High Specificity Research Articles

The first 2-year prospective audit of prenatal cell-free deoxyribonucleic screening using single nucleotide polymorphisms approach in a single academic laboratory.

We reported a performance during an implementation of prenatal cell-free (cf) DNA screening using single nucleotide polymorphism (SNP) approach in our accredited laboratory. Prospective audit with prompt intervention was set for the processing of 2,502 samples from singleton pregnancy from August 2017 to July 2019. Risks of trisomy 21 (T21), T18, T13, monosomy X (XO), and other sex chromosome aneuploidies (SCAs) were clarified by a proprietary bioinformatics algorithm. Laboratory failure occurred in 192 samples (7.7 %) as a result of inadequate sequencing (n=144), fundamental limitation of the testing (n=19), and obvious human error (n=29). Faulty setting of the calibration curve was the most common human error (n=22/29). After a redraw (n=110), 79 (71.8 %) were settled. Overall, 2,389/2,502 samples (95.5 %) were reportable. Thirty-five samples were high-risk for T21 (n=19), T18 (n=5), T13 (n=1), XO (n=3), and other SCAs (n=7), respectively. Positive predictive values calculated from either prenatal confirmatory tests or postnatal findings were 93.8 % (n=16), 100 % (n=4), 50 % (n=2), and 83.3 % (n=6) for T21, T18, XO, and other SCAs, respectively, with high sensitivity and specificity (>99.9 %). Vanishing twin was detected from 1 out of 4 samples with detected additional haplotypes. An overall performance of SNP-based prenatal cf-DNA screening during our initial implementation can be optimized with proactive approach. The technical know-how was a significant limiting factor for adopting the technology. The lessons learnt may be of interest to the academic laboratory considering adopting their own test instead of sending blood to a testing service of a supplier.

Bioorthogonal Chemistry: Enzyme Immune and Protein Capture for Enhanced LC-MS Bioanalysis.

Immunocapture liquid chromatography-mass spectrometry (IC-LC-MS) bioanalysis has become an indispensable technique across various scientific disciplines, ranging from drug discovery to clinical diagnostics. While traditional immunocapture techniques have proven to be effective, they often encounter limitations in sensitivity, specificity, and compatibility with MS analysis. Chemoenzymatic immunocapture and protein capture (IPC) offers a promising solution, combining the high specificity of antibodies or proteins with the versatility of enzymatic and chemical modifications. This Review explores the foundational principles of chemoenzymatic IPC and examines various modification strategies including bioorthogonal click-chemistry, enzymatic-tagging, and HaloTag/CLIP-tag. Recent advancements in chemoenzymatic IPC techniques have significantly expanded their applicability to a diverse range of biomolecules including small molecules, peptides, RNAs, and proteins. This Review focuses on improvements in analytical performance achieved through these innovative approaches. Moreover, we discuss the broad applications of chemoenzymatic immunocapture in drug discovery, clinical diagnostics, and environmental analysis and explore its potential for future advancements in bioanalysis. We propose a novel solid-phase chemoenzymatic IPC assay (SCEIA) that effectively utilizes bioorthogonal click chemistry and chemoenzymatic approaches for efficient IPC and target analyte release. In summary, chemoenzymatic IPC represents a transformative paradigm shift in IC-LC-MS bioanalysis. By overcoming the limitations of traditional IPC techniques, this approach paves the way for more robust, sensitive, and versatile analytical workflows.

Metagenomic sequencing of CRISPRs as a new marker to aid in personal identification with low-biomass samples.

The high specificity of the human skin microbiome is expected to provide a new marker for personal identification. Metagenomic sequencing of clustered regularly interspaced short palindromic repeats (CRISPRs), which we call metaCRISPR typing, was shown to achieve personal identification accurately. However, the intra-individual variability observed in previous studies, which may be due to poor DNA yields from skin samples, has resulted in non-reproducible results. Furthermore, whether metaCRISPR typing can assist in the forensic human DNA analysis of low-biomass samples, from which the information obtained is insufficient, is unknown. In the present study, we sequenced serially diluted control streptococcal CRISPRs cloned into plasmids to determine the minimum copy number required to obtain reproducible results from metaCRISPR typing. We found that at least 102 copies of CRISPRs are necessary to obtain reproducible results. We then analyzed the skin swab samples using both metaCRISPR typing and human DNA typing. When the DNA extracted from the skin swabs was diluted, no information was obtained from six out of eight samples by human DNA typing. On the other hand, beta diversity indices of spacer sequences compared with reference samples were below 0.8 for three out of six samples, for which no information was obtained from human DNA analysis, indicating that the spacers observed in these samples were similar to those in the references. These results indicate that metaCRISPR typing may contribute to the identification of individuals from whom the samples were obtained, even in cases where human DNA yields are insufficient to perform human DNA analysis.IMPORTANCEPrevious studies have developed new personal identification methods utilizing personal differences in the skin microbiome. However, intra-individual diversity of skin microbiome may preclude the application of microbiome-based personal identification. Moreover, no study has compared microbiome-based personal identification and practical human DNA analysis. Here, we revealed that the results of metaCRISPR typing, a previously developed microbiome-based personal identification method, are stable if the copy number of the marker gene is sufficient. We then analyzed the skin swab samples using both metaCRISPR typing and human DNA analysis. Our results indicate that metaCRISPR typing may provide additional information for personal identification using low-biomass samples that cannot be used for conventional human DNA analysis.

