High Concordance Research Articles

Comparative analysis of BCR::ABL1 p210 mRNA transcript quantification and ratio to ABL1 control gene converted to the International Scale by chip digital PCR and droplet digital PCR for monitoring patients with chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, leading to the BCR::ABL1 fusion gene and hyper-proliferation of granulocytes. Tyrosine kinase inhibitors (TKIs) are effective, and minimal residual disease (MRD) monitoring is crucial. Digital PCR platforms offer increased precision compared to quantitative PCR but lack comparative studies. Eighty CML patient samples were analyzed in parallel using digital droplet PCR (ddPCR) (QXDx™ BCR-ABL %IS Kit) and chip digital PCR (cdPCR) (Dr. PCR™ BCR-ABL1 Major IS Detection Kit). Overall, qualitative and quantitative agreement was good. Sensitivity analysis showed positive percentage agreement and negative percentage agreement were both≥90 %, and the quadratic weighted kappa index for molecular response (MR) level categorization was 0.94 (95 %CI 0.89, 0.98). MR levels subgroup analysis showed perfect categorical agreement on MR level at MR3 or above, while 35.4 % (17/48) of patient samples with MR4 or below showed discordant categorizations. Overall, Lin's concordance correlation coefficient (CCC) for the ratio of %BCR::ABL1/ABL1 converted to the International Scale (BCR::ABL1 IS) was almost perfect quantitative agreement (Lin's CCC=0.99). By subgroups of MR levels, Lin's CCC showed a quantitative agreement of BCR::ABL1 IS decreased as MR deepened. Both cdPCR and ddPCR demonstrated comparable performance in detecting BCR::ABL1 transcripts with high concordance in MR3 level or above. Choosing between platforms may depend on cost, workflow, and sensitivity requirements.

Development of a triplex RT-qPCR assay for simultaneous quantification of Japanese encephalitis, Murray Valley encephalitis, and West Nile viruses for environmental surveillance.

The co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) has impacted human and animal health in multiple countries worldwide. To facilitate early warnings and surveillance of the presence of these viral infectious agents in the environment, a triplex reverse transcription-quantitative PCR (RT-qPCR) was developed for simultaneous quantification of JEV, MVEV, and WNV in potential hotspots such as piggery and urban wastewater and environmental water samples. The performance of the developed triplex RT-qPCR assay was compared with that of simplex counterparts, all using the same primer and probe sequences. The quantifiable results showed a concordance rate of 93.9%-100% (Cohen's kappa) between the triplex and simplex assays. The mean concentrations of exogenous JEV, MVEV, and WNV using the triplex and simplex RT-qPCR assays were remarkably similar in piggery/urban wastewater and environmental water samples. However, the impacts of the matrix effects (i.e., sample composition and PCR inhibition) of environmental water samples on the accurate quantification of these viruses need to be considered. Taken together, this newly developed triplex RT-qPCR assay of JEV, MVEV, and WNV will allow for a more rapid and cost-efficient sample analysis and data interpretation. The application of the triplex assay for environmental surveillance may be a valuable tool to complement the existing disease and mosquito surveillance approaches used to safeguard the health of both humans and animals.IMPORTANCEThe co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) poses significant threats to human and animal health globally. In this study, a triplex RT-qPCR assay was developed for simultaneous quantification of these viruses in wastewater and environmental water samples. Results demonstrated high concordance and sensitivity of the newly developed triplex RT-qPCR assay compared to simplex assays, indicating its efficacy for environmental surveillance. This cost-effective and rapid assay offers a vital tool for timely monitoring of mosquito-borne viruses in environmental samples, enhancing our ability to mitigate potential outbreaks and safeguard public health.

