Frontiers in Molecular Biosciences

The COVID-19 pandemic is challenging the global supply chain and equipment needed for mass testing with RT-qPCR, the gold standard for SARS-CoV-2 diagnosis. Here, we propose the RT-LAMP assay as an additional strategy for rapid virus diagnosis. However, its validation as a diagnostic method remains uncertain. In this work, we validated the RT-LAMP assay in 1,266 nasopharyngeal swab samples with confirmed diagnosis by CDC 2019-nCoV RT-qPCR. Our cohort was divided, the first (n=984) was used to evaluate two sets of oligonucleotides (S1 and S3) and the second (n=281) to determine whether RT-LAMP could detect samples with several types of variants. This assay can identify positive samples by color change or fluorescence within 40 minutes and shows high concordance with RT-qPCR in samples with CT [≤]35. Also, S1 and S3 are able to detect SARS-CoV-2 with a sensitivity of 68.4% and 65.8%, and a specificity of 98.9% and 97.1%, respectively. Furthermore, RT-LAMP assay identified 279 sequenced samples as positive (99.3% sensitivity) corresponding to the Alpha, Beta, Gamma, Delta, Epsilon, Iota, Kappa, Lambda, Mu and Omicron variants. In conclusion, RT-LAMP is able to identify SARS-CoV-2 with good sensitivity and excellent specificity, including all VOC, VOI, VUM and FMV variants.

ObjectiveTo identify and detect metabolomic markers to differentiate cancer related from other pleural effusions. Material and methodsUn-targeted flow infusion electrospray mass spectrometry was used on a cohort of 100 samples from benign and malignant pleural effusions (12 primary lung cancers, 14 mesotheliomas, 24 other cancers, 25 congestive cardiac failure, 22 parapneumonic effusion, 3 empyema). Standard metabolomic statistical models for analysis were performed. ResultsFive novel markers were identified using univariate and multivariate receiving operator curve analyses using five discriminant features yielding a diagnostic accuracy of above 0.91 (95% CI: 0.839-0.976) with a sensitivity of 0.97 (95% CI 0.952-0.979) and specificity 0.87 (95% CI 0.852-0.894). ConclusionIndividual biomarkers with the highest accuracy (>0.84) were all raised in cancer and are involved in metabolism of sterol-lipids and acyl carnitines linked to {beta}-oxidation. Our markers performed better than those previously published. Further, these published markers failed to achieve their stated accuracies in our data.

The development of the use of DNA aptamers for clinical applications to detect human diseases is at the forefront of research. Synthetic DNA aptamers are easy to generate, inexpensive, highly specific and have been postulated to replace antibodies for research and clinical use. Despite the considerable amount of published work on the use of DNA aptamers for research use, to date they have not been exploited for clinical diagnostics. SARS-CoV-2 virus is a pandemic causing a global disruptive event preventing people from travel, work and leisure activities resulting in a major health crisis, hospital overloads and a high death rate. The detection of SARS-CoV-2 virus in communities is therefore very important, especially for returning normality of life. The current gold standard for detection of SARS-CoV-2 virus is RT-PCR, a technique that is relatively expensive and most importantly with a slow turnaround time between sample procurement and result. This paper describes the development of a rapid, accurate, low-cost, facile to use assay for the detection of the SARS-CoV-2 spike protein in saliva. The assay exploits a simple system based on the use of a gold nanoparticle-aptamer complex, that can be easily produced and distributed, facilitating its deployment to the point-of-need, potentially reaching millions of individuals in resource-limited settings. The proposed colorimetric diagnostic test kit uses a SARS-CoV-2 DNA aptamer adsorbed on gold nanoparticles and salt-induced aggregation to detect the presence of SARS-CoV-2 Spike protein in saliva samples indicated by a color change of the gold absorbance spectrum that can be quantified by a spectrophotometer, linked to a mobile phone for data processing and analysis. The assay parameters were optimized and then tested in a field calibration clinical test in Indonesia. At a research level, a limit of detection of ca. 1.25 nM to synthetic spike protein (S1) was observed and a method to test human saliva samples developed. The DNA aptamer was specific to SARS-CoV-2, with minor cross-reactivity observed with MERS and SARS-CoV-1, but negligible cross-reactivity seen with common cold coronaviruses. A calibration clinical test of patients in Indonesia demonstrated a classification resulting in a > 97% sensitivity and a > 97% specificity compared with saliva RT-PCR test for SARS-CoV-2. Furthermore, the data indicates that anatomical location and sample type (swab vs saliva) can affect RT-PCR results. In conclusion, we have developed the use of a robust and reproducible aptamer-gold nanoparticle assay for clinical diagnostic use based on a colorimetric system that is cheap, simple, rapid, sensitive and can be employed for large scale testing of human populations for SARS-CoV-2 virus.

