Genetic Study of SARS-CoV-2 Non Structural Protein 12 in COVID-19 Patients Non Responders to Remdesivir.

Remdesivir (RDV) was the first antiviral drug approved by the FDA to treat severe coronavirus disease-2019 (COVID-19) patients. RDV inhibits SARS-CoV-2 replication by stalling the non structural protein 12 (nsp12) subunit of the RNA-dependent RNA polymerase (RdRp). No evidence of global widespread RDV-resistance mutations has been reported, however, defining genetic pathways to RDV resistance and determining emergent mutations prior and subsequent antiviral therapy in clinical settings is necessary. This study identified 57/149 (38.3%) patients who did not respond to one course (5-days) (n = 36/111, 32.4%) or prolonged (5 to 20 days) (n = 21/38, 55.3%) RDV therapy by subgenomic RNA detection. Genetic variants in the nsp12 gene were detected in 29/49 (59.2%) non responder patients by Illumina sequencing, including the de novo E83D mutation that emerged in an immunosuppressed patient after receiving 10 + 8 days of RDV, and the L838I detected at baseline and/or after prolonged RDV treatment in 9/49 (18.4%) non responder subjects. Although 3D protein modeling predicted no interference with RDV, the amino acid substitutions detected in the nsp12 involved changes on the electrostatic outer surface and in secondary structures that may alter antiviral response. It is important for health surveillance to study potential mutations associated with drug resistance as well as the benefit of RDV retreatment, especially in immunosuppressed patients and in those with persistent replication. IMPORTANCE This study provides clinical and microbiologic data of an extended population of hospitalized patients for COVID-19 pneumonia who experienced treatment failure, detected by the presence of subgenomic RNA (sgRNA). The genetic variants found in the nsp12 pharmacological target of RDV bring into focus the importance of monitoring emergent mutations, one of the objectives of the World Health Organization (WHO) for health surveillance. These mutations become even more crucial as RDV keeps being prescribed and new molecules are being repurposed for the treatment of COVID-19. The present article offers new perspectives for the clinical management of non responder patients treated and retreated with RDV and emphasizes the need of further research of the benefit of combinatorial therapies and RDV retreatment, especially in immunosuppressed patients with persistent replication after therapy.

Validación de un método seguro y sencillo para la elaboración de secuencias consenso del virus de la inmunodeficiencia humana a partir de los datos de secuenciación masiva 454 / A safe an easy method for building consensus HIV sequences from 454 massively parallel sequencing data

Objetivo: Generar una secuencia consenso a partir de los datos de secuenciación masiva obtenidos en estudios de resistencias a antiretrovirales, que sea representativa de la secuencia Sanger y que sirva para estudios de epidemiología molecular. Material y métodos: En 62 pacientes se obtuvo la secuencia de transcriptasa reversa-proteasa, mediante Sanger (Trugene-Siemens), y NGS (454GSJunior-Roche). Las secuencias consenso NGS se generaron con Mesquite, seleccionando umbrales 10%, 15% y 20%. Para el estudio filogenético se empleó MEGA. Resultados: Utilizando el umbral 10%, 17/62 pacientes presentaron secuencias pareadas NGS-Sanger, con una mediana de bootstrap del 88% (IQR83,5-95,5). La asociación aumenta a 36/62 pacientes y el bootstrap, a 94% (IQR85,5-98), y alcanza el máximo al 20% en 61/62 pacientes, bootstrap 99% (IQR98-100). Conclusión: Mostramos un método seguro para generar secuencias consenso NGS para su uso en estudios de epidemiología molecular procesadas con umbral 20%, de fácil uso y aplicación en los servicios de microbiología clínica (AU)

Objective: To show how to generate a consensus sequence from the information of massive parallel sequences data obtained from routine HIV anti-retroviral resistance studies, and that may be suitable for molecular epidemiology studies. Material and methods: Paired Sanger (Trugene-Siemens) and next-generation sequencing (NGS) (454 GSJunior-Roche) HIV RT and protease sequences from 62 patients were studied. NGS consensus sequences were generated using Mesquite, using 10%, 15%, and 20% thresholds. Molecular evolutionary genetics analysis (MEGA) was used for phylogenetic studies. Results: At a 10% threshold, NGS-Sanger sequences from 17/62 patients were phylogenetically related, with a median bootstrap-value of 88% (IQR83.5-95.5). Association increased to 36/62 sequences, median bootstrap 94% (IQR85.5-98)], using a 15% threshold. Maximum association was at the 20% threshold, with 61/62 sequences associated, and a median bootstrap value of 99% (IQR98-100). Conclusion: A safe method is presented to generate consensus sequences from HIV-NGS data at 20% threshold, which will prove useful for molecular epidemiological studies (AU)

A safe an easy method for building consensus HIV sequences from 454 massively parallel sequencing data. / Validación de un método seguro y sencillo para la elaboración de secuencias consenso del virus de la inmunodeficiencia humana a partir de los datos de secuenciación masiva 454.

