Institut Pasteur Unit\351 de G\351n\351tique Mol\351culaire



 

 The following text has been extracted from the PhD thesis of Timothy Stinear, entitled 'Molecular and Environmental Aspects of Mycobacterium ulcerans. Department of Microbiology, Monash University, Clayton, Australia.



Creation of this page and adaptation of the text by Florence Martin.






    Contents



1. Basic Microbiology

2. Molecular microbiology

         2.1 Molecular taxonomy

         2.2 Diagnosis

         2.3 Molecular epidemiology

3. Clinical presentation

4. Pathology

5. Human immune response to infection

6. Treatment

7. Epidemiology

         7.1 Geographic distribution

               a. Africa

               b. Australia and Pacific region

               c. Papua New Guinea (PNG)

               d. Southeast Asia

               e. Asia and the Americas

               f.Other

         7.2 Age/sex distribution

         7.3 Transmission

         7.4 Seasonality

8. Environmental source

         8.1 Studies in Uganda

         8.2 Studies in the Congo (Kinshasa)

         8.3 Other studies in Africa

         8.4 Papua New Guinea

         8.5 Studies in Australia

9. References

The first detailed clinical descriptions of ulcers caused by Mycobacterium ulcerans have been attributed to Dr Albert Cook, a missionary physician who worked in Uganda in the late 1800s. The images below include his collection of writings and an example of one of his patient reports, depicting several lower leg ulcers. These images have been reproduced from the slide collection of Prof. John A. Hayman.

 

         albertcook
                     albertcook



                     albertcook





1. Basic Microbiology

    Taxonomically and physically, M. ulcerans is a slow growing mycobacterium. By 16S rRNA sequence analysis it has the long version of helix 18 (Escherichia coli positions 451-482) (67) which is usually, but not exclusively, associated with slow growing mycobacteria (51). The generation time from primary culture is about 48 h (79)but secondary cultures exhibit generation times as short as 20 h, depending on the size of the inoculum used for subculture (107). Several phenotypic characteristics permit differentiation of M. ulcerans from other slow growing mycobacteria. Most remarkable is its lack of reactivity with the standard biochemical tests. It grows optimally at 33°C, and poorly or not at all above 35°C. The organism is resistant to isoniazid but most strains are inhibited by the presence of hydroxilamine and p-nitrobenzoate (80). While the growth rate is slow, M. ulcerans will grow readily on most egg-based media with Lowenstein-Jensen medium being optimal. It will also grow easily in Middlebrook 7H9 broth media, supplemented with albumin, dextrose and catalase. The organism will also grow on defined, protein-free, minimal media such as Sauton’s broth or agar provided it is supplemented with Tween 80 (144). Growth has been shown to be optimal under microaerophilic conditions (101).

2. Molecular microbiology

    Disease caused by M. ulcerans is uncommon in most developed countries and for this reason detailed molecular studies of the organism are scarce. Most of the knowledge of M. ulcerans at a molecular level has come only in the last five years, primarily from research focussed on developing tools for either rapid diagnosis or molecular epidemiological investigation.

      2.1 Molecular taxonomy

    One of the first findings from molecular taxonomic classification of M. ulcerans was the discovery of an unexpectedly close genetic relationship with M. marinum . Analysis of the 16S rRNA gene revealed greater than 99.8% sequence identity between the species. The only sequence differences within this gene are two single nucleotides at the 3’ end and the nucleotide at one of these positions is only variant from M. marinum for some strains of M. ulcerans (115). The next most closely related species is M. tuberculosis (98% sequence identity) (143). Biochemically, M. ulcerans has also been found to resemble M. marinum. Both species have identical mycolates and phenolic glycolipids (29), (30). However there are also substantial phenotypic differences (144). M. marinum is relatively fast growing, photochromogenic, and capable of Tween 80 hydrolysis while M. ulcerans is not. M. marinum can also utilise glucose, succinate, pyruvate, acetate and ethanol as sole carbon sources while M. ulcerans can not (144). There are also dramatic differences in the pathologies of the diseases caused by each of these organisms.

