key: cord-0078466-bphnevdk authors: Meumann, Ella M.; Menouhos, Dimitrios; Christofis, Stefanos; Kondambu-Saaka, Kwaku M.; Harbidge, Jaimee; Dakh, Farshid; Freeman, Kevin; Baird, Robert title: Local genomic sequencing enhances COVID-19 surveillance in the Northern Territory of Australia date: 2022-05-23 journal: Pathology DOI: 10.1016/j.pathol.2022.03.005 sha: f760b3c74d1ab2e97e215b8ecdd12402a2f6d989 doc_id: 78466 cord_uid: bphnevdk nan To the Editor, Genomic sequencing has played a critical role in the global public health response to COVID-19. Within days of the first reported cases of pneumonia of unknown aetiology in Wuhan, the novel coronavirus now known as SARS-CoV-2 was identified by metagenomic sequencing and made publicly available, enabling rapid development of sensitive and specific diagnostic tests and development of effective vaccines. 1 Genomic sequencing is now a gold standard component of 'precision public health', with surveillance applications including outbreak investigation, identification of emerging variants of concern, and estimation of transmission dynamics. 2e4 In 2020, the AusTrakka platform developed by the Communicable Disease Genomics Network was launched for data sharing and centralised pathogen genomic analysis across Australia and New Zealand, 5 and all jurisdictions now have capacity to sequence and submit SARS-CoV-2 genomes. SARS-CoV-2 variants of concern associated with increased transmissibility, increased virulence, and/or immune evasion, have been defined by the World Health Organization (WHO). In AprileMay 2021, SARS-CoV-2 lineage B.1.617.2 (Delta) caused a surge in cases in India, subsequently becoming the dominant SARS-CoV-2 lineage globally. 6 In late November 2021 there was a rapid increase in SARS-CoV-2 lineage B.1.1.529 case numbers in Gauteng province, South Africa, displacing Delta as the dominant strain. 7 B.1.1.529 is phylogenetically distinct from other variants of concern and has many mutations predicted to be associated with immune evasion and increased transmissibility. 8 This lineage was designated the Omicron variant of concern by WHO on 26 November 2021. The Northern Territory (NT) is a sparsely populated and geographically remote Australian jurisdiction, with a population of 245,000 and an area of over a million square kilometres. Approximately one-third of the population are Australian First Nations people, almost three-quarters of whom live in remote regions. 9 To mitigate the risk of COVID-19 in the NT, from March 2020 individuals entering the NT from overseas and designated Australian 'hotspot' regions were required to undertake a mandatory period of quarantine. Until 20 December 2021, with few exceptions the requirement was a 14-day period of supervised quarantine at the Howard Springs Quarantine Facility, known as the Centre for National Resilience. Until November 2021, there was no sustained SARS-CoV-2 community transmission in the NT. Territory Pathology has performed SARS-CoV-2 testing for all NT public hospitals, government-operated testing clinics, and quarantine facilities since the beginning of the pandemic. Until recently, genomic sequencing for NT COVID-19 cases was carried out at a reference laboratory in Melbourne; however, the Darwin-Melbourne transit time for samples is 1e4 days. In September 2021 Territory Pathology began SARS-CoV-2 sequencing on site at Royal Darwin Hospital. Here, we report on two months' experience with local SARS-CoV-2 genomic sequencing in the NT, including detection of the Omicron variant of concern, and genomic epidemiological investigation of the first large NT community COVID-19 outbreak. We included all sequenced COVID-19 cases between 3 October 2021 and 3 December 2021 in the study. Testing for SARS-CoV-2 was performed on swabs collected from the oropharynx and bilateral deep nasal passages using real time quantitative polymerase chain reaction (RT-qPCR). Commercial assays including AusDiagnostics (ORF1a and ORF8 targets; AusDiagnostics, Australia), BD MAX (nucleocapsid phosphoprotein genes N1 and N2 targets; BD, USA), Cobas 6800 (ORF1a/b and envelope E gene