Mapping suitability for Buruli ulcer at fine spatial scales across Africa: A modelling study

Buruli ulcer (BU) is a disabling and stigmatising neglected tropical disease (NTD). Its distribution and burden are unknown because of underdiagnosis and underreporting. It is caused by Mycobacterium ulcerans, an environmental pathogen whose environmental niche and transmission routes are not fully understood. The main control strategy is active surveillance to promote early treatment and thus limit morbidity, but these activities are mostly restricted to well-known endemic areas. A better understanding of environmental suitability for the bacterium and disease could inform targeted surveillance, and advance understanding of the ecology and burden of BU. We used previously compiled point-level datasets of BU and M. ulcerans occurrence, evidence for BU occurrence within national and sub-national areas, and a suite of relevant environmental covariates in a distribution modelling framework. We fitted relationships between BU and M. ulcerans occurrence and environmental predictors by applying regression and machine learning based algorithms, combined in an ensemble model to characterise the optimal ecological niche for the disease and bacterium across Africa at a resolution of 5km x 5km. Proximity to waterbodies was the strongest predictor of suitability for BU, followed potential evapotranspiration. The strongest predictors of suitability for M. ulcerans were deforestation and potential evapotranspiration. We identified patchy foci of suitability throughout West and Central Africa, including areas with no previous evidence of the disease. Predicted suitability for M. ulcerans was wider but overlapping with that of BU. The estimated population living in areas predicted suitable for the bacterium and disease was 46.1 million.These maps could be used to inform burden estimations and case searches which would generate a more complete understanding of the spatial distribution of BU in Africa, and may guide control programmes to identify cases beyond the well-known endemic areas.     

Buruli Ulcer Disease and Its Association with Land Cover in Southwestern Ghana

BackgroundBuruli ulcer (BU), one of 17 neglected tropical diseases, is a debilitating skin and soft tissue infection caused by Mycobacterium ulcerans. In tropical Africa, changes in land use and proximity to water have been associated with the disease. This study presents the first analysis of BU at the village level in southwestern Ghana, where prevalence rates are among the highest globally, and explores fine and medium-scale associations with land cover by comparing patterns both within BU clusters and surrounding landscapes.Conclusions/SignificanceOur study highlights the importance of multiple land use changes along the Offin River, particularly mining and agriculture, which might be associated with BU disease in southwestern Ghana. Our study is the first to use both medium- and high-resolution imagery to assess these changes. We also show that older populations (≥ 60 y) appear to be at higher risk of BU disease than children, once BU data were weighted by population age structures.     

Chronic wounds in Sierra Leone: Searching for Buruli ulcer,a NTD caused by Mycobacterium ulcerans,at Masanga Hospital

BackgroundChronic wounds pose a significant healthcare burden in low- and middle-income countries. Buruli ulcer (BU), caused by Mycobacterium ulcerans infection, causes wounds with high morbidity and financial burden. Although highly endemic in West and Central Africa, the presence of BU in Sierra Leone is not well described. This study aimed to confirm or exclude BU in suspected cases of chronic wounds presenting to Masanga Hospital, Sierra Leone.MethodologyDemographics, baseline clinical data, and quality of life scores were collected from patients with wounds suspected to be BU. Wound tissue samples were acquired and transported to the Swiss Tropical and Public Health Institute, Switzerland, for analysis to detect Mycobacterium ulcerans using qPCR, microscopic smear examination, and histopathology, as per World Health Organization (WHO) recommendations.FindingsTwenty-one participants with wounds suspected to be BU were enrolled over 4-weeks (Feb-March 2019). Participants were predominantly young working males (62% male, 38% female, mean 35yrs, 90% employed in an occupation or as a student) with large, single, ulcerating wounds (mean diameter 9.4cm, 86% single wound) exclusively of the lower limbs (60% foot, 40% lower leg) present for a mean 15 months. The majority reported frequent exposure to water outdoors (76%). Self-reports of over-the-counter antibiotic use prior to presentation was high (81%), as was history of trauma (38%) and surgical interventions prior to enrolment (48%). Regarding laboratory investigation, all samples were negative for BU by microscopy, histopathology, and qPCR. Histopathology analysis revealed heavy bacterial load in many of the samples. The study had excellent participant recruitment, however follow-up proved difficult.ConclusionsBU was not confirmed as a cause of chronic ulceration in our cohort of suspected cases, as judged by laboratory analysis according to WHO standards. This does not exclude the presence of BU in the region, and the definitive cause of these treatment-resistance chronic wounds is uncertain.     

