Molecular and phylogenetic characterization of the monkeypox outbreak in the South of Spain.

Background and Aim: Until the May 2022 Monkeypox (MPXV) outbreak, which spread rapidly to many non-endemic countries, the virus was considered a viral zoonosis limited to some African countries. The Andalusian circuit of genomic surveillance was rapidly applied to characterize the MPXV outbreak in the South of Spain. Methods: Whole genome sequencing was used to obtain the genomic profiles of samples collected across the south of Spain, representative of all the provinces of Andalusia. Phylogenetic analysis was used to study the relationship of the isolates and the available sequences of the 2022 outbreak. Results: Whole genome sequencing of a total of 160 MPXV viruses from the different provinces that reported cases were obtained. Interestingly, we report the sequences of MPXV viruses obtained from two patients who died. While one of the isolates bore no noteworthy mutations that explain a potential heightened virulence, in another patient the second consecutive genome sequence, performed after the administration of tecovirimat, uncovered a mutation within the A0A7H0DN30 gene, known to be a prime target for tecovirimat in its Vaccinia counterpart. In general, a low number of mutations were observed in the sequences reported, which were very similar to the reference of the 2022 outbreak (OX044336), as expected from a DNA virus. The samples likely correspond to several introductions of the circulating MPXV viruses from the last outbreak. The virus sequenced from one of the two patients that died presented a mutation in a gene that bears potential connections to drug resistance. This mutation was absent in the initial sequencing before treatment.

Detection of High Level of Co-Infection and the Emergence of Novel SARS CoV-2 Delta-Omicron and Omicron-Omicron Recombinants in the Epidemiological Surveillance of Andalusia.

Recombination is an evolutionary strategy to quickly acquire new viral properties inherited from the parental lineages. The systematic survey of the SARS-CoV-2 genome sequences of the Andalusian genomic surveillance strategy has allowed the detection of an unexpectedly high number of co-infections, which constitute the ideal scenario for the emergence of new recombinants. Whole genome sequence of SARS-CoV-2 has been carried out as part of the genomic surveillance programme. Sample sources included the main hospitals in the Andalusia region. In addition to the increase of co-infections and known recombinants, three novel SARS-CoV-2 delta-omicron and omicron-omicron recombinant variants with two break points have been detected. Our observations document an epidemiological scenario in which co-infection and recombination are detected more frequently. Finally, we describe a family case in which co-infection is followed by the detection of a recombinant made from the two co-infecting variants. This increased number of recombinants raises the risk of emergence of recombinant variants with increased transmissibility and pathogenicity.

Optimización y adecuación de la capacidad diagnóstica para la realización de grandes volúmenes de RT-PCR de SARS-CoV-2 / Optimization and adaptation of the diagnostic capacity for the performance of large volumes of SARS-CoV-2 RT-PCR

Introducción: Este artículo describe la planificación efectuada para adecuar la capacidad diagnóstica de grandes volúmenes de RT-PCR de SARS-CoV-2. Métodos: El análisis y predicción del flujo de trabajo incluyó el número de RT-PCR desde el inicio de la pandemia, con 31.971 registros. La planificación de la capacidad y de las opciones diagnósticas se planteó en base a los posibles escenarios derivados de las predicciones efectuadas. Resultados: De acuerdo con las predicciones obtenidas, se optó por una solución automatizada basada en el empleo de robots OT-2 (Opentrons) para configurar un flujo de trabajo reproducible, que logró una capacidad de procesamiento de 5.640 muestras/día, con un tiempo de respuesta de cuatro horas. Conclusiones: El análisis y predicción del flujo de trabajo, unido al empleo de plataformas basadas en OT-2, proporciona una infraestructura robusta que permite atender con éxito las demandas de pruebas que exige esta pandemia.(AU)

Introduction: In the present manuscript we describe the planning carried out in our hospital to adapt our diagnostic capability to perform large numbers of SARS-CoV-2 RT-PCR. Methods: The analysis and prediction of workflow included the number of RT-PCR per week from the beginning of the pandemic, with a total of 31971 determinations. The planning phase was developed based on the different scenarios previously predicted. Results: According to the predictions obtained, an automated custom solution was chosen, based on the use of the OT-2 open-source liquid-handling robots (Opentrons), to design a reproducible workflow that achieved a production capacity of 5640 samples/day, with a time of response of four hours per procedure. Conclusions: The analysis and prediction of workflow, along with the use of the robotic platforms OT-2, provided a robust structure to deal with the high demand of determinations that this pandemic requires.(AU)

Assessing the Impact of SARS-CoV-2 Lineages and Mutations on Patient Survival.

