A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.
Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.
Human noroviruses are a leading cause of epidemic and sporadic acute gastroenteritis worldwide 1,2,3. The virus is extremely contagious and transmission occurs through direct person to person interaction or indirectly through contact with contaminated food, water or environmental surfaces. Noroviruses can be shed for extended periods and prolonged survival of the virus on environmental surfaces has been documented 1,2,3. During peak shedding, billions of virus particles are released per gram of feces, and vomit also contains a sufficient number of viral particles to cause infection 4,5,6,7,8,9,10. In addition, transfer of the virus between inanimate surfaces and human skin can occur easily 2,11,12. Hence, monitoring of environmental contamination may assist in outbreak investigations and in assessing the effectiveness of clean-up and disinfection procedures.
Several environmental sampling protocols have been described for the detection of rotavirus, coliphage MS2, feline calicivirus (FCV), and bacteriophage P22 13,14,15,16. However, the validation conditions described in these studies, including fast desiccation (<1 hr) and small surface areas (25 x 100 cm2), may not adequately represent field settings. In addition, the expected low contamination levels of environmental surfaces require protocols that are able to detect very few virus particles.
We developed a macrofoam-based surface sampling method for the detection and typing of norovirus. This method has been validated during several norovirus outbreaks. The protocol includes 1) how to collect swab samples from environmental surfaces (2) how to best maintain integrity of the samples during collection and shipping to the laboratory, and 3) laboratory testing and typing of norovirus.
1. Swab Sampling in the Field
2. Storage and Transport of Swabs to the Laboratory
3. Virus Concentration, Viral RNA Extraction and Purification
NOTE: All centrifugation steps use a table top centrifuge at 5,000 x g for 5 min at room temperature, unless stated otherwise. Be extra careful when working with the universal nucleic acid extraction (UNEX) buffer. Wear goggles or face shield.
4. Midi Column Viral Nucleic Acid Extraction
5. Concentration of Viral Nucleic Acid Using RNA Clean and Concentrator Kits
6. Mutiplex RT-qPCR Detection of Genogroup I, and II Noroviruses, and Coliphage MS 2 (Supplementary Table 2)
7. Quantification of Norovirus in Swab Samples
8. Genotyping of Real-time RT-PCR Positive Samples by Hemi Nested Conventional PCR Amplification
Figure 1 presents a flowchart of the swab sampling protocol. This protocol consists of four main steps; 1) sample collection, 2) sample storage and transportation, 3) viral RNA purification and concentration and 4) RT-qPCR assay and genotyping.
Figure 1: Flow chart of the final protocol for environmental surface sampling of norovirus Please click here to view a larger version of this figure.
Table 1 summarizes the results from 34 swab samples that were collected from a cruise ship that had reported cases of suspected norovirus gastroenteritis during a voyage. Swab samples were extracted and tested in duplicate for norovirus by multiplex real-time RT-qPCR. Seventeen (18.5%) samples tested positive including 8 samples from surfaces in cabins where passengers with norovirus symptoms had stayed and 9 from common areas on the ship. The number of genomic copies per sample were calculated from the Ct values (which ranged from 16 to 31) using a standard curve of a norovirus GII.7 RNA transcript. The median number of genome copies from swab samples in cabins was 3.6 log10(range: 2.4 – 4.5 log10 RNA copies), which was significantly higher than the genome copies from common areas (range: 1.2 – 2.1 log10 genome copies) (P <0.001). Four (23.5%) of the 17 positive swab samples were able to be genotyped, and all samples had identical GII.1 sequences.
Location environmental swap collecteda | Sample Point Description | Average Ct value (# positive of the total number of samples tested) | Genotype | Norovirus RNA copy number per sampled areac |
Atrium | Handrails | 34.3 (1/2) | GII | 16 |
Cabin A | Toilet seat | 31.4 (2/2) | GII.b | 31,217 |
Cabin A | Faucet | 37.5 (1/2) | GII | 491 |
Cabin A | Door handle | 35.0 (2/2) | GII | 2,675 |
Cabin A | Remote control | 38.6 (2/2) | GII.1b | 233 |
Cabin B | Toilet seat | 33.5 (2/2) | GII.1b | 986 |
Lido | Ice Cream Machine | 34.2 (2/2) | GII | 16 |
Lido | Table Condiments | 35.2 (1/2) | GII | 15 |
Lido | Table Top | 35.3 (1/2) | GII | 14 |
Pizzeria | Counter surface | 35.7 (1/2) | GII | 14 |
Main 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 |
Cabin C | Faucet and door handle | 31.6 (2/2) | GII.1b | 26,458 |
Cabin C | Telephone | 36.4 (2/2) | GII | 1,035 |
Cabin C | Keyboard | 33.0 (2/2) | GII | 1,317 |
Medical center | Clipboard | 36.0 (2/2) | GII | 113 |
aCabin A, B and C has been occupied by individuals who had been clinically ill with viral gastoenteristis symptoms. | ||||
bFour of the 17 GII-positive swab samples could be genotyped. | ||||
cRNA copies were caluated based on a standard curve of norovirus GII.7 RNA transcripts. | ||||
Note: above date were slightly modified from the original article6. |
Table 1: Results from 34 swab samples that were collected from a cruise ship that had reported cases of suspected norovirus gastroenteritis during a voyage.