In-vitro Activity of Avermectins against Mycobacterium ulcerans

Mycobacterium ulcerans causes Buruli ulcer (BU), a debilitating infection of subcutaneous tissue. There is a WHO-recommended antibiotic treatment requiring an 8-week course of streptomycin and rifampicin. This regime has revolutionized the treatment of BU but there are problems that include reliance on daily streptomycin injections and side effects such as ototoxicity. Trials of all-oral treatments for BU show promise but additional drug combinations that make BU treatment safer and shorter would be welcome. Following on from reports that avermectins have activity against Mycobacterium tuberculosis, we tested the in-vitro efficacy of ivermectin and moxidectin on M. ulcerans. We observed minimum inhibitory concentrations of 4–8 μg/ml and time-kill assays using wild type and bioluminescent M. ulcerans showed a significant dose-dependent reduction in M. ulcerans viability over 8-weeks. A synergistic killing-effect with rifampicin was also observed. Avermectins are well tolerated, widely available and inexpensive. Based on our in vitro findings we suggest that avermectins should be further evaluated for the treatment of BU.     

Activities of rifampin, Rifapentine and clarithromycin alone and in combination against mycobacterium ulcerans disease in mice

Background

Treatment of Mycobacterium ulcerans disease, or Buruli ulcer (BU), has shifted from surgery to treatment with streptomycin(STR)+rifampin(RIF) since 2004 based on studies in a mouse model and clinical trials. We tested two entirely oral regimens for BU treatment, rifampin(RIF)+clarithromycin(CLR) and rifapentine(RPT)+clarithromycin(CLR) in the mouse model.

Methodology/Principal Findings

BALB/c mice were infected in the right hind footpad with M. ulcerans strain 1059 and treated daily (5 days/week) for 4 weeks, beginning 11 days after infection. Treatment groups included an untreated control, STR+RIF as a positive control, and test regimens of RIF, RPT, STR and CLR given alone and the RIF+CLR and RPT+CLR combinations. The relative efficacy of the drug treatments was compared on the basis of footpad CFU counts and median time to footpad swelling. Except for CLR, which was bacteriostatic, treatment with all other drugs reduced CFU counts by approximately 2 or 3 log₁₀. Median time to footpad swelling after infection was 5.5, 16, 17, 23.5 and 36.5 weeks in mice receiving no treatment, CLR alone, RIF+CLR, RIF alone, and STR alone, respectively. At the end of follow-up, 39 weeks after infection, only 48%, 26.4% and 16.3% of mice treated with RPT+CLR, RPT alone and STR+RIF had developed swollen footpads. An in vitro checkerboard assay showed the interaction of CLR and RIF to be indifferent. However, in mice, co-administration with CLR resulted in a roughly 25% decrease in the maximal serum concentration (Cmax) and area under the serum concentration-time curve (AUC) of each rifamycin. Delaying the administration of CLR by one hour restored Cmax and AUC values of RIF to levels obtained with RIF alone.

Conclusions/Significance

These results suggest that an entirely oral daily regimen of RPT+CLR may be at least as effective as the currently recommended combination of injected STR+oral RIF.     