OBJECTIVES: More than two years into the COVID-19 pandemic, SARS-CoV-2 still remains a global public health problem. Successive waves of infection have produced new SARS-CoV-2 variants with new mutations for which the impact on COVID-19 severity and patient survival is uncertain. METHODS: A total of 764 SARS-CoV-2 genomes, sequenced from COVID-19 patients, hospitalized from 19th February 2020 to 30 April 2021, along with their clinical data, were used for survival analysis. RESULTS: A significant association of B.1.1.7, the alpha lineage, with patient mortality (log hazard ratio (LHR) = 0.51, C.I. = [0.14,0.88]) was found upon adjustment by all the covariates known to affect COVID-19 prognosis. Moreover, survival analysis of mutations in the SARS-CoV-2 genome revealed 27 of them were significantly associated with higher mortality of patients. Most of these mutations were located in the genes coding for the S, ORF8, and N proteins. CONCLUSIONS: This study illustrates how a combination of genomic and clinical data can provide solid evidence for the impact of viral lineage on patient survival.

Optimization and adaptation of the diagnostic capacity for the performance of large volumes of SARS-CoV-2 RT-PCR.

INTRODUCTION: In the present manuscript we describe the planning carried out in our hospital to adapt our diagnostic capability to perform large numbers of SARS-CoV-2 RT-PCR. METHODS: The analysis and prediction of workflow included the number of RT-PCR per week from the beginning of the pandemic, with a total of 31971 determinations. The planning phase was developed based on the different scenarios previously predicted. RESULTS: According to the predictions obtained, an automated custom solution was chosen, based on the use of the OT-2 open-source liquid-handling robots (Opentrons), to design a reproducible workflow that achieved a production capacity of 5640 samples/day, with a time of response of four hours per procedure. CONCLUSIONS: The analysis and prediction of workflow, along with the use of the robotic platforms OT-2, provided a robust structure to deal with the high demand of determinations that this pandemic requires.

Excretion and viability of SARS-CoV-2 in feces and its association with the clinical outcome of COVID-19.

The main objective was to evaluate the viability of the SARS-CoV-2 viral particles excreted in stools. In addition, we aimed to identify clinical factors associated with the detection of SARS-CoV-2 RNA in feces, and to determine if its presence is associated with an unfavorable clinical outcome, defined as intensive care unit (ICU) admission and/or death. A prospective multicenter cohort study of COVID-19 adult patients, with confirmed SARS-CoV-2 infection by RT-PCR assay in nasopharyngeal (NP) swabs admitted to four hospitals in Spain, from March 2020 to February 2021. Sixty-two adult COVID-19 patients had stool samples collected at admission and/or during the follow up, with a total of 79 stool samples. SARS-CoV-2 RNA was detected in stool samples from 27 (43.5%) out of the 62 patients. Replicative virus, measured by the generation of cytopathic effect in cell culture and subsequent RT-PCR confirmation of a decrease in the Ct values, was not found in any of these stool samples. Fecal virus excretion was not associated with the presence of gastrointestinal symptoms, or with differences in the evolution of COVID-19 patients. Our results suggest that SARS-CoV-2 replicative capacity is null or very limited in stool samples, and thus, the fecal-oral transmission of SARS-CoV-2 as an alternative infection route is highly unlikely. In our study, the detection of SARS-CoV-2 RNA in feces at the beginning of the disease is not associated with any clinical factor nor with an unfavorable clinical outcome.

Highly accurate whole-genome imputation of SARS-CoV-2 from partial or low-quality sequences.

