Accelerating SARS-CoV-2 genomic surveillance in a routine clinical setting with nanopore sequencing.

BACKGROUND: SARS-CoV-2 genomic analysis has been key to the provision of valuable data to meet both epidemiological and clinical demands. High-throughput sequencing, generally Illumina-based, has been necessary to ensure the widest coverage in global variant tracking. However, a speedier response is needed for nosocomial outbreak analyses and rapid identification of patients infected by emerging VOCs. An alternative based on nanopore sequencing may be better suited to delivering a faster response when required; however, although there are several studies offering side-by-side comparisons of Illumina and nanopore sequencing, evaluations of the usefulness in the hospital routine of the faster availability of data provided by nanopore are still lacking. RESULTS: We performed a prospective 10-week nanopore-based sequencing in MinION in a routine laboratory setting, including 83 specimens where a faster response time was necessary. The specimens analyzed corresponded to i) international travellers in which lineages were assigned to determine the proper management/special isolation of the patients; ii) nosocomial infections and health-care-worker infections, where SNP-based comparisons were required to rule in/out epidemiological relationships and tailor specific interventions iii) sentinel cases and breakthrough infections to timely report to the Public Health authorities. MinION-based sequencing was compared with the standard procedures, supported on Illumina sequencing; MinION accelerated the delivery of results (anticipating results 1-12 days) and reduced costs per sample by 28€ compared to Illumina, without reducing accuracy in SNP calling. CONCLUSIONS: Parallel integration of Illumina and nanopore sequencing strategies is a suitable solution to ensure both high-throughput and rapid response to cope with accelerating the surveillance demands of SARS-CoV-2 while also maintaining accuracy.

Early SARS-CoV-2 Reinfections Involving the Same or Different Genomic Lineages, Spain.

Centers for Disease Control and Prevention guidelines consider SARS-CoV-2 reinfection when sequential COVID-19 episodes occur >90 days apart. However, genomic diversity acquired over recent COVID-19 waves could mean previous infection provides insufficient cross-protection. We used genomic analysis to assess the percentage of early reinfections in a sample of 26 patients with 2 COVID-19 episodes separated by 20-45 days. Among sampled patients, 11 (42%) had reinfections involving different SARS-CoV-2 variants or subvariants. Another 4 cases were probable reinfections; 3 involved different strains from the same lineage or sublineage. Host genomic analysis confirmed the 2 sequential specimens belonged to the same patient. Among all reinfections, 36.4% involved non-Omicron, then Omicron lineages. Early reinfections showed no specific clinical patterns; 45% were among unvaccinated or incompletely vaccinated persons, 27% were among persons <18 years of age, and 64% of patients had no risk factors. Time between sequential positive SARS-CoV-2 PCRs to consider reinfection should be re-evaluated.

Sensitive Instrumental Method for Quantitative Determination of High-Brominated Flame Retardants in Human Serum Samples.

BACKGROUND: High-brominated flame retardants (BFRs) can be released into the environment from consumer products, such as electric and electronic equipment, and enter the human body by different pathways. Because of their toxicity and the regulations, it is very relevant to know their levels and trends in human samples. However, chromatographic serum analysis of some of these compounds represents nowadays a challenge in the general population. OBJECTIVE: To optimize and validate an instrumental method based on gas chromatography coupled to mass spectrometry, which, together with a simple sample preparation procedure, allows the analysis of decabromodiphenyl ether (BDE-209), decabromodiphenyl ethane (DBDPE), and tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA-DBPE) in human serum samples from the general population. METHOD: To minimize the high degradation during instrumental analysis, GC parameters such as injection volumes, carrier flow rates, and column lengths were assessed and optimized. This instrumental approach in combination with solid-phase extraction (SPE) followed by multilayer silica gel column purification allowed satisfactory analysis using only 1 mL of serum. RESULTS: The performance of the complete method was evaluated at three spiking levels, 0.01, 0.05, and 0.2 ng/mL. Recoveries in the range 87-108% were obtained whereas the relative standard deviation in interday measurements, were, in general, lower than 19%. Limits of detection were in the range of 0.0045-0.0070 ng/mL. The optimized procedure was successfully applied to the determination of the investigated pollutants in real human samples of general population. CONCLUSIONS: The proposed method could contribute to the inclusion of these environmental pollutants in human biomonitoring (HBM) studies, increasing the knowledge of levels and trends in the general population. HIGHLIGHTS: GC-MS parameters optimization to minimize instrumental analytes degradation. Successful application to human serum samples from the general population. Tetrabromobisphenol A bis(2,3-dibromopropyl ether) human serum levels are reported for the first time.