ObjectiveThe present investigation explored the disease spectrum of SPN ([&le;]2cm) and attempted to establish the potential predictive models for non-benign small pulmonary nodules (NBSPN) identified from CT screening in a real-world perspective study. MethodsA retrospective analysis was conducted on 6166 patients with SPN detected via chest CT scans at Fuzhou University Affiliated Provincial Hospital from January 2017 to September 2022. R language and SPSS software were used for data analyses. ResultsOf the 6,166 patients with SPN, 954 (15.47%) had their diagnosis confirmed pathologically. Among these nodules, NBSPN accounted for 88.7%. In NBSPN, the precursor glandular lesion (PGL) accounted for 14.18%, including 5.83% with atypical adenomatous hyperplasia (AAH) and 94.17% with adenocarcinoma in situ (AIS). Lung adenocarcinoma (LUAD) accounted for 85.82%, of which 32.07% were microinvasive adenocarcinoma (MIAD) and 67.93% invasive adenocarcinoma (IAD). Using the ROC model, none of the classical clinical factors had predictive value to the nature of SPN, including age, smoking, underlying conditions, CEA level, family history etc. Interestingly, the risk factors for NBSPN predicted by the ROC model include: female (OR: 1.842, 95%CI: 1.086-3.125, P=0.024), pure ground-glass opacities(pGGO) (OR: 5.243, 95%CI: 2.640-10.411, P<0.001), and part-solid (PS) (OR: 5.643, 95%CI: 1.970-16.167, P=0.001) in chest imaging, collectively delivering a highly significant AUC value at 0.748 in training set and 0.799 in validation set for distinguishing NBSPN from BSPN. ConclusionConventional clinical data are not sufficient to differentiate NBSPN from BSPN. Factors with high predictive values for NBSPN include female, pGGO and PS.

Screening for colorectal cancer (CRC) using plasma methylation is challenging due to the low abundance of cell-free DNA (cfDNA). Therefore, the development of signal amplification assays based on appropriate markers is essential to increase sensitivity. In this study, we employed an epigenome-wide approach for de novo identification differentially methylated CpGs (DMCs) common to CRC and adenoma using 17 public datasets. A sense-antisense and dual MGB probe (SADMP) assay was then developed. Subsequently, the biomarkers were validated in 712 plasma samples based on SADMP. A total of 2237 DMCs showed overlap between CRC and adenoma. Of these, 75 were hypomethylated in 30 other non-CRC cancers. Following LASSO regression, WBC validation and primer/probe design evaluation, NTMT1 and MAP3K14-AS1 were identified as the most informative candidate biomarkers. At preset template concentrations, the SADMP assay for NTMT1 or MAP3K14-AS1 could reduce the cycle threshold by 1. The NTMT1 and MAP3K14-AS1 dual-target SADMP assay demonstrated a sensitivity of 84.8% for CRC (stage I: 75.0%), a sensitivity of 32.0% for advanced adenomas (AA), and a specificity of 91.5% in controls. The dual-target assay showed high performance for CRC early detection in plasma, suggesting that it may serve as a promising noninvasive tool for CRC screening.

BackgroundEndobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is commonly used to diagnose and stage lung cancer. In real-life practice, limitations are seen with cytology samples from EBUS-TBNA. Obtaining adequate samples can be challenging when faced with necrotic lesions with low cellular yield and the evolving landscape of targeted therapy, necessitating additional samples for comprehensive testing. Hence, transbronchial mediastinal cryobiopsy guided by endobronchial ultrasound (EBUS-TBMC) has emerged as a promising approach for obtaining larger tissue samples. In retrospective review, our aim is to present our early experience regarding the feasibility of performing EBUS-TBMC, employing a similar approach to EBUS-TBNA, followed by the outcomes of our procedures. We include a step-by-step explanation and some recommendations to conduct a successful EBUS-TBMC. MethodSingle center retrospective analysis to evaluate the feasibility and utility of EBUS-TBMC cases over six months from July to December 2022. Results36 EBUS-TBMC procedures on 30 patients. Moderate sedation was used in 80% of cases. Majority (83.4%) of the patients had biopsy of a single lesion with a median of 3 cryobiopsies (Interquartile range 3-4). The median cryo-activation time was 6 seconds (Interquartile range 6-8). EBUS-TBMC demonstrated a positive yield of 86.1% with an overall diagnostic yield of 83.3%. Mild bleeding occurred in six biopsies (16.7%) which did not required further intervention. No other major complications were observed. ConclusionEBUS-TBMC is a safe and effective alternative to EBUS-TBNA. Histology samples obtained through EBUS-TBMC have the potential to increase confidence in diagnosing and staging lung cancer, thereby alleviating concerns about tissue inadequacy.

ObjectiveTo evaluate the diagnostic efficiency of different methods in detecting COVID-19 to provide preliminary evidence on choosing favourable method for COVID-19 detection. MethodsPubMed, Web of Science and Embase databases were searched for identifing eligible articles. All data were calculated utilizing Meta Disc 1.4, Revman 5.3.2 and Stata 12. The diagnostic efficiency was assessed via these indicators including summary sensitivity and specificity, positive likelihood ratio (PLR), negative LR (NLR), diagnostic odds ratio (DOR), summary receiver operating characteristic curve (sROC) and calculate the AUC. Results18 articles (3648 cases) were included. The results showed no significant threshold exist. EPlex: pooled sensitivity was 0.94; specificity was 1.0; PLR was 90.91; NLR was 0.07; DOR was 1409.49; AUC=0.9979, Q*=0.9840. Panther Fusion: pooled sensitivity was 0.99; specificity was 0.98; PLR was 42.46; NLR was 0.02; DOR was 2300.38; AUC=0.9970, Q*=0.9799. Simplexa: pooled sensitivity was 1.0; specificity was 0.97; PLR was 26.67; NLR was 0.01; DOR was 3100.93; AUC=0.9970, Q*=0.9800. Cobas(R): pooled sensitivity was 0.99; specificity was 0.96; PLR was 37.82; NLR was 0.02; DOR was 3754.05; AUC=0.9973, Q*=0.9810. RT-LAMP: pooled sensitivity was 0.98; specificity was 0.99; PLR was 36.22; NLR was 0.04; DOR was 751.24; AUC=0.9905, Q*=0.9596. Xpert Xpress: pooled sensitivity was 0.99; specificity was 0.97; PLR was 27.44; NLR was 0.01; DOR was 3488.15; AUC=0.9977, Q*=0.9829. ConclusionsThese methods (ePlex, Panther Fusion, Simplexa, Cobas(R), RT-LAMP and Xpert Xpress) bear higher sensitivity and specificity, and might be efficient methods complement to the gold standard.

Background & AimsCell-free DNA (cfDNA) has advanced cancer genetic profiling through liquid biopsy. While plasma is traditionally the primary source, emerging evidence highlights urinary cfDNA as a novel and noninvasive alternative. This study aimed to comprehensively assess transrenal DNA (trDNA) as a novel noninvasive biomarker source in HCC patients, compared to blood-based liquid biopsy. Approach & ResultsHBV DNA was used as a biomarker for trDNA. HBV-targeted and HCC-focused next generation sequencing (NGS) and whole genome sequencing (WGS) were used to compare fragment insert-sizes, the genome coverage, and germline genotyping accuracy. Urinary cfDNA overall exhibited a predominantly mononucelosomal pattern similar to plasma cfDNA, but with shorter fragments, broader size distribution and a more pronounced 10-bp periodicity. In contrast, trDNA were shorter and more variable among all patients. In HCC patients, trDNA was even shorter, with distinct 4-mer end motifs, compared to non-HCC trDNA. Higher concentrations of HCC-distinctive 4-mer end motif and TP53 mutations were found in urine compared to plasma. The overall genome coverage breadth by WGS was similar between urine and plasma cfDNA, with a higher fraction of covered cancer-associated mutation hotspots in urine cfDNA. In 101 HCC patients, there was a 78% overall concordance of HCC-associated mutations (TP53, CTNNB1, and hTERT) and in select 15 patients, 97% overall position-level concordance by targeted NGS between plasma and urine cfDNA. ConclusionUrine cfDNA has comparable features with distinct characteristics to plasma cfDNA and is a promising tool for liver cancer studies.

