RU2743687C1 - Method of monitoring covid-19 morbidity with the use of wastewater analysis - Google Patents

Abstract

FIELD: virology.

SUBSTANCE: invention relates to the field of virology. Disclosed is a method for monitoring COVID-19 morbidity. According to the method, it’s necessary to determine a correction factor (m) representing a lower limit for determining the morbidity rate and monitoring COVID-19 morbidity. To determine m, aliquots of fecal samples of patients with a confirmed diagnosis of COVID-19 are taken and diluted with model wastewater to obtain samples № 1 .... No p. Samples № 1 .... No p undergo a serial dilution until the lower limit of determination of the real-time RT-PCR method is reached. After that the arithmetic mean value of the maximum degrees of dilution is averaged and calculated. The correction factor m is defined as the ratio of 100 to the arithmetic mean. RNA is isolated from wastewater samples with the purpose of monitoring. The samples are analyzed for the presence of SARS-CoV-2 viral particles. If the result is negative, the morbidity rate is 0%. If the result is positive, serial dilutions of wastewater samples are prepared until the lower limit of determination of the real-time RT-PCR method is reached. After that the maximum degree of dilution of wastewater (k) is determined. The value ​​of the morbidity rate of COVID-19 (N,%) is calculated in accordance with the formula N = k * m.

EFFECT: improved accuracy of results and reduced duration of sample analysis.

1 cl, 2 ex, 5 dwg

Description

The claimed invention relates to virology as well as measuring or testing methods using enzymes or microorganisms to monitor the incidence of COVID-19 using wastewater analysis.

As of the filing date of this application, the number of reported cases of COVID-19 in the world is growing, and some countries are reporting a second wave of the pandemic. Therefore, to make management decisions in the countries, regions and municipalities affected by the pandemic, accurate and up-to-date information about the epidemiological situation is required. To test for the presence of a disease with the COVID-19 virus, a whole range of methods is currently used based on the determination of viral particles in biological fluids and body tissues, for example, in saliva, a swab from the nose, nasopharynx and / or oropharynx, in bronchial lavage water obtained using fibrobronchoscopy (bronchoalveolar lavage), nasopharyngeal aspirate, sputum, lung biopsy or autopsy, whole blood, serum or antibodies in the blood cannot provide relevant information on the number of COVID-19 cases in the community, since simultaneous testing of the entire community is technically impossible, information obtained from testing of certain groups is not operational, besides, mass testing involves significant material costs, difficulties in the selection, delivery and storage of samples.

Further in the text, the applicant provides the terms that are necessary to facilitate an unambiguous understanding of the essence of the declared materials and to exclude contradictions and / or controversial interpretations when performing an examination on the merits.

COVID-19 is a potentially severe acute respiratory infection caused by the SARS-CoV-2 coronavirus

SARS-CoV-2 is an enveloped single-stranded RNA virus belonging to the genus Betacoronavirus, subgenus Sarbecovirus, causing a dangerous infectious disease - COVID-19

SARS-CoV-2 positive - for example, a sample, analysis, dilution, for which the presence of SARS-CoV-2 viral particles has been established by the RT-PRC-RT method.

SARS-CoV-2-negative - for example, a sample, analysis, dilution, for which the presence of SARS-CoV-2 viral particles has not been established by the RT-PRC-RT method.

PCR is a molecular biology method that allows a significant increase in small concentrations of certain fragments of nucleic acid (DNA) in a biological material (sample).

RT-PCR - real-time polymerase chain reaction (or quantitative PCR), which is a laboratory method based on the PCR method, which allows to determine not only the presence of a target nucleotide sequence in a sample, but also to measure the number of its copies.

RT-PCR-RT is a method of one-step reverse transcription reaction, combined polymerase chain reaction in real time, which allows to amplify a specific fragment of ribonucleic acid (RNA) and measure the number of its copies.

Dilution - suspensions or solutions obtained by mixing a certain volume of the original sample with a multiple volume of the diluent.

Serial dilutions - suspensions or solutions obtained by mixing a certain volume of the initial suspension with a multiple volume of the diluent and repeating this procedure until a series of multiple dilutions is obtained, suitable for further analysis

Smallest dilution - the smallest dilution of wastewater in which the presence of SARS-CoV-2 viral particles is detected by any certified method.

Wastewater dilution algorithm - a developed sequence of actions for the preparation of serial dilutions of samples

Commercial kit is a ready-to-use certified kit that includes all reagents, consumables, instructions for carrying out the work for which this commercial kit is intended.

The incidence rate of COVID-19 is the proportion of the population in a given area that is infected with COVID-19 caused by the SARS-CoV-2 virus, expressed as a percentage of the total population living in the area

The lower limit of detection is the lowest value that allows you to determine the method used for the analysis.

Correction factor is the lower limit of morbidity determination using selected commercial RNA extraction and RT-PCR-RT kits.

Excrement - human feces and urine.

COVID-19 is caused by SARS-CoV-2 viral particles, characterized by a long incubation period and various course of the disease, from severe to asymptomatic.

As of the filing date of this application, the main monitoring of the COVID-19 epidemiological situation in the world is testing patients with a characteristic set of symptoms using the PCR method and statistical analysis of the results.

Diagnostics by PCR is based on determining the sequence of SARS-CoV-2 RNA encoding gene N, gene E, Orf1ab gene, RdRp gene or fragments of these genes. In diagnostic laboratories at Dhow submission of application materials for the diagnosis of COVID-19 by PCR, commercial kits are mainly used, for example, TaqMan 2019-nCoV Assay Kit v1 (Thermo Fisher Scientific, USA), Coronavirus (COVID-19) CE IVD (Primerdesign Ltd, UK), GSD NovaPrime® SARS-CoV-2 (COVID-19) (Gold Standard Diagnostics Corp, USA), Labotaq SL, (Spain); Biosan, (Latvia); GeneProod, (Czech Republic), which are available worldwide and simplify the analysis procedure.

