Trends of Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates Between 2012 and 2023: Results From an Open Italian Cohort.

BACKGROUND: Neisseria gonorrhoeae (Ng) is a public health priority because of the rapid evolution of antimicrobial resistance, the emergence of antibiotic resistance, and the absence of a vaccine against Ng. The aim of this study was to investigate trends in the minimum inhibitory concentration and resistance (R) or reduced susceptibility (DS) of Ng cases to ceftriaxone (CRO), azithromycin (AZM), tetracycline (TET), benzylpenicillin (PenG), and ciprofloxacin (CIP) during a 10-year period. METHODS: We conducted a retrospective analysis on an open cohort of Ng cases diagnosed on rectal, urethral, and pharyngeal samples at San Raffaele Scientific Institute, between September 2012 and February 2023. Minimum inhibitory concentrations of antibiotics were determined by gradient-test strips. Bivariate linear regression models were applied on logarithmic minimum inhibitory concentrations values; Cochran-Armitage test was used to determine a linear trend in the proportions of resistant strains. RESULTS: A total of 436 Ng isolates from 352 individuals were analyzed. Minimum inhibitory concentrations of CRO and PenG reduced over time ( P < 0.001, P = 0.030), AZM increased ( P = 0.001), and CIP and TET did not change ( P = 0.473, P = 0.272). The percentages of resistant strains were as follows: PenG, 89.9%; TET, 90.8%; CIP, 48.2%; AZM, and 4.4%. CRO-DS strains were 8.7%, and only 1 case of CRO-R was identified. The proportion of resistant strains increased over time for AZM ( P = 0.007), TET ( P = 0.001), and CIP ( P < 0.001), whereas it decreased for PenG ( P < 0.001) and CRO-DS/R strains ( P < 0.001). CONCLUSIONS: Ng strains showed high susceptibility to CRO, although we identified cases of DS/R and observed high levels of susceptibility to AZM. Overall, the recommended primary regimen for Ng treatment was confirmed to be effective.

Identification of a novel mutation involved in colistin resistance in Klebsiella pneumoniae through Next-Generation Sequencing (NGS) based approaches.

The spread of multidrug-resistant (MDR) K. pneumoniae carbapenemase-producing bacteria (KPC) is one of the most serious threats to global public health. Due to the limited antibiotic options, colis- tin often represents a therapeutic choice. In this study, we performed Whole-Genome Sequencing (WGS) by Illumina and Nanopore platforms on four colistin-resistant K. pneumoniae isolates (CoRKp) to explore the resistance profile and the mutations involved in colistin resistance. Mapping reads with reference sequence of the most com- mon genes involved in colistin resistance did not show the presence of mobile colistin resistance (mcr) genes in all CoRKp. Complete or partial deletions of mgrB gene were observed in three out of four CoRKp, while in one CoRKp the mutation V24G on phoQ was identified. Complementation assay with proper wild type genes restored colistin susceptibility, validating the role of the amino acid substitution V24G and, as already described in the literature, confirming the key role of mgrB alterations in colistin resistance. In conclusion, this study allowed the identification of the novel mutation on phoQ gene involved in colistin resistance phenotype.

Impact of surgical timing on survival in patients with infective endocarditis: a time-dependent analysis.

The purpose of this study was to evaluate the impact of surgical timing on survival in patients with left-sided infective endocarditis (IE). This was a retrospective study including 313 patients with left-sided IE between 2009 and 2017. Surgery was defined as urgent (US) or early (ES) if performed within 7 or 28 days, respectively. A multivariable Cox regression analysis including US and ES as time-dependent variables was performed to assess the impact on 1-year mortality. ES was associated with a better survival (aHR 0.349, 95% CI 0.135-0.902), as US (aHR 0.262, 95% CI 0.075-0.915). ES and US were associated with a better prognosis in patients with left-sided IE.

Salvage heart transplantation for Mycoplasma hominis prosthetic valve endocarditis: A case report and review of the literature.