Ultra-Widefield and Early Treatment Diabetic Retinopathy Study 7-Field Grading of Diabetic Retinopathy

High concordance in diabetic retinopathy (DR) outcomes between 7-field (7F) and ultra-widefield (UWF) images would allow for combining longitudinal assessments based on the 2 modalities both in clinical studies and clinical care. To compare 7F and UWF imaging with regard to DR severity and the associations of DR severity with risk factors, such as hemoglobin A1c, age, diabetes duration, and sex. This cross-sectional study describes the outcomes of the randomized clinical Diabetes Control and Complications Trial (DCCT) and its subsequent observational study, the Epidemiology of Diabetes Interventions and Complications (EDIC) study. Of the 1441 participants with type 1 diabetes in the DCCT, 1375 were enrolled in the EDIC study. Of the 1171 participants who were active between March 2019 and December 2021, 200° UWF color imaging and 7F fundus photographs were obtained for 785 participants once at the same visit. Central graders assessed 7F-UWF with a 7F template masking the retinal periphery and the full UWF image (UWF-global). Data were analyzed from January 2022 to March 2023. Hemoglobin A1c was assessed quarterly during the DCCT and annually during the EDIC study using high-performance liquid chromatography. Retinopathy was determined independently for all imaging as mild, moderate, or severe nonproliferative DR (SNPDR) using the Early Treatment Diabetic Retinopathy Study (ETDRS) grading scale for the 7F images and the global ETDRS grading scale for the UWF images. Panretinal and focal photocoagulation were self-reported or based on scarring location and pattern observed during grading. Proliferative DR (PDR) was defined by observed neovascularization or evidence of panretinal photocoagulation. Among the 785 participants included in this study, 420 (53%) were male and 365 (47%) were female. The mean (SD) age was 61 (7) years. DR grading between UWF-7F and 7F imaging was correlated for all outcomes, including for severe outcomes, such as SNPDR (κ, 0.73; concordance, 96%), PDR (κ, 0.74; concordance, 97%), scatter photocoagulation (κ, 0.97; concordance, 99%), and focal photocoagulation (κ, 0.71; concordance, 98%). Most DR severity scores were within 1 step (1410 of 1529 [92%]), and 3% (51 of 1529) were more than 2 steps apart (κ, 0.45; 95% CI, 0.42-0.49; weighted κ, 0.63; 95% CI, 0.60-0.67) on the ETDRS severity scale. DR severity assessed within the UWF-global area was higher compared to 7F (median [IQR] UWF-global score, 3 [2-3] vs median 7F level score, 2.0 [1-3]; P < .001), although the 2 modalities were correlated (1225 of 1508 [81%] 1-step agreement; weighted κ, 0.41). Standard ETDRS 7F and UWF evaluations of DR were comparable for ETDRS severity levels as previously reported by Diabetic Retinopathy Clinical Research Retina Network reports. In addition, these evaluations of DR were comparable for DCCT/EDIT study outcomes and major study conclusions, suggesting that use of UWF imaging is not likely to introduce relevant measurement biases in future longitudinal studies. ClinicalTrials.gov Identifiers: NCT00360815.

Clonal background and routes of plasmid transmission underlie antimicrobial resistance features of bloodstream Klebsiella pneumoniae

Bloodstream infections caused by the opportunistic pathogen Klebsiella pneumoniae are associated with adverse health complications and high mortality rates. Antimicrobial resistance (AMR) limits available treatment options, thus exacerbating its public health and clinical burden. Here, we aim to elucidate the population structure of K. pneumoniae in bloodstream infections from a single medical center and the drivers that facilitate the dissemination of AMR. Analysis of 136 short-read genome sequences complemented with 12 long-read sequences shows the population consisting of 94 sequence types (STs) and 99 clonal groups, including globally distributed multidrug resistant and hypervirulent clones. In vitro antimicrobial susceptibility testing and in silico identification of AMR determinants reveal high concordance (90.44–100%) for aminoglycosides, beta-lactams, carbapenems, cephalosporins, quinolones, and sulfonamides. IncF plasmids mediate the clonal (within the same lineage) and horizontal (between lineages) transmission of the extended-spectrum beta-lactamase gene blaCTX-M-15. Nearly identical plasmids are recovered from isolates over a span of two years indicating long-term persistence. The genetic determinants for hypervirulence are carried on plasmids exhibiting genomic rearrangement, loss, and/or truncation. Our findings highlight the importance of considering both the genetic background of host strains and the routes of plasmid transmission in understanding the spread of AMR in bloodstream infections.

An atlas of genetic effects on the monocyte methylome across European and African populations.

