The cyanobacterial lectin, microvirin-N, enhances the specificity and sensitivity of lipoarabinomannan-based TB diagnostic tests.

Tuberculosis (TB) is one of the top ten causes of death globally, despite being treatable. The eradication of TB disease requires, amongst others, diagnostic tests with high specificity and sensitivity that will work at the point of care (POC) in low-resource settings. The TB surface glycolipid antigen, mannose-capped lipoarabinomannan (ManLAM) currently serves as the only POC molecular diagnostic biomarker suitable for use in low cost immunoassays. Here, we demonstrate the high affinity and exceptional specificity of microvirin-N (MVN), a 14.3 kDa cyanobacterial lectin, toward H37Rv TB ManLAM and utilize it to develop a novel on-bead ELISA. MVN binds to ManLAM with sub-picomolar binding affinity, but does not bind to other variants of LAM expressed by non-pathogenic mycobacteria - a level of binding specificity and affinity that current commercially available anti-LAM antibodies cannot achieve. An on-bead ELISA was subsequently developed using MVN-functionalized magnetic beads which allows for the specific capture of ManLAM from human urine with a limit of detection (LOD) of 1.14 ng mL-1 and no cross-reactivity when tested with PILAM, a variant of LAM found on non-pathogenic mycobacteria.

Reconstruction of Perioral Defects After Mohs Micrographic Surgery or Excision: A Systematic Review of the Literature.

BACKGROUND: Although many repair methods for postsurgical lip defects have been described, the literature lacks a comprehensive review of these methods. OBJECTIVE: To perform a systematic review of lip defect repair methods after Mohs surgery or excisions. MATERIALS AND METHODS: Terms related to perioral anatomy, Mohs surgery and excision, and reconstruction were used to search 8 databases. Articles were included if they reported postsurgical lip repair data for 4 or more patients, were in English, and were published from 2004 onward. Two reviewers screened all titles and abstracts, followed by the full texts of the remaining articles. Data were then extracted including author specialties, study design, demographic, tumor, and defect information, surgical procedures, outcomes, and complications. RESULTS: Forty-two studies were eligible, including a randomized trial, 25 case series, and 16 cohort studies. Most were written by dermatologic or plastic surgeons, and most studies were small, with an average subject number of 61. Very few studies used structured outcome measures. Many repair methods were described, the most common of which were linear closures and various flaps. CONCLUSION: Many repair methods for lip defects have been published, but overall, the quality of the available evidence is low.

Periocular Mohs Reconstruction by Lateral Canthotomy With Inferior Cantholysis: A Retrospective Study.

BACKGROUND: Most eyelid defects after Mohs micrographic surgery are referred to oculoplastic surgery or plastic surgery for reconstruction, but growing evidence suggests the safety of such repairs performed by dermatologic surgeons is equivalent if not better. Lateral canthotomy with inferior cantholysis may be used by the dermatologic surgeon to reconstruct larger lower eyelid defects. OBJECTIVE: To demonstrate lateral canthotomy with inferior cantholysis performed by the dermatologic surgeon can result in safe, functionally and cosmetically acceptable surgical outcomes. MATERIALS AND METHODS: An institutional review board-approved retrospective study of repairs performed by a single dermatologic surgeon between January 2013 and August 2019. Patient demographics, operative and follow-up notes were reviewed. Two cosmetic dermatologists assessed aesthetic results based on final follow-up photographs using a visual analogue scale. RESULTS: Eight cases were included in the analysis. Seventy-five percent of patients were men, with a mean age of 74.1 years old. All tumors were basal cell carcinoma; the mean defect size was 2.4 cm2. No serious complications or postoperative interventions occurred. The median cosmetic score was 85.6 ± 11.5. CONCLUSION: Dermatologic surgeons can safely perform repairs of lower eyelid defects with lateral canthotomy with inferior cantholysis, achieving satisfactory functional and cosmetic outcomes.

Multifocal infantile hemangiomas associated with concomitant giant hepatic rapidly involuting congenital hemangioma.

We present a 9-day-old girl with multifocal cutaneous and hepatic infantile hemangiomas as well as a hepatic rapidly involuting congenital hemangioma. These two distinct vascular tumors have rarely been reported to co-occur. We additionally review the sonographic features that distinguish a hepatic congenital hemangioma from the hepatic infantile hemangioma.

