A Major Role for Mammals in the Ecology of Mycobacterium ulcerans

Background

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a destructive skin disease found predominantly in sub-Saharan Africa and south-eastern Australia. The precise mode(s) of transmission and environmental reservoir(s) remain unknown, but several studies have explored the role of aquatic invertebrate species. The purpose of this study was to investigate the environmental distribution of M. ulcerans in south-eastern Australia.

Methodology/Principal Findings

A range of environmental samples was collected from Point Lonsdale (a small coastal town southwest of Melbourne, Australia, endemic for BU) and from areas with fewer or no reported incident cases of BU. Mycobacterium ulcerans DNA was detected at low levels by real-time PCR in soil, sediment, water residue, aquatic plant biofilm and terrestrial vegetation collected in Point Lonsdale. Higher levels of M. ulcerans DNA were detected in the faeces of common ringtail (Pseudocheirus peregrinus) and common brushtail (Trichosurus vulpecula) possums. Systematic testing of possum faeces revealed that M. ulcerans DNA could be detected in 41% of faecal samples collected in Point Lonsdale compared with less than 1% of faecal samples collected from non-endemic areas (p<0.0001). Capture and clinical examination of live possums in Point Lonsdale validated the accuracy of the predictive value of the faecal surveys by revealing that 38% of ringtail possums and 24% of brushtail possums had laboratory-confirmed M. ulcerans skin lesions and/or M. ulcerans PCR positive faeces. Whole genome sequencing revealed an extremely close genetic relationship between human and possum M. ulcerans isolates.

Conclusions/Significance

The prevailing wisdom is that M. ulcerans is an aquatic pathogen and that BU is acquired by contact with certain aquatic environments (swamps, slow-flowing water). Now, after 70 years of research, we propose a transmission model for BU in which terrestrial mammals are implicated as reservoirs for M. ulcerans.     

Family Relationship,Water Contact and Occurrence of Buruli Ulcer in Benin

Background

Mycobacterium ulcerans disease (Buruli ulcer) is the most widespread mycobacterial disease in the world after leprosy and tuberculosis. How M. ulcerans is introduced into the skin of humans remains unclear, but it appears that individuals living in the same environment may have different susceptibilities.

Objectives

This study aims to determine whether frequent contacts with natural water sources, family relationship or the practice of consanguineous marriages are associated with the occurrence of Buruli ulcer (BU).

Design

Case control study.

Setting

Department of Atlantique, Benin.

Subjects

BU-confirmed cases that were diagnosed and followed up at the BU detection and treatment center (CDTUB) of Allada (Department of the Atlantique, Benin) during the period from January 1st, 2006, to June 30th, 2008, with three matched controls (persons who had no signs or symptoms of active or inactive BU) for age, gender and village of residence per case.

Main Outcomes Measured

Contact with natural water sources, BU history in the family and the practice of consanguineous marriages.

Results

A total of 416 participants were included in this study, including 104 cases and 312 controls. BU history in the family (p<0.001), adjusted by daily contact with a natural water source (p = 0.007), was significantly associated with higher odds of having BU (OR; 95% CI = 5.5; 3.0–10.0). The practice of consanguineous marriage was not associated with the occurrence of BU (p = 0.40). Mendelian disorders could explain this finding, which may influence individual susceptibility by impairing immunity.

Conclusion

This study suggests that a combination of genetic factors and behavioral risk factors may increase the susceptibility for developing BU.     

Phase Change Material for Thermotherapy of Buruli Ulcer: A Prospective Observational Single Centre Proof-of-Principle Trial

Background

Buruli ulcer (BU) is an infection of the subcutaneous tissue leading to chronic necrotizing skin ulcers. The causative pathogen, Mycobacterium ulcerans, grows best at 30°C–33°C and not above 37°C. We explored the safety, tolerability and efficacy of phase change material (PCM), a novel heat application system for thermotherapy of BU.

