AbstractDonovanosis is a chronic ulcerative disease of the genitals and surrounding regions with endemic foci in socially disadvantaged populations throughout the world. In recent years there has been a resurgence of interest in this disease due to the association of genitoulcerative disease with an increased risk of HIV-1 transmission and acquisition. The Australian government has a target to eradicate donovanosis by 2003.
Diagnosis is problematic as the causative organism, Calymmatobacterium granulomatis, cannot be cultured on conventional microbiological media and, until recently, has relied almost exclusively on the detection of typical histological features in tissue smears and biopsies. There is general consensus that the sensitivity of histology for the diagnosis of donovanosis is in the range 60-80%. More recently, successful culture has been reported using fresh human monocytes and a human epithelial cell line though the utility of these techniques for routine and rapid diagnosis is limited.
In contrast, molecular diagnostic techniques are of great utility in the detection of unculturable organisms. The main objective of this study was therefore to develop a sensitive and specific molecular diagnostic test for donovanosis and to then incorporate this test into a colorimetric detection system for use in routine diagnostic laboratories. To this end DNA was extracted from specimens clinically suggestive of donovanosis and a 604-bp region of the phoE gene was amplified and sequenced. Results of this aspect of the study showed that there were only two unique base changes distinguishing C. granulomatis from subspecies of Klebsiella pneumoniae. Extension of the sequence data to the almost complete phoE gene (1001-bp) resulted in no additional changes being observed.
In order to clarify the relationship between C. granulomatis and the Klebsiella genus sequence data was extended to include the 16S rRNA (1088-bp),gdhA (376-bp) and iluK (772-bp) genes. DNA similarity was shown to be in the range 98.6- 100% across these four genes. Tree topologies and associated confidence levels of branching resulting from the use of a number of phylogenetic inferencing methods confirmed that C. granulomatis has a close phylogenetic relationship with the three subspecies of K. pneumoniae and it is proposed that C. granulomatis be reclassified as Klebsiella granulomatis comb. nov.
As a further result of the phylogenetic aspect of the study it was shown that K. granulomatis appears to be missing the Klebsiella sucrose regulon. This enabled the development of a first-generation diagnostic PCR for donovanosis based on the presence of phoE and absence of the highly conserved scrA. However, this test was limited by the fact that it relied on a phoE+/scrA- result for positive diagnosis and so would give an indeterminate result if other Klebsiella were contaminating the lesion. A second-generation diagnostic PCR was subsequently developed based on the finding that the two unique base changes in the K. granulomatis phoE gene eliminated Haelll restriction sites. Through the use of a capture oligonucleotide covalently linked to tosyl-activated magnetic beads it was possible to modify the PCR-Haelll digest so that it could articulate with EIA-based PCR diagnostic systems for use in routine diagnostic laboratories. This test is now ready to be taken up in validation trials.
Finally, the results of the study enabled the question of the habitat and transmission of K. granulomatis to be revisited. An analysis of the literature within the context of these results is strongly suggestive of the presence of an enteric asymptomatic carrier state that needs to be taken into consideration if the Australian government is serious about eradicating donovanosis from this country.
Note: Please note that appendix 1 - 7 available in hard copy only.
|Date of Award||Apr 2002|
|Supervisor||Charles Webb (Supervisor)|