Patient 1

Patient 1 was a previously healthy, 26-year-old Peruvian man who was evaluated in July 1985 for a 3-month history of periorbital pain, headaches, and periodic generalized tonic-clonic seizures. The initial neurologic examination was nonfocal, but subsequent computerized tomography scan showed a 5x5-cm enhancing mass in the left frontoparietal region associated with midline shift.

Figure 1

Figure 1. Hematoxylin and eosin–stained sections from patient 1 (panels A, B) and patient 2 (panels C, D). Note the predominantly lymphohistocytic infiltrate forming large granulomas (A, original magnification 100x); well-formed giant cells...

At the time of surgical biopsy, frozen section histology raised the possibility of a high-grade astrocytoma or lymphoma, prompting resection of a 3x3-cm well-circumscribed left frontal lobe mass. Final examination of pathologic specimens showed granulomas with multinucleated giant cells (Figure 1). Bacterial, fungal, and acid-fast bacilli stains were negative. Based on these pathologic findings and concern that the patient may have had tuberculosis, treatment with isoniazid, rifampin, and ethambutol was begun. Serologic tests for Histoplasma capsulatum, Blastomyces dermatidis, Coccidioides immitis, and Paracoccidioides brasiliensis were negative. Toxoplasma gondii serologic titers were weakly positive at 1:64.

On postoperative day 39, fungal tissue cultures became positive for a Brucella spp., preliminarily identified as B. melitensis. The patient’s antimicrobial treatment was changed to tetracycline and rifampin and was continued for 2 months. An initial serologic titer for Brucella was positive at 1:160 by tube agglutination assay. A follow-up serologic titer obtained in January 1986 was negative (<1:20).

Three months before his initial evaluation, the patient had immigrated to the United States from Lima, Peru. His diet included regular consumption of unpasteurized cow or goat cheese (queso fresco) and occasional consumption of raw shellfish (ceviche). He denied eating other raw or significantly undercooked meat. He had frequently swum in the Pacific Ocean from December through March but recalled no direct contact with marine mammals.

Patient 2

Figure 2

Figure 2. Axial MRI (magnetic resonance imaging) from patient 2 obtained when first seen in Peru (April 21, 2001), before surgical biopsy in the United States (September 21, 2001), and 7 months after...

Patient 2 was a 15-year-old Peruvian boy seen in September 2001 with a 1-year history of headaches, nausea, vomiting, and progressive deterioration in visual function. A magnetic resonance imaging (MRI) scan performed in April 2001 showed several large, irregular enhancing mass lesions involving the left occipital and parietal lobes (Figure 2). He had come to the United States in September for further evaluation. On neurologic examination, the patient had a right homonymous hemianopsia, optic nerve atrophy, and major visual impairment (left eye—20/100; right eye—20/200). Repeat MRI showed several irregular areas of enhancement in the left parietooccipital area associated with marked brain edema, left-to-right shift, and mass effect (Figure 2).

The patient was taken to surgery, where a firm avascular mass was found beneath a 0.5-cm layer of softer gliotic cortical tissue. Frozen sections showed lymphohistiocytic infiltrates with granuloma formation. Specimens for cultures and histopathologic examination were obtained. Bacterial, fungal, and acid-fast bacilli stains were negative. Final histopathologic examination showed numerous granulomas with multinucleated giant cells but no organisms (Figure 1).

Serologic test results for Brucella, T. gondii, and Taenia soleum were negative. On postoperative day 8, mycobacteria cultures (BacT/ALERT MP; Organon Teknika Corp., Durham, NC) showed growth of a gram-negative coccobaccillus, later confirmed as Brucella spp. Treatment with rifampin, doxycycline, and intravenous gentamicin was begun. After 1 week, the gentamicin was discontinued per current recommendations, and trimethoprim-sulfamethoxazole was started. Follow-up imaging 7 months later demonstrated resolution of the enhancing areas and edema, with residual areas of brain atrophy (Figure 2). The patient’s vision improved, but some visual acuity deficits persisted. Anti-brucella therapy continued for 1 year.

