Using neural networks to autonomously assess adequacy in intraoperative cholangiograms.

BACKGROUND: Intraoperative cholangiography (IOC) is a contrast-enhanced X-ray acquired during laparoscopic cholecystectomy. IOC images the biliary tree whereby filling defects, anatomical anomalies and duct injuries can be identified. In Australia, IOC are performed in over 81% of cholecystectomies compared with 20 to 30% internationally (Welfare AIoHa in Australian Atlas of Healthcare Variation, 2017). In this study, we aim to train artificial intelligence (AI) algorithms to interpret anatomy and recognise abnormalities in IOC images. This has potential utility in (a) intraoperative safety mechanisms to limit the risk of missed ductal injury or stone, (b) surgical training and coaching, and (c) auditing of cholangiogram quality. METHODOLOGY: Semantic segmentation masks were applied to a dataset of 1000 cholangiograms with 10 classes. Classes corresponded to anatomy, filling defects and the cholangiogram catheter instrument. Segmentation masks were applied by a surgical trainee and reviewed by a radiologist. Two convolutional neural networks (CNNs), DeeplabV3+ and U-Net, were trained and validated using 900 (90%) labelled frames. Testing was conducted on 100 (10%) hold-out frames. CNN generated segmentation class masks were compared with ground truth segmentation masks to evaluate performance according to a pixel-wise comparison. RESULTS: The trained CNNs recognised all classes.. U-Net and DeeplabV3+ achieved a mean F1 of 0.64 and 0.70 respectively in class segmentation, excluding the background class. The presence of individual classes was correctly recognised in over 80% of cases. Given the limited local dataset, these results provide proof of concept in the development of an accurate and clinically useful tool to aid in the interpretation and quality control of intraoperative cholangiograms. CONCLUSION: Our results demonstrate that a CNN can be trained to identify anatomical structures in IOC images. Future performance can be improved with the use of larger, more diverse training datasets. Implementation of this technology may provide cholangiogram quality control and improve intraoperative detection of ductal injuries or ductal injuries.

The importance of using WHO International Standards to harmonise SARS-CoV-2 serological assays.

The COVID-19 pandemic led to the rapid development of tests to diagnose SARS-CoV-2 infection and ascertain the prevalence of infection, along with the formulation of various treatments and vaccines. Globally, over 220 anti-SARS-CoV-2 serological assays have been developed for laboratory use, and many of these assays are currently used to assess immune responses against SARS-CoV-2. However, because these assays were independently developed by different manufacturers with different target antigens, immunoglobulin detection, technologies, and data reporting approaches, the results are not directly comparable, making it challenging to draw conclusions regarding immune responses at the population level. With deficiencies in assay validation, standardisation, and harmonisation, the inability to use and compare large datasets is becoming a major issue as serological data continue to increase. To help in addressing this issue, WHO established the first International Standard for the anti-SARS-CoV-2 immunoglobulin in late 2020. In this Personal View, we define the WHO International Standard for the anti-SARS-CoV-2 immunoglobulin, summarise the uses of primary versus secondary serology standards, recommend the use of such standards for data harmonisation, and list guidance and resources for using serology standards to improve data comparability.

Standardised quantitative assays for anti-SARS-CoV-2 immune response used in vaccine clinical trials by the CEPI Centralized Laboratory Network: a qualification analysis.

BACKGROUND: Accurate quantitation of immune markers is crucial for ensuring reliable assessment of vaccine efficacy against infectious diseases. This study was designed to confirm standardised performance of SARS-CoV-2 assays used to evaluate COVID-19 vaccine candidates at the initial seven laboratories (in North America, Europe, and Asia) of the Coalition for Epidemic Preparedness Innovations (CEPI) Centralized Laboratory Network (CLN). METHODS: Three ELISAs (pre-spike protein, receptor binding domain, and nucleocapsid), a microneutralisation assay (MNA), a pseudotyped virus-based neutralisation assay (PNA), and an IFN-γ T-cell ELISpot assay were developed, validated or qualified, and transferred to participating laboratories. Immune responses were measured in ELISA laboratory units (ELU) for ELISA, 50% neuralisation dilution (ND50) for MNA, 50% neutralisation titre (NT50) for PNA, and spot-forming units for the ELISpot assay. Replicate assay results of well characterised panels and controls of blood samples from individuals with or without SARS-CoV-2 infection were evaluated by geometric mean ratios, standard deviation, linear regression, and Spearman correlation analysis for consistency, accuracy, and linearity of quantitative measurements across all laboratories. FINDINGS: High reproducibility of results across all laboratories was demonstrated, with interlaboratory precision of 4·1-7·7% coefficient of variation for all three ELISAs, 3·8-19·5% for PNA, and 17·1-24·1% for MNA, over a linear range of 11-30 760 ELU per mL for the three ELISAs, 14-7876 NT50 per mL for PNA, and 21-25 587 ND50 per mL for MNA. The MNA was also adapted for detection of neutralising antibodies against the major SARS-CoV-2 variants of concern. The results of PNA and MNA (r=0·864) and of ELISA and PNA (r=0·928) were highly correlated. The IFN-γ ELISpot interlaboratory variability was 15·9-49·9% coefficient of variation. Sensitivity and specificity were close to 100% for all assays. INTERPRETATION: The CEPI CLN provides accurate quantitation of anti-SARS-CoV-2 immune response across laboratories to allow direct comparisons of different vaccine formulations in different geographical areas. Lessons learned from this programme will serve as a model for faster responses to future pandemic threats and roll-out of effective vaccines. FUNDING: CEPI.

