University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES DETECTION OF MYCOLACTONE BY FLUORESCENT THIN LAYER CHROMATOGRAPHY (f-TLC) FOR THE DIAGNOSIS OF BURULI ULCER AKOLGO GIDEON ATINGA JULY 2015 i University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES DETECTION OF MYCOLACTONE BY FLUORESCENT THIN LAYER CHROMATOGRAPHY (f-TLC) FOR THE DIAGNOSIS OF BURULI ULCER BY AKOLGO GIDEON ATINGA (ID. NO. 10274146) THIS THESIS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL CHEMISTRY JULY 2015 i University of Ghana http://ugspace.ug.edu.gh Declaration I, hereby declare that this submission is my own work towards the award of MPhil Chemistry in the Department of Chemistry, University of Ghana and that, to the best of my knowledge, it contains no material previously published by another person nor material which has been accepted for the award of any other degree of the University, except where due acknowledgement has been made in text. AKOLGO GIDEON ATINGA (10274146) ………………………………………… …………………………… Student’s Signature Date Certified by: DR. RICHARD K. AMEWU ………………………………………… …………………………… Principal Supervisor’s Signature Date DR. RAPHAEL KWAKU KLAKE ………………………………………… …………………………… Co-supervisor’s Signature Date ii University of Ghana http://ugspace.ug.edu.gh Abstract Buruli ulcer (BU) is a bacterial infectious disease of the subcutaneous fat caused by Mycobacterium ulcerans, resulting in chronic, devastating, necrotizing consequences. M. ulcerans produces a polyketide-derived macrolide toxin called mycolactone, required for the organism’s virulence. The disease manifests initially as a painless pre-ulcerative subcutaneous nodule, a plaque or as a rapidly progressing oedema. The oedema, the severe form breaks down to form characteristic ulcers with undermined edges which can progress to large necrotic lesions that, if untreated can extend to 15% of patient’s skin surface. With the recent introduction of antibiotic therapy by WHO, more emphasis has been placed on early diagnosis of BU. All forms of Buruli ulcer (papules, nodules, plagues, oedema and ulcers) however extensive, respond well to antibiotic treatment. However, antibiotics can only be administered upon adequate diagnosis of the disease, therefore, the earlier the diagnosis, the better. Acid-fast smear microscopy, culture, polymerase chain reaction (PCR), and histopathology are employed for the confirmation of BU. However, numerous challenges are encountered ranging from dedicated facilities and specialized equipment (culture or PCR), limited sensitivity (smear microscopy), to very demanding and time-consuming nature of both histopathology and culture of M. ulcerans in which results are available only after 6 – 12 weeks. The quest for a cheap and less time-consuming technique with the potential of being developed into a point-of-care diagnosis technique led to the discovery of the fluorescent-Thin Layer Chromatography (f-TLC) method. In this study, the f-TLC method was employed to detect the biomarker (mycolactone A/B) reported to be biosynthetically restricted to M. ulcerans and homogeneously distributed within infected tissue. Clinical evaluation of the diagnostic potential of the f-TLC technique in comparison to the gold standard PCR method in 50 suspected BU patients admitted into various health facilities: Agogo, Tepa, Dunkwa, Amasaman and Obom was iii University of Ghana http://ugspace.ug.edu.gh undertaken. Among the suspected cases (n = 50), the sensitivity of f-TLC test was 77% and the specificity was 63%. The positive predictive value (PPV) and the negative predictive value (NPV) of the test were 69% and 71% respectively at their respective 95% CI. Preliminary exploration of the possibility of using urine from BALB/c mice infected with M. ulcerans disease showed that the mycolactone could be detected by the f-TLC method. Urine sampling therefore could potentially be a non-invasive, more convenient and co-operative sampling technique for patients suspected of BU. iv University of Ghana http://ugspace.ug.edu.gh Dedication To my family, who supported me in all ways throughout this programme. To my mother, Azeyeta Akolgo who inspires me to always work harder. More importantly, I dedicate this work to my lovely sister, Azumah Akolgo who has helped me to discover my true potential. Gideon A Akolgo v University of Ghana http://ugspace.ug.edu.gh Acknowledgements My gratitude first of all is to Almighty God for seeing me through this research. Also, my profound gratitude goes to my family and friends for the support and encouragement all through my programme. I wish to thank my supervisors: Dr. Richard Amewu for guiding me through this research and Dr. Raphael Klake for your valuable criticisms and encouraging comments that has made this thesis a success. Their support has been invaluable. I thank Dr. Kingsley Asiedu for making it possible for my travel to Harvard University as part of my research work. I thank Prof. Kishi and his entire group for their invaluable suggestions that has made my research work a success and also for making my stay in the USA pleasurable. I am grateful to the following for assisting my research in various ways: Dr. Richard O. Phillips, Dr. Edwin Ampadu, Dr. Joseph Tuffour, Dr. Anthony Ablordey, Dr. Albert Paintsil, Prof. Yoshito Kishi, Thomas Spangenberg, Sudheer Vaddela, Dr. Isaac Lamptey, Dr. Naa Addison, Prof. Dorothy Yeboah-Manu, Mr. Eric Koka, Auntie Pat, Auntie Dorothy, Samuel Aboagye, Daniel, Emma, Mr. Maxwell Quartey, Ken Badu, Mr. Bob Essien. Not forgetting all the nurses in the various centres who have contributed tremendously to this work. I also thank Emelia Ansu and her entire family for facilitating my stay in Cambridge, Massachusetts. In fact, the list is endless. I am highly indebted to everyone who has contributed to the success of this work. I am grateful to the entire staff of Department of Chemistry, University of Ghana who contributed immensely to my intellectual development during my undergraduate studies leading to my MPhil programme. To my colleagues in the department, it’s been fun working together; I thank you all for the support. vi University of Ghana http://ugspace.ug.edu.gh Table of Contents Declaration ...................................................................................................................................... ii Abstract .......................................................................................................................................... iii Dedication ....................................................................................................................................... v Acknowledgements ........................................................................................................................ vi Table of Contents .......................................................................................................................... vii List of Figures ................................................................................................................................. x List of Tables ................................................................................................................................. xii List of Abbreviations .................................................................................................................... xiii CHAPTER ONE ............................................................................................................................ 1 1 Introduction .............................................................................................................................. 1 1.1 Background ....................................................................................................................... 1 1.2 Research questions ........................................................................................................... 5 1.3 Aim ................................................................................................................................... 5 1.4 Objectives ......................................................................................................................... 6 1.5 Importance of the Study ................................................................................................... 6 1.6 Justification for the study ................................................................................................. 7 CHAPTER TWO ........................................................................................................................... 9 2 Literature review ...................................................................................................................... 9 2.1 History .............................................................................................................................. 9 2.2 The causative organism of Buruli ulcer .......................................................................... 10 2.3 Epidemiology and transmission of M. ulcerans infection .............................................. 13 2.4 Pathology and Immunology of M. ulcerans disease ....................................................... 16 2.5 Mycolactone: A polyketide toxin from Mycobacterium ulcerans required for virulence… ................................................................................................................................ 18 2.6 Stages of Mycobacterium ulcerans disease .................................................................... 23 2.6.1 Papules .................................................................................................................... 23 2.6.2 Nodules .................................................................................................................... 24 2.6.3 Plaques .................................................................................................................... 25 vii University of Ghana http://ugspace.ug.edu.gh 2.6.4 Oedemas .................................................................................................................. 26 2.6.5 Ulcers ...................................................................................................................... 27 2.7 Categories of M. ulcerans disease .................................................................................. 28 2.8 Sampling techniques for laboratory diagnosis of M. ulcerans disease ........................... 29 2.8.1 Biopsy (punch or surgical) ...................................................................................... 29 2.8.2 Swabs ...................................................................................................................... 30 2.8.3 Fine needle aspiration (FNA) .................................................................................. 31 2.9 Laboratory diagnosis of BU ........................................................................................... 32 2.10 Modes of treatment of M. ulcerans disease .................................................................... 39 2.10.1 Surgical treatment ................................................................................................... 39 2.10.2 Chemotherapy of M. ulcerans disease .................................................................... 39 2.10.3 Consideration of an all oral regimen for M. ulcerans disease ................................. 42 2.10.4 Experimental treatments .......................................................................................... 43 CHAPTER THREE .................................................................................................................... 47 3 Methodology .......................................................................................................................... 47 3.1 Sampling of clinical specimens ...................................................................................... 47 3.2 Synthetic mycolactone A/B ............................................................................................ 48 3.3 Confirming the presence of mycolactone in samples ..................................................... 48 3.3.1 f-TLC Analysis of Swab/FNA Samples .................................................................. 48 3.3.2 f-TLC Analysis of spiked urine Samples ................................................................ 50 3.4 M. ulcerans infected BALB/c mice studies .................................................................... 51 3.4.1 M. ulcerans inoculum .............................................................................................. 51 3.4.2 Experimental animals .............................................................................................. 51 3.4.3 Metabolic cage ........................................................................................................ 52 3.4.4 Urine sample collection ........................................................................................... 53 3.4.5 f-TLC analysis of urine samples from M. ulcerans infected mice .......................... 54 3.5 Polymerase chain reaction (PCR) ................................................................................... 54 3.6 Identification of mycolactone A/B by physical properties on chromatography and mass spectrometry .............................................................................................................................. 54 3.6.1 Characteristic behaviour of mycolactone A/B in Thin Layer Chromatography (TLC)…. ................................................................................................................................ 54 3.6.2 Mass spectrometry (MS) confirmation of mycolactone A/B structure ................... 55 viii University of Ghana http://ugspace.ug.edu.gh 3.7 Estimating the detection limit ......................................................................................... 56 3.8 Enhancing the sensitivity ................................................................................................ 56 3.8.1 UV Absorption spectra ............................................................................................ 56 3.9 Statistical analysis ........................................................................................................... 57 CHAPTER FOUR ....................................................................................................................... 58 4 Results and discussion ........................................................................................................... 58 4.1 Introduction .................................................................................................................... 58 4.2 Thin layer chromatography (TLC) ................................................................................. 61 4.3 Laboratory confirmed BU cases of clinical samples ...................................................... 62 4.4 Measures of diagnostic accuracy of f-TLC compared to PCR in BU diagnosis ............ 66 4.4.1 Overall Sensitivity and specificity of f-TLC technique .......................................... 66 4.4.2 Sensitivity and specificity of the optimized procedure for f-TLC detection of mycolactone A/B in swab and FNA samples ........................................................................ 67 4.5 UV absorption of spectra analysis .................................................................................. 72 4.6 Urine sampling ............................................................................................................... 76 4.6.1 Detection of mycolactone A/B in urine samples using f-TLC technique ............... 77 4.6.2 Mass Spectrometry Studies ..................................................................................... 79 CHAPTER FIVE ......................................................................................................................... 85 5 Conclusion and recommendations ......................................................................................... 85 5.1 Conclusion ...................................................................................................................... 85 5.2 Recommendations .......................................................................................................... 86 References .................................................................................................................................... 88 Appendices ................................................................................................................................. 100 ix University of Ghana http://ugspace.ug.edu.gh List of Figures Figure 2.1: Molecular structure of mycolactone showing a core cyclic lactone ring and polyketide-derived highly unsaturated acyl side chains. ............................................................... 20 Figure 2.2: Properties of mycolactone. (A)Thin layer chromatography of lipid extracts from M. ulcerans cultures showing mycolactone, the major lipid species in ASL with an Rf of 0.23 in a solvent phase of chloroform, methanol, water (90:10:1). (B) Mass spectrometry showing mycolactone A/B with a peak mass-to-charge ratio (m/z) of 765.5 (George et al., 1999). .......... 20 Figure 2.3: Circular representation of pMUM001. Source: (Stinear et al., 2004). ....................... 22 Figure 2.4: Papule (Courtesy: Dr J. Hayman, Australia) .............................................................. 24 Figure 2.5: Nodule (Courtesy: Dr K. Asiedu, Ghana) .................................................................. 24 Figure 2.6: Plaque (Courtesy: Dr A. Chauty, AFRF, Benin) Figure 2.7: Plaque (Courtesy: Dr K. Asiedu, Ghana) ......................................................................................................................... 25 Figure 2.8: Plaque (Courtesy: Dr A. Paintsil, Ghana) ................................................................... 25 Figure 2.9: Oedema (Courtesy: Dr K. Asiedu, Ghana) ................................................................. 26 Figure 2.10: Oedema (Courtesy: Dr K. Asiedu, Ghana) ............................................................... 26 Figure 2.11: Large Ulcer (Courtesy: Dr A. Tiendrebéogo, Nigeria) ............................................. 27 Figure 2.12: Large Ulcer (Courtesy: Professor H. Assé, Côte d'Ivoire) ....................................... 28 Figure 2.13: Ziehl-Neelsen stained smear from a Buruli ulcer showing extracellular acid-fast bacilli. (Photo: Wayne Meyers) .................................................................................................... 34 Figure 2.14: Molecular structures of some anti-mycobacterial drugs used in Chemotherapy of M. ulcerans disease with their stereochemistry .................................................................................. 40 Figure 2.15: A photographic example of the stages of healing by clay: destroyed tissue (a) is easily removed by one treatment with CsAr02 to expose raw muscle and bone (b). The progression of healing is shown (c, d) with daily treatments of CsAg02. After 3–4 months, the infection is healed with soft, supple scarring and a return of normal motor function (e). ............ 45 Figure 3.1: Experimental protocol. Step a: an acetone solution of mycolactone A/B (10 ng) is applied to a dye-free silica gel C60 TLC plate and eluted with 5/4/1 CHCl3–Hexanes–MeOH as mobile phase; mycolactone A/B is not detected with conventional methods (photograph I). Step x University of Ghana http://ugspace.ug.edu.gh b: the eluted TLC plate is briefly warmed on a hot plate to evaporate the solvents and, while warm, quickly immersed into a 0.1 M acetone solution of 2-naphthylboronic acid. Step c: the TLC plate is heated to ~100 ºC for 60 s. Step d: the TLC plate is irradiated with a UV lamp (8 W) with a 365 nm filter, after its reverse side has been cleaned with acetone, to detect mycolactone A/B as a green-yellow fluorescent spot. .................................................................. 50 Figure 3.2: BALB/c mouse placed in a metabolism cage at the NMIMR. Photograph by Gideon Akolgo. .......................................................................................................................................... 53 Figure 4.1: Age and sex distribution of 50 suspected case patients who presented to the study hospitals between August 2014 and April 2015. ........................................................................... 61 Figure 4.2: 7 spots of synthetic mycolactone A/B in serial amounts from 10 ng to 100 ng respectively were spotted and examined under UV light with a 365 nm filter. The detection limit on the TLC was 20 ng (corresponding to 2 µL of mycolactone A/B). ......................................... 62 Figure 4.3: Comparison of results of f-TLC and PCR from swab and FNA samples .................. 64 Figure 4.4: Measures of diagnostic accuracy using f-TLC of data stratified according to diagnostic specimen type .............................................................................................................. 69 Figure 4.5: The proposed scheme for the reaction between (1) and naphthylboronic acid to form the corresponding naphthylboronate (2)........................................................................................ 72 Figure 4.6: TLC pictures showing background spots overshadowing the mycolactone spot ....... 73 Figure 4.7: Adducts formed with mycolactone A/B; (A) – adduct formed with the 2- naphthylboronic acid; (B) – adduct formed with the 4-nitrobenzaldehyde................................... 74 Figure 4.8: Absorption spectra in MeOH: Green: 4-nitrobenzaldehyde (λmax 250; Abs = 3.9210). Blue: 2-naphthylboronic acid (λmax 270; Abs = 2.9999) Red: Mycolactone A/B (λmax 355; Abs = 2.8729). .......................................................................................................................................... 75 Figure 4.9: Structure of mycolactone A/B showing the pentaenoate chromophore (red) ............. 76 Figure 4.10: TLC profiles of spiked non-infected human urine (A) and urine samples from non- infected and infected SPF BALB/c mice (B and C respectively) (Photo-enhanced using Adobe Photoshop CS 6) ............................................................................................................................ 78 Figure 4.11: The proposed fragmentation of mycolactone (precursor ion m/z 765) to produce fragment ions A and B .................................................................................................................. 80 Figure 4.12: Positive-ion ESI-micrOTOF-QII high-resolution mass spectrum (HRMS) of Mycolactone A/B (A – E) in a scan range 200-1200 m/z. ............................................................ 83 xi University of Ghana http://ugspace.ug.edu.gh List of Tables Table 4.1: Study participants, clinical information, and diagnostic results .................................. 59 Table 4.2: Results of f-TLC test for mycolactone and PCR from swabs and fine-needle aspirations (FNAs) ........................................................................................................................ 63 Table 4.3: Comparison of results of f-TLC versus PCR in FNA and Swab samples ................... 65 Table 4.4: Fisher’s Comparative Sensitivity and specificity test of fluorescent TLC mycolactone test and Gold Standard PCR .......................................................................................................... 67 Table 4.5: Comparison of Sensitivity, Specificity, PPV and NPV in Swab and FNA samples .... 68 Table 4.6: Formula, identity and mass accuracy with their respective error of mycolactone A/B on HRMS micrOTOF-Q II mass spectrometer ............................................................................. 