Search Results (222)

Background/Objectives: Non-fermenting Gram-negative bacilli (NFGNB) represent a significant clinical challenge due to their intrinsic and acquired resistance, particularly in immunocompromised patients. Infections cause by NFGNB are associated with high morbidity and mortality, especially among patients with cystic fibrosis and hematologic malignancies. This study aimed to assess the in vitro susceptibility of clinically relevant NFGNB isolates to two newer antibiotics, cefiderocol and aztreonam/avibactam, and an established antibiotic, trimethoprim/sulfamethoxazole. Methods: This retrospective, monocentric study analysed 94 NFGNB isolates (30 Pseudomonas aeruginosa, 30 Acinetobacter sp., 24 Stenotrophomonas maltophilia, and 10 Burkholderia cepacia complex). Susceptibility testing for cefiderocol, aztreonam/avibactam, and trimethoprim/sulfamethoxazole was conducted using gradient strip method. MIC values were interpreted using EUCAST breakpoints, ECOFFs, or alternative criteria when necessary. Results: All S. maltophilia isolates were susceptible to cefiderocol (FCR) and aztreonam/avibactam (A/A) based on ECOFFs, with one strain resistant to trimethoprim–sulfamethoxazole (COT). Burkholderia cepacia complex strains also showed high susceptibility to FCR, with only one isolate exceeding the ECOFF for A/A, and 20% resistant to COT. All Acinetobacter sp. isolates were susceptible to FCR; however, most MIC values clustered at or just below the ECOFF value. In P. aeruginosa, one isolate was resistant to FCR, and three isolates (10%) were resistant to A/A. Interestingly, confirmed carbapenemase producers remained susceptible to both FCR and A/A. Most A/A MIC values for P. aeruginosa were just below the ECOFF. Conclusions: Cefiderocol and aztreonam/avibactam demonstrated promising in vitro activity against clinically relevant NFGNB, including carbapenem-resistant strains. These findings support their potential role as therapeutic options for difficult-to-treat infections, particularly in immunocompromised patients. Full article

Background: Cefiderocol (FDC) presents challenges in antimicrobial susceptibility testing (AST). The reference standard is the broth microdilution (BMD) method with iron-depleted cation-adjusted Mueller-Hinton broth (ID-CAMHB). Still, it is cumbersome for routine clinical laboratory use, while variable accuracy has been reported with available commercial systems. Variability in interpretive criteria and areas of technical uncertainty (ATUs) further complicate assessments. Methods: This review and expert opinion presents: (1) an overview of non-susceptibility to FDC and then delves into the performance of current FDC AST methods for Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii complex; (2) a practical decision framework to guide clinical microbiologists in making informed choices. Results and Conclusions: For Enterobacterales, including carbapenem-resistant Enterobacterales (CRE), and Pseudomonas aeruginosa, we propose disk diffusion (DD) as a preliminary screening tool to classify isolates as susceptible (S) or resistant (R). Confirmatory testing using the UMIC^® FDC system or the ID-CAMHB BMD method is recommended for R isolates. In cases of discrepancy, repeating the test with ID-CAMHB BMD is advised. Additionally, isolates falling within the ATU during DD testing should be retested using the UMIC^® system or ID-CAMHB BMD. For A. baumannii complex, since EUCAST breakpoints have not been defined yet, we propose a stepwise framework based on the first DD result: isolates with inhibition zones < 17 mm are considered non-susceptible and should be confirmed with standard BMD. Those between 17 and 22 mm require retesting with a commercial BMD method, with further confirmation recommended if S isolates with zones ≥ 23 mm may be considered S without additional testing. Full article

