Phylogenetic analysis of Calymmatobacterium granulomatis based on 16S rRNA gene sequences.
Calymmatobacterium granulomatis is the aetiological agent of granuloma inguinale - a chronic granulomatous genital infection - and is morphologically similar to members of the genus Klebsiella. This study determined the 16S rRNA gene sequence of C. granulomatis and the taxonomic position of the organism in relation to the genus Klebsiella. Genomic DNA was extracted from C. granulomatis-infected monocytes and from frozen and formalin-fixed paraffin wax-embedded tissue biopsy specimens from patients with histologically proven granuloma inguinale. The 16S rDNA was amplified by PCR with broad range oligonucleotide primers. The amplified DNA fragments were cloned into pMOS vector, digested with Bam HI and Pst1 restriction endonucleases, hybridised with a gram-negative bacterial probe (DL04), sequenced in both directions by the automated ALF DNA sequencer, verified on an ABI Prism 377 automated sequencer and analysed with DNASIS and MEGA software packages. Sequence analysis revealed DNA homology of 99% in C. granulomatis from the different sources, supporting the belief that the bacteria in the culture and the biopsy specimens belonged to the same species, although there was some diversity within the species. Phylogenetically, the strains were closely related to the genera Klebsiella and Enterobacter with similarities of 95% and 94% respectively. C. granulomatis is a unique species, distinct from other related organisms belonging to the gamma subclass of Proteobacteria. (+info)
Phylogenetic evidence for reclassification of Calymmatobacterium granulomatis as Klebsiella granulomatis comb. nov.
By sequencing a total of 2089 bp of the 16S rRNA and phoE genes it was demonstrated that Calymmatobacterium granulomatis (the causative organism of donovanosis) shows a high level of identity with Klebsiella species pathogenic to humans (Klebsiella pneumoniae, Klebsiella rhinoscleromatis). It is proposed that C. granulomatis should be reclassified as Klebsiella granulomatis comb. nov. An emended description of the genus Klebsiella is given. (+info)
A colorimetric detection system for Calymmatobacterium granulomatis.
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 differentiation, 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. (+info)
A serological test for granuloma inguinale.
OBJECTIVES: An indirect immunofluorescence technique applied to paraffin embedded tissue sections of lesions containing Donovan bodies was evaluated as a serological test for the diagnosis of granuloma inguinale. METHODS: Sera from patients with proven granuloma inguinale, other sexually acquired genital ulcerations and blood donors from areas where granuloma inguinale is rarely encountered as well as from disease-endemic regions were tested. Sera were tested either unabsorbed or following absorption with whole Klebsiella pneumoniae bacteria. RESULTS: Using unabsorbed sera at a dilution of 1:160 the test was found to have a sensitivity of 100%, specificity of 98%, positive predictive value (PPV) of 89% and negative predictive value (NPV) of 100%. There proved to be no advantage in preabsorbing sera with K. pneumoniae antigen. CONCLUSIONS: In the absence of culture methods for Calymmatobacterium granulomatis, an indirect immunofluorescence technique may prove valuable for the diagnosis of individual cases of granuloma inguinale and as an epidemiological tool in studies of the disease. (+info)
Genital ulcer disease in women in Durban, South Africa.