RNA Aptamer That Specifically Binds to Mycolactone and Serves as a Diagnostic Tool for Diagnosis of Buruli Ulcer

BackgroundBuruli ulcer (BU) is a subcutaneous skin disease listed among the neglected tropical diseases by the World Health Organization (WHO). Early case detection and management is very important to reduce morbidity and the accompanied characteristic disfiguring nature of BU. Since diagnosis based on clinical evidence can lead to misdiagnosis, microbiological confirmation is essential to reduce abuse of drugs; since the anti-mycobacterial drugs are also used for TB treatment. The current WHO gold standard PCR method is expensive, requires infrastructure and expertise are usually not available at the peripheral centers where BU cases are managed. Thus one of the main research agendas is to develop methods that can be applied at the point of care. In this study we selected aptamers, which are emerging novel class of detection molecules, for detecting mycolactone, the first to be conducted in a BUD endemic country.MethodsAptamers that bind to mycolactone were isolated by the SELEX process. To measure their affinity and specificity to mycolactone, the selected aptamers were screened by means of isothermal titration calorimetry (ITC) and an enzyme-linked oligonucleotide assay (ELONA). Selected aptamers were assessed by ELONA using swab samples from forty-one suspected BU patients with IS2404 PCR and culture as standard methods. ROC analysis was used to evaluate their accuracy and cutoff-points.ResultsFive out of the nine selected aptamers bound significantly (p< 0.05) to mycolactone, of these, three were able to distinguish between mycolactone producing mycobacteria, M. marinum ({"type":"entrez-nucleotide","attrs":{"text":"CC240299","term_id":"30566962","term_text":"CC240299"}}CC240299, Israel) and other bacteria whilst two others also bounded significantly to Mycobacterium smegmatis. Their dissociation constants were in the micro-molar range. At 95% confidence interval, the ROC curve analysis among the aptamers at OD450 ranged from 0.5–0.7. Using this cut-off for the ELONA assay, the aptamers had 100% specificity and sensitivity between 0.0% and 50.0%. The most promising aptamer, Apt-3683 showed a discernible cleavage difference relative to the non-specific autocatalysis over a 3-minute time course.ConclusionThis preliminary proof-of-concept indicates that diagnosis of BUD with RNA aptamers is feasible and can be used as point of care upon incorporation into a diagnostic platform.     

Genetic Diversity of Staphylococcus aureus in Buruli Ulcer

Background

Buruli ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. Previous studies have shown that wounds of BU patients are colonized with M. ulcerans and several other microorganisms, including Staphylococcus aureus, which may interfere with wound healing. The present study was therefore aimed at investigating the diversity and topography of S. aureus colonizing BU patients during treatment.

Methodology

We investigated the presence, diversity, and spatio-temporal distribution of S. aureus in 30 confirmed BU patients from Ghana during treatment. S. aureus was isolated from nose and wound swabs, and by replica plating of wound dressings collected bi-weekly from patients. S. aureus isolates were characterized by multiple-locus variable number tandem repeat fingerprinting (MLVF) and spa-typing, and antibiotic susceptibility was tested.

Principal Findings

Nineteen (63%) of the 30 BU patients tested positive for S. aureus at least once during the sampling period, yielding 407 S. aureus isolates. Detailed analysis of 91 isolates grouped these isolates into 13 MLVF clusters and 13 spa-types. Five (26%) S. aureus-positive BU patients carried the same S. aureus genotype in their anterior nares and wounds. S. aureus isolates from the wounds of seven (37%) patients were distributed over two different MLVF clusters. Wounds of three (16%) patients were colonized with isolates belonging to two different genotypes at the same time, and five (26%) patients were colonized with different S. aureus types over time. Five (17%) of the 30 included BU patients tested positive for methicillin-resistant S. aureus (MRSA).

Conclusion/Significance

The present study showed that the wounds of many BU patients were contaminated with S. aureus, and that many BU patients from the different communities carried the same S. aureus genotype during treatment. This calls for improved wound care and hygiene.     