Background: Copy number variants of uncertain significance (CNVus) from chromosome microarray analysis (CMA) presents unresolved challenges for clinical geneticists, genetic counselors, and patients. We performed a systematic reevaluation of reported CNVus and reanalysis of selected CNVus by whole genome sequencing (WGS) to assess the diagnostic value and clinical impact on CNVus reclassification. Methods: We retrospectively reviewed 5277 consecutive pediatric cases by CMA from the Yale Clinical Cytogenetics Laboratory over a 13-year period. Reevaluation was performed on all reported CNVus following current ACMG/ClinGen guidelines. Reanalysis by WGS was applied to selected cases for reclassification of CNVus. Results: A total of 567 CNVus from 480 cases were reported, which accounted for 9.1% of pediatric cases. A total of 4 CNVus in 4 cases (0.8%, 4/480) were reclassified to pathogenic/likely pathogenic CNVs (pCNVs/lpCNVs); while 23 CNVus in 23 cases (4.8%, 23/480) were reclassified to benign/likely benign CNVs (bCNVs/lbCNVs). The overall rate of reclassification was 5.6%. WGS performed on selected cases further defined breakpoints and ruled out additional causative genetic variants. Conclusions: The results from this study demonstrated the diagnostic value of periodic reevaluation of CNVus and reanalysis by WGS in an interval of 3–5 years and provided evidence to support standardized laboratory reevaluation and reanalysis. Full article

The excessive use of antimicrobials drives the emergence of multidrug resistance (MDR) in bacterial strains, which harbor resistance genes to survive under diverse drug pressures. Such resistance can result in life-threatening infections. The predominance of MDR Pseudomonas spp. poses significant challenges to public health and environmental sustainability, particularly in ecosystems affected by human activities. Characterizing MDR Pseudomonas spp. is crucial for developing effective diagnostic tools and biosecurity protocols, with broader implications for managing other pathogenic bacteria. Strains were diagnosed through 16S rRNA PCR and sequencing, complemented by phylogenetic analysis to evaluate local and global evolutionary connections. Antibiotic susceptibility tests revealed extensive resistance across multiple classes, with MIC values surpassing clinical breakpoints. This study examined the genetic diversity, resistance potential, and phylogenetic relationships among Pseudomonas aeruginosa strain DG2 and Pseudomonas fluorescens strain FM3, which were isolated from solid waste dump sites (n = 30) and dairy farms (n = 22) in West Bengal, India. Phylogenetic analysis reveals distinct clusters that highlight significant geographic linkages and genetic variability among the strains. Significant biofilm production under antibiotic exposure markedly increased resistance levels. RAPD-PCR profiling revealed substantial genetic diversity among the isolates, indicating variations in their genetic makeup. In contrast, SDS-PAGE analysis provided insights into the protein expression patterns that are activated by stress, which are closely linked to MDR. This dual approach offers a clearer perspective on their adaptive responses to environmental stressors. This study underscores the need for vigilant monitoring of MDR Pseudomonas spp. in anthropogenically impacted environments to mitigate risks to human and animal health. Surveillance strategies combining phenotypic and molecular approaches are essential to assess the risks posed by resilient pathogens. Solid waste and dairy farm ecosystems emerge as critical reservoirs for the evolution and dissemination of MDR Pseudomonas spp. Full article

Peters anomaly (PA) is a rare congenital disorder within the anterior segment dysgenesis (ASD) spectrum, characterized by corneal opacity, iridocorneal adhesions, and potential systemic involvement. The genetic basis of PA and related syndromes are complex and incompletely understood. This study investigates novel genetic variants and their clinical impact in two unrelated individuals diagnosed with PA spectrum disorder. Whole-exome sequencing (WES), long-range PCR, and breakpoint analysis were applied to identify pathogenic variants. In the first patient, a heterozygous ~1.6 Mb deletion was detected, spanning the genes PEX2 and ZFHX4 (GRCh37 chr8:g.76760782_78342600del). The second patient carried a heterozygous FOXC1 variant (NM_001453.3:c.310A>G), classified as likely pathogenic. Both variants were confirmed by Sanger sequencing and considered de novo, as they were not present in the biological parents. Clinical evaluations revealed phenotypic variability, with the first patient displaying both ocular and systemic anomalies as in a Peters plus-like syndrome phenotype, while the second patient had isolated ocular manifestations as in a PA type 1 phenotype. These findings expand the genetic landscape of PA, underscoring the importance of comprehensive genomic analysis in subclassifying ASD disorders. Further studies are needed to elucidate the functional consequences of these variants and improve diagnostic and therapeutic strategies. Full article