      2.2 Diagnosis

    The first PCR method described for detection of M. ulcerans was based on amplification of a region of the 16S rRNA gene (113). The technique required hybridisation of the PCR amplicon with an oligonucleotide probe to ensure specificity. However because of the near complete identity between M. ulcerans and M. marinum within this gene, the test was unable to discriminate between the species. Similarly, a nested PCR test, designed within a region of groEL (65 kDa heat shock gene) (128)was also unable to distinguish between M. ulcerans and M. marinum because of the very high sequence identity within this gene (112). The discovery of a highly repeated AluI fragment in M. ulcerans that was apparently species-specific provided an ideal target for a diagnostic PCR test (130). In comparison with culture it was a superior test with increased sensitivity and importantly rapidity (55). Further identification and characterization of this repeated sequence revealed it to be a new insertion sequence (IS). PCR screening of mycobacteria demonstrated that M. ulcerans also contains IS6110 (81). This element is widely used for diagnosis and genotyping of M. tuberculosis (136). It has also been shown to be important in driving the evolution of epidemic clones of M. tuberculosis . The significance of IS6110 in M. ulcerans is unknown.

      2.3 Molecular epidemiology

    Early taxonomic studies of M. ulcerans were confounded by the lack of reactivity of the organism to nearly all the standard biochemical tests used to distinguish between slow growing mycobacteria. While some drug sensitivity and other biochemical differences were observed between isolates from different regions these were not sufficient to provide a sound basis for sub-species differentiation (102), (145). Strain analysis using immunodiffusion techniques were useful in confirming that African, Mexican, Malaysian and Australian strains belonged to the same species but the test produced no observable antigenic variation for sub-species determination (137).
    Restriction fragment length polymorphism (RFLP) analysis with a probe derived from the polymorphic GC- rich repeat sequence (PGRS) region of M. tuberculosis displayed strain-specific patterns of multiple bands in M. ulcerans (69). This provided the first evidence of the existence of discrete geographic subtypes or genotypes of M. ulcerans and simultaneously confirmed the presence of multiple copies of the PGRS locus in M. ulcerans. The PGRS are sequence motifs that belong to the PE family of glycine-rich proteins (117). Analysis of the M. tuberculosis genome indicates that one tenth of the coding potential of the chromosome is occupied by this and one other family of glycine-rich proteins (PPE family) (24). The function of these proteins is unknown, although it has been speculated that they may be important virulence determinants for the bacterium either by providing a source of antigenic variation via strand slippage or recombination, or by inhibition of MHC class I antigen processing and presentation pathways (24). Given the apparent high copy number of the PE genes in M. ulcerans, it is likely that these proteins may also play an important role in the pathogenesis of this organism.
    Analysis of the 3’ region of the 16S rRNA gene from different strains of M. ulcerans also revealed a correlation between sequence type and geographic origin. Five alleles of the 16S rRNA gene were discovered that corresponded to isolates originating from Africa, Australia/Southeast Asia, Japan, Surinam, and Mexico (115). This data fitted well with the results of the PGRS probe and together these data suggested that M. ulcerans exists as a clonal population within a geographic region.

3. Clinical presentation

    The initial lesion is usually a painless nodule or papule, occasionally leading to diffuse oedema. Eventually the skin ulcerates, under which there is an extensive zone of necrotic subcutaneous fat. There can be extensive lateral destruction of the subcutaneous fat (panniculus) beneath the intact epidermal layer and this causes the characteristic undermining of the dermal edges of the ulcer (61). The lesion is usually painless. In some cases the necrotizing panniculitis is associated with diffuse oedema. The presentation of M. ulcerans disease appears to vary between strains from different regions. In Africa the most common presentation is a small, mobile skin nodule that enlarges over days to weeks. In Australia, patients first notice a small papule with no nodular stage. Lesions have been known to heal spontaneously but most progress to ulceration (59). A classification scheme has been proposed which identifies five different presentations of the disease, namely (a) nodule/papule, (b) plaque, (c) oedema, (d) disseminated, (e) mixed (9).
   Healing of large areas of necrosis after surgical treatment or self resolution is characterized by the formation of granulomatous tissue at the base of the ulcer then epithelialization around the edges. Scars form slowly, akin to a severe burn, with muscle atrophy, joint contractures and lymphoedema (71), (142). While the morbidity is high, infection with M. ulcerans is rarely fatal.
   Ulcers most commonly occur on the extensor surfaces of limbs (18), (35), (56), (109), (135), (148). In Australia, ulcers are most common on the anterior aspect of the lower leg (135). However, any external surface may be vulnerable with reports of lesions on the face, buttock, eye, thorax, and abdomen (18), (26), (96), (125), (149)