A One Health approach revealed the long-term role of Mycobacterium caprae as the hidden cause of human tuberculosis in a region of Spain, 2003 to 2022.

IntroductionMycobacterium caprae is a member of the Mycobacterium tuberculosis complex (MTBC) not routinely identified to species level. It lacks specific clinical features of presentation and may therefore not be identified as the causative agent of tuberculosis. Use of whole genome sequencing (WGS) in the investigation of a family microepidemic of tuberculosis in Almería, Spain, unexpectedly identified the involvement of M. caprae.AimWe aimed to evaluate the presence of additional unidentified M. caprae cases and to determine the magnitude of this occurrence.MethodsFirst-line characterisation of the MTBC isolates was done by MIRU-VNTR, followed by WGS. Human and animal M. caprae isolates were integrated in the analysis.ResultsA comprehensive One Health strategy allowed us to (i) detect other 11 M. caprae infections in humans in a period of 18 years, (ii) systematically analyse M. caprae infections on an epidemiologically related goat farm and (iii) geographically expand the study by including 16 M. caprae isolates from other provinces. Integrative genomic analysis of 41 human and animal M. caprae isolates showed a high diversity of strains. The animal isolates' diversity was compatible with long-term infection, and close genomic relationships existed between isolates from goats on the farm and recent cases of M. caprae infection in humans.DiscussionZoonotic circulation of M. caprae strains had gone unnoticed for 18 years. Systematic characterisation of MTBC at species level and/or extended investigation of the possible sources of exposure in all tuberculosis cases would minimise the risk of overlooking similar zoonotic events.

Rapid Identification of Relevant Microbial Strains by Identifying Multiple Marker Single Nucleotide Polymorphisms via Amplicon Sequencing: Epidemic Monkeypox Virus as a Proof of Concept.

Despite the proven value of applying genomic data for epidemiological purposes, commonly used high-throughput sequencing formats are not adapted to the response times required to intervene and finally control outbreaks. In this study, we propose a fast alternative to whole-genome sequencing (WGS) to track relevant microbiological strains: nanopore sequencing of multiple amplicons including strain marker single nucleotide polymorphisms (SNPs). As a proof a concept, we evaluated the performance of our approach to offer a rapid response to the most recent public health global alarm, the monkeypox virus (MPXV) global outbreak. Through a multisequence alignment, a list of 42 SNPs were extracted as signature makers for this outbreak. Twenty primer pairs were designed to amplify in a multiplex PCR the regions including 22 of these SNPs. Amplicon pools were sequenced in a MinION device, and SNPs were called in real time by an in-house bioinformatic pipeline. A total of 120 specimens (95 MPXV-PCR positive, Ct values from 14 to 39) were selected. In 67.37% of the positive subset, all 22 SNPs were called. After excluding low viral load specimens, in 92% of samples ≥11 outbreak SNPs were called. No false positives were observed in any of the 25 negative specimens. The total turnaround time required for this strategy was 5 hours, and the cost per sample was 14 euros. Nanopore sequencing of multiple amplicons harboring signature SNPs escapes the targeting limitations of strain-specific PCRs and offers a powerful alternative to systematic WGS, paving the way to real-time genomic epidemiology and making immediate intervention possible to finally optimize transmission control. IMPORTANCE Nanopore sequencing of multiple amplicons harboring signature single nucleotide polymorphisms (SNPs) escapes the targeting limitations of strain-specific PCRs and offers a powerful alternative to systematic whole-genome analysis, paving the way to real-time genomic epidemiology and making immediate intervention possible to finally optimize transmission control.

A Small Change With a Twist Ending: A Single Residue in EGF-CFC Drives Bilaterian Asymmetry.

