Potential value of a rapid syndromic multiplex PCR for the diagnosis of native and prosthetic joint infections: a real-world evidence study.

Introduction: The BIOFIRE Joint Infection (JI) Panel is a diagnostic tool that uses multiplex-PCR testing to detect microorganisms in synovial fluid specimens from patients suspected of having septic arthritis (SA) on native joints or prosthetic joint infections (PJIs). Methods: A study was conducted across 34 clinical sites in 19 European and Middle Eastern countries from March 2021 to June 2022 to assess the effectiveness of the BIOFIRE JI Panel. Results: A total of 1527 samples were collected from patients suspected of SA or PJI, with an overall agreement of 88.4 % and 85 % respectively between the JI Panel and synovial fluid cultures (SFCs). The JI Panel detected more positive samples and microorganisms than SFC, with a notable difference on Staphylococcus aureus, Streptococcus species, Enterococcus faecalis, Kingella kingae, Neisseria gonorrhoeae, and anaerobic bacteria. The study found that the BIOFIRE JI Panel has a high utility in the real-world clinical setting for suspected SA and PJI, providing diagnostic results in approximately 1 h. The user experience was positive, implying a potential benefit of rapidity of results' turnover in optimising patient management strategies. Conclusion: The study suggests that the BIOFIRE JI Panel could potentially optimise patient management and antimicrobial therapy, thus highlighting its importance in the clinical setting.

Is Silver Addition to Scaffolds Based on Polycaprolactone Blended with Calcium Phosphates Able to Inhibit Candida albicans and Candida auris Adhesion and Biofilm Formation?

Candida spp. periprosthetic joint infections are rare but difficult-to-treat events, with a slow onset, unspecific symptoms or signs, and a significant relapse risk. Treatment with antifungals meets with little success, whereas prosthesis removal improves the outcome. In fact, Candida spp. adhere to orthopedic devices and grow forming biofilms that contribute to the persistence of this infection and relapse, and there is insufficient evidence that the use of antifungals has additional benefits for anti-biofilm activity. To date, studies on the direct antifungal activity of silver against Candida spp. are still scanty. Additionally, polycaprolactone (PCL), either pure or blended with calcium phosphate, could be a good candidate for the design of 3D scaffolds as engineered bone graft substitutes. Thus, the present research aimed to assess the antifungal and anti-biofilm activity of PCL-based constructs by the addition of antimicrobials, for instance, silver, against C. albicans and C. auris. The appearance of an inhibition halo around silver-functionalized PCL scaffolds for both C. albicans and C. auris was revealed, and a significant decrease in both adherent and planktonic yeasts further demonstrated the release of Ag+ from the 3D constructs. Due to the combined antifungal, osteoproliferative, and biodegradable properties, PCL-based 3D scaffolds enriched with silver showed good potential for bone tissue engineering and offer a promising strategy as an ideal anti-adhesive and anti-biofilm tool for the reduction in prosthetic joints of infections caused by Candida spp. by using antimicrobial molecule-targeted delivery.

First case of Chryseobacterium gallinarum bloodstream infection: a diagnostic and therapeutic challenge for an emerging pathogen.

Chryseobacterium spp. belongs to the Flavobacteriaceae family and is a rod-shaped gram-negative, glucose non-fermenting, non-motile bacterium ubiquitous in the environment. In humans, Chryseobacterium may be responsible for infections such as urinary tract infections (UTI) and ventriculitis with a pathogenic burden increasing in recent years. Chryseobacterium gallinarum was isolated for the first time in 2014 in a pharyngeal scrape sample of chicken and, until now, only one case of human UTI has been described in a pregnant 20-year-old Indian patient. Herein, we report the first case of bloodstream infection caused by C. gallinarum in a 67-year-old female burn patient, correctly identified by 16S-rRNA sequencing and successfully treated with cefepime and fosfomycin.

Herpes Virus Infection in Lung Transplantation: Diagnosis, Treatment and Prevention Strategies.

