Repurposing of Tuberculosis Drug Candidates for the Treatment of Mycobacterium ulcerans Disease.

Buruli ulcer (BU) is a chronic necrotizing skin disease caused by Mycobacterium ulcerans. Historically, the disease was treated by surgical excision of the skin lesions, until an 8-week combination therapy of rifampicin and streptomycin was introduced in 2004. This treatment modality was effective and reduced recurrence rates. Rifampicin is the most efficacious antibiotic for the treatment of BU and, should rifampicin-resistant M. ulcerans strains emerge, there is currently no replacement for it. As for mycobacterial diseases in general, there is a pressing need for the development of novel, fast-acting drugs. Under market economy conditions, repurposing of new tuberculosis drug candidates is the most promising avenue for alternative BU treatments. Our drug repurposing activities have led to the identification of several actives against M. ulcerans. In particular, the cytochrome bc1 complex inhibitor telacebec (Q203) is a promising drug candidate for the treatment of BU in Africa and Australia. While an active cytochrome-bd oxidase bypass limits the potency of the cytochrome-bc1-specific inhibitor telacebec against M. tuberculosis, classical lineage M. ulcerans strains rely exclusively on cytochrome-bc1 to respire. Hence, telacebec is effective at nanomolar concentration against M. ulcerans, and a high treatment efficacy in an experimental mouse infection model indicates that treatment of BU could be substantially shortened and simplified by telacebec.

Key Contributions by the Swiss Tropical and Public Health Institute Towards New and Better Drugs for Tropical Diseases.

Thanks to its expertise in clinical research, epidemiology, infectious diseases, microbiology, parasitology, public health, translational research and tropical medicine, coupled with deeply rooted partnerships with institutions in low- and middle-income countries (LMICs), the Swiss Tropical and Public Health Institute (Swiss TPH) has been a key contributor in many drug research and development consortia involving academia, pharma and product development partnerships. Our know-how of the maintenance of parasites and their life-cycles in the laboratory, plus our strong ties to research centres and disease control programme managers in LMICs with access to field sites and laboratories, have enabled systems for drug efficacy testing in vitro and in vivo, clinical research, and modelling to support the experimental approaches. Thus, Swiss TPH has made fundamental contributions towards the development of new drugs - and the better use of old drugs - for neglected tropical diseases and infectious diseases of poverty, such as Buruli ulcer, Chagas disease, food-borne trematodiasis (e.g. clonorchiasis, fascioliasis and opisthorchiasis), human African trypanosomiasis, leishmaniasis, malaria, schistosomiasis, soil-transmitted helminthiasis and tuberculosis. In this article, we show case the success stories of molecules to which Swiss TPH has made a substantial contribution regarding their use as anti-infective compounds with the ultimate aim to improve people's health and well-being.

Efficacy of an Acid-Oxidizing Solution against Mycobacterium ulcerans.

For the treatment of chronic wounds, acid-oxidizing solutions (AOSs) with broad-spectrum microbicidal activity without disturbing granulation tissue formation have been developed. We found AOSs to efficiently kill Mycobacterium ulcerans, the causative agent of Buruli ulcer, which is able to survive harsh decontamination treatments. Topical AOS treatment of Buruli ulcer lesions may support the recommended antibiotic therapy (oral rifampin and clarithromycin), prevent contamination of the environment by the mycobacteria, and control secondary infections, which are a prevalent wound management problem in resource-poor settings where Buruli ulcer is endemic.

Competitive ELISA for the Detection and Quantification of Mycobacterium ulcerans Mycolactone.

Lipids and other hydrophobic analytes are difficult to quantify by routine immunoassays due to the need to use aqueous buffers. Here, we describe an ELISA protocol suitable for the detection of mycolactone, the polyketide toxin of Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU). Given that mycolactone is unique to this species and has been found in all M. ulcerans lineages, the assay herein described has the potential to be useful both as a research tool and as a diagnostic test, even in low-resource BU endemic regions. Furthermore, the triethanolamine buffer described here may also be useful in the specific detection of other lipid analytes by ELISA.

An Antigen Capture Assay for the Detection of Mycolactone, the Polyketide Toxin of Mycobacterium ulcerans.

Mycolactone is a cytotoxin responsible for most of the chronic necrotizing pathology of Mycobacterium ulcerans disease (Buruli ulcer). The polyketide toxin consists of a 12-membered lactone ring with a lower O-linked polyunsaturated acyl side chain and an upper C-linked side chain. Mycolactone is unique to M. ulcerans and an immunological Ag capture assay would represent an important tool for the study of Buruli ulcer pathogenesis and for laboratory diagnosis. When testing sets of mycolactone-specific mouse mAbs, we found that Abs against the hydrophobic lower side chain only bind mycolactone immobilized on a solid support but not when present in solution. This observation supports previous findings that mycolactone forms micellar structures in aqueous solution with the hydrophobic region sequestered into the inner core of the aggregates. Although an Ag capture assay typically requires two Abs that recognize nonoverlapping epitopes, our search for matching pairs of mAbs showed that the same mAb could be used both as capture and as detecting reagent for the detection of the mycolactone aggregates. However, the combination of a core-specific and a core/upper side chain-specific mAb constituted the most sensitive ELISA with a sensitivity in the low nanogram range. The results of a pilot experiment showed that the sensitivity of the assay is sufficient to detect mycolactone in swab samples from Buruli ulcer lesions. Although the described capture ELISA can serve as a tool for research on the biology of mycolactone, the assay system will have to be adapted for use as a diagnostic tool.

