Rapid and Affordable High Throughput Screening of SARS-CoV-2 Variants Using Denaturing High-Performance Liquid Chromatography Analysis

Mutations in the receptor binding domain (RBD) of SARS-CoV-2 alter the infectivity, pathogenicity, and transmissibility of new variants of concern (VOCs). In addition, those mutations cause immune escape, undermining the population immunity induced by ongoing mass vaccination programs. There is an urgent need for novel strategies and techniques aimed at the surveillance of the active emergence and spread of the VOCs. The aim of this study was to provide a quick, cheap and straightforward denaturing high-performance liquid chromatography (DHPLC) method for the prompt identification of the SARS-CoV-2 VOCs. Two PCRs were designed to target the RBD region, spanning residues N417 through N501 of the Spike protein. Furthermore, a DHPLC screening analysis was set up. The screening consisted of mixing the unknown sample with a standard sample of a known variant, denaturing at high temperature, renaturing at room temperature followed by a 2-minute run using the WAVE DHPLC system to detect the heteroduplexes which invariably form whenever the unknown sample has a nucleotide difference with respect to the standard used. The workflow was able to readily detect all the variants including B.1.1.7, P.1, B.1.585 B.1. 617.2 and lineages at a very affordable cost. The DHPLC analysis was robust being able to identify variants, even in the case of samples with very unbalanced target concentrations including those samples at the limit of detection. This approach has the potential of greatly expediting surveillance of the SARS-CoV-2 variants

Importance of external quality assessment for SARS-CoV-2 antigen detection during the COVID-19 pandemic

Background: Antigen testing has become an essential part of fighting the ongoing COVID-19 pandemic. With the continual increase in available tests, independent and extensive comparative evaluations using data from external quality assessment (EQA) studies to evaluate test performance between different users are required.Objectives: An EQA scheme was established to assess the sensitivity of antigen tests and the potential impact of circulating SARS-CoV-2 strains on their performance.Study design: Panels were prepared for three challenges in 2021 containing inactivated SARS-CoV-2-positive samples of various genetic strains (including variants of concern, VOCs) at different concentrations, and negative samples. Data was analysed based on qualitative testing results in relation to the antigen test used. Results: Participants registered for each individual challenge in any combination. In total, 258 respondents from 27 countries worldwide were counted submitting 472 datasets. All core samples were correctly reported by 76.7 to 83.1% at participant level and by 73.5 to 83.8% at dataset level. Sensitivity differences could be shown in viral loads and SARS-CoV-2 strains/variants including the impact on performance by a B.1.1.7-like mutant strain with a deletion in the nucleoprotein gene. Lateral flow rapid antigen tests showed a higher rate of false negatives in general compared with automated point-of-care tests and laboratory ELISA/immunoassays.Conclusions: EQA schemes can provide valuable data to inform participants about weaknesses in their testing process or methods and support ongoing assay evaluations for regulatory approval or post-market surveillance

Molecular Approach for the Laboratory Diagnosis of Periprosthetic Joint Infections

The incidence of total joint arthroplasty is increasing over time since the last decade and expected to be more than 4 million by 2030. As a consequence, the detection of infections associated with surgical interventions is increasing and prosthetic joint infections are representing both a clinically and economically challenging problem. Many pathogens, from bacteria to fungi, elicit the immune system response and produce a polymeric matrix, the biofilm, that serves as their protection. In the last years, the implementation of diagnostic methodologies reduced the error rate and the turn-around time: polymerase chain reaction, targeted or broad-spectrum, and next-generation sequencing have been introduced and they represent a robust approach nowadays that frees laboratories from the unique approach based on culture-based techniques

Bacterial vaginosis: epidemiologic, clinical and diagnostic updates

Background and aims. Bacterial vaginosis (BV) is one the more frequently identified genital syndrome among childbearing aged women. The basic condition that generates this condition is a modification in the vaginal microbiota. The aim of this paper is to briefly review the current status of the art of BV and to report the results of a pilot study performed with an innovative PCR based technique. vaginae has been identified in 20 samples by the molecular approach and Lactobacillus spp. was identified in 19 samples (by culture) and in 32 by PCR. The overall diagnosis of BV was made in 9 patients by standard techniques and in 7 by applying the molecular approach. (Cohen\u2019s kappa test: 0,84). The findings of this study clearly demonstrate that the joint use of the routine cul- ture-based techniques with the multiplex PCR methods amplifies by far the sensitivity of the overall diagnostic workflow of BV

Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in Wuhan, China, in late 2019 and is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) represents the gold standard for diagnostic assays even if it cannot precisely quantify viral RNA copies. Thus, we decided to compare qRT-PCR with digital polymerase chain reaction (dPCR), which is able to give an accurate number of RNA copies that can be found in a specimen. However, the aforementioned methods are not capable to discriminate if the detected RNA is infectious or not. For this purpose, it is necessary to perform an endpoint titration on cell cultures, which is largely used in the research field and provides a tissue culture infecting dose per mL (TCID50/mL) value. Both research and diagnostics call for a model that allows the comparison between the results obtained employing different analytical methods. The aim of this study is to define a comparison among two qRT-PCR protocols (one with preliminary RNA extraction and purification and an extraction-free qRT-PCR), a dPCR and a titration on cell cultures. The resulting correlations yield a faithful estimation of the total number of RNA copies and of the infectious viral burden from a Ct value obtained with diagnostic routine tests. All these estimations take into consideration methodological errors linked to the qRT-PCR, dPCR and titration assays