A commercial set of reagents PCR-RV-2019-nCov has been developed in Russia, based on the detection of fragments of the Orf1ab gene, which is officially registered and used in laboratory conditions [https://www.syntol.ru/catalog/nabory-reagentov-dlya- ptsr-v-realnom-vremeni / nabor-reagentov-ptsr-rv-2019-ncov.html].

From the investigated state of the art, the applicant identified an invention demonstrating the use of PCR methods for diagnosing COVID-19 disease under RF patent 2731390 "Test system and method for detecting RNA of viral particles of SARS-COV-2 coronavirus, virus - the causative agent of 2019 COVID-19 coronavirus disease, by the method of polymerase chain reaction in real time (Variants) ". The essence is a test system for detecting the RNA of the SARS-CoV-2 virus by carrying out a polymerase chain reaction in real time, comprising a primer SBT0017 having a nucleotide sequence 5'-GCTGGCACAGACTTAGAAGGT-3 ', a primer SBT0018 having a nucleotide sequence 5'-AGCAGCAGTACA ', and a probe SBT0019 having the sequence 5'-GTTGACAGGCAAACAGCACAAGCAGCTG-3', as well as for positive control of the passage of all stages of the assay, including the primer SBT0014 specific to the human housekeeping gene GAPDH having the nucleotide sequence 5'-TCCAAAATCAAG primer-SBTG ' having the nucleotide sequence 5'-TGATGACCCTTTTGGCTCCC-3 'and the SBT0016 probe having the sequence 5'-CGTCTTCACCACCATGGAGAAGGCTG-3', or comprising the human housekeeping gene specific ACTCCB primer SBT0011 having the nucleotide CGTCT sequence 5'-CGTCT ' SBT0012 having the nucleotide sequence 5'-AGGGTGAGGA TGCCTCTCTT-3 ', and probe SBT0013 having the nucleotide sequence 5'-GATGGTGGGCATGGGTCAGAAGGATTC-3'. (Option 1).

A test system for detecting SARS-CoV-2 virus RNA by real-time polymerase chain reaction, comprising primer SBT003 having a nucleotide sequence 5'-GTTCGCATTCAACCAGGACAG-3 ', primer SBT004 having a nucleotide sequence 5'-ACCTTCTAAGCTGTGCC, and probe SBT0026, having the sequence 5'-CTGGTGTTTACCAATGTGCTATGAGGCCC-3 ', as well as for positive control of the passage of all stages of the analysis, including the specific to the human housekeeping gene GAPDH primer SBT0014 having the nucleotide sequence 5'-TCCAAAATCA, nucleotide sequence SBTG' sequence 5'-TGATGACCCTTTTGGCTCCC-3 ', and probe SBT0016 having the sequence 5'-CGTCTTCACCACCATGGAGAAGGCTG-3', or including the human housekeeping gene specific ACTB primer SBT0011, having the nucleotide sequence 5'-CGTCC-3 ' nucleotide sequence 5'-AGGGTGAGGATGCCTCTCTT-3 ', and probe SBT0013 having the nucleotide sequence 5'-GATGGTGGGCATGGGTCAGAAGGATTC-3 '. (Option 2).

A method for detecting the RNA of the SARS-CoV-2 virus, including the isolation of RNA from biological material, the synthesis of cDNA and the PCR-RT, characterized in that the synthesis of cDNA is carried out at 45 ° C for 30 minutes, after which MMLV revertase is inactivated by heating at 95 ° C for from 2 to 5 minutes, then the obtained cDNA is amplified in RT-PCR with the participation of a test system specific to the viral gene 3CLpro according to claim 1 or 2, and as a control in the same or parallel, but with the same sample, cDNA is amplified housekeeping gene GAPDH with the participation of a pair of primers and a probe or cDNA of the housekeeping gene ACTB with the participation of a pair of primers and a probe, while the RT-PCR temperature regime includes the following stages: heating at 95 ° C for 2 to 5 minutes, combined with inactivation of MMLV revertase, and from 45 to 49 amplification cycles 95 єС - from 5 to 15 s, 55 єС - from 20 s to 1 min, and fluorescence detection is carried out during each cycle at a temperature of 55 єС via HEX / VIC channels for viral infection and FAM for the housekeeping gene sequence, with the results interpreted based on the appearance of a characteristic kinetic curve and its intersection with the threshold line.

The known technical solution is used in the claimed technical solution as a tool for detecting the presence of RNA virus particles SARS-CoV-2.

There are known methods for monitoring the epidemiological situation of COVID-19, which include data on the number of cases based on the analysis of the clinical picture of the patient's disease by medical workers and the diagnosis. Among the diagnostic signs, groups of symptoms are distinguished: common - fever, dry cough, fatigue, less common - sore throat, diarrhea, conjunctivitis, headache, loss of taste or smell, skin rash or discoloration of fingers or toes, and rare, usually observed in severe cases, shortness of breath or shortness of breath, chest pain or pressure, loss of speech or movement [Coronavirus Disease (COVID-19) Situation Reports…; La Rosa et al., 2020; Mao et al., 2020]. Based on the diagnoses made by such a method and diagnoses established by the PCR method, a statistical analysis of the data is carried out and the level of morbidity is established, which makes it possible to track the epidemiological situation.

A common disadvantage of the known methods is that they make it possible to estimate the incidence rate for only a part of the population who sought medical help due to the development of symptoms.

Accordingly, the method based on the analysis of the patient's clinical picture does not allow, in principle, to establish the number of asymptomatic patients. And the PCR-based morbidity pool under this approach also contains more data on symptomatic people.

Moreover, in 18-31% of people, the disease is asymptomatic (Haramoto. Et al., 2020). The use of mass diagnostics using the PCR method could compensate for the above disadvantages, but conducting mass tests for a large number of people is associated with a huge cost of resources, including material, and time.

In addition, in the mode of mass testing, diagnostic laboratories are overloaded, which, among other things, leads to an increase in the time for performing analyzes.

Accordingly, when monitoring the epidemiological situation, data on the test results are not provided in a timely manner, which leads to a decrease in the effectiveness and efficiency of monitoring.

