F. tularensis is a highly infectious bacterium. Hence, isolation ofthe bacteria from clinical specimens is not attempted in rou-tine laboratories because culture poses a danger to laboratory workers. The plates must be sealed and handled by using a bio-safety level (BSL)-2 facility that is essential for handling the cul-ture plates and BSL-3 facility for isolation and identification of F. tularensisisolates.
Sputum, pleural fluid, wounds, blood, lymph node biopsy samples, and gastric washings are the specimens collected for culture, but isolation rate is very low. CSF may show a mild elevation of protein concentration or pleocytosis.
Gram staining of the aspirates from ulcer exudates or from infected lymph nodes is not useful, because F. tularensis is extremely small and stains very faintly. Detection of antigen directly in clinical specimens by direct fluorescent antibody staining, using fluorescein-labeled antibodies directed against the organism, is more sensitive and specific method for diagnosis of the condition. The test is yet to be available for wide use.
Blood cultures in specific medium (such as cysteine– glucose–blood agar) are usually negative unless the cultures are incubated for a week or longer. Aspirates of lymph nodes or draining sinuses are usually positive if the cultures are incu-bated for 3 days or longer. The identification of F. tularensis is confirmed by testing with specific antiserum containing anti-bodies against Francisella.
Diagnosis of tularemia is usually based on serology. Serodiagnosis depends on demonstration of serum anti-bodies by tularemia tube agglutination test and ELISA. Tularemia tube agglutination test is the most commonly used serological test. An agglutination titer greater than 1:160 is considered presumptively positive. Diagnosis is con-firmed by a fourfold increase in titer of the serum samples collected 2 weeks apart. The test shows cross-reactivity with Brucella species, Proteus Ox-19, Salmonella species, and Yersiniaspecies.
Capture ELISA using specific monoclonal antibodies raised against LPS of the virulent forms of F. tularensis is a sensitive method for detection of serum antigen.