Feasibility of a dried blood spot strategy for serological screening and surveillance to monitor elimination of Human African Trypanosomiasis in the Democratic Republic of the Congo.

In recent years, the number of reported Human African Trypanosomiasis (HAT) cases caused by Trypanosoma brucei (T.b.) gambiense has been markedly declining, and the goal of 'elimination as a public health problem' is within reach. For the next stage, i.e. interruption of HAT transmission by 2030, intensive screening and surveillance will need to be maintained, but with tools and strategies more efficiently tailored to the very low prevalence. We assessed the sequential use of ELISA and Immune Trypanolysis (ITL) on dried blood spot (DBS) samples as an alternative to the traditional HAT field testing and confirmation approach. A cross-sectional study was conducted in HAT endemic and previously endemic zones in Kongo Central province, and a non-endemic zone in Haut Katanga province in the Democratic Republic of the Congo (DRC). Door-to-door visits were performed to collect dried blood spot (DBS) samples on filter paper. ELISA/T.b. gambiense was conducted followed by ITL for those testing positive by ELISA and in a subset of ELISA negatives. In total, 11,642 participants were enrolled. Of these, 11,535 DBS were collected and stored in appropriate condition for ELISA testing. Ninety-seven DBS samples tested positive on ELISA. In the endemic zone, ELISA positivity was 1.34% (95%CI: 1.04-1.64). In the previously endemic zone and non-endemic zone, ELISA positivity was 0.34% (95% CI: 0.13-0.55) and 0.37% (95% CI: 0.15-0.60) respectively. Among the ELISA positives, only two samples had a positive ITL result, both from the endemic zone. One of those was from a former HAT patient treated in 2008 and the other from an individual who unfortunately had deceased prior to the follow-up visit. Our study showed that a surveillance strategy, based on DBS samples and centralized testing with retracing of patients if needed, is feasible in DRC. ELISA seems well suited as initial test with a similar positivity rate as traditional screening tests, but ITL remains complex. Alternatives for the latter, also analyzable on DBS, should be further explored.

Mycobacterium ulcerans Population Genomics To Inform on the Spread of Buruli Ulcer across Central Africa.

Buruli ulcer is a neglected tropical disease of skin and subcutaneous tissue caused by infection with the pathogen Mycobacterium ulcerans Many critical issues for disease control, such as understanding the mode of transmission and identifying source reservoirs of M. ulcerans, are still largely unknown. Here, we used genomics to reconstruct in detail the evolutionary trajectory and dynamics of M. ulcerans populations at a central African scale and at smaller geographical village scales. Whole-genome sequencing (WGS) data were analyzed from 179 M. ulcerans strains isolated from all Buruli ulcer foci in the Democratic Republic of the Congo, The Republic of Congo, and Angola that have ever yielded positive M. ulcerans cultures. We used both temporal associations and the study of the mycobacterial demographic history to estimate the contribution of humans as a reservoir in Buruli ulcer transmission. Our phylogeographic analysis revealed one almost exclusively predominant sublineage of M. ulcerans that arose in Central Africa and proliferated in its different regions of endemicity during the Age of Discovery. We observed how the best sampled endemic hot spot, the Songololo territory, became an area of endemicity while the region was being colonized by Belgium (1880s). We furthermore identified temporal parallels between the observed past population fluxes of M. ulcerans from the Songololo territory and the timing of health policy changes toward control of the Buruli ulcer epidemic in that region. These findings suggest that an intervention based on detecting and treating human cases in an area of endemicity might be sufficient to break disease transmission chains, irrespective of other reservoirs of the bacterium.IMPORTANCE Buruli ulcer is a destructive skin and soft tissue infection caused by Mycobacterium ulcerans The disease is characterized by progressive skin ulceration, which can lead to permanent disfigurement and long-term disability. Currently, the major hurdles facing disease control are incomplete understandings of both the mode of transmission and environmental reservoirs of M. ulcerans As decades of spasmodic environmental sampling surveys have not brought us much closer to overcoming these hurdles, the Buruli ulcer research community has recently switched to using comparative genomics. The significance of our research is in how we used both temporal associations and the study of the mycobacterial demographic history to estimate the contribution of humans as a reservoir in Buruli ulcer transmission. Our approach shows that it might be possible to use bacterial population genomics to assess the impact of health interventions, providing valuable feedback for managers of disease control programs in areas where health surveillance infrastructure is poor.