The patient lived in a small town 7 hours from Lima and had not traveled outside of the country before coming to the United States. His diet included regular consumption of queso fresco and occasional ceviche but no other raw meats. He reported no direct contact with marine mammals and seldom visited the Peruvian coast.

Histopathology

Both cases demonstrated well-developed, granulomatous inflammation with accompanying astrogliosis. Patient 1 showed dense perivascular lymphoplasmacytic infiltrates with nonnecrotizing granulomas containing epithelioid and spindle-shaped macrophages and occasional well-formed giant cells (Figure 1). Tissue Gram, Fite, and silver methenamine stains showed no definite organisms. Patient 2 showed florid granulomatous inflammation with large areas of epithelioid and spindle-shaped histiocytes containing foci of necrosis surrounded by dense chronic inflammation (Figure 1). Giemsa, Steiner, Warthin-Starry, tissue Gram, Grocott methenamine silver, and Fite stains did not detect organisms. Giant cells were not observed in patient 2. Masson trichrome stain showed accompanying marked fibrosis.

Molecular Microbiology

Presumptive Brucella isolate 01A09163 was isolated from the brain biopsy of patient 2 and was submitted to the Microbial Diseases Laboratory (the microbiology reference laboratory for the State of California) for laboratory confirmation. Identification of Brucella spp. was confirmed by a 5′-exonuclease assay that targets a 223-bp region of the bcsp31 gene. Isolates confirmed as Brucella were then further classified as B. abortus, B. melitensis, or B. suis biovar 1 by the real-time PCR assays described by Redkar et al. (11). Strain 01A09163 tested negative by these assays, indicating that this isolate likely represented a different species of Brucella.

A review of Microbial Diseases Laboratory records showed a second Brucella strain, 85A05748, associated with an intracerebral granuloma. This strain was isolated in 1985 from the brain lesion of patient 1. Given the clinical similarities in these two cases, strain 85A05748 was tested by PCR. Like strain 01A09163, strain 85A05748 was negative in the B. abortus, B. melitensis, and B. suis biovar 1 assays.

To aid in the identification of these strains to the species level, a portion of the omp2a was amplified and sequenced. Between the two strains, the omp2a sequences were identical and indicated that the two strains were likely of the same species. A BLAST search of the National Center for Biotechnology Information databases established that the omp2a sequence of these two strains was identical to the omp2a sequence derived from Brucella sp. B2/94 (GenBank accession no. AF300819), a marine mammal strain of Brucella isolated from a common seal (7). Compared to other Brucella strains, one or more polymorphisms were noted in the omp2a sequence of 01A09163 and 85A05748. This observation suggested that strains 01A09163 and 85A05748 were most closely related to a marine mammal strain, rather than a terrestrial strain, of Brucella.

Figure 3

Figure 3. Amplification of bp26 from marine mammal and terrestrial strains of Brucella. The amplification products were electrophoresed on a 1% agarose gel and stained with ethidium bromide. Lane 1, strain 01A09163; lane...

To verify that strains 01A09163 and 85A05748 were genetically related to strains of Brucella derived from marine mammals, we performed a PCR assay targeting the bp26 gene (12). Marine mammal strains of Brucella possess an IS711 element immediately downstream of bp26, whereas the sequence of terrestrial strains does not. Consequently, amplification of the region surrounding bp26 yields a much larger DNA fragment for marine mammal strains as compared to the amplification product produced by terrestrial strains of Brucella. As shown in Figure 3, amplification of the bp26 gene from strains 01A09163 and 85A05748 yielded a 1,900-bp product that was diagnostic for marine mammal strains of Brucella. Amplification of bp26 from a terrestrial strain, B. abortus, produced the expected 1,024-bp product.