Lung transplantation is an ultimate treatment option for some end-stage lung diseases; due to the intense immunosuppression needed to reduce the risk of developing acute and chronic allograft failure, infectious complications are highly incident. Viral infections represent nearly 30% of all infectious complications, with herpes viruses playing an important role in the development of acute and chronic diseases. Among them, cytomegalovirus (CMV) is a major cause of morbidity and mortality, being associated with an increased risk of chronic lung allograft failure. Epstein-Barr virus (EBV) is associated with transformation of infected B cells with the development of post-transplantation lymphoproliferative disorders (PTLDs). Similarly, herpes simplex virus (HSV), varicella zoster virus and human herpesviruses 6 and 7 can also be responsible for acute manifestations in lung transplant patients. During these last years, new, highly sensitive and specific diagnostic tests have been developed, and preventive and prophylactic strategies have been studied aiming to reduce and prevent the incidence of these viral infections. In this narrative review, we explore epidemiology, diagnosis and treatment options for more frequent herpes virus infections in lung transplant patients.

Antiviral Approach to Cytomegalovirus Infection: An Overview of Conventional and Novel Strategies.

Human cytomegalovirus (HCMV) is a herpesvirus capable of establishing a lifelong persistence in the host through a chronic state of infection and remains an essential global concern due to its distinct life cycle, mutations, and latency. It represents a life-threatening pathogen for immunocompromised patients, such as solid organ transplanted patients, HIV-positive individuals, and hematopoietic stem cell recipients. Multiple antiviral approaches are currently available and administered in order to prevent or manage viral infections in the early stages. However, limitations due to side effects and the onset of antidrug resistance are a hurdle to their efficacy, especially for long-term therapies. Novel antiviral molecules, together with innovative approaches (e.g., genetic editing and RNA interference) are currently in study, with promising results performed in vitro and in vivo. Since HCMV is a virus able to establish latent infection, with a consequential risk of reactivation, infection management could benefit from preventive treatment for critical patients, such as immunocompromised individuals and seronegative pregnant women. This review will provide an overview of conventional antiviral clinical approaches and their mechanisms of action. Additionally, an overview of proposed and developing new molecules is provided, including nucleic-acid-based therapies and immune-mediated approaches.

Four-Component Recombinant Protein-Based Vaccine Effectiveness Against Serogroup B Meningococcal Disease in Italy.

Importance: Population-based data on the 4-component recombinant protein-based (4CMenB) vaccine effectiveness and reduction in incidence rate ratios (IRRs) are continuously needed to assess vaccine performance in the prevention of serogroup B invasive meningococcal disease (IMD). Objective: To assess the effectiveness and reduction in IRRs associated with the 4CMenB vaccine in the pediatric population in 6 regions in Italy. Design, Setting, and Participants: This retrospective cohort screening study and case-control study included data from children aged younger than 6 years in 6 highly populated Italian regions from January 1, 2006, to January 1, 2020. Participants included children younger than 6 years diagnosed with serogroup B IMD without predisposing factors. Data were collected from regional surveillance and vaccination registries and were analyzed from September 2021 to January 2022. Exposures: Routine 4CMenB vaccination, per regional vaccination programs. Main Outcomes and Measures: The main outcome was the effectiveness of the 4CMenB vaccine in the prevention of serogroup B IMD in the population of children aged younger than 6 years in 6 Italian regions. The percentages of vaccine effectiveness (VE) were obtained through the concomitant use of a screening method and a case-control study. Secondary outcomes were the comparison of effectiveness results obtained using the 2 different computational methods, the description of serogroup B IMD incidence rates, and reduction in IRRs before and after 4CMenB introduction, as a proxy for vaccine impact. Results: The cohort screening study included a resident population of 587 561 children younger than 6 years in 3 regions with similar surveillance protocols, and the matched-case controls study assessed a resident population of 1 080 620 children younger than 6 years in 6 regions. Analyses found that 4CMenB VE in fully immunized children was 94.9% (95% CI, 83.1%-98.4%) using the screening method and 91.7% (95% CI, 24.4%-98.6%) using the case-control method. Overall reduction in IRR was 50%, reaching 70% in regions with early-start vaccination schedules. The case-control method involving 6 highly-populated Italian regions included 26 cases and 52 controls and found an estimated VE of 92.4% (95% CI, 67.6%-97.9%) in children old enough for the first vaccine dose and 95.6% (95% CI, 71.7%-99.1%) in fully immunized children. VE was more than 90% for partially immunized children. Even in regions where the first dose was administered at age 2 months, almost 20% of unvaccinated cases were among infants too young to receive the first 4CMenB dose. Conclusions and Relevance: This screening cohort study and matched case-controls study found high effectiveness of 4CMenB vaccination and greater reduction in IRR for early-start vaccination schedules in preventing invasive serogroup B meningococcal disease. The high proportion of children too young to be vaccinated among unvaccinated cases suggests that starting the vaccination even earlier may prevent more cases. Screening and case-control methods provided similar estimates of VE: either method may be used in different study settings, but concomitant use can provide more robust estimates.