These shortcomings lead to distortion and unreliability of the results of monitoring the epidemiological situation and, as a result, cannot be considered as the basis for making management decisions adequate to the situation.

An alternative to mass testing of the population is the analysis of wastewater for the content of SARS-CoV-2 viral particles. It is known that the source of viral particles in wastewater is the excrement of sick people (Wu et al., 2020; Wurtzer et al., 2020; Medema et al., 2020; Ahmed et al., 2020; Haramoto. Et al., 2020) ...

A monitoring method based on the analysis of wastewater by the content of biomarkers (psychoactive substances, hormones, toxicants, viral particles, pathogenic bacteria, antibiotics and antibiotic-resistant bacteria) is actively used in practice and is called "wastewater based epidemiology" or "wastewater epidemiology" (WBE) ...

This approach has found its application in China to control drug trafficking (Mao et al., 2020), to control the spread of antibiotic resistance in bacteria in European countries, the United States and Australia (Sims and Kasprzyk-Hordern, 2020). Thanks to the use of modern methods of analytical chemistry and molecular biology, including PCR methods, this method is characterized by high specificity in relation to the biomarker.

Accordingly, a method for analyzing wastewater for the content of SARS-CoV-2 viral particles is one of the WBE options. The advantages of the WBE method lie in its scalability and the virtual absence of restrictions on the size of the tested sample, since in order to obtain information on the number of cases, it is necessary to correctly organize the collection of wastewater samples at the selected community level - from an individual multi-storey building to a city or region. The WBE allows for “real-time” monitoring of the population because, depending on the type of biomarker, the time to obtain results ranges from a few minutes to a maximum of two days (Sims and Kasprzyk-Hordern, 2020).

Accordingly, the WBE method allows obtaining operational information about the epidemiological situation, including for large samples ("Water, sanitation, hygiene and waste disposal for SARS-CoV-2, the virus that causes COVID-19"; Mao et al., 2020) ...

Additional advantages are associated with the absence of the need to organize mass testing of people, respectively, to ensure the safety of a large number of medical personnel, increase the burden on diagnostic laboratories, and depend on the mentality and consciousness of the population.

Thanks to these advantages, the cost and the accuracy of monitoring are significantly reduced, including by obtaining information about asymptomatic patients. At the same time, it was shown that SARS-CoV-2 viral particles are present in the feces of infected people, including in the case of an asymptomatic course of the disease, in an amount of up to 10 ¹⁰ copies / g (Wu et al., 2020; La Rosa et al., 2020 ; Kitajima et al., 2020), which is sufficient for their determination by PCR (Mao et al., 2020) in the feces of a sick person with COVID-19. At this level, the reliability, accuracy and specificity of the tests carried out are not inferior to traditional methods, for example, analysis using a pharyngeal swab by PCR (Kitajima et al., 2020).

Moreover, it is known that virus particles are also detected in wastewater from settlements with an infected population, and it is believed that the advantages of this approach allow determining the presence of SARS-CoV-2 viral particles if the incidence of the tested population exceeds 0.03% ( Ahmed et al., 2020).

For example, SARS-CoV-2 viral particles were detected in wastewater in the Netherlands, USA, Australia and Italy, even before the first case of COVID-19 was recorded in the study area (Wu et al., 2020; Medema et al. , 2020; Ahmed et al., 2020; La Rosa et al., 2020).

Despite the natural decrease in the amount of viral particles in wastewater compared to their amount in the excrement of a patient with COVID-19, it was found that the amount of viral particles in wastewater is 10 ² -10 ⁴ copies / l (Wu et al., 2020; Wurtzer et al., 2020; Medema et al., 2020; Ahmed et al., 2020; Haramoto. et al., 2020).

The ratio of the number of viral particles in the wastewater and in the excrement of a patient with COVID-19 is the basis for determining the incidence rate in the WBE method.

The source chosen by the applicant as a prototype is a technical solution for the implementation of the WBE method for determining the number of infected COVID-19 is the publication "First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community "(" First Confirmed Detection of SARS-CoV-2 in Raw Wastewater in Australia: Proof of Concept for COVID-19 Wastewater Surveillance in the Community ") (by Warish Ahmed et al.) [Https: // www.sciencedirect.com/science/article/pii/S0048969720322816]. The essence of the prototype is the quantitative determination of SARS-CoV-2 in wastewater, which makes it possible to track the number of infected people using the WBE method. In the prototype SARS-CoV-2 RNA was concentrated and isolated from a wastewater sample from a sewage treatment plant in Australia, the amount of viral RNA copy was counted using RT-PCR-RT. Based on the established amount of SARS-CoV-2 RNA in wastewater and prior art data on the amount of SARS-CoV-2 RNA in the feces of people infected with COVID-19, the number of people infected with COVID-19 living in the test area was determined. , and whose domestic wastewater is collected at a treatment plant in the tested area. Given the information on the distribution of COVID-19 cases and modeling using the Monte Carlo method, the prototype authors were able to estimate the average range of the number of infected people 171-1090 people. when the population of the tested area is 600,000 people. As a result of comparing the data obtained with the official statistics on the incidence rate in the region, which includes the tested region, the authors of the prototype conclude that it is in good agreement with clinical observations. The incidence rate is calculated using the formula:

Thus, the lower limit of detection for the prototype method is 0.03%, which allows the authors to draw a conclusion about the applicability of their proposed WBE method for monitoring the epidemiological situation of COVID-19.

The prototype describes the following sequence of actions for the WBE:

- carry out the selection of untreated wastewater;

- Concentration of wastewater samples is carried out;

- carry out the isolation of RNA;

- samples are analyzed for the presence of RNA of SARS-CoV-2 viral particles using PCR-based tests with the introduction of an external control - Oncorhynchus keta (10 ⁴ units / reaction);

- in the case of a positive result for the presence of SARS-CoV-2 viral particles, the prevalence of infection is assessed, taking into account the results of the modeling performed in the Oracle Crystal Ball (version 11.1.2.4.600, Redwood City, California).

