key: cord-0927479-aiwzfknd
authors: Ahmed, Warish; Bivins, Aaron; Smith, Wendy J.M.; Metcalfe, Suzanne; Stephens, Mikayla; Jennison, Amy V.; Moore, Frederick A.J.; Bourke, Jayden; Schlebusch, Sanmarie; McMahon, Jamie; Hewitson, Glen; Nguyen, Son; Barcelon, Jean; Jackson, Greg; Mueller, Jochen F.; Ehret, John; Hosegood, Ian; Tian, Wei; Wang, Haofei; Yang, Lin; Bertsch, Paul; Tynan, Josh; Thomas, Kevin V.; Bibby, Kyle; Graber, Tyson E.; Ziels, Ryan; Simpson, Stuart L.
title: Detection of the Omicron (B.1.1.529) variant of SARS-CoV-2 in aircraft wastewater
date: 2022-01-17
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2022.153171
sha: 549e813440552cec8fc7c25b424edcd4e275c20b
doc_id: 927479
cord_uid: aiwzfknd

On the 26th of November 2021 the World Health Organization (WHO) designated the newly detected B.1.1.529 lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) the Omicron Variant of Concern (VOC). The genome of the Omicron VOC contains more than 50 mutations, many of which have been associated with increased transmissibility, differing disease severity, and potential to evade immune responses developed for previous VOCs such as Alpha and Delta. In the days since the designation of B.1.1.529 as a VOC, infections with the lineage have been reported in countries around the globe and many countries have implemented travel restrictions and increased border controls in response. We putatively detected the Omicron variant in an aircraft wastewater sample from a flight arriving to Darwin, Australia from Johannesburg, South Africa on the 25th of November 2021 via positive results on the CDC N1, CDC N2, and del(69–70) RT-qPCR assays per guidance from the WHO. The Australian Northern Territory Health Department detected one passenger onboard the flight who was infected with SARS-CoV-2, which was determined to be the Omicron VOC by sequencing of a nasopharyngeal swab sample. Subsequent sequencing of the aircraft wastewater sample using the ARTIC V3 protocol with Nanopore and ATOPlex confirmed the presence of the Omicron variant with a consensus genome that clustered with the B.1.1.529 BA.1 sub-lineage. Our detection and confirmation of a single onboard Omicron infection via aircraft wastewater further bolsters the important role that aircraft wastewater can play as an independent and unintrusive surveillance point for infectious diseases, particularly coronavirus disease 2019.

Johannesburg, South Africa on the 25 th of November (https://www.abc.net.au/news/2021-11-29/nt-covid-outbreak-katherine-traveller-positive-for-omicron/100657690), and within days Australia began reporting cases of community transmission in New South Wales (NSW) and

Australian Capital Territory (ACT).

The surveillance of wastewater from passenger aircraft may afford an efficient means of monitoring VOCs, especially in the context of emerging VOCs for which introduction via travel is a concern. Early in the COVID-19 pandemic, SARS-CoV-2 RNA was successfully detected in wastewater from passenger aircraft following international flights (Ahmed et al., 2020 J o u r n a l P r e -p r o o f Journal Pre-proof and N2 RT-qPCR, the ALOD is defined as the minimum GC number with a 95% probability of detection and determined as previously described (Verbyla et al., 2016) .

To minimize RT-qPCR contamination, RNA extraction and RT-qPCR setup were performed in separate laboratories. A method negative control was included for each batch of wastewater samples. A reagent negative control was also included during RNA extraction to account for any contamination during extraction. For each RT-qPCR run, triplicate negative controls were included. All method, reagent and RT-qPCR negative controls were negative for the targets analyzed.

Confirmation of the RT-qPCR detected variant strain was accomplished by two multiplex tiling PCR sequencing approaches; the ARTIC Network V3 scheme with Nanopore, and ATOPlex amplicon sequencing. For Nanopore sequencing, short amplicon fragments specific for SARS-CoV-2 spanning the full genome were generated using the nCOV-2019 primer set V3 Both sequencing approaches suggested the detection of Omicron VOC in sample A12.

However, in this sample, the consensus genome obtained using the ARTIC V3 protocol was less complete (~61%) in comparison with that of ATOPlex (i.e., 99%). This resulted in comparatively fewer mutations detected by the ARTIC V3 protocol, particularly in the S gene (Supplementary Table ST2 cases onboard international flights (all of which were negative by pre-flight testing) (Ahmed et al., 2021) . And in the current study, a single Omicron infection onboard an international flight was detected and corroborated by wastewater analysis with RT-qPCR and genomic sequences.

 The application of routine monitoring of aircraft wastewater at airports requires further method optimization for trace detections and rapid on-site analysis rather than transporting samples to a centralized laboratory. Furthermore, collecting wastewater samples from aircraft is tedious and the success of this mode of surveillance largely dependent on the toileting behavior of passengers, the details of which cannot be easily surveyed or quantified.

 Further research is needed to identify suitable sample matrices such as air sampling or surface swab sampling to determine whether these matrices are more appropriate than wastewater. A combination of sampling approaches and high-throughput analyses at major airport hubs may have the potential to provide rapid and scalable information about importations of potential variants to public health agencies.

J o u r n a l P r e -p r o o f Phylogenetic tree and constellations haplotyping of Australian Omicron sequences clustered with aircraft wastewater Omicron sequence (A12). Omicron mutations were highlighted in blue, ambiguous mutations were highlighted in yellow, and red is the reference strain (MN908947.3)

Detection of SARS-CoV-2 RNA in commercial passenger aircraft and cruise ship wastewater: a surveillance tool for assessing the presence of COVID-19 infected travellers

Wastewater surveillance demonstrates high predictive value for COVID-19 infection on board repatriation flights to Australia

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We thank Qantas ground staff for collecting wastewater samples.

The authors have declared no conflicts of interest.

The authors did not receive any funding for this project. 

 Table 1 Detection of SARS-CoV-2 and variant Omicron RNA in aircraft wastewater samples collected from flights landed at Darwin International Airport (DRW) andSydney International Airport (SYD) J o u r n a l P r e -p r o o f