4. Pathology

    The pathology and histopathology of M. ulcerans infection have been extensively described and reviewed (59). Histological examination of the skin lesions shows extensive destruction of adipocytes. Large numbers of acid-fast bacilli are observed in high concentration within this necrotic tissue and inflammatory cells are sparse (61). Unusually for a mycobacterial infection, the bacteria are rarely seen within macrophages and appear to maintain an exclusively extracellular location during infection (61), (70). As the disease progresses all elements of the skin are damaged including nerves and blood vessels. Another unusual observation is the extension of necrotic tissue beyond the areas containing visible bacilli. This prompted early investigators to suggest that M. ulcerans may elaborate a toxin (25).
   Studies of the effects of M. ulcerans culture filtrates on murine fibroblasts indicated the presence of cytopathic activity. Cell fractionation studies of M. ulcerans identified cytotoxic activity in the culture filtrate and the cytoplasmic fractions when these preparations were injected intradermally into a guinea pig model (76). Importantly, the pathology of the lesions was similar to those seen in human cases of ulcerative disease. Further analysis suggested that the toxin was a high molecular weight phopholipoprotein-polysaccharide complex (66). However later work demonstrated that the toxic activity was linked to an acetone-soluble, low molecular weight lipid compound (45). This was subsequently shown to be a polyketide-derived macrolactone, named mycolactone (46). Inoculation of the guinea pig model with 100 mg of mycolactone reproduced the necrosis, the lack of inflammatory response and other pathological characteristics of M. ulcerans infection (46). Phospholipases have also been identified in M. ulcerans although the role these enzymes have in pathogenesis is yet to be determined (50). A recent study of M. ulcerans culture filtrates in an adipose cell model has suggested that additional factors such as lipoprotein complexes may be involved in pathogenesis (33).
   There is no widely accepted animal model for M. ulcerans infection although many have been proposed including rats, mice, rabbits (86), possums (44), calves (121), fruit bats (20), guinea pigs (76), anole lizards (87), koalas (89), and nine-banded armadillos (154). The guinea pig appears to be the most useful model, as intradermal inoculation with M. ulcerans will reproduce most of the pathology seen in human infection including, contiguous necrosis, ulceration and an absence of purulent exudate (46), (76). However there is some question as to whether this animal is a faithful model for the human immune response to M. ulcerans infection. One report describes a relative abundance of inflammatory cells in the guinea pig lesion whereas in the human disease, inflammatory cells are generally scarce (76).

