Successful outcomes with oral fluoroquinolones combined with rifampicin in the treatment of Mycobacterium ulcerans: an observational cohort study

Background

The World Health Organization currently recommends combined streptomycin and rifampicin antibiotic treatment as first-line therapy for Mycobacterium ulcerans infections. Alternatives are needed when these are not tolerated or accepted by patients, contraindicated, or neither accessible nor affordable. Despite in vitro effectiveness, clinical evidence for fluoroquinolone antibiotic use against Mycobacterium ulcerans is lacking. We describe outcomes and tolerability of fluoroquinolone-containing antibiotic regimens for Mycobacterium ulcerans in south-eastern Australia.

Methodology/Principal Findings

Analysis was performed of prospectively collected data including all primary Mycobacterium ulcerans infections treated at Barwon Health between 1998 and 2010. Medical treatment involved antibiotic use for more than 7 days; surgical treatment involved surgical excision of a lesion. Treatment success was defined as complete lesion healing without recurrence at 12 months follow-up. A complication was defined as an adverse event attributed to an antibiotic that required its cessation. A total of 133 patients with 137 lesions were studied. Median age was 62 years (range 3–94 years). 47 (34%) had surgical treatment alone, and 90 (66%) had combined surgical and medical treatment. Rifampicin and ciprofloxacin comprised 61% and rifampicin and clarithromycin 23% of first-line antibiotic regimens. 13/47 (30%) treated with surgery alone failed treatment compared to 0/90 (0%) of those treated with combination medical and surgical treatment (p<0.0001). There was no difference in treatment success rate for antibiotic combinations containing a fluoroquinolone (61/61 cases; 100%) compared with those not containing a fluoroquinolone (29/29 cases; 100%). Complication rates were similar between ciprofloxacin and rifampicin (31%) and rifampicin and clarithromycin (33%) regimens (OR 0.89, 95% CI 0.27–2.99). Paradoxical reactions during treatment were observed in 8 (9%) of antibiotic treated cases.

Conclusions

Antibiotics combined with surgery may significantly increase treatment success for Mycobacterium ulcerans infections, and fluoroquinolone combined with rifampicin-containing antibiotic regimens can provide an effective and safe oral treatment option.     

Secondary Buruli Ulcer Skin Lesions Emerging Several Months after Completion of Chemotherapy: Paradoxical Reaction or Evidence for Immune Protection?

Background

The neglected tropical disease Buruli ulcer (BU) caused by Mycobacterium ulcerans is an infection of the subcutaneous tissue leading to chronic ulcerative skin lesions. Histopathological features are progressive tissue necrosis, extracellular clusters of acid fast bacilli (AFB) and poor inflammatory responses at the site of infection. After the recommended eight weeks standard treatment with rifampicin and streptomycin, a reversal of the local immunosuppression caused by the macrolide toxin mycolactone of M. ulcerans is observed.

Methodology/Principal Findings

We have conducted a detailed histopathological and immunohistochemical analysis of tissue specimens from two patients developing multiple new skin lesions 12 to 409 days after completion of antibiotic treatment. Lesions exhibited characteristic histopathological hallmarks of Buruli ulcer and AFB with degenerated appearance were found in several of them. However, other than in active disease, lesions contained massive leukocyte infiltrates including large B-cell clusters, as typically found in cured lesions.

Conclusion/Significance

Our histopathological findings demonstrate that the skin lesions emerging several months after completion of antibiotic treatment were associated with M. ulcerans infection. During antibiotic therapy of Buruli ulcer development of new skin lesions may be caused by immune response-mediated paradoxical reactions. These seem to be triggered by mycobacterial antigens and immunostimulators released from clinically unrecognized bacterial foci. However, in particular the lesions that appeared more than one year after completion of antibiotic treatment may have been associated with new infection foci resolved by immune responses primed by the successful treatment of the initial lesion.     