Multiple Introductions and Recent Spread of the Emerging Human Pathogen Mycobacterium ulcerans across Africa.

Buruli ulcer (BU) is an insidious neglected tropical disease. Cases are reported around the world but the rural regions of West and Central Africa are most affected. How BU is transmitted and spreads has remained a mystery, even though the causative agent, Mycobacterium ulcerans, has been known for more than 70 years. Here, using the tools of population genomics, we reconstruct the evolutionary history of M. ulcerans by comparing 165 isolates spanning 48 years and representing 11 endemic countries across Africa. The genetic diversity of African M. ulcerans was found to be restricted due to the bacterium's slow substitution rate coupled with its relatively recent origin. We identified two specific M. ulcerans lineages within the African continent, and inferred that M. ulcerans lineage Mu_A1 existed in Africa for several hundreds of years, unlike lineage Mu_A2, which was introduced much more recently, approximately during the 19th century. Additionally, we observed that specific M. ulcerans epidemic Mu_A1 clones were introduced during the same time period in the three hydrological basins that were well covered in our panel. The estimated time span of the introduction events coincides with the Neo-imperialism period, during which time the European colonial powers divided the African continent among themselves. Using this temporal association, and in the absence of a known BU reservoir or-vector on the continent, we postulate that the so-called "Scramble for Africa" played a significant role in the spread of the disease across the continent.

Simple, Rapid Mycobacterium ulcerans Disease Diagnosis from Clinical Samples by Fluorescence of Mycolactone on Thin Layer Chromatography.

INTRODUCTION: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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%). CONCLUSIONS: 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.

Effectiveness of routine BCG vaccination on buruli ulcer disease: a case-control study in the Democratic Republic of Congo, Ghana and Togo.

BACKGROUND: 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. METHODOLOGY: 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. PRINCIPAL FINDINGS: 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. CONCLUSIONS: 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.

Insertion sequence element single nucleotide polymorphism typing provides insights into the population structure and evolution of Mycobacterium ulcerans across Africa.

Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans. In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans. Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the "pan-African clade" were found to be widespread throughout Africa, while the ISE-SNP types of the "Gabonese/Cameroonian clade" were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types.

Effect of a control project on clinical profiles and outcomes in buruli ulcer: a before/after study in Bas-Congo, Democratic Republic of Congo.

BACKGROUND: Buruli ulcer (BU) is a necrotizing bacterial infection of skin, subcutaneous tissue and bone caused by Mycobacterium ulcerans. Although the functional impairment caused by BU results in severe suffering and in socio-economic problems, the disease remains largely neglected in Africa. The province of Bas-Congo in Democratic Republic of Congo contains one of the most important BU foci of the country, i.e. the Songololo Territory in the District of Cataractes. This study aims to assess the impact of a BU control project launched in 2004 in the Songololo Territory. METHODS: We used a comparative non-randomized study design, comparing clinical profiles and outcomes of the group of patients admitted at the General Reference Hospital (GRH) of the "Institut Médical Evangélique" (IME) of Kimpese 3 years before the start of the project (2002-2004) with those admitted during the 3 years after the start of the project (2005-2007). RESULTS: The BU control project was associated with a strong increase in the number of admitted BU cases at the GRH of IME/Kimpese and a fundamental change in the profile of those patients; more female patients presented with BU, the proportion of relapse cases amongst all admissions reduced, the proportion of early lesions and simple ulcerative forms increased, more patients healed without complications and the case fatality rate decreased substantially. The median duration since the onset of first symptoms however remained high, as well as the proportion of patients with osteomyelitis or limitations of joint movement, suggesting that the diagnostic delay remains substantial. CONCLUSION: Implementing a specialized program for BU may be effective in improving clinical profiles and outcomes in BU. Despite these encouraging results, our study highlights the need of considering new strategies to better improve BU control in a low resources setting.