Methodology/Principal Findings

In a prospective observational single centre proof-of-principle trial in Ayos/Cameroon, six laboratory reconfirmed patients with ulcerative Buruli lesions received 28–31 (ulcers ≤2 cm) or 50–55 (ulcers >2 cm) days of thermotherapy with the PCM sodium acetate trihydrate as heat application system. This PCM is widely used in commercial pocket heat pads, it is easy to apply, rechargeable in hot water, non-toxic and non-hazardous to the environment. All patients enrolled in the trial completed treatment. Being completely mobile during the well-tolerated heat application, acceptability of the PCM bandages was very high. In patients with smaller ulcers, wounds healed completely without further intervention. Patients with large defects had skin grafting after successful heat treatment. Heat treatment was not associated with marked increases in local inflammation or the development of ectopic lymphoid tissue. One and a half years after completion of treatment, all patients are relapse-free.

Conclusions/Significance

Our reusable PCM-based heat application device appears perfectly suited to treat BU in endemic countries with limited resources and infrastructure.

Trial Registration

Controlled-Trials.com ISRCTN88392614     

Contribution of the Community Health Volunteers in the Control of Buruli Ulcer in Bénin

Background

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. Usually BU begins as a painless nodule, plaque or edema, ultimately developing into an ulcer. The high number of patients presenting with ulcers in an advanced stage is striking. Such late presentation will complicate treatment and have long-term disabilities as a consequence. The disease is mainly endemic in West Africa. The primary strategy for control of this disease is early detection using community village volunteers.

Methodology/Principal Findings

In this retrospective, observational study, information regarding Buruli ulcer patients that reported to one of the four BU centers in Bénin between January 2008 and December 2010 was collected using the WHO/BU01 forms. Information used from these forms included general characteristics of the patient, the results of diagnostic tests, the presence of functional limitations at start of treatment, lesion size, patient delay and the referral system. The role of the different referral systems on the stage of disease at presentation in the hospital was analyzed by a logistic regression analysis. About a quarter of the patients (26.5%) were referred to the hospital by the community health volunteers. In our data set, patients referred to the hospital by community health volunteers appeared to be in an earlier stage of disease than patients referred by other methods, but after adjustment by the regression analysis for the health center, this effect could no longer be seen. The Polymerase Chain Reaction (PCR) for IS2404 positivity rate among patients referred by the community health volunteers was not systematically lower than in patients referred by other systems.

Conclusions/Significance

This study clarifies the role played by community health volunteers in Bénin, and shows that they play an important role in the control of BU.     

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.     

Sero-epidemiology as a tool to screen populations for exposure to Mycobacterium ulcerans

Background

Previous analyses of sera from a limited number of Ghanaian Buruli ulcer (BU) patients, their household contacts, individuals living in BU non-endemic regions as well as European controls have indicated that antibody responses to the M. ulcerans 18 kDa small heat shock protein (shsp) reflect exposure to this pathogen. Here, we have investigated to what extent inhabitants of regions in Ghana regarded as non-endemic for BU develop anti-18 kDa shsp antibody titers.

Methodology/Principal Findings

For this purpose we determined anti-18 kDa shsp IgG titers in sera collected from healthy inhabitants of the BU endemic Densu River Valley and the Volta Region, which was so far regarded as BU non-endemic. Significantly more sera from the Densu River Valley contained anti-18 kDa shsp IgG (32% versus 12%, respectively). However, some sera from the Volta Region also showed high titers. When interviewing these sero-responders, it was revealed that the person with the highest titer had a chronic wound, which was clinically diagnosed and laboratory reconfirmed as active BU. After identification of this BU index case, further BU cases were clinically diagnosed by the Volta Region local health authorities and laboratory reconfirmed. Interestingly, there was neither a difference in sero-prevalence nor in IS2404 PCR positivity of environmental samples between BU endemic and non-endemic communities located in the Densu River Valley.

Conclusions

These data indicate that the intensity of exposure to M. ulcerans in endemic and non-endemic communities along the Densu River is comparable and that currently unknown host and/or pathogen factors may determine how frequently exposure is leading to clinical disease. While even high serum titers of anti-18 kDa shsp IgG do not indicate active disease, sero-epidemiological studies can be used to identify new BU endemic areas.     

A Genomic Approach to Resolving Relapse versus Reinfection among Four Cases of Buruli Ulcer

Background

Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes.

Methodology

We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin.

Principal Findings

The findings suggest that after surgical treatment—without antibiotics—the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse.

Conclusions

To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development.     

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.     

