The only available vaccine that could be potentially beneficial against mycobacterial diseases contains live attenuated bovine tuberculosis bacillus (Mycobacterium bovis) also called Bacillus Calmette-Guérin (BCG). Even though the BCG vaccine is still widely used, results on its effectiveness in preventing mycobacterial diseases are partially contradictory, especially regarding Buruli Ulcer Disease (BUD). The aim of this case-control study is to evaluate the possible protective effect of BCG vaccination on BUD. The present study was performed in three different countries and sites where BUD is endemic: in the Democratic Republic of the Congo, Ghana, and Togo from 2010 through 2013. The large study population was comprised of 401 cases with laboratory confirmed BUD and 826 controls, mostly family members or neighbors. After stratification by the three countries, two sexes and four age groups, no significant correlation was found between the presence of BCG scar and BUD status of individuals. Multivariate analysis has shown that the independent variables country (p = 0.31), sex (p = 0.24), age (p = 0.96), and presence of a BCG scar (p = 0.07) did not significantly influence the development of BUD category I or category II/III. Furthermore, the status of BCG vaccination was also not significantly related to duration of BUD or time to healing of lesions. In our study, we did not observe significant evidence of a protective effect of routine BCG vaccination on the risk of developing either BUD or severe forms of BUD. Since accurate data on BCG strains used in these three countries were not available, no final conclusion can be drawn on the effectiveness of BCG strain in protecting against BUD. As has been suggested for tuberculosis and leprosy, well-designed prospective studies on different existing BCG vaccine strains are needed also for BUD. After tuberculosis and leprosy, Buruli Ulcer Disease (BUD) is the third most common human mycobacterial disease. The only available vaccine that could be potentially beneficial against these diseases is BCG. Even though BCG vaccine is widely used, the results on its effectiveness are partially contradictory, probably since different BCG strains are used. The aim of this study was to evaluate the possible protective effect of BCG vaccines on BUD. The present study was performed in three different countries and sites where BUD is endemic: in the Democratic Republic of the Congo, Ghana, and Togo from 2010 through 2013. The large study population was comprised of 401 cases with laboratory confirmed BUD and 826 controls, mostly family members or neighbors. Considering the three countries, sex, and age, the analysis confirmed that the BCG vaccination did not significantly decrease the risk for developing BUD or for developing severe forms of BUD. Furthermore, the status of BCG vaccination was also not significantly related to duration of BUD or to time to healing of lesions. In our study, we could not find any evidence of a protective effect of routine BCG vaccination on BUD.

Buruli ulcer can cause disfigurement and long-term loss of function. It is underdiagnosed and under-reported, and its current distribution is unclear. We aimed to synthesise and evaluate data on Buruli ulcer prevalence and distribution. We did a systematic review of Buruli ulcer prevalence and used an evidence consensus framework to describe and evaluate evidence for Buruli ulcer distribution worldwide. We searched PubMed and Web of Science databases from inception to Aug 6, 2018, for records of Buruli ulcer and Mycobacterium ulcerans detection, with no limits on study type, publication date, participant population, or location. English, French, and Spanish language publications were included. We included population-based surveys presenting Buruli ulcer prevalence estimates, or data that allowed prevalence to be estimated, in the systematic review. We extracted geographical data on the occurrence of Buruli ulcer cases and M ulcerans detection from studies of any type for the evidence consensus framework; articles that did not report original data were excluded. For the main analysis, we extracted prevalence estimates from included surveys and calculated 95% CIs using Byar's method. We included occurrence records, reports to WHO and the Global Infectious Diseases and Epidemiology Network, and surveillance data from Buruli ulcer control programmes in the evidence consensus framework to grade the strength of evidence for Buruli ulcer endemicity. This study is registered with PROSPERO, number CRD42018116260. 2763 titles met the search criteria. We extracted prevalence estimates from ten studies and occurrence data from 208 studies and five unpublished surveillance datasets. Prevalence estimates within study areas ranged from 3·2 (95% CI 3·1-3·3) cases per 10 000 population in Côte d'Ivoire to 26·9 (23·5-30·7) cases per 10 000 population in Benin. There was evidence of Buruli ulcer in 32 countries and consensus on presence in 12. The global distribution of Buruli ulcer is uncertain and potentially wider than currently recognised. Our findings represent the strongest available evidence on Buruli ulcer distribution so far and have many potential applications, from directing surveillance activities to informing burden estimates. AIM Initiative.