81 xii University of Ghana http://ugspace.ug.edu.gh List of Abbreviations AFB Acid-fast bacilli AMK Amikacin BU Buruli ulcer CI Confidence Intervals CLR Clarithromycin DNA Deoxyribonucleic acid DTH Delayed-type hypersensitivity ESI Electrospray ionization FN False negative FNA Fine needle aspiration FP False positive f-TLC fluorescent Thin Layer Chromatography HIV Human Immunodeficiency virus HRMS High-resolution mass spectrum IFN-γ gamma interferon IL-1 Interleukin-1 IS2404 Insertion sequence 2404 IS2606 Insertion sequence 2606 L-J Löwenstein-Jensen m/z mass-to-charge ratio MLSA Multi-locus sequence analysis MM Mycobacterium marinum MPM Mycolactone-producing mycobacteria MS Mass spectrometry MU Mycobacterium ulcerans NF-κB Nuclear Factor kappa-light-chain-enhancer of activated B cells NMIMR Noguchi Memorial Institute for Medical Research NPV Negative predictive value PBS Phosphate-buffered saline PCR Polymerase chain reaction PPV Positive predictive value Rf Retention factor RIF Rifampin SPF Specific pathogen-free SPX Sparfloxacin STPK Serine/threonine protein kinase STR Streptomycin TH1 T-cell helper-1 TH2 T-cell helper-2 TH17 T-cell helper-17 TLC Thin layer chromatography TNF Tumour necrosis factor xiii University of Ghana http://ugspace.ug.edu.gh TN True negative TP True positive UV Ultraviolet VNTR Variable-Number Tandem Repeats Vpp Volt peak to peak WHO World Health Organization ZN Ziehl-Neelsen xiv University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1 Introduction 1.1 Background Buruli ulcer (BU) is a bacterial infectious disease of the subcutaneous fat. The disease is caused by Mycobacterium ulcerans, and occurs less frequently but with chronic, devastating, necrotizing consequences resulting in tissue damage. It is the third commonest mycobacterial disease, after tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae) (WHO, 2012), but the most poorly understood of the three diseases. It is predominant among children in rural West Africa (WHO, 2000). M. ulcerans produces a polyketide-derived macrolide toxin called mycolactone, whose synthetic enzymes are encoded on a giant plasmid which is required for the organism’s virulence. Mycolactone induces necrosis and ulceration by its cytotoxic and immunosuppressive properties (Bozzo et al., 2010; Walsh et al., 2005). Buruli ulcer is classified as a neglected tropical disease. Currently, it is known to manifest in at least 35 countries and is common in some marshy tropical and subtropical regions, particularly in Africa (Ghana, Benin, Ivory Coast, and Uganda), where it is frequent in children between the ages of 5 and 15 years (Wansbrough-Jones and Phillips, 2006). Despite the endemic nature of this disease in tropical regions, it has received little attention as compared to the other two mycobacterial infections namely Mycobacterium tuberculosis and Mycobacterium leprae until over the past decade when the World Health Organization (WHO) recognized it as a global health problem. This led to the establishment of the WHO Global Buruli Ulcer Initiative in 1998 with the aim of seeking partnerships in research and control, and also to coordinate global control and 1 University of Ghana http://ugspace.ug.edu.gh research efforts (Wansbrough-Jones and Phillips, 2006). This initiative prompted major researchers all over the world to research into this endemic disease and share information in areas of epidemiology, control measures, and research into transmission, pathogenesis, molecular biology, and treatment (WHO, 2000). Notwithstanding the extensive research following the establishment of the Buruli Ulcer Initiative in 1998, the mode of transmission of the ulcer still remains unknown, even though various researches link the transmission of the mycobacterium to insects such as mosquitoes (Johnson et al., 2007) and aquatic insects such as beetles and water bugs (Marsollier et al., 2002). The disease occurs in stages with initial manifestations as painless pre-ulcerative subcutaneous nodules, a plaque or as a rapidly progressing oedema, the severe form of the disease which breaks down to form characteristic ulcers with undermined edges which can progress to large necrotic lesions that, if untreated, can extend to 15% of a patient’s skin surface (WHO, 2000). With the institution of antibiotic treatment, WHO has defined lesions sizes with cross- sectional diameter of less than 5 cm as category I, 5-15 cm as category II, and more than 15 cm, lesions on important sites (eye, breast, and genitalia), or multiple lesions as category III. M. ulcerans infection can be self-limiting, but scar tissue and contractures in joints leave patients with functional limitations and can result in social stigma (Stienstra et al., 2005, 2002). Interestingly, Buruli ulcer lesions are described as relatively painless (Goto et al., 2006; Marion et al., 2014) and a murine study by En et al in 2008 demonstrated that mycolactone is responsible for this phenomenon (En et al., 2008). However, clinical observations revealed that some patients experience considerable pain during their treatment - especially during wound care and physiotherapy (Alferink et al., 2015; de Zeeuw et al., 2015; Onoe et al., 2012). The absence 2 University of Ghana http://ugspace.ug.edu.gh of pain which is characteristic of most BU cases often lead patients to underestimate the severity of the disease and therefore do not seek early diagnosis and possible treatment. This sometimes results in severe sequelae leading to limb amputation (En et al., 2008). Surgical interventions to remove all infected tissue has been the traditional curative approach to BU cases until the recent introduction of antibiotic therapy consisting of a combination of rifampicin and streptomycin treatments (WHO, 2012). All forms of Buruli ulcer (papules, nodules, plaques, oedema and ulcers) however extensive, respond well to antibiotic treatment, although large lesions may be slow to heal and require surgery such as debridement, skin grafting and scar revision. However, antibiotics can only be administered upon accurate diagnosis of the disease, therefore, the earlier the diagnosis, the better. Many BU patients however report late to health facilities for diagnosis which hinders the treatment of the disease. Various reasons have been attributed to the late presentation of BU cases and they include but not limited to the following; (1) the disease itself – nodules can go unnoticed and when the ulcer appears, it progresses slowly, is painless, and the patient does not have systemic symptoms; (2) beliefs and stigma regarding the origin of the disease (namely witchcraft) making affected individuals reluctant to seek help; (3) limited geographic, financial, and cultural access to medical care and (4) fear of surgery and anaesthesia (Sizaire et al., 2006; Stienstra et al., 2002). Standard laboratory diagnostic tests available for confirmation of BU are smear microscopy for detection of acid-fast bacilli (AFB), M. ulcerans isolation by culture, histopathology, and polymerase chain reaction (PCR) for detection of the insertion sequence IS2404 (Beissner et al., 2012). Although both histopathology and culture for M. ulcerans (slow growth over 8 – 12 weeks) are technically very demanding and time-consuming diagnostic approaches, the fast and less demanding smear microscopy has limited sensitivity. Therefore, 3 University of Ghana http://ugspace.ug.edu.gh IS2404 PCR has become the gold standard diagnostic tool (Phillips et al., 2009b), even though PCR facilities are located in cities which are far away from endemic communities. Its routine use in resource-poor settings is also limited by the high costs and need for sophisticated laboratory infrastructure. Therefore, PCR tests are usually not performed at the treatment facilities but in bulk at national reference laboratories or outside the endemic country at international reference laboratories (Eddyani et al., 2008). New tools for disease diagnosis that can be implemented at the district hospitals is therefore a priority for WHO working group for diagnosis. Formerly, punch biopsy specimens were considered suitable diagnostic samples for laboratory confirmation of BU (Herbinger et al., 2009). However, due to the invasive character of the sample collection method, a consensus has been reached by stakeholder groups that in the interest of the patient, FNA can replace punch biopsies for non-ulcerative lesions and may serve as an alternative for ulcerative lesions in cases where scarred edges prevent the collection of swabs. Regarding PCR assessment of FNA from non-ulcerative lesions, results from three recent studies, one from Ghana (Phillips et al., 2009b) and two from Benin (Cassisa et al., 2010; Eddyani et al., 2009) respectively report sensitivities around 90%, similar to the sensitivity for punch biopsy specimens according to the findings of Eddyani et al (2009). Also, equal sensitivities were determined for microscopy of FNA samples (corresponding to 65%), as reported by Eddyani et al. and punch biopsy specimens from non-ulcerative lesions. For ulcerative lesions, available data suggest that for both diagnostic tests, swabs are clearly superior to tissue samples (Cassisa et al., 2010). Histopathological studies have shown that in tissues, mycolactone is widely distributed compared to the causative organism (Sarfo et al., 2010b). Therefore, the detection of mycolactone may serve as a useful rapid point-of-care diagnostic tool. For the detection of mycolactone or its 4 University of Ghana http://ugspace.ug.edu.gh related molecules, different methods have been employed including mass spectrometry, cytotoxicity assays, or thin-layer chromatography (TLC) (Daniel et al., 2004). The major mycolactone is mycolactone A/B (Demangel et al., 2009). Total synthesis of the mycolactones was demonstrated (Song et al., 2007) and synthetic mycolactone A/B has been made available for research purposes. 1.2 Research questions The research seeks to answer the following research questions; 1. Can the mycolactone in M. ulcerans infected tissues be detected using fluorescent TLC (f- TLC)? 2. How sensitive is the boron-assisted staining of f-TLC compared to the gold standard PCR diagnostic tool? 3. Is it possible to improve the sensitivity of the technique through other compounds? 4. Can mycolactone be detected in urine samples? Can the mycolactone in the urine samples be detected using mass spectrometry? 1.3 Aim The aim of the proposed study is to evaluate the performance of f-TLC against standard IS2404 PCR and to determine its usefulness as a treatment monitoring tool. 5 University of Ghana http://ugspace.ug.edu.gh 1.4 Objectives The objectives of this project are to; 1. Evaluate mycolactone as a potential biomarker for M. ulcerans disease. 2. Evaluate the performance of f-TLC against standard IS2404 PCR to:  Estimate the overall sensitivity and specificity of the f-TLC compared to PCR  Measure the positive predictive value and the negative predictive value of the f-TLC assay compared with IS2404 PCR; and  Estimate the sensitivity and specificity of the f-TLC compared to PCR by the type of sample (swabs or FNAs). 3. Explore urine sampling as a potential non-invasive alternative to FNAs and swabs by;  Identification of mycolactone A/B by its recognized physical properties on chromatography and mass spectrometry 1.5 Importance of the Study The research work seeks to employ f-TLC in the detection of mycolactone, the causative toxin responsible for Buruli ulcer, evaluate its sensitivity and specificity and compare it to the gold standard IS2404 PCR diagnostic tool. The problem of contamination is very unlikely to occur in the f-TLC method as compared to PCR. A major challenge of the f-TLC is background spots which affects the sensitivity of the method, however, Spangenberg and Kishi have improved the TLC 6 University of Ghana http://ugspace.ug.edu.gh method by the reduction of background spots. This has been made possible by developing a boronate-assisted fluorescent-TLC (f-TLC) method in which there is a marked reduction of background spots associated with the TLC method (Spangenberg and Kishi, 2010a). The TLC plate is developed by immersion in a boronic acid acetone solution that binds to mycolactone and fluoresces on excitation by ultraviolet light (Converse et al., 2014). Interestingly, this method is specific to detect human mycolactones, but not fish or frog mycolactones, as well as some unknown contaminants. Currently, where PCR facilities are readily available, a number of samples may have to be accumulated before they are processed leading to a turnaround time of up to one week. The f-TLC is a relatively simple and inexpensive method, compared to PCR. The results can be visualized with UV light. The method has been demonstrated in infected mice tissues and was used to monitor the response to antibiotic treatment as mycolactone levels decrease during treatment (Converse et al., 2014). 1.6 Justification for the study Buruli ulcer occurs in remote rural communities that are poorly covered by national health surveillance systems and as a consequence, they are largely unnoticed. Many of the affected populations do not seek health services because of financial constraints, beliefs that treatment does not work, fear of surgery and anaesthesia, or superstition and stigma (Stienstra et al., 2002). Mycolactone plays a critical role in bacterial pathogenicity, as shown by the necrotic lesions induced by intradermal injection of the purified molecule (George et al., 1999). M. ulcerans bacilli remain essentially localized in peripheral subcutaneous tissues, but there is evidence from animal studies that mycolactone diffuses from infectious foci into the blood, where it accumulates in 7 University of Ghana http://ugspace.ug.edu.gh mononuclear cell subsets (Hong et al., 2008a). There is therefore the need to develop a useful rapid point-of-care diagnostic tool for the detection of mycolactone which is the toxin responsible for virulence of the Mycobacterium ulcerans. Recent research efforts have therefore been focused on direct detection of mycolactones in tissues by f-TLC (Converse et al., 2014; Spangenberg and Kishi, 2010a; Zhang et al., 2011). 8 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2 Literature review 2.1 History Buruli ulcer is primarily an infection of the subcutaneous fat caused by the environmental pathogen Mycobacterium ulcerans. The disease manifests initially as a painless pre-ulcerative subcutaneous nodule, or a plaque or as a rapidly progressing oedema. The oedema, the severe form consequently breaks down to form characteristic ulcers with undermined edges which can progress to large necrotic lesions that, if untreated can extend to 15% of patient’s skin surface (WHO, 2000). Sir Albert Cook was the first to describe large cutaneous ulcers in 1897 (Cook, 1970), and later Kleinschmidt in the 1920s, indicating that the disease was present in Uganda and northeast Zaire (now Democratic Republic of the Congo) at those times. The first report of the disease in Africa came from Zaire in the late 1950s but there are suggestions that the disease had been in Zaire since 1935 (Meyers et al., 1974a; Wansbrough-Jones and Phillips, 2005). However, the first detailed description of the disease was in 1948 by MacCallum and Colleagues; they established the aetiology in a small group of patients in Australia, although contact with their first patient was in 1940 in Australia. Several notable periods of intensive study of the disease happened during the 20th century, initiated by the description of the first culture of M. ulcerans from a leg ulcer in a child from Bairnsdale, Australia by MacCallum. At this point, it was described as a Bairnsdale ulcer. After initial failed attempts at cultivation, the culture of M. ulcerans was achieved fortuitously when an incubator broke down, the optimal temperature for culture of M. ulcerans ranged from 30 – 33 °C (Maccallum et al., 1948). 9 University of Ghana http://ugspace.ug.edu.gh During the 1960s and 1970s, the Uganda Buruli Group studied the epidemiology of the disease and noted new cases in recent refugees from Rwanda gathered in an area close to the Nile (Clancey et al., 1962; Uganda Buruli Group, 1970). Large number of cases were found in the Buruli County (now known as the Nakasongola district) near Lake Kyoga which led to the coinage of the name “Buruli ulcer” by the Ugandan Buruli Group which has been favoured since then (Lunn et al., 1965). Zaire was another area where workers, often missionaries associated with charities for leprosy, were managing cases of Buruli ulcer; in 1965, the observation that there was no inflammation around clumps of acid-fast bacilli in necrotic subcutaneous fat led to the suggestion that M. ulcerans secretes a toxin that causes tissue damage (Connor and Lunn, 1965; Krieg et al., 1974). 2.2 The causative organism of Buruli ulcer The causative organism of Buruli ulcer is Mycobacterium ulcerans. A distinguishing characteristic of the microorganism is the production of mycolactone, a cytotoxic, fibrinolytic, and/or immunosuppressive toxin, required for the organism’s virulence. It is responsible for tissue necrosis and apoptosis, and causes vast cutaneous ulcerations (Bozzo et al., 2010; Walsh et al., 2005). It is a polyketide-derived macrolide, the genes of which are located on a plasmid known as pMUM001 (Stinear et al., 2005). Various types of mycolactones have been identified in particular geographical distributions of M. ulcerans (George et al., 1999; Sarfo et al., 2010b). The molecule is composed of an invariant core comprising a 12-membered lactone ring and side-chain that is esterified to a highly unsaturated acyl side chain, the latter structure varying amongst different MU strains (Hong et al., 2008b). MU strains from Africa, Australia and China produce variants named 10 University of Ghana http://ugspace.ug.edu.gh mycolactones A/B, C, and D, respectively whilst Mycobacterium liflandii (ML), a pathogen of frogs, produces mycolactone E, and the fish pathogens (Mycobacterium pseudoshottsii and Mycobacterium marinum DL240490 (DL) and others) produce mycolactone F (Fidanze et al., 2001; George et al., 1999; Judd et al., 2004; Kim and Kishi, 2008; A Mve-Obiang et al., 2003; Mve-Obiang et al., 2005; Song et al., 2007). Despite the multiple species names given to mycolactone-producing mycobacteria (MPM), multi locus sequence analysis (MLSA) of all these strains indicates they share greater than 98% nucleotide identity (Yip et al., 2007). M. ulcerans is an acid-fast, slow-growing environmental mycobacterium that falls into a group of closely related mycobacterial pathogens which comprise of the M. marinum complex (Stinear et al., 2000a). A third member of the M. marinum complex was recently uncovered with the discovery that other mycobacteria, not associated with Buruli ulcer also produce mycolactone. These mycobacteria, recovered from diseased fish and frogs around the world, have been given various species names including M. marinum (fish), Mycobacterium pseudoshottsii (fish) and Mycobacterium liflandii (frogs) (Käser et al., 2009; Mve-Obiang et al., 2005; Ranger et al., 2006). However, detailed phylogenetic studies of more than fifty (50) M. ulcerans species, other mycolactone-producing mycobacteria (MPM), and M. marinum strains, based on multi-locus sequence analysis (MLSA) of chromosomal and pMUM sequences and studies of large DNA InDel polymorphisms, indicate that all MPM are highly related and could have evolved from the same common M. marinum progenitor (Demangel et al., 2009; Käser et al., 2009). Like M. ulcerans, the fish and frog strains have a large virulence plasmid and accumulation of multiple copies of insertion sequences, IS2404 and IS2606, but the pattern of DNA deletion and pseudogene accumulation seems to be different: only those strains that cause BU had reduced genomes. M. ulcerans has evolved into five InDel haplotypes that separate into two distinct lineages: (i) the 11 University of Ghana http://ugspace.ug.edu.gh "classical" lineage including the most pathogenic genotypes – those that come from Africa, Australia and South East Asia; and (ii) an "ancestral" M. ulcerans lineage comprising strains from Asia (China/Japan), South America and Mexico. The ancestral lineage is genetically closer to the progenitor M. marinum in both RD composition and DNA sequence identity, whereas the classical lineage has undergone major genomic rearrangements (Käser et al., 2009; Yip et al., 2007). Subsequent studies have used the collective term mycolactone-producing mycobacteria (MPM) when describing M. ulcerans and these bacteria, as they all produce a form of mycolactone. The MPM can be distinguished from M. ulcerans by the type of mycolactone they produce and the fact that they do not exhibit the same high level of genome reduction and decay as M. ulcerans (Yip et al., 2007). For instance, in Japan, BU is caused by Mycobacterium ulcerans subspecies. shinshuense, not by M. ulcerans (Nakanaga et al., 2011). A comprehensive analysis of both the genotypic and biochemical profiles reveal similarities in both M. ulcerans subsp. Shinshuense and M. ulcerans infections from M. marinum infection. The most important test for early and differential diagnoses for distinguishing both infections from M. marinum infection involves IS2404 by PCR detection. Also, another similarity is the 16S rRNA gene sequences of M. ulcerans subsp. shinshuense and M. ulcerans. The species in the M. marinum complex can be considered as single based on the fact that they share over 97% identity in the 16sRNA gene sequence (Stinear et al., 2007). This is consistent with the gene sequence reported by Portaels et al. and is useful for the rapid detection of M. ulcerans and discriminates M. marinum and M. shinshuense from M. ulcerans (Nakanaga et al., 2007; Portaels et al., 1996). PCR targeting of pMUM001 reveal that all M. ulcerans subsp. shinshuense isolates lack the band representing the serine/threonine protein kinase (STPK) gene. This is suggestive of the fact that M. ulcerans subsp. shinshuense and M. ulcerans have undergone small but conservative mutation(s) in their sequences. The PCR test is 12 University of Ghana http://ugspace.ug.edu.gh also a fine tool applied for detection of a virulent plasmid and for differential diagnosis of M. ulcerans and M. ulcerans subsp. Shinshuense (Nakanaga et al., 2007). Evolutionary paths of these related mycobacterial species indicate that the rpoB gene show more similarity to M. marinum and M. pseudoshottsii than to M. ulcerans (Käser et al., 2007). The Japanese M. ulcerans subsp. shinshuense isolates and the Chinese strain of M. ulcerans presumably belong to the same cluster, based on genetic analyses such as microarray-based comparative genomic hybridization (Käser et al., 2007) and comparative sequence analysis of polymorphic Variable-Number Tandem Repeats (VNTR). Their genomes were distinctly different from those of M. ulcerans strains that originated in other geographic regions. However, one of the VNTR loci can be used to distinguish between the Chinese and Japanese strains (Ablordey et al., 2005). Pidot et al. (2008) described the clear difference between the two strains by analyzing virulent plasmid genes and the resulting mycolactone production, noting that the Japanese strain produces mycolactone A/B, while the Chinese strain produces a unique mycolactone D (Pidot et al., 2008). The common characteristic features of these species is slow growth rates and low optimal growth temperatures (Garrity, 2001). For instance, M. ulcerans can be isolated from primary lesions after a 5 – 8 week incubation period, although up to 6 months may be required (WHO, 2000; Yeboah-Manu et al., 2004). 2.3 Epidemiology and transmission of M. ulcerans infection The epidemiology of BU is poorly understood and outbreaks are sporadic and unpredictable; however, it has long been observed that Buruli ulcer is associated with tropical and humid areas in Africa where there are numerous patches of ground water or slow moving rivers. 