Performance Evaluation of BD Phoenix and MicroScan WalkAway Plus for Determination of Fosfomycin Susceptibility in Enterobacterales.

BACKGROUND: Fosfomycin is an old bactericidal drug that has gained increasing interest in the last decade for its potential use in multi-drug resistant gram-negative infections. However, evidence on fosfomycin susceptibility testing reports a poor correlation between commercial methods vs. reference agar dilution (AD) for Enterobacterales (EB). The study aimed at assessing the performance of two automated systems for the determination of fosfomycin susceptibility in EB clinical isolates. METHODS: Fosfomycin susceptibility testing results of two collections of 100 non-duplicate clinical EB strains obtained using two different platforms (BD Phoenix and MicroScan WalkAway Plus) were compared with those obtained by AD. Categorical agreement (CA), major error (ME) and very major error (VME) rates were calculated. RESULTS: BD Phoenix exhibited a 6.9% rate of false-resistant results and achieved a CA of 69%, whereas MicroScan WalkAway Plus achieved 3.7% of false-resistant results and 72% of CA. Both automated systems showed poor detection of resistant isolates, with 49.1% and 56.2% of false-susceptible results for BD Phoenix and Microscan WalkAway Plus, respectively. CONCLUSIONS: Overall, agar dilution remains the most suitable method for routine laboratory antimicrobial susceptibility testing of fosfomycin on Enterobacterales strains, given the poor performance of automated systems. The application of both automated systems, in the clinical laboratories reporting of fosfomycin, should be reviewed in light of the accuracy results falling below the acceptable threshold.

Application of FT-IR Spectroscopy for Mycobacterium abscessus complex subspecies differentiation.

Mycobacterium abscessus complex (MABSC) subspecies differentiation improves patients' therapy and outcome. Fourier-Transform-Infrared Spectroscopy (FT-IRS) was applied for subspecies discrimination of 15 strains on different media: Löwenstein-Jensen showed the best resolution power; Linear Discriminant Analysis model differentiated M. abscessus susbsp. abscessus from M. abscessus subsp. massiliense. FT-IRS has a potential role in rapidly MABSC subspecies identification.

Implementation of Chromatic Super CAZ/AVI® medium for active surveillance of ceftazidime-avibactam resistance: preventing the loop from becoming a spiral.

Acquired resistance towards ceftazidime-avibactam (CAZ-AVI) is increasingly reported. Several mechanisms can be involved, but mutations in the Ω-loop region of ß-lactamases are the most described. Herein, we assessed the implementation of Chromatic Super CAZ/AVI® medium in rectal swab surveillance cultures in a geographic area with endemic distribution of KPC-producing Klebsiella pneumoniae. Routine rectal swabs collected from the intensive care unit (ICU) and non-ICU patients were screened for carbapenemase-producing Enterobacterales (CPE), carbapenem-resistant Gram-negative organisms (CR-GN) and CAZ-AVI-resistant organisms by Chromatic CRE and Super CAZ/AVI® media. Among the 1839 patients screened, 146 (7.9%) were found to be colonized by one or more CPE and/or CR-GN isolates during hospitalization. Overall, among colonized patients the most common bacteria encountered were KPC-producing Enterobacterales (n = 60; 41.1%), carbapenem-resistant Pseudomonas aeruginosa (n = 41; 28.1%) and carbapenem-resistant A. baumannii (n = 34; 23.3%). Among patients colonized by KPC-producing Enterobacterales, thirty-five (58.3%) had CAZ-AVI-resistant strains. A 30.5% rate of faecal carriage of CAZ-AVI-resistant KPC-producing K. pneumoniae, substantially higher than that of susceptible isolates (2.8%), was observed in the COVID-19 ICU. Prevalence of faecal carriage of metallo-ß-lactamase-producing organisms was low (0.5% and 0.2% for Enterobacterales and P. aeruginosa, respectively). Chromatic Super CAZ/AVI® medium showed 100% sensitivity in detecting CPE or CR-GN isolates resistant to CAZ-AVI regardless of both MIC values and carbapenemase content. Specificity was 86.8%. The Chromatic Super CAZ/AVI® medium might be implemented in rectal swab surveillance cultures for identification of patients carrying CAZ-AVI-resistant organisms to contain the spread of these difficult-to-treat pathogens.