Availability and use of Standards in vaccine development.

Reference materials are critical in assay development for calibrating and assessing their suitability. The devasting nature of the COVID-19 pandemic and subsequent proliferation of vaccine platforms and technologies has meant that there is even a greater need for standards for immunoassay development, which are critical to assess and compare vaccines' responses. Equally important are the standards needed to control the vaccine manufacturing processes. Standardized vaccine characterization assays throughout process development are essential for a successful Chemistry, Manufacturing and Controls (CMC) strategy. In this perspective paper, we advocate for reference material incorporation into assays and their calibration to International Standards from preclinical vaccine development through control testing and provide insight into why this is necessary. We also provide information on the availability of WHO international antibody standards for CEPI-priority pathogens.

A Systematic Review of the Variability in Performing and Reporting Intraprostatic Prostate-specific Membrane Antigen Positron Emission Tomography in Primary Staging Studies.

Context: Prostate cancer (PCa) remains one of the leading causes of cancer-related deaths in men worldwide. Men at risk are typically offered multiparametric magnetic resonance imaging and, if suspicious, a targeted biopsy. However, false-negative rates of magnetic resonance imaging are consistently 18%; therefore, there is growing interest in improving the diagnostic performance of imaging through novel technologies. Prostate-specific membrane antigen (PSMA) positron emission tomography (PET) is being utilised for PCa staging and, more recently, for intraprostatic tumour localisation. However, significant variability has been observed in how PSMA PET is performed and reported. Objective: In this review, we aim to evaluate how pervasive this variability is in trials investigating the performance of PSMA PET in primary PCa workup. Evidence acquisition: Following the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines, we performed an optimal search in five different databases. After removing duplicates, 65 studies were included in our review. Evidence synthesis: Studies dated back as early as 2016, with numerous different source countries. There was variation in the reference standard for PSMA PET, with some using biopsy specimens or surgical specimens, and in some cases, a combination of the two. Similar inconsistencies were noted when studies selected histological definitions of clinically significant PCa, while some omitted their definition altogether. The most significant variations in performing PSMA PET were the radiotracer type, dose, acquisition time after injection, and the PET camera being utilised. Substantial variation in the reporting of PSMA PET was noted, with no consistency in defining what constitutes a positive intraprostatic lesion. Across 65 studies, four different definitions were used. Conclusions: This systematic review has highlighted considerable variation in obtaining and performing a PSMA PET study in the context of primary PCa diagnosis. Given the discrepancy in how PSMA PET was performed and reported, it questions the homogony of studies from centre to centre. Standardisation of PSMA PET is required for this to become a consistently useful and reproducible modality in the diagnosis of PCa. Patient summary: Prostate-specific membrane antigen (PSMA) positron emission tomography (PET) is being utilised for staging and localisation of prostate cancer (PCa); however, there is significant variability in performing and reporting PSMA PET. Standardisation of PSMA PET is required for results to be consistently useful and reproducible for the diagnosis of PCa.

VelcroVax: a "Bolt-On" Vaccine Platform for Glycoprotein Display.

Having varied approaches to the design and manufacture of vaccines is critical in being able to respond to worldwide needs and newly emerging pathogens. Virus-like particles (VLPs) form the basis of two of the most successful licensed vaccines (against hepatitis B virus [HBV] and human papillomavirus). They are produced by recombinant expression of viral structural proteins, which assemble into immunogenic nanoparticles. VLPs can be modified to present unrelated antigens, and here we describe a universal "bolt-on" platform (termed VelcroVax) where the capturing VLP and the target antigen are produced separately. We utilize a modified HBV core (HBcAg) VLP with surface expression of a high-affinity binding sequence (Affimer) directed against a SUMO tag and use this to capture SUMO-tagged gp1 glycoprotein from the arenavirus Junín virus (JUNV). Using this model system, we have solved the first high-resolution structures of VelcroVax VLPs and shown that the VelcroVax-JUNV gp1 complex induces superior humoral immune responses compared to the noncomplexed viral protein. We propose that this system could be modified to present a range of antigens and therefore form the foundation of future rapid-response vaccination strategies. IMPORTANCE The hepatitis B core protein (HBc) forms noninfectious virus-like particles, which can be modified to present a capturing molecule, allowing suitably tagged antigens to be bound on their surface. This system can be adapted and provides the foundation for a universal "bolt-on" vaccine platform (termed VelcroVax) that can be easily and rapidly modified to generate nanoparticle vaccine candidates.