13 University of Ghana http://ugspace.ug.edu.gh Minor endemic foci have been reported in Australia, tropical areas in Southeast Asia (Malaysia, Papua New Guinea, and Sri Lanka) and Latin America (Guyana, Mexico, Peru), but major foci with the largest number of patients with Buruli ulcer have been detected in sub-Saharan Africa (Hayman, 1991). There are reports from European countries, Canada, America, and Australia attributed to travellers visiting friends or relatives in BU-endemic countries who manifested BU upon return to a non-disease-endemic area (Faber et al., 2015; Thomas et al., 2014). Environmental and ecological studies show that the disease is most common in populations living near rivers, swamps and wetlands (Raghunathan et al., 2005). Proximity to, or contact with, slowly flowing or stagnant watercourses is therefore a recognized risk factor (Aiga et al., 2004; Hayman, 1991). In Uganda, Rwandan refugees who had not been susceptible to the disease in their own country started to develop lesions after being placed in the Kinyara refugee settlement, located adjacent to swampy regions near the Nile River. However, new cases ceased to occur when they were dispersed to other areas (Anon, 1971). These clinical observations in Uganda supports the theory of the acquisition of M. ulcerans infection in riverine regions (Lunn et al., 1965). In Ghana, the first probable case of Buruli ulcer was reported in the Greater Accra Region in 1971. The presence of additional cases were also considered likely along the tributaries of the Densu River (Bayley, 1971). Amofah et al. report that the major endemic foci where BUD is highly prevalent in Ghana are Amansie West, Asante Akim North, and Upper Denkyira districts (Amofah et al., 2002). These districts are characterized by abundant rivers, streams, swamps, and environmental changes due to logging and mining. Several different districts associated with particular rivers have high prevalence of the disease but it is hardly seen in adjacent districts (Amofah et al., 2002). However, the disease may also occur in temperate climates; the area around Melbourne, Southeast Australia appears to be one of the few foci of the disease in a temperate 14 University of Ghana http://ugspace.ug.edu.gh climate (Johnson et al., 1996; Veitch et al., 1997). In temperate southeastern Australia, outbreaks of M. ulcerans infection occur in localized areas, but few patients report direct contact with environmental water other than the ocean. This led to the proposal that aerosols from contaminated water may cause human infections (Hayman, 1991). However, these low-lying disease-endemic areas also harbour large populations of mosquitoes, and it has been established that transmission by mosquitoes offers a partial explanation for the outbreak at Point Lonsdale and possibly at other sites in southeastern Australia (Johnson et al., 2007). Also, a recent publication on cases diagnosed in Japan from 1980 to 2010 in different regions of Honshu Island revealed BU cases despite the fact that there was no evidence of patient contact with an aquatic environment. It was revealed that the BU cases recorded in Japan were induced by Mycobacterium ulcerans subsp. shinshuense, and not by M. ulcerans (Nakanaga et al., 2011). These findings suggest a unique combination of external environmental conditions, including land cover type, temperature and many more being responsible for M. ulcerans transmission and, therefore, BU incidence. In Ghana, a national case search performed in 1999 yielded a crude national BU prevalence rate of 20.7/100,000 and hence demonstrated that BU is the second most common mycobacterial infection in the country after tuberculosis (Amofah et al., 2002). Point prevalence estimates have varied between regions, but have been reported to be as high as 150 – 280/ 100,000 population in some highly endemic districts in Ghana (Amofah et al., 2002). In southern Benin, a study has reported detection rates of 21.5/100,000 per year, higher than for either tuberculosis or leprosy (Debacker et al. 2004). Similar prevalence rates have also been reported from Cote d’Ivoire. The peak age group in West Africa studies is 5 to 15 years with an almost equal gender distribution, although Buruli ulcer can affect any age group (Debacker et al., 2004). In Japan, the median age 15 University of Ghana http://ugspace.ug.edu.gh is 40 to 57 years (Nakanaga et al., 2007), whereas in Australia, the median age is 50 to 66 years (Veitch et al., 1997). The role of living agents such as aquatic insects or mosquitoes as reservoirs and vectors of M. ulcerans have been proposed but remains controversial. For instance, the transmission by mosquitoes offers a partial explanation for the outbreak at Point Lonsdale and possibly at other sites in Southeastern Australia (Johnson et al., 2007). This is not the situation in Africa, although there is a close genetic relationship of Australian isolates of M. ulcerans with strains from humans with BU in Africa (Yip et al., 2007). Extensive use of PCR in West Africa revealed that water bugs such as Naucoris cimicoides (Marsollier et al., 2002), and small fish gave positive signals that could be localized to their salivary glands. Portaels and colleagues (Portaels et al., 2008) reported the first direct isolation of M. ulcerans from nature in 2008 from a water strider, an aquatic insect that does not bite humans. The bacterium has been detected by PCR in other species, including snails which harbour the species and act as passive hosts (Marsollier et al., 2004) but the mode of transmission is yet to be elucidated. Person-to-person transmission has rarely been reported. Only two cases have been reported of human-to-human transmission; one involved a surgeon who developed a Buruli ulcer lesion on his hand after having done plastic surgery on a patient with Buruli ulcer disease in Ghana (Debacker et al., 2002; Exner and Lemperle, 1987). 2.4 Pathology and Immunology of M. ulcerans disease The unique pathology of BU is primarily attributed to two (2) properties of M. ulcerans: optimal growth at temperatures (30 °C – 33 °C) slightly below the core body temperature, and the production of the plasmid-encoded macrolide toxin, mycolactone (George et al., 1999; Stinear et 16 University of Ghana http://ugspace.ug.edu.gh al., 2004; Walsh et al., 2011). The temperature requirement of M. ulcerans favours the development of lesions in cooler tissues, especially the skin and subcutaneous tissue. Mycolactone destroys tissues by apoptosis and necrosis and suppresses host immune responses (Silva et al., 2009). The predominant pattern in the pathology of M. ulcerans disease is that in early, pre- ulcerative and ulcerative lesions, large numbers of extra-cellular mycobacteria are seen, with extensive necrosis and very little inflammatory response, but with no granuloma formation. In surgical specimens resected in later stages during healing, bacilli are scanty or even absent, with granuloma formation (Guarner et al., 2003; Hayman and McQueen, 1985; Maccallum et al., 1948). Okenu et al. demonstrated for the first time that the humoral immune response during M. ulcerans infection may be useful for the confirmation of BU. Many healthy individuals in Buruli ulcer- endemic areas show specific immune responses to M. ulcerans (Okenu et al., 2004; Schipper et al., 2007), suggesting that, in analogy with leprosy and tuberculosis, the disease develops only in a limited proportion of those infected with M. ulcerans (Exner and Lemperle, 1987). Further evidence that a cellular immune response may protect individuals with Buruli ulcer is provided by case reports that describe disseminated, overwhelming M. ulcerans disease in patients co-infected with HIV (van der Werf et al., 1999). Infected individuals from several geographical areas (Africa and Australia) where BU is endemic have also been shown to have reproducibly high antibody responses to M. ulcerans antigens (Dobos et al., 2000; Gooding et al., 2002; Okenu et al., 2004). Mycolactone profoundly suppresses elements of innate and adaptive cell-mediated immunity, thereby enhancing progression of BU. Mycolactone inhibits macrophages, monocytes, B cells, and T cells at least, in part, by inhibiting production of interleukin IL-1, IL-2, IL-6, IL-8, IL-10, tumor necrosis factor α, and interferon-γ (IFN-γ) (Boulkroun et al., 2010; Torrado et al., 17 University of Ghana http://ugspace.ug.edu.gh 2010). The immunosuppressive effects of mycolactone extend beyond skin lesions to circulating leukocytes and lymphoid organs (Hong et al., 2008a). The clinical and histopathological features of BU suggest an immunologic spectrum of host responses over time, which may be relevant for vaccine strategies. Early progressive ulcers generate abundant IL-10 with little inflammation (TH2 response) and numerous, often extracellular, M. ulcerans within areas of coagulation necrosis. Necrosis reflects mycolactone- induced death of tissue and inflammatory cells. In contrast, mature or resolving BU lesions, especially under treatment with antibiotics, contain IFN-γ within granulomatous inflammation, organizing lymphoid aggregates, and typically intracellular M. ulcerans, consistent with a TH1, delayed-type hypersensitivity (DTH) response (Kiszewski et al., 2006; Schutte and Pluschke, 2009; Silva et al., 2009). DTH in these patients, but not those with early BU or uninfected persons, is verified by skin test reactivity against burulin, a protein derivative of M. ucerans (Stanford et al., 1975). Several research groups, using different models, have observed that in early stages of disease, specific immune protection seems to be lost. Almost 30 years ago, Stanford et al. observed that cell-mediated immune response, as evidenced by skin testing using burulin, showed low responses in initial stages of Buruli ulcer disease that appeared to improve over time, in later stages of the disease (Stanford et al., 1975). Gooding et al. described similarly low specific immune protection in early Buruli ulcer in Australian patients but this effect seemed to persist after patients were cured (Gooding et al., 2002). 2.5 Mycolactone: A polyketide toxin from Mycobacterium ulcerans required for virulence Most pathogenic bacteria produce toxins that play important role(s) in disease. However, there has been no evidence thus far for any toxin identified for Mycobacterium tuberculosis and 18 University of Ghana http://ugspace.ug.edu.gh Mycobacterium leprae which happen to be the most recognized mycobacterial infections. The only mycobacterial pathogen for which there is any evidence of toxin production is Mycobacterium ulcerans, the causative agent of Buruli ulcer. In 1965, it was suggested that the extensive necrosis surrounding clumps of M. ulcerans in infected human tissue might be due to a diffusible substance (Connor and Lunn, 1965) and subsequently it was found that M. ulcerans culture filtrate could produce similar lesions after injection in guinea pig skin (Krieg et al., 1974). The possible presence of a toxin in M. ulcerans was hypothesized for a number of years prior to the isolation and characterization by Small and co-workers in 1999 (George et al., 1999). It was not until the late 1990s that the toxin was partly purified and its chemical structure defined. Subsequently the toxin was characterized as a 743 Da molecule consisting of a 12-membered ring macrolide with two polyketide-derived side-chains (George et al., 1999). George et al. (1999) identified an initial molecule on thin layer chromatography (TLC) with a light yellow, ultraviolet-active component with a retention factor (Rf) of 0.23 in a solvent system containing chloroform, methanol and water (90:10:1) as shown in (Figure 2.2 A). The compound was further purified by reversed phase high-performance liquid chromatography and subjected to structural analysis. Mass spectral analysis of the molecule under microspray conditions showed peaks at m/z 765 (strong), 743 (weak), and 725 (medium) (Figure 2.2 B). Accurate mass measurement of the peak at m/z 765 with formula C44H70O9Na, the sodium adduct [M + Na]+ of the molecule was observed at 765.4912; and that for the peak at m/z 725 had formula C44H69O8 and corresponded to the dehydrated protonated molecular ion [M+H – H2O] +, also observed at 725.4988. These analyses gave the formula C44H70O9, for the compound. The compound was further identified by two-dimensional nuclear magnetic resonance spectral analysis 19 University of Ghana http://ugspace.ug.edu.gh as the polyketide-derived 12-membered ring macrolide (Figure 2.1). The toxin was named mycolactone to reflect its mycobacterial source and chemical structure (George et al., 1999). Figure 2.1: Molecular structure of mycolactone showing a core cyclic lactone ring and polyketide-derived highly unsaturated acyl side chains. Figure 2.2: Properties of mycolactone. (A)Thin layer chromatography of lipid extracts from M. ulcerans cultures showing mycolactone, the major lipid species in ASL with an Rf of 0.23 in a solvent phase of chloroform, methanol, water (90:10:1). (B) Mass spectrometry showing mycolactone A/B with a peak mass-to-charge ratio (m/z) of 765.5 (George et al., 1999). The toxin seems to have cytotoxic, analgesic and immunosuppressive activities. This is evident because despite their abundance and extensive tissue damage, there is a remarkable absence of an acute inflammatory response to the bacteria, and the lesions are often painless (Hong et al., 2008b). However, its mode of action is unclear, but, in a guinea pig model of the disease, 20 University of Ghana http://ugspace.ug.edu.gh injection of purified mycolactone reproduces the natural pathology, and mycolactone negative variants are avirulent, implying a key role for the toxin in pathogenesis (George et al., 1999). This was further confirmed in 1974 by Read et al. who reported that a sterile filtrate of M. ulcerans had a cytopathic effect on cultured murine fibroblasts (Read et al., 1974) and more recently, Pimsler et al. reported that a sterile filtrate of M. ulcerans had immunosuppressive properties (Pimsler et al., 1988). M. ulcerans produces a heterogeneous mixture of mycolactone variants characterized by mass spectrometry as mycolactone A, B, C, and D. The most biologically active variants A and B are produced by African strains, whereas mycolactone C is the dominant type among M. ulcerans strains from Australia and mycolactone D in those from Asia (Wansbrough-Jones and Phillips, 2006). There is some evidence of a correlation between the potency of mycolactone variants in vitro and their clinical virulence (Armand Mve-Obiang et al., 2003). Mycolactone exhibits a specific cytopathic effect on murine L929 fibroblasts characterized by cell rounding within 24 hours and cell cycle arrest in the G0/G1 phase of the cell cycle, followed by apoptosis in 72 hours(George et al., 2000). Mycolactone can also inhibit pro-inflammatory cytokines such as TNF- α (Coutanceau et al., 2005) and IFN-γ (Kiszewski et al., 2006; Yeboah-manu et al., 2006). Paradoxically, cytokine-induced nuclear factor κB (NF-κB), a pro-inflammatory transcription factor, is activated by mycolactone (Pahlevan et al., 1999). M. ulcerans also induces a persistent inflammatory response in mouse and human skin. Human tissue biopsies have shown that pro- inflammatory cytokines are expressed at various stages of the infection, suggesting that the immune response to M. ulcerans is similar to the responses seen with other mycobacteria (Phillips et al., 2006; Schipper et al., 2007). Pretreatment of a J774 macrophage cell line with partly purified mycolactone inhibited induction of tumour necrosis factor (TNF) α and interleukin 10 in response 21 University of Ghana http://ugspace.ug.edu.gh to lipopolysaccharide stimulation. Production of interleukin 2 from activated T lymphocytes was blocked and TNF-induced nuclear factor κB activation was abrogated, leading the authors to suggest that mycolactone causes local immune suppression in tissues (Pahlevan et al., 1999). During characterization of the full genome sequence of M. ulcerans it became apparent that the cluster of genes encoding giant polyketide synthases and polyketide modifying enzymes necessary for mycolactone synthesis are carried on two identical copies of a 174-kb plasmid known as pMUM001, separate from the main DNA sequence (Stinear et al., 2004). Figure 2.3: Circular representation of pMUM001. Source: (Stinear et al., 2004). Mycobacterium ulcerans (MU) and Mycobacterium marinum (MM) share over 98% DNA mutual sequence identity, and they occupy aquatic environments, and both cause cutaneous infections (Stinear et al., 2000a). This suggests that M. ulcerans is probably derived from M. marinum after acquisition of the plasmid (Stinear et al., 2000b). However, M. marinum produces 22 University of Ghana http://ugspace.ug.edu.gh a granulomatous intracellular lesion, typical for pathogenic mycobacteria and totally distinct from Buruli ulcer in which M. ulcerans are mainly found extracellularly (Stinear et al., 2004). 2.6 Stages of Mycobacterium ulcerans disease BU is primarily a disease of the skin. It presents in two different major active forms: non- ulcerative (papules, nodules, plaques, and oedematous forms) and ulcerative forms (Pszolla et al., 2003; WHO, 2012). This neglected tropical disease primarily affects the skin and evolves in four stages. In the majority of cases, it starts insidiously as painless nodules, plaques, papules or oedema (van der Werf et al., 1999). In stage two, large skin ulcers with ill-defined edges develop (Guarner et al., 2003). In stage three, spontaneous granulomatous healing often occurs (van der Werf et al., 1999). In stage four, permanent severe functional limitations, including destructive lesions with extensive scarring and contractures of the limbs, may occur due to tissue fibrosis (Stienstra et al., 2005). 2.6.1 Papules M. ulcerans disease typically starts as a painless papule that may go unnoticed, most commonly on the extremities, and may then expand into a plaque or widespread indurated oedema and subsequently ulcerate (van der Werf et al., 1999). A papule is a painless, raised skin lesion < 1 cm in diameter, normally with the surrounding skin being reddened. Papular lesions are commonly seen in Australia and may initially be confused with an insect bite. 23 University of Ghana http://ugspace.ug.edu.gh Figure 2.4: Papule (Courtesy: Dr J. Hayman, Australia) 2.6.2 Nodules A nodule is a lesion < 3 cm in diameter that extends from the skin into the subcutaneous tissue. It is usually firm and painless but may be itchy, and the surrounding skin may be discoloured in comparison with adjacent areas. Nodules are commonly seen in Africa. Early nodular lesions are occasionally confused with boil, lipoma, ganglion, lymph node tuberculosis, onchocerciasis- related nodules or other subcutaneous infections, such as fungal infections. Figure 2.5: Nodule (Courtesy: Dr K. Asiedu, Ghana) 24 University of Ghana http://ugspace.ug.edu.gh 2.6.3 Plaques A plaque is a firm, painless, elevated, lesion > 3 cm in diameter with ill-defined edges. The skin over the lesion may be reddened or otherwise discoloured. Figure 2.6: Plaque (Courtesy: Dr A. Chauty, AFRF, Benin) Figure 2.7: Plaque (Courtesy: Dr K. Asiedu, Ghana) Figure 2.8: Plaque (Courtesy: Dr A. Paintsil, Ghana) 25 University of Ghana http://ugspace.ug.edu.gh 2.6.4 Oedemas Oedema is the severe form of the disease. The oedematous form is a diffuse, extensive, usually non-pitting swelling. The affected area has ill-defined margins, is firm and painless and involves part or all of a limb or other parts of the body. The colour of the skin may be changed over the affected area. The disease may be accompanied by low-grade fever. Figure 2.9: Oedema (Courtesy: Dr K. Asiedu, Ghana) Figure 2.10: Oedema (Courtesy: Dr K. Asiedu, Ghana) 26 University of Ghana http://ugspace.ug.edu.gh 2.6.5 Ulcers All the above forms may progress to ulcers after a variable time (as short as 4 weeks). Some of the largest ulcers follow from the oedematous form. Oedema may also develop around an already formed ulcer, leading to rapid extension. When fully developed, Buruli ulcer is a painless, deep ulcer extending into the subcutaneous fatty tissue. It has undermined edges where the overlying skin may be necrotic. The floor of the ulcer may have a white, cotton wool-like appearance due to necrotic slough. Untreated ulcers are painless, unless there is secondary bacterial infection. When there is more than one ulcer and the ulcers are close together, they often communicate beneath normal looking skin and could extend over a considerable distance (WHO, 2012). Figure 2.11: Large Ulcer (Courtesy: Dr A. Tiendrebéogo, Nigeria) 27 University of Ghana http://ugspace.ug.edu.gh Figure 2.12: Large Ulcer (Courtesy: Professor H. Assé, Côte d'Ivoire) 2.7 Categories of M. ulcerans disease In addition to the standard classification of the disease into non-ulcerative and ulcerative forms, there was the introduction of an additional classification of Mycobacterium ulcerans disease by WHO with the institution of antibiotic treatment, based on lesion size (WHO, 2008). The three categories of lesion are: Category I: a single lesion < 5 cm in diameter. Most category I lesions heal completely with antibiotic treatment. Category II: a single lesion measuring 5 – 15 cm in diameter. Some category II lesions heal completely with antibiotic treatment. Category III: a single lesion > 15 cm in diameter, multiple lesions, lesion(s) at a critical site (eye, breast, genitalia) and osteomyelitis. Category III ulcers are usually managed, in addition to antibiotics, by surgery (debridement and skin grafting) to achieve an acceptable rate of healing, 28 University of Ghana http://ugspace.ug.edu.gh but the optimal timing is not yet known. Multiple small lesions and lesions at critical sites may heal with antibiotics alone, and careful consideration should be given to avoid surgery. 2.8 Sampling techniques for laboratory diagnosis of M. ulcerans disease Sampling is very important in the diagnosis of BU in endemic areas. This was supported by a study by Siegmund et al. in 2007 when they found a great variation in positivity rates of bacteriological tests between BU treatment centres. They noticed that the quality of the clinical diagnosis and of sampling specimens differed between centres, and hence strongly influence positivity ratio. Siegmund et al. therefore recommended the development of training sessions on the different sampling techniques for the confirmation of BU: tissue or swab specimens from ulcerative lesions and FNA from non-ulcerative lesions (Siegmund et al., 2007). Three techniques are used to collect specimens: biopsy (punch or surgical), swabs and fine- needle aspirations (FNAs) and these are all suitable diagnostic samples (Eddyani et al., 2008; Etuaful et al., 2005; Warden et al., 2003). 2.8.1 Biopsy (punch or surgical) Before the use of antibiotics in the treatment of BU, surgery was the mainstay of management of M. ulcerans disease. However, surgically removed tissues for microbiological diagnosis of Buruli ulcer is becoming unavailable in recent times as a result of the decreasing importance of surgery as a therapeutic intervention. Surgery therefore, has been replaced by tissue 29 University of Ghana http://ugspace.ug.edu.gh obtained from punch biopsy for the confirmation of the clinical diagnosis of BU (Phillips et al., 2005). Punch biopsy or surgical biopsy may be used under the following circumstances or when the diagnosis is in the direct interest of the patient; for example, when swabs and fine-needle aspiration have been tried or abandoned. Surgical biopsy may be preferable when larger diagnostic sample specimens are required for histopathological analyses. However, the technique is invasive but with the issuance of guidelines by WHO in 2004 for the use of streptomycin (15 mg/kg of body weight) and rifampin (10 mg/kg) for 8 weeks, there has been a compelling need for a cheap and minimally invasive way of obtaining clinical samples, especially for pre-ulcerative lesions of Buruli ulcer, in order to reach a treatment decision (Phillips et al., 2009b). Due to the invasive character of this sample collection method, a consensus has been reached by stakeholder groups that in the interest of the patient, FNA and swabbing can replace punch biopsies for non-ulcerative lesions and ulcerative lesions with undermined edges respectively. 