Improvements in metagenomic virus detection by simple pretreatment methods

Highlights • Methods assessed for non-viral nucleic acid depletion from clinical samples.• SARS-CoV-2, human papillomavirus (HPV) and molluscum contagiosum virus (MCV) detection used as test of concept.• DNase I treatment followed by filtration dramatically improved virus detection efficiency.• Presenting a metagenomic workflow with Nanopore sequencing for prompt RNA and DNA virus detection.• The Delta variant of SARS-CoV-2, HPV-genotype and a co-infection of HPV and MCV-1 were correctly identified with this workflow. Early detection of pathogens at the point of care helps reduce the threats to human and animal health from emerging pathogens. Initially, the disease-causing agent will be unknown and needs to be identified;this often requires specific laboratory facilities. Here we describe the development of an unbiased detection assay for RNA and DNA viruses using metagenomic Nanopore sequencing and simple methods that can be transferred into a field setting. Human clinical samples containing the RNA virus SARS-CoV-2 or the DNA viruses human papillomavirus (HPV) and molluscum contagiosum virus (MCV) were used as a test of concept. Firstly, the virus detection potential was optimized by investigating different pretreatments for reducing non-viral nucleic acid components. DNase I pretreatment followed by filtration increased the proportion of SARS-CoV-2 sequenced reads > 500-fold compared with no pretreatments. This was sufficient to achieve virus detection with high confidence and allowed variant identification. Next, we tested individual SARS-CoV-2 samples with various viral loads (measured as CT-values determined by RT-qPCR). Lastly, we tested the assay on clinical samples containing the DNA virus HPV and co-infection with MCV to show the assay's detection potential for DNA viruses. This protocol is fast (same day results). We hope to apply this method in other settings for point of care detection of virus pathogens, thus eliminating the need for transport of infectious samples, cold storage and a specialized laboratory.

Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations.

Multiple mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) increase transmission, disease severity, and immune evasion and facilitate zoonotic or anthropozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K, and N501Y, occurred in the SARS-CoV-2 spike glycoprotein in combinations that allow the simultaneous detection of VOCs. Here, we present two flexible reverse transcription-quantitative PCR (RT-qPCR) platforms for small- and large-scale screening (also known as variant PCR) to detect these mutations and schemes for adapting the platforms to future mutations. The large-scale RT-qPCR platform was validated by pairwise matching of RT-qPCR results with whole-genome sequencing (WGS) consensus genomes, showing high specificity and sensitivity. Both platforms are valuable examples of complementing WGS to support the rapid detection of VOCs. Our mutational signature approach served as an important intervention measure for the Danish public health system to detect and delay the emergence of new VOCs. IMPORTANCE Denmark weathered the SARS-CoV-2 crisis with relatively low rates of infection and death. Intensive testing strategies with the aim of detecting SARS-CoV-2 in symptomatic and nonsymptomatic individuals were available by establishing a national test system called TestCenter Denmark. This testing regime included the detection of SARS-CoV-2 signature mutations, with referral to the national health system, thereby delaying outbreaks of variants of concern. Our study describes the design of the large-scale RT-qPCR platform established at TestCenter Denmark in conjunction with whole-genome sequencing to report mutations of concern to the national health system. Validation of the large-scale RT-qPCR platform using paired WGS consensus genomes showed high sensitivity and specificity. For smaller laboratories with limited infrastructure, we developed a flexible small-scale RT-qPCR platform to detect three signature mutations in a single run. The RT-qPCR platforms are important tools to support the control of the SARS-CoV-2 endemic in Denmark.

Improvements in metagenomic virus detection by simple pretreatment methods.

Early detection of pathogens at the point of care helps reduce the threats to human and animal health from emerging pathogens. Initially, the disease-causing agent will be unknown and needs to be identified; this often requires specific laboratory facilities. Here we describe the development of an unbiased detection assay for RNA and DNA viruses using metagenomic Nanopore sequencing and simple methods that can be transferred into a field setting. Human clinical samples containing the RNA virus SARS-CoV-2 or the DNA viruses human papillomavirus (HPV) and molluscum contagiosum virus (MCV) were used as a test of concept. Firstly, the virus detection potential was optimized by investigating different pretreatments for reducing non-viral nucleic acid components. DNase I pretreatment followed by filtration increased the proportion of SARS-CoV-2 sequenced reads > 500-fold compared with no pretreatments. This was sufficient to achieve virus detection with high confidence and allowed variant identification. Next, we tested individual SARS-CoV-2 samples with various viral loads (measured as CT-values determined by RT-qPCR). Lastly, we tested the assay on clinical samples containing the DNA virus HPV and co-infection with MCV to show the assay's detection potential for DNA viruses. This protocol is fast (same day results). We hope to apply this method in other settings for point of care detection of virus pathogens, thus eliminating the need for transport of infectious samples, cold storage and a specialized laboratory.