Heart transplantation (HT) has been rarely performed in patients with infective endocarditis (IE) and is considered a "last resort" procedure. Orthotropic HT with bicaval technique was performed in a man with culture-negative endocarditis. Mycoplasma hominis was later detected using 16S ribosomal DNA PCR from surgically removed valve tissue. Literature review and previous results are summarized. HT may be considered as salvage treatment in selected patients with intractable IE. In cases when there is no growth in culture, 16S ribosomal DNA PCR sequencing can be used to identify the pathogen in excised valvular tissue. Mycoplasma spp. is extremely uncommon and difficult to diagnose cause of infective endocarditis (IE). There are no proposed or defined criteria for heart transplantation (HT) in patients with refractory IE, and HT has been rarely performed in this setting. We report a case of M hominis prosthetic valve endocarditis diagnosed by 16S ribosomal DNA PCR in a patient who underwent a salvage HT. We reviewed in the literature other cases of IE caused by Mycoplasma spp.

New Antimicrobials for Gram-Positive Sustained Infections: A Comprehensive Guide for Clinicians.

Antibiotic resistance is a public health problem with increasingly alarming data being reported. Gram-positive bacteria are among the protagonists of severe nosocomial and community infections. The objective of this review is to conduct an extensive examination of emerging treatments for Gram-positive infections including ceftobiprole, ceftaroline, dalbavancin, oritavancin, omadacycline, tedizolid, and delafloxacin. From a methodological standpoint, a comprehensive analysis on clinical trials, molecular structure, mechanism of action, microbiological targeting, clinical use, pharmacokinetic/pharmacodynamic features, and potential for therapeutic drug monitoring will be addressed. Each antibiotic paragraph is divided into specialized microbiological, clinical, and pharmacological sections, including detailed and appropriate tables. A better understanding of the latest promising advances in the field of therapeutic options could lead to the development of a better approach in managing antimicrobial therapy for multidrug-resistant Gram-positive pathogens, which increasingly needs to be better stratified and targeted.

Beauveria bassiana Keratitis: A Case Series and Review of Literature.

BACKGROUND: Beauveria bassiana is a filamentous fungus commonly used as an insecticide that rarely causes keratitis. METHODS: Patients affected by Beauveria bassiana keratitis were retrospectively recruited at San Raffaele Hospital (Milan, Italy) between 2020 and 2022. All subjects underwent comprehensive ophthalmic evaluation, including in vivo confocal microscopy (IVCM) and microbiologic examination of corneal scrapings. Beauveria bassiana was identified using 18S rDNA targeted PCR. RESULTS: Four eyes of four patients (51 ± 8.8 years old) were evaluated. The main risk factors were soft contact lens wear (75%) and trauma with vegetative matter (50%). A superficial infiltrate was displayed in the majority of patients. Three cases (75%) showed hyphae on IVCM. All patients showed clinical improvement after topical antifungal therapy, although mostly through a combination of two antifungals (75%). One patient with a deeper infection required a systemic antifungal agent after one month of topical therapy. All cases required debridement to reduce the microbial load and enhance drug penetration. All patients experienced keratitis resolution following medical treatment (average: 3.3 months). CONCLUSIONS: The identification of risk factors and the early diagnosis of Beauveria bassiana keratitis are fundamental in order to avoid its penetration in the deeper corneal stromal layers. Topical antifungal drugs, possibly accompanied by ulcer debridement, may be a successful treatment if instilled from the early phases of the disease.

Levofloxacin prophylaxis vs no prophylaxis in patients with neutropenia within an endemic country for carbapenem-resistant GNB.

Fluoroquinolone prophylaxis's (FQ-P) usefulness in patients with neutropenia is controversial. In recent decades, Italian epidemiological data has shown worrisome rates of FQ resistance. A single-center cohort study on 136 autologous stem cell transplantations (ASCTs) and 223 allogeneic hematopoietic stem cell transplantations (allo-HSCTs) was performed from January 2018 to December 2020. Piperacillin/tazobactam was the first-line therapy for febrile neutropenia (FN). Since February 2019, FQ-P has been omitted. We evaluated the day +30 posttransplant cumulative incidence function (CIF) of gram-negative bacteria pre-engraftment bloodstream infections (PE-BSIs) and any changes in antimicrobial resistance, FN, and infection-related mortality (IRM). In ASCTs, ≥1 FN episode occurred in 74.3% of transplants, without differences among groups (P = .66). CIF of gram-negative bacteria PE-BSI was 10.1%, with a significant difference according to FQ-P (0% [LEVO-group] vs 14.1% [NO-LEVO-group], P = .016). CIF of IRM was 0% in both groups. In allo-HSCTs, ≥1 FN episode occurred in 96.4% of transplants, without differences among groups (P = .72). CIF of gram-negative bacteria PE-BSI was 28%, significantly higher without FQ-P (14.7% [LEVO-group] vs 34.4% [NO-LEVO-group], P = .003). CIF of IRM was 5%, superimposable in both groups (P = .62). Comparing antimicrobial resistance among gram-negative bacteria of allo-HSCT setting, in the group without FQ-P, a significantly higher proportion of pathogens was susceptible to piperacillin/tazobactam (71% vs 30%, P = .026), FQ (49% vs 10%, P = .03), and carbapenems (95% vs 50%, P = .001). FQ-P discontinuation increased gram-negative bacteria PE-BSI but did not impact IRM, both in the ASCT and allo-HSCT settings; importantly, it concurred to significantly decrease antimicrobial resistance in gram-negative bacteria.