2.8.2 Swabs For ulcerated lesions, swab samples can be collected by swabbing the surface beneath the entire undermined edges of the ulcerative lesion. Physicians or experienced health workers can perform this technique. In general, most patients report to the clinics with ulcers so this technique is widely applicable in every setting. However, every effort should be made to minimize pain and bleeding, and proper training provided to health workers to perform this technique. 30 University of Ghana http://ugspace.ug.edu.gh Swabs have been demonstrated to be less traumatic for the patients as compared to the more invasive punch biopsy technique. For ulcerative lesions, available data suggest that, swabs are clearly superior to tissue samples (Cassisa et al., 2010; Herbinger et al., 2010) and that there are no significant differences in sensitivities between the use of FNA samples and that of punch biopsy specimens (Eddyani et al., 2009; Phillips et al., 2009b). Furthermore, the study by Herbinger et al. among 73 ulcerative cases recorded sensitivities of 75.0% for swabs, 55.6% for FNA samples, 66.2% for punch biopsy specimens and 30.0% for surgically excised tissue respectively using PCR. The sensitivities of microscopy were 46.4% for swabs, 22.2% for FNA samples, 37.5% for punch biopsy specimens, and 20.0% for surgically excised tissue (Herbinger et al., 2010). This indicates that the sensitivities for both tests were better in swab samples compared to the other sampling techniques. 2.8.3 Fine needle aspiration (FNA) Fine-needle aspiration (FNA) is mainly used to obtain samples from clinically-diagnosed non-ulcerative lesions (nodule, plaque and oedema). This technique is necessary in up to 30% of patients depending on the setting and is simple enough to be applied more widely in the field. Only physicians or experienced health workers should perform this technique; ongoing training and regular supervision should be provided to health workers to improve their skills. Recently, WHO recommended fine-needle aspiration (FNA) as a minimally invasive method for non-ulcerative lesions as well as for ulcerative lesions where scarring of edges prevents collection of swab samples (Warden et al., 2003). It may also be used in some ulcerative lesions where it is difficult to take swabs because of healing edges. For non-ulcerated lesions, samples obtained by FNA have now replaced punch biopsies, as these are less-invasive and less traumatic 31 University of Ghana http://ugspace.ug.edu.gh for the patient. FNA could be valuable for the follow-up of the infection, during and after treatment with anti-mycobacterial drugs. Compared to punch biopsy specimens, FNAs include less risk of ulceration or the possibility of subsequent reactivation of healing lesions since it is a minimally invasive procedure. Very recently, Phillips et al. also reported the use of FNA for the diagnosis of BU (Phillips et al., 2009b). Results by Eddyani et al. show that the minimally invasive FNA technique offers enough sensitivity to be used for the diagnosis of BU in non-ulcerative lesions (Eddyani et al., 2009). Summarily, FNA is a simple, fast, accurate, painless, and inexpensive method of sampling which may be used for diagnosing M. ulcerans infection by PCR, particularly in patients presenting early-stage non-ulcerative lesions (Cassisa et al., 2010). However, extreme care should be exercised when performing fine-needle aspiration around the head and neck area especially around the eyes and the genitalia. Where necessary, an expert clinician should perform this technique in order to minimize any unintended damage to important organs or structures. 2.9 Laboratory diagnosis of BU Recently, major advances have been made in the treatment of the BU disease with antibiotics (Etuaful et al., 2005; Wansbrough-Jones and Phillips, 2006). The introduction of antibiotic therapy as first-line treatment of Buruli ulcer highlights the importance of laboratory confirmation of diagnosis. There are various tools used in BU diagnosis. Currently available diagnostic laboratory tests for Buruli ulcer disease include the following methods: (1) direct smear examination with Ziehl-Neelsen (ZN) staining to detect the presence of acid-fast bacilli (AFB); (2) culture on Löwenstein-Jensen medium at 32 ºC; (3) histopathology; (4) polymerase chain 32 University of Ghana http://ugspace.ug.edu.gh reaction (PCR) and more recently (5) Loop mediated isothermal amplification (LAMP) and (6) flourescent thin layer chromatography (f-TLC). (1) Direct smear examination with Ziehl-Neelsen (ZN) staining to detect the presence of acid-fast bacilli (AFB): Swab samples from undermined edges of an ulcer, biopsy tissue obtained surgically or punch biopsies smears are prepared for microscopic examination from decontaminated material (a slide) and stained with the Ziehl-Neelsen stain for the presence of acid- fast bacilli (AFB) (WHO, 2012). Mycobacterium ulcerans stains red (acid-fast bacilli, AFB) in the Ziehl-Neelsen staining procedure (Figure 2.13) (Portaels et al., 1997) but this method has low sensitivity; Eddyani et al. reported the sensitivity of direct smear examination of 64.6% (Eddyani et al., 2008), Cassisa et al. in 2010 report an even much lower sensitivity in smears in direct smear examination in FNA samples (9.9%) as compared to 50.7% in swab samples (Cassisa et al., 2010). Yeboah-Manu et al. compared the sensitivity of Ziehl-Neelsen staining with the gold standard IS2404 PCR, and the sensitivity and specificity of microscopy with 100 concentrated specimens were determined to be 58.4% and 95.7%, respectively (Yeboah-Manu et al., 2011). It may be a useful test tool in ulcerative stages (Werf et al., 2005) in which a smear can be done from a swab, fine-needle aspirates or a biopsy. It is the simplest diagnostic technique and it presents rapid results hence Ziehl-Neelsen staining is the only method that could be easily implemented in the field. It is therefore mostly presented to endemic communities in sub-Saharan Africa. Direct smear examination with Ziehl-Neelsen staining offers in addition, the benefit of being performed easily at the local level, with less expensive materials and equipment. This is confirmed by a report by Yeboah-Manu et al. in which the materials needed to perform two tests (ZN smear microscopy and the gold standard IS2404 PCR) were compared. By combining direct smear microscopy with PCR stepwise, the cost of analysis was reduced significantly to less than 33 University of Ghana http://ugspace.ug.edu.gh 50% of the cost if the samples were analyzed by PCR alone. Also, optimization from swab specimen and concentration of bacterial suspensions before smearing, led to an improvement of the detection rate of acid-fast bacilli by microscopy after Ziehl–Neelsen staining (Yeboah-Manu et al., 2011). However, the major disadvantages of the technique have to do with the need for trained personnel and external quality assurance as well as low sensitivity (WHO, 2007). Figure 2.13: Ziehl-Neelsen stained smear from a Buruli ulcer showing extracellular acid- fast bacilli. (Photo: Wayne Meyers) (2) Culture of M. ulcerans: M. ulcerans belongs to the large group of environmental mycobacteria. It is a slowly growing acid-fast and alcohol-fast microorganism that is best cultured in egg yolk-enriched Löwenstein-Jensen (L-J) medium at 32 °C (pH 5·4 – 7·4). Attempts to culture the microorganism from clinical specimens fail in over half of all cases. Standard techniques require much time for isolation and identification (Werf et al., 2005). This method is difficult, expensive, has a low sensitivity, and results are available only after 6 – 12 weeks). There is also the need for a sophisticated laboratory setup and highly trained personnel. 34 University of Ghana http://ugspace.ug.edu.gh (3) Histopathological examination: Histopathology has been described to be useful specially for demonstration of granuloma for specimens such as aspirates/biopsies from bone marrow, liver or lymph nodes (Katoch and Mohan Kumar, 2001). Histopathology has been reported with a sensitivity of about 90%, the results are fairly rapid (about 4 minutes) and it offers a useful approach in establishing differential diagnosis and monitoring unexpected response to treatment. The challenges of histology have to do with the necessity for a sophisticated laboratory and need for trained personnel. Histopathology is expensive to perform and invasive sampling procedures such as biopsies are required. (4) Polymerase chain reaction (PCR): PCR is currently the method of choice and hence the gold standard test for BU and targets the IS2404 and IS2606 insertion elements, which have multiple copies in the M. ulcerans genome. Standard and real-time polymerase chain reaction (PCR) techniques have been used to identify M. ulcerans, primarily by detecting two M. ulcerans insertion sequences (IS2404 and IS2606), in the environment in Australia and West Africa (Ross et al., 1997). This test allows for quick detection, and has high sensitivity and specificity for M. ulcerans infection (>90%). A modified PCR method targeting the insertion sequence IS2404 of M. ulcerans, had a sensitivity of 98 to 100% compared with the combination of microscopy (42%), culture (49%), and histology (82%) when applied to 4- mm punch biopsy samples (Phillips et al., 2009b). Specificity of the IS2404 target in patient samples is because other environmental mycobacterium will not be present in patient samples as they are in environmental samples (Portaels et al., 2001). Nevertheless, the targets of PCR, IS2404 and IS2606, may be present in other pathogenic mycobacteria, such as M. marinum; so clinical features or variable number of tandem repeat assays may discriminate M. ulcerans from other species (Stragier et al., 2007). PCR can be performed on a number of different 35 University of Ghana http://ugspace.ug.edu.gh samples, such as fine needle aspirates (FNAs) from pre-lesion nodules, swabs from ulcerous lesions and infected tissue. However, it is highly sensitive when applied to swabbed material from ulcers and to biopsies and FNAs of non-ulcerative lesions (Herbinger et al., 2009). The technique offers results within 24 hours (Wansbrough-Jones and Phillips, 2006) therefore it is the most useful technique for reaching a treatment decision provided that it is done in a laboratory with high standards to avoid false-positive results (Phillips et al., 2009b). This technology however, requires specialist equipment, training and infrastructure that are only available in tertiary laboratories. Other technical difficulties presented by the method despite its high sensitivity and rapid analysis have to do with cold chain requirement, stable electric supply and qualified laboratory staff. The issue of cold chain could be addressed or overcome by the recent development of a dry reagent-based PCR (Siegmund et al., 2007). (5) Loop mediated isothermal amplification (LAMP): LAMP is a more recent diagnostic approach of M. ulcerans disease even though it has been used in the molecular diagnosis of a number of diseases, such as influenza, malaria, human African trypanosomiasis (sleeping sickness) and tuberculosis. LAMP is a novel nucleic acid amplification method for molecular detection and identification (Notomi et al., 2002). The principle of LAMP involves autocycling strand displacement DNA synthesis in the presence of Bst DNA polymerase with high strand displacement activity under isothermal conditions between 60-65°C within 60 minutes (Wang et al., 2008). LAMP is a highly specific assay due to the recognition of target DNA by 4 to 6 independent sequences with an amplification efficiency equivalent to that of PCR-based methods (Nagamine et al., 2002; Notomi et al., 2002; Poon et al., 2004). LAMP could potentially be used as a point-of-care test for M. ulcerans disease because the problem 36 University of Ghana http://ugspace.ug.edu.gh of carry over contamination is reduced (Jayawardena et al., 2007) and second the assay is less affected by a number of inhibitors of conventional PCR (Kaneko et al., 2007). (6) Detection of mycolactone using fluorescent Thin Layer Chromatography (f-TLC): Despite the tremendous advances in the development of various laboratory diagnostic tools as discussed above, BU is still mainly usually diagnosed on the basis of clinical symptoms. This is because of the numerous challenges encountered ranging from dedicated facilities and specialized equipment as required for the identification of M. ulcerans by means of culture or PCR, limited sensitivity of smear microscopy and the very demanding and time-consuming nature of both histopathology examination and culture of M. ulcerans in which results are available only after 6 – 12 weeks. There is therefore an urgent need for the development of a cost and time effective method, early point-of-care tool which is ideally simple enough for field-use in remote areas, to detect M. ulcerans infection to ensure accurate diagnosis in order to administer a planned antibacterial therapy to Buruli ulcer patients. Hence, it has become necessary to establish an alternative diagnostic approach with the need for better biomarkers that allows for the early identification of patients in order to control the emergence of BU (Phillips et al., 2009a). Interestingly, M. ulcerans presents a distinctive feature in its ability to produce a macrocyclic polyketide called mycolactone A/B which appears to be biosynthetically restricted to M. ulcerans and homogeneously distributed within the infected tissue (Hong et al., 2008a; Sarfo et al., 2010b). Daniel et al. further evidenced strongly that mycolactone A/B production is not common among mycobacteria and suggest that mycolactone may be unique to M. ulcerans (Daniel et al., 2004). Mycolactone plays a critical role in bacterial pathogenicity, as shown by the necrotic lesions induced by intradermal injection of the purified molecule (George et al., 37 University of Ghana http://ugspace.ug.edu.gh 1999). In a murine model, it has recently been demonstrated that the decline in tissue mycolactone concentration during antibiotic therapy tracked closely with reduction in colony forming units of M. ulcerans and with resolution of footpad swelling, highlighting the close association between mycolactone and pathogenesis of M. ulcerans disease (Sarfo et al., 2013). M. ulcerans bacilli remain essentially localized in peripheral subcutaneous tissues, but there is evidence from animal studies that mycolactone diffuses from infectious foci into the blood, where it accumulates in mononuclear cell subsets (Hong et al., 2008a). There is also evidence to the effect that mycolactone diffuses far beyond its infection site (Guarner et al., 2003). Based on the knowledge of the unique features of mycolactone, it is important particularly to make mycolactone, an attractive biomarker for the detection of M. ulcerans and also for monitoring clinical response to antibiotic treatment for M. ulcerans disease (Sarfo et al., 2014). Fortunately, mycolactone A/B is known to behave well in thin-layer chromatography (TLC), and could potentially meet the need, except for the sensitivity of detection. This led to the improvement of the promising TLC method as a diagnostic tool by Spangenberg and Kishi which employs a naphthylboronate-assisted fluorogenic chemosensor that can detect as low as 2 ng of mycolactone A/B in a semi-quantitative manner (Spangenberg and Kishi, 2010a). In the fluorescent-TLC (f-TLC) method, the TLC plate is developed by immersion in a boronic acid acetone solution that binds to mycolactone and fluoresces on excitation by ultraviolet light. The method results in plates in which there is a marked reduction of background spots thereby enhancing sensitivity. Relying on the excitation/emission of the pentaenoate chromophore, this method allows us to detect all of the mycolactones originating from the human pathogen M. ulcerans, but not mycolactones from the fish pathogen M. marinum or frog pathogen M. liflandii. 38 University of Ghana http://ugspace.ug.edu.gh 2.10 Modes of treatment of M. ulcerans disease 2.10.1 Surgical treatment BU was traditionally managed by surgical excision with or without skin grafting to remove all infected tissue, including a margin of healthy tissue. This was regarded as the most effective treatment measure for BU cases. However, several factors hinder surgery as the treatment of choice. These include the following; rural areas in most endemic countries often lack adequate surgical facilities and expertise, and prolonged hospitalization stretches the limited bed capacity of health centres, further reducing the number of patients who can be admitted for treatment. In addition, the cost of surgical treatment is far beyond the means of those most severely affected (Asiedu and Etuaful, 1998; Grietens et al., 2008). There are also risks associated with surgery and problems with infections, including HIV. In addition, there are reports of increased recurrence between 6 and 17% of BU after surgery (Amofah et al., 1998; Debacker et al., 2005; Revill et al., 1973). In general, surgery is performed under general or regional anaesthesia. These and other factors call for the employment of other methods in the management of M. ulcerans disease with the aim of resolving some of the challenges posed by surgical excision as a treatment method for BU cases. 2.10.2 Chemotherapy of M. ulcerans disease Antimicrobial therapy was initially thought to be ineffective until experiments in the mouse footpad model demonstrated the efficacy of combinations composed of an aminoglycoside and rifampin (RIF) (Bentoucha et al., 2001; Dega et al., 2000; Lefrançois et al., 2007). Also, recent evidence suggest that rifampicin, amikacin and clarithromycin may promote healing in pre- ulcerative and early ulcerative lesions, but they are often not effective in extensive lesions (Portaels 39 University of Ghana http://ugspace.ug.edu.gh et al., 2000). In vitro studies demonstrated the susceptibility of M. ulcerans to several antimicrobials, including rifampin (RIF), clarithromycin (CLR), streptomycin (STR), amikacin (AMK), sparfloxacin (SPX), and clofazimine (Portaels et al., 2000; Revill et al., 1973; Thangaraj et al., 2000). Rifampicin (RIF) Clarithromycin (CLR) Streptomycin (STR) Amikacin (AMK) Figure 2.14: Molecular structures of some anti-mycobacterial drugs used in Chemotherapy of M. ulcerans disease with their stereochemistry 40 University of Ghana http://ugspace.ug.edu.gh Antibiotic therapy has been recommended by WHO as the treatment of choice for all forms of BU cases. With increasing BU incidence and limited surgical resources in Africa, supported by experimental and encouraging preliminary human data, (Etuaful et al., 2005), the WHO advocated a provisional antibiotic regimen for BU, comprising oral rifampin (10 mg/kg) plus intramuscular streptomycin (15 mg/kg), both given daily for 8 weeks under supervision (WHO, 2004). Amikacin (15 mg/kg) can be substituted for streptomycin, administered intramuscularly or intravenously. Important contraindications and side effects for these drugs are described elsewhere (WHO, 2004). However, anti-mycobacterial drugs are relatively effective during the pre-ulcerative stage of the disease, antibiotic treatment requires optimization, and surgical excision with skin grafting remains the only alternative for advanced lesions (Phillips et al., 2009a). Early detection and early antibiotic treatment are therefore essential for obtaining the best results and minimizing the disabilities associated with Buruli ulcer. Since the introduction of antibiotic treatment, recurrence rates of 0 – 2% have been reported and the requirement for surgical intervention has diminished (Chauty et al., 2007; Sarfo et al., 2010a). However, surgical treatment without antibiotics records recurrence rates varying from 16% to 28% (Kibadi et al., 2009); recurrences further inflate treatment costs and undermine patients’ confidence in conventional surgical treatment. Treatment with rifampin-clarithromycin or moxifloxacin-clarithromycin for 8 weeks also displayed promising bactericidal activity against Mycobacterium ulcerans in mice; none of the mice treated with rifampin-clarithromycin relapsed, whereas 59% of those treated with moxifloxacin-clarithromycin relapsed after treatment was stopped (Ji et al., 2008). Subsequent clinical experience has confirmed that 2 months of streptomycin (STR) and RIF is the treatment 41 University of Ghana http://ugspace.ug.edu.gh of choice for all forms of BU, although adjunctive surgery may be necessary to heal large ulcers (Chauty et al., 2007; Etuaful et al., 2005). Physiotherapy should be considered as an important adjunct in cases of surgery to prevent contractures (Asiedu et al., 2000a). 2.10.3 Consideration of an all oral regimen for M. ulcerans disease Significant progress has been made with the demonstration of the efficacy of rifampicin plus streptomycin (RIF + STR) chemotherapy (Etuaful et al., 2005). Its routine implementation has dramatically improved healing while reducing the frequency of relapses (Chauty et al., 2007) hence it is currently the recommended regimen for treating BU. However, it has significant disadvantages in that, streptomycin is an injectable drug and may cause ototoxicity and nephrotoxicity, and the 2-month duration of therapy poses an obstacle to treatment completion. Chan-Tompkins reported that vestibular toxicity occurs in 0.5% – 5% of patients and nephrotoxicity occurs in 5% – 10% in patients treated with streptomycin (Chan-Tompkins, 1995). Because of difficulties of providing daily RIF + STR treatment at the peripheral health centres, a simpler strategy is to modify the treatment regimen to an efficacious oral treatment in order to decentralize care at local level. One possible approach is to develop entirely oral regimens such as the combination of RIF with clarithromycin (Ji et al., 2008; Sarfo, 2014) and/or regimens capable of treating BU in 1 month or less, which does not require patients to be treated daily at the health centre. A recently published randomized controlled trial from Ghana showed no significant difference in the proportion of patients who achieved cure after receiving the World Health Organization (WHO) recommended 8-week course of RIF + STR chemotherapy, compared with the proportion who achieved cure after receiving a treatment consisting of 4 weeks of RIF + STR 42 University of Ghana http://ugspace.ug.edu.gh followed by 4 weeks of rifampicin + clarithromycin (RIF + CLR) (Nienhuis et al., 2010; Sarfo, 2014). Clarithromycin would therefore be a potential replacement for streptomycin since it could be more easily administered, better accepted by patients, and contribute to limiting the number of injections in the developing world. 2.10.4 Experimental treatments Other experimental treatments have been proposed, including heat treatment (Meyers et al., 1974b), hyperbaric oxygen (Krieg et al., 1979), medicinal clay, phenytoin powder (Adjei et al., 1998), and nitric oxide releaseing creams (Phillips et al., 2004). (a) Heat treatment: continuous local heating to 40 ºC (e.g. by circulating water jackets) promotes healing even without excision, but must be applied constantly for 4 – 6 weeks. In addition, it is believed heat treatment improves blood flow, antibiotic penetration and phagocytosis (Goutzamanis and Gilbert, 1995; Meyers et al., 1974b). However, this type of treatment is often not practicable in many endemic areas. Heat treatment was proposed long ago, and seems simple and affordable, but has not been shown to have a large beneficial effect; in fact, the first report made mention of several recurrences (Meyers et al., 1974b). (b) Hyperbaric oxygen therapy: M. ulcerans grows best at lower oxygen tension. Hyperbaric oxygen treatment alone has inhibited lesions in a murine model of M. ulcerans infection, but not in a patient in which it was used as an adjunct to rifampicin and heat treatment (Krieg et al., 1979; Meyers et al., 1974b). The case for hyperbaric oxygen treatment in M. ulcerans infection has its proponents but large-scale implementation in endemic regions is unlikely to be feasible. 