key: cord-341543-gcnph9gf
authors: Kuryntseva, P.; Karamova, K.; Fomin, V.; Selivanovskaya, S.; Galitskaya, P.
title: A simplified approach to monitoring the COVID-19 epidemiologic situation using waste water analysis and its application in Russia
date: 2020-09-23
journal: nan
DOI: 10.1101/2020.09.21.20197244
sha: 
doc_id: 341543
cord_uid: gcnph9gf

The number of registered cases of COVID-19 is increasing in the world, and some countries are reporting a second wave of the pandemic. Accurate and real time information about epidemiological situation is therefore urgently needed for managing decisions in the countries, regions and municipalities which are affected. Massive testing of viral presence in human saliva, a smear from the nose, nasopharynx and / or oropharynx, bronchial lavage water obtained by fibrobronchoscopy (bronchoalveolar lavage), as well as from (endo) tracheal, nasopharyngeal aspirate, sputum, biopsy or autopsy material of the lungs, whole blood, serum or antibodies presence in blood cannot give relevant information about the COVID-19 infection rate in the community since simultaneous testing of the whole community is not technically possible, the information obtained in testing of specific groups is retarded and, in addition, such testing is expensive. The alternative to mass testing of the population is the testing of wastewater that could contain SARS-CoV-2 particles originating from excreta. Such testing has several limitations connected with the particularities of the testing procedure. In the present study, a modified approach for detection of COVID-19 infection rate using wastewater analysis has been developed. The approach includes i) the creation of a calibration curve on the basis of the serial dilution of excreta collected from people who are infected with COVID-19 and ii) the analysis of wastewater samples and their serial dilutions but the approach excludes usage of concentration techniques before wastewater sample analysis as well as usage of external control in RT-PCR reactions for calculation of numbers of viral particles. The minimum infection rate that can be detected using this approach is 0.01%. The approach developed was used to investigate wastewater from eleven sewage inspection chambers in the city of Kazan (Russia). It was demonstrated that the average infection rate of people using these sewers was over 0.4% in July 2020.

by fibrobronchoscopy (bronchoalveolar lavage), as well as from (endo) tracheal, nasopharyngeal 23 aspirate, sputum, biopsy or autopsy material of the lungs, whole blood, serum or antibodies 24 presence in blood cannot give relevant information about the COVID-19 infection rate in the 25 community since simultaneous testing of the whole community is not technically possible, the 26 information obtained in testing of specific groups is retarded and, in addition, such testing is 27 expensive. The alternative to mass testing of the population is the testing of wastewater that 28 could contain SARS-CoV-2 particles originating from excreta. Such testing has several 29 limitations connected with the particularities of the testing procedure. 30 In the present study, a modified approach for detection of COVID-19 infection rate using 31 wastewater analysis has been developed. The approach includes i) the creation of a calibration 32 curve on the basis of the serial dilution of excreta collected from people who are infected with minimum infection rate that can be detected using this approach is 10-2%. The approach 37 developed was used to investigate wastewater from eleven sewage inspection chambers in the 38 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 23, 2020. . https://doi.org/10.1101/2020.09.21.20197244 doi: medRxiv preprint Introduction 42 The WHO announced that the COVID-19 outbreak had become a pandemic on 12th March 2020 43 WHO 2020). COVID-19 is caused by the respiratory SARS-CoV-2 virus with a long incubation 44 period, and very different courses of disease -from severe to asymptomatic. To the 15 th of 45 September 2020, over 29 million coronavirus cases have been reported in the world, and over 1 46 million of them in Russia, which has 4 th highest amount of cases in the world 47 (https://www.worldometers.info).