Comparison of Three Different Commercial Methods for Fosfomycin Susceptibility Testing in Pseudomonas aeruginosa.

The lack of internationally approved breakpoint and a handy susceptibility testing reduces fosfomycin usefulness against Pseudomonas aeruginosa. Previous works defined low or moderate agreement between commercial methods and the reference method (agar dilution [AD]). In particular, data lack about testing against P. aeruginosa. We compared disk diffusion (DD), E-test (ET), and automated broth microdilution (BMD) to AD by testing 150 P. aeruginosa isolates. We obtained better categorical agreement (CA) for DD and ET for minimal inhibitory concentration >128 mg/L (84.7% and 92.7%, respectively), but with high very major error (VME). BMD had the lowest VME rate (2/42), but with 64% CA and 52/108 major errors. We cannot define a method comparable to AD. Larger studies, as well as the definition of a breakpoint value are needed for fosfomycin against P. aeruginosa.

In vitro activity of cefiderocol against ceftazidime-avibactam susceptible and resistant KPC-producing Enterobacterales: cross-resistance and synergistic effects.

PURPOSE: To assess the in vitro activity of cefiderocol (CFDC) against a collection of both ceftazidime-avibactam (CZA) susceptible and resistant KPC-producing Enterobacterales (KPC-EB) isolates. Secondly, to assess its synergistic activity in combination with different antibiotics. METHODS: One hundred KPC-EB isolates were tested: 60 CZA susceptible and 40 CZA resistant. Among them, 17 pairs of CZA susceptible and resistant KPC-producing Klebsiella pneumoniae (KPC-Kp) isolates were collected from 17 distinct patients before and after CZA treatment, respectively. CFDC susceptibility was evaluated by both broth microdilution (lyophilized panels; Sensititre; Thermo Fisher) and disk diffusion testing. Results were interpreted using EUCAST breakpoints. Synergistic activity of CFDC in combination with CZA, meropenem-vaborbactam, imipenem, and amikacin against six characterized KPC-Kp strains, before and after acquisition of CZA resistance, was evaluated using gradient diffusion strip crossing method. RESULTS: CFDC resistance rate was significantly higher in CZA resistant EB subset than in the susceptible one (p < 0.001): 82.5% vs 6.7%. MIC50 and MIC90 values were 0.25 and 2 mg/L, 8 and 64 mg/L in CZA-susceptible and CZA-resistant subset, respectively. KPC-Kp isolates harboring KPC-D179Y or KPC-Δ242-GT-243 variants showed CFDC MICs ranging from 4 to 64 mg/L. CFDC showed in vitro synergistic effect mostly with CZA, against both CZA susceptible and resistant isolates, resulting in a synergy rate of 66.7%. CONCLUSIONS: CZA resistance mechanisms in KPC-EB impair the in vitro activity of CFDC, often leading to co-resistance. CFDC in combination with the new ß-lactamases inhibitors might represent a strategy to enhance its activity.

Carbapenemase detection testing in the era of ceftazidime/avibactam-resistant KPC-producing Enterobacterales: A 2-year experience.