43 University of Ghana http://ugspace.ug.edu.gh (c) Medicinal clay: Medicinal and therapeutic use of mineral products has impacted human health for thousands of years, and pure clay minerals, such as smectite and illite, are nanomaterials of geological origin (Haydel et al., 2008). Clay minerals can affect bacterial metabolism indirectly by altering the physicochemical properties of a specific environment or directly through surface interactions (Stotzky, 1986). French green clays have recently been shown to heal Buruli ulcer. Assessing the antibacterial properties of these clays could provide an inexpensive treatment for Buruli ulcer and other skin infections. Line Brunet de Courssou, a French humanitarian working in the Ivory Coast of Africa, observed the suffering of many tribal communities where mostly women and children were afflicted with Buruli ulcer. Growing up in France, she had used a local green clay on wounds and experienced rapid healing from application of a clay paste to bug bites, stings, and cuts. She imported French clay to the Ivory Coast in an attempt to treat the skin infections of the tribal communities, and she carefully documented (photographically) the healing effects of clay on patients suffering from Buruli ulcer (Brunet de Courrsou, 2002; Williams et al., 2004). There are two different clay minerals that have been demonstrated to have antibacterial properties: CsAg02, an iron rich smectite and illite clay mineral enriched with magnesium and potassium, and CsAr02, an iron-rich smectite and illite clay mineral enriched with calcium (Williams et al., 2004). One of the French green clays promoted bacterial growth, while the other substantially or completely killed bacteria. By trial and error, Brunet de Courssou developed a treatment protocol for Buruli ulcer. The stages of healing (Figure 2.15) show that after one day of treatment with one green clay (CsAr02), the therapeutic properties of the clay are demonstrated with the initiation of rapid, non-surgical debridement of the destroyed tissue. When the edges of the skin showed signs of damage (purplish 44 University of Ghana http://ugspace.ug.edu.gh blood vessels), the second green clay (CsAg02) was administered. Extended treatment with the CsAg02 clay resulted in continued tissue regeneration and wound healing. After several months of daily clay applications, the infection healed with soft, supple scarring and return of normal motor function (Brunet de Courrsou, 2002; Williams et al., 2004). Despite the observed therapeutic benefits of treating Buruli ulcer with the French clays, an approved treatment with clay depends on scientific validation of the observed effect and testing for potentially harmful side effects. Figure 2.15: A photographic example of the stages of healing by clay: destroyed tissue (a) is easily removed by one treatment with CsAr02 to expose raw muscle and bone (b). The progression of healing is shown (c, d) with daily treatments of CsAg02. After 3–4 months, the infection is healed with soft, supple scarring and a return of normal motor function (e). (d) Phenytoin powder: Phenytoin (diphenylhydantoin), a hydantoin derivative, has been used in the treatment of a wide range of medical conditions including epilepsy, muscle disorders and other related types of pain (Adjei et al., 1998). There is evidence that phenytoin has a beneficial role in wound repair (Smith et al., 1988). Its ability to reduce pain and promote healing suggests that it may be useful in treating chronic cutaneous ulcers, including trophic ulcers of lepromatous origin, venous stasis decubitus and diabetic ulcers (Modaghegh et al., 1988). Phenytoin powder appeared promising in accelerating the healing process of ulcerative Buruli ulcer lesions and decreasing the risk of hypertrophic scarring. In a small randomized controlled trial, ulcer surface reduction of more than 50% was observed more frequently in patients treated with phenytoin powder (72%) compared with placebo (35%), especially in young people (<30 years) and ulcers of less than 30 cm in diameter (Klutse et al., 2006). 45 University of Ghana http://ugspace.ug.edu.gh (e) Nitric oxide releasing creams: These creams have been studied in 37 people with a clinical diagnosis of Buruli ulcer, and a beneficial clinical response was shown (Phillips et al., 2004). The disadvantage of all such localized treatments is that the antimicrobial effect may be incomplete. Recurrent infections are problematic, especially in immuno-compromised hosts and in patients with disseminated disease, as well as in those who have developed M. ulcerans osteomyelitis, which is not uncommon in Benin (Portaels et al., 2004). As patients with lesions involving joints are prone to develop contractures, these individuals may benefit most from physiotherapy. 46 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3 Methodology Between August 2014 and April 2015, patients with suspected BU disease were routinely recruited from BU endemic districts of Ghana; Agogo, Tepa, Dunkwa, Amasaman and Obom Health Centres. 3.1 Sampling of clinical specimens Biological samples (swabs and fine needle aspirations) were collected and handled according to WHO guidelines (WHO, 2007). Patients were recruited from treatment centres in Amasaman Hospital and Obom Health Centre if they had a skin lesion suspected to be caused by M. ulcerans infection. After the patients had given their informed consent, the diagnosis was confirmed by obtaining two fine needle aspirations (FNAs) and two swab samples from non- ulcerative lesions or from the undermined edge of ulcers respectively. These were tested by PCR for the IS2404 insertion sequence of M. ulcerans (Phillips et al., 2009b, 2005) and also examined for the presence of mycolactone by f-TLC. FNA/Swab samples taken in the centres for the detection of mycolactone were stored immediately in 1.5 mL absolute ethanol in polypropylene microcentrifuge tube with a screw cap. Samples were immediately capped, wrapped in aluminium foil and transported to the laboratory for analysis. 47 University of Ghana http://ugspace.ug.edu.gh 3.2 Synthetic mycolactone A/B Synthetic mycolactone A/B was generously provided by Professor Yoshito Kishi, Harvard University, USA. 3.3 Confirming the presence of mycolactone in samples 3.3.1 f-TLC Analysis of Swab/FNA Samples The ethanol (EtOH) solvent in which the samples were stored was transferred using a disposable glass pipet (VWR Pasteur pipet) and filtered through another Pasteur pipette containing a cotton plug into a glass vial A (VWR 66011-041). The sample container was then rinsed with 1 mL ethyl acetate (EtOAc) and added to the vial labelled A through the cotton plug. The solvent in vial A was evaporated using a rotary evaporator to complete dryness under reduced pressure. 0.1 mL (1:1)-hexanes/ether solution was then added to vial A, the vial was rinsed, and only the liquid was transferred to a clean vial B (VWR 66011-020) using a micro-syringe (100 μL from VWR 60376-241, 1075N syringe). The micro-syringe was used in order to avoid the transfer of solid contaminating materials which were responsible for much of the fluorescent background impurities. All solids were then left on the walls of the vial. Vial B was then air-dried completely using Argon gas. 50 μL of hexanes/ether (1:1) solution was added to the air-dried vial B using a 50 μL micro-syringe. 10 μL and 5 μL of the sample solution was taken using 1-5 μL calibrated disposable pipets, Drummond (VWR 53432-604) and spotted onto a 3.3 × 6.6 cm fluorescent-dye free TLC plate (TLC Silica gel 60, EMD Millipore, purchased from VWR). The sample was then spotted alongside standardised mycolactone A/B. Sample spots were cautiously made as small as possible on the TLC plate and air was blown after each spot to dry the solvent quickly to avoid spreading. Also, 40 ng (4 μL)-standard mycolactone A/B solution was spotted. Note: [Standard 48 University of Ghana http://ugspace.ug.edu.gh mycolactone A/B solution for TLC is 10 ng/μL and this was made from 0.1 mg/mL ampule of synthetic mycolactone A/B (a gift of Professor Yoshito Kishi, Harvard University, USA). 0.1 mL of the ampule solution was taken into another Glass vial (VWR 66011-020) and 0.9 mL ethyl acetate (EtOAc) was added to give the required 10 ng/μL mycolactone A/B standard solution]. A co-spot was also made by spotting 10 μL human sample solution plus 40 ng (4 μL) standard mycolactone A/B solution at the same spot on the TLC plate. The plates were eluted using TLC developing chamber (VWR 80066-466) with CHCl3-Hexanes-MeOH (5:4:1 vol/vol/vol) solution to a solvent front, 0.2 cm below the top of the TLC plate. (Note: Fresh elution solution was made every day before use), the plate was then air-dried, and quickly dipped into a 0.1 M 2- naphthylboronic acid solution stain once, then the plate was heated at 100 °C using heating plate for 60 seconds, before wiping the glass back with acetone on a paper towel. The plate was then place on a UV lamp with a 365 nm filter (365nm UV LED 40 W). Fluorescent spot intensity at Rf ̴ 0.23 from the patient sample was compared to that of the standard mycolactone A/B spots in order to confirm the presence and/or absence and to estimate the amount of mycolactone A/B in the sample. TLC photos were taken in the dark (Cannon EOS, M mode, ISO 300, 4 second) and subjected to Photoshop resolution enhancement (Adobe Photoshop CS 6). TLC-pictures thus obtained serve for recording purposes, although the sensitivity with eye-analysis is better [Note: 0.1 M of the 2-naphthylboronic acid solution in acetone was prepared by weighing 1.72 gram of the 2-naphthylboronic acid and dissolving in 100 mL acetone, kept in a glass jar]. 49 University of Ghana http://ugspace.ug.edu.gh Figure 3.1: Experimental protocol. Step a: an acetone solution of mycolactone A/B (10 ng) is applied to a dye-free silica gel C60 TLC plate and eluted with 5/4/1 CHCl3–Hexanes–MeOH as mobile phase; mycolactone A/B is not detected with conventional methods (photograph I). Step b: the eluted TLC plate is briefly warmed on a hot plate to evaporate the solvents and, while warm, quickly immersed into a 0.1 M acetone solution of 2-naphthylboronic acid. Step c: the TLC plate is heated to ~100 ºC for 60 s. Step d: the TLC plate is irradiated with a UV lamp (8 W) with a 365 nm filter, after its reverse side has been cleaned with acetone, to detect mycolactone A/B as a green-yellow fluorescent spot. 3.3.2 f-TLC Analysis of spiked urine Samples 50 ng of mycolactone in Ethanol (5 μL) was added to 5 mL of urine, this was mixed well with shaking. The urine and mycolactone mixture was then passed through a 18C column, the column was washed with 0.3 mL water. The combined urine and water was disposed of. Subsequently, 18C column was washed with 0.2 mL ethanol in order to dissolve the mycolactone remaining in the column. It was then collected in a new clean vial. The ethanol was then evaporated 50 University of Ghana http://ugspace.ug.edu.gh by gently air blowing with Argon gas. After evaporating the sample with ethanol to complete dryness, 10 μL of ethyl acetate (EtOAc) was introduced into the vial, and this was spotted on the TLC similarly as with the FNA and swab samples. 3.4 M. ulcerans infected BALB/c mice studies 3.4.1 M. ulcerans inoculum The M. ulcerans inoculum was prepared from subcultures of primary isolates from a patient in Ghana. The M. ulcerans subcultures were examined by the Ziehl-Neelson’s acid-fast bacilli (AFB) staining method and confirmed by gold standard PCR. Suspensions of M. ulcerans in phosphate-buffered saline (PBS) were prepared and quantified by the McFarland nephelometric standards. Ten bacterial suspensions equivalent to the 10 McFarland standards were used as inocula. 3.4.2 Experimental animals Six-week old specific pathogen-free (SPF) BALB/c mice produced at the Noguchi Memorial Institute for Medical Research (NMIMR) were used for the study. The mice were divided into groups, and each group was inoculated in the right footpad with 30 µL of the inoculum equivalent to one McFarland standard. The animals were observed daily for post inoculation changes until they die or euthanized at the appearance of lesions. The hind limbs were measured weekly to assess the severity of oedematous lesions. Control mice were not injected. Mice showed post inoculation changes such as localized erythema, foot/thigh oedema, foot necrosis, fluid 51 University of Ghana http://ugspace.ug.edu.gh exudation, scab formation and ulcers. Cultures of all inoculated limbs were positive for M. ulcerans. 3.4.3 Metabolic cage The metabolic cage consists of an upper chamber made of transparent, gnaw-proof polycarbonate with a collection funnel, a small plastic shield, a lower chamber, collecting tubes, and a simple stand (Kurien et al., 2004). A feed chamber is located outside the cage and the size of the feed chamber is designed to prevent mice to sleep or nest inside. The feed chamber contains a drawer that is easy to pull out to simplify filling with minimum disturbance of the animal. This drawer is usually designed to hold feed pellets to prevent the animal from dragging feed into the cage. The device is designed to avoid contamination of the urine and effectively separate urine and faeces into collection tubes outside of the cage (Kurien et al., 2004). The construction of the feeding chamber and drawer prevents urine from getting contaminated with feed. The water bottle is calibrated, located outside the cage and is made of polycarbonate. Under the water bottle there is a calibrated spillage collecting tube which prevents water from entering the cage and contaminating the urine. The spillage collecting tube is calibrated and enables the investigator to calculate the actual water intake of the animals. The cage has grid floor and the urine flows down in the middle of a funnel under the cage to the urine collection tube which is graded in cubic centimetres. Faeces roll down on the side of the funnel into a specific faeces tube that can be removed from outside the cage to prevent disturbance of the animal. All parts of the device can be disassembled and cleaned with warm soapy water or detergent solution and sterilized to prevent any contamination (Figure 3.2). The time the animal is placed in the metabolic cage is normally 24 hours or longer. 52 University of Ghana http://ugspace.ug.edu.gh Figure 3.2: BALB/c mouse placed in a metabolism cage at the NMIMR. Photograph by Gideon Akolgo. 3.4.4 Urine sample collection Metabolism cage housed BU infected mice were fed with food pellets with identical nutrient composition and caloric density to the cage with the control group’s pellets. Food pellets and water were provided ad libitum throughout the entire experiment. Urine from the metabolism cages were collected and stored in the fridge prior to analysis. Urine volumes ranged from 5 to 10 ml per 24 h for three mice that were kept in each cage overnight. However, as the infection progresses, urine volumes reduced because of the inability of animals to eat and drink properly. When needed, the separation funnels and grating of the metabolism cages were changed. The feed in the dispensers was enough to last for 12 hours or possibly more, but since running low on available feed could be a potential stressor, the feed was refilled more frequently. 53 University of Ghana http://ugspace.ug.edu.gh 3.4.5 f-TLC analysis of urine samples from M. ulcerans infected mice The urine samples which were collected from the M. ulcerans infected mice in the metabolic cages as described above were monitored weekly for the mycolactone A/B content using the f-TLC technique as described for the spiked urine sample earlier. 3.5 Polymerase chain reaction (PCR) In order to compare the specificity and sensitivity of the f-TLC mycolactone technique, all samples were subjected to the gold standard conventional PCR. This was however conducted independently at the Noguchi Memorial Institute for Medical Research by a different technician in order to avoid bias. 3.6 Identification of mycolactone A/B by physical properties on chromatography and mass spectrometry 3.6.1 Characteristic behaviour of mycolactone A/B in Thin Layer Chromatography (TLC) Standard Synthetic mycolactone A/B which is a positive control was spotted onto a 3.3 × 6.6 cm fluorescent-dye free TLC plate (TLC Silica gel 60, EMD Millipore, purchased from VWR) using 1-5 μL calibrated disposable pipets, Drummond (VWR 53432-604). The plate was then eluted with an elution mixture of CHCl3-Hexanes-MeOH (5:4:1 vol/vol/vol) solution to a solvent front, 0.2 cm below the top of the TLC plate and the retention factor (Rf) was measured. 54 University of Ghana http://ugspace.ug.edu.gh 3.6.2 Mass spectrometry (MS) confirmation of mycolactone A/B structure The aim of mass spectrometric analysis of synthetic mycolactone A/B was to determine the fragmentation patterns of its structure. Mass measurement was performed using a micrOTOF- Q II mass spectrometer (BrukerDaltonik, Germany). The data analysis was done using Bruker Compass DataAnalysis 4.0 software (BrukerDaltonik, Germany). High-resolution mass spectrometric (HRMS) analysis was performed using a Bruker MicroTOF-QII spectrometer with an electrospray ionization (ESI) source and run in positive mode with a capillary potential of 4.5 kV. The scan range was 100–1200 m/z. High purity nitrogen was used as the nebulizing (5.8 psi) and drying gas (7 L min–1, 200 ºC). Argon was applied as the collision gas, and the collision energy was set to 10 eV to provide some structural information and to focus ion flux. An internal calibration was performed using a sodium formate solution (1.0 mg mL–1) before and after each acquisition. The sample was introduced into the spectrometer by direct infusion at a constant flux of 3.0 mL min–1. The micrOTOF-QII MS parameters were optimised to the following: both funnel 1 and 2 were 200.0 Vpp (volt peak to peak); hexapole Rf was 100.0 Vpp; quadrupole ion energy was 7.0 eV. Mass spectra were acquired with an MS1 spectral rate of 2 Hz, an MS2 rate of 3 Hz, and the collision Rf was stepped from 200 Vpp to 600 Vpp (increments of 100 V) during each MS2 event in order to maximize observed fragment ions. MS/MS- experiments were accomplished in Auto-MS. The isolation width was set to 5 m/z and the collision energy to 35 eV. Characteristic ions in mycolactone A/B MicroTOF-QII MS ESI+ spectra were m/z 765.5 [M+Na]+, 429.3 for the core lactone ring and 359.2 for the polyketide side chains. 55 University of Ghana http://ugspace.ug.edu.gh 3.7 Estimating the detection limit Also, a measure of the detection limit of mycolactone was monitored using the TLC technique. A 6 × 6.6 cm fluorescent-dye free TLC plate (TLC Silica gel 60, EMD Millipore, purchased from VWR) was marked with 7 spots and serial dilutions of purified synthetic mycolactone A/B from 1 µL to 10 µL corresponding to 10 ng to 100 ng respectively were spotted carefully using 1-5 μL calibrated disposable pipets onto TLC plates with air drying to determine the detection limit for the system. It was then eluted with a CHCl3-Hexanes-MeOH (5:4:1 vol/vol/vol) solution to a solvent front, 0.2 cm below the top of the TLC plate. The eluted TLC plate was briefly warmed on a hot plate to evaporate the solvents, and while warm, it was quickly immersed into a 0.1 M acetone solution of the 2-naphthylboronic acid. The TLC plate was then heated to 100 °C for ~ 60 seconds. The glass side of the TLC plate was wiped clean using acetone on a paper towel, and then the TLC plate was irradiated with an 8 W UV lamp 365/302 nm with a 365 nm filter. Mycolactone A/B is detected as a green-yellow fluorescent spot. A photograph was taken and subjected to Photoshop CS 6 enhancement (this can be seen in Figure 4.2). 3.8 Enhancing the sensitivity 3.8.1 UV Absorption spectra The individual absorbance values of mycolactone A/B, 2-naphthylboronic acid and 4- nitrobenzaldehyde were recorded in MeOH on a Shimadzu UV mini-1240 spectrophotometer from 190 nm to 460 nm wavelength. The measured absorbance values were plotted against the wavelength, λ (nm) using Microsoft Excel to obtain the UV spectra of these compounds. 56 University of Ghana http://ugspace.ug.edu.gh 3.9 Statistical analysis GraphPad Prism 5 and Excel software tools were used to analyse the data obtained. Fisher’s measures of diagnostic tests such as Sensitivity, Specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) with their respective 95% Confidence Intervals (CI) of the f- TLC method were measured against the gold standard PCR test using the GraphPad Prism 5 software. 57 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4 Results and discussion 4.1 Introduction Early diagnosis of Buruli ulcer (BU) facilitates rapid and appropriate antibiotic treatment of patients in BU endemic communities. The diagnosis of BU relies primarily on conventional PCR method. This is because PCR targeting the insertion sequence IS2404 is highly sensitive. This has been demonstrated by various researches, for instance, regarding sensitivities of PCR, results from three recent studies, one from Ghana (Phillips et al., 2009b) and two from Benin (Cassisa et al., 2010; Eddyani et al., 2009) respectively report sensitivities around 90%. A comparative study of the sensitivity of different diagnostic methods for the laboratory diagnosis of Buruli ulcer disease by Herbinger et al.(2009) also reported an overall sensitivity of 85% in PCR, far higher than that reported for the other techniques (Herbinger et al., 2009). Owing to the high sensitivities of PCR, it has ultimately become a rapid tool for the diagnosis of BU and therefore provides sufficient evidence to commence anti-mycobacterial treatment. It is regarded as the gold standard and the recommended technique for laboratory confirmation of Mycobacterium ulcerans disease. The high sensitivity of PCR notwithstanding, the method is expensive and limited to well-resourced laboratories. Owing to these challenges, a study was instituted to find an alternative and the f-TLC is one such method (Converse et al., 2014; Spangenberg and Kishi, 2010a). The f-TLC method is employed in this study on clinical samples. 58 University of Ghana http://ugspace.ug.edu.gh Table 4.1 shows study participants with their clinical information and diagnostic results of 50 patients suspected to be BU cases in the Agogo, Tepa, Dunkwa ( Ashanti region - Ghana), and Amasaman and Obom Health Centres (Greater Accra region - Ghana). The clinical and epidemiological information of the suspected BU patients were derived from laboratory data entry forms (Appendix A). FNA and swab samples from the 50 suspected BU cases were subjected to conventional IS2404 PCR and fluorescent TLC (f-TLC) analyses. Table 4.1: Study participants, clinical information, and diagnostic results Code Results No. Health facility Age (yrs) Sex (M/F) Type of sample PCR f-TLC 001 Agogo 11 M Swab Pos Neg 002 Agogo 6 M FNA Pos Neg 003 Tepa 14 M Swab Pos Pos 004 Dunkwa 13 M Swab Pos Pos 005 Agogo 15 F Swab Pos Neg 006 Agogo 11 F Swab Pos Pos 007 Agogo 9 F FNA Pos Pos 008 Agogo 12 F FNA Pos Pos 009 Agogo 11 M Swab Pos Pos 010 Agogo 42 F Swab Pos Pos 011 Agogo 3 F FNA Pos Pos 012 Tepa 52 F Swab Pos Pos 013 Agogo 8 M Swab Pos Neg 014 Agogo 8 F FNA Pos Pos 015 Agogo 35 M FNA Pos Pos 016 Agogo 22 F FNA Neg Neg 017 Agogo 7 M FNA Neg Neg 018 Agogo 41 F FNA Neg Neg 019 Agogo 71 F FNA Neg Neg 020 Amasaman 41 M Swab Neg Pos 021 Obom 21 F Swab Pos Pos 022 Amasaman 6 M Swab Pos Pos 023 Amasaman 11 M FNA Pos Pos 024 Obom 62 F Swab Neg Pos 025 Amasaman 16 M Swab Neg Neg 026 Amasaman 9 M Swab Neg Pos 59 University of Ghana http://ugspace.ug.edu.gh 027 Obom 50 F Swab Neg Pos 028 Obom 55 F Swab Neg Neg 029 Amasaman 10 F Swab Pos Pos 030 Amasaman 20 F FNA Neg Neg 031 Obom 4 M Swab Neg Neg 032 Obom 10 M Swab Neg Neg 033 Obom 10 M Swab Neg Neg 034 Obom 8 F FNA Neg Neg 035 Obom 14 F Swab Neg Pos 036 Obom 14 M Swab Neg Pos 037 Obom 13 M Swab Neg Neg 038 Amasaman 40 F Swab Neg Pos 039 Amasaman 5 F Swab Pos Pos 040 Amasaman 39 F Swab Pos Pos 041 Amasaman 38 M Swab Pos Neg 042 Obom 7 F Swab Neg Pos 043 Obom 4 F FNA Neg Neg 044 Obom 10 M FNA Neg Pos 045 Amasaman 52 F Swab Pos Pos 046 Amasaman 37 M Swab Neg Neg 047 Amasaman 16 F Swab Pos Pos 048 Amasaman 35 F Swab Pos Pos 049 Amasaman 50 F Swab Pos Neg 050 Amasaman 25 M FNA Neg Neg Neg – negative test result; Pos – positive test result; M – male; F – female; FNA – fine needle aspiration; PCR – Polymerase Chain Reaction; f-TLC – Fluorescent Thin Layer Chromatography Out of these 50 patients who reported to the various clinics with BU suspected cases, 22 (44%) were males and 28 (56%) were females with an age range of 4 – 71 years, mean of 22.46 years and the median of 14 years. 