Detection of Mycobacterium ulcerans in the environment predicts prevalence of Buruli ulcer in Benin

Background

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU). In West Africa there is an association between BU and residence in low-lying rural villages where aquatic sources are plentiful. Infection occurs through unknown environmental exposure; human-to-human infection is rare. Molecular evidence for M. ulcerans in environmental samples is well documented, but the association of M. ulcerans in the environment with Buruli ulcer has not been studied in West Africa in an area with accurate case data.

Methodology/Principal Finding

Environmental samples were collected from twenty-five villages in three communes of Benin. Sites sampled included 12 BU endemic villages within the Ouheme and Couffo River drainages and 13 villages near the Mono River and along the coast or ridge where BU has never been identified. Triplicate water filtrand samples from major water sources and samples from three dominant aquatic plant species were collected. Detection of M. ulcerans was based on quantitative polymerase chain reaction. Results show a significant association between M. ulcerans in environmental samples and Buruli ulcer cases in a village (p = 0.0001). A “dose response” was observed in that increasing numbers of M. ulceran- positive environmental samples were associated with increasing prevalence of BU cases (R² = 0.586).

Conclusions/Significance

This study provides the first spatial data on the overlap of M. ulcerans in the environment and BU cases in Benin where case data are based on active surveillance. The study also provides the first evidence on M. ulcerans in well-defined non-endemic sites. Most environmental pathogens are more broadly distributed in the environment than in human populations. The congruence of M. ulcerans in the environment and human infection raises the possibility that humans play a role in the ecology of M. ulcerans. Methods developed could be useful for identifying new areas where humans may be at high risk for BU.     

The “Buruli Score”: Development of a Multivariable Prediction Model for Diagnosis of Mycobacterium ulcerans Infection in Individuals with Ulcerative Skin Lesions,Akonolinga, Cameroon

Background

Access to laboratory diagnosis can be a challenge for individuals suspected of Buruli Ulcer (BU). Our objective was to develop a clinical score to assist clinicians working in resource-limited settings for BU diagnosis.

Methododology/Principal Findings

Between 2011 and 2013, individuals presenting at Akonolinga District Hospital, Cameroon, were enrolled consecutively. Clinical data were collected prospectively. Based on a latent class model using laboratory test results (ZN, PCR, culture), patients were categorized into high, or low BU likelihood. Variables associated with a high BU likelihood in a multivariate logistic model were included in the Buruli score. Score cut-offs were chosen based on calculated predictive values. Of 325 patients with an ulcerative lesion, 51 (15.7%) had a high BU likelihood. The variables identified for the Buruli score were: characteristic smell (+3 points), yellow color (+2), female gender (+2), undermining (+1), green color (+1), lesion hyposensitivity (+1), pain at rest (-1), size >5cm (-1), locoregional adenopathy (-2), age above 20 up to 40 years (-3), or above 40 (-5). This score had AUC of 0.86 (95%CI 0.82–0.89), indicating good discrimination between infected and non-infected individuals. The cut-off to reasonably exclude BU was set at scores <0 (NPV 96.5%; 95%CI 93.0–98.6). The treatment threshold was set at a cut-off ≥4 (PPV 69.0%; 95%CI 49.2–84.7). Patients with intermediate BU probability needed to be tested by PCR.

Conclusions/Significance

We developed a decisional algorithm based on a clinical score assessing BU probability. The Buruli score still requires further validation before it can be recommended for wide use.     

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.     

Burden of Mycobacterium ulcerans Disease (Buruli Ulcer) and the Underreporting Ratio in the Territory of Songololo,Democratic Republic of Congo

Background

Cutaneous infection by Mycobacterium ulcerans, also known as Buruli ulcer (BU), represents the third most common mycobacterial disease in the world after tuberculosis and leprosy. Data on the burden of BU disease in the Democratic Republic of Congo are scanty. This study aimed to estimate the prevalence rate and the distribution of BU in the Songololo Territory, and to assess the coverage of the existing hospital-based reporting system.

Methods

We conducted a cross-sectional survey (July–August 2008) using the door-to-door method simultaneously in the two rural health zones (RHZ) of the Songololo Territory (RHZ of Kimpese and Nsona-Mpangu), each containing twenty health areas. Cases were defined clinically as active BU and inactive BU in accordance with WHO-case definitions.