5. Human immune response to infection

    The human immune response to M. ulcerans infection has been little studied. Unfortunately parallels with the well-examined response to M. tuberculosis infection cannot be made, as M. ulcerans appears to utilise a markedly different pathological process despite the close antigenic and genetic relationship between these species. Successful pulmonary disease induced by M. tuberculosis infection is dependent on the relationship between cell-mediated immunity (CMI) and the response of the host (31). M. tuberculosis favours an intracellular environment for growth and in particular with in non-activated macrophages. Thus it appears that it is an inability to mount an effective CMI response that leads to a delayed type hypersensitivity-induced caseous necrosis seen in M. tuberculosis infection. This necrosis occurs as the immune response removes bacteria by indiscriminate destruction of infected, non-activated macrophages and surrounding tissue (31).
    From the few studies that have been reported so far it appears that the human immune response to infection with M. ulcerans is substantially different to infection with M. tuberculosis. The most obvious difference is the immunopathological observations that demonstrate the absence of immune inflammatory cells within or proximal to the site of M. uclerans infection during the early and acute phases of disease (25), (26), (35), (59), (61).
    Studies of the effect of the M. ulcerans lipid toxin, mycolactone, have shown that it appears to act indirectly on suppression of production of IL-2 and TNF and thus down regulates the Th1 type pro-inflammatory cytokine response (100). This result correlates with an earlier report that described the suppression of IFN-g in Con-A stimulated murine lymphocytes after exposure to a crude M. ulcerans culture filtrate (106). A suppressed pro-inflammatory immune response to M. ulcerans infection during the necrotizing phase of the disease would also explain the general wellness reported by Buruli ulcer patients. Serum surveys and stimulation of lymphocytes from patients with healed ulcers have shown that patients do mount an immune response to the organism as evidenced by antibody production and a strong Th2 cytokine bias (52). However these patients also demonstrated marked, systemic Th1 cell anergy to mycobacterial antigens. In contrast, lymphocytes from exposed control subjects who had not had ulcers produced primarily Th1 cytokines to M. ulcerans challenge with a significant bias to this arm of the immune response (52). It is not clear yet whether these observations indicate a genetic defect of the host or a capability of the organism to permanently suppress a Th1 response.
    Attempts have also been made towards immunoprophylaxis. Fenner and Leach (41), (43)found that Mycobacterium bovis BCG absorbed all antibodies to M. ulcerans strains from sera produced against them. This suggested a close relationship between M. ulcerans and BCG as well as the potential for BCG to offer cross-protection against infection with M. ulcerans. In a mouse model, BCG offered complete protection against small inocula of M. ulcerans but reduced protection against larger doses. These laboratory findings seemed to translate to field uses of BCG. A trial of the BCG vaccine was undertaken at the Kinyara refugee camp in Uganda. It was found that either a positive tuberculin skin test or BCG vaccination offered short-term protection and appeared to delay the onset of ulcerative disease by 2-3 months (147). It was also found that BCG offered only low protection (18%) in areas of high Buruli ulcer incidence compared with 74% protection in low-incidence areas, fitting with the hypothesis of a high dose overwhelming host immunity. Later a skin test reagent called Burulin was produced by 0.2 m m filtration of sonicated M. ulcerans cell lysates. This polyvalent antigen preparation was used to try and index a DTH response to M. ulcerans infection. Testing in Uganda and the Ivory Coast showed that in the early pre-ulcerative phase of disease, Burulin skin testing is negative (34), (139). Burulin also cross-reacted with BCG and M. tuberculosis infection, limiting the usefulness of this test for disease diagnosis or prevalence surveys. A serologic response to three M. ulcerans culture filtrate antigens in a study of 39 patients and 21 healthy controls indicated approximately 65% positivity in patients at all stages of disease compared with 37% positivity in healthy controls (34). This result suggested that serum screening might have diagnostic value however 30% of non-M. ulcerans exposed M. tuberculosis patients were also positive for at least one of the three specified protein antigens. It could not be determined from this work if the high rate of serum positivity in the healthy controls was due to sub-clinical M. ulcerans infection or exposure to other mycobacterial antigens. As observed with Burulin skin testing, cross-reactivity would probably reduce the usefulness of this approach.

6. Treatment

    Despite the in vitro sensitivity of M. ulcerans to a range of drugs such as streptomycin (40), diphenylsulphone (103), clofazimine (84), clarithromycin (116), ciprofloxacin, sparfloxacin, ofloxacin, amikacin and rifampicin (141), wide surgical excision of the ulcer remains the only effective treatment. Other therapies have been promoted including hyberbaric oxygen (77) and local heat treatment (48) however the evidence for their efficacy is limited.

