Traditionally, cephalothin susceptibility results were used to predict the susceptibility of additional cephalosporins; however, in 2013-2014, the Clinical and Laboratory Standards Institute (CLSI) revisited this practice and determined that cefazolin is a more accurate proxy than cephalothin for uncomplicated urinary tract infections (uUTIs). Therefore, a cefazolin surrogacy breakpoint was established to predict the susceptibility of seven oral cephalosporins for Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis in the context of uUTIs. Clinical microbiology laboratories face several operational challenges when implementing the cefazolin surrogacy breakpoint, which may lead to confusion for the best path forward. Here, we review the historical context and data behind the surrogacy breakpoints, review PK/PD profiles for oral cephalosporins, discuss challenges in deploying the breakpoint, and highlight the limited clinical outcome data in this space.

BACKGROUND: Large deletions and duplications within the low-density lipoprotein receptor (LDLR) gene make up approximately 10% of LDLR pathogenic variants found in Czech patients with familial hypercholesterolemia. The goal of this study was to test the hypothesis that all probands with each rearrangement share identical breakpoints inherited from a common ancestor and to determine the role of Alu repetitive elements in the generation of these rearrangements.
METHODS: The breakpoint sequence was determined by PCR amplification and Sanger sequencing. To confirm the breakpoint position, an NGS analysis was performed. Haplotype analysis of common LDLR variants was performed using PCR and Sanger sequencing.
RESULTS: The breakpoints of 8 rearrangements within the LDLR gene were analysed, including the four most common LDLR rearrangements in the Czech population (number of probands ranging from 8 to 28), and four less common rearrangements (1-4 probands). Probands with a specific rearrangement shared identical breakpoint positions and haplotypes associated with the rearrangement, suggesting a shared origin from a common ancestor. All breakpoints except for one were located inside an Alu element. In 6 out of 8 breakpoints, there was high homology (≥ 70%) between the two Alu repeats in which the break occurred.
CONCLUSIONS: The most common rearrangements of the LDLR gene in the Czech population likely arose from one mutational event. Alu elements likely played a role in the generation of the majority of rearrangements inside the LDLR gene.

UNASSIGNED: Recombination serves as a common strategy employed by RNA viruses for their genetic evolution. Extensive genomic surveillance during the COVID-19 pandemic has reported SARS-CoV-2 Recombinant strains indicating recombination events during the viral evolution. This study introspects the phenomenon of genome recombination by tracing the footprint of prominent lineages of SARS-CoV-2 at different time points in the context of on-going evolution and emergence of Recombinants.
UNASSIGNED: Whole genome sequencing was carried out for 2,516 SARS-CoV-2 (discovery cohort) and 1,126 (validation cohort) using nasopharyngeal samples collected between the time period of March 2020 to August 2022, as part of the genomic surveillance program. The sequences were classified according to the different lineages of SARS-CoV-2 prevailing in India at respective time points.
UNASSIGNED: Mutational diversity and abundance evaluation across the 12 lineages identified 58 Recombinant sequences as harboring the least number of mutations (n = 111), with 14 low-frequency unique mutations with major chunk of mutations coming from the BA.2. The spontaneously/dynamically increasing and decreasing trends of mutations highlight the loss of mutations in the Recombinants that were associated with the SARS-CoV-2 replication efficiency, infectivity, and disease severity, rendering them functionally with low infectivity and pathogenicity. Linkage disequilibrium (LD) analysis revealed that mutations comprising the LD blocks of BA.1, BA.2, and Recombinants were found as minor alleles or as low-frequency alleles in the LD blocks from the previous SARS-CoV-2 variant samples, especially Pre-VOC. Moreover, a dissipation in the size of LD blocks as well as LD decay along with a high negative regression coefficient (R squared) value was demonstrated in the Omicron and BA.1 and BA.2 lineages, which corroborated with the breakpoint analysis.
UNASSIGNED: Together, the findings help to understand the evolution and emergence of Recombinants after the Omicron lineages, for sustenance and adaptability, to maintain the epidemic spread of SARS-CoV-2 in the host population already high in immunity levels.

The rapid emergence of multi-drug resistant Gram-negative pathogens has driven the introduction of novel β-lactam combination agents (BLCs) to the antibiotic market: ceftolozane-tazobactam, ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, cefiderocol, and sulbactam-durlobactam. These agents are equipped with innovative mechanisms that confer broad Gram-negative activity, notably against certain challenging carbapenemases. While their introduction offers a beacon of hope, clinical microbiology laboratories must navigate the complexities of susceptibility testing for these agents due to their diverse activity profiles against specific β-lactamases and the possibility of acquired resistance mechanisms in some bacterial isolates. This review explores the complexities of these novel antimicrobial agents detailing the intricacies of their application, providing guidance on the nuances of susceptibility testing, interpretation, and result reporting in clinical microbiology laboratories.

