Validation of nRT-PCR

The 14 known NLV positive specimens associated with 4 different outbreaks of NLV infection were detected by nRT-PCR. Eight were first and second round positive and 6 were second round positive. The 4 adult specimens known to be NLV RT-PCR negative (Central Public Health Laboratory, Colindale, and London) and the 4 specimens from children with rotavirus infection were confirmed negative by nRT-PCR.

Virological investigation of non-outbreak specimens

Faecal specimens from 532 adult hospitalised patients who were not involved in outbreak situations were investigated at the same time as specimens from outbreak situations. These were included and used as protocol controls. Of these 530 were negative by nRT-PCR and 2 were repeatedly positive.

Virological investigation of outbreaks

Outbreak 1 was associated with a roll-on-roll-off ferry ship. Vomiting and diarrhoea affected more than 14 crew out of a total of 53 over a period of several days. No passengers were affected. Eight of 10 faecal specimens were positive for NLV by nRT-PCR. All 10 specimens were negative for viruses by EM. An environmental inspection of the ship revealed no concerns about food preparation and handling but identified poor sanitation in the crew facilities and sleeping quarters.

Outbreak 2 was in a country hotel and involved staff and the guests of 5 wedding functions that had taken place over a 6-day period. Vomiting and diarrhoea affected individuals with an onset within 72 hours of their meal. The case attack rate varied from 8.3% to 52% depending on the function attended. In total at least 172 guests and 14 staff were affected giving an overall attack rate of 33%. We tested 17 specimens from symptomatic staff and guests. Fourteen were positive by nRT-PCR and 2 were positive by EM.

Outbreak 3 involved 23 children and 2 staff of a nursery school. The index case was identified as a child who had been ill at home 24 hours before vomiting in the nursery school sandpit. We received specimens from 12 children with 7 testing positive.

Outbreak 4 involved 23 cases of vomiting and diarrhoea among guests and staff of a city hotel. On investigation a member of staff had vomited in the hotel kitchen during the preparation of the meals. Three catering staff with symptoms were tested by nRT-PCR and they were all positive.

Outbreak 6 which occurred in a restaurant also involved a member of the kitchen staff being taken ill during the preparation of the meals. Seven cases of vomiting and diarrhoea among diners and staff were reported and investigated. All 7 were positive by nRT-PCR for NLV.

Outbreak 7 took place in a large hospital. The outbreak was reported to have affected 70 patients and staff from 4 different ward areas (maternity, medical, elderly and surgical). The hospital was closed to all new admissions. During the first nine days of the outbreak 58 specimens were examined and 14 were positive (from 13 patients). The perception in the hospital was that the outbreak continued for a further 14 days. During this time we tested 58 specimens which were all negative.

Outbreak 10 was spread over 4 wards of a large hospital. There were 16 cases positive out of 58 tested during a 3 week period of March 1999. Although the last positive case in this outbreak was on March 29 and the outbreak was declared over in early April we continued to receive specimens from this hospital for the investigation of gastroenteritis. Over the next 9 months of 1999 we received and tested 171 specimens from patients in this hospital. These additional specimens were all negative.

Outbreak 11 was confined to a medical ward in a district general hospital and involved 21 patients and 9 staff. There symptoms were vomiting and diarrhoea which affected them over the course of an 8 day period. We tested 14 patients with 8 positive and 3 members of staff with one positive. The index case in this outbreak was almost certainly infected in the community before entering hospital. Two of the patients affected, including the index case, reported similar illness among family members.

Outbreak 15 occurred in a nursing home for elderly adults where 28 of the 33 residents and 7 members of staff were affected by vomiting and diarrhoea. We received and tested only 2 specimens from the outbreak and both were positive.

Outbreak 17 spread to 4 wards over a 3 week period during which we tested specimens from 39 patients with 9 positive. The perception in the hospital was that the outbreak continued for a further 4 weeks with diarrhoea being the most frequent symptom. We tested a further 39 specimens in this period and all were negative. A further outbreak occurred in this hospital 2 months later (outbreak 22).

Outbreak 20 developed 2 days after the index case became ill with nausea, vomiting and diarrhoea. In total 13 patients and 3 staff were affected giving a 63% attack rate. We tested 5 specimens and 2 were positive.

Outbreak 27 involved 485 hotel guests and their families over the new year holiday period. The hotel guests were responsible for a large number of secondary cases among family members when they returned home after the holiday. Twelve specimens from hotel guests were submitted for virological investigations and 6 were positive.

Outbreak 28 occurred in one wing of a large hospital. Forty-two specimens were tested over a 6 day period with 12 positives. A further 25 specimens were then tested over the following 9 days and all were negative. The outbreak came to our attention via a telephone call from an infection control doctor on a Friday afternoon. Approximately 24 staff and patients were affected with vomiting and diarrhoea and the index case appeared to be a patient who was admitted 3 days previously and had vomited profusely in the ward. This index case was subsequently shown to be positive. We agreed to test 8 samples on call on Friday evening. This involved specimen extraction and first round RT-PCR on Friday evening with second round nested PCR and gel electrophoresis on Saturday morning. Six of the 8 specimens were positive. On the Saturday morning cases of diarrhoea were reported in the main hospital block and for optimal management of the outbreak it was agreed to test 4 specimens from representative cases within the main hospital block. These were set up as before with the final result available on Sunday. All 4 proved negative and on the basis of these results it was decided not to close any of the wards in the main hospital block.