Neutralisation Hierarchy of SARS-CoV-2 Variants of Concern Using Standardised, Quantitative Neutralisation Assays Reveals a Correlation With Disease Severity; Towards Deciphering Protective Antibody Thresholds.

The rise of SARS-CoV-2 variants has made the pursuit to define correlates of protection more troublesome, despite the availability of the World Health Organisation (WHO) International Standard for anti-SARS-CoV-2 Immunoglobulin sera, a key reagent used to standardise laboratory findings into an international unitage. Using pseudotyped virus, we examine the capacity of convalescent sera, from a well-defined cohort of healthcare workers (HCW) and Patients infected during the first wave from a national critical care centre in the UK to neutralise B.1.1.298, variants of interest (VOI) B.1.617.1 (Kappa), and four VOCs, B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta), including the B.1.617.2 K417N, informally known as Delta Plus. We utilised the WHO International Standard for anti-SARS-CoV-2 Immunoglobulin to report neutralisation antibody levels in International Units per mL. Our data demonstrate a significant reduction in the ability of first wave convalescent sera to neutralise the VOCs. Patients and HCWs with more severe COVID-19 were found to have higher antibody titres and to neutralise the VOCs more effectively than individuals with milder symptoms. Using an estimated threshold for 50% protection, 54 IU/mL, we found most asymptomatic and mild cases did not produce titres above this threshold.

WHO International Standard for evaluation of the antibody response to COVID-19 vaccines: call for urgent action by the scientific community.

The first WHO International Standard and International Reference Panel for anti-SARS-CoV-2 immunoglobulin were established by the WHO Expert Committee on Biological Standardization in December, 2020. The WHO International Antibody Standards are intended to serve as global reference reagents, against which national reference preparations or secondary standards can be calibrated. Calibration will facilitate comparison of results of assays (eg, of the neutralising antibody response to candidate COVID-19 vaccines) conducted in different countries. Use of these standards is expected to contribute to better understanding of the immune response, and particularly of the correlates of protection. This Personal View provides some technical details of the WHO Antibody Standards for SARS-CoV-2, focusing specifically on the use of these standards for the evaluation of the immune response to COVID-19 vaccines, rather than other applications (eg, diagnostic or therapeutic). The explanation with regard to why rapid adoption of the standards is crucial is also included, as well as how funders, journals, regulators, and ethics committees could drive adoption in the interest of public health.

Analysis of Serological Biomarkers of SARS-CoV-2 Infection in Convalescent Samples From Severe, Moderate and Mild COVID-19 Cases.

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study is to identify biomarkers of humoral immunity that could be used to differentiate severe from mild or asymptomatic SARS-CoV-2 infections. Some of these biomarkers could be used to define CoP in further serological studies using samples from vaccination breakthrough and/or re-infection cases. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (IU) for virus neutralisation assays or in Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG/IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and an electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD/S antibodies. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

Interpretation, confidence and application of the standardised terms: Identified, Probable, Possible, Exclude and Insufficient in forensic odontology identification.

Forensic odontology identification scales are used to express certainty of identifications of deceased persons. These standardized scales are assumed to convey unambiguous expert opinions and facilitate communication between forensic odontologists and end users. However, to date no studies have investigated how the experts interpret and use these scales. Forensic odontology identification scales are used to express certainty of identifications of deceased persons. These standardized scales are assumed to convey unambiguous expert opinions and facilitate communication between forensic odontologists and end users. However, to date no studies have investigated how the experts interpret and use these scales. This paper aims to examine the interpretation of the DVISYS forensic identification scale and choices of the levels in the scale subsequent to, and derived from, comparison of pairs of dental radiographs by extending the analysis of the data collected in the study by Page and Lain et. al. 2017. The studied variables: self-reported confidence, forced binary decision of match and non-match, choice of level in the DVISYS scale (Identified, Probable, Possible, Insufficient and Exclude) were further analysed in this study using mixed models for relationships between the choices of level in the identification scale and the fundamental beliefs of likelihood of identification. The results of this further analysis showed that the reported confidence of the decisions was correlated to the difficulty of cases, and as confidence decreased the use of less definitive terms ('Probable', 'Possible' and 'Insufficient') increased. 'Probable' and 'Possible' were used mainly in underlying beliefs below that of 'Identified' whereas 'Insufficient' was used mainly to convey a sublevel of 'Exclude'. The use of 'Insufficient' in this study was not consistent with the prescribed definition of the term. The participants of the original study were not aware of the difficulty grading of the cases nor were required to grade them, however the reported confidence was systematically correlated to difficulty. Furthermore, indicated confidence level was correlated with choice of level on the scale in general, but the interpretation of the definition and application of the terms varied. The findings reported here contribute to the foundational knowledge of factors governing the interpretation and application of the DVISYS forensic odontology identification scale and suggest that this scale may need to be modified.