7. Epidemiology

      7.1 Geographic distribution

    Since the first description of M. ulcerans disease from the Bairnsdale region of southeast Australia (86)cases have been reported from at least 27 countries worldwide (63). The distribution is predominantly tropical and most frequent throughout equatorial Africa. Endemic areas are generally close to water sources such as rivers, swamps and lakes although there have been several reports from areas not associated with large water masses (19). A striking characteristic of the disease in all regions is its highly focal distribution such that clustering of cases does not necessarily correlate with the population distribution along a watercourse (120). The emergence of new endemic areas is often associated with recent environmental disturbance such as flooding (11), (60).

            a. Africa

   In 1950 a single case was reported from the Toro area in the Congo-Kinshasa (152). However the disease was probably endemic in this country prior to this as there had been anecdotal reports of M. ulcerans-like lesions as early as 1940 (90). In the Congo, cases have been reported in six of the eight regions in that country. During the 1970s large numbers of cases were reported from the Songololo zone in the Bas-Zaïre region (108). Reports of ulcers caused by a M. ulcerans -like organism also began to emerge from the Buruli County in neighbouring Uganda where the causative organism was named M. buruli and the disease was named Buruli ulcer (22), (23). The name M. buruli was never officially adopted as comparisons with M. ulcerans showed the two organisms to be sufficiently similar to be considered the same species (121). By 1972 over 1500 cases of Buruli ulcer had been reported from several regions in Uganda. All regions were within close proximity to the River Nile (11). M. ulcerans is now widespread throughout central and West Africa. In addition to the Congo and Uganda, cases have been reported in Congo-Brazzaville (104), Nigeria (39), (53), (75), (82), (97), Cameroon (123), Angola (10), (134), Central African Republic (49), Benin (95), Togo (92), (6), (16), (151), Ivory Coast, Sierra Leone (47), Liberia (155), Gabon (18), and Burkina Faso (99). The true incidence of the disease in each of these countries is unknown although it is quite clear that throughout West Africa incidence has increased dramatically and catastrophically in the last 15 years (2), (5), (6), (28), (88), (94).


         b. Australia and Pacific region

    The first report of disease in Australia was the late 1930s in the Gippsland region of Victoria (4). Since that time, there has continued to be a small number of cases identified from that area each year (58), (64), (65), including a Koala colony on a small island in the Gippsland Lakes (93). However during the mid 1980s there was a dramatic shift westward with confirmed cases occurring within the vicinity of Melbourne (61), (73). Most notably was the outbreak at Phillip Island in 1993, a holiday settlement 80 km south east of Melbourne, where 29 cases were identified over a three-year period (153). Prior to 1993 there had been no cases of M. ulcerans disease attributable to that area. Between 1990 and 1998 there were 66 culture-confirmed cases of disease identified in Victoria of which 32 cases were outside the known endemic areas of Gippsland and Phillip Island. At least eleven but perhaps fifteen of these cases were attributable to the Frankston/Langwarrin area and about another 17 were sporadic (37). Despite the recent outbreak at Phillip Island, the disease remains rare in southeast Australia.


    There have also been several reports of M. ulcerans disease from different areas of Queensland. In the late 1950s and 1960s a few cases were identified in the Beerwah district, north of Brisbane (1), (78)and also Maryborough and Rockhampton (7), (8). The highest incidence of ulcerative disease in Australia comes from the Douglas Shire in far north Queensland. Detailed epidemiological analysis in the Douglas Shire has revealed an ongoing history of the disease since at least 1950 with characteristic sub-foci of infection occurring within the region (135). A recent report from this area strongly suggested that the incidence of M. ulcerans disease has increased significantly in the past two years (72).
    There have been no reports of cases originating in any of the other Australian states. A small series of cases have been reported from Darwin and also Croker Island off the Northern Territory coast (119). There have also been likely cases of infection in the Republic of Kiribati (19).


         c. Papua New Guinea (PNG)

    M. ulcerans infections were first recognized in PNG in 1957. It became an important focus of the disease with 172 cases recorded by 1972 (68), (122). Settlements along the Kumasi and Sepik rivers have been associated with the majority of cases although sporadic cases have been reported throughout PNG (85). At least one case has been reported from the small island of Daru off the southwestern coast (125). There have been no recent reports of disease from this country.


         d. Southeast Asia

   Cases have been described from Malaysia (105) and Northern Sumatra (133).


         e. Asia and the Americas

    A single case has been reported from both Japan (146)and China (38). There has been one report of suspected M. ulcerans infection from Sri-Lanka. Diagnosis was retrospective and based only on clinical descriptions of the lesions (132). Three cases have been reported from North America however two had recently returned from Nigeria (39), (82)and the third had returned from Bolivia (82). M. ulcerans foci have also been described in Mexico (3), Bolivia (82), Surinam (109), and French Guayana (54).


         f.Other

    Single cases, linked to recent travel from known endemic areas, have been reported in Northern Ireland and Germany (32), (150).


