A total of 215 peripheral blood specimens was collected from patients in four comprehensive health centers over a period of 18 months. Of these blood specimens, 165 were from patients who presented with clinical signs compatible with brucellosis. Clinical diagnosis was made by the physicians in the Badia of Jordan, a rural area in the north east of Jordan inhabited by recently settled Bedouins. These centers receive patients from 40 villages with a population exceeding 12,000 inhabitants. The diagnosis of brucellosis is established based on the presence of compatible signs and symptoms with the demonstration of specific antibodies at significant titer or seroconversion. The physicians are aware of the zoonotic nature of the disease because of the continuous reporting of the disease in sheep, goats and in humans in this area. Sixty (36.4%) of the samples were collected after adequate antibiotic treatment was started.
A questionnaire was completed for each subject at the time of specimen collection to record demographic and other relevant information such as the clinical history, symptoms and physical signs, contact with animals, drinking unpasteurized milk, and homemade dairy products.
The duration of symptoms prior to diagnosis was 1 to 16 weeks. Cases with clinical symptoms less than two months were considered as acute cases, those that lasted more than 6 months before treatment was initiated were considered as chronic cases. A relapse was considered to be either a positive blood culture two months to one year after completing the treatment or the reappearance of compatible symptoms not otherwise explained together with an increase in the previous serological titers. All patients had fever during the course of the disease with other symptoms such as muscle pain, wrist arthritis.
Treatment of recent cases is by a combination of a daily intramuscular injection of one gram of streptomycin for 14 days and doxycycline for a month. In the chronic cases, a combination of doxycycline and rifampin is given to the patient for one month according to the internationally accepted treatment regimens . The treatment was repeated when the symptoms persist and antibody titer was still high two weeks after concluding the treatment.
Samples were obtained from 50 subjects, composed of patients randomly selected among patients from the same region attending the same health centers. None of these patients was currently diagnosed or had a history of brucellosis.
The study was approval by the University ethics committee.
The serological tests were carried out by the laboratories of the health centers. The serologic diagnosis was established by Rose Bengal agglutination test. A titer of 1/160 was considered positive.
Five to ten ml peripheral blood samples were withdrawn and immediately inoculated under aseptic conditions in broth media (Bloodgrow®, Medical Wire & Equipment C. Ltd, Corsham, Wiltshire, England) or diphasic blood culture bottles (Hemoline performance diphasique bioMerieux, Marcy l'Etoile, France). Cultures were incubated at 37°C for 30 days in the presence of 5% CO2 and were periodically checked for growth. Subcultures on blood agar plates were performed in a blind manner at 10, 20, 30 days, they were recorded as negative after the last negative subculture. Brucella spp. were identified using standard methods .
DNA was extracted from blood specimens using a commercial purification system (Wizard Genomic DNA Purification Kit, Promega, Madison, WI) according to the manufacturer's instructions for DNA purification from blood. Final pellets were resuspended in 50 l of TE (10 mM Tris, I mM EDTA, pH 7.2).
The Brucella DNA-Detect PCR Kit (Vita-Tech International Inc., CAN), including the reagents and oligonucleotid primers designed for the direct amplification of the genus Brucella, was used for the detection of Brucella spp. in blood samples. The primers used in the kit were genus-specific primer pair designed to amplify a highly conserved region within the 16S rRNA of the genus Brucella .
The size of the amplification products was a 905 fragment of the rRNA gene. The reaction mixture contained 5 μl of 10 × PCR buffer, 1 μl of the primer mix, 2 μl of dNTP mix (5 mM each), 3 μl of 25 mM MgCl2, 1 unit of Taq DNA polymerase, 5 μl of sample DNA in a volume of 50 μl. The same mixtures were used with the positive control provided by the kit. The mixture with no DNA sample, and DNA free water were used as negative controls to monitor contamination.
The reaction was performed in a thermal cycler (Gene Amp PCR System 9700, Perkin Elmer, Norwalk, Con.). The cycling conditions were an initial denaturation at 95°C for 5 min, template denaturation at 95°C for 30s, annealing at 54°C for 90s, and primer extension at 72°C for 90s for a total of 35 cycles, with a final extension at 72°C for 6 min. A sample was considered positive when the size of the DNA band matched with that of the po sitive control (905 bp). All standard precautions recommended for prevention of contamination with DNA and amplicons were undertaken. Twelve microliters of the PCR product were run by electrophoresis in a 2% agarose gel in 1 × TBE buffer (Promega, Madison, Wis.), and gels were stained with ethidium bromide (2 μg/ml).
PCR amplification products were detected by visualization of the bands under UV light.
The means, ranges, percentages of positive samples, specificity, sensitivity and positive predictive value of PCR were calculated.