Differentiation of severe from non-severe mitral stenosis with dimensionless index and its prognostic implications in patients with rheumatic mitral stenosis

Abstract Introduction Accurate identification of the severity of rheumatic mitral stenosis (MS) commonly relies on measurement of mitral valve area (MVA) by transthoracic echocardiography (TTE). The dimensionless index (DI) of mitral valve (MV) was recently shown to be useful in identifying severity of degenerative MS. It is uncertain if DI MV is useful in rheumatic MS. Purpose We aimed to validate the DI MV in rheumatic mitral stenosis, identify threshold values of the DI MV for the accurate identification of severe and non-severe rheumatic MS, and study its association with clinically significant outcomes. Methods We studied DI MV in 406 cases of rheumatic MS, with 174 cases in a derivation cohort , 121 cases in a TTE validation cohort, and 111 cases in a transoesophageal echocardiography (TEE) validation cohort. Outcome data were collected in the derivation and TTE validation cohorts. DI MV was calculated by dividing the left ventricular outflow tract pulsed-wave Doppler time-velocity integral (TVI) by the MV continuous-wave Doppler TVI. Results In the derivation cohort, ROC analysis showed that MV DI was significantly associated with MS severity (AUC = 0.838, 95% CI, 0.780-0.897, p<0.001) but no threshold value was able to identify severe MS (MVA ≤ 1.5 cm2) with both sensitivity and specificity greater than 80%. DI MV ≤ 0.34 had the best combination of sensitivity and specificity (78.5% and 75.8% respectively). However, with an approach using two threshold values, namely DI MV ≤ 0.25 to identify severe MS and DI MV ≥ 0.40 to identify non-severe MS, both values showed a high specificity of 93.7%. When tested in the validation cohorts, these values have similar high specificity for identifying severe (93.8%) and non severe MS (91.4%). DI MV was univariately significant (HR = 0.075, 95% CI 0.0215-0.378, p=0.002) in Cox regression analysis for a composite outcome of death and MV intervention. Multivariate analysis incorporating age, sex, DI MV, pulmonary artery systolic pressure (PASP) and left ventricular ejection fraction (LVEF) showed that age, DI MV and PASP were independently associated with the composite outcome. Conclusion While not a replacement for MVA in the assessment of MS severity, the DI MV can rule-in or rule-out severe MS with a high specificity and is useful as a complementary tool in the integrative assessment of MS severity. DI MV is independently associated with composite outcomes of death and MV intervention.Receiver operating characteristic curveSensitivity and Specificity of DI MV

Imaging PD-L1 in the brain-Journey from the lab to the clinic.

Immune checkpoint inhibitors (ICPIs) have proven to restore adaptive anti-tumor immunity in many cancers; however, no noteworthy therapeutic schedule has been established for patients with glioblastoma (GBM). High programmed death-ligand 1 (PD-L1) expression is associated with immunosuppressive and aggressive phenotypes in GBM. Presently, there is no standardized protocol for assessing PD-L1 expression levels to select patients and monitor their response to ICPI therapy. The aim of this study was to investigate the use of 89Zr-DFO-Atezolizumab to image the spatio-temporal distribution of PD-L1 in preclinical mouse models and in patients with newly diagnosed GBM treated with/without neoadjuvant Pembrolizumab. The immunoreactivity, binding affinity, and specificity of 89Zr-DFO-Atezolizumab were confirmed in vitro. Mice-bearing orthotopic GBM tumors or patients with newly diagnosed GBM treated with/without Pembrolizumab were intravenously injected with 89Zr-DFO-Atezolizumab, and PET/CT images were acquired 24, 48, and 72 hours in mice and at 48 and 72 post-injection in patients. Radioconjugate uptake was quantified in the tumor and healthy tissues. Ex vivo immunohistochemistry (IHC) and immunophenotyping were performed on mouse tumor samples or resected human tumors. 89Zr-DFO-Atezolizumab was prepared with high radiochemical purity (RCP > 99%). In vitro cell-associated radioactivity of 89Zr-DFO-Atezolizumab corroborated cell line PD-L1 expression. PD-L1 in mouse GBM tumors was detected with high specificity using 89Zr-DFO-Atezolizumab and radioconjugate uptake correlated with IHC. Patients experienced no 89Zr-DFO-Atezolizumab-related side effects. High 89Zr-DFO-Atezolizumab uptake was observed in patient tumors at 48 hours post-injection, however, the uptake varied between patients treated with/without Pembrolizumab. 89Zr-DFO-Atezolizumab can visualize distinct PD-L1 expression levels with high specificity in preclinical mouse models and in patients with GBM, whilst complementing ex vivo analysis.