Asymmetries are essential for proper organization and function of organ systems. Genetic studies in bilaterians have shown signaling through the Nodal/Smad2 pathway plays a key, conserved role in the establishment of body asymmetries. Although the main molecular players in the network for the establishment of left-right asymmetry (LRA) have been deeply described in deuterostomes, little is known about the regulation of Nodal signaling in spiralians. Here, we identified orthologs of the egf-cfc gene, a master regulator of the Nodal pathway in vertebrates, in several invertebrate species, which includes the first evidence of its presence in non-deuterostomes. Our functional experiments indicate that despite being present, egf-cfc does not play a role in the establishment of LRA in gastropods. However, experiments in zebrafish suggest that a single amino acid mutation in the egf-cfc gene in at least the common ancestor of chordates was the necessary step to induce a gain of function in LRA regulation. This study shows that the egf-cfc gene likely appeared in the ancestors of deuterostomes and "protostomes", before being adopted as a mechanism to regulate the Nodal pathway and the establishment of LRA in some lineages of deuterostomes.

First importations of SARS-CoV-2 P.1 and P.2 variants from Brazil to Spain and early community transmission.

INTRODUCTION: SARS-CoV-2variants of concern (VOC) have been described in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P.1). Among them, the most scarce information has been obtained from the P.1 variant and more data on its global presence and about its spreading dynamics are needed. METHODS: Whole genome sequencing was performed prospectively on travellers arriving from Brazil and on a random selection of SARS-CoV-2 positive cases from our population. RESULTS: In this study we report the first SARS-CoV-2 P.1 and P.2 variants exported from Brazil to Spain. The case infected with the P.1 variant, who had only stayed in Rio de Janeiro, required hospitalisation. The two P.2 cases remained asymptomatic. A wider distribution for P.1 variant beyond the Brazilian Amazonia should be considered. The exportation of the P.2 variant, carrying the E484K mutation, deserves attention. One month after the first description of P.1 and P.2 importations from Brazil to Madrid, these variants were identified circulating in the community, in cases without a travel history, and involved in household transmissions CONCLUSION: Whole genome sequencing of SARS-CoV-2 positive travellers arriving from Brazil allowed us to identify the first importations of P.1 and P.2 variants to Spain and their early community transmission.

First importations of SARS-CoV-2 P.1 and P.2 variants from Brazil to Spain and early community transmission / Primeras importaciones de las variantes SARS-CoV-2 P.1 y P.2 de Brasil a Madrid y transmisión comunitaria

Introduction: SARS-CoV-2variants of concern (VOC) have been described in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P.1). Among them, the most scarce information has been obtained from the P.1 variant and more data on its global presence and about its spreading dynamics are needed.Methods: Whole genome sequencing was performed prospectively on travellers arriving from Brazil and on a random selection of SARS-CoV-2 positive cases from our population.Results: In this study we report the first SARS-CoV-2 P.1 and P.2 variants exported from Brazil to Spain. The case infected with the P.1 variant, who had only stayed in Rio de Janeiro, required hospitalisation. The two P.2 cases remained asymptomatic. A wider distribution for P.1 variant beyond the Brazilian Amazonia should be considered. The exportation of the P.2 variant, carrying the E484K mutation, deserves attention. One month after the first description of P.1 and P.2 importations from Brazil to Madrid, these variants were identified circulating in the community, in cases without a travel history, and involved in household transmissionsConclusion: Whole genome sequencing of SARS-CoV-2 positive travellers arriving from Brazil allowed us to identify the first importations of P.1 and P.2 variants to Spain and their early community transmission.

Introducción: Se han descrito «variantes de preocupación» (VOC) de SARS-CoV-2 en el Reino Unido (B.1.1.7), Sudáfrica (B.1.351) y Brasil (P.1). Entre ellas, se dispone de información más escasa para la variante P.1 y se necesitan más datos sobre su presencia global y sobre su dinámica de expansión.Métodos: Se realizó secuenciación del genoma completo de forma prospectiva de SARS-CoV-2 en viajeros procedentes de Brasil y en una selección aleatoria de casos positivos de SARS-CoV-2 de nuestra población.Resultados: En este estudio reportamos las primeras variantes de SARS-CoV-2 P.1 y P.2 exportadas desde Brasil a España. El caso infectado por la variante P.1, que solo había permanecido en Río de Janeiro, requirió hospitalización. Los 2 casos de la variante P.2 permanecieron asintomáticos. Se debe considerar una distribución más amplia para la variante P.1 más allá de la Amazonía brasileña. La exportación de la variante P.2, que porta la mutación E484K, merece asimismo atención adicional. Un mes después de la primera descripción de las importaciones de P.1 y P.2 de Brasil a Madrid, se identificaron estas variantes circulando en la comunidad, en casos sin antecedentes de viaje, e implicadas en transmisiones domiciliarias.Conclusión: La secuenciación de genoma completo de viajeros positivos para SARS-CoV-2 procedentes de Brasil nos permitió identificar las primeras importaciones de variantes P.1 y P.2 a España y su transmisión comunitaria precoz.