The disadvantages of the prototype in comparison with the declared technical solution are:

- insufficient accuracy of monitoring results. The lower limit for determining the incidence rate for the prototype is 0.03%, for the claimed technical solution - 0.01%;

- long duration of the sample analysis procedure as part of monitoring the incidence of COVID-19. For example, the duration of the analysis of 150 samples for the prototype is 2145 minutes, for the claimed technical solution - 1170 minutes. (see Table 4 in Fig. 4)

- the higher cost of analyzing samples as part of monitoring the incidence of COVID-19. For example, the cost of analyzing 50 samples for a prototype is 300,388.43 rubles, for the claimed technical solution - 183,343.47 rubles. (see Table 5 in Fig. 5).

The purpose and technical result of the claimed technical solution is to eliminate the shortcomings of the prototype by developing a method for monitoring the incidence of COVID-19 using wastewater analysis, which allows:

- to improve the accuracy of monitoring results by lowering the value of the lower limit for determining the level of morbidity;

- reduce the duration of the sample analysis procedure as part of monitoring the incidence of COVID-19;

- to reduce the cost of analyzing samples as part of monitoring the incidence of COVID-19.

The essence of the claimed technical solution is a method for monitoring the incidence of COVID-19 using wastewater analysis, which consists in the fact that

at stage 1, the correction factor m is determined, which is the lower limit for determining the incidence rate using selected commercial kits for RNA extraction and RT-PCR-RT, for which:

prepare model waste water,

collecting feces from p patients with a confirmed diagnosis of COVID-19 based on clinical data and the results of a pharyngeal smear diagnosis by PCR,

Aliquots of samples of feces from p patients are taken and diluted with model waste water in a ratio that ensures the content of excrements in the composition of the samples at the level of the average content of excrements in the average runoff from 1 person per day, the mixture is stirred mechanically, samples No. 1 are obtained .... No. p,

then serial dilutions of samples No. 1 .... No. p are carried out, while at each stage of serial dilution, samples No. 1 ... No. p are diluted 2-4 times and tested by RT-PCR-RT for the presence of SARS-CoV-2 virus particles using commercial kits for RNA extraction and RT-PCR-RT reaction, the dilution series is continued until the lower limit of detection by RT-PCR-RT method is reached,

then the values of the maximum degrees of dilution are averaged for samples No. 1 ... No. p, at which the lower limit of determination of the RT-PCR-RT method is reached, then the arithmetic mean value of the maximum degrees of dilution is calculated, which is then used to calculate the value of the correction factor m,

determine the correction factor m as the ratio of 100 to the arithmetic mean of the maximum dilution rates of SARS-CoV-2-positive sample of a model wastewater sample containing excrement from patients with COVID-19;

then, in phase 2, the incidence of COVID-19 is monitored, for which

determine the places of wastewater sampling and the number of wastewater samples, depending on the purpose and size of the tested sample to monitor the incidence of COVID-19 in the population,

sampling wastewater,

isolate RNA from wastewater samples using the commercial RNA isolation kits used in step 1,

analyze samples for SARS-CoV-2 viral particles using commercial RT-PCR-RT kits used in Step 1,

in case of a negative result, the wastewater sample is considered SARS-CoV-2 negative, and, accordingly, the incidence rate is 0%,

in case of a positive result, the wastewater sample is recognized as SARS-CoV-2-positive, then serial dilutions of wastewater samples are prepared, at each stage of serial dilution, the samples are diluted 2-4 times and tested by RT-PCR-RT for the presence of SARS virus particles -CoV-2 using commercial kits for RNA extraction and RT-PCR-RT reaction used in stage 1, a series of dilutions is prepared until the lower limit of determination of the RT-PCR-RT method is reached, the maximum degree of dilution k of wastewater is determined,

then the value of N is calculated - the incidence rate of COVID-19 according to the formula N = k * m, where N is the incidence rate of COVID-19 in%, k is the maximum dilution rate of wastewater, m is a correction factor for the value of the lower limit of the RT-PCR method -RV.

The claimed technical solution is illustrated in Fig. 1 - Fig. 5.

Figure 1 shows Table 1, which presents the data of ten patients with a confirmed diagnosis of COVID-19 based on clinical data and the results of a pharyngeal swab by PCR, collected within 24 hours, 5-8 days after the registration of the first symptoms.

Figure 2 shows Table 2, which shows the data for determining the lower limit of the incidence rate and the correction factor m.

Figure 3 shows Table 3, which shows the detection of SARS-CoV-2 RNA in waste water samples.

Figure 4 shows Table 4, which shows a comparison of the duration of the monitoring procedure for the prototype and the claimed technical solution.

Figure 5 shows Table 5, which shows a comparison of the cost of analyzes for the prototype and the claimed technical solution.

To achieve the declared technical result:

- the stage of sample concentration is excluded from the analysis procedure according to the prototype. This improves the accuracy of determining the number of SARS-CoV-2 viral particles and, as a result, improves the accuracy of monitoring results by lowering the lower limit for determining the incidence level, since the method of sample concentration significantly affects the accuracy of the analysis of the number of SARS-CoV-2 viral particles in wastewater samples - depending on the conditions, method and implementation of this stage, its accuracy varies in a very wide range - from 10 to 80% (Hata et al.; Medema et al., 2020; Haramoto et al., 2020; Kitajima et al. ., 2020). Also, the concentration procedure is one of the most costly in terms of its time and cost of consumables, which significantly increases the duration and cost of sample analysis within the framework of monitoring the incidence of COVID-19 (see Table 4 and 5 in Figures 4 and 5);

- the stage of introducing an external control when isolating RNA, when carrying out RT and PCR-RT reactions is excluded from the analysis procedure according to the prototype. This improves the accuracy of determining the number of SARS-CoV-2 viral particles and, as a result, improves the accuracy of monitoring results by lowering the value of the lower limit for determining the incidence rate.