Background/Objectives: Carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are challenging multidrug-resistant pathogens. This study evaluated the in vitro susceptibility of CRE and CRPA blood isolates from Korea to novel β-lactam/β-lactamase inhibitor combinations: ceftolozane/tazobactam (C/T), ceftazidime/avibactam (CZA), imipenem/cilastatin/relebactam (IMR), and meropenem/vaborbactam (MEV). Methods: Blood isolates of CRE (n = 55) and CRPA (n = 65) collected between September 2017 and September 2022 in a Korean tertiary hospital were included. Carbapenemase production was determined using phenotypic and molecular methods. In vitro susceptibility to C/T, CZA, IMR, and MEV was determined primarily by broth microdilution using current CLSI/EUCAST breakpoints. Clinical characteristics and in-hospital mortality were retrospectively reviewed. Results: Among non-carbapenemase-producing (non-CP) CRPA isolates (n = 47), susceptibility rates were 83.0% to C/T and 70.2% to CZA. For KPC-producing CRE isolates (n = 28), susceptibility rates were high to CZA (92.9%), IMR (82.1%), and MEV (96.4%). However, non-CP CRE isolates (n = 22) showed low susceptibility to C/T (18.2%) but high susceptibility to CZA (100%), IMR (81.8%), and MEV (95.5%). CRE infections were associated with higher rates of hematologic malignancy, immunosuppression, and in-hospital mortality (63.6% vs. 18.5% for CRPA, p < 0.001). Conclusions: The susceptibility of CRE and CRPA to novel β-lactam/β-lactamase inhibitors varies significantly by species and carbapenemase production. CZA, IMR, and MEV showed promising activity against KPC-producing CRE. These findings can inform empirical therapy and stewardship efforts in Korea. Full article

Candida auris (C. auris) is an emerging multidrug-resistant fungal pathogen recognized by the World Health Organization (WHO) as a critical global health threat. Its rapid transmission, high mortality rate, and frequent misidentification in clinical laboratories present significant challenges for diagnosis and infection control. This review provides a comprehensive overview of current and emerging diagnostic methods for C. auris detection, including culture-based techniques, biochemical assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and molecular diagnostics such as PCR and loop-mediated isothermal amplification (LAMP). We evaluate each method’s sensitivity, specificity, turnaround time, and feasibility in clinical and surveillance settings. While culture remains the diagnostic gold standard, it is limited by slow turnaround and phenotypic overlap with related species. Updated biochemical platforms and MALDI-TOF MS with expanded databases have improved identification accuracy. Molecular assays offer rapid, culture-independent detection. Antifungal susceptibility testing (AFST), primarily using broth microdilution, is essential for guiding treatment, although standardized breakpoints remain lacking. This review proposes an integrated diagnostic workflow and discusses key innovations and gaps in current practice. Our findings aim to support clinicians, microbiologists, and public health professionals in improving early detection, containment, and management of C. auris infections. Full article

Background: Campylobacter causes gastroenteritis worldwide with increasing antimicrobial resistance. Furazolidone (FZD) shows potential in resource-poor areas but needs further study. We aimed to assess the in vitro susceptibility of Campylobacter spp. to FZD, ciprofloxacin (CIP), and erythromycin (ERY) in a high-risk pediatric cohort and to evaluate the clinical relevance of resistance patterns using inhibitory quotient (IQ) pharmacodynamics. Methods: A two-phase prospective study (2012–2013, 2014–2015) was conducted at a tertiary pediatric hospital in Lima, Peru. Stool samples from children ≤24 months were cultured on selective media, with Campylobacter isolates identified via conventional bacteriological methods. Antimicrobial susceptibility was determined using Kirby–Bauer disk diffusion and regression-derived minimum inhibitory concentrations (MICs). IQ analysis correlated inhibition zones with therapeutic outcomes. Results: Among 194 Campylobacter isolates (C. jejuni: 28%; C. coli: 72%), resistance to CIP declined from 97.7% (2012–2013) to 83% (2014–2015), while ERY resistance rose from 2.3% to 9.4% (p= 0.002). No FZD resistance was observed, with mean inhibition zones of 52 ± 8 mm (2012–2013) and 43 ± 10.5 mm (2014–2015). MICs for FZD were predominantly <0.125 μg/mL, and all susceptible isolates demonstrated favorable IQ outcomes. Multidrug resistance (≥2 drugs) increased to 6.2% (2014–2015), though all MDR strains retained FZD susceptibility. CLSI and EUCAST breakpoints showed concordance for ERY (p = 0.724) but discordance for CIP (p = 0.022 vs. 0.008). Conclusions: FZD exhibits sustained in vitro efficacy against Campylobacter spp., even among MDR strains, contrasting with escalating fluoroquinolone and macrolide resistance. Full article

