients Infected by HCV Genotype 4.

Direct-acting antiviral drugs to cure infections with Hepatitis C virus (HCV) achieve a sustained virological response (SVR) in more than 90% of adult patients. At present, clinical trials are ongoing and real-life data are still limited in children. Herein, we report two cases of pediatric patients treated with fixed-dose combination of sofosbuvir/ledipasvir, already approved to treat HCV4 genotype. Both young girls achieved SVR even though HCV4 isolates carried L28M and M31L NS5A resistance-associated substitutions (RASs). Therefore, possible effects of these RASs merit further study, especially in children.