Number of samples Positive Brucella culture
Blood culture isolates other than Brucella
Characteristics of the patients and the control groups. Control Group (33)
Positive antibrucella antibodies+
Positive serological tests other than Brucella
Only 14 samples were collected before treatment; ** see text; +antibody
titers range from 160-2560.
Comparison of the results of PCR-based assay with those of
conventional microbiological techniques for the diagnosis of brucellosis.
No. of samples
Positive by PCR
Positive by blood culture
Positive by Serology
Brucella group (15) Sensitivity
Control group (33)
*Only the 14 samples that were collected before treatment were included in the calculation; **all were from the occupationally exposed group; +this was
from the febrile illness group;
occupationally exposed person.
++calculated after exclusion of asymptomatic
Materials and Methods
A total of 50 peripheral blood samples were obtained from 33 controls and from 15 consecutive patients with brucellosis diagnosed in the Infectious Disease Department at Dammam Central Hospital and Maternity and Children Hospital, over a six-month period. There were eight males and seven females, aged between 6 and 65 years. One patient gave three samples (accounting for a total of 17 samples), corresponding to initial infection, convalescent phase and suspected relapse. Two additional samples from the patients with brucellosis could only be obtained after treatment was begun during their follow-up in the outpatient clinic.
All patients had fever with arthralgia and/or backache, with a mean duration of symptoms of about four months (range 1-13). They all gave a positive history of animal contact or ingestion of unpasteurized dairy products. In eight of these patients, brucellosis was confirmed by culture and high titer anti-brucella antibodies that were performed at the time of diagnosis. For the others, diagnosis was based on compatible clinical picture and high-titer antibodies. High titer was defined as a titer of
1:160 by the standard tube agglutination (STA) method.
The 33 control blood samples (Table 1) were obtained from the following subgroups: 1) 19 patients with febrile illnesses due to factors other than Brucella etiology (four cases of bacteremia due to Salmonella, Staphylococcus
DIAGNOSIS OF ACUTE HUMAN BRUCELLOSIS
aureus, Staphylococccus epidermidis and Pseudomonas and 15 cases of backache and/or arthralgia with or without fever in which no particular pathogen could be isolated). In all these patients, blood cultures for Brucella were negative, while repeated serological tests using febrile agglutinins showed a titer of Salmonella paratyphoid of 1:320 in one patient and a titer of 1:160 for Brucella in another one. The latter was a sickler presenting with backache due to vaso-occlusive crisis with no history of exposure to animals or animal products. Repeated serology in this patient did not show titer increment of the anti- brucella antibody. 2) Nine asymptomatic patients who were occupationally exposed to Brucella, with or without history of brucellosis in the previous 2 to 4 years. Two of them showed low-titer anti-Brucella (1:40) antibodies on repeated occasions. 3) Five healthy volunteers with no history of brucellosis or exposure to animals. From all the patients and controls, three specimens were collected simultaneously and were sent for culture, serology and molecular methods. The majority of the specimens were processed within 24 hours.
Microbiological and Serological Techniques
Blood cultures were processed with BACTEC 9240 and BACTEC NR660 (Becton Dickinson, USA), incubated for six weeks and subcultured weekly. Suspected colonies were identified by colonial morphology, gram-staining and standard biochemical and serological methods; typing was not available. Serological tests were done using standard tube agglutination method3 and SAS Febrile Antigens (USA).
Sample Processing for PCR
DNA was prepared using the method described by Miller et al.6 with a slight modification. Briefly, a minimum of 2 mL of blood collected in a citrated tube was resuspended in 5 mL erythrocyte lysis solution (5 mM MgCl2, 320 mM sucrose, 12 mM Tris HCl, 1% Triton X- 100 [pH 7.5]), mixed and centrifuged at 15,000 Xg for 2 minutes. Treatment with erythrocyte lysis solution was repeated, followed by treatment with deionized water until the leukocyte pellets lost all reddish coloring. Template DNA was obtained from the leukocytes by adding 400 µL of nucleic lysis buffer (60 mM NH4Cl, 24 mM Na2 EDTA [pH 8]), sodium dodecyl sulfate (1%) and proteinase K 1 mg/mL, followed by mixing and incubation for 2 h at 55°C or overnight at 37°C with slow shaking. A quantity of 100 µL of NaCl (6 M) was then added, followed by centrifugation at 15,000 Xg for 10 min. The supernatant containing total DNA was transferred to a fresh tube and two volumes of cold ethanol was then added and shaken until the DNA precipitated, which was then collected by fishing, using hooks and dissolved in 30 µL double- distilled water. DNA, which was not tested immediately, was stored at –70C until the time of the assay. The
Annals of Saudi Medicine, Vol 20, Nos 3-4, 2000