Epidemiological and genomic findings of the first documented Italian outbreak of SARS-CoV-2 Alpha variant of concern.

From 24 December 2020 to 8 February 2021, 163 cases of SARS-CoV-2 Alpha variant of concern (VOC) were identified in Chieti province, Abruzzo region. Epidemiological data allowed the identification of 14 epi-clusters. With one exception, all the epi-clusters were linked to the town of Guardiagrele: 149 contacts formed the network, two-thirds of which were referred to the family/friends context. Real data were then used to estimate transmission parameters. According to our method, the calculated Re(t) was higher than 2 before the 12 December 2020. Similar values were obtained from other studies considering Alpha VOC. Italian sequence data were combined with a random subset of sequences obtained from the GISAID database. Genomic analysis showed close identity between the sequences from Guardiagrele, forming one distinct clade. This would suggest one or limited unspecified viral introductions from outside to Abruzzo region in early December 2020, which led to the diffusion of Alpha VOC in Guardiagrele and in neighbouring municipalities, with very limited inter-regional mixing.

One-Step, Wash-free, Nanoparticle Clustering-Based Magnetic Particle Spectroscopy Bioassay Method for Detection of SARS-CoV-2 Spike and Nucleocapsid Proteins in the Liquid Phase.

With the ongoing global pandemic of coronavirus disease 2019 (COVID-19), there is an increasing quest for more accessible, easy-to-use, rapid, inexpensive, and high-accuracy diagnostic tools. Traditional disease diagnostic methods such as qRT-PCR (quantitative reverse transcription-PCR) and ELISA (enzyme-linked immunosorbent assay) require multiple steps, trained technicians, and long turnaround time that may worsen the disease surveillance and pandemic control. In sight of this situation, a rapid, one-step, easy-to-use, and high-accuracy diagnostic platform will be valuable for future epidemic control, especially for regions with scarce medical resources. Herein, we report a magnetic particle spectroscopy (MPS) platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biomarkers: spike and nucleocapsid proteins. This technique monitors the dynamic magnetic responses of magnetic nanoparticles (MNPs) and uses their higher harmonics as a measure of the nanoparticles' binding states. By anchoring polyclonal antibodies (pAbs) onto MNP surfaces, these nanoparticles function as nanoprobes to specifically bind to target analytes (SARS-CoV-2 spike and nucleocapsid proteins in this work) and form nanoparticle clusters. This binding event causes detectable changes in higher harmonics and allows for quantitative and qualitative detection of target analytes in the liquid phase. We have achieved detection limits of 1.56 nM (equivalent to 125 fmole) and 12.5 nM (equivalent to 1 pmole) for detecting SARS-CoV-2 spike and nucleocapsid proteins, respectively. This MPS platform combined with the one-step, wash-free, nanoparticle clustering-based assay method is intrinsically versatile and allows for the detection of a variety of other disease biomarkers by simply changing the surface functional groups on MNPs.

Magnetic Particle Spectroscopy with One-Stage Lock-In Implementation for Magnetic Bioassays with Improved Sensitivities.

In recent years, magnetic particle spectroscopy (MPS) has become a highly sensitive and versatile sensing technique for quantitative bioassays. It relies on the dynamic magnetic responses of magnetic nanoparticles (MNPs) for the detection of target analytes in the liquid phase. There are many research studies reporting the application of MPS for detecting a variety of analytes including viruses, toxins, nucleic acids, and so forth. Herein, we report a modified version of the MPS platform with the addition of a one-stage lock-in design to remove the feedthrough signals induced by external driving magnetic fields, thus capturing only MNP responses for improved system sensitivity. This one-stage lock-in MPS system is able to detect as low as 781 ng multi-core Nanomag50 iron oxide MNPs (micromod Partikeltechnologie GmbH) and 78 ng single-core SHB30 iron oxide MNPs (Ocean NanoTech). We first demonstrated the performance of this MPS system for bioassay-related applications. Using the SARS-CoV-2 spike protein as a model, we have achieved a detection limit of 125 nM (equal to 5 pmole) for detecting spike protein molecules in the liquid phase. In addition, using a streptavidin-biotin binding system as a proof-of-concept, we show that these single-core SHB30 MNPs can be used for Brownian relaxation-based bioassays while the multi-core Nanomag50 cannot be used. The effects of MNP amount on the concentration-dependent response profiles for detecting streptavidin were also investigated. Results show that by using a lower concentration/ amount of MNPs, concentration-response curves shift to a lower concentration/amount of target analytes. This lower concentration-response indicates the possibility of improved bioassay sensitivities by using lower amounts of MNPs.

Multiple detection and spread of novel strains of the SARS-CoV-2 B.1.177 (B.1.177.75) lineage that test negative by a commercially available nucleocapsid gene real-time RT-PCR.

Several lineages of SARS-CoV-2 are currently circulating worldwide. During SARS-CoV-2 diagnostic activities performed in Abruzzo region (central Italy) several strains belonging to the B.1.177.75 lineage tested negative for the N gene but positive for the ORF1ab and S genes (+/+/- pattern) by the TaqPath COVID-19 CE-IVD RT-PCR Kit manufactured by Thermofisher. By sequencing, a unique mutation, synonymous 28948C > T, was found in the N-negative B.1.177.75 strains. Although we do not have any knowledge upon the nucleotide sequences of the primers and probe adopted by this kit, it is likely that N gene dropout only occurs when 28948C > T is coupled with 28932C > T, this latter present, in turn, in all B.1.177.75 sequences available on public databases. Furthermore, epidemiological analysis was also performed. The majority of the N-negative B.1.177.75 cases belonged to two clusters apparently unrelated to each other and both clusters involved young people. However, the phylogeny for sequences containing the +/+/- pattern strongly supports a genetic connection and one common source for both clusters. Though, genetic comparison suggests a connection rather than indicating the independent emergence of the same mutation in two apparently unrelated clusters. This study highlights once more the importance of sharing genomic data to link apparently unrelated epidemiological clusters and to, remarkably, update molecular tests.

A Portable Magnetic Particle Spectrometer for Future Rapid and Wash-Free Bioassays.

Nowadays, there is an increasing demand for more accessible routine diagnostics for patients with respect to high accuracy, ease of use, and low cost. However, the quantitative and high accuracy bioassays in large hospitals and laboratories usually require trained technicians and equipment that is both bulky and expensive. In addition, the multistep bioassays and long turnaround time could severely affect the disease surveillance and control especially in pandemics such as influenza and COVID-19. In view of this, a portable, quantitative bioassay device will be valuable in regions with scarce medical resources and help relieve burden on local healthcare systems. Herein, we introduce the MagiCoil diagnostic device, an inexpensive, portable, quantitative, and rapid bioassay platform based on the magnetic particle spectrometer (MPS) technique. MPS detects the dynamic magnetic responses of magnetic nanoparticles (MNPs) and uses the harmonics from oscillating MNPs as metrics for sensitive and quantitative bioassays. This device does not require trained technicians to operate and employs a fully automatic, one-step, and wash-free assay with a user friendly smartphone interface. Using a streptavidin-biotin binding system as a model, we show that the detection limit of the current portable device for streptavidin is 64 nM (equal to 5.12 pmole). In addition, this MPS technique is very versatile and allows for the detection of different diseases just by changing the surface modifications on MNPs. Although MPS-based bioassays show high sensitivities as reported in many literatures, at the current stage, this portable device faces insufficient sensitivity and needs further improvements. It is foreseen that this kind of portable device can transform the multistep, laboratory-based bioassays to one-step field testing in nonclinical settings such as schools, homes, offices, etc.