OBJECTIVE: To show how to generate a consensus sequence from the information of massive parallel sequences data obtained from routine HIV anti-retroviral resistance studies, and that may be suitable for molecular epidemiology studies. MATERIAL AND METHODS: Paired Sanger (Trugene-Siemens) and next-generation sequencing (NGS) (454 GSJunior-Roche) HIV RT and protease sequences from 62 patients were studied. NGS consensus sequences were generated using Mesquite, using 10%, 15%, and 20% thresholds. Molecular evolutionary genetics analysis (MEGA) was used for phylogenetic studies. RESULTS: At a 10% threshold, NGS-Sanger sequences from 17/62 patients were phylogenetically related, with a median bootstrap-value of 88% (IQR83.5-95.5). Association increased to 36/62 sequences, median bootstrap 94% (IQR85.5-98)], using a 15% threshold. Maximum association was at the 20% threshold, with 61/62 sequences associated, and a median bootstrap value of 99% (IQR98-100). CONCLUSION: A safe method is presented to generate consensus sequences from HIV-NGS data at 20% threshold, which will prove useful for molecular epidemiological studies.

[Measles outbreak in Campo de Gibraltar, Cádiz, Spain, during the Period February-July 2008]. / Brote de sarampión en Campo de Gibraltar, Cádiz, durante el período febrero-julio 2008.

BACKGROUND: On the 4th of February 2008, 2 cases of measles, epidemiologically linked (2 members of the crew of the Fast-Ferry Jaime I from the company Balearia, which performs the route Algeciras-Tangier), were notified to the Epidemiological Surveillance Network in Andalusia (SVEA). The aim of this paper is to epidemiologically characterize this population level outbreak detected in the area of Campo de Gibraltar, the vaccine effectiveness and the control measures implemented. METHODS: Descriptive observational study of reported cases. We have analysed the following variables: age, sex, municipality of residence, onset date, virus genotype, groups involved, previous immunization status, interventions, vaccine effectiveness. Information sources are SVEA records, vaccination program and individual digital story (Diraya). Rates 10(5) were calculated according to age group and frequency measurements. To compare vaccine effectiveness, the Chi(2) test was used. RESULTS: We confirmed 155 cases of measles, 88.4% by laboratory techniques. Most affected age groups under 2 years (19%) and from 21 to 40 (51%). The 54.2% male. The 72,14% were not vaccinated. Virus was isolated from imported measles genotype D4. The vaccine efficacy was greater than 99%. CONCLUSIONS: The outbreak of the imported measles virus was confirmed. More than half of the cases were not vaccinated. The decrease in the incidence in vaccinated individuals recommends the necessity of carrying out Catch-Up campaigns to increase the coverage therefore avoiding the appearance of these outbreaks.

Brote de sarampión en Campo de Gibraltar, Cádiz, durante el período febrero- julio 2008 / Measles outbreak in Campo de Gibraltar, Cadiz, Spain, during the period February-July 2008

Fundamento: El 4 de febrero de 2008 se notificó a la Red de VigilanciaEpidemiológica de Andalucía (SVEA), 2 casos de sarampiónvinculados epidemiológicamente, 2 tripulantes del Fast-Ferry Jaime Ide la Compañía Balearia, que realiza la línea de Algeciras - Tánger. Elobjetivo de este trabajo es caracterizar epidemiológicamente el brote deámbito poblacional detectado en la Comarca Campo de Gibraltar, lasmedidas de control implementadas y la efectividad vacunal.Método: Estudio observacional descriptivo de casos notificados.Se analizan las variables edad, sexo, municipio de residencia,sintomatología, fecha de inicio, colectivos implicados, estado vacunalprevio, intervenciones realizadas, genotipo del virus, y efectividadvacunal. Fuentes de información los registros del SVEA, programade vacunas e historia digital individual (Diraya). Se calcularontasas x 105 por grupo de edad y medidas de frecuencia. Para comparaciónde efectividad vacunal se utilizó test Chi2.Resultados: Se confirmaron 155 casos de sarampión, 88.4%por laboratorio. Grupos de edad mas afectados menores de 2 años(19%) y de 21 a 40 (51%). El 54.2% varones. El 72,14% no estabanvacunados. Se aisló virus sarampión Genotipo D4 importado. Laefectividad vacunal era superior al 99%.Conclusiones: Se confirma un brote por virus del sarampiónimportado. Mas de la mitad de los casos no estaban vacunados. Ladisminución de la incidencia en vacunados hace necesario recomendarcampañas de Cacht - Up que aumenten las coberturas para evitarla aparición de estos brotes vacunales(AU)

Background: On the 4th of February 2008, 2 cases of measles,epidemiologically linked (2 members of the crew of the Fast-FerryJaime I from the company Balearia, which performs the route Algeciras- Tangier), were notified to the Epidemiological SurveillanceNetwork in Andalusia (SVEA). The aim of this paper is to epidemiologicallycharacterize this population level outbreak detected inthe area of Campo de Gibraltar, the vaccine effectiveness and thecontrol measures implemented.Methods: Descriptive observational study of reported cases.We have analysed the following variables: age, sex, municipality ofresidence, onset date, virus genotype, groups involved, previousimmunization status, interventions, vaccine effectiveness. Informationsources are SVEA records, vaccination program and individualdigital story (Diraya). Rates 105 were calculated according to agegroup and frequency measurements. To compare vaccine effectiveness,the Chi2 test was used.Results: We confirmed 155 cases of measles, 88.4% by laboratorytechniques. Most affected age groups under 2 years (19%) andfrom 21 to 40 (51%). The 54.2% male. The 72,14% were not vaccinated.Virus was isolated from imported measles genotype D4. Thevaccine efficacy was greater than 99%.Conclusions: The outbreak of the imported measles virus wasconfirmed. More than half of the cases were not vaccinated. Thedecrease in the incidence in vaccinated individuals recommends thenecessity of carrying out Catch-Up campaigns to increase the coveragetherefore avoiding the appearance of these outbreaks(AU)