Mycobacterium ulcerans

Mycobacterium ulcerans (M. Ulcerans) is a slow-growing mycobacterium that classically infects the skin and subcutaneous tissues, giving rise to indolent nonulcerated (nodules, plaques) and ulcerated lesions. After tuberculosis and leprosy, Buruli ulcer is the third most common mycobacteriosis of humans. M. ulcerans grows optimally on routine mycobacteriologic media at 33°C and elaborates a necrotizing immunosuppressive cytotoxin (mycolactone). Large ulcers almost certainly caused by M. ulcerans were first observed by Cook in Uganda in 1897; however, the etiologic agent was not isolated and characterized until 1948 in Australia by MacCallum and associates.

Lesions of M. ulcerans disease have several synonyms (e.g. Bairnsdale or Searle's ulcer). The name Buruli is probably most appropriate for historic reasons, as it is a county of Uganda where important foci of the disease were studied.

Epidemiology and transmission
The source(s) of M. ulcerans in nature is becoming clearer from epidemiologic data and from molecular biologic findings. Because all major endemic foci are in wetlands of tropical or subtropical countries, environmental factors must play an essential role in the survival of the etiologic agent. Koalas and possums are naturally infected animals in Australia. The disease is rarely transmitted from patient to patient. Trauma is probably the most frequent means by which M. ulcerans is introduced into the skin from surface contamination. Individuals of all ages are affected, but the highest frequencies of infection are in children under 15 years of age (Debacker et al. accepted for publication).

The largest known concentrations of patients are in Africa: Congo and Cameroon in Central Africa, Côte d'Ivoire, Ghana and Benin in West Africa. Some Southeast Asian countries (Papua New Guinea) and Australia have major foci, and there have been a few patients reported from South America (French Guyana and Surinam) and Mexico. Focal outbreaks have followed flooding, human migrations, and man-made topographic modifications such as dams and resorts. Deforestation and increased basic agricultural activities may significantly contribute to the recent marked increases in incidences of M. ulcerans infections, especially in West Africa, where the disease is rapidly emerging.

Geographical distribution
Buruli ulcer has been reported from at least 32 countries around the world, mostly in tropical areas:
 * West Africa: Benin, Burkina Faso, Côte d'Ivoire, Ghana, Liberia, Nigeria, Togo, Guinea, Sierra Leone.
 * Other African Countries: Angola, Cameroon, Congo, Democratic Republic of Congo, Equatorial Guinea, Gabon, Sudan, Uganda.
 * Western Pacific: Australia, Papua New Guinea, Kiribati; Americas: French Guyana, Mexico, Peru, Surinam.
 * Asia: China, Malaysia, Japan.

In several of these countries, the disease is not considered to be a public health problem, hence the current distribution and the number of cases are not known. Possible reasons include:
 * the distribution of the disease is often localized in certain parts of endemic countries;
 * Buruli ulcer is not a notifiable disease
 * In most places where the disease occurs, patients receive care from private sources such as voluntary mission hospitals and traditional healers. Hence the existence of the disease may not come to the attention of the ministries of health.

Endemic regions and association with water
In many areas, M. ulcerans infection has only occurred after significant environmental disturbance. In the original paper describing the disease, published in 1948, the first patients from the Bairnsdale District in Victoria presented in 1939 (13). In December 1935, there had been terrible floods in the district, when all road and rail links had been cut and there had been considerable destruction of property. In Uganda, Barker examined cases of M. ulcerans infection (Buruli ulcer disease) occurring in the Busoga District on the east side of the Victoria Nile, north of Lake Victoria. Although cases were known in the other parts of the country, cases were unknown in the district before 1965. Barker postulated that the outbreak was related to the unprecedented flooding of the lakes of Uganda between 1962 to 1964 as a result of heavy rainfall.

