19 November 2007
1 PHLN Summary laboratory definition
1.1.1 Definitive Criteria
- Isolation of Francisella tularensis from wound (ulcer) or aspirates or blood.
1.1.2 Suggestive Criteria
- Detection of F. tularensis by nucleic acid tests (NAT); or
- Gram negative single poorly staining pleomorphic coccobacilli. Characteristic growth on agar plates (cysteine requirement) from patients with clinical features of tularaemia.
Tularaemia is a zoonotic disease caused by the gram-negative coccobacillus Francisella tularensis
, a facultative intracellular pathogen. It was first described in the United States in 1911 and is also known there as “rabbit fever” and “deer fly fever”. Currently four subspecies or biovars are recognized; F. tularensis
(type A formerly known as subsp nearctica
)), F. tularensis
, (type B, formerly known as subsp. paleacrtica) F. tularensis subsp. mediasiatica and F. tularensis
. Two subspecies, type A (F. tularensis
) and type B (F. tularensis
) cause disease in man and have been considered as potential biological weapon agents (3). Type B strains occur across northern Europe and Japan and generally cause mild illness in humans. Type A strains are restricted to defined geographical foci in North America and cause severe disease (3). Neither type A nor B strains have been found naturally in Australia. A strain of Francisella tularensis
, a non-pathogenic species, was recently isolated from a wound in Darwin.
Tularaemia is a debilitating illness, rife amongst wild animals and common in the Rocky Mountains, California, Texas, Oklahoma and Martha’s Vineyard in the US, as well as parts of Eastern Europe (Kosovo), China, Japan, Scandinavia, and Siberia (1, 3, 5). F. tularensis
is a very hardy organism, capable of surviving for weeks and sometimes months in decaying animal corpses (3, 5). Tularaemia is primarily transmitted to humans by ticks, mosquitoes and wild rabbits, although squirrels, sheep, cats, and dogs have also been identified as carriers (3, 5). Whilst tularaemia is a highly infectious disease it is rarely spread directly from person to person (3, 5). Tularaemia is fatal in approximately thirty percent of untreated cases. As a result of its highly infectious nature and its very slow growth patterns, the handling of the organism by laboratory staff represents a considerable risk (particularly in its unidentified state) and in endemic areas F. tularensis
is a significant cause of laboratory-associated infections (2, 3).
Tularaemia characteristically presents as an acute febrile illness. The route of infection plus the host’s response will determine the clinical manifestation of the illness. These may range from an ulcer at the site of cutaneous or mucous membrane inoculation, pharyngitis, ocular lesions, regional lymphadenopathy and pneumonia. Tularaemia is characterised by sudden onset of chills, fever, headache, generalized body aches, coryza, pharyngitis, cough, and chest pain or tightness. Typically the patient will have a temperature of 38–40o
C. The incubation period is 2–10 days. Without treatment, nonspecific symptoms usually persist for several weeks, and sweats, chills, progressive weakness, and weight loss characterise the illness (3).
Antibiotic resistance in wild strains is rare. Streptomycin, followed by gentamicin is the drug of choice (2), but doxycycline or chloramphenicol are acceptable alternatives. Some patients have been successfully treated with ciprofloxacin, but penicillins and cephalosporins are not effective (3, 5).
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The usual mode of infection is through puncture of the skin whilst handling infected material or through the bite of an insect (Dermacentor
ticks are active in summer months) (3). An ulcer will form as the bacteria multiply. From the penetration site the bacteria are transported by the lymphatic system to regional lymph nodes and from there to other sites if the infection is not contained (3). Tularaemia presents in humans primarily as ulceroglandular disease (45–80% of reported cases), as primarily glandular infection (10–25%) and less frequently, as oculoglandular, septic, oropharyngeal and pneumonic forms (3). Despite the existence of a pulmonary form of the disease, human-to-human transmission is unusual (2). Any of the principal forms of tularaemia may be complicated by bacteraemic spread, leading variously to tularaemic pneumonia (common), sepsis (uncommon), and meningitis (rare) (3). Other sources of infection include:
(a) handling infectious animal tissues or fluids,
(b) direct contact or ingestion of contaminated water, food or soil,
(c) inhalation of infective aerosols e.g. handling damp hay.
has been developed as a biological weapon (1). There is evidence that the USSR released a concentrated amount of Francisella tularensis
over German lines near Stalingrad in 1942, resulting in numerous cases on both sides, many of them displaying the pneumonic form of the disease (1). After the war, the former USSR successfully weaponised F. tularensis
and developed multi-drug resistant strains, whilst maintaining or enhancing virulence (1). A biological attack could take the form of aerosol distribution of F. tularensis
(2). Large numbers of pneumonia cases in a short period of time with a massive casualty rate might be the first indication of such an attack. The infectious dose by aerosol is approximately 100 – 500 organisms (1).
