SHORT COMMUNICATION Scand J Infect Dis 29: 415-418, 1997

Waterborne Outbreak of Viral Gastroenteritis MARJA KUKKULA', PERTTI ARSTILA2, MARJA-LIISA KLOSSNER3, LEENA MAUNULA4, CARL-HENRIK V. BONSDORFF4 and PEKKA JAATINENS From the 'National Public Health Institute, Kuopio, the 2Department of Virology, University of Turku, Turku, the "Central Hospital of Satakunta, Pori, the 4Department of Virology, University oJ' Helsinki, and 5Health Care Centre, Noormarkku, Finland

A waterborne epidemic took place in a Finnish municipality in April 1994. Some 1500-3000 people, i.e. 25-50% of the population, had symptomatic acute gastroenteritis. Laboratory findings confirmed adenovirus, a Norwalk-like agent, small round viruses (SRV), and group A and C rotaviruses as causative agents, Norwalk virus being the main cause of the outbreak. The epidemic was most probably associated with contaminated drinking water. The groundwater well, situated in the embankment of a river, was contaminated by polluted river water during the spring flood. A back flow from the river to the well had occurred via a forgotten drainage pipe.

M. Kukkula, DVM, National Public Health Institute, P.O. Box 95, FIN-70701 Kuopio, Finland

INTRODUCTION

The viruses most often found in outbreaks of gastrointesti- nal infections include rotaviruses, adenovirus types 40 and 41, astrovirus and caliciviruses, i.e. Norwalk- and Norwalk- like viruses, small round viruses (SRV) and small-structured round viruses (SSRV). Rota- and adenoviruses are easily diagnosed by ordinary immunoassay (1). Diagnosis of other viruses is based on electron microscopy of stool samples, which has limited ou r knowledge of their true significance.

The vast majority of drinking water-associated outbreaks is of unknown aetiology (2). In Finland, 24 reported water- borne epidemics have occurred between 1980 and 1992 (3). A viral aetiology was found in 17% of epidemics, Campy- lobacterium jejuni in 13% and Salmonella typhimurium in 8%. However, in more than half of the outbreaks the aetiology was unknown (58%). Most of the epidemics oc- curred in countryside villages, holiday and sport resorts, hospitals and garrisons which had their own water works with insufficient water treatment. Disinfection is used in only 10% of Finnish groundwater supplies.

MATERIALS AND METHODS Geographical and demographical description of the community Noormarkku is a municipality with 6300 inhabitants in Southwest- ern Finland. About 80% of the population (5000) live in the village and are served by a municipal water supply. There are 760 schoolchildren in the 4 schools of the village.

The Noormarkku River flows through the village. Upriver on the watershed lies agricultural land and four other municipalities. The waste-water works of each municipality discharge treated, non-disinfected sewage into the river.

Outbreak The first patients became ill on April 11, 1994. The next day, another 37 persons contacted the health care centre reporting symptoms of gastroenteritis. Typical symptoms were abdominal pain, severe vomiting, in some cases high fever, headache, and diarrhoea. The duration of symptoms was usually 2-3 days. A few patients suffered from prolonged diarrhoea. Horizontal spreading (secondary infections) was also observed among those who had not

used the contaminated water. About 1000 people became ill during the first week: school children first, then adults and the elderly. School absence (Fig. 1) during the first week varied from 8 to 31%, compared to the annual mean of 4.5%. The incidence rate in a home for the elderly was 90%. The epidemic was over in 3 weeks. About half of the population, i.e. up to 2500 people, was estimated to have had symptomatic gastroenteritis.

In routine bacteriological examinations, coliform bacteria were found both in raw water and in tap water indicating faecal contamination. Efforts to restrict the outbreak included, among others, informing the population, boiling drinking water and shock chlorination (freely available chlorine 10 mg/l) of the water pipes. Clean drinking water was delivered by fire trucks.

