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Guideline for the Prevention and Control of Norovirus Gastroenteritis Outbreaks in Healthcare Settings, 2011

Q2: What are the best methods to identify a norovirus outbreak in a healthcare setting?

Diagnostic methods – Fecal specimens

PCR

Author, Yr (Ref) Study Design Quality Study Objective Population and Setting N Results Comments Ref ID_Data extracted by
Nordgren, J; 2008 129 Diagnostic Study
 
2,3
To evaluate 2 novel light upon-extension (LUX) RT- PCR assays for norovirus genogroup I and II detection and quantification. 61stool samples from Sweden. 42 samples from Nicaragua. A reference panel of 15 stool samples from Sweden used for external validation of norovirus.

Positive samples
Overall - 99% correlation between LUX RT- PCR and TaqMan RT-PCR.
LUX RT-PCR – 47/103
Conventional PCR – 39/103

TaqMan RT-PCR – 48/103

Swedish samples
LUX RT-PCR and TaqMan RT-PCR – 18/61 (100% correlation).

Nicaraguan samples
LUX RT-PCR –  29/42
TaqMan RT-PCR – 30/42
Conventional PCR – 25/42
IDEIA – 24/42 

Reference panel
LUX RT-PCR correctly identified all (n=11) coded controlled specimens.

Detection level

LUX RT- PCR detected ≤ 101 to 107 genes/reaction, with a theoretical lower limit of ≤ 20,000 viruses/gm of stool.
TaqMan based RT-PCR described by Kageyama, conventional PCR described by Zintz were used as the reference standards for both the Swedish and Nicaraguan samples. IDEIA (DakoCytomation, Copenhagen, Denmark) was used as a reference for the Nicaraguan specimens. Power and sample size not reported. 5115_IL
DeMedici, D; 2007 130 Diagnostic Study
 
1,2, 3
To compare IDEIA, a published RT-PCR, and an RT-boosted-PCR in detecting norovirus in stools collected after the end of a gastroenteritis outbreak.

Samples obtained from an outbreak in Italy in December 2002 where 202 patients developed vomiting and/or diarrhea after eating oysters.

41 stool samples.

Positive samples
ELISA – 6/41
RT-PCR – 6/41
RT-boosted-PCR – 23/41

Results of RT-PCR vs. ELISA (χ2=0.17; p>0.05).

RT-boosted-PCR vs. RT-PCR and ELISA (χ2=15.06 and 13.47; p<0.05 for both).
IDEIA NLV kit (Dako, Ely, UK) Power and sample size not reported. 049_IL
Hymas, W; 2007 131 Diagnostic Study
 
2, 3
To evaluate a novel one step real-time eclipse RTPCR
designed to detect norovirus genogroups I and II compared to conventional CDC TaqMan assay.
29 stool samples and 9 RNA samples provided from Utah and North Carolina.

Correlation between eclipse RT-PCR and TaqMan PCR
97% overall agreement

By genotype:
Genotype I: 100% correlation
Positive by both tests – 4
Negative by both tests – 32

Genotype II: 91% correlation
Positive by both tests – 25
Negative by both tests – 10
Discordant results - 3

1 stool sample was positive by eclipse RT-PCR but negative by TaqMan PCR.

2 samples were positive by eclipse RT-PCR but indeterminate by TaqMan PCR.

Limit of detection and cross reactivity

Sensitivity for GI and GII was approximately 50 copies/reaction.
CDC Taqman assay was the reference standard. Power and sample size not reported. 130_IL
Logan, C; 2007 132 Diagnostic Study
 
2, 3
To test real-time RT-PCR compared to EM in detecting viral gastroenteritis, including norovirus, Sapovirus, and human Astrovirus. Stool samples from pediatric patients with diarrhea and/or vomiting received at a microbiology laboratory in Ireland, from February 2004- April 2005. 140 stool samples from symptomatic patients. 25 stool samples from asymptomatic patients.

Positive results
Enteric viruses were detected in 53/140 (38%) samples by RT-PCR vs. 10/140 (8%) by EM.
Detection of norovirus increased 200% using RT-PCR over EM.
All norovirus samples were genogroup II/4.

Agreement between EM and RT-PCR
norovirus
Positive by both tests – 5
Negative by both tests – 109
Discordant results – 26
4 were positive by EM but negative by RT-PCR.
22 were negative by EM but positive by RT-PCR.