Liquid biopsy comprehensive genomic profiling (LB-CGP) testing is performed on circulating tumor DNA to detect tumor recurrence, predict prognosis, and select therapeutic agents. Pathogenic variants of germline origin in genes associated with hereditary tumor syndrome (HTS) can be simultaneously detected by CGP testing. Nonetheless, it is often challenging to differentiate whether the variants are of somatic or germline origin. The differentiation criteria were primarily based on the variant allele frequencies (VAFs). However, more evidence is needed to establish clear criteria, and it is often difficult to differentiate between variants based on VAF alone. In this study, using the national database of the Center for Cancer Genomics and Advanced Therapeutics, which accumulates real-world data on CGP testing in Japan, we analyzed 169,370 variants detected in 11,399 patients registered with FoundationOne Liquid CDx testing. By extracting the predominantly presumed somatic and germline variants, we established a criterion for VAF that could achieve high specificity and sensitivity. Further investigation into the detection status of other variants led to the development of an algorithm for differentiating somatic/germline variants of genes associated with HTSs. Based on this algorithm, 726 variants were extracted as presumed germline pathogenic variants among the 26 genes with high germline conversion rates in 710 patients in the study. This algorithm should help to discriminate with high accuracy whether the variants detected in LB-CGP tests are of somatic or germline origin, although further analyses are required to confirm the validity of this algorithm. Highlights- The highly accurate VAF criterion for differentiating somatic and germline variants was determined using real-world data from more than 11,000 patients who underwent liquid biopsy CGP testing. - To improve the specificity of the PGPV extraction, an additional criterion was defined: checking the status of other genomic alterations detected. - Criteria for considering information other than VAFs associated with the variant under PGPV consideration were developed to improve the sensitivity of PGPV extraction. - By integrating these criteria and previous evidence on germline conversion rates, a GeMSort algorithm was established. - Based on this algorithm, 726 variants from 710 patients were extracted as PGPVs among 26 hereditary tumor syndrome-associated genes with high germline conversion rates.

BackgroundTuberculous pleuritis (TBP), the extra-pulmonary manifestation of tuberculosis, is the second most common after TB lymphadenitis. Histopathology using biopsy samples is the most sensitive diagnostic procedure for TBP, however the biopsy acquisition is invasive. Therefore, better screening markers for diagnosis using pleural fluid are required. The pathogen biomarkers expressed at the site of infection may play a potential role in designing a newer diagnostic assay. Thus, the current study was planned to look for mycobacterial RNA biomarkers in TBP and to assess their diagnostic utility in pleural fluid. MethodsTBP suspects (n=261) were recruited in the current study. Out of these 45 suspects were excluded and the remaining (n=216) were divided into TBP (n=54) and non-TBP (n=162) groups based on composite reference standard. A whole genome microarray was carried using M.tb RNA from pleural biopsies of TB patients. The data was validated using qRT-PCR and the diagnostic utility of top two highly expressed genes was assessed in pleural fluid of using a real time RT-PCR assay. ResultsOverall, 1856 genes were differentially expressed in microarray of which 1365 were upregulated and 491 were downregulated. After validation of microarray gene expression, two genes namely Rv1586 and Rv2543 were selected for assessment of their diagnostic utility in TBP. The combined analysis for the presence of either of genes in the pleural fluid led to identification of pleural TB patients with 79.6% sensitivity and 93.28% specificity. ConclusionThe transcripts of genes Rv1586 and Rv2543 holds potential for the development of a RNA based molecular diagnostic assay in pleural fluid of TBP patients.