Corticosteroid-Induced Immunosuppression Ultimately Does Not Compromise the Efficacy of Antibiotherapy in Murine Mycobacterium ulcerans Infection

Background

Buruli ulcer (BU) is a necrotizing disease of the skin, subcutaneous tissue and bone caused by Mycobacterium ulcerans. It has been suggested that the immune response developed during the recommended rifampicin/streptomycin (RS) antibiotherapy is protective, contributing to bacterial clearance. On the other hand, paradoxical reactions have been described during or after antibiotherapy, characterized by pathological inflammatory responses. This exacerbated inflammation could be circumvented by immunosuppressive drugs. Therefore, it is important to clarify if the immune system contributes to bacterial clearance during RS antibiotherapy and if immunosuppression hampers the efficacy of the antibiotic regimen.

Methodology/Principal Findings

We used the M. ulcerans infection footpad mouse model. Corticosteroid-induced immunosuppression was achieved before experimental infection and maintained during combined RS antibiotherapy by the administration of dexamethasone (DEX). Time-lapsed analyses of macroscopic lesions, bacterial burdens, histology and immunohistochemistry were performed in M. ulcerans-infected footpads. We show here that corticosteroid-immunosuppressed mice are more susceptible to M. ulcerans, with higher bacterial burdens and earlier ulceration. Despite this, macroscopic lesions remised during combined antibiotic/DEX treatment and no viable bacteria were detected in the footpads after RS administration. This was observed despite a delayed kinetics in bacterial clearance, associated with a local reduction of T cell and neutrophil numbers, when compared with immunocompetent RS-treated mice. In addition, no relapse was observed following an additional 3 month period of DEX administration.

Conclusions/Significance

These findings reveal a major role of the RS bactericidal activity for the resolution of M. ulcerans experimental infections even during immunosuppression, and support clinical investigation on the potential use of corticosteroids or other immunosuppressive/anti-inflammatory drugs for the management of BU patients undergoing paradoxical reactions.     

Mycobacterium Ulcerans Treatment – Can Antibiotic Duration Be Reduced in Selected Patients?

Introduction

Mycobacterium ulcerans (M. ulcerans) is a necrotizing skin infection endemic to the Bellarine Peninsula, Australia. Current treatment recommendations include 8 weeks of combination antibiotics, with adjuvant surgery if necessary. However, antibiotic toxicity often results in early treatment cessation and local experience suggests that shorter antibiotic courses may be effective with concurrent surgery. We report the outcomes of patients in the Barwon Health M. ulcerans cohort who received shorter courses of antibiotic therapy than 8 weeks.

Methodology / Principal findings

A retrospective analysis was performed of all M. ulcerans infections treated at Barwon Health from March 1, 1998 to July 31, 2013. Sixty-two patients, with a median age of 65 years, received < 56 days of antibiotics and 51 (82%) of these patients underwent concurrent surgical excision. Most received a two-drug regimen of rifampicin combined with either ciprofloxacin or clarithromycin for a median 29 days (IQR 21–41days). Cessation rates were 55% for adverse events and 36% based on clinician decision. The overall success rate was 95% (98% with concurrent surgery; 82% with antibiotics alone) with a 50% success rate for those who received < 14 days of antibiotics increasing to 94% if they received 14–27 days and 100% for 28–55 days (p<0.01). A 100% success rate was seen for concurrent surgery and 14–27 days of antibiotics versus 67% for concurrent surgery and < 14 days of antibiotics (p = 0.12). No previously identified risk factors for treatment failure with surgery alone were associated with reduced treatment success rates with < 56 days of antibiotics.

Conclusion

In selected patients, antibiotic treatment durations for M. ulcerans shorter than the current WHO recommended 8 weeks duration may be associated with successful outcomes.     

Detection of Mycolactone A/B in Mycobacterium ulcerans–Infected Human Tissue

Background

Mycobacterium ulcerans disease (Buruli ulcer) is a neglected tropical disease common amongst children in rural West Africa. Animal experiments have shown that tissue destruction is caused by a toxin called mycolactone.