Test characteristics (%) of RT-PCR vs EM
Sensitivity – 55.6
Specificity – 83.2
PPV – 18.5

NPV – 96.5
EM was the reference standard Power and sample size not reported. 008_IL
Menton, JF; 2007 133 Diagnostic Study
 
1, 3
To evaluate a real-time RT PCR and a Reverse Line Blot Hybridization assay developed based on the open reading frame (ORF)1ORF2
region. The assays were validated using a reference stool panel and then used to investigate two outbreaks of gastroenteritis.
Reference stool panel contained 5 genotypes of GI norovirus and 9 genotypes of GII norovirus. 56 samples from two norovirus outbreaks in Irish hospitals in 2005 and 2006.

Level of detection
GI – 107 to 101 molecules of plasmid DNA
GII – 5 x 107 to 5 x 101 molecules of plasmid DNA

Positive results
26/56 samples positive.
All belonged to the GII/4 variant.

Power and sample size not reported. 052_IL
Wolf, S; 2007 134 Diagnostic Study
 
1,2
To evaluate a multiplex real-time RT-PCR that distinguishes between norovirus genogroups I, II, and III and targets the junction between open reading frames 1 and 2 compared to Kageyama real time RT-PCR. Real time RT-PCR assays evaluated against 45 RNA stool samples collected from 2001-2006 known to be positive for norovirus including: 34 human stool samples from New Zealand, 6 raw and 3 treated sewage samples, and single samples of contaminated drinking water and source water. 28 stool samples collected from asymptomatic cattle in May 2006 from farms in New Zealand.

Positive results
Multiplex real time RT-PCR positive for norovirus GI/1, GI/2, GI/3, GI/4, GI/5, GI/6, G1/7, GII/8, GII/10, GII/12, and GII/17 in different matrices (stool samples, treated and raw sewage, source water, and treated drinking water).

Agreement between the multiplex real time RT-PCR vs. Kageyama real time RT-PCR
All samples positive by Kageyama RT-PCR also positive by multiplex RT-PCR.
Norovirus GI – 2/25 (8%) negative by Kageyama RT-PCR but positive by multiplex RT-PCR.
Norovirus GII –  3/17 (18%) negative by Kageyama RT-PCR but positive by multiplex RT-PCR.

Cycle threshold (CT) values
In 16/20 norovirus GI samples and 26/28 norovirus GII samples positive by both assays, CT values for the multiplex assay were on average -2.4 CT U lower than for the Kageyama assay.

Remaining 6 samples had higher CT  values using the multiplex assay:
3/3 GI/3 specimens, on average +3.9 CT  U
1/1 GI/7 specimen, +3.5 CT U
1/1 GII/1 specimen, +3.3 CT U
1/1 GII/12, +1.4 CT U 

Level of detection
Multiplex real-time RT-PCR detects <10 copies/reaction of norovirus GI/1, GII/3, and GIII/1. Calculated efficiency values of the assay were 0.93, 0.90, and 1.04 based on the slopes of the standard curves of 3.59, 3.60, and 3.23.

Kageyama real time RT-PCR compared to the multiplex real time RT-PCR. A new bovine NLV, Bo/NLV/ Norsewood/2006/NZL was identified using multiple real-time RT-PCR. Power and sample size not reported. 068_IL
Yoda, T; 2007 135 Diagnostic Study
 
2, 3
To evaluate a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in comparison to routine RT-PCR. 94 samples from Japan obtained during 2004-2006 which had previously been analyzed for bacterial and enteric viruses.

Agreement between RT-LAMP (OPH) vs. RT-PCR (Eiken)
All results – RT-LAMP (OPH) vs. RT-PCR (Eiken) # positive/# samples
GI/1 – 1/1 vs. 1/1
GI/3 – 7/7 vs. 3/7
GI/4 – 3/3 vs. 3/3
GI/8 – 4/4 vs. 4/4
GI/11 – 2/2 vs. 0/2
GI/12 – 8/8 vs. 2/8
GII/2 – 10/10 vs. 10/10
GII/3 – 10/10 vs. 10/10
GII/4 – 10/10 vs. 10/10
GII/6 – 10/10 vs. 10/10
GII/12 – 2/2 vs. 2/2
GII/1 – 3/5 vs. 4/5
GII/5 – 4/4 vs. 4/4
GII/7 –  3/3 vs. 3/3

Sensitivity tests
All results –No. of copies in clinical sample – sensitivity  RT-LAMP (OPH) vs. sensitivity RT-PCR (Eiken)
GI/3 – 8 x 105 – 8 x 101 vs. 8 x 104
GI/8 – 8 x 104 – 8 x 10-1 vs. 8 x 10-1
GII/2 – 7 x 104 – 7 x 100 vs. 7 x 101
GII/3 – 8 x 103 – 8 x 101 vs. 8 x 103
GII/4 – 5 x 106 – 5 x 101 vs. 5 x 101
GII/6 – 2 x 105 – 2 x 102 vs. 2 x 102