We investigated the relationship between bacterial load in Buruli ulcer (BU) lesions and the development of paradoxical reaction following initiation of antibiotic treatment. This was a longitudinal study involving BU patients from June 2013 to June 2017. Fine needle aspirates (FNA) and swab samples were obtained to establish the diagnosis of BU by PCR. Additional samples were obtained at baseline, during and after treatment (if the lesion had not healed) for microscopy, culture and combined 16S rRNA reverse transcriptase/ IS2404 qPCR assay. Patients were followed up at regular intervals until complete healing. Forty-seven of 354 patients (13%) with PCR confirmed BU had a PR, occurring between 2 and 42 (median 6) weeks after treatment initiation. The bacterial load, the proportion of patients with positive M. ulcerans culture (15/34 (44%) vs 29/119 (24%), p = 0.025) and the proportion with positive microscopy results (19/31 (61%) vs 28/90 (31%), p = 0.003) before initiation of treatment were significantly higher in the PR compared to the no PR group. Plaques (OR 5.12; 95% CI 2.26–11.61; p<0.001), oedematous (OR 4.23; 95% CI 1.43–12.5; p = 0.009) and category II lesions (OR 2.26; 95% CI 1.14–4.48; p = 0.02) were strongly associated with the occurrence of PR. The median time to complete healing (28 vs 13 weeks, p <0.001) was significantly longer in the PR group. Buruli ulcer patients who develop PR are characterized by high bacterial load in lesion samples taken at baseline and a higher rate of positive M. ulcerans culture. Occurrence of a PR was associated with delayed healing. ClinicalTrials.gov NCT02153034. Buruli ulcer is a neglected tropical skin disease caused by the third most common pathogenic mycobacterium: Mycobacterium ulcerans. Paradoxical reaction, a phenomenon observed in some patients is characterised by worsening of existing lesion(s) with attendant pain and occurrence of new lesions during or after antibiotic therapy following an initial period of clinical improvement. This significantly affects treatment outcomes. In this clinical study, tissue samples obtained from patients were subjected to 16S rRNA/ IS2404 qPCR to measure bacterial load. This was to identify a link between bacterial load in BU lesions and the development of paradoxical reactions following initiation of antibiotic treatment. We found that 13% of participants developed PR. Patients who developed PR had higher baseline bacterial load; a higher rate of positive M. ulcerans culture and persistently positive culture during antibiotic treatment. Occurrence of a paradoxical reaction was associated with delayed healing.

Mycobacterium ulcerans infection, known as Buruli ulcer, is a disease of the skin and subcutaneous tissues which is an important but neglected tropical disease with its major impact in rural parts of West and Central Africa where facilities for diagnosis and management are poorly developed. We evaluated fluorescent thin layer chromatography (f-TLC) for detection of mycolactone in the laboratory using samples from patients with Buruli ulcer and patients with similar lesions that gave a negative result on PCR for the IS2404 repeat sequence of M. ulcerans Mycolactone and DNA extracts from fine needle aspiration (FNA), swabs and biopsy specimen were used to determine the sensitivity and specificity of f-TLC when compared with PCR for the IS2404. For 71 IS2404 PCR positive and 28 PCR negative samples the sensitivity was 73.2% and specificity of 85.7% for f-TLC. The sensitivity was similar for swabs (73%), FNAs (75%) and biopsies (70%). We have shown that mycolactone can be detected from M. ulcerans infected skin tissue by f-TLC technique. The technique is simple, easy to perform and read with minimal costs. In this study it was undertaken by a member of the group from each endemic country. It is a potentially implementable tool at the district level after evaluation in larger field studies. Mycobacterium ulcerans infection, known as Buruli ulcer, is a disease that affects the skin and underlying tissues. The organism responsible for the infection produces a potent toxin called mycolactone that causes extensive skin damage. Easy to perform and cheaper techniques are needed for diagnostic confirmation. We have evaluated fluorescent thin layer chromatography (fTLC) for detection of mycolactone in skin samples from patients with Buruli ulcer comparing them with samples from similar non-Buruli ulcer lesions that gave a negative result in the standard polymerase chain reaction (PCR) test for M. ulcerans. Fluorescent TLC had sensitivity of 73.2% and specificity of 85.7% when compared with PCR whether the skin sample was a swab, a biopsy or a fine needle aspirate. This study shows that mycolactone can be detected reliably from M. ulcerans infected skin tissue by the simple, low cost technique of fluorescent thin layer chromatography that could be developed for point of care use. It requires further evaluation in countries where Buruli ulcer disease is endemic.