The era of molecular and other non-culture-based methods in diagnosis of sepsis.

Sepsis, a leading cause of morbidity and mortality throughout the world, is a clinical syndrome with signs and symptoms relating to an infectious event and the consequent important inflammatory response. From a clinical point of view, sepsis is a continuous process ranging from systemic inflammatory response syndrome (SIRS) to multiple-organ-dysfunction syndrome (MODS). Blood cultures are the current "gold standard" for diagnosis, and they are based on the detection of viable microorganisms present in blood. However, on some occasions, blood cultures have intrinsic limitations in terms of sensitivity and rapidity, and it is not expected that these drawbacks will be overcome by significant improvements in the near future. For these principal reasons, other approaches are therefore needed in association with blood culture to improve the overall diagnostic yield for septic patients. These considerations have represented the rationale for the development of highly sensitive and fast laboratory methods. This review addresses non-culture-based techniques for the diagnosis of sepsis, including molecular and other non-culture-based methods. In particular, the potential clinical role for the sensitive and rapid detection of bacterial and fungal DNA in the development of new diagnostic algorithms is discussed.

Cloning and molecular characterization of a human recombinant IgG Fab binding to the Tat protein of human immunodeficiency virus type 1 (HIV-1) derived from the repertoire of a seronegative patient.

A study aiming at cloning and characterizing natural antibodies to human immunodeficiency virus type 1 (HIV-1) targets is described. In particular, we report the molecular cloning of a Fab molecule binding the HIV-1/Tat protein from a seronegative patient. The Fab was characterized for its binding specificity and investigated in regards to its molecular structure. Furthermore, to evaluate the role played by the heavy and light chains in the binding to the antigen, hybrid Fabs were constructed combining the heavy and the light chain of the natural anti-Tat clone with a control high-affinity Fab derived from the repertoire of the same patient. The results indicate that the natural immunoglobulin under study: (i) is a polyreactive antibody of IgG1 isotype, and not an IgM as usually described for anti-HIV natural clones, (ii) shows a pattern of mutations compatible with an antigen-driven mechanisms, (iii) its heavy chain derives from a V-gene subfamily (V3-23) highly represented in fetal life, and (iv) its heavy chain variable region exhibits several characteristics, including an extremely long, hydrophilic CDR3, that are unusual and theoretically important in determining the polyreactive capacity of the molecule.

Quantitation of Bacillus clausii in biological samples by real-time polymerase chain reaction.

A real-time PCR assay targeting the highly specific erm34 sequence of Bacillus clausii DNA was developed and optimized. The quantitative assay showed a sensitivity level of 10(2) CFU/microl of sample. The method may represent a useful tool for monitoring the role of B. clausii as probiotic in vivo.

Direct sequencing of Scedosporium apiospermum DNA in the diagnosis of a case of keratitis.

The present report describes the diagnostic strategy followed in a case of keratomycosis. Together with conventional methods, a molecular strategy that involved the direct sequencing of an amplified portion of the genome encompassing the internal transcribed spacer 1 and 2 regions and sequence analysis was used. The data highlight the diagnostic role of molecular techniques, in parallel with conventional methods, in the management of ocular infections of fungal aetiology.

Cost-effectiveness of blood culture and a multiplex real-time PCR in hematological patients with suspected sepsis: an observational propensity score-matched study.