targets; Roche, USA), and Xpert (E and N2 targets; Cepheid, USA) are in use at Territory Pathology. Genomic sequencing was performed on one sample for each imported quarantine case, on at least one case per household for community cases, and on all community cases without a known epidemiological link to a previous COVID-19 case; sequencing was only undertaken if RT-qPCR cycle thresholds were <30 on both targets on the AusDiagnostics platform. Genomic sequencing was performed using the COVIDSeq Assay kit (Illumina, USA) using ARTIC version 3 primers (https://github.com/artic-network/artic-ncov2019/ tree/master/primer_schemes/nCoV-2019) and 75 bp paired end reads on the Illumina iSeq 100. Sequence data were uploaded to the Illumina Basespace cloud, and analysed with the DRAGEN COVID Lineage pipeline v3.5.4 to generate consensus sequences and assign lineages (pangolin v3.1.16; pangoLEARN v1.2.97). 10 Short read sequence files were uploaded to the NCBI Sequence Read Archive (BioProject PRJNA817869) and consensus sequences were uploaded to GISAID (https://www.gisaid.org/). The NT SARS-CoV-2 genomes were uploaded to AusTrakka for real time surveillance, and for this report were included in a phylogenetic analysis in the context of publicly available Australian SARS-CoV-2 genomes. To select context genomes, all 5,212 sequences from Australia between 3 October 2021 and 3 December 2021 with 5% missing or ambiguous base calls were downloaded from GISAID on 4 December 2021, and 386 of these were randomly selected for inclusion. In addition, all eight available Australian sequences belonging to lineage B.1.1.529 (Omicron) were included. The SARS-CoV-2 genomes were aligned to the Wuhan-Hu-1 reference genome using MAFFT v7.464.11, 11 and problematic sites (https://raw.githubusercontent.com/W-L/ ProblematicSites_SARS-CoV2/master/problematic_sites_ sarsCov2.vcf, updated 27 October 2021) were masked from the alignment. Phylogenetic analysis was undertaken using IQ-TREE v1.6.12 12 using a generalised time reversible model with four gamma categories and 1,000 ultrafast bootstrap replicates. The phylogenetic tree was annotated using the ggtree (https://bioconductor.org/packages/release/ bioc/html/ggtree.html) package in R v4.0.2 (https://cran.rproject.org/bin/windows/base/). The study was approved by the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research (approval number 2020e3737). There were 22 notified imported COVID-19 cases in domestic and international quarantine during the study period, and 15 cases had samples sequenced. Except for one case, these belonged to B.1.617.2 (Delta) and were distributed throughout the phylogenetic tree (Fig. 1) . On 25 November 2021, a flight arrived in Darwin carrying 20 passengers from Johannesburg, South Africa. On day one of quarantine testing, one passenger tested positive to SARS-CoV-2 by RT-qPCR. This individual was asymptomatic, had nucleocapsid IgG detected in July 2021 indicating past SARS-CoV-2 infection, and had received two doses of the Pfizer mRNA vaccine in August and September 2021. We tested this sample (NT-RDH-37) on all platforms in use at Territory Pathology, and there were no target failures (Table 1 ). Genomic sequencing revealed that the infecting SARS-CoV-2 lineage was B.1.1.529 (sublineage BA.1; Omicron). The NT-RDH-37 sequence had 94.11% coverage, and <10Â coverage for five amplicons ( Table 1 ). The consensus sequence was uploaded to GISAID on 29 November 2021 (accession EPI_ISL_6825551), 3 days after Omicron had been declared a variant of concern, and at the time was the third publicly available B.1.1.529 sequence from the Oceania region and the 139th B.1.1.529 sequence on GISAID. Compared to the reference genome there were 30 amino acid substitutions detected in the spike protein (Table 1) . There were 60 community COVID-19 cases notified during the study period. On 3 November 2021 a positive SARS-CoV-2 RT-qPCR result was detected in a symptomatic Darwin resident with no history of recent interstate or overseas travel. On testing the contacts of this case, who lived between Darwin and the town of Katherine, 320 km to the south, a