Microbiology For bacteriological analysis, stool samples were obtained from 35 acute cases with gastrointestinal symptoms. The median age of these cases was 37 years; 4 of them were children (1-12 years) and 5 elderly persons (78-87 years). The specimens were cultured for enteropathogenic E. coli, E. coli, Salmonella, Shigella, Campy- lobacter and Yersinia. They were also tested for Giardia and Cryptosporidium.

A standard solid-phase enzyme immunoassay (EIA) was applied for rota-and adenoviruses using polyclonal anti-adenovirus and anti-rotavirus antisera (I). The rotavirus-EIA finds group A-ro- taviruses, but is unable to detect groups B and C.

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Fig. 1. Daily school absence among 760 children, The annual mean is 4.5%.

0 1997 Scandinavian University Press. ISSN 0036-5548

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416 M. Kukkula et al. Scand J Infect Dis 29

For electron microscopy (EM) 19 stool samples were available. Viruses were identified from faecal suspensions on grids negatively stained with potassium phosphotungstate using Phillips 410 and Jeol microscopes without any concentration step. Three samples positive in EM but negative in EIA were submitted to RNA analysis by homogenous 7.5% polyacrylamide gel electrophoresis (PAGE) (4) and stained by silver staining (5).

Seven paired sera were investigated by in-house group A and C rotavirus antibody assays. Simian group A Rotavirus SA-I 1 grown in MA-104 cells and porcine group C rotavirus AmC-1 grown in swine testicular (ST) cell cultures were used as antigens. The indirect EIA-Assay was performed by first coating the microplates with rabbit hyperimmune serum aginst either group A or group C rotavirus.

The same sera were also tested against Norwalk virus antibodies by ELISA with baculovirus-expressed Norwalk particles as antigen (6) (kind gift of Dr Mary Estes).

Water samples were tested bacteriologically for coliforms, E. coli, Salmonella, Campylobacterium and Yersinia. One water sam- ple was analysed virologically by using concentration, electron microscopy and cell cultures (GMK, A549 and RD cell lines). A sample of 20 1 tap water was first concentrated with Minitan-sys- tem (Millipore), continued with a two-phase concentration.

RESULTS

Examination of water system and water quality Drinking water supply of the village came from 2 ground- water wells, situated on a river embankment. The only treatment was adjustment of pH with sodium hydroxide. There was no protection zone around the wells. Microbio- logical quality (coliforms as indicator) of the water samples had been good until the epidemic.

In the winter of 1994 there was twice as much snow as usual in Finland. Rapid melting of snow started in the beginning of April. An ice dam was formed just downriver from the groundwater wells. A record high flood enabled a back flow of the river water into 1 of the wells via a drainage pipe between the well and the river, the existence of which had been forgotten.

During the spring flood the river water was contaminated from 2 probable sources: the capacity of waste-water works, situated upriver from the wells, was overloaded, and about half of the municipal waste-water had to be dis- charged untreated. In addition, manure on the fields in the watershed area flooded to the river with the run-off water.

Microbiological findings No bacteriological pathogen was found in water or human samples with the exception of a Yersinia enterocolitica-pos- itive stool specimen (Table I). Specimens from 35 cases were tested by EIA for rota- and adenovirus antigen. Two adenovirus- and 4 rotavirus-positive specimens were found. In addition, 3 rotavirus-positive samples were seen by EM. One of these EIA - /EM + specimens had an RNA pattern typical of group C rotavirus in PAGE analysis.

SRV were detected by EM in 5 cases. One of these patients also showed a diagnostic rise in IgG antibody titres against Norwalk virus. One more diagnostic rise in IgG

Table I. Results from microbiological analyses of stool and serum samples

Positive findings Microbe No. of samples No. (%)

EPEC 8 0 0 Salmonella 33 0 0 Shigella 33 0 0

0 0 Campylobacter 33 Yersinia 33 1 3 Cryptosporidium 14 0 0

0 0 Giardia 14 2 6 Adenovirus 35

Rotavirus 35 7 20 Small round viruses 19 5 26 Norwalk (serum pairs) 7 2 29

antibody titres against Norwalk was found among the 7 patients tested in serology, but none against rotavirus. The positive findings are summarized in Table I.

