key: cord-0933897-5oh1x3wg
authors: Ahmed, F.; Islam, M. A.; Kumar, M.; Hossain, M.; Bhattachariya, P.; Islam, M. T.; Hossen, F.; Hossain, M. S.; Uddin, M. M.; Islam, M. N.; Bahadur, N. M.; Alam, M. D. u.; Reza, H. M.; Jakariya, M.
title: First detection of SARS-CoV-2 genetic material in the vicinity of COVID-19 isolation centre through wastewater surveillance in Bangladesh
date: 2020-09-18
journal: nan
DOI: 10.1101/2020.09.14.20194696
sha: c79facd726b3d5fac8400a237920098088504b87
doc_id: 933897
cord_uid: 5oh1x3wg

In the course of a COVID-19 pandemic, 0.33 million people got infected in Bangladesh, we made the first and successful attempt to detect SARS-CoV-2 viruses' genetic material in the vicinity wastewaters of an isolation centre i.e. Shaheed Bhulu Stadium, situated at Noakhali. The idea was to understand the genetic loading variation, both temporal and distance-wise in the nearby wastewater drains when the number of infected COVID-19 patients is not varying much. Owing to the fact that isolation center, in general, always contained a constant number of 200 COVID-19 patients, the prime objective of the study was to check if several drains carrying RNA of coronavirus are actually getting diluted or accumulated along with the sewage network. Our finding suggested that while the temporal variation of the genetic load decreased in small drains over the span of 50 days, the main sewer exhibited accumulation of SARS-CoV-2 RNA. Other interesting finding displays that probably distance of sampling location in meters is not likely to have a significant impact on gene detection concentration, although the quantity of the RNA extracted in the downstream of the drain was higher. These findings are of immense value from the perspective of wastewater surveillance of COVID-19, as they largely imply that we do not need to monitor every wastewater system, and probably major drains monitoring may illustrate the city health. Perhaps, we are reporting the accumulation of SARS-CoV-2 genetic material along the sewer network i.e. from primary to tertiary drains. The study sought further data collection in this line to simulate conditions prevailed in the most of south Asian country and to shed further light on the temporal variation and decay/accumulation processes of the genetic load of the SARS-COV-2.

CC-BY-ND 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 (which was not certified by peer review) preprint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 18, 2020. . https://doi.org/10.1101/2020.09.14.20194696 doi: medRxiv preprint

In the course of a COVID-19 pandemic, 0.33 million people got infected in Bangladesh, we made 62 the first and successful attempt to detect SARS-CoV-2 viruses' genetic material in the vicinity 63 wastewaters of an isolation centre i.e. Shaheed Bhulu Stadium, situated at Noakhali. The idea was 64 to understand the genetic loading variation, both temporal and distance-wise in the nearby interesting finding displays that probably distance of sampling location in meters is not likely to 72 have a significant impact on gene detection concentration, although the quantity of the RNA 73 extracted in the downstream of the drain was higher. These findings are of immense value from 74 the perspective of wastewater surveillance of COVID-19, as they largely imply that we do not need 75 to monitor every wastewater system, and probably major drains monitoring may illustrate the city 76 health. Perhaps, we are reporting the accumulation of SARS-CoV-2 genetic material along the 77 sewer network i.e. from primary to tertiary drains. The study sought further data collection in this 78 line to simulate conditions prevailed in the most of south Asian country and to shed further light 79 . CC-BY-ND 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 18, 2020. Italy, ORF1ab assay and N protein assay was used to detect SARS-CoV-2 RNA from untreated 93 wastewater and river water samples (Rimoldi et al., 2020) . However, the wastewater surveillance 94 of COVID-19 (WWSoC-19) has been mostly focused on wastewater treatment plants around the 95 globe, and there is slow progress on ambient waters and especially in the sewer system. This is 96 critical because several developing countries like India, Bangladesh, Pakistan, and others do not 97 have proper wastewater systems even in their class-I cities. As a result, the policymakers in these 98 countries are in confusion pertaining to the national scale implementation of WWSoC-19. 99 100 Further, while the infectivity issues of SARS-CoV-2 RNA present in the wastewater as well as 101 ambient environmental waters are not yet neglected or proved in the scholarly world, the public 102 . CC-BY-ND 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 18, 2020. . https://doi.org/10.1101/2020.09.14.20194696 doi: medRxiv preprint around the world are very skeptical about the wastewaters coming from the isolation centers 103 mainly that are not equipped with wastewater treatment systems. There have been some decay 104 reports of genetic loading of SARS-CoV-2 (Ahmed et al., 2020b, Kumar et al., 2020b) in the 105 wastewater system, but sewer systems are not yet investigated. In addition, there is a lack of 106 explicit understanding of either decay or accumulation of Covid-19 genetic material along with the 107 sewer systems with distance and the networking from small drain to larger drains followed by the 108 canal and main sewer system. CC-BY-ND 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 18, 2020. . https://doi.org/10.1101/2020.09.14.20194696 doi: medRxiv preprint from the sewage network in the isolation centre at Noakhali, Bangladesh. The aim of the study 125 was to: i) understand the genetic load in the vicinity of the isolation centre with an almost constant 126 number of COVID-19 patients. ii) analyse the distance impact on genetic loading and tracing the 127 decay/accumulation of the same along with the sewage network, iii) and temporal variation in 128 genetic material shedding from the infected patient. was selected based on the fact that Shaheed Bhulu Stadium is the largest detention Centre for 134 COVID-19 patients at Noakhali district, Bangladesh. This facility has been established to 135 accommodate more than two hundred COVID-19 positive patients for isolation purposes but kept 136 around 200 patients all the time during the monitoring period. This preliminary study has been 137 carried out with samples collected from the three different drains that are coming out of the 138 stadium and can be considered as primary drains, which connects to a canal (secondary drainage 139 system) and eventually meets the main sewer system (tertiary drains). 140