30 (60%) of them were in the age group 0–19 years (Appendix B and Figure 4.1). This supports previous findings in West Africa studies that the disease affects predominantly children between the ages of 5 to 15 years with an almost equal gender distribution even though it involves all age groups (Debacker et al., 2004). Prevalence rates can be as high as 22%, as has been recorded in endemic communities in Ghana (Asiedu et al., 2000b). 60 University of Ghana http://ugspace.ug.edu.gh Age and Sex distribution of 50 suspected case patients 12 10 8 6 Male 4 Female 2 0 0-9 10-19 20-29 30-39 40-49 50-59 60 → Age group (yrs) Figure 4.1: Age and sex distribution of 50 suspected case patients who presented to the study hospitals between August 2014 and April 2015. 4.2 Thin layer chromatography (TLC) Mycolactone is a light yellow UV-active lipid with a retention factor (Rf) of 0.23 in chloroform- methanol-water (90:10:1, vol/vol/vol) (George et al., 1999). The retention factor (Rf) value of 0.23 was also obtained using purified synthetic mycolactone A/B with fluorescent thin layer chromatography (f-TLC) technique; a modification of the TLC method previously reported (George et al., 1999). In the new method, the elution solvent was a mixture of chloroform-hexane- methanol (5: 4: 1, vol/vol/vol) (Converse et al., 2014). Figure 4.2 shows the profile of the sensitivity of the f-TLC method for the synthetic mycolactone. It was observed that the f-TLC method is highly sensitive and the mycolactone could be detected to a limit of 20 ng. Also, TLC 61 No. of case patients University of Ghana http://ugspace.ug.edu.gh photos of samples analyzed using the f-TLC method were taken and subjected to resolution enhancement (Adobe Photoshop CS 6) as shown in Appendices D & E. Figure 4.2: 7 spots of synthetic mycolactone A/B in serial amounts from 10 ng to 100 ng respectively were spotted and examined under UV light with a 365 nm filter. The detection limit on the TLC was 20 ng (corresponding to 2 µL of mycolactone A/B). 4.3 Laboratory confirmed BU cases of clinical samples The WHO has adopted the PCR as the gold standard for the diagnosis of BU and all other diagnostic tools available are compared to it. The overall positive case confirmation rate by PCR was 52.0% (26/50), and 58.0% (29/50) were confirmed using the f-TLC criteria (Table 4.2). Only FNA and swabs were used in the study with 16 out of the 50 representing 32% sampled by FNA while 34 (68%) were swabs. Of the 26 PCR positively confirmed BU cases, 19 (73.1%) were 62 University of Ghana http://ugspace.ug.edu.gh swab samples while 7 (26.9%) were FNAs. From the total of 29 samples that tested positive by the f-TLC method, 22 (75.9%) were swab samples whilst 7 (24.1%) were FNA samples. Similarly, of the 24 samples that tested negative for PCR, 15 (62.5%) were swabs and 9 (37.5 %) were FNAs. 12 (57.1%) of the negatively tested samples in the f-TLC analysis were swabs whilst 9 (42.9%) were FNAs (Figure 4.3). Table 4.2: Results of f-TLC test for mycolactone and PCR from swabs and fine-needle aspirations (FNAs) No. of specimens with indicated results PCR Type of specimen/sampling method Positive Negative Total Swab (n = 34) 19 15 34 FNA (n = 16) 7 9 16 Total (Swab + FNA) (n=50) 26 24 50 No. of specimens with indicated results f-TLC Type of specimen/sampling method Positive Negative Total Swab (n = 34) 22 12 34 FNA (n = 16) 7 9 16 Total (Swab + FNA) (n=50) 29 21 50 f-TLC – fluorescent Thin Layer Chromatography; PCR – Polymerase Chain Reaction 63 University of Ghana http://ugspace.ug.edu.gh f-TLC and PCR from swab and FNA samples 25 22 20 19 Swab FNA 15 15 12 10 9 9 7 7 5 0 f-TLC Positive f-TLC Negative PCR Positive PCR Negative f-TLC vrs PCR Results Figure 4.3: Comparison of results of f-TLC and PCR from swab and FNA samples Out of the 26 IS2404 PCR confirmed positive suspected BU cases; 20 of them, representing 76.9% were also confirmed by the f-TLC technique. These were the true positives (TP), and out of 24 PCR negative results, 15 of them were also confirmed by f-TLC giving a 62.5% true negative (TN) value. However, 6 (23.1%) of PCR confirmed positives were f-TLC negative and 9 (37.5%) of PCR negative samples gave f-TLC positive results representing false negative (FN) and false positive (FP) results respectively. Also, FNAs gave a true positive (TP) value of 85.7% (6/7), false positive (FP) value of 11.1% (1/9), false negative value of 14.3% (1/7) and true negative value of 88.9% (8/9). For swab samples, values of 73.7% (14/19) true positive (TP), 53.3% (8/15) false positive (FP), 26.3% (5/19) false negative (FN) and 46.7% (7/15) true negative (TN) were obtained (Table 4.3). 64 No. of specimens with indicated reults University of Ghana http://ugspace.ug.edu.gh Table 4.3: Comparison of results of f-TLC versus PCR in FNA and Swab samples Gold Standard PCR method FNA samples Clinical trial f-TLC result PCR Positive PCR Negative Total f-TLC Positive 6 1 7 f-TLC Negative 1 8 9 Total 7 9 16 Swab samples PCR Positive PCR Negative Total f-TLC Positive 14 8 22 f-TLC Negative 5 7 12 Total 19 15 34 FNA + Swab samples PCR Positive PCR Negative Total f-TLC Positive 20 9 29 f-TLC Negative 6 15 21 Total 26 24 50 Mycolactone A/B was not detected by f-TLC methodology in six (6) samples that tested positive in the PCR method representing false negatives. Several reasons could account for this observation; first, the organisms might be producing lower amount of the mycolactone below the detection limit (below 20 ng) of the f-TLC method. Secondly, it could also be attributed to non- representative sampling from suspected lesions. For instance, if the FNAs and swabs were taken from different sites for the two analyses, it is likely that the area of the lesion sampled was not representative (Sarfo et al., 2010b). 65 University of Ghana http://ugspace.ug.edu.gh On the other hand, there are nine (9) false positive cases. Whereas mycolactone A/B appears to be biosynthetically restricted to M. ulcerans and homogeneously distributed within the infected tissue (Hong et al., 2008a; Sarfo et al., 2010b), the mycobacterium M. ulcerans is found almost exclusively extracellularly in ulcerated cutaneous and subcutaneous tissues (Cosma et al., 2003). This could account for the detection of the mycolactone in the case of the f-TLC method even though the same sample may be negative in the PCR analysis. Another possible interpretation of the discordance between the two techniques could be due to low M. ulcerans DNA concentration hence the PCR targeting the insertion sequence of IS2404 may not detect the DNA sequence even though the f-TLC detects mycolactone A/B. Mycolactone A/B could therefore be used as a marker to detect M. ulcerans infection and/or diagnose Buruli ulcer. Mycolactone A/B from the natural source is often contaminated with various unknown compounds, including mycolactone congeners (Kishi, 2011) and hence could result in false-negatives. There are two possible basic mechanisms by which M. ulcerans may produce these mycolactone congeners, one of which is the possibility of a heterogeneous mixture of mycolactones being produced by differential β-keto processing on a type I polyketide synthase. The other possibility is by p450 hydroxylases through the modification of the polyketide backbone (Armand Mve-Obiang et al., 2003). 4.4 Measures of diagnostic accuracy of f-TLC compared to PCR in BU diagnosis 4.4.1 Overall Sensitivity and specificity of f-TLC technique The Fisher’s exact test of accuracy of f-TLC as compared to the gold standard IS2404 PCR method is shown in Table 4.4. Using the total number of suspected BU cases as the study group (n = 50), the sensitivity of the f-TLC criteria for the diagnosis of patients with BU was 76.9% at a 66 University of Ghana http://ugspace.ug.edu.gh 95% CI of 56.4 – 91.0. The specificity was 62.5% (95% CI, 40.6 – 81.2), PPV was 69.0% (95% CI, 49.2 – 84.7), and NPV was 71.4% (95% CI, 47.8 – 88.7). Table 4.4: Fisher’s Comparative Sensitivity and specificity test of fluorescent TLC mycolactone test and Gold Standard PCR Gold Standard Measures of Diagnostic Method Accuracy (%) New tool PCR PCR Sensitivity Specificity PPV NPV Positive Negative (%) (%) (%) (%) f-TLC 0.769 0.625 0.690 0.714 Positive 20 (TP) 9 (FP) (76.9) (62.5) (69.0) (71.4) f-TLC 56.4 – 91.0 40.6 – 81.2 49.2 – 84.7 47.8 – 88.7 Negative 6 (FN) 15 (TN) [95% CI] [95% CI] [95% CI] [95% CI] TP – True Positive = test positive in actually positive cases; FP – False Positive = test positive in actually negative cases ; FN – False Negative = test negative in actually positive cases; TN – True Negative = test negative in actually negative cases; PPV – Positive Predictive Value; NPV – Negative Predictive Value; CI – Confidence Interval 4.4.2 Sensitivity and specificity of the optimized procedure for f-TLC detection of mycolactone A/B in swab and FNA samples Table 4.5 compares the test results for f-TLC method in both swab and FNA samples after the samples have been stratified according the method of sampling. For FNA samples (n = 16), the sensitivity was determined to be 85.7% (95% CI, 42.1 – 99.6), specificity was 88.9% (95% CI, 51.8 – 99.7). The PPV and NPV for FNA samples were also 85.7% and 88.9% at their respective 95% CIs. For swab samples (n = 34), the sensitivity, specificity, PPV and NPV were 73.7% (95% CI, 48.8 – 90.9), 46.7% (95% CI, 21.3 – 73.4), 63.6% (95% CI, 40.7 – 82.8) and 58.3% (95% CI, 27.7 – 84.8) respectively. 67 University of Ghana http://ugspace.ug.edu.gh Table 4.5: Comparison of Sensitivity, Specificity, PPV and NPV in Swab and FNA samples Sensitivity Specificity PPV NPV (%) (%) (%) (%) FNA 85.7 88.9 85.7 88.9 (n = 16) 42.1 – 99.6 51.8 – 99.7 42.1 – 99.6 51.8 – 99.7 [95% CI] [95% CI] [95% CI] [95% CI] Swab 73.7 46.7 63.6 58.3 (n = 34) 48.8 – 90.9 21.3 – 73.4 40.7 – 82.8 27.7 – 84.8 [95% CI] [95% CI] [95% CI] [95% CI] Total (FNA + Swab) 76.9 62.5 69.0 71.4 (n = 50) 56.4 – 91.0 40.6 – 81.2 49.2 – 84.7 47.8 – 88.7 [95% CI] [95% CI] [95% CI] [95% CI] PPV – Positive Predictive Value; NPV – Negative Predictive Value; CI – Confidence Interval After the data were stratified by diagnostic specimen type, analysis revealed that the sensitivity, specificity, PPV and NPV values of the f-TLC were generally much higher in FNA samples than that of the swab samples. However, the sensitivities in FNA, swab and Overall (FNA + Swab) were 85.7%, 73.7% and 76.9% respectively indicating that there was no significant difference in their values. FNA samples were highly specific, and also had the highest predictive values; PPV and NPV at 85.7% and 88.9% respectively while swabs recorded the least specific value of 46.7%. The PPV and NPV of swab samples were also the least recorded (63.6% and 58.3% respectively) (Figure 4.4 A – D). 68 University of Ghana http://ugspace.ug.edu.gh Sensitivity Specificity 100 100 80 85.7 80 88.9 73.7 76.9 60 60 62.5 40 40 46.7 20 20 0 0 FNA Swab FNA + Swab FNA Swab FNA + Swab Figure 4.4 A Figure 4.4 B Positive Predictive Negative Predictive Value (PPV) Value (NPV) 100 100 80 85.7 80 88.9 60 69 60 71.4 63.6 58.3 40 40 20 20 0 0 FNA Swab FNA + Swab FNA Swab FNA + Swab Figure 4.4 C Figure 4.4 D Figure 4.4: Measures of diagnostic accuracy using f-TLC of data stratified according to diagnostic specimen type One consideration of the measures of accuracy model in diagnostic studies is a compromise on whether it is more important to use a model with a high PPV or a high sensitivity. A high PPV indicates that, when the model predicts high incidence rate, high incidence is very likely to actually occur. Therefore, it is very appropriate and important to have high PPV in instances where disease 69 University of Ghana http://ugspace.ug.edu.gh prevention and mitigation resources are limited (Buczak et al., 2014). However, it must be stated that samples for the study were obtained from different locations and hence could impact the PPV and NPV values. In this study, the overall sensitivity and negative predictive values were high (occurring at 76.9% and 71.4% respectively), indicating that the predictions of presence of the mycolactone A/B which is a marker for Mycobacterium ulcerans is more likely to be true. On the other hand, specificity and positive predictive values were lower; 62.5% and 69% respectively; indicating predictions of absence of mycolactone A/B are likely to be incorrect. The f-TLC method is more sensitive than the other techniques aside PCR. For instance, from the results obtained, the method yielded an overall sensitivity of 76.9%. This was significantly higher than that of microscopic examination (57%) and culture (51%) (Herbinger et al., 2009). Also, the f-TLC technique is convenient, less time-consuming, simple and requires low technical expertise. The technique, therefore, is amenable to point-of-care diagnosis in rural health facilities where the disease is prevalent. Smear microscopy for the detection of acid-fast bacilli is a relatively faster and less demanding diagnostic approach; however, it is less sensitive. For instance, Dorothy-Manu et al. (2011) reported a sensitivity value of 57.1% for microscopy (Yeboah-Manu et al., 2011). The most sensitive and specific method of detection is culture at 32 °C on microbiological media but it is time-consuming requiring up to 8 weeks for detection and up to 12 weeks for quantification in a mouse model (Sarfo et al., 2013). It is also prone to contamination. Here, detection of mycolactone A/B, the specific marker of M. ulcerans using the f-TLC technique, required very little manipulation of the samples and results were achieved within 1 hour after sample collection. Furthermore, the f-TLC method using FNA and swab samples produced visibly detectable results within a very short period of time; results can be obtained within 1 hour. Generally, the 70 University of Ghana http://ugspace.ug.edu.gh time involved from the sample treatment to obtaining the result was considerably lower as compared to the conventional PCR. Additionally, conventional IS2404 PCR method is estimated at a unit cost of approximately $11.24 (Yeboah-Manu et al., 2011) for diagnostic analysis. Based on the assessment of the materials, equipment and supplies needed to perform the f-TLC, the unit cost for the analysis of samples is expected to be lower than that of the PCR (the actual costing is yet to be done). The PCR technique also requires elaborate infrastructure, expertise, and costly reagents that are not commonly available in most of the BU treatment facilities. For these reasons with the accompanying high sensitivities associated with the f-TLC technique, it is of utmost importance for the applicability of the method in resource-poor settings. The use of this technique in low-resource settings eliminates the requirements for time-consuming and expensive PCR method. The fluorescent TLC (f-TLC) technique offers several advantages over existing diagnostic tools and could potentially emerge as useful point-of-care tool for rapid diagnosis of Mycobacterium ulcerans disease. Despite the numerous observed benefits of the f-TLC method, a number of factors have also been identified that could affect the outcome of the analysis including the reliability of the sample collection, the medium of storage of samples and more importantly, the time from sample collection to processing. As a result, WHO has formulated standard protocols for collection and handling of samples in various forms. Converse et al. (2014) also recommended that samples collected for f-TLC analysis should be stored and transported in absolute ethanol and analyzed immediately. This is because mycolactone is stably preserved in absolute ethanol for at least 3 weeks (Converse et al., 2014). 71 University of Ghana http://ugspace.ug.edu.gh 4.5 UV absorption of spectra analysis Deciphering the functional interactions of mycolactone A/B is of fundamental importance to the understanding, and ultimately, improving the sensitivity of the f-TLC technique which has the great potential to lead to the control of the Mycobacterium ulcerans disease. Mycolactone A/B is known to have an absolute configuration of the 1,3-diol moiety. 2-naphthylboronic acid selectively reacts with the 1,3-diol(s) in the mycolactone A/B structure resulting in the improvement of the fluorescent intensity. Spangenberg & Kishi have established this reaction pathway copiously by the preparation of several boronic esters from their corresponding 1,3-diols using 2-naphthylboronic acid (Spangenberg and Kishi, 2010b), one of which is represented in Figure 4.5 below. Spangenberg & Kishi, 2010 Figure 4.5: The proposed scheme for the reaction between (1) and naphthylboronic acid to form the corresponding naphthylboronate (2) In Figure 4.5, the diol (1) reacted with the 2-naphthylboronic acid to produce (2). The transformation from 1,3-diols to the corresponding 2-naphthylboronates was confirmed by 1H and 72 University of Ghana http://ugspace.ug.edu.gh 13C NMR spectra in which chemical shift changes were observed in the transformation. These were further confirmed with HRMS in the ESI positive mode (Spangenberg and Kishi, 2010b). One major challenge of the f-TLC method is the occurrence of background fluorescence from human tissue. This sometimes overshadows the mycolactone spot leading to ambiguity in deciding whether the sample being tested is positive or negative (Figure 4.6). Figure 4.6: TLC pictures showing background spots overshadowing the mycolactone spot In the quest to improve upon the f-TLC method, literature search for a substitute for the naphthylboronic acid suggest that 4-nitrobenzaldehyde couples with 1,3-diol systems (Jafari and Khodabakhshi, 2012) to form similar adducts as the 2-naphthylboronic acid used by Kishi and co- workers (Figure 4.7). Coupled with the fact that the use of the aldehyde could lead to a much cheaper process (benzaldehyde is seven (7) times cheaper than the 2-naphthylboronic acid), the fluorescence spot of the 4-nitrobenzaldehyde is more intense and obvious compared to that of the naphthylboronic acid, the aldehyde was therefore selected. This suggest that the benzaldehyde adduct potentially, could be easier to observe on the TLC plate. The intensity of the fluorescent 73 University of Ghana http://ugspace.ug.edu.gh adduct spot will be much stronger and the background overlaps, as with the boronic acid, could be differentiated from the mycolactone adduct spot. Figure 4.7: Adducts formed with mycolactone A/B; (A) – adduct formed with the 2- naphthylboronic acid; (B) – adduct formed with the 4-nitrobenzaldehyde. The suitability of the use of the 4-nitrobenzaldehyde was further investigated by conducting UV studies to ascertain whether the benzaldehyde will absorb in the same region as the naphthylboronic acid. UV absorption data were obtained using a Shimadzu UV mini-1240 spectrophotometer (Appendix C). In Figure 4.8 (A – D), the absorption spectra obtained in MeOH from individual absorbance values are represented with mycolactone A/B (red) and 2- naphthylboronic acid (blue) as controls for the 4-nitrobenzaldehyde (green). Strong absorptions were observed at λmax 250 (Abs = 3.9210) for 4-nitrobenzaldehyde, λmax 270 (Abs = 2.9999) for 2- naphthylboronic acid and λmax 355 (Abs = 2.8729) for mycolactone A/B respectively. The strong absorption (abs = 3.9210) for the 4-nitrobenzaldehyde could be ascribed to ππ* excitations involving the nitro and benzene groups (Leyva et al., 2011). 74 University of Ghana http://ugspace.ug.edu.gh Figure 4.8 A Figure 4.8 B Figure 4.8 C Figure 4.8 D Figure 4.8: Absorption spectra in MeOH: Green: 4-nitrobenzaldehyde (λmax 250; Abs = 3.9210). Blue: 2-naphthylboronic acid (λmax 270; Abs = 2.9999) Red: Mycolactone A/B (λmax 355; Abs = 2.8729). The absorption spectra of the benzaldehyde and the naphthylboronic acid were observed in the same region which suggest that the benzaldehyde adduct will absorb at the same wavelength as the naphthylboronic acid adduct. Also, the mycolactone structure is composed of a hexadecanoic acid backbone with a pentaenoate chromophore (shown in red) (Figure 4.9). Previous studies have demonstrated that 75 University of Ghana http://ugspace.ug.edu.gh setting a filter at 365nm, irradiation at this wavelength results in the selective excitation of the pentaenoate chromophore of the mycolactone A/B resulting in the emission of the yellow fluorescent spot (Spangenberg and Kishi, 2010b). Figure 4.9: Structure of mycolactone A/B showing the pentaenoate chromophore (red) We therefore anticipate that the adduct formation with the 4-nitrobenzaldehye will not prevent the excitation of the pentaenoate moiety hence the resulting adduct will absorb in the same region as that of the boronic acid. However, these initial attempts to confirm the hypothesis that 4-nitrobenzaldehyde can be used as an alternative to the 2-naphthylboronic acid in enhancing the fluorescent spot and reducing the background interference on the TLC plate were inconclusive thus far and investigations are still ongoing to address the situation. 4.6 Urine sampling FNAs and swabs are examples of sampling techniques currently used for the diagnosis of BU. They present relatively high sensitivities but are not devoid of issues, for instance; the local population may not be very co-operative. Sampling may also be non-representative which may result in some false negative results. As a result there is the need to search for a non-invasive 76 University of Ghana http://ugspace.ug.edu.gh sampling procedure in which the marker (mycolactone A/B or its metabolites) can be detected and hence used for the diagnosis of Buruli ulcer. It is well known that urine normally does not contain macromolecules (Lucena et al., 1998). For this reason, urine is used in the diagnosis of many diseases by tracing the pathogen responsible for the disease or some markers in the urine that give an indication that a particular disease was present. It was thus considered that if the mycolactone could be detected in the urine of the patients, it will be non-invasive and wholly representative. 4.6.1 Detection of mycolactone A/B in urine samples using f-TLC technique The possibility of using urine samples for the f-TLC method was explored because; ethically, people are more willing to give urine samples in the clinic. Also, urine sampling is simple and the overall cost is expected to be low. Other advantages of using urine samples coupled with the f-TLC method are simple operation, high specificity and it may come with higher sensitivity. In addition, patients will find the urine sampling method more convenient than the other methods, for example, there will be no pain associated with urine sampling whilst the FNA and swab sampling is usually painful. To begin with, fresh human non-infected urine samples were therefore spiked with synthetic mycolactone and ran with the f-TLC technique. The preliminary results obtained showed that mycolactone could be detected by the f-TLC method (Figure 4.10 A). This observation was corroborated by using urine samples from SPF BALB/c mice infected with M. ulcerans disease. Again the mycolactone was detected by the f-TLC technique (Figure 4.10 B – C, Appendix E). 