Post-COVID-19 syndrome. SARS-CoV-2 RNA detection in plasma, stool, and urine in patients with persistent symptoms after COVID-19.

BACKGROUND: There is a paucity of knowledge on the long-term outcome in patients diagnosed with COVID-19. We describe a cohort of patients with a constellation of symptoms occurring four weeks after diagnosis causing different degrees of reduced functional capacity. Although different hypothesis have been proposed to explain this condition like persistent immune activation or immunological dysfunction, to date, no physiopathological mechanism has been identified. Consequently, there are no therapeutic options besides symptomatic treatment and rehabilitation. METHODS: We evaluated patients with symptoms that persisted for at least 4 weeks after COVID-19. Epidemiological and clinical data were collected. Blood tests, including inflammatory markers, were conducted, and imaging studies made if deemed necessary. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) in plasma, stool, and urine were performed. Patients were offered antiviral treatment (compassionate use). RESULTS: We evaluated 29 patients who reported fatigue, muscle pain, dyspnea, inappropriate tachycardia, and low-grade fever. Median number of days from COVID-19 to positive RT-PCR in extra-respiratory samples was 55 (39-67). Previous COVID-19 was mild in 55% of the cases. Thirteen patients (45%) had positive plasma RT-PCR results and 51% were positive in at least one RT-PCR sample (plasma, urine, or stool). Functional status was severely reduced in 48% of the subjects. Eighteen patients (62%) received antiviral treatment. Improvement was seen in most patients (p = 0.000) and patients in the treatment group achieved better outcomes with significant differences (p = 0.01). CONCLUSIONS: In a cohort of COVID-19 patients with persistent symptoms, 45% of them have detectable plasma SARS-CoV-2 RNA. Our results indicate possible systemic viral persistence in these patients, who may benefit of antiviral treatment strategies.

Systematic Genomic and Clinical Analysis of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfections and Recurrences Involving the Same Strain.

Estimates of the burden of severe acute respiratory syndrome coronavirus 2 reinfections are limited by the scarcity of population-level studies incorporating genomic support. We conducted a systematic study of reinfections in Madrid, Spain, supported by genomic viral analysis and host genetic analysis, to cleanse laboratory errors and to discriminate between reinfections and recurrences involving the same strain. Among the 41,195 cases diagnosed (March 2020-March 2021), 93 (0.23%) had 2 positive reverse transcription PCR tests (55-346 days apart). After eliminating cases with specimens not stored, of suboptimal sequence quality, or belonging to different persons, we obtained valid data from 22 cases. Of those, 4 (0.01%) cases were recurrences involving the same strain; case-patients were 39-93 years of age, and 3 were immunosuppressed. Eighteen (0.04%) cases were reinfections; patients were 19-84 years of age, and most had no relevant clinical history. The second episode was more severe in 8 cases.

Overlapping prison/community tuberculosis outbreaks in Costa Rica revealed by alternative analysis of suboptimal material.

Costa Rica has a low incidence of tuberculosis. Thus, identifying transmission hotspots is key to implement interventions. A tuberculosis outbreak was suspected in a prison in Costa Rica. Given the suboptimal quality of the samples received in our laboratory in Madrid, we applied alternative schemes for their analysis. In the first scheme, we bypassed the standard approach of applying systematic mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and used a strain-specific polymerase chain reaction (PCR) that allowed identifying a cluster involving six cases (C1). The second scheme followed the canonical MIRU-VNTR path coupled with a whole-genomic amplification step, by which a second unsuspected overlapping cluster (C2), was detected in the same prison. These findings justified the implementation of a surveillance programme adapted to local resources based on a tailored multiplex allele-specific oligonucleotide (ASO)-PCR targeting C1 and C2. Presence of the C2 strain at a different prison was determined. ASO-PCR was applied extensively and alerted to the active circulation of one of the strains within and beyond prisons. Our study shows that alternative methodological strategies may provide useful data in settings with lack of resources for performing systematic standard molecular epidemiology programmes and/or with suboptimal material for analysis.