In Nigeria, cases have occurred among Caucasians living on the campus of University of Ibadan only after 1965, when a small stream flowing through the campus was dammed to make artificial lake. The first case reported in Côte d'Ivoire was a French boy of seven years who lived with his parents beside lake Lake Kossou, an artificial lake in the center of the country. In Liberia, cases have been reported in the north of the country following the introduction of swamp rice to replace upland rice. This introduction has been associated with construction of dams on the Mayor river and extended wetlands. In Papua New Guinea, the infection occurs mainly in relation to the Sepik and Kumusi rivers; in the later areas, the disease is known as the "Kumusi ulcer" (20). The disease occurred after flooding and devastation, which followed the eruption of Mount Lamington in 1951. Reid described how older people living in the villages blamed the volcano for the disease. The recent outbreak of the disease on Philip Island, Victoria (21), was initially associated with the building of a roadway, inadvertently forming marshlands at the headwaters of an estuary, which was divided by the construction. Again in Australia, the recent increase in the number of cases between 1991 and 1994 in Victoria was associated with the use of recycled wastewater to irrigate a golf course.

A recent visit to Papua New Guinea has not identified any case along the Fly River, that country's largest river, despite significant environmental disturbance due to mining operations in the headwaters. It is clear that other factors must be responsible apart from simple disturbance, one of these must be formation of new water areas where the water is stagnant or only slow moving. A delay between one or three years occurs between the environmental changes and the first patients appearing. Severe flooding has occurred again in the last few days in the Bairnsdale District of Australia, exceeding the severity of the floods of 1935. It will be interesting to see if this disaster is again followed by increased numbers of patients with M. ulcerans infection.

Seasonal variation
A series of epidemiological studies show the existence of seasonal variation in the appearance of Buruli ulcer cases. It seems that the number of cases augments during dry periods or after inundations (22) (23). These conditions are probably favorable for the development of M. ulcerans, because of the concentration of possible vectors in areas that are frequently visited by humans.

Reservoir(s) and mode(s) of transmission
The disease often occurs in close proximity to water bodies, but no specific activities that bring people into contact with water have been identified (i.e. fetching of water, fishing, washing, bathing, etc). The mode of transmission of Buruli ulcer is not entirely known. Recent evidence suggests that insects may be involved in the transmission of the infection. These insects are aquatic bugs belonging to the genus Naucoris (family Naucoridae) and Diplonychus (family Belostomatidae). Trauma is probably the most frequent means by which M. ulcerans is introduced into the skin from surface contamination. The initial trauma can be as slight as a hypodermic needle puncture or as severe as gunshot or exploding land mine wounds. Other studies have suggested aerosol spread but these are not proven. In Australia, animals such as koalas and possums are naturally infected. Epidemiological evidence has not clearly supported person-to-person transmission. However, Muelder & Nourou found that 10 out of 28 patients had relatives whom had also had the disease, and cautioned against the dismissal of person-to-person transmission. Given the number of patients who shed large numbers of bacilli from their wounds and live in very close contact with relatives, more cases should have been observed. The cases reported by Muelder & Nourou could perhaps have been exposed to a common source of infection.

After considering the various suspected agents, Portaels et al. proposed the hypothesis that human beings as well as domestic and wild animals could be contaminated or infected by biting insects such as water bugs. Aquatic bugs are cosmopolite insects found throughout temperate and tropical regions especially rich in freshwater. They represent about 10% of all species of Hemiptera associated with water and belong to two series of the suborder Heteroptera: the Nepomorpha, which include four superfamilies whose members spend most of their time under water, and the Naucoridea, which include a single family, the Naucoridae, whose members are commonly termed creeping water bugs.

Whether found in temperate countries like France or tropical ones like Ivory Coast, aquatic bugs exhibit the same way of life, preying, according to their size, on mollusks, snails, young fish, and the adults and larvae of other insects that they capture with their raptorial front legs and bite with their rostrum. These insects can inflict painful bites on humans as well. In the Ivory Coast, where Buruli ulcer is endemic, the water bugs are present in swamps and rivers, where human activities such as farming, fishing, and bathing take place. Present findings describing the experimental transmission of M. ulcerans from water bugs to mice are in good agreement with the possibility of this mode of transmission to humans by bites.