3 Laboratory diagnosis/tests
Francisella tularensis is a fastidious bacterium requiring cysteine for growth. It will not grow on MacConkey agar. Whilst it may at first grow on standard sheep blood agar, it will fail to grow on subsequent passage unless the medium has been supplemented with cysteine (5).
3.1.1 MediaFrancisella tularensis
will grow on 5% sheep blood agar, chocolate agar, Thayer Martin agar or BCYE (3, 5). However, as noted above, best growth on blood agar will be obtained on cysteine-supplemented media. Growth will also occur in standard blood culture media. Plates should be firmly taped shut and clearly label to prevent inadvertent opening (3, 5).
3.1.2 Suitable specimens
A. Samples from acutely ill patients
- Blood for culture
- Tissue biopsy or material scraped from an ulcer (swabs are acceptable)
- Aspirates of tissue at site of lesion
NB. Samples should be labelled as “High Risk” and forwarded to a PHLN laboratory for culture. Samples that are transported immediately may be stored at room temperature, but if a delay is anticipated they should be cooled to
B. Samples from post-mortem
- Blood from a vein (if possible)
- Aspirate from lesion
- Scraping from ulcer site
NB. Extensive post-mortem examination is discouraged in cases of suspected tularaemia because of the risk of aerosolising F. tularensis
present in body fluids, drips, etc. (3).
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3.1.3 Test sensitivity
No quantitative data available.
On agar plates supplemented with cysteine (Chocolate agar, Thayer-Martin, BCYE or supplemented SBA), growth will be too small to be seen after 24 hours. At 48 hours, colonies will still be very small (1–2 mm), white to grey to bluish-grey, opaque, flat, with an entire edge, smooth with a shiny surface. Plates may be incubated aerobically or in 5% CO2 (4, 5). However, the growth of this organism is not enhanced by CO2
3.1.4 Test specificity
The slow growth of F. tularensis
and the requirement for cysteine provide clues to the identification of this organism. A stained smear demonstrating poorly staining pleomorphic coccobacilli that stain as single cells should increase suspicion when suitable symptomology is evident (4).
3.1.5 Predictive values
A negative culture does not exclude F. tularensis, especially if there is some doubt over the age and storage in transit of the specimen. A positive culture in the absence of symptoms should be treated with utmost suspicion and should not lead to an automatic diagnosis of tularaemia.
3.1.6 Suitable acceptance criteria
On blood agar, growth is invisible after 24 hours at 37o
C. After 48 hours or longer incubation, colonies range in size from 1–2 mm in diameter, are white to grey to bluish-grey in appearance, and are opaque, flat, with an entire edge and smooth with a shiny surface; colonies are not haemolytic. There is no growth on MacConkey or EMB agar. Subcultures on standard sheep blood agar will fail to grow because of the cysteine requirement. There is little or no haemolysis on blood agar. Cultures should be kept for at least 10 days before discarding (3, 4, 5). The organism grows poorly at 28o
3.1.7 Suitable internal controls
Blood agar is a fairly consistent product, but all batches must be validated for suitable performance using a documented internal quality control system. The consistent growth of fastidious and non-fastidious Gram-negative bacilli should be indicative of suitable performance.
3.1.8 Suitable test validation criteria
Biochemical identification should not be attempted with commercial test systems, firstly because they are unreliable, and secondly because of the risk of transmission to staff (4, 5). The organism can only be identified to the “suspect” stage in Australian laboratories with currently available reagents.
3.1.9 Suitable external QC program
There is at present no external quality control program for laboratory detection of F. tularensis
and related species.
3.2 Identification of Francisella tularensis
There are two main steps likely to occur in the aetiological diagnosis of tularaemia:
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- Diagnostic laboratories – In the event of a patient returning from a tularaemia-endemic area or a deliberate biohazard release, clinical samples should go to a routine diagnostic microbiology laboratory for analysis. As soon as there is any suspicion of the organism being Francisella tularensis, all culture work should cease. The cultures should be forwarded immediately to a PHLN laboratory by arrangement with senior PHLN lab staff. Likewise, a request to culture for Francisella tularensis should result in automatic referral of specimens to a PHLN laboratory.
- Reference (PHLN) laboratories – Samples as described above or, where a presumptive diagnosis has already been made and clinical samples are being collected based on this diagnosis, these samples should be referred to a PHLN laboratory for culture and confirmation.