The accuracy of SARS-CoV-2 viral particle counting using the external control application procedure depends on the type of external control. It is known from the prior art that the external control is not SARS-CoV-2 viral particles, but mouse hepatitis virus particles, MS2 coliphage (ATCC 15597-B1), tobacco mosaic virus, Pseudomonas F6 phage and mouse norovirus (Hata et al. ; Medema et al., 2020; Haramoto et al., 2020; Kitajima et al., 2020).

The accuracy of determining the amount of SARS-CoV-2 viral particles ranges from 8.5 to 71.6%, depending on the external control used and the concentration method (Hata et al .; Medema et al., 2020; Haramoto et al., 2020; Kitajima et al., 2020).

As of the filing date of this application, an external control representing standardized SARS-CoV-2 virus particles with a known concentration has not been identified by the applicant from the prior art.

By eliminating the stage of introducing external control, which introduces an additional uncontrolled error in the COVID-19 monitoring results, an increase in the accuracy of monitoring results is achieved. At the same time, the cost of monitoring is reduced due to the decrease in the cost of consumables used in the procedure for introducing external control in the process of RNA extraction (see Table 5 in Fig. 5).

- added a step for determining the correction factor m, which is the lower limit for determining the incidence rate using selected commercial kits for RNA extraction and RT-PCR-RT. The correction factor m is defined as the ratio of 100 to the maximum dilution ratio of a SARS-CoV-2 positive wastewater model sample containing feces from patients with confirmed COVID-19 diagnosis based on clinical data and results from a throat swab by PCR.

The introduced stage leads to an increase in the accuracy of determining the level of morbidity, since the calculation using the correction factor m does not depend on the introduction of external control. The step is carried out once for a model wastewater sample containing excrement from patients with a confirmed diagnosis of COVID-19, therefore its introduction does not lead to a significant increase in the duration and cost of sample analysis in the framework of monitoring the incidence of COVID-19 (see Tables 4 and 5 in Fig. 4 and 5).

- added a step for determining the maximum dilution of k wastewater samples taken for monitoring by serial dilutions of wastewater samples and determining the presence of SARS-CoV-2 virus particles in them. The duration of the procedure and the cost of this stage does not exceed the total value of the duration and cost of the excluded stages of sample concentration and external control (see Table 4 and 5 in Figs. 4 and 5).

- the stage of calculating N - the incidence rate of COVID-19 has been changed in comparison with the prototype.

This step allows you to translate the result of determining the presence of SARS-CoV-2 into the value of N - the incidence rate of COVID-19 in% in relation to the total population, with the value of k - the maximum dilution rate of wastewater and adjusting for the value of the lower limit of the RH-PCR method -РВ - correction factor m. The calculation is carried out using a specially developed formula N = k * m.

The following is a sequence of actions of the main stages of the claimed method.

Step 1. Determination of the correction factor m, which is the lower limit for determining the incidence rate using selected commercial kits for RNA extraction and RT-PCR-RT .

At Stage 1, the following sequence of actions is carried out according to the claimed method:

1– prepare model waste water, in accordance with the procedure (see Example 2);

2− collect excrement from patients (p is the number of patients) with a confirmed diagnosis of COVID-19 based on clinical data and the results of a pharyngeal smear diagnosis by PCR (hereinafter referred to as patients with COVID-19);

3– take aliquots of samples of feces from p patients and dilute them with model waste water in a ratio that ensures the content of excrements in the composition of the samples at the level of the average content of excrements in the average runoff from 1 person per day, the mixture is stirred mechanically. For example, 1 g of excrement is mixed with 5 liters of model waste water, and then brought to the average volume of waste from one person at knocks, for example, 50 ml of the resulting mixture is diluted 40 times. Samples # 1 are obtained…. # P, where p is the number of patients;

4− carry out serial dilutions of samples No. 1 ... No. p, at each stage of serial dilution, samples No. 1 ... No. p are diluted 2-4 times and tested by RT-PCR-RT for the presence of SARS-CoV-2 virus particles using commercial kits for RNA extraction and RT-PCR-RT reaction, the dilution series is continued until the lower limit of detection by RT-PCR-RT method is reached;

5− carry out averaging of the values of the maximum degrees of dilution for samples No. 1 ... No. p, at which the lower limit of determination of the RT-PCR-RT method is reached, the arithmetic mean of the maximum degrees of dilution is obtained, which is then used to calculate the value of the correction factor m. Determine the correction factor m as the ratio of 100 to the arithmetic mean of the maximum dilutions of a SARS-CoV-2 positive sample of a model wastewater sample containing excrement from patients with COVID-19.

Stage 2. Monitoring the incidence of COVID-19 .

At Stage 2, the following sequence of actions is carried out according to the claimed method:

6− determine the places of wastewater sampling and the number of wastewater samples, depending on the purpose and size of the tested sample for monitoring the incidence of COVID-19 in the population;

7− carry out sampling of wastewater according to PND F 12.15.1-08;

8– carry out the isolation of RNA from wastewater samples using commercial kits for RNA isolation used in step 1;

9 - Analyze samples for SARS-CoV-2 virus particles using commercial RT-PCR-RT kits used in Step 1;

10 - in case of a negative result, the wastewater sample is considered SARS-CoV-2 negative, and, accordingly, the incidence rate is 0%.

11 - in case of a positive result, the wastewater sample is considered SARS-CoV-2 positive, serial dilutions of wastewater samples are prepared, at each stage of serial dilution, the samples are diluted 2-4 times and tested by RT-PCR-RT for the presence of SARS viral particles -CoV-2 using commercial kits for RNA extraction and RT-PCR-RT reaction used in stage 1, a series of dilutions is prepared until the lower limit of determination by RT-PCR-RT method is reached, the maximum degree of wastewater dilution k is determined;

12− then calculate the value of N - the incidence of COVID-19 using a specially developed formula N = k * m, where N is the incidence of COVID-19 in%, k is the maximum dilution of wastewater, m is a correction factor for the value of the lower the limit of RT-PCR-RT method;

Further, the applicant gives examples of the implementation of the claimed technical solution.

Example 1. Conducting Stage 1 - determining the correction coefficient m, which is the lower limit for determining the incidence rate using selected commercial kits for RNA extraction and RT-PCR-RT.