The impact of escape mapping for roof line completion during BOX isolation for atrial fibrillation

Abstract Background and Aim The creation of complete roof line (RL) is often challenging due to the presence of epicardial myocardial bundle or fat interposition. The disconnecting the septopulmonary bundle from the Buchmann bundle arrays requires for RL completion. Therefore, the conduction time to the left atrial appendage (LAA) and the high right atrium (HRA) potentials under left superior pulmonary vein pacing (LSPVp), so-called •escape mapping•, should be significantly prolonged after the creation of the transmural RL. This study investigates whether the changes conduction times from LSPVp to LAA and HRA (∆LSPVp-LAA and ∆LSPVp-HRA) between before and after the creation of the line as the assessment of bi-directional RL block. Methods 76 de-novo non-paroxysmal atrial fibrillation (nPAF) patients underwent BOXI strategy ablation. We initially intended single ring protocol BOXI for all patients. The activation times were measured at LAA and HRA under LSPV pacing at baseline and after application for the RL. Then we compared the change of activation time at LAA (∆LSPVp-LAA) and at HRA (∆LSPVp-HRA) between 2 groups; the group that completed BOXI with the first-pass roof line (fp-RL group) and the other group (Nfp-RL group). Results In 47 patients, the first-pass roof line was successfully obtained and in patients was done with additional application for gap(s) on the roof line to complete BOXI. There was a stepwise increase in mean ∆LSPVp-HRA not ∆LSPVp-LAA. Increased LA diameter, lower LVEF, and thinner LA wall were associated with the first-pass roof line. ∆LSPVp-HRA ≥33ms predict roof line block with high specificity and sensitivity. Conclusions Our findings suggest that the changes conduction times between LSPV and HRA during LSPV pacing help in the assessment of RL block.

Cross-species translational paradigms for assessing positive valence system as defined by the RDoC matrix.

Functions associated with processing reward-related information are fundamental drivers of motivation, learning, and goal-directed behavior. Such functions have been classified as the positive valence system under the Research Domain and Criteria (RDoC) criteria and are negatively impacted across a range of psychiatric disorders and mental illnesses. The positive valence system is composed of three comprehensive categories containing related but dissociable functions that are organized into either Reward Responsiveness, Reward Learning, or Reward Valuation. The presence of overlapping behavioral dysfunction across diagnostic mental disorders is in-part what motivated the RDoC initiative, which emphasized that the study of mental illness focus on investigating relevant behavior and cognitive functions and their underlying mechanisms, rather than separating efforts on diagnostic categories (i.e., transdiagnostic). Moreover, the RDoC approach is well-suited for preclinical neuroscience research, as the rise in genetic toolboxes and associated neurotechnologies enables researchers to probe specific cellular targets with high specificity. Thus, there is an opportunity to dissect whether behaviors and cognitive functions are supported by shared or distinct neural mechanisms. For preclinical research to effectively inform our understandings of human behavior however, the cognitive and behavioral paradigms should have predictive, neurobiological, and pharmacological predictive validity to the human test. Touchscreen-based testing systems provide a further advantage for this endeavor enabling tasks to be presented to animals using the same media and task design as in humans. Here, we outline the primary categories of the positive valence system and review the work that has been done cross-species to investigate the neurobiology and neurochemistry underlying reward-related functioning. Additionally, we provide clinical tasks outlined by RDoC, along with validity and/or need for further validation for analogous rodent paradigms with a focus on implementing the touchscreen-based cognitive testing systems.

18F-FDG PET/CT in the diagnosis of infective endocarditis

Abstract Background Increased uptake of 18F-fluorodeoxyglucose (FDG) by positron-emission tomography/computed tomography (PET/CT) is one of the diagnostic criteria for prosthetic valve endocarditis (PVE). However, there is limited data on the diagnostic value of FDG PET/CT in diagnosing native valve endocarditis (NVE) and semi-quantitative parameters of FDG uptake. Purpose The objective of this retrospective study was to evaluate the accuracy of FDG PET/CT and additional value of semi-quantitative measures of FDG uptake in the detection of NVE and PVE. Methods All patients who had undergone FDG PET/CT due to suspected infective endocarditis (IE) at a single tertiary referral center between 2010 and 2020 were identified. Additional patients who underwent FDG PET/CT for detecting an infection focus were included as controls. Clinical reports were evaluated and images were analyzed for maximal standardized uptake value (SUVmax) and target to background ratio (TBR = SUVmax normalized to mean blood pool SUV) around valves. The scan was defined positive based on either clinical reading or previously proposed cut-off values for SUVmax and TBR (>4.2 and >2.1, respectively). The final diagnosis of IE was defined based on all available information in electronic medical records. Results The study cohort included 16 patients with suspected NVE (median age 58 years, 88% male), 24 patients with suspected PVE (median age 72 years, 92% male) and 37 patients who underwent FDG PET/CT for detecting an infectious focus (8 with a prosthetic valve). The final diagnosis was definite IE in 25 (14 PVE), possible IE in 7 (6 PVE), and no IE in 45 (12 with a prosthetic valve). Overall, clinical reading of FDG PET/CT identified 17 (sensitivity 68%) of definite IE with a specificity of 91% and accuracy of 83%. Compared with NVE, the sensitivity of FDG PET/CT was higher in the detection of PVE (46% vs. 86%) whereas specificity was lower (97% vs. 75%) resulting in small difference in accuracy (84% vs. 81%). In PVE, SUVmax had higher sensitivity (93%) than clinical reading, resulting in the same accuracy (81%) despite lower specificity (67%). A combination of either positive clinical reading or positive SUVmax identified all patients with PVE (sensitivity 100%). In NVE, TBR had higher sensitivity (64%) than clinical reading, but accuracy (66%) and specificity (67%) were significantly lower. Conclusions FDG PET/CT shows high sensitivity in the detection of PVE and high specificity in the detection of NVE in a real-world clinical cohort. Semi-quantitative parameters of FDG uptake can improve sensitivity of detecting PVE maintaining diagnostic accuracy. However, in NVE semi-quantitative parameters were associated with a prominent reduction in specificity.