Currently, the basic mechanism for determination of the COVID-19 epidemiological situation in 49 countries is testing symptomatic cases using the PCR (polymerase chain reaction) technique and 50 evaluating the number of positive tests over time. Additional mechanisms include analysis of 51 symptoms such as those which are most common (fever, dry cough, tiredness) and less common 52 symptoms (aches and pains, sore throat, diarrhea, conjunctivitis, headache, loss of taste or smell, 53 a skin rash, or discoloration of fingers or toes) and also serious symptoms (breathing difficulty or 54 shortness of breath, chest pain or pressure, loss of speech or movement) ("Coronavirus"; La Rosa CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 23, 2020. . https://doi.org/10.1101/2020.09.21.20197244 doi: medRxiv preprint or wastewater), RNA extraction from different matrices containing different PCR and RT 79 (reverse transcription) reaction inhibitors, RT and PCR reactions, both with their own efficacy 80 depending on reagents, protocols and external controls used, and on RNA purity. Therefore, the 81 results obtained may contain significant discrepancies. Some authors suggest relying only on 82 qualitative (yes/no), but not on quantitative, information while analyzing COVID-19 83 epidemiological situation using WBE approach (Wu et al., 2020; Haramoto et al., 2020) . 84 Besides, the procedure of SARS-CoV-2 gene copy number estimation described above has 85 limitations in terms of equipment and consumables requirements, which means that the WBE 86 approach cannot be implemented in a daily routine immediately. The objectives of this study were i) to simplify and to improve the WBE approach for SARS- CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 23, 2020. . https://doi.org/10.1101/2020.09.21.20197244 doi: medRxiv preprint particles were excluded from the analysis procedure. In addition, a locally produced and easily 119 available commercial medical kit (Kuzubov, 2020) was used for RT and PCR reactions. We 120 hypothesized that the approach of finding the lowest dilution that can be qualified as coronavirus 

In this study, we aimed to develop a simplified approach for WBE of COVID-19 that can be 178 applied in Russian cities. In order to do this, modelling with feces and urine of coronavirus 179 infected people was conducted. 10 patients with differing ages and symptoms participated in the 180 modelling experiment (Table 2 ). It was found that their urine did not contain virus particles while Table S1 ). (Table 2) .

Modelling presented in Table 2 demonstrates that the method of WBE used can detect a minimal Table S2 ). The infection rate of people whose excreta are collected in the "positive" 208 inspection chamber was then calculated from the lowest dilution found to be virus positive.

The results are presented in Table 3 . As follows from the table, no SARS-CoV-2 particles were 210 found in the Kazan wastewater in March while some were found in July. Four out of eleven 211 initial wastewater samples taken in July were characterized as coronavirus positive. The lowest 212 dilutions of these samples that were also found to be positive were 1:100, 1:150, 1:75 and 1:150 213 for samples 6, 7, 10 and 11, respectively, that corresponds to the 1.00%, 1.50%, 0.75% and 1.5% 214 sickness rate in the residences that use the sewage inspection chamber. Taking into account the 215 number of apartments in the blocks presented in Table 3 can be calculated that the total number of people whose excreta were analyzed in the 219 investigation was 1790, and the number of infected people was 8. That corresponds to 0.44% 220 infection rate in the community.

Interestingly, the 11th sample was taken from the sewage inspection chamber that collects In this study, the approach of serial dilution preparation for wastewater investigation for SARS-234 CoV-2 viral load was suggested. In the modelling experiment with the excreta of ten COVID-19 235 patients, it was demonstrated that the minimal rate of infected people in the community that can 236 be detected by this method is 10-2%. This rate is in range of results presented in the literature.

Thus, it was demonstrated that the number of SARS-CoV-2 virus particles in wastewater ranges 238 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 23, 2020. . https://doi.org/10.1101/2020.09.21.20197244 doi: medRxiv preprint

COVID-19) outbreak: some serious consequences with urban and 318 rural water cycle

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Pavel Perfilov is acknowledged for inspiring this research. 

Manuscript to be reviewed . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted September 23, 2020. . https://doi.org/10.1101/2020.09.21.20197244 doi: medRxiv preprint