A RT-qPCR system using a degenerate probe for specific identification and differentiation of SARS-CoV-2 Omicron (B.1.1.529) variants of concern.

Fast surveillance strategies are needed to control the spread of new emerging SARS-CoV-2 variants and gain time for evaluation of their pathogenic potential. This was essential for the Omicron variant (B.1.1.529) that replaced the Delta variant (B.1.617.2) and is currently the dominant SARS-CoV-2 variant circulating worldwide. RT-qPCR strategies complement whole genome sequencing, especially in resource lean countries, but mutations in the targeting primer and probe sequences of new emerging variants can lead to a failure of the existing RT-qPCRs. Here, we introduced an RT-qPCR platform for detecting the Delta- and the Omicron variant simultaneously using a degenerate probe targeting the key ΔH69/V70 mutation in the spike protein. By inclusion of the L452R mutation into the RT-qPCR platform, we could detect not only the Delta and the Omicron variants, but also the Omicron sub-lineages BA.1, BA.2 and BA.4/BA.5. The RT-qPCR platform was validated in small- and large-scale. It can easily be incorporated for continued monitoring of Omicron sub-lineages, and offers a fast adaption strategy of existing RT-qPCRs to detect new emerging SARS-CoV-2 variants using degenerate probes.

Household transmission of SARS-CoV-2 Omicron variant of concern subvariants BA.1 and BA.2 in Denmark.

SARS coronavirus 2 (SARS-CoV-2) continues to evolve and new variants emerge. Using nationwide Danish data, we estimate the transmission dynamics of SARS-CoV-2 Omicron subvariants BA.1 and BA.2 within households. Among 22,678 primary cases, we identified 17,319 secondary infections among 50,588 household contacts during a 1-7 day follow-up. The secondary attack rate (SAR) was 29% and 39% in households infected with Omicron BA.1 and BA.2, respectively. BA.2 was associated with increased susceptibility of infection for unvaccinated household contacts (Odds Ratio (OR) 1.99; 95%-CI 1.72-2.31), fully vaccinated contacts (OR 2.26; 95%-CI 1.95-2.62) and booster-vaccinated contacts (OR 2.65; 95%-CI 2.29-3.08), compared to BA.1. We also found increased infectiousness from unvaccinated primary cases infected with BA.2 compared to BA.1 (OR 2.47; 95%-CI 2.15-2.84), but not for fully vaccinated (OR 0.66; 95%-CI 0.57-0.78) or booster-vaccinated primary cases (OR 0.69; 95%-CI 0.59-0.82). Omicron BA.2 is inherently more transmissible than BA.1. Its immune-evasive properties also reduce the protective effect of vaccination against infection, but do not increase infectiousness of breakthrough infections from vaccinated individuals.

Household transmission of the SARS-CoV-2 Omicron variant in Denmark.

In late 2021, the Omicron SARS-CoV-2 variant overtook the previously dominant Delta variant, but the extent to which this transition was driven by immune evasion or a change in the inherent transmissibility is currently unclear. We estimate SARS-CoV-2 transmission within Danish households during December 2021. Among 26,675 households (8,568 with the Omicron VOC), we identified 14,140 secondary infections within a 1-7-day follow-up period. The secondary attack rate was 29% and 21% in households infected with Omicron and Delta, respectively. For Omicron, the odds of infection were 1.10 (95%-CI: 1.00-1.21) times higher for unvaccinated, 2.38 (95%-CI: 2.23-2.54) times higher for fully vaccinated and 3.20 (95%-CI: 2.67-3.83) times higher for booster-vaccinated contacts compared to Delta. We conclude that the transition from Delta to Omicron VOC was primarily driven by immune evasiveness and to a lesser extent an inherent increase in the basic transmissibility of the Omicron variant.

A qualitative RT-PCR assay for the specific identification of the SARS-CoV-2 B.1.1.529 (Omicron) Variant of Concern.