Incidental detection of prostate cancer with computed tomography scans.

Prostate cancer (PCa) is the second most frequent type of cancer found in men worldwide, with around one in nine men being diagnosed with PCa within their lifetime. PCa often shows no symptoms in its early stages and its diagnosis techniques are either invasive, resource intensive, or has low efficacy, making widespread early detection onerous. Inspired by the recent success of deep convolutional neural networks (CNN) in computer aided detection (CADe), we propose a new CNN based framework for incidental detection of clinically significant prostate cancer (csPCa) in patients who had a CT scan of the abdomen/pelvis for other reasons. While CT is generally considered insufficient to diagnose PCa due to its inferior soft tissue characterisation, our evaluations on a relatively large dataset consisting of 139 clinically significant PCa patients and 432 controls show that the proposed deep neural network pipeline can detect csPCa patients at a level that is suitable for incidental detection. The proposed pipeline achieved an area under the receiver operating characteristic curve (ROC-AUC) of 0.88 (95% Confidence Interval: 0.86-0.90) at patient level csPCa detection on CT, significantly higher than the AUCs achieved by two radiologists (0.61 and 0.70) on the same task.

Incidental findings in and around the prostate on prostate MRI: a pictorial review.

Prostate MRI has seen rapid growth in use in recent years as an advanced diagnostic modality to detect focal areas of clinically significant prostate cancer, to identify an area for targeted biopsy and to guide management and surveillance. The increase in use has also led to increased diagnosis of incidental lesions arising from structures around the prostate. These incidental findings may be related to the genitourinary system or non- genitourinary system and may have a benign aetiology which needs no additional follow-up, or it may require surveillance and management. The field of view in a multiparametric prostate MRI includes other pelvic organs, neurovascular bundles, bowel, lymph nodes and bones. Being familiar with standard MRI characteristics and a sound knowledge of anatomy of the prostate and surrounding structures can help in distinguishing normal anatomy from pathology. Given that patients undertaking a prostate MRI are usually a cohort with increased anxiety from their known or suspicion of prostate cancer, it is important that radiologists are familiar with these common incidental findings to minimise anxiety to the patient, have a well-informed discussion with the referring clinician and reduce costs associated with unnecessary further testing and follow-up of benign incidental findings. Additionally, being able to diagnose more serious incidental pathologies early can be life-saving and potentially significantly alter patient management.

Passive immunisation of convalescent human anti-Zika plasma protects against challenge with New World Zika virus in cynomolgus macaques.

Zika virus (ZIKV) causes neurological complications in susceptible individuals, highlighted in the recent South American epidemic. Natural ZIKV infection elicits host responses capable of preventing subsequent re-infection, raising expectations for effective vaccination. Defining protective immune correlates will inform viral intervention strategies, particularly vaccine development. Non-human primate (NHP) species are susceptible to ZIKV and represent models for vaccine development. The protective efficacy of a human anti-ZIKV convalescent plasma pool (16/320-14) developed as a candidate reference material for a WHO International Standard was evaluated in macaques. Convalescent plasma administered to four cynomolgus macaques (Macaca fascicularis) intra-peritoneally 24 hrs prior to sub-cutaneous challenge with 103 pfu ZIKVPRVABC59 protected against detectable infection, with absence of detectable ZIKV RNA in blood and lymphoid tissues. Passively immunised anti-ZIKV immunoglobulin administered prior to time of challenge remained present only at very low levels 42 days post-challenge. Absence of de novo antibody responses in passively immunised macaques indicate sterilising immunity compared with naïve challenge controls that exhibited active ZIKV-specific IgM and IgG responses post-challenge. Demonstration that the presence of convalescent anti-ZIKV at levels of 400 IU/mL neutralising antibody protects against virus challenge provides a scientific framework for development of anti-ZIKV vaccines and facilitates regulatory approval.