Coronavirus disease 2019 (COVID-19) is a highly transmissible disease that has affected more than 90% of the countries worldwide. At least 17 million individuals have been infected, and some countries are still battling first or second waves of the pandemic. Nucleic acid tests, especially reverse-transcription polymerase chain reaction (RT-PCR), have become the workhorse for early detection of COVID-19 infection. Positive controls for the molecular assays have been developed to validate each test and to provide high accuracy. However, most available positive controls require cold-chain distribution and cannot serve as full-process control. To overcome these shortcomings, we report the production of biomimetic virus-like particles (VLPs) as SARS-CoV-2 positive controls. A SARS-CoV-2 detection module for RT-PCR was encapsidated into VLPs from a bacteriophage and a plant virus. The chimeric VLPs were obtained either by in vivo reconstitution and co-expression of the target detection module and coat proteins or by in vitro assembly of purified detection module RNA sequences and coat proteins. These VLP-based positive controls mimic SARS-CoV-2 packaged RNA while being non-infectious. Most importantly, we demonstrated that the positive controls are scalable, stable, and can serve broadly as controls, from RNA extraction to PCR in clinical settings.

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BackgroundIn Germany, previous reports have demonstrated transmitted HIV-1 drug resistance mutations (DRM) in 10% of newly diagnosed individuals, affecting treatment failure and the choice of antiretroviral therapy (ART). Here, we sought to understand the molecular epidemiology of HIV DRM transmission throughout the Cologne-Bonn region, an area with one of the highest rate of new HIV infections in Europe (13.7 per 100,000 habitants).MethodsWe analyzed 714 HIV-1 ART naïve infected individuals diagnosed at the University Hospitals Cologne and Bonn between 2001 and 2016. Screening for DRM was performed according to the Stanford University Genotypic Resistance Interpretation. Shared DRM were defined as any DRM present in genetically linked individuals (<1.5% genetic distance). Phylogenetic and network analyses were performed to infer putative relationships and shared DRMs.ResultsWe detected 123 DRMs in our study population (17.2% of all sequences). Prevalence of any DRM was comparable among risk groups and was highest among people from an endemic area (i.e., country with HIV prevalence >1%) (11/51, 21.6%). Nucleoside-and non-nucleoside reverse transcriptase inhibitor (NRTI/NNNRTI) resistance mutations were detected in 49 (7%) and 97 (13.6%) individuals, with the E138A in 29 (4.1%) and K103N in 11 (1.5%) being the most frequent. Frequency of DRM was comparable in clustering and not clustering individuals (17.1% vs. 17.5%). Transmission network analysis indicated that the frequency of DRM in clustering individuals was the highest in PWID (3/7, 42.9%) (Figure 1A). Genetically linked individuals harboring shared DRMs were more likely to live in suburban areas than in Central Cologne (18.8% vs. 8% of clustering sequences with DRM; Figure 1B).ConclusionThe rate of DRMs was exceptionally high in the Cologne/Bonn area. Network analysis elucidated frequent cases of shared DRMs among genetically linked individuals, revealing the potential spread of DRMs and the need to prevent onward transmission of DRM in the Cologne-Bonn area. Disclosures M. Hoenigl, Gilead, Basilea, Merck: Speaker’s Bureau, Research grant and Speaker honorarium.