Biallelic loss-of-function variants in WWOX cause WWOX-related epileptic encephalopathy (WOREE syndrome), which has been reported in 60 affected individuals to date. In this study, we report on an affected individual with WOREE syndrome who presented with early-onset refractory seizures and global neurodevelopmental delay and died at the age of two and a half years.
We present clinical and molecular findings in the affected individual, including biallelic pathogenic variants in the WWOX gene. We employed different molecular approaches, such as whole exome sequencing, quantitative real-time polymerase chain reaction (qPCR), and whole-genome sequencing, to identify the genetic variants. The breakpoints were determined through gap PCR and Sanger sequencing.
Whole exome sequencing revealed homozygous exon 6 deletion in the WWOX gene in the proband. Quantitative real-time PCR confirmed that the parents were heterozygous carriers of exon 6 deletion. However, using whole-genome sequencing, we identified three larger deletions (maternal allele with exon 6-8 deletion and paternal allele with two deletions in proximity one in intron 5 and the other in exon 6) involving the WWOX gene in the proband, with deletion sizes of 13,261 bp, 53,904 bp, and 177,200 bp. The exact breakpoints were confirmed through gap PCR and Sanger sequencing. We found that the proband inherited the discontinuous deletion of intron 5 and exon 6 from the father, and the exons 6-8 deletion from the mother using gap PCR.
Our findings extend the variant spectrum of WOREE syndrome and support the critical role of the WWOX gene in neural development.

BACKGROUND: Using the pencil beam raster scanning method employed at most carbon beam treatment facilities, spots can be moved without interrupting the beam, allowing for the delivery of a dose between spots (move dose). This technique is also known as Dose-Driven-Continuous-Scanning (DDCS). To minimize its impact on HIMAK patient dosimetry, there\'s an upper limit to the move dose. Spots within a layer are grouped into sets, or \"break points,\" allowing continuous irradiation. The beam is turned off when transitioning between sets or at the end of a treatment layer or spill. The control system beam-off is accomplished by turning off the RF Knockout (RFKO) extraction and after a brief delay the High Speed Steering Magnet (HSST) redirects the beam transport away from isocenter to a beam dump.
OBJECTIVE: The influence of the move dose and beam on/off control on the dose distribution and irradiation time was evaluated by measurements never before reported and modelled for Hitachi Carbon DDCS.
METHODS: We conducted fixed-point and scanning irradiation experiments at three different energies, both with and without breakpoints. For fixed-point irradiation, we utilized a 2D array detector and an oscilloscope to measure beam intensity over time. The oscilloscope data enabled us to confirm beam-off and beam-on timing due to breakpoints, as well as the relative timing of the RFKO signal, HSST signal, and dose monitor (DM) signals. From these measurements, we analyzed and modelled the temporal characteristics of the beam intensity. We also developed a model for the spot shape and amplitude at isocenter occurring after the beam-off signal which we called flap dose and its dependence on beam intensity. In the case of scanning irradiation, we measured move doses using the 2D array detector and compared these measurements with our model.
RESULTS: We observed that the most dominant time variation of the beam intensity was at 1 kHz and its harmonic frequencies. Our findings revealed that the derived beam intensity cannot reach the preset beam intensity when each spot belongs to different breakpoints. The beam-off time due to breakpoints was approximately 100 ms, while the beam rise time and fall time (tdecay ) were remarkably fast, about 10 ms and 0.2 ms, respectively. Moreover, we measured the time lag (tdelay ) of approximately 0.2 ms between the RFKO and HSST signals. Since tdelay ≈ tdecay at HIMAK then the HSST is activated after the residual beam intensity, resulting in essentially zero flap dose at isocenter from the HSST. Our measurements of the move dose demonstrated excellent agreement with the modelled move dose.
CONCLUSIONS: We conducted the first move dose measurement for a Hitachi Carbon synchrotron, and our findings, considering beam on/off control details, indicate that Hitachi\'s carbon synchrotron provides a stable beam at HIMAK. Our work suggests that measuring both move dose and flap dose should be part of the commissioning process and possibly using our model in the Treatment Planning System (TPS) for new facilities with treatment delivery control systems with higher beam intensities and faster beam-off control.

Accurate antimicrobial susceptibility testing (AST) and reporting are essential for guiding appropriate therapy for patients and direction for public health prevention and control actions. A critical feature of AST reporting is the interpretation of AST results using clinical breakpoints for reporting as susceptible, susceptible-dose dependent, intermediate, or resistant. Breakpoints are subject to continuous adjustment and updating to best reflect current clinical data. These breakpoint changes can benefit patients and public health only if adopted in a timely manner. A recent survey identified that up to 70% of College of American Pathologists (CAP)-accredited U.S. laboratories and 45% of CAP-accredited laboratories outside the U.S. use various obsolete clinical breakpoints to interpret AST results to guide patient care. The reason for the ongoing use of obsolete breakpoints is multifactorial, including barriers encountered by laboratories, commercial AST device manufacturers, standards development organizations, and regulatory bodies alike. To begin to address this important patient safety issue, CAP implemented checklist requirements for CAP-accredited laboratories to ensure up-to-date clinical breakpoint use. Furthermore, the topic was discussed at the June 2022 American Society for Microbiology Clinical Microbiology Open (CMO) with various stakeholders to identify potential solutions. This minireview summarizes the breakpoint setting process in the U.S. and highlights solutions to close the gap between breakpoint revisions and implementation in clinical and public health laboratories. Solutions discussed include clarification of data requirements and minimum inhibitory concentration only reporting for regulatory clearance of AST devices, clinical data generation to close breakpoints gaps, advocacy, education, and greater dialogue between stakeholders.