At Stage 1, the following sequence of actions is carried out according to the claimed method.

Model waste water is preliminarily prepared. For this, a mixture is prepared containing typical waste for a resident of a given settlement (district, microdistrict, house), for example, of the following composition.

For a volume, for example, 1 liter, take, for example: 0.05 g of toothpaste, 0.25 g of meat soup, 0.5 g of vegetable salad, 0.08 g of shower gel, 0.08 g of sand, 0.03 g of soil, 0.08 g of liquid soap, 0.08 g of dishwashing detergent and 0.03 g of washing powder, the rest is water.

The applicant explains that the specified composition of the model waste water is given to illustrate the implementation of the claimed technical solution and does not limit the scope of patent claims for another composition of the model waste water, since the composition of the waste water does not affect the claimed technical result.

Model waste water is placed in a container, for example, plastic, with a volume of, for example, 10 liters, in an amount of, for example, 5 liters in each container. The number of container units corresponds to the number of examined patients - ten. The applicant explains that the number of patients - ten is taken to illustrate the implementation of the claimed technical solution, which does not limit the scope of patent claims for a different number of patients. At the same time, the applicant believes that the minimum number of patients to determine the correction factor should be at least 5 people.

Next, excrement is collected from p patients (in this example, excrement samples were taken from ten patients with COVID-19, p = 10) with a confirmed diagnosis of COVID-19. Feces were collected from each patient within 24 hours, in a separate container for each patient, 5-8 days after the registration of the first symptoms of the disease (Table 1 in Fig. 1).

Next, the resulting mixture is diluted to simulate the average daily volume of wastewater produced by a person living in an apartment building in Russia, which is 200 liters. This volume corresponds to the average amount of wastewater produced by residents of apartment buildings in the central part of Russia [SNiP 2.04.01-85 Internal water supply and sewerage of buildings, 1985]. For this, aliquots of excrement samples from p patients are taken and diluted with model waste water in a ratio that ensures the content of excrement in the composition of the samples at the level of the average excrement content in the average runoff from 1 person per day, for example, 1 g of excrement was mixed with 5 liters of model waste water. The mixture is stirred mechanically, for example, using an electric drill with a stirring attachment, for example, at a speed of 1000 rpm. Next, a sample is prepared corresponding to the average volume of runoff from one person per knock, for example, 50 ml of the resulting mixture is taken and diluted 40 times.

Samples # 1 are obtained…. # P, where p is the number of patients. The applicant explains that with such preparation of samples No. 1 .... No. p, the viral load in them must correspond to the waste waters of settlements with a 100% degree of morbidity.

Next, prepare serial dilutions of samples No. 1 ... No. p. At each stage of serial dilution, samples No. 1 ... No. p were diluted 2-4 times and tested by RT-PCR-RT for the presence of SARS-CoV-2 viral particles using commercial kits for RNA extraction and RT-PCR reaction. RT, the series of dilutions was continued until the lower limit of detection by RT-PCR-RT method was reached. The lower limit is determined so that the presence of SARS-CoV-2 viral particles in the penultimate dilution is not detected by RT-PCR-RT method. At the same time, one more is done - the last dilution for the reliability of confirming the impossibility of determining the viral particles.

For example, a series of dilutions of samples No. 1 ... No. 10 was prepared from 25% to 0.003% using simulated wastewater until SARS-CoV-2-negative concentration was reached. This does not limit the scope of patent claims for other concentrations, which depends on the sensitivity of commercial RNA isolation kits and commercial RT-PCR-RT assays that will be used for monitoring in each specific laboratory.

Next, the diluted samples No. 1, ... No. 10 were analyzed for the presence of SARS-CoV-2 viral particles using PCR-based tests. The analyzes are carried out immediately after the preparation of samples No. 1, ... No. 10. All analyzes were carried out in three replicates.

RNA was isolated from diluted samples No. 1, ... No. 10 using a commercial viral RNA mini kit (QIAGEN, Germany). For the isolated total RNA, the presence of RNA of viral particles of the SARS-CoV-2 coronavirus was determined by the RT-PCR-RT method using a commercial kit PCR-RV-2019-nCov (Syntol LLC, Russia). For amplification, this commercial kit uses two oligonucleotide primers flanking the Orf1ab gene fragment of the SARS-CoV-2 genome. The kit contains sterile 0.2 ml PCR tubes with lyophilized reaction mixture. The kit allows you to simultaneously detect RNA of the SARS-CoV-2 coronavirus (R6G detection channel) and check the efficiency of nucleic acid isolation, the degree of inhibition of reverse transcription and amplification reactions (FAM and Cy5 detection channels, respectively).

The RT-PCR-RT reaction was carried out using the following temperature program on a CFX-96 thermal cycler (Bio-Rad, USA):

reverse transcription at 50 ° C for 15 minutes,

inactivation at 95 ° C for 5 minutes,

amplification - 50 three-step cycles:

95 ° C for 10 seconds,

58 ° C for 10 seconds,

72 ° C for 20 seconds.

According to the instructions for the PCR-RV-2019-nCov kit, wastewater samples are recorded as positive when Ct in the R6G detection channel is less than 30.

The smallest dilution for all 10 samples in this experiment is shown in Table 2, column 2, line 8).

Next, the values of the maximum degrees of dilution were averaged for samples No. 1 ... No. 10, at which the lower limit of determination of the RT-PCR-RT method was reached, the arithmetic mean value of the maximum degrees of dilution was obtained, which is used to calculate the value of the correction factor m.

The correction factor m was determined as the ratio of 100 to the arithmetic mean of the maximum dilutions of a SARS-CoV-2 positive sample of a model wastewater sample containing excrement from patients diagnosed with COVID-19 (Table 2, column 3, line 8). For this, 100% was divided by 8192. The result is 0.01%.

Thus, the correction factor m, which is the lower limit for determining the incidence rate using the selected commercial kits for RNA extraction and RT-PCR-RT, for this experiment is 0.01%.

