Evaluation of a rapid Loop Mediated Isothermal Amplification (LAMP) test for the laboratory diagnosis of sexually transmitted infections.

Sexually transmitted infections (STIs) have seen a considerable increase in the last years and given the health burden they may represent from both a personal and community perspective, they require surveillance and prevention programmes based on a timely and decentralized diagnosis. In this context, user-friendly rapid molecular tests may represent a good trade-off between diagnostic accuracy, accessibility and affordability. In this study we evaluated the diagnostic performance of a new real-time LAMP (Loop Mediated Isothermal Amplification) method for the rapid detection and differentiation of 7 major sexually transmissible pathogens by analysing real clinical samples (genital and extra-genital matrices) from individuals with suspected STIs. The assay showed good overall diagnostic performances in terms of sensitivity, specificity and concordance with a gold-standard PCR-based molecular method. This assay, not requiring specialised laboratory technicians or expensive instrumentation, but nonetheless capable of guaranteeing accurate results, is within the reach of outpatient settings, obstetrics, and gynaecology clinic, hence ensuring on-field access to early diagnosis.

Evaluation of Biofilm Production and Antifungal Susceptibility to Fluconazole in Clinical Isolates of Candida spp. in Both Planktonic and Biofilm Form.

Candida spp. are an important opportunistic pathogen that can represent a possible cause of severe infections, especially in immunocompromised individuals. The clinical impact of Candida spp. depends, in part, on the ability to form biofilms, communities of nestled cells into the extracellular matrix. In this study, we compared the biofilm formation ability of 83 strains of Candida spp. isolated from blood cultures and other materials, such as respiratory samples, urine, and exudate, and their sensitivity to fluconazole (FLZ). Strains were divided into tertiles to establish cut-offs to classify isolates as low, moderate, or high biofilm producers (<0.26, 0.266-0.839, >0.839) and biofilms with low, moderate, or high metabolic activity (<0.053, 0.053-0.183, >0.183). A non-linear relationship between biofilm production and metabolic activity was found in C. glabrata and C. tropicalis. In addition, the increase in minimum biofilm eradication concentrations (MBEC50) compared to the Minor Inhibitory Concentration (PMIC) of the planktonic form in Candida spp. confirms the role of biofilm in the induction of resistance to FLZ.

The Global Evolutionary History of Orf Virus in Sheep and Goats Revealed by Whole Genomes Data.

Orf virus (ORFV) belongs to the genus Parapoxvirus (Poxviridae family). It is the causative agent of contagious ecthyma (CE) that is an economically detrimental disease affecting small ruminants globally. Contagious ecthyma outbreaks are usually reported in intensive breeding of sheep and goats but they have also been reported in wildlife species. Notably, ORFV can infect humans, leading to a zoonotic disease. This study aims to elucidate the global evolutionary history of ORFV genomes in sheep and goats, including the first genomes from Central America in the analyses. In comparison to the last study on ORFV whole genomes, the database now includes 11 more sheep and goat genomes, representing an increase of 42%. The analysis of such a broader database made it possible to obtain a fine molecular dating of the coalescent time for ORFV S and G genomes, further highlighting the genetic structuring between sheep and goat genomes and corroborating their emergence in the latter half of 20th century.

Cyclosporine A micellar nasal spray characterization and antiviral action against SARS-CoV-2.

The upper airways represent the point of entrance from where Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection spreads to the lungs. In the present work, α-tocopheryl-polyethylene-glycol succinate (TPGS) micelles loaded with cyclosporine A (CSA) were developed for nasal administration to prevent or treat the viral infection in the very first phases. The behavior of the micelles in presence of simulated nasal mucus was investigated in terms of stability and mucopenetration rate, evidencing long-term stability and fast diffusion across the glycoproteins matrix. Moreover, the spray characteristics of the micellar formulation and deposition profile in a silicon nasal model were studied using three nasal spray devices. Results allowed to identify the nasal spray pump (BiVax, Aptar) able to provide the wider and uniform deposition of the nasal cavity. The cyclosporine A micelles antiviral activity against SARS-CoV-2 was tested on the Omicron BA.1 variant using Vero E6 cells with protocols simulating treatment before, during and after the infection of the upper airways. Complete viral inactivation was observed for the cyclosporine-loaded micelles while a very low activity was evidenced for the non-formulated drug, suggesting a synergistic activity of the drug and the formulation. In conclusion, this work showed that the developed cyclosporine A-loaded micellar formulations have the potential to be clinically effective against a wide spectrum of coronavirus variants.

