Environmental Sampling as a Supplement for Detection of Norovirus

Jan Vinjé Ph.D.Head National Calicivirus Laboratory / Director CaliciNet

Division of Viral DiseasesCenters for Disease Control and Prevention

Atlanta, GA, USjvinje@cdc.gov

Annual Public Meeting – Vessel Sanitation Program – Miami, FL – June 22, 2015

• Incubation period: 12-48 hours

• Infect enterocytes in the small intestine and possibly B-cells

• Acute-onset vomiting and/or diarrhea– Watery, non-bloody stools– Abdominal cramps, nausea, low-grade

fever

• Most recover after 12-72 hours– 10-12% seek medical attention; some

require hospitalization and fluid therapy– More severe illness and death possible

in elderly and those with other illnesses

• Up to 30% of infections are asymptomatic

Clinical Disease Characteristics Norovirus Gastroenteritis

Norwalk infected

Normal microvilli

Background NorovirusesDisease Burden: - Most important cause of epidemic and sporadic acute gastroenteritis

- estimated 56,000-71,000 hospitalizations and 570-800 deaths each year- #1 cause of foodborne illness in the US (‘One-a-day’)

Transmission: person to person, food and waterDiscovery virus: by electron microscopy in stool from GI outbreak in 1968 in Norwalk, OH Virus particles: non-enveloped icosahedral ~35 nm viral particlesClassification: family CaliciviridaeGenome: linear positive sense RNA of 7.4 - 7.7 kb in lengthClassification: 7 genogroups, 3 (GI, GII, GIV) including 29 genotypes infect humansAnimal model: no (chimpanzees, swine, humanized mice (?) can be infected)Prevention and : - infection control principles, (hand hygiene, limiting exposure to Control infectious individuals, and environmental decontamination)

- Virus-like particle (VLP)-based norovirus vaccine is performingpromising in clinical trials

©Green K 2013; Pringle et al 2015; Hall et al 2014; Vinjé et al 2015; Barclay et al., 2014; Atmar et al 2011; Bernstein et al 2015

Foodborne11%

Person-to-Person

62%

Other & Unknown

27%

Transmission Route of 3,960 Norovirus Outbreaks Reported to CaliciNet, 2009-2013

Transmission route

Vega et al, 2014 JCM

Food = 16% of OBs with knowntransmission route

Risk factors for foodborne outbreaks

Norovirus Salmonella C. Perfringens

Risk factors

1. Infected worker (63.3%)

2. Bare hand contact (40.4%)

3. Inadequate cleaning (12.5%)

1. Room temp for several hours (33%)

2. Insufficient time or temp during cooking (32%)

3. Raw ingredients contaminated by pathogens from animals or environment (30%)

1. Room temp for several hours (52%)

2. Slow cooling (49%)

3. Insufficient time or temp during reheating (40%)

Lynch et al 2006. Surveillance for foodborne outbreaks-United States, 1998-2002, Morb, Mortal.Wkly. Rep. 55:1-42

Human norovirusPersonal/environmental hygiene

Salmonella and C. PerfringensInadequate storage and

cooking conditionsVS

7

Infe

ctio

usne

ss

Time

Direct transmission

Environmentally-mediated transmission

Vomiting incident

Day 1 Days One to two weeks

Lopman et al., 2012

Direct and indirect transmission potential of norovirus

Origin of environmental contamination

StoolVomitus

(20-50% norovirus positive)

Contaminationmode

Hands, and infrequently aerosol derived (e.g.,

flushing)

direct, aerosol derived, hands

Norovirus concentration Up to 1012 virus Particles/g up to 108 virus particles

per objectContaminated

sitesPrimarily in rest rooms and

adjacent areas Possibly everywhere

Median levels of norovirus

contamination

3.0 log10 RNA copies (1.71-6.0)

3.3 Log10 RNA copies (2.7 to 8.6 Log10 )

Norovirus Diagnostics and Typing Flowchart

Stool / Vomitus specimen

Food/water/environmental sample

Real-time RT-PCR

Positive

Detection

Typing

RNA Extraction :Stool supernatant

+ lysis buffer

+ MS2 virus

Cycle sequencing Compare norovirus sequences with database

PCR purification

Cycle seqpurification

Conventional RT-PCR

Region D (Reg C if neg)