The results are shown in Table 2 in Fig. 2. From the data presented in Table 2, it can be concluded that the claimed method can reveal the minimum incidence of COVID-19, equal to 0.01%.

Example 2. Conducting Phase 2. Conducting monitoring incidence of COVID-19.

At Stage 2, the following sequence of actions was carried out according to the claimed method.

Monitoring of the incidence of the population of COVID-19 according to Example 1 was carried out in an urban settlement.

Determine the sites of wastewater sampling, the frequency and number of wastewater samples, depending on the purpose and size of the tested sample for monitoring the incidence of COVID-19 in the population.

So, in the experiment according to Example 2, wastewater was sampled from several city sewers according to PND F 12.15.1-08. We used 2 series of experimental wastewater samples with a period between sampling of 4 months, thus, series 1 (March 2020) and series 2 (July 2020) were studied in 11 sewers, 10 of which were located in residential areas of the city, and one - in the administrative region. The applicant explains that the number of sampling points (for example, sewers) is given to illustrate the implementation of the claimed technical solution and does not limit the scope of patent claims both for a different number of sampling points and for other wastewater sampling points, since the change in the number and sampling points does not affects the claimed technical result. At the same time, the applicant explains that the amount and location of wastewater collection depend on the monitoring tasks, for example, monitoring the incidence in the village as a whole, or in the area of the village, etc.

Samples from each sewer were collected over 24 hours at 200 ml each hour, mixing in a sterile 5 L plastic container. Between collections, the containers were stored at a reduced temperature on ice (at 4 ° C).

After completion of sampling, the samples were transported at 4 ° C to the laboratory. Isolation of total RNA was performed immediately after delivery of samples to the laboratory using commercial kits for RNA isolation used in step 1. The isolated RNA was analyzed for the presence of SARS-CoV-2 viral particles using commercial kits for carrying out the RT-PCR-RT reaction used on stage 1.

The samples were divided into 2 categories depending on the results of the analysis: SARS-CoV-2-positive and SARS-CoV-2-negative. Samples were labeled according to the result.

If the result is positive, the wastewater samples are considered SARS-CoV-2 positive and serial dilutions are prepared for them with model wastewater. At each stage of serial dilution, samples are diluted 2-4 times and tested by RT-PCR-RT for the presence of SARS-CoV-2 viral particles using commercial kits for RNA extraction and RT-PCR-RT reaction, the dilution series is continued until reaching the lower limit of determination by RT-PCR-RT method, at the maximum degree of dilution of waste water k.

So, according to Example 2, the samples were diluted 5, 25, 50, 75, 100, 150, 200 and 250 times by volume, so that in the penultimate dilution the presence of SARS-CoV-2 viral particles was not established using the OT-method. RT-PCR. At the same time, one more was done - the last dilution for the reliability of confirmation of the impossibility of determining the viral particles. Then, total RNA was isolated from each sample of the dilution series and analyzed for the presence of SARS-CoV-2 viral particles using the RT-PCR-RT method according to Example 1. Each undiluted sample and a sample of the dilution series were analyzed in triplicate.

Next, the value of N is calculated - the incidence of COVID-19 using a specially developed formula N = k * m, where N is the incidence of COVID-19 in%, k is the maximum dilution of wastewater, m is a correction factor for the value of the lower limit of the method RT-PCR-RT.

The results are shown in Table 3 in Fig. 3.

As shown in Table 3, no SARS-CoV-2 particles were detected in Experimental Series 1, and SARS-CoV-2 virus particles were detected in four samples of Experimental Series 2.

The maximum dilution ratio of wastewater for which the presence of SARS-CoV-2 viral particles was determined is 1: 100, 1: 150, 1:75 and 1: 150, thus k = 100, 150, 75, 150, respectively.

Correction factor for the value of the lower limit of the RT-PCR-RT method m = 0.01%.

As a result, for the experiment carried out by the applicant, the incidence of COVID-19 in the corresponding residential areas is:

N ₁ = 100 * 0.01 = 1.00%,

N ₂ = 150 * 0.01 = 1.50%,

N ₃ = 75 * 0.01 = 0.75%

N ₄ = 100 * 0.01 = 1.50%.

The accuracy of monitoring according to the claimed method is 0.01% compared to 0.03% for the prototype (since the prototype detects at least 171 patients out of 600,000 people), that is, the claimed method allows, with sufficient ease of analysis, to identify 1 infected COVID-19 person per 10,000 people, which is 3 times more accurate than the prototype, which confirms the achievement of the declared technical result.

At the same time, the applicant reduced the duration of the procedure and the cost of monitoring as a result of excluding the stages of concentration and external control.

Table 4 in Fig. 4 shows the data on the duration of the monitoring procedure,

From the data shown in Table 4, it can be seen that the duration of the monitoring procedure, for example, when calculated for 150 samples, is 2145 minutes according to the prototype, compared to 1170 minutes for the claimed technical solution, which confirms the achievement of the claimed technical result. At the same time, the difference in the duration of monitoring in comparison with the prototype increases as the number of samples increases, since the duration of the stage of determining the correction factor does not change.

Table 5 in Fig. 5 shows the data on the cost of the monitoring procedure.

From the data given in Table 5, it can be seen that the cost of monitoring, for example, calculated for 50 samples, is 300,388.43 rubles for the prototype. compared to 183,343.47 rubles. the claimed technical solution, which confirms the achievement of the claimed technical result. At the same time, the difference in the cost of monitoring in comparison with the prototype increases with the increase in the number of samples, since the cost of the stage of determining the correction factor does not change.

Based on the above, it can be concluded that the applicant has achieved all the goals and the claimed technical result, namely, the shortcomings of the prototype have been eliminated by developing a method for monitoring the incidence of COVID-19 using wastewater analysis, which made it possible to:

- to improve the accuracy of monitoring results by lowering the value of the lower limit for determining the level of morbidity three times compared with the prototype - from 0.03% to 0.01%;

- to reduce the duration of the procedure for analyzing samples as part of monitoring the incidence of COVID-19 in comparison with the prototype, for example, from 2145 minutes to 1170 minutes when analyzing 150 samples;

- to reduce the cost of analyzing samples as part of monitoring the incidence of COVID-19 in comparison with the prototype, for example, from 300,388.43 rubles. up to 183,343.47 rubles. when analyzing 50 samples.