A streamlined protocol for exercise right heart catheterisation for the diagnosis of heart failure with preserved ejection fraction

Abstract Background Heart failure with preserved ejection fraction (HFpEF) can be a challenging diagnosis. The gold standard diagnostic test is exercise right heart catheterisation (eRHC), with HFpEF confirmed if pulmonary capillary wedge pressure (PCWP) >15mmHg at rest or ≥25mmHg with exercise. However, many centres do not perform eRHC and the current, well-established, nine-minute eRHC protocol can be time consuming and costly. Purpose We sought to determine whether an abbreviated 3-minute eRHC protocol is sufficient for accurate diagnosis of HFpEF, thus allowing simplification, reduced patient risk and costs, and broader uptake of eRHC across centres. Methods Patients undergoing eRHC between 09/09/2008 and 18/07/2023 at a tertiary hospital in Australia were retrospectively analysed. Haemodynamic measurements at three minutes of exercise were correlated with peak measurements. Results Overall cohort of n=532, with a mean age of 64 ± 12 years, and 60% female. Compared to PCWP ≥25mmHg at peak exercise, measurement of PCWP at 3 minutes yielded an area under the receiver-operating characteristic curve of 0.92±0.01, P<0.0001. PCWP ≥25mmHg at 3 minutes has a high specificity (97%) but moderate sensitivity (65%) for diagnosis of HFpEF; PCWP ≥21 mmHg at 3 minutes has good specificity (83%) and sensitivity (85%) for predicting peak PCWP ≥25mmHg. Using the 3-minute protocol with PCWP cut-off of 21mmHg, 86% of participants were correctly diagnosed (Figure 1). There were strong positive correlations in PCWP (R2=0.6922), cardiac output (R2=0.5543) and pulmonary vascular resistance (R2=0.6516) between 3 minutes and peak exercise (Figure 2). Conclusions Abbreviated 3-minute eRHC protocol has good diagnostic potential in HFpEF, with strong correlations in haemodynamics, when compared to peak exercise. This suggests that simplification of the current HFpEF protocol to 3 minutes exercise could be a valid strategy to enable improved uptake and maximise resource utilisation while maintaining diagnostic accuracy.Figure 1.Sankey plot of PCWP valuesFigure 2.Haemodynamics during eRHC

Using anginal symptoms to predict underlying etiology in patients with angina and no obstructive coronary disease (ANOCA)

Abstract Background Patients with angina with no obstructive coronary disease (ANOCA) can present with exertional anginal symptoms or angina at rest. Invasive coronary functional angiography (CFA) is used to diagnose endothelial-dependent and independent coronary microvascular dysfunction (CMD), microvascular spasm, and epicardial spasm. Historically, the type of anginal symptoms has been thought to be suboptimal in predicting the etiology of ANOCA. However, the sensitivity, specificity, predictive value, and correlation of angina type related to CFA abnormalities are unknown. We hypothesize that patients with predominantly exertional angina are more likely to have endothelial-independent and dependent CMD, and patients with angina at rest are more likely to have microvascular or epicardial spasm. Objective To explore sensitivity, specificity, predictive value, and correlation of angina type with underlying etiology of ANOCA as diagnosed by CFA. Methods A prospective registry-based cohort study of consecutive CFA performed in 197 ANOCA patients between 2020 and 2023 using the Doppler-tipped guidewire method. ANOCA was defined as <50% stenosis in any major epicardial artery at time of CFA. Patients were classified based on CFA diagnosis: (1) endothelial-independent CMD (coronary flow reserve [CFR] < 2.5 in response to adenosine), (2) endothelial-dependent CMD (coronary blood flow [CBF] < 50% or no change in vessel diameter in response to 54mcg intracoronary acetylcholine), (3) microvascular (<90% constriction) and epicardial spasm (>90% constriction) to 108 mcg intracoronary acetylcholine. Patients were categorized based on anginal symptoms (exertional angina or at rest angina) documented at cardiologist visit prior to CFA. McNemar’s test and Spearman correlations were performed for both types of angina and the 3 ANOCA underlying diagnoses. Results Among 197 ANOCA patients, the average age was 56.5 ± 11.9 years and 91% were female. Angina at rest had a high specificity (91.3%) but a low sensitivity (25.2%) for the diagnosis of spasm with a PPV of 90.5 (Table 1). There was a significant correlation between angina at rest and spasm (p=0.012). Angina at rest had a similarly intermediate sensitivity and specificity for endothelial independent and dependent CMD. Exertional angina had similar sensitivity, specificity, and a high PPV for endothelial independent and dependent CMD. There was a significant correlation between exertional angina and endothelial dependent CMD (p=0.025). McNemar’s test was statistically significant (p<0.001) for all analyses. Conclusion ANOCA patients with symptoms of angina at rest had a significant correlation with coronary spasms and exertional angina correlates with endothelial-dependent CMD. We demonstrate that angina at rest can be 90% specific for the diagnosis of spasm. Our findings warrant further investigation but suggest that empiric therapy based on angina type is a reasonable first step before invasive testing.

