Browsing by Author "Amewu, R.K"
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Item Alternative boronic acids in the detection of Mycolactone A/B using the thin layer chromatography (f-TLC) method for diagnosis of Buruli ulcer(BMC Infectious Diseases, 2023) Akolgo, G.A.; Partridge, B.M.; Craggs, T.D.; Amewu, R.KBackground Mycobacterium ulcerans is the causative agent of Buruli ulcer. The pathology of M. ulcerans disease has been attributed to the secretion of a potent macrolide cytotoxin known as mycolactone which plays an important role in the virulence of the disease. Mycolactone is a biomarker for the diagnosis of BU that can be detected using the fuorescent-thin layer chromatography (f-TLC) technique. The technique relies on the chemical derivatization of mycolactone A/B with 2-naphthylboronic acid (BA) which acts as a fuorogenic chemosensor. However, back ground interferences due to co-extracted human tissue lipids, especially with clinical samples coupled with the subjectivity of the method call for an investigation to fnd an alternative to BA. Methods Twenty-six commercially available arylboronic acids were initially screened as alternatives to BA using the f-TLC experiment. UV–vis measurements were also conducted to determine the absorption maximum spectra of mycolactone A/B and myco-boronic acid adducts followed by an investigation of the fuorescence-enhancing ability of the boronate ester formation between mycolactone A/B and our three most promising boronic acids (BA15, BA18, and BA21). LC–MS technique was employed to confrm the adduct formation between mycolactone and boronic acids. Furthermore, a comparative study was conducted between BA18 and BA using 6 Polymerase Chain Reaction (PCR) confrmed BU patient samples. Results Three of the boronic acids (BA15, BA18, and BA21) produced fuorescent band intensities superior to BA. Complexation studies conducted on thin layer chromatography (TLC) using 0.1 M solution of the three boronic acids and various volumes of 10 ng/µL of synthetic mycolactone ranging from 1 µL – 9 µL corresponding to 10 ng – 90 ng gave similar results with myco-BA18 adduct emerging with the most visibly intense fuorescence bands. UV–vis absorption maxima (λmax) for the free mycolactone A/B was observed at 362 nm, and the values for the adducts myco-BA15, myco-BA18, and myco-BA21 were at 272 nm, 270 nm, and 286 nm respectively. The comparable experi mental λmax of 362 nm for mycolactone A/B to the calculated Woodward-Fieser value of 367 nm for the fatty acid side chain of mycolactone A/B demonstrate that even though 2 cyclic boronates were formed, only the boronate of the southern side chain with the chromophore was excited by irradiation at 365 nm. Fluorescence experiments have demonstrated that coupling BA18 to mycolactone A/B along the 1,3-diols remarkably enhanced the fuores cence intensity at 537 nm. High-Resolution Mass Spectrometer (HR-MS) was used to confrm the formation of the myco-BA15 adduct. Finally, f-TLC analysis of patient samples with BA18 gave improved BA18-adduct intensities compared to the original BA-adduct. Conclusion Twenty-six commercially available boronic acids were investigated as alternatives to BA, used in the f-TLC analysis for the diagnosis of BU. Three (3) of them BA15, BA18, and BA21 gave superior fuorescence band intensity profles. They gave profles that were easier to interpret after the myco-boronic acid adduct formation and in experi ments with clinical samples from patients with BA18 the best. BA18, therefore, has been identifed as a potential alternative to BA and could provide a solution to the challenge of background interference of co-extracted human tissue lipids from clinical samples currently associated with the use of BA.Item Evaluation of the fluorescent-thin layer chromatography (f-TLC) for the diagnosis of Buruli ulcer disease in Ghana(PLOS ONE, 2022) Amewu, R.K; Akolgo, G.A; Asare, M.E; Abdulai, Z; Ablordey, A.S; Asiedu, KAbstract Background Buruli ulcer is a tissue necrosis infection caused by an environmental mycobacterium called Mycobacterium ulcerans (MU). The disease is most prevalent in rural areas with the highest rates in West and Central African countries. The bacterium produces a toxin called mycolactone which can lead to the destruction of the skin, resulting in incapacitating deformities with an enormous economic and social burden on patients and their caregivers. Even though there is an effective antibiotic treatment for BU, the control and management rely on early case detection and rapid diagnosis to avert morbidities. The diagnosis of Mycobacterium ulcerans relies on smear microscopy, culture histopathology, and PCR. Unfortunately, all the current laboratory diagnostics have various limitations and are not available in endemic communities. Consequently, there is a need for a rapid diagnostic tool for use at the community health centre level to enable diagnosis and confirmation of suspected cases for early treatment. The present study corroborated the diagnostic performance and utility of fluorescent- thin layer chromatography (f-TLC) for the diagnosis of Buruli ulcer. Methodology/Principal findings The f-TLC method was evaluated for the diagnosis of Buruli ulcer in larger clinical samples than previously reported in an earlier preliminary study Wadagni et al. (2015). A total of 449 patients suspected of BU were included in the final data analysis out of which 122 (27.2%) were positive by f-TLC and 128 (28.5%) by PCR. Using a composite reference method generated from the two diagnostic methods, 85 (18.9%) patients were found to be truly infected with M. ulcerans, 284 (63.3%) were uninfected, while 80 (17.8%) were misidentified as infected or noninfected by the two methods. The data obtained was used to determine the discriminatory accuracy of the f-TLC against the gold standard IS2404 PCR through the analysis of its sensitivity, specificity, positive (+LR), and negative (–LR) likelihood ratio. The positive (PPV) and negative (NPV) predictive values, area under the receiver operating characteristic curve Azevedo et al. (2014), and diagnostic odds ratio were used to assessthe predictive accuracy of the f-TLC method. The sensitivity of f-TLC was 66.4% (85/128), specificity was 88.5% (284/321), while the diagnostic accuracy was 82.2% (369/449). The AUC stood at 0.774 while the PPV, NPV, +LR, and–LR were 69.7% (85/122), 86.9% (284/ 327), 5.76, and 0.38, respectively. The use of the rule-of-thumb interpretation of diagnostic tests suggests that the method is good for use as a diagnostic tool. Conclusions/Significance Larger clinical samples than previously reported had been used to evaluate the f-TLC method for the diagnosis of Buruli ulcer. A sensitivity of 66.4%, a specificity of 88.5%, and diagnostic accuracy of 82.2% were obtained. The method is good for diagnosis and will help in making early clinical decisions about the patients as well as patient management and facilitating treatment decisions. However, it requires a slight modification to address the challenge of background interference and lack of automatic readout to become an excellent diagnostic tool. Introduction