Single-tube, nested PCR for the diagnosis of human brucellosis in Kuwait

Abstract

Abstract The polymerase chain reaction (PCR) offers a sensitive and specific way of detecting microbial DNA in clinical samples. The aims of the present study were to develop an assay, based on a single-tube, nested PCR, for identifying Brucella in samples of human blood and then to explore the use of this test in diagnosis. The primers chosen were derived from IS711, the insertion sequence gene found in all species of Brucella. The assay amplified a 52-bp final product which was detected colorimetrically. The PCR was sensitive and specific, giving positive reactions with 14 strains of Brucella from five species. The lower limit of detection in vitro was 30 organisms. There were no false-positive reactions either with a range of bacteria known to evoke serological cross-reactions with Brucella (Vibrio cholerae, Yersinia enterocolitica, Serratia marcescens, Haemophilus influenzae, Pseudomonas aeruginosa and Escherichia coli K12) or with organisms producing similar clinical syndromes (Mycobacterium tuberculosis and Salmonella typhi). The results of a preliminary field trial of the assay in Kuwait indicate that the assay may be a valuable technique in the diagnosis of human brucellosis, meriting further study with larger numbers of cases. All 28 subjects with brucellosis (diagnosed on the basis of typical clinical features and confirmed by positive serology and, in three cases, by positive blood cultures) were PCR-positive whereas 28 healthy controls and 28 patients with febrile illness attributable to infections other than brucellosis were PCR-negative.