National Early Warning Score 2 to Predict ICU Admission of COVID-19 Patients in Bangladeshi Cohort

Objective: To assess the predictive accuracy of the National Early Warning Score 2 (NEWS 2) for early prediction of clinical deterioration (ICU admission) of COVID-19- infected patients in Bangladesh. Methods: Data from 244 patients with COVID-19 infection admitted from July 2021- September 2021, to the Aichi Hospital Ltd, Bangladesh, were retrospectively reviewed. Consecutive adult patients with laboratory-confirmed COVID-19 initially admitted to non- ICU wards were included. Categorical and quantitative variables were expressed as numbers (percentages) and median (interquartile range, IQR). We evaluated the predictive performance of NEWS2 to predict ICU admission by comparing the area under the receiver operating characteristics curve (AUROC) at thresholds 5 and 7 and assessed its association with ICU admission by performing multivariate logistic analyses. STATA conducted all the statistical analyses. Results: Among the included 218 patients, 68 patients were transferred to ICU. The AUROC was 0.96 (Standard error 0.01, 95% confidence interval 0.93-0.98), revealing that NEWS2 at hospital admission was a good predictor of ICU admission. A NEWS2 threshold of 5 had higher sensitivity than a threshold of 7 (72.06% and 29.41%). A point of 7 also had high specificity (99.33% and 63.33%) and a high positive predictive value than a threshold of 5 (95.24% vs. 47.12%). The NEWS2 entries 5 and 7 were related to ICU admission found in multivariate logistic regression analysis. Conclusion: Our results suggest that NEWS2 at hospital admission is a good predictor for ICU admission of COVID-19 patients in Bangladesh. Screening patients at admission with this tool may identify patients at risk of clinical deterioration and help in better management. EWMCJ Vol. 12, No. 1&2, January-July 2024: 60-65

ESC Heart Failure Association age-adjusted natriuretic peptide thresholds for a new diagnosis of heart failure: diagnostic accuracy study

Abstract Background Natriuretic peptide (NP) testing is an important part of the heart failure (HF) diagnostic pathway. The European Society of Cardiology (ESC) HF guidelines currently recommend a NT-proBNP cut-off <125pg/mL to rule out HF in the community.1 However, NP level increases with age and an NT-proBNP above 125pg/mL is common in older people in the general population. The ESC Heart Failure Association (HFA) recently published a practical algorithm for early diagnosis of HF recommending age-specific rule-in NT-proBNP thresholds: ≥125pg/ml for patients aged under 50 years, ≥250pg/ml for patients aged 50-75 years, and ≥500pg/ml for patients over 75 years (Figure 1).2 Purpose Our aim was to assess NT-proBNP test performance for HF diagnosis at the ESC HFA age-specific thresholds. Method We analysed our existing diagnostic accuracy study dataset containing primary care data from the Clinical Practice Research Datalink (CPRD) linked to inpatient Hospital Episode Statistics (HES) inpatient data between 2004 and 2018. NT-proBNP results were taken from the GP record (index test) and the primary outcome (reference standard) was a HF diagnostic code entered in CPRD or HES within six months of the test. NT-proBNP accuracy for HF was assessed by calculating sensitivity, specificity, positive predictive values (PPV), and negative predictive value (NPV). Receiver operating characteristic (ROC) curves were plotted to assess overall test performance. Results In total, 229,580 patients had an NP test and 21,102 (9.2%) were diagnosed with HF. The current ESC NT-proBNP threshold ≥125pg/mL had sensitivity 94.6% (95% confidence intervals [CI] 94.2 to 95.0), specificity 50.0% (95%CI 49.7 to 50.3), PPV 16.4% (16.1 to 16.6), NPV 98.9% (98.8 to 99.0)). Area under the ROC curve was 0.874 (0.871 to 0.877). Age-specific NT-proBNP thresholds performance: Below 50 years (≥125pg/mL): 32,882 people tested, HF prevalence 3.8%. Sensitivity 83.5 % (81.3 to 85.5), specificity 77.6% (77.2 to 78.1), PPV 13.0% (12.2 to 13.7), NPV 99.2% (99.0 to 99.3). 50 to 75 years ≥250pg/mL: 105,771 people tested, HF prevalence 9.8%. Sensitivity 88.5% (87.9 to 89.1), specificity 67.8% (67.5 to 68.0), PPV 23.1% (22.6 to 23.5), NPV 98.2% (98.1 to 98.3). Above 75 years ≥500pg/mL: 16,694 people tested, HF prevalence 17.5%. Sensitivity 84.4% (83.0 to 85.7), specificity 63.5% (62.7 to 64.3), PPV 32.8% (31.7 to 33.9), NPV 95.0% (94.6 to 95.5). Conclusion The age-specific NT-proBNP thresholds in the HFA practical algorithm had higher specificity so fewer people without HF would be referred, reducing demand for echocardiography and cardiology assessment overall meaning people with HF could be seen earlier. However, lower sensitivity means some cases of HF would initially be missed. NT-proBNP was a reliable ‘rule-out’ test at population level. The optimal NT-proBNP threshold will depend on the priorities and capacity of the national healthcare system.