Figure 2 shows the relationship of Ct values determined by RT-qPCR and the ability to sequence these samples. In total, 127 out of 217 swab samples tested positive for GII norovirus by RT-qPCR. The samples displayed a wide range (12-40) of Ct values. In total, 29 (22.8%) of the RT-qPCR positive samples could be genotyped. Swab samples with Ct values below 27 and ranging between 28-31 were genotyped at rates of 100% and 72.2%, respectively. In contrast, only 22.0% and 1.6% of swab samples with Ct values of 32-36 and 37-40, respectively, produced hemi-nested amplicons that could be sequenced successfully.
Figure 2: Results of RT-qPCR screening and sequencing of swab samples that had been collected during confirmed norovirus outbreaks. White bars represent the number of RT-qPCR positive swab samples virus. Human norovirus nucleic acids in those RT-qPCR positive samples were amplified using hemi-nested PCR assay and then sequenced for confirmation of genotyping. Black bars and line represent the number and the percentage of genotype confirmed swab samples, respectively. Please click here to view a larger version of this figure.
Ct value range | Number of RT-qPCR positive samples | Number (%) of sequence confirmed samples a |
20-23 | 4 | 3 (75) |
24-27 | 3 | 3 (100) |
28-31 | 18 | 13 (72.2) |
32-36 | 41 | 9 (22.0) |
37-40 | 61 | 1 (1.6) |
a) hemi nested PCR |
Table 2: RT-qPCR and genotyping results of swab samples.
Sample Number | LOCATION | Description of surface | Surface area (cm2) | Date/time of sample collection | Clean (Y/N) | If cleaned, what disinfectant was used? | Detailed description of surface material and location |
1 | Room# 7-1302 | Toilet Seat | 700 cm2 | 6/26/2016; 9:00 am | No | not applicable | Toilet seat [top surfaces), and hard plastoc |
2 | Room #7-1330 | Telephone handle | 500 cm2 | 6/26/2016; 9:25 am | Yes | 1,000 ppm bleach | Hard plastic, rubber button |
Supplementary Table 1: Example of sample description form.
genogroup / virus | Name oligonucleotide primer/probe | Sequence 5΄→3΄ | Reference |
GI | Cog1F | CGY TGG ATG CGI TTY CAT GA | 17 |
Cog1R | CTT AGA CGC CAT CAT CAT TYA C | 17 | |
G1SKF | CTG CCC GAA TTY GTA AAT GA | 17 | |
G1SKR | CCA ACC CAR CCA TTR TAC A | 17 | |
Ring1E-probe | FAM – TGG ACA GGR GAY CGC – MGBNFQa | This study | |
GII | Cog2F | CAR GAR BCN ATG TTY AGR TGG ATG AG | 17 |
Cog2R | TCG ACG CCA TCT TCA TTC ACA | 17 | |
Ring2-primer | TGG GAG GGC GAT CGC AAT CT | 17 | |
G2SKR | CCR CCN GCA TRH CCR TTR TAC AT | 17 | |
Ring2-probe | Cy5 or QUASAR 670 – TGG GAG GGC GAT CGC AAT CT – BHQ2b | 17 | |
MS2 | MS2F | TGG CAC TAC CCC TCT CCG TAT TCA CG | 18 |
MS2R | GTA CGG GCG ACC CCA CGA TGA C | 18 | |
MS2P-probe | HEX – CAC ATC GAT AGA TCA AGG TGC CTA CAAGC – BHQ1c | 18 | |
aGI TaqMan probe is 5’-labeled with 6-carboxyfluorescein (FAM) and 3’-labeled with MGBNFQ (Minor-groove Binding site) | |||
bGII TaqMan probe is 5’-labeled with Cy5 or Quasar 670 and 3’-labeled with Black Hole quencher; Black Hole Quencher (BHQ) 2 used due to availability, BHQ 3 is preferred. | |||
cMS2 TaqMan probe is 5’-labeled with HEX and 5’-labeled with BHQ1 |
Supplementary Table 2: Oligonucleotide primers and probes information.
