Recent advances of nanotechnology in COVID 19: A critical review and future perspective

The global anxiety and economic crisis causes the deadly pandemic coronavirus disease of 2019 (COVID 19) affect millions of people right now. Subsequently, this life threatened viral disease is caused due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, morbidity and mortality of infected patients are due to cytokines storm syndrome associated with lung injury and multiorgan failure caused by COVID 19. Thereafter, several methodological advances have been approved by WHO and US-FDA for the detection, diagnosis and control of this wide spreadable communicable disease but still facing multi-challenges to control. Herein, we majorly emphasize the current trends and future perspectives of nano-medicinal based approaches for the delivery of anti-COVID 19 therapeutic moieties. Interestingly, Nanoparticles (NPs) loaded with drug molecules or vaccines resemble morphological features of SARS-CoV-2 in their size (60–140 nm) and shape (circular or spherical) that particularly mimics the virus facilitating strong interaction between them. Indeed, the delivery of anti-COVID 19 cargos via a nanoparticle such as Lipidic nanoparticles, Polymeric nanoparticles, Metallic nanoparticles, and Multi-functionalized nanoparticles to overcome the drawbacks of conventional approaches, specifying the site-specific targeting with reduced drug loading and toxicities, exhibit their immense potential. Additionally, nano-technological based drug delivery with their peculiar characteristics of having low immunogenicity, tunable drug release, multidrug delivery, higher selectivity and specificity, higher efficacy and tolerability switch on the novel pathway for the prevention and treatment of COVID 19. © 2022 The Author(s)

Could tear be an alternative specimen for SARS-CoV-2 detection?

Objectives: For a definitive diagnosis of COVID-19, respiratory tract samples are evaluated by polymerase chain reaction (PCR). In our study, PCR using a tear sample was used to diagnose COVID-19, and it was questioned whether it was a screening method. Unlike the general practice, Schirmer strips were used instead of a swab for tear sample collection in this study. In addition, the diagnostic values of serum procalcitonin (PCT), C-reactive protein (CRP), and Neutrophil (NEU) count in predicting COVID-19 disease from tears were also questioned. Method(s): A total of 94 patients who were positive for COVID-19 by PCR test were included in this study. Tear samples were obtained from patients with Schirmer strips, commonly used in eye examination, and studied with the PCR technique. CRP, PCT value, and NEU count were also compared between the positive and negative groups of the PCR. The obtained data were analyzed using the R Studio software, and the results were considered statistically significant for p<0.05. Result(s): Of these patients, 61 (64.9%) tear PCR was negative, and 33 (35.1%) tear PCR was positive. The mean age was 61.72 +/- 17.62 years. The patients were divided into two groups: tear PCR positive and negative. There was no significant age difference between these groups. As a result of ROC Analysis;When serum PCT, CRP, and NEU % values were examined in predicting COVID-19 disease from tears, it was seen that CRP (p=0.027) and especially PCT (p=0.003) values of patients with PCR-positive were significantly higher. Conclusion(s): PCR study on tears collected with Schirmer strips is a different and non-invasive method, but it was concluded that the proposed method could not be used as a screening test. In addition, significantly higher serum PCT values were found in patients with COVID-19 positivity in tears (p<0.05). Copyright © 2022 the author(s), published by De Gruyter.

Positive SARS-CoV-2 RT-qPCR of a nasal swab spot after 30 days of conservation on filter paper at room temperature.

We tested the use of nasal swabs spotted onto filter paper (Whatman 3M) for the molecular diagnosis of SARS-CoV-2 infection. Spots of a positive nasal swab in conservation medium (B.1.177 strain, 21Ct) were still positive (duo E-gene/IP4) after 10, 20, and 30 days of conservation at room temperature, with Ct values of 28, 27, and 26, respectively. Direct spotting of the swab at bedside (omicron strain) still gave a positive result after 10 days in two RT-qPCR systems: 33.7 Ct using duo E-gene/IP4, and 34.8 using a specific Omicron system. Spotting of a dilution range of media spiked with the Delta (strain 2021/FR/0610, lineage B 1.617.2) and Omicron strains (strain UVE/SARS-CoV-2/2021/FR/1514) showed a threshold of 0.04 TCID50 after 10 days of conservation. We show, for the first time, that this simple and low-cost conservation method can be used to store samples for RT-qPCR against SARS-CoV-2 for up to at least 1 month.

