The changing epidemiology of genital ulcer disease in South Africa: has donovanosis been eliminated?

OBJECTIVES: We used an in-house molecular assay for the detection of Klebsiella granulomatis in ulcer specimens collected over a 12-year surveillance period in order to determine whether a diagnosis of donovanosis could be ascribed to genital ulcer disease (GUD) of unknown aetiology in our setting. METHODS: Between 2007 and 2018, a total of 974 genital ulcer specimens with no previously identified sexually transmitted (STI) pathogens were selected from STI aetiological surveys conducted in all nine provinces of South Africa. Giemsa-stained ulcer smears from the same participants had previously been routinely analysed for the presence of typical Donovan bodies within large mononuclear cells. A Klebsiella screening assay targeting the phoE (phosphate porin) gene was used in combination with restriction digest analysis and sequencing to confirm the presence of K. granulomatis. RESULTS: The Klebsiella screening assay tested positive in 19/974 (2.0%) genital ulcer specimens. Restriction digest analysis and nucleotide sequencing of the phoE gene confirmed that none of these specimens was positive for K. granulomatis DNA. Similarly, Donovan bodies were not identified in the Giemsa stained ulcer smears of these specimens. CONCLUSIONS: This is the first study to assess K. granulomatis as a cause of genital ulceration in South Africa over a 12-year surveillance period using molecular methods. The results demonstrate that K. granulomatis is no longer a prevalent cause of GUD in our population.

Extragenital granuloma inguinale mimicking a soft-tissue neoplasm: a case report and review of the literature.

Granuloma inguinale has been reported to mimic squamous cell carcinoma, as well as sexually transmitted diseases. We describe a rare case of extragenital granuloma inguinale that mimicked a soft tissue neoplasm in an 18-year-old pregnant woman who presented with cervical and labial ulcers. An elective cesarean section was performed, at which time a retroperitoneal mass that involved the right ureter was found. At frozen section, the mass had a xanthogranulomatous appearance consistent with a soft-tissue neoplasm. Special stains of the permanent sections revealed the pathognomic features of granuloma inguinale.

Ultrastructure of Calymmatobacterium granulomatis: comparison of culture with tissue biopsy specimens.

The ultrastructural features of cells of Calymmatobacterium granulomatis from monocyte co-cultures and tissue biopsy specimens were compared. In cultures the bacteria were mainly extracellular, i.e., not within membrane-bound vacuoles. The bacterial body was surrounded by a uniformly extensive homogeneous layer with a relatively high electron density. This layer varied considerably in tissue biopsy specimens, having either homogeneously electron-dense or delicate web-like structures with varying density and thickness. In tissue specimens the bacteria were located predominantly within vacuoles of varying sizes in the cytoplasm of the macrophages and, occasionally, extracellularly within the intercellular spaces of the stroma. The bacterial cytoplasm contained ribosomes scattered throughout with electron-dense granules located peripherally. The trilaminar cell-wall structure was typical of a gram-negative organism, comprising an outer membrane, a middle electron-opaque layer and an inner plasma membrane. Surface structures such as fimbriae, flagella and bacteriophages were not identified in specimens from either source.

Growth and cultural characteristics of Calymmatobacterium granulomatis--the aetiological agent of granuloma inguinale (Donovanosis).