OBJECTIVE: To study the microbial aetiology of genital ulcer disease (GUD) in women. DESIGN: Microbial and clinical assessment of genital ulcers in women. SETTING: City Health sexually transmitted diseases clinic, King Edward VIII Hospital, Durban, South Africa. PARTICIPANTS: 100 Zulu women with genital ulceration who had not received antibiotics in the previous two weeks. RESULTS: Syphilis was diagnosed in 40%, genital herpes in 18%, donovanosis (granuloma inguinale) in 16%, chancroid in 14%, lymphogranuloma venereum in 7% and scabies in 2%. No recognised cause was detected in 18%. Secondary syphilis was diagnosed in 21%, primary syphilis in 16% and mixed primary and secondary syphilis in 3%. Multiple infections were detected in 13 women, of whom 12 had syphilis. Bleeding was observed from the ulcers of 59 during swab collection. Three women had HIV-1 antibodies. Neisseria gonorrhoeae was isolated from the ulcers and endocervix of two women and from the endocervix alone in nine. Generalised scabies was diagnosed in 14. CONCLUSIONS: All the major causes of GUD are prevalent in Zulu women in Durban: secondary syphilis was the commonest diagnosis. Donovanosis, which often presents late with large ulcers, and genital herpes are now significant problems. Mixed infections with coexisting syphilis are common. All women in this population with GUD should be treated for syphilis and receive oral antibiotics effective for chancroid and donovanosis. (+info)
Genital ulcer disease in men in Durban, South Africa.
OBJECTIVE: To study the microbial aetiology of genital ulcer disease (GUD) in men. DESIGN: Microbiological and clinical assessment of genital ulcers in men. SETTING: City Health sexually transmitted diseases clinic, King Edward VIII Hospital, Durban, South Africa. PARTICIPANTS: 100 Zulu men with genital ulcers who had not received antibiotics in the previous four weeks. RESULTS: Syphilis was diagnosed in 42%, chancroid in 22%, donovanosis (granuloma inguinale) in 11%, genital herpes in 10% and lymphogranuloma venereum (LGV) in 6%. No pathogens were identified in 24%. Mixed infections were detected in 14 men, in whom 13 had syphilis. Five men had HIV-1 antibodies. Neisseria gonorrhoeae was isolated from the ulcers and urethra in seven men and from the urethra alone in five. Scabies was diagnosed clinically in eight. CONCLUSIONS: All the major causes of GUD are prevalent in Zulu men in Durban. Primary syphilis was the commonest and was invariably present in mixed infections. Donovanosis was under-reported and was associated with a long delay before presentation. In this population, genital ulcers other than superficial lesions should be treated with anti-syphilitic therapy and oral antibiotics effective against chancroid and donovanosis. (+info)
Detection and discrimination of herpes simplex viruses, Haemophilus ducreyi, Treponema pallidum, and Calymmatobacterium (Klebsiella) granulomatis from genital ulcers.
BACKGROUND: Genital ulcer disease (GUD) is commonly caused by pathogens for which suitable therapies exist, but clinical and laboratory diagnoses may be problematic. This collaborative project was undertaken to address the need for a rapid, economical, and sensitive approach to the detection and diagnosis of GUD using noninvasive techniques to sample genital ulcers. METHODS: The genital ulcer disease multiplex polymerase chain reaction (GUMP) was developed as an inhouse nucleic acid amplification technique targeting serious causes of GUD, namely, herpes simplex viruses (HSVs), H. ducreyi, Treponema pallidum, and Klebsiella species. In addition, the GUMP assay included an endogenous internal control. Amplification products from GUMP were detected by enzyme linked amplicon hybridization assay (ELAHA). RESULTS: GUMP-ELAHA was sensitive and specific in detecting a target microbe in 34.3% of specimens, including 1 detection of HSV-1, three detections of HSV-2, and 18 detections of T. pallidum. No H. ducreyi has been detected in Australia since 1998, and none was detected here. No Calymmatobacterium (Klebsiella) granulomatis was detected in the study, but there were 3 detections during ongoing diagnostic use of GUMP-ELAHA in 2004 and 2005. The presence of C. granulomatis was confirmed by restriction enzyme digestion and nucleotide sequencing of the 16S rRNA gene for phylogenetic analysis. CONCLUSIONS: GUMP-ELAHA permitted comprehensive detection of common and rare causes of GUD and incorporated noninvasive sampling techniques. Data obtained by using GUMP-ELAHA will aid specific treatment of GUD and better define the prevalence of each microbe among at-risk populations with a view to the eradication of chancroid and donovanosis in Australia. (+info)