OBJECTIVES: The aim of this study was to develop a RT-PCR assay for the specific detection of the SARS-CoV-2 Omicron Variant of Concern (VOC) as a rapid alternative to sequencing. METHODS: A RT-PCR was designed in silico and then validated using characterised clinical samples containing Omicron (both BA.1 and BA.2 lineages) and the Omicron synthetic RNA genome. As negative controls, SARS-CoV-2 positive clinical samples collected in May 2020, and synthetic RNA genomes of the isolate Wuhan Hu-1 and of the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Iota (B.1.526), Epsilon (B.1.429) and Delta (B.1.617.2) SARS-CoV-2 VOC were used. RESULTS: Experiments performed using as templates the synthetic RNA genomes demonstrate the high specificity of the PCR-method for the SARS-CoV-2 Omicron. Despite the synthetic RNAs were used at high copy numbers, specific signal was mainly detected with the Omicron synthetic genome. Only a non-specific late signal was detected using the Alpha variant genome, but these results were considered negligible as Alpha VOC has been replaced by the Delta and it is not circulating anymore in the world. Using our method, we confirmed the presence of Omicron on clinical samples containing this variant but not of other SARS-CoV-2 lineages. The method is highly sensitive and can detect up to 1 cp of the Omicron virus per µl. CONCLUSIONS: The method presented here, in combination with other methods in use for detection of SARS-CoV-2, can be used for an early identification of Omicron.

Risk of hospitalisation associated with infection with SARS-CoV-2 omicron variant versus delta variant in Denmark: an observational cohort study.

BACKGROUND: Estimates of the severity of the SARS-CoV-2 omicron variant (B.1.1.529) are crucial to assess the public health impact associated with its rapid global dissemination. We estimated the risk of SARS-CoV-2-related hospitalisations after infection with omicron compared with the delta variant (B.1.617.2) in Denmark, a country with high mRNA vaccination coverage and extensive free-of-charge PCR testing capacity. METHODS: In this observational cohort study, we included all RT-PCR-confirmed cases of SARS-CoV-2 infection in Denmark, with samples taken between Nov 21 (date of first omicron-positive sample) and Dec 19, 2021. Individuals were identified in the national COVID-19 surveillance system database, which included results of a variant-specific RT-PCR that detected omicron cases, and data on SARS-CoV-2-related hospitalisations (primary outcome of the study). We calculated the risk ratio (RR) of hospitalisation after infection with omicron compared with delta, overall and stratified by vaccination status, in a Poisson regression model with robust SEs, adjusted a priori for reinfection status, sex, age, region, comorbidities, and time period. FINDINGS: Between Nov 21 and Dec 19, 2021, among the 188 980 individuals with SARS-CoV-2 infection, 38 669 (20·5%) had the omicron variant. SARS-CoV-2-related hospitalisations and omicron cases increased during the study period. Overall, 124 313 (65·8%) of 188 980 individuals were vaccinated, and vaccination was associated with a lower risk of hospitalisation (adjusted RR 0·24, 95% CI 0·22-0·26) compared with cases with no doses or only one dose of vaccine. Compared with delta infection, omicron infection was associated with an adjusted RR of hospitalisation of 0·64 (95% CI 0·56-0·75; 222 [0·6%] of 38 669 omicron cases admitted to hospital vs 2213 [1·5%] of 150 311 delta cases). For a similar comparison by vaccination status, the RR of hospitalisation was 0·57 (0·44-0·75) among cases with no or only one dose of vaccine, 0·71 (0·60-0·86) among those who received two doses, and 0·50 (0·32-0·76) among those who received three doses. INTERPRETATION: We found a significantly lower risk of hospitalisation with omicron infection compared with delta infection among both vaccinated and unvaccinated individuals, suggesting an inherent reduced severity of omicron. Our results could guide modelling of the effect of the ongoing global omicron wave and thus health-care system preparedness. FUNDING: None.

Epidemiological characterisation of the first 785 SARS-CoV-2 Omicron variant cases in Denmark, December 2021.

By 9 December 2021, 785 SARS-CoV-2 Omicron variant cases have been identified in Denmark. Most cases were fully (76%) or booster-vaccinated (7.1%); 34 (4.3%) had a previous SARS-CoV-2 infection. The majority of cases with available information reported symptoms (509/666; 76%) and most were infected in Denmark (588/644; 91%). One in five cases cannot be linked to previous cases, indicating widespread community transmission. Nine cases have been hospitalised, one required intensive care and no deaths have been registered.