Asymptomatic and Symptomatic Excretion of Noroviruses during a Hospital Outbreak of Gastroenteritis

The genomic diversity of noroviruses includes two genogroups (I and II) (2, 18) and a number of genotypes (8), on which formal agreement has yet to be reached. Representatives of genogroup I genotypes include GI-1 (Norwalk/1968/US), GI-2 (Southampton/1991/UK), GI-3 (Desert Shield/1990/SA), and GI-4 (Valetta/1995/MA), while genogroup II genotypes include GII-1 (Hawaii/1971/US), GII-2 (Melksham/1994/UK), GII-3 (Mexico/1989/MX), and GII-4 (Grimsby/1995/UK). Globally, norovirus outbreaks are caused by different strains of norovirus from genogroups I and II, with the genogroup II strains predominating worldwide (3, 11, 19). GII-4 has been the predominant norovirus genotype circulating worldwide in recent years (8, 9, 11, 17).

In a study of individuals attending general practitioners, 31 of 161 (19%) of the patients with gastroenteritis were positive for caliciviruses (noroviruses and sapoviruses), whereas caliciviruses were not detected in samples collected from control patients (attending general practitioners for other medical reasons) with no symptoms of gastroenteritis (1).

Norwalk virus human volunteer infection studies (4, 5) showed that 41 of 50 (82%) of the volunteers became infected and that, of these, 28 of 41 (68%) were symptomatic, 13 of 41 (32%) were asymptomatic (includes five mild, asymptomatic cases), and 8 of 41 (20%) had no symptoms.

There is little information on the asymptomatic carriage of norovirus in adults or children and on its implication in the transmission of noroviruses from these individuals.

The application of a nested PCR assay (7) to test fecal specimens collected from staff in a pediatric tertiary hospital during a norovirus outbreak in 2002 was undertaken to ascertain if noroviruses were present and to determine the diversity of strains cocirculating in these symptomatic and asymptomatic individuals.

Fecal specimens from 6 patients and 3 members of staff that were symptomatic and 87 members of staff and 12 patients who were asymptomatic between 5 June and 24 July 2002 were tested by single-round and nested reverse transcriptase PCR (RT-PCR) for noroviruses. The staff were all advised and provided samples voluntarily. This was a standard practice for controlling a major outbreak that had resulted in ward closure.

Overall, 40 patients and 59 staff members had gastroenteric symptoms, and 12 wards in the hospital had cases in the patients and staff. The staff members who were ill were not allowed to return to work until symptoms had receded, and only a small number of them were ill at work. Five samples from patients who were symptomatic and in whom enteric viruses other than norovirus were identified at the time of infection and one sample from an asymptomatic patient collected during the 5 months preceding the outbreak were included for comparison.

Single-round RT-PCR was performed by using Ni/E3 (6) and SG1/D1 (10) primers and a nested PCR GI/GII/E3 for the first round and Ni/E3 for the second round, as previously described (7). Standard control measures and unidirectional workflow were used for all nested PCRs with the second-round PCR setup performed in a PCR workstation with UV decontamination. PCR amplicons were cloned and sequenced as previously described (13).

Samples from the five patients with symptoms of gastroenteritis, selected from specimens tested in the 5 months preceding the outbreak, were positive for other enteric viruses, including astroviruses (16), sapoviruses (14), or adenovirus. All were positive for the GII-4 (8) Grimsby/1995/UK strain of norovirus by nested PCR only (strains P-B, P-D, P-F, P-G, and P-K) (Fig. 1). One sample from the asymptomatic patient (P11) was also positive for GII-4 norovirus by nested PCR only.

Specimens from a total of 87 asymptomatic staff members were tested and 23 of 87 (26%) were positive according to the nested Ni/E3 PCR. All were GII-4 noroviruses by polymerase region DNA sequencing (Table 1). A total of 4 of 12 (33%) asymptomatic patients were positive by nested Ni/E3 PCR (Table 1).

Two of the staff (S12 and S15) who had symptoms of gastroenteritis had samples collected on 20 and 21 June; neither was positive for the Harrow/2001/UK strain, but they excreted low concentrations of the GII-4 strain (detected by nested PCR only).

One member of the staff (S32) was positive by single-round PCR with SG1/D1 primers, and a GI norovirus strain, Potsdam196/2000/DE, was identified. Five symptomatic patients (P1, P3, P4, P5 and P9) were positive for the norovirus strain Harrow/2001/UK, which was responsible for the outbreak. The outbreak strain was not detected in a sample collected from patient 2, but two other noroviruses, a GII-7 strain Leeds/1990/UK (18) and a GII-4 strain, were detected.

Four outbreak patient strains (P1, P3, P4, and P5) were shown to be Harrow/Mexico recombinants or rGII-3a. Recombinant strains were verified by sequencing a 425-bp region overlapping the ORF1-ORF2 junction, and strain P4-5/2002/UK has 98% identity to a published recombinant NV strain, Oberhausen455/02/DE (GenBank accession no. AF539440 ). Three staff members and three patients had strains that were identified as GII-4 in the ORF1 region (Fig. 1) but also had Mexico/1989/MX at the 5′ end region of the ORF2 (15), suggesting a Grimsby/1995/UK-Mexico/1989/MX recombinant (rGII-3b). A contiguous ORF1/ORF2 sequence could not be obtained, and therefore these may represent dual infections. One asymptomatic patient (P7-36) and one staff member (S11) had GII-4 strains, as confirmed by the contiguous sequencing of the ORF1/ORF2 regions (12).

During the gastroenteritis outbreak it was hospital policy that asymptomatic staff on duty during the outbreak provide fecal samples for virological screening for enteric viruses.

A GII-4 strain was detected by nested PCR in 26% and 33% of asymptomatic staff members and patients, respectively, during the outbreak, suggesting a low level of excretion of the virus.

Although a GII-4 was identified in one symptomatic patient during this hospital outbreak (and in five patients between January and May), it was detected only by nested PCR, and other strains of noroviruses or other enteric viruses were identified by single-round RT-PCR and were considered to be the primary agent responsible for the gastroenteric symptoms. GII-4 strains were not detected by single-round PCR in any patients or staff, and it was concluded that the low-level excretion of the virus did not result in sporadic cases or any GII-4-related outbreaks of gastroenteritis between January and July 2002.

The detection of asymptomatic carriage of noroviruses in patients and/or staff in the hospital may indicate the susceptibility of the staff of a children's hospital to asymptomatic carriage of a norovirus strain prevalent in the community or may indicate that the strict cross-infection control measures in practice at this hospital worked well. It is possible that low-level excretion of these GII-4 strains did not lead to infection or that people exposed to the virus had a preexisting immunity to these GII-4 strains. This study has demonstrated that asymptomatic excretion of noroviruses circulating widely in the population can occur in hospital staff and patients; however, in this case it was not thought to be the cause of nosocomial infections.