Elucidating the genetic architecture of DNA methylation (DNAm) is crucial for decoding the etiology of complex diseases. However, current epigenomic studies often suffer from incomplete coverage of methylation sites and the use of tissues containing heterogeneous cell populations. To address these challenges, we present a comprehensive human methylome atlas based on deep whole-genome bisulfite sequencing (WGBS) and whole-genome sequencing (WGS) of purified monocytes from 298 European Americans (EA) and 160 African Americans (AA) in the Louisiana Osteoporosis Study. Our atlas enables the analysis of over 25 million DNAm sites. We identified 1,383,250 and 1,721,167 methylation quantitative trait loci (meQTLs) in cis -regions for EA and AA populations, respectively, with 880,108 sites shared between ancestries. While cis -meQTLs exhibited population-specific patterns, primarily due to differences in minor allele frequencies, shared cis -meQTLs showed high concordance across ancestries. Notably, cis -heritability estimates revealed significantly higher mean values in the AA population (0.09) compared to the EA population (0.04). Furthermore, we developed population-specific DNAm imputation models using Elastic Net, enabling methylome-wide association studies (MWAS) for 1,976,046 and 2,657,581 methylation sites in EA and AA, respectively. The performance of our MWAS models was validated through a systematic multi-ancestry analysis of 41 complex traits from the Million Veteran Program. Our findings bridge the gap between genomics and the monocyte methylome, uncovering novel methylation-phenotype associations and their transferability across diverse ancestries. The identified meQTLs, MWAS models, and data resources are freely available at www.gcbhub.org and https://osf.io/gct57/ .

Osteosarcoma patient-derived orthotopic xenograft (PDOX) models for identification of novel and effective therapeutics.

228 Background: Osteosarcoma is the most common malignant primary tumor of bone and mainly occurs in young generations. Due to the heterogeneity, rarity, poor response rate to systemic therapy, and metastatic potential of osteosarcoma, individualized precision medicine and novel drug discovery are greatly needed. Toward this goal, we have established the patient-derived orthotopic xenograft (PDOX) mouse model with surgical orthotopic implantation for all major cancers. The PDOX models recapitulate human tumors better than subcutaneous-transplanted xenografts including patient-derived xenograft (PDX). Metastasis is observed to a greater extent in PDOX models due to the intact histology and correct-organ tumor micro-environment of the orthotopically implanted tissue. Therefore, the PDOX model is highly predictive of drug efficacy and useful for identifying effective drugs for tumors in which new drugs are difficult to develop. Methods: We have reported 18 osteosarcoma PDOX studies evaluating approved and experimental drugs since 2017. The present report reviews our research group’s experience with the osteosarcoma-PDOX model, and the power of the PDOX models to identify effective therapeutics. Results: Effective treatment for drug-resistant osteosarcoma includes regorafenib, as monotherapy, and temozolomide-irinotecan, trabectedin-irinotecan, sorafenib-everolimus, sorafenib-palbociclib, and olaratumab-doxorubicin-cisplatin, as combinations. Experimental therapy with recombinant methioninase, tumor-targeting S. typhimurium A1-R, or combinations of these agents and other drugs have shown surprising efficacy in the recalcitrant-osteosarcoma PDOX models. Conclusions: Owing to the high concordance of drug efficacy between patients and their corresponding PDOX models, these models provide improved and personalized treatment options for patients with osteosarcoma. The patient does not need to suffer from the potential drug toxicity and morbidity of ineffective chemotherapies. In an era of growing promise of new treatment and precision medicine, PDOX models can offer a unique opportunity to provide specific and individualized therapy and novel therapeutic options for osteosarcoma patients.

