B11008: The serodiagnosis of infections caused by Verocytotoxin-producing Escherichia coli based on salivary antibodies to E. coli lipopolysaccharide (LPS)

Study Duration: June 2001 to September 2003

Contractor: Health Protection Agency (HPA)

The project relates to human disease caused by a form of enteric bacterium called Escherichia coli (E. coli) which produces a poison (toxin) called 'Verocytotoxin' and these Verocytotoxin-producing E. coli make up a family of bacteria termed - VTEC. Strains of E. coli can be 'typed' based on various bacterial properties and one particular form of VTEC, called O157 (E. coli O157), has been shown to be a cause of serious illness with symptoms including haemolytic uraemic syndrome (HUS). People are diagnosed as being infected with E. coli O157 or other VTEC following the culture of these bacteria from patients' faeces. In the absence of faecal VTEC, a precise diagnosis cannot be made.

Patients infected with E. coli O157 respond to infection by activating their immune system. One of the responses is the production of molecules called antibodies, which appear in the patients' blood during disease and may persist for several months. These antibody molecules are specifically designed to attach to the E. coli O157 causing them to clump together, making it easier for the immune system to clear these bacteria from the body.

These antibody molecules are highly specific for E. coli O157 and can be used to provide evidence that a person has been infected with E. coli O157, by screening the patient's blood for antibodies which bind to the E. coli O157. This procedure has been shown to be highly sensitive and forms the basis of a routine laboratory test. Infections with E. coli O157 occur more frequently in young children and infants, and with the aim of avoiding the need to take a blood sample from these patients, other body fluids were considered as a source of antibodies. Initial studies have demonstrated that a patient's saliva may also contain antibody molecules that bind to E. coli O157, but the incidence of these antibodies and the levels at which they occur during disease are not known. The present project was designed to obtain a better understanding of these salivary antibodies, which bind to E. coli O157 and VTEC, and how they can be used for diagnosis of infections caused by these bacteria.

The aim of the project was to investigate the possibility of using samples of saliva and avoid the need to use samples of blood. A commercial sampling kit (Oracol&#169) was used to obtain saliva from patients, and involved a sponge on a stick placed within a plastic transport tube. Antibodies to E. coli O157 were detected in patients' saliva; however, the levels of these antibodies and how long they persist in a patient are unknown. It was hoped that the project would provide information about the sensitivity and specificity of the antibody tests. Due to the ease of sampling a patient, it was also intended to provide knowledge of when the levels of salivary antibodies were at their highest during the disease process, so that the times when patients should be sampled can be optimised.

By routine E. coli O157 antibody testing using samples of blood, patients can be identified as having antibodies to E. coli O157. These patients were contacted and supplied with saliva sampling kits and requested to submit samples of saliva for research purposes. In anticipation of putative cases of infection with O157, the ten laboratories, which had the highest incidence of antibody-positive patients for a given year, were targeted and equipped with saliva sampling kits and documentation enabling patients/parents to provide their permission for their samples of saliva to be used for the study.

Between 1st June 2001 and 1st September 2003, the Health Protection Agency, Laboratory of Enteric Pathogens (LEP) received 668 samples of blood. Of these, 235 were found to contain antibodies binding to E. coli O157 lipopolysaccharide (LPS) and samples of saliva were requested. Fifty-four samples of saliva were received and 15 were found to contain antibodies to E. coli O157. In addition to these, 27 samples of saliva and 23 blood samples were received from 33 patients involved in an outbreak of E. coli O157 infection. A further 22 samples of saliva only were received from a suspected outbreak of E. coli O157 infection.

To establish the sensitivity of antibody tests, it was essential to acquire samples of saliva from healthy people, and in particular young children. A collaborative study with the Science Museum resulted in the LEP acquiring 425 samples of saliva from children under 11 years of age. Furthermore, 52 samples were obtained from staff at the Health Protection Agency, forming a valuable collection of control saliva.

The study identified 44 patients infected with E. coli who had provided both saliva and blood samples. These were used to determine the incidence of salivary antibodies to E. coli O157. Of the 44 patients with antibodies to E. coli O157 in their blood, 20 (45.5%) also had salivary antibodies binding to this organism. The remaining 24 patients did not have detectable levels of salivary antibodies to E. coli O157. None of the 425 healthy children nor the 52 adult volunteers had salivary antibodies to E. coli O157, demonstrating that the tests being used in this study were unlikely to produce false-positive results.

To determine the stability of salivary antibodies to E. coli O157, when stored on the sampling swabs, normal human saliva was 'spiked' with serum known to contain antibodies to O157 LPS.

The patients' antibodies added to healthy saliva were stable at 4°C for up to 4 weeks. Storage at room temperature resulted in a decline in the levels of antibodies, but sufficient antibodies remained to give a positive reaction when tested. In contrast, antibodies in saliva were comparatively unstable when kits were maintained at 37°C, with antibodies disappearing after 1 week.

From the study it was concluded that patients infected with E. coli O157, may have salivary antibodies to these organisms. The commercial sampling kits were shown to be suitable for collecting samples of saliva and that antibodies in saliva were stable during transport to the testing laboratory. The tests employed were able to detect antibodies in saliva.

Final report is available from the FSA Library and Information centre. To obtain a copy, please contact the Enquiry Desk, Dr Elsie Widdowson Library and Information Services, Food Standards Agency ( Tel: 020 7276 8181/8182 or by e-mail to: library&info@foodstandards.gsi.gov.uk ).

Contact: Email: science@foodstandards.gsi.gov.uk