First importations of SARS-CoV-2 P.1 and P.2 variants from Brazil to Spain and early community transmission.

Introduction: SARS-CoV-2variants of concern (VOC) have been described in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P.1). Among them, the most scarce information has been obtained from the P.1 variant and more data on its global presence and about its spreading dynamics are needed. Methods: Whole genome sequencing was performed prospectively on travellers arriving from Brazil and on a random selection of SARS-CoV-2 positive cases from our population. Results: In this study we report the first SARS-CoV-2 P.1 and P.2 variants exported from Brazil to Spain. The case infected with the P.1 variant, who had only stayed in Rio de Janeiro, required hospitalisation. The two P.2 cases remained asymptomatic. A wider distribution for P.1 variant beyond the Brazilian Amazonia should be considered. The exportation of the P.2 variant, carrying the E484K mutation, deserves attention. One month after the first description of P.1 and P.2 importations from Brazil to Madrid, these variants were identified circulating in the community, in cases without a travel history, and involved in household transmissions. Conclusion: Whole genome sequencing of SARS-CoV-2 positive travellers arriving from Brazil allowed us to identify the first importations of P.1 and P.2 variants to Spain and their early community transmission.

Introducción: Se han descrito «variantes de preocupación¼ (VOC) de SARS-CoV-2 en el Reino Unido (B.1.1.7), Sudáfrica (B.1.351) y Brasil (P.1). Entre ellas, se dispone de información más escasa para la variante P.1 y se necesitan más datos sobre su presencia global y sobre su dinámica de expansión. Métodos: Se realizó secuenciación del genoma completo de forma prospectiva de SARS-CoV-2 en viajeros procedentes de Brasil y en una selección aleatoria de casos positivos de SARS-CoV-2 de nuestra población. Resultados: En este estudio reportamos las primeras variantes de SARS-CoV-2 P.1 y P.2 exportadas desde Brasil a España. El caso infectado por la variante P.1, que solo había permanecido en Río de Janeiro, requirió hospitalización. Los 2 casos de la variante P.2 permanecieron asintomáticos. Se debe considerar una distribución más amplia para la variante P.1 más allá de la Amazonía brasileña. La exportación de la variante P.2, que porta la mutación E484K, merece asimismo atención adicional. Un mes después de la primera descripción de las importaciones de P.1 y P.2 de Brasil a Madrid, se identificaron estas variantes circulando en la comunidad, en casos sin antecedentes de viaje, e implicadas en transmisiones domiciliarias. Conclusión: La secuenciación de genoma completo de viajeros positivos para SARS-CoV-2 procedentes de Brasil nos permitió identificar las primeras importaciones de variantes P.1 y P.2 a España y su transmisión comunitaria precoz.

SARS-CoV-2 superinfection and reinfection with three different strains.

We report a corona virus disease (COVID-19) case with unprecedented viral complexity. In the first severe episode, two different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains (superinfection) were identified within a week. Three months after discharge, the patient was readmitted and was infected in a nosocomial outbreak with a different strain, suffering a second milder COVID-19 episode.

Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy.

