Reinfection rate and disease severity of the BA.5 Omicron SARS-CoV-2 lineage compared to previously circulating variants of concern in the Canary Islands (Spain).

ABSTRACTThe emergence of the Omicron SARS-CoV-2 variant of concern has changed the COVID-19 scenario as this variant is characterized by high transmissibility and immune evasion ability. To evaluate the impact of this variant on the Canary Islands (Spain) population, we determined the reinfection rates and disease severity associated with the Omicron sublineages and the previously circulating variants of concern. We performed a retrospective observational study on 21,745 SARS-CoV-2 viral genomes collected from December 2020 to July 2022 in the Canary Islands (Spain). We compared the reinfection rates between lineages using pairwise proportion and Fisher's exact tests. To assess disease severity, we studied the association of Alpha, Delta, BA.1, BA.2, BA.5, and other risk factors on 28-day hospital mortality using logistic regression and Cox proportional hazard models. We observed 127 bona fide reinfection cases throughout the study period. We found that BA.5 had the highest reinfection rate compared to other lineages (vs. Delta p = 2.89 × 10-25; vs. BA.1 p = 5.17 × 10-11; vs. BA.2 p = 0.002). Among the 1,094 hospitalized patients, multivariate logistic regression showed that Alpha (Odds Ratio [OR] = 0.45, 95% Confidence Interval [CI] = 0.23-0.87, p = 0.02), BA.2 (OR = 0.38, 95% CI = 0.22-0.63, p = 1.91 × 10-4), and BA.5 (OR = 0.30, 95% CI = 0.16-0.55, p = 1.05 × 10-4) had lower 28-day hospital mortality compared to Delta. These results were confirmed by using Cox proportional hazard models. Omicron lineages, and in particular BA.5, were associated with higher reinfection rates and lower disease severity (28-day hospital mortality) than previously circulating variants of concern.

Bioinformatic approaches to draft the viral genome sequence of Canary Islands cases related to the multicountry mpox virus 2022-outbreak.

On July 23, 2022, monkeypox disease (mpox) was declared a Public Emergency of International Concern (PHEIC) by the World Health Organization (WHO) due to a multicountry outbreak. In Europe, several cases of mpox virus (MPXV) infection related to this outbreak were detected in the Canary Islands (Spain). Here we describe the combination of viral DNA sequencing and bioinformatic approaches, including methods for de novo genome assembly and short- and long-read technologies, used to reconstruct the first MPXV genome isolated in the Canary Islands on the 31st of May 2022 from a male adult patient with mild symptoms. The same sequencing and bioinformatic approaches were then validated with three other positive cases of MPXV infection from the same mpox outbreak. We obtained the best results using a reference-based approach with short reads, evidencing 46-79 nucleotide variants against viral sequences from the 2018-2019 mpox outbreak and placing the viral sequences in the new B.1 sublineage of clade IIb of the MPXV classification. This study of MPXV demonstrates the potential of metagenomics sequencing for rapid and precise pathogen identification.

Nanomaterials to combat SARS-CoV-2: Strategies to prevent, diagnose and treat COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the associated coronavirus disease 2019 (COVID-19), which severely affect the respiratory system and several organs and tissues, and may lead to death, have shown how science can respond when challenged by a global emergency, offering as a response a myriad of rapid technological developments. Development of vaccines at lightning speed is one of them. SARS-CoV-2 outbreaks have stressed healthcare systems, questioning patients care by using standard non-adapted therapies and diagnostic tools. In this scenario, nanotechnology has offered new tools, techniques and opportunities for prevention, for rapid, accurate and sensitive diagnosis and treatment of COVID-19. In this review, we focus on the nanotechnological applications and nano-based materials (i.e., personal protective equipment) to combat SARS-CoV-2 transmission, infection, organ damage and for the development of new tools for virosurveillance, diagnose and immune protection by mRNA and other nano-based vaccines. All the nano-based developed tools have allowed a historical, unprecedented, real time epidemiological surveillance and diagnosis of SARS-CoV-2 infection, at community and international levels. The nano-based technology has help to predict and detect how this Sarbecovirus is mutating and the severity of the associated COVID-19 disease, thereby assisting the administration and public health services to make decisions and measures for preparedness against the emerging variants of SARS-CoV-2 and severe or lethal COVID-19.

NanoRTax, a real-time pipeline for taxonomic and diversity analysis of nanopore 16S rRNA amplicon sequencing data.

