SPECIMEN COLLECTION, TRANSPORT, AND PROCESSING

 A definitive diagnosis of brucellosis requires isolation of the organisms in cultures of blood, bone marrow, cerebrospinal fluid (CSF), pleural and synovial fluids, urine, abscesses, or other tissues. If processing will be delayed, the specimen may be held in the refrigerator.

It is essential that the clinical microbiology laboratory be notified whenever brucellosis is suspected:

• To ensure that specimens are cultivated in an appropriate manner for optimum recovery from clinical specimens

 • To avoid accidental exposure of laboratory personnel handling the specimens, because Brucella spp. are considered class III pathogens (specimen labels should indicate that Brucella spp. are a potential pathogen)

Blood for culture can be collected routinely into most commercially available blood culture bottles and the lysis-centrifugation system (Isola tor; Alere, Waltham, MA). For other clinical specimens, no special requirements must be met for collection, transport, or processing.

DIRECT DETECTION METHODS

Direct stains of clinical specimens are not particularly useful for the diagnosis of brucellosis. Conventional and real-time polymerase chain reaction (PCR) assays are reliable and specific means of directly detecting Brucella organisms in clinical specimens. Sensitivity varies among assays, ranging from 50% to 100%. Several gene targets have been used, including a cell surface protein (BCS P31), a periplasmic protein (BP26), 16S rRNA, and transposon insertion sequence 711(IS711). Molecular assays currently are not available in routine laboratory testing; therefore, use of a reference laboratory may be required.

CULTIVATION

Although most isolates of Brucella spp. grow on blood and chocolate agars (some isolates are also able to grow on MacConkey agar), more enriched agars and special incubation conditions generally are needed to achieve optimal recovery of these fastidious organisms from clinical specimens. Brucella agar or infusion base is recommended for specimen types other than blood. The addition of 5% heated horse or rabbit serum enhances growth on all media. Cultures should be incubated in 5% to 10% CO₂ in a humidified atmosphere; inoculated plates are incubated for up to 3 weeks before they are considered negative and discarded.

Commercial blood culture systems (e.g., BacT/Alert, BACTEC, and lysis-centrifugation systems) all have successfully detected brucellae in blood. Other blood culture bottles, such as those with brain-heart infusion and trypticase soy broth, also support the growth of brucellae if the bottles are continuously vented and placed in a CO₂ incubator. Most isolates can be detected within 5 to 7 days using commercial systems. Bottles need not be incubated longer than 10 to 14 days. Culture bottles may not become turbid. All subculture plates should be held for a minimum of 7 days.

On culture, colonies appear small, convex, smooth, translucent, nonhemolytic, and slightly yellow and opalescent after at least 48 hours of incubation (Figure 1).

Fig1. Growth of Brucella spp. on chocolate agar after incubation for 2 days (A) and 4 days (B). 

Rough variants may be seen with B. canis. The colonies may become brownish with age.

APPROACH TO IDENTIFICATION

 Because brucellosis is the most commonly reported laboratory-acquired bacterial infection, all handling and manipulations of suspected Brucella spp. should be performed in a class II or higher biologic safety cabinet. On Gram stain, the organisms are small coccobacilli that resemble fine grains of sand (Figure 2). Brucella spp. are catalase and urease positive, and most strains are oxidase positive. Other nonfermentative gram-negative coccobacilli that may be confused with brucellae are Bordetella, Moraxella, Kingella, and Acinetobacter spp. Brucella spp., however, are nonmotile, urease and nitrate positive, and strictly aerobic. The most rapid test for presumptive identification of Brucella spp. is the particle agglutination test with anti–smooth Brucella serum (Difco Laboratories, Detroit, Michigan).

Fig2. Brucella melitensis with traditional Gram stain (A) and Gram stain with 2-minute safranin counterstain (B) to allow easier visualization of the organism. 

Brucella spp. are differentiated by the rapidity with which an organism hydrolyzes urea, its relative ability to produce hydrogen sulfide (H₂S), its requirements for CO₂, and its susceptibility to the aniline dyes thionine and basic fuchsin (Table 1). For determination of the CO₂ requirement, identical plates of Brucella agar or brain-heart infusion agar should be given equal inocula (e.g., with a calibrated loop) of a broth suspension of the organism to be tested. One plate should be incubated in a candle jar and the other plate in air in the same incubator. Most strains of B. abortus do not grow in air but show growth in the candle jar. Presumptive identification can be reported based on the colony’s morphology and a positive catalase, oxidase, urease, and slide agglutination reaction. Brucella isolates should be sent to state or other reference laboratories for confirmation or definitive identification, because most clinical laboratories lack the necessary media and containment facilities.

Table1. Characteristics of Brucella spp. That Are Pathogenic for Humans

Subtyping of biovars may be performed using a variety of molecular techniques, including pulsed-field electrophoresis, random amplification of polymorphic DNA, amplified fragment length polymorphism, various PCR techniques, and multilocus sequence typing.

SERODIAGNOSIS

Because isolating brucellae is difficult, a serologic test is widely used (e.g., serum agglutination test [SAT] or microplate agglutination [MAT]). This technique detects antibodies to B. abortus, B. melitensis, and B. suis; however, the SAT does not detect B. canis antibodies. An indirect Coombs’ test is performed after the SAT. This test detects nonagglutinating or incomplete antibodies in complicated and chronic cases of brucellosis.

The serology associated with Brucella infection follows the classic antibody response: IgM appears initially, followed by IgG. A titer of 1 : 160 or greater in the SAT is considered diagnostic if this result fits the clinical and epidemiologic findings. The SAT can cross-react with class M immunoglobulins with a variety of bacteria, such as Francisella tularensis and Vibrio cholerae. Enzyme-linked immunosorbent assays (ELISAs) also have been developed. Purified LPS or protein extracts are primarily used in ELISAs. However, currently no reference antigen exists; therefore, it is important to identify the antigen in the commercial antigen when evaluating test results. In patients with neurobrucellosis, ELISA offers significant diagnostic advantages over conventional agglutination methods.

Additional serologic assays are commercially available, including a lateral flow dipstick for screening outbreaks and an immunocapture agglutination method. The immunocapture assay demonstrates sensitivity and specificity similar to a Coombs’ test and is less cumbersome to perform. The dipstick test has a high degree of sensitivity (greater than 90%).

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