In order to understand the distance impact on genetic loading along the drains, we collected 142 samples at various distance i.e. 100m, 200m, 300m, and 400m as presented in Table 1 . Specimens 143

were aseptically collected in a 50 ml sterile falcon tube, transported in the laboratory keeping 144 inside the ice-box, refrigerated at 4 o C during preparatory activities, and were analyzed on the 145 . CC-BY-ND 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 18, 2020. . https://doi.org/10.1101/2020.09.14.20194696 doi: medRxiv preprint same day. Sterile falcon tubes for sampling with identical blanks were analyzed to determine any 146 possible contamination during the transport. All analyses were done at the Microbiology 147

Laboratory of the Department of Microbiology, Noakhali Science and Technology University 148 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 18, 2020. . https://doi.org/10.1101/2020.09.14.20194696 doi: medRxiv preprint shaking for overnight. The next day, the mixture was centrifuged at 13000×g for 90 min. After 165 centrifugation, the supernatant was discarded and the pellet was resuspended in 300 μL RNase 166 free water. This was further used as a sample for RNA isolation. RNA isolation was carried out using 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 18, 2020. . https://doi.org/10.1101/2020.09.14.20194696 doi: medRxiv preprint with the distance of sampling in the vicinity of isolation center with 50 days temporal resolution 184

i.e. in between 10 th July and 29 th August 2020 has been presented in Table 1 . CC-BY-ND 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.

The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.14.20194696 doi: medRxiv preprint Table 1 3 seems carrying the heavier RNA load of SARS-CoV-2 followed by drain 2 and drain 1. Althoguh 216 dissimilarities observed between the primary drainage line i.e. drain samples and secondary 217 drainage system i.e. canal and tertiary drainage system i.e. the main sewer, however, trend of 218 higher genetic material loading in the secondary and tertiary system was also found on twilight 219

tenure. This is a unique finding where gene accumulation has been observed instead of the general 220 expectation of dilution in the larger sewer system. The probable reasoning, other than the 221 accumulation of loading from various drains of the isolation centre, in support of this observation 222 can be the additional contribution of RNA excreted from the asymptomatic patient as well as yet 223

to be diagnosed people into the sewer system. 224 . CC-BY-ND 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.

The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.14.20194696 doi: medRxiv preprint We employed the distance factor in our sampling strategies and the results are presented in Figure  229 3. While drain 3 in general showed the increase in the ORF1ab and N genes, drain 1 was not 230

showing a consistent trend in genetic material loading of SARS-CoV-2 along with the distance. We 231 . CC-BY-ND 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.

The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.14.20194696 doi: medRxiv preprint tend to propose that probably distance in meters is not likely of a critical factor capable of 232 producing a trend. 233 238 Table 2 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 18, 2020. . https://doi.org/10.1101/2020.09.14.20194696 doi: medRxiv preprint internal control, which monitors the presence of PCR inhibitors in test specimens by detecting 269 whether the internal control signal is normal, to avoid a false negative result when used for human 270 RT-PCR experiments. In our experiments with wastewater, few specimens were negative for CY5 271 indicated that the human gene RNase P gene was missing in the samples. Hence it has been noted 272 that the human RNase P gene is more vulnerable to degradation than Covid-19 viral genes. 273

We also refrained to put here any semi-quantitative calculation of gene copies owing to lack of 275 enough replicates, kit designed for the human sample as well as uncertainties involving RT-PCR 276 (Stuart et al 2014) and will induct such calculation in our future study when there will be a method 277

at hand for precise copy calculations using suitable methods. Nevertheless, the bottom line is that which is first of its kind. Apart from this being the first detection report of SARS-CoV-2 RNA in the 289 wastewaters of Bangladesh, the uniqueness of the study has been the tracing the genetic load in 290 . CC-BY-ND 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 18, 2020. . https://doi.org/10.1101/2020.09.14.20194696 doi: medRxiv preprint the vicinity of the isolation center that contains almost the constant number with 200 COVID-19 291 patients, which takes the variable of the number of infected persons out of the equation. This has 292 been the key feature of this study as most of the study reported worldwide has either reported 293 total infected person in the city or country. There has been a complete lack of infected person 294 information that is contributing to the total genetic load to the sampled wastewater. We have 295 found about 75% of our sampled water positive based on ORF1ab gene absence or presence. 296

However, the critical observation has been the temporal variation where small drains showed an 297 easing of the loading of genetic load, the bigger canal, and main sewer city exhibited temporal 298 accumulation of SARS-CoV-2 RNA. On the other hand, the distance of sampling location in meters 299 appears to be insignificant from the perspective of wastewater surveillance of COVID-19. We 300 intend to analyze further samples taken in June, July, and August and preserved to shed further 301 light on the temporal variation and decay/accumulation processes of the genetic load of the SARS-302 COV-2. 303 304

The work did not involve any human subject and animal experiments. Science and Technology University, Bangladesh. 312 . CC-BY-ND 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 18, 2020. . https://doi.org/10.1101/2020.09.14.20194696 doi: medRxiv preprint

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