UNASSIGNED: Existing breakpoint guidelines are not optimal for interpreting antimicrobial resistance (AMR) data from animal studies and low-income countries, and therefore their utility for analysing such data is limited. There is a need to integrate diverse data sets, such as those from low-income populations and animals, to improve data interpretation.
UNASSIGNED: There is very limited research on the relative merits of clinical breakpoints, epidemiological cut-offs (ECOFFs) and normalized resistance interpretation (NRI) breakpoints in interpreting microbiological data, particularly in animal studies and studies from low-income countries.
UNASSIGNED: The aim of this study was to compare antimicrobial resistance in Escherichia coli isolates using ECOFFs, CLSI and NRI breakpoints.
UNASSIGNED: A total of 59 non-repetitive poultry isolates were selected for investigation based on lactose fermentation on MacConkey agar and subsequent identification and confirmation as E. coli using chromogenic agar and uidA PCR. Kirby Bauer disc diffusion was used for susceptibility testing. For each antimicrobial agent, inhibition zone diameters were measured, and ECOFFs, CLSI and NRI bespoke breakpoints were used for resistance interpretation.
UNASSIGNED: According to the interpretation of all breakpoints except ECOFFs, tetracycline resistance was significantly higher (TET) (67.8 -69.5 %), than those for ciprofloxacin (CIPRO) (18.6 -32.2 %), imipenem (IMI) (3.4 -35 %) and ceftazidime (CEF) (1.7 -45.8 %). Prevalence estimates of AMR using CLSI and NRI bespoke breakpoints did not differ for CEF (1.7 % CB and 1.7 % COWT), IMI (3.4 % CB and 4.0 % COWT) and TET (67.8 % CB and 69.5 % COWT). However, with ECOFFs, AMR estimates for CEF, IMI and CIP were significantly higher (45.8, 35.6 and 64.4 %, respectively; P<0.05). Across all the three breakpoints, resistance to ciprofloxacin varied significantly (32.2 % CB, 64.4 % ECOFFs and 18.6 % COWT, P<0.05).
UNASSIGNED: AMR interpretation is influenced by the breakpoint used, necessitating further standardization, especially for microbiological breakpoints, in order to harmonize outputs. The AMR ECOFF estimates in the present study were significantly higher compared to CLSI and NRI.

Chromosomal structural variations (SVs) are a main cause of human genetic disease. Currently, karyotype, chromosomal microarray analysis (CMA), and fluorescent in situ hybridization (FISH) form the backbone of current routine diagnostics (CRD). These methods have their own limitations. CRD cannot identify cryptic balanced SVs and complex SVs even if these techniques were performed either simultaneously or in a sequential manner. Optical genome mapping (OGM) is a novel technology that can identify several classes of SVs with higher resolution, but studies on the applicability of OGM and its comparison with CRD are inadequate for difficult and complicated chromosomal SVs are lacking. Herein, seven patients with definite complicated SVs involving at least two breakpoints (BPs) were recruited for this study. The results of BPs and SVs from OGM were compared with those from CRD. The results showed that all BPs of five samples and partial BPs of two samples were detected by OGM. The undetected BPs were all close to the repeat-rich gap region. Besides, OGM also detected additional SVs including a cryptic balanced translocation, two additional complex chromosomal rearrangement (CCR). OGM yielded the additional information, such as the orientation of acentric fragments, BP positions, and genes mapped in the BP region for all the cases. The accuracy of additional SVs and BPs detected by OGM was verified by FISH panel and next-generation sequencing and Sanger sequencing. Taken together, OGM exhibit a better performance in detecting chromosomal SVs compared to the CRD. We suggested that OGM method should be utilized in the clinical examination to improve the efficiency and accuracy of genetic disease diagnosis, supplemented by FISH or karyotyping to compensate for the SVs in the repeat-rich gap region if necessary.

Antimicrobial susceptibility testing (AST) is an important component of antimicrobial stewardship. This article discusses how AST methods and breakpoints are developed, describes when AST may or may not be useful in clinical practice, and discusses how to interpret AST results from bacterial isolates from cattle, sheep, and goats. Discussion of when AST is not appropriate or when veterinarians should have low confidence in AST results is also included. Applicability of genomic testing for antimicrobial susceptibility is briefly addressed.