3.2.1 Presumptive identification
- Staining – F. tularensis is a small (0.2–0.5 Ám by 0.7–1 Ám) Gram-negative pleomorphic, encapsulated and weakly staining coccobacillus seen mostly as single cells. It does not exhibit the characteristic bipolar staining of Y. pestis. A suspect diagnosis of tularaemia may be made if the direct stain (Gram) made from blood, tracheal or lung aspirates or an ulcer from a patient with compatible clinical symptoms shows small weakly staining Gram negative coccobacilli (3, 4, 5).
- Identification test strips – a biochemical identification of F. tularensis from a suitable sample from a patient demonstrating compatible symptoms provides further suspect evidence.
- PCR protocols targeting the TUL4 and putative PPIase genes for F. tularensis are under development, but have yet to be validated for diagnostic use.
3.2.2 Definitive identification
Confirmation of the identity of suspect F. tularensis requires specialised reagents with limited availability in Australia. These include specific direct fluorescent antibody stain and Nucleic Acid Tests (NAT). A slide agglutination test is commercially available in Australia. Suggestive identification can be achieved with Catalase (weak +ve), Oxidase (-ve), Beta-lactamase (+ve), Urease (-ve) and XV-requirement (-ve) (4). The most common misidentifications with cultures of Francisella tularensis are Haemophilus influenzae (XV-requirement) and Actinobacillus sp. (beta-lactamase –ve), especially when commercial identification systems are used (3).
3.2.3 Predictive values
A negative result in a biochemical test strip does not rule out F. tularensis. Isolates must be confirmed by the methods outlined above.
3.2.4 Suitable test criteria
An isolate that exhibits characteristic weak staining, has characteristic growth on agar, biochemically confirms as F. tularensis and gives a positive specific agglutination test or stains with specific fluorescent antibody stain, with confirmation by NAT (PCR for TUL4 gene and 16S rRNA gene sequencing).
3.2.5 Suitable internal controls
Each batch of reagents tested with positive and negative controls. Results of all QC testing should be recorded and the records maintained.
3.2.6 Suitable validation criteria
Correct reactions exhibited by a recognised control strain of F. tularensis
3.2.7 External QC Program
There is at present no external quality control program for laboratory detection of F. tularensis
and related species.
3.3 Nucleic Acid Detection F. tularensis
is an ideal target for nucleic acid detection because of its fastidious nature and its high infectivity. In house PCR assays have been used successfully to detect F. tularensis
during outbreaks in humans and animals in endemic areas overseas using conventional PCR formats (6). Real time PCR has been shown to be significantly more sensitive than conventional assays for detecting F. tularensis
from animal samples, however there is no data published on its efficacy for human diagnosis. Most assays target the lpnA
gene encoding a 17 kDa lipoprotein which is conserved and cannot be used to differentiate among the subspecies. There is no commercial PCR assay available as yet.
3.4 Antigen Detection
A rapid hand held detection assay and antigen capture enzyme-linked immunosorbent assays (cELISA) have been developed to detect the 17 kDa lipopolysaccharide antigen in specimens and environmental samples. The hand held device has an estimated detection limit of 106
bacteria/ml, the cELISA 103
and PCR 102
(7). The antigen detection assay is available in a limited number of laboratories .
3.5 Serological Diagnosis
Serology for F. tularensis
is not available in Australian laboratories. Serological diagnosis is highly specific and can be useful in culture-negative cases (4). Acute and convalescent sera can be forwarded to the Centers for Disease Control and Prevention, Atlanta, GA, US. Tests available include microagglutination and tube agglutination (4).
- Alibek K with Handelman S. Biohazard. 1999, Random House UK Limited.
- Gilchrist M J R et al. Cumitech 33 – Laboratory Safety, Management, and Diagnosis of Biological Agents Associated with Bioterrorism. 2000 ASM Press.
- Tularaemia – Interim PHLS Guidelines for Action in the Event of a Deliberate Release. 2002 PHLS-CDSC.
- Level A Laboratory Procedures for the Identification of Francisella tularensis. 2001. American Society for Microbiology.
- Basic Laboratory Protocols for the Presumptive Identification of Francisella tularensis.2001. Centres for Disease Control and Prevention, Atlanta, USA.
- Johansson A, Forsman M, Sjostedt A, The development of tools for diagnosis of tularemia and typing of Francisella tularensis. 2004 APMIS 112:898-907.
- Chu MC, Weyant RS. Francisella and Brucella In Manual of Clinical Microbiology 8th Edition 2003. Murray PR et al Eds. ASM Press Top of page