Monkeypox virus genomic accordion strategies.

The 2023 monkeypox (mpox) epidemic was caused by a subclade IIb descendant of a monkeypox virus (MPXV) lineage traced back to Nigeria in 1971. Person-to-person transmission appears higher than for clade I or subclade IIa MPXV, possibly caused by genomic changes in subclade IIb MPXV. Key genomic changes could occur in the genome's low-complexity regions (LCRs), which are challenging to sequence and are often dismissed as uninformative. Here, using a combination of highly sensitive techniques, we determine a high-quality MPXV genome sequence of a representative of the current epidemic with LCRs resolved at unprecedented accuracy. This reveals significant variation in short tandem repeats within LCRs. We demonstrate that LCR entropy in the MPXV genome is significantly higher than that of single-nucleotide polymorphisms (SNPs) and that LCRs are not randomly distributed. In silico analyses indicate that expression, translation, stability, or function of MPXV orthologous poxvirus genes (OPGs), including OPG153, OPG204, and OPG208, could be affected in a manner consistent with the established "genomic accordion" evolutionary strategies of orthopoxviruses. We posit that genomic studies focusing on phenotypic MPXV differences should consider LCR variability.

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.

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.

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.

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.

[Clinical and epidemiological features of pertussis among hospitalized infants in Seville during 2007-2011]. / Características epidemiológicas y clínicas de la tos ferina en los lactantes hospitalizados en Sevilla durante el periodo 2007-2011.

INTRODUCTION: Despite routine pertussis immunization, pertussis burden remains high, especially among infants. The aim of this study was to describe epidemiologic, clinical and outcome features in pediatric patients admitted to a tertiary hospital in Andalusia (Southern Spain) with confirmed Bordetella pertussis infection. METHODS: Retrospective descriptive study based on a review of medical records for all pediatric patients admitted to Hospital Universitario Virgen del Rocío (Sevilla) between January 1, 2007 and December 31, 2011. RESULTS: Overall, 39 patients were diagnosed with pertussis during the study period with significant higher incidence rate in 2011 compared to 2007 (p=0.0003). Eleven children were admitted to the pediatric intensive care unit (ICU) in 2010 and 2011 and two of them died. Patients who were admitted to ICU presented with more atypical disease compared to controls in a conventional ward. They were less likely to have pertussoid cough and clinical diagnosis at admission and had a smaller percentage of lymphocytes. Creactive protein values were higher and they had a longer duration of hospital stay. CONCLUSION: The pertussis incidence rate increased significantly among hospitalized infants during the study period, and was associated with severe morbidity, including unusual complications, and mortality. A higher awareness of the clinical diagnosis of pertussis among infants admitted to ICU is required due to more atypical manifestations, and the risk of sudden deterioration associated to fatal outcome.

[Detection of inducible resistance to clindamycin in cutaneous isolates of Staphylococcus spp. by phenotypic and genotypic methods]. / Detección de resistencia inducible a clindamicina en aislados cutáneos de Staphylococcus spp. por métodos fenotípicos y genotípicos.

INTRODUCTION: Resistance to macrolides, lincosamides and type B streptogramins (MLSB) in Staphylococcus isolates can be due to several mechanisms. The most important are an active efflux mechanism (MSB phenotype) and ribosomal target modification (MLSB phenotype); this latter mechanism confers resistance to all three groups of antimicrobials (MLSB resistance). Expression of MLSB resistance can be constitutive (cMLSB) or inducible (iMLSB). METHODS: A group of 117 erythromycin-resistant Staphylococcus spp. clinical isolates from cutaneous samples were selected from 536 recent clinical isolates of this microorganism. Resistance phenotypes were determined by the double disk diffusion test. Presence of the ermA, ermC, ermB and msrA genes was detected by real time PCR. RESULTS: The MSB phenotype was the most common, comprising 11.2% (7.2% in S. aureus and 23% in CoNS) of the erythromycin-resistant strains. The rate of iMLSB resistance was significantly higher, 7.4% (5.2% in S. aureus and 14% in CoNS), than the rate of cMLSB resistance, 3.2% (1.7% in S. aureus and 7.4% in CoNS). The msrA gene was present in all isolates with the MSB phenotype, and the ermC gene was the most common among clindamycin-resistant strains with the MLSB phenotype (constitutive or inducible). CONCLUSION: The good correlation between the phenotypic (disk-diffusion) and genotypic (real time PCR) methods used allows prediction of the mechanisms of erythromycin and clindamycin resistance, provides insight into the epidemiological differences in their distribution, and is an aid to selecting the most appropriate antimicrobial therapy.