Also in strong support of this hypothesis was the localization of M. ulcerans within the salivary glands of Naucoridae. Local physiological conditions of this niche appear to fit the survival and the replication needs of M. ulcerans but not those of other mycobacteria. Surprisingly, infiltration of the salivary glands of Naucoridae by M. ulcerans does not seem to be accompanied by any tissue damage similar to the ulcerative skin lesions developed by bitten individuals and mediated by the cytotoxic activity of the mycolactone and other toxins produced by M. ulcerans. The inactivation of the latter toxins could be the result of salivary enzymatic activities, which remain to be determined.

Race, age and sex
Buruli ulcer commonly affects poor people in remote rural areas with limited access to health care. The disease can affect all age groups, although children under the age of 15 years (range 2-14 years) are predominantly affected. There are no sex differences in the distribution of cases among children. Among adults, some studies have reported higher rates among women than males (Debacker et al. accepted for publication). No racial or socio-economic group is exempt from the disease. Most ulcers occur on the extremities; lesions on the lower extremities are almost twice as common as those on the upper extremities. Ulcers on the head and trunk accounted for less than 8% of cases in one large series (37).

Clinical manifestations
Lesions are usually single and begin as firm, painless, non-tender, movable, subcutaneous nodules 1 to 2 cm in diameter or as small papules. Many patients complain of itching in the lesion. In 1 or 2 months, the nodule may become fluctuant and ulcerates, with an undermined edge that often extends 15 cm or more. The skin adjacent to the lesion, and often that of the entire corresponding limb, may be indurated by edema. Ordinarily, no regional lymphadenopathy or systemic manifestations are noted. There is, however, speculation that massive disseminated lesions may cause systemic toxic effects. Ulcers may remain small and heal without treatment, or may spread rapidly, undermining the skin over large areas, even an entire leg, thigh, or arm. Important structures such as the eye, breast, or genitalia are sometimes severely damaged. Most lesions heal spontaneously but without appropriate therapy frequently leave extensive scarring, with deformity. Osteomyelitis may lead to amputation.

Symptoms
After inoculation into the skin, M. ulcerans proliferates extracellular and elaborates a toxin, mycolactone, that enters the cells and causes necrosis of the dermis, panniculus, and deep fascia. Early lesions are closed, but as the necrosis spreads, the overlying dermis and epidermis eventually ulcerate, with undermined edges and a necrotic slough in the base of the ulcer. Histopathologic sections reveal a contiguous coagulation necrosis of the deep dermis and panniculus, with destruction of nerves, appendages, and blood vessels. Interstitial edema is noted. Clumps of extracellular acid-fast bacilli are plentiful and are frequently limited to the base of the ulcer and adjacent necrotic subcutaneous tissue. Bone is sometimes involved, and specific osteomyelitis is well known. Histologically there may be local and regional lymphadenitis with invasion by M. ulcerans. In active lesions, inflammatory cells are conspicuously few, presumably as a result of the immunosuppressive activity of the toxin. With healing, there is a granulomatous response, and the ulcerated area is eventually replaced by a depressed scar. HIV infection does not seem to predispose to Buruli ulcer but may facilitate dissemination of M. ulcerans (38).

Mycolactone
The major virulence determinant in M. ulcerans is a polyketide-derived macrolide: mycolactone. Mycolactone was originally isolated from M. ulcerans 1615, a Malaysian isolate, as a mixture of cis/trans isomers designated mycolactone A and mycolactone B. Identical molecules were also found to be present in two M. ulcerans isolates from the Democratic Republic of Congo (39). More recent evidence shows that M. ulcerans 1615 produces a family of mycolactone congeners which differ primarily in the number of hydroxyl groups and double bonds (40).