OBJECTIVES: The aim of this study was to investigate the prevalence of ceftazidime/avibactam (CZA) resistance among carbapenemase-producing Enterobacterales (CPE) blood culture isolates as well as the performance of the main carbapenemase phenotypic detection methods to identify KPC variants associated with CZA resistance. METHODS: Non-duplicate CPE strains isolated from blood cultures during 2018-2020 were tested for antimicrobial susceptibility. Molecular testing was used to identify carbapenemase-producers. Strains harbouring blaKPC and with a CZA minimum inhibitory concentration (MIC) ≥8 mg/L were investigated by sequencing. Subsequentially, five phenotypic carbapenemase detection methods were evaluated on these strains, namely the modified carbapenem inactivation method (mCIM), Rapidec® Carba NP, the disk diffusion synergy test, NG-Test CARBA® 5 and RESIST-5 O.O.K.N.V. RESULTS: Overall, the CZA resistance rate was high (13.7%) and remained relevant (5.9%) excluding metallo-ß-lactamases-producers. All isolates harbouringblaKPC mutants (n = 8) were associated with reduced carbapenem MICs and negative results by all detection methods based on revelation of enzyme activity. Lateral flow immunoassays failed to detect KPC-31 (n = 4) and KPC-33 (n = 2) but correctly identified KPC-14 (n = 2). Conversely, isolates harbouring wild-type KPC genes (n = 3) were associated with high-level CZA resistance and carbapenem resistance and tested positive by all of the evaluated methods. CONCLUSION: In the era of CZA-based therapies, molecular blaKPC identification followed by a carbapenem hydrolysis-based phenotypic assay could be the most reasonable diagnostic algorithm to detect all KPC-producers and to identify mutants associated with impaired carbapenemase activity and CZA resistance.

Evaluation of BD Phoenix and Microscan WalkAway for determination of fosfomycin susceptibility in Staphylococcus aureus.

We evaluate the performance of BD Phoenix (BD Diagnostic; Hunt Valley, MD) and MiscroScan WalkAway (Beckman Coulter Inc; Carlsbad, CA) systems versus reference agar dilution method for fosfomycin susceptibility determination in 200 Staphylococcus aureus clinical isolates. Both methods exhibit ≤89.0% of categorica agreement and ≥63.3% of very major error. All commercial antimicrobial susceptibility systems show poor concordance with reference method.

An unpleasant souvenir: Endoscopic finding of Trichuris trichiura (Nematoda: Trichuridae).

Whipworms are responsible for up to 500 million cases of trichuriasis worldwide, with higher endemicity in tropical and sub-tropical countries. In non-endemic countries, trichuriasis can be accidentally diagnosed upon colonoscopy, often in the presence of negative microscopy. Here, we describe an incidental diagnosis of trichuriasis in an HIV patient residing in a non-endemic area (i.e., Turin, Italy), six months after his return from Antigua. The species-level diagnosis was made thanks to PCR-based molecular identification of Trichuris sp. following optical microscopy detection. Overall, this case highlights the importance of improving parasitic diseases diagnosis through cutting-edge clinical and laboratory diagnostic tools alongside advanced training of specialists in the area of parasitology.

Comparison of 3 Tfr2-deficient murine models suggests distinct functions for Tfr2-alpha and Tfr2-beta isoforms in different tissues.

Transferrin receptor 2 (TFR2) is a transmembrane protein that is mutated in hemochromatosis type 3. The TFR2 gene is transcribed in 2 main isoforms: the full-length (alpha) and a shorter form (beta). alpha-Tfr2 is the sensor of diferric transferrin, implicated in the modulation of hepcidin, the main regulator of iron homeostasis. The function of the putative beta-Tfr2 protein is unknown. We have developed a new mouse model (KI) lacking beta-Tfr2 compared with Tfr2 knockout mice (KO). Adult Tfr2 KO mice show liver iron overload and inadequate hepcidin levels relative to body iron stores, even though they increase Bmp6 production. KI mice have normal transferrin saturation, liver iron concentration, hepcidin and Bmp6 levels but show a transient anemia at young age and severe spleen iron accumulation in adult animals. Fpn1 is strikingly decreased in the spleen of these animals. These findings and the expression of beta-Tfr2 in wild-type mice spleen suggest a role for beta-Tfr2 in Fpn1 transcriptional control. Selective inactivation of liver alpha-Tfr2 in KI mice (LCKO-KI) returned the phenotype to liver iron overload. Our results strengthen the function of hepatic alpha-Tfr2 in hepcidin activation, suggest a role for extrahepatic Tfr2 and indicate that beta-Tfr2 may specifically control spleen iron efflux.