Binary classification of copy number alteration profiles in liquid biopsy with potential clinical impact in advanced NSCLC

Liquid biopsy has recently emerged as an important tool in clinical practice particularly for lung cancer patients. We retrospectively evaluated cell-free DNA analyses performed at our Institution by next generation sequencing methodology detecting the major classes of genetic alterations. Starting from the graphical representation of chromosomal alterations provided by the analysis software, we developed a support vector machine classifier to automatically classify chromosomal profiles as stable (SCP) or unstable (UCP). High concordance was found between our binary classification and tumor fraction evaluation performed using shallow whole genome sequencing. Among clinical features, UCP patients were more likely to have ≥ 3 metastatic sites and liver metastases. Longitudinal assessment of chromosomal profiles in 33 patients with lung cancer receiving immune checkpoint inhibitors (ICIs) showed that only patients that experienced early death or hyperprogressive disease retained or acquired an UCP within 3 weeks from the beginning of ICIs. UCP was not observed following ICIs among patients that experienced progressive disease or clinical benefit. In conclusion, our binary classification, applied to whole copy number alteration profiles, could be useful for clinical risk stratification during systemic treatment for non-small cell lung cancer patients.

Anti-phosphohistone H3 (PHH3) as a proliferation marker to assess mitotic activity and to grade neuroendocrine neoplasms of hepatopancreaticobiliary (HPB) system.

The world health organization (WHO) classification of neuroendocrine neoplasms (NENs, i.e. neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs)) of the gastrointestinal system involves grading of these tumors by mitotic count (i.e. H and E mitotic index or Haematoxylin and Eosin mitotic index [HE-MI] and Mindbomb E3 ubiquitin protein ligase 1 labelling index (MIB1-LI) into Grade 1 (G1), Grade 2 (G2), or Grade 3 (G3). However, the assessment of HE-MI and MIB1-LI is hindered by several factors that contribute to discordance between these two grading methods. Clinical data demonstrate the dependency of prognosis on grade. The objective of this study was to compare the grading of NENs of the hepatopancreatobiliary (HPB) system using Anti-phosphohistone H3 mitotic index (i.e. PHH3-MI), HE-MI and MIB1-LI. In a cohort of 140 NENs selected from January 2011 to August 2019, the concordance and correlation between HE-MI, MIB1-LI and PHH3-MI grading methods were analysed using Cohen's weighted kappa (κ) statistics and Spearman's correlation (ρ), respectively. Receiver operating characteristic (ROC) curve and cut-off analyses were done to determine optimal PHH3-MI cut-off values to grade NENs. The rates of discordance between HE-MI vs. MIB1-LI, PHH3-MI vs. MIB1-LI and PHH3-MI vs. HE-MI were 52% (κ =0.416), 29% (κ =0.64) and 41% (κ =0.508), respectively. There was a significant correlation between the grading methods. PHH3-MI had good overall sensitivity and specificity at cut-offs 2 and 17 in distinguishing between G1 vs. G2, and G2 vs. G3 tumors, respectively. PHH3 immunolabeling allowed for quick and easy identification of mitotic figures (MF). It had the highest concordance with MIB1-LI. At cut-off values of 2 and 17, there was good overall sensitivity and specificity. The interobserver agreement was excellent.

FAST (fast analytical simulator of tracer)-PET: an accurate and efficient PET analytical simulation tool

Objective. Simulation of positron emission tomography (PET) images is an essential tool in the development and validation of quantitative imaging workflows and advanced image processing pipelines. Existing Monte Carlo or analytical PET simulators often compromise on either efficiency or accuracy. We aim to develop and validate fast analytical simulator of tracer (FAST)-PET, a novel analytical framework, to simulate PET images accurately and efficiently. Approach. FAST-PET simulates PET images by performing precise forward projection, scatter, and random estimation that match the scanner geometry and statistics. Although the same process should be applicable to other scanner models, we focus on the Siemens Biograph Vision-600 in this work. Calibration and validation of FAST-PET were performed through comparison with an experimental scan of a National Electrical Manufacturers Association (NEMA) Image Quality (IQ) phantom. Further validation was conducted between FAST-PET and Geant4 Application for Tomographic Emission (GATE) quantitatively in clinical image simulations in terms of intensity-based and texture-based features and task-based tumor segmentation. Main results. According to the NEMA IQ phantom simulation, FAST-PET’s simulated images exhibited partial volume effects and noise levels comparable to experimental images, with a relative bias of the recovery coefficient RC within 10% for all spheres and a coefficient of variation for the background region within 6% across various acquisition times. FAST-PET generated clinical PET images exhibit high quantitative accuracy and texture comparable to GATE (correlation coefficients of all features over 0.95) but with ∼100-fold lower computation time. The tumor segmentation masks comparison between both methods exhibited significant overlap and shape similarity with high concordance CCC > 0.97 across measures. Significance. FAST-PET generated PET images with high quantitative accuracy comparable to GATE, making it ideal for applications requiring extensive PET image simulations such as virtual imaging trials, and the development and validation of image processing pipelines.