Results

We detected 775 BU patients (259 active and 516 inactive) in a total population of 237,418 inhabitants. The overall prevalence of BU in Songololo Territory was 3.3/1000 inhabitants, varying from 0 to 27.5/1000 between health areas. Of the 259 patients with active BU, 18 (7%) had been reported in the hospital-based reporting system at Kimpese in the 6–8 months prior to the survey.

Conclusion

The survey demonstrated a huge variation of prevalence between health areas in Songololo Territory and gross underreporting of BU cases in the hospital-based reporting system. Data obtained may contribute to better targeted and improved BU control interventions, and serve as a baseline for future assessments of the control program.     

Climate and Landscape Factors Associated with Buruli Ulcer Incidence in Victoria,Australia

Background

Buruli ulcer (BU), caused by Mycobacterium ulcerans (M. ulcerans), is a necrotizing skin disease found in more than 30 countries worldwide. BU incidence is highest in West Africa; however, cases have substantially increased in coastal regions of southern Australia over the past 30 years. Although the mode of transmission remains uncertain, the spatial pattern of BU emergence in recent years seems to suggest that there is an environmental niche for M. ulcerans and BU prevalence.

Methodology/Principal Findings

Network analysis was applied to BU cases in Victoria, Australia, from 1981–2008. Results revealed a non-random spatio-temporal pattern at the regional scale as well as a stable and efficient BU disease network, indicating that deterministic factors influence the occurrence of this disease. Monthly BU incidence reported by locality was analyzed with landscape and climate data using a multilevel Poisson regression approach. The results suggest the highest BU risk areas occur at low elevations with forested land cover, similar to previous studies of BU risk in West Africa. Additionally, climate conditions as far as 1.5 years in advance appear to impact disease incidence. Warmer and wetter conditions 18–19 months prior to case emergence, followed by a dry period approximately 5 months prior to case emergence seem to favor the occurrence of BU.

Conclusions/Significance

The BU network structure in Victoria, Australia, suggests external environmental factors favor M. ulcerans transmission and, therefore, BU incidence. A unique combination of environmental conditions, including land cover type, temperature and a wet-dry sequence, may produce habitat characteristics that support M. ulcerans transmission and BU prevalence. These findings imply that future BU research efforts on transmission mechanisms should focus on potential vectors/reservoirs found in those environmental niches. Further, this study is the first to quantitatively estimate environmental lag times associated with BU outbreaks, providing insights for future transmission investigations.     

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.     

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 Mycobacterium ulcerans by the loop mediated isothermal amplification method

Background

Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) has emerged as an important public health problem in several rural communities in sub-Saharan Africa. Early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. Presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent.

Methodology

We compared conventional and pocket warmer loop mediated isothermal amplification (LAMP) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of M. ulcerans in clinical specimens. The effect of purified and crude DNA preparations on the detection rate of the LAMP assays were also investigated and compared with that of IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR.

Principal Findings

The lower detection limit of both LAMP methods at 60°C was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65°C. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 PCR when crude extracts of clinical specimens were used.

Conclusion/Significance

The LAMP method can be used as a simple and rapid test for the detection of M. ulcerans in clinical specimens. However, obtaining purified DNA, as well as generating isothermal conditions, remains a major challenge for the use of the LAMP method under field conditions. With further improvement in DNA extraction and amplification conditions, the pwLAMP could be used as a point of care diagnostic test for BU     

New Policies,New Technologies: Modelling the Potential for Improved Smear Microscopy Services in Malawi

Background

To quantify the likely impact of recent WHO policy recommendations regarding smear microscopy and the introduction of appropriate low-cost fluorescence microscopy on a) case detection and b) laboratory workload.

Methodology/Principal Findings

An audit of the laboratory register in an urban hospital, Lilongwe, Malawi, and the application of a simple modelling framework. The adoption of the new definition of a smear-positive case could directly increase case detection by up to 28%. Examining Ziehl-Neelsen (ZN) sputum smears for up to 10 minutes before declaring them negative has previously been shown to increase case detection (over and above that gained by the adoption of the new case definition) by 70% compared with examination times in routine practice. Three times the number of staff would be required to adequately examine the current workload of smears using ZN microscopy. Through implementing new policy recommendations and LED-based fluorescence microscopy the current laboratory staff complement could investigate the same number of patients, examining auramine-stained smears to an extent that is equivalent to a 10 minutes ZN smear examination.

