Detection of SARS-CoV-2 on contact surfaces within shared sanitation facilities and assessment of the potential risks for COVID-19 infections

Contamination of contact surfaces with SARS-CoV-2 has been reported as a potential route for the community transmission of COVID-19. This could be a major issue in developing countries where access to basic sanitation is poor leading to the sharing of toilet facilities. In this study, we present the first report of SARS-CoV-2 contamination on key contact surfaces in shared toilets, in the city of Durban, using droplet digital PCR and assessed the probabilistic risks of COVID-19 infections. Approximately, 53-69% of the contact surfaces were contaminated, with SARS-CoV-2 viral loads per cm ranging from 25.9 to 132.69 gc/cm. Toilet seats had the highest contamination per cm. The results suggested that the leading cause of contamination in shared toilets could be the shedding of the viral particles in feces and contaminated hands. We observed a significant reduction in viral loads on the contaminated surfaces after cleaning, showing the potential of effective cleaning on the reduction of contamination of these surfaces. The probabilistic assessment showed a high potential for COVID-19 infections. Touching the internal latch of the toilet cubicle had the highest risk of infections (4.3x10(6.0x10)) when a person uses the toilet once in a day, increasing to 1.0x10(1.4x10) for three uses in a day. The risks estimated in this study were higher than any of the tolerable/acceptable risk figures proposed for COVID-19 from environmental exposure. This calls for the implementation of risk reduction measures, such as strict adherence to wearing face masks, regular washing of hands with soap, and effective and regular cleaning.


INTRODUCTION
The current COVID-19 pandemic has claimed over 943 000 lives and infected another 30 million globally as at 18 th September 2020 1 . The primary mode of transmission of the SARS-CoV-2 virus, the causative agent for COVID-19, is through respiratory droplets 2,3,4,5 . This has led to the implementation of mitigation measures, such as social distancing and the use of face masks 6,7,8,9,10 . Additionally, there are reports of potential transmission through contact with contaminated surfaces 11,12,13 . These are of concern due to the stability/survival of this virus on the surface such as plastic, steel, wood and aluminium 14,15,16 . Their survival on contact surfaces is dependent on the material, for instance, it is reported to persist on plastics for 3-4 days 14,15 , aluminium for 2-3 hours 16 , stainless steel for four days and on glass for two days 14 , all at room temperature. However, Goldman 17 posited that most of these studies reporting on the survival of SARS-CoV-2 or surrogate viruses on fomites exaggerate the potential risks due to the use of unrealistic viral titre. Despite in-depth information on the potential transmission routes of the virus, there is a lack of data on the role of shared sanitation facilities as a possible route of transmission. Although this has been studied within hospital settings 18,19 , the risks posed by shared sanitation facilities outside of the hospital environment have been neglected.
The reported shedding of viral particles, by both symptomatic and asymptomatic individuals, highlights the increased risks from the use of shared sanitation. The World Health Organization (WHO) reports that between 2-27% of COVID-19 patients have diarrhoea 20 , which results in the frequent shedding of this virus in feces. SARS-CoV-2 viral loads of 1.7 × 10 6 -4.1 × 10 7 gc/mL have been reported by Han et al., 21 , and 6.3× 10 6 -1.26× 10 8 gc/g of stool by Lescure et al., 22 . These results show that in circumstances where fecal contamination of surfaces could occur, such as shared sanitation facilities, the risks of COVID-19 infections could be high. This is especially important in slums or informal settlements in developing countries such as South Africa, where a lack of basic sanitation facilities is a significant concern. The World Bank reported that living in cramped conditions within cities has a significant contribution to a high risk of infections with COVID-19 23 .
The risks associated with shared sanitation could be due to the contamination of contact surfaces by infected individuals either via deposition of aerosols or faecal matter or urine contaminations. Additionally, several studies have shown strong evidence in support of the indoor airborne transmission of viruses, especially in crowded and poorly ventilated areas 24, 25, 26,27 , such as shared toilets. For instance, SARS-CoV-2 is reported to survive in aerosols for up to 3 hours 28 , meaning the sharing of toilet facilities could be major risk factor. Therefore, by detecting and quantifying the concentration of SARS-CoV-2 on key contact surfaces within these shared sanitation facilities, the risks of infection could be estimated. The quantitative microbial risks assessment (QMRA) approach has been encouraged as a tool to assess risks associated with bioaerosols, drinking water, reclaimed water and irrigation water 29,30, 31, 32, 33 . This approach has been used in estimation of the risks for COVID-19 infections for wastewater treatment workers 34 and exposure in a market setting 35 .
This research focuses on: (i) the detection of SARS-CoV-2 on key contact surfaces within a shared toilet facility in an informal settlement (slum) in Durban city. This could provide background information on the contamination of such surfaces within similar shared facilities, and (ii) an assessment of the risk of COVID-19 infections due to the use of such shared facilities. The approach presented in this study can be used in developing risks reduction measures aimed at reducing the spread of COVID-19 (and possible other similar outbreaks) via the use of shared toilet facilities.