Background/Objectives: The aetiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), a chronic and severe debilitating disease with a complex phenotype, remains elusive. Associations with infectious diseases and autoimmune and neuropsychiatric disorders have been observed, without the identification of mechanisms. Previous studies suggest that genetic predisposition plays a role, but results are difficult to replicate, with Genome-Wide Association Studies of ME/CFS being challenging due to the relative rareness and heterogeneity of the disorder. Methods: We studied a well-defined Australian patient cohort diagnosed via the International Consensus Criteria, recruited by a specialist ME/CFS clinic. The whole-exome sequences of 77 patients were contrasted against genome variation in the 1000 Genome Project’s genome-matched population. Results: Significant associations with ME/CFS were harboured in genes that belong to the Neuroblastoma Breakpoint Family encoding Olduvai (DUF1220) domains, namely NBPF1 (rs3897177, p-value = 3.15 × 10⁻⁸), NBPF10 (rs1553120233, p-value = 9.262 × 10⁻¹³), and NBPF16 (rs200632836, p-value = 1.04 × 10⁻⁶). Other significantly associated variants were detected in the ATR, RSPH10B, ADGRE5-CD97, and NTRK2 genes, among others. Replication of these results was attempted via a GWAS on raw data from a US cohort, which confirmed shared significant associations with variation identified in the PTPRD, CSMD3, RAPGEF5, DCC, ALDH18A1, GALNT16, UNC79, and NCOA3 genes. Conclusions: These genes are involved in cortical neurogenesis, brain evolution, and neuroblastoma, and have been implicated by several studies in schizophrenia and autism. The sharing of these associations by the two cohorts supports their validity and grants the necessity of future studies to evaluate the implications for ME/CFS aetiology. Full article

The treatment of Mycobacterium avium complex (MAC) remains a clinical challenge as multidrug regimens are needed and may be limited by treatment-related toxicity. The Clinical and Laboratory Standards Institute (CLSI) endorses breakpoints for several agents used for MAC infection treatment. Amikacin has distinct breakpoints for intravenous (IV) therapy and inhaled therapy using amikacin liposome inhalation suspension (ALIS) for MAC pulmonary disease. The purpose of the present retrospective cohort study of MAC pulmonary isolates was to assess the number of amikacin non-susceptible isolates by the IV breakpoints that remain susceptible to the inhaled breakpoints. One isolate per patient per year was assessed and susceptibility was described for amikacin IV, amikacin inhaled, clarithromycin, moxifloxacin, and linezolid per the CLSI. Of the 218 isolates, 94% [204/218] tested as susceptible to amikacin per the IV breakpoints compared with 99.5% [217/218] to the inhaled breakpoints. Of the amikacin IV non-susceptible isolates, 93% [13/14] were susceptible by the inhaled breakpoints. For comparison, clarithromycin was the next most active agent followed by moxifloxacin and linezolid with 97% [211/218], 82% [178/218], and 66% [143/218] of isolates testing as susceptible to each, respectively. These data highlight the importance of laboratories to report both the IV and inhaled amikacin interpretive criteria so that clinicians do not disregard potential therapeutic options for the treatment of MAC pulmonary disease. Full article

