Table 2.

Non-human studies (wastewater studies)

Study	Setting	Methods	Sample source	Sample n/d	Live cultures	Notes
Ahmed 2020	Commercial passenger aircrafts; (i) Los Angeles–Brisbane (arrival on 26 April 2020; 117 px plus crew; duration 13 h and 52 min); (ii) Hong Kong–Brisbane (arrival on 07 May 2020; 19 px plus crew; duration 8 h and 10 min; (iii) New Delhi–Sydney (arrival on 10 May 2020; 185 passengers plus crew; duration 11 h and 23 min	Observational; seven samples were concentrated using the adsorption—extraction method and three samples were concentrated using Amicon® Ultra-15 centrifugal filter (Merck Millipore Ltd). RNA was directly extracted from the electronegative membrane using a combination of two kits (RNeasy PowerWater Kit and RNeasy PowerMicrobiome Kit; Qiagen, Hilden, Germany). The authors used five different RT qPCR assays (targeting different regions of RNA from SARS-CoV-2 genome)	Wastewater; 3 wastewater samples (1 l each) were collected from a valve at the bottom of the vacuum-truck that collects the wastewater tanks of the aircraft immediately after landing. The tanks of the aircraft and the vacuum trucks were emptied but not cleaned between flights	The results showed positive SARS-CoV-2 signals though concentrations were close to the limit of detection	N/A	Cq values of SARS-CoV-2 in RT-qPCR positive samples were near the assay limit of detection ALOD (i.e. amplified between 37 and 40 cycles). The RT-qPCR amplifications were not consistent for all RT-qPCR replicates; Cq values of the positive samples ranged from 36.3 to 39. 0 It is possible that the SARS-CoV-2 RNA detected could be carried over from other flights or residuals left in the vacuum truck
Albastaki 2020	198 commercial aircrafts from 59 airport destinations from all six continents	Observational; viral RNA was concentrated following a modified method, with an initial step of pipetting 10 ml of the wastewater sample through 11-μm pore size, 125-mm diameter cellulose filter (Z240095; Whatman®), followed by centrifuging 1.5 ml of the filtered sample at 4750 g for 30 min. Without disturbing the pellet, 400 μl of the supernatant was later centrifuged at 3500 g for 15 min through MB Spin Column from the DNeasy PowerSoil Kit® (12888-100; Qiagen, Germany), the eluted sample was collected for extraction. Viral RNA was extracted using MagMax Viral/Pathogen Kit (A42352; ThermoFisher Scientific™, MA, US) following the manufacturer manual using KingFisher™ Flex Purification System (5 400 610, ThermoFisher Scientific™, Massachusetts, US). The presence of SARS-CoV-2 RNA was tested using RT-PCR by the detection of three different genes specific to this virus; ORF1ab, N gene and S gene. The adopted qPCR methodology followed TaqPath™ Covid-19 RT-PCR Kit (A48067; ThermoFisher Scientific™, Massachusetts, US), using the manufacturer’s protocol. MS2 is used as an internal standard, and nuclease free water as a negative control. Samples were prepared and set accordingly with the total volume of 25 μl. The reactions were carried out using QuantStudio™ 5 Real-Time PCR System (A34322; ThermoFisher Scientific™, Massachusetts, US). The results were analyzed as instructed in the manual	Wastewater. A dedicated team from Dubai Airports collected samples from arriving aircrafts directly from the excretory valve beneath the airplane, using a big bucket. The wastewater was then transferred into1000-ml LDPE bottles (BNH1000BULK; Azlon®, Staffordshire, UK) and stored at room temperature waiting for processing	Percentage of positive signals showed to be 13.6%; Ct values that ranged from 33 to 36	N/A	Ct = 33–36. 10/16 flights coming from Pakistan were found to be positive for SARS-CoV-2 RNA