Immunofluorescence (IF) microscopy is gaining increased popularity as a pre-genetic diagnostic method in primary ciliary dyskinesia (PCD). Efficient IF-based diagnostics in PCD requires standardization of staining methods and antibodies. Obviously, specificity of the analysis highly depends on the quality of the analyzed material. The nasal epithelium brushing quality depends on the patient health status, but conditions of the slides storage are also very important. We applied automated image analysis to systematically examine the influence of samples storage conditions on the specificity of IF staining with eight polyclonal antibodies routinely used for PCD diagnostics, against epitopes of proteins representing various axoneme structural complexes. Seven various combinations of temperatures and storage times were tested to mimic the procedures of handling epithelial brushing on glass slides: at the clinic, during transport, or after reception at the diagnostic laboratory. Our study revealed that proper slide storage conditions are essential for the reliable PCD diagnosis via IF staining. If microscopic slides are prepared at the diagnostic laboratory, we suggest continuous storage at -80{degrees}C or -20{degrees}C. If the slides are prepared at a collaborating clinic and shipped, we suggest storage at -20{degrees}C or 4{degrees}C. The IF sensitivity to slide storage conditions differs among antibodies targeting various ciliary elements; for example, molecular ruler proteins appear the most sensitive to prolonged slide storage at room temperature. Moreover, to improve the reliability of the IF staining, additional control slides should be used to account for inter-individual differences. Finally, IF results indicative of PCD should be carefully confronted with patients clinical history.

RNA Sequencing (RNA-Seq) can facilitate prompt and precise management of many illnesses, including infection, especially when coupled with PCR. To further optimize RNA-Seq for the purposes of creating a RNA-Seq-informed PCR test, identifying reliable RNA primer targets is paramount. Essential criteria for constructing RNA primer targets include high gene expression or stability of transcripts. We hypothesize that free energy evaluation and motif analysis can demonstrate gene expression level or stability of transcripts to construct ideal RNA primers for RNA-Seq-informed PCR that can expedite the diagnosis and treatment of infection. Whole blood samples were collected from patients diagnosed with COVID-19 in the ICU at Rhode Island Hospital, stored in Paxgene tubes to preserve the integrity of the specimens, and submitted for RNA-Seq by a commercial sequencing service (Azenta/Genewiz). After quality assurance and quality control (QA/QC) measures are performed, we used RNAfold from the ViennaRNA Package to calculate the minimum free energy (MFE) and ensemble free energy (EFE) values to compare the stability among different secondary structures constructed from RNA-Seq read sequences. Energy parameters for calculations were set at 37 degree Celsius and standard physiological conditions at pH 7.4. In our analysis, we utilized multiple statistical tools, such as analysis of variance, Games-Howell post hoc tests, and a negative binomial regression. Of the 676 reads that aligned with SARS-CoV-2, there were 137 unique sequences among all patients. Among the unique read sequences, the average MFE was -30.46 kcal/mol and the average EFE was -32.94 in kcal/mol. There were 6 genes with these unique read sequences, two of which -- nucleocapsid (N) gene and spike (S) gene -- coded for integral structural proteins. Notably, the mean MFE and EFE of N gene were significantly different from ORF1ab gene (p = 2.81e-7; p = 0.005) and ORF6 gene (p = 0.023; p = 0.03). Our motif analysis demonstrated 7 motifs that corresponded to destabilization of the RNA, and a single motif (MEME-28) that corresponded to stabilization of the RNA. Within the nucleocapsid (N) gene, we found that reads from different regions also differed in stability. Our results demonstrate that the stability of reads from different genes varies in SARS-CoV-2 infections. Free energy evaluation and motif analysis are viable approaches to determine RNA structure stability or levels of differential gene expression. Stable transcripts that are highly expressed can be ideal RNA primers to be used in conjunction with PCR to expedite the management of infection.

We are currently facing a frightening increase in COVID-19 patients admitted to the ICU. Aiming at screening for secondary pneumonia, we collected the data of our first twelve ICU patients who underwent bronchoalveolar lavage (BAL). Surprisingly, four were detected with Pneumocystis jirovecii (Pj) DNA and RNA, resulting in Pj prevalence of 17%. Pj is a ubiquitous ascomycetes fungus that thrives at the surface of type-I pneumocytes, specifically in human alveoli, leading to pneumocystosis in immunocompromised patients. Interestingly, none of our patients was immunocompromised per se before admission, while all presented the recognized risk factors for life-threatening COVID-19 infection. Observing such high prevalence in COVID-infected patients was unexpected. Almost all patients developed ARDS and received high-dose steroids to prevent worsening, as suggested by reports from China. In Pj-positive patients requiring steroids, prophylaxis was given to avoid the risk of pneumocystosis and increased lung inflammation that may compromise the outcome.We are strongly convinced that testing deep lung specimens for Pj in severe COVID-19 patients should be recommended and Pj-positive patients treated with steroids, and given anti-Pj prophylaxis. This message is important, given the high mortality rate of COVID-19 patients in the ICU.

