Laboratory Procedures

Risk assessment

Francisella tularensis, particularly type A, is an extremely hazardous pathogen and has been associated with severe infection in exposed laboratory personnel. Although currently regarded as an ACDP category 3 pathogen and covered by existing risk assessments, extreme caution should be exercised when handling such organisms in diagnostic laboratories. Any exposure through leakage or breakage of clinical specimens, from patients known or thought to have tularemia, should be reported immediately to the laboratory safety officer, infection control team and occupation health physician so that a risk assessment can be performed and appropriate action taken.

Receipt of samples

All clinical specimens from patients known or thought to have tularemia should be labelled 'High risk' by the submitting staff and further work, including unpacking, conducted in a properly maintained Class I protective safety cabinet within a containment level 3 facility.

Protection of laboratory staff

All laboratory procedures must be performed in a Containment Level 3 facility using a Class I biological safety cabinet. Under there circumstances there is no indication for antibiotic prophylaxis for laboratory staff unless there is an inoculation injury or spillage releasing aerosols containin the organism.

Any member of laboratory staff, working with specimens or culures of F. tularensis who develops a febrile/ respiratory illness, shouls seek urgent medical attention.

Isolation and identification

In the event of there being a credible risk of tularemia arising from a deliberate release it is recommended that a senior member of the clinical or laboratory staff contact one of the national specialists to discuss processing of specimens, along with appropriate Consultants in Communicable Disease Control and the duty doctor at HPA CfI. The handling of material that is likely to contain viable F. tularensis by inexperienced personnel is strongly discouraged.

Culture

F. tularensis type A is a small (0.2x0.2-0.7µm) encapsulated, pleomorphic Gram-negative bacterium. It can be readily distinguished from the Gram-positive rod Bacillus anthracis and does not show the characteristic bipolar staining of Yersinia pestis. It is a fastidious organism requiring cysteine-enriched media such as cysteine-glucose blood agar, or BCYE Legionella medium, for growth. Positive blood cultures showing small Gram-negative bacilli that fail to grow on conventional media should be subcultured to such media. Small, 1-2mm grey-white colonies, appear after 24-72 hours in CO₂ enriched air at 37ºC although growth may be delayed and cultures should be held for at least 10 days before discarding. (Click here for laboratory images )

The organism is non-motile, is a slow catalase producer, oxidase negative, H₂S positive and produces acid but not gas from glucose, maltose and mannose. It can be differentiated from other Gram-negative organisms except Legionella spp due to cysteine dependence. Legionella spp are motile and do not produce H₂S. Attempts to isolate F. tularensis strains should be avoided in the absence of adequate containment facilities.

F. tularensis type B grows poorly on primary culture and is best isolated by subcutaneous injection into white mice.

Antibiotic sensitivity

This should be performed by a laboratory experienced in handling F. tularensis.

Serology

Serodiagnosis of tularemia can be performed using ELISA. A significant rise in antibody titre demonstrated on samples of acute and convalescent sera (2-4 weeks later) offer a confirmatory diagnosis of tularemia. This would be unlikely to provide useful information for initial management of an outbreak although a single high titre offers a presumptive diagnosis in a patient who has not been previously vaccinated (CDC criteria).

Molecular methods

Primers specific for F. tularensis have been developed and PCR may offer the best means of rapid diagnosis in samples from patients with suspected tularemia. Specimen collection and transport should be discussed with the reference laboratory (see below).

Confirmation

This should be performed at laboratory experienced in handling F. tularensis.

Reference laboratory

All positive isolates and cultures should be sent to the reference laboratory for confirmation. In addition, samples may be sent there directly if local laboratories lack the facilities for dealing with them. All samples and cultures must be packaged appropriately taking care to observe the procedures outlined in transport of clinical specimens. The sender's name and address should be clearly marked. The reference laboratory must be telephoned prior to sending to expect the sample. Samples should be forwarded urgently to:

Dr Tim Brooks
HPA Centre for Emergency Preparedness and Response
Special Pathogens Reference Unit

Porton Down
Salisbury
Wiltshire SP4 0JG

Tel: 01980 612100 (24 hours)

 

Click here for further information on SPRU and referal of specimens and samples

Waste disposal

In the laboratory, surfaces that have been contaminated with F. tularensis should be disinfected with hypochlorite solution (10,000 ppm available chlorine). Hands should be washed after removing gloves. All waste containers should be autoclaved.

Last reviewed: 9 November 2009