Methodology/Principal Findings

A molecule was identified among acetone-soluble lipid extracts from M. ulcerans (Mu)-infected human lesions with chemical and biological properties of mycolactone A/B. On thin layer chromatography this molecule had a retention factor value of 0.23, MS analyses showed it had an m/z of 765.6 [M+Na⁺] and on MS:MS fragmented to produce the core lactone ring with m/z of 429.4 and the polyketide side chain of mycolactone A/B with m/z of 359.2. Acetone-soluble lipids from lesions demonstrated significant cytotoxic, pro-apoptotic and anti-inflammatory activities on cultured fibroblast and macrophage cell lines. Mycolactone A/B was detected in all of 10 tissue samples from patients with ulcerative and pre-ulcerative Mu disease.

Conclusions/Significance

Mycolactone can be detected in human tissue infected with Mu. This could have important implications for successful management of Mu infection by antibiotic treatment but further studies are needed to measure its concentration.     

Good Quality of Life in Former Buruli Ulcer Patients with Small Lesions: Long-Term Follow-up of the BURULICO Trial

Background

Buruli Ulcer is a tropical skin disease caused by Mycobacterium ulcerans, which, due to scarring and contractures can lead to stigma and functional limitations. However, recent advances in treatment, combined with increased public health efforts have the potential to significantly improve disease outcome.

Objectives

To study the Quality of Life (QoL) of former Buruli Ulcer patients who, in the context of a randomized controlled trial, reported early with small lesions (cross-sectional diameter <10 cm), and received a full course of antibiotic treatment.

Methods

127 Participants of the BURULICO drug trial in Ghana were revisited. All former patients aged 16 or older completed the Dermatology Life Quality Index (DLQI) and the abbreviated World Health Organization Quality of Life scale (WHOQOL-BREF). The WHOQOL-BREF was also administered to 82 matched healthy controls. Those younger than 16 completed the Childrens'' Dermatology Life Quality Index (CDLQI) only.

Results

The median (Inter Quartile Range) score on the DLQI was 0 (0–4), indicating good QoL. 85% of former patients indicated no effect, or only a small effect of the disease on their current life. Former patients also indicated good QoL on the physical and psychological domains of the WHOQOL-BREF, and scored significantly higher than healthy controls on these domains. There was a weak correlation between the DLQI and scar size (ρ = 0.32; p<0.001).

Conclusions

BU patients who report early with small lesions and receive 8 weeks of antimicrobial therapy have a good QoL at long-term follow-up. These findings contrast with the debilitating sequelae often reported in BU, and highlight the importance of early case detection.     

Development of highly organized lymphoid structures in Buruli ulcer lesions after treatment with rifampicin and streptomycin

Background

Buruli ulcer caused by Mycobacterium ulcerans is an infection of the subcutaneous tissue leading to chronic necrotising skin ulcers. The pathogenesis is associated with the cytocidal and immunosuppressive activities of a macrolide toxin. Histopathological hallmark of progressing disease is a poor inflammatory response despite of clusters of extracellular bacilli. While traditionally wide excision of the infected tissue was the standard treatment, provisional WHO guidelines now recommend an eight week pre-treatment with streptomycin and rifampicin.

Methodology/Principal Findings

We conducted a detailed immunohistochemical analysis of tissue samples from Buruli patients who received antibiotic treatment. Cellular immune response along with bacterial load and distribution were monitored. We demonstrate that this treatment leads to the development of highly organized cellular infiltration surrounding areas of coagulative necrosis. Diffuse infiltrates, granulomas and dense lymphocyte aggregation close to vessels were observed. Mycobacterial material was primarily located inside mononuclear phagocytes and microcolonies consisting of extracellular rod-shaped mycobacteria were no longer found. In observational studies some patients showed no clinical response to antibiotic treatment. Corresponding to that, one of five lesions analysed presented with huge clusters of rod-shaped bacilli but no signs of infiltration.

Conclusions/Significance

Results signify that eight weeks of antibiotic treatment reverses local immunosuppression and leads to an active inflammatory process in different compartments of the skin. Structured leukocyte infiltrates with unique signatures indicative for healing processes developed at the margins of the lesions. It remains to be analysed whether antibiotic resistance of certain strains of M. ulcerans, lacking patient compliance or poor drug quality are responsible for the absent clinical responses in some patients. In future, analysis of local immune responses could serve as a suitable surrogate marker for the efficacy of alternative treatment strategies.     

Phage Therapy Is Effective against Infection by Mycobacterium ulcerans in a Murine Footpad Model

Background

Buruli Ulcer (BU) is a neglected, necrotizing skin disease caused by Mycobacterium ulcerans. Currently, there is no vaccine against M. ulcerans infection. Although the World Health Organization recommends a combination of rifampicin and streptomycin for the treatment of BU, clinical management of advanced stages is still based on the surgical resection of infected skin. The use of bacteriophages for the control of bacterial infections has been considered as an alternative or to be used in association with antibiotherapy. Additionally, the mycobacteriophage D29 has previously been shown to display lytic activity against M. ulcerans isolates.

Methodology/Principal findings

We used the mouse footpad model of M. ulcerans infection to evaluate the therapeutic efficacy of treatment with mycobacteriophage D29. Analyses of macroscopic lesions, bacterial burdens, histology and cytokine production were performed in both M. ulcerans-infected footpads and draining lymph nodes (DLN). We have demonstrated that a single subcutaneous injection of the mycobacteriophage D29, administered 33 days after bacterial challenge, was sufficient to decrease pathology and to prevent ulceration. This protection resulted in a significant reduction of M. ulcerans numbers accompanied by an increase of cytokine levels (including IFN-γ), both in footpads and DLN. Additionally, mycobacteriophage D29 treatment induced a cellular infiltrate of a lymphocytic/macrophagic profile.

Conclusions/Significance

Our observations demonstrate the potential of phage therapy against M. ulcerans infection, paving the way for future studies aiming at the development of novel phage-related therapeutic approaches against BU.     

Differential Gene Repertoire in Mycobacterium ulcerans Identifies Candidate Genes for Patho-Adaptation

Background

Based on large genomic sequence polymorphisms, several haplotypes belonging to two major lineages of the human pathogen Mycobacterium ulcerans could be distinguished among patient isolates from various geographic origins. However, the biological relevance of insertional/deletional diversity is not understood.

Methodology

Using comparative genomics, we have investigated the genes located in regions of difference recently identified by DNA microarray based hybridisation analysis. The analysed regions of difference comprise ∼7% of the entire M. ulcerans genome.

Principal Findings

Several different mechanisms leading to loss of functional genes were identified, ranging from pseudogenization, caused by frame shift mutations or mobile genetic element interspersing, to large sequence polymorphisms. Four hot spot regions for genetic instability were unveiled. Altogether, 229 coding sequences were found to be differentially inactivated, constituting a repertoire of coding sequence variation in the rather monomorphic M. ulcerans.

Conclusions/Significance

The differential gene inactivation patterns associated with the M. ulcerans haplotypes identified candidate genes that may confer enhanced adaptation upon ablation of expression. A number of gene conversions confined to the classical lineage may contribute to particular virulence of this group comprising isolates from Africa and Australia. Identification of this spectrum of anti-virulence gene candidates expands our understanding of the pathogenicity and ecology of the emerging infectious disease Buruli ulcer.     

Structure-Activity Relationship Studies on the Macrolide Exotoxin Mycolactone of Mycobacterium ulcerans

Background

Mycolactones are a family of polyketide-derived macrolide exotoxins produced by Mycobacterium ulcerans, the causative agent of the chronic necrotizing skin disease Buruli ulcer. The toxin is synthesized by polyketide synthases encoded by the virulence plasmid pMUM. The apoptotic, necrotic and immunosuppressive properties of mycolactones play a central role in the pathogenesis of M. ulcerans.

Methodology/Principal Findings

We have synthesized and tested a series of mycolactone derivatives to conduct structure-activity relationship studies. Flow cytometry, fluorescence microscopy and Alamar Blue-based metabolic assays were used to assess activities of mycolactones on the murine L929 fibroblast cell line. Modifications of the C-linked upper side chain (comprising C12–C20) caused less pronounced changes in cytotoxicity than modifications in the lower C5-O-linked polyunsaturated acyl side chain. A derivative with a truncated lower side chain was unique in having strong inhibitory effects on fibroblast metabolism and cell proliferation at non-cytotoxic concentrations. We also tested whether mycolactones have antimicrobial activity and found no activity against representatives of Gram-positive (Streptococcus pneumoniae) or Gram-negative bacteria (Neisseria meningitis and Escherichia coli), the fungus Saccharomyces cerevisae or the amoeba Dictyostelium discoideum.

Conclusion

Highly defined synthetic compounds allowed to unambiguously compare biological activities of mycolactones expressed by different M. ulcerans lineages and may help identifying target structures and triggering pathways.     

Risk of Buruli ulcer and detection of Mycobacterium ulcerans in mosquitoes in southeastern Australia

Background

Buruli ulcer (BU) is a destructive skin condition caused by infection with the environmental bacterium, Mycobacterium ulcerans. The mode of transmission of M. ulcerans is not completely understood, but several studies have explored the role of biting insects. In this study, we tested for an association between the detection of M. ulcerans in mosquitoes and the risk of BU disease in humans in an endemic area of southeastern Australia.

Methodology/Principal Findings

Adult mosquitoes were trapped in seven towns on the Bellarine Peninsula in Victoria, Australia, from December 2004 to December 2009 and screened for M. ulcerans by real-time PCR. The number of laboratory-confirmed cases of BU in permanent residents of these towns diagnosed during the same period was tallied to determine the average cumulative incidence of BU in each location. Pearson''s correlation coefficient (r) was calculated for the proportion of M. ulcerans-positive mosquitoes per town correlated with the incidence of BU per town. We found a strong dose-response relationship between the detection of M. ulcerans in mosquitoes and the risk of human disease (r, 0.99; 95% CI, 0.92–0.99; p<0.001).

Conclusions/Significance

The results of this study strengthen the hypothesis that mosquitoes are involved in the transmission of M. ulcerans in southeastern Australia. This has implications for the development of intervention strategies to control and prevent BU.     

Empiric Antibiotic Therapy for Staphylococcus aureus Bacteremia May Not Reduce In-Hospital Mortality: A Retrospective Cohort Study

Background

Appropriate empiric therapy, antibiotic therapy with in vitro activity to the infecting organism given prior to confirmed culture results, may improve Staphylococcus aureus outcomes. We aimed to measure the clinical impact of appropriate empiric antibiotic therapy on mortality, while statistically adjusting for comorbidities, severity of illness and presence of virulence factors in the infecting strain.

Methodology

We conducted a retrospective cohort study of adult patients admitted to a tertiary-care facility from January 1, 2003 to June 30, 2007, who had S. aureus bacteremia. Time to appropriate therapy was measured from blood culture collection to the receipt of antibiotics with in vitro activity to the infecting organism. Cox proportional hazard models were used to measure the association between receipt of appropriate empiric therapy and in-hospital mortality, statistically adjusting for patient and pathogen characteristics.

Principal Findings

Among 814 admissions, 537 (66%) received appropriate empiric therapy. Those who received appropriate empiric therapy had a higher hazard of 30-day in-hospital mortality (Hazard Ratio (HR): 1.52; 95% confidence interval (CI): 0.99, 2.34). A longer time to appropriate therapy was protective against mortality (HR: 0.79; 95% CI: 0.60, 1.03) except among the healthiest quartile of patients (HR: 1.44; 95% CI: 0.66, 3.15).

Conclusions/Significance

Appropriate empiric therapy was not associated with decreased mortality in patients with S. aureus bacteremia except in the least ill patients. Initial broad antibiotic selection may not be widely beneficial.     

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.     

The Impact of Healthcare-Associated Infection on Mortality: Failure in Clinical Recognition Is Related with Inadequate Antibiotic Therapy

Purpose

To understand if clinicians can tell apart patients with healthcare-associated infections (HCAI) from those with community-acquired infections (CAI) and to determine the impact of HCAI in the adequacy of initial antibiotic therapy and hospital mortality.

Methods

One-year prospective cohort study including all consecutive infected patients admitted to a large university tertiary care hospital.

Results

A total of 1035 patients were included in this study. There were 718 patients admitted from the community: 225 (31%) with HCAI and 493 (69%) with CAI. Total microbiologic documentation rate of infection was 68% (n = 703): 56% in CAI, 73% in HCAI and 83% in hospital-acquired infections (HAI). Antibiotic therapy was inadequate in 27% of patients with HCAI vs. 14% of patients with CAI (p<0.001). Among patients with HCAI, 47% received antibiotic therapy in accordance with international recommendations for treatment of CAI. Antibiotic therapy was inadequate in 36% of patients with HCAI whose treatment followed international recommendations for CAI vs. 19% in the group of HCAI patients whose treatment did not follow these guidelines (p = 0.014). Variables independently associated with inadequate antibiotic therapy were: decreased functional capacity (adjusted OR = 2.24), HCAI (adjusted OR = 2.09) and HAI (adjusted OR = 2.24). Variables independently associated with higher hospital mortality were: age (adjusted OR = 1.05, per year), severe sepsis (adjusted OR = 1.92), septic shock (adjusted OR = 8.13) and inadequate antibiotic therapy (adjusted OR = 1.99).

Conclusions

HCAI was associated with an increased rate of inadequate antibiotic therapy but not with a significant increase in hospital mortality. Clinicians need to be aware of healthcare-associated infections among the group of infected patients arriving from the community since the existing guidelines regarding antibiotic therapy do not apply to this group and they will otherwise receive inadequate antibiotic therapy which will have a negative impact on hospital outcome.     

Amoebae as Potential Environmental Hosts for Mycobacterium ulcerans and Other Mycobacteria, but Doubtful Actors in Buruli Ulcer Epidemiology

Background

The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, remain unknown. Ecological, genetic and epidemiological information nonetheless suggests that M. ulcerans may reside in aquatic protozoa.

Methodology/Principal Findings

We experimentally infected Acanthamoeba polyphaga with M. ulcerans and found that the bacilli were phagocytised, not digested and remained viable for the duration of the experiment. Furthermore, we collected 13 water, 90 biofilm and 45 detritus samples in both Buruli ulcer endemic and non-endemic communities in Ghana, from which we cultivated amoeboid protozoa and mycobacteria. M. ulcerans was not isolated, but other mycobacteria were as frequently isolated from intracellular as from extracellular sources, suggesting that they commonly infect amoebae in nature. We screened the samples as well as the amoeba cultures for the M. ulcerans markers IS2404, IS2606 and KR-B. IS2404 was detected in 2% of the environmental samples and in 4% of the amoeba cultures. The IS2404 positive amoeba cultures included up to 5 different protozoan species, and originated both from Buruli ulcer endemic and non-endemic communities.

Conclusions/Significance

This is the first report of experimental infection of amoebae with M. ulcerans and of the detection of the marker IS2404 in amoeba cultures isolated from the environment. We conclude that amoeba are potential natural hosts for M. ulcerans, yet remain sceptical about their implication in the transmission of M. ulcerans to humans and their importance in the epidemiology of Buruli ulcer.     

Cellular immunity confers transient protection in experimental Buruli ulcer following BCG or mycolactone-negative Mycobacterium ulcerans vaccination

Background

Buruli ulcer (BU) is an emerging infectious disease caused by Mycobacterium ulcerans that can result in extensive necrotizing cutaneous lesions due to the cytotoxic exotoxin mycolactone. There is no specific vaccine against BU but reports show some degree of cross-reactive protection conferred by M. bovis BCG immunization. Alternatively, an M. ulcerans-specific immunization could be a better preventive strategy.

Methodology/Principal Findings

In this study, we used the mouse model to characterize the histological and cytokine profiles triggered by vaccination with either BCG or mycolactone-negative M. ulcerans, followed by footpad infection with virulent M. ulcerans. We observed that BCG vaccination significantly delayed the onset of M. ulcerans growth and footpad swelling through the induction of an earlier and sustained IFN-γ T cell response in the draining lymph node (DLN). BCG vaccination also resulted in cell-mediated immunity (CMI) in M. ulcerans-infected footpads, given the predominance of a chronic mononuclear infiltrate positive for iNOS, as well as increased and sustained levels of IFN-γ and TNF. No significant IL-4, IL-17 or IL-10 responses were detected in the footpad or the DLN, in either infected or vaccinated mice. Despite this protective Th1 response, BCG vaccination did not avoid the later progression of M. ulcerans infection, regardless of challenge dose. Immunization with mycolactone-deficient M. ulcerans also significantly delayed the progression of footpad infection, swelling and ulceration, but ultimately M. ulcerans pathogenic mechanisms prevailed.

Conclusions/Significance

The delay in the emergence of pathology observed in vaccinated mice emphasizes the relevance of protective Th1 recall responses against M. ulcerans. In future studies it will be important to determine how the transient CMI induced by vaccination is compromised.     

Mycolactone diffuses into the peripheral blood of Buruli ulcer patients--implications for diagnosis and disease monitoring

Background

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established.

Methodology/Principal Finding

Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone.

Conclusions/Significance

Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies.     

Topography and Land Cover of Watersheds Predicts the Distribution of the Environmental Pathogen Mycobacterium ulcerans in Aquatic Insects

Background

An understanding of the factors driving the distribution of pathogens is useful in preventing disease. Often we achieve this understanding at a local microhabitat scale; however the larger scale processes are often neglected. This can result in misleading inferences about the distribution of the pathogen, inhibiting our ability to manage the disease. One such disease is Buruli ulcer, an emerging neglected tropical disease afflicting many thousands in Africa, caused by the environmental pathogen Mycobacterium ulcerans. Herein, we aim to describe the larger scale landscape process describing the distribution of M. ulcerans.

Methodology

Following extensive sampling of the community of aquatic macroinvertebrates in Cameroon, we select the 5 dominant insect Orders, and conduct an ecological niche model to describe how the distribution of M. ulcerans positive insects changes according to land cover and topography. We then explore the generalizability of the results by testing them against an independent dataset collected in a second endemic region, French Guiana.

Principal Findings

We find that the distribution of the bacterium in Cameroon is accurately described by the land cover and topography of the watershed, that there are notable seasonal differences in distribution, and that the Cameroon model does not predict the distribution of M. ulcerans in French Guiana.

Conclusions/Significance

Future studies of M. ulcerans would benefit from consideration of local structure of the local stream network in future sampling, and further work is needed on the reasons for notable differences in the distribution of this species from one region to another. This work represents a first step in the identification of large-scale environmental drivers of this species, for the purposes of disease risk mapping.     

Mycolactone diffuses from Mycobacterium ulcerans-infected tissues and targets mononuclear cells in peripheral blood and lymphoid organs

Background

Buruli ulcer (BU) is a progressive disease of subcutaneous tissues caused by Mycobacterium ulcerans. The pathology of BU lesions is associated with the local production of a diffusible substance, mycolactone, with cytocidal and immunosuppressive properties. The defective inflammatory responses in BU lesions reflect these biological properties of the toxin. However, whether mycolactone diffuses from infected tissues and suppresses IFN-γ responses in BU patients remains unclear.

Methodology/Principal Findings

Here we have investigated the pharmacodistribution of mycolactone following injection in animal models by tracing a radiolabeled form of the toxin, and by directly quantifying mycolactone in lipid extracts from internal organs and cell subpopulations. We show that subcutaneously delivered mycolactone diffused into mouse peripheral blood and accumulated in internal organs with a particular tropism for the spleen. When mice were infected subcutaneously with M. ulcerans, this led to a comparable pattern of distribution of mycolactone. No evidence that mycolactone circulated in blood serum during infection could be demonstrated. However, structurally intact toxin was identified in the mononuclear cells of blood, lymph nodes and spleen several weeks before ulcerative lesions appear. Importantly, diffusion of mycolactone into the blood of M. ulcerans–infected mice coincided with alterations in the functions of circulating lymphocytes.

Conclusion

In addition to providing the first evidence that mycolactone diffuses beyond the site of M. ulcerans infection, our results support the hypothesis that the toxin exerts immunosuppressive effects at the systemic level. Furthermore, they suggest that assays based on mycolactone detection in circulating blood cells may be considered for diagnostic tests of early disease.