ObjectivesConventional lung biopsy for critically ill patients who under invasive mechanical ventilation (IMV) is limited due to high risks of procedure-related complications. We developed a novel technique named bronchus-blocked ultrasound-guided percutaneous transthoracic needle biopsy (BUS-PTNB) to mitigate the risks of biopsy under IMV, but its safety and efficacy have not been prospectively evaluated. MethodsIn this prospective, single-arm trial (Chictr.org, ChiCTR2100054047), invasively ventilated patients with undiagnosed lung opacities were screened and underwent BUS-PTNB after enrollment. The peri-operative conditions, severity of complications, pathological findings, and tissue-based metagenomic next-generation sequencing (mNGS) results were systemically evaluated. ResultsA total of 22 critically ill patients (18 men, mean age, 64.2 years [SD 11.7], APACHE II score, 27.0 [SD 5.6], PaO2/FiO2 ratio, 120.5 [IQR 91.9-169.1]) under IMV were enrolled in the study. Throughout the procedure, there were no significant changes in respiratory rate or PaO2. Biopsy-related complications occurred in 3 patients (13.6%), including pneumothorax (n=1, 4.5%), intrabronchial hemorrhage (n=2, 9.1%), and hemothorax (n=3, 13.6%). Notably, one patient (P10) required a blood transfusion due to hemothorax, which was classified as a severe complication (1/22, 4.5%). Satisfactory biopsy samples were obtained from 21 patients (95.5%) for pathological study and from all 22 patients (100%) for mNGS. ConclusionsThe novel BUS-PTNB is a promising bedside biopsy technique for ICU patients under IMV with acceptable complication risk. This technique may prove instrumental in advancing pathological studies of severe lung diseases. Key points and Clinical Relevance StatementO_ST_ABSQuestionC_ST_ABSWe developed bronchus-blocked ultrasound-guided percutaneous transthoracic needle biopsy (BUS-PTNB) for patients under invasive mechanical ventilation, but its safety and efficacy have not been prospectively evaluated. FindingsPatients vital signs showed no significant fluctuations. Biopsy-related complications occurred in 3 patients out of 22. Satisfactory samples were obtained for pathological study and mNGS. Clinical RelevanceBUS-PTNB is a promising bedside biopsy technique for ICU patients under IMV with acceptable complication risk. This technique may prove instrumental in advancing pathological studies of severe lung diseases.

BackgroundThe most frequent primary intracranial tumor is a meningiomas; however, atypical (WHO grade II) and an plastic (WHO grade III) variants are more aggressive and have increased rates of recurrence and being lethal. The current Histopathological grading is not specific in making predictions. New molecular and genetic profiling has identified key biomarkers potentially used as prognostic refinements, or targets in the personalized medicine strategy. ObjectivesTo describe the molecular and genetic repertoire of atypical and an plastic meningiomas and evaluate their prognostic impact, to inform targeted therapy options. Study designA Retrospective Study. Place and duration of studyDepartment of Neurosurgery MMC Hospital Mardan from jan 2024 to Jan 2025 KPK PAKISTAN MethodsPatient-derived tumor samples were classified as having atypical and an plastic meningiomas and underwent whole exam sequencing, RNA sequencing, and DNA methylation profiling. Key markers (Ki-67, p53, PR) were tested by immunohistochemistry. Molecular alterations were statistically associated with clinical data, such as recurrence and survival. In the bioinformatics analysis, there was one common mutation and signaling pathways. T-tests, Kaplan-Meier survival analysis, and Cox regression modeling statistics were applied to determine statistical significance. ResultsFifty patients (25 atypical and 25 an plastic). Patients were diagnosed at a mean age of 58.4 years (SD +/- 11.6). The ratio between males and females was 1:1.3. There was a major disparity between the two groups in terms of overall survival (p = 0.038), and plastic meningiomas were related to a reduced survival rate. The most common mutations were NF2 (47%), deletions of CDKN2A/B (29%) and TERT promoter mutations (18%). Tumor clustering into specific subgroups based on methylation profiling was found to correlate with prognosis. ConclusionAtypical and an plastic meningiomas differ, with molecular and genetic profiles indicating various changes linked to prognosis. The application of these findings into clinical care can positively affect the risk stratification and the development of targeted therapies. It is reasonable to develop this direction further and analyze the validity of these biomarkers and be able to gauge their usefulness in predicting therapeutic response and survival.