Mycolactone appears to play a key role in the pathogenesis of Buruli ulcer. In vivo studies using a guinea pig model of infection suggest that mycolactone is responsible for both the extensive tissue damage and immunosuppression which accompanies Buruli ulcer (39). The activity of mycolactone on cultured fibroblasts and macrophage cell lines produces a distinct cytopathic phenotype. The earliest effect is cell rounding, which occurs within 10 h after addition of mycolactone to cultured cells. At 36 h, treated cells are arrested in G1 of the cell cycle, and by 72 h, cells begin to die via apoptosis (35).

Bacterial macrolides are produced as secondary metabolites by soil bacteria, particularly bacteria such as Streptomyces and Saccharopolyspora species in the order Actinomycetales (41). Interestingly, a number of related macrolides or congeners are often produced by a single bacterial isolate (42).

Classical approach for identification of mycobacteria
According to the traditional methods, mycobacteria are preliminarily identified by growth rate and pigmentation (43). This preliminary grouping may provide presumptive identification of the organism and directs the selection of key biochemical tests to characterize an unknown mycobacterium (44) (45).

Clinical diagnosis
Because M. ulcerans infection is associated with nonspecific clinical manifestations and indolent course, it is important to consider every nodule or ulcer in an endemic area as a suspected M. ulcerans infection until proven otherwise. A nodule is firm and painless. In the absence of superinfection(s) an ulcer is painless or minimally painful, has the characteristic undermined edge and a whitish-yellow necrotic base. Previous residence in an endemic area should raise the suspicion of M. ulcerans infection.

Laboratory diagnosis

 * Smears from the necrotic base of ulcers stained by the Ziehl-Neelsen method often reveal clumps of acid-fast bacilli (AFB). Appropriately selected biopsy specimens that include the necrotic base and the undermined edge of lesions with subcutaneous tissue are nearly always diagnostic.
 * M. ulcerans can be cultured from many lesions, either from exudates or tissue fragments, but visible growth often requires 6 to 8 weeks incubation at 33°C.

Appropriately selected tissue specimen that include necrotic subcutaneous tissue and the undermined edge of ulcerated lesions are frequently diagnostic. Specimens from skin and subcutaneous tissue from nonulcerated lesions are likewise often diagnostic.

Modern diagnostic techniques
Buruli ulcer is often diagnosed late, when treatment can be very difficult and frustrating. Confirmation by culture takes 6-8 weeks. Rapid diagnostic methods for M. ulcerans infection, as well as methods of rapid identification of the organism in clinical and environmental specimens would be a significant advance in the management of M. ulcerans infection. Screening to detect early infection could guide early intervention.

Polymerase chain reaction (PCR) in diagnosis
There are several PCR methods available that could increase the speed of diagnosis of M. ulcerans infection (46). PCR is relatively expensive compared to microscopy, and is notorious for producing false-positive results in laboratories that lack experience with PCR. In high-prevalence regions such as West Africa, PCR may not be any more rapid than an accurate clinical case definition combined with a smear that shows acid-fast bacilli. In countries such as Australia, where the incidence is low, the great majority of patients who have nodules, papules or skin ulcers do not have M. ulcerans disease. In this situation, PCR is a quicker way of making the diagnosis with a high degree of confidence. The main advantage of PCR is that M. ulcerans disease can be diagnosed within 24 hours. PCR usefulness for mycobacterial infections is generally limited, however, and at present it is recommended that PCR is used as a rapid ancillary test, not as a replacement for culture and histology.

The PCR method developed by Stinear et al. (47) targets a DNA insertion sequence in M. ulcerans. When genomic M. ulcerans DNA is digested with the restriction enzyme AluI, many 1109 base-pair fragments were obtained. These AluI fragments have been shown to be part of a larger 1293 base-pair repeated sequence that, by chance, happened to contain two AluI restriction sites. The sequence has been named IS2404 (Genbank accession number AF003002) (48) (47). It has been recently discovered that IS2404 copies are also present in a large circular plasmid. The total number of IS copies is thus 220. It has been identified in all isolates of M. ulcerans tested to date and has not been found in at least 45 other mycobacterial species, including M. marinum, M. leprae and M. tuberculosis. Recent publications have however demonstrated the presence of IS2404 in M. marinum-like bacteria (Trott et al. accepted for publication) (49).