       7.2 Age/sex distribution

    In Africa the incidence of the disease appears to be highest in children. There is no bias towards either sex in children but disease occurrence is higher in adult females than in men (18), (149). No gender-based difference has been noted in other endemic countries such as Australia or Papua New Guinea (61), (120). Based on the global distribution of M. ulcerans (63), and the lack of observable ethnic bias within endemic zones (23), race does not appear to influence susceptibility to infection.


       7.3 Transmission

    The route of transmission of M. ulcerans has not been determined but two general, and possibly overlapping, hypotheses have been invoked. The first hypothesis maintains that the skin is inoculated directly by host contact with a contaminated environment e.g. soil, water, vegetation, or insect vector. This model would explain the high prevalence of ulcers on the exposed parts of the body such as arms and legs. The contention of the second hypothesis is acquisition of M. ulcerans from the environment by inhalation or ingestion (26), (60), (74). The organism is then disseminated and reactivates in low temperature areas of the body at sites of trauma. There have been numerous reports of M. ulcerans infection following both penetrating skin injuries and soft tissue damage, but it is more common for there to be no record of antecedent trauma (1), (36), (62), (67), (91). These observations fit with either hypothesis however the balance of evidence, in particular the presence of only single lesions, tends to suggest the direct inoculation model is the more likely (60), (74), (110), (120).
    Direct transmission from an infected host has been demonstrated experimentally with possums (44) and does occur in humans but is uncommon (105), (121), (134). The strongest evidence for the lack of direct transmission comes from the study of the large number of cases observed in Uganda at the Kinyara refugee camp (149). They found that ulcerative disease was no more frequent in the relatives living with patients than with controls. Furthermore, when the camp was relocated the incidence of disease halted despite the presence within that population of patients with active ulcerative disease (149).
    The incubation period for M. ulcerans disease is generally considered to be two to three months although it can be as short as two weeks (125). The movement of the Kinyara refugee camp to a non-endemic area permitted the estimation of the incubation period. As there were no further cases of disease amongst the refugees 13 weeks after moving the camp, this was considered the incubation period (149). This time frame is consistent with other estimates based on date of entry of patients into known endemic areas and onset of disease (125). Quiescence for up to seven years has also been demonstrated (118). Studies in animals have shown incubation periods ranging from less than a week to 18 months, influenced by vaccination with BCG and size of the infecting inocula (42), (86).


       7.4 Seasonality

    In Papua New Guinea there was some correlation between season and disease prevalence with a peak incidence reported in the dry season (122). Similar data was obtained from two separate studies in Uganda that suggested that peak transmission of the disease occurred in the low rainfall months between May and September (126), (149).

8. Environmental source

   All attempts to date to identify the environmental source of M. ulcerans using mycobacterial culture methods have been unsuccessful. However, there have been several key epidemiological studies that have sought to infer the environmental source by studying the distribution of ulcers in human populations.