77 University of Ghana http://ugspace.ug.edu.gh Figure 4.10: TLC profiles of spiked non-infected human urine (A) and urine samples from non-infected and infected SPF BALB/c mice (B and C respectively) (Photo-enhanced using Adobe Photoshop CS 6) The TLC profiles obtained showed that the TLC plates of the urine samples were much cleaner and are devoid of the background overlaps of human tissues as observed when swab and FNA samples are used, consequently, eliminating the ambiguity of deciding whether a sample is positive or negative. These preliminary results suggest that urine could be an alternative to swab and FNA hence should be extended to human urine samples from patients. These provide sufficient evidence to test this technique in urine samples collected from the field, especially from low prevalence areas, in order to define sensitivity and specificity in comparison to the diagnostic tests currently in use, and also to compare to the other sampling techniques such as FNAs and swabs. The application of urine samples coupled with the f-TLC technique to the diagnosis of Buruli ulcer could resolve the foremost difficulty regarding obtaining clinical samples, especially 78 University of Ghana http://ugspace.ug.edu.gh for pre-ulcerative lesions of Buruli ulcer, in order to reach a treatment decision. This is particularly important and helpful since early diagnosis is paramount for antibiotic treatment. Therefore, the urine sampling method could potentially be a major advantage in diagnosis of M. ulcerans disease and subsequently function as a follow up tool for monitoring treatment. 4.6.2 Mass Spectrometry Studies In Section 4.6.1, it was demonstrated that mycolactone could be detected in urine samples using the f-TLC method. This is an important development and is likely to have a huge impact on the diagnosis of BU in the clinic and has a potential of developing into a point-of-care diagnostic tool. It is therefore important that all the necessary techniques are applied to ascertain that the spot seen on the TLC is indeed that of the mycolactone adduct. In this section, the target was to use mass spectrometry to confirm the molecular mass of the spot corresponding to the mycolactone adduct. Also, the mycolactone is prone to esterase hydrolysis and it is expected that either the metabolites or the parent ion will be detected in the MS in urine samples. The molecular ion with a m/z of 765.5 [M + Na]+, the conserved core lactone ring and fatty acid side chain with m/z 429.3 and 359.2 respectively have been observed. These are similar to those previously reported (Hong et al., 2005, 2003; Sarfo et al., 2010b). The aim of the mass spectrometric analysis was to confirm the fragmentation patterns of the mycolactone A/B structure. The mass spectrometry should also be employed to the urine samples to confirm the fragmentation patterns as has been established for the synthetic mycolactone A/B sample. The ESI-HRMS spectrum of synthetic mycolactone A/B was investigated using a MicrOTOF-QII spectrometer in the positive ion mode. The base peak m/z 765.4960 corresponding 79 University of Ghana http://ugspace.ug.edu.gh to the molecular ion of mycolactone A/B, C44H70 O9Na [M + Na] + and 725.4942 corresponding to the dehydrated protonated molecular ion [M + H - H2O] + were identified. A small peak m/z 743.5068, corresponding to [M + H]+ was also identified. The fragmented ions of most significance from the mycolactone A/B were m/z 429 (ion A) and 359 (ion B) corresponding to the lactone core [C25H42O4Na] + and polyketide side chain [C +19H28O5Na] respectively (Figure 4.12). The proposed fragmentation routes for generating these ions are shown in Figure 4.11. The formation of ions A and B follow a pathway caused by the cleavage of the ester bond attached to the polyketide side chain. Figure 4.11: The proposed fragmentation of mycolactone (precursor ion m/z 765) to produce fragment ions A and B The error limit when these observed m/z values were compared to calculated m/z value was within the range of 0.0025 – 0.0202 ppm (Table 4.6). The fission of the ester bond to produce the core lactone ring and fatty acid side chain with their respective m/z values as observed are 80 University of Ghana http://ugspace.ug.edu.gh identical to those previously reported and identified in the MSMS spectrum of mycolactone A/B (Hong et al., 2003). Table 4.6: Formula, identity and mass accuracy with their respective error of mycolactone A/B on HRMS micrOTOF-Q II mass spectrometer Formula Identity Observed Calculated Error m/z m/z (ppm) [M + Na]+ sodium adduct of the 765.4960 765.4918 0.0042 C44H70 O9Na molecular ion [M + H]+ protonated molecular ion 743.5068 743.5093 0.0025 C44H71O + 9 [M + H - H2O] + dehydrated protonated 725.4942 725.4987 0.0045 C + 44H69O8 molecular ion [C H + 25 42O4Na] core lactone ring 429.3012 429.2975 0.0165 [C19H28O5Na] + polyketide side chain 359.2031 359.1829 0.0202 81 University of Ghana http://ugspace.ug.edu.gh 82 University of Ghana http://ugspace.ug.edu.gh Figure 4.12: Positive-ion ESI-micrOTOF-QII high-resolution mass spectrum (HRMS) of Mycolactone A/B (A – E) in a scan range 200-1200 m/z. 83 University of Ghana http://ugspace.ug.edu.gh Ultimately, the goal was to analyze the urine samples using mass spectrometry and look out for the molecular ion corresponding to either the mycolactone or to the fragmentations observed with the synthetic mycolactone, as seen in the spectra in Figure 4.12. 84 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5 Conclusion and recommendations 5.1 Conclusion Laboratory confirmation of BU cases is necessary for antibiotic treatment of the disease. Four methods are commonly used for laboratory confirmation of M. ulcerans infection: direct smear examination, polymerase chain reaction (PCR), culture and histopathology. Even though WHO adopted the PCR as the gold standard for early detection and diagnosis of BU, the method has its limitations. Notably, elaborate infrastructure, high level of technical expertise required, high cost of reagents, non-availability at peripheral health facilities, etc. In this study, the performance of the fluorescent TLC (f-TLC) technique was evaluated as a diagnostic tool against the gold standard IS2404 PCR method. The f-TLC method was found to be more sensitive than the other techniques except PCR. For instance, from the results obtained, the method yielded an overall sensitivity of 76.9%. This was significantly higher than that of microscopic examination (57%) and culture (51%) (Herbinger et al., 2009). Apart from the high sensitivity attributed to f-TLC method, it also has added potential advantages including easy use, minimal cost, requires low technical expertise, less time-consuming (where results are obtained after 1 hr) and ability to detect mycolactone to the limit of 20 ng. The f-TLC method, therefore, has the potential of being developed to a point-of-care tool for the rapid diagnosis of M. ulcerans as well as monitoring clinical response to antibiotic treatment for the disease. It is well known that urine normally does not contain macromolecules (Lucena et al., 1998) and is non-invasive. In this preliminary studies using urine samples from M. ulcerans infected BALB/c mice, encouraging results were obtained. The results from the study showed that the mycolactone was only detected 85 University of Ghana http://ugspace.ug.edu.gh in week three post infection of the animals and no background fluorescence overlaps were observed. Summarily, the f-TLC method shows promise as a diagnostic tool at point-of-care facilities in BU endemic communities. This new method requires very little manipulation of BU samples, and presents results within an hour with high specificity and sensitivity. Sample confirmation can be done visually with the naked eye. 5.2 Recommendations Although this study has established that the f-TLC detection of mycolactone A/B could potentially be employed as a diagnostic and treatment monitoring tool for BU owing to its high sensitivity, much can still be done to further improve the technique and help to better optimize the technique as a point-of-care diagnostic tool for BU. The following suggestions can therefore be helpful for future research, relying primarily on mycolactone detection using the f-TLC technique;  Combine two swab specimens or FNA samples from the same lesion together to increase the mycolactone concentration in order to facilitate easier detection.  Investigate further the reaction of the 4-nitrobenzaldehyde with the mycolactone in order to enhance the fluorescence spot in order to distinguish between the background noise and the mycolactone.  Extend the urine analysis to clinical samples and compare the results with the gold standard PCR.  Mass spectrometry should be performed on urine samples since it is the most specific technique. 86 University of Ghana http://ugspace.ug.edu.gh  It is also recommended that in future studies, representative samples should be sent to a reference laboratory to establish the veracity of the results obtained by both the PCR and f- TLC techniques as a Quality Control measure. 87 University of Ghana http://ugspace.ug.edu.gh References Ablordey, A., Hilty, M., Stragier, P., Swings, J., Portaels, F., 2005. Comparative nucleotide sequence analysis of polymorphic variable-number tandem-repeat loci in Mycobacterium ulcerans. J. Clin. Microbiol. 43, 5281–5284. Adjei, O., Evans, M., Asiedu, A., 1998. Phenytoin ulcer in the treatment of Buruli. Trans R Soc Med Hyg 92, 108–9. Aiga, H., Amano, T., Cairncross, S., Adomako, J., Domako, J.A., Nanas, O.-K.K., Coleman, S., 2004. Assessing water-related risk factors for buruli ulcer: a case-control study in ghana. Am. J. Trop. Med. Hyg. 71, 387–392. Alferink, M., de Zeeuw, J., Sopoh, G., Agossadou, C., Abass, K.M., Phillips, R.O., Loth, S., Jutten, E., Barogui, Y.T., Stewart, R.E., van der Werf, T.S., Stienstra, Y., Ranchor, A. V, 2015. Pain Associated with Wound Care Treatment among Buruli Ulcer Patients from Ghana and Benin. PLoS One 10, e0119926. Amofah, G., Asamoah, S., Afram-Gyening, C., 1998. Effectiveness of excision of pre- ulcerative Buruli lesions in field situations in a rural district in Ghana. Trop. Doct 28, 81 – 83. Amofah, G., Bonsu, F., Tetteh, C., Okrah, J., Asamoa, K., Asiedu, K., Jonathan, A., 2002. Buruli ulcer in Ghana: results of a national case search. Emerg Infect Dis 8, 167 – 70. Anon, 1971. Epidemiology of Mycobacterium ulcerans infection (Buruli ulcer) at Kinyara, Uganda. Trans R Soc Trop Med Hyg 65, 763 – 75. Asiedu, K., Etuaful, S., 1998. Socioeconomic implications of Buruli ulcer in Ghana: A three-year review. Am. J. Trop. Med. Hyg. 59, 1015–1022. Asiedu, K., Scherpbier, R., Raviglione, M., 2000a. Buruli ulcer. Geneva: WHO [WWW Document]. WHO/CDS/GBUI/2000.1http://www.who.int/gtb-buruli/. Asiedu, K., Scherpbier, R., Raviglione, M., 2000b. Buruli ulcer . Mycobacterium ulcerans Infection . World Health Organization: Geneva 9 – 12. Bayley, A., 1971. Buruli ulcer in Ghana. BMJ 2, 401–2. Beissner, M., Symank, D., Phillips, R.O., Amoako, Y.A., Awua-Boateng, N.-Y., Sarfo, F.S., Jansson, M., Huber, K.L., Herbinger, K.-H., Battke, F., Löscher, T., Adjei, O., Bretzel, G., 2012. Detection of viable Mycobacterium ulcerans in clinical samples by a novel combined 16S rRNA reverse transcriptase/IS2404 real-time qPCR assay. PLoS Negl. Trop. Dis. 6, 1756. Bentoucha, A., Robert, J., Dega, H., Lounis, N., Jarlier, V., Grosset, J., 2001. Activities of new macrolides and fluoroquinolones against Mycobacterium ulcerans infection in mice. Antimicrob. Agents Chemother. 45, 3109–12. Boulkroun, S., Guenin-Macé, L., Thoulouze, M.-I., Monot, M., Merckx, A., Langsley, G., Bismuth, G., Di Bartolo, V., Demangel, C., 2010. Mycolactone suppresses T cell responsiveness by altering both early signaling and posttranslational events. J. Immunol. 184, 1436–1444. Bozzo, C., Tiberio, R., Graziola, F., Pertusi, G., Valente, G., Colombo, E., Small, P.L.C., Leigheb, G., 2010. A Mycobacterium ulcerans toxin, mycolactone, induces apoptosis in primary human keratinocytes and in HaCaT cells. Microbes Infect. 12, 1258–63. 88 University of Ghana http://ugspace.ug.edu.gh Brunet de Courrsou, L., 2002. Study Group Report on Buruli Ulcer Treatment with Clay. Fifth WHO Advisory Group Meeting on Buruli Ulcer, Geneva, Switzerland. WHO. Buczak, A.L., Baugher, B., Babin, S.M., Ramac-Thomas, L.C., Guven, E., Elbert, Y., Koshute, P.T., Velasco, J.M.S., Roque, V.G., Tayag, E. a., Yoon, I.K., Lewis, S.H., 2014. Prediction of High Incidence of Dengue in the Philippines. PLoS Negl. Trop. Dis. 8. Cassisa, V., Chauty, A., Marion, E., Ardant, M.F., Eyangoh, S., Cottin, J., Aubry, J., Koussemou, H., Lelièvre, B., Férec, S., Tekaia, F., Johnson, C., Marsollier, L., 2010. Use of fine-needle aspiration for diagnosis of Mycobacterium ulcerans infection. J. Clin. Microbiol. 48, 2263–2264. Chan-Tompkins, N., 1995. Toxic effects drug interactions of anti-mycobacterial therapy. Clin Dermatol 13, 223 – 233. Chauty, A., Ardant, M.-F., Adeye, A., Euverte, H., Guédénon, A., Johnson, C., Aubry, J., Nuermberger, E., Grosset, J., 2007. Promising clinical efficacy of streptomycin-rifampin combination for treatment of buruli ulcer (Mycobacterium ulcerans disease). Antimicrob. Agents Chemother. 51, 4029–4035. Clancey, J., Dodge, R., Lunn, H.F., 1962. Study of a mycobacterium causing skin ulceration in Uganda. Ann Soc Belg Med Trop 42, 585–590. Connor, D., Lunn, H., 1965. Mycobacterium ulcerans infection (with comments on pathogenesis). Int J Lepr. 33 (suppl), 698 – 709. Converse, P.J., Xing, Y., Kim, K.H., Tyagi, S., Li, S.Y., Almeida, D. V., Nuermberger, E.L., Grosset, J.H., Kishi, Y., 2014. Accelerated Detection of Mycolactone Production and Response to Antibiotic Treatment in a Mouse Model of Mycobacterium ulcerans Disease. PLoS Negl. Trop. Dis. 8, 52. Cook, A., 1970. Mengo Hospital Notes, 1897, Makerere Medical School Library. Br Med J 2, 378–9. Cosma, C.L., Sherman, D.R., Ramakrishnan, L., 2003. The secret lives of the pathogenic mycobacteria. Ann Rev Microbiol 57, 641 – 676. Coutanceau, E., Marsollier, L., Brosch, R., Perret, E., Goossens, P., Tanguy, M., Cole, S.T., Small, P.L.C., Demangel, C., 2005. Modulation of the host immune response by a transient intracellular stage of Mycobacterium ulcerans: the contribution of endogenous mycolactone toxin. Cell. Microbiol. 7, 1187–96. Daniel, A.K., Lee, R.E., Portaels, F., Small, P.L.C., 2004. Analysis of Mycobacterium Species for the Presence of a Macrolide Toxin, Mycolactone. Infect. Immun. 72, 123–132. de Zeeuw, J., Alferink, M., Barogui, Y.T., Sopoh, G., Phillips, R.O., van der Werf, T.S., Loth, S., Molenbuur, B., Plantinga, M., Ranchor, A. V., Stienstra, Y., 2015. Assessment and Treatment of Pain during Treatment of Buruli Ulcer. PLoS Negl. Trop. Dis. 9, 1–10. Debacker, M., Aguiar, J., Steunou, C., Zinsou, C., Meyers, W.M., Portaels, F., 2005. Buruli ulcer recurrence, Benin. Emerg. Infect. Dis 11, 584 – 589. Debacker, M., Aguiar, J., Steunou, C., Zinsou, C., Meyers, W.M., Scott, J.T., Dramaix, M., Portaels, F., 2004. Mycobacterium ulcerans disease: role of age and gender in incidence and morbidity. Trop. Med. Int. Health 9, 1297–1304. 89 University of Ghana http://ugspace.ug.edu.gh Debacker, M., Zinsou, C., Aguiar, J., Meyers, W., Portaels, F., 2002. Mycobacterium ulcerans disease (Buruli ulcer) following human bite. Lancet 360, 1830. Dega, H., Robert, J., Bonnafous, P., Jarlier, V., Grosset, J., 2000. Activities of Several Antimicrobials against Mycobacterium ulcerans Infection in Mice. Antimicrob. Agents Chemother. 44, 2367–2372. Demangel, C., Stinear, T.P., Cole, S.T., 2009. Buruli ulcer: reductive evolution enhances pathogenicity of Mycobacterium ulcerans. Nat. Rev. Microbiol. 7, 50–60. Dobos, K.M., Spotts, E. a., Marston, B.J., Horsburgh, C.R., King, C.H., 2000. Serologic response to culture filtrate antigens of Mycobacterium ulcerans during Buruli ulcer disease. Emerg. Infect. Dis. 6, 158–164. Eddyani, M., Debacker, M., Martin, A., Aguiar, J., Johnson, C.R., Uwizeye, C., Fissette, K., Portaels, F., 2008. Primary culture of Mycobacterium ulcerans from human tissue specimens after storage in semisolid transport medium. J. Clin. Microbiol. 46, 69–72. Eddyani, M., Fraga, A.G., Schmitt, F., Uwizeye, C., Fissette, K., Johnson, C., Aguiar, J., Sopoh, G., Barogui, Y., Meyers, W.M., Pedrosa, J., Portaels, F., 2009. Fine-needle aspiration, an efficient sampling technique for bacteriological diagnosis of nonulcerative Buruli ulcer. J. Clin. Microbiol. 47, 1700–1704. En, J., Goto, M., Nakanaga, K., Higashi, M., Ishii, N., Saito, H., Yonezawa, S., Hamada, H., Small, P.L.C., 2008. Mycolactone is responsible for the painlessness of Mycobacterium ulcerans infection (Buruli ulcer) in a murine study. Infect. Immun. 76, 2002–2007. Etuaful, S., Carbonnelle, B., Grosset, J., Lucas, S., Horsfield, C., Phillips, R., Evans, M., Ofori- Adjei, D., Klustse, E., Owusu-Boateng, J., Amedofu, G.K., Awuah, P., Ampadu, E., Amofah, G., Asiedu, K., Wansbrough-Jones, M., 2005. Efficacy of the combination rifampin-streptomycin in preventing growth of Mycobacterium ulcerans in early lesions of Buruli ulcer in humans. Antimicrob. Agents Chemother. 49, 3182–6. Exner, K., Lemperle, G., 1987. Buruli-Ulkus—Nekrotisierende Infektion an der Hand eines Plastischen Chirurgen. Handchir Mikrochir Plast Chir 19, 230–32. Faber, W.R., Jong, B. De, Vries, H.J.C. De, Zeegelaar, J.E., 2015. Buruli Ulcer in Traveler from Suriname, South America, to the Netherlands 21, 497–499. Fidanze, S., Song, F., Szlosek-Pinaud, M., Small, P., Kishi, Y., 2001. Complete structure of the mycolactones. J Am Chem Soc 123, 10117–10118. Garrity, G., 2001. Bergey’ s Manual of Systematic Bacteriology; In: Garrity GM, ed. New York: Springer-Verlag. George, K.M., Chatterjee, D., Gunawardana, C., Welty, D., Hayman, J., 1999. Mycolactone : A Polyketide Toxin from Mycobacterium ulcerans Required for Virulence 283. George, K.M., Pascopella, L., Welty, D.M., Small, L.C., 2000. A Mycobacterium ulcerans toxin, mycolactone, causes apoptosis in guinea pig ulcers and tissue culture cells. Control 68, 877– 883. Gooding, T.M., Johnson, P.D.R., Smith, M., Kemp, A.S., Robins-browne, R.M., 2002. Cytokine Profiles of Patients Infected with Mycobacterium ulcerans and Unaffected Household Contacts. Infect Immun 70, 5562–5567. 90 University of Ghana http://ugspace.ug.edu.gh Goto, M., Nakanaga, K., Aung, T., Hamada, T., Yamada, N., Nomoto, M., Al., E., 2006. Nerve Damage in Mycobacterium ulcerans -Infected Mice Probable Cause of Painlessness in Buruli Ulcer 805–811. Goutzamanis, J., Gilbert, G., 1995. Mycobacterium ulcerans infection in Australian children: report of eight cases and review. Clin. Infect. Dis. 21, 1186 – 1192. Grietens, K.P., Boock, A.U., Peeters, H., Hausmann-Muela, S., Toomer, E., Ribera, J.M., 2008. “It is me who endures but my family that suffers”: Social isolation as a consequence of the household cost burden of buruli ulcer free of charge hospital treatment. PLoS Negl. Trop. Dis. 2, 1–7. Guarner, J., Bartlett, J., Spotts Whitney, E. a., Raghunathan, P.L., Stienstra, Y., Asamoa, K., Etuaful, S., Klutse, E., Quarshie, E., Van der Werf, T.S., Van der Graaf, W.T. a, King, C.H., Ashford, D. a., 2003. Histopathologic features of Mycobacterium ulcerans infection. Emerg. Infect. Dis. 9, 651–656. Haydel, S.E., Remenih, C.M., Williams, L.B., 2008. Broad-spectrum in vitro antibacterial activities of clay minerals against antibiotic-susceptible and antibiotic-resistant bacterial pathogens. J. Antimicrob. Chemother. 61, 353–61. Hayman, J., 1991. Postulated epidemiology of Mycobacterium ulcerans infection. Intern. J. Epidemiol. 20, 1093 – 1098. Hayman, J., McQueen, A., 1985. The pathology of Mycobacterium ulcerans infection. Pathology 17, 594–600. Herbinger, K.-H., Adjei, O., Awua-Boateng, N.-Y., Nienhuis, W.A., Kunaa, L., Siegmund, V., Nitschke, J., Thompson, W., Klutse, E., Agbenorku, P., Schipf, A., Reu, S., Racz, P., Fleischer, B., Beissner, M., Fleischmann, E., Helfrich, K., van der Werf, T.S., Löscher, T., Bretzel, G., 2009. Comparative study of the sensitivity of different diagnostic methods for the laboratory diagnosis of Buruli ulcer disease. Clin. Infect. Dis. 48, 1055–1064. Herbinger, K.-H., Beissner, M., Huber, K., Awua-Boateng, N.-Y., Nitschke, J., Thompson, W., Klutse, E., Agbenorku, P., Assiobo, A., Piten, E., Wiedemann, F., Fleischmann, E., Helfrich, K., Adjei, O., Löscher, T., Bretzel, G., 2010. Efficiency of fine-needle aspiration compared with other sampling techniques for laboratory diagnosis of Buruli ulcer disease. J. Clin. Microbiol. 48, 3732–3734. Hong, H., Coutanceau, E., Leclerc, M., Caleechurn, L., Leadlay, P.F., Demangel, C., 2008a. Mycolactone diffuses from Mycobacterium ulcerans-infected tissues and targets mononuclear cells in peripheral blood and lymphoid organs. PLoS Negl. Trop. Dis. 2, 1–8. Hong, H., Demangel, C., Pidot, S.J., Leadlay, P.F., Stinear, T., 2008b. Mycolactones: immunosuppressive and cytotoxic polyketides produced by aquatic mycobacteria. Nat. Prod. Rep. 25, 447–454. Hong, H., Gates, P.J., Staunton, J., Stinear, T., Cole, S.T., Leadlay, P.F., Spencer, J.B., 2003. Identification using LC-MSn of co-metabolites in the biosynthesis of the polyketide toxin mycolactone by a clinical isolate of Mycobacterium ulcerans. Chem. Commun. (Camb). 88, 2822–2823. Hong, H., Stinear, T., Skelton, P., Spencer, J.B., Leadlay, P.F., 2005. Structure elucidation of a novel family of mycolactone toxins from the frog pathogen Mycobacterium sp. MU128FXT 91 University of Ghana http://ugspace.ug.edu.gh by mass spectrometry. Chem. Commun. (Camb). 4306–4308. Jafari, F., Khodabakhshi, S., 2012. Mg(HSO4)2/SiO2 as a Highly Efficient Catalyst for the Green Preparation of 2-Aryl-1,3-Dioxalanes/Dioxanes and Linear Acetals. Org. Chem. Int. 1–5. Jayawardena, S., Cheung, C.Y., Barr, I., Chan, K.H., Chen, H., Guan, Y., Peiris, J.S., Poon, L.L., 2007. Loop-mediated isothermal amplification for influenza A (H5N1) Virus. Emerg Infect Dis 13, 2007. Ji, B., Chauffour, A., Robert, J., Jarlier, V., 2008. Bactericidal and sterilizing activities of several orally administered combined regimens against Mycobacterium ulcerans in mice. Antimicrob. Agents Chemother. 52, 1912–1916. Johnson, P., Veitch, M., Leslie, D., Flood, P., Hayman, J., 1996. The emergence of Mycobacterium ulcerans near Melbourne. Med J Aust 164, 76–78. Johnson, P.D.R., Azuolas, J., Lavender, C.J., Wishart, E., Stinear, T.P., Hayman, J.A., Brown, L., Jenkin, G.A., Fyfe, J.A.M., 2007. Mycobacterium ulcerans in Mosquitoes Captured during Outbreak of Buruli Ulcer , Southeastern Australia 13. Judd, T., Bischoff, A., Kishi, Y., Adusumilli, S., Small, P., 2004. Structure determination of mycolactone C via total synthesis. Org Lett 2004 6, 4901–4904. Kaneko, H., Kawana, T., Fukushima, E., 2007. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods 70, 499–501. Käser, M., Hauser, J., Small, P., Pluschke, G., 2009. Large sequence polymorphisms unveil the phylogenetic relationship of environmental and pathogenic mycobacteria related to Mycobacterium ulcerans. Appl. Environ. Microbiol. 75, 5667–5675. Käser, M., Rondini, S., Naegeli, M., Stinear, T., Portaels, F., Certa, U., Pluschke, G., 2007. Evolution of two distinct phylogenetic lineages of the emerging human pathogen Mycobacterium ulcerans. BMC Evol. Biol. 7, 177. Katoch, V., Mohan Kumar, T., 2001. Atypical mycobacterial infections. In: Sharma SK, editor. Tuberculosis, 1st ed. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd. Kibadi, K., Mputu-Yamba, J., Mokassa, B., Al., E., 2009. Relapse after surgical treatment of Mycobacterium ulcerans infection (Buruli ulcer): study of risk factors in 84 patients in the Democratic Republic of the Congo. Médécine Trop. 69, 471–474. Kim, H., Kishi, Y., 2008. Total synthesis and stereochemistry of mycolactone F. J Am Chem Soc 130, 1842–1844. Kishi, Y., 2011. Chemistry of mycolactones, the causative toxins of Buruli ulcer. Proc. Natl. Acad. Sci. U. S. A. 108, 6703–6708. Kiszewski, a E., Becerril, E., Aguilar, L.D., Kader, I.T. a, Myers, W., Portaels, F., Hernàndez Pando, R., 2006. The local immune response in ulcerative lesions of Buruli disease. Clin. Exp. Immunol. 143, 445–451. Klutse, E., Adjei, O., Ampadu, E., Arthur, L., 2006. Management of Buruli ulcer cases with topical application of phenytoin powder. In: WHO. Report of the 6th WHO Advisory Group Meeting on Buruli ulcer. 