Laboratory Diagnosis, Treatment and Prevention of Francisella tularensis

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Laboratory diagnosis of Francisella tularensis

Specimen

Scrapings from infected ulcers, lymph node biopsies, and sputum, whole blood.
Serum is generally collected from all patients early in disease and during convalescence.
To minimize the loss of viable organisms, samples should be transported to the laboratory within 24 hours.
If specimens are to be held longer than 24 hours, specimens should be refrigerated in Amie’s transport medium.
F. tularensis should remain viable for up to 7 days stored at ambient temperature in Amie’s medium.
Specimens for molecular testing should be placed in guanidine isothiocyanate buffer.

Laboratory diagnosis of Francisella tularensis

Gram staining of clinical material is of little use with primary specimens unless the concentration of organisms is high, as in swabs from wounds or ulcers, tissues, and respiratory aspirates.
Gram negative coccobacilli are seen in Gram staining procedure.
A more sensitive and specific approach is direct staining of the clinical specimen with fluorescein labeled antibodies directed against the organism.
Fluorescent antibody stains and immunohistochemical stains are commercially available for direct detection of the organism in lesion smears and tissues and are typically available in reference laboratories.

The organism is strictly aerobic and is enhanced by enriched media containing sulfhydryl compounds (cysteine, cystine, thiosulfate or IsoVitaleX) for primary isolation.
Two commercial media for cultivation of the organism are available: glucose cystine agar and cystine-heart agar, both require the addition of 5% sheep or rabbit blood.
However, F. tularensis can grow on chocolate agar or buffered charcoal yeast extract (BCYE) agar, media supplemented with cysteine that are used in most laboratories.
They are slow-growing organisms and require 2 to 4 days for maximal colony formation.
They are weakly catalase positive and oxidase negative.
Identification is done by growth on chocolate agar but not blood agar (blood agar is not supplemented with cysteine) is also helpful.
The identification is confirmed by demonstrating the reactivity of the bacteria with specific antiserum (i.e., agglutination of the organism with antibodies against Francisella).

Because of the risk of infection to laboratory personnel and other inherent difficulties with culture, diagnosis of tularemia is usually accomplished serologically by whole-cell agglutination (febrile agglutinins or newer enzyme-linked immunosorbent assay techniques).
Serum antibody detection is useful for all forms of tularemia.
After the initial specimen, a convalescent sample should be collected at 14 days and preferable up to 3 to 4 weeks after the appearance of symptoms.
Tularemia is diagnosed in most patients by the finding of a fourfold or greater increase in the titer of antibodies during the illness or a single titer of 1:160 or greater.
However, antibodies (including IgG, IgM, and IgA) can persist for many years, making it difficult to differentiate between past and current disease.

Conventional and real-time polymerase chain reaction (PCR) assays have been developed to detect F. tularensis directly in clinical specimens.
Of significance, several patients with clinically suspected tularemia with negative serology and culture had detectable DNA by PCR.

The organism is susceptible to aminoglycosides, and streptomycin is the drug of choice.
Gentamicin is a possible alternative and now considered as drug of choice.
Doxycycline and ciprofloxacin can be used to treat mild infections.
Doxycycline and chloramphenicol also have been used, although these two agents have been associated with a higher rate of relapse after treatment.
F. tularensis strains produce β-lactamase, which renders penicillins and cephalosporins ineffective.
Fluoroquinolones appear promising for treatment of severe tularemia.

Preventing bites from ticks, flies, as well as mosquitos.
Refrain from drinking untreated water.
Wearing protective clothing and using insect repellents reduce the risk of exposure
If working with cultures of F. tularensis in the laboratory, wearing a gown, impermeable gloves, mask, and eye protection is must.
Prompt removal of the tick can prevent infection.
Persons who have a high risk of exposure (e.g., exposure to an infectious aerosol) should be treated with prophylactic antibiotics.