Mohs Micrographic Surgery at Challenging Anatomical Sites.

BACKGROUND: Mohs micrographic surgery (MMS) is often the treatment of choice for skin cancer removal as it maximizes normal tissue sparing and can be paired with a reconstructive approach that optimizes function and cosmesis. Many tumors on the eyelid, nose, ear, and genitals are particularly well suited for MMS but can be challenging for the dermatologic surgeon. OBJECTIVE: To review the complex anatomy, as well as the authors' approach to executing and interpreting Mohs layers, at each of these anatomical sites. METHODS: A review of the literature on MMS of the eyelid, nose, ear, and genitals was performed using the PubMed database and relevant search terms. CONCLUSION: These sites present potential pitfalls for tumor resection and reconstruction, but with the proper technique, the dermatologic surgeon can minimize tumor recurrence and MMS complications. Warning signs for potentially difficult tumor resection can signify when an interdisciplinary approach is warranted.

Metal Affinity-Enabled Capture and Release Antibody Reagents Generate a Multiplex Biomarker Enrichment System that Improves Detection Limits of Rapid Diagnostic Tests.

Multi-antigen rapid diagnostic tests (RDTs) are highly informative, simple, mobile, and inexpensive, making them valuable point-of-care (POC) diagnostic tools. However, these RDTs suffer from several technical limitations-the most significant being the failure to detect low levels of infection. To overcome this, we have developed a magnetic bead-based multiplex biomarker enrichment strategy that combines metal affinity and immunospecific capture to purify and enrich multiple target biomarkers. Modifying antibodies to contain histidine-rich peptides enables reversible loading onto immobilized metal affinity magnetic beads, generating a novel class of antibodies coined "Capture and Release" (CaR) antibody reagents. This approach extends the specificity of immunocapture to metal affinity magnetic beads while also maintaining a common trigger for releasing multiple biomarkers. Multiplex biomarker enrichment is accomplished by adding magnetic beads equipped with CaR antibody reagents to a large sample volume to capture biomarkers of interest. Once captured, these biomarkers are magnetically purified, concentrated, and released into a RDT-compatible volume. This system was tailored to enhance a popular dual-antigen lateral flow malaria RDT that targets Plasmodium falciparum histidine-rich protein-II (HRPII) and Plasmodium lactate dehydrogenase (pLDH). A suite of pLDH CaR antibody reagents were synthesized, characterized, and the optimal CaR antibody reagent was loaded onto magnetic beads to make a multiplex magnetic capture bead that simultaneously enriches pLDH and HRPII from Plasmodium falciparum parasitized blood samples. This system achieves a 17.5-fold improvement in the dual positive HRPII/pan-pLDH detection limits enabling visual detection of both antigens at levels correlating to 5 p/µL. This front-end sample processing system serves as an efficient strategy to improve the sensitivity of RDTs without the need for modifications or remanufacturing.

Magnetically-enabled biomarker extraction and delivery system: towards integrated ASSURED diagnostic tools.

Diagnosis of asymptomatic malaria poses a great challenge to global disease elimination efforts. Healthcare infrastructure in rural settings cannot support existing state-of-the-art tools necessary to diagnose asymptomatic malaria infections. Instead, lateral flow immunoassays (LFAs) are widely used as a diagnostic tool in malaria endemic areas. While LFAs are simple and easy to use, they are unable to detect low levels of parasite infection. We have developed a field deployable Magnetically-enabled Biomarker Extraction And Delivery System (mBEADS) that significantly improves limits of detection for several commercially available LFAs. Integration of mBEADS with leading commercial Plasmodium falciparum malaria LFAs improves detection limits to encompass an estimated 95% of the disease reservoir. This user-centered mBEADS platform makes significant improvements to a previously cumbersome malaria biomarker enrichment strategy by improving reagent stability, decreasing the processing time 10-fold, and reducing the assay cost 10-fold. The resulting mBEADS process adds just three minutes and less than $0.25 to the total cost of a single LFA, thus balancing sensitivity and practicality to align with the World Health Organization's ASSURED criteria for point-of-care (POC) testing.

Accuracy of virtual assessment in hand trauma.