Omicron Sub-Lineage BA.5 and Recombinant XBB Evasion from Antibody Neutralisation in BNT162b2 Vaccine Recipients

The recent emergence of a number of new SARS-CoV-2 variants resulting from recombination between two distinct parental lineages or sub-lineages within the same lineage has sparked the debate regarding potential enhanced viral infectivity and immune escape. Among these, XBB, recombinant of BA.2.10 and BA.2.75, has caused major concern in some countries due to its rapid increase in prevalence. In this study, we tested XBB escape capacity from mRNA-vaccine-induced (BNT162b2) neutralising antibodies compared to B.1 ancestral lineage and another co-circulating variant (B.1.1.529 BA.5) by analysing sera collected 30 days after the second dose in 92 healthcare workers. Our data highlighted an enhanced and statistically significant immune escape ability of the XBB recombinant. Although these are preliminary results, this study highlights the importance of immune escape monitoring of new and forthcoming variants and of the reformulation of existing vaccines

An Enhanced Dissolving Cyclosporin-A Inhalable Powder Efficiently Reduces SARS-CoV-2 Infection In Vitro

: This work illustrates the development of a dry inhalation powder of cyclosporine-A for the prevention of rejection after lung transplantation and for the treatment of COVID-19. The influence of excipients on the spray-dried powder's critical quality attributes was explored. The best-performing powder in terms of dissolution time and respirability was obtained starting from a concentration of ethanol of 45% (v/v) in the feedstock solution and 20% (w/w) of mannitol. This powder showed a faster dissolution profile (Weibull dissolution time of 59.5 min) than the poorly soluble raw material (169.0 min). The powder exhibited a fine particle fraction of 66.5% and an MMAD of 2.97 µm. The inhalable powder, when tested on A549 and THP-1, did not show cytotoxic effects up to a concentration of 10 µg/mL. Furthermore, the CsA inhalation powder showed efficiency in reducing IL-6 when tested on A549/THP-1 co-culture. A reduction in the replication of SARS-CoV-2 on Vero E6 cells was observed when the CsA powder was tested adopting the post-infection or simultaneous treatment. This formulation could represent a therapeutic strategy for the prevention of lung rejection, but is also a viable approach for the inhibition of SARS-CoV-2 replication and the COVID-19 pulmonary inflammatory process

Evaluation of STANDARDTM M10 SARS-CoV-2, a Novel Cartridge-Based Real-Time PCR Assay for the Rapid Identification of Severe Acute Respiratory Syndrome Coronavirus 2

Since the beginning of the pandemic, SARS-CoV-2 has caused problems for all of world’s population, not only in terms of deaths but also in terms of overloading healthcare facilities in all countries. Diagnosis is one of the key aspects of controlling the spread of SARS-CoV-2, and among the current molecular techniques, real-time PCR is considered as the gold standard. The availability of tests that allow for the rapid and accurate identification of SARS-CoV-2 is therefore of considerable importance. Moreover, if these tests allow for even minimal intervention by the operator, any risk of contamination is reduced. In this study, the performances of the new STANDARDTM M10 SARS-CoV-2 (SD Biosensor Inc., Suwon, Korea) rapid molecular test, which incorporates the above-mentioned features, were characterized. The clinical and analytical performances measured by testing different variants circulating in Italy of STANDARDTM M10 SARS-CoV-2 were compared to the test already on the market and recognized as the gold standard: Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, CA, USA). The results obtained between the two tests are largely comparable, suggesting that STANDARDTM M10 SARS-CoV-2 can be used with excellent results in the fight against the global spread of SARS-CoV-2

Genomic and Temporal Analysis of Deletions Correlated to qRT-PCR Dropout in N Gene in Alpha, Delta and Omicron Variants

Since the first SARS-CoV-2 outbreak, mutations such as single nucleotide polymorphisms (SNPs) and insertion/deletions (INDELs) have changed and characterized the viral genome sequence, structure and protein folding leading to the onset of new variants. The presence of those alterations challenges not only the clinical field but also the diagnostic demand due to failures in gene detection or incompleteness of polymerase chain reaction (PCR) results. In particular, the analysis of understudied genes such as N and the investigation through whole-genome next generation sequencing (WG-NGS) of regions more prone to mutate can help in the identification of new or reacquired mutations, with the aim of designing robust and long-lasting primers. In 48 samples of SARS-CoV-2 (including Alpha, Delta and Omicron variants), a lack of N gene amplification was observed in the genomes analyzed through WG-NGS. Three gene regions were detected hosting the highest number of SNPs and INDELs. In several cases, the latter can interfere deeply with both the sensitivity of diagnostic methodologies and the final protein folding. The monitoring over time of the viral evolution and the reacquisition among different variants of the same mutations or different alterations within the same genomic positions can be relevant to avoid unnecessary consumption of resources