STUDY AREA AND SAMPLING
Two peri-urban informal settlements located within the eThekwini municipality of South Africa were selected for this study. A total of eight (8) shared toilets, referred to as community ablution blocks (CABs), were investigated, four in each settlement. The contact surfaces selected included the following: cistern handle, toilet seat, floor surface in front of the toilet, internal pull latch of cubicle door and tap in wash hand basin ( Figure 1). These were selected based on recommendations made in previous studies 36,37,38 . A total of 68 swab samples were taken, with additional six (6) fecal samples that were intentionally sampled for analysis.
Sampling was done twice in September 2020 when the reported active clinical cases were low in South Africa. The swab samples were taken according to the methodology proposed by Park et al., 37 . Briefly, the swab was moistened with PCR grade nuclease free water moved across the sampling area horizontally, vertically and diagonally. An area of approximately, 50cm 2 was swabbed for the toilet seat and toilet floors, 20cm 2 for the cistern handle and internal latch and 30cm 2 for the tap handle. The swab area was determined based on the available area of these contact surfaces.
Swabs were placed in a 400uL PCR-grade nuclease free water and transported to the lab on ice. The personnel carrying out the sampling were fully clothed in personal protective equipment (face masks, shields, lab coats, gloves and face shields).

Molecular detection of SARS-CoV-2
Upon arrival at the laboratory, each tube containing the swab was vortexed for 10s and the swab carefully removed from the tube, pressing gently against the side of the tube to remove excess water. The swab was then discarded and disposed of as biohazard waste. Two approaches were used in the detection of the viral RNA in the samples. An initial direct detection without RNA extraction was done. This involved using 5 uL of the solution as a template for the molecular analysis. The second approach involved the extraction of the RNA as described below.

RNA extraction:
Nucleic acid was extracted directly from 140 µl of swab solution using the QiAmp Viral RNA MiniKit (Qiagen, Hilden, Germany) according to manufacturer's instructions. RNA was eluted in 80 µl of sterile nuclease free water and then quantified using the Implen Nanophotometer® NP 80 (Implen GmbH, Munich, Germany). In the absence of a viral control strain, the suitability of a concentration method to be used in our study was determined by assessing the

Risk of COVID-19 infection from the use of shared sanitation: A case of the community ablution blocks
The quantitative microbial risk assessment (QMRA) approach was used for the health risk assessment. According to Haas et al. 39 the QMRA approach involves a sequence of interrelated steps: a) hazard identification; b) exposure assessment; c) dose-response assessment and d) risk characterization. This will the first report for assessing risks from the use of sanitation facilities despite the widespread understanding that sanitation facilities may facilitate its spread.

Hazard Identification:
The SARS-CoV-2 virus is the hazard of choice for this assessment.
To date two papers has reported a risks assessment for SARS-CoV-2 using the quantitative microbial risk assessment approach 34,35 . However, the potential risks of infection with this virus from contact surfaces has been established.
Exposure assessment: Contact surfaces are recognized as important routes for the spread of infectious diseases, mainly through surface-hand interactions. These surfaces sometimes referred to as fomites, have been associated with outbreaks in cruise ships, restaurants, nursing homes, schools, daycare centres and gyms 40,41,42,43 . Therefore, the main exposure scenario considered in this study is hand contamination as a result of contact with the surfaces monitored. To assess the dose of the SARS-CoV-2 virus ingested via this route Figure 2 presents the process flow. These are adopted for the SARS-CoV-2 because SARS-CoV-2 and SARS-CoV have the same cell receptor (angiotensin-converting enzyme 2 (ACE2) and a similar cellular tropism 46,47 .
These dose-response parameters have been used in assessing the risks of COVID-19 infections for workers in wastewater treatment plants 34 .
The dose d was based on the concentration of the viral RNA detected by the ddPCR analysis.
This accounted for the fraction of the viral particles that are transferred from the contact surfaces to the mouth/lips or eyes. A two-step process was used to calculate the dose; 1. The efficiency of viral transfer from the contact surface to the hand was accounted for by assuming that 2 cm 2 of the surface will be touched with a transfer efficiency as presented in Table 1. 2. The potential of transfer of the viral particle on the hands to the mouth/lips or eyes. Table 1 presents the information used to ascertain the concentration of the SARS-CoV-2 virus transferred from the contact surface to the hands and subsequently from the hands to the mouth/lips or eyes. Table 1: Input information for determination of dose of SARS-CoV-2 transferred from contact surfaces to mouth/lips or eyes.