Detection of mixed Mycobacterium tuberculosis (MTB) infections is essential, particularly when resistance mutations are present in minority bacterial populations that may affect patients' disease evolution and treatment. Whole-genome sequencing (WGS) has extended the amount of key information available for the diagnosis of MTB infection, including the identification of mixed infections. Having genomic information at diagnosis for early intervention requires carrying out WGS directly on the clinical samples. However, few studies have been successful with this approach due to the low representation of MTB DNA in sputa. In this study, we evaluated the ability of a strategy based on specific MTB DNA enrichment by using a newly designed capture platform (MycoCap) to detect minority variants and mixed infections by WGS on controlled mixtures of MTB DNAs in a simulated sputum genetic background. A pilot study was carried out with 12 samples containing 98% of a DNA pool from sputa of patients without MTB infection and 2% of MTB DNA mixtures at different proportions. Our strategy allowed us to generate sequences with a quality equivalent to those obtained from culture: 62.5× depth coverage and 95% breadth coverage (for at least 20× reads). Assessment of minority variant detection was carried out by manual analysis and allowed us to identify heterozygous positions up to a 95:5 ratio. The strategy also automatically distinguished mixed infections up to a 90:10 proportion. Our strategy efficiently captures MTB DNA in a nonspecific genetic background, allows detection of minority variants and mixed infections, and is a promising tool for performing WGS directly on clinical samples. IMPORTANCE We present a new strategy to identify mixed infections and minority variants in Mycobacterium tuberculosis by whole-genome sequencing. The objective of the strategy is the direct detection in patient sputum; in this way, minority populations of resistant strains can be identified at the time of diagnosis, facilitating identification of the most appropriate treatment for the patient from the first moment. For this, a platform for capturing M. tuberculosis-specific DNA was designed to enrich the clinical sample and obtain quality sequences.

SARS-CoV-2 B.1.1.7 Decline Is Not Driven by the Introduction of a More Successful Variant.

The SARS-CoV-2 variant of concern (VOC) Delta (B.617.2 lineage) displaced the predominant VOC Alpha (B.1.1.7 lineage) in the United Kingdom. In Madrid, recent start of the decline of predominant VOC Alpha suggested an equivalent phenomenon. However, 11 different variants, none overrepresented in frequency, occupied progressively over a period of 7 weeks the niche previously dominated by VOC Alpha. Only after these 7 weeks, VOC Delta started to emerge. Viral competition due to the entry of VOC Delta is not the major force driving the start of VOC Alpha decline in Madrid. IMPORTANCE Our data indicate that the dynamics of SARS-CoV-2 VOCs turnover in our setting differ from those proposed for other countries. A systematic genomic analysis, updated on a weekly basis, of representative randomly selected samples of SARS-CoV-2 circulating variants allowed us to define a lapse of 7 weeks between the start of VOC Alpha decline and the final emergence of VOC Delta. During this period, VOC Alpha showed a sustained decline, while 11 VOCs, variants of interest (VOIs), and other identified variants, none overrepresented, occupied the niche left by VOC Alpha. Only after these 7 weeks, emergence of VOC Delta occurred, indicating that viral competition involving VOC Delta was not the exclusive direct driving force behind the starting of VOC Alpha decline.

High within-host diversity found from direct genotyping on post-mortem tuberculosis specimens in a high-burden setting.

OBJECTIVES: To characterize the clonal complexity in Mycobacterium tuberculosis (MTB) infections considering factors that help maximize the detection of coexisting strains/variants. METHODS: Genotypic analysis by Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeats (MIRU-VNTR) was performed directly on 70 biopsy specimens from two or more different tissues involving 28 tuberculosis cases diagnosed post-mortem in Mozambique, a country with a high tuberculosis burden. RESULTS: Genotypic data from isolates collected from two or more tissues were obtained for 23 of the 28 cases (82.1%), allowing the analysis of within-patient diversity. MIRU-VNTR analysis revealed clonal diversity in ten cases (35.7%). Five cases showed allelic differences in three or more loci, suggesting mixed infection with two different strains. In half of the cases showing within-host diversity, one of the specimens associated with clonal heterogeneity was brain tissue. CONCLUSIONS: Direct MTB genotyping from post-mortem tissue samples revealed a frequent within-host Mycobacterium tuberculosis diversity, including mixed and polyclonal infections. Most of this diversity would have been overlooked if only standard analysis of respiratory specimens had been performed.

Expression Pattern of Nitric Oxide Synthase during Development of the Marine Gastropod Mollusc, Crepidula fornicata.

Nitric Oxide (NO) plays a key role in the induction of larval metamorphosis in several invertebrate phyla. The inhibition of the NO synthase in Crepidula fornicata, a molluscan model for evolutionary, developmental, and ecological research, has been demonstrated to block the initiation of metamorphosis highlighting that endogenous NO is crucial in the control of this developmental and morphological process. Nitric Oxide Synthase contributes to the development of shell gland, digestive gland and kidney, being expressed in cells that presumably correspond to FMRF-amide, serotoninergic and catecolaminergic neurons. Here we identified a single Nos gene in embryonic and larval transcriptomes of C. fornicata and studied its localization during development, through whole-mount in situ hybridization, in order to compare its expression pattern with that of other marine invertebrate animal models.

Different dynamics of mean SARS-CoV-2 RT-PCR Ct values between the first and second COVID-19 waves in the Madrid population.

SARS-CoV-2 RT-PCR cycle threshold values from 18,803 cases (2 March-4 October) in Madrid define three stages: (i) initial ten weeks with sustained reduction in viral load (Ct: 23.4-32.3), (ii) stability with low viral loads (Ct: 31.9-35.5) in the next nine weeks and (iii) sudden increase with progressive higher viral loads until reaching stability at high levels in the next twelve weeks, coinciding with an increased percentage of positive cases and reduced median age. These data indicate differential virological/epidemiological patterns between the first and second COVID-19 waves in Madrid.

Inference of Active Viral Replication in Cases with Sustained Positive Reverse Transcription-PCR Results for SARS-CoV-2.

The purpose of this study was to detect coronavirus disease 2019 (COVID-19) cases with persistent positive reverse transcription-PCR (RT-PCR) results for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which viable virus can be inferred due to the presence of subgenomic (SG) viral RNA, which is expressed only in replicating viruses. RNA remnants purified from diagnostic nasopharyngeal specimens were used as the templates for RT-PCR-specific detection of SG E gene RNA. As controls, we also detected viral genomic RNA for the E gene and/or a human housekeeping gene (RNase P). We assessed the samples of 60 RT-PCR-positive cases with prolonged viral SARS-CoV-2 shedding (24 to 101 days) since the first diagnostic RT-PCR. SG viral RNA was detected in 12/60 (20%) of the persistent cases, 28 to 79 days after the onset of symptoms. The age range of the cases with prolonged viral shedding and the presence of SG RNA was quite wide (40 to 100 years), and the cases were equally distributed between males (42%) and females (58%). No case was HIV positive, although seven were immunosuppressed. According to the severities of the COVID-19 episodes, they were mild (40%), intermediate (20%), and severe (40%). In a percentage of persistent SARS-CoV-2 PCR-positive cases, the presence of actively replicating virus may be inferred, far beyond diagnosis. We should not assume a universal lack of infectiousness for COVID-19 cases with prolonged viral shedding.

Probing recalcitrant problems in polyclad evolution and systematics with novel mitochondrial genome resources.

For their apparent morphological simplicity, the Platyhelminthes or "flatworms" are a diverse clade found in a broad range of habitats. Their body plans have however made them difficult to robustly classify. Molecular evidence is only beginning to uncover the true evolutionary history of this clade. Here we present nine novel mitochondrial genomes from the still undersampled orders Polycladida and Rhabdocoela, assembled from short Illumina reads. In particular we present for the first time in the literature the mitochondrial sequence of a Rhabdocoel, Bothromesostoma personatum (Typhloplanidae, Mesostominae). The novel mitochondrial genomes examined generally contained the 36 genes expected in the Platyhelminthes, with all possessing 12 of the 13 protein-coding genes normally found in metazoan mitochondrial genomes (ATP8 being absent from all Platyhelminth mtDNA sequenced to date), along with two ribosomal RNA genes. The majority presented possess 22 transfer RNA genes, and a single tRNA gene was absent from two of the nine assembled genomes. By comparison of mitochondrial gene order and phylogenetic analysis of the protein coding and ribosomal RNA genes contained within these sequences with those of previously sequenced species we are able to gain a firm molecular phylogeny for the inter-relationships within this clade. Our phylogenetic reconstructions, using both nucleotide and amino acid sequences under several models and both Bayesian and Maximum Likelihood methods, strongly support the monophyly of Polycladida, and the monophyly of Acotylea and Cotylea within that clade. They also allow us to speculate on the early emergence of Macrostomida, the monophyly of a "Turbellarian-like" clade, the placement of Rhabditophora, and that of Platyhelminthes relative to the Lophotrochozoa (=Spiralia). The data presented here therefore represent a significant advance in our understanding of platyhelminth phylogeny, and will form the basis of a range of future research in the still-disputed classifications within this taxon.