RESUMEN
BACKGROUND: Sequencing of SARS-CoV-2 from rapid diagnostic tests (RDTs) can bolster viral genomic surveillance efforts; however, approaches to maximise and standardise pathogen genome recovery from RDTs remain underdeveloped. We aimed to systematically optimise the elution of genetic material from RDT components and to evaluate the efficacy of RDT sequencing for outbreak investigation. METHODS: In this laboratory and cohort-based study we seeded RDTs with inactivated SARS-CoV-2 to optimise the elution of genomic material from RDT lateral flow strips. We measured the effect of changes in buffer type, time in buffer, and rotation on PCR cycle threshold (Ct) value. We recruited individuals older than 18 years residing in the greater Boston area, MA, USA, from July 18 to Nov 5, 2022, via email advertising to students and staff at Harvard University, MA, USA, and via broad social media advertising. All individuals recruited were within 5 days of a positive diagnostic test for SARS-CoV-2; no other relevant exclusion criteria were applied. Each individual completed two RDTs and one PCR swab. On Dec 29, 2022, we also collected RDTs from a convenience sample of individuals who were positive for SARS-CoV-2 and associated with an outbreak at a senior housing facility in MA, USA. We extracted all returned PCR swabs and RDT components (ie, swab, strip, or buffer); samples with a Ct of less than 40 were subject to amplicon sequencing. We compared the efficacy of elution and sequencing across RDT brands and components and used RDT-derived sequences to infer transmission links within the outbreak at the senior housing facility. We conducted metagenomic sequencing of negative RDTs from symptomatic individuals living in the senior housing facility. FINDINGS: Neither elution duration of greater than 10 min nor rotation during elution impacted viral titres. Elution in Buffer AVL (Ct=31·4) and Tris-EDTA Buffer (Ct=30·8) were equivalent (p=0·34); AVL outperformed elution in lysis buffer and 50% lysis buffer (Ct=40·0, p=0·0029 for both) as well as Universal Viral Transport Medium (Ct=36·7, p=0·079). Performance of RDT strips was poorer than that of matched PCR swabs (mean Ct difference 10·2 [SD 4·3], p<0·0001); however, RDT swabs performed similarly to PCR swabs (mean Ct difference 4·1 [5·2], p=0·055). No RDT brand significantly outperformed another. Across sample types, viral load predicted the viral genome assembly length. We assembled greater than 80% complete genomes from 12 of 17 RDT-derived swabs, three of 18 strips, and four of 11 residual buffers. We generated outbreak-associated SARS-CoV-2 genomes using both amplicon and metagenomic sequencing and identified multiple introductions of the virus that resulted in downstream transmission. INTERPRETATION: RDT-derived swabs are a reasonable alternative to PCR swabs for viral genomic surveillance and outbreak investigation. RDT-derived lateral flow strips yield accurate, but significantly fewer, viral reads than matched PCR swabs. Metagenomic sequencing of negative RDTs can identify viruses that might underlie patient symptoms. FUNDING: The National Science Foundation, the Hertz Foundation, the National Institute of General Medical Sciences, Harvard Medical School, the Howard Hughes Medical Institute, the US Centers for Disease Control and Prevention, the Broad Institute and the National Institute of Allergy and Infectious Diseases.
Asunto(s)
COVID-19 , Genoma Viral , Prueba de Diagnóstico Rápido , SARS-CoV-2 , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Estudios de Cohortes , COVID-19/diagnóstico , COVID-19/virología , COVID-19/epidemiología , Prueba de Ácido Nucleico para COVID-19/métodos , Prueba de COVID-19/métodos , Genoma Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificaciónRESUMEN
Background: Households are a major setting for SARS-CoV-2 infections, but there remains a lack of knowledge regarding the dynamics of viral transmission, particularly in the setting of widespread pre-existing SARS-CoV-2 immunity and evolving variants. Methods: We conducted a prospective, case-ascertained household transmission study in the greater Boston area in March-July 2022. Anterior nasal swabs, along with clinical and demographic data, were collected for 14 days. Nasal swabs were tested for SARS-CoV-2 by PCR. Whole genome sequencing was performed on high-titer samples. Results: We enrolled 33 households in a primary analysis set, with a median age of participants of 25 years old (range 2-66); 98% of whom had received at least 2 doses of a COVID-19 vaccine. 58% of households had a secondary case during follow up and the secondary attack rate (SAR) for contacts infected was 39%. We further examined a strict analysis set of 21 households that had only 1 PCR+ case at baseline, finding an SAR of 22.5%. Genomic epidemiology further determined that there were multiple sources of infection for household contacts, including the index case and outside introductions. When limiting estimates to only highly probable transmissions given epidemiologic and genomic data, the SAR was 18.4%. Conclusions: Household contacts of a person newly diagnosed with COVID-19 are at high risk for SARS-CoV-2 infection in the following 2 weeks. This is, however, not only due to infection from the household index case, but also because the presence of an infected household member implies increased SARS-CoV-2 community transmission. Further studies to understand and mitigate household transmission are needed.