Granuloma inguinale is a chronic destructive granulomatous disease of the genitalia. The clinical diagnosis is often unreliable and the definitive diagnosis is based on the visualisation of 'Donovan bodies' in tissue smears or biopsy specimens. The organism implicated in its aetiology, Calymmatobacterium granulomatis, was reported to have been cultured > 30 years ago, but little is known about the organism because of its fastidious nature and the difficulty in culturing it. Twenty-two biopsy specimens from female patients with clinical and laboratory-confirmed granuloma inguinale were treated with amikacin 10 mg/L and inoculated in a monocyte co-culture system with peripheral blood mononuclear cells (PBMC) from a single donor and autologous sera. The method was subsequently modified by pretreatment of specimens with vancomycin 5 mg/L and metronidazole 10 mg/L in addition to amikacin 10 mg/L for the purpose of decontamination, pooled blood donor PBMC and by the use of heat-inactivated fetal calf serum instead of autologous serum for culture. This modified method was used to culture additional biopsy specimens and genital ulcer scrapings from female and male patients, respectively. All monocyte co-cultures were examined by a rapid Giemsa (RapiDiff) stain and by an indirect immunofluorescence test with immune sera. Representative cultures were examined by transmission electron microscopy. C. granulomatis was successfully isolated in pure culture by the monocyte co-culture system from four biopsy specimens and 14 genital ulcer scrapings. The cultured organisms were visible both intra- and extra-cellularly and were extremely pleomorphic, with characteristic single and biopolar condensation. The numbers of the organisms increased after each passage. All positive cultures showed bright fluorescence when tested with immune sera. Transmission electron microscopy of the cultured bacteria demonstrated a typical gram-negative cell wall consisting of an outer membrane, middle electron opaque layer and an inner plasma membrane. The capsule was thick and electron dense. Numerous electron dense granules were present within the cytoplasm.

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.

Biology of sexually transmitted diseases.

The clinical concept of sexually transmitted diseases describes a common mode of transmission of pathogens spanning the full spectrum of medical microbiology. These organisms have few common biologic characteristics aside from their ability to colonize or invade particular anatomic sites. New information on the biology of sexually transmitted pathogens has led to improved diagnostic and therapeutic strategies. Understanding that patients with sexually transmitted pathogens may be asymptomatic or may be simultaneously infected with multiple organisms has important implications for epidemiologic control of these diseases.

Chancroid and granuloma inguinale.

Haemophilus ducreyi is a fastidious pathogen that can be routinely cultured with the appropriate media and incubation environment. Prostituted women appear to be the usual reservoir. In Africa, chancroid is emerging as the major risk factor for acquisition of HIV-1 following heterosexual intercourse. Despite the emergence of resistance to a number of antimicrobial agents, H. ducreyi remains susceptible to ceftriaxone, erythromycin, and ciprofloxacin. Control and eradication of outbreaks of chancroid have been successful on several occasions in Western societies; strategies to control epidemic genital ulcer disease are required in developing countries. Granuloma inguinale is caused by a small, gram-negative rod, which has never been well-characterized. The presence of Donovan bodies is a specific and sensitive diagnostic characteristic. Very little is known about the epidemiology of the disease. Specific treatment programs using trimethoprim-sulfamethoxazole or erythromycin seem to be quite effective. Control strategies have not been adequately investigated.

Laboratory techniques used in the diagnosis of chancroid, granuloma inguinale, and lymphogranuloma venereum.

The preceding discussion summarized the historical, modern, and evolving laboratory techniques for the diagnosis of chancroid, GI, and LGV. The correct diagnosis of these sexually transmitted diseases, which are uncommon in the United States and Europe but often endemic in Africa, Asia, and South American, can usually be made if appropriately selected laboratory techniques are used to confirm the presumptive clinical impression.

Donovanosis: an update.

Donovanosis has been ignored for many years until recently. The condition still has a limited geographical distribution. A significant epidemic of donovanosis has been identified in KwaZulu/Natal, South Africa where it may be a risk factor for acquiring HIV in men. After a gap of more than 30 years, the organism was cultured by researchers in Durban, South Africa and Darwin, Australia. Polymerase chain reaction (PCR) techniques for donovanosis were developed soon after, most recently using a colorimetric detection system. Similarities between the causative organism, Calymmatobacterium granulomatis and Klebsiella spp. were confirmed. A proposal that the organism be reclassified under the genus Klebsiella has been put forward. Azithromycin has been confirmed as the drug of choice but is yet to be accepted universally because of cost issues. Treatment in patients with significant HIV induced immune deficiency may need to be prolonged. A donovanosis eradication programme is underway amongst the aboriginal community in Australia. Elsewhere, management through current syndromic guidelines for genital ulcers are yet to be validated in areas where donovanosis is endemic. PCR testing should enable further recognition of donovanosis and lead to more concerted efforts in disease control and possible eradication.