Buruli Ulcer in Cameroon: The Development and Impact of the National Control Programme

BackgroundCameroon is endemic for Buruli ulcer (BU) and organised institutional BU control began in 2002. The objective was to describe the evolution, achievements and challenges of the national BU control programme (NBUCP) and to make suggestions for scaling up the programme.MethodsWe analysed collated data on BU from 2001 to 2014 and reviewed activity reports NBUCP in Cameroon. Case-detection rates and key BU control indicators were calculated and plotted on a time scale to determine trends in performance. A linear regression analysis of BU detection rate from 2005–2014 was done. The regression coefficient was tested statistically for the significance in variation of BU detection rate.Conclusion/SignificanceAlthough institutional BU control Cameroon only began 30 years after the first cases were reported in 1969, a number of milestones have been attained. These would serve as stepping stones for charting the way forward and improving upon control activities in the country if the major challenge of resource allocation is dealt with.     

Local and regional re-establishment of cellular immunity during curative antibiotherapy of murine Mycobacterium ulcerans infection

Background

Buruli ulcer (BU) is a neglected necrotizing disease of the skin, subcutaneous tissue and bone, caused by Mycobacterium ulcerans. BU pathogenesis is associated with mycolactone, a lipidic exotoxin with cytotoxic and immunosuppressive properties. Since 2004, the World Health Organization recommends the treatment of BU with a combination of rifampicin and streptomycin (RS). Histological analysis of human tissue samples suggests that such antibiotic treatment reverses the mycolactone-induced local immunosuppression, leading to increased inflammatory infiltrations and phagocytosis of bacilli.

Methodology/Principal Findings

We used a mouse model of M. ulcerans footpad infection, followed by combined RS treatment. Time-lapsed analyses of macroscopic lesions, bacterial burdens, histology and immunohistochemistry were performed in footpads. We also performed CFU counts, histology and immunohistochemistry in the popliteal draining lymph nodes (DLN). We observed a shift in the cellular infiltrates from a predominantly neutrophilic/macrophagic to a lymphocytic/macrophagic profile in the infected footpads of antibiotic-treated mice. This shift occurred before the elimination of viable M. ulcerans organisms, which were ultimately eradicated as demonstrated by the administration of dexamethasone. This reduction of bacillary loads was accompanied by an increased expression of inducible nitric oxide synthase (NOS2 or iNOS). Predominantly mononuclear infiltrates persisted in the footpads during and after treatment, coincident with the long persistence of non-viable poorly stained acid-fast bacilli (AFB). We additionally observed that antibiotherapy prevented DLN destruction and lymphocyte depletion, which occurs during untreated experimental infections.

Conclusions/Significance

Early RS treatment of M. ulcerans mouse footpad infections results in the rapid elimination of viable bacilli with pathogen eradication. However, non-viable AFB persisted for several months after lesion sterilization. This RS regimen prevented DLN destruction, allowing the rapid re-establishment of local and regional cell mediated immune responses associated with macrophage activation. Therefore it is likely that this re-establishment of protective cellular immunity synergizes with antibiotherapy.     

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.     

Pain Associated with Wound Care Treatment among Buruli Ulcer Patients from Ghana and Benin

Buruli ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. People living in remote areas in tropical Sub Saharan Africa are mostly affected. Wound care is an important component of BU management; this often needs to be extended for months after the initial antibiotic treatment. BU is reported in the literature as being painless, however clinical observations revealed that some patients experienced pain during wound care. This was the first study on pain intensity during and after wound care in BU patients and factors associated with pain. In Ghana and Benin, 52 BU patients above 5 years of age and their relatives were included between December 2012 and May 2014. Information on pain intensity during and after wound care was obtained during two consecutive weeks using the Wong-Baker Pain Scale. Median pain intensity during wound care was in the lower range (Mdn = 2, CV = 1), but severe pain (score > 6) was reported in nearly 30% of the patients. Nevertheless, only one patient received pain medication. Pain declined over time to low scores 2 hours after treatment. Factors associated with higher self-reported pain scores were; male gender, fear prior to treatment, pain during the night prior to treatment, and pain caused by cleaning the wound. The general idea that BU is painless is incorrect for the wound care procedure. This procedural pain deserves attention and appropriate intervention.     