Background: The study of microbial communities and their applications have been leveraged by advances in sequencing techniques and bioinformatics tools. The Oxford Nanopore Technologies long-read sequencing by nanopores provides a portable and cost-efficient platform for sequencing assays. While this opens the possibility of sequencing applications outside specialized environments and real-time analysis of data, complementing the existing efficient library preparation protocols with streamlined bioinformatic workflows is required. Results: Here we present NanoRTax, a Nextflow pipeline for nanopore 16S rRNA gene amplicon data that features state-of-the-art taxonomic classification tools and real-time capability. The pipeline is paired with a web-based visual interface to enable user-friendly inspections of the experiment in progress. NanoRTax workflow and a simulated real-time analysis were used to validate the prediction of adult Intensive Care Unit patient mortality based on full-length 16S rRNA sequencing data from respiratory microbiome samples. Conclusions: This constitutes a proof-of-concept simulation study of how real-time bioinformatic workflows could be used to shorten the turnaround times in critical care settings and provides an instrument for future research on early-response strategies for sepsis.

Evaluation of a whole-exome sequencing pipeline and benchmarking of causal germline variant prioritizers.

Most causal variants of Mendelian diseases are exonic. Whole-exome sequencing (WES) has become the diagnostic gold standard, but causative variant prioritization constitutes a bottleneck. Here we assessed an in-house sample-to-sequence pipeline and benchmarked free prioritization tools for germline causal variants from WES data. WES of 61 unselected patients with a known genetic disease cause was obtained. Variant prioritizations were performed by diverse tools and recorded to obtain a diagnostic yield when the causal variant was present in the first, fifth, and 10th top rankings. A fraction of causal variants was not captured by WES (8.2%) or did not pass the quality control criteria (13.1%). Most of the applications inspected were unavailable or had technical limitations, leaving nine tools for complete evaluation. Exomiser performed best in the top first rankings, while LIRICAL led in the top fifth rankings. Based on the more conservative top 10th rankings, Xrare had the highest diagnostic yield, followed by a three-way tie among Exomiser, LIRICAL, and PhenIX, then followed by AMELIE, TAPES, Phen-Gen,  AIVar, and VarNote-PAT. Xrare, Exomiser, LIRICAL, and PhenIX are the most efficient options for variant prioritization in real patient WES data.

Tracing the trajectories of SARS-CoV-2 variants of concern between December 2020 and September 2021 in the Canary Islands (Spain).

Several variants of concern (VOCs) explain most of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic waves in Europe. We aimed to dissect the spread of the SARS-CoV-2 VOCs in the Canary Islands (Spain) between December 2020 and September 2021 at a micro-geographical level. We sequenced the viral genome of 8,224 respiratory samples collected in the archipelago. We observed that Alpha (B.1.1.7) and Delta (B.1.617.2 and sublineages) were ubiquitously present in the islands, while Beta (B.1.351) and Gamma (P.1/P.1.1) had a heterogeneous distribution and were responsible for fewer and more controlled outbreaks. This work represents the largest effort for viral genomic surveillance in the Canary Islands so far, helping the public health bodies in decision-making throughout the pandemic.

Nanopore sequencing and its application to the study of microbial communities.

Since its introduction, nanopore sequencing has enhanced our ability to study complex microbial samples through the possibility to sequence long reads in real time using inexpensive and portable technologies. The use of long reads has allowed to address several previously unsolved issues in the field, such as the resolution of complex genomic structures, and facilitated the access to metagenome assembled genomes (MAGs). Furthermore, the low cost and portability of platforms together with the development of rapid protocols and analysis pipelines have featured nanopore technology as an attractive and ever-growing tool for real-time in-field sequencing for environmental microbial analysis. This review provides an up-to-date summary of the experimental protocols and bioinformatic tools for the study of microbial communities using nanopore sequencing, highlighting the most important and recent research in the field with a major focus on infectious diseases. An overview of the main approaches including targeted and shotgun approaches, metatranscriptomics, epigenomics, and epitranscriptomics is provided, together with an outlook to the major challenges and perspectives over the use of this technology for microbial studies.

NanoCLUST: a species-level analysis of 16S rRNA nanopore sequencing data.

SUMMARY: NanoCLUST is an analysis pipeline for the classification of amplicon-based full-length 16S rRNA nanopore reads. It is characterized by an unsupervised read clustering step, based on Uniform Manifold Approximation and Projection (UMAP), followed by the construction of a polished read and subsequent Blast classification. Here, we demonstrate that NanoCLUST performs better than other state-of-the-art software in the characterization of two commercial mock communities, enabling accurate bacterial identification and abundance profile estimation at species-level resolution. AVAILABILITY AND IMPLEMENTATION: Source code, test data and documentation of NanoCLUST are freely available at https://github.com/genomicsITER/NanoCLUST under MIT License. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Increasing SARS-CoV-2 RT-qPCR testing capacity by sample pooling.