BACKGROUND: The current SARS-CoV-2 pandemic has emphasized the utility of viral whole-genome sequencing in the surveillance and control of the pathogen. An unprecedented ongoing global initiative is producing hundreds of thousands of sequences worldwide. However, the complex circumstances in which viruses are sequenced, along with the demand of urgent results, causes a high rate of incomplete and, therefore, useless sequences. Viral sequences evolve in the context of a complex phylogeny and different positions along the genome are in linkage disequilibrium. Therefore, an imputation method would be able to predict missing positions from the available sequencing data. RESULTS: We have developed the impuSARS application, which takes advantage of the enormous number of SARS-CoV-2 genomes available, using a reference panel containing 239,301 sequences, to produce missing data imputation in viral genomes. ImpuSARS was tested in a wide range of conditions (continuous fragments, amplicons or sparse individual positions missing), showing great fidelity when reconstructing the original sequences, recovering the lineage with a 100% precision for almost all the lineages, even in very poorly covered genomes (<20%). CONCLUSIONS: Imputation can improve the pace of SARS-CoV-2 sequencing production by recovering many incomplete or low-quality sequences that would be otherwise discarded. ImpuSARS can be incorporated in any primary data processing pipeline for SARS-CoV-2 whole-genome sequencing.

SARS-CoV-2 viral load in nasopharyngeal swabs is not an independent predictor of unfavorable outcome.

The aim was to assess the ability of nasopharyngeal SARS-CoV-2 viral load at first patient's hospital evaluation to predict unfavorable outcomes. We conducted a prospective cohort study including 321 adult patients with confirmed COVID-19 through RT-PCR in nasopharyngeal swabs. Quantitative Synthetic SARS-CoV-2 RNA cycle threshold values were used to calculate the viral load in log10 copies/mL. Disease severity at the end of follow up was categorized into mild, moderate, and severe. Primary endpoint was a composite of intensive care unit (ICU) admission and/or death (n = 85, 26.4%). Univariable and multivariable logistic regression analyses were performed. Nasopharyngeal SARS-CoV-2 viral load over the second quartile (≥ 7.35 log10 copies/mL, p = 0.003) and second tertile (≥ 8.27 log10 copies/mL, p = 0.01) were associated to unfavorable outcome in the unadjusted logistic regression analysis. However, in the final multivariable analysis, viral load was not independently associated with an unfavorable outcome. Five predictors were independently associated with increased odds of ICU admission and/or death: age ≥ 70 years, SpO2, neutrophils > 7.5 × 103/µL, lactate dehydrogenase ≥ 300 U/L, and C-reactive protein ≥ 100 mg/L. In summary, nasopharyngeal SARS-CoV-2 viral load on admission is generally high in patients with COVID-19, regardless of illness severity, but it cannot be used as an independent predictor of unfavorable clinical outcome.

Optimization and adaptation of the diagnostic capacity for the performance of large volumes of SARS-CoV-2 RT-PCR. / Optimización y adecuación de la capacidad diagnóstica para la realización de grandes volúmenes de RT-PCR de SARS-CoV-2.

INTRODUCTION: In the present manuscript we describe the planning carried out in our hospital to adapt our diagnostic capability to perform large numbers of SARS-CoV-2 RT-PCR. METHODS: The analysis and prediction of workflow included the number of RT-PCR per week from the beginning of the pandemic, with a total of 31971 determinations. The planning phase was developed based on the different scenarios previously predicted. RESULTS: According to the predictions obtained, an automated custom solution was chosen, based on the use of the OT-2 open-source liquid-handling robots (Opentrons), to design a reproducible workflow that achieved a production capacity of 5640 samples/day, with a time of response of four hours per procedure. CONCLUSIONS: The analysis and prediction of workflow, along with the use of the robotic platforms OT-2, provided a robust structure to deal with the high demand of determinations that this pandemic requires.

Gentamicin as Empirical Treatment in Hemodialysis Patients: Safety, Pharmacokinetics, and Pharmacodynamics.

The aim of this study was to describe the safety profile and pharmacokinetic/pharmacodynamic parameters in end-stage renal disease patients who received gentamicin as empirical treatment in catheter-related bacteremia when they showed infection signs, regardless of the timing of the next HD. Patients received gentamicin 3 mg/kg before blood culture extraction when they showed infection signs and regardless of the timing of next hemodialysis session. Serum concentrations were collected after the gentamicin administration (peak level) and before the next HD (trough level). Toxicities and adverse drug events were registered. The main pharmacokinetic/pharmacodynamic goal for Gram-negative infections was peak:minimum inhibitory concentration (MIC) ≥10. Sixteen patients were included. Nephrotoxicity was not assessed in this population, and no ototoxicity was found. According to microbial isolation and gentamicin susceptibility, the value of peak:MIC was 5.4 ± 2.0. The administration of gentamicin in these conditions was safe. Estimated pharmacokinetic values were consistent with previous studies and appropriate according to peak:MIC goal for Gram-negative organisms with MIC ≤1 mg/L.