Diagnostic polymerase chain reaction for donovanosis.

Donovanosis is an important cause of genital ulceration that occurs in geographic clusters throughout the world. Diagnosis is problematic, as the causative organism, Calymmatobacterium granulomatis, cannot be cultured on conventional microbiological media. Until recently, diagnosis has relied almost exclusively on the detection of typical histological features in tissue smears and biopsies [1]. More recently, successful culture has been reported by use of fresh human monocytes [2] and, in our laboratory, by use of a human epithelial cell line [3]. We report herein the development of the first PCR for the detection of C. granulomatis, the first major advance in the diagnosis of this disease since 1905 [4]. C. granulomatis and human Klebsiella species are ultrastructurally, morphologically, and antigenically indistinguishable [5]. Previous analysis of the phosphate porin gene (phoE) sequence from C. granulomatis, Klebsiella pneumoniae, Klebsiella ozaenae, and Klebsiella rhinoscleromatis showed that there were two base changes unique to C. granulomatis [6]. We have found that these base changes eliminate HaeIII restriction sites. Swab or biopsy specimens were obtained from patients with a clinical diagnosis of donovanosis who attended remote area health centers. Total DNA was extracted from 14 specimens from patients with clinically diagnosed donovanosis (9 histologically confirmed), reference isolates of K. rhinoscleromatis, K. ozaenae, and K. pneumoniae, and 8 samples from patients with unrelated genital conditions (herpes genitalis, syphilis, gonorrhea, human papillomavirus infection, and mixed anaerobic infection) [7]. Eleven DNA extracts from patients with donovanosis were subjected to amplification of a 700-bp region of the Klebsiella phoE gene (5*-CTATGACAGCAAGGATGGCGA-3* and 5*-CAGFigure 1. PCR products from Klebsiella pneumoniae, Klebsiella ACCGAAGTCGAACTGATACTG-3*), encompassing the two ozaenae, and Klebsiella rhinoscleromatis isolates and from a patient unique base changes of C. granulomatis. Products of the expected with a clinical diagnosis of donovanosis. The figure shows a 2% size amplified from the Klebsiella isolates and all donovanosis low-melting-point ethidium bromide–stained agarose gel of HaeIIIsamples were subsequently digested with HaeIII. As expected from digested PCR products (700 bp). Bands marked as Ç40-bp consist the sequence data, we were able to distinguish the C. granuloof products ranging from 33 to 45 bp. The band marked as 87 bp matis–derived phoE product from that of the Klebsiella isolates also consists of an 86-bp band for the Klebsiella isolates. Lanes 1 by the presence of a 167-bp fragment resulting from the eliminated and 6, FX174 HaeIII markers (New England Biolabs, Beverly, MA); HaeIII restriction sites (figure 1). No products were obtained for lane 2, K. pneumoniae; lane 3, K. ozaenae; lane 4, K. rhinoscleromatis; lane 5, specimen from a patient with donovanosis. the 8 samples from patients with unrelated genital conditions. We then targeted the 167-bp differential product of C. granulomatis. The 14 donovanosis and 3 Klebsiella DNA samples were amplified with primers targeting a 188-bp region of the Klebsiella articulate with EIA-based PCR diagnostic systems. In the KlebphoE gene (5*-GATCTGACCCTGCAGTACC-3* and 5*-TCCsiella isolates, the cleavage of the fragment containing the capture GCTTTCGAACCCTGG-3*) and digested with HaeIII. All 8 samoligonucleotide target region from the biotinylated fragment will ples from patients with unrelated genital conditions were negative give a negative result in the EIA-based assay, whereas the intact with this primer set. Band sizes expected from the sequence data fragment of C. granulomatis will give a positive result. were obtained for all 14 donovanosis samples and were again This PCR requires further validation in a larger sample of paclearly distinguishable from those of the Klebsiella samples. tients in other settings, but it should have an important place in By use of a 5*-biotinylated oligonucleotide primer, it should the rapidly developing array of diagnostics available for genitoulnow be possible to modify the PCR-HaeIII digest so that it can cerative disease, as swabs can now be used.