Statistical analysis
Descriptive statistics to represent the mean and standard deviation were performed with Excel (Microsoft Corporation, USA). Comparison of viral load between the different contact surfaces was performed using the Kruskal-Wallis Test, comparison between two data categories (such as comparing viral load on cleaned and uncleaned surfaces) was done using the Mann Whitney Test. Comparative statistical analysis were all performed with GraphPad Prism Version 7 (GraphPad Software, CA, USA). All the QMRA modelling was done with @Risk Version 7.5 (Palisade Corporation, USA) addon to Excel.

Prevalence of contamination
The chance of contamination on the contact surfaces varied over the two sampling events. The highest prevalence of contamination of 68.8% was observed for the tap handle, followed by the toilet floor with the internal latch giving the least prevalence of contamination (53.3%) ( Figure 3). Despite the observed difference, there was no statistically significant difference in the prevalence (p value ≥ 0.05). Out of the six (6) faecal samples purposefully taken, five (5) were positive for SARS-CoV-2 representing a prevalence of about 83.3%

Concentration of SARS-CoV-2 on contact surfaces before and after cleaning
Per cm 2 swabbed, the concentration of SARS-CoV-2 was highest on the toilet seats (132.7 (±252.7) gc/cm 2 ), followed by the cistern handle (72.2 (±75.3) gc/cm 2 ) and internal latch (51.5 (±42.9) gc/cm 2 ). The differences in the concentration between the different contact surfaces were statistically significant (p value ≤ 0.05). Cleaning reduced the concentration on these contact surfaces, with significant (p value ≤ 0.05) reduction on the toilet seat, cistern handle, internal latch and toilet floors. For instance, after cleaning, the viral load on the toilet seats was 2.1(±0.6) gc/cm 2 ( Figure 4). However, there was no significant reduction in the SARS-CoV-2 viral load on the tap handles after cleaning, as shown in Figure 4. Additionally, the standard deviation in the viral load measured on the cleaned and uncleaned contact surfaces indicated a trend. The variation on the uncleaned surfaces was consistently higher than the mean values calculated; however, on the cleaned surfaces, the standard deviations were lower than the mean values (Table S1: Appendix 1). The mean viral load in the positive faecal samples was 381.3 (±266.1) gc/g.

Comparison of direct quantification and quantification via extracted RNA
Detection of the SARS-CoV-2 on the swab without an initial RNA extraction step presented higher prevalence compared with the prevalence observed using the extracted RNA. For instance, via direct sample analysis, the highest prevalence was observed for cistern handle (83.3%) with a corresponding prevalence of 37.5% when the viral RNA was extracted first before analysis. Similar trends were observed, where prevalence was consistently lower when the RNA was extracted. The only exception were swab samples from the floor, where prevalence via analysis of extracted RNA was higher (66.7%) compared to direct detection (50%) ( Figure 5A). Contact surface

Uncleaned Cleaned
There was a similar trend in the difference in the viral load when these two approaches (direct quantification and quantification via extracted RNA) were used. For instance, via direct quantification without RNA extraction, 244.9 (±339.6) g/cm 2 was recorded on the toilet seats, however when the RNA was extracted the concentrations were 21 (±15.8) gc/cm 2 . These differences are statistically significant, indicating consistently lower concentrations when the RNA was extracted from the samples prior to analysis. However, as observed with the prevalence, the only exception was the floor swab samples ( Figure 5B) 15 | P a g e

Potential risks of infection with COVID-19 from use of the shared toilets
The probability of infection with COVID-19 as a result of exposure to the SARS-CoV-2 virus on these contact surfaces varied considerably, driven mainly by the difference in the viral loads described above. The magnitude of the risks was similar for contact with all the surfaces (10 -2 ), however the highest risks were observed for contact with the internal latch of the toilet cubicles. It was estimated that approximately four (4) out of every 100 people using the toilet who touch the internal latch could potentially be infected with COVID-19. The lowest risks were determined for contact with the toilet floors, where an estimated eight (8) out of a 1000 people exposed may be infected ( Table 2). These estimates were made based on a single exposure event. However, considering that these toilet facilities are the only source of sanitation services within the communities studied, providing both access to potable water and sanitation multiple exposures within a day were considered. Use of the toilet facilities twice or three times in a day was observed to increase the risks of infections with COVID-19. For instance, multiple contact with the internal latch within a day resulted in an increase in the risks from 4.3x10 -2 (6.0x10 -4 ) for a single exposure to 1.0x10 -1 (1.4x10 -3 ). This means that for every 10 people who use the toilet facility twice or three times in a day at least one of them will be infected. Similar significantly increased risks were observed for all the other contact surfaces ( Table 2).