Outbreak 30 involved 47 identified cases out of approximately 750 people who attended several functions at a regimental reunion which was held over a weekend. We investigated 11 specimens, 9 of which were positive. The first case occurred within 16 hours of the suspected meal and the last identified case occurred 48 hours later.

Outbreak 31 involved 3 groups of teenagers who were attending a residential leisure centre specialising in sports activities. Out of 94 teenagers present 61 had symptoms of vomiting and diarrhoea giving an attack rate of 65%. Six specimens were investigated virologically for nRT-PCR and 4 were positive. An index case could not be identified but common to all 3 groups was that they were engaged in water sports activities in seawater. The seawater subsequently was shown to be contaminated with raw sewage.

Specimens and patients

Faecal specimens were received at the Regional Virus Laboratory from patients with gastroenteritis. The specimens were prepared in 10% suspensions in Eagles medium with Earles salts, centrifuged at 4,000g for 10 min and the clear supernatant either stored at -20°C or tested before freezing. For nRT-PCR the specimens were further prepared using the QIAGEN DNA Blood extraction kit. This kit is the standard extraction method for PCR and RT-PCR in this laboratory and it copurifies RNA and DNA. The QIAGEN protocol was adapted to use a vacuum manifold developed in house and RNA was eluted from the spin columns using 50μl of nuclease free water. The extracts were tested by nRT-PCR immediately or stored at -20°C until tested the following day.

Nested multiplex RT-PCR (nRT-PCR)

The nRT-PCR was developed after evaluating various combinations of published primers against EM positive faeces from outbreaks of gastroenteritis associated with NLV infection [3, 17, 18]. The primers were specific for the polymerase gene of NLV. The primers selected were chosen for their broad range specificity and sensitivity against NLV and are listed in Table 1. The outer primer set yielded a product of 333 bp and the nested inner pair yielded a product of 114 bp.

The nRT-PCR was developed using Access RT-PCR (Promega) for the first round and conventional PCR for the second round in 10 μl volumes. The nRT-PCR was systematically optimised with respect to primer and magnesium ion concentrations in the presence of 0.25 mM cresol red and 15% sucrose. The latter were added to facilitate direct gel loading. The optimum primer and magnesium ion concentrations for first and second round are given in the respective mastermixes below. The reverse transcription step in the first round was also optimised. It was found that 48°C/10 min were sufficient for this step in the protocol. The optimum annealing temperature for both first and second rounds was found to be a broad temperature band within the range 30°C to 54°C. An annealing temperature of 37°C was selected which is similar to that used by others for NLV RT-PCR.

For first round nRT-PCR 2 μl of extracted sample RNA was added to 8μl of master-mix to give a final volume of 10 μl. The first round final 10 μl volume contained Access RT-PCR buffer (Promega), 0.6 μM outer primers A and B, 1.5 mM MgS04, 1 unit AMV, 1 unit Tfl, 0.2 mM NTP, 0.25 mM cresol red and 15% sucrose. The nRT-PCR first round cycling conditions were as follows; 48°C/10 min, 94°C/3 min followed by 35 cycles of 94°C/30 s, 37°C/30 s and 72°C/30 s. This was followed by 72°C 5 min and finally a hold step at 20°C.

For second round PCR 0.2 μl of first round product was added to 9.8μl of second round mastermix to give a final volume of 10 μl. The second round contained Tris buffer, 0.25 units TAQ in storage buffer B (Promega), 0.2 mM NTP, 3.5 mM MgCl₂, 0.2 μM nested primers C and D, 0.25 mM cresol red and 15% sucrose. The second round PCR cycling conditions were as follows; 94°C/2 min followed by 25 cycles of 94°C/30 s, 37°C/30 s and 72°C/30 s. This was followed by 72°C 5 min and finally a hold step at 20°C.

Mastermixes for first and second round nRT-PCR were made up in batches, labelled with a unique identifier, aliquoted and stored at -70°C. For use an aliquot of mastermix was thawed and appropriate volumes were dispensed into PCR tubes.

The nRT-PCR tests were routinely run on either GeneAmp 2400 and 9700 thermal cyclers. Both first and second round products were run on 2.5% agarose gels in TAE buffer for 45 mins in a 15 cm gel at 75 volts. The gels were stained with ethidium bromide (1 μg/ml) for 30 min and photographed using a Polaroid land camera.

Validation and application of nRT-PCR

The nRT-PCR was developed and validated using a panel of 14 faecal specimens, which were positive for NLV by EM. They were representative specimens from 4 different outbreaks, which had been collected over a 3-year period. Eight faecal specimens were also tested as negative controls. Four of these were from adults known to be NLV RT-PCR negative (Central Public Health Laboratory, Colindale, and London) and 4 were from children with rotavirus infection. The nRT-PCR was used in parallel with EM on clinical specimens from the first 2 outbreaks of gastroenteritis associated with NLV infection. Thereafter it was used without comparison with EM.

Electron microscopy

Two millilitre volumes of 10% faecal specimens were centrifuged at 40,000 rpm for one hour and the resulting pellet was resuspended in 25 μl distilled water. The suspension was then adsorbed onto a glow discharged formvar coated EM grid for a minimum of 3 hours and stained with 2% methylamine tungstate for 10 s. The grid was viewed in a Phillips CM 10 EM at a magnification of 45,000 for approximately 10 min.