ObjectiveThis study aimed to evaluate the diagnostic performance of plasma methylated SEPT9 (mSEPT9) gene combined with carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA199), fecal occult blood test (FOBT), red blood cell distribution width (RDW), and inflammation-related indices from complete blood count (CBC) for colorectal cancer (CRC). MethodsA prospective study was conducted on 188 patients pathologically diagnosed with CRC (CRC group) and 693 control subjects with gastrointestinal symptoms but non-CRC diagnoses (control group) admitted to Hunan Provincial Peoples Hospital from January 01, 2024 to December 31, 2024. Data on mSEPT9, CEA, CA199, FOBT, and CBC were collected. Binary logistic regression analysis was used to establish a predictive model for CRC risk factors, and receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic performance of each index. ResultsThe positive rates of mSEPT9 and FOBT in the CRC group were significantly higher than those in the non-CRC group (P<0.001). The levels of CEA, CA199, RDW-CV, RDW-SD, neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) were significantly higher in the CRC group than in the control group (P<0.001), while the lymphocyte-to-monocyte ratio (LMR) was significantly lower (P<0.001). Univariate logistic regression analysis showed that mSEPT9, CEA, CA199, FOBT, RDW-CV, RDW-SD, NLR, and PLR were independent predictive risk factors for CRC. Multivariate regression analysis indicated that patients with positive or elevated mSEPT9, CEA, CA199, FOBT, RDW-CV, and PLR were more likely to have CRC. The combined model of mSEPT9, CEA, CA199, FOBT, RDW-CV, and PLR demonstrated an impressive area under the ROC curve (AUC) of 0.939, with a sensitivity of 0.920 and specificity of 0.839, highlighting excellent screening efficacy for CRC. ConclusionA screening model incorporating mSEPT9, CEA, CA199, FOBT, RDW-CV, and PLR provides valuable insights for CRC diagnosis.

BackgroundHypervirulent Klebsiella pneumoniae (hvKp) infections have mainly been described in Asia. Two patients in the age group of 30 to 50 years presented within a two month period to a tertiary referral hospital in Texas with septic shock, hepatic abscess and septic thrombophlebitis. Blood cultures were positive for Klebsiella pneumoniae (isolates 2020CK-00441 and 2021CK-00720 respectively). MethodsWhole genome sequencing was performed using paired-end Illumina MiSeq reads for both isolates. Nanopore sequencing to obtain a closed genome was performed for 2020CK-00441. Results2020CK-00441 belonged to ST23 type while 2021CK-00720 was a ST65 type isolate. Kleborate analyses predicted with high confidence that both the isolates were hvKp. Phylogenetic analyses showed that the two strains are not closely related to each other or to any other known hvKp isolates. Both the isolates had yersiniabactin, colibactin, aerobactin and salmochelin producing loci which likely confer these isolates hvKp phenotype. 2020CK-00441 had a unique pK2044 like plasmid. ConclusionsHvKp strains capable of causing devastating metastatic septic infections have emerged in Texas. These isolates are unique when compared to other hvKp strains of the world. Country wide surveillance and whole genome sequencing of these strains is essential to prevent a major public health emergency in USA.