Examination of river, as well as tap water, showed E. coli (10- 160 CFU/l) and other coliforms indicating faecal con- tamination of the water source. Analyses for Salmonella, Campylobacterium and Yersinia were negative. No virus was detected in the water sample.

DISCUSSION

The epidemic was most probably associated with contami- nated tap water. This conclusion is based on the clinical findings, the descriptive epidemiology and the fact of water quality failure (7). The epidemic had multiple aetiologies, as shown in Table I. Although Norwalk viruses or Nonvalk- like viruses were indicated only in 5/24 cases, it can be speculated that they were the main culprit in the outbreak. The epidemic fulfils mainly the Kaplan criteria for Nor- walk-like virus infections: stool cultures negative for bacte- rial pathogens, mean duration of illness 12-60 h, vomiting in 2 50% of patients, and mean incubation period of 24-48 h, if known (8). It has been shown that a single infected person may transmit the virus to thousands of other indi- viduals (9).

Interesting was the discovery of group C rotavirus in a 12-year-old patient. Retrospectively, the cases with ro- tavirus (including group c ) were among those with an anomalous course of illness with prolonged diarrhoea. Con- trary to the laboratory confirmation of calici viruses, the diagnosis of rota- and adenoviruses is easy, and one would expect higher proportions of positive findings in this mate- rial if they were the main aetiological agents.

Laboratory comfirmation of the aetiological agent of viral waterborne outbreaks has been very limited until now. Nowadays, calici viruses have been detected as a causative agent in many food or waterborne outbreaks by RT-PCR methods. When the samples of this epidemic were analysed,

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Scand J Infect Dis 29 Waterborne viral gastroenteritis 417

however, no calici virus PCR mehods (10, 11) were yet available in our laboratory. Antigen assays like rNV (12, 13) and rMX EIAs (14) are also reported to be sensitive, but they are very genogroup-specific and are not commercially available. The PCR-based detection methods for adenoviruses in water samples have also advanced (1 5). Concentration of virus particles from larger water samples is still an inadequately solved problem.

The significance of viruses as the cause of gastroenteritis is often underestimated by physicians and environmental health inspectors because specimens are not routinely taken for virological investigations. However, the absence of bac- terial pathogens or indicators does not ensure the absence of viruses.

In addition, the significance of drinking water as the vehicle of transmission of infectious diseases has histori- cally been underestimated. For example, as late as in 1940 poliovirus was believed to be transmitted via the nasal route (16). Some Canadian researchers have recently claimed, based on case-control trials, that one-third of sporadic gastroenteritis cases may be caused by treated drinking water which meets bacteriological quality stan- dards, even though the raw water has been polluted by faecal bacteria (1 7).

The presence of human enteric viruses in surface or ground water is always a result of human faecal pollution. The concentration of enteric viruses in sewage is decreased by waste-water treatments. Reduction varies according to the used treatment processes (18, 19), but without disinfec- tion, a proportion of viruses always survives. Viruses in sewage effluent have been reviewed for instance, by Feachem et al. (20) and Rao et al. (21). Certain coxsack- ievirus and echovirus serotypes have been the most com- mon enterovirus isolates in regular screening of Finnish sewage specimens (22).

The cold winter climate of Scandinavia enables the sur- vival of enterovirus in river water. In Alaska, it has been demonstrated that 34% of enteric viruses survived in an ice-covered river flowing 317 km in 7 days (23). En- teroviruses can survive in the soil at 4°C for at least 180 days (24).

It is questionable if current drinking water standards and monitoring methods ensure safe drinking water. The regu- lar monitoring program at Noormarkku water works in 1994 was in accordance with the decision of the Finnish Ministry of Social Affairs and Health (25), which imple- ments the Directive of the European Economic Community (80/778/EEC). The monitoring, however, failed to detect the pollution until the epidemic was a fact.

ACKNOWLEDGEMENTS The authors thank Drs Matti Jahkola and Mirja Stenvik, National Public Health Institute, Helsinki, for virological analyses of water samples.

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Submitted January 3, 1997; accepted May 23, 1997

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