Utility of semithin sections in the diagnosis of donovanosis.

The relative merits of tissue smears, paraffin sections and semithin plastic embedded sections in the diagnosis of donovanosis were assessed. In three cases reported negative or doubtful for the organisms by the former two methods, semithin plastic sections provided definite diagnosis. The latter method revealed in addition the organisms in epidermis and perivascular locations. From amongst the three stains: toluidine blue, Leishman's and thionine azure II basic fuchsin, the last mentioned was found to be the method of choice for demonstrating the organisms differently from the host cells.

An ultrastructural study of donovanosis.

In an ultrastructural study undertaken on donovanosis, the inflammatory response was studied along with the fine structure of causative organisms, Calymmatobacterium granulomatis. Macrophages were seen to be activated and showed presence of numerous filopodia and increase in the number of lysosomes and rough endoplasmic reticulum. Many cells showed vacuoles of varying size in the cytoplasm some of which contained the organisms. Other cells seen included plasma cells, polymorphonuclear cells and sparse lymphocytes. In one case few multinucleated giant cells and dendritic cells with long cytoplasmic processes making cell-cell contact with other inflammatory cells were seen. Such cells have not been described in donovanosis so far. The organisms showed surface structures like pili and vesicles, the role of which is yet to be clearly understood.

Cytologic identification of Donovan bodies in granuloma inguinale.

The cytologic findings in two histologically confirmed cases of granuloma inguinale are presented. The Papanicolaou staining method used in routine cytology screening proved to be suitable for the detection of the characteristic Donovan bodies in smears of the vulvar lesions.

Squamous cell carcinoma complicating donovanosis not a thing of the past!

Donovanosis causes granulomatous ulceration of genitalia and neighbouring sites with little tendency to heal spontaneously. It is uncommonly seen nowadays in sexually transmitted infection clinics of north India. The present case is reported for its scarcity and to make clinicians aware of this disease which may rarely accompany carcinoma.

Chancroid, lymphogranuloma venereum, granuloma inguinale, genital herpes simplex infection, and molluscum contagiosum.

Chancroid, lymphogranuloma venereum, and granuloma inguinale may be considered as tropical venereal diseases. These diseases were a major diagnostic and therapeutic challenge in past centuries. Currently, patients with these bacterial infections that are endemic to the tropics occasionally consult with dermatologists in temperate climates. Due to the increasing frequency of travel to the tropics for tourism and work, as well as the increasing number of immigrants from these areas, it is important for dermatologists practicing in temperate climates to be familiar with the dermatologic manifestations of such infections, to be prepared to diagnose these diseases, and to treat these patients. All three "tropical" infections respond well to prompt and appropriate antimicrobial treatment, although herpes progenitalis still cannot be cured, and the number of people infected keeps growing; moreover, genital herpes can be transmitted by viral shedding before and after the visual signs or symptoms. Acyclovir, valacyclovir, and famciclovir can shorten outbreaks and make them less severe or even stop them from happening. There is currently no etiologic treatment for molluscum contagiosum, and the majority of treatment options are mechanical, causing a certain degree of discomfort. The molluscum contagiosum virus, unlike the other infectious agents mentioned, does not invade the skin.

Cutaneous metastases of rectal mucinous adenocarcinoma mimicking granuloma inguinale.

A 46-year-old man complained of ulcerovegetative lesions in the anogenital region, which he had noted one month prior to presentation. The patient had a history of travel to African countries. Therefore, the ulcerovegetative lesions of the patient were suspected to be granuloma inguinale (GI). Calymmatobacterium granulomatis was not observed in the direct examination of scrapings collected from the base of the ulcerovegetative lesion. Instead, a histological examination revealed cutaneous metastasis of mucinous adenocarcinoma of the rectum. Therefore, a diagnosis of GI was eliminated. As the patient did not report his history of rectal cancer and had travelled to African countries, we had primarily focused on the diagnosis of GI.