Component | Volume per reaction (µl) | Final concentration |
2x RT-PCR Buffer* | 12.5 | 1x |
Nuclease-free water* | 1.08 | n/a |
Detection Enhancer* | 1.67 | n/a |
Cog 1F (10 µM) | 1 | 400 nM |
Cog 1R (10 µM) | 1 | 400 nM |
Ring 1E-probe (10 µM) | 0.5 | 200 nM |
Cog 2F (10 µM) | 1 | 400 nM |
Cog 2R (10 µM) | 1 | 400 nM |
Ring 2-probe (10 µM) | 0.5 | 200 nM |
MS2F (10 µM) | 0.25 | 100 nM |
MS2R (10 µM) | 0.25 | 100 nM |
MS2P-probe (10 µM) | 0.25 | 100 nM |
2x RT-PCR enzyme * | 1 | 1x |
Master Mix volume | 22 | |
* included in the real time RT-PCR kit |
Supplementary Table 3: Master mix for multiplex real-time GI/GII/MS2 norovirus RT-PCR
Type of control | Result interpretation | ||
Sample | Negative control | Should be negative | |
Positive control | Should be positive | ||
Negative sample | Sample is negative if each Ct value is undetectable for GI/GII | ||
Positive sample | Ct values (GI/GII) of both replicates is < 38; this (arbitrarily) cut-off needs to be determined experimentally for each realtime PCR platform and kit | ||
Tentative positive sample | Sample is tentatively positive if Ct value (GI/GII) of one replicate is <38 | ||
Internal process control (MS2) | Samples with a threshold cycle (Ct) value of ≥32 for MS2 should be retested undiluted and 1/10 diluted. | ||
Parameter | Acceptable value | ||
Standard curve using norovirus RNA transcripts | R2 | >0.97 | |
Efficiency | 90% to 115% |
Supplementary Table 4: Controls to include in each RT-qPCR test for detection of norovirus in environmental swab samples.
Component | Final concentration | vol/rxn (µl) |
5x RT-PCR buffer | 1x | 5.00 |
dNTP mix (10 mM) | 0.4 mM | 1.00 |
Enzyme mix (RT and Taq) | 1.00 | |
Forward primera | 0.5 µM | 0.50 |
Reverse primer a | 0.5 µM | 0.50 |
Rnase inhibitor (20 U/µl) | 20 U | 1.00 |
Rnase-free water | 11.00 | |
Total Volume | 20.00 | |
aforward and reverse primer sets for GI and GII group are Cog1F + G1SKR, and Cog2F + G2SKR, respectively. | ||
Second round RT-PCR | ||
Component | Final concentration | vol/rxn (µl) |
5x RT-PCR buffer | 1x | 5.00 |
dNTP mix (10 mM) | 0.4 mM | 1.00 |
Enzyme mix (RT and Taq) | 1.00 | |
Forward primerb | 0.5 µM | 0.50 |
Reverse primerb | 0.5 µM | 0.50 |
Rnase inhibitor (20 U/µl) | 20 U | 1.00 |
Rnase-free water | 14.00 | |
Total Volume | 23.0 | |
bforward and reverse primer sets for GI and GII group are G1SKF + G1SKR, and Ring2 primer + G2SKR, respectively. | ||
Note: above informaion was slightly modified from the original article19 |
Supplementary Table 5: Hemi-nested RT-PCR Master mix.
Phase I | Field Sampling | 1. Check macrofoam swab kits (e.g., expiration date, tube leakages, and swab wetness) |
2. Swab surface (limit surface area to ≤100 inch2 (645 cm2)) | ||
3. Place swabs into the transport tubes, and tighten the caps securely to prevent leaking during shipping | ||
4. Place swab kit in a ziplock-bag | ||
Phase II | Sample transport and storage | 1. Sampled swabs should be stored -70 °C (or -20 °C) |
2. Transporting swab samples to laboratory at 0-4 °C (cold-packs) in an insulated container and ship within 48 hr of collecting swabs | ||
Phase III | RNA etraction | 1. Check lysis buffer (e.g., expiration data) |
2. Add MS2 as an internal control into lysis buffer before adding 100% ethanol | ||
RNA purification and concentration | 3. Clean lab bench and small equipment using RNA RNase removal solution | |
4. Change gloves frequently during steps to avoid a RNA cross-contamination | ||
Phase IV | RT-PCR Assay (QA/QC) | 1. Include quantified norovirus transcript RNAs (GI and GII) for each RT-qPCR assay to control for variations in Ct values for each PCR run |
2. Use absence of MS2 signal to monitor presence of PCR inhibitors |
Supplementary Table 6: Checklist for CDC's environmental sampling procedure.
Noroviruses have a 50% human infectious dose between 18 and 103 virus particles20. Therefore, even low-level contamination of surfaces may pose a public health risk. Several aspects of the swab sampling protocol were evaluated including: 1) different swab materials, 2) storage condition swabs during transport, 3) viral RNA concentration, and 4) coliphage MS2 as internal extraction control.