Buruli ulcer (BU) caused by Mycobacterium ulcerans is effectively treated with rifampicin and streptomycin for 8 weeks but some lesions take several months to heal. We have shown previously that some slowly healing lesions contain mycolactone suggesting continuing infection after antibiotic therapy. Now we have determined how rapidly combined M. ulcerans 16S rRNA reverse transcriptase / IS2404 qPCR assay (16S rRNA) became negative during antibiotic treatment and investigated its influence on healing. Fine needle aspirates and swab samples were obtained for culture, acid fast bacilli (AFB) and detection of M. ulcerans 16S rRNA and IS2404 by qPCR (16S rRNA) from patients with IS2404 PCR confirmed BU at baseline, during antibiotic and after treatment. Patients were followed up at 2 weekly intervals to determine the rate of healing. The Kaplan-Meier survival analysis was used to analyse the time to clearance of M. ulcerans 16S rRNA and the influence of persistent M ulcerans 16S rRNA on time to healing. The Mann Whitney test was used to compare the bacillary load at baseline in patients with or without viable organisms at week 4, and to analyse rate of healing at week 4 in relation to detection of viable organisms. Out of 129 patients, 16S rRNA was detected in 65% of lesions at baseline. The M. ulcerans 16S rRNA remained positive in 78% of patients with unhealed lesions at 4 weeks, 52% at 8 weeks, 23% at 12 weeks and 10% at week 16. The median time to clearance of M. ulcerans 16S rRNA was 12 weeks. BU lesions with positive 16S rRNA after antibiotic treatment had significantly higher bacterial load at baseline, longer healing time and lower healing rate at week 4 compared with those in which 16S rRNA was not detected at baseline or had become undetectable by week 4. Current antibiotic therapy for BU is highly successful in most patients but it may be possible to abbreviate treatment to 4 weeks in patients with a low initial bacterial load. On the other hand persistent infection contributes to slow healing in patients with a high bacterial load at baseline, some of whom may need antibiotic treatment extended beyond 8 weeks. Bacterial load was estimated from a single sample taken at baseline. A better estimate could be made by taking multiple samples or biopsies but this was not ethically acceptable. Buruli ulcer (BU) caused by Mycobacterium ulcerans is effectively treated with rifampicin and streptomycin for 8 weeks but some lesions take several months to heal. We have shown previously that some slowly healing lesions contain the M. ulcerans toxin, mycolactone, suggesting continuing infection after completion of antibiotic therapy. In the present study we have determined how soon M. ulcerans was killed during antibiotic treatment using the M. ulcerans 16S rRNA assay combined with qPCR for IS2404 to detect live bacilli in clinical samples and investigated its influence on healing. This assay is more sensitive than culture for the organism. Using samples collected from one hundred and twenty-nine BU patients prior to antibiotic treatment, viable organisms were detected by culture in 34% but the 16S rRNA assay was positive in 65%. The 16S rRNA remained positive in 78% of patients with unhealed lesions at 4 weeks, 52% at 8 weeks, 23% at 12 weeks, and 10% at week 16. Lesions with positive 16S rRNA after antibiotic treatment also contained a higher number of bacteria at baseline, had a lower rate of healing at week 4 and took a longer time to heal compared with those in which the organism was undetectable at baseline or by week 4. Positive 16S rRNA was less likely in ulcerative compared with nodular forms of disease 4 weeks after antibiotic treatment. It may be possible to shorten the treatment to 4 weeks in patients with low numbers of bacteria at baseline. Since persistent infection appears to contribute to slow healing, some patients with a high bacterial load at baseline may need antibiotic treatment for longer than 8 weeks.

As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents. Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays. Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level. Buruli ulcer disease (BUD) mainly occurs in remote rural areas of Sub-Saharan Africa, affects skin and soft tissue, and may lead to severe disabilities. Therefore, early diagnosis and treatment with antimycobacterial therapy are essential whereby the WHO recommends laboratory confirmation of 70% of the cases. As the current diagnostic gold standard (polymerase chain reaction [PCR]) is restricted to third-level laboratories, development of confirmatory point-of-care (POC) tests for BUD applicable at primary health care level has become a research priority to bring diagnosis closer to where the patients are. Loop-mediated isothermal amplification (LAMP) has been selected by the WHO as one of the promising candidate technologies for POC tests. The aim of this study was to establish and validate a LAMP assay applying lyophilized reagents which are stable at ambient temperature, thus avoiding the need for cold-chains. The results from this study suggest that the assay provides a valuable alternative to other PCR tests as currently used for laboratory confirmation of BUD.