January 21, 2021

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8th International Workshop on Decentralized Wastewater Treatment in Asia

分散型汚水管理分野における水系感染症対策
Wastewater Epidemiology for Novel
Coronavirus in Japan
Hiroyuki Katayama, Professor,
Dept. of Urban Engineering, School of Eng.,The University of Tokyo

Environmental Public
Health Engineering
Laboratory

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Importance of disinfection
Water Supply• Water contaminatedwith pathogens posed agreat risk to human

Rainfall & runoff

ClPathogen
contamination(US EPA 2004)• Water disinfection: inactivate pathogens so that they are
not harmful to humans and animals

Wastewater
treatment plant

3Chlorine Reactions with Ammonia
NH3 + HOCl -> NH2Cl + H2O
NH2Cl + HOCl -> NHCl2 + H2O
NHCl2 + HOCl -> NCl3 + H2O•Free chlorine can react with organic nitrogen in the water
to form chloramines.
• The chloramines are not very effective against viruses andare a weaker disinfectant.
“combined available chlorine”,“combined chlorine residual”

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CT value for 2-log reduction

Chloramine is not effective to viruses

5OzoneModerately effective to inactivate Cryptosporidium
Highly reactive
Residual ozone then enough virus inactivation→ Need ozone destruction of off-gas→ Combination with activated carbon

Disinfection by-product: Bromate anion (BrO3-) :drinking water quality guideline <0.01mg/L
Depends on temperature

Coronavirus
enveloped viruses are usually unstablein water than non-enveloped virus
a positive-sense single-stranded RNAgenome

The genome size of coronaviruses ranges from approximately 27
to 34 kilobases, the largest among known RNA viruses
The name coronavirus is derived from the Latin corona, meaning"crown"Coronaviruscause respiratory tract infections
typically mild, such as the common cold,SARS, MERS, and COVID-19 can be lethal
COVID-19 is named SARS-CoV-2Outbreak

Virus typeDeaths

2003 severe acute respiratory syndrome outbreak

SARS-CoV774[31]2012 Middle East respiratory syndrome coronavirusoutbreak

MERS-CoVOver 400[32]2015 Middle East respiratory syndrome outbreak inSouth Korea

MERS-CoV36[33]2018 Middle East respiratory syndrome outbreak

MERS-CoV41[34]

2019–20 coronavirus outbreak

SARS-CoV-2

Over 3000[35]SARS-CoV is sensitive to Cl2

SARS-CoV is weaker than E. coli

SARS-CoV2 in waterCause less risk than other enteric virus
If properly managed, then water sectors are not posing risk
Water tap may be a possible spreading sourceAnother issue: monitoring of COVID in the community.

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PCR : Game changerBefore PCR• Medical microbiology, Virology• Culture methods• Emphasis on identifying viruses

After PCR• From 1990s, Applied to environmental samples• Active enrollment of Researchers
in Environmental Engineering
Public health• Importance of Quantitative results

Microbiology

Pathogens inWater

Environmental
engineeringIntroduction12Viral quantification as a vital process to implement WBE
 Concept of wastewater-based epidemiology (WBE)

Virusconcentration

SARS-CoV-2(Wikipedia)

Clinicalsurveillance

Viral RNA areexcreted in feces(Wölfel et al., 2020)

Capturing infections trend

Detection of SARS-CoV-2 RNA → Presence of infected people in the catchment.

 Detection process
Primary concentration

Previous studies: Optimized for non-enveloped viruses in most cases
RNA Extraction

RT-qPCR･One-step･Two-step

Research gaps
Are the virus concentration methods applicable to the recovery of enveloped viruses?

方法

WBE on Norovirus in Japan
Environmental Surveillance of Norovirus Genogroups I and II
for Sensitive Detection of Epidemic Variants(Kazama et al. Appl Environ Microbiol. 2017)

Virus concentration go up before increase of patients -> Possibility of early warning?
Same approach for SARS-CoV-2?