OBJECTIVES: Limited testing capacity has characterized the ongoing coronavirus disease 2019 (COVID-19) pandemic in Spain, hampering timely control of outbreaks and opportunities to reduce the escalation of community transmission. This study investigated the potential to use sample pooling, followed by one-step retrotranscription and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) to increase testing capacity for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). METHODS: Various pool sizes (five, 10 and 15 samples) were evaluated prior to RNA extraction followed by standard RT-qPCR for the diagnosis of COVID-19. The pool size achieving reproducible results with individual sample testing was subsequently used to assess nasopharyngeal samples in a tertiary hospital in August 2020. RESULTS: A pool size of five samples had higher sensitivity compared with pool sizes of 10 and 15 samples, showing a mean cycle threshold (Ct) shift of 3.5 [standard deviation (SD) 2.2] between the pooled test and positive samples in the pool. Next, a pool size of five was used to test a total of 895 pools (4475 prospective samples) using two different RT-qPCR kits. The Real Accurate Quadruplex corona-plus PCR Kit (PathoFinder) reported the lowest mean Ct shift [2.2 (SD 2.4)] between the pool and individual samples. This strategy enables detection of individual positive samples in positive pools with Ct of 16.7-39.4. CONCLUSIONS: Grouping samples into pools of five for RT-qPCR resulted in an increase in SARS-CoV-2 testing capacity with minimal loss of sensitivity compared with testing each sample individually.

Sensitivity of different RT-qPCR solutions for SARS-CoV-2 detection.

OBJECTIVES: The ongoing COVID-19 pandemic continues to impose demands on diagnostic screening. In anticipation that the recurrence of outbreaks and the measures for lifting the lockdown worldwide may cause supply chain issues over the coming months, this study assessed the sensitivity of a number of one-step retrotranscription and quantitative polymerase chain reaction (RT-qPCR) solutions to detect SARS-CoV-2. METHODS: Six different RT-qPCR alternatives were evaluated for SARS-CoV-2/COVID-19 diagnosis based on standard RNA extractions. The one with best sensitivity was also assessed with direct nasopharyngeal swab viral transmission medium (VTM) heating; thus overcoming the RNA extraction step. RESULTS: A wide variability in the sensitivity of RT-qPCR solutions was found that was associated with a range of false negatives from 2% (0.3-7.9%) to 39.8% (30.2-50.2%). Direct preheating of VTM combined with the best solution provided a sensitivity of 72.5% (62.5-81.0%), in the range of some of the solutions based on standard RNA extractions. CONCLUSIONS: Sensitivity limitations of currently used RT-qPCR solutions were found. These results will help to calibrate the impact of false negative diagnoses of COVID-19, and to detect and control new SARS-CoV-2 outbreaks and community transmissions.

Estimation of parasite age and synchrony status in Plasmodium falciparum infections.

Human malaria parasites have complex but poorly understood population dynamics inside their human host. In some but not all infections, parasites progress synchronously through the 48 h lifecycle following erythrocyte invasion, such that at any one time there is a limited spread of parasites at a particular time (hours) post-invasion. Patients presenting with older parasites, and with asynchronous infections, have been reported to have higher risks of fatal outcomes, associated with higher parasite biomass and multiplication rates respectively. However, practical tools to assess synchrony and estimate parasite age post-invasion in patient samples are lacking. We have developed a novel method based on three genes differentially expressed over the parasite intra-erythrocytic lifecycle, and applied it to samples from patients with uncomplicated malaria attending two health clinics in Ghana. We found that most patients presented with synchronous infections, and with parasites within 12 h of erythrocyte invasion. Finally we investigated if clinical features such as fever and parasite density could act as predictors of parasite age and synchrony. The new method is a simple and practicable approach to study parasite dynamics in naturally-infected patients, and is a significant improvement on the subjective microscopical methods for parasite staging in vivo, aiding patient management.

Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples.

OBJECTIVES: The gold-standard COVID-19 diagnosis relies on detecting SARS-CoV-2 using RNA purification and one-step retrotranscription and quantitative PCR (RT-qPCR). Based on the urgent need for high-throughput screening, we tested the performance of three alternative, simple and affordable protocols to rapidly detect SARS-CoV-2, bypassing the long and tedious RNA extraction step and reducing the time to viral detection. METHODS: We evaluated three methods based on direct nasopharyngeal swab viral transmission medium (VTM) heating before the RT-qPCR: a) direct without additives; b) in a formamide-EDTA (FAE) buffer, c) in a RNAsnapTM buffer. RESULTS: Although with a delay in cycle threshold compared to the gold-standard, we found consistent results in nasopharyngeal swab samples that were subject to a direct 70°C incubation for 10 min. CONCLUSIONS: Our findings provide valuable options to overcome any supply chain issue and help to increase the throughput of diagnostic tests, thereby complementing standard diagnosis.

Detection of human disease conditions by single-cell morpho-rheological phenotyping of blood.

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis.