In Vitro Assessment of Drug Activity Against Mycobacterium ulcerans.

As acknowledged by the Clinical and Laboratory Standards Institute (CLSI), there is an insufficient evidence base on which to recommend a standard method for antimicrobial susceptibility testing against M. ulcerans. The agar proportion method has been recognized as the standard method for susceptibility testing against Mycobacterium tuberculosis complex (MTBC) isolates for decades (Woods GL, Engenack NL, Lin G, Turnidge JD (2018) CLSI standards: guidelines for health care excellence. Susceptibility testing of mycobacteria, Nocardia spp., and other aerobic Actinomycetes, 3rd edn. Clinical and Laboratory Standards Institute Copyright©2018 Clinical and Laboratory Standards Institute, Wayne (PA)). While it is more labor-intensive and requires larger amounts of drug or compound than broth-based testing, we recommend the agar proportion method for determination of minimum inhibitory concentrations against M. ulcerans. Herewith we present the method we implemented in our laboratory over the last 2 decades.

Diagnosis delay and duration of hospitalisation of patients with Buruli ulcer in Nigeria.

BACKGROUND: Delayed diagnosis of Buruli ulcer can worsen clinical presentation of the disease, prolong duration of management, and impose avoidable additional costs on patients and health providers. We investigated the profile, delays in diagnosis, duration of hospitalisation, and associated factors among patients with Buruli ulcer in Nigeria. METHODS: This was a prospective cohort study of patients with Buruli ulcer who were identified from a community-based survey. Data on the patients' clinical profile, delays in diagnosis and duration of hospitalisation were prospectively collected. RESULTS: Of 145 patients notified, 125 (86.2%) were confirmed by one or more laboratory tests (81.4% by PCR). The median age of the patients was 20 years, 88 (60.7%) were >15years old and 85 (58.6%) were females. In addition, 137 (94.5%) were new cases, 119 (82.1%) presented with ulcers and 110 (75.9%) had lower limb lesions. The mean time delay to diagnosis was 50.6 (±101.9) weeks. The mean duration of hospitalisation was 108 (±60) days. Determinants of time delay to diagnosis were higher disease category (p=0.001) and laboratory confirmation of disease (p=0.02). Determinants of longer hospitalisation were; multiple lesions (p=0.035), and having functional limitation at diagnosis and undertaking surgery (p=0.003). CONCLUSIONS: Patients with Buruli ulcer have very long time delays to diagnosis and long hospitalisation during treatment. This calls for early case-finding and improved access to Buruli ulcer services in Nigeria.

Nontuberculous Mycobacterial Disease in Children - Epidemiology, Diagnosis & Management at a Tertiary Center.

BACKGROUND: There are limited data on the epidemiology, diagnosis and optimal management of nontuberculous mycobacterial (NTM) disease in children. METHODS: Retrospective cohort study of NTM cases over a 10-year-period at a tertiary referral hospital in Australia. RESULTS: A total of 140 children with NTM disease, including 107 with lymphadenitis and 25 with skin and soft tissue infections (SSTIs), were identified. The estimated incidence of NTM disease was 0.6-1.6 cases / 100,000 children / year; no increasing trend was observed over the study period. Temporal analyses revealed a seasonal incidence cycle around 12 months, with peaks in late winter/spring and troughs in autumn. Mycobacterium-avium-complex accounted for most cases (77.8%), followed by Mycobacterium ulcerans (14.4%) and Mycobacterium marinum (3.3%). Polymerase chain reaction testing had higher sensitivity than culture and microscopy for acid-fast bacilli (92.0%, 67.2% and 35.7%, respectively). The majority of lymphadenitis cases underwent surgical excision (97.2%); multiple recurrences in this group were less common in cases treated with clarithromycin and rifampicin compared with clarithromycin alone or no anti-mycobacterial drugs (0% versus 7.1%; OR:0.73). SSTI recurrences were also less common in cases treated with two anti-mycobacterial drugs compared with one or none (10.5% versus 33.3%; OR:0.23). CONCLUSIONS: There was seasonal variation in the incidence of NTM disease, analogous to recently published observations in tuberculosis, which have been linked to seasonal variation in vitamin D. Our finding that anti-mycobacterial combination therapy was associated with a reduced risk of recurrences in patients with NTM lymphadenitis or SSTI requires further confirmation in prospective trials.

[EVALUATION STANDARD OF EXTERNAL QUALITY ASSESSMENT PROGRAMME FOR DRUG SUSCEPTIBILITY TESTING OF MYCOBACTERIUM TUBERCULOSIS IN JAPANESE LABORATORIES: PROFICIENCY TESTING IN 2004-2010].

OBJECTIVE: To analyze the results of the external quality assessments (EQA) for anti-tuberculosis drug susceptibility testing (DST) and to set-up its rational passing criterion. METHOD: Each participating laboratory in EQA performed DST, and the sensitivity, specificity, agreement (efficiency) and kappa coefficient were calculated from the results. We analysed the data of seven EQA results for DST from 2004 to 2010. RESULTS: A total of 20, 20, 10, 5, 10, 10, and 10 strains of M. tuberculosis with known susceptibility were sent to each participating laboratory in 2004, 2005, 2006, 2007, 2008, 2009, and 2010, respectively. The total of participating laboratories was 564. Each laboratory was asked to perform DST with its routine methods and reported 25,100 test results in these seven years. The laboratories showed relatively high specificity than sensitivity, and an improving sensitivity through the years. Sixteen laboratories participated the EQA continuously, and the sensitivity and specificity to isoniazid (INH), rifampicin (RFP), streptomycin (SM) and ethambutol (EB) were 0.999 (95% CI 0.992-1.000) and 0.998 (95% CI 0.991-1.000), 0.985 (95% CI 0.973-0.992) and 0.997 (95% CI 0.989-0.999), 0.932 (95% CI 0.912-0.948) and 0.977 (95% CI 0.962-0.986), and 0.965 (95% CI 0.947-0.977) and 0.978 (95% CI 0.966-0.986), respectively. DISCUSSION: The analyses revealed that the accuracy of DST for INH and RFP was highly maintained throughout the years. However, SM showed a high unevenness of performance quality and required situational considerations for evaluation. In conclusion, the EQA for DST would require a minimum number of 10 strains for each assessment, and INH and RFP should show over 95% of sensitivity and specificity with over 90% of efficiency to SM and EB as passing remark.

Methods used in preclinical assessment of anti-Buruli ulcer agents: A global perspective.

Buruli ulcer (BU) caused by Mycobacterium ulcerans is the third most common chronic mycobacterial infection in humans. Approximately 5000 cases are reported annually from at least 33 countries around the globe, especially in rural African communities. Even though anti-mycobacterial therapy is often effective for early nodular or ulcerative lesions, surgery is sometimes employed for aiding wound healing and correction of deformities. The usefulness of the antibiotherapy nonetheless is challenged by huge restrictive factors such as high cost, surgical scars and loss of income due to loss of man-hours, and in some instances employment. For these reasons, more effective and safer drugs are urgently needed, and research programs into alternative therapeutics including investigation of natural products should be encouraged. There is the need for appropriate susceptibility testing methods for the evaluation of potency. A number of biological assay methodologies are in current use, ranging from the classical agar and broth dilution assay formats, to radiorespirometric, dye-based, and fluorescent/luminescence reporter assays. Mice, rats, armadillo, guinea pigs, monkeys, grass cutters and lizards have been suggested as animal models for Buruli ulcer. This review presents an overview of in vitro and in vivo susceptibility testing methods developed so far for the determination of anti-Buruli ulcer activity of natural products and derivatives.

Maximizing microscopy as a diagnostic tool in peripheral health centres of BU endemic areas in Ghana.

BACKGROUND: Buruli ulcer (BU) disease, a skin condition caused by Mycobacterium ulcerans (M. ulcerans) is endemic in remote rural areas. Disease diagnosis on clinical basis alone can be misleading, requiring definitive diagnosis based on laboratory tests. Resource constraints in BU endemic areas make microscopy for the detection of acid fast bacilli (AFB) an important and useful method. It is rapid, user-friendly, convenient and cheap. Despite its usefulness, its performance is relatively low. This study investigated modifications of the current method aimed at improving its performance. Forty (IS) 2404 polymerase chain reactions (PCR) positive BU samples were processed by eight physical (centrifugation and overnight sedimentation) and chemical (phenol ammonium sulphate and sodium hypochlorite) modifications of the current direct method. Assessments were based on standard AFB evaluation coupled with in house criteria; positivity (P), clarity and contrast (C) release of bacilli from specimen (R). Overall AFB positivity rate was 64% (409/640). Each protocol had 80 smears. The percentage positivity (P) for the conventional method was 58% (46/80) smears. The highest positivity rate of 57/80 (%) was by protocol 7 (5% phenol in 4% ammonium sulphate (PhAS) and concentrated by overnight gravitational sedimentation). The least positivity rate at 35% (28/80) was by protocol 1 (smears from direct application of swab tips). The differences in performance between the two chemical tested; 5% phenol in 4% ammonium sulphate (PhAS) and 3.5% NaHOCl was significant (p<0.05). The differences between the two physical methods were however not significant (p>0.05). This study concluded that BU samples treated with a solution of 5% phenol in 4% ammonium sulphate and concentrated by either centrifugation or overnight sedimentation is useful for maximizing AFB detection by bright field microscopy. This can be useful in rural health facilities with resource constraints.

The puzzle of Buruli ulcer transmission, ethno-ecological history and the end of "love" in the Akonolinga district, Cameroon.

The "One World One Health Initiative" has attended little to the priorities, concepts and practices of resource-poor communities confronting disease and the implications of these concerns for its biomedical, ecological and institutional approach to disease surveillance and control. Using the example of Buruli ulcer (BU) and its bacterial etiology, Mycobacterium ulcerans, in south-central Cameroon, we build on debates about the contributions of "local knowledge" and "alternative models" to biomedical knowledge of disease transmission. BU's mode of transmission remains poorly understood. Our approach employs ethno-ecological histories - local understandings of the putative emergence and expansion of a locally important, neglected disease. We develop these histories from 52 individual and small group interviews, group discussions, and participant-observation of daily and seasonal activities, conducted in 2013-2013. These histories offer important clues about past environmental and social change that should guide further ecological, epidemiological research. They highlight a key historical moment (the late 1980s and 1990s); specific ecological transformations; new cultivation practices in unexploited zones that potentially increased exposure to M. ulcerans; and ecological degradation that may have lowered nutritional standards and heightened susceptibility to BU. They also recast transmission, broadening insight into BU and its local analog, atom, by emphasizing the role of social change and economic crisis in its emergence and expansion.

Epidemiology and management of Buruli ulcer.

Buruli ulcer (Mycobacterium ulcerans infection) is a neglected tropical disease of skin and subcutaneous tissue that can result in long-term cosmetic and functional disability. It is a geographically restricted infection but transmission has been reported in endemic areas in more than 30 countries worldwide. The heaviest burden of disease lies in West and Sub-Saharan Africa where it affects children and adults in subsistence agricultural communities. Mycobacterium ulcerans infection is probably acquired via inoculation of the skin either directly from the environment or indirectly via insect bites. The environmental reservoir and exact route of transmission are not completely understood. It may be that the mode of acquisition varies in different parts of the world. Because of this uncertainty it has been nicknamed the 'mysterious disease'. The therapeutic approach has evolved in the past decade from aggressive surgical resection alone, to a greater focus on antibiotic therapy combined with adjunctive surgery.

Multicenter external quality assessment program for PCR detection of Mycobacterium ulcerans in clinical and environmental specimens.

BACKGROUND: Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a necrotizing disease of the skin, soft tissue and bone. PCR is increasingly used in the diagnosis of BU and in research on the mode of transmission and environmental reservoir of M. ulcerans. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to evaluate the performance of laboratories in detecting M. ulcerans using molecular tests in clinical and environmental samples by implementing sequential multicenter external quality assessment (EQA) programs. The second round of the clinical EQA program revealed somewhat improved performance. CONCLUSIONS/SIGNIFICANCE: Ongoing EQA programs remain essential and continued participation in future EQA programs by laboratories involved in the molecular testing of clinical and environmental samples for M. ulcerans for diagnostic and research purposes is strongly encouraged. Broad participation in such EQA programs also benefits the harmonization of quality in the BU research community and enhances the credibility of advances made in solving the transmission enigma of M. ulcerans.

Rapid, serial, non-invasive assessment of drug efficacy in mice with autoluminescent Mycobacterium ulcerans infection.

BACKGROUND: Buruli ulcer (BU) caused by Mycobacterium ulcerans is the world's third most common mycobacterial infection. There is no vaccine against BU and surgery is needed for patients with large ulcers. Although recent experience indicates combination chemotherapy with streptomycin and rifampin improves cure rates, the utility of this regimen is limited by the 2-month duration of therapy, potential toxicity and required parenteral administration of streptomycin, and drug-drug interactions caused by rifampin. Discovery and development of drugs for BU is greatly hampered by the slow growth rate of M. ulcerans, requiring up to 3 months of incubation on solid media to produce colonies. Surrogate markers for evaluating antimicrobial activity in real-time which can be measured serially and non-invasively in infected footpads of live mice would accelerate pre-clinical evaluation of new drugs to treat BU. Previously, we developed bioluminescent M. ulcerans strains, demonstrating proof of concept for measuring luminescence as a surrogate marker for viable M. ulcerans in vitro and in vivo. However, the requirement of exogenous substrate limited the utility of such strains, especially for in vivo experiments. METHODOLOGY/PRINCIPAL FINDING: For this study, we engineered M. ulcerans strains that express the entire luxCDABE operon and therefore are autoluminescent due to endogenous substrate production. The selected reporter strain displayed a growth rate and virulence similar to the wild-type parent strain and enabled rapid, real-time monitoring of in vitro and in vivo drug activity, including serial, non-invasive assessments in live mice, producing results which correlated closely with colony-forming unit (CFU) counts for a panel of drugs with various mechanisms of action. CONCLUSIONS/SIGNIFICANCE: Our results indicate that autoluminescent reporter strains of M. ulcerans are exceptional tools for pre-clinical evaluation of new drugs to treat BU due to their potential to drastically reduce the time, effort, animals, compound, and costs required to evaluate drug activity.

A quick and cost effective method for the diagnosis of Mycobacterium ulcerans infection.

BACKGROUND: Buruli ulcer (BU), a neglected tropical skin disease caused by Mycobacterium ulcerans, has been reported in over 30 countries worldwide and is highly endemic in rural West and Central Africa. The mode of transmission remains unknown and treatment is the only alternative to disease control. Early and effective treatment to prevent the morbid effects of the disease depends on early diagnosis; however, current diagnosis based on clinical presentation and microscopy has to be confirmed by PCR and other tests in reference laboratories. As such confirmed BU diagnosis is either late, inefficient, time consuming or very expensive, and there is the need for an early diagnosis tool at point of care facilities. In this paper we report on a simple, quick and inexpensive diagnostic test that could be used at point of care facilities, in resource-poor settings. METHODS: The methodology employed is based on the loop mediated isothermal amplification (LAMP) technique. Four sets of Primers, targeting the mycolactone encoding plasmid genome sequence of M. ulcerans were designed. The BU-LAMP assay was developed and tested on five M. ulcerans strains from patients in Ghana and two American Type Culture Control (ATCC) reference isolates; Ghana #970321 (D19F9) and Benin #990826 (D27D14). We also tested the assay on other closely related, mycolactone-producing mycobacterial strains; M. marinum 1218, M. marinum DL240490, M. liflandii and M. pseudoshotsii, as well as experimentally infected laboratory animal and clinical samples. RESULTS: The results revealed a high specificity of the BU-LAMP assay for selectively detecting M. ulcerans. Compared to the conventional IS-2404 PCR, the new assay is cheaper and simpler and ten times more sensitive. Test results can be obtained within 1 hour. CONCLUSIONS: This study indicates that the BU-LAMP assay could be suitable for early disease diagnosis and application in low-resource health facilities.

Combining PCR with microscopy to reduce costs of laboratory diagnosis of Buruli ulcer.

The introduction of antibiotic therapy as first-line treatment of Buruli ulcer underlines the importance of laboratory confirmation of clinical diagnosis. Because smear microscopy has very limited sensitivity, the technically demanding and more expensive IS2404 diagnostic polymerase chain reaction (PCR) has become the main method for confirmation. By optimization of the release of mycobacteria from swab specimen and concentration of bacterial suspensions before smearing, we were able to improve the detection rate of acid-fast bacilli by microscopy after Ziehl-Neelsen staining. Compared with IS2404 PCR, which is the gold standard diagnostic method, the sensitivity and specificity of microscopy with 100 concentrated specimens were 58.4% and 95.7%, respectively. We subsequently evaluated a stepwise laboratory confirmation algorithm with detection of AFB as first-line method and IS2404 PCR performed only with those samples that were negative in microscopic analysis. This stepwise approach reduced unit cost by more than 50% to $5.41, and the total costs were reduced from $917 to $433.

Health services for Buruli ulcer control: lessons from a field study in Ghana.

BACKGROUND: Buruli ulcer (BU), caused by Mycobacterium ulcerans infection, is a debilitating disease of the skin and underlying tissue. The first phase of a BU prevention and treatment programme (BUPaT) was initiated from 2005-2008, in the Ga-West and Ga-South municipalities in Ghana to increase access to BU treatment and to improve early case detection and case management. This paper assesses achievements of the BUPaT programme and lessons learnt. It also considers the impact of the programme on broader interests of the health system. METHODS: A mixed-methods approach included patients' records review, review of programme reports, a stakeholder forum, key informant interviews, focus group discussions, clinic visits and observations. PRINCIPAL FINDINGS: Extensive collaboration existed across all levels, (national, municipality, and community), thus strengthening the health system. The programme enhanced capacities of all stakeholders in various aspects of health services delivery and demonstrated the importance of health education and community-based surveillance to create awareness and encourage early treatment. A patient database was also created using recommended World Health Organisation (WHO) forms which showed that 297 patients were treated from 2005-2008. The proportion of patients requiring only antibiotic treatment, introduced in the course of the programme, was highest in the last year (35.4% in the first, 23.5% in the second and 42.5% in the third year). Early antibiotic treatment prevented recurrences which was consistent with programme aims. CONCLUSIONS: To improve early case management of BU, strengthening existing clinics to increase access to antibiotic therapy is critical. Intensifying health education and surveillance would ultimately increase early reporting and treatment for all cases. Further research is needed to explain the role of environmental factors for BU contagion. Programme strategies reported in our study: collaboration among stakeholders, health education, community surveillance and regular antibiotic treatment can be adopted for any BU-endemic area in Ghana.

Rapid assessment of antibacterial activity against Mycobacterium ulcerans by using recombinant luminescent strains.

Mycobacterium ulcerans causes Buruli ulcer, an emerging infectious disease for which antimicrobial therapy has only recently proven to be beneficial. The discovery and development of new drugs against M. ulcerans are severely impeded by its very slow growth. Recombinant bioluminescent strains have proven useful in drug development for other mycobacterial infections, but the ability of such strains to discriminate bacteriostatic from bactericidal activity has not been well demonstrated. We engineered recombinant M. ulcerans strains to express luxAB from Vibrio harveyi. In drug susceptibility tests employing a wide range of antimicrobial agents and concentrations, the relative light unit (RLU) count measured in real time was a reliable surrogate marker for CFU counts available 3 months later, indicating utility for the rapid determination of drug susceptibility and discrimination of bacteriostatic and bactericidal effects. A second important finding of this study is that the addition of subinhibitory concentrations of the ATP-binding cassette transporter inhibitor reserpine increases the susceptibility of M. ulcerans to tetracycline and erythromycin, indicating that drug efflux may explain at least part of the intrinsic resistance of M. ulcerans to these agents.

Highly sensitive, operationally simple, cost/time effective detection of the mycolactones from the human pathogen Mycobacterium ulcerans.

A boronate-assisted fluorogenic chemosensor in a solid phase is developed, selectively to detect the mycolactones produced by the human pathogen Mycobacterium ulcerans.

A stepwise approach to the laboratory diagnosis of Buruli ulcer disease.

OBJECTIVE: In view of technical and financial limitations in areas of endemicity, the current practice and recommendations for the laboratory diagnosis of Buruli ulcer disease (BUD) may have to be reconsidered. We reviewed diagnostic results in order to explore options for a modified, more practicable, cost-effective and timely approach to the laboratory diagnosis of BUD. METHODS: Diagnostic specimens from 161 clinically diagnosed BUD patients from four different treatment centres in Ghana were subjected to laboratory analysis. The positivity rates of the laboratory assays were compared. RESULTS: The number of laboratory-confirmed clinically diagnosed BUD cases with one positive confirmative test was 20% higher than that with two positive confirmative tests. The specificity of microscopy (MIC) and PCR was 96.6% and 100%, respectively. Subsequent analysis of specimens from surgically excised pre-ulcerative tissue-by-tissue MIC and tissue PCR rendered 65% laboratory-confirmed BUD cases. Subsequent analysis of diagnostic swabs from ulcerative lesions by swab smear MIC and swab PCR rendered 70% of laboratory-confirmed BUD cases. CONCLUSIONS: The specificity of the diagnostic tests used in this study suggests that one positive diagnostic test may be considered sufficient for the laboratory confirmation of BUD. Subsequent application of different diagnostic tests rendered a laboratory confirmation of 65% pre-ulcerative and of 70% ulcerative lesions. Implementation of a stepwise, subsequent analysis of diagnostic specimens will result in considerable cost saving compared with simultaneous testing of specimens by several diagnostic assays.