From Biowaste to Lab-Bench: Low-Cost Magnetic Iron Oxide Nanoparticles for RNA Extraction and SARS-CoV-2 Diagnostics.

The gold standard for diagnostics of SARS-CoV-2 (COVID-19) virus is based on real-time polymerase chain reaction (RT-PCR) using centralized PCR facilities and commercial viral RNA extraction kits. One of the key components of these kits are magnetic beads composed of silica coated magnetic iron oxide (Fe2O3 or Fe3O4) nanoparticles, needed for the selective extraction of RNA. At the beginning of the pandemic in 2019, due to a high demand across the world there were severe shortages of many reagents and consumables, including these magnetic beads required for testing for SARS-CoV-2. Laboratories needed to source these products elsewhere, preferably at a comparable or lower cost. Here, we describe the development of a simple, low-cost and scalable preparation of magnetic nanoparticles (MNPs) from biowaste and demonstrate their successful application in viral RNA extraction and the detection of COVID-19. These MNPs have a unique nanoplatelet shape with a high surface area, which are beneficial features, expected to provide improved RNA adsorption, better dispersion and processing ability compared with commercial spherical magnetic beads. Their performance in COVID-19 RNA extraction was evaluated in comparison with commercial magnetic beads and the results presented here showed comparable results for high throughput PCR analysis. The presented magnetic nanoplatelets generated from biomass waste are safe, low-cost, simple to produce in large scale and could provide a significantly reduced cost of nucleic acid extraction for SARS-CoV-2 and other DNA and RNA viruses.

A DNA biosensors-based microfluidic platform for attomolar real-time detection of unamplified SARS-CoV-2 virus.

The emergence of the coronavirus 2019 (COVID-19) arose the need for rapid, accurate and massive virus detection methods to control the spread of infectious diseases. In this work, a device, deployable in non-medical environments, has been developed for the detection of non-amplified SARS-CoV-2 RNA. A SARS-CoV-2 specific probe was designed and covalently immobilized at the surface of glass slides to fabricate a DNA biosensor. The resulting system was integrated in a microfluidic platform, in which viral RNA was extracted from non-treated human saliva, before hybridizing at the surface of the sensor. The formed DNA/RNA duplex was detected in presence of SYBR Green I using an opto-electronic system, based on a high-power LED and a photo multiplier tube, which convert the emitted fluorescence into an electrical signal that can be processed in less than 10 min. The limit of detection of the resulting microfluidic platform reached six copies of viral RNA per microliter of sample (equal to 10 aM) and satisfied the safety margin. The absence of non-specific adsorption and the selectivity for SARS-CoV-2 RNA were established. In addition, the designed device could be applicable for the detection of a variety of viruses by simple modification of the immobilized probe.

A novel paper-based lysis strip for SARS-CoV-2 RNA detection at low resource settings.

Infectious respiratory diseases such as COVID-19 are serious and global concerns from the past to the present. To isolate the spread of infectious diseases even in the absence of a health system, a simple, inexpensive, reliable, sensitive, and selective molecular diagnosis platform for Point of Care Test (POCT) is required. Especially, the nucleic acid extraction step is difficult to perform out of laboratory. Here, we propose a paper-based lysis (PBL) strip for nucleic acid extraction, especially in low-resource settings (LRS). PBL strips are suitable for isolating RNA from viruses with biological interference and inhibitors. We optimized the buffer compositions and membranes of the strip. A simple preparation method using a PBL strip could obtain an eluent for downstream inspection within 20 min. Overall, 104 copies/swaps were detected for 20 min for amplification in combination with Reverse Transcription Loop-Mediated Amplification (RT-LAMP).

Single-walled carbon nanotubes-based RNA protection and extraction improves RT-qPCR sensitivity for SARS-CoV-2 detection.