Community-acquired infections caused by Enterobacterales are a growing public health concern, particularly in border regions where patient mobility may influence resistance patterns. Antimicrobial resistance (AMR) surveillance is critical for establishing local treatment guidelines. The aim of this study was to investigate AMR rates in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis obtained from community-acquired infections in Mexicali between 2019 and 2021. A retrospective study was conducted, analyzing 2871 Enterobacterales isolates (E. coli, K. pneumoniae, P. mirabilis). Species identification and antimicrobial susceptibility testing were performed using MALDI-TOF and VITEK 2 systems, interpreted according to CLSI and EUCAST breakpoints. ESBL production was detected in 37.6% of E. coli, 27.7% of K. pneumoniae, and none of the P. mirabilis isolates. Among ESBL producers, ciprofloxacin resistance reached 90.4% in E. coli and 81.0% in K. pneumoniae, indicating a significant level of co-resistance. Carbapenem-resistant K. pneumoniae (n = 13) and one E. coli isolate were also identified, all from community-acquired infections. Resistance patterns varied by infection site, with UTIs accounting for the majority of isolates. The high rates of ESBLs and co-resistance to ciprofloxacin among Enterobacterales highlight the urgent need for targeted AMR surveillance and site-specific empirical treatment strategies. Full article

Background: Clarithromycin is a commonly used macrolide antibiotic. Infection is a major source of mortality and morbidity in critical care units. Pharmacokinetics may vary during critical illness and suboptimal antimicrobial exposure has been shown to be associated with treatment failure. The pharmacokinetics of intravenous clarithromycin in critical illness have not previously been described. Methods: Pharmacokinetic, clinical and demographic data were collected from critically ill adults receiving intravenous clarithromycin. Drug concentrations were measured using high-performance liquid chromatography/mass spectrometry. Population pharmacokinetic analysis was performed using NONMEM version 7.5.1. Allometric weight scaling was added, and periods of renal replacement therapy were excluded a priori. Simulations of 10,000 patients were performed to assess pharmacokinetic–pharmacodynamic (PKPD) target attainment. Results: The analysis included 121 samples taken from 19 participants. A two-compartment model was found to provide the best fit. The addition of covariates did not improve model fit. There was no evidence of auto-inhibition in this population. Population parameter estimates of clearance and volume of distribution were lower than previously reported, with high interindividual variability. Simulations suggested reasonable pharmacokinetic–pharmacodynamic (PKPD) target attainment with current dosing regimens for most organisms that clarithromycin is used to treat with known clinical breakpoints. Conclusions: To our knowledge, this is the first study to describe the pharmacokinetics of intravenous clarithromycin in humans. Although our simulations suggest reasonable target attainment, further investigation into appropriate PKPD targets and clinical breakpoints for clarithromycin may enable dosing optimisation in this population. Full article

Background/Objectives: Pathogenic recessive GJB2 variants are the main genetic cause of non-syndromic sensorineural hearing loss. However, following GJB2 testing, a significant proportion of deaf patients are only found to be heterozygous carriers of pathogenic GJB2 alleles. Five large deletions not affecting GJB2 but encompassing a minimal common 62 kb region within the neighbouring CRYL1 gene have been described to cause loss of cis GJB2 expression and, as a result, produce hearing loss when in trans with pathogenic GJB2 variants. We describe the identification and characterization of a novel deletion of this type in deaf patients from northwestern Spain. Methods: We used panel NGS sequencing to detect the deletion, MLPA to validate it, whole-genome sequencing to map its breakpoints, PCR + Sanger sequencing to finely characterize it and triple-primer PCR to screen for it. Results: We identified a novel 200 kb deletion spanning the whole CRYL1 gene in two unrelated deaf patients from Asturias (in northwestern Spain) who were heterozygous for the pathogenic GJB2 c.35delG variant. Although the large deletion was absent from gnomAD v4.1.0 and 2052 local control alleles, screening for it in 20 additional deaf carriers of monoallelic pathogenic GJB2 variants detected it in another patient from Galicia (also in northwestern Spain). The novel deletion, termed del(200 kb)insATTATA, explained hearing loss in 3/43 (7%) deaf patients from our cohort that were otherwise heterozygous for pathogenic GJB2 variants. Conclusions: This work highlights the importance of comprehensively testing all genomic regions known to be clinically relevant for a given genetic condition, including thorough CRYL1 CNV screening for DFNB1A diagnostics. Full article