Characterization of myocardial ischemia-reperfusion injury in a pig model with novel CW T1r and RAFF2 MRI methods in 1.5T

Abstract Introduction Myocardial ischemia-reperfusion injury (IRI) occurs after myocardial infarction when reperfusion aggravates the initial damage in myocardium by vast infiltration of inflammatory cells and elevated oxidative stress in myocardium that may lead to cardiac remodeling, heart failure and even death [1]. Magnetic resonance imaging (MRI) can accurately determine the anatomy of the heart and to characterize myocardial tissue. Novel endogenous rotating frame MRI methods, including continuous wave (CW) T1r and relaxation along a fictitious field with rank 2 (RAFF2), have been shown to characterize MI and other CVD in mouse and human myocardium with high sensitivity and specificity [2,3]. CW T1r and RAFF2 contrasts are occurring during radio frequency (RF) pulse (in kHz range) making them to be sensitive to slow molecular motions [2]. Comparing to conventional endogenous T1 and T2 methods, these are occurring after the RF pulse (in Larmor frequency (MHz) range) and therefore, these are non-selectively sensitive to different correlation times [4]. Golden standard to image MI is to use contrast agent (CA) in late gadolinium enhancement (LGE) method [2,4]. Using CA has drawbacks and therefore, there is an urgent need for optimizing the endogenous MRI methods in clinical practice. Purpose To characterize myocardial IRI in a pig model and to determine the sensitivity of both novel and conventional cardiac MRI methods. Methods IRI operation was done in 7 pig (30-35 kg) by occluding the LAD with a balloon catheter for 50 minutes. Imaging was done with 1.5T (MAGNETOM Aera) at 3 days and 28 days after the operation. Pig hearts were imaged with conventional native T1, T2, LGE, and with novel CW T1r and RAFF2 methods in both time points. These were imaged before CA injection and LGE was done 25 minutes after CA injection. Relaxation time mappings were done in a pixel-by-pixel manner with monoexponentially fitting. Region-of-interest (ROI) analysis was done to determine the relaxation times in IRI and remote areas. LGE was used to confirm the IRI areas in the myocardium. Relative relaxation time difference (RRTD)-values were calculated with the function of (((Relaxation Time of IRI area) – (Relaxation Time of Remote Area))/ (Relaxation Time of Remote Area)) to determine the contrast difference between IRI and remote areas. Results Relaxation times were elevated in IRI areas compared to remote areas (Figure 1A). Novel rotating frame RRTD-values were significantly higher than RRTD-values in conventional methods. This means that novel methods have larger contrast difference between IRI and remote areas compared to conventional methods, which indicates that novel methods are sensitive to characterize IRI area (Figure 1B). The location of MI areas was similar in both novel rotating frame and conventional methods compared to LGE (Figure 2). Conclusion Novel rotating frame relaxation time methods are sensitive to characterize the IRI area in pig model.Relaxation times, RRTD-valuesRelaxation times maps and LGE

Salivary antigens rPagSP02 and rPagSP06 are a reliable composite biomarker for evaluating exposure to Phlebotomus argentipes in Sri Lanka

Phlebotomus argentipes is the established vector of leishmaniasis in the Indian sub-continent. Antibodies to sand fly salivary antigens are biomarkers for vector-host exposure in leishmaniasis-endemic regions. Ph. argentipes transmits Leishmania donovani in Sri Lanka, primarily causing cutaneous leishmaniasis (CL). Our study compared the performance of salivary gland homogenate (SGH) from a lab-reared local strain of Ph. argentipes females to a composite recombinant salivary biomarker (rPagSP02 + rPagSP06) in a CL-endemic population. Sera from 546 healthy individuals, 30 CL patients, and 15 non-endemic individuals were collected. Western blot analysis of Ph. argentipes SGH identified immunogenic bands between 15 kDa and 67 kDa, with bands of predicted molecular weight ∼of 15 kDa (SP02) and ∼28–30 kDa (SP06) as the major antibody targets. Indirect ELISAs using SGH or rPagSP02 + rPagSP06 antigens showed high sensitivity (96.7%) and specificity (100%), detecting comparable seropositivity in endemic populations. rPagSP02 + rPagSP06 exhibited enhanced discriminatory ability, supported by a strong positive correlation (r = 0.869) with SGH. Our findings indicate that the composite rPagSP02 + rPagSP06 salivary biomarker effectively identifies Ph. argentipes exposure in individuals living in Sri Lanka, showing promising potential for use in surveillance. These findings should be further validated to confirm the epidemiological applications in leishmaniasis-endemic regions.