We evaluated the costs and clinical outcomes of episodes of suspected sepsis in hematological patients. A propensity score-matched study was planned, comparing a retrospective cohort managed with standard assays and a prospective cohort managed with the addition of a molecular assay. Diagnostic procedures and therapy were considered as costs variables. The primary clinical endpoint was sepsis-related mortality, whereas the length of each suspected sepsis episode was investigated as a secondary endpoint. A total of 137 and 138 episodes in the prospective and the retrospective cohorts were studied, respectively; 101 pairs of highly matched episodes were analyzed, evidencing a trend of higher mortality in the retrospective cohort. No difference in length of suspected sepsis episode was observed. Significant savings were observed in the prospective cohort, especially due to reduced costs in antifungal therapy. The apparently more expensive molecular assay favored a more rational use of economic resources without influencing, and probably improving, the clinical outcome.

Potential role of the detection of enterobacterial DNA in blood for the management of neonatal necrotizing enterocolitis.

We present three cases of pre-term low-weight infants with suspected necrotizing enterocolitis (NEC) [one eventually recognized as a connatal cytomegalovirus (CMV) infection], microbiologically monitored using a molecular assay detecting bacterial and fungal DNA in blood. The detection of DNA from enteric pathogens in blood was interpreted as a sign of ongoing perforation, and represented a useful complement in the management of the presented cases. Moreover, these cases suggest the opportunity for larger future studies to assess the possible role of a molecular approach in the close monitoring of infants with suspected NEC or with other conditions at-risk for intestinal perforation.

Isolation and genotyping of Acanthamoeba strains from corneal infections in Italy.

Acanthamoeba keratitis (AK) is a corneal disease caused by members of a genus of free-living amoebae and is associated predominantly with contact lens (CL) use. This study reports 16 cases of culture-proven AK diagnosed in northern Italy. Genotype identification was carried out with a PCR assay based on sequence analysis of the 18S rRNA gene, and sensitivity and specificity were evaluated in comparison with traditional parasitological techniques. A 405 bp region of the 18S rRNA gene (ASA.S1) including diagnostic fragment 3 (DF3) was amplified using the genus-specific primers JDP1 and JDP2. Genotype assignment was based on phenetic analysis of the ASA.S1 subset of the nuclear small-subunit rRNA gene sequence excluding the highly variable DF3 region. Phylogenetic analysis was also performed on the sequences obtained. All patients complained of monolateral infection; 11 (68.75%) admitted improper CL disinfection. In 14/16 (87.5 %) subjects, corneal scrapings were stained with calcofluor white and haematoxylin and eosin and, in ten cases (62.5 %), microscopy was positive for Acanthamoeba cysts. In vitro culture on 3 % non-nutrient agar plates was obtained in all cases (100 %), whereas cloning and axenic growth were positive for 14 amoebic stocks (87.5 %). PCR analysis had 100 % sensitivity and specificity compared with in vitro axenic culture, showing positive amplification from 15 isolates. All Acanthamoeba strains belonged to the T4 genotype, the main AK-related genotype worldwide. These results confirmed the importance of a complete diagnostic protocol, including a PCR assay, for the clinical diagnosis of AK on biological samples. Genotyping allowed inclusion of all isolates in the T4 group, thus demonstrating the prevalence of this genotype in northern Italy.

Modulation of epitope-specific anti-hepatitis C virus E2 (anti-HCV/E2) antibodies by anti-viral treatment.

The dynamic features of three specific anti-hepatitis C virus (HCV) antibody subpopulations directed against different conformational epitopes of the viral E2 protein (HCV/E2) have been evaluated in patients with primary and persistent HCV infection; the three subpopulations are present in patients infected with different HCV genotypes and have shown a different activity using a pseudovirus neutralization assay (antibodies e301 and e137 exhibiting high neutralizing activity, while antibody e509 enhancement of HCV infectivity). In sequential samples from five patients with primary HCV infection and different virological outcome, all samples tested negative with the single exception of the e509 antibody in a patient not clearing the virus. In sequential samples from 28 patients with persistent infection under treatment with pegylated interferon-alpha plus ribavirin (14 sustained virological responders and 14 non-responders), the therapy did not selectively influence titers of the two neutralizing antibody subpopulations; otherwise, a net increase of the e509 antibody subpopulation related to enhancement of HCV infectivity was observed in non-responders, but not in sustained virological responders (P = 0.0156). This increase was not related to the trend of total anti-HCV/E2 response. The data indicate that a specific antibody response against these epitopes is elicited only late during the infection, thus not influencing virus clearance during primary infection, and that a selective increase of the antibody subpopulation enhancing virus infectivity is observed only in the cohort of patients not responding to antiviral therapy.