Monitoring COVID-19 transmission risks by RT-PCR tracing of droplets in hospital and living environments

SARS-CoV-2 environmental contamination occurs through droplets and biological fluids released in the surroundings from patients or asymptomatic carriers. Surfaces and objects contaminated by saliva or nose secretions represent a risk for indirect transmission of COVID-19. We assayed surfaces from hospital and living spaces to identify the presence of viral RNA and the spread of fomites in the environment. Anthropic contamination by droplets and biological fluids was monitored by detecting the microbiota signature using multiplex RT-PCR on selected species and massive sequencing on 16S-amplicons. A total of 92 samples (flocked swab) were collected from critical areas during the pandemic, including indoor (3 hospitals and 3 public buildings) and outdoor surfaces exposed to anthropic contamination (handles and handrails, playgrounds). Traces of biological fluids were frequently detected in spaces open to the public and on objects that are touched with the hands (>80%). However, viral RNA was not detected in hospital wards or other indoor and outdoor surfaces either in the air system of a COVID-hospital, but only in the surroundings of an infected patient, in consistent association with droplets traces and fomites. Handled objects accumulated the highest level of multiple contaminations by saliva, nose secretions and faecal traces, further supporting the priority role of handwashing in prevention. In conclusion, anthropic contamination by droplets and biological fluids is widespread in spaces open to the public and can be traced by RT-PCR. Monitoring fomites can support evaluation of indirect transmission risks for Coronavirus or other flu-like viruses in the environment.

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The copyright holder for this preprint this version posted August 25, 2020. . https://doi.org/10.1101/2020.08.22.20179754 doi: medRxiv preprint 6 SARS-CoV-2 was clearly detected in several studies, even when surveillance and sanitation were 106 accurately performed (Wu et al., 2020;Wang et al. 2020). 107 Finally, when approaching the question of the presence of SARS-CoV-2 in the environment, we 108 need to consider that the pathogen is conveyed by biological fluids. Therefore, the possibility of  (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The overall aim of this study was to search for both SARS-CoV-2 and fomites in hospitals and 129 public buildings, to evaluate the possibility of monitoring by RT-PCR fomites and biofluid 130 contamination, as a novel indicator of hygiene as well as a candidate marker for indirect 131 transmission risks for COVID-19. CoV-2 RNA, whereas anthropic contamination was assessed searching for biological fluids of 143 nose, mouth, gut through their microbiota traces by RT-PCR and/or NGS (Table 1) Surface sampling was carried out after their use and prolonged exposure to human presence (>4h).  (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  154 All samples in UTM were heat inactivated at 56°C for 5 minutes to reduce the risk of accidental 155 transmission of SARS-CoV-2 to laboratory personnel. Nucleic acids were purified and extracted 156 using the eMag automated nucleic acid sample extraction system (bioMérieux, Marcy l'Etoile, 157 France). Briefly, total nucleic acids were extracted from UTM using an input sample volume of 158 200 ml into 2,000 ml of easyMag lysis buffer using B protocol to a final eluted volume of purified 159 nucleic acids of 50 ml. TaqPath 1-step reverse transcriptase quantitative PCR (RT-qPCR) master (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

SARS-CoV-2 detection
The copyright holder for this preprint this version posted August 25, 2020. . https://doi.org/10.1101/2020.08.22.20179754 doi: medRxiv preprint gene (N or Orf1ab) with a Ct values ≤40 were considered inconclusive; and 3) no fluorescent 177 signals or over the 40th Ct in ORF1ab and N genes were considered not specific and reported as 178 negative for SARS-CoV-2 RNA. The declared LoD is 500 RNA copies/ml.  (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted August 25, 2020.   Positive samples were those where ≥1 positive indicator (CT ≤35) was found in at least 2 mixes. 215 Conversely, a microbial indicator was considered negative when over the CT ≥39 threshold. were obtained using Ba27F and Ba338R primers containing overhang adapters, as previously 221 described (40, 41). Tagged PCR products were generated using primer pairs with unique barcodes 222 through two-step PCR. In this strategy, target primers containing overhang adapters were used in using primers-containing barcodes. Each amplification reaction had a total volume of 25 μL,   (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted August 25, 2020.  Table 2). The presence of fomites appeared largely diffused in indoor and 268 outdoor areas exposed to human crowding or frequently touched with hands. Floors and walls were 269 less contaminated than handles or buttons. Droplets DNA traces were detected in most of surfaces 270 and almost 10% of sampled points displayed a multiple contamination from different biological 271 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted August 25, 2020. . https://doi.org/10.1101/2020.08.22.20179754 doi: medRxiv preprint 13 fluids. Correlation between selected bacterial species and biological fluids in droplets and fomites 272 was confirmed (p-value <0.01) (Table 3)   The stethoscope used on the patient was positive, too. The low frequency (<4%) of positive 291 samples in comparison with other studies (20-30%) can be associated to differences in the 292 sampling strategy or to a lower sensitivity of the method (Liu, Y., 2020); moreover, it depends on 293 the epidemiological scenario when the study was carried out, at time when reopening of activities 294 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted August 25, 2020. by NGS showed not only the wide presence of fomites on several surfaces exposed to anthropic 339 contamination, but also the inhabiting microorganisms or those from other environmental sources. 340 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted August 25, 2020. . https://doi.org/10.1101/2020.08.22.20179754 doi: medRxiv preprint feasibility and effectiveness, but we did not made comparisons with different sequences, indicators 387 or reaction conditions for RT-PCR. The same concern can be raised for the NGS approach, which 388 was adopted for the analysis of 16S rDNA amplicon sequencing, following standard protocols. A 389 whole genome analysis would have been more informative. However, it would have also been   (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In addition to searching for SARS-CoV-2 in the environment, the possibility to detect fomites by (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint this version posted August 25, 2020.  (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
657 All rights reserved. No reuse allowed without permission.
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