Functional Mechanism and Clinical Implications of lncRNA LINC-PINT in Delayed Fracture Healing

Background Fracture healing can be impeded or even compromised by various factors, resulting in a growing number of patients suffering. The lncRNA LINC-PINT has garnered attention for its latent role in enhancing fracture healing, but its specific functions in this process remain unclear. Objectives The primary objective of this study is to investigate the clinical relevance and underlying molecular mechanisms of LINC-PINT in delayed fracture healing (DFH), while also assessing its potential as an early diagnostic biomarker. Materials and methods The expression levels of LINC-PINT were measured in the serum of DFH patients and those with normal fracture healing using RT-qPCR. In MC3T3-E1 cells, the study investigated the influence on the expression of differentiation-related protein, cell viability, and apoptosis through the modulation of LINC-PINT and miR-324-3p. To elucidate the targeting relationship between LINC-PINT, miR-324-3p, and BMP2, a dual-luciferase reporter assay was employed. Results The findings revealed a significant downregulation of LINC-PINT expression in DFH patients. LINC-PINT showed high sensitivity and specificity as a diagnostic marker for DFH. In MC3T3-E1 cells, LINC-PINT overexpression markedly enhanced the expression levels of ALP, OCN, Runx2, and OPN, improved cell viability, and inhibited apoptosis. LINC-PINT negatively regulated miR-324-3p, and the effects of LINC-PINT were counteracted by miR-324-3p. LINC-PINT was found to regulate BMP2 by targeting miR-324-3p. Conclusion LINC-PINT could serve as an early diagnostic biomarker for DFH and slow the progression of DFH by modulating BMP2 through the targeted regulation of miR-324-3p. This research presents new molecular targets for the diagnosis and treatment of DFH.

Novel parameters for micra transcatheter pacing system implantation on the right ventricular mid-septum

Abstract Background No procedural parameters for Micra transcatheter pacing system (Micra TPS) implantation on the right ventricular mid-septum have been established though it is encouraged for safety. Purpose We aimed to investigate procedural predictors for Micra TPS implantation on the ventricular mid-septum. Methods This is a single-center, retrospective and observational study. Eighty-two patients who underwent Micra TPS implantation were enrolled. The implantation sites of the ventricular septum were classified into basal, mid and apical-septum in the right anterior oblique 35° image. Predictors for mid-septal implantation were evaluated using univariable and multivariable logistic regression analysis which involved explanatory variables considered clinically important. The explanatory variables included the angle between the axis of a device cup of a delivery catheter and the tangent of the ventricular septum in the left anterior oblique 45° image (defined as angle θ). The diagnostic cut-off value of the significant predictor for mid-septal Micra implantation was assessed using receiver operating characteristic (ROC) curve analysis. Results Micra TPS was implanted on the ventricular mid-septum in 61 patients (74.4%), the apical-septum in 21 patients (25.6%), and the basal-septum in no patients. In univariable logistic regression analysis, each angle θ and α-loop shape appearance of a delivery catheter was significantly associated with mid-septal implantation (Odds ratio [OR], 1.1; 95% confidential interval [CI], 1.02-1.10; P=0.002, and OR, 4.6; 95% CI, 1.63-13.6; P=0.005, respectively). After multivariable adjustment, both angle θ and α-loop shape appearance were independently associated with mid-septal implantation (adjusted OR, 1.1; 95% CI, 1.01-1.09; P=0.010, and adjusted OR, 3.3; 95% CI, 1.01-11.4; P=0.048, respectively). Because no Micra TPS was implanted on the basal-septum, an ROC curve for binary outcome of mid-septal and apical-septal implantation was drawn. The ROC curve showed a cut-off value of angle θ for Micra TPS implantation on the ventricular mid-septum was 22° with high sensitivity and specificity at 80.0% and 70.0% (area under the curve= 0.781). Conclusion The angle θ ≧ 22°and the α-loop shape appearance of a delivery catheter may be useful predictors for Micra TPS implantation on the ventricular mid-septum.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae (and Its Resistance to Ciprofloxacin): Validation of a Molecular Biology Tool for Rapid Diagnosis and Treatment

Background and Objectives: Neisseria gonorrhoeae and Chlamydia trachomatis can cause similar clinical syndromes and may coexist in infections. In emergency medicine, empirical treatment targeting both pathogens is often employed, potentially contributing to antibiotic resistance. Gonococcal resistance has emerged against first-line antimicrobials, necessitating prior testing for fluoroquinolone susceptibility. Certest Biotec developed two molecular diagnostic products for simultaneous detection: VIASURE C. trachomatis & N. gonorrhoeae Real-Time PCR Detection Kit and VIASURE Neisseria gonorrhoeae Ciprofloxacin-Resistant Real-Time PCR Detection Kit. To evaluate these products, clinical performance assessments were conducted at the Clinical Microbiology Laboratory of Miguel Servet University Hospital in Zaragoza, Spain. Results and Conclusions: Both VIASURE assays under study demonstrated high clinical sensitivity and specificity compared to reference molecular assays and Sanger sequencing. These kits offer an accurate diagnosis, facilitating appropriate treatment choices while addressing concerns about emerging antibiotic resistance. Methods: A total of 540 clinical samples from 540 patients already characterized as positive or negative for CT and NG by a molecular assay and by antibiotic susceptibility testing for ciprofloxacin using a concentration gradient diffusion method were used for the clinical evaluation. In cases where sensitivity results were unavailable, conventional PCR and Sanger sequencing were employed.