Background and Objectives: Secondary bacterial pneumonia can substantially worsen the clinical trajectory of patients hospitalized for Coronavirus Disease 2019 (COVID-19). This study aimed to characterize bacterial superinfections in COVID-19, including pathogen profiles, resistance patterns, inflammatory responses, severity scores, and ICU admission risk. Methods: In a retrospective cohort design, we reviewed 141 patients admitted to a single tertiary-care hospital between February 2021 and December 2024. A total of 58 patients had laboratory-confirmed bacterial superinfection by sputum, bronchoalveolar lavage, or blood cultures (superinfection group), whereas 83 had COVID-19 without any documented bacterial pathogens (COVID-only group). We collected detailed microbiological data from sputum, bronchoalveolar lavage (BAL), and blood cultures. Antibiotic sensitivity testing was performed using standard breakpoints for multidrug resistance (MDR). Inflammatory markers (C-reactive protein, procalcitonin, neutrophil-to-lymphocyte ratio, and systemic immune-inflammation index) and the severity indices Acute Physiology and Chronic Health Evaluation (APACHE) II, Confusion, Urea, Respiratory rate, Blood pressure (CURB), and National Early Warning Score (NEWS) were measured at admission. Primary outcomes included intensive care unit (ICU) admission, mechanical ventilation, and mortality. Results: Patients in the superinfection group showed significantly elevated inflammatory markers and severity scores compared to the COVID-only group (mean APACHE II of 17.2 vs. 13.8; p < 0.001). Pathogens most frequently isolated from sputum and BAL included Klebsiella pneumoniae (27.6%) and Pseudomonas aeruginosa (20.7%). Multidrug-resistant strains were documented in 32.8% of isolates. The superinfection group had higher ICU admissions (37.9% vs. 19.3%; p = 0.01) and more frequent mechanical ventilation (25.9% vs. 9.6%; p = 0.01). Mortality trended higher among superinfected patients (15.5% vs. 7.2%; p = 0.09). A total of 34% of the cohort had prior antibiotic use, which independently predicted MDR (aOR 2.6, p = 0.01). The presence of MDR pathogens such as Klebsiella pneumoniae (OR 2.8), Pseudomonas aeruginosa (OR 2.5), and Staphylococcus aureus (OR 2.1) significantly increases the risk of ICU admission. Conclusions: Bacterial superinfection exacerbates inflammation and worsens outcomes in COVID-19 patients, such as a higher risk of ICU admission. Full article

Accurate genetic diagnosis is essential for appropriate treatment in cystic fibrosis (CF). Large copy number variants like duplications in the CFTR gene are rare and often classified as variants of uncertain significance (VUSs) due to unknown characteristics of the inserted material, complicating diagnosis and treatment decisions. We identified a previously uncharacterized exon 22 duplication (CFTRdup22) in the CFTR gene in two anamnestically unrelated people with CF, both exhibiting a mild phenotype. Initial classification as a VUS was based on standard genetic testing. We employed a custom next-generation sequencing (NGS) panel to determine the exact breakpoints of the duplication and conducted mRNA sequencing to confirm its effect on splicing. DNA and RNA analyses allowed for precise breakpoint determination, confirming that the duplication was in tandem and the reading frame remained intact. This, as well as a residual CFTRdup22 function of ~30% as measured via intestinal current measurement, is consistent with a clinically milder CF phenotype. Collectively, the precise characterization of the variants’ breakpoints, localization and orientation enabled us to reclassify the variant as likely pathogenic. This study highlights the importance of advanced genetic techniques, such as NGS and breakpoint analysis, in accurately identifying CF-causing variants. It underscores the importance of a comprehensive approach and persistence when suspecting a specific genetic condition. This can aid in reclassifying VUSs, providing a definitive diagnosis for the affected family and enabling appropriate therapeutic interventions, including the use of CFTR modulators. Full article