Development of an ELISA for the quantification of mycolactone, the cytotoxic macrolide toxin of Mycobacterium ulcerans.

Mycolactones, macrolide cytotoxins, are key virulence factors of Mycobacterium ulcerans, the etiological agent of the chronic necrotizing skin disease Buruli ulcer. There is urgent need for a simple point-of-care laboratory test for Buruli ulcer and mycolactone represents a promising target for the development of an immunological assay. However, for a long time, all efforts to generate mycolactone-specific antibodies have failed. By using a protein conjugate of a truncated non-toxic synthetic mycolactone derivative, we recently described generation of a set of mycolactone-specific monoclonal antibodies. Using the first mycolactone-specific monoclonal antibodies that we have described before, we were able to develop an antigen competition assay that detects mycolactones. By the systematic selection of a capturing antibody and a reporter molecule, and the optimization of assay conditions, we developed an ELISA that detects common natural variants of mycolactone with a limit of detection in the low nanomolar range. The mycolactone-specific ELISA described here will be a very useful tool for research on the biology of this macrolide toxin. After conversion into a simple point-of-care test format, the competition assay may have great potential as laboratory assay for both the diagnosis of Buruli ulcer and for the monitoring of treatment efficacy.

Transmission of Hepatitis B and D Viruses in an African Rural Community.

According to the World Health Organization (WHO), an estimated 257 million people worldwide are chronically infected with hepatitis B virus (HBV), with approximately 15 million of them being coinfected with hepatitis D virus (HDV). To investigate the prevalence and transmission of HBV and HDV within the general population of a rural village in Cameroon, we analyzed serum samples from most (401/448) of the villagers. HBV surface antigen (HBsAg) was detected in 54 (13.5%) of the 401 samples, with 15% of them also containing anti-HDV antibodies. Although Cameroon has integrated HBV vaccination into their Expanded Program on Immunization for newborns in 2005, an HBsAg carriage rate of 5% was found in children below the age of 5 years. Of the 54 HBsAg-positive samples, 49 HBV pre-S/S sequences (7 genotype A and 42 genotype E sequences) could be amplified by PCR. In spite of the extreme geographical restriction in the recruitment of study participants, a remarkable genetic diversity within HBV genotypes was observed. Phylogenetic analysis of the sequences obtained from PCR products combined with demographic information revealed that the presence of some genetic variants was restricted to members of one household, indicative of intrafamilial transmission, which appears to take place at least in part perinatally from mother to child. Other genetic variants were more widely distributed, reflecting horizontal interhousehold transmission. Data for two households with more than one HBV-HDV-coinfected individual indicate that the two viruses are not necessarily transmitted together, as family members with identical HBV sequences had different HDV statuses. IMPORTANCE This study revealed that the prevalence of HBV and HDV in a rural area of Cameroon is extremely high, underlining the pressing need for the improvement of control strategies. Systematic serological and phylogenetic analyses of HBV sequences turned out to be useful tools to identify networks of virus transmission within and between households. The high HBsAg carriage rate found among children demonstrates that implementation of the HBV birth dose vaccine and improvement of vaccine coverage will be key elements in preventing both HBV and HDV infections. In addition, the high HBsAg carriage rate in adolescents and adults emphasizes the need for identification of chronically infected individuals and linkage to WHO-recommended treatment to prevent progression to liver cirrhosis and hepatocellular carcinoma.

Development of Dengue Virus Serotype-Specific NS1 Capture Assays for the Rapid and Highly Sensitive Identification of the Infecting Serotype in Human Sera.

Dengue fever can be caused by one of four distinct dengue virus (DENV) serotypes that cocirculate in many parts of the world. Point of care serotype-specific nonstructural protein-1 (NS1) capture assays for the rapid serotyping of DENV in human sera would greatly support epidemiological surveillance and potentially also prognosis in individual patients. To ensure both serotype specificity and broad coverage of variants within serotypes, we have applied an innovative approach for the generation and selection of serotype-specific anti-NS1 mAbs. To elicit mAbs against conformational epitopes, NMRI mice were immunized with living HEK 293 transfectants expressing the native target Ags in multiple display on the cell surface. For each serotype, three different NS1 sequence variants were sequentially used for immunization of mice, hybridoma selection, and capture assay development, respectively. Selection of optimal combinations of capturing and detecting mAbs yielded highly sensitive and specific NS1 serotyping ELISAs (st-ELISAs) for the four serotypes. st-ELISA testing of 41 dengue patient sera showed a 100% concordance with the serotype determined by serotype-specific reverse transcriptase real-time quantitative PCR. The respective NS1 variants could be detected for ∼10 d after the onset of illness. Ab-dependent enhancement of DENV infections may be associated with a specific range of pre-existing anti-DENV serological Ab titers. Testing of patient sera with the developed st-ELISAs will not only be useful for epidemiological studies and surveillance, but it may also help to develop and validate assays that can distinguish protective versus enhancing Ab responses for risk assessment for the development of severe dengue disease in individual patients.