Neutrophil-Mimetic Upconversion Photosynthetic Nanosystem Derived from Microalgae for Targeted Treatment of Thromboembolic Stroke.

Thromboembolic stroke constitutes the majority of brain strokes, resulting in elevated mortality and morbidity rates, as well as significant societal and economic burdens. Although intravenous thrombolysis serves as the standard clinical treatment, its narrow therapeutic window and the inflammatory response induced by tissue plasminogen activator (tPA) administration limit its efficacy. In the initial stages of stroke, the abrupt cessation of blood flow leads to an energy metabolism disorder, marked by a substantial decrease in adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) levels, causing irreversible damage to neural cells. In this study, we introduce a neutrophil-mimetic, microalgae-derived upconversion photosynthetic nanosystem designed for targeted treatment of thromboembolic stroke. This system features upconversion nanoparticles coated with a thylakoid membrane and wrapped in an activated neutrophil membrane, further decorated with ROS-responsive thrombolytic tPA on its surface. The neutrophil-mimetic design facilitates high targeting specificity and accumulation at the thrombus site after intravenous administration. Upon exposure to elevated levels of reactive oxygen species (ROS) at the thrombus location, the nanosystem promptly demonstrated potent thrombolytic efficacy through the surface-modified tPA. Furthermore, near-infrared II (NIR-II) laser irradiation activated the generation of ATP and NADPH, which inhibited inflammatory cell infiltration, platelet activation, oxidative stress, and neuronal injury. This constructed nanoplatform not only showcases exceptional targeting efficiency at the stroke site and controllable release of the thrombolytic agent but also facilitates ATP/NADPH-mediated thrombolytic, anti-inflammatory, antioxidative stress, and neuroprotective effects. Additionally, it offers valuable insights into the potential therapeutic applications of microalgae-based derivatives in managing thromboembolic stroke.

Pericardial adhesion and performance of hemodynamically significant constrictive pericarditis using cine magnetic resonance feature tracking.

To investigate the diagnostic performance of cine magnetic resonance feature tracking (FT) for pericardial adhesions and hemodynamically significant constrictive pericarditis (CP). This retrospective study included patients who underwent cardiac magnetic resonance (CMR) imaging between 2011 and 2021. Twenty-four patients (median age, 73 years; seven females) suspected of having CP and undergoing CMR were selected (CP group). Age-matched 24 participants with heart failure (HF) and 24 healthy volunteers were also analyzed. The FT method assessed septal bounce, the presence of a dip or plateau pattern, and pericardial adhesions. The diagnostic performance, including the area under the receiver operating characteristic curve (AUC) for hemodynamically significant CP, was evaluated against catheterization results, and the adhesion detection in the combined CP and HF control was based on cine tagging or surgical records. Septal bounce was detected in 54% (13/24) of the patients by FT and 38% (9/24) of the patients by visual evaluation in the CP group but not in the HF or normal group (p < 0.001). To detect hemodynamically significant CP, the sensitivity and specificity of each finding were as follows: septal bounce, 85% (11/13) and 82% (9/11); adhesion, 100% (13/13) and 9% (1/11); dip, 77% (10/13) and 36% (4/11); and plateau, 69% (9/13) and 46% (5/11), respectively. The sensitivity and specificity of pericardial adhesion detection using FT and visual evaluation were 96% (23/24) and 100% (24/24), respectively, and the AUCs were 0.998 and 0.999, respectively. CMR-FT demonstrates high sensitivity and specificity for diagnosing hemodynamically significant CP and pericardial adhesions.

Myocarditis prognostic score: a new risk assessment tool in patients with myocarditis

Abstract Background Myocarditis is an inflammatory disease of the myocardium with multiple causes and evolutions. The aim of our study was to design a prognostic multiparametric score in patients with myocarditis, to identify those at higher risk of adverse cardiovascular events (AE). Methods A prospective study was performed enrolling 98 patients with myocarditis: 72 M, 26 F; median age 27 [IQR 20–40]. Patients were divided into 2 groups: complicated (CM) and uncomplicated myocarditis (UM). Six months after hospital admission, cardiac magnetic resonance (CMR) and cardiological consultation were repeated. AE (death, hospitalization for heart failure, heart transplant, ICD implantation, heart failure development) were evaluated at 6 months and after 3 years. Results We found 67 UM and 31 CM. AE were significantly higher in patients with complicated myocarditis. We found a significant correlation between AE and reduced left ventricular ejection fraction (LVEF) at hospital admission, increased GLS, septal LGE at CMR, longer persistence time of increased troponin, LGE extension progression or persistence 6-month CMR. A myocarditis prognostic score was developed on the basis of these parameters. A score ≥ 5 showed higher sensitivity (100%) and specificity (87%) - AUC 1, to identify AE in patients with myocarditis. A score between 3 and 4 showed high sensitivity but low specificity. A score ≤ 2 was associated with low probability of events. Conclusion Our study confirms the high probability of AE in patients with CM and it suggests a myocarditis prognostic score to identify patients at higher risk of AE.