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Initial Idea on WBE
Safe
Zone

Pos

Neg

Pos

Infection
Neg

Pos
Neg

Pos

Neg
Information can be usedfor restriction or activity
level control in the area

Limitation of wastewater-based epidemiology
SamplingInterpritation

 Highly fluctuated virus emission in feces

Excreted/ dayhttps://twitter.com/psychedonwata/status/12
https://doi.org/10.1038/s41586-020-2196-x 82757230945763330/photo/1(Wölfel et al. 2020. Nature )･Possible false negative in case of small number infection･Limitation of WBE should be discussed

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Report from oversea
Presence of SARS-Coronavirus-2 RNA in Sewage and Correlation with Reported COVID-19
Prevalence in the Early Stage of the Epidemic in The Netherlands(Medema et al. ES&T letters. 2020)･Correlation between number of patients and SARS-CoV-2 level･Variation ← Stability of concentration method, difference of PCR method

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Other report in Japan 1

First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and riverwater in Japan(Haramoto et al. Sci. Total Environ. 2020)

Detected from treated wastewater in Yamanashi Prefecture

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Other report in Japan 2
Identification of SARS-CoV-2 in wastewater in Japanby multiple molecular assays -implication for wastewater-based epidemiology (WBE)

Hata et al. Science of The Total Environment
Volume 758, 1 March 2021, 143578.

In Toyama and Kanazawa, PCR positive before outbreakDaily newly confirmed positive in Tokyo
1200
1000
8006004002000

Out of 13 million population in Tokyo district.
7-day average in the middle of the month, except for Jan 5th19

20Detection of SARS-CoV-2 RNA bypolyethylene glycol precipitation followed by acid
guanidinium thiocyanate-phenol-chloroform extraction

from municipal wastewater in Tokyo, Japan

Dept. of Urban Engineering, School of Eng.,The University of TokyoMS2

φ6Raw sewage

Primary concentration

qPCR-basedrecoveryUFEMVPEG×RNA Extraction

QIAamp Viral
RNA Mini KitTRIzol

PhD studentShotaro Torii,H Furumai, H Katayama

Results and discussions

Whole process recovery

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Comparison of whole process recovery
 Materials and methods
MS2Raw sewage

φ6 Results and discussion

Whole process
recoveryRNA Extraction

φ6: an enveloped virus,
surrogate for SARS-CoV-2
Primary concentration

Primary concentration

UFEMV

PEG×RNA Extraction

QIAamp Viral
RNA Mini Kit

TRIzolqPCR-based
recoveryMS2

φ6

PEG+TRIzol provided the highest recovery for φ6, followed by UF+QIAampResults and discussions

22Detection of SARS-CoV-2 RNA from raw sewage
 Weekly sampling
Grab sample of raw sewage

 Occurrence in raw sewage
Datelog10 Wa

SLOQb(copies/mL)

SARS-CoV-2

June 30-0.41

16.1

Neg

-0.76

38.5

Neg

-0.79

40.6

Pos-0.65

27.5

Neg

0.04

6.0

Pos-0.29

13.2

Neg

-1.14

86.6

Neg

-0.34

13.5

Neg

-0.36

14.3

Pos-0.26

11.3

Pos-0.2

10.4

Neg

-0.68

31.3

Neg

July 7

In June and July 2020
July 16

 Processing

July 22
July 29

Frozen
40 mL

PEG Modified RT-qPCR
TRIzolAugust 5

Pos indicates positive for the target while Neg indicates negative
a : Log whole process recovery b : Sample limit of quantification (SLOQ)Of the 12 grab samples, 4 were positive for SARS-CoV-2 by RT-qPCR

SummaryWastewater-based
epidemiology is useful tool
to monitor COVID-19 in acommunity
SARS-CoV-2 RNA wasdetected from wastewaterin Japan23

Shift of Idea on WBE
At first
It is possible for WBE to detect earlier than human cases.
Rapid detection system is needed.
• Right after arrival of sample, how long does it take?

We want positive data.

• For news value• To convince the method is OK

After first wave
WBE is meaningful for society because of low number of infection.
It is impossible to detect earlier than human PCR.
We need efficient workflow for WBE.
• Many samples from many places, mostly negative, but constantly monitor.
• When positive then we need action. How?

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COVID-19 Taskforce in JSWE
Wastewater sectors are not positive to provide samples at first.
Japan Society for Water Environment launched COVID-19
Taskforce in May 2020.
Many wastewater works started to cooperate with researchers.
Most of the members belongs to International Water Association(IWA) specialist group on Health-related Water Microbiology
Ministry of Land, Infrastructure, Transport and Tourism is now
supporting its activity.
We published a laboratory manual draft for detection of novel
coronavirus from wastewater in Dec 202025