Background and study aimsPancreatic cysts are common incidental findings, with an estimated prevalence of 13-15% in imaging done for other reasons. It is difficult to identify cysts with malignant potential. Diagnosis often relies on collection of cyst fluid, but tissue sampling using micro-forceps may allow for a more reliable diagnosis and higher yield of DNA for next-generation sequencing (NGS). Patients and methods24 patients referred for endoscopic ultrasound were recruited. Biopsies were taken using micro-forceps and the AmpliSeq Cancer Hotspot panel was used for NGS, a PCR assay targeting several hotspots within 50 genes, including GNAS, KRAS and VHL. ResultsThe concentration of DNA extracted from 24 cyst wall samples was significantly higher than in the 9/24 available matched cyst fluid samples. Cyst wall biopsy was able to diagnose 19/24 cysts (5 high risk, 6 intraductal papillary mucinous neoplasm and 4 benign). The sensitivity, specificity and diagnostic accuracy for standard of care was 66.6%, 50% and 63.1% respectively and for standard of care with NGS was 100%, 50% and 89.4% respectively. ConclusionsCyst wall biopsy performs well in diagnosing cysts but was inadequate in 5/24 patients. NGS data correlates well with histology and may aid in diagnosis and risk stratification of pancreatic cysts.

PurposeTo investigate the usefulness of quantitative evaluation of a width of lateral contour on a lateral split-view computed tomographic air-contrast enema (CT enema) image for diagnosing the invasion depth of colorectal cancer (CRC) located on a colorectal fold. MethodsThe cases of 22 patients with 22 fold-located CRCs (12 early CRCs and 10 advanced CRCs) who underwent a pretherapeutic computed tomographic colonography were retrospectively examined. T1-stage CRCs were classified into two categories according to the Japanese guideline: T1a-stage, carcinoma invading the superficial submucosa (< 1000 um); and T1b-stage, carcinoma invading the deeper submucosa ([&ge;] 1000 um). The maximum width of a lateral contour of the colorectal fold on which the CRC was located, i.e., the gap distance between the two adjacent haustrations, was calculated from the lateral split-view CT enema image by two gastrointestinal radiologists. These values were compared between the intramucosal / T1a CRCs and the T1b / more deeply invading CRCs. The inter-rater intraclass correlation coefficients were also evaluated for reliability. ResultsThe maximum widths of a lateral contour of the colorectal fold were significantly higher in the T1b / more deeply invading CRCs than in the intramucosal / T1a CRCs (p<0.0001). The optimum cut-off value of the maximum width of a lateral contour of the colorectal fold for differentiating the former from the latter was 6.0 mm, with a sensitivity and specificity of 100% and 100%, respectively. The inter-rater intraclass correlation coefficient for measurement of a lateral contour of the colorectal fold was 0.958. ConclusionsWe demonstrated for the first time that quantitative evaluation of a width of lateral contour using a lateral split-view CT enema image can improve the diagnostic accuracy of the invasion depth for CRCs located on a colorectal fold.

Structural variations (SVs) play a key role in the pathogenicity of hematological malignancies. Standard-of-care (SOC) methods such as karyotyping and fluorescence in situ hybridization (FISH), employed globally for the past three decades have significant limitations in the resolution or the number of recurrent aberrations that can be simultaneously assessed, respectively. Next-generation sequencing (NGS) based technologies are now widely used to detect clinically significant sequence variants but are limited in their ability to accurately detect SVs. Optical genome mapping (OGM) is an emerging technology enabling the genome-wide detection of all classes of SVs at a significantly higher resolution than karyotyping and FISH. OGM neither requires cultured cells nor amplification of DNA and hence addresses the limitations of culture and amplification biases. This study reports the clinical validation of OGM as a laboratory developed test (LDT), according to CLIA guidelines, for genome-wide SV detection in different hematological malignancies. In total, 68 cases with hematological malignancies (of various subtypes), 27 controls and two cancer cell lines were used for this study. Ultra-high molecular weight DNA was extracted from the samples, fluorescently labeled, and run on the Bionano Genomics Saphyr system. A total of 207 datasets, including replicates, were generated and 100% could be analyzed successfully. Sample data were then analyzed using either disease specific or pan-cancer specific BED files to prioritize calls that are known to be diagnostically or prognostically relevant. Accuracy, precision, PPV and NPV were all 100% against standard of care results. Sensitivity, specificity, and reproducibility were 100%, 100% and 96%, respectively. Following the validation, 11 cases were run and analyzed using OGM at three additional sites. OGM found more clinically relevant SVs compared to SOC testing due to its ability to detect all classes of SVs at much higher resolution. The results of this validation study demonstrate OGMs superiority over traditional SOC methods for the detection of SVs for the accurate diagnosis of various hematological malignancies.