Conclusions/Significance

Combined implementation of the new WHO recommendations on smear microscopy and LED-based fluorescence microscopy could result in substantial increases in smear positive case-detection using existing human resources and minimal additional equipment.     

Experimental Infection of the Pig with Mycobacterium ulcerans: A Novel Model for Studying the Pathogenesis of Buruli Ulcer Disease

Background

Buruli ulcer (BU) is a slowly progressing, necrotising disease of the skin caused by infection with Mycobacterium ulcerans. Non-ulcerative manifestations are nodules, plaques and oedema, which may progress to ulceration of large parts of the skin. Histopathologically, BU is characterized by coagulative necrosis, fat cell ghosts, epidermal hyperplasia, clusters of extracellular acid fast bacilli (AFB) in the subcutaneous tissue and lack of major inflammatory infiltration. The mode of transmission of BU is not clear and there is only limited information on the early pathogenesis of the disease available.

Methodology/Principal Findings

For evaluating the potential of the pig as experimental infection model for BU, we infected pigs subcutaneously with different doses of M. ulcerans. The infected skin sites were excised 2.5 or 6.5 weeks after infection and processed for histopathological analysis. With doses of 2×10⁷ and 2×10⁶ colony forming units (CFU) we observed the development of nodular lesions that subsequently progressed to ulcerative or plaque-like lesions. At lower inoculation doses signs of infection found after 2.5 weeks had spontaneously resolved at 6.5 weeks. The observed macroscopic and histopathological changes closely resembled those found in M. ulcerans disease in humans.

Conclusion/Significance

Our results demonstrate that the pig can be infected with M. ulcerans. Productive infection leads to the development of lesions that closely resemble human BU lesions. The pig infection model therefore has great potential for studying the early pathogenesis of BU and for the development of new therapeutic and prophylactic interventions.     

Prevalence of Buruli Ulcer in Akonolinga Health District,Cameroon: Results of a Cross Sectional Survey

Background

Buruli ulcer (BU) is a chronic, indolent necrotizing disease of the skin and underlying tissues caused by Mycobacterium ulcerans, which may result in functional incapacity. In 2002, Médecins Sans Frontières (MSF) opened a BU programme in Akonolinga Hospital, Cameroon, offering antibiotic treatment, surgery and general medical care. Six hundred patients have been treated in the project to date. However, due to the nature of the disease and its stigmatization, determining the exact prevalence and burden of disease is difficult and current estimates may not reflect the magnitude of the problem. The objectives of this survey were to estimate the prevalence of BU in the health district of Akonolinga, describe the geographic extension of the highly endemic area within the health district, and determine the programme coverage and its geographical distribution.

Methodology/Principal Findings

We conducted a cross-sectional population survey using centric systematic area sampling (CSAS). A 15×15 km grid (quadrats of 225 km²) was overlaid on a map of Akonolinga district with its position chosen to maximize the area covered by the survey. Quadrats were selected if more than 50% of the quadrat was inside of the health district. The chiefdom located closest to the centre of each quadrat was selected and Buruli cases were identified using an active case finding strategy (the sensitivity of the strategy was estimated by capture-recapture). WHO-case definitions were used for nodules, plaque, ulcer, oedema and sequelae. Out of a total population of 103,000 inhabitants, 26,679 were surveyed within the twenty quadrats. Sensitivity of the case finding strategy was estimated to be 84% (95%CI 54–97%). The overall prevalence was 0.47% (n = 105) for all cases including sequelae and 0.25% (n = 56) for active stages of the disease. Five quadrats had a high prevalence of >0.6% to 0.9%, 5 a prevalence >0.3% to 0.6% and 10 quadrats <0.3%. The quadrats with the high prevalence were situated along the rivers Nyong and Mfoumou. Overall coverage of the project was 18% (12–27%) for all cases and 16% (9–18%) for active cases, but was limited to the quadrats neighbouring Akonolinga Hospital.

Conclusions/Significance

Prevalence was highest in the area neighbouring the Nyong River. Coverage was limited to the area close to the hospital and efforts have to be made to increase access to care in the high prevalence areas. Use of the CSAS method was particularly useful for project planning and to identify priority areas of intervention. An added benefit of the method is that the survey procedure incorporated an awareness campaign, providing information about the disease and treatment to the population.     

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.