Locally Confined Clonal Complexes of Mycobacterium ulcerans in Two Buruli Ulcer Endemic Regions of Cameroon

Background Mycobacterium ulcerans is the causative agent of the necrotizing skin disease Buruli ulcer (BU), which has been reported from over 30 countries worldwide. The majority of notified patients come from West African countries, such as Côte d’Ivoire, Ghana, Benin and Cameroon. All clinical isolates of M. ulcerans from these countries are closely related and their genomes differ only in a limited number of single nucleotide polymorphisms (SNPs).ConclusionsOur comparative genomic analysis revealed that M. ulcerans clones diversify locally by the accumulation of SNPs. Case isolates coming from more recently emerging BU endemic areas, such as the Mapé river basin, may be less diverse than populations from longer standing disease foci, such as the Nyong river basin. Exchange of strains between distinct endemic areas seems to be rare and local clonal complexes can be easily distinguished by whole genome sequencing.     

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.     

Interferon-γ Is a Crucial Activator of Early Host Immune Defense against Mycobacterium ulcerans Infection in Mice

Buruli ulcer (BU), caused by infection with Mycobacterium ulcerans, is a chronic necrotizing human skin disease associated with the production of the cytotoxic macrolide exotoxin mycolactone. Despite extensive research, the type of immune responses elicited against this pathogen and the effector functions conferring protection against BU are not yet fully understood. While histopathological analyses of advanced BU lesions have demonstrated a mainly extracellular localization of the toxin producing acid fast bacilli, there is growing evidence for an early intra-macrophage growth phase of M. ulcerans. This has led us to investigate whether interferon-γ might play an important role in containing M. ulcerans infections. In an experimental Buruli ulcer mouse model we found that interferon-γ is indeed a critical regulator of early host immune defense against M. ulcerans infections. Interferon-γ knockout mice displayed a faster progression of the infection compared to wild-type mice. This accelerated progression was reflected in faster and more extensive tissue necrosis and oedema formation, as well as in a significantly higher bacterial burden after five weeks of infection, indicating that mice lacking interferon-γ have a reduced capacity to kill intracellular bacilli during the early intra-macrophage growth phase of M. ulcerans. This data demonstrates a prominent role of interferon-γ in early defense against M. ulcerans infection and supports the view that concepts for vaccine development against tuberculosis may also be valid for BU.     

On the mechanism of bromouracil-induced mutagenesis

Bromouracil (BU)-induced mutagenesis of λC17 am o8 phage, in relation to the recombination systems of phage (red) or bacteria (rec), was studied. The mutations investigated were am → am⁺. For efficient BU-induced mutagenesis, red or recA genes as well as bacterial lex gene functions, known to be involved in UV-induced mutagenesis, were required. This suggests a common mechanism or some common step(s) in UV- and BU-induced mutagenesis. Moreover, a several-fold increase was observed in the number of mutants induced by BU in the excision-repair-deficient strain (uvrA), implying that incorporated BU induces some premutational lesions that are recognized and repaired by excision-repair enzymes. A hypothesis on the possible mechanism of BU-induced mutagenesis is proposed, which assumes a common mechanism for UV- and BU-induced mutagenesis, involving recombination repair processes. Incorporation of a tautomeric or ionized form of BU is considered only as a premutational change in DNA activating the dark-repair mechanisms in cells. The observation that BU enhances the frequency of recombination in λ phages also supports teh idea that recombination processes are involved in BU-induced mutagenesis.     