Comparative assessment of MScanFit MUNE and quantitative EMG in amyotrophic lateral sclerosis diagnosis: A prospective study

ObjectiveMotor Unit Number Estimation (MUNE) techniques are crucial in assessing lower motor neuron loss. MScanFit MUNE (MScanFit) is a novel tool which estimates MUNE values from compound muscle action potential (CMAP) scans by considering the probabilistic nature of motor unit firing. We conducted a prospective study to evaluate the diagnostic utility of MScanFit compared to quantitative electromyography (qEMG) in ALS patients. MethodsWe enrolled 35 patients diagnosed with amyotrophic lateral sclerosis (ALS) and 14 healthy controls, assessing qEMG and MScanFit MUNE in abductor pollicis brevis, abductor digiti minimi and tibialis anterior muscles. ResultsWe found higher sensitivity of qEMG in detecting abnormalities compared to MScanFit, with a high concordance rate between the two techniques. Notably, a few muscles exhibited abnormal MUNE but normal qEMG findings, suggesting a potential complementary role for MScanFit in ALS diagnosis. Neurophysiological parameters from MScanFit showed good correlations with qEMG measures. Subclinical neurophysiological involvement was observed in muscles with normal strength, emphasizing the importance of sensitive diagnostic tools. ConclusionMScanFit demonstrated validity in distinguishing ALS patients from healthy subjects and correlated well with qEMG parameters. SignificanceOur study confirmed the diagnostic utility of MScanFit MUNE in ALS, highlighting its role as a supplementary diagnostic tool.

At-home specimen self-collection as an additional testing strategy for chlamydia and gonorrhoea: a systematic literature review and meta-analysis

IntroductionChlamydia trachomatis (Ct) and Neisseria gonorrhoeae (Ng) infections are often asymptomatic; screening increases early detection and prevents disease, sequelae and further spread. To increase Ct and Ng testing, several countries...

Comprehensive genomic profiling by liquid biopsy captures tumor heterogeneity and identifies cancer vulnerabilities in patients with RAS/BRAFV600E wild-type metastatic colorectal cancer in the CAPRI 2-GOIM trial

Emerging evidence supports tumor tissue-based comprehensive genomic profiling (CGP) in metastatic colorectal cancer (mCRC). Data on liquid biopsy-based circulating tumor DNA (ctDNA) CGP are scarce and mainly retrospective. Prospective comparison between the two tests is not currently available. The CAPRI 2-GOIM trial investigates efficacy and safety of ctDNA-driven, cetuximab-based, sequence of three treatment lines in patients with RAS/BRAFV600E wild type (WT) mCRC, as determined by local laboratory. Before first-line therapy, CGP is performed with FoundationOne (F1) CDx and F1 Liquid (F1L) CDx (324 genes) on tumor tissue DNA and plasma ctDNA, respectively. For 2/207 (0.96%) patients, no ctDNA was detected by F1L CDx. No patient displayed tumor fraction (TF) below 1%, whereas elevated ctDNA (TF≥10%) was detected among 140/205 (68.3%) patients. 1013 genomic variants were identified. F1L CDx found KRAS, NRAS or BRAFV600E alterations in 19 patients, whose tumors were classified as RAS/BRAFV600E WT by local laboratory. Both F1 CDx and F1L CDx were available for 164/205 (80%) patients. Concordance of 61.4% between the two tests was observed. Concordance increased to 72.7% for F1L CDx with TF ≥10%. Concordance for genes potentially involved in anti-epidermal growth factor receptor (EGFR) resistance was found in 137/164 (83%) patients, increasing to 91.5% for F1L CDx with TF ≥10%. A higher number of genomic alterations was detected by F1L CDx compared with F1 CDx, including 6 cases with KRAS and NRAS alterations. Overall, 109/205 (53.2%) patients displayed at least one actionable genomic alteration (I to IIIB), according to the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT). Baseline liquid biopsy-based CGP is feasible, it has high concordance with tumor tissue-based CGP, it could better recapitulate tumor heterogeneity, and it is clinically informative by identifying additional actionable genomic alterations in approximately half of RAS/BRAFV600E WT mCRC patients.