The false-negative result of nucleic acid testing is an important cause of continued spread of COVID-19, while SARS-CoV-2 RNA degradation during transportation and nucleic acid extraction can lead to false-negative results. Here, we investigated that single-walled carbon nanotubes (SCNTs) could protect RNA from degradation for at least 4 days at room temperature. By constructing magnetism-functionalized SCNTs (MSCNTs), we developed a method that enabled protection and simple extraction of SARS-CoV-2 RNA, and the RNA-bound MSCNTs can be directly used for reverse transcription polymerase chain reaction (RT-qPCR) detection. The experimental results showed that 1 µg of MSCNTs adsorbed up to 24 ng of RNA. Notably, the MSCNTs-based method for extracting SARS-CoV-2 RNA from simulated nasopharyngeal swabs and saliva samples with mean recovery rates of 103% and 106% improved the sensitivity of RT-qPCR detection by 8-32 fold in comparison to current common methods. This improvement was largely attributable to the protection of RNA, enabling increased RNA load for downstream assays.

Technical Note: RNA Extraction Alternative Method for SARS-CoV-2 Molecular Diagnosis.

The devastating COVID-19 outbreak posed serious challenges for the diagnostics laboratories, facing global shortage of reagents and equipment. This study aimed at evaluating an additional RNA extraction method respect to those already recommended by WHO and CDC. A new protocol for RNA extraction from nasopharyngeal swab was set up, adapting a Qiagen kit, and validated on a set of 96 clinical samples. The analysis showed a sensitivity of 94% and a specificity of 97%, but considering samples with Ct<36.5, the sensitivity and the specificity increased to 100%. The adapted method was also able to detect samples with very low viral load (Ct>38), indicating that the two approaches can be considered equivalent for the SARS-CoV-2 diagnostics. This extraction method can help in increasing the throughput for SARS-CoV-2 molecular test, even in a low automation setting.

Wastewater Sequencing-An Innovative Method for Variant Monitoring of SARS-CoV-2 in Populations.

The SARS-CoV-2 outbreak has already affected more than 555 million people, and 6.3 million people have died. Due to its high infectivity, it is crucial to track SARS-CoV-2 outbreaks early to prevent the spread of infection. Wastewater monitoring appears to be a powerful and effective tool for managing epidemiological situations. Due to emerging mutations of SARS-CoV-2, there is a need to monitor mutations in order to control the pandemic. Since the sequencing of randomly chosen individuals is time-consuming and expensive, sequencing of wastewater plays an important role in revealing the dynamics of infection in a population. The sampling method used is a crucial factor and significantly impacts the results. Wastewater can be collected as a grab sample or as a 24 h composite sample. Another essential factor is the sample volume, as is the method of transport used. This review discusses different pretreatment procedures and RNA extraction, which may be performed using various methods, such as column-based extraction, TRIzol, or magnetic extraction. Each of the methods has its advantages and disadvantages, which are described accordingly. RT-qPCR is a procedure that confirms the presence of SARS-CoV-2 genes before sequencing. This review provides an overview of currently used methods for preparing wastewater samples, from sampling to sequencing.

Useful molecular tools for facing next pandemic events: Effective sample preparation and improved RT-PCR for highly sensitive detection of SARS-CoV-2 in wastewater environment.

Viral pandemics can be inevitable in the next future. Considering SARS-CoV-2 pandemics as an example, there seems to be a need to develop a surveillance system able to monitor the presence of potential pathogenic agents. The sewage and wastewater environments demonstrated to be suitable targets for such kind of analysis. In addition, it is important to have reliable molecular diagnostic tools and also to develop a robust detection strategy. In this study, an effective sample preparation procedure was selected from four options and combined with a newly developed improved RT-PCR. First, a model viral system was constructed, containing a fragment of the SARS-CoV-2 gene encoding for the Spike protein. The encapsidated S RNA mimic (ESRM) was based on the plum pox virus (PPV) genome with the inserted targeted gene fragment. ESRM was used for seeding wastewater samples in order to evaluate the viral recovery of four different viral RNA concentration/extraction methods. The efficiency of individual approaches was assessed by the use of a quantitative reverse transcription PCR (qRT-PCR) and by a one-step single-tube nested quantitative reverse transcription PCR (OSN-qRT-PCR). For the detection of viruses in wastewater samples with low viral loads, OSN-qRT-PCR assay produced the most satisfactory results and the highest sensitivity.