Mpox outbreak among men who have sex with men in Amsterdam and Rotterdam, the Netherlands: no evidence for undetected transmission prior to May 2022, a retrospective study.

Since May 2022, over 21,000 mpox cases have been reported from 29 EU/EEA countries, predominantly among men who have sex with men (MSM). The Netherlands was the fourth most affected country in Europe, with more than 1,200 cases and a crude notification rate of 70.7 per million population. The first national case was reported on 10 May, yet potential prior transmission remains unknown. Insight into prolonged undetected transmission can help to understand the current outbreak dynamics and aid future public health interventions. We performed a retrospective study and phylogenetic analysis to elucidate whether undetected transmission of human mpox virus (hMPXV) occurred before the first reported cases in Amsterdam and Rotterdam. In 401 anorectal and ulcer samples from visitors to centres for sexual health in Amsterdam or Rotterdam dating back to 14 February 2022, we identified two new cases, the earliest from 6 May. This coincides with the first cases reported in the United Kingdom, Spain and Portugal. We found no evidence of widespread hMPXV transmission in Dutch sexual networks of MSM before May 2022. Likely, the mpox outbreak expanded across Europe within a short period in the spring of 2022 through an international highly intertwined network of sexually active MSM.

Standardization of SARS-CoV-2 Nucleic Acid Amplification Techniques by Calibration and Quantification to the First WHO International Standard for SARS-CoV-2 RNA.

Clinical decision-making regarding isolation of SARS-CoV-2 patients is usually based on semiquantitative cycle-threshold (Ct) values without standardization. However, not all molecular assays produce Ct values, and there is ongoing discussion about whether Ct values can be safely used for decision-making. In this study, we standardized two molecular assays which use different nucleic acid amplification techniques (NAAT): the Hologic Aptima SARS-CoV-2/Flu (TMA) and Roche Cobas 6800 SARS-CoV-2 assays. We calibrated these assays against the first WHO international standard for SARS-CoV-2 RNA by using linear regression of log10 dilution series. These calibration curves were used to calculate viral loads for clinical samples. Clinical performance was assessed retrospectively using samples collected between January 2020 and November 2021, including known positives of the wild-type SARS-CoV-2 virus, the VOCs (alpha, beta, gamma, delta, and omicron) and quality control panels. Linear regression and Bland-Altman analysis showed good correlations for SARS-CoV-2 between Panther TMA and Cobas 6800 when standardized viral loads were used. These standardized quantitative results can benefit clinical decision-making and standardization of infection control guidelines.

Classification and specific primer design for accurate detection of SARS-CoV-2 using deep learning.

In this paper, deep learning is coupled with explainable artificial intelligence techniques for the discovery of representative genomic sequences in SARS-CoV-2. A convolutional neural network classifier is first trained on 553 sequences from the National Genomics Data Center repository, separating the genome of different virus strains from the Coronavirus family with 98.73% accuracy. The network's behavior is then analyzed, to discover sequences used by the model to identify SARS-CoV-2, ultimately uncovering sequences exclusive to it. The discovered sequences are validated on samples from the National Center for Biotechnology Information and Global Initiative on Sharing All Influenza Data repositories, and are proven to be able to separate SARS-CoV-2 from different virus strains with near-perfect accuracy. Next, one of the sequences is selected to generate a primer set, and tested against other state-of-the-art primer sets, obtaining competitive results. Finally, the primer is synthesized and tested on patient samples (n = 6 previously tested positive), delivering a sensitivity similar to routine diagnostic methods, and 100% specificity. The proposed methodology has a substantial added value over existing methods, as it is able to both automatically identify promising primer sets for a virus from a limited amount of data, and deliver effective results in a minimal amount of time. Considering the possibility of future pandemics, these characteristics are invaluable to promptly create specific detection methods for diagnostics.

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.

BACKGROUND: The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. AIM: We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. METHODS: Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. RESULTS: The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive - Global (EVAg), a European Union infrastructure project. CONCLUSION: The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.

Performance evaluation of the Panther Fusion® respiratory tract panel.

BACKGROUND: Respiratory tract infections are among the most common infections during winter season. Rapid diagnostics is required for clinical decision making regarding isolation of patients and appropriate therapy. OBJECTIVES: The aim of this study was to evaluate the analytical and clinical performance characteristics of the Panther Fusion® respiratory panel using published laboratory-developed real-time PCR assays (LDT). STUDY DESIGN: Analytical sensitivity of Panther Fusion® Flu A/B/RSV was assessed by testing dilutions of cell culture isolates. Clinical performance assessment included the complete Panther Fusion® respiratory panel (Flu-A/B/RSV, PIV 1-4 and AdV/hMPV/RV) and consisted of a retrospective and a prospective study-arm. The retrospective evaluation included 201, stored (-80 °C) samples collected between February 2006 and January 2017. Prospective evaluation was performed on 1045 unselected pretreated respiratory tract samples from patients presented to our hospital between November 2017 and May 2018. RESULTS: Analytical sensitivity was generally slightly lower for the Panther Fusion® assays. Clinical specificity and sensitivity was between 96 %-100 % and 71.9 %-100 %, respectively. Discrepant results were found in 146 samples of which 88 samples tested LDT positive / Panther Fusion® negative and 58 samples were LDT negative / Panther Fusion® positive. A total of ten discrepant samples with Ct-values <30 were sequenced to confirm the presence of 7 RV-C not-detected by LDT and 1 RV-A and 2 ADV-2 not detected by Panther Fusion®. CONCLUSIONS: The Panther Fusion® provides a random-access system with continuous loading and much shorter sample-to-answer times compared to LDT, albeit with a slightly less clinical sensitivity compared to the LDT.

The effect of vagus nerve stimulation on cardiorespiratory parameters during rest and exercise.

PURPOSE: Vagus nerve stimulation (VNS) has been successfully applied to reduce seizure frequency in numerous patients with epilepsy. However, various side effects, including dyspnea and bradycardia have been reported, that appear exercise related in some patients. This pilot study aims to obtain insight in the cardiorespiratory effects of VNS during both rest and exercise. METHODS: Patients with a VNS device who experience side effects during exercise are compared with patients without side effects. Respiratory and cardiac parameters measured during rest and exercise include heart rate, breathing frequency and tidal volume. RESULTS: Sixty-two episodes of VNS in five patients with and five patients without side effects were recorded. In addition, five control subjects have been measured. During rest, all subjects showed stable values for the cardiorespiratory parameters. During the first minutes of exercise, heart rate, breathing frequency and tidal volume increased. Thereafter, a steady state was reached again for all subjects. During VNS episodes, eight out of 10 patients showed a small but consistent decrease in heart rate, along with an increase in breathing frequency in eight out of nine patients. Tidal volumes decreased during VNS episodes. These effects, induced by VNS, occurred during both rest and exercise. Magnitude of these effects varied between patients, but was not necessarily related to the intensity of the experienced side effects. CONCLUSION: This pilot study shows that VNS causes an increase in breathing frequency and a decrease in tidal volume and heart rate in the majority of patients, during both rest and exercise.