2011-SP-00012011-SP-0002GII_4_Minerva_USA06 (2006b)GII_4_NewOrleans (NO1805_USA09)GII_4_NewOrleans (NSW001P_AUS08)2010-SP-00012010-SP-0002GII_4_NewOrleans (Apeldoorn_NLD07)GII_4_NewOrleans (NO1500_USA08)2010-SP-00102010-SP-0011GII_4_Osaka_JPN07 (Riviera)GII_1_Hawaii_USA71

Currently Available Surface Sampling Methods

Fiber tipped swabs o Very Easy to use, and cost effectiveo Cotton swab (health care settings)1

o Polyester swabs (food industry)2

o polyester-tipped swab for clinical sampling of influenza virus3

Use of antistatic wipes to investigate norovirus surface contamination in restaurants 4

Little information on virus sampling performance Poor translation of lab results into field sampling performance 25- 50 cm2 of test surface area in laboratory vs broad range of

surface areas in the field1 Clinical Microbiology Procedure Handbook, Vol III2 Compendium of methods for the microbiological examination of foods3 http://www.cdc.gov/h1n1flu/specimencollection.html4 Boxman et al 2010 J of Food Protection

Comparison of Swab Materials

Fiber tipped swabs Macrofoam swabs Antistatic wipe

Cotton Rayon Polyester

Swab ValidationDrying

Moistening of swabs

SwabbingElution

22

0

10

20

30

40

50

60

70

Cotton

Rayon

Polyester

Macrofoam

Vir

al R

NA

rec

over

y (%

)

0 1 8 24 48Drying time (h)

Effect of Drying Time on Norovirus RecoveryFrom 4 Different Swab Materials

Park et al., 2015 AEM

Size of Surface Area of Frequently touched surfaces

25.8 cm2

58.1 cm2

103.2 cm2

161.3cm2

645.2 cm2

Sink faucet handles, doorknob, and Computer mouse (206.5-258.1 cm2 )

Computer keyboard (387.1 cm2)

Surface area of toilet seat (645 cm2 )

Swab validation tests

Norovirus recovery from Different Sized Surface areas using 4 different swab

materials

26 58.1 130.2 161.3

Vir

al R

NA

rec

over

y (%

)

Surface area (cm2)

0

10

20

30

40

50

Cotton

Rayon

Polyester

Macrofoam

Park et al., 2015 AEM

Recovery of Norovirus from Larger Surface Areas:Comparison of Macrofoam Swab with Antistatic Wipe

0.1

1

10

100Macrofoam

Antistatic wipe

161.3 645.0

Vir

us r

ecov

ery

(%)

Surface area (cm2)

Park et al., 2015 AEM

Detection limit of Swab sampling of Larger Sized Surface Areas

Stainless steel (645 cm2): Toilet seat (700 cm2):

0

10

20

30

40

50

2 3 4 5 6

Vir

al R

NA

rec

over

y (%

)

GII.4 RNA titer(10Log RNA copy #/coupon)

Swabs collected after 48 h of dryingPark et al., 2015 AEM

Validation study in the field

18

Based on

Previous VSP Ops Manual Current FDA Model Food Code WHO Guide to Ship Sanitation Extensive References

Norovirus on Cruise Ships

The Vessel Sanitation Program (VSP) at CDC assists the cruise ship industry to prevent and control the introduction, transmission, and spread of gastrointestinal (GI) illnesses on cruise ships.