Post-surgical assessment of excised tissue from patients with Buruli ulcer disease: progression of infection in macroscopically healthy tissue.

OBJECTIVE: The current standard of treatment of Buruli ulcer disease (BUD) is surgical excision of lesions. Excision size is determined macroscopically assuming the complete removal of all infected tissue. However, dissemination of infection beyond the excision margins into apparently healthy tissue, possibly associated with recurrences, cannot be excluded in this way. To assess the central to peripheral progression of Mycobacterium ulcerans infection and the mycobacterial infiltration of excision margins, excised tissue was examined for signs of infection. METHODS: 20 BUD lesions were excised in general anaesthesia including all necrotic and subcutaneous adipose tissue down to the fascia and at an average of 40 mm into the macroscopically unaffected tissue beyond the border of the lesion. Tissue samples were subjected to PCR and histopathology. RESULTS: Although the bacillary load decreased from central to peripheral, M. ulcerans infection was detected throughout all examined tissue specimens including the peripheral segments as well as excision margins of all patients. During the post-operative hospitalization period (averaging 2 months) no local recurrences were observed. CONCLUSION: Available data suggest a correlation of surgical techniques with local recurrences. The results of this study indicate the unnoticed early progression of mycobacterial infection into macroscopically healthy tissue. Thus, the removal of all infected tissue cannot always be verified visually by the surgeon. Provided that long-term follow up of patients with positive excision margins will establish the clinical relevance of these findings, on-site laboratory assessment of excised tissue in combination with follow up may contribute to reduce recurrence rates.

[Mycobacterium ulcerans disease (Buruli ulcer): surgical treatment of 102 cases in the Democratic Republic of Congo]. / Ulcère à Mycobacterium ulcerans: prise en charge chirurgicale dans 102 observations en Republique Démocratique du Congo.

This report describes the preliminary results of surgical treatment of 102 patients presenting Buruli ulcer (BU) over the 5-year period from January 1, 2000 to January 1, 2005. The overall purpose is to improve therapeutic management of BU in the Democratic Republic of Congo. The main disease features were the same as those described in the literature. Diffuse mixed ulcerative forms were the most common in the hospital and at the health care center. Infection by Mycobacterium ulcerans was confirmed by microbacteriological analysis and histological study. Surgical removal of the BU was performed with primary suture, protective dressing, or skin grafting. Local care consisted of application of an aqueous solution of chloramine-metronidazole-nitrofurandoine daily after debridement. Skin grafting was performed with or without protective dressing. Preliminary results with a follow-up of 12 months showed healing in 62 cases, recurrece in 22, and unknown outcome in 18. Although surgical treatment was feasible in poor rural facilities, the cost depending on clinical form is high and recurrence is frequent. These findings undersscore the importance of early detection and treatment with antimycobacterials.

Mycobacterium ulcerans treatment costs, Australia.

Mycobacterium ulcerans gives rise to severe skin ulceration that can be associated with considerable illness. The cost of diagnosis, treatment, and lost income has never been assessed in Australia. A survey of 26 confirmed cases of the disease in Victoria was undertaken. Data were collected on demographic details, diagnostic tests, treatment, time off work, and travel to obtain treatment. All costs are reported in Australian dollars in 1997-98 prices. The cost varies considerably with disease severity. For mild cases, the average direct cost is 6,803 Australian dollars, and for severe cases 27,681 Australian dollars. Hospitalization accounts for 61% to 90% of costs, and indirect costs amount to 24% of the total per case. M. ulcerans can be an expensive disease to diagnose and treat. Costs can be reduced by early diagnosis and definitive treatment. Research is needed to find cost-effective therapies for this disease.