      8.1 Studies in Uganda

   The abrupt cessation of cases following the relocation of the 2500 refugees from Kinyara to Kyangwali provided convincing evidence that M. ulcerans was acquired via contact with some aspect of the local environment (149). The Kinyara settlement was geographically compact, approximately 5.5 km x 2.5 km, and only 4 km from the River Nile yet the river was visited by few of the 2500 residents. This important observation of close proximity to the river, but not necessarily contact with it was found in another Ugandan survey (13). The incidence of cases was highest in women and highest in those areas of the camp closest to the River Nile. Lesions on children occurred anywhere on the body while on men it was predominantly on the lower leg and on women it was arms, legs and buttocks. It was the women who collected domestic water from bore holes and also marshes and streams to the north and south of the camp. It was also reported that the women at Kinyara who lived closest to the Nile had a higher rate of infections than those living away from it but that incidence in men was more variable. This fitted with the more confined pattern of movement of women around the camp compared with the men who tended to wander all over. From these observations it was suggested that the infection maybe acquired close to the ground and perhaps by contact with contaminated vegetation (149). An insect vector was considered unlikely because of the anatomical sites of the lesions and the gender bias among the adult cases (149).
    A second epidemiological investigation in the Busoga district, an area approximately 150 km from Kinyara, showed 87% of the 82 cases studied occurred within swampy lowlands to the north of the district around the edge of Lake Kyoga (13). All cases were less than 32 km from the River Nile. Interestingly, the disease was not known in this area before 1965. This led to the hypothesis that the unprecedented flooding in the region between 1962 and 1964, which raised water levels in Lake Victoria 12 — 13 metres, caused an extension of swampy areas in which M. ulcerans could flourish (13). In support of a causal link between flooding and the appearance of M. ulcerans disease, there were very few cases reported in the drier, upland areas of the district. In addition, the upland areas had a higher population density than the swampy lands to the south.
    The link between swamps and M. ulcerans was further highlighted in a year-long survey during 1970 of 572 cases across Uganda that showed all but 8 cases were in the vicinity of swampy lowland areas (11). It was proposed that M. ulcerans infection occurred by contact with M. ulcerans-contaminated swamp grasses and that the introduction or proliferation of M. ulcerans in a region was related to the expansion of swamps and associated flora (13).
    To test these hypotheses extensive sampling of the environment was undertaken at Kinyara and in surrounding districts with attempts to detect M. ulcerans by mycobacterial culture techniques. Samples of water, soil, fish and rodents were collected from around the River Nile but all were negative (127). Among the 700 rodents tested, many had mycobacterial infections but none were M. ulcerans. Thousands of the small biting fly (Simulium griseicollis), aquatic snails, water, sand, mosquitoes, and reptiles from the vicinity of the Nile at Kinyara were also tested but all were negative (127). Many of the samples, including the insect specimens, recovered mycobacteria but these tended to be fast growing species such as M. fortuitum which overgrew the culture medium (127). A range of biting insects, collected in a separate study of the disease in the Madi district, were tested for M. ulcerans but all were negative (83).
    In another survey, 323 plant specimens were collected across three endemic districts in Uganda. The highest numbers of mycobacteria were recovered from plant material in permanent swamps but none of these were M. ulcerans (14). It was observed that the grass species, Echinocloa pyramidalis was predominant in endemic areas compared to the non-endemic (14) and so efforts were made to detect M. ulcerans by culture from this grass (138). A total of 792 grass specimens were collected from endemic areas and 264 samples were collected from non-endemic regions. One hundred and eighty five mud samples were also collected. A wide variety of mycobacteria were cultured from all sample types but not M. ulcerans (138).
    Other epidemiological investigations in Uganda such as case control studies of patient activities revealed no differences between patients and controls (15). This suggested that development of disease may be related to the intensity of exposure and an individual’s response to mycobacterial infection. A study of Tsetse control workers indicated that there was a higher incidence of disease in families using swamps as domestic water sources rather than bores, thus reinforcing the link between M. ulcerans and swamps (12).

      8.2 Studies in the Congo (Kinshasa)

    In the M. ulcerans-endemic areas of the Songololo and Kasongo regions in the Congo-Kinshasa (also called the Democratic Republic of Congo and Zaïre) many thousands of water, mud, soil, plant and fish specimens were collected (109). A total of 956 mycobacterial cultures were obtained which included M. avium. M. scrofulaceum, M. fortuitum, M. terrae, M. nonchromogenicum, M. gordonae, M. parafortuitum, M. asiaticum and M. marinum. However, M. ulcerans was not isolated (109). Fish were a common source of mycobacteria in this study and it was speculated that they may be a reservoir for M. ulcerans (109).

      8.3 Other studies in Africa

    In Nigeria, infections were reported among 14 residents of the University of Ibadan living near a shallow 10 acre lake that had been recently created by damming a small stream that ran through the southern end of the campus (97). Physical contact with this lake was thought not to have occurred in the majority of cases and many patients were convinced that their lesions developed from insect bites (97). Examination of soil, water and snails from the lake for M. ulcerans were negative (98).