Hepatic expression of hemochromatosis genes in two mouse strains after phlebotomy and iron overload.

BACKGROUND AND OBJECTIVES: Iron homeostasis is tightly regulated in mammals according to the needs of erythropoiesis and the iron stores present. This regulation is disrupted in hereditary hemochromatosis (HH), a genetic disorder characterized by increased intestinal iron absorption, leading to iron overload. The genes coding for HFE, transferrin receptor 2 (TFR2), ferroportin (SLC40A1 or FPN1), hepcidin (HEPC) and hemojuvelin (HJV or RGMC) are responsible for different types of genetic iron overload. All these genes are highly expressed in the liver and their protein products are likely components of a single hepcidin-related pathway. In order to gain insights into the molecular relationship among the HH proteins we evaluated the hepatic expression of HH genes in conditions of iron restriction or overload. DESIGN AND METHODS: Data were obtained after phlebotomy, to activate the erythroid regulators and following parenteral iron dextran loading, to activate the store regulators, in two mice strains (C57BL/6 and DBA/2). HH genes and proteins expression were analyzed by quantitative real time polymerase chain reaction and by Western blotting, respectively. RESULTS: Hepc RNA was reduced after phlebotomy and increased in iron overload. A statistically significant reduction of hepatic Fpn1 RNA expression was observed after phlebotomy; this effect was more evident in the DBA/2 strain. Fpn1 increased in C57BL/6 mice, but not in the DBA/2 ones in parenteral iron loading. Fpn1 protein did not change substantially in either condition. Hfe, Rgmc and Tfr2 expression was not influenced by phlebotomy. In parenteral iron overload, Tfr2 gene and protein expression decreased concomitant to the increase in Hepc, while Hfe RNA remained constant. INTERPRETATION AND CONCLUSIONS: Our results indicate that regulation of hepatic Fpn1 differs from that reported for duodenal Fpn1. Furthermore, taken the differences in gene expression in dietary overload (increased Hfe but not Tfr2), distinct roles are suggested for Hfe and Tfr2 in Hepc activation.

Screening selected blood donors with biochemical iron overload for hemochromatosis: a regional experience.

BACKGROUND AND OBJECTIVES: Hemochromatosis is a genetic disorder characterized by progressive iron overload which leads to early abnormalities of iron parameters (increased transferrin saturation =TS and serum ferritin=SF) and late clinical complications. The disease is prevalently due to C282Y and H63D mutations in the HFE gene, but additional molecular defects are present in a minority of patients. DESIGN AND METHODS: From January to December 2002 we screened first time blood donors of Piedmont, a region of North-western Italy, for TS>45%. Individuals with TS>45% underwent a second fasting check, SF assessment and molecular tests, investigating 12 hemochromatosis-associated molecular defects. RESULTS: A total of 13,998 subjects were screened; 868 (6.2%) had TS>45% and were recalled. Four hundred and eight-six underwent molecular testing. In this selected population allele frequencies of C282Y, H63D and S65C were 6.8%, 22.4% and 1.0%, respectively. No rare mutations were detected, except E168Q in HFE. When measured during fasting, TS was significantly higher in C282Y homozygotes and H63D/C282Y heterozygotes (p<0.05) than in wild type subjects, but not in H63D homozygotes. Hyperferritinemia was documented in 32 cases, 9 with wild type genotype. Mean age, body mass index (BMI) and alcohol intake were higher in this group than in individuals with normal SF. INTERPRETATION AND CONCLUSIONS: This study is an example of a large, two-step hemochromatosis screening with moderate effort and low cost, that enriches basal C282Y allele frequency by about three-fold. Screening based on genotyping only subjects found to have a TS>45% is feasible but, in order to be cost effective should be based on the identification of the two prevalent mutations even in an area where several forms of hemochromatosis have been reported.