Working Station for field sampling

19

Sampling Surfaces in Public Areas - 1

20

21

Sampling Surfaces in Public Areas - 2

22

Sampling Surfaces in Public Areas - 3

Sampling High Contact Surfaces in Case Cabins

23

24

Sampling High Contact Surfaces in Case Cabins

High Contact Objects in Restaurants - 1

25

26

High Contact Objects in Restaurants - 2

27

High Contact Objects in Restaurants - 3

Flow chart of final protocol for environmental surface sampling of norovirus

Use pre-moistened macrofoam swab (e.g., ENVIROMAX Puritan, Guildford, ME)

RNA extractionAdd 2 ml of UNEX buffer (Microbiologics, St Cloud, MN) plus MS2 (internal extraction control) to swab

Incubate 10 min at room temperature to lyse the virusAdd 2 ml of 100% ethanol

RNA purification and concentration: Load 4 ml of lysed sample on HiBind RNA Midi column (Omega Bio-Tek, Norcross, GA)

Concentrate 250 µl of RNA to 25 µl on RNA Clean & Concentrator ™-5 (Zymo Research, Irvine, CA)

Norovirus detection by Taqman™ realtime RT-PCR

Norovirus genotyping by sequencing

Transport swab (at 0-4°C or frozen) to laboratory within 48 hr of collecting samples

Swab inanimate surface (≤ 700 cm2) and place swab back in transport tube

Report results

Sample Area Sample Point DescriptionAverage Ct value

(# positive / total samples tested)

GenotypeNorovirus RNAcopy number per

sampled areab

Atrium Handrails 34.3 (1/2) GII 16GI cabin A toilet seat 31.4 (2/2) GII.1a 31,217GI cabin A Hand sink faucet 37.5 (1/2) GII 491GI cabin A door handle 35.0 (2/2) GII 2,675GI cabin A remote control 38.6 (2/2) GII.1a 233GI cabin B toilet seat 33.5 (2/2) GII.1a 986

Lido Dispense handles of ice Cream Machine 34.2 (2/2) GII 16

Lido Table Condiments 35.2 (1/2) GII 15Lido Table Top 35.3 (1/2) GII 14

Pizzeria Counter surface 35.7 (1/2) GII 14Main Galley Fun-time Machine 37.1(1/2) GII 64

Vending Machine Touchable Surfaces 38.8 (1/2) GII 18

Crew lounge Keyboard Surface and Mouse 36.8 (1/2) GII 80

GI Cabin C faucet and door handle 31.6 (2/2) GII.1a 26,458GI Cabin C telephone 36.4 (2/2) GII 1,035GI Cabin C keyboard 33.0 (2/2) GII 1,317

Medical center Clipboard 36.0 (2/2) GII 113

Norovirus Positive Swab Samples from surfaces on a Cruise Ship with passengers with viral gastroenteritis symptoms

a Of 17 GII positive swab samples, only four samples could be genotyped. b RNA copy numbers were estimated based on a standard curve of GII.7 RNA transcripts (Y=-(3.1±0.3) X + (39.6 ± 2.0), R2 ≥ 0.98) Park et al., 2015 AEM

Conclusions

Macrofoam swabs demonstrate highest recovery of norovirus from inanimate surfaces highest virus recovery from larger surface areas

High level of surface contamination in cabins of people with norovirus symptomso Low level of contamination of surfaces in public areas

Are cleaning practices effective?

Passengers

Public spaces

Passengers

Public spaces

ConclusionsWide distribution of norovirus on environmental surfaces on

a cruise ships with reported norovirus symptomso 33% (8/24) (case cabins) and 13% (9/68) (common settings)

A strong association between level of environmental contamination and norovirus infection o 2-4 log norovirus titers in case cabins vs 1-2 log in common areas

Toilet seats had highest positivity rate (50%)

Data from 4 ships with norovirus outbreaks: cleaning practices / reagents need to be re-assessed

AcknowledgementsNational Calicivirus Laboratory, CDCBen Anderson, PhD student UGALeslie Barclay MPHPreeti Chhabra PhDNikki Collins MSVeronica Costantini PhDTherese Cromeans PhDMarta Diez-Valcarce PhDNicole Gregoricus MSPHMonica MotonGeun Woo Park PhDThomas Yeargin MS

Vessel sanitation program, CDCCDR Aimee TreffilettiJill ShugartLCDR Amy Freeland PhDCAPT Charles Otto, CAPT Jaret Ames

Cruise linesMario Hrsak – Carnival CruisesManny Rivas – Celebrity Cruises

– Royal Caribbean

Questions?

Infectivity and Persistence

Norovirus can remain infectious for ≥61 days and detectable for >3 years in groundwater

Seitz et al. 2011 AEM