92 University of Ghana http://ugspace.ug.edu.gh http://whqlibdoc.who.int/hq/2003/WHO_CDS_CPE_GBUI_2003.8.pdf (accessed Jan 18, 2015). Krieg, R., Hockmeyer, W., Connor, D., 1974. Toxin of Mycobacterium ulcerans. Production and effects in guinea pig skin. Arch Dermatol 110, 783 – 88. Krieg, R., Wolcott, J., Meyers, W., 1979. Mycobacterium ulcerans infection: treatment with rifampin, hyperbaric oxygenation, and heat. Aviat Sp. Env. Med 50, 888–92. Kurien, B.T., Everds, N.E., Scofield, R.H., 2004. Experimental animal urine collection: a review. Lab. Anim. 38, 333–361. Lefrançois, S., Robert, J., Chauffour, A., Ji, B., Jarlier, V., 2007. Curing Mycobacterium ulcerans infection in mice with a combination of rifampin-streptomycin or rifampin- amikacin. Antimicrob. Agents Chemother. 51, 645–50. Leyva, V., Corral, I., Schmierer, T., Gilch, P., González, L., 2011. A comparative analysis of the UV/Vis absorption spectra of nitrobenzaldehydes. Phys. Chem. Chem. Phys. 13, 4269– 4278. Lucena, W.A., Dhalia, R., Abath, F.G.C., Nicolas, L., Regis, L.N., Furtado, A.F., 1998. Diagnosis of Wuchereria bancrofti infection by the polymerase chain reaction using urine and day blood samples from amicrofilaraemic patients. Trans. R. Soc. Trop. Med. Hyg. 92, 290–293. Lunn, H.F., Connor, D.H., Wilks, N.E., Barnley, G.R., Kamunvi, F., J. K. Clancey, J.K., Bee, J.D., 1965. Buruli (mycobacterial) ulceration in Uganda (a new focus of Buruli ulcer in Madi district, Uganda): report of a field study. East Afr Med J 42, 275–288. Maccallum, P., Tolhurst, J.C., Buckle, G., Sissons, H.A., 1948. A new mycobacterial infection in man. J. Pathol. Bacteriol. 60, 93–122. doi:10.1002/path.1700600111 Marion, E., Song, O., Christophe, T., Babonneau, J., Fenistein, D., Letournel, F., Henrion, D., Clere, N., Paille, V., Gue, N.C., Gersbach, P., Altmann, K., Stinear, T.P., Comoglio, Y., Sandoz, G., 2014. Mycobacterial Toxin Induces Analgesia in Buruli Ulcer by Targeting the Angiotensin Pathways. Cell 157, 1565–1576. Marsollier, L., Robert, R., Aubry, J., André, J. Saint, Kouakou, H., Legras, P., Manceau, A., Mahaza, C., Andre, J. Saint, Carbonnelle, B., 2002. Aquatic Insects as a Vector for Mycobacterium ulcerans Aquatic Insects as a Vector for Mycobacterium ulcerans 68, 4623. Marsollier, L., Se, T., Aubry, J., Merritt, R.W., Andre, J. Saint, Legras, P., Manceau, A., Chauty, A., Carbonnelle, B., Cole, S.T., 2004. Aquatic Snails , Passive Hosts of Mycobacterium ulcerans 70, 6296–6298. Meyers, W., Connor, D.H., McCullough, B., Bourland, J., Moris, R., Proos, L., 1974a. Distribution of Mycobacterium ulcerans infections in Zaire, including the report of new foci. Ann. Soc. Belg. Med. Trop. (1920). Meyers, W., Shelly, W., Connor, D., 1974b. Heat treatment of Mycobacterium ulcerans infections without surgical excision. Am J Trop Med Hyg 23, 924–929. Modaghegh, S., Ghoraian, M.A., Moshkgou, M.&, Reziazadeh, A., 1988. The effect of phenytoin on healing of war and non-war intractable wounds. Med. J. Islam. Repub. Iran 2, 81–86. 93 University of Ghana http://ugspace.ug.edu.gh Mve-Obiang, A., Lee, R., Portaels, F., Small, P., 2003. Heterogeneity of mycolactones produced by clinical islates of Mycobacterium ulcerans: implications for virulence. Infect Immun 71, 774– 783. Mve-Obiang, A., Lee, R.E., Portaels, F., Small, P.L.C., 2003. Heterogeneity of mycolactones produced by clinical isolates of Mycobacterium ulcerans: Implications for virulence. Infect. Immun. 71, 774–783. Mve-Obiang, A., Lee, R.E., Umstot, E.S., Trott, K.A., Grammer, T.C., Parker, J.M., Ranger, B.S., Grainger, R., Mahrous, E.A., Small, P.L.C., 2005. A newly discovered mycobacterial pathogen isolated from laboratory colonies of Xenopus species with lethal infections produces a novel form of mycolactone, the Mycobacterium ulcerans macrolide toxin. Infect. Immun. 73, 3307–3312. Nagamine, K., Hase, T., Notomi, T., 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16, 223–229. Nakanaga, K., Hoshino, Y., Yotsu, R.R., Makino, M., Ishii, N., Al., E., 2011. Nineteen cases of Buruli ulcer diagnosed in Japan from 1980 to 2010. J. Clin. Microbiol. 49, 3829–3836. Nakanaga, K., Ishii, N., Suzuki, K., Tanigawa, K., Goto, M., Okabe, T., Imada, H., Kodama, A., Iwamoto, T., Takahashi, H., Saito, H., 2007. “Mycobacterium ulcerans subsp. shinshuense” isolated from a skin ulcer lesion: Identification based on 16S rRNA gene sequencing. J. Clin. Microbiol. 45, 3840–3843. Nienhuis, W.A., Stienstra, Y., Thompson, W.A., Awuah, P.C., Abass, K.M., Tuah, W., Awua- Boateng, N.Y., Ampadu, E.O., Siegmund, V., Schouten, J.P., Adjei, O., Bretzel, G., van der Werf, T.S., 2010. Antimicrobial treatment for early, limited Mycobacterium ulcerans infection: a randomised controlled trial. Lancet 375, 664–672. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T., 2002. Loop-mediated isothermal amplification of DNA. Mol Cell Probes 16, 223–229. Okenu, D.M.N., Ofielu, L.O., Easley, K.A., Guarner, J., Spotts Whitney, E.A., Raghunathan, P.L., Stienstra, Y., Asamoa, K., van der Werf, T.S., van der Graaf, W.T.A., Tappero, J.W., Ashford, D.A., King, C.H., 2004. Immunoglobulin M Antibody Responses to Mycobacterium ulcerans Allow Discrimination between Cases of Active Buruli Ulcer Disease and Matched Family Controls in Areas Where the Disease Is Endemic. Clin. Vaccine Immunol. 11, 387–391. Onoe, H., Yotsu, R., Nakanaga, K., Hoshino, Y., Ishii, N., Takeuchi, T., 2012. Buruli ulcer accompanied by pain in a Japanese patient Invasive behavior of small diameter melanomas 869–870. Pahlevan, a a, Wright, D.J., Andrews, C., George, K.M., Small, P.L., Foxwell, B.M., 1999. The inhibitory action of Mycobacterium ulcerans soluble factor on monocyte/T cell cytokine production and NF-kappa B function. J. Immunol. 163, 3928–3935. Phillips, R., Adjei, O., Lucas, S., Benjamin, N., 2004. Pilot Randomized Double-Blind Trial of Treatment of Mycobacterium ulcerans Disease ( Buruli Ulcer ) with Topical Nitrogen Oxides. Society 48, 2866–2870. Phillips, R., Horsfield, C., Kuijper, S., Lartey, A., Tetteh, I., Nyamekye, B., Awuah, P., Nyarko, K.M., Lucas, S., Kolk, a H.J., Etuaful, S., 2005. Sensitivity of PCR Targeting the IS 2404 94 University of Ghana http://ugspace.ug.edu.gh Insertion Sequence of Mycobacterium ulcerans in an Assay Using Punch Biopsy Specimens for Diagnosis of Buruli Ulcer Sensitivity of PCR Targeting the IS 2404 Insertion Sequence of Mycobacterium ulcerans in an Assay 43, 3650–3656. Phillips, R., Horsfield, C., Mangan, J., Laing, K., Etuaful, S., Awuah, P., Nyarko, K., Butcher, P., Lucas, S., 2006. Cytokine mRNA Expression in Mycobacteriam ulcerans -Infected Human Skin and Correlation with Local Inflammatory Response 74, 2917–2924. Phillips, R., Sarfo, F.S., Guenin-mace, L., Wansbrough-jones, M., Albert, M.L., Demangel, C., 2009a. Immunosuppressive Signature of Cutaneous Mycobacterium ulcerans Infection in the Peripheral Blood of Patients with Buruli Ulcer Disease 200, 1675–1684. Phillips, R., Sarfo, F.S., Osei-Sarpong, F., Boateng, A., Tetteh, I., Lartey, A., Adentwe, E., Opare, W., Asiedu, K.B., Wansbrough-Jones, M., 2009b. Sensitivity of PCR targeting Mycobacterium ulcerans by use of Fine-needle aspirates for diagnosis of Buruli ulcer. J. Clin. Microbiol. 47, 924–926. Pidot, S.J., Hong, H., Seemann, T., Porter, J.L., Yip, M.J., Men, A., Johnson, M., Wilson, P., Davies, J.K., Leadlay, P.F., Stinear, T.P., 2008. Deciphering the genetic basis for polyketide variation among mycobacteria producing mycolactones. BMC Genomics 9, 462. Pimsler, M., Sponsler, T., Meyers, W., 1988. Immunosuppressive properties of the soluble toxin from Mycobacterium ulcerans. J. Infect Dis 157, 577. Poon, L.L., Leung, C.S., Tashiro, M., Chan, K.H., Wong, B.W., Yuen, K.Y., Guan, Y., Peiris, J.S., 2004. Rapid detection of the severe acute respiratory syndrome (SARS) coronavirus by a loop-mediated isothermal amplification assay. Clin Chem 50, 1050–1052. Portaels, F., Aguiar, J., Debacker, M., Guédénon, A., Meyers, W.M., 2004. Mycobacterium bovis BCG Vaccination as Prophylaxis against Mycobacterium ulcerans Osteomyelitis in Buruli Ulcer Disease. Infect Immun 72, 62–65. Portaels, F., Aguiar, J., Fissette, K., Fonteyne, P. a., De Beenhouwer, H., De Rijk, P., Guédénon, A., Lemans, R., Steunou, C., Zinsou, C., Dumonceau, J.M., Meyers, W.M., 1997. Direct detection and identification of Mycobacterium ulcerans in clinical specimens by PCR and oligonucleotide-specific capture plate hybridization. J. Clin. Microbiol. 35, 1097–1100. Portaels, F., Fonteyne, P. a., De Beenhouwer, H., De Rijk, P., Guédénon, a., Hayman, J., Meyers, W.M., 1996. Variability in 3′ end of 16S rRNA sequence of Mycobacterium ulcerans is related to geographic origin of isolates. J. Clin. Microbiol. 34, 962–965. Portaels, F., Johnson, P., Meyers, W., 2001. Buruli ulcer: diagnosis of Mycobacterium ulcerans disease. In: Portaels F, Johnson P, Meyers WM, eds. Buruli ulcer: a manual for health care providers. WHO/CDS/GBUI, Geneva, Switz. 4–92. Portaels, F., Meyers, W.M., Ablordey, A., Castro, A.G., Chemlal, K., de Rijk, P., Elsen, P., Fissette, K., Fraga, A.G., Lee, R., Mahrous, E., Small, P.L.C., Stragier, P., Torrado, E., Van Aerde, A., Silva, M.T., Pedrosa, J., 2008. First cultivation and characterization of Mycobacterium ulcerans from the environment. PLoS Negl. Trop. Dis. 2. Portaels, F., Traore, H., Ridder, K.D.E., 2000. In Vitro Susceptibility of Mycobacterium ulcerans to Clarithromycin 42, 2070–2073. Pszolla, N., Sarkar, M.R., Strecker, W., Kern, P., Kinzl, L., Meyers, W.M., 2003. Buruli Ulcer : A Systemic Disease 37. 95 University of Ghana http://ugspace.ug.edu.gh Raghunathan, P.L., Whitney, E.A.S., Asamoa, K., Stienstra, Y., Taylor, T.H., Amofah, G.K., Ofori-adjei, D., Dobos, K., Guarner, J., Martin, S., Pathak, S., Klutse, E., Etuaful, S., Graaf, W.T.A. Van Der, Werf, T.S. Van Der, King, C.H., Tappero, J.W., Ashford, D.A., 2005. Risk Factors for Buruli Ulcer Disease ( Mycobacterium ulcerans Infection ): Results from a Case-Control Study in Ghana 30333, 1445–1453. Ranger, B.S., Mahrous, E.A., Mosi, L., Adusumilli, S., Lee, R.E., Colorni, A., Rhodes, M., Small, P.L.C., 2006. Globally distributed mycobacterial fish pathogens produce a novel plasma-encoded toxic macrolide, mycolactone F. Infect. Immun. 74, 6037–6045. Read, J.K., Heggie, C.M., Meyers, W.M., Connor, D.H., 1974. Cytotoxic Activity of Mycobacterium ulcerans. Infect. Immun. 9, 1114–1122. Revill, W.D., Morrow, R.H., Pike, M.C., Ateng, J., 1973. A controlled trial of the treatment of Mycobacterium ulcerans infection with clofazimine. Lancet ii, 873 – 877. Ross, B., Marino, L., Oppedisano, F., Edwards, R., Robins-Browne, R., Johnson, P., 1997. Development of a PCR assay for rapid diagnosis of Mycobacterium ulcerans infection. J Clin Microbiol 35, 1696 – 1700. Sarfo, F.S., 2014. The kinetics of mycolactone in relation to the microbiological, clinical and immunological responses to antibiotic therapy for mycobacterium ulcerans disease. Kwame Nkrumah University of Science and Technology. Sarfo, F.S., Converse, P.J., Almeida, D. V, Zhang, J., Robinson, C., Wansbrough-Jones, M., Grosset, J.H., 2013. Microbiological, histological, immunological, and toxin response to antibiotic treatment in the mouse model of Mycobacterium ulcerans disease. PLoS Negl. Trop. Dis. 7, e2101. Sarfo, F.S., Phillips, R., Asiedu, K., Ampadu, E., Bobi, N., Adentwe, E., Lartey, A., Tetteh, I., Wansbrough-Jones, M., 2010a. Clinical efficacy of combination of rifampin and streptomycin for treatment of Mycobacterium ulcerans disease. Antimicrob. Agents Chemother. 54, 3678–3685. Sarfo, F.S., Phillips, R.O., Rangers, B., Mahrous, E. a., Lee, R.E., Tarelli, E., Asiedu, K.B., Small, P.L., Wansbrough-Jones, M.H., 2010b. Detection of mycolactone A/B in Mycobacterium ulcerans - Infected human tissue. PLoS Negl. Trop. Dis. 4, e577. Sarfo, F.S., Phillips, R.O., Zhang, J., Abass, M.K., Abotsi, J., Amoako, Y. a, Adu-Sarkodie, Y., Robinson, C., Wansbrough-Jones, M.H., 2014. Kinetics of mycolactone in human subcutaneous tissue during antibiotic therapy for Mycobacterium ulcerans disease. BMC Infect. Dis. 14, 202. Schipper, H.S., Rutgers, B., Huitema, M.G., Etuaful, S.N., Westenbrink, B.D., Limburg, P.C., Timens, W., van der Werf, T.S., 2007. Systemic and local interferon-gamma production following Mycobacterium ulcerans infection. Clin. Exp. Immunol. 150, 451–459. Schutte, D. and, Pluschke, G., 2009. Immunosuppression and treatment-associated inflammatory response in patients with Mycobacterium ulcerans infection (Buruli ulcer). Expert Opin Biol Ther 9, 187–200. Siegmund, V., Adjei, O., Nitschke, J., Thompson, W., Klutse, E., Herbinger, K.H., Thompson, R., van Vloten, F., Racz, P., Fleischer, B., Loescher, T., Bretzel, G., 2007. Dry reagent- based polymerase chain reaction compared with other laboratory methods available for the 96 University of Ghana http://ugspace.ug.edu.gh diagnosis of Buruli ulcer disease. Clin. Infect. Dis. 45, 68–75. Silva, M.T., Portaels, F., Pedrosa, J., 2009. Pathogenetic mechanisms of the intracellular parasite Mycobacterium ulcerans leading to Buruli ulcer. Lancet Infect. Dis. 9, 699–710. Sizaire, V., Nackers, F., Comte, E., Portaels, F., 2006. Mycobacterium ulcerans infection: control, diagnosis, and treatment. Lancet Infect Dis 6, 288–296. Smith, B.H., Bogoch, S., Dreyfus, J., 1988. The Broad Range of Clinical Use of Phenytoin. New York Dreyfus Med. Found. 89–120. Song, F., Fidanze, S., Benowitz, A.B., Kishi, Y., 2007. Total Synthesis of Mycolactones A and B. Tetrahedron 63, 5739–5753. Spangenberg, T., Kishi, Y., 2010a. Highly sensitive, operationally simple, cost/time effective detection of the mycolactones from the human pathogen Mycobacterium ulcerans. Chem. Commun. (Camb). 46, 1410–1412. Spangenberg, T., Kishi, Y., 2010b. Highly sensitive, operationally simple, cost/time effective detection of the mycolactones from the human pathogen Mycobacterium ulcerans. Chem. Commun. (Camb). 46, 1410–1412. Stanford, J.L., Revill, W.D., Gunthorpe, W.J., Grange, J.M., 1975. The production and preliminary investigation of Burulin, a new skin test reagent for Mycobacterium ulcerans infection. J. Hyg. (Lond). 74, 7–16. doi:10.1017/S0022172400046659 Stienstra, Y., Graaf, W.T.A. Van Der, Asamoa, K., Van Der Werf, T.S., 2002. Beliefs and attitudes toward Buruli uler in Ghana 67, 207–213. Stienstra, Y., Van Roest, M.H.G., Van Wezel, M.J., Wiersma, I.C., Hospers, I.C., Dijkstra, P.U., Johnson, R.C., Ampadu, E.O., Gbovi, J., Zinsou, C., Etuaful, S., Klutse, E.Y., Van Der Graaf, W.T.A., Van Der Werf, T.S., 2005. Factors associated with functional limitations and subsequent employment or schooling in Buruli ulcer patients. Trop. Med. Int. Heal. 10, 1251–1257. Stinear, T., Pryor, M.J., Porter, J.L., Cole, S.T., 2005. Functional analysis and annotation of the virulence plasmid pMUM001 from Mycobacterium ulcerans. 2005. Microbiology 151, 683 – 692. Stinear, T., Seemann, T., Pidot, S., Frigui, W., Reysset, G., Garnier, T., Meurice, G., Simon, D., Bouchier, C., Ma, L., Tichit, M., Porter, J.L., Ryan, J., Johnson, P.D.R., Davies, J.K., Jenkin, G.A., Small, P.L.C., Jones, L.M., Tekaia, F., Laval, F., Daffé, M., Parkhill, J., Cole, S.T., 2007. Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans , the causative agent of Buruli ulcer 192–200. Stinear, T.P., Davies, J.K., Jenkin, G.A., Hayman, J.A., Oppedisano, F., Johnson, P.D.R., 2000a. Identification of Mycobacterium ulcerans in the environment from regions in southeast Australia in which it is endemic with sequence capture-PCR. Appl. Environ. Microbiol. 66, 3206–3213. Stinear, T.P., Jenkin, G. a., Johnson, P.D.R., Davies, J.K., 2000b. Comparative genetic analysis of mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence. J. Bacteriol. 182, 6322–6330. Stinear, T.P., Mve-Obiang, A., Small, P.L.C., Frigui, W., Pryor, M.J., Brosch, R., Jenkin, G. a, 97 University of Ghana http://ugspace.ug.edu.gh Johnson, P.D.R., Davies, J.K., Lee, R.E., Adusumilli, S., Garnier, T., Haydock, S.F., Leadlay, P.F., Cole, S.T., 2004. Giant plasmid-encoded polyketide synthases produce the macrolide toxin of Mycobacterium ulcerans. Proc. Natl. Acad. Sci. U. S. A. 101, 1345– 1349. Stotzky, G., 1986. Influence of soil mineral colloids on metabolic processes, growth, adhesion, and ecology of microbes and viruses. In: Huang PM, Schnitzer M, eds. Interactions of Soil Minerals with Natural Organics and Microbes. Wisconsin Soil Soc. Am. Inc. 305 – 427. Stragier, P., Ablordey, A., Durnez, L., Portaels, F., 2007. VNTR analysis differentiates Mycobacterium ulcerans and IS2404 positive mycobacteria. Syst. Appl. Microbiol. 30, 525–530. Thangaraj, H.S., Adjei, O., Allen, B.W., Portaels, F., Evans, M.R., Banerjee, D.K., Wansbrough- Jones., M.H., 2000. In vitro activity of ciprofloxacin, sparfloxacin, ofloxacin, amikacin and rifampicin against Ghanaian isolates of Mycobacterium ulcerans. J. Antimicrob. Chemother 45, 231 – 233. Thomas, B.S., Bailey, T.C., Bhatnagar, J., Ritter, J.M., Emery, B.D., Jassim, O.W., Hornstra, I.K., George, S.L., 2014. Mycobacterium Ulcerans Infection Imported from Australia to Missouri, USA, 2012. Emerg. Infect. Dis. 20, 1876–1879. Torrado, E., Fraga, A.G., Logarinho, E., Martins, T.G., Carmona, J. a, Gama, J.B., Carvalho, M. a, Proença, F., Castro, A.G., Pedrosa, J., 2010. IFN-gamma-dependent activation of macrophages during experimental infections by Mycobacterium ulcerans is impaired by the toxin mycolactone. J. Immunol. 184, 947–955. Uganda Buruli Group, 1970. Clinical features and treatment of pre-ulcerative Buruli lesions (Mycobacterium ulcerans infection). Report II. Br. Med. J. 2, 390–393. van der Werf, T.S., van der Graaf, W.T., Tappero, J.W., Asiedu, K., 1999. Mycobacterium ulcerans infection. Lancet 354, 1013–1018. Veitch, M.G., Johnson, P.D., Flood, P.E., Leslie, D.E., Street, a C., Hayman, J. a, 1997. A large localized outbreak of Mycobacterium ulcerans infection on a temperate southern Australian island. Epidemiol. Infect. 119, 313–318. Walsh, D.S., Meyers, W.M., Portaels, F., Lane, J.E., Service, D., Army, E., Gordon, F., 2005. High rates of apoptosis in human Mycobacterium ulcerans culture-positive Buruli ulcer skin lesions 73, 410–415. Walsh, D.S., Portaels, F., Meyers, W.M., 2011. Buruli ulcer: Advances in understanding mycobacterium ulcerans infection. Dermatol. Clin. 29, 1–8. doi:10.1016/j.det.2010.09.006 Wang, L., Li, L., Alam, M.J., Geng, Y., Li, Z., Yamasaki, S., Shi, L., 2008. Loop-mediated isothermal amplification method for rapid detection of the toxic dinoflagellate Alexandrium, which cause algal blooms and poisoning of shellfish. FEMS Microbiol Lett 282, 15–21. Wansbrough-Jones, M., Phillips, R., 2006. Buruli ulcer: emerging from obscurity. Lancet 367, 1849–1858. Wansbrough-Jones, M., Phillips, R., 2005. Buruli ulcer. Br. Med. J. 330, 1402–1403. Warden, V., Hurley, A.C., Volicer, L., 2003. Development and psychometric evaluation of the 98 University of Ghana http://ugspace.ug.edu.gh Pain Assessment in Advanced Dementia (PAINAD) scale. J. Am. Med. Dir. Assoc. 4, 9–15. Werf, T.S. Van Der, Stienstra, Y., Johnson, R.C., Phillips, R., Adjei, O., Fleischer, B., Wansbrough-jones, M.H., Johnson, P.D.R., Portaels, F., Graaf, W.T.A. Van Der, Asiedu, K., 2005. Public Health Reviews Mycobacterium ulcerans disease 020099, 785–791. WHO, 2012. Treatment of mycobacterium ulcerans disease (buruli ulcer): guidance for health workers. WHO, 2008. WHO Weekly Epidemiological Record no. 17 83, 145–156. WHO, 2007. Guidance on sampling techniques for laboratory-confirmation of Mycobacterium ulcerans infection ( Buruli ulcer disease ). WHO, 2004. Provisional guidance on the role of specific antibiotics in the management of Mycobacterium ulcerans disease (Buruli ulcer). (WHO/CDS/CPE/GBUI/2004). Geneva (Switzerland): World Health Organization. World Health. WHO, 2000. Buruli ulcer- diagnosis of Mycobacterium ulcerans disease. Geneva: World Health Organisation , Geneva, Switzerland. 92 p. Williams, L., Holland, M., Eberl, D. et al., 2004. Killer clays! Natural antibacterial clay minerals. Miner. Soc Bull 139, 3 – 8. Yeboah-Manu, D., Asante-Poku, A., Asan-Ampah, K., Ampadu, E.D.E., Pluschke, G., 2011. Combining PCR with microscopy to reduce costs of laboratory diagnosis of buruli ulcer. Am. J. Trop. Med. Hyg. 85, 900–904. Yeboah-Manu, D., Bodmer, T., Owusu, S., Ofori-adjei, D., Pluschke, G., Mensah-quainoo, E., 2004. Evaluation of Decontamination Methods and Growth Media for Primary Isolation of Mycobacterium ulcerans from Surgical Specimens Evaluation of Decontamination Methods and Growth Media for Primary Isolation of Mycobacterium ulcerans from Surgical Specimens 42, 5875–5877. Yeboah-manu, D., Peduzzi, E., Mensah-quainoo, E., Asante-poku, A., Ofori-adjei, D., Pluschke, G., Daubenberger, C.A., 2006. Systemic suppression of interferon-responses in Buruli ulcer patients resolves after surgical excision of the lesions caused by the extracellular pathogen Mycobacterium ulcerans 1150–1156. Yip, M.J., Porter, J.L., Fyfe, J. a M., Lavender, C.J., Portaels, F., Rhodes, M., Kator, H., Colorni, A., Jenkin, G. a., Stinear, T., 2007. Evolution of Mycobacterium ulcerans and other mycolactone-producing mycobacteria from a common Mycobacterium marinum progenitor. J. Bacteriol. 189, 2021–2029. Zhang, T., Li, S.-Y., Converse, P.J., Almeida, D. V, Grosset, J.H., Nuermberger, E.L., 2011. Using bioluminescence to monitor treatment response in real time in mice with Mycobacterium ulcerans infection. Antimicrob. Agents Chemother. 55, 56–61. 99 University of Ghana http://ugspace.ug.edu.gh Appendices Appendix A: BU data entry form BU04 Appendix B: Age and Sex distribution of 50 suspected case patients No. of case patients Age group (yrs) Male Female Total 0-9 6 7 13 10-19 11 6 17 20-29 1 3 4 30-39 3 2 5 40-49 1 3 4 50-59 0 5 5 60 → 0 2 2 Total 22 (44%) 28 (56%) 50 (100%) 100 University of Ghana http://ugspace.ug.edu.gh Appendix C: UV absorption data of three compounds obtained in MeOH on a Shimadzu UV mini-1240 spectrophotometer from 190 nm to 460 nm. λ nm Absorbance 4-nitrobenzaldehyde 2-naphthylboronic acid Mycolactone A/B 190 0.0259 -0.0127 0.5930 195 0.2068 0.0259 0.5461 200 1.6799 -0.0172 0.5120 205 0.1113 0.0692 0.8561 210 0.1522 0.1113 0.8444 215 2.0999 0.1522 0.9901 220 2.7372 0.1422 0.9406 225 3.0109 0.1245 1.2799 230 2.7994 0.1653 1.2799 235 3.4203 0.1692 1.1891 240 3.6892 0.2720 1.0614 245 3.9016 0.3217 1.0525 250 3.9210 0.4104 0.6954 255 3.4362 0.5190 0.5990 260 2.7999 1.0259 0.5390 265 2.7372 1.2068 0.4265 270 2.4362 2.9999 0.3043 275 2.4819 0.1113 0.2231 280 2.5154 0.1522 0.2316 285 2.3693 0.1422 0.2908 290 1.8341 0.1422 0.4198 295 1.2302 0.1245 0.4198 300 0.7650 0.0743 0.5962 305 0.4373 0.0695 0.4018 310 0.1245 0.0648 0.5701 315 0.1934 0.0603 0.5984 101 University of Ghana http://ugspace.ug.edu.gh 320 0.1422 0.0566 0.6018 325 0.2068 0.0516 0.7563 330 0.1357 0.0475 1.0018 335 0.2705 0.0436 1.7618 340 0.3256 0.0405 2.4941 345 0.4128 0.0350 2.3636 350 0.4258 0.0328 2.3420 355 0.4948 0.0310 2.8729 360 0.1113 0.1932 2.7274 365 0.1522 0.1556 2.8060 370 0.1422 0.1169 2.0566 375 0.1245 0.0890 2.0516 380 0.1934 0.0607 1.0475 385 0.1422 0.0404 0.9436 390 0.2068 0.0282 0.9005 395 0.1357 0.0371 0.6350 400 0.2705 0.0189 0.2328 405 0.3256 0.0087 0.1310 410 0.4128 0.0035 0.1291 415 0.4258 -0.0002 0.0283 420 0.2184 -0.0033 0.0275 425 0.0991 -0.0054 0.0273 430 0.0316 -0.0078 0.0269 435 0.0006 -0.0099 0.0266 440 -0.0132 -0.0122 0.0260 445 -0.0199 -0.0139 0.0250 450 -0.0229 -0.0151 0.0239 455 -0.0254 -0.0149 0.0228 460 -0.0272 -0.0160 0.0220 102 University of Ghana http://ugspace.ug.edu.gh Appendix D: TLC pictures of some of the 50 suspected Clinial samples. TLC-photos were taken and subjected to resolution enhancement (Adobe Photoshop CS 6) 103 University of Ghana http://ugspace.ug.edu.gh 104 University of Ghana http://ugspace.ug.edu.gh 105 University of Ghana http://ugspace.ug.edu.gh 106 University of Ghana http://ugspace.ug.edu.gh Appendix E: TLC pictures of M. ulcerans Infected SPF BALB/c mice taken weekly with a control sample. TLC-photos were taken and subjected to resolution enhancement (Adobe Photoshop CS 6). 107