The results of RT-LAMP correlated well to RT-PCR.
EC NLV GI and GII detection kits (Eiken Chemical Co., Ltd.) Power and sample size not reported. 167_IL
Antonishyn, NA; 2006 136 Diagnostic Study
 
1,2
To evaluate a one-step real time multiplex RT-PCR compared to conventional PCR. 150 stool samples from cases of acute nonbacterial gastroenteritis between November 2004-March 2005. 50 archived samples used to compare TaqMan PCR with a separate RT using random primers or a single-step RTPCR.
100 samples used to compare sensitivity of multiplex PCR with conventional RTPCR

Agreement between one-step multiplex RT-PCR vs. conventional PCR
Both tests positive - 59
Both tests negative - 27
Discordant results – 14
14 were negative by conventional RT-PCR but positive using one-step real-time RT-PCR.

Sensitivity of multiplex RT-PCR 19% higher than manual extraction with conventional RT-PCR
Power and sample size not reported 223_IL
Trujillo, A; 2006 18 Diagnostic Study
 
1,2
To compare the test characteristics of Taqman RT-PCR with conventional RT-PCR for the detection of GI, GII and GIV strains Stool specimens from sporadic cases and outbreaks of gastroenteritis. Water samples from outbreaks of gastroenteritis in the US. 92 stool samples and 33 water
samples

Test characteristics of Taqman RT-PCR vs. conventional RT-PCR
Stool specimens
TP – 65
TN – 27
FP – 0
FN – 0
By means of serially diluted norovirus RNA transcripts, a potential detection limit of < 10 transcript copies per reaction mixture was observed with the GII assay and a potential detection limit of < 10 transcript copies per reaction mixture was observed with the GI assay.

Water specimens
8/33 specimens were found to be positive. No test characteristics were reported

Power and sample size not reported 4225_RA
Hohne, M; 2004 137 Diagnostic Study
 
1,2
To evaluate a one-tube RTPCR
method, which would prevent the product carryover, in comparison to an in-house RT-PCR.
70 positive stool samples from outbreaks in Germany and 34 European samples collected over a 4 year period (1997-2000).

Positive detection by one-tube RT-PCR of previously identified positive stool samples
Overall 93% detection including isolates of 4 different GGI and 7 different GGII genotypes.

German outbreaks – 66/70 (94.3%) samples were positive including those of 6 different GGII genotypes and 2 different GGI genotypes.

European samples –31/34 (91%) samples were positive including those of 4 different GGI genotypes and 7 different GGII genotypes. 
Samples had previously been diagnosed positive via PCR or EM. 3090_IL
Rohayem, J; 2004 138 Diagnostic Study
 
1,2
To evaluate a single-step multiplex RT-PCR compared to simplex RTPCR
for norovirus, Astrovirus, and Adenovirus.
460 stool samples from infants or children in Germany with non-Rotavirus acute gastroenteritis during 14 months (March 1997 to May 1998): 257 archived samples 203 rotavirus-negative samples collected prospectively

Detection limit of the multiplex RT-PCR
Detection limit of 102 copies for norovirus and Astrovirus RNA transcript, and adenovirus plasmid DNA.

Positive tests
Retrospective collection (n=257)
norovirus:
Simplex RT-PCR – 17 (6.6%)
Multiplex RT-PCR – 17 (6.6%)

IDEIA Astrovirus and norovirus genogroup I and II, Dako, Germany. Acute gastroenteritis defined as ≥ 1 episode of diarrhea (watery or loose stools in a 24 hour period), with vomiting and/or other symptoms (fever, nausea, abdominal pain, and/or cramps). 668_IL
Schmid, M; 2004 139 Diagnostic Study
 
1,2
To evaluate a real-time RT PCR assay on the LightCycler (LC) with SYBR Green detection and melting curve analysis (Tm) compared to RIDASCREEN. 52 stool samples from Germany between January-April 2003: 38 from patients in gastroenteritis outbreaks 14 single sporadic
cases in children <5 years of age 13.1% were < 10 years of age, 39.5% between 1060
years, and 47.4% were > 60 years old

Positive cases
Antigen ELISA – 18/52 (34.6%) samples positive
Real-time PCR and nPCR – 26/52 (50%) samples positive 

Agreement between real-time PCR, antigen ELISA, and nPCR
Positive by all three tests – 9
Negative by all three tests – 17
Positive by real-time PCR and nPCR but negative by ELISA – 17
Positive by ELISA but negative by real-time PCR and nPCR – 9

100% correlation between real-time PCR and nPCR.