A virtual clinic was developed from an existing telemedicine system to manage hand trauma in the Queen Victoria Hospital, East Grinstead, UK, during the first wave of the COVID-19 pandemic. This study evaluates the accuracy of the assessments made and makes comparisons to the traditional face-to-face clinic. The accuracy of assessment was analysed by comparing diagnosis with findings at surgery. One hundred and eighty-nine virtual assessments conducted by telephone with photographic data or by video were compared to 129 face-to-face assessments conducted prior to the pandemic. There was no difference in the accuracy of virtual and face-to-face clinics for patients treated surgically (p=0.27); treatment was correctly predicted for 87% of the virtual group and 78% of the face-to-face group. However, fewer virtual assessments led to a surgical outcome (p=0.0064); 68% of the virtual group had surgical outcomes compared to 82% of the face-to-face group. Most face-to-face assessments were done by a specialty trainee compared to a range of clinicians in the virtual clinic. Accuracy of assessment among trainees was significantly associated with experience for the virtual (p=0.045) but not face-to-face clinics (p=0.94). Virtual assessment by video versus telephone plus photographs were similarly accurate. We conclude that virtual triage serves as a successful alternative to face-to-face appointments. It is robust and succeeds in reducing footfall to the hospital site in line with recent social distancing measures against COVID-19. We have shown that video conferencing triage is no better than telemedicine triage with telephone and photographs.

A phase synchrony measure for quantifying dynamic functional integration in the brain.

The temporal coordination of neural activity within structural networks of the brain has been posited as a basis for cognition. Changes in the frequency and similarity of oscillating electrical potentials emitted by neuronal populations may reflect the means by which networks of the brain carry out functions critical for adaptive behavior. A computation of the phase relationship between signals recorded from separable brain regions is a method for characterizing the temporal interactions of neuronal populations. Recently, different phase estimation methods for quantifying the time-varying and frequency-dependent nature of neural synchronization have been proposed. The most common method for measuring the synchronization of signals through phase computations uses complex wavelet transforms of neural signals to estimate their instantaneous phase difference and locking. In this article, we extend this idea by introducing a new time-varying phase synchrony measure based on Cohen's class of time-frequency distributions. This index offers improvements over existing synchrony measures by characterizing the similarity of signals from separable brain regions with uniformly high resolution across time and frequency. The proposed measure is applied to both synthesized signals and electroencephalography data to test its effectiveness in estimating phase changes and quantifying neural synchrony in the brain.

Application of mass transfer theory to biomarker capture by surface functionalized magnetic beads in microcentrifuge tubes.

In many diagnostic assays, specific biomarker extraction and purification from a patient sample is performed in microcentrifuge tubes using surface-functionalized magnetic beads. Although assay binding times are known to be highly dependent on sample viscosity, sample volume, capture reagent, and fluid mixing, the theoretical mass transport framework that has been developed and validated in engineering has yet to be applied in this context. In this work, we adapt this existing framework for simultaneous mass transfer and surface reaction and apply it to the binding of biomarkers in clinical samples to surface-functionalized magnetic beads. We discuss the fundamental fluid dynamics of vortex mixing within microcentrifuge tubes as well as describe how particles and biomolecules interact with the fluid. The model is solved over a wide range of parameters, and we present scenarios when a simplified analytical expression would be most accurate. Next, we review of some relevant techniques for model parameter estimation. Finally, we apply the mass transfer theory to practical use-case scenarios of immediate use to clinicians and assay developers. Throughout, we highlight where further characterization is necessary to bridge the gap between theory and practical application.

Rapid concentration and elution of malarial antigen histidine-rich protein II using solid phase Zn(II) resin in a simple flow-through pipette tip format.

Rapid diagnostic tests (RDTs) designed to function at the point of care are becoming more prevalent in malaria diagnostics because of their low cost and simplicity. While many of these tests function effectively with high parasite density samples, their poor sensitivity can often lead to misdiagnosis when parasitemia falls below 100 parasites/µl. In this study, a flow-through pipette-based column was explored as a cost-effective means to capture and elute more Plasmodium falciparum histidine-rich protein II (HRPII) antigen, concentrating the biomarker available in large-volume lysed whole blood samples into volumes compatible with Plasmodium falciparum-specific RDTs. A systematic investigation of immobilized metal affinity chromatography divalent metal species and solid phase supports established the optimal design parameters necessary to create a flow-through column incorporated into a standard pipette tip. The bidirectional flow inherent to this format maximizes mixing efficiency so that in less than 5 min of sample processing, the test band signal intensity was increased up to a factor of twelve from HRPII concentrations as low as 25 pM. In addition, the limit of detection per sample was decreased by a factor of five when compared to the RDT manufacturer's suggested protocol. Both the development process and commercial viability of this application are explored, serving as a potential model for future applications.