For this:

- the stage of concentration of samples was excluded from the standard procedure of analysis according to the prototype, which led to an increase in the accuracy of monitoring. At the same time, the duration and cost of analysis of samples in the framework of monitoring the incidence of COVID-19 has been reduced (see Table 4 and 5 in Figs. 4 and 5).

- the stage of external control during the isolation of RNA, carrying out RT and PCR-RT reactions for quantitative counting of viral particles was excluded from the standard analysis procedure according to the prototype, which led to an increase in the monitoring accuracy. At the same time, the cost of analyzing samples in the framework of monitoring the incidence of COVID-19 has been reduced as a result of reducing the cost of consumables (see Table 5 in Fig. 5).

- added a stage for determining the correction factor m, which is the lower limit for determining the incidence rate using selected commercial kits for RNA extraction and RT-PCR-RT, which led to an increase in the monitoring accuracy, since the calculation using the correction factor m does not depend on the introduction of an external control. The step is carried out once for a model wastewater sample containing excrement from patients with a confirmed diagnosis of COVID-19, therefore its introduction does not lead to a significant increase in the duration and cost of sample analysis in the framework of monitoring the incidence of COVID-19 (see Table 4 in Fig. 4) ... In this case, the difference in the duration and cost of monitoring in comparison with the prototype increases with the increase in the number of samples, since the cost of the stage of determining the correction factor m does not change.

- added a step for determining the maximum dilution of k wastewater samples taken for monitoring by serial dilutions of wastewater samples and determining the presence of SARS-CoV-2 virus particles in them. The duration of the procedure and the cost of this stage does not exceed the total value of the duration and cost of the excluded stages of sample concentration and external control (see Table 4 and 5 in Figs. 4 and 5).

- the stage of calculating N - the incidence rate of COVID-19 has been changed in comparison with the prototype. This step allows you to translate the result of determining the presence of SARS-CoV-2 into the value of N - the incidence rate of COVID-19 in% in relation to the total population, with the value of k - the maximum dilution rate of wastewater and adjusting for the value of the lower limit of the RH-PCR method -РВ - correction factor m. The calculation is carried out using a specially developed formula N = k * m.

The claimed technical solution meets the "novelty" requirement of patentability for inventions, since as of the date of submission of the application materials by the applicant, no sources have been identified from the studied prior art with a set of features identical to the set of features of the claimed technical solution.

The claimed technical solution meets the requirement of patentability "inventive step" for inventions, since the combination of the claimed features provides for obtaining technical results that are not obvious to a specialist, exceeding the technical result of the prototype.

The claimed technical solution meets the condition of patentability "industrial applicability" for inventions, since The claimed method can be carried out using known reagents using standard equipment and known techniques and allows, with sufficient ease of analysis, to identify 1 infected person with COVID-19 per 10,000 people. Moreover, the claimed method can be adapted for any type of wastewater, climatic conditions, commercial kits for DNA / RNA extraction and commercial kits for the RT-PCR reaction and is more economical.

Claims (16)

A method for monitoring the incidence of COVID-19 using wastewater analysis, which consists in at stage 1, the correction factor m is determined, which is the lower limit for determining the incidence rate using selected commercial kits for RNA extraction and RT-PCR-RT, for which: prepare model waste water, collecting feces from p patients with a confirmed diagnosis of COVID-19 based on clinical data and the results of a pharyngeal smear diagnosis by PCR, Aliquots of samples of feces from p patients are taken and diluted with model waste water in a ratio that ensures the content of excrements in the composition of the samples at the level of the average content of excrements in the average runoff from 1 person per day, the mixture is stirred mechanically, samples No. 1 are obtained .... No. p, then serial dilutions of samples No. 1 .... No. p are carried out, while at each stage of serial dilution, samples No. 1 ... No. p are diluted 2-4 times and tested by RT-PCR-RT for the presence of SARS-CoV-2 virus particles using commercial kits for RNA extraction and RT-PCR-RT reaction, the dilution series is continued until the lower limit of detection by RT-PCR-RT method is reached, then the values of the maximum degrees of dilution are averaged for samples No. 1 ... No. p, at which the lower limit of determination of the RT-PCR-RT method is reached, then the arithmetic mean value of the maximum degrees of dilution is calculated, which is then used to calculate the value of the correction factor m, determine the correction factor m as the ratio of 100 to the arithmetic mean of the maximum dilution rates of SARS-CoV-2-positive sample of a model wastewater sample containing excrement from patients with COVID-19; then, in phase 2, the incidence of COVID-19 is monitored, for which determine the places of wastewater sampling and the number of wastewater samples, depending on the purpose and size of the tested sample to monitor the incidence of COVID-19 in the population, sampling wastewater, isolate RNA from wastewater samples using the commercial RNA isolation kits used in step 1, analyze samples for SARS-CoV-2 viral particles using commercial RT-PCR-RT kits used in Step 1, in case of a negative result, the wastewater sample is considered SARS-CoV-2 negative and, accordingly, the incidence rate is 0%, in case of a positive result, the wastewater sample is recognized as SARS-CoV-2-positive, then serial dilutions of wastewater samples are prepared, at each stage of serial dilution, the samples are diluted 2-4 times and tested by RT-PCR-RT for the presence of SARS virus particles CoV-2 using commercial kits for RNA extraction and RT-PCR-RT reaction used in step 1, a series of dilutions is prepared until the lower limit of detection of RT-PCR-RT method is reached, the maximum dilution rate k of wastewater is determined, then the value of N is calculated - the incidence rate of COVID-19 according to the formula N = k * m, where N is the incidence rate of COVID-19 in%, k is the maximum dilution rate of wastewater, m is a correction factor for the value of the lower limit of the RT-PCR method -RV.

Images