PCR methods that have been developed are based on the 16S rRNA gene (50), the hsp65 gene (31), or the insertion sequence IS2404 (48). In 1999, Guimaraes-Peres et al. (51) evaluated two nested PCRs: the nested IS2404-based PCR and the nested 16S rRNA gene-based PCR. IS2404-based PCR was positive only with M. ulcerans isolates and the closely related M. shinshuense. The 16S rRNA gene-based PCR was positive not only for these two strains but also for M. marinum. The use of IS2404-based PCR as a detection method for M. ulcerans showed better sensitivity and specificity, required less time, and was less costly than the 16S rRNA gene-based PCR (52).

To date, it has been established that PCR has a specificity of 100% and a sensitivity of 96% compared with culture.

PCR for detecting M. ulcerans in environmental samples
M. ulcerans belongs to the group of occasional pathogens. Most species belonging to this group are found almost everywhere in nature, and may become pathogenic under special circumstances. Some of them have rarely (e.g. M. malmoense) or never (M. ulcerans) been isolated from the environment. The epidemiological profiles of the diseases they cause, however, suggest that they are present in nature (53). Recently, M. ulcerans has been detected by molecular biological techniques in water samples collected in Australia (54) (55) and in bugs collected from roots of aquatic plants in swamps in endemic regions of Benin and Ghana (24). M. ulcerans was, however, not recovered by culture from these environmental samples.

PCR is not inhibited by the presence of culturable organisms. Unfortunately, PCR is exquisitely sensitive to inhibition by many compounds such as humic and fulvic acids, which are ubiquitous in the environment and are not removed by standard DNA extraction protocols. The first confirmation that M. ulcerans was present in environmental water samples was obtained in 1997 (54), by combining the highly sensitive and specific IS2404 PCR with a method that separated sample DNA from naturally occurring inhibitors of PCR.

Three different strategies have now been used to overcome inhibition in environmental samples from M. ulcerans endemic regions. The first of these is gel chromatography. Environmental water samples are concentrated and subjected to homogenization with glass beads, followed by heat and alkaline lysis to release DNA. Total extracted DNA is then run through gel chromatography columns that separate DNA from contaminants on the basis of size (48). Although relatively simple, the method is cumbersome and time-consuming. The second method uses paramagnetic beads linked to M. ulcerans antibodies to capture whole cells and separate them from contaminants in a magnetic field (immunomagnetic separation) (31). Antibodies are raised in laboratory animals. Captured cells are washed to remove inhibitors and then DNA is released by standard methods prior to PCR. The third approach also uses paramagnetic beads, but here the beads are linked to M. ulcerans-specific oligonucleotide probes, which capture IS2404 DNA that has been released from M. ulcerans by homogenization and alkaline lysis. The immobilized DNA is washed to remove inhibitors and used directly as a template for IS2404 PCR. The latter two methods each have limitations and advantages, but offer superior detection sensitivity and are less time-consuming than gel chromatography.

DNA fingerprinting techniques for M. ulcerans
Molecular typing methods may be categorized into three broad groups on the basis of the type of macromolecules targeted for sub-typing, i.e. methods based on fatty acids, proteins and nucleic acids. Actually, the genotypic typing methods (DNA fingerprinting) that evaluate differences at the DNA level are used more commonly and have emerged as revolutionary tools for epidemiological studies.

The use of DNA fingerprinting for the identification of M. tuberculosis has greatly improved understanding of the epidemiology of tuberculosis: transmission routes of different strains have been recognized (56); outbreaks of multidrug-resistant strains have been detected early; and the relative importance of reinfection versus reactivation can now be elucidated (57).

Various molecular methods for fingerprinting of M. ulcerans are now being developed to facilitate studies on the epidemiology of Buruli ulcer.

