J. Med. Microbiol. - Vol. 45 (1996), 192-199 0 1996 The Pathological Society of Great Britain and Ireland MOLECULAR I DENTI FICATION Identification of Bartonella henselae and B. quintana 16s rDNA sequences by branch-, genus- and species-specific amplification C. DAUGA, I. MIRAS and I? A. D. GRIMONT Unite des Enterobacteries, Institut National de la Sante et de la Recherche Medicale, Unite 389, lnstitut Pasteur, F-75724 Paris Cedex 15, France Given the controversy surrounding the aetiology of cat scratch disease and the association of both Bartonella henselae and B. quintana with bacillary angiomatosis, a method for the direct detection in clinical samples of 16s rRNA from the Proteobacteria alpha subgroup was developed. The primary structure of amplified 16s rDNA was determined by cloning and sequencing. Three sequences were identified: one corresponded exactly to GenBank accession number M73229 (B. henselae); the second was related to, but distinct from, GenBank accession number 211684 (referred to as ‘B. henselae variant’); and a third sequence was identical with GenBank accession number M73228 (B. quintana). No sequence corresponding to Aflpia spp. was found. To speed identification and reduce the cost of analysis, a nested amplification method for B. henselae and B. quintana was devised. These techniques were applied to DNA extracted from 30 unfixed lymph node biopsies, two liver biopsies and 36 node pus samples from patients with suspected cat scratch disease, and from 17 skin biopsies from AIDS patients with suspected bacillary angiomatosis. B. henselae or B. henselae variant sequences were found in 42 (62%) of 68 samples from suspected cat scratch disease. B. quintana was not associated with cat scratch disease, but a B. quintana sequence was found in seven (41%) of 17 samples from suspected bacillary angiomatosis patients. B. henselae 16s rDNA sequences were not found in bacillary angiomatosis specimens. Introduction Cat scratch disease (CSD) has been known for 45 years as an infectious disease transmitted to man by cats [l]. This relatively common cause of subacute regional lymphadenopathy is diagnosed currently on the basis of three of four criteria: contact with or scratch from a cat; negative conventional bacteriolo- gical results; positive skin test with heated purulent lymph node material from a patient with CSD; gram- negative bacilli visualised by Warthin Starry silver stain in lymph node tissues [2,3]. The precise identification of the aetiological agent has been the source of controversy. Various bacteria have been successively incriminated [4,5], including Afipia felis and Rochalimaea henselae [6,7], but at the present time R. henselae seems to be the only causal agent of CSD in the USA [8,9]. Received 5 Dec. 1995; revised version accepted 22 Jan. 1996. Corresponding author: Dr C. Dauga. The AIDS epidemic has allowed the emergence of infections that were previously unknown or rare. Among these infections is bacillary epitheloid angio- matosis (BA). This debilitating systemic disease is characterised by vascular lesions that are generally cutaneous and less frequently hepatic, splenic, pulmonary or cerebral [ 10, 1 11. Molecular methods have implicated R. quintana in this disease, a bacterium described long ago as causing trench fever [9-121. Both R. henselae and R. quintana have been isolated from bacillary angiomatosis in the USA [3-7, 13, 141. Isolation of the causative bacterium from CSD and BA is tedious and often fails. Molecular methods now allow the detection of non-cultivable bacteria, based on 16s rRNA sequence comparisons [15]. Rapidly expanding international data bases now contain c. 3500 bacterial 16s rRNA sequences. Use of these sequences has allowed the detection and identification of bacteria without culture [16]. The CSD and BA agents belong to the alpha subgroup