further three COVID-19 cases were diagnosed between 4 and 8 November 2021. Samples from these four cases underwent genomic sequencing. The SARS-CoV-2 genomes from all four cases belonged to B.1.617.2 (Delta), and formed a genomic cluster within a clade predominantly comprising Victorian SARS-CoV-2 genomes (Fig. 1) . On 15 November 2021, a positive SARS-CoV-2 RT-qPCR result was detected from a swab collected in the Katherine District Hospital Emergency Department 2 days previously. On the same day, a positive RT-qPCR result was detected by Cepheid Xpert in the clinic at Robinson River, a remote First Nations community of w300 individuals located w500 km southeast from Katherine. Neither of these cases had recently left the NT, and both had no known epidemiologic links to previous COVID-19 cases. Phylogenetic analysis revealed that these two cases belonged to the same genomic cluster as the initial four community cases, confirming a single introduction event (Fig. 1) . To 3 December 2021, a further 54 COVID-19 cases were diagnosed across the communities of Katherine, Robinson River, Binjari, and Lajamanu. All 23 sequenced SARS-CoV-2 genomes from the outbreak belonged to the same genomic cluster (Fig. 1) . Here, we have described our experience with local SARS-CoV-2 genomic sequencing in a sparsely populated, geographically remote Australian jurisdiction. The workflow associated with the kit we are using includes RNA-to-cDNA conversion, SARS-CoV-2 DNA amplification, library preparation, sequencing, lineage assignment, and consensus sequence generation; to have all steps integrated into one kit with an established protocol has reduced the lead-in time needed to commence this work. The option of uploading sequences to the cloud to run analysis pipelines using a graphical user interface has enabled our molecular scientists to generate consensus sequences without having coding skills. We still rely on centralised bioinformatics support through AusTrakka for phylogenetic analysis; however, the 1e4 day delay associated with specimen transport is circumvented by local sequencing. This enabled us to detect an emergent variant within 3 days of it being declared a variant of concern by the WHO. It has also led to timely genomic epidemiological investigation of local community cases, with associated strengthening of the relationship between the laboratory and the public health unit coordinating surveillance and response. Future challenges include increasing throughput, which will require some automation and a sequencing platform with greater capacity, and broadening our scope to other pathogens of public health concern. A new coronavirus associated with human respiratory disease in China Surveillance for SARS-CoV-2 variants of concern in the Australian context Genomics-informed responses in the elimination of COVID-19 in Victoria, Australia: an observational, genomic epidemiological study Revealing COVID-19 transmission in Australia by SARS-CoV-2 genome sequencing and agent-based modeling AusTrakka: fast-tracking nationalized genomics surveillance in response to the COVID-19 pandemic Increased transmissibility and global spread of SARS-CoV-2 variants of concern as at Network for Genomic Surveillance in South Africa. Briefing on COVID-19 and vaccination programme developments 25 Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa Northern Territory economy: population Assignment of epidemiological lineages in an emerging pandemic using the pangolin tool MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies Table 1 Features of the NT-RDH-37 (EPI_ISL_6825551) Omicron specimen and genome Feature RT-qPCR cycle thresholds AusDiagnostics: ORF1a 28 Roche Cobas: ORF1a/b 30.29, E 31.00 Amplification by ARTIC v3 primers Amplicons with <10Â sequencing coverage: 64 Acknowledgements: We gratefully acknowledge the AusTrakka and Microbiological Diagnostic Unit Public Health Laboratory teams for their generosity in SARS-CoV-2 sequencing and analysis support. We are also grateful to the Northern Territory Centre for Disease Control and Royal Darwin Hospital Department of Infectious Diseases teams