Integrating single-cell transcriptomics and machine learning to predict breast cancer prognosis: A study based on natural killer cell-related genes.

Breast cancer (BC) is the most commonly diagnosed cancer in women globally. Natural killer (NK) cells play a vital role in tumour immunosurveillance. This study aimed to establish a prognostic model using NK cell-related genes (NKRGs) by integrating single-cell transcriptomic data with machine learning. We identified 44 significantly expressed NKRGs involved in cytokine and T cell-related functions. Using 101 machine learning algorithms, the Lasso + RSF model showed the highest predictive accuracy with nine key NKRGs. We explored cell-to-cell communication using CellChat, assessed immune-related pathways and tumour microenvironment with gene set variation analysis and ssGSEA, and observed immune components by HE staining. Additionally, drug activity predictions identified potential therapies, and gene expression validation through immunohistochemistry and RNA-seq confirmed the clinical applicability of NKRGs. The nomogram showed high concordance between predicted and actual survival, linking higher tumour purity and risk scores to a reduced immune score. This NKRG-based model offers a novel approach for risk assessment and personalized treatment in BC, enhancing the potential of precision medicine.

Performance Characteristics of Next-Generation Sequencing–Based Engraftment Monitoring and Microchimerism Detection in Allogeneic Hematopoietic Cell Transplantation: A Practical Approach for Clinical Assay Validation

Chimerism analysis by next-generation sequencing (NGS) is an emerging method for engraftment monitoring after allogeneic hematopoietic cell transplantation. A high-sensitivity method is required for the detection of microchimerism (<1% chimerism), which may have clinical utility in early relapse detection, allograft monitoring in organ transplantation, and other allogeneic cellular therapies (such as microtransplantations). As more clinical laboratories adopt this method, a thorough assessment of performance is needed. This study evaluated one such NGS-based assay that uses both single-nucleotide polymorphisms and insertions/deletions as genetic markers. An assessment of accuracy, linearity, sensitivity, and reproducibility was performed. Analytical sensitivity was 0.2% donor for single donor and 0.5% donors for double donors. The assay showed a high degree of reproducibility over a full range of chimerism. Comparison to short-tandem-repeat (STR) PCR showed high concordance; yet <5% chimerism was consistently detected by NGS, but not by STR-PCR. Comparison to real-time quantitative PCR showedhigh concordance, but with lower correlation in the midrange (40% to 60% chimerism). Overall, the assay showed consistent performance with high sensitivity and accuracy compared with STR-PCR and real-time quantitative PCR across a full range of chimerism in the setting of single-donor and multidonor transplantations. In addition, criteria for quality metrics were established for sequencing performance and data analysis and considerations made for clinical laboratory validation of NGS-based chimerism assay andanalysis software.

A Droplet Digital Polymerase Chain Reaction-Based Tool to Aid in Melanoma Diagnosis.

