The COVID-19 pandemic is driven by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) that emerged in 2019 and quickly spread worldwide. Genomic surveillance has become the gold standard methodology used to monitor and study this fast-spreading virus and its constantly emerging lineages. The current deluge of SARS-CoV-2 genomic data generated worldwide has put additional pressure on the urgent need for streamlined bioinformatics workflows. Here, we describe a workflow developed by our group to process and analyze large-scale SARS-CoV-2 Illumina amplicon sequencing data. This workflow automates all steps of SARS-CoV-2 reference-based genomic analysis: data processing, genome assembly, PANGO lineage assignment, mutation analysis and the screening of intrahost variants. The pipeline is capable of processing a batch of around 100 samples in less than half an hour on a personal laptop or in less than five minutes on a server with 50 threads. The workflow presented here is available through Docker or Singularity images, allowing for implementation on laptops for small-scale analyses or on high processing capacity servers or clusters. Moreover, the low requirements for memory and CPU cores and the standardized results provided by ViralFlow highlight it as a versatile tool for SARS-CoV-2 genomic analysis.

https://www.mdpi.com/1999-4915/14/2/217/pdf?version=1642941200

ViralFlow: A Versatile Automated Workflow for SARS-CoV-2 Genome Assembly, Lineage Assignment, Mutations and Intrahost Variant Detection

/pdf/microfluidics-based-biosensing-platforms-emerging-frontiers-1ao184qv.pdf

Microfluidics-Based Biosensing Platforms: Emerging Frontiers in Point-of-Care Testing SARS-CoV-2 and Seroprevalence

Rapid, highly sensitive, and accurate virus circulation monitoring techniques are critical to limit the spread of the virus and reduce the social and economic burden. Therefore, point-of-use diagnostic devices have played a critical role in addressing the outbreak of COVID-19 (SARS-CoV-2) viruses. This review provides a comprehensive overview of the current techniques developed for the detection of SARS-CoV-2 in various body fluids (e.g., blood, urine, feces, saliva, tears, and semen) and considers the mutations (i.e., Alpha, Beta, Gamma, Delta, Omicron). We classify and comprehensively discuss the detection methods depending on the biomarker measured (i.e., surface antigen, antibody, and nucleic acid) and the measurement techniques such as lateral flow immunoassay (LFIA), enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), microarray analysis, clustered regularly interspaced short palindromic repeats (CRISPR) and biosensors. Finally, we addressed the challenges of rapidly identifying emerging variants, detecting the virus in the early stages of infection, the detection sensitivity, selectivity, and specificity, and commented on how these challenges can be overcome in the future. 

https://doi.org/10.1016/j.trac.2022.116750

Current trends in COVID-19 diagnosis and its new variants in physiological fluids: Surface antigens, antibodies, nucleic acids, and RNA sequencing

https://doi.org/10.1016/j.biopha.2022.113538

Recent advances in point of care testing for COVID-19 detection

Since the onset of the COVID-19 pandemic, over 610 million cases have been diagnosed and it has caused over 6.5 million deaths worldwide. The crisis has forced the scientific community to develop tools for disease control and management at a pace never seen before. The control of the pandemic heavily relies in the use of fast and accurate diagnostics, that allow testing at a large scale. The gold standard diagnosis of viral infections is the RT-qPCR. Although it provides consistent and reliable results, it is hampered by its limited throughput and technical requirements. Here, we discuss the main approaches to rapid and point-of-care diagnostics based on RT-qPCR and isothermal amplification diagnostics. We describe the main COVID-19 molecular diagnostic tests approved for self-testing at home or for point-of-care testing and compare the available options. We define the influence of specimen selection and processing, the clinical validation, result readout improvement strategies, the combination with CRISPR-based detection and the diagnostic challenge posed by SARS-CoV-2 variants for different isothermal amplification techniques, with a particular focus on LAMP and recombinase polymerase amplification (RPA). Finally, we try to shed light on the effect the improvement in molecular diagnostics during the COVID-19 pandemic could have in the future of other infectious diseases.

/pdf/the-future-of-point-of-care-nucleic-acid-amplification-i6z5vfgd.pdf

The Future of Point-of-Care Nucleic Acid Amplification Diagnostics after COVID-19: Time to Walk the Walk

Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

The COVID-19 pandemics unfolded due to the widespread SARS-CoV-2 transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric RT-LAMP (reverse transcription loop-mediated isothermal amplification) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 {+/-} 2.7 viral genomic copies/L when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65 {o}C for 30 min. When compared to RT-qPCR, up to Ct value 32, RT-LAMP presented 97% (87.4-99.4% 95% CI) sensitivity and 100% (86.2-100%) specificity for SARS-CoV-2 RNA detection targeting N gene. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern (VOC) and variants of interest (VOI), such as variants occurring in Brazil named P.1, P.2, B.1.1.374 and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipments, infrastructure and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

/pdf/clinical-validation-of-colorimetric-rt-lamp-a-fast-highly-15hl77fyv7.pdf

Clinical validation of colorimetric RT-LAMP, a fast, highly sensitive and specific COVID-19 molecular diagnostic tool that is robust to detect SARS-CoV-2 variants of concern

Flávia de S. Rocha

Papers

Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

Clinical validation of colorimetric RT-LAMP, a fast, highly sensitive and specific COVID-19 molecular diagnostic tool that is robust to detect SARS-CoV-2 variants of concern