    Twenty-two cases of Buruli ulcer were confirmed in the vicinity of the River Ogooue at Lambaréné, Gabon (18). Fifty-three water samples of 500 ml — 1000 ml volumes were collected from this river, concentrated by membrane filtration and tested by culture for M. ulcerans. Mycobacteria were recovered from these samples but not M. ulcerans (18).

      8.4 Papua New Guinea

    In PNG it was hypothesised that the erruption of Mt Lamington in 1951 and subsequent major flooding preceded a large number of cases that occurred along the Kumasi River (120). In 1966 samples of water, fish, riverine vegetation, canoes and utensils were collected in the Kumasi area. Mycobacteria were detected but not M. ulcerans (122).

      8.5 Studies in Australia

    Flooding was also thought to have been a factor influencing the prevalence of the disease in southeast Australia. There was severe flooding in the Gippsland region in 1935 and it was in the following few years that the first recognized cases occurred (58). There have been several attempts to detect M. ulcerans from regions in which it is endemic in southeast Australia. In the Bairnsdale region of Gippsland, insectivorous bats were considered possible sources of M. ulcerans . In one survey, 40 bats (Miniopteris schreibersii) were examined for M. ulcerans with negative results (17). Attempts to infect the bats with M. ulcerans were also unsuccessful (17). Between 1980 and 1985 11 koalas from Raymond Island were diagnosed with ulcers caused by M. ulcerans (93). Raymond Island is a small island (750 ha) in the brackish Gippsland lakes. This unusual occurrence prompted an epidemiological survey of the island and the collection of soil samples to try and identify M. ulcerans. Fifty samples were processed by mycobacterial culture methods and all samples were negative (93).
   An interesting hypothesis was proposed to explain the global distribution of M. ulcerans, in particular the presence of the organism in temperate southeast Australia. It was suggested that M. ulcerans was a ‘survivor’ of the rainforest microbiota of Gondwana Land (57). Evidence for this hypothesis was based on the observation that its global distribution mimicked that of the Gondwanian flora (57). However, subsequent cases of M. ulcerans on a pacific atoll, and in Japan and China cast some doubt on this theory.
   Beginning in September 1992 and ending in December 1995, there were 29 cases of M. ulcerans disease in the small seaside town of Cowes on Phillip Island, 80 km south east of Melbourne (73). Before 1992 there were no recorded cases of infection from this island. The cases clustered within a narrow zone 2 km x 1 km to the eastern end of the township and epidemiological studies revealed a number of potential environmental sources. These sources included a recently formed swamp and a golf course with a small, spring-fed dam that was supplemented with recycled waste water (73). Initially it was thought that swamp may have been the primary source of infection however works carried out in 1994 that drained this area were followed by a shift in the epicentre of cases towards the golf course in the following year (153). A large series of water samples were collected in 1994 and 1995 and attempts were made to culture M. ulcerans. Many mycobacteria were recovered but all samples tested negative for M. ulcerans (129). At this time a PCR method for detection of M. ulcerans was developed and applied to the sample concentrates used for culture as well as another set of samples taken from the golf course and the swamp (129). Five positive PCR results were obtained from water samples collected from the swamp, the golf course dam and from the pump used to distribute water around the golf course (129). Water samples collected from other sites on Phillip Island were PCR negative (129). Modifications were subsequently made to the golf course irrigation system and these changes coincided with a rapid decline in cases (153). No cases have been reported from this area since September 1998 (37). The PCR data provided the first direct evidence of the environmental presence of M. ulcerans. In this instance it seemed that golf course irrigation system was contaminated with M. ulcerans and that the organism was being distributed throughout the local environment via aerosols created by the sprinkler network. Further investigation of the golf club system and the surrounding environs was required.
   All the attempts to detect M. ulcerans in the environment based on culture methods have failed. However given the positive findings using DNA-based detection, it seems quite likely that negative results from previous studies were probably a reflection of the poor sensitivity of culture for detection of this mycobacteria rather than the demonstration of its absence in the environment.

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