Management of Hyperglycemia in Diabetic Patients with Hematologic Malignancies During Dexamethasone Therapy

ObjectiveTo compare the response to different insulin regimens for management of hyperglycemia in diabetic patients with hematologic malignancies who are receiving dexamethasone.MethodsA retrospective analysis was conducted to determine whether a basal bolus insulin (BBI) regimen with detemir and aspart is superior to a sliding scale regular insulin (SSI) regimen for management of hyperglycemia in hospitalized diabetic patients receiving dexamethasone.ResultsForty patients with hematologic malignancies were treated with intravenous (8 to 12 mg/day) or oral (40 mg/day) dexamethasone for 3 days. The average blood glucose (BG) level was 301 ± 57 mg/dL in the SSI group (n = 28) and 219 ± 51 mg/dL in the BBI group (n = 12) (P <.001). The BBI regimen resulted in an average BG reduction of 52 ± 82 mg/dL throughout the course of dexa-methasone therapy, while the SSI regimen produced an increase in the mean daily BG level of 128 ± 77 mg/dL (P <.001). On the last day of dexamethasone administration, the insulin requirement was 49 ± 29 units/day in the SSI group and 122 ± 39 units/day in the BBI group (P <.001). Three patients in the SSI group developed diabetic ketoacidosis or hyperosmolar hyperglycemia during steroid therapy. No hypoglycemia was observed in either group. The length of stay and infection rates were similar between groups.ConclusionBasal and bolus insulin regimen is an effective and safe approach for managing dexamethasone-induced hyperglycemia in hospitalized patients with hematologic malignancies. (Endocr Pract. 2013;19:231-235)     

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.     

Mycobacterium ulcerans Persistence at a Village Water Source of Buruli Ulcer Patients

Buruli ulcer (BU), a neglected tropical disease of the skin and subcutaneous tissue, is caused by Mycobacterium ulcerans and is the third most common mycobacterial disease after tuberculosis and leprosy. While there is a strong association of the occurrence of the disease with stagnant or slow flowing water bodies, the exact mode of transmission of BU is not clear. M. ulcerans has emerged from the environmental fish pathogen M. marinum by acquisition of a virulence plasmid encoding the enzymes required for the production of the cytotoxic macrolide toxin mycolactone, which is a key factor in the pathogenesis of BU. Comparative genomic studies have further shown extensive pseudogene formation and downsizing of the M. ulcerans genome, indicative for an adaptation to a more stable ecological niche. This has raised the question whether this pathogen is still present in water-associated environmental reservoirs. Here we show persistence of M. ulcerans specific DNA sequences over a period of more than two years at a water contact location of BU patients in an endemic village of Cameroon. At defined positions in a shallow water hole used by the villagers for washing and bathing, detritus remained consistently positive for M. ulcerans DNA. The observed mean real-time PCR Ct difference of 1.45 between the insertion sequences IS2606 and IS2404 indicated that lineage 3 M. ulcerans, which cause human disease, persisted in this environment after successful treatment of all local patients. Underwater decaying organic matter may therefore represent a reservoir of M. ulcerans for direct infection of skin lesions or vector-associated transmission.     

Effect of Shuangjinlian mixture on oral ulcer model in rat

ObjectiveTo observe the effect of common clinical drug Shuangjin Lian mixture on rats with oral ulcer and discuss its mechanism.MethodsSodium carboxymethyl cellulose caused leukocyte aggregation in rats, observed the anti-inflammatory effect of Shuangjin mixture. 3 mm * 3 mm size ulcer surface in the oral cavity of rats was caused by 90% phenol solution, to observe the therapeutic effect and anti-inflammatory effect of Shuangjinlian mixture on rats with oral ulcers.ResultThe low, middle and high dose Shuangjinlian mixture can inhibits the accumulation of white blood cells caused by sodium carboxymethyl cellulose in rats significantly (P < 0.01). And reduce the degree of edema and hyperaemia around the ulcer tissue significantly (P < 0.01), improve the ulcer healing probability, and reduce the level of TNF-α, VEGF levels and increase IL-2 level in the serum of rats with oral ulcers significantly (P < 0.01). Pathological examination showed that the lesion of ulcer tissue in each treatment group was obviously alleviated.ConclusionShuangjinlian mixture had anti-inflammatory effect and was effective for the prevention and treatment of oral ulcer in rats.