Detecting copy number variations (CNVs) at certain loci can aid in the diagnosis of histologically ambiguous melanocytic neoplasms. Droplet digital polymerase chain reaction (ddPCR) is a rapid, automated, and inexpensive method for CNV detection in other cancers, but not yet melanoma. To evaluate the performance of a 4-gene ddPCR panel that simultaneously tests for ras responsive binding element protein 1 (RREB1) gain; cyclin-dependent kinase inhibitor 2A (CDKN2A) loss; MYC proto-oncogene, bHLH transcription factor (MYC) gain; and MYB proto-oncogene, transcription factor (MYB) loss in melanocytic neoplasms. One hundred sixty-four formalin-fixed, paraffin-embedded skin samples were used to develop the assay, of which 65 were used to evaluate its performance. Chromosomal microarray analysis (CMA) data were used as the gold standard. ddPCR demonstrated high concordance with CMA in detecting RREB1 gain (sensitivity, 86.7%; specificity, 88.9%), CDKN2A loss (sensitivity, 80%; specificity, 100%), MYC gain (sensitivity, 70%; specificity, 100%), and MYB loss (sensitivity, 71.4%; specificity, 100%). When one CNV was required to designate the test as positive, the 4-gene ddPCR panel distinguished nevi from melanomas with a sensitivity of 78.4% and a specificity of 71.4%. For reference, CMA had a sensitivity of 86.2% and a specificity of 78.6%. Our data also revealed interesting relationships with histology, namely (1) a positive correlation between RREB1 ddPCR copy number and degree of tumor progression; (2) a statistically significant correlation between MYC gain and nodular growth; and (3) a statistically significant correlation between MYB loss and a sheetlike pattern of growth. With further validation, ddPCR may aid both in our understanding of melanomagenesis and in the diagnosis of challenging melanocytic neoplasms.

Insights into mycobacteriome composition in Mycobacterium bovis-infected African buffalo (Syncerus caffer) tissue samples

Animal tuberculosis significantly challenges global health, agriculture, and wildlife conservation efforts. Mycobacterial cultures are resource-intensive, time-consuming, and challenged by heterogeneous populations. In this study, we employed a culture-independent approach, using targeted long-read-based next-generation sequencing (tNGS), to investigate the mycobacterial composition in 60 DNA samples extracted from Mycobacterium bovis infected culture-confirmed African buffalo tissue. We detected mycobacterial DNA in 93.3% of the samples and the sensitivity for detecting Mycobacterium tuberculosis complex (MTBC) was 91.7%, demonstrating a high concordance of our culture-independent tNGS approach with mycobacterial culture results. In five samples, we identified heterogenous mycobacterial populations with various non-tuberculous mycobacteria, including members of the Mycobacterium avium complex (MAC), M. smegmatis, and M. komaniense. The latter Mycobacterium species was described in South Africa from bovine nasal swabs and environmental samples from the Hluhluwe-iMfolozi Park, which was the origin of the buffalo samples in the present study. This finding suggests that exposure to environmental mycobacteria may confound detection of MTBC in wildlife. In conclusion, our approach represents a promising alternative to conventional methods for detecting mycobacterial DNA. This high-throughput technique enables rapid differentiation of heterogeneous mycobacterial populations, which will contribute valuable insights into the epidemiology, pathogenesis, and microbial synergy during mycobacterial infections.

DeepQR: single-molecule QR codes for optical gene-expression analysis

Abstract Optical imaging and single-molecule imaging, in particular, utilize fluorescent tags in order to differentiate observed species by color. The degree of color multiplexing is dependent on the available spectral detection window and the ability to distinguish between fluorophores of different colors within this window. Consequently, most single-molecule imaging techniques rely on two to four colors for multiplexing. DeepQR combines compact spectral imaging with deep learning to enable 4 color acquisition with only 3 spectral detection windows. It allows rapid high-throughput acquisition and decoding of hundreds of unique single-molecule color combinations applied here to tag native RNA targets. We validate our method with clinical samples analyzed with the NanoString gene-expression inflammation panel side by side with the commercially available NanoString nCounter system. We demonstrate high concordance with “gold-standard” filter-based imaging and over a four-fold decrease in acquisition time by applying a single snapshot to record four-color barcodes. The new approach paves the path for extreme single-molecule multiplexing.

Comparison of the Direct Identification and Short-Term Incubation Methods for Positive Blood Cultures via MALDI-TOF Mass Spectrometry.