DISCUSSION
Contact surface contamination within the toilet facilities was widespread (Figure 3 (Table 2). It is worth noting that the estimated risks from contact with all the contact surfaces were all within the same magnitude (10 -2 ). This shows that despite the significant difference in viral load per cm 2 between the different contact surfaces, the risks did not differ significantly. A manageable risk of 1.17×10 -3 has been recommended by Zhang et al., 35 , meaning 1 person out of a thousand being infected is acceptable. In contrast Zanetti et al., 34 derived a tolerable risk of infection for SARS-CoV-2 to be 5.5×10 −4 per person per year (pppy), setting a very high tolerable/acceptable risk figure.
Considering both one-time and multiple exposures, the risks estimates from our study are much higher than these recommended tolerable/acceptable risks figure. The risks estimated from this study were higher compared with the risks of COVID-19 infections for customers within a market 35 . However, the estimates were similar to data published by Zanetti et al., 34 for workers in wastewater treatment plants (2.6×10 −3 to 1.3×10 −2 ). The comparative risks estimate from our study and for the wastewater treatment workers is largely due to the high viral loads measured on the contact surfaces and wastewater. The most fundamental assumption in these risks estimates is that the viral particles detected are infectious. Reports have shown that SARS-CoV-2 viral particles shed in feces may still be infectious 72,73,74 , however this is inconclusive due to the varying reports on their survival in the environment. It is also important to consider that the potential risk can be high due to the frequent use of these facilities by the communities.
The contact time is very short due to a high population that rely on these facilities and the SARS-CoV-2 virus is reported to be survice on surfaces from a few hours 14 , to four days 14,15 .
Cleaning could potentially reduce the risks of infection, however, in our study, we observed that despite the significant reduction in viral load after cleaning on almost all the surfaces, the potential of infections with COVID-19 was still high. The reduction in risks of infections was not commensurate with the decrease in viral load. However, it must be noted that we assumed a worst-case scenario where a gene copy is considered an infectious viral particle. This could potentially result in over estimation of the associated risks, because the detection and quantification were based on viral RNA and inactivated viruses may still yield positive results.
Tuladhar et al., 75 found residual bacterial and viral contamination on surfaces after cleaning, which means the detection of the SARS-CoV-2 on the contact surfaces after cleaning could be residual viral particles. Therefore, the estimated risks on the contact surfaces after cleaning could be much lower. However, to ensure maximum protection for users of these shared toilets and other facilities with similar characteristics, other risks reduction interventions should be considered.

CONCLUSIONS
We established in this study that key contact surfaces within shared toilets investigated in this study were contaminated with SARS-CoV-2, with the highest prevalence of contamination on the tap and cistern handles. This shows areas of high hand contact had the highest possibility of being contaminated, indicating that uncleaned hands may be the main source of contamination. However, based on viral load per cm 2 , the most contaminated surface is the toilet seat, the shedding of SARS-CoV-2 virus in feces and urine could be the main reason for this high concentration. We also showed that the presence and quantity of SARS-CoV-2 on contact surfaces could be determined directly without an RNA extraction step using ddPCR, which can potentially reduce the cost associated with such analysis. Cleaned contact surfaces had significantly lower viral load compared to the uncleaned surfaces except for the tap handle, this shows that the potential risks of infection with COVID-19 due to contact with these surfaces could be reduced with effective and regular cleaning. We determined that the use of the shared toilets could potentially cause COVID-19 infections, with risks estimates higher than any tolerable/acceptable risk figures published.

RECOMMENDATION/ RISK REDUCTION INTERVENTIONS
The observed risks of infections associated with the use of the shared toilets call for the introduction of additional measures to protect public health, especially in developing countries where a large population is relying on community toilets. Some of these risk reduction measures are: 1. Frequent and effective cleaning: Cleaning of the shared toiles is currently done once a day, due to the high contamination found on the key contact surfaces we recommend that cleaning be carried out at least twice. For instance, Tuladhar et al., 75  4. Face masks: Aerosols are easily generated during flushing and these may remain suspended for a while, therefore the use of face masks could provide an additional layer of protection.

ACKNOWLEDGEMENT
We acknowledge the financial support from the South African Research Chair Initiative