Until recently, only the performance of swabs made from cotton, polyester, nylon and antistatic wipe) had been evaluated and proposed for field use13,14,15,21,22. Of these, cotton swabs have been recommended by the ISO 15216 protocol for detection of norovirus and hepatitis A virus from food preparation surfaces and fomites23. However, there are limited data on the testing performance of cotton swabs under field conditions with large high-touch surfaces such as doorknobs, computers, and toilet seats that have been frequently implicated in the transmission of noroviruses24,25. Additionally, the size of these environmental surfaces exceeds the capacity of fiber-tipped swabs. In our study, we demonstrated that macrofoam swabs, which have a larger swab head than the fiber-tip swabs, have higher recovery rates for human norovirus when sampling environmental surfaces with an area up to 700 cm2.
Noroviruses in solution are relatively unstable at ambient temperature and require at least refrigeration. Therefore, shipping to the laboratory should occur at 0-4 °C within 48 h after collection of the swabs. The volume of sample that can typically be tested in a PCR reaction is much smaller than that of the amount of sample after elution from the swab (3-5 μL vs. 5-10 mL) which, without concentration, significantly reduces the positivity rate. In environmental virology, polyethylene glycol (PEG) has been used successfully to concentrate viruses from large volumes of environmental waters. However, volumes up to 10 mL can be easily extracted and concentrated using Midi spin columns to volumes as low as 250 μL, and further concentrated 10-fold with high recovery using spin columns.
Although RT-qPCR is the gold standard for sensitive detection and quantification of norovirus, the sensitivity of these assays can be easily affected by the presence of PCR inhibitors that are co-extracted from the swab samples. Therefore, inclusion of an extraction control such as coliphage MS2 virus particles, in a multiplex PCR format that detects both norovirus as well as MS2, helps to monitor the presence of PCR inhibition and to assess variation among different experiments.
The test variables of a laboratory validation method are critical to determine the discriminatory power of a test method and are further translated into the method's likely field sampling performance. Taking real world field condition (e.g., delayed sampling, larger surface areas of up to 700 cm2) into account, the detection limit for norovirus was 3.4 log10(stainless steel) and 4 log10(toilet seat) copies norovirus per surface sampled6. Consequently, negative swab results are not definitive that no viral contamination has occured. Clinical and epidemiological information of norovirus illness always trumps environmental findings2,6.
Our data from a cruise ship showed that high-touch surfaces such as telephones, computer keyboards, and door handles tested positive for norovirus in cabins where people with reported viral gastroenteritis had stayed. These surfaces either became contaminated through direct contact or indirectly through droplets produced during toilet flushing or vomiting episodes6,7,8,9,10.
In conclusion, detection of norovirus on environmental surfaces using this newly developed swab protocol may assist in determining the level of environmental contamination during outbreaks, detect virus when clinical samples are not available and in monitoring of the effectiveness of cleaning practices. Fiber tipped swab (cotton and rayon) based surface sampling methods have been widely used to investigate other enteric viruses (e.g. rotavirus, and adenovirus)27,28,29. Considering the higher recovery efficiency for norovirus of macrofoam swabs over those fibers tipped swabs6, we expected that the macrofoam swabs will be able to detect other enteric viruses as well.
The authors have nothing to disclose.
The authors have no acknowledgements.
Generic name for kits | ||||
Macrofoam swab | Premoistened EnviroMax Swab kit | Puritan | 2588060PFUW | |
RNA Lysis buffer | CDC UNEX buffer | Microbiologics | Cat No MR0501 | |
RNA extraction spin column | Midi column | Omega Biotek | Cat No R6664-02 | |
RNA purification spin column | Zymol RNA Clean and Concentrator kit | Zymo Research | Cat No R1016 | |
Real time RT-PCR kit | AgPath kit One-Step RT-PCR Kit | Life Technologies | Cat No 4387391 | |
Conventional RT-PCR kit | Qiagen one step RT-PCR kit | Qiagen kit | Cat No 210212 | |
Gel extraction kit | Qiagen QIAquick gel extraction kit | Qiagen kit | Cat No 28704 or 28706 | |
Coliphage MS2 | ATCC | Cat No 15597-B1 | ||
RNA run-off transcripts | Bacteriophage MS2 (ATCC No. 15597-B1) can be cultivated using Escherichia coli (E.coli) Famp (ATCC No. 700891). | |||
Realtime PCR platform | Applied Biosystems | Model ABI 7500 | GI and GII RNA run off transcripts were quantified spectrophotometrically at A260, diluted in diethyl pyrocarbonate-treated water to 1 × 106 copies/ μl, and stored at −80°C with 1.0 U /μl RNasin (Promega, Madison, WI). | |
Optical 96-well reaction plate | Thermo Scientific | Cat No 4316813 | ||
MicroAmp Clear Adhesive Film | Thermo Scientific | Cat No 4306311 |