Timely pathogen identification in bloodstream infections is crucial for patient care. A comparison is made between positive blood culture (BC) pellets from serum separator tubes using a direct identification (DI) method and colonies on agar plates from a short-term incubation (STI) method with a matrix-assisted laser desorption/ionization Biotyper for the evaluation of 354 monomicrobial BCs. Both the DI and STI methods exhibited similar identification rates for different types of bacteria, except for Gram-positive and anaerobic bacteria. The DI method's results aligned closely with the STI method's results for Enterobacterales, glucose-non-fermenting Gram-negative bacilli (GNB), and carbapenem-resistant Enterobacterales. The DI method exhibited high concordance with the conventional method for GNB identification, achieving 88.2 and 87.5% accuracy at the genus and species levels, respectively. Compared with the STI method, the DI method showed a less successful performance for Gram-positive bacterial identification (50.5 vs. 71.3%; p < 0.01). The DI method was useful for anaerobic bacterial identification of slow-growing microorganisms without any need for colony growth, unlike in the STI method (46.7 vs. 13.3%; p = 0.04). However, both methods could not identify yeast in positive BCs. Overall, the DI method provided reliable results for GNB identification, offering many advantages over the STI method by significantly reducing the turnaround time and enabling quicker pathogen identification in positive BCs.

Prognostic Utility of the Flow Cytometry and Clonality Analysis Results for Feline Lymphomas.

Feline lymphoma, a prevalent cancer in cats, exhibits varied prognoses influenced by anatomical site and cellular characteristics. In this study, we investigated the utility of flow cytometry and clonality analysis via PCR for antigen receptor rearrangement (PARR) with respect to characterizing the disease and predicting prognosis. For this purpose, we received fine needle aspirates and/or blood from 438 feline patients, which were subjected to flow cytometry analysis and PARR. We used a subset of the results from patients with confirmed B- or T-cell lymphomas for comparison to cytological or histological evaluation (n = 53). Using them as a training set, we identified the optimal set of flow cytometry parameters, namely forward scatter thresholds, for cell size categorization by correlating with cytology-defined sizes. Concordance with cytological sizing among this training set was 82%. Furthermore, 90% concordance was observed when the proposed cell sizing was tested on an independent test set (n = 24), underscoring the reliability of the proposed approach. Additionally, lymphoma subtypes defined by flow cytometry and PARR demonstrated significant survival differences, validating the prognostic utility of these methods. The proposed methodology achieves high concordance with cytological evaluations and provides an additional tool for the characterization and management of feline lymphoproliferative diseases.

Parallel comparison of ocular metrics in non-human primates with high myopia by LS900, ultrasonography and MRI-based 3D reconstruction

We investigate the ocular dimensions and shape by using Lenstar900 (LS900), A-scan ultrasonography, and Magnetic Resonance Imaging (MRI) in highly myopic Macaca fascicularis. The ocular dimensions data of LS900, A-scan ultrasonography and MRI was assessed from 8 eyes (4 adult male cynomolgus macaque) with extremely high myopia (≤-1000DS) and compared by means of coefficients of concordance and 95% limits of agreement. Multiple regression analysis was performed to explore the associations between ocular biometry, volume, refraction and inter-instrument discrepancies. Test-retest reliability of three measurements of ocular parameters at two time points was almost equal (intraclass correlation = 0.831 to 1.000). The parallel-forms reliability of three measurements was strong for vitreous chamber depth (VCD) (coefficient of concordance = 0.919 to 0.981), moderate for axial length (AL) (coefficient of concordance = 0.486 to 0.981), and weak for anterior chamber depth (ACD) (coefficient of concordance = 0.267 to 0.621) and lens thickness (LT) (coefficient of concordance = 0.035 to 0.631). The LS900 and MRI systematically underestimated the ACD and LT comparing to A-scan ultrasonography (P < 0.05). Notably, the average AL on LS900 displayed a significant correlation with those on MRI (r = 0.978, P < 0.001) and A-scan ultrasonography (r = 0.990, P < 0.001). Almost 4/5 eyeballs were prolate. The mean eyeball volume positively correlated with AL (r = 0.782, P = 0.022), the width (r = 0.945, P = 0.000), and the length (r = 0.782, P = 0.022) of eyeball, while negatively correlated with SER (r = −0.901, P = 0.000). In conclusion, there was a high inter-instrument concordance for VCD with LS900, A-scan ultrasonography and MRI, while ACD and LT were underestimated with LS900 compared to A-scan ultrasonography, and the LS900 and A-scan ultrasonography could reliably measure the AL. MRI further revealed an equatorial globe shape in extremely myopic non-human primates.