A colorimetric detection system for Calymmatobacterium granulomatis

Abstract

Objective: To incorporate the first polymerase chain reaction (PCR) assay for Calymmatobacterium granulomatis into a colorimetric detection system for use in routine diagnostic laboratories. Methods: A capture oligonucleotide specific for the Klebsiella phoE gene was covalently linked to tosyl activated magnetic beads. Biotinylated phoE PCR products obtained from 14 positive specimens from patients with donovanosis and isolates of Klebsiella pneumoniae, K rhinoscleromatis, and K ozaenae were cleaved with HaeIII for the purpose of diVerentiation, captured by the prepared beads, and subjected to standard EIA detection methodology. Eight samples from unrelated genital conditions underwent the same procedure. It was anticipated from the sequence data that the biotinylated fragment would be cleaved from the capture oligonucleotide target region in the three Klebsiella phoE products (that is, a negative colorimetric result) while the entire fragment of interest would remain intact in the positive C granulomatis phoE products (that is, a positive colorimetric result). Results: All 14 positive specimens from patients with donovanosis gave strong colorimetric readings with this detection system. Isolates of K pneumoniae, K rhinoscleromatis, K ozaenae, and the eight specimens from unrelated genital conditions were negative. Conclusion:The successful development of a colorimetric detection system for C granulomatis incorporating two levels of specificity enables the molecular diagnosis of this condition to be undertaken by routine diagnostic laboratories. This should have an important role in the Australian government's campaign to eradicate donovanosis by 2003 though the test still needs to undergo trials and be validated using a larger number of samples from geographically diverse parts of the world in order to ascertain the generalisability of the methodology. (Sex Transm Inf 2000;76:134-136)

Detection and Discrimination of Herpes Simplex Viruses, Haemophilus ducreyi, Treponema pallidum, and Calymmatobacterium (Klebsiella) granulomatis from Genital Ulcers

“…changes in C. granulomatis that eliminated HaeIII restriction sites and differentiated this bacterium from other members of the Enterobacteriaceae family. Whereas Carter et al [7,10] used known positive biopsy samples to validate PCR sensitivity, together with a small number of genital swabs obtained from patients with other conditions to check specificity, we applied a synthetic control and examined DNA from numerous related microorganisms to extend the assay validation data, particularly for H. ducreyi and C. granulomatis, for which limited positive clinical material was available. Limited sequence data is publicly available for C. granulomatis strains; therefore, future studies should aim to sequence the phoE target to ensure the stability of the nucleotide polymorphism over time and around the world.…”

“…Multiplex PCR makes best use of limited amounts of clinical material when multiple targets are under investigation, and it improves cost-effectiveness [27]. C. (Klebsiella) granulomatis has not been included in previously reported multiplex PCR assays investigating GUD, despite the existence of a robust PCR technique [7,10].…”

Donovanosis: an update

“…A diagnostic PCR was developed based on the observation that 2 unique base changes in the phoE gene eliminate HaeIII restriction sites enabling clear differentiation from closely related species of Klebsiella 16 . Carter et al have further re® ned their PCR assay into a colorimetric detection system for use in diagnostic laboratories 17 . Firstly this involved linking a capture oligonucleotide speci® c for the Klebsiella phoE gene to tosyl activated magnetic beads.…”

European guideline for the management of tropical genito-ulcerative diseases

“…These techniques are not available at the majority of (reference) laboratories . Alternative diagnostic laboratory methods Ð a PCR-based technique for routine diagnosis of C. granulomatis has been reported 25 . However, this test needs further validation using a suf® cient number of samples from different geographical areas in order to ascertain its value for diagnosing donovanosis.…”