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.

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.

SARS-CoV-2 coinfection in immunocompromised host leads to the generation of recombinant strain.

OBJECTIVES: Recombination related to coinfection is a huge driving force in determining the virus genetic variability, particularly in conditions of partial immune control, leading to prolonged infection. Here, we characterized a distinctive mutational pattern, highly suggestive of Delta-Omicron double infection, in a lymphoma patient. METHODS: The specimen was characterized through a combined approach, analyzing the results of deep sequencing in primary sample, viral culture, and plaque assay. RESULTS: Bioinformatic analysis on the sequences deriving from the primary sample supports the hypothesis of a double viral population within the host. Plaque assay on viral culture led to the isolation of a recombinant strain deriving from Delta and Omicron lineages, named XS, which virtually replaced its parent lineages within a single viral propagation. CONCLUSION: It is impossible to establish whether the recombination event happened within the host or in vitro; however, it is important to monitor co-infections, especially in the exceptional intrahost environment of patients who are immunocompromised, as strong driving forces of viral evolution.

Viral Population Heterogeneity and Fluctuating Mutational Pattern during a Persistent SARS-CoV-2 Infection in an Immunocompromised Patient.

Literature offers plenty of cases of immunocompromised patients, who develop chronic and severe SARS-CoV-2 infections. The aim of this study is to provide further insight into SARS-CoV-2 evolutionary dynamic taking into exam a subject suffering from follicular lymphoma, who developed a persistent infection for over 7 months. Eight nasopharyngeal swabs were obtained, and were analyses by qRT-PCR for diagnostic purposes. All of them were considered eligible (Ct < 30) for NGS sequencing. Sequence analysis showed that all sequences matched the B.1.617.2 AY.122 lineage, but they differed by few mutations identifying three genetically similar subpopulations, which evolved during the course of infection, demonstrating that prolonged replication is paralleled with intra-host virus evolution. These evidences support the hypothesis that SARS-CoV-2 adaptive capacities are able to shape a heterogeneous viral population in the context of immunocompromised patients. Spill-over of viral variants with enhanced transmissibility or immune escape capacities from these subjects is plausible.

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.

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.

Mutational induction in SARS-CoV-2 major lineages by experimental exposure to neutralising sera.

The ongoing evolution of SARS-CoV-2 and the emergence of new viral variants bearing specific escape mutations responsible for immune evasion from antibody neutralisation has required a more accurate characterisation of the immune response as one of the evolutive forces behind viral adaptation to a largely immunised human population. In this work, culturing in the presence of neutralising sera vigorously promoted mutagenesis leading to the acquisition of known escape mutations on the spike as well as new presumptive escape mutations on structural proteins whose role as target of the neutralizing antibody response might have been thus far widely neglected. From this perspective, this study, in addition to tracing the past evolution of the species back to interactions with neutralising antibody immune response, also offers a glimpse into future evolutive scenarios.

Organic Electrochemical Transistors as Versatile Tool for Real-Time and Automatized Viral Cytopathic Effect Evaluation.

In-vitro viral studies are still fundamental for biomedical research since studying the virus kinetics on cells is crucial for the determination of the biological properties of viruses and for screening the inhibitors of infections. Moreover, testing potential viral contaminants is often mandatory for safety evaluation. Nowadays, viral cytopathic effects are mainly evaluated through end-point assays requiring dye-staining combined with optical evaluation. Recently, optical-based automatized equipment has been marketed, aimed at the real-time screening of cell-layer status and obtaining further insights, which are unavailable with end-point assays. However, these technologies present two huge limitations, namely, high costs and the possibility to study only cytopathic viruses, whose effects lead to plaque formation and layer disruption. Here, we employed poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (Pedot:Pss) organic electrochemical transistors (OECTs) for the real-time, electrical monitoring of the infection of cytolytic viruses, i.e., encephalomyocarditis virus (EMCV), and non-cytolytic viruses, i.e., bovine coronavirus (B-CoV), on cells. OECT data on EMCV were validated using a commercially-available optical-based technology, which, however, failed in the B-CoV titration analysis, as expected. The OECTs proved to be reliable, fast, and versatile devices for viral infection monitoring, which could be scaled up at low cost, reducing the operator workload and speeding up in-vitro assays in the biomedical research field.

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.