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II.

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2(+) (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 µm magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

Use of the activated partial thromboplastin time for heparin monitoring.

The objectives of the present study were to evaluate the relationship between heparin concentration and activated partial thromboplastin time (aPTT) results, define a heparin concentration-derived therapeutic range for each aPTT instrument, compare aPTT- and heparin concentration-guided dosage adjustment decisions, and compare laboratory- and bedside aPTT-guided decisions. In phase 1, 102 blood samples were analyzed for bedside and laboratory aPTTs and heparin concentration (used to establish aPTT therapeutic range). In phase 2, 100 samples were analyzed in the same manner. Correlations for aPTT compared with heparin ranged from 0.36 to 0.82. Dosage adjustment decisions guided by the aPTT agreed with those based on heparin concentration 63% to 80% of the time. Laboratory and bedside aPTT dosage adjustment decisions agreed 59% to 68% of the time. The correlation of aPTT with heparin concentration and agreement between aPTT- and heparin-guided decisions vary with the aPTT instrument. Decisions guided by laboratory aPTT results often disagree with decisions guided by bedside aPTT results.

Correlation of activated clotting time and activated partial thromboplastin time to plasma heparin concentration.

STUDY OBJECTIVE: To determine the correlation between activated clotting time (ACT) or activated partial thromboplastin time (aPTT) and plasma heparin concentration. DESIGN: Two-phase prospective study. SETTING: University-affiliated community hospital. PATIENTS: Thirty patients receiving continuous-infusion intravenous heparin. INTERVENTIONS: Measurement of ACT, aPTT and plasma heparin concentrations. MEASUREMENTS AND MAIN RESULTS: Linear and log linear correlations were determined between clotting time tests and heparin concentrations. Linear correlations yielded r values of 0.58 for ACT (p=0.008) and 0.89 for aPTT (p=0.0001). Log linear correlations yielded r values of 0.60 for ACT (p=0.005) and 0.88 for aPTT (p=0.0001). A decision analysis was performed to determine possible consequences of dosage adjustments based on either test in relationship to the decision based on plasma heparin concentration. The decision analysis based on ACT disagreed with corresponding decisions based on plasma heparin concentration in 15 of 30 patients; 13 disagreements may have increased the risk of bleeding, and the other 2 may have increased the risk of thrombosis. Decisions based on aPTT disagreed with corresponding decisions based on plasma heparin concentration in 13 of 30 patients; 2 disagreements may have increased the risk of bleeding, and the other 11 may have increased the risk of thrombosis. CONCLUSION: There are significant statistical linear and log linear correlations between both clotting time tests and plasma heparin concentrations, with aPTT showing stronger correlation than ACT. However, decisions regarding heparin therapy based on ACT may increase a patient's risk of bleeding, whereas decisions based on aPTT may increase the risk of thrombus progression or rethrombosis.

Different heparin lots: Does it matter?

OBJECTIVE: To assess the impact of heparin lot on the correlation between heparin concentration and activated partial thromboplastin time (aPTT), the aPTT therapeutic range, and the heparin level. DESIGN: Retrospective analysis of data from 2 previous studies. SETTING: Teaching institution with 929 beds. PATIENTS: Ninety-five patients receiving heparin with 5 different lots (study 1) and 35 patients receiving heparin with 3 different lots (study 2). MAIN OUTCOME MEASURES: Laboratory-based aPTT and heparin level by anti-factor Xa analysis. Standard heparin curves were created for each lot. Each patient's heparin level was determined off each standard curve. RESULTS: Correlations between heparin concentration and aPTT ranged from 0.87 to 0.89 (study 1) and 0.86 to 0.87 (study 2). Slopes of regression lines were not significantly different. Therapeutic ranges generated from lot-specific heparin levels were similar. Average bias in heparin levels from varying lots ranged from 0.005 to 0.036 units/mL. CONCLUSIONS: The recommendation to reevaluate the aPTT therapeutic range with each new lot of heparin requires further evaluation.