Test characteristics compared to nested PCR
ELISA – sensitivity 9/26 (34.6%) and specificity 17/26 (65.3%)
Real-time PCR – sensitivity 26/26 (100%) and specificity 26/26 (100%)

Difference in sensitivity between ELISA and real-time PCR (34.6% vs. 100%; p<0.001)

PCR-based procedures are more sensitive and specific than antigen ELISA.
RIDASCREEN Norwalk-like virus kit (R-Biopharm, Darmstadt, Germany) and well-established nested PCR used as reference standards. 655_IL
Vinje J, 2003 140 Diagnostic Study
 
1,2
To evaluate the performance of 5 different RT-PCR assays for the detection of norovirus in an international collaborative study.

5 laboratories in 5 countries in the European consortium tested stool specimens collected over a 4 year period (1997 to 2000) from both outbreaks and sporadic cases of gastroenteritis and had previously been tested by RT-PCR and EM.

91 stool samples – 82 norovirus positive and 9 controls

Overall characteristics
Norovirus detected by at least 1 RT-PCR assay in 69 (84%) of the samples that originally tested positive.
Overall sensitivity: 52-73% overall
Overall sensitivity by genotype: 54-100% for genogroup I vs. 58-85% for genogroup II
Overall sensitivity by test: p1 67% vs. p2 59% vs. p5 52% vs. p6 73% vs. p13 60%

64% of false-negative results in a set of diluted stools (n=20) that may have lost quality upon storage. Sensitivity improved when these samples were excluded.
No single assay was best although the p1 assay demonstrated the most satisfactory overall performance.

Sensitivity  by genotype
GI genotype: p1 100%, p2 54%, p5 85%, p6 92%, p13 85%
GII genotype: p1 75%, p2 75%, p5 58%, p6 85%, p13 69%

PCR assays: Laboratory p1 use primer pair JV12-JV13
Laboratory  p2 use NVp110 followe by PCR with the primers NVp110, Ni, an NVp69
Laboratory p5 used two RT-PCR assays with E3-Ni an E3-Ando primer pairs respectively
Laboratory p6 use nested RT-PCR assay format

Laboratory p13 use single tube RT-PCR targeting the 3’ en of ORF1 (region B)
IL_836
Tatsumi, M; 2002 141 Diagnostic Study
 
1,2
To determine the sensitivity and specificity of RT-PCR-ELISA for detecting Norwalk virus when compared with conventional PCR                                                              Children aged 2 months to 14 years (mean age 28.7 months) admitted with acute gastroenteritis. Study was conducted in Japan. 93 children; 154 stool samples

Test characteristics
All 46 stool specimens that were positive for viruses other than Norwalk by RT-PCR-Southern hybridization were identified as such by RT-PCR-ELISA

All 30 stool specimens that were positive for Norwalk virus by RT-PCR-Southern hybridization were identified as such by RT-PCR-ELISA

In terms of detection limits, the sensitivity of RT-PCR-ELISA was the same as that of conventional PCR with Southern hybridization and was 10-100 times more sensitive than the conventional PCR.

In 93 other stool specimens from hospitalized patients, 20% samples were found to be positive with RT-PCR-ELISA and 13% were found to be positive with conventional PCR.
Power and sample size not reported 911_RA
O’Neill, H; 2001 142 Diagnostic Study
 
1

To assess the use of nRT-PCR in detecting norovirus

31 outbreaks in various settings including nursing homes, small district hospitals, large general hospitals, a ferry ship, hotels, restaurants and staff canteens. Study was conducted in the UK.

Total N not reported

Number of samples positive for norovirus (follow-up not reported)
All results number positive/number tested; percentage positive
Ferry ship – 8/10; 80 (All 10 specimens negative for virus by EM)
Country hotel – 14/17; 82 (2 positive by EM)
Nursery school – 7/12; 50
City hotel – 3/3; 100
Restaurant – 8/32; 25
Restaurant – 7/7; 100
Large hospital – 14/116; 12
Psychiatric hospital – 27/35; 77
Restaurant – 5/5; 100
Large hospital – 16/58; 27
Medical ward – 9/17; 53
District hospital – 8/32; 25
Medical ward – 3/5; 60
Nursing home – 2/2; 100
Nursing home – 2/2; 100
Large Hospital – 7/37; 19
District hospital – 2/2; 100
Care of elderly ward – 9/12; 75
Nursing home – 2/5; 40
Hotel – 8/10; 80
Hotel – 6/12; 50
Large area hospital – 12/67; 18
Hotal – 3/3; 100
Regimental reunion – 9/11; 82
Leisure center – 4/6 - 66

Power and sample size not reported

Simultaneous testing with EM was done only for the first two outbreaks
983_RA

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