RESUMO
In the past few years NGS has become the technology of choice to replace animal-based virus safety methods and this has been strengthened by the recent revision to the ICHQ5A virus safety chapter. Here we describe the validation of an NGS method using an agnostic analysis to detect and identify RNA virus and actively replicating DNA virus contaminants in cell banks. We report the results of the validation of each step in the sequencing process that established quality criteria to ensure consistent sequencing data. Furthermore, the validation of the analysis algorithm designed to identify virus specific sequences is described along with steps undertaken to ensure the integrity of the sequencing data from generation to analysis. Lastly, the validated sequencing and analysis systems were used to establish a limit of detection (LOD) for model viruses in cells that are commonly used in biomanufacturing. The LOD from these studies ranged from 1E+03 to 1E+04 genome copies and were dependent on the virus type with little variability between the different cell types. Thus, the validation of the NGS method for adventitious agent testing and the establishment of a general LOD for cell-based samples provides a suitable alternative to traditional virus detection methods.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Humanos , Linhagem Celular , Limite de Detecção , Vírus/genética , Vírus/isolamento & purificação , Vírus de RNA/genética , Vírus de RNA/isolamento & purificação , Vírus de DNA/genéticaRESUMO
INTRODUCTION: In January 2019, the West Virginia Bureau for Public Health detected increased HIV diagnoses among people who inject drugs in Cabell County. Responding to HIV clusters and outbreaks is 1 of the 4 pillars of the Ending the HIV Epidemic in the U.S. initiative and requires activities from the Diagnose, Treat, and Prevent pillars. This article describes the design and implementation of a comprehensive response, featuring interventions from all pillars. METHODS: This study used West Virginia Bureau for Public Health data to identify HIV diagnoses during January 1, 2018-October 9, 2019 among (1) people who inject drugs linked to Cabell County, (2) their sex or injecting partners, or (3) others with an HIV sequence linked to Cabell County people who inject drugs. Surveillance data, including HIV-1 polymerase sequences, were analyzed to estimate the transmission rate and timing of infections using molecular clock phylogenetic analysis. Federal, state, and local partners designed and implemented a comprehensive response during January 2019-October 2019. RESULTS: Of 82 people identified in the outbreak, most were male (60%), were White (91%), and reported unstable housing (80%). In a large molecular cluster containing 56 of 60 (93%) available sequences, 93% of inferred transmissions occurred after January 1, 2018. HIV testing, HIV pre-exposure prophylaxis, and syringe services were rapidly expanded, leading to improved linkage to HIV care and viral suppression. CONCLUSIONS: Evidence of rapid transmission in this outbreak galvanized robust collaboration among federal, state, and local partners, leading to critical improvements in HIV prevention and care services. HIV outbreak response requires increased coordination and creativity to improve service delivery to people affected by rapid HIV transmission.