DNA sequencing
Direct comparison of some genomic DNA sequences of bacterial strains is the best means of quantitatively determining whether two strains are similar or different. Portaels et al. have analyzed the 3’-terminal region of the 16S rRNA gene sequence of 17 strains of M. ulcerans from Africa, Australia and America (58). This analysis has revealed three subgroups that vary according to the continent of origin. Later, a fourth subgroup was discovered in China and Japan confirming the existence of an Asian type (59).

RFLP based on insertion sequences
Insertion sequences (IS) are mobile genetic elements that are usually present in numerous copies within a bacterial genome. These elements can be used as probes, and because the number and location of IS elements vary, each strain will have a unique banding pattern. Molecular analysis of M. ulcerans has revealed two insertion sequences: IS2404 and IS2606 (47). Southern blot analysis to detect IS2404 and IS2606 shows inconclusive RFLP patterns between different strains. Due to the high number of copies of both elements, the banding patterns are difficult to interpret, limiting the value of the Southern blot method to type M. ulcerans isolates (47).

RFLP based on pTBN12 plasmid
Jackson et al. have used pTBN12, a well-defined plasmid, as a probe with AluI restriction fragments (60). The probe was able to distinguish 11 RFLP patterns.

Pulsed field gel electrophoresis (PFGE)
PFGE permits the generation of simplified chromosomal restriction fragment patterns without having to resort to probe hybridization methods. In this method, restriction enzymes that cut DNA infrequently are used to generate large fragments of chromosomal DNA, which are then separated by special electrophoretic procedures. Preliminary results showed that M. ulcerans genomes produce three different profiles according to the three geographical origins of the strains (Type I: Africa, Type II: Australia and Type III: North America) (61)

Amplified fragment length polymorphism (AFLP)
The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA (62). This technique involves three steps: restriction of DNA and ligation of oligonucleotides and adaptors; selective amplification of sets of restriction fragments; and gel analysis of the amplification fragments. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gel. AFLP typing results in a clear distinction of M. marinum from M. ulcerans, but interspecies differentiation is not trustworthy (63)

PCR typing methods
PCR is another molecular method that has become increasingly important for epidemiological studies. The technique detects and amplifies small amounts of DNA; 10-100 copies of the templates are enough to perform DNA amplification. Thus, PCR can be used to type organisms that grow slowly on laboratory media, such as M. tuberculosis (64). PCR also can be used to detect and type pathogens in patients whose culture are negative because they have been treated. Moreover, PCR can be used to amplify the DNA from organisms that are present in tissues preserved in formalin (65) and from non-cultivable organisms (e.g. M. leprae).

PCR of repetitive chromosomal elements (Rep-PCR)
Rep-PCR is a modification of the PCR technique that is more suitable for epidemiological purposes than conventional PCR. In this case, the primers are directed towards repetitive chromosomal elements such as IS6110 in M. tuberculosis and the ERIC sequence in other bacteria (60). In M. ulcerans, the genomic sequence between the IS2404 elements has been amplified. The profiles produced by this technique categorized the strains into three subgroups related to the three different endemic regions (Africa, Australia and North America).

Ribotyping
This method involves amplification of a known sequence cut by restriction enzymes, and compares restriction fragments of amplified DNA from different strains. Using this technique, the M. ulcerans genome has been found to produce three different restriction profiles related to the origin of the strains.

Treatment
Preulcerative lesions are excised, and the skin is closed primarily. Ulcers are widely excised, and skin grafts applied. Continuous local heating to 40°C (e.g. by circulating water jackets) promotes healing, even without excision. Amputation of limbs is sometimes necessary. Appropriate physiotherapy is important when contracture deformities are likely to develop. Recent studies demonstrated some efficacy of rifampin and streptomycin in treating early lesions, without surgery (Chauty et al, unpublished results). Rifampin promotes healing of pre-ulcerative lesions or early ulcers but is often not effective for extensive lesions.

Prevention
No effective prophylactic measures have been demonstrated. The Bacille of Calmette-Guérin’ (BCG) vaccination may produce protection or delay onset of lesions for approximately 6 months (66) and may protect against severe forms of the disease such as osteomyelitis (67).